Journal of Immunological Methods, 147 (1992) 167-172 167 © 1992 Elsevier Science Publishers B.V. All rights reseIVed 0022-1759/92/$05.00

JIM 06249

Direct micro titre plate enzyme immunoassay of testosterone in unextracted serum

Tarun K. Dhar and Esahak Ali Indian Institute of Chemical Biology, 4, Raja s. C. MulJick Road, Calcutta-7()() 032, Indill

A method for enzyme immunoassay of testosterone in serum has been developed which does not require extraction of the serum with organic solvents. The release of testosterone from the binding proteins is achieved by heating the serum at 70°C for 30 min in an alkaline buffer. The results correlated well (r = 0.96) with those of a radioimmunoassay using \25I-Iabelled testosterone and with enzyme immunoassay with prior extraction of samples (r = 0.98). The detection limit of the assay is 1 pg per well and the tum around time for 36 samples is 3.5 h. The procedure is simple and well suited for routine analysis.

Key words: Testosterone; Direct enzyme immunoassay

Introduction

The determination of serum or plasma testos­terone is clinically useful for diagnosis and man­agement of several endocrine disorders. Radioim­munoassay is the most widely used method for testosterone assay. To overcome the problems associated with the use of radioisotopes several enzyme immunoassay methods for testosterone have been developed in the recent years (Tateishi et aI., 1977; Joshi et aI., 1979; Elder and Lewis, 1985; Marcus and Dumford, 1985; Rajkowski et aI., 1989). However, all these methods require extraction of samples with an organic solvent. Such methods are laborious and costly with re­spect to operators time. Here we describe a rapid and sensitive method for assay of serum testos­terone which does not require any solvent extrac­tion. While this work was in progress a direct

Correspondence to: E. Ali, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta-700 032, India.

method for estimation of salivary testosterone was reported (Howard et aI., 1989) but its suit­ability for use in serum samples had not been investigated. The major problem in development of a direct enzyme immunoassay of testosterone has been the strong binding of the hormone to some serum proteins especially sex hormone binding globulins (Ismail, 1976). We have found that the problem can be eliminated by heating serum samples at 70°C in alkaline buffer. To our knowledge no similar method based on sensitive double antibody technique (Meyer and Guven, 1986) has so far been reported for testosterone.

Materials and methods

Materials

Flat bottomed 96 well polystyrene microtitre plates were from Costar, Cambride, MA, USA. The automatic microtitre plate reader was from Bio-Rad Laboratories, Richmond, USA. The RIA

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kit for testosterone determination with 1Z51_

labelled testosterone was from Diagnostics Sys­tem Laboratories, Webster, TX, USA. Testos­terone, dicyclohexylcarbodiimide, N-hydroxysuc­cinimide, (aminooxy)acetic acid, morpholine, Sephadex G-50, horseradish peroxidase type VI (EC 1.11.1.7), polyoxyethylene sorbitan monolau­rate (Tween 20), 3,3',5,5' -tetramethylbenzidine were purchased from Sigma, St. Louis, MO, USA. Serum controls were from Indo Medix, Friendswood, USA. All other chemicals and buffer salts were of analytical grade. Testos­terone-3-carboxymethyloxime was synthesized by the method of Samanta and Ali (1990).

Buffers Coating buffer was Na 2HP04/KHzP04 (50

mmol/l, pH 7.6). Washing buffer was coating buffer containing 0.5 ml of Tween 20/1. Post­coating buffer was coating buffer containing 5 g of bovine serum albumin (BSA) /1. Assay buffer (pH 7.5) contained 50 mmol of Na zHP04/

NaH zP04 , 150 mmol of NaCl, 2.5 g of BSA and 0.1 g of thimerosal/I. The buffer for standards was prepared by dissolving 9 g of NaCl. 50 g of BSA, 5 g of human serum albumin, 16.5 g of human l'-globulin and 0.1 g of thimerosal in 1 litre of sodium phosphate (50 mmol/l, pH 7.6). The solution was centrifuged for 5 min. Alkaline buffer was NaHC03/Na 2C03 (250 mmol/l, pH 9.6). Enzyme assay buffer was sodium acetate/ citric acid (100 mmol/l, pH 3.95).

