山羊血清中CD58含量的检测
摘要
本研究利用酶消化法结合柱层析法制备了绵羊红细胞表面活性蛋白CD58;用弗氏佐剂法和淋巴细胞吸附法免疫家兔;用方阵滴定法确定包被抗原最适工作浓度,检测抗血清,建立间接酶联免疫吸附法(ELISA),用间接ELISA测定所制备的抗体效价,并建立了检测山羊血清中CD58的间接竞争抑制性ELISA方法,检测了乏情期与发情期山羊血清中CD58的含量。获得以下结果:
1.酶消化法结合柱层析法制备的绵羊红细胞表面活性蛋白CD58,玫瑰花环抑制试验检测结果抑制率为62.01±2.06%。SDS–PAGE显示制备的CD58提取物中,存在3种分子量大小的蛋白质。
2.比较弗氏佐剂法和淋巴细胞吸附法2种免疫方法,淋巴细胞吸附法获得了针对CD58的特异性抗体,抗体效价达到1︰1×105,说明利用细胞表面受体与配体作用免疫动物,获取抗体的方式是可行的。
3.建立了抗CD58抗体测定的间接ELISA方法。方阵滴定法确定包被原最适工作浓度为10.0μg/mL,最适包被液为pH 9.6,0.05 mol/L的CBS;辣根过氧化物酶标记羊抗兔IgG(酶标二抗)最适稀释倍数为l∶2 000,1.0%明胶封闭。重复性好,批间重复的变异系数(CV)2.48 % ~7.78 %。
4.建立了测定山羊血清中CD58的间接竞争抑制性ELISA,线性范围20.0 ~ 500 000.0 ng/mL,回归方程Y = -0.207X + 1.159 (R2 = 0.988),该方法灵敏度为1.76 ng/mL。统计分析显示乏情期与发情期山羊血清中CD58含量差异显著(P﹤0.05)。
试验证明,淋巴细胞吸附法与间接ELISA方法,制备CD58的特异性抗体和测定山羊血清中CD58含量是可行的,并利用间接ELISA方法测定了乏情期与发情期山羊血清中CD58含量,为进一步研究CD58在山羊不同生理时期的作用提供了理论依据。
In this study, the surfactant protein CD58 of sheep red blood cells (SRBC) was prepared with enzymatic digestion and column chromatography. Rabbits were immunised with the methods of Freund's adjuvant and lymphocyte adsorption. The titer of anti-CD58 serum was tested with the establishment indirectly enzyme linked immnosorbent assay (ELISA). The optimised working concentrations of coated antigen and antibody were performed with phalanx titration. The inhibition of indirect competitive ELISA methods was established to detect the level of CD58 in goat serum, and used to detect the goat serum levels of CD58 estrous cycle and anestrus period. The results were as follows:
1. The preparation of CD58 was tested with Rosette Inhibition Test, its inhibition rate was the percentage of 62.01±2.06. There were three kinds of proteins in the CD58 preparation from SDS-PAGE.
2. Compared to Freund's adjuvant, the method of lymphocyte adsorption was obtained specific antibodies anti-CD58, antibody level was 1︰1×105, the method of using cell surface receptor and ligand to immune animals to prepare antibody is feasible.
3. The indirect ELISA method, coated antigen 10.0μg·mL-1 CD58 dissolved in carbonate buffer solution (CBS, pH9.6, 0.05 mol·L-1), with HRP-labelled goat anti-rabbit IgG diluted by PBS (pH7.4, 0.1 mol·L-1) in l∶2 000 as a secondary antibody, 1.0% gelatinum as a blocking agent, was developed to detect antibody against CD58. The method’s reproducibility was good results and its coefficient of variability (CV) was 2.48% ~ 7.78%.
4. Establishment the indirect competitive ELISA to test the level of CD58 in goat serum, the linear range 20.0~500 000.0 ng·mL-1, regression equation Y = 0.207X-1.159 (R2 = 0.988), the lowest concentration of CD58 is 1.76 ng·mL-1, which can be detected theoretically. Statistical analysis indicated that the anestrus and the estrus period goat serum CD58 content is significant differences (P <0.05).
