Estimation of serum antibody to subgingival species using checkerboard immunoblotting

Abstract

Measurement of serum antibody to subgingival bacterial species has been useful in discriminating possible periodontal pathogens and in assessing the host's immune response to subgingival species. An immunoassay system was developed to measure the level of serum antibody to multiple subgingival species in multiple serum samples on a single nitrocellulose membrane. The principle steps of the assay are the following: 1) binding of antigens from bacterial preparations and protein A in parallel lanes on nitrocellulose membranes; 2) incubation of known concentrations of human immunoglobulin as well as various dilutions of serum from patients in lanes perpendicular to the antigen lanes; 3) incubation of the membrane with a peroxidase-conjugated second antibody; 4) detection of positive reactions by enhanced chemiluminescence. Emitted light was captured on a photographic film in which the positive reactions appeared as squares at the intersections of antigens with appropriate antibody. Antibody was quantified using a laser densitometer to compare 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 antibody in as many as 45 serum samples against up to 45 bacterial species. The mean and standard error of the coefficients of variation for replicates within an assay averaged 7.3 +/- 2.3%. Coefficients of variation of the assay run on different days for serum antibody to a range of subgingival species averaged 10.1 +/- 2.1%. Checkerboard immunoblotting is a simple and rapid immunoassay to permit quantification and/or screening of antibody to multiple subgingival species or antigens in multiple serum samples.

Association of Systemic Oxidative Stress with Suppressed Serum IgG to Commensal Oral Biofilm and Modulation by Periodontal Infection

“…Although these titers reflect whole-bacterial titers, a high degree of specificity occurs in organism-specific IgG without significant cross-reactivity across various microbes under the stringency conditions of the immunobinding (57). Thus, the effect of oxidative stress on IgG responses appears to reflect a suppression of IgG across a wide range of potential antigenic stimuli.…”

Oxidative Stress and IgG Antibody Modify Periodontitis-CRP Association

“…The IgG clusters (i.e., other, orange, and red) were directed against microbial species associated with health, gingivitis, and periodontitis, respectively (Sakellari et al 1997). The models included demographic, behavioral, and medical variables identified in the χ 2 models for serum levels of CRP, as well as plaque and pocket depth, and an interaction term for pocket depth and serum 8-isoprostane.…”

“…Samples were assayed for serum CRP levels using the high-sensitivity method described previously (Slade et al 2003). Samples were assayed for IgG antibody levels directed against the 16 periodontal organisms using the checkerboard immunoblotting technique described by Sakellari et al (1997).…”

Periodontal Disease and Coronary Heart Disease

“…Serum samples were frozen at Ϫ80°C, transported on dry ice to our laboratory, and stored in aliquots at Ϫ80°C. The samples were assayed for IgG antibody levels directed against the aforementioned periodontal organisms with the use of the checkerboard immunoblotting technique described by Sakellari et al 37 See the online-only Data Supplement for additional details.…”