Human IL-22 Kit (HICA)

Principle of the assay:

This kit utilizes a homogeneous immuno chemiluminescence assay (HICA) based on the double-antibody sandwich principle for the detection of cytokine concentrations. The operation is simple and requires no washing steps.

The detection system consists of two types of microspheres: acceptor microspheres conjugated with antibody 1 specific to the target protein, and donor microspheres conjugated with streptavidin, along with biotin-labeled antibody 2. During the reaction, the target protein binds with the antibodies and microspheres to form an immune complex, bringing the two types of microspheres into close proximity. When the distance between the donor and acceptor microspheres is less than 200 nm, singlet oxygen generated by light excitation can transfer to the acceptor microspheres, triggering chemiluminescence. Conversely, if the target protein is absent, the microspheres remain too far apart, resulting in no signal.

By measuring the intensity of the chemiluminescent signal, quantitative analysis of the target protein can be achieved. This method offers advantages such as simplicity, rapid reaction, and high sensitivity.

Note: It is recommended to use microplates (with384or96wells, white, shallow wells) for this assay.

I. Sample Requirements
1. To remove impurities from the sample, centrifuge the sample prior to detection (1000 ×g, 10minutes).
2. When detecting target proteins in serum samples, the standard should be diluted with negative serum that has a background value below the limit of detection, and a standard curve should be established accordingly to calculate the actual concentration.
3. If the measured concentration of a sample exceeds the highest value of the standard curve, appropriately dilute the sample and retest to ensure the results fall within the valid measurement range.

II. Detection Workflow

2.1 Standard Curve Preparation: Reconstitute the lyophilized standard with 50μL of deionized water, then dilute with standard buffer. The recommended dilution scheme is shown in the table below:

2.2 Detection Workflow:Workflow 1 and Detection Workflow 2 are suitable for the vast majority of assays,Detection Workflow3 is applicable for testing matrices containing RPMI medium.

Note:

* Prior to testing, pre-mix R1A, R1F, and R2 at a volume ratio of 1:1:1. Use the mixture within 1 hour after preparation.

* If testing RPMI samples, you may additionally add RPMI enhancer solution (Catalog No.: UA086145). Pre-mix 50 μL of R3 with 100 μL of RPMI enhancer solution before adding, which significantly improves reagent performance.

III.Performance Testing

3.1 Example of Complete Standard Curve:

3.2 Validation of Performance Parameters:

LOD:Repeat detection of standard C1 twenty times, calculate the mean signal and SD. Calculate the concentration corresponding to mean signal + 2 × SD using the standard curve; this value is the Limit of Detection (LOD).

•Detection Range:0~1000000 pg/mL.

•Quantification Range:15.8~30000 pg/mL.

Precision (Precision):
Intra-assay Precision (Intra-Precision):On the same microplate within a single experiment, each of the known low, medium, and high concentration samples is tested in replicate 10 times to evaluate the intra-assay precision of the assay. Standards and samples are detected using different workflows. The coefficient of variation (%CV) for all concentration samples is less than 10%, indicating good reproducibility within the same batch of tests.

Inter-assay Precision (Inter-Precision):Samples of known low, medium, and high concentrations are tested separately across 5 independent experiments. Standards and samples are both detected using Detection Workflow 1 with 5 replicates set up. The test results for all concentration samples show low coefficients of variation (%CV < 10%), indicating good reproducibility between different batches of tests.

Accuracy (Recovery):

High and low concentration quality control products are mixed at a ratio of 1:9 and tested for recovery rate. All values fall within 80%-120%, indicating that the method has good accuracy.

Specificity: The following proteins were diluted to 0.1μg/mL using standard buffer, and cross-reactivity rates were tested.

Reagent R3 should be protected from light during use. Sampling and incubation are recommended to be performed under green light (<100 LUX).

Recalibration is required for each test. Each concentration point of the standard should be tested in at least duplicate wells, and the calculation should be performed using a four-parameter (weight 1/Y²) fitting method.

Temperature and time should be carefully controlled during incubation. Microplates should be covered with film, and a microplate reader with ALPHA functionality is recommended.

The dilution matrix of the calibrator should be consistent with the sample matrix. The reconstituted solution should be used within 2 hours.

Components from different reagent kit batches must not be mixed.