Journal of Immunological Methods, 147 (1992) 167-172 © 1992 Elsevier Science Publishers B.V. All rights reseIVed 0022-1759/92/$05.00
167
JIM 06249
Direct micro titre plate enzyme immunoassay of testosterone in unextracted serum Tarun K. Dhar and Esahak Ali s. C. MulJick Road, Calcutta- 7()() 032, Indill
Indian Institute of Chemical Biology, 4, Raja
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
168
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 2 HP04 /KH zP04 (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 NaHC0 3 /Na 2C0 3 (250 mmol/l, pH 9.6). Enzyme assay buffer was sodium acetate/ citric acid (100 mmol/l, pH 3.95).
Methods
Antisera preparation
Antiserum against testosterone was raised in New Zealand White rabbits using testosterone-3O-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 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.
Testosterone-3-carboxymethyloxime was covalently 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.
169
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 standards 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.
Radioimmunoassay Radioimmunoassay was carried out by the direct 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 7075°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.
170 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
0.40 0.05 18 12.5
3.20 0.18 12 5.6
7.90 0.54 18 6.8
0.58 0.096 10 16.5
3.76 0.25 10 6.6
5.5 0.5 10 9
Intra-assay Mean SO n CV%
lnterassay
o 2~~.,;-'-_--:::"-::':-----L.',..-----1.'_ _ _' 00'01
0.5
20
50
20.0
Mean SO n CV%
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 testosterone 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
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 samples 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. % Recovery
Testosterone nglml Endogenous
Added
Calculated
Found
6.5
5.0
2.5
0.5
11.5 9.0 7.0
12.0 8.4 7.5
104.35 93.33 107.14
5.0 2.5
7.0 4.5
7.0 4.3 2.7
100.00 95.55 108.00
2.0
0.5
2.5
171 200
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 factor
Expected ng/ml
1/2 1/4 1/8 1/16 1/32
11.00 5.50 2.75 1.37 0.69 0.35
Observed ng/ml
Recovery %
E ..... '"c
-~
16.0 o
0
•
12.0
(I) (I)
«
I-
u
8.0
ILl
5.50 2.70 1.30 0.70 0.40
100.00 98.18 94.89 101.45 114.28
Q:
is
4.0
0.0 0.0
4.0
8.0
12.0
16.0
EXTRACTED ASSAY (ng 1m!)
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.
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.
172
References Abraham, G.E. (1969) Solid phase radioimmunoassay of oestradiol 17fJ. J. C1in. Endocrinol. Metab. 29, 866. Elder, PA and Lewis, J.G. (1985) An enzyme-linked immunosorbent assay (ELISA) for plasma testosterone. J Steroid Biochem. 22, 635. Gallati, V.H. and Pracht, I. (1985) Horseradish peroxidase: Kinetic studies and optimization of the peroxidase activity determination with the substrate H 2 0 2 and 3,3',5,5'-tetramethylbenzidine. J. C1in. Chern. Clin. Biochem. 23, 453. 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. Ismail, A.A.A. (1976) Testosterone. In: J.A. Loraine and E.T. Bell (Eds.), Hormone assays and their clinical application. Churchill Livingstone, Edinburgh, p. 581. Joshi, V.M., Shah, H.P. and Sudhama, S.P. (1979) A sensitive and specific enzyme immunoassay for serum testosterone. Steroids 34, 35. Marcus, G.J. and Dumford, R. (1985) A simple enzyme-linked immunosorbent assay for testosterone. Steroids 46, 975.
Mattox, V.R., Litwiller, R.D. and Nelson, A.N. (1979) A comparison of procedures for attaching steroidal glucosiduronic acid in bovine serum albumin. J. Steroid Biochem. to, 167. Meyer, H.H.D. and Guven, B. (1986) Improvement ofmicrotitration plate enzyme immunoassays for steroid determination by a second antibody technique. J. Steroid Biochem_ 25 (suppJ.), 139. Ra;kowski, K.M., Hanquez C., Bauzoumou, A. and Cittanova. N. (1989) A competitive microtitre plate enzyme immunoassay for plasma testosterone using polyc1onal antitestosterone immunoglobulin. Clin. Chim. Acta 183, 197_ Samanta, A.K. and Ali, E. (I990) Enzyme immunoassay of testosterone using nitrocellulose discs as the solid phase. J. Clin. Chem. Clin. Biochem. 28, 943. Tateishi, K., Yamamoto, H., Ogihara, T. and Hayashi, C. (1977) Enzyme immunoassay of serum testosterone. Steroids 30, 25. Vermeulen, A. (1979) The androgens. In: C.H. Grey and V.H.T. James (Eds.), Hormones in Blood, Vol. 3. Academic Press, New York, p. 356.
