A Direct (Non-Competitive) Immunoassay for Gentamicin Residues with an Optical Biosensor

Using sensor chip immobilized anti-gentamicin monoclonal antibodies in an optical biosensor (BIACORE 3000) resulted in the first single antibody based and label free noncompetitive immunoassay for the direct detection of residues of a low molecular weight compound in a food product. Calibration curves for gentamicin (Mw 466 Da) in buffer and milk showed 50% binding at 20 and 35 ng ml^-1, respectively, which is well below the maximum residue limit of 100 ng ml^-1.     

Development and validation of a biosensor-based immunoassay for progesterone in bovine milk

We have developed a rapid automated immunoassay, using the BIACORE surface plasmon resonance (SPR) biosensor, to measure progesterone in bovine milk. The assay was designed as an inhibition assay with progesterone covalently immobilised to the carboxymethyl dextran matrix of a CM5 sensor chip. A fixed amount of monoclonal anti-progesterone antibody 39C5H7 was mixed 9:1 with the sample and the amount of free antibody was then determined using biomolecular interaction analysis (BIA) by injection of the mixture over the immobilised progesterone sensor surface. The assay was designed to cover the concentration range 0.5 to 50 ng/ml. The limit of detection (LOD) was 3.56 ng/ml. Reproducibility of the assay was very good with both intra-assay and inter-assay coefficients of variation <5%. As results become available within minutes of injection and the procedure involves fully automated instrumentation, we believe that this BIA assay for progesterone in milk could be used in-line in the milking parlour and, thus, provide an important tool for reproductive management of dairy cattle to detect heat and predict pregnancy.     

Enzyme immunoassay for the detection of streptomycin and dihydrostreptomycin in milk

Polyclonal antisera against streptomycin were prepared by using a streptomycin‐oxime derivative coupled to bovine serum albumin for the immunization of rabbits. The specificity and sensitivity of these antibodies were tested in a competitive assay using a streptomycinenzyme conjugate (prepared by coupling a streptomycin‐hydrazone derivative to horseradish peroxidase) in a double antibody solid phase technique. The only detectable cross‐reactivity of the assay system with other aminoglycoside antibiotics and other substances similar in structure was shown to be with dihydrostreptomycin of about 148.7%. The detection limit in buffer solution was 0.6 ng ml^‐1 for streptomycin and 0–4 ng ml^‐1 for dihydrostreptomycin. Employing rapid sample preparation procedures, streptomycin and dihydrostreptomycin were detected in milk at levels as low as 6 and 0.8 ng ml^‐1respectively.     

Rapid detection of streptomycin and Dihydrostreptomycin in milk by enzyme‐linked immunofiltration assay

Using a membrane‐based immunochemical technique, a simple and rapid method for the detection of streptomycin and dihydrostreptomycin in milk has been developed. This enzyme‐linked immunofiltration assay has a detection limit in milk of 2 ng ml^‐1 for dihydrostreptomycin and 5 ng ml^‐1 for streptomycin. No sample preparation was needed, and the total assay procedure was completed within 10 min.     

Development of a One Step Strip Test for the Detection of (Dihydro)streptomycin Residues in Raw Milk

The one step strip test described is a competitive immunoassay in which the detector reagent consists of colloidal gold particles coated with affinity purified monoclonal anti(dihydro)streptomycin antibodies. The capture reagent in the assay is a streptomycin- bovine serum albumin conjugate which is immobilised on the lateral flow membrane of the test device. In the test procedure, three drops of raw milk are brought into the sample well of the test device and allowed to migrate over the membrane. The more analyte present in the sample, the more effectively it will compete with the streptomycin immobilised on the membrane for binding to the limited amount of antibodies of the detector reagent. A sufficient amount of (dihydro)streptomycin in the sample will thus prevent the binding of the detector reagent to the streptomycin immobilised on the membrane, resulting in disappearance of the test line in the read out zone. Using raw milk samples, spiked with either streptomycin or dihydrostreptomycin, complete disappearance of the test line was obtained with 160 ng ml^-1 and 190 ng ml^-1, respectively. This demonstrates that the test is applicable for screening raw milk samples for the presence of (dihydro)streptomycin residues at MRL level (EU: 0.2 mg (dihydro)streptomycin kg^-1 milk). The major advantages of the one step strip test are that results can be obtained within 10 min and that all reagents are included in the test device.     

