Anti-testosterone Antibody Research Articles

Competitive light-initiated chemiluminescent assay: using 5-α-dihydrotestosterone-BSA as competitive antigen for quantitation of total testosterone in human sera.

This paper described a homogeneous method, light-initiated chemiluminescent assay (LICA), for quantitation of total testosterone in human sera. The assay was bead based and built on a competitive-binding reaction format, in which 5-α-dihydrotestosterone (5-α-DHT) competed with the testosterone in serum samples in binding with biotinylated anti-testosterone antibody. The more testosterone in the serum sample, the less 5-α-DHT that bonded with biotinylated anti-testosterone antibodies. 5-α-DHT was coupled with emission beads (doped with thioxene derivatives and Eu(III) as a chemiluminescence emitter) via bovine serum albumin as a linker. Once streptavidin-coated sensitizer beads (modified with phthalocyanine as a photosensitizer) were added, the streptavidin/biotin reaction between 5-α-DHT-bound anti-testosterone antibody and sensitizer beads could bring emission and sensitizer beads together, which allowed energy transfer from sensitizer bead to emission bead. As such, an exciting light (680nm) impinging on the sensitizer beads led to light emission at 520-620nm by emission beads. The strength of the emitted light was inversely proportional to the testosterone in serum sample. The detection range of this assay was from 13.3 to 1200ng/dL. The coefficient variation for intra- and inter-assay was lower than 15%. The recovery of this method ranged from 95.5 to 105.9% for different samples. Moreover, the LICA assay was highly specific with low cross-reactivity and interference. The concentration of testosterone from 58 serum samples analyzed by the LICA method significantly correlated (y = 0.97x + 1.87, R2 = 0.970, p < 0.001) with those obtained with the SIEMENS Centaur Xp System. Graphical abstract ᅟ.

Complement C3 and anti-testosterone antibody in the 150 kDa Bali cattle (Bos sondaicus/javanicus) subordinate follicular fluid peptide observed using MALDI-TOF/TOF-MS

Objective : This study aimed to elucidate the proteome profile of the 150 kDa protein isolated from the subordinate follicle of Bali cattle ( Bos sondaicus / javanicus ). Some researchers have revealed several factors in the follicular subordinate with a 150 kDa protein substance, which play important roles in the bovine ovulation. Materials and methods : In the present study, subordinate follicles (~ 5 mm in diameter) were collected from 10 female Bali cattle from slaughterhouses in Taliwang, Sumbawa of West Nusa Tenggara Province, Indonesia. The follicular liquid was pooled; fractionated using SDSPAGE 10%, the 150 kDa band was sliced and then analyzed using MALDI-TOF/TOF-MS. Results : Mascot search results significantly revealed the presence of four species of proteins/peptides. Of the four peptides, two were predominant i.e. complement C3 and anti-testosterone antibody, which both were 100% identical to complement C3 and anti-testosterone antibody of Bos Taurus cattle. Conclusion : Complement C3 and anti-testosterone antibody are present in the follicle fluid of Bos sondaicus/javanicus cows. These findings are novel in Bali cattle follicles. http://doi.org/10.5455/javar.2018.e240

Quantitative proteomic profiling of bovine follicular fluid during follicle development.

Bovine follicular fluid (FF) constitutes the microenvironment of follicles and includes various biologically active proteins. We performed a study involving 18 healthy nonlactating Holstein cows to determine the protein expression profile of FF at key stages of follicular development. Follicles were individually aspirated in vivo at predeviation (F1 ∼ 7.0 mm), deviation (F1 ∼ 8.5 mm), postdeviation (F1 ∼ 12.0 mm), and preovulatory stages of follicle development, which were confirmed by measurement of follicular estradiol and progesterone concentrations. The FFs from nine cows were selected for proteomic analysis. After albumin depletion, triplicates of pooled FF were reduced, alkylated, and digested with trypsin. The resulting peptides were labeled with TMTsixplex and quantified using liquid chromatography-mass spectrometry/mass spectrometry. A total of 143 proteins were identified and assigned to a variety of biological processes, including response to stimulus and metabolic processes. Twenty-two differentially (P<0.05) expressed proteins were found between stages indicating intrafollicular changes over development, with expected deviation time critical to modulate the protein expression. For instance, high concentrations of follistatin, inhibin, serglycin, spondin-1, fibrinogen, and anti-testosterone antibody were found during early stages of follicular development. In contrast, apolipoprotein H, alpha-2-macroglobulin, plasminogen, antithrombin-III, and immunoglobulins were increased after deviation. Among the differentially abundant proteins, 19 were found to be associated with steroidogenesis. Pathway analysis identified proteins that were mainly associated with the acute phase response signaling, coagulation system, complement system, liver/retinoid X receptor activation, and biosynthesis of nitric oxide and reactive oxygen. The differentially expressed proteins provide insights into the size-dependent protein changes in the ovarian follicle microenvironment that could influence follicular function.

