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.
Mixed-mode chromatography sorbents n-hexylamine HyperCel™ (HEA) and phenylpropylamine HyperCel™ (PPA) were evaluated for the study of adsorption of catalase from two different sources. Various parameters such as buffer composition, ionic strength and pH were investigated to study the mechanism of interaction of commercially available pre-purified catalase from Bovine liver, purified catalase from black gram (Vigna mungo) and crude extract of black gram containing catalase with these mixed-mode ligands. A simple and economical screening protocol for identifying optimal buffer conditions for adsorption and desorption of catalase was established with micro volumes of the sorbent in batch mode. With HEA HyperCel, it was observed that pre-purified catalase from both bovine liver and black gram was completely retained at pH 7.0, irrespective of the presence or absence of NaCl in the adsorption buffer, whereas the catalase from crude extract of black gram was completely retained only in the presence of 0.2 M salt in the adsorption buffer. The elution of catalase from both the sources was accomplished by lowering the pH to 4.5 in absence of salt. In case of PPA HyperCel, catalase from both the sources was very strongly adsorbed under different buffer conditions studied, and elution did not yield a significant catalase activity. From the screening experiments, it could be concluded that the interaction of catalase with HEA HyperCel could be dominated by hydrophobic forces with minor contributions from ionic interaction and with PPA HyperCel, it could be a combination of different non-covalent interactions acting on different loci on the surface of the protein.
Fishes counteract certain microbial attacks in water by producing antimicrobial proteins/peptides in their skin surface. The present study focused on screening the bactericidal activity of skin and skin mucus extracts of Catla catla and Channa striatus. The bactericidal activity was assessed against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris, Aeromonas hydrophila, Staphylococcus aureus and Bacillus coagulans by disc diffusion method. The minimal inhibitory concentration was also determined. Protein profiles in skin and skin mucus extracts were analyzed by SDS-PAGE. Samples from both fishes showed antibacterial activity. Detailed analysis of individual protein and peptide would throw light on their medicinal importance to be used against pathogenic microbes.
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