Radioimmunoassay - Clinical Chemistry

Transcription

CLINICALCHEMISTRY, Vol. 19, No.2, (1973) 146-186
Radioimmunoassay
D. S. Skelley,
L. P. Brown,
and P. K. Besch
Principles
of radioimmunoassay
are discussed.
Methods are outiined for preparing radiolabeied antigenic compounds and characterizing antibodies. The
various techniques
for separating
bound and free antigen are also reviewed.
Commercial
suppliers of
various components
needed for radioimmunoassay
are listed. Also presented is a selected list of normal
values for hormonal
from humans.
and nonhormonal
substances
Additionai Keyphrases: competitive
protein-binding
analassay
#{149}cross-reactivity
antigenic
radiolabeling
#{149} antibody
induction
separation
techniques
#{149}analytical
considerations
normal human values
#{149}
technical variations of AlA
ysis
#{149}
immunoradiometric
The technique
of RIA,1 first developed
for the
measurement
of hormones
(see reviews 1-20), has expanded
durjng the past decade to include the detection of other biological
agents. The chemist
can well
appreciate
the fact that antisera
or complete
commercial kits are now available
for clinical use and are
revolutionizing
the entire
field
of investigational
analyses.
Sources
for the purchase
of complete
RIA
kits or antisera
for either clinical or research
ise are
listed in Table 1.
All RIA procedures
are based on the original
observation
by Berson
and Yalow (19, 21-25) that low
concentrations
of antibodies
to the antigenic
hormone, insulin,
could be detected
by their ability to
bind radiolabeled
(3I)
insulin. Unknown
concentraFrom the Department
of Obstetrics
and Gynecology,
Baylor
College of Medjcine, and The Reproductive Research Laboratory,
St. Luke’s Episcopal
Hospital,
Texas Medical Center, Houston,
Texas 77025.
‘Nonstandard
abbreviations
used: ifiA, radioimmunoassay,
also referred to as radioimmune
assay, competitive
radioassay,
radiostereoassay,
or immune radiometric
analysis; Ag, antigen;
Ab,
antibody;
CPBA,
competitive
thyroxine-binding
globulin;
(H)FSH,
follicle-stimulating
(human)
luteinizing
human
hormone;
chorionic
TSH,
protein
ACTH,
hormone;
gonadotropin;
bovine
serum
albumin;
EDTA,
HSA,
assay;
TBG,
HCG,
ethylenediaminetetraace-
human
placental
serum
PTH,
parathyroid
hormone;
Prog,
albumin;
RSA,
CEA,
1g.
progesterone;
DHEA, dehydroepiandrosterone;
E,, estrone; E2, estradiol-17$;
E3, estriol; Aldo, aldosterone;
PGF2a, prostaglandin
F2a; cAMP
and cGMP, cyclic AMP and guanosine 3’-5’-cyclic monophosphate; aFP, alpha fetoprotein; and LSD, lysergic acid diethylamide; and cpm, counts per minute.
146
CLINICAL CHEMISTRY, Vol. 19, No.2, 1973
BOUND
Ag
#{149}
Ag
#{149}
Ab
II
human
lactogen);
rabbit serum albumin; T3, triodothyronine;
T4, thyroxine;
carcinoembryonic
antigen; HAA, hepatitis-associated
antigen;
linmunoglobulin;
The literature
on radioimmunologic
procedures
has given rise to a new set of terms that are often
confusing
to the novice.
RIA is one form of “displacement
analysis”
(27)
or “saturation
analysis”
(human)
hormone;
hormone; HCS(HPL),
(human
Terminology
FREE
hormone;
(H)LH,
thyroid-stimulating
tic acid; (H)GH, (human) growth
chorionic
somatomammotrophin
BSA,
binding
adrenocorticotropic
tions of antigen
may be determined
by taking advantage of the observation
that the radiolabeled
hormone molecules
or “tracer”
(Ag*I) compete
physicochemically
with the nonlabeled
hormone
molecules
(Agil)
(either
standards
(S) or unknowns
(U)) for
binding
sites on the antibodies
(Abifi)
(25, 26) (see
Figure 1). Moreover,
the assay requires
that behavior
between
the standard
and unknown
antigen be identical in their ability to displace
labeled antigens
from
a
labeled
antigen-antibody
immune
complex
(Ag*AbIV),
but not identical
behavior
between
the
tracer and the unknown
or standard
antigen.
When
increasing
amounts
of unlabeled
antigen
(Agil) are
added to the assay, the limited
binding
sites of the
antibody
(Abifi)
are progressively
saturated
and the
antibody
can bind less of the radiolabeled
antigen
(Ag*I). The antibody
solution,
or antiserum,
js diluted to allow about 50% of the tracer dose of Ag*I to
be bound
in the absence
of unlabeled
standard
or
unknown
antigen.
If there is no nonspecific
inhibition of the immunochemi#{231}al reaction,
a diminished
binding
of labeled
antjgen
offers evidence
for the
presence
of unlabeled
antigen.
After an incubation
of
the three
essential
components
(Ag*I,
Agil,
and
AbilI),
the antigen-antibody
complexes,
or bound
antigens
(Ag*AbIV
and AgAbVI)
are separated
from
the free antigens
(AgV and Ag*Vll) and the radioactivity of either or both phases is measured.
,IV
/
AgAb
V
llt::
F
AgAb
F/I
I
I
II
IV
V
VI
VII
Ag.
Ag
Ab
Ag’Ab
Ag
Ag Ab
Ag’
Ag
*
Ag
VII
Labeled Antigen (e.g. Hormone Xl
Unlabeled Antigen (e.g. Hormone Xl Standard IS) or Unknavn (UI (H,)
Antibody to Ag land Ag’)
Labeled Antigen Bound to Antibody )B)
Free Unlabeled Antigen
Unlabed Antigen Bound to Antibody
Freel.abeled Antigen IF’)
Fig. 1. Diagram
showing
the various
components
of AlA
Table 1. Sources of Antisera and Radioimmunoassay Materials
Antigen
Isotope
ACTH
Aldosterone
Corn-
Method of
Separation5
Sampl&’
Sensitivity
1251
P
DCC
10 pg/mI
1251
S
DA
1-2 pg/mI
DCC
5 pg
3H
Supplier
ment
Amersham/Searle
2626 S. Clearbrooke Drive
Arlington Heights, III. 60005
Inter Science Institute
2000 Cotner Avenue
Los Angeles, Calif. 90025
Sorin
Radioisotopic
Saluggia (Vercelli)
National Institute of Arthritis
and Metabolic Diseases
Hormone Distribution Officer
DCC
c,d
Office of the Director
Bldg. 31-9A47
National Institute of Health
Bethesda, Md. 20014
P, U
(NH4)2S04
C
U
Gelatin
Antibodies,
C
Inc.,
Route 1, Box 1482
Davis, Calif. 95616
P. S
Endocrine ServicesProducts Division
18418 Oxnard
C
Street
Tarzana, Calif. 91356
Research Plus Steroid
p, 5
C
Laboratories
P.O. Box 571
Denville, N.J. 07834
Spectrum Medical
c
Industries
P.O. Box 60916 Terminal
Annex
Androstenedione
Angiotensin I
(Renin7
1251
1251
P, S
P(EDTA)
P, S
DCC
DCC
0.5 ng/mI
0.05 ng
Endocrine Services
Sorin
Schwarz/Mann
Mountain
1251
P(EDTA)
DA
0.03 ng
1251
P
DCC
10 pg/mI
(Renin)
Avenue
Orangeburg, N.Y. 10962
Clinical Assays, Inc.
237 Binney Street
Cambridge, Mass. 0’2142
New England Nuclear
Biomedical Assay
Laboratories, Inc.
15 Harvard
Angiotensin
I
1251
P
Charcoal
1 ng/ml
Street
Worcester, Mass. 01608
E. R. Squibb & Sons, Inc.
Radiopharmaceutical
New Brunswick,
Angiotensin
CEA
II
1251
DCC
Dept.
N.J. 08903
Sorin
Cordis Laboratories
P.O. Box 684
Miami,
1 ng/ml
Corticosterone
C
C
Fla. 33137
P, S
Spectrum Medical
Industries
Endocrine Services
C
P, S
Research Plus Steroid Labs
C
c
Cortisol
Cortisone
CLINICAL CHEMISTRY, Vol. 19, No. 2, 1973
147
Table 1. continued
Antigen
Method of
Separation5
Corn-
Isotope
Sarnpl&’
1125;
P, E, U
0.05-0.10
Schwarz/Mann
U, H
2.0 pmol
Collaborative Research, Inc.
Cyclic AMP
S,
125l
sensitivity
pmol
Supplier
mint
e
Research Products Division
1365 Main Street
Waltham, Mass. 02154
Cyclic GMP
Deoxycorticosterone
Collaborative Research, Inc.
Research Plus Steroid Labs
“
P. S
DA
P, S
Endocrine Services
C
1251
0.05 pmol
Deoxycortisol
DHEA
Dihydrotestosterone
Digitoxin
3H
3H
P, S
P, S
S
DCC
DCC
DCC
4.0 ng/ml
5.0 ng/ml
0.5 ng
3H
P
Albumin-coated
charcoal
DCC
DCC
0.5 ng/mI
3H, 1251
ii,
125;
P. S
P. S
5.0 ng/mI
5 ng/ml
S
DCC
1.0 ng/ml
P,S
P, S
DCC
1.Ortg/mI
0.5 ng/ml
P, S
S
P
0.5 ng/ml
0.1 ng/mI
0.05 ng/mI
P,S
DCC
DCC
Albumin-coated
charcoal
DCC
Sorin
Kallestad Laboratories, Inc.
4005 Vernon Avenue
Minneapolis, Minn. 55416
Bioware, Inc.
P.O. Box 8152
Wichita, Kan. 6728
Meloy Laboratories
Biological Products Division
6715 Electronics Dr.
Springfield, Va. 22151
Schwarz/Mann
Kallestad Laboratories, Inc.
4005 Vernon Avenue
Minneapolis, Minn. 55416
Wien Laboratories
New England Nuclear
S
SP-Ab(tube)
1.Ong/mI
O.2ng/ml
0.1 ng/ml
Sorin
Bioware, Inc.
Curtis Nuclear Corp.
0.1 ng/ml
Meloy Laboratories
5 ng/mI
3H
Digoxin
3H,
3H
1251
3H
3H
3H
3H,
1251
1251
Schwarz/Mann
Wien Laboratories, Inc.
41 Honeyman Drive
Succasunna, N.J. 07876
New England Nuclear
c
c
1948 East 46th St.
3H
EstradioI-17f
Estriol
DCC
P
P, S
p, 5
(NH4)2S04
P. S
p, S
1251
S
Hepatitis B Antigen
1251
S
148
CLINICAL
(NH4)2SO4
A
Gastrin
HAA
DCC
SP-Ab(tube)
1251
CHEMISTRY,
Vol. 19, No.2,
Endocrine Services
Laboratories
Spectrum Medical Industries
Endocrine Services
Laboratories
P
P, S
p, S
Estrone
Fibrinopeptide
S
1973
C
C
C
c
C
C
Spectrum Medical Industries
Endocrine Services
Laboratories
C
Spectrum
c
Medical
Industries
Sorin
Abbott Laboratories
North Chicago, III. 60064
Abbott Laboratories
C
C
C
g
Table 1. continued
Antigen
HCG
Isotope
125;
HFSH
1251
HGH
1251
1251
DA
1.5 mIU/ml
Serono Immunochemicals
607 Boylston Street
Boston, Mass. 02116
P. 5
DA
1 mlU
Calbiochem
1.5mlU/mI
P.O. Box 12087
San Diego, Calif. 92112
Serono
U
P, S
S. E
1251
1251
S
DA
DA
DA
0.1 ng
1.0-5.0 ng/mI
DA
Filtration
Corn.
ment
Supplier
Sensitivity
s, U
p, S
S
125;
Method of
Separation5
Sample#{176}
0.5 ng/mI
1.0 ng/ml
h
NIH
Schwarz/Mann
d,i
Collaborative
Research,
Inc.
