Opposite behaviors of reactive metabolites of tienilic acid and its isomer toward liver proteins: use of specific anti-tienilic acid-protein adduct antibodies and the possible relationship with different hepatotoxic effects of the two compounds.

Tienilic acid (TA) is responsible for an immune-mediated drug-induced hepatitis in humans, while its isomer (TAI) triggers a direct hepatitis in rats. In this study, we describe an immunological approach developed for studying the specificity of the covalent binding of these two compounds. For this purpose, two different coupling strategies were used to obtain TA-carrier protein conjugates. In the first strategy, the drug was linked through its carboxylic acid function to amine residues of carrier proteins (BSA-N-TA and casein-N-TA), while in the second strategy, the thiophene ring of TA was attached to proteins through a short 3-thiopropanoyl linker, the corresponding conjugates (BSA-S-5-TA and betaLG-S-5-TA) thus preferentially presenting the 2, 3-dichlorophenoxyacetic moiety of the drug for antibody recognition. The BSA-S-5-TA conjugate proved to be 30 times more immunogenic than BSA-N-TA. Anti-TA-protein adduct antibodies were obtained after immunization of rabbits with BSA-S-5-TA (1/35000 titer against betaLG-S-5-TA in ELISA). These antibodies strongly recognized the 2, 3-dichlorophenoxyacetic moiety of TA but poorly the part of the drug engaged in the covalent binding with the proteins. This powerful tool was used in immunoblots to compare TA or TAI adduct formation in human liver microsomes as well as on microsomes from yeast expressing human liver cytochrome P450 2C9. TA displayed a highly specific covalent binding focused on P450 2C9 which is the main cytochrome P450 responsible for its hepatic activation in humans. On the contrary, TAI showed a nonspecific alkylation pattern, targeting many proteins upon metabolic activation. Nevertheless, this nonspecific covalent binding could be completely shifted to a thiol trapping agent like GSH. The difference in alkylation patterns for these two compounds is discussed with regard to their distinct toxicities. A relationship between the specific covalent binding of P450 2C9 by TA and the appearance of the highly specific anti-LKM2 autoantibodies (known to specifically recognize P450 2C9) in patients affected with TA-induced hepatitis is strongly suggested.

Studies of the reactivity of artificial peroxidase-like hemoproteins based on antibodies elicited against a specifically designed ortho-carboxy substituted tetraarylporphyrin.

The temperature and pH dependence as well as the selectivity of the peroxidase activity of a complex associating a monoclonal antibody 13G10 with its iron(III)-alpha,alpha,alpha,beta-mesotetrakis(ortho-carboxyphenyl) porphyrin (Fe(ToCPP)) hapten have been studied and compared to those of Fe(ToCPP) alone. It first appears that the peroxidase activity of the 13G10-Fe(ToCPP) complex is remarkably thermostable and remains about 5 times higher than that of Fe(ToCPP) alone until at least 80 degrees C. Secondly, this complex is able to use not only H2O2 as oxidant but also a wide range of hydroperoxides such as alkyl, aralkyl and fatty acid hydroperoxides and catalyze their reduction 2-6-fold faster than Fe(ToCPP) alone. It is also able to catalyze the oxidation by H202 of a variety of reducing cosubstrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB) and 3,3'-dimethoxybenzidine 3-8-fold faster than Fe(ToCPP) alone, the bicyclic aromatic ABTS and TMB being the best reducing cosubstrates. Finally, a pH dependence study, between pH 4.6 and 7.5, of the oxidation of ABTS by H2O2 in the presence of either 13G10-Fe(ToCPP) or Fe(ToCPP) shows that Km(H2O2) values vary very similarly for both catalysts, whereas very different variations are found for the k(cat) values. With Fe(ToCPP) as catalyst the k(cat) value remains constant around 100 min(-1) whereas with the 13G10-Fe(ToCPP) complex, it increases sharply below pH 5 to reach 540 min -1 at pH 4.6. This could be due to the participation of a carboxylic acid side chain of the antibody protein, as a general acid-base catalyst, to the heterolytic cleavage of the O-O bond of H2O2 leading to the highly reactive iron(V)-oxo intermediate in the peroxidase mechanism. Accordingly, the modification of the carboxylic acid residues of antibody 13G10 by glycinamide leads to a 50% decrease of the peroxidase activity of the 13G10-Fe(ToCPP) complex.

