抗弓形虫速殖子单克隆抗体的制备与特性研究

STUDY ON THE PREPARATION AND CHARATERIZATION OF A MONOCLONAL ANTIBODY TO TOXOPLASMA GONDII TACHYZOITES

目的制备抗弓形虫速殖子单克隆抗体（Mab），并对其特性进行分析，探讨其在弓形虫病诊断中的作用。方法利用杂交瘤技术制备抗弓形虫速殖子Mab。用间接ELLSA法测定活性，以琼脂糖免疫双扩散鉴定亚类，通过SDSPAGE和Westemblot分析该Mab，所识别的抗原分子量，以IFA确定抗原位点在虫体的定位，并对其保护性及特异性进行分析。通过Mab-ELISA法检测弓形虫抗原，以研究其在弓形虫病血清学诊断中的意义。结果抗弓形虫MabF7C8H12属IgG1亚类，识别抗原的分子量为16．5和24kDa。以固定的完整虫体进行IFA，未显示荧光。竞争抑制试验显示50％抑制率时的抗原浓度为40μg／ml。将RH株速殖子与Mab腹水进行孵育后接种子小鼠腹腔，并不能延长小鼠的平均死亡时间。以Mab-ELISA法检测人工溶于PBS和正常人血清中的弓形虫RH株速殖子抗原，最小检出量为0．78μg／ml和1．5μg／ml。RH株速殖子103／只感染小鼠，分别于第6d和第8d能检出循环抗原。结论抗弓形虫速殖子MabF7C8H12是研究弓形虫和弓形虫病的很好探针，其应用前景值得进一步研究。

Aim To prepare and identify monoclonal antibody (Mab) specific for Toxoplasma gondii tachyzoites.Method The Mab specific for Toxoplasma gondii tachyzoite were prepared via hybridoma technique.Indirect ELLSA was used to determine the activity of the Mab.Agarose double immuodiffusion test was performed to confirm subclass and SDS-PAGE & westem blot were used to analysis molecular weight of the antigen(s) recignized by the Mab.IFA was used to identify the epitope of Toxoplasma gondii tachyzoites.The protection and specificity of the Mab were analysed at same time.The Mab was tested in Mab-ELISA method to detect Toxoplasma gondii antigen.Results A Mab F7C8H12 specific for T. gondii was produced.It belongs to IgG1 subclass.Molecular weight of the antigens recognized by the Mab was 6.5 and 24 kDa.IFA did not show fluorescence in intact tachyzoite.Inhibition test showed that the inhibition rate was 50% when the concetration of the antigen was 40μg/ ml.After the RHI strain tachyzoites were incubated with Mab ascites, mice were injected with the tachyzoites through peritoneum.The - showed that the mean dead time of mice were not delayed . T. gondii antign mixed with PBS and noed human serum was detected by Mab-ELISA, the sensitivity was 0.78μg/ml and 1.5μg/ml respectively.When mice were infected withT.gondii RH strain tacyzoites, 103/mouse P.i., circulating antigen could be detected in 6 day and 8 day.Conclusion The Mab (F7C8H12) to T. gondii tachyzoites is an excellent probe for studying T. gondii and toxoplasmosis.