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

Bartholomew N Ondigo,Lyticia A Ochola,George O Ayodo,Severin O Gose,Ayub V Ofulla,Benjamin M Ho,Chandy C John,Gregory S Park

doi:10.1186/1475-2875-11-427

Bartholomew N Ondigo, Lyticia A Ochola + Show 6 more

Open Access

https://doi.org/10.1186/1475-2875-11-427

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Abstract

BackgroundMultiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA.MethodsAntibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared.ResultsOptimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA.ConclusionWith optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.

Highlights

Multiplex cytometric bead assay (CBA) have a number of advantages over enzyme linked immunosorbent assay (ELISA) for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens
Plasma samples For validation and standardization of the multiplex assay of antibodies to P. falciparum antigens, a plasma pool made of 30 plasma samples from adults living in a Ugandan area of seasonal malaria transmission [13] and seven plasma samples from North American individuals never exposed to malaria was used
Optimal amount of antigen for multiplex CBA To determine optimal amounts of antigen for testing, 612,500 beads were coupled with differing amounts of antigens (Table 1)

Summary

Introduction

Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Most studies that have determined antibody responses to Plasmodium falciparum antigens and vaccine candidates in human plasma samples have used enzyme linked immunosorbent assay (ELISA) [1,2]. The Luminex100 has two lasers; one laser beam excites the internal colored dyes for classification of the bead sets, while the other laser excites the reporter fluorochrome phycoerythrin (PE) [4,5]. Through classification of the bead set, various bead sets are distinguished, which correspond to up to 100 different analytes that the machine can quantitate, while the amount of analyte present in the plasma, serum or supernatant is quantified by excitation of the reporter fluorochrome [6]

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