CLIN. CHEM. 30/4, 538-541 (1984)
Measurement of Choriogonadotropin by Chemiluminescence Immunoassay
and Immunochemiluminometric Assay: 1. Use of Isoluminol Derivatives
G. J. Barnard,’ J. B. Kim,”4 J. L Brockelbank,’ W. P. CoIlins,” B. Gaier,2 and F. Kohen2
We have developed immunoassays, monitored by the detection of chemiluminescence, for measuring choriogonadotropin in human urine. These methods involve the use of
derivatives of isoluminol and include: (a) a labeled antigen
with a second antibody covalently linked to polyacrylamide
beads as the solid-phase reagent (i.e., solid-phase chemiluminescence immunoassay); and (b) an excess concentration
of a specific antibody passively adsorbed onto the walls of
polystyrene tubes and a labeled antibody of different specificity (i.e., two-site immunochemiluminometric assay). After the
respective binding reactions, the solutions are aspirated, the
antigen- or antibody-bound fractions are washed twice with
500 L of buffer, sodium hydroxide (2 mol/L; 200 L) is
added, and the mixture is incubated for 60 mm at 60 #{176}C.
After
cooling, the label is oxidized with microperoxidase/hydrogen
peroxide and the resulting chemiluminescence signal is
measured for 10 s. We have evaluated the methods in terms
of their sensitivity, precision, and clinical utility, and we
compare results with values obtained by radioimmunoassay.
Additional
Keyphrases:hormones
urine
say compared
cutoff value
pregnancy
radioimmunoas-
is secreted by the
and is detectable in
the peripheral
circulation
of the mother shortly after implantation.
Renal clearance rates of hCG have been reported, and a discrete
immunoreactive
degradation
product of
the /3-subunit has been isolated from urine and characterized (1). Early tests for hCG based upon hemagglutination
and latex agglutination
have been supplemented
with radioimmunoassays
(RIAs) in which specific antibodies directed against the (3-subunit are capable of detecting pregnancy
a few days after implantation
(2). Recently, several RIA kits
have become available commercially
and have been evaluated (3). Despite this increasing
availability
and usefulness
of RIA, however, there are still several drawbacks, including: (a) the brief radioactive
half-life and radiolysis
of the
labeled reagent;
(b) health
hazards associated
with the
preparation,
use, and disposal of radioactive
compounds;
(c)
legislative and emotional biases against the use of radioactivity; (d) the increasing
trend towards the development
of
Human
trophoblast
tests
choriogonadotropin
of the developing
plasma, urine, or saliva in which the antigens
are labeled
with derivatives of isoluminol (10, 11); these methods are as
sensitive, precise, and accurate as the corresponding RIAs.
In addition, some immunoassays
have involved the use of
luminol and isoluminol
derivatives
of proteins (12, 13).
Using protein-isoluminol
conjugates prepared with a hemisuccinamide
derivative of aminobutylethyl
isoluminol
(ABEl), we have developed a solid-phase CIA and a two-site
immunochemiluminometric
assay (zc.s) for the measurement of hCG. In these assays we use: (a) a labeled antigen
(hCG-ABEI)
in a competitive
binding
assay,
and (b) a
labeled antibody (rabbit anti-hCG IgG-ABEI) in the immunometric assay. Here, we describe and evaluate the methods,
assess their clinical utility for the early detection of pregnancy, and discuss the advantages
and limitations
of each.
(hCG)5
embryo
for use outside the laboratory; and (e) the difficulty in
RIA procedures
in isolated communities.
Sever-
establishing
‘Department of Obstetrics and Gynaecology, Diagnostics Research Unit, King’s College School of Medicine, Denmark Hill,
London SE5 8RX.
2Department of Hormone Research, The Weizmann Institute of
Science, Rehovot, Israel.
Address correspondence to this author.
Present address: Dept. of Animal Product Science, College of
Animal Husbandry, Kon-Kuk Univ., Seoul, Korea.
5Nonstandard
abbreviations: hCG, human choriogonadotropin;
CIA, chemiluminescence immunoassay; ict, immunochemiluminometric assay; ABEl, aminobutylethyl isoluminol; ABEl-H, hemisuccinainide derivative of ABEl (see Fig. 1).
Received October 7, 1983; accepted January 10, 1984.
538
al proposed alternatives
to RIA have been reviewed (4) and
various nonisotopic immunoassays for the measurement of
hCG in serum, plasma, or urine by use of colorimetry (5, 6)
or fluorometiy
(7) have been described.
