Human IL-2 Kit (HICA)

Test Principle:

This kit employs a homogeneous immuno chemiluminescence assay (HICA) based on the double-antibody sandwich method 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 upon light excitation can transfer to the acceptor microsphere and trigger chemiluminescence. Conversely, if the target protein is absent, the distance between the microspheres is too large, 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 (384- or 96-well plates, white, with shallow wells) for this assay.

I. Sample Requirements
1. To remove impurities from the sample, centrifuge the sample before testing (1000 ×g, 10 minutes).
2. When detecting target proteins in serum samples, the standard should be diluted using negative serum with a background value below the lower 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, it must be appropriately diluted and retested to ensure the results fall within the valid measurement range.

II. Detection Procedure

2.1 Preparation of Standard Gradient Samples Reconstitute the lyophilized standard with 50μL of deionized water, then dilute using the standard buffer. The recommended dilution scheme is shown in the table below (adjustable according to specific conditions):

2.2 Detection Procedure: Procedure 1 and Procedure 2 are suitable for the vast majority of tests, while Procedure 3 is suitable for samples containing Biotin, such as RPMI medium.

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

* 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 Performance Parameter Validation:

LOD: Repeat the detection of Standard C1 20 times, 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-Precision: On the same plate in a single experiment, samples with known low, medium, and high concentrations are each tested 10 times to evaluate the intra-assay precision. Standards and samples are tested using different procedures. The coefficient of variation (%CV) for all concentration samples is below 10%, indicating good reproducibility of this method within the same batch.

Inter-Precision: Samples with known low, medium, and high concentrations are tested in 5 independent experiments. Standards and samples are both tested using Procedure 1 with 5 replicates. The test results for all concentration samples show a low coefficient of variation (%CV＜10%), indicating good reproducibility of this method across different batches.

Accuracy (Recovery):

High-concentration and low-concentration quality control samples are mixed at a 1:9 ratio and tested for recovery rate. The recovery rates are all between 80%-120%, indicating good accuracy of this method.

Specificity: The following proteins are diluted to 3μg/mL using standard buffer to test the cross-reactivity rate.

Traceability: The reference standard NIBSC 86/500 was tested, where 1IU/mL≈105pg/mL.

Reagent R3 must be protected from light. Sampling and incubation should be performed under green light (<100 LUX).

Recalibration is required for each test, with at least duplicate wells for each standard concentration point. A four-parameter (weighting: 1/Y²) fitting calculation should be applied.

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

The dilution matrix for calibrators should match the test samples. Reconstituted calibrators must be used within 2 hours.

Components from different reagent kit lots must not be mixed.