Preparation For Anti-idiotypic Antibodies To Avian Influenza Virus Subtype H9 And Primary Analysis To Their Immunogenicity

Avian influenza, as an acute and highly contagious disease caused by avian influenza virus (AIV), has occurred in many countries and regions worldwide. Since 1990s, mildly pathogenic avian influenza (MPAI) caused by AIV subtype H9N2 has appeared in some regions of China. Although the dangers brought by this subtype are not as serious as highly pathogenic avian influenza (HPAI), these viruses have caused considerable economic losses in poultry. In order to overcome some shortcomings with the whole virus inactivated vaccine used in fields and the whole virus antigen used in laboratory diagnosis at present, a kind of antibody called anti-idiotypic antibody (anti-Id or Ab2) which can mimic the AIV antigen was aimed to prepared and studied, with the anticipation for it to be used in avian influenza prevention and laboratory diagnosis.High titer serum and immunoglobulin G (IgG) were prepared from specific pathogen free (SPF) chickens immunized with inactivated AIV subtype H9N2. Rabbits were immunized with this chicken anti-H9N2-AIV IgG and high titer serum were collected. The rabbit anti-Ids (RAb2s) were isolated with anti-isotypic and anti-allotypic antibodies to be absorbed by aldahated red blood cells binding unimmunized chicken IgG. Results indicated that the anti-isotypic and anti-allotypic antibodies could be absorbed completely in this way. The RAb2s bound to chicken anti-H9N2-AIV IgG specifically, and inhibited its neutralization activity to H9N2 AIV. A competitive test showed the RAb2s competed with H9N2 AIV to bind anti-H9N2-AIV IgG. So it can be concluded that "internal image" Ab2s were included among the RAb2s.After RAb2s were prepared and identified, the immunological characteristic of one kind of monoclonal Ab2 (mAb2) to H9 AIV secreated by one hybridoma was studied. Results demonstrated that the mAb2 was able to inhibit the binding of hemagglutinin to anti-H9-AIV antibodies and to induce SPF chickens to generate hemagglutination inhibition (HI) antibodies, indicating this anti-idiotypic antibody bore the internal image of hemagglutinin on avian influenza virus.1-week-old SPF chickens were inoculated with RAb2s and mAb2 oil emulsion vaccine respectively, with chickens injected with whole virus inactivated vaccine or PBS as controls. Chickens were detected for antibodies against H9N2 AIV once a week after vaccination and challenged with H9N2 AIV 4 weeks after vaccination. Results showed both the Ab2 vaccine teams were induced to generate antibodies to H9N2 AIV, which were able to partially protect SPF chicken embryos from being infected by H9N2 AIV. The results of virus isolation from cloaca demonstrated partial challenged chickens were held to discharges virus in team RAb2s and team mAb2. The experiments indicated both the Ab2s were well immunogenic to chickens, able to induced chickens to generate antibodies against homologous virus, decrease virus discharge to environment from chickens, reduce morbidity rate and lessen symptom in challenged chickens.In order to explore the feasibility for Ab2 to be used as surrogate of virus antigen for detecting serum antibodies (Abl), agar gel precipitation (AGP) test and indirect ELISA were carried out to detect chicken serum with H9N2 AIV and Ab2, individually. In AGP test, 70% coincidence rate was achieved between the AGP with Ab2 as antigen and that with standard AIV antigen. In ELISA, 90% coincidence rate was achieved between the ELISA with mAb2 as antigen and that with standard AIV antigen. Positive detectable rate by mAb2 ELISA (8/10) is higher than HI test (7/10) and AGP test (5/10). The results indicated Ab2 can be used as surrogate of virus antigen which has the risk to pollute environment for detecting Abl in some cases.In a word, RAb2s which have the similar antigenicity to H9N2 AIV and mAb2 which bears the internal image of hemagglutinin on H9N2 AIV were proved to be able to induce chickens to generate antibodies against homologous virus which can partially protect chickens from discharging virus after challenged by homologous virus and to be used as surrogate of virus antigen for detecting serum antibodies against AIV. This research proved once again the rationality for Immune Network Theory and supplemented the prevention and survey techniques in MPAI, which were the reference to control the other infectious diseases in Human and animals.