As another alternative,
chemiluminescent
derivatives
of
isoluminol have been evaluated for their potential as labels
in monitoring specific protein-binding
reactions (8, 9). We
have developed and evaluated several chemiluminescence
immunoassays
(CIAs) for the measurement
of haptens in
CLINICAL CHEMISTRY, Vol. 30, No. 4, 1984
Materials and Methods
Materials
Reagents.
A hemisuccinamide
derivative of aminobutylethyl isoluminol
(ABEl-H; 6-[n-(4-aminobutyl)-N-ethyl]amino-2,3-dihydrophthalazine-1,4-dione)
was kindly donated by Dr. T. Lovgren, Wallac Oy, Turku, Finland. The hCG
(Canfield CR123) was obtained from Dr. G. Bialy, National
Institutes of Health, Bethesda, MD. Rabbit antibodies to the
(3-subunit of hCG were donated by Amersham International
plc, Amersham, Bucks., U.K., and RIA kits for measuring
hCG (Amerlex kit; IM 2091) were purchased
from the same
source. An IgG fraction of the antibody
was prepared
by
affinity chromatography
on Protein A-Sepharose
CL-4B as
already reported (10). Microperoxidase
(MP-11), bovine serum albumin
(Cohn Fraction V), and Sepharose-Protein
A
were from Sigma London Chemical Co. Ltd., Poole, Dorset,
U.K. Sheep anti-rabbit IgG covalently
linked to polyacrylainide beads (“hnmunobeads”)
was from Bio-Rad Laboratories Ltd., Watford, Herts., U.K., and was reconstituted
by
the addition of 50 mL of phosphate buffer (pH 7.5). All other
reagents were obtained from Hopkin and Williams Ltd.,
Romford, Essex, U.K.
Buffers. We used two assay buffers. The first was a coating
buffer (barbital,
70 mmollL, pH 9.6), prepared by dissolving
14.4 g of sodium barbital
in 1 L of doubly distilled water
containing 1 g of sodium azide. The other was a phosphate
buffer (100 mmolJL, pH 7.5), prepared by dissolving 3.06 g of
NaH2PO2
2H2O and 11.6 g of Na2HPO4 in 1 L of doubly
distilled water containing
5 g of bovine serum albumin, 9 g
of NaCl, 1 g of sodium aside, and 1.66 g of EDTA. Microperoxidase was dissolved in phosphate
buffer and the stock
.
solution
(1 mg/mL), stored at 4 #{176}C,
was stable for at least
three months. The working solution of microperoxidase
was
prepared
by diluting the stock solution 50-fold. The oxidant
solution was prepared by adding 100 L of 300 g/L hydrogen
peroxide solution to 10 mL of doubly distilled water.
Preparation
of chemlluminescent
reagents.
We prepared
the hCG-ABEI conjugate in two steps. Initially, an Nhydroxysuccinimide
ester of ABEl-H was prepared in dry
dimethylformamide
in the presence of carbodiimide and Nhydroxysuccinimide
(Figure 1). Then we added an aliquot (6
1zL;300 nmol) of the activated ester (in dimethylformamide)
to a solution of hCG (Canfield 119: 0.6 mg, 15 nmol)
dissolved in 1 mL of phosphate buffer (50 mmol/L, pH 8).
The reaction mixture was stirred and dialyzed overnight at
4#{176}C
against the pH 8 phosphate buffer. The resulting
conjugate was purified by ion-exchange chromatography
on
diethylauninoethyl-Sephacel
prepared in the pH 8 phosphate
buffer, and eluted with a gradient of NaCl (from 50 to 500
mmol/L).
We checked the protein content of all fractions
eluted from the column by using a spectrophotometer
and by
measuring
chemiluminescence
activity. Fractions containing the labeled hormone were assessed for immunoreactivity in a double-antibody
RIA with I-labeled
hCG as the
competing label. Incorporation
of 3 mol of ABEl per mole of
hCG did not significantly
affect immunoreactivity,
whereas
6 mol of ABEl per mole of hCG increased
chemiluminescence but decreased
immunoreactivity.
Consequently,
we
used the preparation
containing 3 mol of label per mole of
hCG.
