Substrate, method and kit for in-vitro quantitative determination of activity of vWF-CP enzyme

A technology for quantitative determination and enzymatic activity, applied in the biological field, can solve the problems of difficult to cater for large-scale TTP screening, complex testing technology process, and long time-consuming.

Active Publication Date: 2017-04-19
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 a fragment 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 i

Method used

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

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Experimental program
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Embodiment 1

[0031] Embodiment one substrate screening

[0032] The present invention provides a screening method and result of a substrate for quantitative determination of vWF-CP enzyme activity in vitro, wherein the experimental process includes the following steps:

[0033] 1. Substrate preparation

[0034] 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.

[0035] 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 R16...

Embodiment 2

[0074] Example 2 In vitro quantitative assay kit and method of use of vWF-CP enzyme activity

[0075] The present invention provides a kit for in vitro quantitative determination of vWF-CP enzyme activity, the components of which are shown in Table 6.

[0076] Table 6 Kits for quantitative determination of vWF-CP enzyme activity in vitro

[0077]

[0078] The use method of above-mentioned kit comprises the following steps:

[0079] S1. Washing: Take out the ELISA strip for one use, wash 3 times with diluted reagent C, 200 μl / well, and control to dry.

[0080] S2. Add substrate: Dilute reagent A and diluted reagent B 1:100, add to the plate, 100 μl / well, make duplicate wells, and set blank wells at the same time (except for washing, color development and stop solution, others are not added. ), incubated at 37°C in the dark for 1 hour, washed 3 times with reagent C, 200 μl / well, and allowed to dry.

[0081] S3, sample addition:

[0082] 1) Well of standard substance I: ad...

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Abstract

The invention relates to the biological field, and especially relates to a substrate, a method and a kit for in-vitro quantitative determination of the activity of vWF-CP enzyme. The substrate comprises a vWF functional area amino acid sequence. The end N and the end C of the vWF functional area amino acid sequence are respectively combined with a HIS label amino acid sequence and a FLAG label amino acid sequence. The method comprises the following steps: the holes of an ELISA plate are coated with an antibody in advance; and then, the substrate, a sample to be tested, an ELISA antibody, and chromogenic agent are added, and the absorbance of liquid in the holes of the ELISA plate is measured using an ELISA reader. The kit comprises an ELISA plate, a substrate, diluent, washing liquid, buffer solution, an enzyme-labeled antibody, chromogenic solution and stop solution, or comprises an ELISA plate, an antibody, a substrate, diluent, washing liquid, buffer solution, an enzyme-labeled antibody, chromogenic solution and stop solution. The substrate, the method and the kit for in-vitro quantitative determination of the activity of vWF-CP enzyme have the advantages of high sensitivity, high accuracy, simple operation and low price, and are suitable for broad patient screening.

Description

technical field [0001] The invention relates to the field of biology, in particular to a substrate, method and kit for quantitatively measuring vWF-CP enzyme activity in vitro. 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. In clinical practice, patients with typical...

Claims

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Application Information

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IPC IPC(8): G01N33/535
CPCG01N33/535
Inventor 林毅然王季武黄海燕
Owner 吴江华药生物技术有限公司
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