抗卵巢癌相关抗原CA125单抗及其磁酶诊断试剂的制备

Preparation of monoclonal antibody against ovary cancer antigen 125 and its magnetic enzyme diagnostic kit

目的制备卵巢癌相关抗原CA125（cancer antigen 125）磁酶检测试剂,并进行验证及初步应用。方法应用CA125抗原免疫BALB/c小鼠,通过细胞融合,筛选效价高、稳定性分泌抗体的杂交瘤细胞株后,接种小鼠腹腔,收集腹水,制备抗CA125单抗,并对其亚型、特异性、相对亲和力、抗原结合位点及抗原决定簇进行鉴定。通优化反应条件制备CA125抗原磁酶检测试剂,并进行验证后,将其应用于50份女性健康体检者及100份卵巢癌患者血清的检测。结果共获得3株阳性杂交瘤细胞株,分别命名为4B6、2G10、1C2;1C2、4B6和2G10单抗的抗体亚型分别为Ig G2a、Ig G1和Ig G1,轻链均为κ链,相对亲和力依次为2G10〉4B6〉1C2,与AFP、CA50、CEA、CA199、CRP、CA242和小牛血清未发生交叉反应,1C2单抗识别位点与2G10和4B6不同,且3株单抗的抗原决定簇均在CA125蛋白部分上。确定该磁酶检测的反应条件为：包被抗体按100μg/ml,37℃包被1 h;20%小牛血清37℃封闭1 h或4℃封闭过夜;酶标抗体最佳反应时间为45 min。当CA125浓度在10~500 U/ml时,与对应的A450值线性关系良好,R2=0.992;该检测试剂最低检测极限约10 U/ml,灵敏度高;与人血清白蛋白、小牛血清、CA50、CEA、AFP、CRP、CA199和CA242均未发生反应,特异性高;于37℃放置4 d的CA125 A450值与2~8℃相比,差异无统计学意义（P〈0.05）;批内及批间变异系数均〈10%,重复性较好;高、中、低3个浓度的CA125抗原的回收率在94.45%~105.21%之间,准确性高。50份健康体检者血清中CA125阳性率为4%,100份卵巢癌患者血清中CA125阳性率为88%。结论已成功制备了卵巢癌相关抗原CA125磁酶检测试剂,该试剂具有应用于卵巢癌患者的诊断价值。

Objective To prepare,verify and preliminarily apply the magnetic enzyme diagnostic kit for ovary cancer antigen（CA）125. Methods BALB / c mice were immunized with CA125,based on which the high titer hybridoma cell strain stably secreting antibody was collected by cell fusion and inoculated i.p. to mice. The ascites of mice were collected,with which monoclonal antibody（Mc Ab） against CA125 was prepared and identified for subtype,specificity,relative affinity,antigen binding site and antigen determinant. Magnetic enzyme diagnostic kit was prepared by optimization of reaction condition then verified and used for determination of 50 serum samples from healthy women and 100 from patients with ovary cancer. Results Three positive hybridoma cell strains were obtained and named as 4B6,2G10 and1C2,of subtypes Ig G2 a,Ig G1 and Ig G1,respectively. All the light chains of the Mc Abs were κ chains. In the order of relative affinities,the Mc Abs were 2G10,4B6 and 1C2,which showed no cross reactions with AFP,CA50,CEA,CA199,CRP,CA242 and calf serum. The recognition site of Mc Ab 1C2 was different from those of 2G10 and 4B6.However, all the antigen determinants of the three Mc Abs were on CA125 protein. The reaction condition for determination by the magnetic enzyme kit was as follows： coating antibody at a concentration of 100 μg / ml was incubated at 37 ℃ for 1 h; 20% calf serum was blocked at 37 ℃ for 1 h or at 4 ℃ overnight; the optimal reaction time of enzymelabeled antibody was 45 min. CA125 concentration at a range of 10 ~ 500 U / ml showed good linearity to the corresponding A450,with a R2 of 0. 992. The minimum detection limit of the kit was about 10 U / ml,indicating a high sensitivity. No reactions with human serum albumin,calf serum,CA50,CEA,AFP,CRP,CA199 and CA242 were observed,indicating a high specificity. The A450 value of CA125 stored at 37 ℃ for 4 d showed no significant difference with that at 2 ~ 8 ℃（P 〈 0. 05）,indicating high stability. Both intra- and inter-coefficients were less than 10%,indicating a high reproducibility. The recovery rates of CA125 at high,moderate and low concentrations were 94. 45% ~105. 21%,indicating a high accuracy. The positive rate of CA125 was 4% in 50 serum samples of healthy women,while was 88% in 100 serum samples of patients with ovary cancer. Conclusion The magnetic enzyme diagnostic kit for CA125 was successfully prepared,which might be used for the diagnosis of ovary cancer.