Preparation Of Immunomagnetic Beads And Preliminary Application In Poultry As Nano-adjuvant

Immunomagnetic Beads (IMB) were magnetic microspheres coated with antibody or ligand which could absorb antigen or receptor specifically.IMB has been widely used in biological and medical field. In this study, magnetic beads were prepared by the chemical co-precipitation method. Then the beads were coated with GST antibody. So we got IMB-GSTAb beads which were able to capture GST-VP2and GST-HN fusion protein. The IMB-GSTAb beads binding GST-VP2and GST-HN were used for chicken immunization test so that the biological safety and immunological enhancement of IMB-GSTAb could be assayed. Animal experiments show that the IMB has a certain degree of immune enhancement and good bio-security. This study provided useful information in looking for a simple, safe, effective and cheaper adjuvant, and also provided a new approach for vaccine production and the prevention of poultry disease.1. Preparation of anti-GST polyclonal antibodyGST protein was extracted by culturing BL21harboring plasmid pGEX-KG, and then purified by Glutathione-Sepharose4B affinity chromatography columns. GST protein was coated onto the surface of the beads, used to immune chickens, ELISA serum titer of1.6×104, proof that the beads have immune enhancement. Chicken anti-GST polyclonal antibody was purified by acid-ammonium sulfate. Western blot test proved that the homemade GST antibody with biological activity and specificity.2. Preparation of IMB-GSTAbFe3O4nanoparticle was synthesis by chemical coprecipitation. Surface Modification made the surface with the-CHO group. The average particle size is50-100nm, and the concentration is mg/mL.IMB-GSTAb beads were obtained by coupling GST antibody to magnetic beads and were used to capture GST-VP2and GST-HN fusion protein. The optimal conditions for beads coupling with antibodies were selected by adjusting and changing the parameters of buffer solution, pH values, reaction time and temperature, and the effects of beads coupling with antibodies were evaluated by using ultraviolet spectrophotometry and SDS-PAGE. The optimal condition was as the following:buffer solution0.1M TBS, Ph6.0, temperature37℃and reacting for3h in the shaker. The maximum capacities of IMB capturing GST-Ab was45-50μg/mg. The beads were blocked for2h at37℃by5%nonfat milk. The optimal reaction condition of IMB-GSTAb capturing GST-VP2and GST-HN were at0.01MPBS (pH7.4/6.8)37℃, for1h. The maximum capacities of IMB-GSTAb capturing GST-VP2was15μg/mg, The maximum capacities of IMB-GSTAb capturing GST-HN was12μg/mg.3. Application in Poultry as nano-adjuvantOne week chickens were immuned with IMB-Ab-VP2and IMB-Ab-HN were. Compared with the purified protein and inactivated vaccine. Test the antibody titer by ELISA. Proved that the IMB vaccine has certain immune effects.