Active site topology of artificial peroxidase-like hemoproteins based on antibodies constructed from a specifically designed ortho-carboxy-substituted tetraarylporphyrin.

The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)-alpha,alpha,alpha,beta-meso-tetrakis(ortho-carboxyphenyl)porph yrin [alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin]], and which exhibit in the presence of this alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] cofactor a peroxidase activity. A comparison of the dissociation constants of the complexes of 13G10 and 14H7 with various tetra-aryl-substituted porphyrin has shown that: (a) the central iron(III) atom of alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] is not recognized by either of the two antibodies; and (b) the ortho-carboxylate substituents of the meso-phenyl rings of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] are essential for the recognition of the porphyrin by 13G10 and 14H7. Measurement of the dissociation constants for the complexes of 13G10 and 14H7 with the four atropoisomers of (o-COOHPh)4-porphyrinH2 as well as mono- and di-ortho-carboxyphenyl-substituted porphyrins suggests that the three carboxylates in the alpha, alpha, beta position are recognized by both 13G10 and 14H7 with the two in the alpha, beta positions more strongly bound to the antibody protein. Accordingly, the topology of the active site of 13G10 and 14H7 has roughly two-thirds of the alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] cofactor inserted into the binding site of the antibodies, with one of the aryl ring remaining outside. Three of the carboxylates are bound to the protein but no amino acid residue acts as an axial ligand to the iron atom. Chemical modification of lysine, histidine, tryptophan and arginine residues has shown that only modification of arginine residues causes a decrease in both the binding of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O-O bond of H2O2. In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.

Hemoabzymes. Different strategies for obtaining artificial hemoproteins based on antibodies.

Besides existing models of chemical or biotechnological origin for hemoproteins like peroxidases and cytochromes P450, catalytic antibodies (Abs) with a metalloporphyrin cofactor represent a promising alternative route to catalysts tailored for selective oxidation reactions. A brief overview of the literature shows that, until now, the first strategy for obtaining such artificial hemoproteins has been to produce antiporphyrin Abs, raised against various free-base, N-substituted, Sn-, Pd-, or Fe-porphyrins. Four of them exhibited, in the presence of the corresponding Fe-porphyrin cofactor, a significant peroxidase activity, with kcat/K(m) values of 10(2) to 5 x 10(3)/M/s. This value remained low when compared to that of peroxidases, probably because neither a proximal ligand of the Fe, nor amino acid residues participating in the catalysis of the heterolytic cleavage of the O-O bond of H2O2, have been induced in those Abs. This strategy has been shown to be insufficient for the elaboration of effective models of cytochromes P450, because only one set of Abs, raised against meso-tetrakis(para-carboxyvinylphenyl)porphyrin, was reported to catalyze the nonstereoselective oxidation of styrene by iodosyl benzene using a Mn-porphyrin cofactor, and attempts to generate Abs having binding sites for both the substrate and the metalloporphyrin cofactor, using as a hapten a porphyrin covalently linked to the substrate, were not successful. A second strategy is then proposed, which involves the chemical labeling of antisubstrate Abs with a metalloporphyrin. As an example, preliminary results are presented on the covalent linkage of an Fe-porphyrin to an antiestradiol Ab, in order to obtain semisynthetic catalytic Abs able to catalyze the selective oxidation of steroids.

Artificial peroxidase-like hemoproteins based on antibodies constructed from a specifically designed ortho-carboxy substituted tetraarylporphyrin hapten and exhibiting a high affinity for iron-porphyrins.

