抗脊髓灰质炎病毒Ⅰ型D抗原单克隆抗体的制备及其初步应用

Preparation and preliminary application of monoclonal antibody against D antigen of poliovirus typeⅠ

目的制备脊髓灰质炎病毒(Poliovirus)Ⅰ型D抗原单克隆抗体,并建立双抗体夹心ELISA检测方法。方法采用Vero细胞培养脊髓灰质炎病毒Ⅰ型,柱层析纯化病毒抗原,SDS-PAGE鉴定病毒蛋白,HPLC分析抗原纯度,Western blot分析抗原的反应原性。以纯化的脊髓灰质炎病毒Ⅰ型D抗原免疫BALB/c小鼠,进行细胞融合,筛选可分泌脊髓灰质炎病毒I型D抗原单克隆抗体的杂交瘤细胞株。单克隆抗体经纯化后,采用间接ELISA法检测小鼠腹水抗体效价、单抗的特异性及相对亲和力。分别以纯化的单抗作为捕获抗体和酶标抗体,进行配对试验,建立用于脊髓灰质炎病毒Ⅰ型D抗原检测的双抗体夹心ELISA方法,确定方法的最佳线性范围,并验证方法的特异性。结果纯化的脊髓灰质炎病毒Ⅰ型D抗原经SDS-PAGE分析,共显示4条蛋白条带,相对分子质量分别为约40 000、35 000、32 000和28 000;经HLPC分析,纯度达99%;小鼠免疫血清可与相对分子质量约为35 000的VP1蛋白发生特异性反应。共筛选出5株可稳定分泌脊髓灰质炎病毒I型D抗原单克隆抗体的阳性杂交瘤细胞株,纯化的单抗仅与D抗原发生反应;2H1和3B5株单抗的效价分别为2×107和2×106,且亲和力高于其他3株单抗。采用2H1和3B5株单抗配对,可以特异性检测D抗原,而与C抗原不反应;D抗原最佳线性范围为4.300～0.071 DU/ml,R2为0.994 6。该法仅可检出脊髓灰质炎病毒Ⅰ型D抗原,而与脊髓灰质炎病毒Ⅱ型、Ⅲ型D抗原和脊髓灰质炎病毒Ⅰ型C抗原、EV71抗原、Vero细胞培养上清不发生交叉反应。结论制备了脊髓灰质炎病毒Ⅰ型D抗原单克隆抗体,建立了可用于脊髓灰质炎病毒Ⅰ型D抗原特异性检测的双抗体夹心ELISA方法。

Objective To prepare monoclonal antibody （McAb） against D antigen of poliovirus type I and develop a double antibody sandwich ELISA method. Methods Poliovims type I was cultured in Vero cells, from which viral antigen was purified by column chromatography, and analyzed for viral protein by SDS-PAGE, for purity by HPLC method and for reactogenicity by Western blot. BALB/c mice were immunized with purified D antigen of poliovirus type I , based on which the hybridoma cell strains secreting McAbs were screened by cell fusion. The secreted MeAbs were purified, and determined for titer, specificity and relative affinity by indirect ELISA. Match test was performed using the purified McAbs as capture and detection antibodies respectively, base on which a double antibody sandwich ELISA method for D antigen of poliovirus type I was developed, determined for the optimal linear range and verified for specificity. Results Purified D antigen of poliovirus type I showed four bands on SDS-PAGE profile, with relative molecular masses of about 40 000, 35 000, 32 000 and 28 000 respectively, and reached a purity of 99%. Murine immune sera showed specific reaction with the VP1 protein with a relative molecular mass of about 35 000. A total of five hybridoma cell strains stably secreting McAbs against D antigen of poliovirus type I were screened, and the purified McAbs showed only reaction with D antigen. McAbs 2H1 and 3B5 reached titers of 2 ~ 107 and 2 ~ 106 respectively, of which the affinities were higher than those secreted by the other three hybridoma cell strains, McAb 2H1 matched to McAb 3B5 showed specific reaction with D antigen while no reaction with C antigen. The optimal linear detection range of D antigen by the developed ELISA method was 4. 300 ~ 0. 071 DU/ml, with a R2 value of 0. 994 6. The D antigen of poliovirus type I was detected by the developed method, while no cross reactions with D antigens of poliovirus types ]I andre, the C antigen of poliovirus type ｜ , EVT1 antigen or culture supernatant of Vero cells were observed. Conclusion The McAbs against D antigen of poliovirus type I were prepared, based on which a double antibody sandwich ELISA method suitable for specific detection of the said antigen was developed.