Technical Manual for Human Interleukin-22 (IL-22) ELISA Detection Kit

BS-1649 Human Interleukin-22 (IL-22) ELISA Kit

1、 Product Overview

This kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to quantitatively detect the concentration of interleukin-2 (IL-22) in human serum, plasma, or cell culture supernatant. IL-22 is a cytokine secreted by activated T cells, NK cells, etc. It plays an important role in inflammatory response, tissue repair, and immune regulation. This reagent kit has the characteristics of high sensitivity, high specificity, and good reproducibility, and is suitable for scientific research experiments and clinical testing.

2、 Detection principle

Coated antibody: Anti human IL-22 monoclonal antibody is pre coated on a microplate and binds to IL-22 in the sample to form a solid-phase complex.

Biotin labeled antibody: Adding biotin labeled anti human IL-22 antibody, it binds with IL-22 to form a sandwich structure.

Enzyme conjugate: Add horseradish peroxidase (HRP) labeled streptavidin and specifically bind to biotin.

Color reaction: Add substrate (TMB) for color development, and the color depth is proportional to the concentration of IL-22.

Termination and measurement: Add termination solution to terminate the reaction, measure the absorbance (OD value) at 450nm wavelength, and calculate the sample concentration through the standard curve.

3、 Composition of reagent kit

Component specifications and quantities

Pre packaged microporous plate 96 holes/plate 1 piece

Standard product (freeze-dried) 2ng/vial 2 vials

Standard diluent 10mL 1 bottle

Biotin labeled antibody 6mL 1 bottle

HRP labeled streptavidin 6mL 1 bottle

Washing solution (20 x) 50mL 1 bottle

TMB 11mL 1 bottle

Termination solution (2M H ₂ SO ₄) 6mL 1 bottle

4、 Sample requirements

Serum/plasma: After collection, let it stand at room temperature for 30 minutes, centrifuge (2000 × g, 15 minutes) to obtain the supernatant, and store it at -20 ℃ or -80 ℃ to avoid repeated freezing and thawing.

Cell culture supernatant: Centrifuge to remove cell debris, immediately detect or package for storage.

Caution: Avoid preservatives such as sodium azide (NaN ∝) or EDTA in the sample to avoid affecting the test results.

5、 Operation steps

Standard dilution: Dissolve the freeze-dried standard in standard dilution solution to 2ng/mL, and dilute it in multiples (such as 2ng/mL, 1ng/mL, 0.5ng/mL, 0.25ng/mL, 0.125ng/mL, 0.0625ng/mL). The 0ng/mL well is used as a blank control.

Sample addition: Add 100 μ L of standard or sample to each well and incubate at 37 ℃ for 90 minutes.

Washing: Discard the liquid in the hole and wash the plate three times with detergent, soaking for 1 minute each time.

Add biotin labeled antibody: Add 100 μ L to each well and incubate at 37 ℃ for 60 minutes.

Enzyme conjugate: Add 100 μ L to each well and incubate at 37 ℃ for 30 minutes.

Color development and termination: Add 90 μ L TMB to each well, avoid light for 15 minutes, and add 50 μ L termination solution.

Measurement: Read the OD value at a wavelength of 450nm, draw a standard curve, and calculate the sample concentration.

6、 Result calculation

Draw a standard curve with standard concentration as the x-axis and OD value as the y-axis.

According to the OD value of the sample, multiply the corresponding concentration on the standard curve by the dilution factor (if the sample is not diluted, multiply by 1).

It is recommended to use professional software such as ELISA Calc for curve fitting and concentration calculation.

7、 Performance indicators

Sensitivity: The detection limit is 0.015ng/mL.

Precision: Within batch coefficient of variation (CV)<10%, between batch CV<15%.

Linear range: 0.0625-2ng/mL, correlation coefficient (R ²)>0.99.

Recovery rate: spiked recovery is performed on high, medium, and low concentration samples, with a recovery rate ranging from 85% to 115%.

Specificity: Does not cross react with cytokines such as IL-10, IL-17, IL-23, etc.

8、 Precautions

Reagent storage: Unopened reagent kits are stored at 2-8 ℃ and have a shelf life of 12 months; After opening, standard samples, biotinylated antibodies, and HRP markers need to be packaged and stored separately to avoid repeated freeze-thaw cycles.

Operation standard: Avoid generating bubbles during pipetting, and ensure that the liquid in the hole is completely discarded during washing.

Safety protection: The termination liquid contains strong acid, and gloves and goggles should be worn during operation.

Quality control: Double hole retesting is required for each batch of experiments, and repeated testing is necessary when the OD value is abnormal.

9、 Common problem analysis

Possible causes and solutions for the problem phenomenon

Standard curve, linear deviation, standard dilution error, re prepare standard to ensure dilution ratio

The sample OD value is too high, and the sample concentration exceeds the detection range. Dilute the sample and retest

The color development is too light or the enzyme conjugate inactivation test reagent has no color development. Replace the reagent with a new one

After washing, there is residual liquid in the hole. If the washing frequency is insufficient or the force is not enough, increase the washing frequency and pat the hole plate dry with force

Note: The above is for reference only. This reagent is for scientific research use only and is not considered as actual data. Experiments must strictly follow the instructions or consult technical teachers.

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