| 1. Gold-labeling of monoclonal antibody against the nucleoprotein (NP) of avian influenza virus (AIV)Colloidal gold suspension was obtained by boiled Tetrachloroauric acid (Trihydrate, HAuCl4·3H2O) mixed with trisodium citrate. The particles were detected under electronic microscope. The results showed that the mean diameter of the particle was about 15.95nm,standard deviation was 1.04% and coefficient of variation was 6.53%. The colloidal gold solution could be labeled with protein.Monoclonal antibody against the nucleoprotein of avian influenza virus (AIV-NP-7B4) was purified by saturated ammonium sulfate (SAS) precipitation and affinity chromatography. The purified McAb was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The result indicated that it was very pure and contained little other protein. Then the McAb was mixed with colloidal gold solution to produce immuno-gold particles. The results showed that there was halo around the gold particle under electronic microscope. The immuno-gold particles could react specifically with AIV antigen in immunology assay.2. Establishment of Gold Immuno-chromatographic Assay for Detection of Avian Influenza VirusA gold immuno-chromatographic assay (GICA) for detection of AIV was developed with the conjugate pad, purified rabbit-anti-mouse IgG, and McAb to AIV NP. The optimal concentration was 35ul/cm for immunogold solution on the conjugate pad, 0.6ul/cm(2.62mg/ml)for T line and 0.6ul/cm(2.02mg/ml)for the C line. No cross-reaction was observed when NDV, IBDV, GPV or ARV was detected with the strips, which showed the specificity of the assay is excellent. Comparing the GICA with HA test, the results showed the GICA was more sensitive. It was positive to 2-9 of allantoic fluid of AIV, which HA was 2-6. The average variant coefficient within assay was 8.63% and the maximum variant coefficient between assays was 9.85%. The strips could be sealed to preserve more than 6 months at 4℃.3. The preliminary application of Gold Immunochromatographic Assay for Detection of Avian Influenza VirusClinical samples, which included 962 organic samples, 83 serum and 216 cloacal swabs from Jiangsu, Anhui, Shandong and Henan provinces, were detected by the strips based on the GICA. 225 of 1261 clinical samples showed positive (17.84%). The positive rates of tissue samples (from high to low rates) were ovary, pancreas, glandular stomach, lungs, liver, spleen and kidney. Compared with double-antibody sandwich ELISA (DAS-ELISA), 370 of 1255 clinical samples were positive (29.48%) which clinical samples included 957 organic samples, 82 sera and 216 cloacal swabs. The positive rate of the tissue sample (from high to low rates) were lungs, ovary, spleen, liver, glandular stomach, kidney and pancreas. However there was little difference between the results from strips and DAS-ELISA for cloacal swab, ovary, glandular stomach and serum. The results indicated that cloacal swab and lung were optimal sample in the strips assay. The samples, identified positive in virus isolation, detected with the strips showed 92.3% (12/13) of the coincidence. Our results in conclusion indicated that GICA was specific, sensitive, quick and convenient. The strips will be function in the prevention and control of AIV. |
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