Development Of An Inmunochromatographic Lateral Flow Test Strip For Detection Of All Subtype Avian Influenza Virus

The result of our research about the base of M protein monoclonal antibody, using the method of double antibody sandwich colloidal gold immunochromatographic analysis, construct that polyclonal antibody as detection line to capture antibody and rabbit antimouse as quality control to capture antibody to detect all subtype Avian influenza virus. After optimized every reactive condition, the best operating conditions is: the diameter of stable,well-distributed,single dispersal colloidal gold is 35nm in 100mL system;the concentration of labeling McAb is 0.2M;the most optimation pH between colloidal gold and McAb is above or equal isoionic point; the most optimizational concentration between colloidal gold and o.1M McAb is 100μm: 8μm; the concentration of purifying M McAb and Caprine polybody is 1.0mg/mL,9.6mg/mL applying caprylic acid ammonium sulfate.The result also should be helpful for further understanding the avian influenza virus and constructing AIV rapid diagnosis method.We had packed test strip maked use of the method of double antibody sandwich colloidal gold immunochromatographic analysis, only demonding insert test strip into the treat specimen, the result appear after 5 min. When detection line and quality control appear two red line at the same time, the result is positive, but only quality control appear a red line, the result is negative. We undertaked the cross-reactivity to NDV,APV,MDV,EDSV,IBDV,ILTV,IBV,AIV 15 subtype (Chicken embryo allantoic fluid),distilled water,allantoic fluid and blank, the result manifested : there is positive result on testing every AIV subtype, but no conspicuous cross-reactivity on testing above-mentioned avian virus and difference compare to negative control. That is to say this method has more specificity to every AIV subtype.The hemagglutinin is 1:1024 H5N1,H7N7 and H9N2 subtype chicken embryo allantoic fluid positive than the dilution of times,compare to HA, the method which our lab set up could test H5N1virus HA titer above 1:16,H7N7 virus HA titer above 1:8,H9N2 virus HA titer above 1:16. Random different batches and lease out the test strip carries on the H5N1 virus repeatability test, the test result showed that the preservation of three months within the test strip good effect, have no specificity.The results showed that the preservation period:the preparated gold labaled antibody could maintain at 4 degree, the gold labaled tray should store at 4 degree protecting from light with sealing up and DES. The result of keeping time appeared: the super ordinary gold labeled antibody could hold above 6 months at 4 degree condition, furthermore the effect is fine; the gold labeled tray also stored at 4 degree above 6 months, and the mount of release retained above 80%.