定向偶联制备三肽囊素人工抗原及其多克隆抗体的研制

摘要

为研制三肽囊素(Bursin,KHG-NH2,BS)抗体,采用EDC法将三肽囊素分子赖氨酰(K)上的氨基与载体蛋白(BSA、OVA)定向偶联,分别制备免疫抗原(BSA-KHG-NH2)和检测抗原(OVA-KHG-NH2).以BSA-KHG-NH2免疫5只BALB/c小鼠,经4次免疫后,均产生了较高的抗体水平,其中5号小鼠血清抗体效价达1∶125 000.免疫小鼠血清多克隆抗体与检测抗原的结合不仅能被三肽囊素阻断,而且能被GKHG-NH2四肽特异性阻断,但不能被KHGK四肽阻断,表明本试验制备的三肽囊素人工抗原是由载体蛋白的羧基与三肽囊素分子赖氨酰(K)上的氨基定向偶联而成.利用小鼠多克隆抗体建立了三肽囊素间接竞争ELISA检测方法,线性范围为15～1 000 ng/mL,IC50为160 ng/mL.%In order to produce polyclonal antibodies against Bursin, and develop immunological detective methods for Bursin (BS) , the amino group on - Lys of Bursin molecules were conjugated to bovine serum albumin ( BSA) or chicken ovalbumin ( OVA) by EDC method. The titer of anti-Bursin antibodies in one BLAB/c mice serum, immunized by BSA-KHG-NH2, was about 1:125000, in which polyclonal antibodies binding to OVA-KHG-NH2 could be inhibited by free Bursin and GKHG-NH2, could not be inhibited by KH-GK peptide. These results indicated that the immunized mice produced specifical antibodies against bursin, and the amino group on-Lys of Bursin were conjugated to BSA or OVA. A competitive indirect ELISA ( CI -ELISA) with the anti - Bursin polyclonal antibodies for detecting Bursin was developed, in which the linear range of the inhibition curve was 15-1 000 ng/mL, and the IC50 was 160 ng/mL.