抗隐孢子虫单克隆抗体研制及其特异性的鉴定

PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST CRYPTOSPORIDIUM PARVUM

目的：制备抗隐孢子虫单克隆抗体（ＭｃＡｂ）并对其特异性进行鉴定。方法：用纯化的人源微小隐孢子虫卵囊免疫ＢＡＬＢ／ｃ小鼠，杂交瘤技术制备ＭｃＡｂ，间接免疫荧光（ＩＦＡ）和免疫印迹试验分析其特性。结果：制备出Ｚ４Ｃ８、Ｚ２Ｂ６、Ｚ３Ｄ７和Ｚ３Ｄ２四株能稳定分泌抗隐孢子虫ＭｃＡｂ的杂交瘤细胞株。经鉴定，Ｚ４Ｃ８和Ｚ３Ｄ７为ＩｇＭ，Ｚ３Ｄ２和Ｚ２Ｂ６为ＩｇＧ１，轻链均为κ链，与多种肠道病原体及肺孢子虫无交叉反应。除Ｚ２Ｂ６在间接免疫荧光试验中识别卵囊壁外，其余均识别隐孢子虫子孢子（ＣＳＰ）。免疫印迹定位表明，四株ＭｃＡｂ分别识别４２条ＣＳＰ抗原中的２０．５ｋＤａ、６０．５ｋＤａ、３３ｋＤａ和９５ｋＤａ４个条带，其中Ｚ４Ｃ８和Ｚ３Ｄ７均识别２０．５ｋＤａ主要抗原带。结论：这四株ＭｃＡｂ在识别ＣＳＰ抗原表位上具有不同的特性，但对隐孢子虫抗原均呈高度特异性反应。

AIM: To develop and characterize the monoclonal antibodies(McAbs) against Cryptosporidium parvum (C.parvum). METHODS: BALB/c mice were immunized with purified oocyst antigens of C.parvum from human isolates and McAbs against C.parvum were prepared by using hybridoma technique. The characterization of McAbs was studied by means of indirect immunofluorescent assay (IFA) and Western blot analysis. RESULTS: Four clones of hybridoma cell lines secreting McAbs against oocyst antigens of C.parvum were obtained. The immunoglobulin subclasses of these four McAbs were identifid as IgM (Z4C8,Z3D7) and IgG1(Z2B6,Z3D2) and their light chains were κ chain. IFA results showed that three McAb (Z4C8, Z3D7 and Z3D2) conjugates produced a diffuse fluorescence in the Cryptosporidium sporozoites (CSP) and one (Z2B6) produced local fluorescence in the oocyst wall of C.parvum. Four McAbs could recognize 4 of 42 polypeptide antigen bands of CSP with molecular weights of 20 5 kDa, 33 kDa,60 5 kDa and 95 kDa, among which both Z4C8 and Z3D7 could recognize 20 5 kDa band that was defined as a principal antigenic protein of CSP. CONCLUSION: These four McAbs possess their own characteristics of recognizing antigenic epitopes on CSP, but react specifically against antigens of C.parvum.