Laboratory requirements for antigen detection kits

Antigen elisa kit experimental operating standards

Antigen elisa kit is generally not paid much attention to the preparation of reagents in clinical laboratories. The usual practice is to remove the reagents from the refrigerator during the experiment. Take it out and use it immediately, ignoring that this approach may affect the problem of insufficient incubation time later, and its direct consequence is false negatives in the detection of some weakly positive specimens. Therefore, the most important thing in preparing reagents for ELISA measurement is to take the kit out of the refrigerator before starting the experiment and leave it at room temperature for more than 20 minutes before performing the measurement, so that the kit can be properly prepared before use. Equilibrium at room temperature. The purpose of this is mainly to enable the temperature in the reaction micropores to reach the required height quickly in the subsequent incubation reaction step to meet the measurement requirements. Secondly, the plate washing solutions in current commercial ELISA kits need to be diluted and prepared from the provided concentrate when used in the laboratory, so the quality of distilled or deionized water used for dilution should be guaranteed. In addition, when the kit uses OPD as the substrate, the substrate solution should be temporarily prepared before the reaction develops color. In current antigen elisa kits, the addition of serum samples is almost the only step that requires the use of a micropipette to add samples. The key points that must be paid attention to when using a micro-injector to add samples are: Do not add the sample too fast, avoid adding the sample to the upper part of the well wall, and avoid splashing and generating bubbles. Adding the sample too fast cannot ensure the accuracy and uniformity of micro-dosing. The non-coated area added to the upper part of the pore wall can easily lead to non-specific adsorption. Spills can contaminate adjacent holes. The presence of bubbles indicates differences in the reaction liquid interface. The addition of reagents in domestic kits is basically done from the dropper bottle. In addition to paying attention to the angle of the drop, the speed of the drop is also very important. If the drop is too fast, it is easy to repeat the drop or add it in two places. The phenomenon between pores will cause non-specific adsorption in the non-coated areas within the pores, resulting in non-specific color development. Therefore, sometimes a sample tested positive this time using the same kit will be tested negative next time, which is often caused by the above-mentioned sample addition and reagent errors. Incubation is the most critical factor affecting the success or failure of the ELISA assay. ELISA is a solid-phase immunoassay. The binding reaction of antigen and antibody is carried out on the solid phase. In order for the antigen or antibody in the liquid phase to combine with the specific antibody or antigen* on the solid phase, the reaction must be certain under certain temperature conditions. time. The time required for incubation is inversely proportional to the temperature, that is, the higher the temperature, the shorter the time required. The most commonly used incubation temperatures for antigen elisa kits are 37°C and room temperature, followed by 43°C and 2-8°C.