Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens

Table 1 Parameters tested in the CBA assay

Experimental condition tested	Values tested	Type and number of plasma samples used	Outcome
Amount of antigen	0.5, 1, 2, 5 and 10 μg	Positive pool (see Methods) in duplicate wells per antigen amount	Different antigens had different optimal amounts (see Results)
Plasma buffer component	BSA, polyvinyl alcohol, polyvinylpyrrolidone concentrations	North American (non-malaria exposed) control pool and positive plasma pool	Buffer B had similar MFI values to Buffer A for positive plasma pool samples, but lower MFI values than Buffer A for NA controls
BSA	BSA, polyvinyl alcohol, polyvinylpyrrolidone concentrations
Polyvinylalcohol	Buffer A (0.1%, 0%, 0%) vs. Buffer B (1%, 0.5%, 0.8%)
Polyvinylpyrrolidone	Buffer A (0.1%, 0%, 0%) vs. Buffer B (1%, 0.5%, 0.8%)
Plasma dilution	1:100, 1:200, 1:400, 1:1,000, 1:2,000 and 1:4,000	30 samples from persons from a malaria endemic area, 7 North American control samples and duplicate positive pool plasma samples	Optimal 1:100 or 1:200
Assay format	Monoplex (each Ag)	8 malaria endemic plasma samples	Multiplex and monoplex gave similar values
Assay format	Multiplex (10 different Ags)	8 malaria endemic plasma samples	Multiplex and monoplex gave similar values
Number of microspheres per reaction	5000 beads/analyte/well, 1000 beads/analyte/well	3 North American, 16 malaria endemic samples	1000 and 5000 beads/analyte/well gave similar values
Reproducibility	0 day	Positive pool, 2 North American and 3 malaria endemic samples	Highly reproducible results
	7 days later

Parameters optimized in the multiplex cytometric bead based assay (CBA) assay with a set of variables investigated, description of samples used and standardized values that gave optimal results.

ISSN: 1475-2875