Human IL-17A Kit (HICA)

Test Principle:

This kit utilizes 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 types of 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 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, the target protein can be quantitatively analyzed. This method is characterized by its simplicity, rapid reaction, and high sensitivity.

Note: It is recommended to use microplates (384or96well plate, white, shallow wells)

I. Sample Requirements
1. To remove impurities from the sample, centrifuge the sample prior to 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 use this to establish a standard curve for calculating actual concentrations.
3. If the measured concentration of a sample exceeds the highest value on the standard curve, appropriately dilute the sample and retest to ensure the result falls 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: Prior to testing, pre-mix R1 and R2 at a volume ratio of 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 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 plate for 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 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 across 5 independent experiments. Standards and samples are both tested using Detection Protocol 1 with 5 replicates. The results for all concentration samples show low coefficients of variation (%CV < 10%), indicating 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 the method possesses good accuracy.

Specificity: The following proteins were diluted to 0.1μg/mL using Standard Buffer to test the cross-reactivity rate.

•Traceability: Certified Reference Material NIBSC 01/420 was tested; 1 IU/mL ≈ 83 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 two replicate wells should be set for each concentration point of the standard, and a four-parameter (weight 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 of the calibrator should match the test samples. Reconstituted calibrators must be used within 2 hours.

Components from different reagent kit lots must not be mixed.