Chromogen Tetramethylbenzidine was dissolved in

dimethyl sulfoxide (concentration 42 mmol/I) and 8 ml of this solution was added to 1 litre of enzyme assay buffer. Just before use, 1.5 ml of 30 ml/l aqueous hydrogen peroxide was added (Gal­lati and Pracht, 1985).

Standards Testosterone stock standard solution (1 mg/ml

in ethanoD was kept at - 20"C and seven working standards (0.1-20 ng/ml) were prepared by dilu­tion with buffer for standards.

Methods

Antisera preparation Antiserum against testosterone was raised in

New Zealand White rabbits using testosterone-3-O-carboxymethyl oxime-bovine serum albumin as immunogen. Rabbits were given s.c. and i.m. in­jections of approximately 1 mg of the steroid-pro­tein conjugate in 1 ml saline, emulsified with an equal volume of Freund's adjuvant (complete). Booster injections were then given with incom­plete Freund's adjuvants at monthly intervals. After checking the antibody titer by Ouchterlony immunodiffusion in gel, blood was collected by cardiac puncture after 6 months and sera stored at -20°e.

The gamma globulin fraction of this antiserum was obtained by repeated precipitation with am­monium sulfate (50% saturation) followed by dialysis against phosphate buffer (10 mmol/I, pH 7.4) containing 0.9% NaCl. It was passed through BSA-Sepharose 4B immunosorbent column for removal of anti-BSA antibodies. The purified y­globulin fraction was stored at - 20°C (IgG con­centration 16 g/I). Working solutions were made by diluting 25-fold with assay buffer and stored at 4°e. For the second antibody, affinity purified goat anti-rabbit IgG was used (Sigma, cat. no. 4880).

Enzyme conjugation Testosterone-3-carboxymethyloxime was cova­

lently linked to horseradish peroxidase by the activated ester method of Mattox et al. (1979). It was purified by dialysis, followed by chromatogra­phy on Sephadex G-25. The molar ratio of testos­terone to protein in the enzyme conjugate as determined from the ratio of absorbances at 255 and 403 nm of the conjugate by the method of Samanta and Ali (1990) was 5.0. The conjugate is stable for at least 24 months when stored at - 20°C in presence of 20 mg of BSA/ml of enzyme conjugate (concentration 1 g/D. Working solutions prepared by 500-fold dilution of the conjugate in assay buffer, is stable for at least 12 months, when stored at 4°C.

Immunoassay reagent Just before the assay, working solutions of the

anti-testosterone antibody (20 ILl) and the en­zyme conjugate (150 ILl) were mixed with 10 ml of assay buffer.

Coating of microtitre plates Each well of the microtitre plate was coated

with second antibody using 200 1.1.1 of 5 ILg/ml IgG solution in coating buffer. After 2 h incuba­tion at room temperature the wells were washed with washing buffer three times. The plates were then incubated with post-coating buffer (200 ILl) for 2 h at 37°C and washed again three times with washing buffer. Coated plates were stable for more than six months if lyophilized for 20 min (after two washes with 0.1 % thimerosal in water) and stored at 4°C.

Heat treatment of serum samples Serum samples and standards were diluted

two-fold with alkaline buffer. It was heated in a water bath at 70-75°C for 30 min, vortexed and used directly for assay.

Extraction of serum samples Serum samples or standards (100 ILl) were

extracted with 2 ml of diethyl ether by vigorous shaking. The separated organic layer was then evaporated on a steam bath in 12 X 75 mm glass tubes and the residues redissolved in 200 ILl of assay buffer.

