Journal of Immunological Methods, 147 (1992) 181-188 181 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00

JIM 06203

Sandwich immunoassay of small molecules

II. Effects of heterology and development of a highly sensitive and specific enzyme immunoassay for testosterone

Judhajit Sengupta, Tarun K. Dhar and Esahak Ali Indian Institute of Chemical Biology, Calcutta-700032, India

(Received 12 August 1991, accepted 29 October 1991)

The effect of dimer heterology in the sandwich immunoassay of testosterone was studied using symmetrical and asymmetrical dimers prepared from testosterone-3-(O-carboxymethyl)oxime and 4-(carboxymethyl-mercapto)testosterone. The effect of antibody heterology was studied using antibodies against 3-carboxymethyl oxime and 17-hemisuccinate derivatives of the steroid and an asymmetrical dimer prepared from the same two derivatives. Using antibody against the 3-carboxymethyl oxime and the dimer of 4-(carboxymethylmercapto)testosterone, the sensitivity of direct enzyme immunoassay of testosterone in serum by this method was 0.5 pg/well and the cross-reactivity of 5a-dihydrotestosterone was 5%.

Key words: Sandwich immunoassay; Testosterone dimer; Heterology; Testosterone-3-(O-carboxymethyl)oxime; 4-(Carboxymethyl­mercapto) testosterone; Testosterone-17-hemisuccinate; Cross-reactivity; 5a-Dihydrotestosterone

Introduction

In the preceding paper (Ali et aI., 1992) we have described a new principle for enzyme im­munoassay of small molecules, wherein a syn­thetic bis-analogue of the hapten forms a sand­wich complex with the immobilised antibody and the same antibody labeled with an enzyme. The formation of this sandwich complex is inhibited

Corresporulence to: E. Ali, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta-700032, India. Abbreviations: s, singlet; d, doublet; t, triplet; m, multiplet; TLC, thin layer chromatography; DCC, dicyclohexylcarbodi­imide; DMF, dimethylformamide; DASP, double-antibody solid phase; TNBS, trinitrobenzene sulphonic acid; BSA, bovine serum albumin; RIA, radioimmunoassay; HRP, horseradish peroxidase.

by the free hapten in a concentration dependent manner. Using the dimeric testosterone deriva­tive 2, obtained by condensation of testosterone-3-( O-carboxymethyl)oxime with ethylenediamine it has been shown that considerable increase in sensitivity and specificity could be obtained in the assay of testosterone compared to a DASP assay (Dhar and Ali, 1992) using the same antibody. The dimer used in the earlier report was pre­pared from the same testosterone derivative that was used for raising the antibody used for immo­bilisation and labeling. The technique however permits considerable flexibility in this respect. For example the dimer might be synthesised from a hapten derivative different from the one used in the immunogen (dimer heterology) and using an asymmetrical dimer, different antibodies can be used for immobilisation and labeling. It was

182

o H X0N~Ny"Y

H 0

2.X=Y=A 3.X= A,Y=B 4.X=Y=B 5.X=A,Y=C

N~ I o I

A

.~ I 0 ~R

I. R: -OH 6. R: -NH-CHZ-CHZ-NHZ

c

OR

o R'

7. R = H, R' = -SCHZCOOH 8. R =-COCHZCHZCOOH, R'= H

Fig. 1. Structure of testosterone derivatives and dimers.

considered likely that such variations may lead to still greater sensitivity and/or specificity of the assay (Van Weemen and Schuurs, 1975; Hosoda et aI., 1980, 1981, 1986). Herein we describe the effect of dimer heterology on the testosterone assay using the dimers 3-5 (Fig. 0. The effect of antibody heterology was studied using the dimer 5 and antibody raised against testosterone-I7-hemisuccinate 8. The development of a highly sensitive and specific enzyme immunoassay of testosterone for clinical use using serum based standards is also described here.

Materials and methods

Testosterone-17-hemisuccinate 8 was pre­pared by method of Hosoda etal. (1979). 4-

(carboxymethylmercapto)testosterone 7 was pre­pared from 4,5-epoxytestosterone by methods de­scribed in the literature (Ringold et aI., 1956; Hosoda et aI., 1979). Both the compounds were fully characterized from their proton NMR spec­tra. The homogeneity of all the steroid derivatives synthesised were checked by TLC on silica gel plates with either 5 or 10% methanol in chloro­form as the mobile phase. Nuclear magnetic reso­nance (NMR) spectra were run on a JEOL model FX-l00 spectrometer at 100 MHz using tetra­methylsilane as internal standard.