In order to get catalytic antibodies modelling peroxidases BALB/c mice have been immunized with iron(III)-alpha,alpha,alpha,beta-mesotetrakis-orthocarboxypheny l-porphyrin (Fe-(ToCPP))-KLH conjugates. Monoclonal antibodies have been produced by the hybridoma technology. Three antibodies, 2 IgG1 and 1 IgG2a, were found to bind both Fe(ToCPP) and the free base ToCPPH2 with similar binding constants. None of those antibodies was found to bind tetraphenylporphyrin. Those results suggest that the recognition of Fe(ToCPP) by the antibodies was mainly due to the binding of the carboxylate groups to some amino acid residues of the protein. True Kd values of 2.9 x 10(-9) M and 5.5 x 10(-9) M have been determined for the two IgG1-Fe(ToCPP) complexes. Those values are the best ones ever reported for iron-porphyrin-antibody complexes. UV-vis. studies have shown that the two IgG1-Fe(ToCPP) complexes were high-spin hexacoordinate iron(III) complexes, with no amino acid residue binding the iron, whereas the IgG2a-Fe(ToCPP) complex was a low-spin hexacoordinate iron(III) complex with two strong ligands binding the iron atom. Both IgG1-Fe(ToCPP) complexes were found to catalyze the oxidation of 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) 5-fold more efficiently than Fe(ToCPP) alone whereas the binding of IgG2a to this iron-porphyrin had no effect on its catalytic activity. kcat values of 100 min(-1) and 63 min(-1) and kcat/Km values of 105 M(-1) s(-1) and 119 M(-1) s(-1) have been found respectively for the two IgG1-Fe(ToCPP) complexes.

A catalytic antibody isomerizing a delta 5-3-cétostéroïde.

Antibodies were raised against 3-fluoro-4-aza-estradiol-17-hemisuccinate to elicit abzymes capable of isomerizing delta 5-3-ketosteroids. The hapten was designed to present a planar A ring showing some analogy with the intermediate dienol and a polarity capable of inducing catalytic groups. Antibodies binding the hapten tightly were cloned and purified from either ascites or hybridoma supernatants. Catalytic activity was tested with androst-5-ene-3,17-dione. Among the five different monoclonal antibodies binding the hapten, one proved to enhance significantly the substrate isomerization rate (kcat/kuncat = 830). Initial rates followed Michaelis kinetics and the activity was competitively inhibited by the hapten.

A direct dot-enzyme immunoassay to detect human ovulation.

This paper describes an original dot-enzyme-linked immunosorbent assay (ELISA) for predicting ovulation in women, based on the detection of the pre-ovulatory estrogen peak in urine. A monoclonal anti-estrogen antibody is used which recognizes not only free estrogens but also some of their urinary metabolites (17-glucuro- and sulfo-conjugates) allowing a direct assay on early morning urines. Antigen is immobilized as a spot on a nitrocellulose membrane which is immersed in urine in the presence of this antibody. A peroxidase-labeled second antibody allows the detection of the first antibody bound to the membrane. Antigen and anti-estrogen antibody concentrations are chosen to obtain a maximal enzymatic coloration of spots corresponding to basal urinary estrogen levels and no coloration corresponding to the pre-ovulatory surge. Six menstrual cycles were studied, comparing dot-ELISA results with patterns of: (1) urinary estrogens measured by RIA either directly or after hydrolysis and extraction, and (2) basal body temperatures. The validity of the pre-ovulatory signal obtained and the requirements for an adaptation of this methodology to a reliable home kit are discussed.

A drug release-facilitating mechanism for human serum albumin in reverse micelles: requirement of a structural switch.

We measured the binding of a drug oxyphenylbutazone to the N-terminal peptic fragment of human serum albumin in 0.1 M Tris buffer, pH 8.0 (Kass = 2.4 10(5) M-1) and in reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate and buffer in isooctane (Kass. = 2.7 10(5) M-1). In the absence of any measured change in conformation of the fragment in reverse micelles, the peptide affinity for the drug is not decreased, in contrast to what is observed in intact albumin (HSA) under similar conditions. The interaction and the subsequent unfolding of HSA at the membrane-mimetic interface, constitutes thus a drug release-facilitating mechanism.

Ligand binding at membrane mimetic interfaces. Human serum albumin in reverse micelles.

