mouseELISAThe pre experimental steps of the reagent kit should focus on sample processing, standard dilution, and condition optimization. The following are the key operating procedures:

1Sample preparation

Serum/Plasma: room temperature coagulation10-20Centrifuge in minutes（2000-3000turn/Divide,20Collect the supernatant and avoid sedimentation.

Cell supernatant: After centrifugation, collect the supernatant and detect the intracellular components by repeatedly freezing, thawing, and lysing the cells.

Organizational samples: homogenized and centrifuged, supernatant divided and stored separately.

IIStandard dilution

Add the original standard solution to the diluent and let it stand10Mix well in minutes and dilute in a gradient according to the instructions (e.g1600 IU/mL）.

Suggest conducting a pre experiment to verify the dilution ratio and ensure that the sample concentration is within the detection range.

IIIReagent equilibrium and sample addition

The reagent kit and sample need to be equilibrated at room temperature60Minutes, avoid temperature affecting the reaction.

Avoid bubbles when adding samples, and add standard and sample wells separately15-100MLAdd diluent to blank hole.

4、 Incubation and washing‌

the first timeIncubation（37℃60-120After minutes, wash the board3-5Next time, soak each time1-2minute.

Enzyme labeled antibody incubation（37℃30-60Repeat washing after minutes.

5Color rendering and termination

joinTMBSubstrate light avoidance reaction10-15Minute, stop liquid（50ML/After the termination of the hole15Test within minutesODValue.

6、 Precautions

Pre experiment suggestion: High concentration samples need to be diluted for validation to avoid exceeding the standard curve range.

Repeatability: At least set up duplicate holes to ensure the reliability of the results.

Through these pre experimental steps, you can verify the performance of the reagent kit, determine the optimal operating conditions, and determine whether your sample is suitable for direct formal experiments. Although pre experiments require time and reagents, they can greatly improve the success rate and reliability of the results of formal experiments.‌