To prepare the labeled antibodies to hCG, we added 10 p.L
(100 nmol) of the activated ester (in dimethylformamide)
to
a solution of rabbit polyclonal nonspecific anti-hCG IgG (2
nmo’l in 1 mL of the pH 8 phosphate buffer). The reaction
mixture
was stirred and dialyzed against the pH 8 phosphate buffer overnight
at 4#{176}C.
We purified the labeled
antibody by chromatography on Sephadex G-25 prepared in
the pH 8 phosphate buffer. The label was eluted in the void
volume (3 mL). We added sodium aside (1 g/L) and bovine
serum
albumin
(10 g/L) to the fractions containing
the
labeled antibody. The average incorporation of the label was
6 mol per mole of protein.
Antibody-coated
tubes. Rabbit anti-(3 hCG IgG, in excess,
was suitably diluted in the coating buffer and 200 &L of this
was added to each polystyrene assay tube (FF 0944; Luckham Ltd., Victoria Gardens,
Burgess Hill, Sussex, U.K.).
After an overnight incubation at 4#{176}C,
the coating buffer was
aspirated and discarded and 400 i.L of pH 7.5 phosphate
buffer was added to each tube. After incubation for 30 miii at
22#{176}C
the tubes were stored at 4#{176}C
until required.
were stored at -20 #{176}C
until pregnancy was conFor the clinical evaluation of these methods,
we
measured hCG in samples from each cycle, from the day of
the lutropin peak until a routine pregnancy test gave a
positive result (arbitrarily set at> 50 mt. units of hCG per
literi on three consecutive days.
samples
firmed.
Procedures
CIA. Add 100 L
of sample or standard (range, 8 to 328
of urine from males) in duplicate to the assay
tubes, then add 100 L of 2000-fold diluted anti-/3-hCG
antibody, 100 .tL of hCG-ABEI (10 ng, producing chemilumineseence
of about 100 mV/100 L of phosphate buffer),
and 200 L of Immunobead
reagent. After incubating the
mixture at 22 #{176}C
for 2 h, add 1 mL of phosphate buffer to
each tube and vortex-mix. Centrifuge for 10 mm at 2000 x g
and remove the supernates by aspiration. Repeat the washing step, discarding the supernates,
then add 200 L of 2
molIL sodium hydroxide to each tube and incubate at 60 #{176}C
forGO mm. Cool the tubes to room temperature
and add 100
L of the microperoxidase solution to each assay tube, then
place it in the luminometer.
Initiate the chemiluminescence
reaction by rapidly injecting 100 L of diluted hydrogen
peroxide with an automatic
dispenser
(we used a “Microspenser” from Hook and Tucker Instruments Ltd., New
Addington, Surrey) and measure the light emitted (we used
an LKB Luminometer
Model 1250 and display, kindly
provided by LKB Instruments Ltd., South Croydon, Surrey,
U.K.) for 10 s, sufficient to measure 65% of total light
emission. Calculate results by comparison with a calibration
curve.
IcMA. Add 100 L of sample or standard
(range 0 to 320
int. units/L of urine from males) in duplicate to the antibody-coated tubes, then add 100 tL of phosphate buffer,
incubate at 22 #{176}C
overnight, and aspirate the liquid. Add
200 L of suitably diluted (excess) nonspecific anti-hCG
IgG-ABEI conjugate and incubate the mixture at 37 #{176}C
for 2
h. Again, aspirate the liquid, add 500 jL of phosphate buffer
to each tube, aspirate the liquid, and discard. After repeating this washing step twice more, add 200 L of 2 molIL
sodium hydroxide to each tube and incubate at 60 #{176}C
for 60
mm. Cool to room temperature, then add 100 L of the
microperoxidase solution to the assay tube and place it in
the luminometer. Initiate the chemiluminescence
reaction
by rapidly injecting 100 L of diluted hydrogen peroxide,
measure
the signal for 10 s as described
previously, and
calculate
the results.