The behaviour of human serum albumin in the presence of three chemically distinct ligands: oxyphenylbutazone, dansylsarcosine and hemin, has been compared in buffer and in reverse micelles of isooctane, water, and either sodium bis(2-ethylhexyl)sulfosuccinate or hexadecyl trimethylammonium bromide, systems selected to mimic the membrane-water interface. Upon micellar incorporation, the dansylsarcosine-albumin complex dissociated, as evidenced by fluorescence emission spectroscopy (red shift from 485 nm to 570 nm) and by fluorescence polarization measurements. In contrast, the hemin-albumin complex remained stable in reverse micelles, as judged from the Soret absorption band at 408 nm and the molar absorption coefficient of 8.4 x 10(4) M-1 cm-1. The oxyphenylbutazone to albumin binding curves reveal that while the association constant remained unchanged (Ka approximately 1.0 x 10(5) M-1), only a fraction of the albumin molecules present reacted with the ligand. The results were unaffected by the nature and the concentration of the surfactant. These findings can be interpreted in the light of conformational changes induced in human serum albumin by the large micellar inner surface area. The blue shift of the fluorescence emission maximum from 344 nm in buffer to 327 nm in sodium bis(2-ethylhexyl)sulfosuccinate micelles and the lesser reactivity/accessibility of the fluorophore to oxidation by N-bromosuccinimide, indicate perturbations of the sole tryptophan-214 microenvironment. However, the distance between the indole residue and tyrosine-411 does not seem substantially modified by the 15% decrease affecting the alpha helices of the albumin molecule. It is proposed that the results reported herein reflect the interactions of albumin with a membrane-like interface which generates two protein subpopulations differing in their membrane-surface and ligand affinities. Overall and local conformational changes, originating from this surface-induced effect, may thus constitute a ligand-release facilitating mechanism acting at cellular membrane levels.

Comparison of monoclonal antibodies to estradiol obtained from structurally different immunogens.

The immunization procedure and immunogen characteristics required to optimize the production of anti-steroid monoclonal antibodies have been studied. Five different estradiol-bovine serum albumin conjugates were tested for immunizing mice, as were two different immunization protocols (high and low dose) and the effect of varying the myeloma/spleen cell ratio for cell fusion. Antibody-producing hybridomas, obtained using the spleens of 9 high anti-steroid titre mice, were detected by RIA and EIA. The latter method was less specific than the former for higher affinity anti-estrogen antibodies. All the immunogens elicited anti-estrogen antibodies and the efficiency appeared related to the steroid density on the immunogen rather than the chemical nature of the derivative or the immunization and fusion protocols. Thirty-six anti-estrogen producing hybridomas were detected. Comparison showed that all the immunogens elicited antibodies in a wide range of affinities and specificities. None of the antibodies recognized corticosteroids or progesterone. Cross reactions with testosterone and other estrogens were not clearly related to the nature of the immunogen except that estradiol coupled to the BSA via its carbon 17 yielded antibodies specific for steroids with a non-derivatized phenolic A-ring.

A syngeneic monoclonal anti-idiotypic antibody with internal image properties restricted to a single aldosterone-binding system.

Immunization with a murine anti-aldosterone mAb (AAC) resulted in the isolation of a syngeneic monoclonal anti-idiotypic antibody, LH9G4. LH9G4 bound to Fab fragments of AAC and was affinity-purified on an AAC column. LH9G4 inhibited the binding of aldosterone to AAC in a dose-dependent manner with an apparent dissociation constant of 0.5 nM as determined by competitive inhibition assays in ELISA and RIA. LH9G4 and aldosterone have similar relative affinities for AAC. Kinetic studies and Scatchard plot analysis support a reversible and reciprocal competitive inhibition mechanism between LH9G4 and aldosterone for the paratope of AAC. The possibility of a steric hindrance mechanism was eliminated. No cross-reactivity was seen with six other murine anti-aldosterone mAb, with a rabbit polyclonal antibody or with aldosterone receptor. The anti-idiotypic antibody, defined as a "restricted" internal image of aldosterone, is apparently directed at a private idiotope present in the paratope of AAC but not in binding sites of other aldosterone-binding proteins. Biophysical considerations involving characteristics of nonbonded attractive forces can explain these findings. An advantage of the one-step auto-anti-idiotypic procedure for the generation of Ab2-beta or internal image antibodies is discussed.

