2) reduced incubation time 3) small semen sample volume

EPISCREEN PLUS™ - -GLUCOSIDASE ASSAY (25 TESTS )
2) reduced incubation time
3) small semen sample volume
Update: 12/03/2012, Document ref.: FP09 I87 R01 A.2
4) inclusion of a standard curve
ABBREVIATIONS
5) semen plasma background correction.
CLSI
CV
IU
LOD
LOQ
NAG
OD
PNP
PNPG
WHO
Clinical and Laboratory Standards Institute
Coefficient of variation
International unit
Limit of detection
Limit of quantification
Neutral -glucosidase
Optical density
Para (4)-nitrophenol
Para (4)-nitrophenyl-α-D-glucopyranoside
World Health Organization
Table 1 shows a comparison between Episcreen™, EpiScreen PlusTM,
and the WHO method7.
Table 1: Comparison of Episcreen™ , EpiScreen Plus™ and the WHO assay
for the determination of -glucosidase activity in semen.
Enzyme activity
Total
(neutral +
acid)
EpiScreen
TM
Plus
Neutral
(epididymisspecific)
Incubation
period
4h
2h
2h
The enzymatic activity of at least 25 samples (including sample
background correction) can be assessed with one EpiScreen Plus TM kit.
Standard curve
-
Yes (PNP)
Yes (PNP)
Kit contents:
Reagent 1 (5 ml): reaction buffer (pH 6.8), supplemented with 1% SDS
Background
correction
-
Glucose
Castanospermine
Sample volume
125 µL
20 µL
15 µL
KIT COMPOSITION
Parameter
TM
EpiScreen
WHO
Neutral (epididymisspecific)
Reagent 2 (0.15ml): 50 X substrate solution (PNPG in DMSO)
Reagent 3 (5ml): inhibitor solution (reaction buffer containing glucose)
Reagent 4 (60ml): stopping buffer (0.02M NaOH)
Reagent 5 (1ml): standard stock solution (5 mM PNP)
Reagent 6 (60ml): standard dilution buffer (0.02M NaOH + 0.1% SDS)
MATERIAL NOT INCLUDED WITH THE TEST



Spectrophotometer with 405 nm filter
Warm water bath /reaction tube thermoshaker
Plasticware (96-well microtiter plates, pipette tips, 1.5 ml eppendorf
tubes)
BACKGROUND
The bulk of -glucosidase activity in semen, and more particularly that
of its neutral iso-enzyme, depends on secretion by the epididymis1. In
patients with azoospermia and normal androgen levels in peripheral
blood, NAG activity in semen plasma is a reliable marker of the
epididymal contribution to the ejaculate.
Azoospermic males with bilateral obstruction between the epididymis
and the ejaculatory duct have very low -glucosidase activity in their
seminal plasma2. In contrast, if azoospermia is due to an arrest of
sperm maturation, or obstruction situated between the epididymis and
the rete testis, or in the rete testis itself, -glucosidase activity is
normal. Hence, NAG assessment in seminal plasma of normally
virilized men with azoospermia can differentiate between the major
causes of this condition3, 4.
Low NAG activity in seminal plasma of patients with oligozoospermia
may reflect partial obstruction of the epididymides, associated with
infections or inflammatory disease2, 5. Enzyme activity in patients with
normal sperm concentration is correlated with the result of the Shorrstain of midpiece and tail, reflecting changes The EpiScreen Plus TM
assay may assist in the diagnosis and the management of male
infertility.
ASSAY PRINCIPLE
Under specified conditions (pH=6.8; T=37°C), -glucosidase will
catalyze the conversion of the substrate 4-nitrophenyl-α-Dglucopyranoside (PNPG) to α-D-glucopyranoside and 4-nitrophenol, as
shown below. The yellow colour of the latter product is measured
spectrophotometrically at 405 nm.
EpiScreen PlusTM and the WHO method, have been tested in parallel
on 144 semen plasma samples, obtained from 94 patients / donors and
50 vasectomized men (negative control), respectively. Method
comparison demonstrated that both assays yield identical results 8. The
EpiScreen PlusTM method differs slightly from the WHO method: a)
reaction buffer (pH 6.8) composition, and b) the use of glucose instead
of castanospermine as inhibitor for blank correction.
Inhibition of -glucosidase by glucose, a process called product
inhibition, is a pH-dependent phenomenon and can be explained by
glucose binding to the monosaccharide binding site of -glucosidase9.
