Mouse IL-2 Kit(HICA)

Test Principle:

This kit utilizes a homogeneous immuno chemiluminescence assay (HICA) for the detection of cytokine concentrations. The operation is simple and requires no washing steps.

The detection system includes two types of microspheres: acceptor microspheres conjugated with antibody 1 targeting the protein of interest, 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 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 distance between the microspheres is too large, and no signal is produced.

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: Microplates (384- or 96-well, white, shallow well)

I. Sample Requirements
1. To remove impurities from the sample, the sample must be centrifuged before detection (1000 ×g, 10 minutes).
2. When detecting target proteins in serum samples, the Standard should be diluted with negative serum that has a background value below the detection limit, 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 on the standard curve, it must be appropriately diluted and re-detected to ensure the results fall within the valid measurement range.

II. Detection Protocol

2.1 Standard Gradient Sample Preparation Reconstitute the lyophilized Standard with 50μL of deionized water, then dilute using Standard Buffer. The recommended dilution scheme is shown in the table below:

2.2 Detection Protocol:

* Note: Before testing, pre-mix R1 and R2 at a volume ratio of 1:1, and use within 1 hour after mixing.

III.Performance Testing

3.1 Example of Complete Standard Curve:

3.2 Performance Parameter Validation:

LOD: Detect the Standard C1 20 times repeatedly, calculate the mean signal and SD, and use the standard curve to calculate the concentration corresponding to mean signal + 2×SD, which is the Limit of Detection (LOD).

Precision (Precision):
Intra-assay Precision (Intra-Precision): On the same well plate in a single experiment, each of the known low, medium, and high concentration samples is detected 10 times repeatedly to evaluate the intra-assay precision of the test. Both standards and samples are detected using different protocols. The coefficient of variation (%CV) for the detection results of samples at each concentration is less than 10%, indicating that this method has good reproducibility within the same batch of tests.

Inter-assay Precision (Inter-Precision): Samples of known low, medium, and high concentrations are detected separately across 5 independent experiments. Standards and samples are both detected using Detection Protocol 1 with 5 replicates set up. The detection results for samples at each concentration show a low coefficient of variation (%CV < 10%), indicating that this method has good reproducibility between different batches of tests.

Accuracy (Recovery):

The recovery rate was tested after mixing high and low concentration quality controls at a ratio of 1:9. All values fell within 80%-120%, indicating that this method has good accuracy.

Specificity: The following proteins were diluted to 0.03μg/mL using Standard Buffer to test cross-reactivity rates.

•Traceability: Tested against reference material NIBSC 88/656, where 1 IU/mL ≈ 50pg/mL.

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

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

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

The diluent matrix for calibrators should be consistent with the test samples, and they should be used within 2 hours after reconstitution.

Components from different reagent kit lots must not be mixed.