Puzari Upasana, Khan Mojibur R, Mukherjee Ashis K
Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Sciences, Tezpur University, Tezpur, 784028, Assam, India.
Division of Life Sciences, Institute of Advanced Study in Science and Technology, Vigyan Path Garchuk, Paschim Boragaon, Guwahati, 781035, Assam, India.
Toxicon X. 2024 Aug 18;23:100203. doi: 10.1016/j.toxcx.2024.100203. eCollection 2024 Sep.
Indian red scorpion is responsible for substantial mortality in India and Sri Lanka; however, no specific diagnostic method is available to detect the venom of this scorpion in envenomed plasma or body fluid. Therefore, we have proposed a novel, simple, and rapid method for detecting venom (MTV) in the plasma of envenomed animals using polyclonal antibodies (PAb) raised against three modified custom peptides representing the antigenic epitopes of K (Tamapin) and Na (α-neurotoxin) channel toxins, the two major MTV toxins identified by proteomic analysis. The optimum PAb formulation containing PAb 1, 2, and 3 in proportion (1:1:1, w/w/w) acted synergistically, demonstrating significantly higher immunological recognition of MTV than anti-scorpion antivenom (developed against native toxins) and individual antibodies against peptide immunogens. The PAb formulation could detect MTV optimally in envenomed rat plasma (intravenous and subcutaneous routes) at 30-60 min post-injection. The acetonitrile precipitation method developed in this study to augment the MTV detection sensitivity enriched the low molecular mass peptide toxins in envenomed rat plasma, which was ascertained by mass spectrometry analysis. The gold nanoparticles conjugated PAb formulation, characterised by biophysical techniques such as Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM), demonstrated their interaction with low molecular mass MTV peptide toxins in envenomed rat plasma. This interaction results in the accumulation of the gold nanoparticles, thus leading to signal change in absorbance spectra that can be discerned within 10 min. From a standard curve of MTV spiked plasma, the quantity of MTV in envenomed rat plasma could be determined by gold nanoparticle-PAb formulation conjugate.
印度红蝎子在印度和斯里兰卡造成了大量死亡；然而，目前尚无特定的诊断方法可用于检测中毒血浆或体液中这种蝎子的毒液。因此，我们提出了一种新颖、简单且快速的方法，用于使用针对三种修饰的定制肽产生的多克隆抗体（PAb）来检测中毒动物血浆中的毒液（MTV），这三种肽代表了通过蛋白质组学分析鉴定出的两种主要MTV毒素——K（塔马平）和Na（α-神经毒素）通道毒素的抗原表位。含有PAb 1、2和3按比例（1:1:1，w/w/w）的最佳PAb制剂具有协同作用，与抗蝎毒血清（针对天然毒素开发）和针对肽免疫原的单个抗体相比，对MTV的免疫识别能力显著更高。该PAb制剂可在注射后30 - 60分钟在中毒大鼠血浆（静脉内和皮下途径）中最佳地检测MTV。本研究开发的乙腈沉淀法提高了MTV检测灵敏度，富集了中毒大鼠血浆中的低分子量肽毒素，这通过质谱分析得以确定。通过傅里叶变换红外光谱（FTIR）和透射电子显微镜（TEM）等生物物理技术表征的金纳米颗粒偶联PAb制剂，证明了它们与中毒大鼠血浆中低分子量MTV肽毒素的相互作用。这种相互作用导致金纳米颗粒的积累，从而导致吸光度光谱中的信号变化，在10分钟内即可辨别。根据MTV加标血浆的标准曲线，可通过金纳米颗粒 - PAb制剂偶联物测定中毒大鼠血浆中MTV的含量。
