人肥大细胞类胰蛋白酶的免疫测定

Immunoassay of tryptase from human mast cells.

作者信息

Wenzel S, Irani A M, Sanders J M, Bradford T R, Schwartz L B

出版信息

J Immunol Methods. 1986 Jan 22;86(1):139-42. doi: 10.1016/0022-1759(86)90277-2.

Abstract

A sandwich ELISA was developed for the measurement of tryptase. The assay utilizes the mouse monoclonal anti-tryptase antibody, termed G5 (IgG2b kappa) in the solid phase and monospecific goat IgG anti-tryptase antibody together with tryptase in the fluid phase. The immunoassay will quantify 0.1 ng-5.6 ng of tryptase per 100 microliters of sample solution to within 0.1 ng. Intra-assay coefficients of variation were determined at 0.3 ng, 1.0 ng and 3.0 ng of tryptase per assay, respectively, to be 19%, 7% and 4% with buffer and 10%, 4%, and 4% in the presence of 20% plasma. Inter-assay coefficients of variation at the same respective levels of tryptase were 22%, 18% and 15% with buffer and 18%, 11% and 14% with 20% plasma. Net absorbance values obtained with a standard amount of tryptase in buffer alone and up to 50% (v/v) normal human citrate-treated plasma were within 10% of one another, indicating nearly complete detection of tryptase added to plasma. This represents the first sensitive immunoassay for a preformed mediator specific for human mast cells.

摘要

开发了一种用于测量类胰蛋白酶的夹心酶联免疫吸附测定法（ELISA）。该测定法在固相使用名为G5（IgG2b κ）的小鼠抗类胰蛋白酶单克隆抗体，在液相使用单特异性山羊抗类胰蛋白酶IgG抗体和类胰蛋白酶。该免疫测定法可将每100微升样品溶液中的0.1纳克至5.6纳克类胰蛋白酶定量至误差在0.1纳克以内。分别在每次测定0.3纳克、1.0纳克和3.0纳克类胰蛋白酶时测定批内变异系数，在缓冲液中分别为19%、7%和4%，在存在20%血浆时分别为10%、4%和4%。在相同类胰蛋白酶水平下，批间变异系数在缓冲液中分别为22%、18%和15%，在20%血浆中分别为18%、11%和14%。仅在缓冲液中以及在高达50%（v/v）正常人枸橼酸盐处理血浆中加入标准量类胰蛋白酶所获得的净吸光度值彼此相差在10%以内，表明加入血浆中的类胰蛋白酶几乎能被完全检测到。这代表了针对人肥大细胞特异性预形成介质的首个灵敏免疫测定法。