胶原包被条件对血管性血友病因子胶原结合法测定FⅧ制品中血管性血友病因子的影响

Effect of coating condition on collagen binding assay for von Willebrand factor in FⅧ concentrate

目的通过考察不同胶原包被条件对血管性血友病因子(von willebrand factor,vWF)与胶原结合的影响,建立vWF胶原结合法(vWF collagen binding assay,vWF:CBA)检测FⅧ制品中vWF活性。方法将胶原Ⅲ分别按以下4组条件包被ELISA板:不同pH(2.8、4.0、5.2、6.4、7.4、8.8、9.8)的缓冲液,不同盐浓度(0、0.05、0.1、0.15、0.2、0.3、0.4、0.6 mol/L NaCl)的缓冲液,不同胶原Ⅲ浓度(5、2.5、1、0.5、0.25、0.125μg/ml),不同包被温度(4、25、37℃)。以5个不同稀释度的vWF标准品为样品,进行vWF:CBA法测定,每种包被条件下绘制1条标准曲线,同时检测每种包被条件下方法的重复性。将标准曲线线性和方法重复性较好的几组条件做为最佳包被条件,验证该包被条件下vWF:CBA法的特异性、重复性,获得标准曲线范围。结果当胶原Ⅲ溶解于含0.15 mol/L NaCl,pH 6.4的磷酸缓冲液中,在4℃条件下以2.5μg/ml包被时,vWF与胶原的结合效率较高。将该包被条件下的vWF:CBA法用于测定冷沉淀上清及FⅧ制品中vWF活性,具有较好的特异性和重复性。结论不同的包被条件对胶原结合法测定vWF活性具有较大的影响,因此,在采用胶原结合法测定FⅧ制品中vWF活性时,应对包被条件进行优化。

Objective To develop a von Willebrand factor（vWF）collagen binding assay（vWF：CBA）for determination of vWF activity in FⅧ concentrate by investigating the effect of various conditions for collagen coating on binding of vWF to collagen. Methods ELISA microplate was coated with collagen Ⅲ by using the buffers at various pH values（2. 8,4. 0,5. 2,6. 4,7. 4,8. 8 and 9. 8）,various salt concentrations（0,0. 05,0. 1,0. 15,0. 2,0. 3,0. 4 and 0. 6 mol / L NaCl）,various collagen Ⅲ concentrations（5,2. 5,1,0. 5,0. 25 and 0. 125 μg / ml）and various temperatures（4,25 and 37 ℃）. The vWF standard at five dilutions were used as the samples and determined by vWF：CBA,as a result one standard curve was plotted under each coating condition. Reproducibility of the method under each coating condition was investigated. The conditions under which the linearities of standard curve and the reproducibility of method were better than the others were selected as the optimal coating condition for verification of specificity and reproducibility of VWF：CBA to obtain the range of standard curve. Results When the ELISA microplate was coated with 2. 5 μg / ml human collagen Ⅲ dissolved in the buffer containing 0. 15 mol / L NaCl at pH 6. 4 and 4 ℃,the binding capability of vWF to collagen was higher than those under other conditions. The vWF：CBA was used for determination of vWF activities in the supernatant of cryoprecipitation and in F Ⅷ concentrate under the optimized condition,and showed high specificity and reproducibility. Conclusion The coating condition showed significant effect on the determination of activity of vWF by collagen binding assay,which should be optimized before determination.