Our experimental data have shown a dose-dependent reduction of
enzyme activity. The glucose concentration in EpiScreen PlusTM was
optimized to yield identical inhibition as castanospermine.
The WHO advises to apply only two internal quality control samples for
blank correction. Our experiments have demonstrated that the
background variance of semen samples is quite large (+/- 20%).
Therefore, we recommend preparing a negative control for each semen
plasma sample in order to allow correct and reproducible background
correction.
ASSAY PERFORMANCE
All validation parameters have been calculated based on the latest
CLSI guidelines10, 11.
Measuring range: 2.32-144 mU/ml
Sensitivity: 96.0 % (vasectomized/normozoospermic)
Specificity: 93.6 % (vasectomized/normozoospermic)
Within-device Precision:
Low pool: 0.96 mIU/ml
High pool: 3.70 mIU/ml
Intra-assay CV: 3.08 %
Inter-assay CV: 10.52%
Cutoff: 6.35 mIU/ml
19.98 mIU/ejaculate (if corrected for ejaculate volume)
STORAGE AND SHELF LIFE
PNPG + α-glucosidase  α-D-glucopyranoside + PNP (yellow)
As the reaction buffer contains SDS, the acid form of -glucosidase
(originating from the prostate), is selectively inhibited. This allows
specific determination of neutral enzyme activity6.
ENZYME ACTIVITY DEFINITION
Alpha-glucosidase activity is expressed as IU per liter (or mIU per ml).
One unit is able to liberate 1 µmolar of PNP from PNPG per minute
under specified conditions (pH=6.8; T=37°C)7.
ASSAY CHARACTERISTICS
EpiScreen PlusTM has been designed to assess neutral enzyme activity.
The main advantages of the new kit are:
1) assessment of neutral activity
EpiScreen PlusTM must be stored at 2-8°C, protected from (sun)light,
and remains stable for 24 months.
PRE-USE CHECKS
Do not use the product if seal of the container is opened or defect when
the product is delivered. When stored at 4°C, precipitation may occur in
reagent 1 but disappears by prewarming to 37°C.
SPECIMEN TYPE
The assay can be performed on fresh or frozen/thawed semen and
semen plasma samples.
METHOD
Before use, allow reagents 1, 2, and 3 to warm up to 37°C for 30min.
We recommend a thermoregulated hot water bath or reaction tube
thermoshaker for optimal heating of the samples. DO NOT incubate in
an air incubator as this may impair assay outcome.
Perform the following steps:
1. For each sample to be analyzed:
- make reaction solution: 3µl of reagent 2 in 147µl of reagent 1
- make inhibitor solution: 3µl of reagent 2 in 147µl of reagent 3
2. Pipette 20µl of each sample into two 1.5ml Eppendorf tubes
3. Add 130µl reaction solution to one reaction vessel and 130µl inhibitor
solution to the other.
4. Vortex and incubate for exactly 2h at 37°C
5. During incubation, prepare the standard curve. Make the highest
standard (200 µM) by dissolving 100 µl of reagent 5 in 2400µl of
reagent 6. Use this solution to prepare the other standards, as
indicated in table 2. Reagent 6 alone serves as 0 standard (blank).
Table 2: Standard curve of PNP
200 µM Standard (µl)
Reagent 6 (µl)
Final concentration (µM)
375
125
150
250
250
100
125
375
50
25
475
10
0
500
0
6. After 2h, stop incubation by adding 1ml of reagent 4 and vortex.
7. Pipette 200 µl of all standards / samples into a microtitre plate.
8. Read the absorbance at 405nm.
DATA INTEGRATION
STANDARD CURVE
Subtract the blank OD value (0-standard) from the standard OD values
(delta OD). Plot the delta OD values (Y-axis) against the standard
concentrations (X-axis) and perform a linear regression to calculate the
slope. Coefficient of determination (R2) should be at least 0.99. An
example is shown below.
Standard curve PNP
y = 0.0097x
R² = 0.9999
2.500
OD 405 nm
2.000
1.500
1.000
0.500
0.000
0
50
100
150
200
250
PNP Concentration (µM)
UNKNOWN ENZYME ACTIVITY
Subtract the blank OD value (0-standard) from the OD value of each
sample and its corresponding background (glucose) control. Next,
subtract the background delta OD from the sample delta OD. Calculate
the corresponding PNP concentration by dividing the backgroundcorrected OD value with the slope of the standard curve. Finally,
enzyme activity (mIU/ml) is obtained by multiplying the PNP value with
0.479 (see below).