167
JIM 06249
Direct micro titre plate enzyme immunoassay of testosterone in unextracted serum Tarun K. Dhar and Esahak Ali s. C. MulJick Road, Calcutta- 7()() 032, Indill
Indian Institute of Chemical Biology, 4, Raja
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
168
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 2 HP04 /KH zP04 (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 NaHC0 3 /Na 2C0 3 (250 mmol/l, pH 9.6). Enzyme assay buffer was sodium acetate/ citric acid (100 mmol/l, pH 3.95).
Methods
Antisera preparation
Antiserum against testosterone was raised in New Zealand White rabbits using testosterone-3O-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 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.
Testosterone-3-carboxymethyloxime was covalently 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.
169
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 standards 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.
Radioimmunoassay Radioimmunoassay was carried out by the direct 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 7075°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.
170 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
0.40 0.05 18 12.5
3.20 0.18 12 5.6
7.90 0.54 18 6.8
0.58 0.096 10 16.5
3.76 0.25 10 6.6
5.5 0.5 10 9
Intra-assay Mean SO n CV%
lnterassay
o 2~~.,;-'-_--:::"-::':-----L.',..-----1.'_ _ _' 00'01
0.5
20
50
20.0
Mean SO n CV%
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 testosterone 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
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 samples 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. % Recovery
Testosterone nglml Endogenous
Added
Calculated
Found
6.5
5.0
2.5
0.5
11.5 9.0 7.0
12.0 8.4 7.5
104.35 93.33 107.14
5.0 2.5
7.0 4.5
7.0 4.3 2.7
100.00 95.55 108.00
2.0
0.5
2.5
171 200
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 factor
Expected ng/ml
1/2 1/4 1/8 1/16 1/32
11.00 5.50 2.75 1.37 0.69 0.35
Observed ng/ml
Recovery %
E ..... '"c
-~
16.0 o
0
•
12.0
(I) (I)
«
I-
u
8.0
ILl
5.50 2.70 1.30 0.70 0.40
100.00 98.18 94.89 101.45 114.28
Q:
is
4.0
0.0 0.0
4.0
8.0
12.0
16.0
EXTRACTED ASSAY (ng 1m!)
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.
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 Abraham, G.E. (1969) Solid phase radioimmunoassay of oestradiol 17fJ. J. C1in. Endocrinol. Metab. 29, 866. Elder, PA and Lewis, J.G. (1985) An enzyme-linked immunosorbent assay (ELISA) for plasma testosterone. J Steroid Biochem. 22, 635. Gallati, V.H. and Pracht, I. (1985) Horseradish peroxidase: Kinetic studies and optimization of the peroxidase activity determination with the substrate H 2 0 2 and 3,3',5,5'-tetramethylbenzidine. J. C1in. Chern. Clin. Biochem. 23, 453. 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. Ismail, A.A.A. (1976) Testosterone. In: J.A. Loraine and E.T. Bell (Eds.), Hormone assays and their clinical application. Churchill Livingstone, Edinburgh, p. 581. Joshi, V.M., Shah, H.P. and Sudhama, S.P. (1979) A sensitive and specific enzyme immunoassay for serum testosterone. Steroids 34, 35. Marcus, G.J. and Dumford, R. (1985) A simple enzyme-linked immunosorbent assay for testosterone. Steroids 46, 975.
Mattox, V.R., Litwiller, R.D. and Nelson, A.N. (1979) A comparison of procedures for attaching steroidal glucosiduronic acid in bovine serum albumin. J. Steroid Biochem. to, 167. Meyer, H.H.D. and Guven, B. (1986) Improvement ofmicrotitration plate enzyme immunoassays for steroid determination by a second antibody technique. J. Steroid Biochem_ 25 (suppJ.), 139. Ra;kowski, K.M., Hanquez C., Bauzoumou, A. and Cittanova. N. (1989) A competitive microtitre plate enzyme immunoassay for plasma testosterone using polyc1onal antitestosterone immunoglobulin. Clin. Chim. Acta 183, 197_ Samanta, A.K. and Ali, E. (I990) Enzyme immunoassay of testosterone using nitrocellulose discs as the solid phase. J. Clin. Chem. Clin. Biochem. 28, 943. Tateishi, K., Yamamoto, H., Ogihara, T. and Hayashi, C. (1977) Enzyme immunoassay of serum testosterone. Steroids 30, 25. Vermeulen, A. (1979) The androgens. In: C.H. Grey and V.H.T. James (Eds.), Hormones in Blood, Vol. 3. Academic Press, New York, p. 356.