Testosterone enzyme immunoassay To each well of a coated plate 50 ILl of stan­

dards or serum samples were transferred in du­plicate and 100 ILl of immunoassay reagent was added. The plate was covered and incubated at 37°C for 2 h. The wells were then washed three times with 300 ILl portions of washing buffer. Then, 150 ILl of chromogen solution was added and incubated in the dark at room temperature. After 30 min, the reaction was stopped by adding 50 ILl of 2 moljl sulphuric acid to each well in the same sequence as the chromogen solution was added. The contents of the wells were mixed by swirling for 1 min and the absorbance mea­sured at 450 om with a microplate reader.

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Radioimmunoassay Radioimmunoassay was carried out by the di­

rect method without extraction according to the kit manufacturers protocol. Briefly, 50 ILl of sam­ple or standard was incubated with 125I-Iabelled testosterone reagent in antibody coated tubes at 37°C for 60 min. The solutions were then de­canted and the radioactivity bound to the tubes measured in a gamma counter.

Results

Assay conditions The conditions of coating of microplates with

second antibody, the dilutions of antiserum and enzyme conjugate to be used and temperature of incubations were optimised in preliminary experi­ments. The antibody and the enzyme conjugate can be added individually or in one-step as a premixed solution without effecting the sensitiv­ity. The one-step technique was used for conve­nience.

For accurate estimation of testosterone in serum samples, three control samples with low, medium and high levels of testosterone were as­sayed after heating at different temperatures in acidic and alkaline buffer. The target values of testosterone in these samples were obtained by RIA. Heating the control samples at 37°C or 60°C in presence of equal volume of glycine-HO buffer (200 mmoljl, pH 2.3) or NaHC03-Na2C03 buffer (250 mmoljl, pH 9.6) gave testosterone values much lower than the target values. Heat­ing in glycine-HCI buffer above 60°C led to pre­cipitation of the serum samples. The best result was obtained by heating the samples with equal volume of the carbonate buffer (pH 9.6) at 70-75°C for 30 min. Under this condition the values obtained for all the three controls correlated well with the target values.

As the vehicle for preparation of standards both charcoal stripped serum and the albumin and globulin solution described here (buffer for standards) gave good results for all the three control sera. The latter medium was selected because of its better stability and reproducibility.

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o 2~~. ,;-' -_--:::"-::' :-----L.' ,..-----1.' ___ ' 00'01 0.5 20 50 20.0

SERUM TESTOSTERONE (ng/mll

Fig. 1. Standard curve for testosterone assay in serum. Each point represents the mean and standard deviation of four measurements in duplicate. Plates were coated with second antibody at a concentration of 5 "g/ml. Anti-testosterone antibody and enzyme conjugate were used at a dilution of

1112,500 and 1/33,000 respectively.

Analytical variables Sensitivity. Fig. 1 depicts an optimised testos­

terone standard cUIVe covering the range from 0.1 to 20 nglml. This range is broad enough to measure accurately the wide range of testos­terone in serum. The smallest amount of testos­terone distinguisable from blank by twice the SO was 1 pg per well.

Precision. Intra- and interassay variabilities in three serum samples covering low, medium and high concentrations of testosterone are shown in Table I.

Specificity. The specificity of the antiserum used was assessed by the present EIA procedure for several steroids structurally related to testos­terone and the cross reactivity calculated accord­ing to the procedure of Abraham (1969). The results were similar to those reported earlier (Samanta and Ali, 1990), with negligible cross-re­activity for all steroids except S-a-dihydro­testosterone which had a cross-reaction of 27%.

Accuracy. To investigate the analytical recovery in the

method, two serum samples containing different

TABLE I

PRECISION OF THE ASSAY

Three serum samples were assayed in replicates by direct EIA. For the interassay variation each sample was measured four times in duplicate on to different days.

Testosterone concentration (ng/ml)

Low Medium High

Intra-assay Mean 0.40 3.20 7.90 SO 0.05 0.18 0.54 n 18 12 18 CV% 12.5 5.6 6.8

lnterassay Mean 0.58 3.76 5.5 SO 0.096 0.25 0.5 n 10 10 10 CV% 16.5 6.6 9

levels of endogenous testosterone were assayed after spiking with different amounts of testos­terone. The analytical recoveries ranged from 93 to 108% (Table 11).

