Training Manual

Training Manual
BioTracking LLC
1150 Alturas Drive, Suite 105
Moscow, Idaho 83843
Phone:
208-882-9736
Fax:
208-882-1490
Email: [email protected]
Website: www.biotracking.com
Section 1 – What are BioPRYN and PSPB?
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INTRODUCTION
This manual will teach you to perform the BioPRYN test in your own laboratory. It is
intended to be specific to the BioPRYN enzyme-linked immunosorbent assay (ELISA)
and not for other types of ELISA tests. You will be instructed in care of test materials
and samples that enter your facility and specific and precise means for obtaining a correct
test result. It is imperative that every sample be handled with care, assigned to the correct
position in test wells, recorded correctly for use by the owner/manager and the laboratory
and that results are reported in a timely manner without error. Technicians must become
proficient in laboratory procedures such that careless and inappropriate mistakes do not
occur.
BioTracking LLC sells tests for pregnancy under the trade name of BioPRYN®. It is a
sandwich ELISA test. More information can be found at www.biotracking.com. This is
a test that is conducted in the laboratory and not on the farm. Managers of animals are
required to send samples by mail or other currier to the laboratory.
History of PSPB and Similar Proteins
The BioPRYN test detects the presence of pregnancy-specific protein B (PSPB) in the
blood of pregnant animals. PSPB is an antigenic protein fraction specific to, and isolated
from, the placenta (at any point in placental development) that can be physically and/ or
biochemically separated (eg. Gel filtration chromatography, isoelectric focusing) from α
Fetoprotein (also called Pregnancy Specific Protein A). The procedure for purifying
PSPB and producing antibodies specific to the placenta using this preparation was
originally published in 1982 (Butler et al., 1982). PSPB is a complex mixture composed
of a number of glycosylated and non-glycosylated proteins. Antibodies raised against
PSPB recognize antigenic epitopes specific to placental tissue. These epitopes include
molecules of various molecular weights and isoelectric points (Sasser and Ruder, 1987;
Sasser et al., 1989). Some of these molecules can be found in the peripheral circulation
of pregnant animals and used for pregnancy detection (Sasser et al, 1986). Due to the
presence of Pregnancy Associated Glycoproteins (PAGs) in placental tissue, the PSPB
fraction is made up of a number PAGs. The presence, concentration and relative
abundance of PAGs in the PSPB fraction is likely dependent on the gestational age of the
placenta used for preparation due to differential expression of PAGs (Green et al., 2000).
During certain gestational ages PSPB contains PAG-1 (Lynch et al., 1992), but PSPB is
not specifically PAG-1 and may not contain PAG-1 depending on the time of placental
harvesting.
To summarize: PSPB is a mixture of several proteins in the aspartic acid protease family
of proteins from the placenta. The PSPB family of proteins has a variety of differing
amino acid sequences and post-translational modifications resulting in differences in
molecular weight and iso-electric properties of each member of the family. This proteinisolate was found in the early 1980’s by R. Garth Sasser while doing research at the
University of Idaho. It was shown to be useful as a marker for pregnancy in cattle and
deer and two publications were presented to this effect in 1986. This was the first assay
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for pregnancy in cattle and deer based upon blood analysis. The anti-PSPB sera will
cross-react with forms of PSPB in a number of ruminant animals. However, for accurate
categorization of pregnancy status it is important that the correct BioPRYN test is chosen
for the application (BioPRYN for cattle, BioPRYN for bison and buffalo, BioPRYN for
goats and sheep, BioPRYNWILD for ruminants such as elk, deer and moose). BioPRYN
for cattle and BioPRYN for sheep and goats are currently available in kit form for use onsite by our affiliate labs. Special pricing of the other tests allows the affiliate labs to
access the particular market segment while utilizing the BioTracking lab services.
The BioPRYN test is based on PSPB forms isolated by Sasser and thus is based on the
original placental aspartic acid protease fraction. Two proteins with biochemical,
immunological and molecular similarities to the original PSPB have been isolated after
direct work with the Sasser laboratory and subsequently given different names by the
founding groups. They were named pregnancy-specific protein 60 (PSP60) and
pregnancy associated glycoprotein (PAG).
A patent was obtained by the University of Idaho for use of PSPB in detection of
pregnancy in ruminant animals (Sasser and Hamilton, 1987). The PSPB protein
preparation had several molecular weight and isoelectric variants of proteins (Sasser and
Ruder, 1987 & Sasser et al., 1989). Thus the polyclonal antiserum for detection of
pregnancy (Sasser et al., 1996) was against many of these variants rather than just one
protein.
Tests based upon two other protein preparations have been described by European
laboratories. Both were isolated following published protocols used for the identification
of PSPB and are also members of the aspartic acid protease family of proteins. One
(PAG) was reported by Zoli et al. (1991) in Belgium after discussions and presentations
to this lab by Sasser and was subsequently shown by Roberts et al. (University of
Missouri) to contain a specific protein (PAG-1) with the identical nucleotide sequence to
a form of PSPB. The other preparation was pregnancy serum protein 60, PSP60, and was
isolated in France with the collaboration of Sasser (sabbatical leave) leading to a joint
publication (Camous et al., 1988). It is offered to breeders as a test for pregnancy. Both
have been developed as a radioimmunoassay and as ELISA tests. The PAG test is said to
be offered commercially in Germany.
The University of Missouri gene clone for PSPB was named PAG-1. This clone (Xie et
al., 1991) was exactly the same as the clone we identified for PSPB (Lynch et al. 1992).
The University of Missouri has a monoclonal ELISA assay for PAG.
The Assay
The NEW BioPRYN ELISA is recommended for detection of pregnancy in cattle at or
greater than 28 days post breeding and 73 days since last calf. The assay can be
completed in a minimum assay time of 6.5 hours, but is most often completed by
applying samples on day 1 and reporting the results in the morning/ early afternoon of
day 2. This antigen-capture, or “sandwich,” Enzyme-Linked Immuno Sorbent Assay
(ELISA) detects Pregnancy Specific Protein B (PSPB) in bovine sera/ plasma. Serum
Page | 3
PSPB binds to antibodies coated in the wells and is detected by secondary binding of a
labeled antibody. Binding of the labeled antibody con u ate is detected by the addition
of the Enhancer and , , , -tetramethylbenzidine (TMB) and is quantified by the
subsequent color development. A strong color indicates binding and substrate reactivity
of the labeled antibody conjugate to the bound PSPB, and is a positive indication of
pregnancy. Weak color development indicates little or no binding of the labeled antibody
conjugate due to the absence of PSPB in the sample, and is a negative indication of
pregnancy. Standards of known concentrations of PSPB are run with each assay and are
used to determine the optical density values for assigning Pregnant / Not Pregnant ranges.
The BioPRYNQK ELISA is recommended for detection of pregnancy in cattle at or
greater than 28 days post breeding and 90 days since last calf. This assay can be
completed with a minimum assay time of 2.5 hours.
The assay is a “sandwich ELISA” test. The test is performed in wells of a polystyrene
micro-titer plate containing 96 test wells. The wells are coated with antiserum to PSPB.
The blood-serum/-plasma sample is added to the test well with Sample Buffer (#1) and
any PSPB present will bind to the antibody and will be sequestered from the serum. For
BioPRYN, the recommended incubation time is overnight (minimum of 4 hours) at room
temperature. For BioPRYNQK , the incubation time is one hour at room temperature. The
sample is washed from the well leaving either the bound antibody-PSPB complex, or
unbound antibody.
Sample with PSPB
Sample without PSPB
Page | 4
Next, buffer containing an antibody (Detector #2) is added and incubated for one hour at
room temperature. During this time, the second antibody, also against PSPB, will bind to
free epitopes on the previously sequestered PSPB. The plate wells are washed again
leaving PSPB sandwiched between two antibodies. If no PSPB is present, the second
antibody conjugate cannot bind.
A horseradish peroxidase enzyme (HRP) labeled molecule that recognizes the second
antibody (Enhancer #3) is then added and incubated for one hour. During this time, the
Enhancer will bind to second antibody on the previously sequestered PSPB. The plate
wells are washed again leaving PSPB sandwiched between two antibodies and the
attached HRP. If no PSPB is present, the Enhancer cannot bind.
Finally, the TMB Substrate (#4) is added to the well and incubated for 15 minutes. The
substrate is stabilized tetramethyl benzidine or TMB. The intensity of the blue color is
dependent on the amount of HRP present which, in turn, is dependent on the amount of
PSPB in the sample. After 15 minutes Stop Solution (#5) is added to the well to stop the
reaction and the plate is read at 650 nm.
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Below is a completed plate using the BioPRYN assay. Each round well contains an
individual standard or sample. The first column contains the standards for determining
pregnancy status. The intensity of blue color reflects the amount of PSPB in each sample
with a darker color indicating a higher level of PSPB.
Website for video of reaction of a sandwich ELISA http://www.sumanasinc.com/webcontent/animations/content/ELISA.html
Below is a figure that demonstrates the expected differences in BioPRYN OD across
bovine estation. These data are based on the “avera e” cow. Due to physiolo ical
differences between cows, it is not possible to predict calving dates with a high level of
accuracy based on the OD. However, it is possible to identify an abnormal OD for a
given stage in gestation that could be useful for management decisions.
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Reason for Care in Testing
Testing for presence of PSPB with the BioPRYN test for pregnancy must be precise and
correct. If errors occur, it can lead to extreme expenses for both the laboratory and
livestock owner. Correct assignment of samples by computer and matching placement
into test plate wells is critical. We take extreme care during BioPRYN training to help
assure that mistakes do not happen. If a series of samples is placed just one position in
error, all or many cows in that series will be mistakenly identified for pregnancy status.
One of the biggest potential problems is to place a pregnant cow into the OPEN category.
With intense reproductive management of cows, such as in dairy or embryo transfer
programs, open cows are enrolled into re-synchronization programs to induce immediate
heat and ovulation and subsequent re-breeding or embryo transfer. The hormones used,
specifically prostaglandin F2 alpha, will cause embryo death/ resorption or fetal abortion.
If loss occurs early near 30 to 40 days, fetal membranes may be observed to attest to loss.
If death occurs after 40 days, aborted fetuses are readily observed. Loss at this time is
expensive for two reasons.
1. It takes 84 days to reestablish a pregnancy in well managed herds with a
pregnancy rate (PR) of 22%. In less well managed herds more days are
required (PR 14% = 137 days). At a cost of $2.50 per day that a cow is not
pregnant after 90 days in milk, as these cows would be, embryo or fetal loss
would cost $210 or $ 4 at PR’s of 22 or 14% respectively. Expense of
semen, hormones and labor are additional. If a veterinary procedure resulted
in loss of 5% of the conceptuses, in a 1000 cow herd that would be 50
conceptuses and at the above PR%, loss would be from $10,500 to $17,150. It
is imperative to not have losses due to BioPRYN.
Cost of a lost embryo/fetus in days open at herd pregnancy rates (PR) from 14% to
30%. Cows have the opportunity to rebreed each 21 days.
PR %
14
18
22
26
30
Cumulative Days to 98%
conception for 100 cows
OPEN Days/Cow
to 98% conception
Total $Cost/Cow
at $2.50/Day Open
546
378
336
273
231
137
103
84
69
58
$343
$258
$210
$173
$145
2. Semen and embryos can be obtained from very valuable animals at high
expense. Incorrect assignment of pregnancy status can lead to loss of
embryos/fetuses. Some have a value exceeding $5,000 to $10,000. Losses should
not be a result of careless management of the BioPRYN test.
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Personnel For BioPRYN:
Other examples of where mistakes can occur in administering the BioPRYN test can
surely be cited. It is imperative that personnel understand the need for care and precision.
Those incapable of paying attention to detail should be excused from running this test.
Recognize that some people competent in other ways cannot do this work.
Accuracy of BioPRYN
The BioPRYN test is very accurate. Although ultrasonography can equal the ELISA test
in accuracy, it requires a very skilled operator to obtain consistent and reliable results, in
addition to being a time-consuming procedure.
a.
