ovalbumin and Antiphospholipid-Syndrome

The use of olaquindox (OLA) as an additive in animal feedstuffs has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for determination of OLA in animal feed samples was developed. OLA was activated by N'N-carbonyldiimidazole and coupled with bovine serum albumin (BSA) and ovalbumin (OVA). It was found that the sensitivity and specificity of the two antisera were very similar, with the IC(50) values of 16 ng mL(-1) and 19 ng mL(-1), respectively. Cross-reactivity was less than 35% for four structurally related compounds and no recognition of five other antibiotics was observed. The better antiserum I was selected for further experiments, for example testing stability, solvent effect, accuracy, and precision. The IC(50) value for eight standard curves was in the range 12-18 ng mL(-1) and the LOD at a signal-to-noise ratio of 3 (S/N = 3) was 0.31 +/- 0.11 ng mL(-1). The ELISA tolerated 5% methanol without significant influence on IC(50) value. The recoveries of spiked OLA in five different animal feed types including auxin, pig complex feed, fish complex feed, broiler concentrated feed, and pig premix feed were in the range 88.3-119.0% and the intra-assay relative standard deviation (RSD) was within 4.7-33.5% (n = 3). The ELISA for unspiked feed samples was confirmed by high-performance liquid chromatography (HPLC), with a high correlation coefficient of 0.9862 (n = 5). The proposed ELISA could be a feasible quantitative/screening method for OLA analysis in feed samples with the properties of high sensitivity, specificity, simplicity of sample pretreatment, high sample throughput, and low expense.

Topics: Animal Feed; Animals; Antibodies; Antigens; Antiphospholipid Syndrome; Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Male; Models, Molecular; Ovalbumin; Quinoxalines; Rabbits; Sensitivity and Specificity; Serum Albumin, Bovine; Spectrophotometry, Ultraviolet; Vaccines, Synthetic

Previous studies have shown the importance of somatic mutations and arginine residues in the complementarity-determining regions (CDRs) of pathogenic anti-double-stranded DNA (anti-dsDNA) antibodies in human and murine lupus, and in studies of murine antibodies, a role of mutations at position 53 in V(H) CDR2 has been demonstrated. We previously demonstrated in vitro expression and mutagenesis of the human IgG1 monoclonal antibody B3. The present study was undertaken to investigate, using this expression system, the importance of the arginine residue at position 53 (R53) in B3 V(H).. R53 was altered, by site-directed mutagenesis, to serine, asparagine, or lysine, to create 3 expressed variants of V(H). In addition, the germline sequence of the V(H)3-23 gene (from which B3 V(H) is derived) was expressed either with or without arginine at position 53. These 5 new heavy chains, as well as wild-type B3 V(H), were expressed with 4 different light chains, and the resulting antibodies were assessed for their ability to bind to nucleosomes, alpha-actinin, cardiolipin, ovalbumin, beta(2)-glycoprotein I (beta(2)GPI), and the N-terminal domain of beta(2)GPI (domain I), using direct binding assays.. The presence of R53 was essential but not sufficient for binding to dsDNA and nucleosomes. Conversely, the presence of R53 reduced binding to alpha-actinin, ovalbumin, beta(2)GPI, and domain I of beta(2)GPI. The combination B3 (R53S) V(H)/B3 V(L) bound human, but not bovine, beta(2)GPI.. The fact that the R53S substitution significantly alters binding of B3 to different clinically relevant antigens, but that the alteration is in opposite directions depending on the antigen, implies that this arginine residue plays a critical role in the affinity maturation of antibody B3.

Topics: Amino Acid Sequence; Antiphospholipid Syndrome; Arginine; Binding Sites; DNA Primers; Germ-Line Mutation; Humans; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Lupus Erythematosus, Systemic; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Ovalbumin; Reproducibility of Results