In a dilution recovery test, a serum sample was diluted 2-32-fold with the 0 nglml testosterone standard and assayed. Comparison of the ob­seIVed and expected concentrations are shown in Table III.

Validation of the method. Testosterone concentrations in 62 serum sam­

ples were measured both by RIA and the present

TABLE II

SPIKE RECOVERY TESTS

Two serum samples were spiked with known amounts of testosterone and then assayed by direct EtA procedure. Recorded values depicts the mean of two measurements in duplicate.

Testosterone nglml % Recovery

Endogenous Added Calculated Found

6.5 5.0 11.5 12.0 104.35 2.5 9.0 8.4 93.33 0.5 7.0 7.5 107.14

2.0 5.0 7.0 7.0 100.00 2.5 4.5 4.3 95.55 0.5 2.5 2.7 108.00

TABLE III

LINEARITY OF DILUTION

A serum sample containing 11 ng/ml of endogeneous testos­terone was diluted serially with zero testosterone standard and assayed by direct EIA procedure. Recorded values de­picts the mean of two measurements in duplicate.

Dilution Expected Observed Recovery factor ng/ml ng/ml %

11.00 1/2 5.50 5.50 100.00 1/4 2.75 2.70 98.18 1/8 1.37 1.30 94.89 1/16 0.69 0.70 101.45 1/32 0.35 0.40 114.28

direct EIA (Fig. 2). The equation of correlation was:

Test'EIA = 0.037 ng/ml + (0.91 x Test.RIA); r - 0.96

Fig. 3 shows the correlation obtained for testosterone determination in 30 serum samples with and without prior extraction. The equation of correlation was:

Test.DireCt "" 0.382 ng/ml + (0.93 x Test.Extnocted); r == 0.98

The results indicate that a direct non-extrac­tion assay of testosterone in serum samples was feasible.

20.0

16.0

E 12.0 ..... 0-c

~ 8.0

4.0

4.0 8.0 120 16.0 RIA (ng/m!)

Fig. 2. Correlation between serum testosterone values of 62 samples estimated by RIA and by direct EIA. The solid line

represents the best fit obtained by regression analysis.

171

200

16.0 E ..... o 0

'" c - 12.0 ~

• (I) (I) « I- 8.0 u ILl Q:

is 4.0

0.0 0.0 4.0 8.0 12.0 16.0

EXTRACTED ASSAY (ng 1m!)

Fig. 3. Correlation between serum testosterone values of 30 samples estimated by the present procedure with and without a solvent extraction step. The solid line represents the best fit

obtained by regression analysis.

Discussion

The present direct method for ELISA of serum testosterone provides several practical advantages rendering it suitable for routine application in the clinical laboratory. The use of microtitre plate, low cost second antibody, single immunological incubation period and ability to measure testos­terone in serum samples without prior extraction with organic solvent facilitates relatively rapid tum around time. Consequently more than 200 samples can be handled by one technician per working day. As most of the testosterone in blood is bound to sex-hormone-binding globulin and albumin (Vermeulen, 1979), it is necessary to release the hormone before assay. This can be done by heating serum samples at 70-7SoC for 30 min in the presence of NaHC03/Na2C03 buffer. Comparison of our data with results obtained by direct RIA and also by extracting samples demonstrates that interference due to serum pro­teins are fairly eliminated after denaturation. Thus the present method provides a convenient non-isotopic alternative to RIA for testosterone assay. We think that the method of direct estima­tion may also be applicable for EIA of other steroid hormones such as progesterone and estra­diol.

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References

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Howard. K., Kane. M., Madden, A., Gosling, J.P. and Fottrell, P.F. (1989) Direct solid phase enzyme immunoassay of testosterone in saliva. C1in. Chern. 35, 2044.

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