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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

Validation of an enzyme-linked immunosorbent assay for the quantification of human IgG directed against the repeat region of the circumsporozoite protein of the parasite Plasmodium falciparum

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Frederic Clement, Vincent Dewar, Eva Van Braeckel, Isabelle Desombere, Marianne Dewerchin, Christine Swysen, Marie-Ange Demoitié, Erik Jongert, Joe Cohen, Geert Leroux-Roels, Pierre Cambron
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-384) contains supplementary material, which is available to authorized users.

Competing interests

The study was funded by GlaxoSmithKline Biologicals SA (GSK). VD, MD, CS, MAD, EJ, JC and PC are employees of GSK. JC, MAD and PC own shares and/or options to shares in GSK. In addition, JC is a designated inventor on patented malaria vaccines, but does not hold a patent for a malaria vaccine. GLR has received fees from GSK for consultancy and lectures on different vaccine topics, and for travel and accommodations for the participation to a congress. The other authors declare no potential conflict of interest.

Authors’ contributions

EJ, FC, GLR, JC and MAD were involved in the design of the study. CS, EJ, EVB, FC, GLR, ID, MAD, PC contributed to the development and/or review of the study protocol. FC, VD and MD made substantial contribution to the method selection and development. EVB, GLR, PC recruited subjects for clinical trials. EVB, FC and GLR participated to the acquisition of data. CS, EJ, EVB, FC, GLR, ID, JC, MAD, PC analysed and interpreted the results. All authors contributed to the development of the manuscript, were involved in finalizing the manuscript, read and approve the final version.

Abstract

Background

Several pre-erythrocytic malaria vaccines based on the circumsporozoite protein (CSP) antigen of Plasmodium falciparum are in clinical development. Vaccine immunogenicity is commonly evaluated by the determination of anti-CSP antibody levels using IgG-based assays, but no standard assay is available to allow comparison of the different vaccines.

Methods

The validation of an anti-CSP repeat region enzyme-linked immunosorbent assay (ELISA) is described. This assay is based on the binding of serum antibodies to R32LR, a recombinant protein composed of the repeat region of P. falciparum CSP. In addition to the original recombinant R32LR, an easy to purify recombinant His-tagged R32LR protein has been constructed to be used as solid phase antigen in the assay. Also, hybridoma cell lines have been generated producing human anti-R32LR monoclonal antibodies to be used as a potential inexhaustible source of anti-CSP repeats standard, instead of a reference serum.

Results

The anti-CSP repeats ELISA was shown to be robust, specific and linear within the analytical range, and adequately fulfilled all validation criteria as defined in the ICH guidelines. Furthermore, the coefficient of variation for repeatability and intermediate precision did not exceed 23%. Non-interference was demonstrated for R32LR-binding sera, and the assay was shown to be stable over time.

Conclusions

This ELISA, specific for antibodies directed against the CSP repeat region, can be used as a standard assay for the determination of humoral immunogenicity in the development of any CSP-based P. falciparum malaria vaccine.
Zusatzmaterial
Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 5
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Literatur
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