Identification of serum protein markers for early diagnosis of pregnancy in buffalo.

Improper or delayed pregnancy diagnosis has significant impact over animal production, particularly in buffaloes which inherently suffer from several reproductive inefficiencies. Thus the present study has undertaken to identify serum protein markers pertaining to early pregnancy diagnosis in buffaloes. Serum samples were collected from 10 pregnant Murrah Buffalo heifers at weekly intervals from days 0-35 post-artificial insemination and from 12 inseminated non-pregnant cyclic buffalo heifers on days 0, 7, 14 and 21. Two-dimensional gel electrophoresis and densitometric analysis revealed the presence of five protein spots showing average density fold change of ≥4 during early pregnancy. Mass spectrometry analysis identified these up-regulated proteins as anti-testosterone antibody light chain, apolipoprotein A-II precursor, serum amyloid A, cytokeratin type II, component IV isoform 1, which are have established roles in embryogenesis, but over-expression of the fifth identified protein immunoglobulin lambda light chain in pregnancy has been elucidated as a novel finding in the current study. Further, with bioinformatics analysis, potential antigenic B-cell epitopes were predicted for all these five proteins. An antibody cocktail-based approach involving antibodies against all these five up-regulated entire proteins or their epitopes could be developed for early detection of pregnancy in buffaloes. © 2016 Japanese Society of Animal Science.

Castration inhibits biliary proliferation induced by bile duct obstruction: novel role for the autocrine trophic effect of testosterone

Increased cholangiocyte growth is critical for the maintenance of biliary mass during liver injury by bile duct ligation (BDL). Circulating levels of testosterone decline following castration and during cholestasis. Cholangiocytes secrete sex hormones sustaining cholangiocyte growth by autocrine mechanisms. We tested the hypothesis that testosterone is an autocrine trophic factor stimulating biliary growth. The expression of androgen receptor (AR) was determined in liver sections, male cholangiocytes, and cholangiocyte cultures [normal rat intrahepatic cholangiocyte cultures (NRICC)]. Normal or BDL (immediately after surgery) rats were treated with testosterone or antitestosterone antibody or underwent surgical castration (followed by administration of testosterone) for 1 wk. We evaluated testosterone serum levels; intrahepatic bile duct mass (IBDM) in liver sections of female and male rats following the administration of testosterone; and secretin-stimulated cAMP levels and bile secretion. We evaluated the expression of 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3, the enzyme regulating testosterone synthesis) in cholangiocytes. We evaluated the effect of testosterone on the proliferation of NRICC in the absence/presence of flutamide (AR antagonist) and antitestosterone antibody and the expression of 17β-HSD3. Proliferation of NRICC was evaluated following stable knock down of 17β-HSD3. We found that cholangiocytes and NRICC expressed AR. Testosterone serum levels decreased in castrated rats (prevented by the administration of testosterone) and rats receiving antitestosterone antibody. Castration decreased IBDM and secretin-stimulated cAMP levels and ductal secretion of BDL rats. Testosterone increased 17β-HSD3 expression and proliferation in NRICC that was blocked by flutamide and antitestosterone antibody. Knock down of 17β-HSD3 blocks the proliferation of NRICC. Drug targeting of 17β-HSD3 may be important for managing cholangiopathies.

Measurement of testosterone in seminal plasma, saliva and serum by solid-phase enzymeimmunoassay.

When testosterone concentrations in serum, saliva and seminal plasma were measured with a solid-phase enzymeimmunoassay in which antitestosterone antibody was bound to CNBr-Sepharose-4B, we found the ratios of testosterone in saliva and seminal plasma to that in serum were 2.5 +/- 0.2% and 5.19 +/- 0.42%. Testosterone level in saliva could be an index of free serum testosterone. In addition, we found a correlation between sperm motility and testosterone level in seminal plasma when sperm motility was measured with a transmembrane migration method. This enzymeimmunoassay could be applied to clinical studies of testosterone.

