Production and characterization of antibodies against microcystins.

Antibodies against a microcystin (MCYST) leucine-arginine variant (MCYST-LR) were demonstrated 4 weeks after immunization of rabbits with either MCYST-LR-polylysine- or MCYST-LR-ethylenediamine-modified bovine serum albumin. A radioimmunoassay (RIA), a direct competitive enzyme-linked immunosorbent assay (ELISA), and an indirect competitive ELISA were developed for characterization of the antibodies. Indirect ELISA and RIA revealed that MCYST-LR-ethylenediamine-bovine serum albumin was a better immunogen. Competitive RIA and direct ELISA revealed that the antibodies had good cross-reactivities with an MCYST-arginine-arginine variant (MCYST-RR), MCYST-LR, an MCYST-tyrosine-arginine variant (MCYST-YR), and nodularin (NODLN); but they had lower reactivities with variants MCYST-leucine-tyrosine (MCYST-LY) and MCYST-leucine-alanine (MCYST-LA). The antibodies did not cross-react with ozonolyzed MCYST-LR. The concentrations causing 50% inhibition of binding of reduced MCYST-LR to the antibodies by MCYST-RR, MCYST-LR, MCYST-YR, NODLN, MCYST-LA, and MCYST-LY in the RIA were 43, 105, 112, 503, 671, and 1,920 ng/ml, respectively. The concentrations causing 50% inhibition of binding of MCYST-LR-horseradish peroxidase to the antibodies by MCYST-RR, MCYST-LR, MCYST-YR, NODLN, MCYST-LY, and MCYST-LA in the ELISA were 1.75, 2.2, 3.4, 4.6, 50, and 114 ng/ml, respectively.     

Cross-Reactivity and Neutralizing Ability of Monoclonal Antibodies against Microcystins

Monoclonal antibodies (MAbs) against the microcystin-leucine-arginine variant (MCYST-LR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. The specificity of the MAbs and their ability to neutralize the toxin were investigated by an indirect enzyme-linked immunosorbent assay (ELISA) and by a neutralizing test in mice, respectively. All MAbs reacted with MCYST-LR and also with the microcystin-arginine-arginine variant (MCYST-RR), 3, 7-didesmethylmicrocystin (MCYST-3, 7-dDMLR) and 7-desmethylmicrocystin (MCYST-7-DMLR). Furthermore, the antibodies reacted with cell-extracts of toxic and non-toxic M. aeruginosa strains. The MAbs can apparently recognize the common configuration, but not the variant-specific structure, in the microcystin molecules. The non-toxic strains apparently contain some substance(s) related antigenically to microcystin. The in vivo toxin-neutralizing ability of MAbs was minimal.     

Generation of antibodies reactive with fumonisins B1, B2, and B3 by using cholera toxin as the carrier-adjuvant.

Murine polyclonal antibodies reactive with fumonisins B1, B2, and B3 were produced after a novel immunization procedure with cholera toxin as both a hapten carrier protein and adjuvant. Immunization of mice with two 7.5-micrograms doses of fumonisin B1-cholera toxin conjugate without adjuvant resulted in the production of fumonisin B1-specific antibodies in all mice within 15 days when intraperitoneal, subcutaneous, and intravenous routes were used. In contrast, conventional immunization procedures with fumonisin B1-bovine serum albumin conjugates with and without Freund's adjuvant were largely ineffective. Fumonisin antibodies could be readily mass-produced in ascites fluid by using cholera toxin as a carrier-adjuvant. A competitive indirect enzyme-linked immunosorbent assay (ELISA) was devised whereby immobilized fumonisin B1-ovalbumin and free fumonisin B1 competed for antibody binding. The detection limit for fumonisin B1 in the ELISA was 100 ng/ml. The antiserum cross-reacted with fumonisins B2 and B3 but not with the hydrolyzed backbone of fumonisin B1 and tricarballylic acid. Concentrations of fumonisins B1, B2, and B3 required for 50% binding inhibition were 260, 300, and 650 ng/ml, respectively. These polyclonal antibodies should find wide usage in the ELISA for fumonisins in foods, feeds, and tissues.     

Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development.

Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices.     

Comparison of Solid-Phase Radioimmunoassays for Baculoviruses

The sensitivity and cross-reaction of four solid-phase radioimmunoassays (RIA) for Trichoplusia ni nuclear polyhedrosis virus containing singly enveloped virions were investigated. The detection limits of each assay were as follows: Indirect RIA, 5 ng of dissolved polyhedron antigen; direct RIA, 50 ng; indirect sandwich RIA, 200 ng; and direct sandwich RIA, 300 ng. The indirect and indirect sandwich RIAs showed considerable cross-reaction with other baculovirus antigens, but the direct and direct sandwich RIAs showed cross-reaction with only one closely related baculovirus. When microtiter plates used for the solid phase were pretreated with bovine serum albumin, nonspecific binding of labeled antibodies was reduced to a minimum. Antibodies prepared by an immunoadsorption procedure showed greater specific binding than antibodies prepared by ammonium sulfate precipitation of the immunoglobulin fraction. Highly contaminated antigen could not be detected by the indirect RIA, but the direct sandwich RIA was unaffected by antigen contamination. Antigen making up 0.0025% (wt/wt) of a sample of bird droppings could be detected by the direct sandwich RIA.     

Production and characterization of antibody against deoxyverrucarol.

Immunization of rabbits with deoxyverrucarol (DOVE) conjugated to bovine serum albumin resulted in antibodies bound with either tritiated DOVE or diacetoxyscirpenol (DAS), but not with T-2 toxin. The affinity of antibodies with DOVE was found to be much higher than with DAS. When [3H] DOVE was used as a marking ligand in the competitive radioimmunoassay, the concentrations causing 50% inhibition of binding radioactivities by unlabeled DOVE, verrucarol, verrucarin A, and 4-monoacetoxyscirpenol were found to be 0.32, 1,070, 9,500, and 10,000 ng per assay, respectively. T-2 toxin, 15-monoacetoxyscirpenol, and deoxynivalenol gave less than 20% inhibition at 10 micrograms per assay. However, when [3H] DAS was used as the marking ligand, the concentrations causing 50% inhibition by DOVE, DAS, and verrucarol were found to be in the 50 to 60 ng per assay range. The antibodies are thus highly specific to DOVE rather than a common trichothecene backbone. The possible use of this antiserum for assay of macrocyclic trichothecenes is discussed.     

Generation of antibodies reactive with fumonisins B1, B2, and B3 by using cholera toxin as the carrier-adjuvant.

Murine polyclonal antibodies reactive with fumonisins B1, B2, and B3 were produced after a novel immunization procedure with cholera toxin as both a hapten carrier protein and adjuvant. Immunization of mice with two 7.5-micrograms doses of fumonisin B1-cholera toxin conjugate without adjuvant resulted in the production of fumonisin B1-specific antibodies in all mice within 15 days when intraperitoneal, subcutaneous, and intravenous routes were used. In contrast, conventional immunization procedures with fumonisin B1-bovine serum albumin conjugates with and without Freund's adjuvant were largely ineffective. Fumonisin antibodies could be readily mass-produced in ascites fluid by using cholera toxin as a carrier-adjuvant. A competitive indirect enzyme-linked immunosorbent assay (ELISA) was devised whereby immobilized fumonisin B1-ovalbumin and free fumonisin B1 competed for antibody binding. The detection limit for fumonisin B1 in the ELISA was 100 ng/ml. The antiserum cross-reacted with fumonisins B2 and B3 but not with the hydrolyzed backbone of fumonisin B1 and tricarballylic acid. Concentrations of fumonisins B1, B2, and B3 required for 50% binding inhibition were 260, 300, and 650 ng/ml, respectively. These polyclonal antibodies should find wide usage in the ELISA for fumonisins in foods, feeds, and tissues.     

Isolation and characterization of a variety of microcystins from seven strains of the cyanobacterial genus Anabaena.

