N. S. Jayaprakash

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

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.

Production of Human Anti-Glycophorin-A Monoclonal Antibodies and Their Purification by Pseudoaffinity Chromatography Using a Convective Interaction Media Monolithic Column

Monoclonal antibodies (MAbs) against glycophorin-A (GPA) could be used in identifying MN blood groups, detecting specific markers of erythroid differentiation, and studying parasite interactions. Large-scale production of MAbs in bioreactors demands an efficient and rapid separation technology. The present study describes the production of a human anti-GPA monoclonal antibody and its purification using a pseudo-bioaffinity L-histidine-convective interaction media (CIM) monolithic column. Hybridomas were generated by fusion of mouse myeloma cell line (Sp2/0) and spleen cells from the mouse immunized with Triton X-100 solubilized RBC membrane proteins. Hybridomas producing antibodies specific to commercial glycophorin-A were screened by indirect enzyme-linked immunosorbent assay (ELISA). The antibodies produced by the stable clones were found to be IgG1 with kappa light chain. Purification of IgG1 MAbs from the cell culture supernatant carried out with a CIM-EDA-histidine disk resulted in high specific activity with purification fold of 8.3 in the fraction eluted with MOPS buffer containing 0.2 M NaCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA showed that the antibodies obtained were highly pure, with high antigen-binding efficiency. The results indicate that faster separation and efficient recovery of high-purity anti-GPA MAbs could be achieved by using CIM-EDA-histidine disk.

Development and Characterization of Monoclonal Antibodies Against Nitro-166Tyrosine of High-Density Lipoprotein: Apolipoprotein A1

Apolipoprotein A1 (ApoA1) of the high-density lipoprotein (HDL) plays a cardinal role in alleviating atherosclerosis in various ways. Its role in reverse cholesterol transport is preeminent. However, the ApoA1 undergoes oxidation under chronic inflammatory conditions and these oxidations are mediated by myeloperoxidase. It has been reported that the oxidation of the amino acids such as methionine, tyrosine, and tryptophan residues at specific sites of ApoA1 renders it not only dysfunctional but also proinflammatory and proatherogenic. Thus, assessing the quality of ApoA1 and, in turn, that of HDL in circulating blood can serve as an early diagnostic tool for cardiovascular diseases (CVDs). In this study, we developed monoclonal antibodies (mAbs) specific to modified ApoA1 with its tyrosine residue at the 166th position nitrated to 3-nitrotyrosine. A 20 amino acid peptide around the modification of interest was designed using an antigenicity prediction tool. The peptide was custom synthesized with ovalbumin as conjugate and used as an antigen to immunize BALB/c mice. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells with spleen cells from the immunized mouse. A hybridoma clone 2E5B7, thus developed and characterized, was found to secrete mAb of the desired specificity and sensitivity against nitrated 166Tyrosine. The lowest concentration of the antigen that could be detected by the mAb with confidence was 15 ng. The mAb was able to detect nitrated 166Tyrosine peptide ovalbumin conjugate antigen spiked in human plasma with high specificity. The generated mAb could be potentially used in immuno-based diagnostic systems to screen the quality of HDL and in turn assess CVD risks in humans.

Diagnostic potential of monoclonal antibodies developed against C-terminal polypeptide of P. falciparum Histidine Rich Protein2 (PfHRP2) in malaria infected patients from India

Abstract Malaria, caused by Plasmodium falciparum has become a major health burden in most tropical and developing countries. P. falciparum Histidine Rich Protein2 (PfHRP2), which exhibits polymorphism, is being widely used as a diagnostic marker. Recently, we reported the development of monoclonal antibodies against conserved C-terminal 105 amino acids of PfHRP2 for malaria diagnosis. Now, in this study, the diagnostic performance of two anti-C-terminal PfHRP2 mAbs (b10c1 and Aa3c10) were evaluated with 100 blood samples from clinically identified malaria patients from seven different geographical centers in India. Sandwich ELISA, polymerase chain reaction (PCR) and statistical tools were used for the evaluation of the performance of the anti-C-terminal PfHRP2 mAb. These mAbs detected P. falciparum (mean OD value 1.525 ± 0.56) malaria with great accuracy with no cross reactivity with P. Plasmodium vivax (mean OD value 0.285 ± 0.051) and normal healthy control samples (mean OD value 0.185 ± 0.06) in Sandwich ELISA assay. The samples which were RDT negative for P. falciparum were also reactive in Sandwich ELISA with mean OD value of (1.303 ± 0.532). The amount of PfHRP2 antigen in the patients’ blood sample was quantified and categorized into three distinct groups having the HRP2 antigen in high, intermediate and low amounts. The presence of Pfhrp2 gene was also confirmed by PCR analysis. The sensitivity and specificity of the mAb were found to be 95 and 96% respectively. These data strongly suggest that the anti-C-terminal PfHRP2 mAbs b10c1 and Aa3c10 have merits for improvising the existing malarial diagnostics.