Browsing by Author "Banerjee, Siddharth"
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Item Detection of pyrogenicity on medical grade polymer materials using rabbit pyrogen, LAL and ELISA method(JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, 2011)The objective of the study is to detect the pyrogenicity of five medical grade gelatinous polymer materials, intended for the manufacturing of capsule for pharmaceutical applications, by an indigenously developed ELISA, LAL and rabbit pyrogen assays. The ELISA methodology includes the incubation of the sample extract with blood from a healthy donor at 37 degrees C. Any pyrogen present in the extract induces the IL-1 beta which can be determined by ELISA. The rabbit pyrogen and LAL assays were performed as per standards. The result of the ELISA method indicated that all the materials extract induced high level of IL-1 beta as a marker for pyrogenicity. The rise in temperature of rabbit pyrogen was above 0.5 degrees C in all materials extract. LAL assay induced an endotoxin level above 0.5 EU. All the five polymer materials were found pyrogenic in all the assays. The ELISA method is very sensitive because the lowest limit of detection was 10 pg/ml endotoxin. Hence it can be concluded that the ELISA method will be an added advantage for the quality control release of a batch of medical products and improving the existing methodologies in the context of reduction and replacement in the use of animal models. (C) 2011 Elsevier B.V. All rights reserved.Item Inflammatory response to pyrogens determined by a novel ELISA method using human whole blood(JOURNAL OF IMMUNOLOGICAL METHODS, 2011)Presence of pyrogens on implants, medical devices, drugs and biological materials compromise on the biosafety and poses a major health hazard in therapeutics. Detection of pyrogenic contamination has so far been done with either in vivo rabbit pyrogen assay or Limulus Amoebocyte Lysate (LAL) methods, each of which having their distinct advantages and disadvantages. An indigenously developed ELISA method quantifying the pro-inflammatory response triggered by pyrogens on human whole blood is demonstrated for its versatility to detect the pyrogenic response to gram-negative, gram-positive bacteria, chemical and biological pyrogens. The method was used to test and quantitate the pyrogen levels in polymeric biomaterials. Unlike the existing pyrogen test procedures, this assay is adapted to detect all pyrogens, besides yielding faster, sensitive and quantifiable data, thereby reduce/replace animal experimentation. The method also provided insight into the possible correlation between variable blood profile among individuals and their role in determining inflammatory response to different pyrogenic stimuli. (C) 2011 Elsevier B.V. All rights reserved.Item Investigation of interleukin-1 beta release from cryopreserved blood stimulated with endotoxin(CRYOBIOLOGY, 2011)The feasibility of an indigenously developed ELISA method to determine cytokine response to wide spectrum of pyrogenic stimuli utilizing fresh human whole blood is limited by the availability of healthy donors. The possibility of using cryopreservation of pooled human blood for detection of cytokine response to lipopolysaccharide is explored in this study. The effect of cryopreservation on blood parameters, cellular morphology and cytokine response were compared with that of the pooled fresh blood and cryopreserved blood from single and multiple donors. In vitro pyrogenic stimulation with 0.5 and 5 EU of LPS was monitored on fresh and cryopreserved pooled blood from single and multiple donors. The release of IL-1 beta was quantitated by Sandwich ELISA (1, 10, 25,45 and 75 days) after storage. The results indicated that the cryopreserved blood displayed enhanced IL-1 beta release on stimulation with LPS, when compared to fresh blood. The maximum release of IL-1 beta level was observed at 2 h when 5 EU of LPS was treated with pooled fresh blood which is similar to that of fresh blood. After 75 days storage of pooled cryopreserved blood the IL-1 beta release was maximum at 9 and 15 h when treated with 5 and 0.5 EU of LPS. Observations of the study suggest that cryopreserved pooled blood is an economically and experimentally viable alternative to fresh blood. This investigative study promises short term storage and regular supply of non-allergic, pathogen free human blood for the detection of interleukin-1 beta for the evaluation of in vitro pyrogenicity. (C) 2011 Elsevier Inc. All rights reserved.Item The cytoplasmic C2A domain of synaptotagmin shows sequence specific interaction with its own mRNA(BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2008)Synaptotagmin-1 (Syt1) is essential in Ca2+-dependent neurotransmitter release, but its expression regulation is unknown. Here we report that the cytoplasmic Syt1 fragment forms ribonucleoprotein complex by interacting with the 3' untranslated region (3'UTR) of its own mRNA. Two protein-binding domains, GU(15) repeat and GUCAAUG, within the Syt 3'UTR and the C2 domains in Syt1, especially C2A, are essential in this ribonucleoprotein complex formation. Furthermore, in in vitro assay the translation efficiency of Syt1 mRNA was downregulated in presence of 3'UTR. These results demonstrate for the fist time that the soluble fraction of Syt1 can interact with its own mRNA in a highly sequence specific manner. (C) 2008 Elsevier Inc All rights reserved.