Sorin
Curtis Nuclear Corp.
1948 East 46th St.
NIH
d,J
Abbott Laboratories
P, S, U
0.025 ng/mI
Arnel Products Co.
2101 AvenueZ
C
P.O. Box 159
Brooklyn, N.Y. 11235
The Sylvana Corp.
22 East Willow Street
Milburn, N.J. 07041
HLH
1251
P, S
S, U
DA
DA
0.5 mlU
1.5mlU/ml
Calbiochem
Serono
DA
HPL (HCS)
DA
1251
DA
1.0 ng
0.1-1.0 ng/ml
5.0 ng/ml
1251
P, S
Alcohol
1.0 ig/ml
125;
P
DCC
0.5 pg/mI
S
DA
2 MIU
Calbiochem
NIH
S
SP-Ag
10 U/mI
Pharmacia
Inc.
HTSH
IgE
NIH
P, S
S
P, S
1251
1251
1251
Schwarz/Mann
Collaborative
Sorin
Research,
Inc.
d
d,J
Laboratories,
Cordis Labs
1251
1251
1251
1251
S
P, U, H
S
P
1251
1251
1251
P
SP-Ag
DA
Ethanol
DA
Amberlite
DCC
DA
3 tzU/ml
1.0.U
resin
Fuglebakkevej
Copenhagen,
Denmark
Sorin
Sorin
Sorin
Ambersham/Searle
Curtis
10U/ml
10 fLU/mI
10 fLU/mI
20 izU/ml
DA
1251
Cappel Laboratories,
Inc.
Downington,
Pa. 19335
Amersham/Searle
Microfiltration
1251
P, S. U
0.025 ng/mI
Arnel Products
P, 5, U
(2.5 fLU/mI)
1014 moles
Collaborative
DA
C
Pharmacia Laboratories
Schwarz/Mann
Novo Research Institute
1251
LSD
d,i
NIH
Pharmacia Laboratories,
Inc.
800 Centennial Avenue
Piscataway,
N.J. 08854
New England Nuclear
Immunoglobulins
(lgG, IgA, 1gM,
lgD, IgE, IgG subgroups, Bence
Jones proteins)
Insulin
C,t
C
Co.
Research,
Inc.
149
Table 1. continued
Antigen
isotope
Lupus
erythematosus
3H or
C14
Morphine
3H,
1251
Sample#{176}
Method of
Separation5
P, S
(N H4) 2504
P, 5, U
(NH4)2S04
3H
P. S
DCC
(NH4) 2S04
5 ng/mI
6Ong/mI(3H)
Roche Diagnostics
Hoffmann-LaRoche,
Nutley, N.J. 07110
0.5 ng/mI
30 pg
Endocrine Services
Laboratories
P. S
p, 5
Prostaglandin
E
Fia
F2a
3H
3H
3H
5, E
S, E
5, E
3H
3H,
S
Testosterone
1251
Inc.
40 pg
100 pg
300 pg
(NH4) 2S04
DCC
DCC
C
Sorirt
DCC
3H
m
Virgo Reagents
Electronucleonics
Laboratories
4905 Del Ray Avenue
Bethesda, Md. 20014
(1251)
Ouabain
Progesterone
Cornmint
Supplier
Sensitivity
50 pg
P, S
p, 5
C
C
Wien Laboratories
Sorin
Cappel Laboratories, Inc.
Endocrine Services
Laboratories
0.5 ng/ml
Bioware,
Thyroxine
(T4)
1251
P, S. U,
DA
0.01 fLg/ml
Pantex
P.O. Box 6007
Inglewood, Calif. 90301
Triiodothyronine
1251
P, 5, U,
DA
0.25 ng/mI
Pantex
C
C
C
C
Inc.
(T3)
#{176}
P, plasma; U, urine; E. ethylenediaminetetraacetic
acid; S. serum; E,
extract; H, homogenate
“DCC. dextran-coated
charcoal; DA. double antibody; SP-Ab, solidphase antibody; SP-Ag, solid-phase antigen.
C Supplies antisera
only.
id For research Investigation only.
e Measured
as succinyl cyclic AMP tyrosine methyl ester (SCAMPTME).
I Measured as succinyl Cyclic GMP tyrosine methyl ester (SCGMPTME).
S Antibody labeled with 1251.
(28-30) [terms originating
with the insulin
(31, 32)
and thyroxine
(33) assaysl,
which specifies
that a
limited
quantity
of antibody
(AbilI)
is added to an
excess of antigen
(Ag*I and Agil). It is furthermore
assumed
that the two forms of the antigen
compete
for binding
sites on the antibody
according
to the
law of mass action (34). The antigen (Age! and Agil)
may also be referred
to as the first molecule
and the
antibody
as the second
molecule
or “specific
reactor.” “Radio-ligand
binding
analysis”
(35) defines
the second
molecule
as a protein
or a nonprotein
chelating
agent and the first orsmaller
molecule
(the
ligand) as the source of radioactivity
(Ag*I).
However,
the “specific
reactor”
or second molecule
may take the form of an enzyme
[e.g., folic acid reductase
(36,
37)],
in which
case the method
is
150
“Antigen supplied unlabeled.
‘Unlabeled antigen, antibody and standard available.
J Unlabeled antigen and antibody available.
Measured as Prostaglandin B1.
‘Antibodies are also available for HCG, HLH, HPL, T3, digoxin, digitoxin,
hydrocortisone,
deoxycorticosterone,
glucagon, angiotensin I and Ii,
testosterone, estrone, estradiol-1 7, estriol, aldosterone, progesterone.
pregnenolone,
prednisone, prednisolone, thyroxine, equllin, and equllenin.
m For description of method see T. Pincus, P. H. Schur, J. A. Rose, ,J. L.
Decker, and N. Talal, Measurement of serum DNA-binding-activity
In
systemic lupus erythematosus. New Eng!. J. Med. 281,701(1969).
termed
“radioenzymatic”
(36).
Other
examples
of
the second molecule
are the intrinsic
factor (or transcobalamin)
(28, 29, 38-42) and the plasma
proteins
that
specifically
bind steroids
(43) or thyronines
(44). The term “competitive
protein-binding
analysis” (CPBA)
(45) refers to the latter case, in which
the ligand is bound specifically
by a protein.
Many
of the competitive
protein-binding
assays utilize normal circulating
plasma
proteins,
such as the globulins, or tissue receptor proteins.
Specific non-immunologic
globulins
used in CPBA
include
corticosteroid-binding
globulin-isolated
from humans
(46-60)
or dogs (53, 61-65)-to
measure human
corticoid
and progestin
concentrations;
thyroxine-binding
globulin
(TBG)
to
measure
human
thyroxine
(33, 44, 66-72); progesterone-bind-
ing protein of guinea pig plasma to measure progesterone (73-75);
and the human sex steroid-binding
globulin,
or testosterone-binding
protein,
for the
assay of androgens
(76-83)
and estrogens
(84-86).
Other sources of binding proteins
include plasma
from pregnant rats for the assay of estrogens (74, 87),
and plasma from pregnant women for the measurement of androgens
(88-91) and estrogens
(92). The
CPBA technique
has also been applied to the assay
of aldosterone
(93),
cAMP
(94, 95), erum
folate
(96),
antimicrosomal
and antithyroglobulin
antibodies (97, 98), and the analogs of Vitamin D (99,
100). To measure steroid and thyronine
concentrations in plasma samples, one must reniove any competing analogs and binding proteins
before assay.
Disadvantages
of these binding proteins are their relative instability
and limited range of affinities. The
term “radioinmunoassay”
(26) defines the second
molecule as an antibody,
while the term CPBA is
now only reserved for assays in which plasma proteins are used as the second molecule (see reviews
80, 101-104).
RIA has several
advantages
over CPBA.
For
CPBA, deproteinization
of the sample is necessary.
Although it is cheaper and quicker to set up a CPBA
for a few analyses, it remains less costly to perform
hundreds
or thousands
of assays by RIA, even
though it may take months to develop a suitable antiserum. The main advantage
of RIA over CPBA is
that RIA is potentially
more sensitive,
owing to
higher affinity constants,
and the nature of the “specific reactor”
(the antibody)
in RIA offers greater
specificity.
For this reason, radioimmunoassays
are
being developed to replace former methods of CPBA.
The use of tissue receptors (i.e., cellular binding
proteins)
as “specific reactors”
shows promise as
better techniques
are developed for receptor extraction and purification.
This recent modification
of
RIA is a natural outgrowth of studies on the mechanism of hormone action, which show that most peptide hormones activate an adenyl cylase at a specific
receptor site on the target cell plasma membrane
(105). Assay systems have been reported that make
use of ACTH tissue (adrenal cortical) receptors (106,
107); estrogen
(uterine cytosol) receptors (35, 108118);
and fractions
of interstitial
cells (from rat
testes) capable of binding [1311]-LH (119), [125I]LH
and HCG (120, 121), and cyclic AMP receptors (95,
112, 122).
These radioreceptor
assays are advantageous, in that relatively short times are required to attain maximum
sensitivity
and the specificity has a
biologic rather than an immunologic basis.
In another modification
of the RIA, termed the
“immunoradiometric
assay”
(123),
the antibody
(Abifi) is radiolabeled
instead of the antigen (Ag*I).
This method has several advantages,
one being the
low assay “blank”
values. Furthermore,
the antibodies have a uniform general structure,
a high molecular weight, and high stability,
and the relative
ease of their being tagged overcomes some of the difficulties inherent in satisfactorily
radiolabeling
low-
molecular-weight
antigens
(124,
125). More of the
unlabeled
antigen (Agli) is allowed to react in the
im’munoradiometric
procedure
than in RIA. Moreover, the labeled antibody can be stored on an immunoadsorbent
under proper conditions (126) for up
to six months and can be repurified. The immunoradiometric method has been used to assay insulin (127),
human growth ,hormone (128), calcitonin
(129),
and
human luteinizing hormone (130).
Although each of the procedures
described differs
according to the nature of the “specific reactor,” or
the position of the radioactive
label, the interaction
of the various components
in each assay can be characterized by similar kinetic reactions and mathematical interpretations.
Basic Kineticsof RIA
The exact nature of the antigen-antibody
interaction remains unknown, but it is generally accepted
that the reaction (Figure 1) reaches an equilibrium
during the incubation
procedure,
according to the
following formula (17):
Ag + Ab
AgAb
(1)
where k is the rate constant for association and k1 is
the rate constant
for dissociation.
According to the
law of mass action, if K equals the equilibrium,
or
affinity constant2 (K = k/k1), then
K[Ag][Ab]
[AgAb]
At equilibrium,
the ratio of the bound
free antigen (F) is
B/F
(2)
antigen
(B) to
[AgAb]/[Ag]
(3)
where the brackets symbolize molar concentration.
It is assumed that the equilibrium
constants
for
the labeled antigens, as well as for the standards
and
unknowns, are equal. However, as Ekins has pointed
out (132), this may not really be the case. Varying
conditions
(e.g., during the incubation)
may affect
each equilibrium
constant differently,
and the antibodies may discriminate
between labeled and unla;
beled antigens.
Under the conditions
of an RIA experiment,
the
components
[Ag] and [Ab] are related to the equilibrium constant by observing that:
[Ag]
[Ag,
=
-
AgAb]
where Ag1 is the initial concentration
[Ab] = [Ab,
AgAb]
Ab1 is the initial concentration
-
where
Therefore,
(4)
of antigen,
(5)
of antibody.
the ratio of the bound and free antigens
2LabeledK by Odell et at.
CLINICAL
and
is
(131).
