Human IL-1β Kit (HICA)

Testing Principle:

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

The detection system consists of two types of microspheres: the acceptor microspheres are conjugated with antibody 1 targeting the protein of interest, while the donor microspheres are conjugated with streptavidin. Additionally, biotin-labeled antibody 2 is used. 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, the singlet oxygen generated upon 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: The recommended plate for use is a microplate (384-well or96-well plate, white, shallow well)

I. Sample Requirements
1. To remove impurities from the sample, the sample needs to be centrifuged 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 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 of the standard curve, it must be appropriately diluted and retested to ensure the result falls within the valid measurement range.

II. Detection Procedure

2.1 Standard Gradient Sample Preparation 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:

2.2 Detection Procedure: Procedure 1 and Procedure 2 are applicable to the vast majority of detections. Procedure 3 is suitable for detecting samples containing Biotin, such as RPMI culture 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 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 Verification of Performance Parameters:

LOD: Detect standard C1 repeatedly 20 times, calculate the mean signal and SD. Calculate the concentration corresponding to the mean signal +2×SD using the standard curve; this value is defined as the Limit of Detection (LOD).

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

Inter-assay Precision: Samples of known low, medium, and high concentrations are detected separately across 5 independent experiments. Both standards and samples are tested using Detection Procedure 1 with 5 replicates. The detection results for samples at each concentration show a low coefficient of variation (%CV < 10%), indicating that the method has good reproducibility between 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 the method has good accuracy.

Specificity: The following proteins were diluted to 30ng/mL using standard buffer, and cross-reactivity rates were tested.

Traceability: Tested against reference material NIBSC 86/680. 1IU/mL≈5.86pg/mL.

Reagent R3 must 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 with at least duplicate wells, and the calculation should be performed using a four-parameter (weight 1/Y²) fitting method.

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

The dilution matrix of the calibrator should be consistent with the test samples, and it must be used within 2 hours after reconstitution.

Components from different reagent kit batches must not be mixed.