The antibody against testosterone-3-< O-car­boxymethyl)oxime, its conjugation with HRP, buffers, the assay method and other reagents were the same as described in the preceding paper. The radioimmunoassay kit was purchased from Diagnostics System Laboratories, Webster,

Texas, USA and the assay performed as per manufacturer's protocol described in the preced­ing paper (Dhar and Ali, 1992).

Preparation of compound 6 To a stirred suspension of testosterone-3-<O­

carboxymethyl)oxime 1 (200 mg, 0.5 mmoI) in 15 ml dry methylene chloride was added 300 mg of DCC and the mixture was stirred until the turbid­ity disappeared. Ethylenediamine 020 mg, 2.0 mmoI) was then added in one portion. The reac­tion was allowed to proceed at room tempera­ture. After 3 h, the solution was filtered to re­move the separated urea. Evaporation of the filtrate yielded a white solid which was redis­solved in 5 ml warm methylene chloride. The solution was allowed to stand at 4°C for 30 min when additional dicyclohexylurea separated which was removed by filtration. The process was re­peated once more to remove a further crop of dicyclohexylurea. The final filtrate was concen­trated and chromatographed on a neutral alu­mina column 0.5 cm X 20 cm). Elution with 2% methanol in chloroform gave 35 mg 06%) of the monomer 6. IH NMR (CDCI 3/D20) 8: O.SO sOS-Me), LOS, 1.12 two X s09-Me, syn and anti), 2.84 t(CH 2-NH 2 , J = 6 Hz), 3.40 t(CH 2-NHCO, J = 6 Hz), 3.64 t07-H, J = 8 Hz), 4.54 s(O-CH 2-

CO), 5.76 s(H-4, antD, 6.44 s(H-4, syn).

Preparation of the dimer 3 To 15 mg of 4-(carboxymethylmercapto)

testosterone 7 (0.039 mmoI) in 300 #£1 of dry DMF was added, 10 mg (0.079 mmoI) of N-hy­droxysuccinimide, 20 mg (0.079 mmoI) of DCC and the reaction was allowed to proceed at 4°C overnight. The supernatant was added dropwise to a solution of 15 mg of 6 (0.04 mmoI) in 1 ml DMF containing a drop of saturated sodium bi­carbonate solution. After 3 h stirring at room temperature the DMF was removed under vac­uum. The residue was treated with 5 ml chloro­form and filtered. The chloroform soluble part was chromatographed on a silica gel column 0.5 em X 20 em). Elution with 1% methanol in chlo­roform gave the desired dimer 3 in 39% yield (11 mg). The crude product was crystallised from chloroform pet ether and dried under vacuum. [alo + 137.8° (MeOH); UV: A~~H 245.8 nm (E

183

33,579), 307 nm (E 21,310); IH NMR (CDCI 3/D20) 8: 0.78, 0.80 two X s08-Me of A and B), 1.08s, 1.1Os09-Me, syn and anti of A), 1.24 s09-Me of B), 3.22 s(-S-CH 2-), 3.44 m(NH­CH 2-Ckh-NH), 3.64 t(H-17, J = 9 Hz), 4.54 s(-O­CH 2-CO), 5.76 s(H-4 anti of A), 6.48 s(H-4 syn of A).

Preparation of the dimer 4 Compound 4 was prepared by condensation of

4-(carboxymethylmercapto)testosterone (150 mg, 0.4 mmoI) with 24 mg (0.4 mmoI) ethylenedi­amine in presence of (160 mg, 0.78 mmoI) DCC in 6 ml dry methylene chloride by the same procedure as described for compound 6. The product was chromatographed over silica gel (1.5 em X 20 cm). Elution with 1-2% methanol in chloroform yielded 24 mg 06%) of the dimer 4. m.p, 129°C; [a]o + 63.49° (MeOH); UV: A~:?H 245 nm (€ 20,000), 307 nm (E 3746); IR (KBr) v~: 3600-3400 (NH, OH), 1660 (C = 0), 1635 (C = N) cm- l ; IH NMR (CDCI 3/D20) 8: 0.88 s08-Me), 1.24 s09-Me), 3.26 s(-S-CH 2CO), 3.40 s(NH-CH 2-C!:h), 3.64 m(17-H).

