Human IL-17F Kit (HICA)

Principle of the Assay:

This kit employs a homogeneous immuno chemiluminescence assay (HICA) based on the double-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: 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 remains 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: Microplates are recommended for use (384or96-well plates, white, shallow wells)

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 having 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 result falls within the valid measurement range.

II. Detection Workflow

2.1 Preparation of Standard Gradient Samples Reconstitute the lyophilized standard with 50μL of deionized water, then dilute with standard buffer. The recommended dilution scheme is shown in the table below:

2.2 Detection Workflow:Workflow 1 and Detection Workflow 2 are applicable to the vast majority of detections,Detection Workflow 3 is suitable for testing matrices containing RPMI culture medium.

Note:

* Prior to testing,pre-mix R1A, R1Fand R2 at a volume ratio of 1:1:1, 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.PerformanceTesting

3.1 Example of a Complete Standard Curve:

3.2 Verification of Performance Parameters:

LOD:Repeat detection of 20times for standard C1, calculate the mean signal and SD, use the standard curve to calculate the concentration corresponding to the mean signal +2×SD, which is the Limit of Detection (LOD).

•DetectionRange:0~300000 pg/mL.

•QuantificationRange:3.34~30000 pg/mL.

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

Inter-assay Precision (Inter-Precision):Samples of known low, medium, and high concentrations are tested separately across 5independent experiments. Standards and samples are both detected using Detection Workflow 1with 5replicates set up. The test results for samples at each concentration show low coefficients of variation (%CV < 10%), indicating that this 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 this method has good accuracy.

Specificity: The following proteins were diluted to 0.1μg/mLusing standard buffer, and cross-reactivity rates were tested.

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 (weight 1/Y²) fitting calculation should be applied.

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 match the test samples. Reconstituted calibrators must be used within 2 hours.

Components from different reagent kit lots must not be mixed.