Measurement of Choriogonadotropin by ... - Clinical Chemistry

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.

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and renal

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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.

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