Characterization of monoclonal antibodies to aflatoxin M1 and molecular modeling studies of related aflatoxins.

Aflatoxin M1 (AFM1) and seven structural analogs were used to investigate the correlation between antibody binding and the conformational and electronic properties of these molecules. Mice were immunized with AFM1-BSA and hybridomas secreting anti-AFM1 antibodies were isolated and characterized. The cross-reactivities of seven structurally similar aflatoxins were determined by competition enzyme-linked immunosorbent assay (cELISA). In an effort to correlate antibody binding with three-dimensional properties of the analogs, all of the aflatoxins (and the immunogen) were modeled and global energy minima were determined using molecular, mechanical and quantum mechanical methods. The results demonstrate that, for these molecules, loss of optimum structure and introduction of steric hindrance in the portion of the molecule that would fit into the antibody binding site are more important to binding than simply loss of a determinant group. Molecular computational techniques can give reasons for the wide variation in IC50 values observed between structural analogs and can be used as a tool for determining which conformational and electronic properties of molecules are most important for antibody binding.

Immunosorbents coupled on-line with liquid chromatography for the determination of fluoroquinolones in chicken liver.

Four fluoroquinolones were analyzed in fortified chicken liver using an automated, on-line immunoaffinity extraction method. The fluoroquinolones were extracted from the liver matrix using an immunoaffinity capture column containing anti-sarafloxacin antibodies covalently cross-linked to protein G. After interfering liver matrix components had been washed away, the captured fluoroquinolones were automatically eluted directly onto a reversed phase column. Liquid chromatographic analyses were performed by isocratic elution using 2% acetic acid/acetonitrile (85:15) as the mobile phase and an Inertsil phenyl column with fluorescence detection at excitation and emission wavelengths of 280 and 444 nm, respectively. No significant interferences from the sample matrix were observed, indicating good selectivity with the immunoaffinity column. Overall recoveries from fortified liver samples (20, 50, and 100 ng/g) ranged between 85.7 and 93.5% with standard deviations of <5%. The limit of quantification for each fluoroquinolone was 1 ng/mL. The limits of detection, based on a signal-to-noise ratio of 5:1, were 0.47, 0.32, 0.87, and 0.53 ng/mL for ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin, respectively.

Development of a monoclonal antibody-based cELISA for the analysis of sulfadimethoxine. 1. Development and characterization of monoclonal antibodies and molecular modeling studies of antibody recognition.

Sulfonamide antibiotics are used to treat a variety of bacterial and protozoan infections in cattle, swine, and poultry. Current residue methods for the analysis of sulfonamides in animal-based food products include bioassays, chromatographic methods (HPLC, GLC), and immunoassays. Most immunoassays have employed highly specific polyclonal antibodies. In this paper, we describe the isolation of monoclonal antibodies against sulfadimethoxine (SDM) that vary in their sensitivities and cross-reactivities against a large number of sulfonamides. The most sensitive monoclonal antibody, designated SDM-18, exhibits an IC(50) value for SDM of 1.53 ppb. Another monoclonal antibody, designated SDM-44, exhibits IC(50) values for six sulfonamides well below the established threshold level of 100 ppb for animal tissues. Molecular modeling studies of the cross-reactive drugs suggest that, depending on the monoclonal antibody, both steric and electronic features govern antibody binding. Due to the diversity of these monoclonal antibodies, it should be possible to design both compound- and class-specific monoclonal antibody-based immunoassays.

Development of a monoclonal antibody-based cELISA for the analysis of sulfadimethoxine. 2. Evaluation of rapid extraction methods and implications for the analysis of incurred residues in chicken liver tissue.

Several rapid extraction methods were evaluated for use with a monoclonal antibody-based competitive inhibition ELISA (cELISA) to detect sulfadimethoxine (SDM) in chicken liver tissue. These methods included extraction of the samples with (1) aqueous buffer with or without ultrafiltration, (2) acetonitrile/water, (3) methanol/water, or (4) acetone. The organic extraction methods were evaluated with or without solvent evaporation prior to dilution into assay buffer for the cELISA. The aqueous-based extraction methods were compatible with the cELISA. However, of the organic extraction methods, only the acetone liver extract with solvent evaporation prior to analysis was compatible with the cELISA. The cELISA method coupled to aqueous- or acetone-based sample extraction as well as an HPLC method was evaluated for the analysis of chicken liver tissues fortified with SDM at levels from 0.2 to 0.025 ppm. Mean SDM recoveries for the HPLC method and for the cELISA method using samples prepared by aqueous extraction, aqueous extraction and ultrafiltration, or acetone extraction, evaporation, and reconstitution were 68.9, 95.7, 60.1, and 52.5%, respectively. For the analysis of samples obtained from an SDM incurred residue study, HPLC and cELISA analysis of the same organic extract gave results that were highly correlated (R(2) = 0.976; p < 0.0001). However, results obtained from the analysis of aqueous extracts by cELISA did not correlate well with those obtained by HPLC (R(2) = 0.61, p > 0. 0006). This was attributed to the coextraction of cross-reactive SDM-related residues that were not quantified by the HPLC method. The presence of these residues should be considered during data interpretation when ELISA methods coupled with rapid aqueous extraction of samples are used in SDM residue monitoring programs.

