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Preparation of ELISA kit of human tissue kallikrein

A kallikrein and kit technology, which can be used in measurement devices, instruments, scientific instruments, etc., can solve the problems of high price, complicated methods, and complicated calculations.

Inactive Publication Date: 2010-03-17
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the method is complicated, the calculation is cumbersome, it is easy to produce non-specific color development, resulting in large error in the results, and the price is expensive, which does not meet China's national conditions, and there is no such kit in China.

Method used

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  • Preparation of ELISA kit of human tissue kallikrein
  • Preparation of ELISA kit of human tissue kallikrein
  • Preparation of ELISA kit of human tissue kallikrein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Preparation of Human Tissue Kallikrein ELISA Kit

[0036] 1. Reagents

[0037] (1) Coating buffer (PH9.60.05M carbonate buffer):

[0038] Na2CO3 1.59 g

[0039] NaHCO3 2.93 g

[0040] Add distilled water to 1000ml;

[0041] (2) Washing buffer (PH7.4PBS): 0.15M

[0042] KH2PO4 0.2 g

[0043] Na2HPO4·12H2O 2.9g

[0044] NaCl 8.0g

[0045] KCl 0.2 g

[0046] Tween-20 0.1% 0.5ml add distilled water to 1000ml;

[0047] (3) Specimen diluent:

[0048] Bovine serum albumin (BSA) 0.1 g plus washing buffer to 100 ml;

[0049] (4) Standard product:

[0050] Human tissue kallikrein fusion protein, self-produced for this experiment;

[0051] (5) Biotinylated secondary antibody:

[0052] Biotin-labeled anti-human tissue kallikrein monoclonal antibody, self-produced by our laboratory;

[0053] (6 enzyme-labeled avidin:

[0054] HRP-labeled streptavidin, produced by Wuhan Boster Biological Co., Ltd.;

[0055] (7) Stop solution (2M H2SO4):

[0056] 178.3ml of distilled ...

Embodiment 2

[0103] Comparison with foreign kits

[0104] The sensitivity range of foreign kits is 0.4ng / ml-25ng / ml; the sensitivity of this kit is 0.3ng / ml-500ng / ml, and its detection range is higher than that of foreign kits. The method used in foreign kits is the indirect competitive ELISA method, and its calculation is cumbersome. However, the method used in this kit is the double-antibody sandwich method, and the calculation is more intuitive and simple.

Embodiment 3

[0106] This kit was used to detect the HK1 content in the plasma of 124 normal people, and each group had three duplicate wells, which were repeated at intervals of 6 months. The within-group difference was 1.7% and the between-group difference was 8%. The plasma content of HK1 in normal people is 252±93ng / ml.

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Abstract

The invention relates to a preparation of an ELISA kit of human tissue kallikrein. The technical scheme is achieved as follows: the ELISA kit for testing the content of human tissue kallikrein is characterized in that: (a) the method adopted by the kit is a double antibody sandwich method; (b) a biotin-aviden amplification system is added; (c) the standard substance is pronucleus-expressed and purified human tissue kallikrein; (d) the coated antibody is a polyclonal antibody for resisting the human tissue kallikrein, and a detection antibody is a monoclonal antibody of biotinylation; and (e) the sensitivity is 0.3ng / ml to 500ng / ml. The kit has the characteristics of high sensitivity, strong specificity and favorable repeatability. By adopting the expressed fusion protein as the standard substance, the correlation coefficient of a standard curve is as high as 0.99, and by adopting a batch of human plasma as samples to be tested, both the difference in a group and the difference among groups are lower than 10 percent.

Description

technical field [0001] The invention relates to a fusion protein of human tissue kallikrein, which is an ELISA kit for detecting the content of human tissue kallikrein. Background technique [0002] Human tissue kallikrein (HK1) is a kind of serine protease, which can promote the release of vasoactive peptide by low-molecular kininogen to exert a series of biological activities, named KLK1. In human and animal models of hypertension, cardiovascular disease and kidney disease, the reduction of HK content can be seen. We found that HK1 can lower blood pressure, improve insulin resistance, and improve renal function in transgenic animal models. The level of HK1 may have important predictive value in the occurrence and development of human cardiovascular disease and kidney disease. Therefore, the establishment of an efficient detection kit for the determination of HK1 content is of great significance in both clinical and scientific research. [0003] In view of the predictive...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 汪道文张琴
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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