阅读说明：本技术 消除纤维蛋白原干扰的化学发光法试剂盒配方 (Chemical luminescence method kit formula for eliminating fibrinogen interference ) 是由 李立和 魏志斌 刘宝阳 于 2019-10-30 设计创作，主要内容包括：本发明公开了一种消除纤维蛋白原干扰的化学发光法试剂盒配方,属于利用可见光,通过测试反应的结果产生颜色变化来测试材料的方法,属于免疫分析技术领域。本发明的技术方案是：化学发光法样品稀释液中含有含有纤溶酶,在进行磁颗粒化学发光法测定中不受纤维蛋白的干扰,特别是肾衰患者,消除了高浓度的纤维蛋白造成磁颗粒化学发光法反应、磁颗粒分离和洗涤不完全等造成的测定偏差,其使用方法和范围与原有的化学发光法相同,不会增加实验人员的负担,经济方便易行,是一种准确性更高的检测方法。(The invention discloses a formula of a chemiluminescence method kit for eliminating fibrinogen interference, belongs to a method for testing materials by using visible light and generating color change through a test reaction result, and belongs to the technical field of immunoassay. The technical scheme of the invention is as follows: the chemiluminescence method sample diluent contains plasmin, is not interfered by fibrin during the measurement of the magnetic particle chemiluminescence method, particularly for renal failure patients, eliminates the measurement deviation caused by the reaction of the magnetic particle chemiluminescence method, incomplete separation and washing of magnetic particles and the like due to high-concentration fibrin, has the same use method and range as the original chemiluminescence method, does not increase the burden of experimenters, is economic, convenient and easy, and is a detection method with higher accuracy.)

1. A chemical luminous method reagent box formula for eliminating fibrinogen interference is characterized in that a sample diluent contains fibrinolysin, the interference of endogenous fibrin in serum is eliminated in the magnetic particle chemical luminous method determination, especially for renal failure patients, the magnetic particle chemical luminous method reaction, magnetic particle separation and incomplete washing and other causes larger deviation due to high-concentration fibrin.

2. The chemiluminescence method kit formula for eliminating fibrinogen interference according to claim 1, characterized in that 8000U of plasmin is dissolved in per liter of Tris-HCl 150mmol (pH6.0) buffer solution in sample diluent, 200 μ L of Proclin-300 preservative, and the final concentration of plasmin in the reaction system is 7800U/L or more.

3. The formulation of the chemiluminescence method kit for eliminating fibrinogen interference of claim 1, wherein the concentration of Tris-HCl buffer solution in the sample dilution is150 mmol/L, and the pH value is 6.0 + -0.2.

Technical Field

The present invention pertains to a method of assay comprising an enzyme; or a method for testing materials by using visible light and generating color change through a test reaction result, in particular to a kit formula for eliminating interference of a chemiluminescence method by using plasmin.

background

Chemiluminescence immunoassay (CLIA) is a quantitative analysis method commonly used in laboratories, which combines a chemiluminescence determination technology with high sensitivity and high specificity immunoreaction, is used for detection and analysis technologies of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs and the like, has fluorescence specificity, does not need exciting light, avoids the influence of exciting light stray light in fluorescence analysis, improves sensitivity, and is a method for quantitative analysis.

The magnetic beads used for chemiluminescence immunoassay are various in types, and comprise magnetic agarose microspheres, magnetic silica microspheres, magnetic polyacrylamide/polyacrylic acid polymer microspheres, magnetic polystyrene microspheres and the like, and the microsphere particles have the characteristics of strong hydrophilicity, easy elution under mild conditions, no enzyme inactivation or protein denaturation and the like.

the kidney diseases include nephritis, nephrotic syndrome, and secondary renal changes caused by various etiology. Changes such as hypoproteinemia, blood concentration, increased blood viscosity, etc. occur in renal insufficiency and failure, and the use of hormones and diuretics, which cause the blood to be in a hypercoagulable state, manifested as an increase in plasma fibrinogen (Fbg) levels. When blood is centrifugally separated and measured by a chemiluminescence method, serum is in a jelly shape and forms a coated particle with magnetic particles in a reagent of the chemiluminescence method, and the reaction, separation and washing of the chemiluminescence method are influenced, so that a result has larger deviation.

Plasmin (plasmin) is a proteolytic enzyme capable of specifically degrading fibrin gel, and is a peptide chain containing 790 amino acid residues, and the N-terminal is glutamic acid. Activated plasmin forms two peptide chains linked by two pairs of disulfide bonds. The light chain is the C-terminal part of the propeptide chain, contains 230 amino acid residues in total, is similar to trypsin in structure, and the active site of the enzyme is positioned in the light chain. The N-terminus of the heavy chain is lysine or valine, and the C-terminus is arginine at which the peptide bond is cleaved upon activation. The heavy chain portion has a structure very similar to the N-terminal A and S-peptide fragments of prothrombin and consists of 5 similar cyclic structures, and plasmin releases 5 corresponding degradation fragments A, B, C, D, E during stepwise degradation of fibrin. A. B, C is a small molecule and D, E is a large molecule. D. The molecular weights of the two E fragments were 80000 and 48000, respectively. Fragment D is about twice the molar weight of fragment E, and also gives the intermediates "X" and "Y" fragments of higher molecular weight. It is presumed that the degradation of fibrin is roughly as follows, that fibrin is degraded into "X" fragments, and small molecule fragments "A" and "B" are released, which correspond to about 40-50 amino acid residues at the N-terminal part of the β -peptide chain and a loose part at the C-terminal part of the α -peptide chain of fibrin, respectively. The fragment X is further degraded into a fragment D and a fragment Y, the fragment D is equivalent to the main body at the C end of the fibrin monomer, the fragment E is equivalent to the middle main body part of the fibrin monomer and comprises the structure of a disulfide bridge, and the fragment C is the structure of a middle spiral region connecting the main body parts at the N end and the C end of the fibrin.

In order to solve the problem that the serum fibrinogen magnetic particles form wrapped particles due to renal insufficiency in the existing magnetic particle chemiluminescence method measurement, and the result has larger deviation caused by influencing chemiluminescence method reaction, separation, washing and the like, the invention designs a soluble reaction system formed by adding plasmin into a reagent diluent and degrading fibrinogen by using the plasmin, which is beneficial to chemiluminescence method reaction, magnetic particle separation and washing and does not interfere normal population measurement.

compared with the prior art, the invention has the following advantages: the sample diluent prepared by the invention contains plasmin, is not interfered by the content of endogenous fibrin in serum during the measurement of a magnetic particle chemiluminescence method, particularly for renal failure patients, and is a detection method with higher accuracy, wherein the interference of fibrinogen in the magnetic particle chemiluminescence method can be eliminated by the method disclosed by the invention, and the application method and the range of the method are the same as those of the original chemiluminescence method, so that the burden of experimenters cannot be increased, and the method is economical, convenient and easy to implement.

the specific implementation mode is as follows:

The present invention is described in further detail below by way of example using the Anagraph A2000PLUS chemiluminescence analyzer for parathyroid hormone.