Standardization of enzyme-linked immunosorbent assay to detect anti- Porphyromonas gingivalis-peptidylarginine-deiminase antibodies

Abstract

Objective: our aim was to standardize an indirect ELISA immunoenzymatic assay for the detection of serum anti-PPAD antibodies in adolescents with juvenile systemic lupus erythematosus (jSLE). Material and Methods: serum of 50 patients, 25 with juvenile systemic lupus erythematosus (group A) (mean age 16.1 ± 1.6 years) and 25 healthy subjects (mean age 15.2 years ± 2.3 years) (group B) were analyzed. The method for anti-PPAD antibodies detection was chronologically performed by addi-tion of: (1) diluted PPAD peptide (sensitization step), (2) bovine serum albumin (BSA) (blocking step), (3) serum from each participating patient and (4) diluted biotinylated monoclonal antibody anti-human IgG. The final steps consisted of the addition of (5) horseradish peroxidase-conjugated streptavidin protein, (6) hydrogen peroxide and (7) a chromogenic substance. Finally, (8) a stop solution was added to stop the reaction and the plaque was read in a spectrophotometric reader at 450nm. Results: serum anti-PPAD antibodies were detected in both groups without any difference between them. Conclusion: the detection of serum anti-PPAD antibodies was achieved in adolescents with juvenile systemic lupus erythematosus using the methodology proposed by the study.