Reasons for the high background of the new Trinder's reagent TODB

In the use of the new Trinder's reagent TODB, high background is a common problem that affects the detection results, while insufficient sealing and incomplete plate washing are key factors that are easily overlooked during operation. These two issues can cause an abnormal increase in the color background due to non-specific binding and interference from residual substances. Below is a detailed analysis and solution.

Inadequate closure: the 'behind the scenes' driving force behind non-specific combinations

The function of the blocking step is to block the non binding sites on the surface of the reaction carrier (such as an enzyme-linked immunosorbent assay plate). If the sealing is not sufficient, the substrates, enzyme conjugates, and other components in the TODB reagent will randomly adsorb onto the surface of the carrier, forming a color signal without the participation of the target substance, directly increasing the background value.

specific reason

1. Insufficient sealing time: Generally speaking, sealing requires placing at 37 ℃ for 60 minutes or at room temperature for 120 minutes. If the time is shortened to less than 30 minutes, the active sites on the surface of the carrier cannot be completely covered by the blocking solution (such as BSA, skim milk powder), and those hydrophobic areas that are not blocked will actively adsorb protein components in the TODB reaction system, resulting in background coloration.

2. Too low concentration of blocking solution: When the effective ingredients in the blocking solution are insufficient, such as when the original 5% BSA drops to 1%, a tight protective film cannot be formed between molecules. Components such as horseradish peroxidase in TODB reagent will stick to the inner wall of the plate pore through hydrophobic interactions, and non-specific reactions will occur with the substrate during the reaction.

3. Failure of sealing solution: If the sealing solution is repeatedly frozen and melted, or stored for more than its expiration date, the protein components inside will deteriorate and lose their sealing function, resulting in many "exposed" sites on the surface of the carrier, which become the source of background signals.

Incomplete board washing: the "stacking effect" of residual substances

The main function of plate washing is to remove unbound free reagents (such as unbound antibodies, TODB precursor substances). If the plate washing is not thorough, residual substances will continue to participate in color development in subsequent reactions, allowing background interference to accumulate.

Specific Reason

1. Too few plate washes: Conventional testing requires washing the plate 3-5 times. If it is reduced to less than 2 times, the residual free enzyme complexes in the well cannot be completely removed, and the reaction will react with the TODB substrate, resulting in additional color development.

2. Too much detergent residue: After washing the board, if the holes are not inverted on the absorbent paper and patted dry, there will be more detergent residue in each hole, and some components inside will disrupt the balance of the TODB reaction system. At the same time, residual enzyme conjugates will diffuse into the adjacent holes with the liquid, causing cross contamination and raising the background.

3. There is a problem with the washing machine: When the needle of the washing machine is blocked or the pressure is insufficient, the corners of the plate holes will become areas that cannot be washed. The residual TODB reagent will crystallize after drying and participate in the reaction when dissolved again, causing the local background color to darken.

Summary: Operational details determine the effectiveness of background control

Although sealing and washing plates are routine steps, their quality directly affects the background level of TODB reagents. In practical use, it is recommended to adopt the method of "extending the sealing time+increasing the number of plate washes", combined with measures such as checking the concentration of the sealing solution and regularly calibrating the washing machine, which can significantly reduce the background value and provide assurance for the accuracy of the detection results.

Desheng specializes in producing more than ten new Trinder's reagents, including TODB. After more than ten years of research and development, it can ensure that TODB appears as a powder with a purity of up to 99.5%, strong water solubility, and stable performance to ensure the accuracy of experimental results. Desheng has a place in the market for in vitro diagnostic kit raw materials with high-quality products, and is deeply trusted and supported by customers at home and abroad. If you have any relevant intentions, please click on the official website for consultation!