Increasing the Throughput of in vitro Assays to Measure the Function of Antibodies to Pneumococci


Matthew L. Clarke1, Robert L. Burton2, Moon H. Nahm2, and Jeeseong Hwang1


1Optical Technology Division, NIST Gaithersburg, MD 20899

2Departments of Pathology and Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294 USA


Quantitation of functional antibodies to pneumococcus as a surrogate marker of protection is important to monitor vaccine efficacy.† The multiplexed opsonophagocytic killing assay (MOPA) is widely accepted for the determination of antibody functional capacity where the pneumococci are incubated with anti-serum, complement, and phagocytes.† MOPA requires counting the number of surviving colonies; however manual counting of the potentially thousands of colonies present on each plate is not practical.† While commercial counting systems exist, they can be prohibitively expensive for small laboratories, and may not handle the high-throughput requirements of MOPA.† We detail the validation and standardization of low-cost imaging techniques, such as digital cameras and document scanners.† We describe and make freely available software (NISTís Integrated Colony Enumerator, NICE) that we developed for the colony enumeration required by MOPA. NICE successfully counted plates as part of the assay with the mean difference between NICE and manual counting being less than 3%.†††††