If a cow has serum PSPB concentration that falls into the category of
cows that are not pregnant (open) when test at 28 days post breeding or
later, the test is over 99% correct in placement into this category.
Rarely is a pregnant cow placed incorrectly into this category. The
breeder is safe to inject estrous or ovulation recycling hormones without
fear of aborting an embryo. One of these hormones is prostaglandin F2
Alpha and it will cause luteolysis (death of the corpus luteum on the
ovary and a drop in progesterone) and will induce abortion. Serum
progesterone is needed to maintain pregnancy and when it is gone,
abortion occurs.
b.
If a cow has serum PSPB concentration that falls into the category of
cows that are pregnant, the test is 91 to 95% correct in placement into
that category. Some cows placed here will be open at the time of follow
up testing. This is most likely due to the high rate of embryonic death
between 30 and 45 days of gestation. In many cases BioPRYN was
correct at the time of sampling but following embryo death and the
clearance of PSPB from the serum, the follow-up call determined the
animal to be open.
c.
The following Table 1 shows results of testing for PSPB in cattle that
were slaughtered. Blood and uteri were collected at slaughter. Uteri
were examined for presence of an embryo/fetus and blood was tested
for PSPB by a radioimmunoassay. Uterine examination is the only
precise way to be certain that the cow is pregnant and is the very best
way to evaluate another method of testing for accuracy. Two
diagnostic terms are sensitivity and specificity. The PSPB test had 99%
sensitivity, showing that rarely was a pregnant cow placed into the open
category. The specificity was 94.7% showing that some open cows
were placed into the pregnant category. Some cows had dead embryos
and they were listed as open. The assay detected residual PSPB and
placed them into the pregnant category.
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Accuracy of Blood Test for PSPB
28 to 60 Days After Breeding
378 Cattle Examined at Slaughter
--UTERINE --
--BLOOD --
Status
Number
Number
% of
uterine
OPEN
187
177
94.7
(Specificity)
PREGNANT
191
189
99
(Sensitivity)
Below is an accuracy table taken from a poster presented at the 2011 ADSA conference
(Branen et al. 2011 ADSA conference poster #M63). Over 900 dairy cows from 10
different farms in California were used for the study. Sensitivity and specificity were
determined by categorizing the samples at T1 (26-30 days since last heat (DSLH)) and
comparing that result with true value determined at 7-10 days later (T2) by matching
rectal palpation and BioPRYN result. BioPRYN showed very high sensitivity as early as
26 DSLH with 100% detection of pregnancies by 28 DSLH. The specificity which is
slightly lower than the slaughter study from above can be directly attributed to embryonic
loss occurring at or near the T1 sampling.
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The Physiology of PSPB
PSPB is produced in the membranes of the embryo/fetus. The accompanying picture
shows a 32 day old bovine embryo within the membranes. A micrograph of placental
tissue shows the binucleated cells and the red stain within the cytoplasm is specific for
PSPB. Thus it is shown that PSPB is synthesized by these cells. The binucleated cells
are migratory and move to the surface epithelium of the uterus. Once there, the cells
deposit the PSPB into tissues and subsequently enters the circulation of the mother.
Binucleated cells begin to be formed during the third week of gestation. Although some
heifers had PSPB in the circulation as early as 15 days after AI, research shows that in
order to detect PSPB in all pregnant animals one must wait until 28 days after AI. Below
is a graph that shows the increase of PSPB (as measured by BioPRYN OD) in the
“avera e” cow between 1 -50 days post breeding. Most cows show an increase in PSPB
between 20-24 days post breeding and achieve a level of PSPB that allows for highly
accurate categorization by 28 days post breeding.
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Residual PSPB after Calving
The next graph shows the decay in amount of PSPB (as measured by BioPRYN OD) in
the circulation of cows after calving. The half-life of PSPB is between 4-7 days.
Because of this rate of clearance of PSPB from serum, it takes approximately 73 days
after calving for PSPB to be non-detectable by BioPRYN and 90 days post calving if you
are using BioPRYNQK. This is rarely a problem when testing for a new pregnancy. If a
cow is inseminated at 45 days postpartum and PSPB testing occurred 28 days later, she
will be at 73 days postpartum and residual PSPB will not contribute to the test result.
Some cattle are bred after a 60 day voluntary waiting period (VWP) while others have
shorter periods. Most ovulation synchronization programs have breeding after 70 days
postpartum and of course this will pose no problem for testing since testing will occur
after 100 days postpartum.
Residual PSPB after Embryo Death
If an embryo dies when PSPB concentration is low in serum, such as during days 30 to 75
after AI, the residual PSPB is below the Pregnant category threshold after 3 to 7 days.
Even though the half-life has not changed, the level was low enough to be gone within
this time period. Of course as concentration increases as it does from 80 days until term
if fetal death occurs, it will take longer for residual PSPB to decline to non detectable
levels. However, in dairy operations, most of the testing is done within the 45 days after
AI and thus residual PSPB will be gone such that a sample taken one week after death
will have non detectable amounts of PSPB. This allows indication of fetal death when a
decrease in BioPRYN signal is measured during the 30 to 75 day window.
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Below is a figure that shows the BioPRYN OD for normal gestation between 15 and 50
days post breeding and the modeled change in BioPRYN OD with an embryonic loss at
28 days post breeding. The second figure shows modeled pregnancy loss at 120 days
post breeding.
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References
Butler JE, Hamilton WC, Sasser RG, Ruder CA, Hass GM, Williams RJ: Detection and
partial characterization of two bovine pregnancy-specific proteins. Biol Reprod 26:925933, 1982.
Camous S, Sharpigny G, Guillomot M, Martal J, Sasser RG: Purification of one bovine
pregnancy-specific protein by high performance liquid chromatography (HPLC). Proc.
Bard Workshop. Maternal Recognition of Pregnancy and Maintenance of the Corpus
Luteum, Jerusalem, Abstract 2, 1988.
Lynch RA, Alexander BM, Sasser RG: The cloning and expression of the pregnancyspecific protein B (bPSPB) Gene. Biol Reprod 46(Suppl. 1):72, 1992.
Sasser RG, Crock J., Ruder-Montgomery CA: Characteristics of pregnancy-specific
protein B in Cattle. J Reprod Fertil 37:109-113 (Suppl.), 1989.
Sasser RG, Ruder CA: Detection of early pregnancy in domestic ruminants. J Reprod
Fertil 34:261-271, 1987.
Sasser RG, Ruder CA, Ivani KA, Butler JE, Hamilton WC: Detection of pregnancy by
radioimmunoassay of a novel pregnancy-specific protein in serum of cows and a profile
of serum concentrations during gestation. Biol Reprod 35:936-942, 1986.
Sasser, RG, Hamilton WC: U.S. Pat. No. 4,554,256. Novel antigen associated with
early detection of mammalian pregnancy. November 19, 1985.
Sasser RG, Hamilton WC: U.S. Pat. No. 4,705,748. Antigen associated with early
detection of mammalian pregnancy. A continuation of U.S. Pat. No. 4,554,256,
November 10, 1987.
Xie S, Low BG, Nagel RJ, Kramer KK, Anthony RV, Zoli AP, Beckers J-F, Roberts RM:
Identification of the major pregnancy-specific antigens of cattle and sheep as inactive
members of the aspartic proteinase family. Proc Natl Acad Sci USA 88:10247-10251,
1991.
Zoli AP, Beckers J-F, Wouters-Ballman P, Closset J, Falmagne P, Ectors F: Purification
and characterization of a bovine pregnancy-associated glycoprotein. Biol Reprod 45: 110, 1991.
Page | 13
Section 2 –Objectives of Training and
Quality Expectations
Page | 14
INTRODUCTION
The BioTracking training team has been designed to support and promote the mission,
values, and company skills and techniques to the affiliate laboratory personnel. Our
desire is to provide the best possible service to the cattle industry in blood pregnancy
detection. BioTracking highly values quality and repeatability of results. We strive for
these goals in our own laboratory, and expect you will want to do the same in your
laboratory. The following Purpose and Objectives and Quality Control Guidelines will
help you in achieving an understanding of our expectations and achieve your own quality
goals.
Purpose and Objectives
BioTracking training equips and enables affiliate laboratory personnel to do the
following:










Describe the importance of the laboratory ELISA test in detecting pregnancy in
cattle.
Understand how the ELISA test works and the importance of ensuring an accurate
test is provided to the client.
Perform accurate and reliable laboratory procedures for the ELISA cattle
pregnancy test.
o Unpack and properly store BioPRYN kits when they arrive from
BioTracking
o Use and calibration of required laboratory equipment for ELISA testing
o Mixing and / or storage of reagents.
o Sample intake procedures – unpacking, sample preparation, and log-in
o ELISA plate loading – pipetting technique, reagents, QC – double checks
o ELISA plate washing
o Experience with all ELISA techniques
o Sample preparation for plate reading to include TMB step
o Analyzing output data and preparing the client report
o Reporting of data – phone, fax, email, mail
Describe the importance of Quality Control (QC) and interpret quality control
data.
Evaluate the QC data to improve laboratory performance.
Describe approaches for monitoring and measuring test performance.
Use of BioTracking software programs.
Follow the laboratory safety precautions.
Identify and know how to obtain equipment and supplies required for the ELISA
cattle test.
Develop and implement an effective marketing strategy for their laboratory.
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External Quality Control Guidelines
The purpose of the BioTracking External Quality Control (EQC) is to determine the
proficiency and accuracy of affiliate laboratories currently using the BioPRYN® assay for
cattle pregnancy detection. This will be accomplished by assaying a variety of samples
provided by BioTracking LLC and comparing the results obtained by the affiliate
laboratory with those of the corporate laboratory. All reported results will be confidential
between BioTracking, LLC and each affiliate laboratory.
Objectives of Quality Control
1.
To provide a measure of the accuracy performing the BioPRYN® test.
2.
To give confidence and reliability to analytical results and to aid in the
detection of laboratory techniques which may lead to inaccurate results.
3.
To provide a measure of the quality of performance of individual affiliate
laboratories.
4.
To encourage the adoption of internal Quality Control procedures by each
affiliate laboratory, or to supplement such procedures already in place.
5.
To provide a method for affiliate laboratories to assure their clientele that the
laboratory personnel are competent in performing the BioPRYN® assay.
6.
To ensure that all producers are receiving accurate and consistent BioPRYN®
results from any affiliate laboratory.
7.
To act as an educational stimulus to strive for continued excellence in
performance.
Advantages and Limitations
1. Since the affiliate laboratories do not know the composition of the EQC samples
the results should provide an objective measure of performance. As with all
quality control methods, affiliate laboratories must not give the EQC samples
special treatment.
2. EQC is not a substitute for internal quality control because testing is too
infrequent. Because EQC is performed once or twice per year, the laboratory
should not use exclusively the results of the EQC testing to calibrate equipment or
to evaluate laboratory protocols.
Page | 16
Requirements
1. In order for the EQC procedures to achieve the intended goal, all affiliate
laboratories must participate.
2. All results of the EQC procedure will be confidential and not published with any
laboratory identity. All results and recommendations will be shared individually
with each laboratory as needed.
3. The scientific basis of an EQC scheme, and all the methods for analyzing the
results, must be made clear to all participants, so the affiliate laboratories have
confidence in the results. This is required so laboratories can compare and
evaluate the EQC results with the results from their internal quality control.
4. The EQC results must be returned in a timely manner as to reflect the
performance level of the affiliate laboratory, and what adjustments to procedures,
if any, must be made.
External Quality Control (EQC) Process
Each affiliate laboratory will be required to provide an annual update outlining changes
in personnel, physical facilities, or equipment that could affect the reliability and
accuracy of the BioPRYN® pregnancy test.
Steps of the EQC process:

The affiliate laboratory manager/supervisor will receive the quality control
procedures and the rationale for using these procedures.

