Human IL-12p70 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 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 upon light excitation can transfer to the acceptor microspheres, triggering chemiluminescence. Conversely, if the target protein is absent, the microspheres remain too far apart, resulting in no signal.

By measuring the intensity of the chemiluminescent signal, quantitative analysis of the target protein can be achieved. This method is characterized by its simplicity, rapid reaction, and high sensitivity.

Note: Recommended plates are microplates (384or96-well plates, white, flat-bottom)

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, dilute the standard using negative serum with a background value below the limit of detection, and use this to establish a standard curve for calculating actual concentrations.
3. If the measured concentration of a sample exceeds the highest value of the standard curve, appropriately dilute and retest 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 deionized water, then dilute with standard buffer standard. The recommended dilution scheme is shown in the table below:

2.2 Detection Workflow:Workflow 1 and Detection Workflow 2 are suitable for most assays,Detection Workflow3 is suitable for testing in RPMI culture medium matrix.

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 Evaluation

3.1 Complete Standard Curve Example:

3.2 Performance Parameter Validation:

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

•Detection Range:0~300000 pg/mL.

•Quantification Range:1.99~30000 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 repeatedly detected 10 times to evaluate the intra-assay precision. Standards and samples are tested using different workflows. The coefficient of variation (%CV) for all concentration samples is below 10%, indicating good reproducibility within the same batch.

Inter-assay Precision (Inter-Precision):Samples of known low, medium, and high concentrations are tested in 5 independent experiments. Standards and samples are both tested using Assay Workflow 1 with 5 replicates. All concentration samples show low coefficients of variation (%CV < 10%), indicating good reproducibility across different batches.

Accuracy(Recovery):

High and low concentration QC samples are mixed at a ratio of 1:9 and tested for recovery rate. Both fall within 80%-120%, indicating that this method has good accuracy.

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

Reagent R3 should 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 replicates of each standard concentration should be tested, and a four-parameter (weighted 1/Y²) fitting calculation should be applied.

Temperature and time should be strictly 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. The reconstituted solution should be used within 2 hours.

Components from different reagent kit batches must not be mixed.