Assessment of urinary total testosterone production by a highly sensitive time‐resolved fluorescence immunoassay

Radioimmunoassay (RIA) that involves purification of the analyte by organic solvent extraction is widely used. Although the extraction RIA is reliable when properly validated, it is time consuming and radioactive, we measure urinary total testosterone with a highly sensitive rapid and accurate time-resolved fluorescence immunoassay (TRFIA) method. High affinity antitestosterone antibody and Eu(3+) labeled Donkey antisheep IgG as tracers were used. The assay was evaluated for specificity, sensitivity, analytical recovery, precision and dilution linearity by the TRFIA method on urine samples. A satisfactory standard curve for testosterone TRFIA has been developed with good sensitivity (5.1 pmol/L). The validity of the assay for urinarytotal testosterone was confirmed by the good correlation between the results obtained by TRFIA (X) and those RIA (Y) (Y=0.075+0.971X, R=0.992). Specificity, analytical recovery, precision and dilution linearity studies were determined and all found to be satisfactory. Male urinary total testosterone excretion ranged from 64.00 to 374.11 nmol/24 hr, which was about four times more than the range for women urinary testosterone excretion (14.16-100.65 nmol/24 hr), which suggests that a direct, reliable, easy to automate, highly sensitive and specific TRFIA type assay for the measurement of total testosterone in urine samples has been developed.

Development and validation of a simple and direct ELISA method for the determination of conjugated (glucuronide) and non-conjugated testosterone excretion in urine

Background Several methods are now available to estimate urinary testosterone levels that can only be performed in established big laboratories using GC/MS techniques. In clinical practice or for research projects, an inexpensive method that does not require skilled technicians would be useful. A simple, rapid and accurate ELISA method has been developed and applied in our laboratory to measure urinary non-conjugated and total testosterone. Methods High affinity anti-testosterone antibody and HRP-Donkey anti-sheep IgG (Horse Radish Peroxidase) as enzyme tracer were used to develop the ELISA method. The assay was evaluated for specificity, sensitivity, parallelism, accuracy and imprecision by the established methods on samples obtained from healthy volunteers. The results from the direct ELISA were compared to those after enzyme hydrolysis plus solvent extraction and HPLC or commercial kits. Results A satisfactory standard curve for the direct testosterone ELISA has been developed with good sensitivity. Cross-reactivity values of anti-testosterone antibody with major interfering steroids were minimal except for testosterone-3-glucuronide (58.8%). The validity of urinary testosterone assay was confirmed by the good correlation between the results obtained by the direct ELISA and those after enzyme hydrolysis and solvent extraction ( Y = 0.987 X + 0.398, R 2 = 0.97). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Urinary testosterone excretion values obtained by our direct ELISA from healthy volunteers were generally in agreement with those published by other workers. Male urinary total testosterone excretion (non-conjugated and testosterone glucuronide) ranged from 177.9 to 865.3 nmol/day, which was about 3–6 times more than the range for women urinary testosterone excretion (34.5–308.8 nmol/day). Conclusion A direct, reliable, easy to perform, sensitive and highly specific ELISA type assay for the measurement of total testosterone in urine samples (conjugated and non-conjugated) has been developed. The novel features of the assay are that it does not require an initial extraction step or involve time consuming procedures such as chromatography. A simple method has also been developed to measure non-conjugated urinary testosterone excretion after solvent extraction alone.

Analysis of the binding energies of testosterone, 5alpha-dihydrotestosterone, androstenedione and dehydroepiandrosterone sulfate with an antitestosterone antibody.

Molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) free energy calculations were used to study the binding of testosterone (TES), 5alpha-dihydrotestosterone (5ADHT), androstenedione (AND), and dehydroepiandrosterone sulfate (DHEAS) to the monoclonal antitestosterone antibody 3-C(4)F(5). The relative binding free energy of TES and AND was also calculated with free energy perturbation (FEP) simulations. The antibody 3-C(4)F(5) has a relatively high affinity (3 x 10(8) M(-1)) and on overall good binding profile for testosterone but its cross-reactivity with DHEAS has been the main reason for the failure to use this antibody in clinical immunoassays. The relative binding free energies obtained with the MM-PBSA method were 1.5 kcal/mol for 5ADHT, 3.8 kcal/mol for AND, and 4.3 kcal/mol for DHEAS, as compared to TES. When a water molecule of the ligand binding site, observed in the antibody-TES crystal structure, was explicitly included in MM-PBSA calculations, the relative binding energies were 3.4, 4.9, and 5.4 kcal/mol for 5ADHT, AND, and DHEAS, respectively. The calculated numbers are in correct order but larger than the corresponding experimental energies of 1.3, 1.5, and 2.6 kcal/mol, respectively. The fact that the MM-PBSA method reproduced the relative binding free energies of DHEAS, a steroid having a negatively charged sulfate group, and the neutrally charged TES, 5ADHT, and AND in satisfactory agreement with experiment shows the robustness of the method in predicting relative binding affinities. The 800-ps FEP simulations predicted that the antibody 3-C(4)F(5) binds TES 1.3 kcal/mol tighter than AND. Computational mutagenesis of selected amino acid residues of the ligand binding site revealed that the lower affinities of AND and DHEAS as compared to TES are due to a combined effect of several residues, each contributing a small fraction to the tighter binding of TES. An exception to this is Tyr99H, whose mutation to Ala lowered the binding of DHEAS 0.7 kcal/mol more than the binding of TES. This is probably due to the hydrogen bonding interaction formed between the OH group of Tyr99H and the sulfate group of DHEAS. Computational mutagensis data also showed that the affinity of the steroids to the antitestosterone antibody 3-C(4)F(5) would be enhanced if Trp47H were repositioned so that it would make more extensive contacts with the bound ligands. In addition, the binding of steroids to antitestosterone, antiprogesterone, and antiestradiol antibodies is discussed.

Structural insights into steroid hormone binding: the crystal structure of a recombinant anti-testosterone Fab fragment in free and testosterone-bound forms.

The monoclonal anti-testosterone antibody (3-C(4)F(5)) has a relatively high affinity (3 x 10(8) m(-1)) with an overall good specificity profile. However, the earlier characterized binding properties have shown that both the affinity and specificity of this antibody must be improved if it is intended for use in clinical immunoassays. In this paper, the crystal structures of the recombinant anti-testosterone (3-C(4)F(5)) Fab fragment have been determined in the testosterone-bound and free form at resolutions of 2.60 and 2.72 A, respectively. The high affinity binding of the (3-C(4)F(5)) Fab is mainly determined by shape complementarity between the protein and testosterone. Only one direct hydrogen bond is formed between the hydroxyl group of the testosterone D-ring and the main-chain oxygen of Gly100(J)H. The testosterone is deeply bound in a hydrophobic pocket, and the close shape complementarity is mainly formed by the third complementarity-determining regions (CDR) of the heavy and light chain. Comparison of the bound structure with the free structure indicates conformational changes in the protein upon testosterone binding. The conformational changes of the side chains of two residues Glu95H and Tyr99H in the CDR-H3 are particularly essential for the binding. Interesting similarities in the binding of different steroids were also observed upon comparison of the available structures of anti-steroid antibodies.

Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs

Background: We have previously reported specificity improvement of an anti-testosterone monoclonal antibody (3-C4F5) by random mutagenesis of the third complementarity determining regions (CDR3s) and by phage display selection. Objectives: Here we extend the mutagenesis strategy to the other CDRs and select the mutant libraries using two different approaches in order to further fine-tune the binding properties of this recombinant Fab fragment. Study design: To improve the affinity the new mutant libraries were selected by using limiting, decreasing concentrations of biotinylated testosterone (TES) in solution and capturing the binders on streptavidin-coated microtiter plate. The specificity was improved by preincubating the mutant libraries in solution with a high concentration of the most problematic cross-reacting steroid, dehydroepiandrosterone sulfate (DHEAS). Results: In two different light chain CDR1 mutant clones isolated from the affinity pannings, the relative TES affinity was increased over 10-fold while the cross-reactivities to related steroids were preserved at the same level as in the parental combined CDR3 mutant clone. New heavy chain CDR1 and light chain CDR2 mutants showing slightly decreased cross-reactivities were isolated from specificity selections. By combining compatible mutant CDRs together we were able to create a Fab fragment with over 12-fold higher relative TES affinity and significantly lower cross-reactivity to DHEAS when compared to the original monoclonal antibody 3-C4F5. Conclusions: Our results demonstrate that a high-affinity and selective recombinant Fab fragment working over a wide TES concentration range with clinical samples could be generated by CDR mutagenesis and phage display selection.