Preparation of the dimer 5 To 17 mg of testosterone-17-hemisuccinate 8

(0.05 mmoI) in 500 #£1 of dry DMF was added, 12 mg (0.1 mmoI) of N-hydroxysuccinimide and 25 mg (0.1 mmoI) of DCC and the reaction was allowed to proceed at 4°C overnight. The super­natant was added dropwise to a solution of 20 mg (0.05 mmoI) of 6 in 1 ml dry DMF containing a drop of saturated sodium bicarbonate solution. After 3 h stirring at room temperature the DMF was removed under vacuum. The residue was treated with 5 ml chloroform and filtered. The chloroform soluble part was chromatographed us­ing an alumina column (1.5 cm X 18 cm). Elution with 2% methanol in chloroform gave the desired dimer 5 in 40% yield. IH NMR (CDCI 3/D20 8: 0.78, 0.83 s08-Me), 1.08s, 1.13 s09-Me) 3.46 m(NH-CH 2-CH 2-NH), 3.70 m{17-H of A), 4.51 s(-O-CH 2-CO), 4.58 m{17-H of C), 5.75 s(H-4 anti of A and H-4 of C), 6.43 s(H-4 syn of A).

Standards Testosterone stock standard solution (1

mg/mI) in ethanol was kept at - 20°C and seven

184

working standards (0.1 ng to 20 ng/mI) were prepared by dilution in charcoal stripped serum.

Preparation of anti-testosterone-17-hemisuccinate Testosterone-17-hemisuccinate was conjugated

to bovine serum albumin by method of Mattox et al. (1979). A solution of 15 mg of the hemisucci­nate in 500 JLI of DMF was treated with 16 mg of Dee and 10 mg of N-hydroxysuccinimide overnight at 4°C. The activated ester solution was allowed to react with 165 mg BSA in 2 ml of sodium bicarbonate solution (0.13 mol/I) for 3 h. The reaction mixture was then dialysed against phosphate buffer, 50 mmol/I, pH 7.6 and puri­fied by column chromatography on Sephadex G-50 (1.5 em x 40 cm) using the same buffer as the mobile phase. Protein fractions were pooled, Iyophilised and stored at - 20°C. The number of steroid molecules coupled to BSA was estimated by measuring the number of free amino groups by TNBS method (Habeeb, 1966).

Immunisation procedure was same as that adopted for testosterone-3-(O-carboxymethyJ) oxime-BSA conjugate (Dhar and Ali, 1992). Satis­factory antibody titers were obtained after 5 months. Blood was collected by cardiac puncture and the antibody was obtained by repeated pre­cipitation with ammonium sulphate (50% satura­tion). The gamma globulin fraction obtained by ammonium sulphate precipitation was dialysed against 10 mmol/I, pH 7.6, phosphate buffer containing 0.9% Naa. It was passed through BSA-Sepharose 4B immunoadsorbent column (1.3 em x 4 em for 20 ml serum) for removal of anti­BSA antibodies using the same buffer and then the antibody titer checked by OuchterJony im­munodiffusion test.

Results and diseussion

The effect of dimer concentration on the for­mation of the immobilised sandwich complex at zero testosterone concentration was studied for dimers 2-4 under identical conditions using the same immobilised antibody and enzyme-con­jugate and similar bell-shaped curves were ob­tained (Fig. 2). It may be noted that the maxi­mum 00 obtained for the dimer 2 shown in Fig.

1.5

1.3

1.1

E c::

0 I/') 0.9 v III U c:: 0

.J:l 0.7 ... 0 III

.J:l <t

0.5

0.3

10 20 40 100200 400 1000 Dimer(pg/well)

Fia. 2. Effect of dimer concentration on measured 00 at zero testosterone concentration: 2 (-0-), 3 (-. -). 4 (- 0 -) and 5 (-e-). Immobilised antibody (t/3000 dilution) used for dimers 2. 3 and 4 was against testosterone-3..(O;;arbox'y­methyl)oxime and for 5 was against testosterone-17-hemisuc­cinate. Labeled antibody used in all cases was against testos-

terone-3..(O-carboxymethyJ)-oxime at 1/166 dilution.

2 is higher than the one depicted in Fig. 3 of the preceding paper. This is due to different amounts of enzyme used in the two studies. As can be seen from Fig. 2, the maxima of the bell-shaped curve shifted to lower dimer concentration (about 50 pg/well) for the dimer 3 compared to 2. This maxima has further shifted to 40 pg/well for the dimer 4. The shifting of the maxima is accompa­nied by significant decrease of color intensity obtainable in case of 3 and 4 compared to 2. These results can be explained by the decreased binding of the immobilised and the labeled anti­body to the heterologous dimers 3 and 4, as a fraction of the antibody specific against the frag­ment A and the spacer will not be able to recog­nize the heterologous fragment B of the dimers 3 and 4. The effect of dimer 5 was studied imIDo-