CHEMISTRY,
Vol. 19, No. 2, 1973
151
B/F
[AgAb]/[Ag1
=
5
AgAb]
-
RB/F
which can be rearranged
(B/F)[Ag,
to
AgAb]
-
=
[AgAb]
or to
[AgAb]
Combining
K([Ag,]
-
[Ag,]B/F/(B/F
=
quations
+
1)
(6)
2, 4, 5, and 6 will give
[Ag,](B/F))/(B/F
#{247}
1)
(Ab
[Ag,](B/F))/(B/F
+ 1)
-
=
#{247}
1)
[Ag](B/F)/(B/F
which rearranged
(B/F)2
+
B/F[1
will give
+
KAg
-
KAb]
-
KAb,
=
0
This produces
the equation
of an hyperbole,
as
shown in Figure 2. The relation between B/F and
the concentration
of the antigen is described by the
curve PQR that approaches,
at its upper end, an asymptote STU, the slope of which is -K. A change in
the B/F ratio is proportional
to a change in the concentration of bound antigen, the proportionality
constant being
K. The ratio of the antibody bound (B)
to free (F) labeled antigen (i.e., B/F) decreases as
the concentration
of the unlabeled antigen (Agli) increases. Therefore,
a change in the B/F is greatest
when the antigen concentration
is small compared to
the antibody
concentration.
At Q, B/F = K [Ab1]
and at T, B/F = (KAb1
1).
Changes in the antibody equilibrium
constant K
markedly affect the sensitivity of the assay, as shown
in Figure 3, in which four curves are drawn, each
with an arbitary
K. Compounds
reacting with the
same antibody site, but with different energy levels,
will produce different dilution curves that are neither
superimposable
nor parallel when plotted on a logarithmic scale (8). Figure 4 illustrates the experimental curves derived from five antisera
(to ACTH),
with the abscissa labeled as per cent of initial B/F
for comparison.
The phenomenon
of increasing
slopes of B/F
curves seen with low concentrations
has been attributed to the bivalent properties of the antibodies.
It
may be possible to increase the sensitivity of a RIA
by using this portion of the curve.
-
Fig. 2. Relation between
mone (17)
RB/F and concentration
of hor-
K
AB
100
01
10
I
I
RB/F
0.5
-
Calculation
Midgley has presented (15) four graphical methods
for the possible analyses of RIA data (Figure 5). In
graph D, the sigmoidal curve is linearized by plotting the logit of the response variable Y vs. the log of
the dose X, where
3Sirnilar mathematical
approaches have been derived by Berson and Yalow (2, 6, 18, 133), Ekins et a!. (8, 30, 134, 135), Feldman and Rodbard (136), Scatchard (137) and others (138-139).
152
CLINICAL
CHEMISTRY,
Vol. 19, No.2.
1973
10
0.1
5
CONCENTRATION OF HORMONE
Fig. 3. Effect of antibody equilibrium constant, K, on the
sensitivity of radioimmunoassay-theoretical
curves with
arbitrary constants (17)
logit
Y
log[X/(100
=
and where b = slope and a
“inverse” equation
Y
Y)]
-
=
y intercept
antilog[logit(Y)
=
(a + b)log,,X
=
-
(140).
The
a]/b
may also be used for dose interpolation.
By algebraic
manipulation,
this latter equation has been shown
(141) to be equivalent
to
X
=
e#{176}5
[Y/(100
-
Y)1
and consequently
suitable for automation
on a programmable
desk-top calculator.
Note that graphs c
and d permit easy determination
of “parallelism,”
and that only the logit transformation
d provides
parallel straight lines for statistical
analysis. While
the logit transformation
allows for linearity, it does
U-
0
C
4.0
hormone
Unlobeled
Fig. 4. Variation
ferent antisera
8.0
6.0
(e.g., ng ACTH)
in the slopes of inhibition curves with dif-
to the same antigen
(11)
formation,
(b) its slope should be significant,
(c) the
slopes of the standard
and unknown curves should
be parallel, and (d) the order of the standards
in an
assay should be randomized
with the unknown samples to avoid bias. For example, when multiple dilutions are prepared from a plasma containing
a high
concentration
of antigen, these dilutions can be compared with the curve prepared from the standards.
If
the points at either end of the curve diverge significantly, then one can assume that there is not complete immunological
identity between plasma samples and standards.
The ultimate requirement
for any RIA is a reliable
antiserum.
Such reliability
exists when there is an
equal competition
between the standard on unknown
antigen
(Agil) with the tracer antigen (Ag*I) for
binding sites on the antibody (Ab1II) (Figure 1). Five
main criteria are used to assess the reliability of RIA:
precision,
sensitivity,
specificity,
accuracy,
and reproducibility.
Reliability Assessment
Precision
ib
20.
30.
40
0,
H1
*
CmS
x
Fig. 5. Four possible
x
graphical
methods
of analyzing
RIA
data
Theoretical inhibition curves for a standard (S) and an unknown (U) with
a quarter the immunologic activity of the standard are presented for
comparison
(15)
so at the expense of an increased non-uniformity
of
residual variance,
or scatter around the regression
line (heteroscedasticity).
Moreover,
this heteroscedasticity
increases at the upper and lower ends of
the curves in d. However, if the variance in Y is
known the variance in logit (Y) can be determined,
and one can then use the reciprocal of this variance
as a weight to perform a weighted “least squares” regression analysis of the linear curve of logit Y vs. X
(15).
The presentation
of statistical
controls for both
RIA and CPBA has been evaluated
(15, 102, 142149) and a number
of calculator
and computer programs have been written for the analysis, description, and simulation
of equilibrium
behavior of the
complex immunoreactive
systems involving antigens
and antibodies
(14, 15, 140, 146, 148, 150-156). The
theoretical
models enable one to predict how changes
in the assay will affect the precision, sensitivity,
or
accuracy of the procedure.
Midgley points out (15)
that, for optimal statistical
validation
by use of the
logit transformation
d, (a) the standard
curve
should be free of curvature
after appropriate
trans-
Precision has been defined by Midgley (15) as “the
extent to which a given set of measurements
of the
same sample agrees with the mean of that set,” i.e.,
the amount of variation
in the estimation
of unlabeled antigen (Agil). This can be estimated
by cal.
culating the index of precision,
or precision coefficient (A) (1), which is defined as the ratio between
the standard
deviation of logit Y for each Y value
and the slope (b) of the regression line between the
mean values of logit Y and the corresponding
log X
(14).
Therefore,
“precision”
incorporates
not only
the coefficient of variation,
but also the associated
slope of the assay curve and the Y value at which the
variation is measured (15).
The indices of precision (A) of RIA compare very
well with those of bioassay. For example, values of
0.0309 have been reported for ACTH (138), 0.0160.056 for insulin (157), and 0.029 for growth hormone
(158).
Precision depends on the extent of change in B/F
with fractional changes in antigen (Agli) concentration, but, unlike sensitivity,
maximum
precision is
independent
of K (19). Maximum
precision is attained when [Ab] is sufficiently
high to make K [Ab]
>> B/F and when B = B/F(Ag)
[Ab] (6). When
measuring high concentrations
of hormones (such as
glandular
extracts)
one desires maximum
precision
rather than sensitivity in RIA.
-
Sensitivity
and
‘Titer”
While “precision”
may be used to describe the
ability of an assay system to distinguish
between two
hormone concentrations
at any point on the response
curve, “sensitivity”
may be used to restrict one of
these points to a zero amount or concentration
(135,
145). Therefore, “sensitivity”
may be defined as “the
smallest
amount
of unlabeled
antigen (Agli) that
CLINICAL
CHEMISTRY,
Vol. 19, No.2,
1973
153
can be distinguished
from no antigen” (15). This is
very important
when concentrations
of substances
are being measured in the circulating blood.
However, sensitivity has been defined by others in
terms of the slope of the response curve. In this case,
the sensitivities
of different assays can also be compared by determining
the relationship
between Y
and its variance, i.e., calculating
the error from the
regression equation of Y vs. variance (Y) for Y = 1.0
(15). Furthermore,
Ekins uses the following formula
to compute “experimental
sensitivities:”
Sensitivity
-j
.4
z
I.-
0
p..
z
I’j
mean difference between duplicate
of (B/F)
1.1 x slope of response curve
at (B/F)
estimates
=
For review of this definition controversy,
see discussions by Ekins et al. and Yalow and Berson (19, 30,
67, 134, 135, 159, 160).
In Figure 6, three standard
curves are shown for
the assay of an antigenic hormone at three dilutions
of the same antiserum.
Sensitivity
is greatest with
higher dilutions
of the antibody
(curve A), but a
wider range of standard
antigen values are covered
when a more concentrated
antibody is used (curve
C). In contrast to precision, maximum
sensitivity
is
dependent
on K and is attained when the concentration of Ag*I is negligible and as Agil approaches
0
(133).
Moreover, a decrease of 50% in the B/F ratio from
an initial value of 1.0 can be more accurately determined than can a similar percentage
decrease from
an initial value of 10. In the former case, there would
be a decrease from 50% (B/F = 1) to 33% (B/F =
0.5), whereas in the latter case, there would be a decrease from 91% (B/F = 10) to 83% (B/F = 5) (4).
Therefore, to obtain a very high sensitivity,
it is advisable to use a dilution of antiserum
that will bind
about 50% of the smallest amount of AgdI in the absence of unlabeled antigen (Agil) (4).
The optimal concentration
of the antibodies in the
assay, or the final dilution used, is referred to as the
“titer.” The titer can be determined
by measuring
the ability of serial dilutions of antisera to bind a
trace amount of radiolabeled
antigen (Ag*I). However, as Berson and Yalow point out (4), the “titer”
should not be made the basis for selection of antisera. Most antisera are used in very high dilutions,
and the only advantage
of a high “titer” is the large
number of assays that can be performed with a small
volume of the antiserum (4). Because each antiserum
contains multiple or heterogenous ,antibodies
reacting with various equilibrium
constants,
K, the potential sensitivity
of the assay will depend on those
antibodies
that react with the highest energy and
that are present in a suitable concentration
to be
utilizable
(4). However, the use of a too highly concentrated
antiserum
will decrease the precision
if
highly concentrated
antibodies
with low affinity
begin to bind a significant
fraction of the labeled
antigen(Ag*I)
(133).
154
II.
Am/buy
Ui/aIIM
A 1/250.000
B l,’IOO,OOo
C i/i;ooo
CLINICAL
CHEMISTRY,
Vol. 19, No.2,
1973
Fig. 6. Standard
at three dilutions
curves for the assay of hormone
of the same antiserum (17)
(ng/ml)
The sensitivity of the initial part of the curve may
be increased by using minimal amounts of tracer antigen. This is accomplished
by using tracer doses of
Ag*I with maximum specific activity and radioactive
measurements
with maximum
efficiencies. The sensitivity can also be generally improved by increasing
the volumes of the incubation
mixture and performing the assay at low ionic strengths. It has also been
shown that the sensitivity
can be enhanced by adding the labeled antigen (Ag*I) after a pre-incubation
of the unlabeled
antigen and antibody (161). Rodbard has presented
a model whereby, in certain defined conditions
of the assay procedure,
one may
predict the error at any point of the dose-response
curve, which may in turn be used with the slope to
predict both sensitivity
and precision (146). When
one wishes to measure fairly high antigen concentrations, the emphasis of the assay design is placed on
precision,
because one can work with an antiserum
that is not as highly specific as when low concentrations of antigens are being measured.
In the latter
case, the sensitivity of the assay is extremely critical.
Although the sensitivity
particularly
depends on the
choice of the antiserum
and on the specific antigenicity of the labeled antigen1 it ultimately
depends
on the equilibrium,
or affinity constant,
K, which
characterizes
the energy of the antigen-antibody
reaction.
One of the great advantages
of RIA over
other clinical procedures
is the high sensitivity
resulting from the nature of the antigen-antibody
interaction.
Because of this sensitivity,
RIA can easily
measure antigenic concentrations
in the range micro-,
nano-, or picograms.
Specificity
Specificity has been defined as “the extent of freedom from interferences
by substances
other than the
one intended to be measured”
(15). The specificity of
the antibody
for the antigen is influenced
by: (a)
heterogenicity,
i.e., the absence of purely specific antibodies;
(b) cross-reaction
with other antigens
or
metabolic
fragments
that retain
immunoreactive
sites; and (c) possible interferences
of the antigenantibody reaction owing to the presence of low-molecular-weight
substances
that may alter the environment of the reaction.
