人血管性血友病因子胶原结合活性检测方法的建立与验证

Development and verification for determination of the vWF collagen binding activity in human plasma

目的建立检测人血管性血友病因子（yon Willebrand factor，vwF）胶原结合活性（collagen-binding assay，CBA）的酶联免疫吸附测定法，验证后用于vwF纯化工艺的探索。方法用微量滴定板做载体，以人Ⅲ型胶原包被酶标板．HRP标记的人抗vWF为检测抗体，通过筛选不同的包被条件、胶原质量浓度及检测抗体含量确定其反应参数，建立人vwF胶原结合活性检测方法，同时验证该方法的准确度、精密度、特异性、选择性及样品稳定性，并初步应用于vWF纯化工艺的探索。结果使用pH9．5碳酸盐缓冲液包被胶原，包被质量浓度为20μg／mL，检测抗体稀释度为1：4000，国际标准品的线性范围为51．500～3．218mIU／mL，R^2≥0．999。定量上限为51．500mlU／mL，回收率在81．60％～105．08％；定量下限为3．218IU／mL，回收率在90．50％～108．18％，cy均≤5．0％；高、中、低浓度质控样品的回收率在80．3％～109．6％范围内：批内CV值≤8．0％，批间cy值≤6．4％。当NaCl浓度在55～350mmol／L、CaCl2浓度≤125mmol／L时，定量上限的活性回收率在80％～120％范围内；当vWF／FⅧ〉0．1时，质控样品准确度在100％±25％范围内：vWF标准品在室温放置2h的回收率在88．06％～87．21％，于-80℃反复冻融的回收率在80．55％．103．1l％范围内，于-20℃反复冻融的回收率在55．28％～79．55％范围内。结论建立了人vWF胶原结合实验，该方法具有良好的准确度、精密度、选择性和特异性，可用于vWF纯化工艺样品中vwF的胶原结合活性检测。

Objective To develop and verify a collagen-binding enzyme-linked immunoassay for detection and purification of yon Willebrand factor（vWF） and related activity. Methods The assay is based on measurement of the quantity of vWF molecules bound to collagen. Microplate was coated with human type III collagen and HRP-labeled human anti-vWF was used as the detection antibody. The optimal coating buffer, concentrations of collagen and detection antibody were optimized in order to develop the assay. The verification was carried out in accuracy, precision, specificity, selectivity and stability of the assay, which was used in a preliminary detection of vWF collagen binding activity in a purification. Results The opti- mal parameters were determined such as, coating buffer/carbonate buffer pH 9.5, the concentration for collagen 20 μg/ mL, the best dilution 1 ： 4 000 for detection antibody. The four-parameter logistic range of standard curve was 51.500-- 3.218 mIU/mL, R2≥ 0.999. The upper limit of quantitation was 51.500 mIU/mL, in which recovery was in 95.8%-- 103.2% ,CV≤5.0%; the lower limit of quantitation was 3.218 IU/mL, in which recovery was in 90.5%--108.18%, CV≤ 5.0%. Intra,assay aceuraey for samples with high, moderate and low contents was in 80.3%--109.6%, intra-assay preei- sion was ≤ 8%, preeision between-run was ≤6.4%. The reeovery of sample adding experiment was between 80% and 120%with NaC1 from 55 to 350 mmol/L, CaC12≤125 mmol/L. When vWF/FVIM was more than 0.1, the recovery was in a range 100%±20%. After thawing at room temperature for 2 h, the recovery was in 88.06%-87.21%. After in a freezing-thawing cycles at -80℃, the recovery was in 80.55%-103.11%, and after in a same cycles at -20 ℃, the recovery was in 55.28%-- 79.55%.Conclusion The developed assay showed a high accuracy, precision, selectivity, specificity and reproducibility, which could be used for the detection of vWF collagen binding activity in samples in a vWF purification.