The detection of two plasmodium falciparum metabolic enzymes using chicken antibodies.

Abstract

Three protein targets are used in malaria rapid diagnostic tests (RDTs). These are Plasmodium falciparum histidine rich protein 2, Plasmodium lactate dehydrogenase and aldolase. A thrust of research in RDTs is to improve on their specificity and sensitivity. In this study the current diagnostic target, P. falciparum lactate dehydrogenase (PƒLDH) was compared to a new target glyceraldehyde-3-phosphate dehydrogenase (PƒGAPDH) that was identified based on transcriptional data. These proteins are conserved amongst all Plasmodium species, with minor amino acid sequence variations which were evaluated as possible species-specific peptide epitopes for PƒLDH: LISDAELEAIFDRC and PƒGAPDH: CADGFLLIGEKKVSVFA; CAEKDPSQIPWGKCQV, where common peptides were identified as pan-malarial epitopes for pLDH: APGKSDKEWNRDDLC and pGAPDH: CKDDTPIYVMGINH. The chosen peptides were located on the surface of their predicted 3D crystal structure models. Antibodies were raised against these peptides in chickens (IgY) and affinity purified. PƒLDH and PƒGAPDH were recombinantly expressed in E. coli BL21(DE3) cells and their coding inserts confirmed by sequencing. The recombinant proteins were detected in Western blots with specific anti-His₆ tag antibodies at approximately 35 kD (PƒLDH ~ 36 kD and PƒGAPDH ~ 39 kD) which compared with their expected values. Both recombinant proteins were found to form tetramers in solution and were used to raise IgY antibodies for comparison of Pheroids™ and Freund’s adjuvants. Pheroids™, like Freund’s appeared to exhibit a depot effect, however Freund’s adjuvant gave higher affinity purified IgY yields. The anti-recombinant and anti-peptide IgY specifically detected their respective recombinant and native antigens and did not cross-react with other human blood proteins. Immunoprecipitation detected higher levels of PƒGAPDH to PƒLDH in P. falciparum culture lysates. A double antibody sandwich ELISA detected 17.3 ng/ml PƒLDH and 138.5 ng/ml PƒGAPDH at 1% parasitemia in in vitro cultures, however this needs to be further evaluated. These findings suggest PƒGAPDH to be at least as good a protein target as PƒLDH for malaria diagnosis and further trials using it as a target in an RDT format should be considered.