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Method for in-vitro quantitative determination of vWF-CP enzyme activity, detection kit and preparation method of detection kit

A detection kit and quantitative determination technology, applied in measuring devices, instruments, disease diagnosis, etc., can solve problems such as difficulty in catering to large-scale TTP screening, complex testing technology process, and long time consumption

Active Publication Date: 2017-04-26
吴江华药生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the previously reported literature used GST-vWF73-His as a substrate, (Zhou W, Tsai HM. An enzyme immunoassay of ADAMTS13distinguishes patients with thrombotic thrombocytopenic purpura from normal individuals and carriers of ADAMTS13 mutations.Thromb) or fragments of the A2 region as a substrate ( Haemost 2004;91: 806–11Whitelock JL, Nolasco L, Bernardo A, Moake J, Dong JF, Cruz MA. ADAMTS-13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant vWF-A2 domain as substrate. JThromb Haemost 2004;2 :485–91.), the molecular weight of this recombinant protein is relatively large, the testing process is complicated and time-consuming, it is difficult to meet the needs of large-scale TTP screening, and it is not suitable for the clinical needs of emergency diagnosis and treatment of patients in the emergency room

Method used

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  • Method for in-vitro quantitative determination of vWF-CP enzyme activity, detection kit and preparation method of detection kit
  • Method for in-vitro quantitative determination of vWF-CP enzyme activity, detection kit and preparation method of detection kit
  • Method for in-vitro quantitative determination of vWF-CP enzyme activity, detection kit and preparation method of detection kit

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Experimental program
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Embodiment 1

[0062] The preparation of embodiment one vWF-CP substrate

[0063] A preferred embodiment of the present invention provides a method for preparing a vWF-CP substrate, comprising the following steps:

[0064] S1. Obtaining the target gene fragment, the target gene fragment can encode the vWF-CP substrate, the substrate includes the amino acid sequence of the vWF functional region, and the N-terminal and C-terminal amino acid sequences of the vWF functional region are respectively combined with tag amino acid sequences, The amino acid sequence of the vWF functional region is the sequence including the enzyme cutting sites Y1605 to M1606 of vWF-CP for vWF.

[0065] According to existing studies, the amino acid sequence of the vWF functional region can be selected from D1459 to R1668 of vWF and includes a sequence of restriction sites Y1605 to M1606, such as D1459 to R1668, or E1554 to R1668, or D1587 to R1668 of vWF, or The sequence from D1596 to R1668, wherein the minimum funct...

Embodiment 2

[0084] Embodiment 2 test strip preparation

[0085] This embodiment provides a method for preparing a test strip for quantitative determination of vWF-CP enzyme activity in vitro. The test strip includes sample pads, gold standard pads, NC membranes, and water-absorbing pads assembled in sequence. The NC membrane is provided with detection lines and The quality control line, the gold label pad, the detection line and the quality control line are respectively coated with gold label antibody, T line antibody and C line antibody, wherein,

[0086] T-line antibody: anti-FLAG tag mouse monoclonal antibody;

[0087] C-line antibody: secondary antibody goat anti-mouse IgG;

[0088] Gold-labeled antibody: colloidal gold-labeled anti-HIS tag mouse monoclonal antibody;

[0089] Gold-labeled antibody diluent: PBS solution (pH7.4) containing 3% sucrose and 1% BSA.

[0090] S1. Preparation of T-line antibody streaking solution: dilute T-line antibody to 1.5ug / ul with PBS; preparation of...

Embodiment 3

[0094] The kit composition of embodiment three in vitro quantitative assay vWF-CP enzymatic activity

[0095] This embodiment provides a kit for in vitro quantitative determination of vWF-CP enzyme activity, the composition of which includes:

[0096] vWF-CP enzyme relative activity colloidal gold detection test paper card (30 parts / box, the test paper strip in embodiment two), room temperature preservation;

[0097] Standard 1 (1ml) (the product with vWF-CP enzyme relative activity of 0), store at -20°C;

[0098] Standard 2 (1ml) (the product with 100% relative activity of vWF-CP enzyme), stored at -20°C;

[0099] vWF-CP substrate (recombinant protein in Example 1) (1ml), stored at -20°C;

[0100] Working solution (10ml) (buffer solution containing divalent metal ions), stored at 4°C;

[0101] Stop solution (2ml) (contains a buffer capable of complexing divalent metal ions), stored at 4°C;

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Abstract

The invention relates to a method for in-vitro quantitative determination of vWF-CP enzyme activity, a detection kit and a preparation method of the detection kit. The method comprises the steps of test paper strip preparation, reaction liquid preparation, chromogenic reaction, enzyme activity calculation and the like. The detection kit includes a test paper strip, a standard substance 1, a standard substance 2, a vWF-CP substrate, a working solution and a stop solution; a gold labeled antibody on the test strip is a colloidal gold labeled anti-vWF-CP substrate antibody, a T line antibody is an anti vWF substrate antibody, a C line antibody is a gold labeled antibody resistant antibody, and the gold labeled antibody and the T line antibody are combined with different loci on the vWF-CP substrate; the standard substance 1 and the standard substance 2 are respectively products having high vWF-CP enzyme relative activity and low vWF-CP enzyme relative activity; the working solution is a buffer solution containing divalent metal ions; the stop solution is a buffer solution containing ions which can be complexed with the divalent metal ions. The preparation method of the detection kit comprises test paper strip preparation, standard substance preparation, vWF-CP substrate preparation, working solution and stop solution preparation and the like. The method and the detection kit are fast in detection, high in sensitivity and good in accuracy.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for quantitatively measuring vWF-CP enzyme activity in vitro, a detection kit and a preparation method. Background technique [0002] Thrombotic Thrombocytopenic Purpura (TTP) is a severe disseminated thrombotic microangiopathy characterized by microangiopathic hemolytic anemia, decreased consumption of platelet aggregation, and microthrombosis causing organ damage (such as the kidney, central nervous system, etc.) According to early reports, the incidence of TTP is 0.2 to 1 / 100,000. With the gradual recognition of this disease in clinical practice, the incidence of TTP has an upward trend in recent years. At present, no relevant epidemiological statistics have been seen in China. Most of the patients are women, and the disease can occur at any age, but the most common age of onset is mostly 20 to 60 years old, and there is no regional or racial difference. Clinica...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N33/558G01N33/536
CPCG01N33/536G01N33/558G01N33/573G01N2333/988G01N2800/222
Inventor 林毅然王季武黄海燕
Owner 吴江华药生物技术有限公司