The affiliate laboratory shall complete an external quality control procedure that
will consist of all affiliate laboratories analyzing the same unknown samples.

The affiliate laboratory shall document proficiency in the performance of the
analysis of the unknown samples on an ongoing basis. Assessment of proficiency
shall be based on objective data, using blind samples of appropriate number and
composition (PSPB standards, and serum from open, pregnant, and repeat status
samples). These samples should be well defined.
Page | 17

The quality control procedure shall contain descriptive information such that an
experienced technician can perform the test without reference to other information
sources. It shall include:
a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
l.
A description of checks and double checks that are used to ensure
that a sample submitted to the laboratory is the same sample in the
plate well that is reported to the producer. This could be a
description of the process and/or a sample of the form(s) used.
A description of reagents and quantities clearly presented
A list and description of use for those reagents that must be
supplied by the affiliate laboratory
Samples of mixed pregnancy status or PSPB concentrations
8 standards
Any safety considerations
A list of, and specifications for, equipment, materials, and
reagents, including software
Conditions for acceptance of blood serum as fit for testing
Conditions for sample identification, collection, handling,
transportation and storage
Conditions for sample preparation
A description of the control standards and their acceptance limits
Checks to be made prior to beginning the assay (i.e. equipment
checks and calibrations)
EQC Classification of Affiliate Laboratories
There will be two types of affiliate laboratory EQC classification:

Meets or exceeds standards
This classification indicates an affiliate laboratory is capable of meeting
the essential requirements and guidelines established for an affiliate
laboratory.

Does not meet standards
This classification indicates that an affiliate laboratory does not meet the
essential requirements and guidelines.
The statistical results will be confidential between BioTracking, LLC and each affiliate
laboratory. If a lab does not meet standards, the affiliate lab will contact BioTracking
for further training. Upon completion of additional training the affiliate lab will have an
opportunity to be re-evaluated for a meets or exceeds standards classification.
Page | 18
Section 3 – Intake of Samples
Page | 19
INTRODUCTION
Intake of samples is the first step in the BioPRYN test. Once your lab begins receiving
samples, it is necessary to have a good system of entering customer information and
keeping track of samples. The following is the method used at BioTracking.
Method of Intake
1. Unpack all boxes/packages, checking to make sure the customer has provided all
the necessary information (address, etc.) and that all of the tubes are present.
2. If the customer sent a check, indicate that on the submittal form in the allotted
space in red pen. Attach the check to the submittal form.
3. Assi n a “lo number” to each set of samples, numbering the submittal form and
the first and last sample of each group. Also write this log number on the memo
portion of the check.
4. Load all samples in numerical order (and by log number) in the centrifuge racks
and centrifuge at 1200-1500 RPMs for five minutes in the centrifuge.
5. Unload the centrifuged samples and arrange
them in order. At BioTracking, we use a box
divided into 10x10 squares, organizing the first
10 rows of eight, and the 11th row
perpendicular to the other 10. See picture to
the right.
6. The sample number on each tube is now ready to be checked (to assure positions
match) with the printed plate grid. After verifying the numbers, samples are
ready to be loaded into the plate.
Tips/ Troubleshooting for Sample Intake

Make sure that all of the customer’s information is completed on a submittal form
before throwing away their box/packaging. This could save time when logging
in.

Producers will sometimes send an extra tube, or a tube that is not indicated on
their submittal form. It is best to call the customer in this situation to determine
how to proceed.
Page | 20

If there are two tubes labeled with the same animal ID, verify the numbers with
the submittal form and check for tube numbers, if present. Call the customer
when in doubt.

If there is a tube with very little sample, spin the sample anyway. A decision can
then be made whether to add sample buffer to a tube that has little to no serum
after spinnin . Be sure to add a note to a customer’s manifest indicatin a
minimal sample. If a minimal sample scores as open on a report, the customer
should resubmit that sample for an accurate test. If the minimal sample scores
pregnant, then no further testing is necessary.
Page | 21
Section 4 – Preparation for Performing the
BioPRYN Test and the Creation of
Manifests and Loading Grids
Page | 22
INTRODUCTION
As your client base grows, the number of samples and plates you process per day will
grow as well. In order to store, manage, and find reports, manifests, and plate readings it
is important to set up a file system that will allow you to track your client information.
This section will give you an overview of preparing for running the BioPRYN test
electronically and train you how to set up and utilize BioTrackin ’s electronic pro ram
for calculating results, CowPIE. This section will also give some guidelines on the
process used at BioTracking to manage these data files.
Start by setting up a BioPRYN folder on your server or hard drive. This will be used to
initially store all of the information, files, and utilities on the CD you received at the
training program.
Setting Up the Working Folders
To begin, open up your hard drive by left-clicking on Start on the taskbar
then left-click on My Computer and double left-click on Local Disc C: to open your C
drive.
Page | 23
If you are storing your BioPRYN data on a network server you would double-click on the
applicable network drive. See your system administrator if you are not sure which drive
to use.
These instructions will use the C-drive for illustration, but the process will be the same if
you are storing your data on a server. Once you have opened your hard drive, left-click
on the File tab (located at the top left of your screen), select New, then left-click on
Folder. Your screen should look like this:
While the New Folder is still highlighted, you can rename it to BioPRYN by typing
BioPRYN. This will be the folder into which all of your subsequent files will be stored.
Open the BioPRYN folder and, using the same procedure as used for creating the
BioPRYN folder, create and name two additional folders: Blank Client Manifests and
Page | 24
Cattle Client Logs. These will be the initial folders you will be using. At this time you
can also transfer all of the files from the BioTracking CD into the BioPRYN folder.
If you are running other test such as BVD, goat and sheep pregnancy, CAE testing etc,
you may want to set up individual folders for these assays as well. When you are
finished, your screen should look like this:
Transfer the Blank Farm Manifest.csv file on your CD into the Blank Client Manifests
folder. If you put a ` in front of the name (`Blank Farm Manifest) it will always be at the
top of the file list.
As indicated by the name of the file, it is important you do not change the format of the
file in any way (except to add client information and sample data). We suggest you make
a back-up copy of this file in case you are unable to recover from a serious error. You
can do this by right-clicking on the file, left-click on Copy, right-click to open a selection
window, then left-click on Paste. Your window should look like this:
Page | 25
You can now use the Blank Manifest template file to create all of the Blank Client
Manifests you will use in running the BioPRYN pregnancy detection system. This
process is covered in the Creating Client Manifests section of this manual.
Daily Folders
Your day-to-day working folders containing completed Client Manifests, Grid sheets and
reports will be stored in the daily folders located in the Cattle Client Logs main folder.
At BioTracking, we use the day the test is started to name the daily folder. For example,
if samples arrive on May 5, 2008, the client logs, grids, plate readings and report folder
would be kept in the 5 May 2008 folder located in the May 2008 folder which in turn is
located in the main 2008 Folder:
Page | 26
Sometimes it is easier to navigate between a number of folders by displaying the main
folder and the sub-folder in a split screen. This is done by left-clicking on the Folders
icon.
This will open up the split window for back-and-forth navigation:
Page | 27
An example of a completed daily folder is shown below, containing the Reports folder,
each of the loading grids, client manifests, and plate readings created that day:
We strongly encourage you to make a backup copy of each main folder in the event of a
catastrophic problem which may make file recovery difficult or impossible.
Page | 28
Creating Manifests and Loading Grids
The BioPRYN utility (New CowPIE ver.2.jar) is a program designed specifically to
process BioPRYN pregnancy ELISA data and to produce an electronic report of the
pregnancy results. The utility accepts input of two types; client manifests and plate
readings. Manifests are .csv spreadsheet files containing a group of animals (usually
those from one customer), their well positions, customer information, etc. Plate readings
are .txt files containing optical density data for one ELISA plate. The utility quickly
processes these input data and outputs pregnancy reports in html or spreadsheet format.
This utility will be described more fully in Section 6. This part deals with how to create
manifests and loading grids.
Manifests
The manifest file is a spreadsheet form that lists a group of samples and specifies their
positions on an ELISA plate. It also stores customer information and other relevant data.
CowPIE will output one report for each manifest file. A manifest can include any
number of samples, which may occupy wells in more than one ELISA plate.
In order for a manifest to be recognized, it must be formatted correctly. Included on your
BioTracking CD is a template manifest (“Blank Manfest.csv”) to fill out (Fig 1). We put
the template manifest into our “Blank Client Manifests” folder at the be innin of this
section.
Fig 1. The Blank Manifest Template
Page | 29
If you want to see the complete field in each column, you can position the cursor between
any of the Columns designations (such as A and B), left-click and hold and drag the
cursor to the right (note that the file will not keep these types of edits once saved it will
go back to the original CSV format):
There are two types of field in the manifest: essential fields, which must be filled in
order for the program to read the manifest, and non-essential fields, which contain
information that will be printed on the report, but can be left blank.
The following are essential fields which must be filled. Note that all data is entered in
Column B:
DATE RECEIVED
CLIENT
LOGIN
PLATE FILENAME
WELL ID
ANIMAL ID
PLATE FILENAME : This field contains the filename of the plate reading that the utility
will associate with the samples listed (see section III). This must be the complete
filename, including the extension, and is CaSe SenSitIvE.
ANIMAL ID : This field holds the name or number used to identify a sample: usually
an ear tag number, tube label, or animal name or number.
Page | 30
WELL NUMBER : These are integers, 1-88, which links the adjacent ANIMAL ID to a
sample well on the ELISA plate. Column A on the plate, which contains standards and
controls, does not have any corresponding well numbers. The wells in column B, from
top to bottom, are referenced by well numbers 1 through 8; column C, 9 through 16, and
so on.
The non-essential fields would be Contact, Home Address, Report notes (see below),
Phone, FAX, or email, and Days Post Breedin (DPB). The field for “Cattle – samples”
in B25 can be left blank, but we encourage you to include the number of samples tested
since this serves as a final check in the reporting phase (see below).
Report notes allows you to add information pertinent to the report such as a minimal
amount of sample, broken or missing tubes, etc. These entries can be added directly after
the “REPORT NOTES:” in B14 and continued in B1 . There is not much field space to
add comments, so be brief and concise:
Generally, we include only the phone, FAX, or email address information designated for
sending the report, and delete the fields not used. This eliminates some “dead space” in
the final report. This is a personal decision, and some labs like to have all the contact
information present in the report.
Unused fields to be deleted (highlighted)
After deletion
Page | 31
The tube numbers, well positions, animal ID and (if provided) Days Post Breeding data
are entered in the remaining fields (the columns have been expanded for clarity).
Generally the tube number will be consecutive from 1 to the last tube sent in by the
client. Usually the client will number the tubes in sequential order, followed by the cow
ID number or name. The well ID indicates the number of the well corresponding to the
position of the sample on the plate.
The following is typical Animal/Well list from a manifest.
Page | 32
The TUBE NUMBER and WELL ID do not have to be listed in ascending or sequential
order. However, a blank line will cause the program to ignore the animals/wells listed
below it.
Acceptable input
Unacceptable input (blank line in Row 33)
A group of samples may span more than one plate. In order to indicate this in the
manifest, skip exactly one row after the final sample in the first plate, and start with
another PLATE FILENAME field.
Page | 33
The first six samples from this group are at the end of plate 1457. They continue on plate
1458. This can be repeated for as many plates as necessary. Don’t for et to put the new
plate number in the PLATE FILNAME field!
We save a copy of the client information into the Blank Client Manifest folder under their
last name or farm name, so the next time there will be a template for that farm ready to
go. This blank manifest will have their name and contact information filled out. The rest
will be filled out with each new set of samples. Once we have filled out the form for that
client that day we save a copy of the filled out manifest in that day’s folder.
Page | 34
Preparing the Well Loading Grids
The loading grids are the output of an EXCEL-based file showing the placement of the
control samples and each test sample in the 8 X 12 ELISA plate. These are made
utilizing a copy-and-paste operation from the completed Farm Manifest.
