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

Gowrisankar Rajam1, George Carlone1, Ellie Kim1

  • 1Meningitis and Vaccine Preventable Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, 30329, USA.

Biologicals : Journal of the International Association of Biological Standardization
|November 22, 2018
PubMed

Insights

A new multiplex assay quantifies antibodies to five pertussis antigens simultaneously. This method, the microsphere based multiplex antibody capture assay (MMACA), is sensitive, specific, and requires minimal sample volume, aiding pediatric pertussis research.

Area of Science:

  • Immunology
  • Vaccinology
  • Biotechnology

Background:

  • Pertussis remains a significant public health concern despite widespread vaccination.
  • Current methods for quantifying antibodies against Bordetella pertussis (Bp) antigens are laborious and require large sample volumes.
  • Existing assays are challenging for pediatric samples due to limited serum availability.

Purpose of the Study:

  • To develop and validate a novel assay for simultaneous quantification of antibodies against multiple Bp antigens.
  • To overcome the limitations of traditional ELISA for pediatric pertussis serology.
  • To establish a sensitive, specific, and robust method for Bp antibody detection.

Main Methods:

  • Development of a microsphere based multiplex antibody capture assay (MMACA) for a 5-plex quantification.
  • Assay included pertussis toxin, pertactin, filamentous hemagglutinin, fimbrial antigens 2/3, and adenylate cyclase toxin.
  • Utilized a calibrated reference standard, QC reagents, and SAS-based data analysis.

Main Results:

  • MMACA demonstrated excellent standard curve fit (R² ≥0.99) and acceptable precision (median CV 16.0%-22.8%).
  • The assay showed high accuracy (1.9%-23.8% %E) and linearity (slopes 0.93-1.02).
  • Low serum volume (5µL), rapid turnaround time (<3h assay, <1min data), and minimal lot-to-lot variation (<12.3%) were achieved.

Conclusions:

  • MMACA is a validated, sensitive, specific, robust, and rugged method for simultaneous anti-Bp antibody quantification.
  • The assay is suitable for use with serum, plasma, and dried blood spots (DBS).
  • MMACA offers a significant advancement for pertussis serological studies, especially in resource-limited settings and pediatric populations.

Keywords:
Assay validationLuminexMultiplex serologyPertussisSAS

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