Study On The Methods Of Membran Adsorption-elution And Immunomagnetic Beads Adsorption For Concentration Of Enteroviruses

Objiective: To optimize membrane adsorbtion-elution method of concentrate enteroviruses. To prepare poliovirus-Ⅰpolyclonal antibody, combine with magnetic-beads to adsorb and separate poliovirus-Ⅰin water, discuss the feasibility of this method.Method: Adjusted pH and the Al3 + concentration of water samples. Compared the recovery rate of enterovirus in the above-mentioned different conditions, according to the level of recovery to choose the best conditions for membrane adsorbtion-elution method to initial concentrate enteroviruses. Prepared different concentrations of PEG6000 solution, compared the concentration level of virus recovery, selected the appropriate concentration of secondary enrichment for enteroviruses. Then chose different membranes and different elutes for sequential test, compared the recovery rate of every combinations. Injected purified poliovirus-Ⅰsubcutaneously at different points of Japanese white rabbit's back and then repeated the injection for 3 times. Then collected antiserum, tested the titer and the antibody concentration by indirect ELISA method and nucleic acid protein measurement. Coated the antibody to magnetic beads to make immunomagnetic beads. Used immunomagnetic beads to adsorb and separate poliovirus-Ⅰin water.Results: While pH=4.0,virus recovery was 70.81%, higher than the other pH conditions'. Adding AlCl3 into water can increase the virus recovery from 55.51% to 76.73%. Virus recovery and concentration of PEG6000 had the same change trends, the appropriate concentration of PEG6000 was 120g/l. In this concentration, the virus recovery can upto 85.9%. Adorbed viruses by mixed cellulose ester membrane and elute by H2SO4/NaOH can recover 75.17% viruses, better than the other combinations. The titer of antiserum for indirect-ELISA was 1:8000 and the concentration of antibody was 12.87mg/ml. Coated the antibody to magnetic beads, the magnetic bead's saturation adsorption capacity was 143.5μg/mg. In 100-fold diluted virus solution, immunomagnetic beads can recover 63.67% viruses.Conclusion: Multivalent cations can increase the viruseses recovery, when pH=4.0 viruses had the highest recovery. Adorbed viruses by mixed cellulose ester membrane and eluted by H2SO4/NaOH can recover more viruses than the other combinations. Appropriate concentration of PEG6000 was 120g/l, most of the virus can be deposited in this condition. Successful prepared polyclonal antibody against the polio virus. Construction of immunomagnetic beads had an unsatisfactory result . Finally, the immunomagnetic beads was applied to the water-rich concentration of the virus, although less than the effect of membrane adsorption elution method, but the recovery rate was basically satisfied.