Begin by opening the Grid template provided (Plate grid for New.xls). After entering the
data into and savin the client’s manifest, the animal ID’s are copied by hi hli htin the
entries in column C of the manifest (Fig 1), pressing Ctrl + C, positioning the cursor at
position C26 of the New Grid (Fi 2) and pastin , by pressin Ctrl + V. The animal ID’s
should show up in column C and should also be visible in the grid (Fig 3). Although it is
not necessary, you can add the LOGIN number of the client to the first Animal ID in the
grid as an aid to help track sample location (Fig 4).
Fi 1. Hi hli ht and Copy (Ctrl + C) the Animal ID’s in the Manifest
Save the Grid in the current folder for the day using the plate number. For example, if
the plate number is 1501, save the Grid as 1501.xls.
Continue copying and pasting from each successive manifest until the grid is filled (Figs
5, 6). Note: there are a maximum of 88 client samples that can be loaded in each Grid.
When all the samples have been positioned in the Grid, save it to the folder for the
current day and print out a copy for use in loading the samples. Make sure you only
print page 1, otherwise you will get a printout of all the samples copied into cells 26 –
133 in the EXCEL file.
Page | 35
Fig 2. Select cell C 26
Fi
Fig 3. Copy Animal ID (Ctrl + V)
. Continue addin Animal ID’s
Fig 4. Add LOG number
Fi 6. Animal ID’s displayed in the Grid
Page | 36
A completed grid ready for printing and loading samples.
BioTracking LLC
NEW BioPRYN Test Grid
Sample Logs ____________________________________
1
Date ______________
Plate # ____36259____ Checked _____
Standards Lot# ______
Signature ____________________
2
3
1
A
Std A
11111- 3196
Std A
4709
Std B
942
Std B
3655
Std C
3929
Std C
2462
Std D
1135
Std D
3869
2
B
10
12
6
F
14
8
H
22
15
16
942
30
23
24
3714
Rack Check ________
38
31
46
39
32
2382
54
47
40
55
23483
48
3351
56
4092
Detector ___________
MF Check ________
78
71
86
4851
79
4968
72
4744
85
3650
1091
2475
64
100
77
70
63
84
23314
9106
4264
876
76
69
62
83
4205
4851
23628
3078
75
68
61
82
4744
3650
3261
1383
4581
3859
3676
53
74
67
60
81
2475
23314
3332
23576
4419
4768
3699
52
45
66
59
12
73
4205
23536
608
6518
1292
4225
23616
44
37
58
51
11
65
23722
4786
784
2787
3782
848
3626
36
29
50
43
10
57
2550
3388
3532
3441
3552
3241
4709
28
21
42
35
9
49
610
1081
463
3679
3869
11111- 3196
7
G
20
13
34
27
8
41
23332
1110
3714
1135
3552
26
19
7
33
23134
3552
2462
3679
5
E
18
11
6
25
3679
3929
3714
4
D
5
17
3655
3626
3
C
4
9
3241
87
9106
80
23315
88
1091
Enhancer ___________
Incubation Times:
Sample:
Detector:
Enhancer:
Substrate:
Date/Time on: _______________
Date/Time on: _______________
Date/Time on: _______________
Date/Time on: _______________
Date/Time off: _______________
Date/Time off: _______________
Date/Time off: _______________
Date/Time off: _______________
Total time: __________________
Total time: __________________
Total time: __________________
Total time: __________________
Note that there are several sign-off areas for double-checking the loading order and
ensuring the samples are loaded in the correct wells on the correct plate:
1. Rack Check. With the racked samples and the grid, pick up each tube and check
that the Animal ID matches the position on the grid with the position in the rack. Write
any discrepancies on the grid in red pen. When all the samples have been checked,
initial the blank next to the Rack Check;
2. Plate # Check. Before loading the samples, make sure the plate number matches
the number written on the loading grid;
3. Manifest (MF) Check. If any errors are noted or encountered while loading the
plate, indicate them in the appropriate well on the grid in red ink. After the plate has
been loaded, go back and check manifests for errors. For example, if sample 181 in
well F4 is added to well G4, note the error (and that the sample originally to be loaded
in well G4 will need to be loaded in F4) and change the well designation in the
manifest. This can be done one of two ways: either the Animal ID can remain
unchanged in the manifest, and the well designations are changed (Fig 7); or the well
desi nations remain unchan ed and the Animal ID’s are chan ed (Fi 8). Verify other
information at this time: such as the client contact info, plate #, well ids, log # and date.
Page | 37
Original manifest
Fig 7. Manifest corrected by
changing well numbers
Fig 8. Manifest corrected by
by changing Animal ID
numbers
There are also other areas on the Loading Grid in which you can indicate the times of
addition and removal of the sample, Detector, Enhancer and the TMB substrate. The lot
number of the standards used should be written in the appropriate place on the grid.
Plate Readings
The CowPIE program is configured to recognize certain data formats which your optical
density reader software may export.
The filename needs to coordinate with the filename listed in the corresponding manifests.
In order to avoid confusion, it is recommended that these filenames be made up of the
plate number followed by a lower-case extension (“12 4.txt”)
If the specific format exported by your plate reader software is incompatible with
CowPIE, it will have to be modified for your use. Contact BioTracking for more
information.
If you are using Gen 5 software with a BioTek reader the following procedure can be
used to be able to export a usable text file for CowPIE.
1.
2.
3.
4.
5.
6.
7.
Open protocol in Gen 5 program used for reading BioPRYN plates.
Click on protocol tab at top of screen.
Choose file export builder.
Under matrix choose 650 (or the name of the data file with plate reading data)
Click add
Right click on newly added file in right hand column
Choose edit
Page | 38
8. Change Name to [Plate:M650]
9. Click data
10. Change title to a semicolon ;
11. Click OK
12. Click on protocol tab at top of screen
13. Choose Protocol options
14. Click on file export settings
15. Include: Headings, Matrix column and row numbers
16. Choose separator semicolon ;
17. Exported text file can be directly used for analysis with New cowPIE ver. 2
Page | 39
Section 5 – How to Perform the BioPRYN
ELISA Assay
Page | 40
INTRODUCTION
The following tutorial discusses how to perform the BioPRYN ELISA test. The tutorial
is meant to inform users about each step in the assay and the specific skills required to
complete each task. This will be given to you along with the training materials. Please
refer to your most recent protocol received with each kit shipment for any updated
information or changes.
BioPRYN Tutorial
The following is a step-by-step tutorial for running the BioPRYN assay. The first section
is specific to the New BioPRYN assay and the second section is specific to BioPRYNQK.
The tutorial is meant to inform users about each step in the assay and the specific skills
required to complete each task. Helpful tips are included with each section to improve
performance. A troubleshooting guide is included at the end of this section for specific
problems that may arise in the running of the assay. Please refer to the most recent
BioPRYN protocol found with each kit for specific volumes, concentrations, times,
storage conditions, etc. Improvements and changes to the assay may require the
protocol to be changed from time to time.
Section 1: General Information about the New BioPRYN ELISA
The NEW BioPRYN ELISA is recommended for detection of pregnancy in cattle at or
greater than 28 days post breeding and 73 days since last calf. The assay can be
completed in a minimum assay time of 6.5 hours, but is most often completed by
applying samples on day 1 and reporting the results in the morning/ early afternoon of
day 2. This antigen-capture, or “sandwich,” Enzyme-Linked ImmunoSorbent Assay
(ELISA) detects Pregnancy Specific Protein B (PSPB) in bovine sera/ plasma. Serum
PSPB binds to antibodies coated in the wells and is detected by secondary bindin of a
labeled antibody. Bindin of the labeled antibody con u ate is detected by the addition
of the Enhancer and , , , -tetramethylbenzidine (TMB) and is quantified by the
subsequent color development. A strong color indicates binding and substrate reactivity
of the labeled antibody conjugate to the bound PSPB, and is a positive indication of
pregnancy. Weak color development indicates little or no binding of the labeled antibody
conjugate due to the absence of PSPB in the sample, and is a negative indication of
pregnancy. Standards of known concentrations of PSPB are run with each assay and are
used to determine the optical density values for assigning Pregnant / Not Pregnant ranges.
Definitions for Your Reference specific to New BioPRYN assay
•
•
•
Standard A- red standard placed in position A1 and B1 of the plate
Standard B- dark orange standard placed in position C1 and D1 of the plate
Standard C- light orange standard placed in position E1 and F1 of the plate
Page | 41
•
•
•
•
•
Standard D- light orange to clear standard placed in position G1 and H1 of the
plate
Sample Buffer #1 – Red buffer used to wet the sample wells and serve as a
buffering agent for the serum samples, added in Step 1 of the assay.
Detector Solution #2-Blue buffer added in the second step of the assay after a
wash step
Enhancer solution #3- Green buffer added in the third step of the assay after a
wash step
TMB substrate solution #4- Colorless buffer in amber bottle added in the last
step of the assay (after a wash step). Do not expose TMB to direct light or
oxidizing agents. TMB substrate solution is extremely sensitive once outside of its
container; avoid excessive handling.
•
•
Stop solution #5- Yellow buffer used to stop the color reaction after 15 minutes.
20 x wash buffer (W)– Concentrated wash buffer. Working concentration is
made by diluting 1 part 20 x wash buffer with 19 parts deionized water. To make
1 liter of the working concentration add 50 milliliters of 20 x to 950 milliliters of
water. Wash buffer is used to wash plates after Step 1, Step 2 and Step 3of the
assay.
Required Equipment
For optimum BioPRYN assay performance several pieces of laboratory equipment are
recommended. These include:
–
–
–
–
–
–
Centrifuge
Absorbance plate reader
Shaker (Optional)
Incubator/ shaker (Optional)
Plate washer
Stir bar mixer (Optional)
Please contact BioTracking for recommendations on preferred laboratory equipment.
Equipment may vary. Each lab must understand and maintain their specific equipment
for use with the BioPRYN assay.
Pipettes
Handheld pipettes are the most fundamental piece of equipment for the BioPRYN assay.
Accurate transfer of samples and solutions is critical to providing accurate results to our
customers. All personnel running the BioPRYN assay must be able to choose the
appropriate pipetter for each job, have precise pipetting technique and be able to monitor
accurate pipetting performance. Labs should carefully maintain pipetters and re-calibrate
as necessary.
Page | 42
Make certain you choose the right size of pipette for the job. Performing the assay
requires the use of different adjustable volume pipetters. We also encourage the use of
adjustable 8- or 12-channel mulitpipetters for addition of the buffers. Choose the right
pipette for the job. Use the Mid-volume (50 µL to 200 µL) – Pipetman P200 or
Finnpipette 20-200 µl for addition of serum samples, standards, or buffers to individual
wells. When using the Large-volume (200 µL to 1000 µL) – Pipetman P1000 or
Finnpipete 100-1000 µL Do NOT use dispensing any volume less than 200 µL (samples,
standards, TMB etc.).
Reminder: choose the right pipette! The choice of pipetter depends on the volume of
solution being prepared. If you are adding 50 ul use the P20. If you are making a
pipetting larger volume of a particular reagent (for example 150 mL of Sample) you
would want to use the P200 to dispense 150 µL rather than using the P20 to dispense 50
µL three times.
Other pipetters may be utilized as well. Multichannel pipetters are used to dispense the
same volume of reagent to multiple wells (for example adding Sample Buffer to a full
plate of 96 wells). One multichannel pipetter capable of dispensing 50 µL up to 300 µL
can be used for the four different solutions used in the assay.
Use of Pipette
To accurately measure and dispense solutions, pipetters must be used with the
appropriate size and style of pipette tip. The pipette tip should firmly seat on the
pipetter and draw a consistent volume. The single- and multi-channel pipetters have 3
positions for the plun er: the fully extended “rest” position; the “fill” position; and the
fully depressed “expel” position. If you are performing multiple dispensing of the same
solution (for example the Standards) you can fill the pipet tip, expel the solution by fully
depressin the plun er, then releasin the plun er to the “fill” position. Be careful if you
use this technique to avoid the common error of refillin the pipet tip from the “expel”
position. Many of the buffers contain a surface-breaking surfactant (Tween 20). To
avoid foaming of the solution, do not depress the plunger with the tip immersed in the
solution.
