Malaria is still one of the world’s most deadly diseases today despite the huge investment in combating the disease. Although P. falciparum is the most lethal of all Plasmodium species, P. vivax is the most widespread and common and thus, responsible for the greatest burden of the disease outside Africa
[1, 15] and results in close to more than half of the worldwide malaria cases
. According to WHO, product testing has shown remarkable improvement in test quality over time, and more high quality tests are being procured over time
. Although there is significant improvement in the quality of these assays, only a few that target malaria parasite antigens have really worked to expectation. The major malaria antigens targeted by these RDTs are P. falciparum specific HRP-2, pLDH and pan-specific aldolase. The ability of these assays to differentiate the various Plasmodium forms is another difficulty. It is, therefore, prudent to develop monoclonal antibodies that can sufficiently differentiate the two most common Plasmodium species, P.vivax and P. falciparum.
The high homology among the Plasmodium species confirmed the assertion that many regions in Plasmodium aldolase gene are completely or highly conserved
. These regions may contribute to determining the authentic common antigenic epitopes among these strains of malaria parasite which can assist in the development of drugs targeting these sites. This common epitopes can be exploited for the development of pan-specific mAbs against Plasmodium species. In this study, the cloning and expression of soluble recombinant P. vivax specific aldolase antigen and its application in the production of high affinity mAbs for malaria diagnosis is described.
The recombinant antigen was used in immunizing rabbit and mice for the production of polyclonal and monoclonal antibodies respectively. The high titers observed in all immunized animals after the second booster immunization are an indication of the immunogenicity of the recombinant protein. Rabbit anti-PvALDO polyclonal antibodies were used in a novel antibody-capture ELISA for the screening of P. vivax specific mAbs. With this screening model, it is possible to screen out very good antibodies that could probably not be detected by the traditional indirect ELISA used by most researchers for hybridoma screening. Three mAbs 14C7, 15F1 and 5H7 were selected after a number of sub-cloning and limiting dilutions. mAb 15F1 had extremely low titer when indirect ELISA was used for antibody screening, but very good titer value with the newly developed antibody-capture method. This means that mAbs with similar characteristics have a greater probability of being rejected when applying the traditional indirect ELISA method of hybridoma screening (Table
1). The reason for this phenomenon is unknown but might probably have been due to overshadowing of the epitopes of the antigen during coating. Selected mAbs were used in the establishment of immunochromatographic test strips for evaluation of assay sensitivity and specificity. All antibodies were of the IgG1 class (Table
2). mAbs 15F1 and 5H7 could favourably pair-up as capture and detection antibodies respectively in immunochromatographic assay for the detection of both recombinant and native aldolase in human blood samples.
Plasmodium vivax positive samples (n=60), P. falciparum positive samples with no mixed infections (n=20), P. malariae samples (n=2) and healthy uninfected blood samples (n=108) were evaluated with the immunochromatographic test strips versus microscopic examination. Among the 60 P. vivax samples, one false negative was observed for samples with parasite densities < 500 parasites/μl (Table
3). This false negative P. vivax sample had a parasitaemia density of 54 parasites/μl. The extremely low level of parasitaemia might have accounted for the inability of the test strip to detect this blood samples. The other 59 P. vivax positive samples had parasitaemia density range between 120 to 14,220 parasites/μl. This result indicates that samples with parasitaemia below a detectable range of about 100 parasites/μl are very likely to be undetected. The disparity in the level of sensitivity of the test strip to actively circulating P. vivax blood aldolase antigen and its recombinant form might probably be due to the a stronger affinity of these antibodies to the active enzyme. Overall sensitivity and specificity of the immunochromatographic assay were 98.33% (59/60) and 99.23% (129/130), respectively at a 95% CI and Kappa statistics of 0.9757, P<0.005 (Table
4), an indication of a strong agreement between this test and standard methods used in malaria diagnosis. The only observed false positive sample was a P. falciparum infected patient that might have been infected with both strains of the parasite. Plasmodium malariae samples were also negative. This assay showed high specificity for the P. vivax aldolase and not the P. falciparum or human blood forms. Because of the scarcity and the difficulty in obtaining the other Plasmodium species (P. ovale, P. knowlesi), only the P. vivax, P. falciparum and P. malariae were tested. The high sensitivity and specificity observed in this assay makes it a favourable alternative to the low sensitivities observed in other commercial RDTs in the detection of P. vivax. Previous studies have also observed decreased levels of sensitivity in pLDH-specific RTDs for non-P. falciparum (P. vivax) at parasite densities above 5,000 parasites/μl and higher rate of false negative results in P. vivax infections