Human IL-10 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 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 simple operation, rapid reaction, and high sensitivity.

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

I. Sample Requirements
1. To remove impurities from the sample, it must be centrifuged 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 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 Standard Gradient Sample Preparation 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:Protocol 1 and Detection Protocol 2 are applicable to most assays. Detection Protocol 3 is intended for samples containing Biotin, such as RPMI culture medium.

* Note: Prior to testing,pre-mixR1A, R1Fand R2 at a volume ratio of 1:1:1.Use the mixture within 1 hour after preparation.

* 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 Complete Standard Curve:

3.2 Validation of Performance Parameters:

LOD:The Standard C1 was detected repeatedly 20 times. The mean signal and SD were calculated. The concentration corresponding to mean signal + 2×SD was determined using the standard curve, which represents the Limit of Detection (LOD).

Precision (Precision):
Intra-assay Precision (Intra-Precision): On the same plate in a single experiment, samples at known low, medium, and high concentrations were each tested 10 times to evaluate the intra-assay precision of the assay. Both Standards and samples were detected using different protocols. The coefficient of variation (%CV) for all concentration levels was below 10%, indicating good reproducibility within the same batch of tests.

Inter-assay Precision (Inter-Precision): Samples at known low, medium, and high concentrations were tested across 5 independent experiments. Both Standards and samples were analyzed using Detection Protocol 1 with 5 replicates. The results for all concentration levels showed low coefficients of variation (%CV < 10%), demonstrating good reproducibility between different batches of tests.

Accuracy (Recovery):

High and low concentration quality control (QC) samples were mixed at a ratio of 1:9 and their recovery rates were tested. All values fell within 80%-120%, indicating that the method possesses good accuracy.

Specificity: The following proteins were diluted to 30ng/mL using Standard buffer to test cross-reactivity rates.

Traceability: Tested against reference material NIBSC 93/722, where 1 IU/mL ≈ 81 pg/mL.

Reagent R3 must be protected from light. Sample addition and incubation are recommended to be performed under green light (<100 LUX). Each test requires recalibration. A minimum of 2 replicate wells should be set for each concentration point of the standard, and the calculation should be performed using a four-parameter (weighting: 1/Y²) fitting method. 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 that of the test samples. Reconstituted calibrators must be used within 2 hours. Components from different reagent kit lots must not be mixed.