Improvement of estradiol enzymoimmunoassay, using a monoclonal antibody and an avidin/biotin amplification system.

A microtitre plate enzyme immunoassay for estradiol, using a purified monoclonal antibody covalently bound to peroxidase and a small amount of immobilized immunogen, was optimized. Decreasing the antibody concentration to 2 X 10(-10) M (Kd/5) gave optimum estradiol detectability. The enzymatic signal was, however, very low in this assay. A 14-fold enhancement could be obtained using an avidin-biotin system in which several biotin molecules are conjugated with the antibody, providing multiple sites for binding by an avidin-enzyme complex. Further reagent concentration optimization gave an assay in which a range of 2 to 140 pg estradiol/well could be assayed simply and reproducibly.

A competitive microtitre plate enzyme immunoassay for plasma aldosterone using a monoclonal antibody.

A competitive microtitre plate enzyme immunoassay for plasma aldosterone was developed using an immobilised aldosterone-bovine serum albumin conjugate and a monoclonal anti-aldosterone preparation, followed by the use of enzyme-labelled second antibodies to determine the degree of competition. The quantity of immobilised aldosterone-protein conjugate was adjusted to give optimum assay sensitivity with respect to the antibodies used. The lower limit of detection of aldosterone (55 fmol) was much less than that of an ELISA for aldosterone, using identical reagents but with an excess of immobilised aldosterone-protein conjugate, and up to 1400 fmol could be determined. Aldosterone levels in small amounts of male and female plasma could be assayed with good reproducibility.

Murine monoclonal antibody against aldosterone: production, characterization and use for enzymoimmunoassay.

Hybridomas secreting monoclonal antibodies to aldosterone were obtained by fusion of myeloma cells and spleen cells from Balb/c mice immunized with aldosterone-3-carboxylmethyloxime-bovine serum albumin. A monoclonal antibody was purified from ascites fluid and characterized. An affinity constant of 1.61 x 10(9) M-1 has been measured and no cross-reactivity with tetrahydroaldosterone (THA), cortisol, cortisone, corticosterone, deoxycorticosterone (DOC), dehydroepiandrosterone (DHA), progesterone and estrone, could be detected. A peroxidase conjugated-antibody (1.5 mole of enzyme per mole of antibody) was obtained and used for microwell enzyme immunoassay and Immun-Blot assay. The high affinity and specificity of this antibody should make the direct determination of aldosterone in biological fluids possible at concentrations as low as 5 x 10(-10) M.

Enzyme-linked immunosorbent assay (ELISA) for steroid hormones with polyclonal and monoclonal antibodies: an assay for urinary aldosterone.

The development of non-competitive microtitre plate enzyme-linked immunosorbent assays for steroids was investigated using immobilised steroid as immunosorbent and enzyme-labelled second antibodies. An assay for testosterone with polyclonal anti-testosterone immunoglobulins was optimised with respect to a number of parameters but remained unsatisfactory for clinical assays. The results were applied to developing an aldosterone assay using monoclonal anti-aldosterone immunoglobulins. The latter method was used for the determination of urinary aldosterone and the results are compared with those obtained by a classical radioimmunoassay. Problems concerned with the use of sulphur-containing proteins and with the presence of low affinity antibodies in polyclonal preparations are discussed.

The use of disulfonatonaphthalimide fluorescent dyes for the fluorescence polarization immunoassay of steroids.

A series of fluorescent disulfonatonaphthalimide derivatives of testosterone and estriol have been synthesized and their fluorescent properties investigated. The fluorescence lifetimes of these derivatives were higher than that of the unreacted fluorescent dye while the quantum yields were of the same order. The compounds were therefore compared in terms of their utilizability in steroid fluorescence polarization immunoassays. The assay sensitivity and precision with each compound is discussed in terms of the position, type, and length of the chemical "bridge" linking the steroid to the fluorescent dye. It is proposed that these fluorescent labels are highly appropriate to this type of immunoassay.

