ABSTRACT
@#Objective To develop and verify a double antibody sandwich ELISA detection method for the determination of ovalbumin(OVA),in order to determine the OVA content in influenza vaccines.Methods The rabbit anti-OVA polyclonal antibody was used as coating antibody and HRP labeled rabbit anti-OVA polyclonal antibody as detection antibody to develop a double antibody sandwich ELISA for OVA.The antibody concentration(2,1,0.5 and 0.25 pg/mL) for coating,enzyme-labeled antibody concentration(0.5,0.25 and 0.125 μg/mL),and kinds of blocking reagent(blocking with1% BSA,blocking with 2% BSA,blocking with 1% BSA and 1% sucrose,and blocking with 2% BSA and 2% sucrose,using nonblocking as control) were optimized,and the Cut-off value was determined as the judgment standard.The developed method was verified for the linear range and detection limit,specificity,repeatability and accuracy.Results The optimum detection conditions were as follows:the concentration of coating antibody was 1 μg/mL,the concentration of enzyme-labeled antibody was 0.25 μg/mL;The blocking reagent was 2% BSA and 2% sucrose;The Cut-off value was 0.051 66.The linear range of the method was 5～0.313 ng/mL,and the detection limit was 0.078 ng/mL;It did not react with influenza virus,bovine serum albumin(BSA) and Vero cell supernatant;The intraplate and interplate coefficient of variation(CV) were between 2.562%～13.887% and 4.000%～16.497% respectively;The coincidence rate between the results of this method and the Germany SERAMUN OVA quantitative test kit ranged from 93.79% to 107.05%.Conclusion The developed OVA double antibody sandwich ELISA has good specificity,repeatability,linear range and accuracy with convenience and lower cost,which might be used for the quantitative detection of OVA.
ABSTRACT
Objective To establish a simple animal model of cough variant asthma(CVA)through sensitizing Brown-Norway(BN)rats with ovalbumin(OVA). Methods A total of 36 BN rats were randomly divided into three groups, including the normal control group,the model control group and the montelukast group. BN rats in the model group and the montelukast group were intraperitoneally administered with 2.0 mg of OVA and 100 mg of Al(OH)3,and the same volume of sterile saline was given to the normal group by intraperitoneal injection. Boosting was carried out by intraperitoneal administration with 0.01 mg of OVA and 100 mg of Al(OH)33 weeks later,and the rats in the normal group were injected with the same dose of physiological saline. Three weeks later,the actively sensitized BN rats were challenged with aerosolized OVA for 7 times on alternative days,and the rats in the normal group were treated with sterile saline instead of OVA. At the same time, the montelukast group was given 1.3 mg/kg of montelukast 30 minutes before atomization by intragastric administration once a day for 2 weeks,and the normal group and the model group were given the same volume of water. The tests of cough sensitivity to capsaicin and bronchial responsiveness were performed 24 h after the last administration. Results Compared with the normal group, the times of coughing(P< 0.01)and the lung resistance(RL)(P< 0.05)in the model group were significantly increased,while the lung compliance(Cdyn)was significantly decreased(P< 0.05). There was a significant difference(P < 0.05)in the times of coughing caused by capsaicin between the model group and the montelukast group. Compared with the model group,RLin the montelukast group was decreased significantly(P< 0.05), and Cdynwas increased significantly(P< 0.05). Conclusions This rat model of CVA is similar to a variety of clinical features of CVA and is easy to operate. Thus it can be used as an effective animal model of CVA.