抗H9N2 AIV HA单克隆抗体的制备及其在胶体金层析检测中的初步应用

Preparation of Monoclonal Antibodies against HA of H9N2 AIV and Its Preliminary Application in Colloidal Gold Immunochromatographic Assay

用基因疫苗pcDNA-H9和纯化H9N2亚型AIV免疫Balb/C小鼠,细胞融合后用HI和ELISA方法筛选出5株抗H9亚型AIV HA的特异性单克隆抗体H9-01(2E6),H9-02(3B1),H9-03(3H11),H9-04(1A4),H9-05(2G9)。各株单抗亚型测定的结果为H9-01,H9-02和H9-05均为IgG2b,H9-03为IgG1,H9-04为IgG2a,轻链的亚型均为kappa链。经特异性和与同型病毒的反应谱检测,它们均是HA特异性单克隆抗体。用制备的抗H9亚型AIV HA的单克隆抗体做成胶体金层析检测试纸条,可以检测到200个EID50的H9亚型AIV,为监测该亚型AIV试剂盒的进一步商品化奠定了基础。

Balb/C mice were immunized with pcDNA--H9 gene vaccine or purified H9N2 subtype AIV. After fusion of the splenocytes obtained from the immunized mice with myeloma cells, five hybridoma cell lines including H9-01（2E6）, Hg--02（3B1）, Hg--03（3H11）, Hg--04（1A4） and Hg-- 05 （2G9） which persistently secrete specific monoclonal antibodies against H9 subtype AIV, were finally selected by HI and ELISA. Testing for the subgroup of the monoclonal antibodies re- vealed that Hg--01,Hg--02 and H9--05 belonged to the subgroup IgG2b,Hg--03 to IgG1,Hg-- 04 to IgG2a,respectively. All of their light chains are of the kappa subtype. Specificity and homologous virus reaction spectrum testing showed that they were all HA--specific monoclonal anti- bodies. A colloidal gold immunochromatographic assay was developed for the detection of H9 AIV by using these monoclonal antibodies as diagnostic agent, and 200 EID50 H9 AIV could be detected by this method. Our results provided useful data for further developing the diagnostic kit to detect the virus in practical application.