1.6.--------------,

1.4

1.2 E c: o If) v 1.0 III u c: o .0 ~0.8 1l «

0.6

0.4

0.2 10 0.1 0.5. Testosterone ng/ml

Fig. 3. Standard curve for testosterone enzyme-immunoassay by sandwich method using antibody against testosterone-3-(O-carboxymethyl)oxime for dimer 2 (-e-) and 4 (-0-). Serum used 5 "I, dimer concentrations 100 pg/well; enzyme­conjugate dilutions: 1/250 for 2 and 1/100 for 4; antibody

dilution 1/1000 in both cases.

bilising antibody against testosterone-17-hemisuc­cinate. The labeled antibody was however same as that used for the other dimers. The compari­son of the data for the dimer 5 is difficult as the immobilised antibody in this case was different. However, the high concentration of dimer re­quired for maximum binding and the high color developed under this condition can be explained by the fact that each part of the dimer has a homologous antibody either in the immobilised or in the labeled component of the system. As ex­pected, no color was obtained with dimer 5 when both the immobilised and the labeled antibody was against 1. Qualitatively, it can be inferred that binding of the steroid fragment C is stronger than that of A towards their respective homolo­gous antibodies. It can also be inferred that higher amounts of immobilised antibody and/or en­zyme-conjugate will be required to obtain compa-

185

rable color intensity at zero testosterone level in case of dimers 3 and 4 compared to 2.

The testosterone dimers used in the present study was prepared by usual conjugation methods employing carbodiimide and the yields are not high. However, the reaction conditions were not optimised. It is interesting to note that this type of dime ric steroids have been prepared earlier and their hormonal activities studied (Kuhl and Taubert, 1974).

Since development of a sensitive and specific enzyme immunoassay of testosterone for clinical use was a major goal of the study, we have decided at this stage to study the displacement curves with all the four dimers using serum based standards. The optimum dilution of the immo­bilised antibody under this condition was found to be around 1/1000 for all the dimers. The best curves were obtained with the dimers 2 and 4, albeit at different labeled antibody concentra­tions. As can be seen from Fig. 3 testosterone can be estimated upto 0.1 ng/ml using 5 JoLI of the serum which corresponds to an absolute sensitiv­ity of 0.5 pg of testosterone (1.73 X to- 15 mol) per well. To estimate the same level of testos­terone, the DASP method (Dhar and Ali, 1992) required 25 JoLI of the serum corresponding to 2.5 pg/well. Thus the ultimate sensitivity obtained by the sandwich enzyme immunoassay method is five times more than that obtained by the DASP method using the same antibody. Samples in all cases were heated at 7CJ>C for 0.5 h at pH 9.6, a

TABLE I

CROSS-REACTIVITIES OF STRUCTURALLY RE­LATED STEROIDS DURING SANDWICH IMMUNOAS­SAY OF TESTOSTERONE

Steroid Cross-reactivity (%)

Dimerused

3 4 5

Testosterone 100.00 100.00 100.00 5a-dihydrotestosterone 14.00 4.8 18.00 Progesterone 0.61 0.9 1.00 J3-estradiol 0.1 0.03 1.00 17a-hydroxyprogesterone 0.16 0.09 0.15 Dehydroisoandrosterone 0.006 0.002 Corticosterone 0.125 0.14 0.02 Hydrocortisone 0.10 0.117

186

condition which was found to be suitable for release of testosterone from binding proteins for direct assay (Dhar and Ali, 1992).

Specificity The effect of the dimer heterology on the

cross-reactivities of 5a-dihydrotestosterone and related steroids were studied for the three dimers (3-5) and compared with the values obtained with dimer 2 (Ali et aI., 1992). As can be seen from Table I, the cross-reactivities of 5a-dihydro­testosterone obtained with the dimers 3 and 5 are similar (14% and 18%) to that obtained with the dimer 2 reported in the preceding paper. However, there is a dramatic decrease in the cross-reactivity for the dimer 4 (5%) which is about 20% of the cross-reactivity obtained by the DASP assay.

Reproducibility The intra- and interassay variations in the

testosterone assay using the dimers 2 and 4 are shown in Table II.