Analysis of the binding of monoclonal and polyclonal antibodies to the glycoproteins of antigenic variants of human respiratory syncytial virus by surface plasmon resonance

The surface glycoproteins of human respiratory syncytial virus (hRSV), F and G, are the major protective antigens of the virus. Both are antigenically variable, although to different degrees, but the role of antigenic variation in the pathogenesis of hRSV disease has not been fully evaluated. Assessment of immunity to different virus strains is difficult with conventional antibody assays where differing properties of the virus antigens, other than antigenicity, may influence the outcome of the assay. Here, we have developed BIAcore surface plasmon resonance based assays for antibodies to the glycoproteins of hRSV which allow valid comparison of antibody titres against multiple hRSV strains. Glycoproteins from a number of lineages of hRSV sub-group A were captured from lysates of infected cells onto the dextran coated surface of a BIAcore sensor chip via primary monoclonal antibodies (MAbs) to conserved epitopes. For the G glycoprotein, primary MAbs were conjugated directly to the dextran of the sensor chip via free amide groups. For the F glycoprotein, direct conjugation was found to inactivate the MAb and primary MAb was immobilised on the chip via rabbit anti-mouse Fc antibody fragments in an indirect system. Using monoclonal antibodies as secondary MAbs, the glycoproteins in both systems were shown to exhibit a sub-set of conserved and variable epitopes, with some epitopes of both sorts being unavailable, presumably blocked by the primary antibody. Polyclonal anti-hRSV sera raised against viruses of different genotype bound equally to both F and G glycoproteins from homologous and heterologous viruses suggesting that mice immunised systemically with lysates of cells infected with recent isolates of virus do not respond well to genotype specific epitopes.     

Competitive ELISA employing monoclonal antibodies specific for dothistromin

Dothistromin (DOTH) is a fungal toxin occurring in Pinus radiata needles contaminated with Dothistroma pini. Monoclonal antibodies (MAbs) of high affinity were prepared against DOTH and incorporated into competitive ELISAs. MAbs were secreted by hybri‐doma prepared from mice immunized with DOTH conjugated, through the aromatic ring of the anthraquinone, to bovine serum albumin. DOTH could be quantitated at 2–60 ng ml^‐1 (0.1–2 ng/assay) and at 8–300 ng ml^‐1 (0.4–15 ng/assay) using peroxidase‐labelled MAb 10C12A5 and MAb 6A2D4 respectively in indirect competitive ELISA. The corresponding limits of detection of DOTH were < 300 pg and 1 ng ml^‐1 respectively. The furan ring was an important structure in the epitope recognized by both MAbs.     

Characterization of ultra-high affinity monoclonal antibodies with a dimeric, symmetrical antigen: inhibition of the receptor recognition site of nerve growth factor.

We generated a family of ultra-high affinity monoclonal antibodies (MAb) which inhibit competitively the binding of nerve growth factor (NGF) to its receptor. Preliminary experiments indicated that the dissociation constants (Kd) of some of the MAb:NGF complexes were substantially less than 0.1 nM. Conventional methods, such as ELISA and radioimmunoassays (RIA), were not sufficiently sensitive to measure the Kds of these MAb. Therefore, experimental conditions were developed to determine binding constants for these very high affinity MAb. The experiments establish that the Kds for our anti-NGF MAb range from 2.6 nM to 39 fM. Additionally, the inhibition of NGF binding to NGF-receptor by MAb is fully consistent with a purely competitive model but is not consistent with a model allowing the formation of a ternary complex of NGF, MAb, and NGF-receptor. One MAb, M4, immunoprecipitates NGF indicating interaction between each protomer of the NGF dimer and individual MAb molecules. We also evaluated the effects of mild denaturing conditions on the binding and biological activity of NGF and on recognition by the MAbs. Guanidine HCl or heat treatment of NGF resulted in only small, but significant, changes in binding or biological activity, in parallel with changes in recognition by the MAbs. However, binding, biological activity, and recognition by six of seven MAbs were completely eliminated by beta-mercaptoethanol reduction. Thus, our results are consistent with the MAbs interacting with the receptor recognition site on the surface of the NGF molecule. The high affinity MAbs will serve as sensitive probes of structural elements of NGF responsible for binding and biological activity.     

