title: Novel monoclonal antibody against truncated C terminal region of Histidine Rich Protein2 (PfHRP2) and its utility for the specific diagnosis of malaria caused by Plasmodium falciparum.
abstract: An accurate diagnosis of malarial infection is an important element in combating this deadly disease. Malaria diagnostic test including, microscopy and other molecular tests are highly sensitive but too complex for field conditions. Rapid detection tests for P. falciparum infection using monoclonal antibodies (mAbs) against highly polymorphic PfHRP2 (Histidine Rich Protein2) are still most preferred test in field conditions, but with limitations such as specificity, and sensitivity leading to false positive and false negative results. To overcome these limitations, we carried out bioinformatics analysis PfHRP2 and PfHRP3 and found that the C-terminal region of PfHRP2 (~105 amino acids) displayed relatively lower sequence identity with PfHRP3. This C-terminal region of PfHRP2 contained unique peptide repeats and was found to be conserved in various isolates of P. falciparum. Moreover, this region was also found to be highly antigenic as predicted by antigenicity propensity scores. Thus we constructed a cDNA clone of the truncated PfHRP2 (recPfHRP2-T3) coding for C-terminal 105 amino acids and expressed it in E. coli and purified the polypeptide to homogeneity. The purified recPfHRP2-T3 was used as an antigen for development of both polyclonal and monoclonal antibody (mAb). The mAbs b10c1 and Aa3c10 developed against recPfHRP2-T3 was found to efficiently recognize recombinant PfHRP2 but not PfHRP3. In addition, the above mAbs reacted positively with spent media and serum sample of P. falciparum infection recognizing the native PfHRP2. The affinity constant of both the clones were found to be 10(9) M(-1). Quantitatively, both these clones showed ~4.4 fold higher reactivity with P. falciparum infected serum compared to serum from healthy volunteers or P. vivax infected patient samples. Thus these anti-C-terminal PfHRP2 mAbs (Aa3c10 and b10c1) display a very high potential for improvising the existing malarial diagnostic tools for detection of P. falciparum infection especially in areas where PfHRP2 polymorphism is highly prevalent.
authors.affiliation: Centre for Bio Separation Technology (CBST), VIT University, Vellore 632 014, India.
authors.forename: Reena
authors.initials: R
authors.lastname: Verma
authors.affiliation: Centre for Bio Separation Technology (CBST), VIT University, Vellore 632 014, India.
authors.forename: N S
authors.initials: NS
authors.lastname: Jayaprakash
authors.affiliation: Centre for Bio Separation Technology (CBST), VIT University, Vellore 632 014, India.
authors.forename: M A
authors.initials: MA
authors.lastname: Vijayalakshmi
authors.affiliation: Centre for Bio Separation Technology (CBST), VIT University, Vellore 632 014, India. Electronic address: krishnan.v@vit.ac.in.
authors.forename: Krishnan
authors.initials: K
authors.lastname: Venkataraman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000913:Antibodies, Protozoan / Q000276:immunology / Q000378:metabolism; D000918:Antibody Specificity; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006825:Hybridomas; D016778:Malaria, Falciparum / Q000175:diagnosis*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
chemical_list: D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; C514457:HRP-2 antigen, Plasmodium falciparum; D015800:Protozoan Proteins; D011994:Recombinant Proteins
keywords: Anti-PfHRP2 monoclonal antibody; Histidine Rich Protein3 (PfHRP3); Malaria diagnosis; Plasmodium falciparum; Plasmodium falciparum Histidine Rich Protein2 (PfHRP2); Plasmodium vivax