Example:
1.
Assay data: ODsample = 0.845; OD sample + glucose = 0.06;
standard curve slope = 0.0097
2.
Semen plasma background correction = 0.845 – 0.06 = 0.785
3.
Concentration PNP = 0.785 / 0.0097 = 80.93 µM
4.
Enzyme activity = 80.93 x 0.479 = 38.76 mIU/ml
Calculated enzyme activity can be multiplied with ejaculate volume, to
evaluate enzyme activity in the whole ejaculate.
Note:
The standard curve consists of distinct points between 0-200 µM, as
most semen samples lie within this range. Based on our experimental
data, linearity is guaranteed up to 300 µM. If desired, the operator can
alter the curve by starting at 300 µM, corresponding to an enzyme
activity of 144 mIU/ml. If unknown samples have higher activity, we
advise to dilute and retest to confirm experimental outcome.
CORRECTION FACTOR
This factor is obtained by taking into account sample dilution and
incubation time (120 min). In this assay, one starts with 20 µL of semen
sample, which is finally diluted to 1150 µL (20 µL semen sample + 130
µL reaction buffer + 1000 µL stop buffer). This results in a dilution factor
of 57.5. An enzyme unit is defined as the formation of PNP per minute.
Therefore, one has to divide the end result with 120 in order to
implement time factor.
PRECAUTIONS
This test is an aid in the diagnosis and, as for other biological tests;
interpretation of the results must be performed within the framework of
clinical findings and data of history taking. Other causes of insufficient
epididymal secretion must be excluded, such as hypo-androgenism or
severe testicular atrophy.
The material safety datasheet (MSDS) is available on our website.
BIBLIOGRAPHY
1. Cooper TG, Yeung CH, Nashan D, Jöckenhovel F, and Nieschlag
E. (1990) Improvement in the assessment of human epididymal
function by the use of inhibitors in the assay of alpha-glucosidase in
seminal plasma. Int. J. Androl., 13: 297-305.
2. Guerin JF, Ben Ali H, Rollet J, Souchier C, and Czyba JC. (1986)
Alpha-glucosidase as a specific epididymal enzyme marker. Its
validity for the etiologic diagnosis of azoospermia. J. Androl., 7:
156-162.
3. Casano R, Orlando C, Caldini AL, Barni T, Natali A, and Serio M.
(1987) Simultaneous measurement of seminal L-carnitine, alpha 14-glucosidase and glycerylphosphorylcholine in azoospermic and
oligospermic patients. Fertil. Steril., 47: 324-328.
4. Cooper TG, Yeung CH, Nashan D, and Nieschlag E. (1988)
Epididymal markers in human infertility. J. Androl., 9: 91-101.
5. Haidl G, Badura B, Hinsch KD, Ghyczy M, Gareiss J, Schill WB.
(1993) Disturbances of sperm flagelle due to failure of epididymal
maturation and their possible relationship to phospholipids. Hum.
Reprod., 7: 1070-1073.
6. Paquin R, Chapdelaine P, Dubé JY, Tremblay RR (1984) Similar
biochemical properties of human seminal plasma and epididymal
alpha-1,4-glucosidase. J. Androl., 5: 227-282.
7. WHO laboratory manual for the examination and processing of
human semen, 5th edition. Measurement of neutral alphaglucosidase in seminal plasma. pp. 134-136.
8. Eertmans F (2012) White paper: EpiScreen PlusTM for assessment
of neutral -glucosidase activity in semen: an update. FertiPro NV.
9. Yao X, Mauldin R, Byers L. (2003) Multiple sugar binding sites in αglucosidase. Biochim. Biophys. Acta, 1645: 22-29.
10. Shrivastava A, Vipin B Gupta VB (2011) Methods for the
determination of limit of detection and limit of quantitation of the
analytical methods. Chron. Young Sci. 2: 21-25.
11. Chesher D. (2008) Evaluating Assay Precision. Clin Biochem. Rev.,
29: S23-S
TECHNICAL SUPPORT
FERTIPRO NV
Industriepark Noord 32, 8730 Beernem, Belgium
http://www.fertipro.com - E-mail: [email protected]
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