Hepatotoxins (microcystins) from seven freshwater Anabaena strains originating from three different Finnish lakes and one lake in Norway were isolated by high-performance liquid chromatography and characterized by amino acid analysis and fast atom bombardment mass spectrometry. All strains produced three to seven different microcystins. A total of 17 different compounds were isolated, of which 8 were known microcystins. The known compounds identified from six strains were MCYST (microcystin)-LR, [D-Asp3]MCYST-LR, [Dha7]MCYST-LR, [D-Asp3,Dha7]MCYST-LR, MCYST-RR, [D-Asp3]MCYST-RR, [Dha7]MCYST-RR, and [D-Asp3,Dha7]MCYST-RR. With the exception of MCYST-LR and [D-Asp3]MCYST-LR, this is the first time that isolation of these toxins from Anabaena strains has been reported. Three of the strains produced one to three toxins as minor components which could not be identified. Anabaena sp. strain 66 produced four unidentified toxins. The other Anabaena strains always contained both MCYST-LR and MCYST-RR and/or their demethyl variants. Quantitative differences between toxins within and between strains were detected; at times MCYST-LR and at other times MCYST-RR or demethyl derivatives thereof were the most abundant toxins found in a strain.     

Immunoenzyme method for detecting microbial producers of palytoxin

A competitive ELISA using the intact toxin as a coating antigen for detecting palytoxin was developed. This immunoassay allows palytoxin (PTX) to be determined in the range of 6-250 ng/ml. In sensitivity, this determination is comparable with RIA but is three times inferior to ELISA using monoclonal antibodies. Inhibition experiments using some toxins of marine invertebrates proved the serological specificity of the palytoxin binding to antibodies. Both the indirect and competitive ELISA were used to find PTX-producing bacteria among 420 isolates of sea bacteria. It was found that gram-negative bacteria Aeromonas sp. and Vibrio sp. associated with toxic samples of the soft coral Palythoa sp. produced compounds antigenically related to PTX.     

Isolation and characterization of a variety of microcystins from seven strains of the cyanobacterial genus Anabaena.

Hepatotoxins (microcystins) from seven freshwater Anabaena strains originating from three different Finnish lakes and one lake in Norway were isolated by high-performance liquid chromatography and characterized by amino acid analysis and fast atom bombardment mass spectrometry. All strains produced three to seven different microcystins. A total of 17 different compounds were isolated, of which 8 were known microcystins. The known compounds identified from six strains were MCYST (microcystin)-LR, [D-Asp3]MCYST-LR, [Dha7]MCYST-LR, [D-Asp3,Dha7]MCYST-LR, MCYST-RR, [D-Asp3]MCYST-RR, [Dha7]MCYST-RR, and [D-Asp3,Dha7]MCYST-RR. With the exception of MCYST-LR and [D-Asp3]MCYST-LR, this is the first time that isolation of these toxins from Anabaena strains has been reported. Three of the strains produced one to three toxins as minor components which could not be identified. Anabaena sp. strain 66 produced four unidentified toxins. The other Anabaena strains always contained both MCYST-LR and MCYST-RR and/or their demethyl variants. Quantitative differences between toxins within and between strains were detected; at times MCYST-LR and at other times MCYST-RR or demethyl derivatives thereof were the most abundant toxins found in a strain.     

Production and characterization of antibody against aflatoxin Q1.

Antibodies against aflatoxin Q1 (AFQ1) were obtained from rabbits after immunization of either AFQ1-hemisuccinate or AFQ2a conjugated to bovine serum albumin. Both radioimmunoassay and enzyme-linked immunosorbent assaY (ELISA) were used for the determination of antibody titers and specificities. Antibodies obtained from rabbits after immunization with AFQ1-hemisuccinate-bovine serum albumin had the highest affinity to aflatoxin B1, whereas antibodies obtained from rabbits after immunization with AFQ2a-bovine serum albumin bound most effectively with AFQ2a. AFQ2a antibody was selected for the subsequent direct and indirect ELISA for the detection of AFQ1 in biological fluids. When AFQ2a-peroxidase and AFQ2a antibody were used, direct ELISA was able to detect as low as 2 ppb (ng/ml) of AFQ1 spiked in the urine samples that had been subjected to a Sep-Pak cleanup treatment. In indirect ELISA in which the antigen (AFQ2a-bovine serum albumin) was coated to the solid phase followed by reaction with rabbit antibody and goat anti-rabbit immunoglobulin G-peroxidase conjugate, 50-fold less antibody was used without sacrificing sensitivity. Recoveries of AFQ1 added to urine samples (2 to 40 ppb) were 46.3 to 73% and 65.8 to 85.8% for direct and indirect ELISA, respectively.     

