In the present study, monoclonal antibodies (MAbs) against recombinant histidine-rich protein (rHRP3) were developed and assessed for their potential in detection of Plasmodium falciparum HRP3. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells and spleen cells from the mouse immunized with purified rHRP3. Three MAbs (IgG1 isotype) specific to rHRP3 were established and characterized by enzyme-linked immunosorbent assay (ELISA) and immunoblotting for sensitivity and specificity. Purification of MAbs from hybridoma cell culture supernatant and PAbs from rabbit anti-serum were carried out using Phenylpropylamine (PPA) HyperCel™ sorbent. The MAbs were able to detect rHRP3 and the HRP3 from P. falciparum spent medium. Sandwich ELISA was developed to quantify HRP3 in the spent medium of P. falciparum culture. The generated MAbs could be potentially used in immuno-based diagnostic systems for the detection of P. falciparum HRP. © 2013 Mary Ann Liebert, Inc.

Jayaprakash N S

Centre for Bio-Separation and Technology

Vellore Campus

Palani B

Ranjini S.S

Vijayalakshmi M.A.

Vellore Institute of Technology (VIT) is a private university located in&nbsp;Tamil Nadu, India. Founded in 1984, as Vellore Engineering College, the institution offers 20 undergraduate, 34 postgraduate, four integrated and four research programs. It has campuses in Vellore, Amravati, Bhopal and Chennai.

VIT is one of the top ranked private universities in India according to NIRF, THE and QS Rankings.&nbsp;Govt. of India has recognized&nbsp;VIT, Vellore as an&nbsp;Institution of Eminence. This has allowed VIT to take independent quality initiatives and move up in world ranking.

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VIT University

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

HDL-ApoA1 plays a pivotal role in the prevention of atherosclerosis and cardiovascular diseases. ApoA1 purification from blood plasma has always remained tedious, involving multiple steps, large volumes of plasma and substantial loss in the final yield of pure ApoA1. In this study, a two-step method has been developed and optimized for the purification of ApoA1 from plasma. Plasma was first subjected to 60% ammonium sulphate (NH4)2SO4 precipitation and subsequently, ApoA1 was recovered using mixed mode chromatographic sorbent, HEA HyperCel™. ApoA1 was found to be enriched in 60% (NH4)2SO4 supernatant that was dialyzed and injected onto HEA sorbent with 50 mM phosphate buffer pH 7.4. The bound proteins were eluted by decreasing the pH in step-gradient from pH 7.4 to pH 4.0 and subsequently to pH 3.5 using 50 mM sodium acetate buffer. Gel electrophoresis showed elution of homogeneous apoA1 at pH 3.5, with purity and yield of 63%. An interesting feature of this approach is that the purified ApoA1 was monomeric with a mass of 28,079.30 Da as confirmed by MS analysis. This simple and efficient method of purification of apoA1 serves as an alternative method which can be combined with traditional approaches and has a great potential for biochemical and clinical studies. © 2017 Elsevier B.V.

Journal of Chromatography B

Efficient purification of Apolipoprotein A1 (ApoA1) from plasma by HEA HyperCel™: An alternative approach

Adjuvants play an important role in eliciting immune responses and subsequent generation of antibodies with high specificity. Recently, poly(N-isopropylacrylamide) (PNiPAAm), also known as a "smart" polymer, has been proposed as a potential adjuvant for making antibodies and vaccines. This material exhibits efficient delivery, protection against degradation, and preservation of antigen epitopes. In this work, we used both CFA and smart polymer to develop a highly specific murine monoclonal antibody (mAb) against recombinant truncated histidine rich protein2 (HRP2) of Plasmodium falciparum. Our results indicate that the mAbs developed using these adjuvants were able to recognize recombinant HRP2 and native PfHRP2 protein from spent medium. The mAbs generated against recombinant truncated HRP2 showed better sensitivity to the antigen and importantly mAbs generated using PNiPAAm adjuvant were in the range of 108-109 M-1. The mAbs generated using PNiPAAm are very efficient and sensitive in detecting HRP2. To the best of our knowledge, this is the first report of such comparison having been made between these two adjuvants and we propose that the smart polymer has huge potential as an alternative to CFA. Additionally, we discuss the utility of the mAbs generated through PNiPAAm for specific diagnosis of malaria caused by P. falciparum. © 2015 WILEY-VCH Verlag GmbH &amp; Co. KGaA, Weinheim.

Biotechnology Journal

Adjuvant poly(N-isopropylacrylamide) generates more efficient monoclonal antibodies against truncated recombinant histidine-rich protein2 ofPlasmodium falciparumfor malaria diagnosis

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 109 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. © 2015 Elsevier Inc.

Experimental Parasitology

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

Journal of Molecular Recognition

Study of catalase adsorption on two mixed-mode ligands and the mechanism involved therein

Study of the mechanism of interaction of antibody (IgG) on two mixed mode sorbents

Acta Tropica

Genetic deletion of HRP2 and HRP3 in Indian Plasmodium falciparum population and false negative malaria rapid diagnostic test

Journal of Clinical Microbiology

Identification of Optimal Epitopes for Plasmodium falciparum Rapid Diagnostic Tests That Target Histidine-Rich Proteins 2 and 3

PLoS ONE

Transcription and Expression of Plasmodium falciparum Histidine-Rich Proteins in Different Stages and Strains: Implications for Rapid Diagnostic Tests

Development, Purification, and Characterization of Monoclonal Antibodies Against Recombinant Histidine-rich Protein 3 of Plasmodium falciparum

Journal	Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Publisher	Mary Ann Liebert Inc
ISSN	2167-9436
Open Access	0