TABLE II

INTRA-ASSAY AND INTERASSAY VARIATION DUR-ING SANDWICH ENZYME IMMUNOASSAY OF SERUM TESTOSTERONE

Dimer used Testosterone n CV% (ng/ml)

Mean SD

Intra-assay 2 0.29 0.034 12 11.72

2.86 0.27 12 9.44 7.19 0.20 12 2.78 Interassay 0.58 0.058 6 10.00 3.06 0.266 6 8.69 6.75 0.47 6 6.96

Intra-assay 4 0.32 0.045 12 14.06

3.50 0.38 12 10.85 5.52 0.36 13 6.52 Interassay 1.1 0.14 8 12.72 3.52 0.197 8 5.59 6.16 0.833 8 13.52

<t !fl

16.0

iil 8.0 :c u

~ z ~ 4.0

o. 0 "'---'-----''--~---'---'---'----' 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

RIA(ng/ml)

Fig. 4. Correlation between serum testosterone values esti­mated by RIA and by sandwich enzyme immunoassay method using dimer 2. Solid line represents the best fit obtained by

regression analysis.

Validation of the assay The testosterone values obtained for 51 serum

samples using dimer 2 was compared with those obtained by a commercial RIA kit. The correla­tion is shown in Fig. 4. The correlation coefficient was 0.957 and the regression equation is y = 0.915x + 0.494, where x = RIA value and y = value obtained by sandwich assay. The data for 42 samples done by the present method using the dimer 4 and by the DASP method was also compared (Fig. 5). The agreement between the two sets of values was excellent (r = 0.985, n = 42) with a regression equation being y = 0.983x + 0.178, where y = value obtained by sandwich as­say and x = value by DASP assay.

Although appreciable enhancement of the as­say sensitivity was obtained by the sandwich method, the major advantage lies in the increased specificity. The cross-reactivity of 5a-dihydro­testosterone towards antisera raised against testosterone derivatives obtained by functional­ization at different positions of the steroid nu­cleus was studied by several groups. As can be seen from Table III, the cross-reactivity varies between 27% to 90% except for some C-1S, C-19 and ell derivatives. The low cross-reactivity of

24.0

E 20.0 "­CJI c:

~ :J w :I: U

~ o Z <t (J')

160

12.0

8.0

40 .. OO~~~~~~--~~--~~--~~

00 2.0 4.06.0 8.010012014016.018.0200 ELISA (ng/mll

Fig. 5. Correlation between serum testosterone values esti­mated by DASP method and by sandwich enzyme immunoas­say method using dimer 4. Solid line represents the best fit

obtained by regression analysis.

TABLE III

CROSS-REACTIVITY OF 5a-DIHYDROTESTOS­TERONE TOWARDS ANTIBODY RAISED AGAINST DIFFERENT TESTOSTERONE DERIVATIVES

Testosterone derivative % Cross- References used in the immunogen reactivity

3-carboxymethyloxime 6S 3-carboxymethyloxime 46.5 3-carboxymethyloxime 27~5 3-carboxymethyloxime 27 3-carboxymethyloxime 33 3-carboxymethyloxime 27 3-carboxymethyloxime 28 4-O-hemisuccinate 64 4-O-hemiglutarate 62 613-carboxymethyl 47 6a-carboxymethyl 95 613-carboxymethyl 75 7a-S-carboxymethyl 42 7a-S-carboxyethyl 55 7a-carboxymethyl 41 7/J-carboxymethyl 52 lla-O-hemisuccinate 27,f:A) lla-hemisuccinate 15 15a-carboxymethyl 3.2 1513-carboxyethylmercapto 1.8 15a-O-hemisuccinate 22

15a-carboxymethyl

1513-carboxymethyl

1713-hemisuccinate 19-O-carboxymethyl ether

4.46-7.70 2.13-4.26

15.7,37 6.7

Hillier et al., 1973 Castro et aI., 1974 Bosch et aI., 1974 Hosoda et aI., 1979 Elder and Lewis, 1985 Dhar and Ali, 1992 Samanta and Ali, 1990 Hosoda et aI., 1979 Hosoda et aI., 1979 Riley et aI., 1972 Jones and Mason, 1975 Jones and Mason, 1975 Weinstein et aI., 1972 Weinstein et aI., 1972 Duval et aI., 1980 Duval et aI., 1980 Bosch et aI., 1974 Hillier et aI., 1973 Condom et aI., 1977 Rao et a., 1976 Nambara and Hosoda,

1977

Miyake et aI., 1982

Miyake et aI., 1982 Bosch et aI., 1974 Rao et aI., 1978

187

5a-dihydrotestosterone obtained with the dimer 4 in this study is therefore quite remarkable.

We believe that the sandwich assay described here will be applicable to other cross-reacting small molecules which need to be estimated in sub-picomolar amounts.

Acknowledgement

We thank Council of Scientific and Industrial Research (India) for Research Fellowship to one of the authors (J .S.).

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