A Rapid Inhibition Micro ELISA for Detecting Antibodies toPlasmodium FalciparumSporozoites in Human Blood

Abstract

A sensitive and specific micro ELISA, named MONOPLATE ELISA, for the detection of antibodies against P. falciparum sporozoites was developed. It can be applied to many kinds of samples including serum, plasma, whole blood, eluted bloodspot and mosquito bloodmeal as well. The method makes use of a single microtiter plate and the chemically synthesized (Asn-Ala-Asn-Pro)20 (NANP20) antigen both as coating material and as competitive (binding) inhibitor in the samples. The specific value of each sample is obtained as the absorbance difference between the uninhibited and the fully inhibited sample. Using appropriate conditions, the results can be evaluated by simple visual inspection of the plate, without any instrument. A rapid procedure, where the incubation times for sample and conjugate are just 15 minutes, is also described. When unknown samples from a P. falciparum endemic area were tested, a close correlation was found between our results and those obtained with the only commercial ELISA kit now available (Sclavo S.p.A). For screening purposes, as many as 48 samples per plate can be tested by this method.

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

“…Different multiples of NANP-repeats have been used in immunoassays, from (NANP) 3 to (NANP) 40 [23-26]. Although anti-CSP repeats antibodies can be detected using repeats of different multiples, it has been observed in a comparative analysis that results of higher multiples of NANP-repeats better correlated with each other than with those of lower multiples and that R32LR-based and (NANP) 40 -based ELISAs had higher sensitivity than (NANP) 3 -based ELISA [24].…”

“…The Asn-Ala-Asn-Pro (NANP) sequence is the major B-cell epitope of the P.falciparum CSP [22], and NANP-based peptides have already been widely used in immunoassays aimed at detecting anti-CSP antibodies [23-26]. Recombinant proteins composed of 15 NANP repeats and an Asn-Val-Asp-Pro (NVDP) oligopeptide, NVDP(NANP) 15 , 30 NANP repeats, [NVDP(NANP) 15 2 , or 45 NANP repeats, [NVDP(NANP) 15 3 , were shown to induce antibodies that bind to the natural CSP on P. falciparum sporozoites and to block the invasion of human hepatocytes by the parasite [27,28].…”

A Peptide-BasedPlasmodium falciparumCircumsporozoite Assay To Test for Serum Antibody Responses to Pre-Erythrocyte Malaria Vaccines

“…Indeed, we have shown highly reproducible results between two different laboratories. Since the first report of using NANP repeat peptides as CS antigen (23), various versions ranging from 3 to 40 repeats have been used, mainly as a diagnostic antibody ELISA for epidemiological studies (2,(9)(10)(11)15). A thorough comparison of the peptide antigen with protein antigens and whole parasite as antigen (immunofluorescence assay) has been performed by Del Giudice et al (4).…”

Niveaux d’anticorps contre la protéine circumsporozoïtique dePlasmodium falciparumet leur utilisation en tant qu’indicateurs épidémiologiques de la transmission du paludisme en RFI des Comores

“…Les éluats des spots sanguins ont été testés en technique ELISA comme décrit par Nuzzolo et al (1990). L'absorbance a été mesurée par lecture spectrophotométrique à une densité optique (DO) de 450 nm de longueur d'onde.…”