Hetereogeneity.
A particular
antigen will induce
the formation
of multiple,
or heterogenous
antibodies. The production
of an “homologous”
antiserum-i.e.,
antibodies
entirely specific for the particular antigen-has
not been reported.
The antigen
will combine with the multiple antibodies to various
degrees,
depending
on the respective
equilibrium
constants,
K, generated.
“Heterogenicity”
refers not
only to the multiple antibodies produced, but also to
the variable number and location of the binding sites
on a single type of antibody. This property of heterogenicity has led to the discovery of multiple forms of
insulin (162-164),
parathyroid
hormone (165), and
gastrin (166-168).
The development
of better techniques for increased antisera purification
is helping
to solve the problem of heterogenicity.
Cross-reactivity.
Two different antigens from the
same species may cross-react.
For example, there is
partial or complete cross-reactivity
between the antigenic determinants
of the glycoproteins
FSH, LH,
and often TSH. Before one can assay for FSH, the
specificity must be increased by preliminary
absorption of any binding sites specific for LH (HCG) by
the use of suitable amounts of HCG (169-175).
Cross-reactivity
can also occur between multiple
hormones,
their precursors,
derivatives,
fragments,
or metabolites
and also between different types of
steroid molecules.
For example, the desoctapeptide
form of insulin is biologically inactive (176), but retains some immunologic
reactivity
(177). To remove
these cross-reacting
antigenic
analogs, the chemist
must purify the sample containing the antigen (AgH)
before incubating
it with the antibody and be certain
that other antigens remaining
in the sample do not
cross-react
with the antibody. Boyd adds to plasma
samples a small amount
of a diatomaceous
earth,
which extracts angiotensin
H, and after centrifugation elutes the vasoactive
agent from the silicate
with ammonia (178). When raw serum or plasma are
to be assayed, a high degree of antibody specificity is
essential, but as methods become available for efficiently purifying the antigen in the sample, antisera
with a lower specificity may be used. Furthermore,
if
one is not interested
in separating
antigenic or haptenic compounds
that are closely related, both in
structure
and biological
action, one may wish to
work with an antiserum
that is specific to a group of
antigens (e.g., the estrogens).
When evaluating
the
specificity of an antiserum,
it is essential
that the
antigenic
material
in the sample being assayed behaves similarly to the antigenic standards.
This can
be determined
by making certain that different concentrations
of the sample antigen (or different dilutions) react to produce the same curve as does the
standard.
Cross-reactivity
may also exist to varying degrees
between the same antigens in two different species.
Cross-reactivity
does not exist when antibodies
are
specific only for the antigens from the species in
which the antibodies
are formed. There is occasionally this lack of cross-reactivity
with polypeptide
hormones because of slight differences in the primary
structure
(amino acid sequence);
the nonpeptide
hormones
have identical
structures,
regardless
of
species source. This absence of cross-reactivity
has
been termed “species specificity.”
In the ideal RIA the same antigen(s)
are used not
only as tracer
(Ag*I), standards,
and unknown
(Agil), but to produce the antibodies (Abifi), all of
which are derived from the same species. Such an
“homologous”
system is mandatory
(although often
difficult to achieve) with polypeptide
hormones having a high degree of species specificity. For example,
growth hormone, a protein molecule larger than insulin, exhibits a high immunologic
species specificity
and should be tested
in an homologous
system
(179-184).
However, when cross-reactivity
does exist,
it is often more efficient to work with antigen-antibody components
from various species, as found in
the “heterologous”
system (185). Such heterologous
systems include the RIA for insulin, ACTH, gastrin,
and other polypeptide
hormones (2, 5, 177, 186-189).
For example,
human
insulin
cross-reacts
almost
completely with pork insulin antibodies and radiolabeled pork insulin. Likewise, human anti-pork insulin sera cannot distinguish
between pork insulin,
beef insulin, desoctapeptide
beef insulin, and human
insulin (190). The difference of only one amino acid
in the C-terminal
of the insulin B chain (133, 191,
192) does not significantly
affect the nature of the
antigenic reaction.
Chemically
unrelated
drugs and hormones-such
as thyroxine
and diazepam
(193)-may
also crossreact, and the clinical chemist should anticipate
additional reports of this type of cross-reactivity
in the
future. Antisera prepared from antigens derived from
urine and pituitary
from the same animal may not
always cross-react.
For example,
Franchimont
has
noted (194) that antisera to pituitary
FSH will bind
serum and urinary FSH identically,
but that antisera to urinary FSH will only react with urinary FSH.
The problem of specificity must be considered separately for each type of antigen being measured. We
emphasize that by the nature of the RIA technique,
specificity depends not on the sites responsible
for
biologic activity per se, but on those antigenic sites
responsible for immunochemical
activity (4). Examples of these differences include RIA for ACTH (195)
and for the gonadotropins
FSH, LH, and HCG
(196-197).
Nonspecific
interference
(non-aritigenic
cross-reactivity). Nonspecific factors in biologic fluids modify the rate of the primary antigen-antibody
reaction.
These include such environmental
factors as the
ionic strength, pH (see discussion to ref. 138), heparin, urea, excessive
bilirubin
concentration,
high
temperatures,
and the composition
of the incubating
buffer medium, all of which may alter the affinity or
CLINICAL
CHEMISTRY,
Vol. 19, No.2,
1973
155
type of contamination
is found in the routine RIA of
avidity of the antigen-antibody
reaction. For examdigoxin
(205,
206).
ple, losses in insulin reactivity
of as much as 25%
may occur when samples are left at room temperature for 24 h, as compared to the reactivity of corresponding samples stored at 4#{176}C
for 24 h, or at -23#{176}C Accuracy
as long as nine months (198). In most protein-pro“Accuracy”
refers to the extent to which a given
tein interactions,
the affinity constant K (or K0) demeasurement
or measurements
of a substance agrees
creases as the temperature
increases from 0#{176}
to 37#{176}C;
with the standard measured value of that substance.
therefore, most RIA procedures should either be conHowever, until the precise chemical structure
and
ducted at lower temperatures
or for short periods of
properties
of the antigens
(especially
those of the
time at 37#{176}C.
Hemolysis will also cause the destrucprotein hormones)
is known, one can only approxition of insulin (199) and present a source of error in
mate this “true” value by evaluating a relative accuthe RIA of digoxin and digitoxin, which may be corracy. One method of evaluating the relative accuracy
rected by treatment
with a hypochlorite
bleach
in a RIA would be to use the results of the corre(200).
Antigen standards
and unknowns should be prepared in antigen-free
plasma containing the same ingredients in the same diluent. If antigen-free
plasma
of the same species is not available,
plasma
(or
serum) from a non-cross-reacting
species may be
used. In addition, old plasma samples should also be
checked for possible antigenic
damage caused by
proteolytic
enzymes,
which
are especially effective in
metabolizing
peptide hormones.
One type of inhibition
of the antigen-antibody
reaction appears to be identical to serum complement (201) and can be eliminated
by appropriate
dilutions,
by heating,
or by adding EDTA to the
buffer medium.
The use of heparin,
however, appears to cause results to vary (138) and the use of
EDTA in collecting blood for angiotensin
II assays
presents a major problem of nonspecific
inhibition,
which may be reversed by the use of divalent ions.
Assay of urine also presents a special problem in certain RIA cases and care must be taken to ensure that
the varying salt concentrations
do not affect the
assay.
The term “nonspecific
binding”
refers to the
amount of Ag*I bound to plasma or serum proteins
in the absence of antibody (AbIII). This can be determined
by running a blank with each assay. The
problem of high blanks cannot be overstressed;
it has
presented
one of the major problems in RIA procedures. Appropriate
steps should be taken whenever
blanks are high (see discussion to ref. 202).
One should also be aware of the problem of nonspecific binding of polypeptides
to the walls of test
tubes. In some cases, this binding can be prevented
or minimized
by using plastic or siliconized
glass
tubes or by coating the tubes with serum albumin
(203). In RIA of bradykinin,
the inner walls of the
tubes are coated with muramidase
to prevent adsorption
of this nonapeptide
(204). One may also
wish to check for parallel activities by running assays with unlabeled
antigen added to water or plasma blanks. One should also be aware of possible radioactive
contamination
of the samples being assayed. The presence of radioisotopes
in the plasma
because of various diagnostic tests will seriously affect the accurate quantitation
of radioactivity
in either the bound or free phases. An example of this
156
CLINICAL CHEMISTRY, Vol. 19. No. 2. 1973
sponding bioassay as a reference point. However, the
net biological potency of a sample does not always
correlate well with the definitive antigen concentration on a molar basis. In addition, one must remember that the validity of the RIA method requires only
that the antigen being measured
and the standard
behave identically
in the immune reaction. Therefore, it is difficult to assess whether ostensibly the
same antigens, used in an assay, will have the same
antibody-antigen
reaction energies when each may
have been isolated from a different animal or even a
different species.
The use of urinary extracts (174, 207-211) has produced good correlation between bioassays and RIA of
the pituitary
hormones.
However,
standards
prepared from one source (such as the pituitary)
do not
always show good correlation
between bioassay and
RIA when assayed with samples derived from a different source-for
example, urine or serum. A greater
degree of disparity is found with LH than with FSH,
which suggests that the hormone in urine has lost
more of its immunologic
activity than of its biologic
activity
(212). Furthermore,
it has been demonstrated that the immunologic
methods for the determination of LH measure different activities than do
the biological assays (213-214). Therefore,
it is important to report the source of the standards
used in
each assay and the exact derivation
or definition of
any units used.
Technically
different RIA systems have also been
used with the same antigen to evaluate relative accuracy and have demonstrated
excellent agreement
between the various procedures
(174, 207-209,
211,
215-218). Relative accuracy may also be assessed by
studying
any changes in the activities
of antigen
samples when subjected to a wide variety of physical
and chemical manipulations,
such as gel filtration,
chromatography,
electrophoresis,
enzymatic
digestion (15), and volume dilution.
One should also be
able to demonstrate
good standard antigen recovery
after additions to samples in vitro.
Reproducibility
The main factor influencing
reproducibility,
the
duplication
of the values within or between assays, is
the difference
in individual
techniques
in carrying
out the same operation
(219). This can usually be
decreased to less than 5% without much difficulty in
most assay systems. To establish reproducibility,
one
should perform replicate
determinations
from the
same plasma samples in the same assay or in separate assays.
For a concise description
and specific examples of
how precision, sensitivity,
specificity,
and reproducibility can be individually
evaluated and interpreted,
see the review by Galskov (138).
Radloimmunoassay Techniques
RIA procedures
depend on the production
and purification
of radiolabeled
antigen, the induction
of
antibodies with a high specificity and affinity for the
antigen and a technique
suitable for the separation
of bound and free antigen. Each of these techniques
is derived from well-established
procedures
basic to
immunology
or immunochemistry.
For a more thorough analysis of the technical
details, the clinical
chemist may wish to consult other reviews [e.g., the
treatises
edited by Williams and Chase (220), Day
(34), or Kabat and Mayer (221)J.
Antigenic
Radiolabelirig
The first requirement
for an acceptable
RIA is the
preparation
of a highly purified antigen that can be
radiolabeled
or “tagged” without producing any loss
of immunoreactivity.
Some substances
(e.g., steroids) can be obtained labeled with either 3H or 14C
from commercial
sources (Table 1). Since most polypeptide hormones contain at least one tyrosine residue, they can be labeled with a radioisotope
of iodine (e.g., 1251 or 1311). The radioisotopes
of iodine
have the advantage
of higher specific activities than
can be found with tritium or carbon-14. Midgley has
calculated
(14) that the theoretical
maximum specific activity for a hormone containing
two atoms of
isotope per molecule, in Ci/rnrnol, would be 58.4 for
3H, 4340 for 125!, and 32400 for 131!. However, an extremely
high specific activity
may require iodide
concentrations
(222) that would affect the antigen
and (or) the antibody,
thereby producing changes in
immunoreactivity
(e.g., losses in precision, sensitivity, and specificity).
