Mouse IgG2b Detection Kit [Wide Range] (HICA)

This product is used for the quantitative detection of mouse immunoglobulin G2b (mIgG2b) concentration in buffer solutions, cell culture supernatants, or serum.

The molecular structure of mIgG2b consists of two heavy chains and two light chains. IgG2b is typically an effective complement activator and can efficiently mediate ADCC (antibody-dependent cell-mediated cytotoxicity) and phagocytosis.

This kit employs a homogeneous luminescence-based competitive assay for the detection of mIgG2b concentration. Homogeneous luminescence is an immunoassay method based on energy transfer between donor and acceptor microspheres in close proximity to emit light.

It utilizes acceptor microspheres conjugated with anti-mIgG2b antibodies (R1), biotin-labeled mIgG2b (R2), and donor microspheres conjugated with streptavidin (R3) to measure the concentration of mouse IgG2b.

When the test sample does not contain mIgG2b, R2 and R1 are mixed with the sample, and the biotin-labeled mIgG2b binds to the acceptor microspheres to form an immune complex. This complex then binds to the donor microspheres of R3, forming a luminescent complex. At this point, the distance between the two microspheres is less than 200 nm. Upon excitation, the donor microspheres generate singlet oxygen, which diffuses to the acceptor microspheres, causing them to emit corresponding light.

Conversely, when the test sample contains mIgG2b, the mIgG2b in the sample competitively binds to R1 with R2, forming an immune complex. After adding R3, the formation of luminescent complexes between donor and acceptor microspheres decreases. Upon laser excitation, the light signal generated by the acceptor microspheres correspondingly declines.

By collecting the light signal through the instrument's photosensitive element and using calibrators to fit a standard curve, the concentration of mIgG2b in the sample can be calculated.

Required materials and instruments not provided with this product:

•Luminescence plates/tubes

•Multimode microplate reader equipped with Alpha module

[Sample Requirements]

•Cell culture supernatants should be centrifuged at 1000 ×g for 10 minutes to remove particles and aggregates.

•If the concentration of the test sample exceeds the upper limit of detection, dilution prior to testing is recommended.

[Assay Procedure Reference]

* This kit utilizes a competitive assay format. Detection reagent R2 reacts directly with R1; they must not be pre-mixed before addition. Pay attention to the addition sequence and strictly follow the procedure!

[Calibrator Gradient Sample Preparation]

Use the same matrix as the test samples to reconstitute and prepare calibrator gradient samples. For example, when testing cell culture supernatants, select cell-free culture medium to reconstitute and prepare the calibrator gradient samples.

Reconstitute the lyophilized calibrator with 100 μL of matrix, then dilute the mIgG2b calibrator using the matrix. The recommended dilution scheme is shown in the table below:

Example of a complete standard curve:

[Performance Parameters]

•Limit of Blank (LOB): Test Calibrator C1 in duplicate 20 times, calculate the mean signal and SD. Use the standard curve to calculate the concentration corresponding to Mean Signal - 2×SD, which is defined as the LOB.

•Dynamic Range: 0–1000 μg/mL.

•Repeatability: Test high and low concentration samples in replicate 10 times each, and calculate the coefficient of variation (CV) for concentration.

•Accuracy: Test accuracy control samples and calculate the deviation from the target value.

•Specificity: Dilute the following cross-reactants to 100 μg/mL using calibrator diluent, and test the cross-reactivity rate.

Reagent R3 is light-sensitive. Protect it from light during use, and it is recommended to perform sample loading and incubation under green light (illuminance < 100 LUX).

It is recommended to recalibrate for each test, with 2~3 replicate wells for each standard concentration point.

Four-parameter (weighting 1/Y²) or cubic spline fitting is recommended for calculation.

Pay attention to the requirements for incubation temperature and duration.

For 37°C incubation, it is recommended to use the HiLA homogeneous luminescence analyzer.

Components from different reagent kit batches should not be mixed.