2, operation steps
1. Prepare all required reagents and standards;
2 Remove the microplate from the sealed bag that has been balanced to room temperature, and put the unused slat back into the aluminum foil bag and re-seal it;
3. Add 300uL washing solution to the microplate, let it soak for 30 seconds, discard the washing solution and pat the microplate dry on absorbent paper, please use immediately do not let the microplate dry;
4. Add different concentrations of standard, experimental samples or quality control into the corresponding Wells, 100uL for each well. The Wells were sealed with plate adhesive and incubated at room temperature for 2 hours.
5 Suck the liquid out of the plate and wash the plate using a bottle washer, a multi-channel plate washer, or an automatic plate washer. Add 300uL of washing liquid to each well, and then suck the washing liquid out of the plate. Repeat 3 times. Every time you wash the plate, try to absorb the residual liquid to help you get a good test result. At the end of the last plate wash, please blot all the liquid in the plate or invert the plate and pat all the residual liquid in the absorbent paper;
6. Add 100uL detection antibody to each microwell. Seal the reaction Wells with sealer tape and incubate for 2 hours at room temperature;
7. Repeat the plate washing operation of step 5;
8. Add 100 ULSA-HRP to each microwell and incubate for 20 minutes at room temperature. Be careful to avoid light;
9. Repeat step 5 to wash the plate;
10. Add 100uL of color development solution to each microwell, incubate at room temperature for 5-30 minutes, pay attention to avoid light;
11. Add 50uL of termination solution to each microwell, and the color of the solution in the well will change from blue to yellow. If the color of the solution turns green or the color change is inconsistent, tap the microplate to mix the solution evenly;
12. Within 30 minutes after the termination solution is added, the absorbance value at 450nm is measured using a microplate reader and 540nm or 570nm is set as the correction wavelength. If the dual wavelength correction is not used, the accuracy of the results may be affected;
13 Calculation results: The corrected absorbance values (OD450-OD540/OD570), the compound reading were averaged for each standard and sample, and then the average zero standard OD value was subtracted. Standard curves were created by 4-parameter logic (4-PL) curve fitting using computer software. Alternatively, a curve can be generated by plotting the logarithm of the concentration of the standard against the logarithm of the corresponding OD value, and the best fit line can be determined by regression analysis. This process produces an adequate but less accurate fit to the data. If the sample is diluted, the concentration should be multiplied by the dilution.
Note: The standard curve data provided in are for reference only, and the sample content should be calculated according to the standard curve drawn in the same test.

3. Kit parameters
1. Recovery rate: Different levels of Mouse IL-6 were incorporated into the cell culture medium samples and the recovery rate was determined. Recoveries ranged from 70 to 114%, with an average recovery of 91%.
2. Sensitivity: The minimum detectable dose (MDD) of Mouse IL-6 was generally less than 1.8pg/mL. The lowest detectable value of mouse IL-6 was calculated as the corresponding concentration based on the average of the zero absorbance values of 20 standard curves plus two standard deviations.
3. Calibration: The ELISA kit was calibrated with high purity recombinant Mouse IL-6 protein expressed in E. coli.
4. Linearity: Four different samples were mixed with high concentrations of Mouse IL-6, followed by dilution (1×). Linearity was determined by dilutes the samples to the detection range.