Development and validation of a robust multiplex serological assay to quantify antibodies specific to pertussis antigens.

Despite wide spread vaccination, the public health burden of pertussis remains substantial. Current acellular pertussis vaccines comprise upto five Bordetella pertussis (Bp) antigens. Performing an ELISA to quantify antibody for each antigen is laborious and challenging to apply to pediatric samples where serum volume may be limited. We developed a microsphere based multiplex antibody capture assay (MMACA) to quantify antibodies to five pertussis antigens; pertussis toxin, pertactin, filamentous hemagglutinin and fimbrial antigens 2/3, and adenylate cyclase toxin in a single reaction (5-plex) with a calibrated reference standard, QC reagents and SAS based data analysis program. The goodness of fit (R) of the standard curves for five analytes was ≥0.99, LLOQ 0.04-0.15 IU or AU/mL, accuracy 1.9%-23.8% (%E), dilutional linearity slopes 0.93-1.02 and regression coefficients r = 0.91-0.99. MMACA had acceptable precision within a median CV of 16.0%-22.8%. Critical reagents, antigen conjugated microsphere and reporter antibody exhibited acceptable (<12.3%) lot-lot variation. MMACA can be completed in <3 h, requires low serum volume (5μL/multiplex assay) and has fast data turnaround time (<1 min). MMACA has been successfully developed and validated as a sensitive, specific, robust and rugged method suitable for simultaneous quantification of anti-Bp antibodies in serum, plasma and DBS.