葎草花粉致敏蛋白多克隆抗体的制备及其在筛选致敏蛋白组分的应用

目的制备、鉴定兔抗葎草花粉蛋白多克隆抗体,并初步应用于葎草花粉致敏蛋白组分的筛选。方法用葎草花粉变应原浸液免疫兔,获得高效价抗葎草花粉蛋白抗血清,应用琼脂免疫双扩散、ELISA法以及Western blotting鉴定产生的抗体。同时对葎草花粉过敏性哮喘患者血清进行Western blotting检测。结果兔抗血清效价经ELISA检测,效价>1∶10000,免疫双扩散结果表明抗血清只与葎草花粉变应原形成特异性IgG沉淀线,Western blotting检测兔抗血清IgG可以特异性识别葎草花粉8种蛋白组分,分子质量分别为100、88、76、69、65、55、49、38 ku,患者血清IgG可以识别5种蛋白组分,分子质量分别为100、76、55、493、8 ku。结论制备的兔抗血清具有较高效价和较好的特异性,应用该血清筛选出的葎草花粉致敏蛋白组分与患者血清筛选的组分相似,因此可进一步应用于初筛cDNA表达文库。

Preparation of anti-Humulus pollen protein polyclonal antibody and its application in determining the composition of Humulus pollen allergic protein

Objective To prepare and characterize the rabbit anti-Humulus pollen protein polyclonal antibody,and identify its application in determining the composition of Humulus pollen allergic protein.Methods The polyclonal antibody was obtained by immunizing rabbits with the crude extracts of Humulus pollen.It was primarily identified using indirect ELISA and double immuno-diffusion.The specificity of polyclonal antibody was detected with Western blotting,which applied rabbit antiserum and sera of known Humulus pollen allergic patients.Results The antibody had high specificity to Humulus pollen protein.The IgG-immunoblotting analysis of rabbit antiserum showed that the protein bands of molecular mass were 100,88,76,69,65,55,49 and 38 ku.The results of analyzing 10 sera of known Humulus pollen allergic patients showed that the protein bands of molecular mass were 100,76,55,49 and 38 ku.Conclusion Experimental results show that the proposed method can be used to prepare high titer and specific rabbit anti-Humulus pollen protein polyclonal antibody.It has similar ability in determining the composition of Humulus pollen allergic protein compared with sera of patients.Therefore,it can be used to immunoscreen cDNA library.