With any of the pipetters, it is important to fill and dispense the solutions in a relatively
slow, steady manner. Avoid a rapid, sudden release of the plunger when filling the tip.
This can cause an air bubble to form in the tip, resulting in a lower volume of solution, or
it can pull the reagent up into the barrel of the pipetter. After filling the tip, remove any
residual solution from the outside of the tip by carefully dragging it across the edge of the
container. Also, avoid a too-rapid dispensing of the solution, and give time (one or two
seconds) for the solution to drain from the walls of the tip before expelling the solution.
*You are the best defense against inaccurate pipetting. Monitor your use of pipetters
and get accustomed to how a certain volume looks in the pipette tip. If you are pipetting
and the volume looks wrong, stop and make sure the pipetter is working correctly. 1
Page | 43
milliliter of water should weigh 1 gram. If you have a balance available, test your
pipetter and your pipetting ability.
Additional Important Laboratory Skills
Similar to pipetters, it is important to use the proper sized cylinder for the amount of
solution being measured. Each cylinder you may use is discussed briefly below:
•
Graduated Cylinder. As a rule of thumb, use the cylinder closest in volume for
the amount of solution you are preparing. For example, use a 10 mL cylinder in
the preparation of the 5 mL of Sample Buffer, and a 50 mL cylinder in the
preparation of 20 mL. Increase the size of the cylinder as needed if you are
scaling up the amount of solution being prepared. After measuring all reagents,
transfer the solution to an appropriately sized beaker or conical tube for mixing.
Some cylinders have a detachable stand, and the contents can be carefully mixed
on a vortex mixer.
When utilizing a graduated cylinder, measure liquids from the bottom of the
meniscus, as shown in the picture below.
•
Serological Pipettes. Many laboratories use these instead of small graduated
cylinders for preparing Sample Buffer or TMB. As for the Graduated Cylinders,
transfer the solution to an appropriately sized beaker or conical tube for mixing.
Take care when mixing solutions. Thorough vortexing or mixing the reagent bottle by
hand will ensure a homogenous mix. If you use a multi-channel pipetter and reservoir, it
is temptin to add the rea ents of a solution directly to the reservoir, then “mix” by
shaking the reservoir back and forth. This practice is to be avoided at all costs, since
homogeneity of the resulting solution cannot be assured.
Page | 44
The BioPRYN Assay
There are four steps to the BioPRYN assay. The first step is preparing plates with
Sample Buffer (#1), adding standards, and adding samples. The second step is washing
plates, and adding Detector Solution (#2). The third step is washing plates, and adding
Enhancer Solution (#3). The last step is washing plates, the addition of TMB substrate
solution (#4), the addition of stop solution (#5), and finally reading of plates. These steps
are described in detail below, with tips and notes following each step to provide more
clarity or understanding.
Before starting any part of the assay, check with the protocol to determine solutions and
equipment that will be required for each step. Prepare your work space to accommodate
easy movement and organization to minimize cross-contamination. Most of the kit
solutions are stored in the refrigerator. Please bring all solutions to room temperature
prior to use in the assay. Use dedicated glassware and plastic ware for all reagents.
Use aseptic techniques and measure all materials prior to use to limit contamination
and reagent waste.
Before Starting Assay- Enter farm and cow specific information in individual manifest
files. Use the manifest to enter sample id’s on the NEW grid worksheet tab in the Excel
file “Plate Grid for NEW.xls” and enter plate and plate location on the manifest. Print off
the grid to assist with plate loading as described in the previous manual sections.
Step One
•
Prepare Plate
– Prepare plate by wetting wells with Sample Buffer #1 (50ul)
– Tips / notes
• A multichannel pipetter can be used to fill wells with prescribed
amount of sample buffer
• Full plates can be filled without changing tips. Monitor pipetter to
make sure the same amount of liquid is being brought into pipette
tip and expelled in well.
• Sample buffer should be a reddish-orange color when added to the
wells. An extreme color change of the solution (yellow or pink)
indicates a pH change of the well that could affect results.
Page | 45
•
•
Add Standards A- D (150 ul)
– Controls are placed in the left most column of the assay plate as explained
in the protocol
– Tips / notes
• Proper placement of standards is critical for data analysis using
hand calculations or software.
• Use a new tip for each different standard / control.
• The same tip can be used for loading a single standard. Consistent
volume is the key. Pre-wet the tip (load the standard then expel it)
when you are loading the first replicate.
Add Samples (150 ul)
– Add samples according to loading grid. Check the number on tube with
the number on grid and location on plate.
– Seal the wells with ParaFilm or DuraSeal, cover with a microplate lid, and
incubate according to protocol.
– Tips / notes
• Use a new tip for every sample.
• Watch to make sure you accurately pipette 150 ul. Extreme
hemolysis or viscous samples can cause pipetting errors if
accuracy is not carefully monitored.
• Avoid disturbing the clot when pipetting serum.
• * Do not cross-contaminate. Be careful not to switch caps on tubes
or allow used tips to contact surfaces.
• Record mislabeled tubes on grid.
• Record irregular samples on grid (extreme hemolysis, inadequate
volume).
• Use sample buffer when the serum volume is less than 150 ul.
When preparing a report for a minimum sample, a pregnant result
is a valid call. A Not Pregnant result does not allow a definitive
result and a new sample is required.
• Careful of dried blood flakes from tube dropping into the wells.
Incubate the plates overnight at room temperature (minimum of 4 hours).
Page | 46
BioPRYN plate filled with standards and samples.
bottom, left to right.
Sample 1
Standards
Column
Samples are loaded top to
Samples 1-88
Standard A
Standard B
Standard C
Standard D
Sample 88
Sample 8
Page | 47
Step Two
– Bring all buffers (#2-#5) to room temperature.
– Prepare the wash solution and prime the plate washer (if using). 20X
wash buffer (stored at room temperature) diluted by adding 1 part of
concentrated wash buffer to 19 parts deionized water. See wash buffer
preparation at end of tutorial
– Tips / Notes
• 20X Wash buffer no longer needs to be stored at room
temperature. Check solution for salt crystals or precipitant prior to
diluting to 1X, they should mix back into solution with a gentle
mixing of the bottle
• Assay plates are washed in Steps 2, 3 and 4 of the assay. Enough
1X wash buffer can be made at this time for both wash steps. A
full plate requires a total of 300 ml for all three wash steps (30 ml
per wash, 10 total washes). Additional wash buffer may be
required for priming an automatic plate washer and to account for
void volumes in the washer itself. Each kit contains additional
wash buffer to account for this discrepancy. However, equipment
varies and some optimization of the required wash buffer volumes
will be required of each lab to assure 20X wash buffer is not
wasted.
•
Wash Wells 4 times
– After the overnight incubation, remove the ParaFilm or DuraShield and
wash the plate four times. Save the DuraShield for the next incubation.
• If an automatic washer is used, dump the contents of the wells into
a sink or tub, blot on a paper towel, place the plate on the washer
and wash 4 times with a volume of 00 μl. Set the washer to soak
for 10 seconds and aspirate for 4 seconds between each wash.
Remove any residual wash solution by striking the inverted plate
on a paper towel (blot dry).
• If manual washing is used, dump the contents of the wells into a
sink or tub and then blot on a paper towel. Using a multichannel
pipettor or RepeatPipettor, add 00 μl of 1X wash buffer to each
well and swirl the plate for 10 seconds. Dump out the wash
solution and blot dry on a paper towel. Repeat the washing
procedure 3 more time (4 washes total).
– Tips / Notes
• Some samples may leave a visual residue in the wells. Use a
single channel pipetter and 1X wash buffer to remove the residue.
• Reverse the sequence across the plate between washes to minimize
differences in soak time from one side of the plate to the other.
Page | 48
•
Add Detector #2 for one hour at room temperature
– Add Detector #2 as described in the protocol. Use a single channel or
multi-channel pipetter to add the solution. Cover with fresh ParaFilm or
the DuraSeal from previous steps, and incubate according to current
protocol.
– Tips / Notes
• Full plates can be filled without changing tips. Monitor the
pipetter to make sure the same amount of liquid is being brought
into pipette tip and expelled in the well.
• The Detector solution is BLUE make sure you have added the
correct buffer in this step.
• Timing of this step has been optimized. Please see the most recent
protocol for details of time and temperature.
Sample with PSPB
Sample without PSPB
Incubate plate for one hour at room temperature.
Step Three
•
•
Wash Wells 2 times
– After the incubation, remove the ParaFilm or DuraShield and wash the
plate two times. Save the DuraShield for the next incubation.
• If an automatic washer is used, dump the contents of the wells into
a sink or tub, blot on a paper towel, place the plate on the washer
and wash 2 times with a volume of 00 μl. Set the washer to soak
for 10 seconds and aspirate for 4 seconds between each wash.
Remove any residual wash solution by striking the inverted plate
on a paper towel (blot dry).
• If manual washing is used, dump the contents of the wells into a
sink or tub and then blot on a paper towel. Using a multichannel
pipettor or RepeatPipettor, add 00 μl of 1X wash buffer to each
well and swirl the plate for 10 seconds. Dump out the wash
solution and blot dry on a paper towel. Repeat the washing
procedure 1 more time (2 washes total).
Add Enhancer #3 for one hour at room temperature
Page | 49
– Add Enhancer #3 as described in the protocol. Use a single channel or
multi-channel pipetter to add the solution. Cover with fresh ParaFilm or
the DuraSeal from previous steps, and incubate according to current
protocol.
– Tips / Notes
• Full plates can be filled without changing tips. Monitor the
pipetter to make sure the same amount of liquid is being brought
into pipette tip and expelled in the well.
• The Detector solution is GREEN make sure you have added the
correct buffer in this step.
• Timing of this step has been optimized. Please see the most recent
protocol for details of time and temperature.
Incubate plate for one hour at room temperature.
Step Four
•
Wash Plates
– Wash wells 4 times
• If an automatic washer is used, dump the contents of the wells into
a sink or tub, blot on a paper towel, place the plate on the washer
and wash 4 times with a volume of 00 μl. Set the washer to soak
for 10 seconds and aspirate for 4 seconds between each wash.
Remove any residual wash solution by striking the inverted plate
on a paper towel (blot dry).
• If manual washing is used, dump the contents of the wells into a
sink or tub and then blot. Using a multichannel pipettor or
RepeatPipettor, add 00 μl of 1X wash buffer to each well and
swirl the plate for 10 seconds. Dump out the wash solution and
blot dry on a paper towel. Repeat the washing procedure 3 more
time (4 washes total).
Page | 50
•
Add TMB substrate solution #4
– Add 200 μl of the TMB substrate solution #4 from the amber bottle. After
all of the wells have been filled, gently mix by swirling a few times in
each direction. Incubate at room temperature for 15 minutes.
– Tips / Notes
• Volume differences can affect OD readings. Use care to add a
consistent volume to each well.
• In the presence of HRP, TMB turns blue (as read at 650 nm).
Color change can be monitored visually to confirm progress of the
assay.
• Note which direction the TMB is added to the wells, so that the
stop solution might be added in the same direction and speed.
TMB substrate is clear when first added to the plate.
After 15 minutes, sample wells with PSPB turn blue. While wells with no PSPB
remain clear.
Page | 51
•
Add Stop Solution (#5)
– Add 100 μl of the YELLOW stop solution #5 to each well. After all of
the wells have been filled, gently mix the well contents by swirling a few
times in each direction.
– Tips / Notes
• Add the stop solution in the same order and timing as was done for
the TMB.
• Volume differences can affect OD readings. Use care to add a
consistent volume to each well.
Sample 1
Standards
Column
Samples 1-88
Standard A
Standard B
Standard C
Standard D
Sample 88
Sample 8
Page | 52
•
Read Plates
– Read the plate: Within 30 minutes after adding the stop solution, the
plate should be read on a plate reader at 650 nm.
