岩沙海葵毒素人工抗原的合成及鉴定

Synthesis and identification of conjugate antigen of palytoxin

目的制备岩沙海葵毒素(PTX)的人工抗原和抗血清,为建立PTX的酶联免疫检测方法提供技术基础.方法用小分子succinimidyl 3-(2-pyridyldithio) propionate(SPDP)将PTX巯基化成半抗原PTX-PDP,用2-iminothiolane将大分子载体蛋白质匙眼血蓝蛋白(KLH)巯基化成KLH-SH,再将PTX-PDP和KLH-SH反应合成人工抗原PTX-PDP-KLH免疫雌性6～8周龄Balb/c小鼠,进行多抗的制备.用同样的方法制备酶联免疫反应之包被抗原PTX-PDP-BSA,用于检测血清抗体效价.结果根据紫外检测图谱,PTX的最大吸收波长在264 nm处,KLH及BSA的最大吸收波长在278及279 nm处,而合成的人工抗原PTX-PDP-KLH和PTX-PDP-BSA的最大吸收波长分别在267和273 nm处.抗血清经间接酶联免疫吸附法检测,效价达到1∶3 200,且与正常Balb/c小鼠、昆明种小鼠及兔血清间无交叉反应.结论合成的人工抗原纯度高,具有较好的免疫原性.

Purpose To produce the conjugates of keyhole limpet hemocyanin and chemically defined palytoxin haptens. Methods Palytoxin haptens were produced by derivatization of the primary amino group with succinimidyl 3-（2-pyridyldithio）propionate. Keyhole limpet hemocyanin was thiolated by 2-iminothiolane. Artificial antigens synthesized by conjugating PTX-PDP to KLH-SH were used to immunized Balb/c mice to obtain polyclonal antibodies. PTX-PDP-BSA was produced through the same method and was used to the determination of serum antibody titers. Results According to the pictures of ultraviolet probe, the maximum absorption wave length of PTX was in 264 nm and KLH in 278 nm. However the maximum absorption of PTX-PDP-KLH and PTX-PDP-BSA were in 267 nm and 273 nm respectively. The serum antibody titers reached to 1：3 200. Conclusion The antigen is very effective. The research provides the preparation for producing McAb of PTX and setting up the ELISA method of detecting PTX.