Quantitation of bovine immunoglobulin G2 antibodies binding Staphylococcus aureus, using a murine monoclonal antibody

A murine monoclonal antibody specific for bovine immunoglobulin (Ig) G2 was used to develop an enzyme-linked immunosorbent assay (ELISA) to measure serum antibodies against the bovine pathogen Staphylococcus aureus. Anion exchange chromatography was used to prepare IgG2 from serum taken from a mastitic cow infected with S aureus. Specific-IgG2 antibodies binding S aureus were purified with an affinity column, using a heat-killed, low protein A strain of S aureus (M-10). Purified antibodies did not contain IgG1 or IgM and were composed of greater than 95% heavy and light chains determined on the basis of polyacrylamide gel electrophoresis. Purified IgG2 anti-S aureus antibody was used as a reference in an ELISA that (i) could detect 5.0 ng of IgG2, (ii) was specific for IgG2 antibodies binding S aureus, (iii) was precise within and among different assays, (iv) yielded 112% recovery of the purified standard, and (v) when diluted in nonspecific IgG2, generated a curve that was parallel to the standard when a sample was serially diluted. A field study with cows having elevated California mastitis test scores showed evidence of infection with S aureus, as judged by a 59% increase (P less than 0.01) in IgG2 S aureus-specific antibodies and a 25% increase (P less than 0.05) in total IgG2 antibodies. There were no differences in IgG1 concentrations in plasma. Using indirect immunofluorescence, we also confirmed that bovine polymorphonuclear cells bound IgG2 preferentially over IgG1 or IgM. Measurement of antigen-specific IgG2 antibodies may therefore be useful as an index of specific antibody immunity to mastitis-causing organisms.