Human IL-8 Kit (HICA)

Testing Principle:

This kit employs a homogeneous immuno chemiluminescence assay (HICA) based on the double-antibody sandwich method for detecting the concentration of cytokines. The operation is simple and requires no washing steps.

The detection system consists of 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 types of 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 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, the target protein can be quantitatively analyzed. This method offers advantages such as simplicity of operation, rapid reaction, and high sensitivity.

Note: The recommended plate for use is a microplate (384 or 96 wells, white, shallow wells)

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

II. Detection Protocol

2.1 Preparation of Standard Gradient Samples 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.

* 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 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 defined as the Limit of Detection (LOD).

Precision (Precision):
Intra-assay Precision (Intra-Precision): On the same microplate from a single experiment, each of the known low, medium, and high concentration samples is detected 10 times to evaluate the intra-assay precision of this assay. Both standards and samples are detected using different protocols. The coefficient of variation (%CV) for all concentration samples is below 10%, indicating good reproducibility within the same batch of tests.

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

Accuracy (Recovery):

High and low concentration quality control samples are mixed at a ratio of 1:9 and their recovery rates are tested. All values fall within 80%-120% , indicating that this method possesses good accuracy.

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

Traceability: The reference material NIBSC 89/520 was tested, where 1 IU/mL ≈ 256 pg/mL.

Reagent R3 must be protected from light. It is recommended to perform sample addition and incubation under green light (<100 LUX).

Recalibration is required for each test. At least duplicate wells should be set for each concentration point of the standard, and a four-parameter (weight 1/Y²) fitting calculation should be used.

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 of the calibrator should match the sample matrix. Use within 2 hours after reconstitution.

Components from different reagent kit batches must not be mixed.