7α-Biotinylated testosterone derivatives as tracers for a competitive chemiluminescence immunoassay of testosterone in serum

Ring core-biotinylated testosterone tracers were synthesized with bridges of three different lengths connecting the biotin moiety to the steroid core (7 alpha-Cn-Bio-T, n = 3, 6, or 11). Together with a position 7-specific polyclonal anti-testosterone antibody, we used the 7 alpha-C11-Bio-T tracer to develop a novel, labeled-hapten competitive immunoassay for total testosterone in serum. (The C3 and C6 tracers proved to be not suitable for analogous immunoassays.) Enhanced chemiluminescence signal was generated by use of a second immobilized antibody and a streptavidin-horseradish peroxidase conjugate. The measuring range of the assay is 0.2-20.0 nmol/L, linearity of serial dilutions can be demonstrated, the lower detection limit is 0.125 nmol/L, and the interassay imprecisions are 13-16%. Accuracy determinations in mass spectrometry-controlled reference specimens showed a mean recovery of 95%. In addition, the assay shows low cross-reactivities, demonstrating the favorable specificity of the combination of a "nearly native" tracer with a position analog antibody. The optimized steric structure and the long spacer arm of the biotinylated testosterone tracer make this chemiluminescence assay well-suited for measuring total testosterone concentration in serum.

Synthesis of 17 beta-hydroxyandrost-4-en-3-one-7 alpha-(biotinyl-6-N-hexylamide), a conjugate useful for affinity chromatography and for testosterone immunoassays.

We describe the synthesis of 17 beta-hydroxyandrost-4-en-3-one-7 alpha-(biotinyl-6-N-hexylamide) from 17 beta-hydroxyandrost-4-en-3-one (testosterone) via copper-catalyzed 1,6 Michael addition of a 6-(tertbutyldimethylsilyloxyhexyl) chain to 6-dehydrotestosterone 17 beta-acetate. After chromatographic separation of the 7 alpha-isomer from the alpha / beta mixture and cleavage of the silyl ether, the alcohol was oxidized to the 6-hexanal side chain and then subjected to reductive amination. The resulting primary amine is easily biotinylated using biotinyl-N-hydroxysuccinimide ester. The overall yield for the epimeric 7 alpha-end product was 30%. The absolute configurations of the epimers were investigated by 1H NMR studies by the nuclear Overhauser effect. We introduced a biotin label to the testosterone molecule at ring position 7 in compliance with Landsteiner's principle, which states that antibody specificity is directed primarily at that portion of the hapten furthest from the functional group linking it to the carrier protein. Thus, this negligible alteration in comparison to the structure of the respective testosterone hapten used to elicit antibodies offers the feasibility of applying the testosterone derivative as an optimal immunoadsorbent in affinity chromatography. The 7 alpha-biotinylated testosterone was used to obtain active antitestosterone antibodies from a specific antiserum by affinity chromatography. This was achieved by attaching the biotinylated testosterone to agarose-coupled streptavidin beads. Accordingly, a 3H-testosterone-binding test demonstrated a 20-fold increase in affinity of the purified antibody to the steroid compared to the original antiserum, and a recovery of > 80% could be obtained. The antitestosterone antibody, obtained by that method, is an effective component for use in a competitive immunoassay for testosterone in human sera. An assay configuration is conceivable with the same 7 alpha-biotinylated testosterone employed as tracer in combination with a streptavidin-linked reporter enzyme.

