Hyperimmune equine IgG is widely used as antivenom and anti-rabies agents. This article discusses a membrane based enhanced hybrid bioseparation technique for efficient and scalable purification of equine immunoglobulin G (IgG) from horse serum. This technique is an improved version of a standard hybrid bioseparation technique developed within our group earlier for fractionation of human plasma proteins (Ghosh. 2004. J Membr Sci 237: 109-117). In the presence of a high antichaotropic salt concentration, equine IgG is selectively and reversibly captured within a stirred cell membrane module from horse serum, partly due to precipitation and microfiltration, and partly due to hydrophobic interaction based membrane adsorption, while the impurities are washed out from the device. The reversibly sequestered IgG is then released by lowering the salt concentration which favor both dissolution of the precipitated IgG and desorption of the membrane bound IgG. The enhanced hybrid bioseparation technique improves the IgG recovery from the membrane module by switching from a stirring to non-stirring mode during the IgG release phase. It also reduces membrane fouling by an appropriate pH switch. The effects of operating conditions on equine IgG capture were first systematically studied. The enhanced hybrid bioseparation technique was followed by an ultrafiltration step to remove ammonium sulfate and low molecular weight impurities. The equine IgG purity obtained under optimized conditions was 88% and its recovery was over 90%, both being significantly higher than corresponding values obtained using currently used purification techniques. 相似文献
An acidic phospholipase A2 enzyme (NnPLA2-I) interacts with three finger toxins (cytotoxin and neurotoxin) from Naja naja venom to form cognate complexes to enhance its cytotoxicity towards rat L6 myogenic cells. The cytotoxicity was further enhanced in presence of trace quantity of venom nerve growth factor. The purified rat myoblast cell membrane protein showing interaction with NnPLA2-I was identified as vimentin by LC-MS/MS analysis. The ELISA, immunoblot and spectrofluorometric analyses showed greater binding of NnPLA2-I cognate complex to vimentin as compared to the binding of individual NnPLA2-I. The immunofluorescence and confocal microscopy studies evidenced the internalization of NnPLA2-I to partially differentiated myoblasts post binding with vimentin in a time-dependent manner. Pre-incubation of polyvalent antivenom with NnPLA2-I cognate complex demonstrated better neutralization of cytotoxicity towards L6 cells as compared to exogenous addition of polyvalent antivenom 60–240 min post treatment of L6 cells with cognate complex suggesting clinical advantage of early antivenom treatment to prevent cobra venom-induced cytotoxicity. The in silico analysis showed that 19–22 residues, inclusive of Asp48 residue, of NnPLA2-I preferentially binds with the rod domain (99–189 and 261–335 regions) of vimentin with a predicted free binding energy (ΔG) and dissociation constant (KD) values of ?12.86 kcal/mol and 3.67 × 10?10 M, respectively; however, NnPLA2-I cognate complex showed greater binding with the same regions of vimentin indicating the pathophysiological significance of cognate complex in cobra venom-induced cytotoxicity. 相似文献
Objectives: To study the venom proteome composition of Southern India (SI) Common Krait (Bungarus caeruleus) and immunological cross-reactivity between venom against commercial antivenom.
Methods: Proteomic analysis was done by nano LC-MS/MS and toxins were quantitated by label-free analysis. The immunological cross-reactivity of venom towards polyvalent antivenom (PAV) was assessed by ELISA, Immunoblotting, and immuno-chromatographic methods.
Results: A total of 57 enzymatic and non-enzymatic proteins belonging to 12 snake venom protein families were identified. The three finger toxins (3FTx) (48.3%) and phospholipase A2 (PLA2) (37.6%) represented the most abundant non-enzymatic and enzymatic proteins, respectively. β-bungarotoxin (12.9%), a presynaptic neurotoxin, was also identified. The venom proteome composition is well correlated with its enzymatic activities, reported pharmacological properties, and clinical manifestations of krait envenomation. Immuno-cross-reactivity studies demonstrated better recognition of high molecular weight proteins (>45 kDa) of this venom by PAVs compared to low molecular weight (<15 kDa) toxins such as PLA2 and 3FTxs.