• Line up well A1 on the plate with A1 on the plate reader
• Open 650 nm reading program
• Read plate and save copy of results. **Follow a system of reading
plates that minimizes the likelihood of reading the same plate
twice. Move the plate away from the area of the plate reader to
assure that the plate is not mistakenly read again and recorded as a
reading for a different plate.
– Tips / Notes
• For Molecular Devices scanners running SOFTmax software,
export the reading as a text file for data analysis.
• Click File; click Import/Export then select Export. Choose
the file for the day that the samples were received. Name
the file the same as the plate number, make sure another
file with same name pops up. Click save.
• If you have questions, contact BioTracking for all other plate
reader manufacturers.
• If standard A does NOT reach the minimum OD of 0.6 during the
15 minute incubation time the assay conditions are incorrect and
the assay has failed. Results should not be reported.
Page | 53
SECTION 2: General Information about the BioPRYNQK ELISA
The BioPRYNQK ELISA is recommended for detection of pregnancy in cattle at or
greater than 28 days post breeding and 90 days since last calf. This antigen-capture, or
“sandwich,” Enzyme-Linked ImmunoSorbent Assay (ELISA) detects Pregnancy Specific
Protein B (PSPB) in bovine sera. Serum PSPB binds to antibodies coated in the wells
and is detected by secondary binding of a labeled antibody (Detector). Binding of the
labeled antibody con u ate is detected by the addition of the Enhancer and , , , tetramethylbenzidine (TMB) and is quantified by the subsequent color development. A
strong color indicates binding and substrate reactivity of the labeled antibody conjugate
to the bound PSPB, and is a positive indication of pregnancy. Weak color development
indicates little or no binding of the labeled antibody conjugate due to the absence of
PSPB in the sample, and is a negative indication of pregnancy. Standards of known
concentrations of PSPB are run with each assay and are used to determine the optical
density values for assigning Pregnant / Not Pregnant ranges.
Definitions for Your Reference specific to New BioPRYN assay
• QUICK High Standard- Green standard placed in position A1 to D1 of the plate
• QUICK Low standard- Light green standard placed in position E1 to H1 of the
plate
• Sample Buffer #1 – Red buffer used to wet the sample wells and serve as a
buffering agent for the serum samples, added in Step 1 of the assay.
• QUICK Detector Solution #2-Orange buffer added in the second step of the
assay after the wash step
• QUICK Enhancer solution #3- Red buffer added in the third step of the assay
• QUICK TMB substrate solution #4- Colorless buffer in amber bottle added in
the last step of the assay. Do not expose TMB to direct light or oxidizing agents.
TMB substrate solution is extremely sensitive once outside of its container; avoid
excessive handling.
•
•
QUICK Stop solution #5- Yellow buffer used to stop the color reaction after 15
minutes.
20 x wash buffer (W)– Concentrated wash buffer. Working concentration is
made by diluting 1 part 20 x wash buffer with 19 parts deionized water. To make
1 liter of the working concentration add 50 milliliters of 20 x to 950 milliliters of
water. Wash buffer is used to wash plates after Step 1, Step 2 and Step 3of the
assay.
Required Equipment
For optimum BioPRYN assay performance several pieces of laboratory equipment are
recommended. These include:
– Centrifuge
– Absorbance plate reader
– Shaker (Optional)
– Incubator/ shaker (Optional)
– Plate washer
– Stir bar mixer (Optional)
Page | 54
The BioPRYNQK Assay
There are four steps to the BioPRYNQK assay. The first step is preparing plates with
Sample Buffer (#1), adding standards, and adding samples. The second step is washing
plates, and adding QUICK Detector Solution (#2). The third step is washing plates, and
adding QUICK Enhancer Solution (#3). The last step is washing plates, the addition of
QUICK TMB substrate solution (#4), the addition of QUICK stop solution (#5), and
finally reading of plates. These steps are described in detail below, with tips and notes
following each step to provide more clarity or understanding.
Before starting any part of the assay, check with the protocol to determine solutions and
equipment that will be required for each step. Prepare your work space to accommodate
easy movement and organization to minimize cross-contamination. Most of the kit
solutions are stored in the refrigerator. Please bring all solutions to room temperature
prior to use in the assay. Use dedicated glassware and plastic ware for all reagents.
Use aseptic techniques and measure all materials prior to use to limit contamination
and reagent waste.
Before Starting Assay- Enter farm and cow specific information in individual manifest
files. Use the manifest to enter sample id’s on the Quick grid worksheet tab in the Excel
file “BioPRYN QK Plate rid.xls” and enter plate and plate location on the manifest.
Print off the grid to assist with plate loading as described in the previous manual sections.
Step One
•
Prepare Plate
– Prepare plate by wetting wells with Sample Buffer #1 (50ul)
– Tips / notes
• A multichannel pipetter can be used to fill wells with prescribed
amount of sample buffer
• Full plates can be filled without changing tips. Monitor pipetter to
make sure the same amount of liquid is being brought into pipette
tip and expelled in well.
• Sample buffer should be a reddish-orange color when added to the
wells. An extreme color change of the solution (yellow or pink)
indicates a pH change of the well that could affect results.
Page | 55
•
•
Add Standards High and Low (150 ul)
– Controls are placed in the left most column of the assay plate as explained
in the protocol
– Tips / notes
• Proper placement of standards is critical for data analysis using
hand calculations or software.
• Use a new tip for each different standard / control.
• The same tip can be used for loading a single standard. Consistent
volume is the key. Pre-wet the tip (load the standard then expel it)
when you are loading the first replicate.
• Only use standards with the same lot#. DO NOT MIX LOTS OF
STANDARDS.
• Record the multiplier (X=?) so that you can enter the correct
multiplier for analysis.
Add Samples (150 ul)
– Add samples according to loading grid. Check the number on tube with
the number on grid and location on plate.
– Seal the wells with ParaFilm or DuraSeal, cover with a microplate lid, and
incubate according to protocol.
– Tips / notes
• Use a new tip for every sample.
• Watch to make sure you accurately pipette 150 ul. Extreme
hemolysis or viscous samples can cause pipetting errors if
accuracy is not carefully monitored.
• When loading large numbers of plates using the QK assay, it is
recommended to pre-plate then transfer all of the sample using a
multi-channel pipette.
• Avoid disturbing the clot when pipetting serum.
• * Do not cross-contaminate. Be careful not to switch caps on tubes
or allow used tips to contact surfaces.
• Record mislabeled tubes on grid.
• Record irregular samples on grid (extreme hemolysis, inadequate
volume).
• Use sample buffer when the serum volume is less than 150 ul.
When preparing a report for a minimum sample, a pregnant result
is a valid call. A Not Pregnant result does not allow a definitive
result and a new sample is required.
Incubate the plates for one hour at room temperature.
Page | 56
BioPRYNQK plate filled with standards and samples. Samples are loaded top to
bottom, left to right.
Sample 1
Samples 1-88
Standards
Column
High Standard
Low Standard
Sample 88
Sample 8
BioTracking LLC
BioPRYNQK Plate Grid
Sample Logs ____________________________________
1
Date ______________
Plate # _N35421_______ Checked _____
Standards Lot# ______
Signature ____________________
2
3
1
A
HIGH
11111-4292
HIGH
295
HIGH
3832
HIGH
1383
LOW
1021
LOW
784
LOW
3472
LOW
2778
23332
2
B
10
12
6
F
14
8
H
22
15
16
3317
30
23
24
3132
Rack Check ________
38
31
46
39
32
2802
54
47
40
55
3160
48
4564
56
3343
Detector ___________
MF Check ________
78
71
86
control#6
79
4701
72
4667
85
control#5
4627
1744
64
5064
77
70
63
84
control#4
4629
4520
4464
76
69
62
83
control#3
4606
1483
4485
75
68
61
82
control#2
2737
5655
4901
4157
121
675
53
74
67
60
81
control#1
4224
1046
4516
910
3455
1049
52
45
66
59
12
73
4234
5050
4682
1098
4092
1714
3195
44
37
58
51
11
65
4671
1796
4530
3416
3174
2446
1972
36
29
50
43
10
57
4623
4575
454
3225
3651
2413
3351
28
21
42
35
9
49
4676
2876
4453
4256
1503
204
7
G
20
13
34
27
8
41
2221
23536
4560
97
1531
26
19
7
33
3134
3267
23218
2922
5
E
18
11
6
25
4505
3489
4244
4
D
5
17
3977
4577
3
C
4
9
87
control#7
80
4064
88
control#8
Enhancer ___________
Incubation Times:
Sample:
Detector:
Enhancer:
Substrate:
Date/Time on: _______________
Date/Time on: _______________
Date/Time on: _______________
Date/Time on: _______________
Date/Time off: _______________
Date/Time off: _______________
Date/Time off: _______________
Date/Time off: _______________
Total time: __________________
Total time: __________________
Total time: __________________
Total time: __________________
Page | 57
Step Two
– Bring all buffers (#2-#5) to room temperature.
– Prepare the wash solution and prime the plate washer (if using). 20X
wash buffer (stored at room temperature) diluted by adding 1 part of
concentrated wash buffer to 19 parts deionized water. See wash buffer
preparation at end of tutorial
– Tips / Notes
• 20X Wash buffer no longer needs to be stored at room
temperature. Check solution for salt crystals or precipitant prior to
diluting to 1X, they should mix back into solution with a gentle
mixing of the bottle
• Assay plates are washed in Steps 2, 3 and 4 of the assay. Enough
1X wash buffer can be made at this time for both wash steps. A
full plate requires a total of 300 ml for all three wash steps (30 ml
per wash, 10 total washes). Additional wash buffer may be
required for priming an automatic plate washer and to account for
void volumes in the washer itself. Each kit contains additional
wash buffer to account for this discrepancy. However, equipment
varies and some optimization of the required wash buffer volumes
will be required of each lab to assure 20X wash buffer is not
wasted.
•
•
Wash Wells 4 times
– After the one hour incubation, remove the ParaFilm or DuraShield and
wash the plate four times. Save the DuraShield for the next incubation.
• If an automatic washer is used, dump the contents of the wells into
a sink or tub, blot on a paper towel, place the plate on the washer
and wash 4 times with a volume of 00 μl. Set the washer to soak
for 10 seconds and aspirate for 4 seconds between each wash.
Remove any residual wash solution by striking the inverted plate
on a paper towel (blot dry).
• If manual washing is used, dump the contents of the wells into a
sink or tub and then blot on a paper towel. Using a multichannel
pipettor or RepeatPipettor, add 00 μl of 1X wash buffer to each
well and swirl the plate for 10 seconds. Dump out the wash
solution and blot dry on a paper towel. Repeat the washing
procedure 3 more time (4 washes total).
– Tips / Notes
• Some samples may leave a visual residue in the wells. Use a
single channel pipetter and 1X wash buffer to remove the residue.
• Reverse the sequence across the plate between washes to minimize
differences in soak time from one side of the plate to the other.
Add QUICK Detector #2 for one hour at room temperature
Page | 58
– Add QUICK Detector #2 as described in the protocol. Use a single
channel or multi-channel pipetter to add the solution. Cover with fresh
ParaFilm or the DuraSeal from previous steps, and incubate according to
current protocol.
– Tips / Notes
• Full plates can be filled without changing tips. Monitor the
pipetter to make sure the same amount of liquid is being brought
into pipette tip and expelled in the well.
• The Detector solution is ORANGE make sure you have added the
correct buffer in this step.
• Timing of this step has been optimized. Please see the most recent
protocol for details of time and temperature.
Sample with PSPB
Sample without PSPB
Incubate plate for one hour at room temperature.
Step Three
•
Wash Wells 2 times
– After the incubation, remove the ParaFilm or DuraShield and wash the
plate two times. Save the DuraShield for the next incubation.