mt. units/L
Results
CIA
Sample Collection
Ten healthy female volunteers with regular
menstrual
cycles (25 to 33 days) were recruited
from the infertility
clinic at King’s College Hospital. Each woman was requested to record day 1 of her cycle and her daily basal body
temperature
(to determine the nadir and shift), to collect an
early morning sample of urine for each day throughout the
cycle, and to undergo pelvic ultrasonography at appropriate
intervals to determine the time of follicular rupture. The
,,0
(_o
Figure 2 shows a typical calibration curve (mean ± SD;
six replicates) for hCG as determined by CIA. The minimum
concentration of hCG that could be significantly
distinguished from zero (mean
2 SD), calculated
from three
calibration curves, was 2 ± 0.1 mt. units/L. Within-batch
precision was estimated by the replicate analysis (n = 20) of
a sample of urine with an hCG at about the arbitrary cutoff
value for the pregnancy test (50 mt. units/L). The mean was
52.3 (SD 6.2) mt. units/L, the CV 11.9%. The between-batch
CV for four assays of the same quality-control
sample was
14.1%. The concentrations of hCG in selected samples of
early-morning
urine collected throughout the 10 cycles,
determined
by CIA (y) and by an RIA with an iodinated
tracer (x), correlated well: r = 0.98, n = 6O;y
1.14x -6.78.
-
=
[_o_H2)2
CO-NH-tCH2)4-
Fig. 1. Proposed structure(tentative)of an activated ester of ABEl-H
ICMA
Figure 3 shows calibration curves prepared with the use
of various incubation times (range 2 h to seven days). The
CLINICALCHEMISTRY,Vol. 30, No. 4, 1984
539
zero
7 days
16024 hours
4 hours
2 hours
140120
>
E
-J
10080-
at
HCG
-J
Fig. 3. Calibrationcurves formeasurementof hCG (mt. units/L) by c
aftervariousincubation times
60-
40
I
20
I
35
I
I
I
$0 24-5 47.5 9$
I
$86
HCG
Fig.2. A calibrationcurve formeasurementof hCG (mt.units/L)byCIA
Bars indicateSD of six replicates
minimum concentration of hCG that could be significantly
distinguished
from zero (mean -2 SD) ranged from 1.8 ±
0.4 mt. unitsfL (2-h incubation) to 0.25 ± 0.05 int. units/L
(seven-day
incubation).
Within-batch
precision
for hCG in
urine (overnight incubation) was estimated as for the CIA.
The results were: mean 51.9 (SD 12.9) int. unitsfL and CV
18.1%. The between-batch
CV (four assays) of the same
quality-control
sample was 18.7%. The concentration of
hCG, determined
in selected samples of urine collected
throughout
the 10 cycles by ic
(y) and RIA (x), also
correlated
well: r = 0.97, n = 64; y = 0.978x + 0.63.
Clinical Comparison Studies
We used 50 i.
unitsfL as the cutoff value of hCG for
diagnosis of pregnancy by RIA, CIA, and ic&, expressing
the results for the measurement
of hCG as positive or
negative according
to whether they were above or below
this. Contingency tables established to determine the clinical utility of the alternative immunoassays are summarized
in Table 1.
Discussion
Recently, CIAs have been developed for the measurement
of various haptens, including plasma steroids and their
urinary metabolites (10), serum thyroxin (14), thromboxane
B2 (15), and salivary progesterone (16). In addition, several
attempts
have been made to develop CIAs with use of
proteins (antigens or antibodies)
labeled with derivatives of
luminol and isoluminol (12, 13). However, luminol associated with peptides or proteins has a significantly
lower
quantum
yield. More recent studies (17, 18) show that
protein-isoluminol
conjugates prepared with ABEl-H have
acceptable quantum yields upon oxidation
(17, 18). Using
540
CLINICAL CHEMISTRY, Vol. 30, No. 4, 1984
this derivative, we have prepared hCG-ABEI and a nonspecific anti-hCG IgG-ABEI for use in the development of the
CIA and ICv1A. The stability of each labeled protein is
satisfactory, no significant reduction in activity being discerned over 12 months (18). We have substituted the labeled
hCG in various RIA kits in place of the iodinated derivative
without any significant alteration in sensitivity, specificity,
or antibody dilution. Moreover, the labeled antigen procedure combines an acceptable sensitivity with good economy
of antibody reagent.
In 1968, Miles and Hales (19) introduced an important
new type of immunoassay, immunoradiometric assay (uu)
based on the use of isotopically labeled antibodies (19). Since
then, two-site nmis have been developed for compounds
with more than one antigemc determinant (epitope) (20).
Nomsotopic immunometric assays include immunoenzymometric assay (21), immunofluorometric
assay (22), and immi.mochemiluminometric
assay (23,24). The advantages
of
immunometric
procedures include greater sensitivity,
broader working range of analyte, improved precision, and
shorter incubation times because high concentrations of
reagent can be used. The Ic
we describe here has the first
two characteristics
but assay sensitivity is dependent on
incubation time, perhaps because of the low concentration of
specific antibody bound to the solid-phase. This aspect might
be improved by the introduction of well-matched, highly
specific monoclonal antibodies.
