抗腺病毒3型单克隆抗体的制备及临床应用研究

Preparation and application of monoclonal antibody against adenovirus 3 in diagnosis of keratoconjunctivitis caused by adenovirus

目的制备特异性抗腺病毒3型(ADV-3)的单克隆抗体(mAB),并用于腺病毒性角膜结膜炎的临床实验室诊断。方法用密度梯度超速离心技术纯化ADV-3免疫5只BALB/c小鼠,取脾细胞及骨髓瘤细胞按常规方法融合、筛选、克隆化及腹水注射制备mAb。采用ELISA及免疫组织化学技术进行鉴定。结果经3次克隆化获得了3株可分泌抗ADV-3mAb的杂交瘤细胞株,经多次复苏传代仍能稳定分泌抗体,分别命名为E7、B9和G11。1株(E7)Ig重链为IgG2b,2株(B9、G11)为IgG1,轻链均属于κ型。ELISA效价均>104,相对亲合力E7>B9>G11,相对亲合力常数分别为8.14×109±1.48、2.03×108±0.43和1.63×107±0.15。腹水中mAb滴度为1×105～1×106。用抗腺病毒ADV-3E7检测临床诊断为病毒性角膜结膜炎患者标本30例,阳性者20例,阳性率66.7%,PCR检出阳性者21例,阳性率70%,两种检测方法的阳性检出率差异无统计学意义(P>0.05)。结论3株杂交瘤细胞株分泌的抗体对ADV-3抗原具有高特异性和亲和力,用于腺病毒性角膜结膜炎的临床诊断具有速度快、阳性率高的优点。

Objective Epidemic keratoconjunctivitis is an ophthalmic disease in the worldwide with high infectious characterization. The adenovirus type 3 （ Adv3 ） is recognized to participate in pathogenesis of keratoconjunctivitis. Research on the special monoclonal antibodies against Ade3 is important for prevention and therapy of keratoconjunctivitis, We aimed to prepare monoclonal antibodies（mAb） against Adv3 as a diagnosis tool of keratoconjunctivitis caused by adenovirus. Methods The Adv3 was purified with uhracentrifugation as immunogen to immunize 5 BALB/c mice. The spleen cells were fused with Sp2/0 cells, Clones secreting specific monoclonal antibody was screened by indirect ELISA. After cloning of three stains of hybridoma cells,clones stably producing anti-Adv3 mAb were obtained. Results Ascites titers were 1 ×10^5 to 1 × 10^6. The affinity of mAbs was 8.14 × 10^9 ± 1.48,2.03 × 10^8 ± 0.43 and 1.63 ×10^7 ± 0. 15, respectively. The isotypes of the two mAbs were IgG1 and IgG2b, Thirty samples were parallel to those detected by anti-Adv3 mAb and PCR. The positive detecting rate was 66.7% and 70% ,respectively. There was no statistically significant difference in the positive detecting rate of two methods （ P 〉 0. 05 ）. Conclusion Anti-Adv3 mAbs above mentioned show high specificity,which laid the foundation for the immunological detection of keratoconjunctivitis caused by adenovirus and further clinical treatment.