Recombinant expression of plasmodium falciparum histidine rich protein-2 (PfHRP-2) and characterisation of chicken anti-PfHRP-2 antibodies.

Abstract

The diagnosis of malaria using rapid diagnostic tests (RDTs) targets three Plasmodial proteins namely, Plasmodium falciparum histidine rich protein-2 (PfHRP-2); lactate dehydrogenase (LDH) and aldolase. Early diagnosis is important for malaria control and accurate diagnosis guides treatment. The diagnosis of malaria prioritises the detection of P. falciparum (Pf), the species with the highest mortality. As a result, PfHRP-2-based RDTs are the most commonly used RDTs to detect P. falciparum infections. PfHRP-2 is a Plasmodium falciparum protein expressed during the blood stages of the parasite infection and is abundant in the blood of patients. The PfHRP-2 protein is stable in the blood, urine, saliva and cerebrospinal fluids of patients where it can be detected. Detection of PfHRP-2 in the field is problematic due to gene deletion, PfHRP-2 protein sequence variation, low heat tolerance of mammalian anti￾PfHRP-2 antibodies in RDTs, prozone effect, low quality assurance standards and low sensitivity of RDTs in some cases. False positive results caused by mammalian antibodies reacting with human rheumatoid factor also affect the antibody-based detection of malaria. This limits the diagnosis of P. falciparum malaria and undermines malaria control efforts. Optimising the performance of current PfHRP-2-based RDTs could prevent some of these problems and would strengthen malaria surveillance. Most malaria RDT problems associated with defective or cross-reactive antibodiescould be abated using a different species of antibodies. Chicken IgY antibodies are stable at room temperatures and do not react with human rheumatoid factor. To raise anti- PfHRP-2 chicken IgY antibodies, recombinant histidine rich protein-2 (rPfHRP-2) was expressed in E. coli BL21 (DE3) at 30°C overnight, induced with lactose. The recombinant protein was affinity purified and analysed on SDS-PAGE gels, resolving as a single protein band of 54 kDa. rPfHRP-2 induced high antibody titers after 13-weeks in immunised chickens. Purified IgY antibodies against rPfHRP-2 detected the 54 kDa band, a rPfHRP-2 dimer and trimer which were also detected by human anti-rPfHRP-2 antibodies. The thermal stability of anti-rPfHRP-2 IgY antibodies was assessed by exposure to temperatures resembling the field were RDTs are deployed. IgY antibodies stored at room temperatures for sixteen weeks were shown to be stable and still detected rPfHRP-2 in an ELISA after storage. To determine epitopes or peptides detected by the anti-rPfHRP-2 IgY antibodies, the recombinant protein was cleaved with trypsin and the fragments probed with IgY. Trypsin cleavage of rPfHRP-2 produced fewer fragments than predicted and the fragments resolved larger than their estimated sizes on SDS-PAGE gels. Thefragments were detected by the anti-rPfHRP- 2 IgY antibodies. Chicken IgY antibodies have potential for possible application in malaria diagnostics to detect Plasmodial proteins. The IgY used in this study was stable at room temperatures, and detected trypsin hydrolysed rPfHRP-2 fragments whose identity requires further investigation to better understand the fragment’s behavior and determine epitopes of anti-rPfHRP-2 IgY antibodies.