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 Tyrosine. The lowest concentration of the antigen that could be detected by the mAb with confidence was 15 ng. The mAb was able to detect nitratedTyrosine 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.
Endophytes are microbes that reside inside the plant without affecting the host. A highly active species of extracellular protease production is Bacillus species. This study aims at optimization and partial purification of fibrinolytic protease enzyme production from the isolate Bacillus toyonensis VKB5 sp. isolated from the fruit Actinidia delicosa. Optimization studies on medium components show that the enzyme production can be achieved in alkaline conditions with nutrient sources mannitol, yeast extract, ammonium chloride. The statistical optimization studies using Plackett-Burman and Response surface methodology determines that the interaction of yeast extract with the mannitol and ammonium chloride enhances the enzyme production. Studies on purification stages describe that the protease purified was 7.4 – fold purer that the crude and 68.9% enzyme was recovered comparing the crude. The molecular weight determined was found to be 21.9 kDa using SDS-PAGE. The purity of the protease was analyzed using HPLC. Further studies on the effect of temperature, pH, inhibitors, detergents and metal ions confirm that the enzyme purified can be alkaline serine protease that withstands thermal condition up to 60°C.
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