Production of monoclonal antibodies against recombinant human interferon-gamma: screening of hybridomas without purified antigen

A panel of mouse monoclonal antibodies (MAbs) against the recombinant human gamma interferon (HuIFN-gamma) has been produced for the study of the structure-function relationships of this important lymphokine. Enzyme linked immunosorbent assay (ELISA) is the current method of choice to screen hybridomas for specific MAb production. The purity of the antigen used for screening dictates the specificity of the ELISA. As often is the case in many systems, adequately purified biologically active HuIFN-gamma was not readily available for this purpose. A sandwich ELISA which allowed the use of unpurified HuIFN-gamma for hybridoma screening was developed. A rabbit antiserum against the denatured HuIFN-gamma purified by SDS-PAGE was prepared and the nonspecific binding activity was removed by adsorption to control cell proteins immobilized on Sepharose. The adsorbed immunoglobulin fraction was bound to the ELISA plate: (i) to trap HuIFN-gamma specifically from the whole cell lysate, thus providing specificity for MAb detection, and (ii) to avoid direct adsorption of the HuIFN-gamma to the ELISA plate because others have found that this prevented detection of neutralizing MAb. The sandwich ELISA detected both neutralizing and non-neutralizing MAbs with relatively low false positive reactions. This approach to the development of an ELISA method to screen hybridomas without purified antigen should be applicable to the production of MAbs to other proteins.     

Anti-idiotypic antibodies to bovine herpesvirus-1 inhibit virus infection in cell cultures

Summary A panel of murine monoclonal antibodies (MAbs) to bovine herpesvirus-1 (BHV-1) was prepared. Three of them were neutralizing MAbs and reacted against 130/75/50 kDa, 77 kDa, or 97 kDa glycoproteins (gp). A fourth non-neutralizing MAb recognized the 97 kDa gp. Competition radioimmunoassay demonstrated that each of the four MAbs reacted against a different virus epitope. Anti-idiotypic antibodies (anti-id) to the four MAbs were produced in rabbits and purified by sequential immunoaffinity chromatography. Each anti-id inhibited the binding of its respective MAb to BHV-1 in competitive ELISA and blocked BHV-1 neutralizing activity of the MAb. This inhibition suggested that the anti-ids were specific for the antigen binding site of the MAbs. Treatment of MDBK cells with anti-ids inhibited BHV-1 infection, which suggested that the anti-ids block a cellular component essential for virus infection. Absence of significant cross-reactivity among the anti-ids for heterologous MAbs indicated that they recognized unique determinants on the antigen binding site of the homologous MAb.     

Penicillin-specific antibodies: Monoclonals versus polyclonals in ELISA and in an optical biosensor

Two penicillin-specific monoclonal antibodies mAb 19C9 and mAb 9H3 and the penicillin-specific polyclonal antibodies pAb K2 were evaluated for their use in a competitive ELISA and in the BIAcore™ optical biosensor. In the ELISA, an ampicillin-protein conjugate was used as a coating molecule. For the biosensor assay, ampicillin was immobilized on a CM5 chip. With both monoclonal antibodies and in both test systems, ampicillin, amoxicillin and benzylpenicillin were better recognized than oxacillin, cloxacillin and dicloxacillin. Because the reproducibility was better in the biosensor (CV = 1.6%) than in the ELISA (CV = 8.9%), the limit of detection for ampicillin in buffer solution using mAb 19C9 was lower in the biosensor (46 ng ml^-1) as compared to the ELISA (356 ng ml^-1). Ampicillin can thus be detected below the MRL (50 ng ml^-1) in the biosensor assay but not in the ELISA. Both the ELISA and biosensor assay using the polyclonal antibodies pAb K2 were more sensitive as compared to the assays with the monoclonals. The ELISA using pAb K2 allowed the detection of all tested penicillins below the MRL. In the biosensor assay, ampicillin was also detected below the MRL (IC50 = 10 ng ml^-1). In contrast to the binding of the monoclonals, no spontaneous dissociation was observed after injection of the polyclonal antibodies in the biosensor. Whereas the monoclonals were completely removed from the sensor surface using ampicillin in the buffer solution as regeneration solution, stronger conditions were necessary for the pAb binding.     