The chemistry (223) as well as the procedures and
problems
of iodination
are discussed
in detail
by various investigators
(1, 14, 18, 138, 222, 224-234). It is
important
that the radioactive
iodine be present as
iodide, that the solution have a neutral pH, and that
the resulting
radioactive
preparation
have a high
specific activity with good stability and produce unimpaired
immunoreactivity.
125!
and 131! each possesses distinct advantages
for use in radioiodination.
1251 has a longer
half-life (60 days) than does 131! (8
days). In addition,
125!
has a higher counting efficiency under certain conditions,
can be produced
practically
carrier free, and is relatively inexpensive.
It also has a lower gamma energy than does 1311, and
the absence of fl-radiation
diminishes
the potential
for autodestpiction
of the labeled
antigen
(138).
However, 125! may also produce antigenic
damage
and, because of its longer half-life, it is often necessary to repurify
the isotope. For example, some preparations of [125I]HGH are not used for more than two
weeks after being iodinated
(235). Moreover 1311 is
easy to obtain with a high specific activity (236) and
may be used freshly for each assay. Other considerations in the choice of an isotope include the time required for radiolabeling,
the need to conserve antigen, the time required for radioactive
counting, and
the relative cost of each isotope. Under proper conditions, 125! and 131j may be used together in a simultaneous assay for insulin and HGH (237).
Oxidation
of the labeled Na! is generally
performed by using excess chloramine
T (235, 238),
which results in a high specific radioactivity
with a
low hazard to health. To minimize preparation
damage to the protein (e.g., oxidation of the sulfhydryl
groups), both reaction time and amount of oxidizing
agent used must be limited. While the favored exposure times range from less than 10 s to 2 mm, the
chioramine
T concentration
and the specific antigen
being labeled must also be considered.
For example,
Midgley has found (239) that conditions were optimal (i.e., minimal changes in biological and immunological activity) with 4 g of chloramine
T per g
of hormone (HCG) with a reaction time of 2 mm,
and suggests decreasing the chloramine
T concentration when iodinating the gonadotropins
(173).
In general, iodination
is easy to perform and is
usually carried out at room temperature
at a pH of
7.5 in a phosphate
buffer medium. lodination
may
take longer to complete at lower temperatures
or at
higher pH. Some peptides label well up to a pH of
8.6, but a pH-optimum
curve should be run before a
new antigen is tagged. The reaction is stopped with
excess sodium
metabisulfite.
The tagged antigen
may be damaged during or soon after the iodination
reaction, and the extent of the damage may vary, not
only among the antigens being labeled but among
different batches of the same antigen. The potential
for increasing
the specific activity of smaller molecules is greater when they are first conjugated
to
larger protein molecules, which in turn can be iodinated. For example, specific activity increases when
the gastrin tetrapeptide
is covalently conjugated
to
the random copolymer of glutamic acid, alanine, and
tyrosine
(240), and when the N-terminal
arginyl
group of bradykinin
is conjugated
to a desamino
tyrosinyl moiety (241). In addition, steroids and cardiac glycosides may be radiolabeled
with 125! or 131!
by first conjugating
them to rabbit (14) or bovine
(242) serum albumin or to the methyl ester of tyrosine (243). It is now possible to obtain from commercial sources (see Table 1) a large number of antigens
already labeled with a high specific activity of 3H.
To decrease potential
damage caused by the isotope, the chloramine
T, or the metabisulfite,
the laCLINICAL
CHEMISTRY.
Vol. 19, No. 2, 1973
157
beled antigen must now be separated
as quickly as
possible from the labeled and unlabeled
degraded
antigen, the mineral salts, and the unreacted
isotopic material. In some cases (244), it may be possible
to omit the metabisulfite
and purify the material immediately.
However,
the antigen
may be further
damaged during storage, and the purification
procedure should never be omitted.
The labeled antigen may be partially protected by
appropriate
dilutions of the plasma or by first incubating the plasma with iodoacetamide
(245). Neutron activation
analysis of nonlabeled 127I-insulin offers one method of avoiding internal radiation damage. In this procedure,
insulin is iodinated with 1271
by the standard
Hunter and Greenwood technique,
and after incubation,
dextran-coated
charcoal
is
added to adsorb the free 1271-insulin. The 127insulin
bound to antibody is then subjected to the neutron
activation
technique
[1271
(n,-y)1281]. Although
the
measurement
of the 128J activity generated requires
fast analytical procedures based on gamma-ray
spectrometry, preliminary
reports (246) indicate that the
method may be useful for standardizing
insulin radioimmunoassays.
In addition, a procedure in which
lactoperoxidase
hydrogen
peroxide and Na’251 are
used has been introduced
for the enzymatic radioiodination
of the gonadotropins
HFSH,
HLH, and
HCG to minimize the loss of biological activity usually incurred
with the use of chloramine
T (247,
248).
Purification
of Labeled
Antigen
Various techniques
are available
for separating
and purifying the tagged antigens. Adsorption
column chromatography
on powdered
cellulose
has
been used to eliminate
mineral salts (26, 245). The
free isotope remains in the column while the labeled
hormone
passes through
with the eluting solvent
(e.g., aqueous albumin).
The powdered cellulose adsorbent method is rapid and has been modified for
assays of HGH, parathyroid
hormone, ACTH, LH,
insulin, and other hormones (26, 249-252). The adsorbent “QUSO” (microfine particles of precipitated
silica) has been used in the purification
of parathyroid hormone
(251), ACTH (253), and calcitonin
(11). In one modification
(230), dilute hydrochloric
acid (0.1 mol/liter)
has been substituted
for whole
human plasma as the eluting solvent, thereby minimizing additional antigenic degradation.
For more extensive purification,
one must resort to
a separation
involving dialysis, gel filtration (using a
molecular
sieve), or ion-exchange
chromatography.
In these methods are used the cross-linked
dextrans
(“Sephadexes”)
(236, 254, 255), “Bio-Gel” (14, 239),
DEAE
(diethylaminoethyl)
cellulose
(249, 256),
Dowex resin (1, 9), or anion-exchange
resin (CG-400)
(257). Inorganic
iodine resin has also been used to
adsorb the unreacted
1311
(175). In separation
with
Sephadex,
the degraded
antigen
(often
protein
158
bound), is eluted first, followed successively
by the
purified antigen,
mineral salts, and unreacted
isotope. Pretreatment
of the column with a protein
such as albumin serves to saturate
the adsorption
sites on the Sephadex, thereby reducing the nonspecific binding, which appears to increase with time
(24), and increasing the adsorption of damaged antigen (227). Cellulose
columns
are more useful in
ACTH purifications
because there is less partial adsorption than is found with use of gel filtration. The
molecular
sieve is very useful for purifying
and
isolating
highly reactive labeled HGH (187, 238),
TSH (258, 259), HPL (260), HCG (261, 262), HLH
(263, 264), HFSH (169, 249, 265), or insulin (266). A
cellulose-neurophysine
resin has also been used to
purify labeled vasopressin
(267). The addition of 2mercaptoethanol
has been found useful in stabilizing
the labeled thyrocalcitonin
during purification,
storage, and incubation (268).
Additional
purification
may be necessary,
especially in cases where the initial antigenic preparation
is not pure or when significant antigenic degradation
occurs during radiolabeling.
Starch gel (249, 269)
and polyacrylamide
gel electrophoresis
(for steroidTME conjugates and protein hormones) (14, 24, 173,
187, 239) have been used with various modifications.
In these chromato-electrophoretic
procedures,
the
“pure” tagged antigen remains near the origin, while
the free unreacted
isotope and degraded antigen migrate. The labeled fractions,
located by autoradiography, are eluted with the assay buffer solution or
with excess BSA solution (208). ‘25I-labeled angiotensin may be purified to a greater extent by collecting small serial elutions from paper electrophoresis
(270) rather than by eluting the labeled peptide as a
whole (271). Separation
with charcoal-dextran
does
not resolve the three components
of the radiolabeling
procedure
(i.e., undamaged
and damaged
antigen
and unreacted isotope) (227). Many of the separation
and purification
procedures reported in the literature
use combinations
or variations of the above methods.
After purification,
one should determine the absolute quantity of antigen (Ag*I) required in a particular assay for high sensitivity.
It is important
that
this quantity be kept at a minimum.
Therefore, it is
desirable to produce a high specific activity of the
radiolabeled
antigen.
The specific activity may be estimated
by determining the cpm found in various
stages during
radiolabeling.
By comparing the cpm of the isotope
before labeling with the total cpm of the various labeled fractions
(e.g., labeled antigen and free isotope),
one can determine
the percent
recovery.
Knowing this recovery, and the amount of antigen
and label initially present, one can then estimate the
specific activity of the “tracer.”
Furthermore,
one may wish to check for immunoreactive changes of the antigen by conducting
a preliminary
radioimmunoassay.
This can readily be
done by assaying the radiolabeled
antigen alone and
then diluted with unlabeled
antigen. A decrease in
immunoreactivity
of the tagged antigen would indiof the antigen. The techniques
for antigenic
injeccate that it had suffered some labeling insult during
tions vary according to the routes of administration
the procedure.
-intraperitoneally,
subcutaneously,
intravenously,
If the labeled antigen is to be stored for considerintradermally,
or directly into the lymph nodesable time, it is usually kept at 2-4#{176}C
(e.g., steroids)
dose, and frequency (220), In general, 0.2-2.0 mg of
or it may be quickly frozen for storage at -20#{176}C the antigenic preparation
is injected as a suspension,
(e.g., polypeptides).
After storage the antigen must
although antibodies have been produced with immube checked for changes in immunoreactivity
before
nizations requiring 5-15 mg (276). Moreover, specific
use in an assay. The actual conditions for storing the
antisera to subunits of HCG and testosterone
conjulabeled antigen depend on the particular
antigen. Ingates have been obtained with as little as 20-100 ig
sulin and growth hormone are best stored frozen in
of the immunogen
(277).
Molecules
that are too
an albumin-barbital
diluent,
but parathyroid
horsmall to induce antibody
formation
are treated as
mone, ACTH, or calcitonin are best stored in diluted
haptens; that is to say, it is necessary to link them to
carriers such as proteins or synthetic compounds beacetone-acetic
acid or in albumin (50 g/liter)-phosphate buffer and kept frozen or refrigerated
in silifore they are injected.
conized test tubes (138, 272).
The mineral oil of the adjuvant contains an emulsifying agent (“Aquafor”
or “Arcacel”) which delays
resorption
of the preparation
by the lymphatic
sysThe Antibody
tem. Mycobacteria,
or a bacterial
endotoxin,
are
added
to
produce
sensitization
in
the
animal.
Some
The second prerequisite
for a RIA is the produc(190, 191) prefer to use a less potent adjuvant
to
tion of a suitable antiserum.
The antibodies
are a
avoid antigenic
reactions
with contaminating
progroup of serum proteins that are also referred to as
teins. Moreover, with some polypeptides,
such as ingamma globulins or immunoglobulins.
Most of these
sulin (257) and HGH (278) antibody formation can
immunoglobulins
belong to the IgG class, while the
other classes are termed IgA, 1gM, IgD, and IgE. Bebe induced in the absence of adjuvant.
For the first or primary immunization,
it is best
cause these immunoglobulins
possess not only antito use a high-quality
antigenic preparation.
