| 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 fusion protein GST-HA1 (HA1 means the Avian Influenza Virus protein antigen HA1). The IMB-GSTAb beads binding GST-HA1 were used for chicken immunization test so that the biological safety and immunological enhancement of IMB-GSTAb could be assayed. The animal experiment results showed that IMB-GSTAb beads enhanced the titer of AIV-HA1 vaccine and the beads had no damage to tested chicken. This study provided useful information in looking for a simple, safe, effective and cheaper adjuvant, and also provided a new idea of enhancing the titer of Avian Influenza Virus vaccine.1. Preparation of anti-GST polyclonal antibodyGST protein was extracted by culturing BL21 harboring plasmid pGEX-KG. GST protein purified by Glutathione-Sepharose 4B affinity chromatography columns was used to immune chickens in order to prepare chicken anti-GST polyclonal antibody (pAb). Western blot results showed that the pAb had biological activity. The titer of serum was 1.28×104 by ELISA assay. The highly purified IgG was obtained by using saturated ammonium sulfate and Sephadex G-200 methods. ELISA results showed that the pAb had good reactivity with GST-NS1, GST-NP and GST-HAl fusion proteins.2. Preparation of IMB-GSTAbThe magnetic beads, prepared by chemical co-precipitation, were modified by silane coupling agent and aldehyde reagent in order to get -NH2 terminal beads and-CHO terminal beads. The concentrations of Super-paramagnetic beads (average diameter 30-200 nm) were 15.62 mg/mL before modified and 5.40 mg/mL after modified respectively.IMB-GSTAb beads were obtained by coupling GST antibody to magnetic beads and were used to capture GST-HA1 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,SDS-PAGE analysis and IMB-ELISA assay. The optimal condition was as the following:buffer solution 0.01M PB, pH7.4, temperature 37℃and reacting for 6 h in the shaker. The beads were blocked for 1 h at 37℃by 3% nonfat milk. The optimal reaction condition of IMB-GSTAb capturing GST-HA1 were at 37℃, for 2 h. The maximum capacities of IMB-GSTAb capturing GST-HA1 were 297.2±11.7μg/mL by using -NH2 beads and 299.1±6.9μg/mL by-CHO beads, respectively.3. Detection of the adjuvant effects of IMBTwenty five two-week-old chickens were separated into five groups including one test group (group A) and four control groups B, C, D and E. Chickens from group A to E were inoculated with IMB-GSTAb-Ag(A), IMB-GSTAb (B), GST-HA1 antigen(C), H5N1 inactivated vaccine Re-5 (D) and PBS (E), respectively. The sera were collected at day 7,14,21,28,35 and 42 after inoculation. The titers of sera were assayed by ELISA. The results showed that the antibody titer of group A and D rose faster than others at the first two weeks. the titer of group A was significantly higher than B, C and E at the third week (P<0.01), but after that, the titer of group A began to fall. The results suggested that the IMB-GSTAb beads were able to enhance the immunization of antigen. The results of beads safety test showed that the chickens inoculated with beads had no acute toxicity reaction and significant pathological changes of inherent organs were not discovered. |
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