Preparation Of The Equine Immunoglobulin F?ab'?₂ And Preventive Effect Against Feline Calicivirus

Feline calicivirus?FCV?belongs to the family of the Caliciviridae,Vesivirus virus genus,which can cause upper respiratory diseases in cats.The main clinical symptoms after infection are chronic stomatitis and oral ulcers,which can also cause serious clinical forms of conjunctivitis,lame syndrome and toxic systemic diseases.Vaccination is still the main preventive measure against the disease,however the vaccine can not prevent reinfection of homologous or heterologous FCV variants.There is no specific therapeutic drug for the disease,and specific antibodies are the preferred biological products for treatment.Monoclonal antibodies are not suitable for mass production due to their high cost.Horse anti-serum products are favored for their low cost and mature production process,and have an irreplaceable role in the treatment of certain diseases.Therefore,it is of great significance to carry out research on FCV horse serum antibodies.In this experiment,the FCV CH-JL2 strain with strong pathogenicity and good immunogenicity was used as an immunogen before immunizing horses,thereby preparing high immune equine serum and purified immunoglobulin F?ab'?₂,and evaluating its protection.In this study,the preparation and detection of anti-FCV high immune equine serum and purified immunoglobulin F?ab'?₂ were first carried out.The FCV was concentrated and purified by ultra centrifugation and sucrose density gradient centrifugation.The immunogen was prepared with Freund's adjuvant and to immunize the horse.The virus neutralization antibody titer was monitored by virus neutralization test,and then immunoglobulin IgG was extracted from horse anti-FCV high immune serum by saturated ammonium s?Lfate precipitation and digested with pepsin and purified by Protein-G column.High-purity equine anti-FCV purified immunoglobulin F?ab'?₂ was tested for its concentration,purity,reactivity,and neutralizing antibody titer.The results showed that the equine anti-FCV serum neutralizing antibody titer was the highest after the sixth immunization,which was 1:1230;the optimal digestion condition for purifying IgG was 2 h at 37°C.The protein-G column purified F?ab'?₂ showed a complete protein band around 110KDa by SDS-PAGE analysis,and its purity was 95.236%by thin layer scanning.The concentration of F?ab'?₂ after concentration by ultrafiltration can reach13.5mg/mL;The antibody prepared by IFA assay has good reactivity,and its antibody neutralization titer was 1:1219,indicating that the ability of prepared purified immunoglobulin F?ab'?₂ is strong in vitro to protect infection with FCV.At the same time,the prepared F?ab'?₂was tested for sterility and safety,and no adverse reactions were observed in the injected mice and cats,which proved that the obtained immunoglobulin F?ab'?₂ was safe.Second,the prepared F?ab'?₂ was inoculated into experimental cats to study its protect effect on FCV infection in vivo.According to the animal infection model established in our laboratory,different doses of immunoglobulin F?ab'?₂ were injected subcutaneously into the experimental cats around 24 h,and challenged by intranasal drip with a dose of 0.5mL per cat.The prevention and treatment effect of immunoglobulin F?ab'?₂ on FCV infection was evaluated by observing and monitoring clinical symptoms,body temperature and body weight,comprehensive clinical score,detoxification and viral load,survival rate and histopathological changes.The results showed that the administration of F?ab'?₂ at doses of 2 mg/kg and 10mg/kg 24 h before and after challenge was effective in reducing clinical symptoms;Compared with the control group,the comprehensive clinical scores were significantly different,the survival rate was increased,the detoxification amount and the viral load in the lung and trachea were decreased;the pathological changes were alleviated by histopathological observation.This indicates that the prepared immunoglobulin F?ab'?₂ has certain emergency prevention and treatment effects on the infection of FCV in cats.