Production and characterization of antibody against aflatoxin Q1

Antibodies against aflatoxin Q1 (AFQ1) were obtained from rabbits after immunization of either AFQ1-hemisuccinate or AFQ2a conjugated to bovine serum albumin. Both radioimmunoassay and enzyme-linked immunosorbent assaY (ELISA) were used for the determination of antibody titers and specificities. Antibodies obtained from rabbits after immunization with AFQ1-hemisuccinate-bovine serum albumin had the highest affinity to aflatoxin B1, whereas antibodies obtained from rabbits after immunization with AFQ2a-bovine serum albumin bound most effectively with AFQ2a. AFQ2a antibody was selected for the subsequent direct and indirect ELISA for the detection of AFQ1 in biological fluids. When AFQ2a-peroxidase and AFQ2a antibody were used, direct ELISA was able to detect as low as 2 ppb (ng/ml) of AFQ1 spiked in the urine samples that had been subjected to a Sep-Pak cleanup treatment. In indirect ELISA in which the antigen (AFQ2a-bovine serum albumin) was coated to the solid phase followed by reaction with rabbit antibody and goat anti-rabbit immunoglobulin G-peroxidase conjugate, 50-fold less antibody was used without sacrificing sensitivity. Recoveries of AFQ1 added to urine samples (2 to 40 ppb) were 46.3 to 73% and 65.8 to 85.8% for direct and indirect ELISA, respectively.     

An enzyme linked immunosorbent assay for detecting palytoxin-producing bacteria

A competitive ELISA using the intact toxin as a coating antigen for detecting palytoxin was developed. This immunoassay allows palytoxin (PTX) to be determined in the range of 6–250 ng/ml. In sensitivity, this determination is comparable with RIA but is three times inferior to ELISA using monoclonal antibodies. Inhibition experiments using some toxins of marine invertebrates proved the serological specificity of the palytoxin binding to antibodies. Both the indirect and competitive ELISA were used to find PTX-producing bacteria among 420 isolates of sea bacteria. It was found that gram-negative bacteriaAeromonas sp. andVibrio sp. associated with toxic samples of the soft coralPalythoa sp. produced compounds antigenically related to PTX.     

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of praziquantel concentration in serum

A simple and reproducible enzyme-linked immunosorbent assay (ELISA) for the determination of the concentration of praziquantel in the serum was developed. Since praziquantel has no functional group to conjugate with carrier protein, praziquantel was first converted to a compound with an amino group similar to praziquantel. This compound was then conjugated to bovine serum albumin for use as an immunogen, and to horseradish peroxidase, as enzyme-labeled praziquantel, respectively. The conjugate of praziquantel-bovine serum albumin conjugate was used to raise anti-praziquantel antiserum in mice. The direct competitive ELISA was conducted by simultaneously incubating praziquantel and horseradish peroxidase-labeled praziquantel conjugate with anti-praziquantel antiserum over a second antibody and the enzyme activity of the remaining horseradish peroxidase-labeled praziquantel conjugate was measured. The intra- and inter-assay coefficient of variation was < 10% in the range of 1.0 to 30 ng ml(-1), and the limit of the detection was 0.3 ng ml(-1). The cross reactivities of anti-praziquantel antibody with compounds related to praziquantel were negligible. Using this ELISA, serum levels of praziquantel were easily determined in male Wistar rats up to 8 h following a single intraperitoneal injection at 2 mg kg(-1) of body weight.     