Isolation and characteristics of anti-adenovirus monoclonal antibodies in immunoenzyme and immunofluorescent reactions

New monoclonal antibodies (MAbs) to adenovirus hexon, highly active in ELISA and immunofluorescent analysis, were prepared. According to competitive ELISA, new MAbs differed in their blocking activity and were directed to 2 different hexon epitopes. MAb 3H8 did not modify antigen binding of the rest MAbs labeled with peroxidase (PAb x Pox), and none of unlabeled MAbs suppressed the reaction of MAb x Pox 3H8. MAbs 1E8 7F1, 1E11, and 3B1 reacted with each other but differed by the spectrum and level of competitive inhibition, which indicated that they were directed to different epitopes of adenovirus hexon. Comparison of the specific activity of MAbs 7F1 and 1E8 in direct immunofluorescent detection of adenovirus antigens in infected cell cultures and clinical materials from patients showed a good coincidence (90-97%) of the results with the IMAGEN Adenovirus test (Dako) and with polyclonal FITC conjugates to adenovirus hexon.     

Escherichia coli hemolysin may damage target cell membranes by generating transmembrane pores.

We used mouse monoclonal antibodies (MAbs) to characterize Neisseria gonorrhoeae lipooligosaccharide (LOS). LOSs that bound two or more MAbs in a solid-phase radioimmunoassay usually bound them to different LOS components, as separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); strains with multiple LOS components on SDS-PAGE usually bound more than one MAb. However, the LOS of some strains bound the same MAb to two LOS components with different relative molecular weights, and some individual LOS components bound more than one MAb. LOSs from different strains bound different amounts of the same MAb at saturation, reflecting differences in the quantitative expression of individual LOS components. Not all components recognized by MAbs were stained by silver after periodate oxidation. Treatment with NaOH variously affected epitopes defined by different MAbs. MAb 3F11 completely inhibited and MAb 2-1-L8 partially inhibited the binding of 125I-labeled 06B4 MAb to WR220 LOS and WR220 outer membranes in competitive binding studies. Other MAbs did not compete with the binding of 125I-labeled 06B4 to either antigen. We conclude that a strain of N. gonorrhoeae elaborates multiple LOSs that can be separated by SDS-PAGE and that are antigenically distinct. Epitope expression within these glycolipids is complex.     

Monoclonal antibodies against bacterial glucosamine 6-phosphate synthase: production and use for structural studies.

Fifteen mouse x rat hybridoma cell lines producing rat monoclonal antibodies (MAbs) directed to Escherichia coli Glucosamine 6-P Synthase (GlmS) were established and characterized. Most of them (13/15) are IgG2a while 2 were typed as IgG1. Their Kaff ranged from 1.5 x 10(6) to 9.6 x 10(8) M-1 as determined by Beatty et al. (1). The epitopes recognized by these MAbs were assigned to one of the two catalytical domains of the enzyme (CT1 and CT2) as demonstrated both by ELISA and Western-blotting using purified GlmS proteolytic fragments. The binding of the MAbs on either the native or denatured forms of GlmS, CT1 and CT2 was further analyzed by competitive immunoassay and most of the MAbs were found to bind preferentially to the denatured proteins. The study of the antigenic topography of GlmS by competitive radioimmunoassay demonstrated the existence of at least 10 independent epitopes on GlmS, divided into three groups. The first one (3/15) includes MAbs whose binding was not inhibited by any of the other MAbs. The second group (9/15) is comprised of MAbs that exhibit reciprocal binding inhibitory activity while the third group includes MAbs (3/15) presenting asymmetric inhibitory activity. Finally, since most of the isolated antibodies (10/15) bind to the 27 kDa amino-terminal glutamine binding domain (CT2), the capacity of these MAb to interfere with the associated glutaminase activity was analyzed.     

Analysis of specificity of interaction between synthetic key saccharide components of lipopolysaccharides and monoclonal antibodies to Coxiella burnetii

Two monoclonal antibodies (MAbs) against the lipopolysaccharides (LPSs) of Coxiella burnetii (C.b.) strains Priscilla and Nine Mile were prepared characterized by their interaction with synthetic glycoconjugates representing parts of LPSs of C.b. in virulent phase. Both MAbs were directed against immunodominant epitopes comprising core constituent of LPSs, Kdo (3-deoxy-alpha-D-manno-2-octulo-pyranosylonic acid). ELISA showed that the anti-Nine Mile MAb 4/11 bound preferably to disaccharides (alpha-Kdo (2 --> 4) alpha-Kdo and alpha-Kdo (2 --> 4) alpha-(5d) Kdo), while the anti-Priscilla MAb 1/4/H bound to all conjugates, though with various intensity. On the other hand, immunoelectron microscopy revealed a positive binding of only one glycoconjugate, namely the trisaccharide alpha-Kdo (2 --> 4) alpha-Kdo (2 --> 4) alpha-Kdo-BSA, to both MAbs. In competitive ELISA (cELISA), the anti-Priscilla MAb 1/4/H distinguished the strains Nine Mile and Priscilla, while the anti Nine Mile MAb 4/11 did not.     

