Stable HRP enzymatic enhanced chemiluminescent substrate solution

A technology of enzymatic chemiluminescence and substrate solution, which is applied in the field of immunoassay, can solve the problems of limiting the application of chemiluminescence immunoassay, low luminous intensity, poor stability, etc., and achieves short reaction time, high luminous intensity value and high sensitivity Effect

Active Publication Date: 2012-05-02
BIOBASE BIODUSTRY (SHANDONG) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The luminescent substrates currently on the market have poor stability, low luminous intensity, and high background, which severely limit the application of chemiluminescent immunoassays. To address these problems, we have conducted research on their formulations

Method used

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  • Stable HRP enzymatic enhanced chemiluminescent substrate solution
  • Stable HRP enzymatic enhanced chemiluminescent substrate solution
  • Stable HRP enzymatic enhanced chemiluminescent substrate solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1 Chemiluminescence substrate solution preparation

[0030] Preparation of buffer: 0.1mol / L Tris-HCl buffer, pH8.4. 0.01mol / L PBS buffer, pH7.4, the specific formula is as follows:

[0031] Liquid A (luminescent agent solution):

[0032]

[0033] B liquid (oxidant solution):

[0034]

[0035]

[0036] The following method can be used to prepare 1000mL of liquid A (luminescent agent solution) of chemiluminescence substrate: first, dissolve 0.2417g of 4-(1,2,4-triazol-1-yl)phenol in 10mL of N,N-di In methylformamide, mix well; take 0.1991g of luminol monosodium salt, 0.1100g of p-iodophenol, 1.8610g of edetate disodium salt, 0.0144g of sodium benzoate, and 0.1755g of sodium chloride; -(1,2,4-Triazol-1-yl)phenol solution and various substances were added to Tris-HCl buffer solution, and the volume was adjusted to 1000ml with Tris-HCl buffer solution, mixed well, and stored at 4°C.

[0037] The preparation of 1000 mL of liquid B (oxidizing agent solut...

Embodiment 2

[0038] Example 2 Enzyme-catalyzed chemiluminescent reaction kinetics curve

[0039] Add 10 μL of HRP (10 ng / mL) to the microwell plate, add 50 μL of the luminescent substrate A solution and 50 μL of the luminescent substrate B solution prepared in Example 1 to each well, and quickly detect the luminous intensity (RLU), and continuously detect for 30 minutes, draw the reaction kinetics curve, the results are shown in the attached figure 1 ,from figure 1 It can be seen that the luminescent substrate solution prepared in Example 1, the luminous intensity of the reaction reaches the maximum in about 3 minutes, and then there is a long plateau period. After 30 minutes of detection, it still maintains a high luminous intensity. This shows that the luminescent substrate solution of the present invention has high reaction stability, and can effectively reduce errors when applied to a detection system.

Embodiment 3

[0040] Embodiment 3 Sensitivity research

[0041] Dilute the newly prepared 1mg / mL HRP solution with PBS buffer, and select the concentration from 100ng / mL to 0.001ng / mL for detection. detection concentration), and then add 50 μL of luminescent substrate A solution and 50 μL of luminescent substrate B solution prepared in Example 1 to each well, and detect RLU after standing for 5 minutes. The results of three detections in parallel are represented by S / N, as shown in Table 1 and attached figure 2 shown. From Table 1 and figure 2 It can be seen that the concentration of HRP is positively correlated with the signal-to-noise ratio. With the decrease of the HRP concentration, the signal-to-noise ratio of the luminous intensity is also decreasing. The logarithm of S / N is the ordinate, and the concentration of HRP is the abscissa In the drawing, each corresponding point is almost on a straight line, which shows that the luminescent substrate prepared in Example 1 has a strong ...

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Abstract

The invention discloses a stable HRP enzymatic enhanced chemiluminescent substrate solution. The stable enzymatic enhanced chemiluminescent substrate solution is composed of a luminescent agent solution A and an oxidizing agent solution B, which are mixed in volume ratios of 2:3-3:2. The luminescent agent solution A contains a luminescent agent: luminol or luminol derivatives, and a reinforcing agent: p-iodo-pheno, 4-(1, 2, 4-triazole-1-radical) phenol, etc., and the oxidizing agent solution B contains sodium perborate, hydrogen peroxide, etc. The stable HRP enzymatic enhanced chemiluminescent substrate solution of the invention is applied in an HRP enzymatic reaction with the characteristics of short reaction time and long platform period, can be stably preserved for 2 years at 4-8 EDG C, and is widely used in a kit using HRP as the detection object.

Description

technical field [0001] The invention relates to the field of immunoanalysis, and more specifically relates to a chemiluminescence substrate solution using HRP as an enzymatic reaction detection system. Background technique [0002] Chemiluminescence immunoassay (CLIA) combines the chemiluminescence system with the immune system to detect antibodies or antigens. It has both the specificity of the immune response and the high sensitivity of the chemiluminescence reaction. The specific process is to label the antibody or antigen with a chemiluminescence reaction reagent (which can be a chemiluminescence reagent or catalyst, etc.), and the labeled antibody or antigen and the analyte go through a series of immune reactions and physical and chemical steps, and finally to detect the intensity of the luminescence. Formal determination of the content of the analyte. It overcomes the radioactive hazard and pollution problems caused by the use of radioactive isotopes in radioimmunoass...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N33/52
Inventor 谭柏清王进张泓胡晓飞
Owner BIOBASE BIODUSTRY (SHANDONG) CO LTD
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