• If an automatic washer is used, dump the contents of the wells into
a sink or tub, blot on a paper towel, place the plate on the washer
and wash 2 times with a volume of 00 μl. Set the washer to soak
for 10 seconds and aspirate for 4 seconds between each wash.
Remove any residual wash solution by striking the inverted plate
on a paper towel (blot dry).
• If manual washing is used, dump the contents of the wells into a
sink or tub and then blot on a paper towel. Using a multichannel
pipettor or RepeatPipettor, add 00 μl of 1X wash buffer to each
well and swirl the plate for 10 seconds. Dump out the wash
solution and blot dry on a paper towel. Repeat the washing
procedure 1 more time (2 washes total).
•
Add QUICK Enhancer #3 for one hour at room temperature
Page | 59
– Add QUICK Enhancer #3 as described in the protocol. Use a single
channel or multi-channel pipetter to add the solution. Cover with fresh
ParaFilm or the DuraSeal from previous steps, and incubate according to
current protocol.
– Tips / Notes
• Full plates can be filled without changing tips. Monitor the
pipetter to make sure the same amount of liquid is being brought
into pipette tip and expelled in the well.
• The Detector solution is RED make sure you have added the
correct buffer in this step.
• Timing of this step has been optimized. Please see the most recent
protocol for details of time and temperature.
Incubate plate for one hour at room temperature.
Step Four
•
Wash Plates
– Wash wells 4 times
• If an automatic washer is used, dump the contents of the wells into
a sink or tub, blot on a paper towel, place the plate on the washer
and wash 4 times with a volume of 00 μl. Set the washer to soak
for 10 seconds and aspirate for 4 seconds between each wash.
Remove any residual wash solution by striking the inverted plate
on a paper towel (blot dry).
• If manual washing is used, dump the contents of the wells into a
sink or tub and then blot. Using a multichannel pipettor or
RepeatPipettor, add 00 μl of 1X wash buffer to each well and
swirl the plate for 10 seconds. Dump out the wash solution and
blot dry on a paper towel. Repeat the washing procedure 3 more
time (4 washes total).
– Tips / Notes
• No suggestions
Page | 60
•
Add QUICK TMB substrate solution #4
– Add 200 μl of the TMB substrate solution #4 from the amber bottle. After
all of the wells have been filled, gently mix by swirling a few times in
each direction. Incubate at room temperature for 15 minutes.
– Tips / Notes
• Volume differences can affect OD readings. Use care to add a
consistent volume to each well.
• In the presence of HRP, TMB turns blue (as read at 650 nm).
Color change can be monitored visually to confirm progress of the
assay.
• Note which direction the TMB is added to the wells, so that the
stop solution might be added in the same direction and speed.
TMB substrate is clear when first added to the plate.
After 15 minutes, sample wells with PSPB turn blue. While wells with no PSPB
remain clear.
Page | 61
•
Add Stop Solution (#5)
– Add 100 μl of the YELLOW stop solution #5 to each well. After all of
the wells have been filled, gently mix the well contents by swirling a few
times in each direction.
– Tips / Notes
• Add the stop solution in the same order and timing as was done for
the TMB.
• Volume differences can affect OD readings. Use care to add a
consistent volume to each well.
•
Read Plates
– Read the plate: Within 30 minutes after adding the stop solution, the
plate should be read on a plate reader at 650 nm.
• Line up well A1 on the plate with A1 on the plate reader
• Open 650 nm reading program
• Read plate and save copy of results. **Follow a system of reading
plates that minimizes the likelihood of reading the same plate
twice. Move the plate away from the area of the plate reader to
assure that the plate is not mistakenly read again and recorded as a
reading for a different plate.
– Tips / Notes
• For Molecular Devices scanners running SOFTmax software,
export the reading as a text file for data analysis.
• Click File; click Import/Export then select Export. Choose
the file for the day that the samples were received. Name
the file the same as the plate number, make sure another
file with same name pops up. Click save.
• If you have questions, contact BioTracking for all other plate
reader manufacturers.
• If standard A does NOT reach the minimum OD of 0.6 during the
15 minute incubation time the assay conditions are incorrect and
the assay has failed. Results should not be reported.
Page | 62
Preparation of Buffers / Reagents
•
Preparation of 1X Wash Buffer
– Add 1 part 20X wash buffer to 19 parts water.
• To make 120 ml of 1X wash buffer, add 6 ml of 20X wash buffer
to 114 ml of water. Mix thoroughly.
• Assume 2.5 ml of wash buffer per well to account for the buffer
required in the two wash steps. A full plate requires 240 ml.
• Additional 1X wash buffer will be required when using a plate
washer.
Page | 63
Section 6 – Reporting Results to
Customers Utilizing CowPIE
Page | 64
INTRODUCTION
After manifests are filled, the assay is completed, and the plate reading data have been
saved in an appropriate format, there are three steps to using CowPIE to process data.
They are, in order, 1) Opening plate readings, 2) Merging Manifests, and 3) exporting
reports.
Launch the CowPIE program. At the top of the program window click on the modify
tab. Choose “Edit the Company Header” and fill in your company specific information
that will be included on your BioPRYN reports.
Page | 65
PLATE ANALYSIS Using New CowPIE with BioPRYN
Opening Plate Readings
Under the modify tab at the top of the sheet, select plate type and choose the assay type
that matches your process. For analysis of the new assay choose either “BioPRYN New:
reater than hour sample incubation” (for assays utilizin a sample incubation step
reater than hours) or “BioPRYN New: less than hour sample incubation”.
a. NOTE: When running standard BioPRYN Sheep/ goat or BioPRYN QK
choose “Three 0.0 8 and three 0.019 standards” under plate type.
b. Check to make sure that the thresholds for the standard and QK tests are
correct for your analysis.
i. Standard BioPRYN and BioPRYN Sheep and Goat: multiplier=
0.45, Open But Recheck Threshold= 0.9, Pregnant but Recheck
Threshold= 1.15.
ii. BioPRYN QK: multiplier= 0.1, Open But Recheck Threshold=
0.8, Pregnant but Recheck Threshold= 1.2.
iii. NOTE: The tabs for BioPRYN QK (BioPRYN QK: greater than 3
hour sample incubation and BioPRYN QK: less than 3 hour
sample incubation) are not for use with the current BioPRYN QK
assay.
Page | 66
Find the text files for your plate readings and drag the files into the cowPIE program.
Each plate should show the standards on the left hand side along with a graph of the
standard curve. If the line achieves an R-squared value of greater than 0.985, results for
the plate will be displayed as Normalized OD values in a grid format with color coding
for the appropriate category. Do not drag the manifest files into the program until a good
fit has been achieved for each plate.
a. Check that you have selected the correct test type after you have brought in your
plate readings by confirming the correct multiplier is displayed.
a. BioPRYN New: greater than 5 hour sample incubation- multiplier = 1.0
b. BioPRYN New: less than 5 hour sample incubation- multiplier = 0.9
b. If the minimum R-squared value is not achieved, the grid will show bad line fit
for all wells. Using the graph and the presented standard OD values, adjust
outlier standards (those farthest from the line fit) to bring the R-squared value
up to the required value. If a slight adjustment of one or two standards does
not bring the plate in line, results should not be reported for this plate. Results
should not be reported for any plate in which the following conditions do not
hold once a good line fit has been achieved:
In the example below on of the standard A od readings is 3.229, the other is 1.7. Both
standard B readings are near 1.8. The R-squared value is 0.728. The second standard A
reading is the outlier. This is recognized because of its value so close to standard B.
Outlier standard A
Page | 67
When the outlier standard A is changed to match the other standard A, a good line fit Rsquared value of 0.998 is achieved. Results for all of the samples are now displayed.
Sample 1
Standard A
Standard B
Standard C
Standard D
Sample 88
Samples 1-88
Sample 1
Standards
Column
Samples 1-88
Standard A
Standard B
Standard C
Standard D
Sample 88
Sample 8
Page | 68
Assay Validation:
Standard A OD > 0.6
PSPB standards Column 1
Standard A OD > Standard B OD> Standard C OD> Standard D OD
Wells A-1, B-1 OD > Wells C-1, D-1 OD > Wells E-1, F-1 OD > Wells G-1, H-1 OD
R2 ≥ 0.98
The ideal fit of the regression equation gives an R2 value of 1.0. Standard results should
be adjusted to improve the fit (R2) of the regression when outliers can be identified.
Calculation of the Cutoff Values and Interpretation of the Results:
The OD of each plate is transformed by a least squares regression of the PSPB standard
column to normalize the results of each plate. Outliers should be adjusted to improve the
fit of the regression equation. The transformed result is compared previously determined
threshold values to categorize each sample result. A multiplier is used to improve data
analysis. Results are reported as normalized “BioPRYN OD”.
Optical densities from each plate are categorized in one of three categories as
outlined below:
Normalized “BioPRYN OD” categorization
High threshold = 0.21
Cut-off = 0.15
Low threshold= 0.135
0.21 < Sample
0.15< Sample < 0.21
0.135< Sample < 0.15
Sample < 0.135
PREGNANT
HIGH RECHECK
LOW RECHECK
OPEN
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When the sample incubation time is less than
hour sample incubation”.
hours choose “BioPRYN New: less than
When the plate is open the multiplier box should show 0.9 rather than 1.0 for this plate
type.
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There are three buttons on the bottom of the CowPIE window.
[Update] will apply a modified standard, multiplier, or repeat threshold value. It is only
necessary to update the results if you modify a value.
[Reset] will revert modified standard values to the original value in the plate reading file.
It will not recall an old multiplier, or repeat thresholds. After you have hit Reset you must
still hit Update to have the results reflect the updated values.
[Close] will close the selected plate reading.
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PLATE ANALYSIS Using New CowPIE with BioPRYNQK
Under the modify tab at the top of the sheet, select plate type and choose the assay type
that matches your process. For analysis of BioPRYNQK(or BioPRYN Sheep/ goat) or
BioPRYN QK choose “Three 0.0 8 and three 0.019 standards” under plate type.
Find the text files for your plate readings and drag the files into the cowPIE
program. The following parameters should be displayed at the top of the file.
i.
ii.
iii.
iv.
BioPRYN QK: multiplier= found on the standards used (usually 0.1 or 0.25),
Low Recheck Threshold= 0.8, High Recheck Threshold= 1.2.
BioPRYN Sheep and Goat: multiplier= 0.45, Low Recheck Threshold= 0.9,
High Recheck Threshold= 1.15.
NOTE: The tabs for BioPRYN QK (BioPRYN QK: greater than 3 hour
sample incubation and BioPRYN QK: less than 3 hour sample
incubation) are not for use with the current BioPRYN QK assay.
For BioPRYNQK and BioPRYN sheep/ goat only those standards in wells B1D1 and E1-G1 are used for analysis.
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Below is the cowPIE program with an open plate reading using “Three 0.0585 and
three 0.019 standards” plate type. The Default settings for the Multiplier, Low
Recheck Threshold and High Recheck Threshold are shown.
High Recheck
Low Recheck
Multiplier box
Threshold
Threshold
Multiplier, Low Recheck Threshold and High Recheck Threshold adjusted for
analysis of BioPRYNQK with a multiplier of X=0.25 (found on standard vial used).
0.8
1.2
0.25
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Assay Validation:
High standards – the maximum permissible difference between the replicates for
standards should not exceed 0.1 OD units.
Low standards – the maximum permissible difference between the replicates for
standards should not exceed 0.1 OD units.
Values which fall outside of this ran e should be considered as “outliers” and not
used in the calculation of the cutoff values.
Calculation of the Cutoff Values and Interpretation of the Results:
HS = average of the High Standard OD’s,
LS = average of the Low Standard OD’s
Cutoff OD = [(HS - LS) (X multiplier)] + LS
Note: The X multiplier may change depending upon the batch preparation of the two
cutoff standards. Check the vial for the correct multiplier.
The Open / Low Recheck Threshold Value is calculated by multiplying the Cutoff
OD by 0.8. The Pregnant /High Recheck Threshold Value is calculated by
multiplying the Cutoff OD by 1.2.