In a word, the method of lymphocyte adsorption and indirect ELISA could be used to prepared specific antibodies anti-CD58 and test the content of CD58 in goat serum. The anestrus and the estrus period goat serum CD58 content was detected with indirect ELISA. It would be provided theoretical basis to study the different physiological goats in the further study.
1.酶消化法结合柱层析法制备的绵羊红细胞表面活性蛋白CD58,玫瑰花环抑制试验检测结果抑制率为62.01±2.06%。SDS–PAGE显示制备的CD58提取物中,存在3种分子量大小的蛋白质。
2.比较弗氏佐剂法和淋巴细胞吸附法2种免疫方法,淋巴细胞吸附法获得了针对CD58的特异性抗体,抗体效价达到1︰1×105,说明利用细胞表面受体与配体作用免疫动物,获取抗体的方式是可行的。
3.建立了抗CD58抗体测定的间接ELISA方法。方阵滴定法确定包被原最适工作浓度为10.0μg/mL,最适包被液为pH 9.6,0.05 mol/L的CBS;辣根过氧化物酶标记羊抗兔IgG(酶标二抗)最适稀释倍数为l∶2 000,1.0%明胶封闭。重复性好,批间重复的变异系数(CV)2.48 % ~7.78 %。
4.建立了测定山羊血清中CD58的间接竞争抑制性ELISA,线性范围20.0 ~ 500 000.0 ng/mL,回归方程Y = -0.207X + 1.159 (R2 = 0.988),该方法灵敏度为1.76 ng/mL。统计分析显示乏情期与发情期山羊血清中CD58含量差异显著(P﹤0.05)。
试验证明,淋巴细胞吸附法与间接ELISA方法,制备CD58的特异性抗体和测定山羊血清中CD58含量是可行的,并利用间接ELISA方法测定了乏情期与发情期山羊血清中CD58含量,为进一步研究CD58在山羊不同生理时期的作用提供了理论依据。
In this study, the surfactant protein CD58 of sheep red blood cells (SRBC) was prepared with enzymatic digestion and column chromatography. Rabbits were immunised with the methods of Freund's adjuvant and lymphocyte adsorption. The titer of anti-CD58 serum was tested with the establishment indirectly enzyme linked immnosorbent assay (ELISA). The optimised working concentrations of coated antigen and antibody were performed with phalanx titration. The inhibition of indirect competitive ELISA methods was established to detect the level of CD58 in goat serum, and used to detect the goat serum levels of CD58 estrous cycle and anestrus period. The results were as follows:
1. The preparation of CD58 was tested with Rosette Inhibition Test, its inhibition rate was the percentage of 62.01±2.06. There were three kinds of proteins in the CD58 preparation from SDS-PAGE.
2. Compared to Freund's adjuvant, the method of lymphocyte adsorption was obtained specific antibodies anti-CD58, antibody level was 1︰1×105, the method of using cell surface receptor and ligand to immune animals to prepare antibody is feasible.
3. The indirect ELISA method, coated antigen 10.0μg·mL-1 CD58 dissolved in carbonate buffer solution (CBS, pH9.6, 0.05 mol·L-1), with HRP-labelled goat anti-rabbit IgG diluted by PBS (pH7.4, 0.1 mol·L-1) in l∶2 000 as a secondary antibody, 1.0% gelatinum as a blocking agent, was developed to detect antibody against CD58. The method’s reproducibility was good results and its coefficient of variability (CV) was 2.48% ~ 7.78%.
4. Establishment the indirect competitive ELISA to test the level of CD58 in goat serum, the linear range 20.0~500 000.0 ng·mL-1, regression equation Y = 0.207X-1.159 (R2 = 0.988), the lowest concentration of CD58 is 1.76 ng·mL-1, which can be detected theoretically. Statistical analysis indicated that the anestrus and the estrus period goat serum CD58 content is significant differences (P <0.05).
In a word, the method of lymphocyte adsorption and indirect ELISA could be used to prepared specific antibodies anti-CD58 and test the content of CD58 in goat serum. The anestrus and the estrus period goat serum CD58 content was detected with indirect ELISA. It would be provided theoretical basis to study the different physiological goats in the further study.
引文
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