Antibodies with specificity for native and denatured forms of ovalbumin differ in reactivity between enzyme‐linked immunosorbent assays

Abstract

In this study, polyclonal and monoclonal antibodies to native and denatured chicken ovalbumin (OVA) were produced to compare their dependency on continuous and three-dimensional epitopes. These antibodies were characterized with respect to reactivity to native and denatured OVA by enzyme-linked immunosorbent assay (ELISA) employing surface-bound OVA and streptavidin-capture ELISA to determine whether effects of different coating influence antibody specificity and with respect to epitope specificity by peptide ELISA, using overlapping peptides, covering the complete OVA sequence. Polyclonal antibodies to native OVA reacted strongly with native and denatured OVA in both assays, but did not react with the overlapping peptides. Polyclonal antibodies to denatured OVA reacted strongly with both OVA forms and with several of the overlapping peptides. Monoclonal antibodies to native OVA reacted preferentially with three-dimensional epitopes on native OVA and not with denatured OVA. Monoclonal antibodies to denatured OVA showed reactivity to both OVA forms. Two of these monoclonal antibodies, HYB 94-06 and 94-07, showed reactivity to overlapping peptides and their epitopes were identified as flexible structures constituting amino acids 130-135 and 136-141, respectively. Moreover, comparison of antibody reactivity to N OVA revealed that in the streptavidin-capture ELISA, antibody reactivity was notably reduced compared to ELISA employing surface-bound OVA. Collectively, immunization with native OVA preferentially generates highly specific antibodies reacting with three-dimensional epitopes, whereas immunization with denatured OVA generates antibodies occasionally reacting with continuous epitopes. Moreover, as differences in monoclonal antibody reactivity was found between the two assays, monoclonal antibodies always should be selected by an assay mimicking the desired use of the final antibodies as closely as possible.

Characterization of continuous monoclonal antibody epitopes in the N‐terminus of Ro60

“…In the study by Holm et al, 21 mAbs were generated against native ovalbumin. Of these 21 mAbs, only one showed specific reactivity to a continuous epitope . In contrast, the mAbs SSI‐HYB 358‐02 and TROVE2 reacted specifically with continuous epitopes located to peptides 4 and 1/2, respectively.…”

“…The mAbs SSI‐HYB 353‐01, 353‐02, 353‐05 and 353‐06 were directed to native bovine Ro60, which may explain why the Ab did not show reactivity to the synthetic peptides, and why SSI‐HYB 353‐01 did not stain the Ro60 overexpressing Hep2 cells. In accordance with recent findings by Holm et al and Rubinstein et al abs directed to native protein structures mainly recognize conformational epitopes. In the study by Holm et al, 21 mAbs were generated against native ovalbumin.…”

Critical Differences between Induced and Spontaneous Mouse Models of Graves’ Disease with Implications for Antigen-Specific Immunotherapy in Humans

“…Antibodies to OVA were measured by ELISA as previously described (33). Briefly, ELISA plates were coated with OVA (1μg/ml) in 20mM Tris-HCL (pH 7.5).…”

“…The signal was developed with o-phenylenediamine and the reaction stopped using 20% H 2 SO 4 . The positive control was mouse monoclonal antibody HYB 099-01 obtained from CF1 × BALB/c mice immunized intra-peritoneally with native OVA (33). The “cut-off” point for positivity was established from the binding (mean + 2SD) to OVA-coated ELISA plates of sera from NOD.…”

“…6B). ELISA binding OD values for the monoclonal (McAb) used in each assay are included, namely 3BD10 for h TSHR A-subunit protein (25) and HYB 099-01 for OVA (33); * p=0.017 (Rank sum tests).…”