Cytoadhesins of Mycoplasma hominis.

The mechanism of Mycoplasma hominis adherence to host cells of the urogenital tract was investigated with monoclonal antibodies (MAbs) directed against antigenic surface-localized polypeptides P50, P60, P80, and P100 of cytoadherent M. hominis FBG. A cell enzyme-linked immunosorbent assay was established allowing quantification of cytoadherent mycoplasmas detected by one of the following MAbs: four MAbs directed against P100 (molecular weight, about 100,000), three MAbs against P80, one MAb against P60, and three MAbs against P50. MAb binding to one of the surface proteins resulted in a decrease of mycoplasmal adherence to HeLa cells. To exclude the thesis that this is caused by nonspecific blocking of adherence, P100 and P50 were purified by affinity chromatography and tested instead of intact mycoplasmas in the cell enzyme-linked immunosorbent assay for cytoadherence. Both proteins bound to the surface of the eukaryotic cells. MAb binding to single epitopes of these proteins resulted in inhibition of protein adherence. These experiments strongly suggest that of the four surface-localized proteins at least P100 and P50 are adhesins of M. hominis FBG.     

Antibody interactions with the capsule of Cryptococcus neoformans

Monoclonal antibodies to the encapsulated fungus Cryptococcus neoformans produce different immunofluorescence (IF) patterns after binding to the polysaccharide capsule. To explore the relationship between the IF pattern and the location of antibody binding, two immunoglobulin M (IgM) monoclonal antibodies (MAbs) (12A1 and 13F1) that differ in protective efficacy and IF pattern and one protective IgG1 MAb (2H1) were studied by IF and electron microscopy (EM). Fixing C. neoformans cells in lung tissue for EM resulted in significantly better preservation of the capsule than fixing yeast cells in suspension. The localization of MAbs 12A1 and 13F1 by immunogold EM differed depending on whether the MAb was bound to cells in cut tissue sections embedded in plastic or to cells in solution. In cut tissue sections, MAbs 12A1 and 13F1 bound throughout the capsule, whereas in solution both MAbs bound near the capsule surface. To investigate whether antibody binding to the C. neoformans capsule affected the binding of other primary or secondary reagents, various combinations of MAbs 12A1, 13F1, and 2H1 were studied by direct and indirect IF. The IF pattern and location of binding for MAbs 12A1, 13F1, and 2H1 varied depending on the presence of other capsule-binding MAbs and the method of detection. The results show that (i) binding of MAbs to the C. neoformans polysaccharide capsule can modify the binding of subsequent primary or secondary antibodies; (ii) the IgM MAbs bind primarily to the outer capsule regions despite the occurrence of their epitopes throughout the capsule; and (iii) MAb 2H1 staining of newly formed buds is reduced, suggesting quantitative or qualitative differences in bud capsule.     

Passive protection of suckling infant mice against F41-positive enterotoxigenic Escherichia coli strains by intravenous inoculation of the dams with monoclonal antibodies against F41.

