Dataset Information

Measuring antibody avidity to Plasmodium falciparum merozoite antigens using a multiplex immunoassay approach.

ABSTRACT:

Background

Antibodies (Ab) play a significant role in immunity to Plasmodium falciparum malaria. Usually, following repeated exposure to pathogens, affinity maturation and clonal selection take place, resulting in increased antibody avidity. However, some studies suggest affinity maturation may not occur to malaria antigens in endemic areas. Information on development of antibody avidity is confusing and conflicting, in part, because different techniques have been used to measure avidity. Today, bead-based multiplex immunoassays (MIA) are routinely used to simultaneously quantitate antibody levels to multiple antigens. This study evaluated the feasibility of developing an avidity MIA with 5 merozoite antigens (AMA1, EBA-175, MSP1-42, MSP2, MSP3) that uses a single chaotropic concentration.

Methods

The most common ELISA protocols that used the chaotropic reagents guanidine HCl (GdHCl), urea, and ammonium thiocyanate (NH₄SCN) were adapted to a multiplex MIA format. Then, different concentrations of chaotropes and incubation times were compared and results were expressed as an Avidity Index (AI), i.e., percentage of antibody remaining bound in the presence of chaotrope. Experiments were conducted to (i) identify the assay with the widest range of AI (discriminatory power), (ii) determine the amount of chaotrope needed to release 50% of bound Ab using plasma from adults and infants, and (iii) evaluate assay repeatability.

Results

Overall, 4 M GdHCl and 8 M urea were weaker chaotropes than 3 M NH₄SCN. For example, they failed to release significant amounts of Ab bound to MSP1-42 in adult plasma samples; whereas, a range of AI values was obtained with NH₄SCN. Titration of NH₄SCN revealed that 2 M NH₄SCN gave the widest range of AI for the 5 antigens. Binding studies using plasma from 40 adults and 57 1-year old infants in Cameroon showed that 2.1 M?±?0.32 (mean?±?SD) NH₄SCN (adults) and 1.8 M?±?0.23 M (infants) released 50% of bound Ab from the merozoite antigens.

Conclusions

An avidity MIA is feasible for the 5 merozoite antigens that uses a single concentration (2 M) of NH₄SCN. The assay provides a simple method to quickly obtain information about Ab quantity and quality in the acquisition of immunity to malaria in endemic populations.

SUBMITTER: Taylor DW

PROVIDER: S-EPMC7195780 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Publications

Measuring antibody avidity to Plasmodium falciparum merozoite antigens using a multiplex immunoassay approach.

Taylor Diane Wallace DW Bobbili Naveen N Kayatani Alex A Tassi Yunga Samuel S Kidima Winifrida W Leke Rose F G RFG

Malaria journal 20200501 1

<h4>Background</h4>Antibodies (Ab) play a significant role in immunity to Plasmodium falciparum malaria. Usually, following repeated exposure to pathogens, affinity maturation and clonal selection take place, resulting in increased antibody avidity. However, some studies suggest affinity maturation may not occur to malaria antigens in endemic areas. Information on development of antibody avidity is confusing and conflicting, in part, because different techniques have been used to measure avidity ...[more]

PMID: 32357882

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Project description:Background: Antibody responses have been used to characterise transmission and exposure history in malaria-endemic settings for over a decade. Such studies have typically been conducted on well-standardised enzyme-linked immunosorbent assays (ELISAs). However, recently developed quantitative suspension array technologies (qSAT) are now capable of high-throughput and multiplexed screening of up to hundreds of analytes at a time. This study presents a customised protocol for the Luminex MAGPIX © qSAT using a diverse set of malaria antigens. The aim is to develop a standardised assay for routine serological surveillance that is implementable across laboratories and epidemiological settings. Methods: A panel of eight Plasmodium falciparum recombinant antigens, associated with long- and short-lived antibody responses, was designed for the Luminex MAGPIX © platform. The assay was optimised for key steps in the protocol: antigen-bead coupling concentration, buffer composition, serum sample dilution, and bead storage conditions. Quality control procedures and data normalisation methods were developed to address high-throughput assay processing. Antigen-specific limits of quantification (LOQs) were also estimated using both in-house and WHO reference serum as positive controls. Results: Antigen-specific bead coupling was optimised across five serum dilutions and two positive controls, resulting in concentrations operational within stable analytical ranges. Coupled beads were stable after storage at room temperature (22?C) for up to eight weeks. High sensitivity and specificity for distinguishing positive and negative controls at serum sample dilutions of 1:500 (AUC 0.94 95%CI 0.91-0.96) and 1:1000 (AUC 0.96 95%CI 0.94-0.98) were observed. LOQs were also successfully estimated for all analytes but varied by antigen and positive control. Conclusions: This study demonstrates that developing a standardised malaria-specific qSAT protocol for a diverse set of antigens is achievable, though further optimisations may be required. Quality control and data standardisation methods may also be useful for future analysis of large sero-epidemiological surveys.

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Dataset Information

Measuring antibody avidity to Plasmodium falciparum merozoite antigens using a multiplex immunoassay approach.

Background

Methods

Results

Conclusions

Publications

Measuring antibody avidity to Plasmodium falciparum merozoite antigens using a multiplex immunoassay approach.

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