[Measurement of sulfoconjugates of 5-androsten-3 beta, 17 beta-diol in idiopathic hirsute virilism]. / Mesure des sulfoconjugués de 5-androstène-3 beta, 17 beta-diol dans le virilisme pilaire idiopathique.

The sulphoconjugates of delta 5-C19O2 steroids were determined in 32 women with idiopathic hirsutism (IH). The plasma and urinary levels of 5-androsten-3 beta, 17 beta-diol (delta 5, 3 beta-diol) were 82 +/- 24 ng/ml and 169 +/- 170 micrograms/24 h respectively for the monosulphate ester and 193 +/- 101 ng/ml and 180 +/- 108 micrograms/24 h for the disulphate ester. These levels are statistically higher (p less than 0,001) as compared with the levels observed in women without IH (30 +/- 20 ng/ml and 23 +/- 13 micrograms/24 h for the monosulphate, 78 +/- 40 ng/ml and 48,0 +/- 23,7 micrograms/24 h for the disulphate). There is a correlation between the urinary levels of the monosulphate of delta 5, 3 beta-diol (y) and of the dehydroepiandrosterone sulphate (S-DHA) (x) : y (microgram/24 h) = 0,124x + 16,8; r = 0,93 (p less than 0,01) and n = 32. The plasma levels of S-DHA ranged form 1,250 ng/ml to 7,000 ng/ml in the abnormal population and from 1,000 ng/ml to 2,400 ng/ml in the normal population. A concomitant determination of plasma free androgens showed that 60 % of the dehydroepiandrosterone values were above the upper limits of normal values. Conversely, the testosterone and androstenedione levels were only higher in 20 % and 27 % of cases respectively. These results suggest that the possible importance of the sulphoconjugates of delta 5-C19O2 steroids in the IH pathogeny should not be overlooked.

Interaction between rat alpha 1 fetoprotein and fluorescent derivatives of estrone in relation to the position and type of the fluorescent label.

A series of derivatives of estrone with fluorescent dyes (dansyl or coumarin) coupled at different positions on the steroid molecule, have been synthesized. These derivatives were tested for their quantum yields and their binding properties were determined with respect to rat alpha 1 fetoprotein. Derivatives at C-3 (estrone-3-hemisuccinate-dansyl-cadaverine and estrone-3-dansyl) compete with estrone for binding to the fetal protein; however derivates of estrone at C-6 (estrone-6-carboxymethyloxime-dansyl-cadaverine) and at C-17 (estrone-17-carboxymethyloxime-coumarine, estrone-17-carboxymethyloxime-dansyl-cadaverine and estrone-17-dansyl-hydrazine) compete poorly or not at all. The association constant of the radioactive derivative estrone-3-dansyl [3H] with rat alpha 1 fetoprotein was measured directly: the same number of high affinity binding sites (0.6) as that for estrone was found with an apparent association constant of 3.7 X 10(6) M-1. In addition to the high affinity binding sites, a low affinity class of binding sites was found which corresponds to the binding of the dansyl fraction of the fluorescent steroid derivative.

[3 alpha-hydroxy-5 alpha-androstane-17-one-3-sulphate in hirsute women's plasma (author's transl)]. / Sulfate de 3 alpha-hydroxy-5 alpha-androstane-17-one plasmatique chez la femme hirsute.

The determination of serum androsterone-3-sulphate was carried out by radioimmunoassay with an antiserum specific against 15 alpha-carboxymethyl-androsterone-BSA. The values obtained (mean +/- SD) in ng/ml were 739 +/- 190 (n = 9) for Men; 285 +/- 157 (n = 13) for Women; 677 +/- 464 (n = 131) for hirsute women respectively and were compared to the values of urinary sulphate of dehydroepiandrosterone in the same subjects.

Preparation of dehydroepiandrosterone, testosterone and progesterone antigens through 7-carboxymethyl derivatives: characteristics of the antisera to testosterone and progesterone.

Dehydroepiandrosterone, testosterone and progesterone 7-carboxymethyl derivatives were prepared: 7 alpha and 7 beta epimers were separated and coupled to bovine serum albumin. Preliminary studies of the antisera induced by these antigens showed that they have high affinity and good specificity.