Human IL-6 Kit (HICA)

Principle of the Assay:

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 consists of two types of microspheres: acceptor microspheres conjugated with antibody 1 specific to the target protein, 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 and trigger 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 offers advantages such as simplicity, rapid reaction, and high sensitivity.

Note: The recommended plate for use is a microplate (384 or 96

Note: The recommended plate for use is a microplate (384 or 96-well plate, white, shallow well)

I. Sample Requirements
1. To remove impurities from the sample, the sample must be centrifuged before testing (1000 ×g, 10 minutes).
2. When detecting target proteins in serum samples, the standard should be diluted using negative serum with 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. Testing Procedure

2.1 Preparation of Standard Gradient Samples  
Dissolve 5 μg of lyophilized standard in 100 μL of deionized water. Take 1 μL of the reconstituted lyophilized standard and mix it with 9 μL of standard buffer solution. Then dilute the mixture using standard buffer solution. The recommended dilution scheme is shown in the table below:

2.2 Testing Procedure:

* Note: Before testing, pre-mix R1 and R2 at a volume ratio of 1:1. 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. Performance Testing

3.1 Example of Complete Standard Curve:

3.2 Performance Parameter Validation:

LOD:Repeat the detection of standardC120 times, calculate the mean signal andSD, and use the standard curve to calculate the concentration corresponding tomean signal+2×SD, which is the Limit of Detection (LOD).

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

Inter-Precision: Samples with known low, medium, and high concentrations were tested in 5 independent experiments. Standards and samples were both tested using Testing Procedure 1 with 5 replicates. The test results for all concentration samples showed a low coefficient of variation (%CV < 10%), indicating good reproducibility between different batches.

Accuracy (Recovery):

High and low concentration quality control samples were mixed at a ratio of 1:9 and tested for recovery rate, which was between 80%-120%, indicating good accuracy of the method.

Specificity: The following proteins were diluted to 0.05μg/mL using standard buffer to test the cross-reactivity rate.

Traceability: Tested standard materialNIBSC 89/548, 1 IU/mL ≈ 5 pg/mL.

Reagent R3 must be protected from light during use. It is recommended to perform sample addition and incubation under green light (<100 LUX). Each test requires recalibration. A minimum of 2 replicate wells per concentration point of the standard should be tested, and a four-parameter logistic regression (weighting: 1/Y²) should be used for calculation. Temperature and time must be strictly controlled during incubation. Microplates should be covered with a sealing 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.