和田羊抗苦马豆素抗体间接ELISA检测方法的建立

Establishment Detection Method of Indirect Enzyme- linked Immun osorbent Assay of Anti- Swainsonine Antibody in Hetian Sheep

建立特异性强、敏感性高的和田羊血清抗苦马豆素(Swainsonine,SW)抗体ELISA检测方法。利用自行制备的人工抗原SW-BSA(牛血清白蛋白)免疫接种和田羊,获得SW抗血清后以间接ELISA法检测血清抗体效价,通过分析包被抗原种类、浓度,血清稀释度等指标对检测数据的影响,选择最适条件,确定检测方法。试验表明,以质量浓度为10μg/m L的SW-卵清蛋白(OVA)偶联抗原作为包被抗原,4℃下包被过夜,质量浓度为20 g/L的明胶封闭,酶标二抗的最佳稀释比为1/1 600,试验条件为37℃温育1 h;样品血清最佳稀释比为1/80,试验条件为37℃温育2 h,底物最佳显色时间为30 min。建立的抗SW抗体ELISA检测方法,特异性强、稳定性高,为进一步研究SW人工抗原的免疫原性等奠定了基础。

The purpose of this experiment was to establish an detection methods of indirect enzyme - linked immunosorbent assay to determine the level of antibody against SW with high sensibility and specificity. The Hetian sheep SW antiserum was prepared by inoculating with home - made SW - BSA. SW antiserum was used to detect serum antibody titer by indirect ELISA method using different coating antigen, then to analyze the optimal conditions of the established indirect ELISA, such as type and concentration of coating antigen, different serum dilution, determine best method. The results showed that using SW - OVA as coating antigen was better than any other, the optimal conditions of the method were as follows： the density of coating antigen（ SW- OVA） was 10 μg/mL, the reaction time was 12 h and reaction temperature was 4 ℃ ;the impacting medium gelatine of 20 g/L, the working concentration of enzyme labeled antibody was 1/1 600, the reaction time was 1 h and reaction temperature was 37 ℃ ; the serum dilution was 1/80, the reaction time was 2 h and reaction temperature was 37 ℃ ;the reaction time was 30 min for substrate colouration. This method was verified with high specificity and stability via the determination of the antibody anti - SW in serum of Hetian sheep, thus providing a basic method for further research of SW artificial antigen.