Preparation Of Polyclonal Antibodies Specific Binding With Camel IgG And Its Application In Measurement Of Camel Immunization And Milk Identification

The humoral immune response of camels produced conventional IgG1 and heavy chain IgG2 and IgG3 antibodies(HCAbs).HCAbs are naturally lacking of light chain and CH1 which only exist in camels.At present,the real role of HCAbs in camel humoral immune defense is remain to be resolved.Due to the lack of detection tools to distinguish HCAbs from conventional antibodies,the research in this field is very lacking.HCAb exists not only in camel serum,but also in camel milk.Because of the unique health benefits of camel milk,it has been attracted by people in recent years,but it also cause the problem of camel milk adulteration.These need to develop camel IgG specific detection tools for research and application.Therefore,in this study,total camel IgG and nanobodies containing HCAb hinge sequences were used as immunogens to prepare polyclonal antibodies against camel IgG and HCAbs,in order to establish a specific and sensitive method for evaluation of camel humoral immune response,identification and quantitative analysis of camel milk antibodies.This study includes the following two parts:1.Preparation and determination of polyclonal antibodies against total camel IgG and HCAb.In order to obtain specific detection reagents for camel IgG and heavy chain antibody,we immunized goats and mice with camel total IgG and nanobody Lys3,VHH06 containing IgG2 hinge and VHHA4 containing IgG3 hinge respectively to prepare polyclonal antibodies.The total camel IgG was purified from camel serum by protein A affinity chromatography.The purified IgG showed three heavy chains and one IgG1 light chain band in SDS-PAGE,showing high purity.The purified total camel IgG was emulsified with Freund's adjuvant in a ratio of 1:1 and injected subcutaneously to immunize goat for four times.The titer of goat anti camel IgG was 1:20000.Recombinant Lys3,VHH06 and VHHA4 were soluble expressed in BL21,and purified by Ni ion affinity chromatography.Mice were immunized with the purified nanobodies four times.The titers of Lys3,VHHA4 and VHH06 were 1:409600,1:102400 and1:204800,respectively.Western blot analysis showed that the four kinds of immune sera were specifically bound to the corresponding antigens.In order to facilitate the detection,we used chemical coupling method to couple the anti camel IgG with horseradish peroxidase(HRP).The results showed that the conjugated goat anti camel IgG HRP had high catalytic activity.It can be used as a secondary antibody for detection.2.Specificity analysis of anti camel IgG polyclonal antibody and its application in humoral immunity and camel milk identificationFirstly,ELISA was used to detect the binding of four kinds of polyclonal antibodies to IgG in serum and milk of different animals.The results showed that Goat anti camel IgG HRP polyclonal antibody could specifically bind to camel serum at the dilution of 10~3,10~4,10~5 and 10~6,but not to bovine,mouse and rabbit serum.Mouse anti Lys3 polyclonal antibody could specifically bind to camel serum at 10~3 and 10~4dilutions,but could not bind to camel serum at 10~5and 10~6 dilutions.Mouse anti VHH06 and VHHA4 polyclonal antibodies could specifically bind to camel serum only at serum dilution ratio of 10~3.The results showed that goat anti camel IgG HRP could still specifically bind to camel milk at 100 fold dilution,while other polyclonal antibodies could not bind to camel milk at same dilution.In order to quantitatively analyze the IgG content in camel milk,the purified camel IgG was used to established standard curve,and the IgG content in serum and milk of five camels was detected by ELISA with goat anti camel IgG HRP.The results showed that the average concentration of IgG in camel serum and milk was 10.45±2.143 mg/ml and 1.24±0.066 mg/ml respectively.ELISA was used to detect the total IgG,IgG2 and IgG3 levels of foot-and-mouth disease vaccine and VP1 antigen in the serum of camels after immunization,so as to evaluate the humoral immune response of camels after antigen stimulation.The results showed that both foot-and-mouth disease vaccine and VP1 antigen could induce camels to produce conventional antibodies(IgG)and heavy chain antibodies(IgG2 and IgG3),and the level of heavy chain antibodies produced was lower than that of total IgG,indicating that camels could produce higher levels of conventional antibodies after antigen stimulation.In heavy chain antibody,the levels of IgG3 subtypes produced by the two antigens were significantly higher than that of IgG2(P<0.05).Finally,indirect ELISA was used to detect the content of camel IgG in camel milk mixed with different proportions of cow milk to judge camel milk adulteration.The results showed that with the increasing proportion of camel milk mixed with cow milk,the OD450 value of goat anti camel IgG HRP gradually decreased,and the camel IgG content in camel milk also gradually decreased.The concentration of IgG in camel milk mixed with 0%,20%,40%,60%and 80%of cow milk was 1.270±0.2 mg/ml,0.723±0.23 mg/ml,0.291±0.11 mg/ml,0.122±0.1 mg/ml,0.127±0.1 mg/ml and 0.076±0.02 mg/ml respectively.In conclusion,the goat anti camel IgG polyclonal antibody prepared in this study has high specificity and sensitivity in the detection of camel serum and camel milk IgG.Based on this antibody,we can further develop the detection kit for camel antigen immunity and camel milk identification.