Chicken mim-1 protein, P33, is a heterophil chemotactic factor present in Salmonella enteritidis immune lymphokine.

Lymphokine (ILK) secreted from concanavalin A-stimulated T cells from Salmonella Enteritidis-immune chickens is an undefined mixture of proteins that confers protection against Salmonella infectivity when administered to day-old chicks. It has previously been shown that polyclonal antibodies raised against human granulocyte colony-stimulating factor (GCSF) can neutralize the heterophil activation that is responsible for ILK's protective effect. Western blot analysis of ILK probed with anti-GCSF antibodies detects a prominent protein of mass 33 kDa. We have sequenced the first 20 amino acids of this protein and found it to be identical to residues 24 to 43 of P33, a 326-amino acid protein of unknown function encoded by the chicken mim-1 gene. The primary structure of P33 consists of two 140-residue imperfect repeats that are each homologous to a mammalian neutrophil chemotactic factor termed leukocyte cell-derived chemotaxin 2 (LECT2). We have expressed mim-1 in Escherichia coli and demonstrated in vitro that recombinant P33 is chemotactic for heterophils, the avian equivalent of mammalian neutrophils. We have also constructed a derivative of P33 that consists of residues 33 to 165 (P33[33-165]), the first repeat sequence of P33 that is homologous to LECT2. P33(33-165) is chemotactic for heterophils both in vitro and in vivo, inducing an influx of heterophils into the peritoneum in a response similar to that observed with ILK. These results suggest that P33 functions as a chemotactic factor in chickens and that it plays an active role in ILK-mediated protection against Salmonella infection.

Determination of four fluoroquinolones in milk by on-line immunoaffinity capture coupled with reversed-phase liquid chromatography.

An automated, on-line immunoaffinity extraction method was developed for the analysis of 4 fluoroquinolones in milk: ciprofloxacin, difloxacin, enrofloxacin, and sarafloxacin. This method involves analyte extraction using an immunoaffinity capture column containing anti-fluoroquinolone antibodies coupled on-line with reversed-phase column chromatography. Liquid chromatographic analyses were performed by isocratic elution using a mobile phase of 2% acetic acid-acetonitrile (85 + 15) and an Inertsil phenyl column with fluorescence detection at excitation and emission wavelengths of 278 and 444 nm, respectively. No significant interferences from the sample matrix were observed, indicating good selectivity with the immunoaffinity column. Recoveries from fortified raw milk samples (5-50 ppb of each fluoroquinolone) ranged from 72 to 90%, with standard deviations of < or = 8%.

Determination of fluoroquinolones in serum using an on-line clean-up column coupled to high-performance immunoaffinity-reversed-phase liquid chromatography.

A simple, rapid and reliable method for the simultaneous analysis of the fluoroquinolones ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin in bovine serum has been developed. Upon injection of serum samples, an on-line protein G-linked column was employed to automatically remove serum components that otherwise would interfere with analyses. A high-performance immunoaffinity chromatography (HPIAC) column containing covalently bound anti-sarafloxacin antibodies was then used to capture the fluoroquinolones while allowing the remainder of the serum components to elute to waste. After binding to the HPIAC column, the fluoroquinolones were eluted directly onto a reversed-phase (RP) column for final separation of the compounds prior to fluorescence detection at excitation and emission wavelengths of 280 and 444 nm, respectively. Due to use of a clean-up column in tandem with a highly selective HPIAC column, the only off-line sample preparation required was dilution (10-fold) in phosphate buffered saline (PBS) and passage of the samples through a 0.2-microm filter to remove particulate matter prior to injection. No significant interferences from the sample matrix were observed, indicating good selectivity with the HPIAC column. The method yielded high recoveries from fortified bovine serum that were >95% for all four fluoroquinolones with good reproducibility (C.V. values <7.0%). The on-line, automated method described here provides a simple, sensitive and specific assay for multiresidue detection of fluoroquinolones in serum.

Separation and quantification of two fluoroquinolones in serum by on-line high-performance immunoaffinity chromatography.

To demonstrate that two structurally similar chemicals can be extracted from a complex matrix and then separated from each other on the basis of their relative affinities for an antibody, an automated column-switching system was used, incorporating on-line, high-performance immunoaffinity chromatography (HPIAC). A high-affinity monoclonal antibody (Mab Sara-95) against the fluoroquinolone sarafloxacin was covalently cross-linked to a protein G column and used to capture fluoroquinolones in fortified serum samples. Interference from matrix components adhering nonspecifically to the column was minimized by the insertion of a protein G cleanup column between the injection port and the Mab Sara-95 derivatized HPIAC column. Upon injection, serum samples containing the fluoroquinolones passed through both columns. The cleanup column detained serum components, that otherwise would bind nonspecifically to the HPIAC column, but allowed the fluoroquinolones to pass through unhindered to the HPIAC column. The fluoroquinolones were then eluted from the HPIAC column according to their relative affinities for the antibody, and individual peaks were monitored using fluorescence detection. By using an on-line cleanup column in tandem with an HPIAC column, the fluoroquinolones could be separated from the serum matrix and then separated from each other on the basis of their affinity for Mab Sara-95 without the use of organic solvents or reversed-phase liquid chromatography (RPLC). This method demonstrates true immunoaffinity separation of structurally related compounds in a complex matrix.