犬细小病毒阳性血清的制备、鉴定和初步应用

Preparation,Identification and Preliminary Application of Canine Parvovirus Positive Serum

犬细小病毒(Canine parvovirus,CPV)阳性血清在CPV疫苗制品的效力检验、外源病毒检验和鉴别检验中不可或缺,对CPV相关生物制品的质量控制至关重要,为了制备中和抗体效价高、特异性好的CPV阳性血清,试验用CPV DD株免疫健康易感比格犬2只,经4次免疫后采血分离血清,采用微量细胞中和试验法、免疫荧光法和《中国兽药典》三部附录中无菌检验、支原体检验和外源病毒检验法对血清进行鉴定,将该血清应用在犬类疫苗的外源病毒检验和鉴别检验中,建立了CPV间接免疫荧光检测法(IFA)并应用于疫苗的效力检验(病毒含量测定)中。结果表明制备的CPV阳性血清中和抗体效价高达1∶32768,中和指数>10^(6.2),对犬瘟热病毒、犬副流感病毒和犬腺病毒的中和抗体效价均<1∶4,狂犬病病毒中和抗体效价<0.03 IU/mL。用制备的阳性血清对3种CPV相关活疫苗进行外源病毒检验可完全中和10头份的疫苗毒;对2种CPV相关活疫苗进行鉴别检验,结果符合质量标准规定。利用CPV间接免疫荧光检测法对3种CPV相关活疫苗进行病毒含量测定,结果显示建立的IFA方法对3株疫苗株均有交叉免疫反应,与商品化CPV荧光单抗检测灵敏度差异不大。说明CPV阳性血清中和能力强、特异性好,可用于犬用活疫苗外源病毒检验、鉴别检验和效力检验。

Canine parvovirus(CPV)positive serum is indispensable in the efficacy test,exogenous virus test and identification test of CPV⁃related vaccines,and is crucial for the quality control of CPV⁃related biological products.In order to prepare CPV positive serum with high neutralizing antibody titer and good specificity,two healthy and susceptible beagles were immunized with CPV DD strain for the test.After four times of immunization,we collected blood and separated serum.The serum was identified by virus neutralization test,immunofluorescence assay,and sterility test,mycoplasma test,and exogenous virus test in the third part of the Chinese Veterinary Pharmacopoeia 2020.And then the serum was used in the exogenous virus test and identification test in canine vaccines.An indirect immunofluorescence assay(IFA)for CPV was established and applied to potency test of vaccine.The results showed that the neutralizing antibody titer of CPV positive serum was as high as 1∶32768,the neutralizing index was higher than 106.2,the neutralizing antibody titers of CPV positive serum against canine distemper virus,canine parainfluenza virus and canine adenovirus were all lower than 1∶4,and the neutralizing antibody titer of rabies virus was lower than 0.03 IU/mL.The CPV positive serum can completely neutralize 10 doses of vaccine virus in exogenous virus test for three CPV related live vaccines.The CPV positive serum was used for identification test of two CPV related live vaccines,and the results met the quality standards.IFA for CPV was used to determine the viral titer of three CPV vaccine strains,and the results showed that the IFA method established had cross immune response to the three vaccine strains,which had little difference with the sensitivity of commercial CPV fluorescent monoclonal antibody.All above show that CPV positive serum has strong neutralizing ability and good specificity,and can be used for the efficacy test,exogenous virus test and identification test of canine live vaccines.