Human IL-5 Kit (HICA)

Principle of the Assay:

This kit employs 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 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 remains too large, resulting in no signal.

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 is a microplate (384 or 96 wells, white, shallow well)

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I. Sample Requirements
1. To remove impurities from the samples, centrifuge the samples before testing (1000 ×g, 10 minutes).
2. When detecting target proteins in serum samples, standards should be diluted using negative serum with a background value below the detection limit, and a standard curve should be established to calculate the actual concentration.
3. If the measured concentration of a sample exceeds the highest value of the standard curve, it should be appropriately diluted and retested to ensure the result falls within the valid measurement range.

II. Detection Process

2.1 Preparation of Standard Gradient Samples: Reconstitute the lyophilized standard with 50μL of deionized water, then dilute it with standard buffer. The recommended dilution scheme is as follows:

2.2 Detection Process:

Note:

* Before testing, pre-mix R1A, R1F, and R2 in a volume ratio of 1:1:1. Mix immediately before use.

** For RPMI samples, add RPMI enhancer by pre-mixing 50μL R3 with 100μL RPMI enhancer before adding. This significantly improves reagent performance.

III. Performance Testing

3.1 Example of Complete Standard Curve:

3.2 Performance Parameter Validation: Data marked with * are results with RPMI enhancer added.

LOD: Repeat testing of standard C1 20 times, calculate the mean signal and SD. The concentration corresponding to the mean signal + 2×SD is the limit of detection (LOD).

•Detection range: 0~100000 pg/mL.

•Quantitative range: 0.15~10000 pg/mL.

Precision:
Intra-assay Precision: In the same experiment, repeat testing of low, medium, and high concentration samples 10 times each to evaluate intra-assay precision. Standards and samples were tested using different processes. The coefficient of variation (%CV) for each concentration was below 10%, indicating good reproducibility within the same batch.

Inter-assay Precision: In 5 independent experiments, test low, medium, and high concentration samples. Standards and samples were tested using Detection Process 1 with 5 replicates. The %CV for each concentration was below 10%, indicating good reproducibility across different batches.

Accuracy (Recovery):

Mix high and low concentration controls at 1:9 and test the recovery rate. All recoveries were within 80%-120%, indicating good accuracy.

Specificity: Dilute the following proteins to 0.3μg/mL using standard buffer and test cross-reactivity.

•Traceability: Tested standard material NIBSC 90/586, 1IU/mL≈130 pg/mL.

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

Each test requires recalibration. 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 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 be consistent with the sample to be tested. It should be used within 2 hours after reconstitution.

Components from different reagent kit batches must not be mixed.