We have found that the chemiluminescence
techniques
developed for the measurement
of hCG are at least as
sensitive and specific as conventional
RIA. Both of our
assays still need improvement,
however. Their relatively
poor precision may be due in part to the fact that end-point
measurement is preceded by an incubation at high temperature and alkaline pH. Perhaps the discovery of alternative
Table 1. Clinical Utility of CIA, ICMA, and RIA for
Pregnancy Tests on Daily Samples of EarlyMorning Urine from 10 Cycles
CIA
RIA
Pos
Neg
Total
Pos
Nag
27
0
27
28
37
39
ICMA
Total
29
37
66
Pea
22
18
23
Nag
Total
38
25
46
49
47
72
#{149}
Samples of early-morning urine collectedon the following day gave a
positive result (hCG> 50 mt. unitslL) with each test.
derivatives
with which to label proteins
will
of chemiluininescence
on oxidation
without prolonged incubation under these conditions. At the
present, however, for high sensitivity, the addition of sodium hydroxide before oxidation is obligatory to raise the pH,
dissociate the antibody-bound complex, and cause the physicochemical
nature of the label to change. The conditions for
each assay must be optimized in terms of temperature
and
time, and the concentration of sodium hydroxide. Alternative techniques to enhance sensitivity may be preferable. At
present we add the last reagent (i.e., hydrogen peroxide) by
rapid injection into the assay tube, which is situated in front
of the photo-detector, because the rate of the chemiluminescent reaction is too fast for the reagent to be added outside of
the instrument. Alternative oxidation systems that lead to
constant,
stable light emissions (cf. bioluminescent
reactions) might be preferable.
In 1981, Simpson et al. (13) reported the use of acridinium
esters as labels in immunoassays.
The use of these compounds may increase
quantum
efficiency, avoid serious
quenching
when labels are associated
with proteins or
haptens, and allow simpler oxidation systems involving
alkaline
peroxide alone. In the reports of assays with
protein-acridinium
conjugates
(24, 25), the most highly
active conjugates
of protein and acridinium
ester have
yielded greater specific activities than those obtained from
the equivalent 1201-labeled proteins, and they are stable for
at least 11 months when stored at -20 “C. Currently we
have developed and are evaluating a CIA and an ica&.&for
measurement
of hCG with protein-acridimum
derivatives
(26).
increase
of isoluminol
quantum
yields
J.B.K. was supported by a grant from Wallac Oy, Thrku, Finland
and J.L.B. from the King’s Voluntary Research Trust. Additional
financial support was provided by the Medical Research Council (to
W.P.C.) and from the U.S.A. Binational Science Foundation, Jerusalem (to F.K.). F.K. is the Fanny and Samuel Kay Research
Fellow. We thank LKB-Wallac for the loan of instrumentation.
References
1. Wehman RE, Nisula BC. Metabolic
clearance rates of
J Clin Invest 68, 184-194
and renal
purified human chorionic gonadotrophin.
(1981).
2. Vaitukaitis JL, Braunstein
GD, Ross GT. A radioimmunoasaay
which specifically measures human choriomc gonadotrophin in the
presence of human luteinizing
hormone. Am J Obstet Gynecol 113,
751-758 (1972).
3. Know BS, McKee JWA, Hair P1, France JT. Determination ofchoriogonadotropin in human plasma: Evaluation and comparison
of five “kit” methods. Clin Chem 26, 1890-1895 (1980).
4. Schall RF, Tenoso HJ. Alternatives to radioimmunoassay:
Labels and methods. Clin Chem 27, 1157-1164 (1981).
5. Sekiya T, Furuhashi Y, Goto S, et al. Specific enzyme immunoassay for human chorionic gonadotrophin. Acta End ocri nol (Copenhagen) 97, 562-568 (1981).
6. Joshi UM, Roy R, Sheth AR, Shah HP. A simple and sensitive
color test for the detection of human chorionic gonadotrophin.
Obstet Gynecol 57, 252-254 (1981).
7. Petterson K, Siitari H, Hemmila I, et al. Time-resolved fluoroimmunoassay of human choriogonadotropin. Clin Chem 29, 60-64
(1983).
