Mouse IgG2a Detection Kit [Wide Range] (HICA)

This kit is used for the quantitative detection of mouse immunoglobulin G2a (mIgG2a) concentration in buffer solutions, cell culture supernatants, or serum samples.

Mouse immunoglobulin G2a consists of two heavy chains and two light chains. As a subclass with strong effector functions, mIgG2a can effectively activate the complement system and potently mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis, playing a key role in processes such as antiviral immunity and tumor immune surveillance.

This kit employs a homogeneous luminescence competitive assay for the detection of mIgG2a concentration. Homogeneous luminescence is an immunoassay method based on energy transfer between donor and acceptor beads to produce luminescence.

It uses acceptor beads conjugated with anti-mIgG2a antibody (R1), biotin-labeled mIgG2a (R2), and donor beads conjugated with streptavidin (R3) to measure the concentration of mouse IgG2a.

When the test sample does not contain mIgG2a, R2 and R1 are mixed with the sample, and the biotin-labeled mIgG2a binds to the acceptor beads to form an immune complex, which then binds to the donor beads of R3 to form a luminescent complex. At this point, the distance between the two beads is less than 200 nm. Upon excitation, the donor beads produce singlet oxygen, which diffuses to the acceptor beads, and the acceptor beads generate corresponding emission light upon receiving the energy.

Conversely, when the test sample contains mIgG2a, the mIgG2a in the sample competitively binds to R1 with R2 to form an immune complex. After adding R3, the formation of luminescent complexes between donor and acceptor beads decreases. Upon laser excitation, the light signal produced by the acceptor beads will correspondingly decrease.

By collecting the light signal with the instrument's photosensitive element and using calibrators to fit a standard curve, the concentration of mIgG2a in the test sample can be calculated.

Materials and instruments required but not provided with this product:

•Luminometer/plate reader

•Multifunctional microplate reader with Alpha module

[Sample Requirements]

•Cell supernatant must be centrifuged at 1000 ×g for 10 minutes to remove particles and polymers.

•If the concentration of the sample to be tested exceeds the upper limit of detection, it is recommended to dilute before testing.

[Reference Testing Procedure]

*This kit is for competitive assay. Reagent R2 will react directly with R1, so they must not be pre-mixed before adding; also, pay attention to the order of addition and strictly follow the procedure!

[Calibrator Gradient Sample Preparation]

Reconstitute and prepare calibrator gradient samples using the same matrix as the samples to be tested. For example, if the test sample is cell culture supernatant, use a medium that has not been used to culture cells to reconstitute and prepare the calibrator gradient samples.

Dissolve the lyophilized calibrator in 100 μL of the matrix, then use the matrix todilute the mIgG2a calibrator. The recommended dilution scheme is shown in the following table:

Example of a complete standard curve:

[Performance Parameters]

•Blank Limit: Repeatedly test calibration standard C1 twenty times, calculate the mean signal and SD, then use the standard curve to determine the concentration value corresponding to mean signal minus 2×SD—this is the blank limit.

•Dynamic Range: 0–1000 μg/mL.

•Repeatability: Test high- and low-concentration samples ten times each and calculate the concentration CV.

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

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

The detection reagent R3 is light-sensitive. Avoid exposure to light during use, and it is recommended to perform sample addition and incubation under green light (illuminance < 100 LUX).

It is recommended to recalibrate for each detection, with 2-3 replicate tests for each concentration point of the standard.

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

Pay attention to the required incubation temperature and duration.

Incubation at 37°C is recommended to be performed in the HiLA homogeneous luminescence analyzer.

Components from different reagent kit batches should not be mixed.