Sandwich immunoassay of small molecules: I. Investigation with testosterone as model hapten

A new strategy has been developed for enzyme immunoassay of small molecules. The method is based on the formation and estimation of the complex Ab.H-H.Ab* where Ab and Ab* are immobilised and labeled antibodies respectively and H-H is a synthetic bis-analogue of the hapten H. The amount of the complex formed is inversely related to the concentration of free hapten present. The estimation of the sandwich complex increases the sensitivity and the specificity of the assay compared to conventional assays. Using the N,N'-diamide of testosterone-3-(O-carboxymethyl)oxime with ethylenediamine and a polyclonal rabbit anti-testosterone antibody (for both labeling and immobilisation) a sensitivity of 100 fg/well (3.5 x 10(-16) mol) was obtained for testosterone. The cross-reactivity of 5 alpha-dihydrotestosterone was 16% compared to 27% obtained by conventional competitive assays.

Molecular modelling and site-directed mutagenesis on a bovine anti-testosterone monoclonal antibody.

A three-dimensional (3D) molecular model of the antigen-combining site of a bovine anti-testosterone monoclonal antibody has been constructed. In the model, the CDRs, and a single heavy chain framework region residue (Trp47), associate to form a hydrophobic cavity large enough to accommodate a single molecule of testosterone. Tyr97 of CDR-H3 lies at the bottom of the cavity with its hydroxyl group exposed to solvent. Using the model and data from binding studies, we predicted that the cavity forms the antibody's paratope and on binding testosterone a hydrogen bond is formed between Tyr97 of CDR-H3 and the hydroxyl group on the D-ring of testosterone. This prediction has subsequently been tested by site-directed mutagenesis. An antibody with phenylalanine in place of tyrosine at position 97 in CDR-H3 has its affinity reduced by approximately 800 fold. The reduction in binding energy associated with the reduced affinity has been calculated to be 3.9 kcal/mol which is within the range (0.5-4.0 kcal/mol) expected for the loss of a single hydrogen bond. The model has been used to suggest ways of increasing the antibody's affinity for testosterone.

Enzyme labeling in steroid enzyme immunoassays. The p-nitrophenyl ester method for alkaline phosphatase and glucose oxidase labelings.

The p-nitrophenyl ester method was assessed as an enzyme labeling technique. The active ester of a carboxylated testosterone derivative was treated with alkaline phosphatase and glucose oxidase to give labeled antigens, using various molar ratios of steroid to enzyme. Satisfactory immunoreactivities with an anti-testosterone antibody in an enzyme immunoassay system were obtained with the labeled antigens prepared at pH 8.5 by the use of molar ratios higher than 30 and 10, respectively, in the alkaline phosphatase and glucose oxidase labelings.

A competitive microtitre plate enzyme immunoassay for plasma testosterone using polyclonal anti-testosterone immunoglobulins

An enzyme immunoassay for plasma testosterone was developed based on competition between an immobilised testosterone-casein conjugate and the analyte for polyclonal anti-testosterone immunoglobulins, followed by the use of enzyme-labelled second antibodies to determine the degree of competition. The quantity of immobilised testosterone-casein conjugate was optimised so that the lower affinity antitestosterone antibody populations present did not affect the assay. The assay standard curve covered a range of 11–300 fmol/well. Testosterone levels in small amounts of male and female plasma could be assayed with good reproducibility and correlated well with results obtained by radioimmunoassay. By comparison with an analogous assay using monoclonal antibodies it appears that, given that the assay sensitivity is the most important criterion for choice, the use a polyclonal antiserum with this type of reactive antibody selection is preferable to the use of monoclonal antibodies.

An improved method for isolation of specific antibodies by affinity chromatography; application to an antiserum to testosterone

The specificity of polyclonal antibodies for use in immunoassay procedures can be improved by purification on affinity columns. The standard procedure involves immunoabsorption of the specific antibody populations on the insolubilized antigen, followed by desorption from the latter. However, the desorption process usually results in antibody damage and loss of antibody titer. We therefore designed an affinity chromatography procedure involving immunoabsorption of the main cross-reacting antibody fractions instead of the specific ones, eliminating thereby the desorption step. We exemplify the procedure with the purification of an anti-testosterone antibody.

An enzyme-linked immunoassay of testosterone

An enzyme-linked immunoassay of testosterone is described. The conjugation of testosterone with high specific activity horseradish peroxidase and a highly sensitive assay for this enzyme having previously been studied, here we describe the immobilization of the anti-testosterone antibody and the development of assay conditions permitting the determination of 50 pg to 1.5 ng testosterone in one working day. In this work attention has been paid to keeping the assay method as simple as possible. The method is discussed in terms of other enzyme-linked immunoassays for steroids.