8. Schroeder HE, Vogelhut P0, Carrico RJ, et al. Competitive
protein-binding assay for biotin monitored by chemiluminescence.
Anal Chem 48, 1933-1937 (1976).
9. Schroeder HR, Bogulaski RC, Carrico RJ, et al. Monitoring
specific protein-binding reactions with chemiluminescence. Met/iods Enzymol 57, 424.-445 (1978).
10. Collins WP, Barnard WE, Kim JB, et al. Chemiluminescence
immunoassay of plasma steroids and urinary steroid glucuronides.
In Immunoassays for Clinical Chemistry, WM Hunter, JET Corrie,
Eds., Churchill Livingstone, Edinburgh, 1983, pp 373-397.
11. Barnard GJR, Kim JB, Brockelbank JL, Collins WP. Recent
advances in chemiluminescence
immunoassay.
In Instrumentation
for the Detection of Chemiluminescence,
K Van Dyke, Ed., CRC
Press, Boca Raton, FL, 1983, in press.
12. Simpson JSA, Campbell AM, Ryall MET, Woodhead JS. A
stable chemiluminescence-labelled antibody for immunological assays. Nature (London) 274, 646-647 (1979).
13. Simpson JSA, Campbell AK, Woodhead JS, et al. Chemiluminescence labels in immunoassay. In Bioluminescence and Chemiluminescence. Basic Chemistry and Analytical Applications,
M DeLuca, WD McElroy, Eds., Academic Press, New York, NY, 1981, pp
673-679.
14. Weerasekera DA, Kim JB, Barnard GJ, Collins WP. The
measurement of serum thyroxine by solid-phase chemiluminescence immunoassay. Ann Clin Biochem 20, 100-104 (1983).
15. Weerasekera DA, Koullapis EN, Kim JB, et al. Chemiluminescence immunoassay of thromboxane B2. In Advances in Prostaglandin, Thromboxane and Leucotriene Research 2, B Samuelsson, R
Paoletti, P Ramwell, Eds., Raven Press, New York NY, 1983, pp
185-190.
16. Kim JB, Sallam HN, Barnard G,JR, Collins WP. The measurement of progesterone in saliva by a solid-phase chemiluminescence
immunoassay. J Steroid Biochem, 1984, in press.
17. Cheng P4, Hemmila I, Lovgren T. Development of solid-phase
iinmunoassay using chemiluimnescent IgG conjugates. J Immunol
Methods 48, 159-168 (1982).
18. Brockelbank JL, Barnard G, Kim JB, et al. The measurement
of urinary LH by a solid-phase chemiluroinescence immunoassay.
Ann Clin Biochem, 1984, in press.
19. Miles LEM, Hales CN. Labeled antibodies and immunological
assay systems. Nature (London) 219, 186-189 (1968).
20. Miles LEM. Immunoradiometric
assay (IRMA) and two site
IRMA system (assay for soluble antigens using labeled antibodies).
In Handbook of Radioimmunoassay,
GE Abraham, Ed., Marcel
Dekker, New York, NY, 1977, pp 131-178.
21. Gnemmi E, O’Sullivan MJ, Chieregatti G, et al. A sensitive
immunoenzymometric
assay (IEMA) to quantitate hormones and
drugs. In Enzyme Labelled Immunoassay of Hormones and Drugs,
SB Pal, Ed., Walter de Gruyter, New York, NY, 1978, pp 29-41.
22. Smith, DS, Al-Hakiem 1111,Landon J. A review of fluoroimmunoassay and iimnunofluorometric assay. Ann Clin Biochem 18,
253-274 (1981).
23. Schroeder HE, Hines CM, Osborn DD, et al. Inununochemiluminometric assay for hepatitis B surface antigen. Clin Chem 27,
1378-1384 (1981).
24. Weeks I, Campbell AK, Woodhead JS. Two-site immunochemiluminometric assay (icat.&)for human a1-fetoprotein. Clin Chem 29,
1480-1483
(1983).
25. Weeks I,
Beheshti
I, McCapra F, et al, Acridinium esters as
high-specific-activity labels in immunoassay. Clin Chem 29, 14741479 (1983).
26. Barnard GJ. Chemiluminescence
immuno- and inununochemiluminometric assay. In Alternative Immunoassays, WP Collins, Ed.,
John Wiley and Sons Ltd., Chichester, Sussex, in press.
CLINICAL CHEMISTRY, Vol.30, No.4, 1984
541