Human IL-2R Kit (HICA)

Principle of the Assay:

This kit employs a homogeneous immuno chemiluminescence assay (HICA) based on the dual-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: receptor 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 types of microspheres into close proximity. When the distance between the donor and receptor microspheres is less than 200 nm, singlet oxygen generated by light excitation can transfer to the receptor microspheres and trigger 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 is characterized by its simplicity, rapid reaction, and high sensitivity.

Note: It is recommended to use microplates for the assay (384or96well plates, white, with shallow wells)

I. Sample Requirements
1. To remove impurities from the sample, samples must be centrifuged prior to detection (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 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 retested to ensure the result falls 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: Protocols 1 and 2 are applicable to most assays. Protocol 3 is suitable for the detection of samples containing Biotin, such as RPMI medium.

* Note: Prior to testing, pre-mix R1A, R1F and R2 at a volume ratio of 1:1:1, and use the mixture 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 Validation of Performance Parameters:

LOD: Detect the Standard C1 repeatedly for 20 times, calculate the mean signal and SD, 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 plate within a single experiment, samples at known low, medium, and high concentrations are each detected repeatedly for 10 times to evaluate the intra-assay precision of this assay. Both standards and samples are tested using different protocols. The coefficient of variation (%CV) for all concentration levels is below 10%, indicating that this method has good reproducibility within the same batch of tests.

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

Accuracy (Recovery):

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

Specificity: The following proteins were diluted to 30 n g/mL using Standard Buffer to test cross-reactivity rates.

Traceability: Tested against reference material NIBSC 97/600. 1 IU/mL ≈ 68 pg/mL.

Reagent R3 must be protected from light. Sample addition and incubation should 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, and a four-parameter (weighting 1/Y²) curve fitting should be applied for calculation.

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

The dilution matrix for calibrators should be consistent with the sample matrix. Reconstituted calibrators must be used within 2 hours.

Components from different reagent kit lots must not be mixed.