血浆Ⅳ型胶原酶含量显色测定法的建立及其应用

Development of a chromogenic assay for blood-plasma collagenase Ⅳ and its initial application

目的建立一种显色法检测血浆Ⅳ型胶原酶活性，探讨健康人群血浆中活性形式Ⅳ型胶原酶的参考区间范围。方法以琥珀酰明胶作为酶切底物，采用TNBS显色剂对酶切产生的末端氨基显色，使用酶标仪在405nm处测量其吸光度。通过对其显色剂用量、显色稳定性和最佳检测波长等因素进行探讨，得出测定血浆Ⅳ型胶原酶的最优分析条件，采用回收实验、精密度实验对其进行方法学评价，并与夹心ELISA进行比较。采用该方法检测了112名健康人血浆Ⅳ型胶原酶含量。使用SPSS软件进行统计学分析，建立健康人血浆Ⅳ型胶原酶含量双侧95％可信区间。结果显色法检测血浆Ⅳ型胶原酶，总测定时间〈1．5h，线性范围1．5—10．0mg／L，最低检出限为0．965mg／L，与ELISA法相关性良好（R2=0．9997，P〈0．01），批内变异系数〈3．564％，批间变异系数〈9．821％。健康人血浆Ⅳ型胶原酶含量双侧95％的可信区间为33．38～49．80mg／L。结论血浆Ⅳ型胶原酶含量显色法可以应用于检测血浆中活性形式Ⅳ型胶原酶的含量，并建立了健康人血浆Ⅳ型胶原酶含量参考区间。

Objective To establish a chromogenic assay for blood-plasma collagenase Ⅳ in order to evaluation the reference range of collagenase Ⅳ in the plasma of healthy individuals. Methods The assay was based on measurement of terminal amino group with succinylated gelatin as substrates and TNBS as chromogenic reagent. The optical density of each reaction was determined at 405 nm using a Sunrise microplate reader. Chromatographic and detection conditions were optimized and performance of the method was evaluated by recovery experiments and precision experiments. It was compared with ELISA. The levels of collagenase Ⅳ in 112 health persons＇ plasma were determined and the data were analyzed by SPSS statistical software. Results The whole determing time was within 1.5 h, the linear range of this method was 1.5- 10. 0 mg/L, and the minimum detection limit was 0. 965 mg/L. They were well correlated with ELISA results （R2 = 0. 999 7, P 〈 0. 01 ）. The within-run CV was less than 3. 16% and between-run CV was less than 9. 81%. The 95% confidence interval of collagenase Ⅳ in healthy plasma was 33.38-49. 80 mg/L Conclusion This chromogenic assay for blood-plasma collagenase Ⅳ can be used for measurement of collagenase Ⅳ in blood-plasma and the reference range of collagenase Ⅳ in healthy plasma was established.