Two-component substrate solution

Product Introduction

Currently, the enzyme immunoassay (EIA) technology has been widely applied to the quantitative or qualitative detection and analysis of antigens, haptens or antibodies. Horseradish peroxidase (HRP) and its conjugates are a commonly used enzyme in enzyme-linked immunosorbent assay (ELISA) technology. Since tetramethylbenzidine (TMB) has higher sensitivity than other chromogens and is non-carcinogenic in the chromogenic reaction system of HRP, it has been widely used. TMB is mainly applied to enzyme-linked immunosorbent assay (ELISA), immunoblot hybridization, immunohistochemistry, as well as the detection and analysis of chlorine and hydrogen peroxide. In order to meet the research and development, production of different kit products, as well as scientific research needs, our company has specifically developed TMB chromogenic solutions for different types of HRP-based immunoassays.
Appearance and Structure: Chromogenic Solution A should be a colorless or light yellow transparent liquid, and Chromogenic Solution B should be a colorless transparent liquid. There should be no precipitation, particles, or flocculent substances in both.
It is used in the chromogenic stage of enzyme-linked immunosorbent assay experiments: It can react with the sample to generate a dark blue product.

Usage Method

Solution Addition: After adding the HRP conjugate to the sample wells and incubating for a certain period of time, wash the plate 3-5 times with an appropriate washing solution. Add 50 μl each of Substrate Chromogenic Solution A and Chromogenic Solution B to each well (mix Solution A and Solution B first and then add them to the wells), gently mix them evenly. According to individual experimental needs, incubate in the dark at room temperature (15-25°C) or at 37°C for 10-30 minutes or longer until the color development reaches the expected depth.

Termination: Add an equal volume of 2M hydrochloric acid or sulfuric acid solution to each well to terminate the reaction. The reaction solution in the wells will change from blue to yellow.

Absorbance Reading: Measure the absorbance value of the solution in each well at 450 nm within 15 minutes after terminating the reaction.

Control: The blank control without the HRP-labeled antibody/antigen and the negative control result should be colorless. The positive control and the well with the HRP-labeled antibody/antigen will produce a dark blue product.

Note: If a high reaction background or precipitation occurs, it indicates that the TMB substrate reaction is too intense. To avoid the formation of precipitation, the absorbance can be read immediately after termination; or the primary antibody and/or the HRP conjugate can be further diluted.

Precautions

Experimental supplies should be dedicated to avoid cross-contamination.
TMB is irritating to the human body, so please pay attention to appropriate protection.
For your safety and health, please wear a laboratory coat and disposable gloves when operating.