Subsebody-reaction
sites, but also antigenic determinant
quent or repeated secondary immunizations
can be
sites, the immunoglobulins
themselves
can serve as
antigens when injected into a “foreign” animal. The
performed with a less-pure antigenic preparation
and
antigen-binding
sites appear to reside on the H and
are often done in the absence of a complete adjuvant
(i.e., without
mycobacteria),
especially
when skin
L chains of the IgG molecule.4
reactions become severe. The animal should also be
While the titer, or concentration
of the antibody is
important,
the main criterion for establishing
a suitclosely monitored for hypoglycemia
or hyperadrenoable antiserum
is the energy of interaction
between
corticalism
caused by repeated
injections-or
too
the antigen and antibody,
or the specificity and afrapid absorption-of
insulin or ACTH, respectively.
finity for the antigen being assayed. For most clinical
Secondary
immunizations
enhance
antibody prochemists who are interested
in performing RIA production,
the specificity
of which has been determined in the primary reaction.
For the secondary
cedures, it would be more advantageous
to procure
immunizations,
one should select those animals that
the specific antiserum
from a laboratory or commerproduced a high titer of antibodies
after the initial
cial supplier (Table 1) that has the facilities (i.e., the
immunization,
because these same animals will also
animals) required for the generation
and evaluation
produce high antiserum titers with additional immuof antibodies.
For successful antibody induction,
all
nizations. However, there is a large variability in the
that is required are a few animals, some patience,
titer and cross-reactivity
of antisera when sheep are
and a certain amount of luck. Even though various
immunized
with aldosterone-21 -hem isuccinate or alantisera specific for the same antigen will elicit simidosterone-18,21-dihemisuccinate,
and there is no relar potency estimates in terms of bioassay, the antilationship
between
maximum
titer and previous
sera will not always exhibit similar radioimmunologicross-reactivity
(279).
cal values when assayed for the same antigen (273).
Blood samples
are removed
either by cardiac
The general method of inducing antibody formapuncture
or from the central artery of the ear and
tion is to inject into a number of animals the pure
the serum checked for its particular
titer, specificity,
antigen mixed with “Freund’s adjuvant”
(274, 275)
and affinity of antibodies.
An example of how the
(a mixture of mineral oil, waxes and killed bacilli,
optimal concentration,
or “titer,” of an antiserum
is
that enhances and prolongs the antigenic response).
determined
for a specific assay is illustrated
in FigThe actual animal that one selects (guinea pig, rabure 7 (280). In this case, one measures the ability of
bit, sheep, goat, chicken, or monkey) depends on the
trace levels of radiolabeled
antigen (Ag*I) to bind
volume of antiserum desired and on the “foreign-ness”
antisera (Abifi) at various dilutions.
The appropri4immunoglobulins
have been resolved
into light (L) and large
ate dilution is the one that will bind about half of
(H) polypeptide
chains. The four-chain model for IgG has been
the radiolabeled
antigen (B/F = 1). The antiserum
proposed by R. R. Porter and G. M. Edelman, winners of the 1972
producing
the
sharpest
slope of B/F provides the
Nobel Prize for Physiology or Medicine for their research into the
chemical structures of antibodies.
most sensitive assay. When heterogeneous
antibodies
CLINICAL CHEMISTRY, Vol. 19, No.2,1973
159
#{149}ntiss,um
dilution
4
u..d
for ....y
3
2
102
,o
IO
rscipro$l
l0
of .flti$.,um
Fig. 7. Curve produced with various
for titer (280)
,o
106
dilution
antiserum
dilutions
in
screening
present a problem in evaluating
an antiserum,
certain specific antibodies
may be isolated by affinity
chromatography,
with use of the adsorbents bromoacetyl
cellulose
or beaded
agarose
(“Sepharose”)
(281). For example,
highly specific antiserum
to
HCS have been obtained by coupling the HCS antibodies to a column of beaded agarose (282-284) and
then eluting with a gradient of guanidine-HC1
containing human albumin (285).
For a thorough
analysis
of the antiserum
produced, one may study its ability to neutralize
hormonal activity (14, 286), its immunohistochemical
(14, 287, 288) or immunodiffusion
(289-291) characteristics, or its ability to bind labeled antigen (14).
Specificity
of the antiserum
is studied with substances
similar
to the antigen
being assayed, and
should always include an assessment of the degree of
correlation
between
RIA and bioassay
or other chemical or isotopic means of measurement
(292).
The
larger-molecular-weight
protein
hormones
(e.g., (H)GH, TSH, and HCS) generally elicit a good
antigenic response. It has been more difficult to obtain highly specific
antibodies
for (H)FSH
and
(H)LH, as these two hormones possess biologic and
immunologic
similarities
not only to each other, but
also to HCG and (H)TSH. However, LH contamination in FSH preparations
may be inactivated
by
using a-chymotrypsin
(293) or by adding HCG. This
chorionic
gonadotropin
confers FSH specificity
on
the FSH assay by adsorbing the antibodies
that do
not distinguish
between FSH, LH, and HCG. Moreover, antisera
developed
against the fl-subunit
of
HCG now make it possible to measure this hormone
in the presence of (H)LH (294).
Hapten
Conjugation
Production
compounds.
1000-5000)
160
CLINICAL
of
immunogenicity
The smaller
and octapeptides
CHEMISTRY,
in
nonantigenic
polypeptides
(mol wt
have low immunogeni-
Vol. 19, No.2,
1973
city and one must attach them as haptens to inert
adsorbing
particles,
synthetic
peptides,
or natural
protein molecules to enhance the antigenic response
(270, 295-302).
Conjugation
with human
gamma
globulin will rapidly produce antisera to ACTH, angiotensin II, and deoxycorticosterone
(303). The albumins (human,
bovine, or rabbit),
synthetic
peptides [e.g., polylysine (270, 297)] and polymers [e.g.,
polyvinylpyrrolidone
(304)] are also commonly used
as carriers. For example,
coupling of antigen with
carbodiimide
to serum albumin has elicited antiserum to glucagon (304) and increased the antigenicity
of angiotensin
and bradykinin
(296). The bradykinin
molecule is coupled to poly-L-lysine
via the amino
terminal
arginine groups by a modification
of the
procedure described by Schick and Sanger (305).
Boyd and Peart describe (306) a method of obtaining antibodies
to angiotensin
II, in which they
adsorb the antigen onto fine microparticles
of charcoal (diameter
<30 nm) and inject this preparation
directly into the spleen. The carbon particles act not
only as a transporting
device to the lymphatic drainage, but also to protect against the action of the angiotensinases.
However, some laboratories
have been
able to elicit antiserum
to unconjugated
oxytocin
(307), gastrin (mol wt 2096) (182, 308), and vasopressin (mol wt 1098) (309-311).
Steroids can also act as haptenic groups when coyalently linked to protein carriers by a number of different methods (see reviews 312-316). Steroid derivatives containing
free carboxyl groups may be coupled to the c-amino groups of the lysine residues with
carbodiimide
[1-cyclohexyl-3-(2-morpholinyl-(4)ethyl)carbodiimide
metho-p-toluene-sulfonate}
(313,
31 7-319). One can also take advantage
of the available hydroxyl groups by forming esters with succinic
anhydride
(317, 320) or chlorocarbonate
(315). Examples include the 3- and 17-monosuccinyl
esters of
estradiol-17fl
coupled to BSA (316, 317, 320, 321),
estradiol- 17fl- 1 la-hemisuccinate-BSA
(322), estriol
ring D-hemisuccinate-BSA
(323), estriol-3-monoand trisuccinate-HSA
(324), 1 1-desoxycortisol-21hemisuccinate-BSA
(325), and the succinyl derivatives of lla-hydroxyprogesterone
(326, 327).
It is also possible to conjugate steroids to proteins
by producing oxime derivatives
(317, 328) of the ketone groups by using (0-carboxymethyl)
hydroxylamine.
Examples
include
estradiol-17fl-6-carboxymethyloxime
(CMO)-BSA
(276, 329-332); progesterone-3- (333), 6- (334), or 20- (335)-BSA;
and the
CMO conjugates
of testosterone
(135, 336, 337) and
aldosterone (338-340). The hemisuccinate
and oxime
derivatives
are linked via the amide bond to the camino group in RSA (341) or BSA (220) by using a
“mixed anhydride”
reaction; the chlorocarbonate
derivatives
are coupled by a simple Schotten-Baumann reaction
(314, 328, 342, 343). Considerably
more cross-reaction
occurs between compounds with
structural
differences nearer the site of covalent linkage than in compounds
with the structural
differences distant from the binding sites. For example,
the 4-ene and 5a-dihydro
forms of steroids
the C6 position
more cross-reaction
exhibit
linked
at
than
those with changes in rings A or D, or in those steroids coval#{232}ntly
linked at C-ha
(276). However, it
has been reported that the li-hemisuccinate
conjugates are comparable
to the 6-hemisuccinate
conjugates in their ability to evoke antisera discriminating
between
estrone,
estradiol-17fl,
and estriol (322).
Other steroid haptenic groups serving as suitable antigens include estrone-2and 17-isocyanate
coupled
to HSA (312), the azobenzoyl
derivatives
of estriol
(344) and the carboxylic
acid groups of the steroid
conjugates testosterone-17a-glucuronoside,
estradiol17fl-glucuronoside
and
dehydroepiandrosterone-3glucuronoside
(345), which are covalently linked to
the c-amino groups of the dibasic amino acids in
BSA.
The product
of the carbodiimide
condensation
reaction
(296, 317-319)
and glutaraldehyde
(317,
346, 347) have also been used for the covalent linkage of other haptens or proteins. For example, the
carbodiimide
reaction product has been used to couple vasopressin to BSA (348). Thyroglobulin
has also
been used for the covalent linkage of the haptens lysme, vasopressin
(349), triiodothyronine,
a.nd thyroxine (131, 350). Antisera
have also been developed
against synthetic
thyroid conjugates,
succinyl polylysine-Ta (351), and triiodothyronine
linked to the
serum albumins
by the carbodiimide
reaction (352,
353).
Production
and preservation
of antisera.
Once a
suitable antiserum
has been obtained from an animal, it might be expected that with regular booster
injections of antigen, the animal would continue to
produce antiserum
sufficient for literally thousands
of RIA determinations.
However, the specificity,
affinity, and titer vary with the number of immunizations given, and vary from animal to animal. Therefore, each antiserum
must be studied and evaluated
separately.
Once characterized,
the antisera can be
stored for long periods of time under proper conditions. Repeated
freezing
and thawing
should be
avoided and all antisera should be stored properly
diluted. Reports vary as to the best temperature
for
antiserum
storage,
some workers prefer -80#{176}C,
others -20#{176}C
or -15#{176}C
(316). Once thawed, the sera is
best kept at 4#{176}C
(5). The antisera may be stored diluted with “Rivanol” (2-ethoxy-6, 9-diaminoacridine
lactate). This agent is used to precipitate
all serum
proteins, including the albumins,
a-globulins,
most
of the fl-globulins,
and the immunoglobulins
except
the IgG antibodies
(354). Bacterial
contamination
can also be avoided by adding sodium azide (1 g/
liter) to the antisera (C. Longcope, personal communication).
Separation
of Bound and Free Antigen
The third requirement
for a good RIA is a suitable
method for complete
and rapid separation
of the
bound antigen (Ag*AbIV and AgAbVI) from the free
antigen (AgV and Ag*VII) so that the radioactivity
of either or both of these phases can be accurately
measured.
In addition,
a separation
procedure that
permits further association
and dissociation
of the
reactants
(Ag*I, Agil, and Abifi) will ,seriously impair the effectiveness
of the assay. Regardless of the
method of separation
chosen, it must be reproducible, simple to perform, and economically feasible.
Chromatoelectrophoresis.
The first
method
reported for the separation
of bound and free fractions
was paper chromatoelectrophoresis
(2, 22, 24, 175,
355), combining
earlier-used
techniques
of paper
chromatography
(356) and paper
electrophoresis
(357). A simple electrophoresis
involves the differential migration in an electrical field of the bound and
free antigen on cellulose acetate, and has been used
in the assay of growth hormone (357). Improvements
were then made by using paper that had a high affinity for the free antigen, such as Whatman
3 MM,
Whatman
3 MC, DEAE paper, and Toyo No. 514.
Starch gel (358) and polyacrylamide
(including the
use of discs) (173, 265, 359, 360) have also proved
satisfactory
as carriers. [For a discussion of polyacrylamide
gel electrophoresis
and its application
to
isoelectric focusing, see review by Chrambach
(361).]