Generation of Polyclonal Antibodies of Predetermined Specificity against Pediocin PA-1

Polyclonal antibodies of predetermined specificity for pediocin PA-1 (pedA1) have been generated by immunization of rabbits with a chemically synthesized C-terminal fragment of this bacteriocin (PH2) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of the PH2-KLH-generated antibodies were evaluated by the development of various enzyme-linked immunosorbent assays (ELISAs)—a noncompetitive indirect ELISA (NCI-ELISA), a competitive indirect ELISA (CI-ELISA), and a competitive direct ELISA (CD-ELISA)—and by immunodotting. All immunoassays indicated the existence of pedA1-specific antibodies with high relative affinities and adequate sensitivities in the sera of immunized animals. The limits of detection of pedA1 in MRS medium (Oxoid Ltd., Basingstoke, United Kingdom) were found to be 2.5 μg/ml by immunodotting and 1 μg/ml in the NCI-ELISA. However, the CI-ELISA enhanced the limit of detection of pedA1 to 0.025 μg/ml, while the amount of free pedA1 required for 50% binding inhibition was 10 μg/ml. Moreover, the CD-ELISA increased the affinity of the PH2-KLH-generated antibodies for pedA1; the limit of detection of pedA1 was less than 0.025 μg/ml, and the 50% binding inhibition value was reduced to 0.5 μg of pedA1/ml. All immunoassays and the slot dot assay detected the presence of pedA1 in the supernatant of the producing strain Pediococcus acidilactici 347, with no reactivity or negligible immunoreactivity with the supernatants of other lactic acid bacteria producing or not producing different bacteriocins. The approaches taken for the generation of antibodies and the development of immunoassays could prove useful for the generation and evaluation of antibodies of predetermined specificity for other bacteriocins of interest in the food industry.     

Seasonal changes in serum leptin of the feral raccoon (Procyon lotor) determined by canine-leptin-specific ELISA

Several reports have been published on blood leptin concentrations in feral animals, including members of the Carnivora, using a commercially available multi-species radioimmunoassay (RIA) kit with anti-human leptin antibody. However, we observed weak immunoreactivity between recombinant canine leptin and anti-human leptin antibody, suggesting a limitation in the applicability of the RIA kit for leptin assays in Carnivora species. We tested the applicability of RIA and sandwich enzyme-linked immunosorbent assay (ELISA) with anti-canine leptin antibody to assay blood leptin in the dog (Canis familiaris) and the raccoon (Procyon lotor). When RIA was used for recombinant canine leptin and dog sera, values were much lower than those determined by ELISA at higher concentrations (>10 ng/ml), while rather higher at lower concentrations (<2 ng/ml). A similar discrepancy between the two methods was found for serum leptin concentrations in raccoons. Clear seasonal variations were observed by ELISA, but not by RIA, with high values in autumn (3.46+/-0.45 ng/ml) and low values in spring and summer (0.71+/-0.07 ng/ml). Serum leptin concentrations in raccoons correlated positively with their body weight (r=0.753) and body mass index (r=0.755), corroborating our previous findings of a strong positive correlation between serum leptin concentrations and body fat content in dogs. Thus, the canine leptin ELISA is useful for assays of dog and raccoon leptin, and blood leptin is a good marker of nutritional condition in the species of Carnivora assayed in this study.     

Biosynthesis, characterization and inhibition of leukotriene B4 in human whole blood

We have evaluated the biosynthesis, characterization and inhibition of Leukotriene (LT) B4 in unstimulated and in A23187-stimulated human whole blood. LTB4 was assayed by radioimmunoassay (RIA) both in unextracted serum and after extraction and thin-layer chromatography (TLC). Unstimulated human whole blood allowed to clot at 37 degrees C for 60 min produced only trace amounts of LTB4 (0.16 +/- 0.05 ng/ml, mean +/- SD, n = 3). LTB4-like immunoreactivity (ir-LTB4) detectable in unstimulated serum samples was largely overestimated by direct RIA, most likely because of interfering substance(s) unrelated to cyclooxygenase or lipoxygenase activity. Incubation of human whole blood with A23187 (2-10 microM) resulted in a concentration-dependent stimulation of LTB4 production. At 10 microM A23187, ir-LTB4 was 18 +/- 2.4 ng/ml (mean +/- SEM, n = 28). In A23187-stimulated serum samples, LTB4 concentrations measured by direct RIA correlated in a statistically significant fashion with those measured after extraction and TLC. Nafazatrom added in vitro caused a dose-dependent inhibition of A23187-stimulated ir-LTB4 production with an IC50 of 17 microM.     

