ESTIMATION OF SERUM ANTIBODY TO SUBGINGIVAL SPECIES USING CHECKERBOARD IMMUNOBLOTTING

Measurement of serum antibody to subgingival bacterial species has bee n useful in discriminating possible periodontal pathogens and in asses sing the host's immune response to subgingival species. An immunoassay system was developed to measure the level of serum antibody to multip le subgingival species in multiple serum samples on a single nitrocell ulose membrane. The principle steps of the assay are the following: I) binding of antigens from bacterial preparations and protein A in para llel lanes on nitrocellulose membranes; 2) incubation of known concent rations of human immunoglobulin as well as various dilutions of serum from patients in lanes perpendicular to the antigen lanes; 3) incubati on of the membrane with a peroxidase-conjugated second antibody; 4) de tection of positive reactions by enhanced chemiluminescence. Emitted l ight was captured on a photographic him in which the positive reaction s appeared as squares at the intersections of antigens with appropriat e antibody. Antibody was quantified using a laser densitometer to comp are the signal intensity of unknown samples with the ones generated by known amounts of human immunoglobulin captured on the same membrane. The assay permitted simultaneous screening and/or quantification of an tibody in as many as 45 serum samples against up to 45 bacterial speci es. The mean and standard error of the coefficients of variation for r eplicates within an assay averaged 7.3 +/- 2.3%. Coefficients of Varia tion of the assay run on different days for serum antibody to a range of subgingival species averaged 10.1 +/- 2.1%. Checkerboard immunoblot ting is a simple and rapid immunoassay to permit quantification and/or screening of antibody to multiple subgingival species or antigens in multiple serum samples.