Abstract：
Fish parvalbumins are heat-stable calcium-binding proteins that are highly cross-reactive in causing allergy symptoms in fish-sensitized patients. The reactivities of parvalbumin-specific monoclonal or polyclonal antibodies with parvalbumins of different fish species allowed their application for development of various immunoassays for allergen identification in fish samples. In this study, monoclonal antibodies (MAbs) were generated against two parvalbumins - natural Atlantic cod parvalbumin and recombinant common carp β-parvalbumin expressed in E. coli. Large collections of recombinant parvalbumins and natural allergen extracts of different fish species and other animals were used to identify the specificities of these MAbs using ELISA, Western blot, and dot blot. MAbs demonstrated different patterns of cross-reactivities with recombinant parvalbumins. Their binding affinities were affected by the addition and removal of Ca2+ ions. Moreover, all MAbs showed a broad reactivity with the target antigens in natural fish, chicken, and pork extracts. The ability of two MAbs (clones 7B2 and 3F6) to identify and isolate native parvalbumins from allergen extracts was confirmed by Western blot. Epitope mapping using recombinant fragments of Atlantic cod parvalbumin (Gad m 1) and common carp parvalbumin (Cyp c 1) revealed that 4 out of 5 MAbs recognize parvalbumin regions that contain calcium binding sites. In conclusion, the generated broadly reactive well-characterized MAbs against fish β-parvalbumins could be applied for investigation of parvalbumins of fish and other animals and their detection in allergen extracts.
摘要：
鱼周白蛋白是热稳定的钙结合蛋白，在鱼类致敏患者中引起过敏症状时具有高度交叉反应性。小白蛋白特异性单克隆或多克隆抗体与不同鱼类小白蛋白的反应性，使其可用于开发各种免疫测定法以鉴定鱼类样品中的过敏原。在这项研究中,产生了针对两种小白蛋白的单克隆抗体（MAb）-在大肠杆菌中表达的天然大西洋鳕鱼小白蛋白和重组鲤鱼β-小白蛋白。不同鱼类和其他动物的大量重组副白蛋白和天然过敏原提取物用于使用ELISA鉴定这些MAb的特异性。蛋白质印迹,和斑点印迹。MAb与重组小白蛋白表现出不同的交叉反应模式。它们的结合亲和力受Ca2离子的添加和去除的影响。此外，所有单克隆抗体都与天然鱼类的靶抗原表现出广泛的反应性，鸡肉,和猪肉提取物。通过Western印迹证实了两种MAb(克隆7B2和3F6)从变应原提取物中鉴定和分离天然小白蛋白的能力。使用大西洋鳕鱼小白蛋白（Gadm1）和鲤鱼小白蛋白（Cypc1）的重组片段进行的表位作图显示，5个MAb中有4个识别含有钙结合位点的小白蛋白区域。总之,所产生的针对鱼类β-小白蛋白的广泛反应性良好的MAb可用于鱼类和其他动物的小白蛋白的研究及其在过敏原提取物中的检测。