Radioimmunoassay of nafarelin ([ 6-(3-(2-naphthyl)-D-alanine)]-luteinizing hormone-releasing hormone) in plasma or serum

A procedure which is suitable for the radioimmunoassay (RIA) of nafarelin [( 6-(3-(2-naphthyl)-D-alanine)]-luteinizing hormone-releasing hormone) in plasma or serum at concentrations as low as 50 pg/ml is described. Antiserum was prepared by replacing the pyroglutamyl portion of nafarelin with glutaric acid, coupling the product to keyhole limpet hemocyanin, and immunizing rabbits with the resulting conjugate. At a dilution of 1:30,000 the binding was approximately 50%. The antibodies did not cross react with luteinizing hormone-releasing hormone. For RIA, 125I-labeled analyte was used as the tracer and charcoal was used to separate the free and the bound fractions. No purification of samples was required prior to RIA. Accuracy of the method was assessed by adding known quantities of nafarelin to nafarelin-free plasma and determining the ratio of measured to added analyte. Linear regression analysis for the concentration range 0.050-5.00 ng/ml yielded a regression equation of y = 1.01x - 0.066 and a correlation coefficient of 0.997. At 0.050 ng/ml the CV was 11.3% (interassay). Additional validation was obtained from an in vivo study in which [3H]nafarelin was administered to monkeys and plasma profiles were determined by RIA, by high-performance liquid chromatography (HPLC), and by an HPLC-radiochemical method. The results obtained by RIA agreed well with those obtained by the HPLC methods.     

A case of hypothyroidism with simultaneous presence of stimulating type anti-thyrotropin (TSH) receptor antibodies and anti-thyroxine (T4) autoantibodies.

We have examined a hypothyroid patient with stimulating type anti-thyrotropin (TSH) receptor antibodies and without blocking type anti-TSH receptor antibodies. Although she had high serum TSH (240 microU/ml) and low free triiodothyronine (FT3, 0.49 pg/ml) concentrations, which agree with physical findings of hypothyroidism, she had an unusually high free thyroxine (FT4) concentration (3.56 ng/dl). Incubation of her serum with 125I-T4, followed by precipitation with 12.5% polyethylene glycol (PEG) disclosed a higher binding of 125I-T4 (34.4%) than in normal controls, being 5-7%. In addition, binding of 125I-T4 to her serum gamma-globulin was completely displaced by the addition of unlabelled T4. From these results it was concluded that her serum contained anti-T4 autoantibodies. Treatment with synthetic T4 was begun and her thyroid function was monitored by sensitive TSH radioimmunoassay (RIA) and RIA of FT4 after PEG treatment. Since both sensitive TSH RIA and FT4 RIA results after PEG treatment give results concordant with the physical findings, it was concluded that both of the RIA results are useful for the evaluation of thyroid function in patients with thyroid hormone autoantibodies.     

Development and validation of competitive ELISA to determine follicular fluid progesterone concentrations

A simple, direct and reliable heterologous ELISA was developed and validated to determine follicular fluid progesterone concentrations in 10- to 20-mm antral follicles in heifers. A competitive ELISA was developed, using a policlonal antisera raised in New Zealand white rabbits and horseradish peroxidase as label. Standard curve covered a range between 0 and 1 ng per well. The intra- and inter-assay coefficients of variation (CV) were 6.54 and 8.27%, respectively (n = 10). The low detection limit was 1 pg per well with a sensitivity of 50% binding 83.17 pg per well. Comparison of ELISA with RIA showed a correlation coefficient of 0.98. A total of 34 follicles obtained from heifers in the follicular phase of the estrous cycle were tested, and the mean progesterone concentration was 86.72 +/- 20.39 ng/ml. These results were similar to those previously reported for the same species, age, follicular size and stage of cycle.