Conclusion: The poor recognition of <15 kDa mass SI B. caeruleus venom proteins is of grave concern for the successful treatment of krait envenomation. Therefore, emphasis should be given to improve the immunization protocols and/or supplement of antibodies raised specifically against the <15 kDa toxins of this venom. 相似文献
A potent toxin with phospholipase A2 (PLA2) and hemolytic activity in vitro was purified from the Russell's viper venom of eastern India (RVV-EI). The purified protein (RVV-PFIIc) of 15.3 kDa molecular weight, and a lethal toxicity dose (LD50 i.p.) of 0.1 mg/kg body weight, was the most toxic PLA2 so far reported from the Indian subcontinent. The material also possessed anticoagulant activity as it enhanced the prothrombin induced plasma clotting time in vitro. The PLA2 toxin (RVV-PFIIc) was shown to be different from other PLA2s of RVV in respect to one or more of these parameters e.g. molecular weight, isoelectric pH, in vivo toxicity, specific activity of the enzyme and certain other biological activities. The first 19 amino terminal sequence (NLFQFAEMIVKMTGKEAVH) of RVV-PFIIc showed variable degree of homology (42.1–94.7%) with those of other RVV-PLA2s described in the literature. Antisera raised against RVV-EI or RVV-PFIIc, though completely neutralized the in vivo lethal toxicity of RVV-EI or RVV-PFIIc, failed to inhibit their PLA2 activity in vitro thereby suggesting that in vivo toxicity and in vitro activity of the enzyme may not be directly related. Apart from RVV-PFIIc, at least two other PLA2 isozymes were found to be present in RVV-EI that were distinct from RVV-PFIIc in respect to their molecular, biological as well as serological properties. The significance of these and related data in antivenom therapy is discussed. 相似文献
Echis carinatus envenomation leads to severe tissue necrosis at the bitten site by releasing DNA from immune cells that blocks the blood flow. An earlier report has shown that exogenous DNase 1 offers protection against such severe local tissue necrosis. Tricosanthus tricuspidata is a medicinal plant and the paste prepared from its leaves has been used extensively for the treatment of snakebite-induced tissue necrosis. Most studies including reports from our laboratory focused on plant secondary metabolite as therapeutic molecules against snakebite envenomation. However, the involvement of hydrolytic enzymes including DNase in treating snake venom-induced tissue necrosis has not been addressed. Several folk medicinal plants used against snakebite treatment showed the presence of DNase activity and found to be rich in T. tricuspidata. Further, purified T. tricuspidata DNase showed a single sharp peak in reversed-phase high-performance liquid chromatography (RP-HPLC) with an apparent molecular mass of 17 kDa. T. tricuspidata DNase exhibited potent DNA degrading activity performed using agarose gel electrophoresis, spectrophotometric assay, and DNA zymography. In addition, purified DNase from T. tricuspidata was able to neutralize E. carinatus venom-induced mouse tail tissue necrosis and normalized elevated serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels 30 minutes post venom injection. T. tricuspidata DNase was also able to reverse E. carinatus venom-induced histopathological changes and collagen depletion in mice tail tissue. All these observed pharmacological actions of T. tricuspidata DNase were inhibited by sodium fluoride (NaF). This study provides scientific validation of the traditional use of T. tricuspidata leaf paste in the healing of snakebite-induced tissue necrosis and might be exploited to treat snake venom-induced local toxicity. 相似文献
Snakebite envenoming remains a neglected tropical disease which poses severe health hazard, especially for the rural inhabitants in Africa. In Nigeria, vipers are responsible for the highest number of deaths. Hydrophilic interaction liquid chromatography coupled with LC-MS/MS was used to analyze the crude venoms of Echis ocellatus (Carpet viper) and Bitis arietans (Puff adder) in order to understand their venom proteomic identities. Results obtained revealed that gel-free proteomic analysis of the crude venoms led to the identification of 85 and 79 proteins, respectively. Seventy-eight (78) proteins were common between the two snake species with a 91.8% similarity score. The identified proteins belong to 18 protein families in E. ocellatus and 14 protein families in B. arietans. Serine proteases (22.31%) and metalloproteinases (21.06%) were the dominant proteins in the venom of B. arietans; while metalloproteinases (34.84%), phospholipase A2s (21.19%) and serine proteases (15.50%) represent the major toxins in the E. ocellatus venom. Other protein families such as three-finger toxins and cysteine-rich venom proteins were detected in low proportions. This study provides an insight into the venom proteomic analysis of the two Nigerian viper species, which could be useful in identifying the toxin families to be neutralized in case of envenomation. 相似文献