The free antigen binds firmly to the cathodal end,
while the heat generated by the electric current acts
to increase water evaporation,
and the antigen-antibody immune
complex migrates by hydrodynamic
flow. While this method is most discriminative-and
has been used in the assay of insulin (2, 22, 25, 26,
32), GH (187, 357), glucagon (362, 363), PTH (181),
TSH (364), ACTH (18, 365), and thyrocalcitonin
(366)-it
has a number of disadvantages
that preclude its use. A limited amount of material may be
applied to the adsorbent,
the separation
is both laborious and time consuming,
the equipment
is expensive, it requires radioactive
antigens with higher
specific activities
than do other techniques,
and a
cold room is necessary.
Nonspecific
precipitation
of the immune complex
with various salts or organic solvents has also been
used as a means of separating
bound and free fractions. The antigen-antibody
complex is precipitated
by the salts or solvents under conditions that do not
affect the free antigen. Among the reagents used are:
acetone, sodium sulfate, or ammoriium
sulfate (367)
for HCG (368), arginine vasopressin
(369), insulin
(31), glucagon (370, 371) and androgens (372); alcohol for TSH (373, 374), LH (375-377),
and insulin
(378); and dioxane for gonadotropins,
ACTH, HGH,
insulin (379-381), and calcitonin (382). In one modified procedure, glutathione-activated
ficin (a proteolytic enzyme) is used, which selectively hydrolyzes
the unbound antigen (AgV and AgVII); the degraded
products
are then soluble in trichioroacetic
acid.
After precipitation
of the immune complex by centrifugation,
the radioactivity
in either of the phases
can be measured. This method has been applied successfully to the assay for insulin (383) and HGH
(384), but requires a proper balance of enzyme to antigen in order to avoid either damage to the bound
161
antigen or inadequate
degradation
of the free antigen. Gel filtration
on the cross-linked
dextrans has
also been used in assays for insulin (385) and HGH
(357), but because of the time and space required for
the preparation
of individual
columns, this method
is impractical
for large-scale use. Another modification, the “back titration”
method, has been used for
the precipitation
of insulin (386). In this procedure,
an excess of antiserum
is first pre-incubated
with
unlabeled
insulin,
and the partially
neutralized
serum is then incubated
with an excess of the labeled insulin.
The use of the salts or solvents has the advantage
that the separation
is immediate
and a second incubation is not necessary. However, the chemical precipitation technique
may bring down other proteins,
often causing an incomplete
separation
of the two
fractions.
Bonding of Ag or Ab to a solid phase. The development of various solid-phase
methods provides for
rapid and efficient separation
(see reviews 387 and
388). In the solid-phase
type of assay, the antibody
or antigen is bonded or fixed to an adsorbent,
which
facilitates
separation
or precipitation
of bound or
free fractions. The solid-phase
antibody techniques
make use of antibodies covalently bonded or fixed to
insoluble
polymers,
covalently
cross-linked
to one
another, or physically adsorbed to a plastic. The insoluble carriers or immunosorbents
include bentonite
particles (389, 390), bromacetyl
cellulose (390), the
cross-linked
dextrans
(Sephadex)
(391), or beaded
agarose (Sepharose),
and “Enzacryl AA” (392). The
beaded agarose molecules have a loose structure,
allowing better fixation for large molecules than is afforded by the dextrans, and may be activated by cyanogen bromide to permit a firm binding of the antibody (388). While large amounts of antibodies
are
required in the solid-phase
antibody systems, they
do have the advantage
that all three components
of
an assay (the antibody, the tracer antigen Ag*I, and
the means of separating
the bound and free phases)
are found within one unit. Binding of the antigen is
rapid, virtually irreversible,
and the two phases may
be separated
by simple decantation
or low-speed
centrifugation.
A modification
of this method, the
technique of selective adsorption of the antigen-antibody complexes by cellulose ester (“Millipore”)
filters (393) has been used in the assay of IgA in
human cord serum (394) and cyclic AMP (395). In
addition,
nitrocellulose
membranes
have been used
to bind antibodies
to angiotensin,
digoxin, cyclic
AMP, morphine,
and prostaglandins
B1, F2a, and
Fia (396).
Polymerized
antibodies,
covalently cross-linked
to
one another
with ethyl chloroformate
or glutaraldehyde, provide a practical and accurate method in
assays for estrogens (321, 397), LB (261, 398), and
FSH (171). While the use of polymerized
antibodies
offers speed and reproducibility,
the procedure
requires high-speed
centrifugation
for precipitation
of
complex.
162
CLINICAL
CHEMISTRY,
Vol. 19, No. 2, 1973
The solid-phase
antibody method by physical adsorption includes the use of poly (tetra-fluoroethylene-g-aminostyrene)
(“Protapol”)
powder
(399,
400), “Protapol DI/l,” or polypropylene-coated
discs
(387-404), and polypropylene
or polystyrene
plastic
tubes (261, 387, 400, 405-408). The preparation
of
the antibody-coated
discs or tubes may be affected
by the pH, molarity, temperature,
and protein concentration
of the incubating
solution. However, the
discs or tubes have the advantage
that, once prepared, they may be stored for many months until
needed. In addition, the technique permits many determinations
to be performed rapidly, and it is highly sensitive
and reproducible.
This technique
has
been successfully
applied in assays of insulin (409),
angiotensin
(400), HGH (401, 402, 407, 408, 410,
411), HCS (412), LII (403), HPL (402, 403, 407,
413), estrogens (405, 414, 415), influenza-specific
immunoglobulin
G (146) and subtypes
of Australia
hepatitis B antigen (289). One modification
uses an
acrylamide
gel that serves to entrap the gamma
globulin,
but allows diffusion
of low-molecularweight antigens (417). Another variation of this technique, the “sandwich
solid-phase
RIA,” has been
used for the characterization
of human immunoglobulins (IgG) synthesized
in tissue culture (418). In
this method, pure antigen is first bound to the walls
of a plastic test tube and the antibody is then bound
immunochemically
to the solid-phase
antigen. Unlabeled antigen or labeled antigen (containing
incorporated [3H]leucine)
is then bound to the antibody.
After incubation,
the contents of the tubes are aspirated, the tubes washed, and the entire tube is solubilized and the radioactivity
counted (419).
Separations
by solid-phase
antigen precipitation
have satisfied
the needs for simplicity,
economy,
speed, and reproducibility.
The free (unbound)
antigens are bound to adsorbents,
which then can be
precipitated
by centrifugation.
Powdered
talc, a
magnesium
silicate (304, 420), kaolin (aluminum
silicate) (421), QUSO (microgranules
of silica) (365,
420), Florisil (374, 397), and cellulose powder (127,
422) are some of the simple adsorbents
used in a
number of radioimmunoassays
(for review see ref.
423). Too low a concentration
of plasma protein will
cause some of the antibody to be adsorbed, while an
excess of plasma proteins often leads to decreased
adsorption of the tagged antigen (Ag*I). Therefore, a
balanced proportion of these proteins and the adsorbent is a prerequisite for good separation.
Many separations
have been performed by using
charcoal coated with a dextran, the molecular weight
of which may vary from 10,000-250,000,
depending
on the size of the unbound antigen being adsorbed.
The synthetic
glucose polymer forms a matrix that
allows only the smaller-molecular-weight
particles
(i.e., the unbound
antigen) to be adsorbed by the
charcoal. This simple, rapid, and economical procedure has been used successfully in the RIA of ACTH
(424), LH (425, 426), HGH (427, 428), insulin (205,
429-432), angiotensin
II (433), human calcitonin M
(434), blood kinins (435), and the cardiac glycosides
(243, 436). Ion-exchange
resins such as Amberlite
CG-400 also adsorb unbound antigen and have been
used in assays of ACTH (437), HGH (438), insulin
(439), and glucagon (440), but have not acquired the
popularity
shared by other adsorbents.
In the immunoradiometric
assay for insulin and calcitonin (127129), in which unlabeled
antigens
are allowed to
react with labeled antibodies,
the unreactive
antibodies are removed with the addition of the hormone
coupled to cellulose.
Double-antibody
technique.
A widely used and
practical method for the separation
of the bound and
free antigen is the double-antibody
technique,
in
which a second antibody
is used to precipitate
the
primary
antigen-antibody
complex.
Although
the
use of a second antibody
introduces
an additional
source of error, the advantages
of the method are
that it can be used for practically
any RIA, the separation is complete,
and it may be used with large
volumes of incubating solutions.
Because more of the precipitating
antiserum
is required, the second antibody
is induced in a larger
animal (e.g., sheep or goat) against the gamma globulin (IgG) from the animal used to elicit the first
antibody.
Therefore,
the term “ARGG”
or “antiRGG” defines that the second antibody
is formed
against rabbit gamma globulin. The second antibody
may be concentrated
by a salt or DEAE precipitation before it is used in an assay.
Present approaches
to the double-antibody
separation technique
include the “post-” and “pre-precipitation” methods, both of which depend on when the
first and second antibody are allowed to interact in
terms of antigenic binding. In the “post-precipitated” approach (441), the second antibody is used to
precipitate
complex, while in
the pre-precipitation
method the antigenic material
interacts with the first antibody insolubilized
by previous precipitation
with the second antibody.
The conditions for the “post-precipitation”
method have been discussed in detail (441, 442); these authors emphasize
that the speed of this precipitation
clearly
depends
on the antibody
concentration.
While most double-antibody
procedures require days
for precipitation
to reach equilibrium,
there are rapid
methods that may be used at the expense of sensitivity and specificity
(441). The technique
requires
the use of heat or chelating agents (e.g. EDTA) to inactivate serum complement
(203, 433, 434) and the
equalization
of the human
serum content
of all
samples and standards
at the time of the precipitation (442). The “pre-precipitation”
method, although
not as popular as the “post-precipitation”
approach,
has the advantage
in that the immunoprecipitation
step is not affected by most of the interfering
nonspecific factors (157,445, 446).
Filtration or centrifugation.
After precipitation
of
the immune complex, the precipitate
and the supernatant fluid may be separated
by filtration or centrifugation.
In the filtration
technique,
the precipi-
tating antibody
is used in a high dilution and the
microprecipitate
removed by cellulose acetate filters
(Oxoid and Millipore)
(138, 157, 447, 448). The alternative approach is to add higher concentrations
of
antibody with normal rabbit serum as a carrier and
to centrifuge
the bulky precipitate
formed (203,
449-452). This centrifugation
method
has become
widely popular,
not only because of its simplicity
and high degree of precision, but also because it is
inexpensive.
With suitable conditions and the use of
a computerized
program, a technician
can easily perform a thousand assays each week. The double-antibody technique-first
described for insulin (157, 451,
452), growth hormone (192, 453), LH (239, 263), and
FSH (172, 173, 454, 455)-has
been applied to assays
for other antigenic
materials
found in plasma
in
physiological states (see Table 2).
One should not assume that an antiserum that exhibits good sensitivity,
specificity, and precision in a
RIA with one particular
method of separation can be
readily applied to a second separation
technique.
If
any one of the steps in a procedure
is altered, the
conditions
for a good RIA must be re-evaluated
before the assay can be applied to routine clinical use.
The Incubation
The separation
techniques
described
are an integral component
of the entire RIA. During the incubation period (i.e., the time needed to reach equilibrium), the radiolabeled
antigen (Ag*I) and unlabeled
antigens (Agil) are allowed to compete for binding
sites on the antibody (AbIII). The time required to
achieve equilibrium
varies from a few hours to several days, depending
on the specific antigen being
measured. In some RIA procedures,
it is recommended that the incubation
not be carried to the point of
equilibrium.
As noted, sensitivity
may be improved
by delaying the addition of the labeled antigen (161).
With long incubations,
the antigen may be altered
(“incubation
damage”)
because of prolonged exposure to the high concentration
of plasma proteins. In
general, the hormones ACTH, glucagon, and thyrocalcitonin
are more susceptible
to this type of damage than are the hormones HGH, TSH, LH, and insulin.
