Rapid evaluation of serum and gingival crevicular fluid immunoglobulin G subclass antibody levels in patients with early‐onset periodontitis using checkerboard immunoblotting

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Abstract
A method was developed to evaluate the presence of immunoglobulin G (IgG) subclass (1–4) antibody to Actinobacillus actinomycetemcomitans, serotype b (strain Y4) in patients with early‐onset periodontitis on a single nitrocellulose membrane. Sera from 30 early‐onset periodontitis patients and gingival crevicular fluid samples from 2 patients were collected and tested with four different preparations of A. actinomycetemcomitans (Y4). The principle steps of the assay are: a) binding of the bacterial antigen (Y4) and the anti‐human IgG antibody (capture antibody) in parallel lanes on nitrocellulose membranes; b) incubation of known concentrations of the IgG subclasses 1,2,3 and 4, as well as a dilution of serum and/or gingival crevicular fluid from patients in lanes perpendicular to the antigen lanes; c) incubation of the membranes with the corresponding peroxidase conjugated anti‐human IgG subclass secondary antibody; d) detection of positive signals by enhanced chemiluminescence. The blots were evaluated by visual comparison to a series of blots containing known concentrations of IgG subclasses. The method was used to rapidly screen a relatively large number of patient sera and gingival crevicular fluid samples for IgG subclasses in a cost‐effective assay. The predominant IgG subclass found in early‐onset periodontitis was IgG2.