Study Of Immonumagnetic Bead For Avian Influenza Virus Isolation And Immunological Adjuvant Effects

Magnetic nanoparticle particle is a composite particle that composed of superparamagnetic and macromolecules or other inorganic materials, magnetic property of solid, but also have the fluidity of liquid. The bioactive molecules such as enzymes, antibodies（monoclonal or polyclonal）, nucleic acids or oligonucleotides may be coupled on the magnetic particle surface via the functional linker or the functional groups such as amino, carboxyl, thiol etc. It possessed a lot of advantages of solid reagen, such as good specificity, rapidness, highly efficacy, and repeatability, it is also cost-effective method due to no requirement of expensive instruments during detection. These merits contribute to it wider application in the fields of immunodetection, immunoadsorption, cell detachment and cultivation. This study use Immonumagnetic bead -based isolation techniques and the results were reported as follows.1.Isolate H9N2 subtype of avian influenza with IMB.30-100 nm Nanocrystalline Fe₃O₄ was synthesized by co-precipitation method and then modified consequently by silaniztion and McAb H94C4 （monoclonal antibody of anti-HA of H9N2 subtype of avian influenza virus） to produce immonumagnetic bead （IMB）. The efficiency of coupling McAbH94C4 gets to 127 mg/g and has high specificity and sensitivity. The 200μL prepared IMB can absorbe 100μL H9-AIV virus allantoic fluid（its hemagglutinin price is 2⁸） .The absorbed virus can be eluted by 1%NaCl. It is evident that this method of assay can provide a new approach for rapid and correct isolating H9N2 subtype of avian influenza.2. The immunological adjuvant effects of immonumagnetic beadMagnetic composite particles could be used to immobilize macrobiomolecules. Anti-GST monoclonal antibody was binded onto the magnetic particles by amino terminal groups by glutaraldehyde .The effciency of coupling was analyzed by UV Vis spectrophotometer, the active,specificity and sensitivity of molecules on magnetic particle surface was detected by Enzyme-Linked Imrnunosorbent Assay （ELISA）. We used the prepared immonumagnetic bead to capture fusion protein S4, and then we used the compound to immunized mouse .In the animal experiment, there are five groups（the immonumagnetic bead which has captured the protein, the complete adjuvant group, the protein, the blank and the immonumagnetic bead group which has not captured the protein） . We collected the serum every week and till 35 day, the antibody level of the collected serum was detected by Enzyme-Linked Imrnunosorbent Assay （ELISA）. The results of the ELISA test was analyzed with SPSS. The experimental result indicated: the efficiency of coupling Anti-GST monoclonal antibody gets to 127 mg/g and has high specificity and sensitivity. The protein adsorptive capacity of the MB is 90mg/g . Animal experimental results show that immunomagnetic beads has a role in immune-enhancing, it can induce body to produce antibodies rapidly, premodinantly time ,the antibody level of the immonumagnetic bead group which has captured the protein S4 higher than S4 protein group, blank control group and the immonumagnetic bead group which has not captured the protein, it has significant difference （P <0.05）, but Freund’s adjuvant group was slightly higher than the immonumagnetic bead group which has captured the protein S4, the difference was not significant （P> 0.05）.In this study, we used the IMS to establish H9N2 subtype of avian influenza virus isolation method and confirmed the adjuvant effect of immunomagnetic beads. The study laying a good foundation for the popularization and application in the animal husbandry of immunomagnetic beads .