The damage caused by free radicals, oxidants and
(or) proteolytic
enzymes can be avoided or minimized by incubating
at low temperatures,
avoiding
high plasma concentrations,
or adding proteinase inhibitors,
such as mercaptoethanol,
c-amino-caproic
acid, or aprotinin
(“Trasylol”)
(456). The effects of
Trasylol in increasing the precipitations
has been reported favorable
for glucagon (457), but poor for
ACTH (138). When the damage persists with Trasylol, as occurs with glucagon (458), “damage controls”
incubated
without antiserum
must be included,
to
correct for these losses. Mercaptoethanol
inhibits the
oxidation of labeled hormones containing methionine
during storage, but will also inhibit the antigenantibody reaction when used in concentrations
greater than 5 g/liter (365). Benzamidine
is less expensive
CLINICALCHEMISTRY,
Vol. 19, No.2,
1973
163
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Rodbard et a!. have discussed (147) several guidelines to establishing
quality-control
procedures.
Basic to these measures is the accurate recording of
the specific activity of the tracer antigen (Ag*I) and
the exact amount of radioactivity
used in each assay.
The primary factors include: (a) the 50% intercept,
i.e., the amount of standard
that will bind 50% of
the 0 standard
(B/B0 = 0.5); (b) the percent of the
total counts bound in the absence of unlabeled antigen (25%-50%);
(c) the within-assay
variance (i.e.,
the variation in multiple determinations
of the sample run in the same assay); and (d) the betweenassay variance (i.e., the variation of multiple determinations
of the same sample in different assays).
Acceptable tolerance ranges for each of these parameters should be determined by each laboratory.
0.U)
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assays (459).
QualityControl
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glucagon
the proteolytic
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The RIA technique has been extended to the measurement of other peptide hormones and nonhormonal substances
including drugs, pathological
agents,
viral proteins, and enzymes. Hormones to which this
technique
can be applied include relaxin (519), j3lipotropic hormone (255), secretin (520), bradykinin
(300, 301, 435), alpha-melanocyte
stimulating
hormone (521), pancreozymin-cholecystokinin
(522) and
the arthropod
molting hormone, ecdysterone
(523).
The non-hormonal
substances
include: the enzymes
Cl esterase (524), trypsin, chymotrypsin,
and chymotrypsinogen
(525),
dopamine-.3-hydroxylase
(526),
pepsin and pepsinogen
(527), carbonic anhydrase
I
and II (528), and fructose-1,6-diphosphatase
(529);
the cyclic nucleotides
cAMP, cGMP, cIMP, and
cUMP (530) and thymidine
(531); and the drugs
morphine (532), D-tubocurarlne
(533) and the barbiturate derivatives (534).
Also developed are radioimmunoassays
for various
immunoglobulins
(535-537)
including
the
antiRho(D) content of Rho(D) immune globulin (538),
antithyroglobulin
antibodies
(539), keyhole limpet
hemocyanin
antibody (540), rheumatoid
factor (541),
antibodies in experimental
allergic encephalomyelitis
(542), the IgE antibody to ragweed antigen E (543),
the incorporation
of leucine into the immunoglobulin
IgG (544), antibodies
to type 12 streptococci
(545)
#{149}
and lymphocyte
surface receptors (546). The technique has also been extended
to the proteins
1Cglobulin (547), human fibrinopeptide
A (548), the
vasopressin
binding protein neurophysin
(549), the
synthesis
of serum albumin
by human embryonic
liver cultures (550), properdin (551), and to the polypeptide mouse epidermal growth factor (552).
Additional antigenic materials used in RIA include
allergens (553), paramecium
surface antigens (554),
vaccinia antigens (555), thermostable
adrenal-specific antigen (556), human histocompatibility
antigens
(557), Australia
(hepatitis-associated)
antigen (289,
558),
(512,
the embryonic reversion antigens a-fetoprotein
565) and carcinoembryonic
antigen
(CEA)
(566-569).
There
is currently
much
interest
being
focused on CEA, as this glycoprotein
has been found
present in malignant
entodermal
tissues and in the
plasma of patients with gastrointestinal
tract cancers.
Future
developments
of CEA-RIA
may find it useful
as a diagnostic
tool in early and sensitive
cancer de-
tection.
Advances
in clinical pathology
have also
been made by the reports of radioimmunoassays
for
7S nerve growth factor (570), mammalian
type C
viral proteins (571-572), staphylococcal
B enterotoxin
(573), hepatitis B antigen (574), schistosomiasis
(575)
and rabies binding antibodies
(576). The RIA procedure has also been extended to the measurement
of
vitamin
A (577), urinary antidiuretic
hormone
(578),
cortisol (579), and medroxyprogesterone
acetate (580).
Needless
all areas
to say, the RIA technique
is revolutionizing
of investigational
and clinical science.
Conclusion
While it is difficult to reach definitive conclusions
on such a new and untamed technique as the RIA, a
number of general statements
may be made regarding its distinctive
features.
When compounds
are
able to induce the formation of specific antibodies,
the nature of the antigen-antibody
reaction affords a
method
of analysis
with a high degree of sensitivity,
which can be performed
simply and efficiently
in any
laboratory.
Indeed,
the use of this tool is now commonly accepted
by countless
laboratories
undertaking investigational
and clinical
determinations.
For
example,
RIA has enabled
researchers
to detect and
characterize
new hormonal
forms and to monitor
in
physiological
and pathological
conditions
the concentrations
of a variety
of hormones
and non-hormonal substances
that could not have been demonstrated
by previous
techniques
except with great difficulties.
In addition,
many laboratories
now perform
hundreds
or thousands
of assays each day, greatly
adding
to the increased
efficiency
and economy
demanded
by modern clinical and research
scientists.
The list of compounds
that can be measured
by
RIA grows each day, and includes
many non-hormonal peptides,
drugs, enzymes,
parasitic,
microbial, and viral agents.
It can also be expected
that
methods
will appear
in the future that will improve
and refine the general RIA procedure-e.g.,
decreasing isotopic
damage
of the antigens
or antibodies,
enhancing
the specificity
and affinity
of the antibody-antigen
complex,
improving
the purification
of
the specific antibodies
generated
and simplifying
the
incubation
and separation
techniques
while maintaining accuracy
and reproducibility.
Certain
general
characteristics
of the method
should be emphasized.
While the procedure
is simple
in design, it is important
that each laboratory
establish good quality-control
measures
for the assays and
that any changes
from a standard
procedure
be thoroughly evaluated.
Because
the reactants
are immunospecific,
the
values found always represent immunological
activity, not biological activity. Therefore, it is quite likely
that one of the future changes in the RIA will be in
this particular
area, to substitute
specific biological
receptors
for the antibody
in the assay. This change
will occur when these receptors
can be efficiently
and
economically
exploited
to the same extent
as the
antibody,
while also maintaining
or improving
the
degree of sensitivity.
If similar
modifications
should
evolve into newer techniques,
RIA will remain
as a
landmark
in quantitative
and qualitative
analyses.
Glossary
Adjuvant,
a substance which will enhance the antigenicity of an antigen. Freund’s adjuvant:
“incomplete”mixture of mineral oil and waxes; “complete”-incomplete plus killed tubercie bacilli.
Affinity,
strength of attraction between antibody and
antigen.
Antibody,
usually a gamma globulin or immunoglobulin
that will specifically react with an antigen or hapten.
Antigen, a substance that can induce the formation
an antibody and is able to react with that antibody.
strength
Avidity,
of the antigen-antibody
of
bond after the
immune complex is formed.
Complement,
serum
factors that when activated
may
diminish the strength of the antibody-antigen
reaction.
Double-antibody,
the separation technique whereby a
second antibody,
produced against the IgG of the first animal, is used to precipitate the immune complex.
Hapten, an incomplete
antigen,
which
must
be coupled
to a carrier to form an antigen.
Homologous,
a system in which the same animal c
species is used to produce the radiolabeled
antigen, unlabeled antigen, and antibody.
Heterologous,
a system in which the radiolabeled
antigen, unlabeled antigen, and antibody are respectively
produced in two or more animal
species.
Ligand, (from the Latin, “that which is bound”) usually
refers to the smaller molecule,
Sensitivity, lowest detectable quantity.
Specificity,
the degree of freedom from interfering substances.
Titer, optimal
dilution
of antibodies
effective in an
assay.
Addendum
Since
the
completion
of this
review
(November
1,
a number of new RIA procedures have been reported. A 7-8S highly negatively
charged antibody
1972),
fraction
with
increased
sensitivity
from crude sheep antisera
Sephadex
chromatography
dure
may
be applicable
has been
isolated
to pregnenolone
by QAE(581). This novel proce-
to the isolation
of highly
sen-
sitive antibodies
against steroids and other haptenBSA antigens.
The use of polyethylene
glycol for the separation of
free- and
antibody-bound
insulin,
parathyroid,
growth hormone, and arginine vasopressin
(582) has
now been demonstrated
as effective in the assays of
digoxin and renin (583). The Somogyi precipitation
has also been found to yield an immediate
and nonreversible
separation
of digoxin and other steroid-like
compounds
(584). Zirconyl phosphate
gel will also
produce
assays
highly
of CEA
sensitive,
precise
by precipitating
and
the
CLINICAL CHEMISTRY, Vol.
reproducible
antibody-bound
19,
No.2,
1973
173
antigen (585). Th use of 8-anilino-1-naphthalene
sulfonic acid (ANS) has been found effective in preventing possible interference
in the binding ofT3 antibodies by TBG (559).
Recent developments
include the RIA for human
skin collagenase
(586), placental-type
alkaline phosphatase (587), and the detection
of antihemophilic
factor (AHF, factor VIII) in concentrations
as low as
0.2% in normal
human
plasma
(588). It is also possible to determine
IgG concentrations
in undiluted
cerebrospinal
fluid with a sensitivity of 10 ng/l00 ml
(589). This RIA may provide a useful diagnostic
tool
in differentiating
between central nervous system
systemic
lupus
erythematosus
and
other
causes
of
central
nervous system symptomatology,
such as
steroid-induced
encephalopathy.
The analysis of cerebrospinal
fluid for encephalitogenic basic protein by RIA may provide a useful
diagnostic tool in the detection of multiple sclerosis
(590). Finally, a RIA method has been reported (591)
for the determination
of parotin present in the parotid
gland, submaxillary
gland, serum, and urine of guinea
pigs, and in the urine and saliva of humans.
A number of hormones labeled with 125! are now
available
for RIA from the New England Nuclear
Corp., 575 Albany St., Boston, Mass. 02118. These
include angiotensin
I and H, bradykinin,
glucagon,
HCS, HCG, HGH, and insulin.
The authors
permission
are grateful
to the following
to reproduce
certain
2, 3, and 6), Dr. Gregor
individuals
for their
items:
Dr. John T. Potts,
Jr.,
H. Grant (Figure 4), Dr. A. Rees
(Figures
Midgley, Jr., (Figure 5), Drs. H. Randar Raud and W. D. Odell
(Figure 7), and Drs. R. P. Ekins, J. L. H. O’Riordan
and John T.
Potts, Jr., for the general derivations
of the formulas used in discussing the basic kinetics
of A.
We also thank
Dr. David M.
Mumford
for his review of this manuscript
and to Mrs. C. Knight
and Mrs. E. Lindsey
for their typing assistance.
Support
for the
publication
of this review was made possible
in part by the Roderick A. MacDonald
Fund and by subsidies
from Serono Laboratories, Inter Science
Institute, New England Nuclear (Biomedical
Assay Laboratories),
Becton,
Dickinson
and Co. (Schwarz/Mann
Division),
Clinical
Assays,
Inc.,
Collaborative
Research,
Inc.,
Pharmacia
Laboratories,
Inc., Calbiochem,
and Endocrine
Sciences-Products
Division.
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factor
antigens
in serum

Radioimmunoassay - Clinical Chemistry

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