Ten monoclonal antibodies (MAbs) against five different epitope clusters of adhesion factor F41 (two MAbs per cluster) were tested for protection of infant mice against an oral challenge with F41-positive enterotoxigenic Escherichia coli (ETEC) B2C and B41M. Infant mice suckling dams intravenously inoculated with MAbs were orally challenged, and the survival rates were measured for 12 days after inoculation and challenge. Irrespective of their epitope specificity, all F41 MAbs given in a single dose of 4 mg per dam had a protective effect against both ETEC strains. In contrast, one K99 MAb of the same isotype and given in the same dose as the F41 MAbs did not protect infant mice at all. A reduction in the dose of F41 MAbs to 0.032 mg per dam resulted in a decrease in protection. Two different MAbs against the same epitope cluster were not necessarily equally protective. Combining MAbs two by two, whether the MAbs recognized the same epitope cluster or not, resulted in protective activity essentially similar to that obtained with each MAb separately, without any improvement. Therefore, one MAb against any epitope may be sufficient for protection. Enzyme-linked immunosorbent assay (ELISA) titers of MAbs in the serum of dams were similar, irrespective of the epitope specificity of the MAbs, and gradually decreased from day 1 to day 12 after inoculation. We found a good correlation between colostrum and milk ELISA titers of MAbs and serum ELISA titers of MAbs. Colostrum and milk MAb titers were 10-fold lower than corresponding serum MAb titers and stayed high until day 5 after inoculation. The most protective MAb had the highest ELISA titers in colostrum and milk for the first 5 days after inoculation. ETEC strain B2C colonized the intestines of infant mice suckling MAb-inoculated mothers until day 12 after challenge. Intestinal levels of the challenge strain were high on day 2 but never reached the very high numbers (10(9) to 10(10)) described previously in a diarrheic infant mouse model. MAbs did not eliminate the challenge ETEC strain from the intestines of infant mice.     

Epitopes on the S1 subunit of pertussis toxin recognized by monoclonal antibodies.

To identify the neutralizing epitopes on the S1 subunit (A promoter) of pertussis toxin, we characterized anti-S1 monoclonal antibodies (MAbs) X2X5, 3CX4, and 6FX1. We confirmed by immunoblot analysis that these MAbs bind to the S1 subunit and not to the B oligomer of pertussis toxin and that they recognize different epitopes by a competitive binding enzyme-linked immunosorbent assay. These MAbs had differential abilities to neutralize the lymphocytosis-promoting factor activity of pertussis toxin in mice: 3CX4 and 6FX1 had partial neutralizing abilities, while MAb X2X5 had none. With these MAbs, the epitopes on the S1 subunit were examined by using trypsinized S1 peptides, recombinant truncated S1 molecules, and synthetic peptides. The non-neutralizing MAb X2X5 bound in immunoblots to tryptic peptides of various sizes as small as 1.5 kilodaltons; the neutralizing MAbs 3CX4 and 6FX1 bound only to a 24-kilodalton tryptic peptide band. Immunoblot studies with recombinant truncated S1 molecules demonstrated that amino acid residues 7 to 14 and 15 to 26 play an important role in the binding of neutralizing MAbs and the non-neutralizing MAb, respectively. The binding of these MAbs was not dependent upon the presence of C-terminal amino acid residues 188 to 234. To further define B-cell epitopes, the binding of the MAbs we tested to synthetic peptides representing the entire S1 subunit were examined. Neutralizing MAbs 3CX4 and 6FX1 bound to none of these peptides, further suggesting that these MAbs recognize conformational epitopes. The non-neutralizing MAb X2X5 bound to peptides 11 to 26 and 16 to 30, demonstrating that the major antigenic determinant recognized by this MAb is a linear epitope located within residues 16 to 26.     

Monoclonal antibodies to progesterone: characterization and selection for enzyme immunoassay in bovine milk.

Thirty-one stabile murine monoclonal antibody (MAb) producing cell lines to progesterone were generated by using a short and a long immunization protocol. Long-term immunization with high doses of 11alpha-hydroxyprogesterone-hemisuccinate-bovine serum albumin (11alpha-OH-P-HS-BSA) antigen led to very good antibody response in Balb/c mice. The donor mouse produced antiserum with a high titre of 1/250,000. Eleven MAbs were selected for further characterization since they showed high sensitivities (<35 pg/well to inhibit 50% of the tracer) in bridge homologous enzyme immunoassay (EIA). The results were compared to the donor mouse polyclonal antiserum. The MAbs and the donor mouse antiserum were generally found to be highly specific, when tested with 30 different steroids. Employing MAb 9C11, with affinity constant, K(alpha), to 11alpha-OH-P-HS of 1.1 x 10(10) M(-1), a bridge heterologous microtitre plate EIA for milk progesterone was developed, using the second-antibody coating technique and horseradish peroxidase (HRP) as an enzyme label. The assay is simple and convenient to use, as it permits direct addition of undiluted milk samples, at the same time maintaining high sensitivity, high precision, and a wide range of optical density (OD) values. The major advantage of the assay developed, compared to previously published direct addition milk progesterone immunoassays, is that progesterone concentrations, measured by the EIA, were not influenced by changing milk fat concentrations, even when milk samples containing up to 10% of milk fat were used for analysis.