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Colour-developing agent preparation for enzyme-linked immunoassay in vitro diagnosis agent

An in vitro diagnosis and enzyme-linked immunosorbent technology, which is applied in the direction of material analysis, measuring devices, instruments, etc. through the observation of the influence on chemical indicators, can solve the problem of high background of chromogenic reagents, achieve low color background and simple preparation process , the effect of high color sensitivity

Inactive Publication Date: 2008-01-09
SHANGHAI HUATAI BIOENG IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chromogenic agent prepared directly with commercially available TMB often has a high background

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Prepare 1 L of chromogen with a TMB hydrochloride concentration of 0.04%:

[0030] 1. Add about 500ml of distilled water into the volumetric flask, and then add the following substances in turn, and then add the latter after the former one is dissolved; Tris 2.42g; HCl (36%-38%) 2.4ml; EDTA disodium salt 0.186g ; penicillin sodium 0.05g; hydroxylamine 0.03ml; TMB hydrochloride 0.4g. After dissolving, make up to 1L with distilled water.

[0031] 2. Adjust the pH to 2.5 with HCl.

Embodiment 2

[0033] Prepare 1 L of chromogen with a TMB hydrochloride concentration of 0.06%:

[0034] 1. Add about 500ml of distilled water into the volumetric flask, and then add the following substances in turn, and then add the latter after the former one is dissolved; Tris 2.42g; HCl (36%-38%) 2.4ml; EDTA disodium salt 0.186g ; penicillin sodium 0.05g; hydroxylamine 0.03ml; TMB hydrochloride 0.6g. After dissolving, make up to 1L with distilled water.

[0035] 2. Adjust the pH to 2.5 with HCl.

Embodiment 3

[0037] Use 0.06% TMB hydrochloride solution as a chromogen to detect HBsAg in serum or plasma samples.

[0038] (1) Add 50ul of the sample in sequence to the anti-HBs-coated microwell plate, and make two wells of negative control, one well of positive control, and one well of blank control at the same time, and then add anti-HBs- 50ul of HRP enzyme conjugate solution was incubated at 37°C for 30 minutes.

[0039] (2) Discard the liquid in the well, and wash the plate 5 times with washing solution.

[0040] (3) 50 ul of substrate solution and 50 ul of the color developer prepared in Example 2 of the present invention were added to each well, and incubated at 37° C. for 15 minutes.

[0041] (4) Add 50ul of stop solution to each well to stop the reaction.

[0042] (5) Put the reaction plate at the 450nm wavelength of the enzyme-labeled colorimeter to zero with a blank well, and read the OD value of each well.

[0043] (6) Calculation, Cut Off value = average OD value of the ne...

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PUM

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Abstract

The invention provides the preparation method for developer in the outside diagnose reagent of enzyme immune body. The method of the invention change the form of developer in the outside diagnose reagent of enzyme immune body. It makes the whole process of confect predigest, improves the sensitivity of showing color, increases the stability of developer, and debases the background of showing color. It has great appliance vale for clinic.

Description

Technical field: [0001] The invention belongs to the technical field of biological in vitro diagnostic reagents. Specifically relates to the preparation of a chromogenic agent in an enzyme-linked immunological in vitro diagnostic reagent. Background technique: [0002] Enzyme-linked immunoassay (ELISA) has been widely used in the detection of antigens, antibodies, cytokines, and biochemical substances due to its advantages of rapidity, sensitivity, simplicity, and ease of standardization. There are many detection methods for ELISA reagents, but each method is inseparable from enzyme conjugates and chromogenic reagents. The enzyme most widely used in ELISA reagents is horseradish peroxidase (HRP). 3,3',5,5'-Tetramethylbenzidine (TMB). Compared with other chromogens, TMB has many advantages, such as non-carcinogenicity and good stability. During the detection, HRP catalyzes the peroxide to release oxygen, and the colorless TMB is oxidized into a blue product. After adding ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N21/78
Inventor 周兴华
Owner SHANGHAI HUATAI BIOENG IND
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