Example with X = 0.25:
HS (avera e of the Hi h Standard OD’s):
0.8
LS (avera e of the Low Standard OD’s):
0.3
Cutoff:
[(0.800 – 0.300) (0.25)] + 0.300 = 0.425
Low recheck Threshold:
(0.425 x 0.80) =
0.340
High Recheck Threshold:
(0.425 x 1.20) =
0.510
The Pregnancy Status of each sample is determined by comparing the OD of the sample
to the OD’s of the Threshold Values:
Open:
Low Recheck:
High Recheck:
Pregnant:
OD < 0.340
0.340< OD < 0.425
0.425< OD < 0.510
OD > 0.510
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MAKING REPORTS AFTER ANALYZING PLATES
BioPRYN and BioPRYNQK
Opening Manifest Files
Once you have the plate reading(s) open and checked over, you can open manifest files.
These can be opened in the same way as plate readings; i.e., one-by-one through the file
menu or by highlighting and dragging.
The manifest will not open if:
1) The manifest is not in the correct format
2) The manifest has empty or invalid essential fields
3) None of the plate filenames referenced in the manifest are open in the program.
Note 1: For a manifest that spans two plates, opening only one of those plate readings
will result in CowPIE processing only those samples listed under the open plate reading.
This will be evident in the final report by comparing the number of samples listed under
Assay/Animal and under Number of Samples.
Note 2: If there is a manifest with a very large number of samples ( > ~100), the
Pregnancy Status display may expand to fill the window, covering up buttons and menus.
To fix this temporarily, minimize the entire program and then open it up again.
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Exporting Reports
CowPIE can export report files in either HTML format or as a spreadsheet (.csv file).
HTML reports are attractive and can be convenient emailed to clients, but the data in a
spreadsheet report is easy to manipulate (a spreadsheet is good if, for instance, you want
to sort the data, or your client wishes to copy the data into a dairy management program).
In order to create reports, simply click on the Export menu, and select either All Results
to Excel or All Results to HTML. You can also export reports one at a time using the
buttons at the bottom of the window.
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If you export the report as an EXCEL file you may want to “pretty it up” for the client.
The file exported directly from Cow PIE looks like:
As with most EXCEL formats, you can position the cursor between the column
designations at the top of the page then, holding down the left-click, drag the column one
way or the other to enlarge or reduce the size of the column.
Keep in mind that the report is exported as a *.csv file, so if you change the format of the
file and try to save it, it will return to the original reduced column format. Additionally,
if you email the report in this format, many clients are unable to do anything with it. To
avoid these problems, once you have expanded the columns and made whatever other
modifications you wish (such as highlighting the column headings and centering the data
as illustrated below), save the file in an EXCEL workbook format NOT in a csv format.
The report can then be emailed to the client as a “true” EXCEL workbook attachment.
Page | 77
Modified report in the EXCEL workbook format.
Exporting reports in HTML format requires no modifications, and can be attached
directly to the email. The report exported in the HTML format should look like this:
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If for some reason you need to change or modify the contents of the report, you can do
this in a number of ways depending on your website browser.
Initially, open the report, left-click on File and look for “Edit with Netscape” to appear in
the drop-down window. The report should open in an editable form of Netscape such as:
Then simply position your cursor to the area you want to edit and make the necessary
modifications, in this example Assay/Animal “Black Angus” was chan ed to “Cattle”
from the previous image to this image.
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Left-click on File, then Save (or Save As if you want to change the filename), and the
modified report will appear in the daily folder.
With some browsers, the Edit function may not be available. If this is the case, try rightclicking on the report, select Open With on the drop-down menu, then click on Microsoft
Office Word. This should open the report in a Word format which you can then edit and
save.
These options are particularly useful for those clients whose email rejects any
attachments, and you need to place the report in the body of the email. In some cases you
can simply open the report, left-click and hold down to highlight the text, press Ctrl + C
to copy, then paste into the body of the email. If this does not work for some reason, try
opening the report in one of the two edit modes, and cut and paste from this into the body
Page | 80
of the email. You may need to experiment with these options to see which works for
your clients and the type of email service provider you have.
Troubleshooting
The principle problem encountered with generating reports is the appearance of one or
both of the following error messages when opening manifests in the CowPIE program:
In either case, the error message does not necessarily give you specific information on the
type of error in the manifest, just that one is present. The best approach is to open the
manifest in question and go through it line by line looking for a formatting error. These
errors are generally one of the following:
1. Incomplete or missing information in one or all of the Essential Fields (see p. 2)
2. A formatting error in the Plate Filename. This must match EXACTLY the name
given to the .txt file of the plate reading. If the file is named BT121.txt, the input
in the Manifest must be BT121.txt. Case and spacing are important: Bt121.txt
will not work, nor will BT 121.txt. One error that is easy to overlook is a space
on one side or the other of the “.” precedin the txt.
3. Incorrect spacing in the adding of multiple plates to a manifest (see p. 5).
4. Saving of the manifest as an EXCEL worksheet format instead of the Comma
Separated Values (csv) format.
Sometimes there are no apparent errors in a Manifest and it still will not open. In this
instance, sometime it can be solved by simply opening the Manifiest, clicking on File, the
Save As, and saving it with the same name, making sure the Save as type window shows
the CSV (Comma Delimited) as the file type.
If this does not work, open up the blank manifest for the client, cut and paste all of the
information from the old Manifest into the new one, and save it by overwriting the
original manifest in the daily folder.
Page | 81
Section 7 – Kit Handling and Shipping
Page | 82
INTRODUCTION
Now that you know all about the BioPRYN test, how do you get your hands on the kit to
run the assay in your own laboratory? And what will come with the kit? The following
section will give you more information about these two concepts.
The BioPRYN Kit
The BioPRYN kit comes sold in 5 or 10 plate iterations. The materials in the kit can vary
from this list if shipment is to a foreign country depending upon importation laws. Check
your packing slip for exact kit contents in this situation. Each kit contains the following:
Components of the Assay
A PSPB Standard A
B PSPB Standard B
C PSPB Standard C
D PSPB Standard D
E Antibody coated plates
F SAMPLE BUFFER (RED) # 1
G DETECTOR SOLUTION (BLUE) #2
I ENHANCER SOLUTION (GREEN) #3
J TMB Substrate Solution #4
K STOP SOLUTION (YELLOW) #5
L 20X Wash Buffer concentrate W
Quantity Shipped
1 vial
1 vial
1 vial
1 vial
5 or 10
1 bottle
1 bottle
1 bottle
1 bottle
1 bottle
1 bottle
The BioPRYNQK kit
The BioPRYNQK kit comes sold in 3 or 6 plate iterations. The materials in the kit can
vary from this list if shipment is to a foreign country depending upon importation laws.
Check your packing slip for exact kit contents in this situation. Each kit contains the
following:
Components of the Assay
A PSPB Standard: QUICK High Std Green
B PSPB Standard: QUICK Low Std Light Green
C QUICK Sample Buffer #1
D QUICK Detector Solution #2
E QUICK Enhancer Solution #3
F QUICK TMB solution #4
G QUICK STOP solution #5
H 20X Wash Buffer concentrate
I Antibody coated plates
Quantity Shipped
2 vials
2 vials
1 bottle
1 bottle
1 bottle
1 bottle
1 bottle
1 bottle
3 or 6
Page | 83
Ordering bioPRYN plates
When ordering please order in advance to insure that I have 5 business days to get the
order sent. Most of the time I ship before the required 5 business days, but we are unable
to guarantee we will be able to fulfill your order in less than 5 business days. I understand
at times we will have emergencies. Call me and we can figure something out.
As a reminder, New BioPRYN is sent in kits of five and ten.
My phone number is 208-596-0443, I encourage you to call me anytime to place an
order, check on your order, and let me know when I have made a mistake. If you get my
voicemail please leave me a message and I will call you back as soon as possible.
You may also send me an email at [email protected]. I check this email
every morning. You may order through this email as well, hopefully ensuring that I get
your order in a timely manner.
The fax number for BioTracking is 208.882.1490. You can fax orders using the Plate Kit
Order Form. Should you need to talk to one of the scientists, lab technicians, or can’t et
a hold of me the office number is 208-882-9736.
We ship using FedEx and I usually send out orders on Monday, Tuesday, and Wednesday
with second day delivery. Internationally I usually ship Fridays. If you need an order
with overnight delivery we can do that too, but it does cost quite a bit more.
Please don’t hesitate to call me, and I am lookin forward to workin with you.
Thank you,
Lorri Sasse
Shipping Manager
BioTracking LLC
Page | 84
Ordering Information
To order a kit, please fax the order or call the BioTracking Shipping Manager at the
BioTracking phone number. Orders may also be sent by email, but it is a good idea to
follow up an email with either a fax or a phone call to determine whether the email was
received. Each kit order requires a minimum of five business days for processing and
shipping. Your kit will be shipped by the end of these five days. Kits are only shipped
Monday, Tuesday, or Wednesday using Fed Ex 2nd day. International orders are
usually shipped Friday. Other shipment days will require overnight shipping, paid by
the customer. Please examine your kits upon receipt and call BioTracking with any
questions or concerns.
Tips / Notes
– Upon receipt verify that all kit components are packed and the appropriate
volumes have been provided
– All components of your kit can be stored together at 4°C
– ***Make note of multiplier on standards. Always use standards from a
matching lot number and make sure the appropriate multiplier is used when
analyzing data
– Evaluate integrity of plate packaging and record any problems
Below are examples of a BioPRYN 5 plate kit and a BioPRYNQK 3 plate kit packing list:
Page | 85
PACKING SLIP FOR NEW Plate Kit
Date Packaged February 27, 2012
Packed By
___________________
Item
Description
Amount
Storage
Initials
1.
PSPB Standard A
Lot # 030312
PSPB Standard B
Lot # 030312
PSPB Standard C
Lot # 030312
PSPB Standard D
Lot # 030312
SAMPLE BUFFER (RED) #1
Lot # 030312
DETECTOR SOLUTION (BLUE) #2
Lot # 030312
ENHANCER SOLUTION (GREEN) #3
Lot # 030312
TMB Substrate Solution #4
Lot # 030312
STOP SOLUTION (YELLOW) #5
Lot# 030312
Wash Buffer W
Lot # 030312
Antibody Coated Microtiter Strip Plates
1 Vial
+4O C
_______
1 Vial
+4O C
_______
1 Vial
+4O C
_______
1 Vial
+4O C
_______
1 bottle
+ 4O C
_______
1 bottle
+ 4O C
_______
1 bottle
+4O C
_______
1 bottle
+ 4O C
_______
1 bottle
+ 4O C
_______
1 bottle
Room Temp
_______
+ 4O C
_______
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Plate #’s: ( N-N )
12.
Assay Protocol, Excel File and Instructions
_______
Page | 86
PACKING SLIP FOR Quick Plate Kit
Date Packaged March 1, 2012
Packed By
Item
Description
Amount
1. PSPB Standard: Quick High Std. 2 Vials
Lot #180112
2. PSPB Standard: Quick Low Std.
2 Vials
Lot #180112
3. Quick Sample Buffer #1
1 bottle
Lot # 300911
4. Quick Detector Solution #2
1 bottle
Lot # 170212
5. Quick Enhancer Solution #3
1 bottle
Lot# 170212
6. QUICK TMB Substrate #4
1 bottle
Lot# 010112
7. Quick STOP Solution #5
1 bottle
Lot#010112
8. Wash Buffer W
1 bottle
Lot # 010112
9. Microtiter Plates coated with Anti-PSPB serum
___________________
Storage
Initials
+4O C
_______
+4O C
_______
+ 4O C
_______
+4O C
_______
+ 4O C
_______
+ 4O C
_______
+ 4O C
_______
Room Temp
_______
+ 4O C
_______
Plate #’s: ( Q414-Q422 )
_______
Assay Protocol and Instructions
Note: The multiplier X for Lot #180112 is 0.25
Page | 87