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Mouse IgG2a Detection Kit [Wide Range] (HICA)

Mouse IgG2a Detection Kit [Wide Range] (HICA)

Catalog Number: UA086084 Brand: UA BIOSCIENCE
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Regular price $1,500 USD
Regular price Sale price $1,500 USD
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Product Details

Product Specification


Stability & Storage

Store at 2~8°C protected from light for 12 months;
After reconstitution, the calibrator can be aliquoted and stored at -20°C, avoiding repeated freeze-thaw cycles.

Background

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.

Components

Name

Active Ingredient

Content

mIgG2aDetection ReagentR1

Receptor Microspheres Conjugated with Anti-Mouse IgG2a Antibody

2mL/bottle × 1

mIgG2aDetection ReagentR2

Biotin-Labeled Mouse IgG2a

2mL/bottle × 1

mIgG2aDetection ReagentR3

Donor Microspheres Conjugated with Streptavidin

5mL/bottle × 1

mIgG2a Calibrator

Mouse IgG2a

100μg,Lyophilized Powder

mIgG2a Calibrator Buffer

BSA

2mL/bottle × 1


Protocol

Required materials and instruments not provided with this product:

Luminescent plate/strips

Multimode 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 sample concentration exceeds the detection limit, dilution is recommended before testing.

 

【Detection Procedure for Reference】

Detection Procedure

Detection Procedure (37°C)

Detection Procedure (Room Temperature)

Step 1:

2μL calibrator/sample + 4μL R2* + 4μL R1 + 10μL R3,light-protected/green light

2μL calibrator/sample + 4μL R2* + 4μL R1 + 10μL R3,light-protected/green light

Step 2:

​Incubate at 37°C for 30 minutes,light-protected/green light

​Incubate at room temperature for 60 minutes,light-protected/green light

Read

Instrument reading

Instrument reading


 

* This kit uses a competitive assay. The detection reagent R2 reacts directly with R1, so they cannot be pre-mixed. Follow the procedure strictly and pay attention to the order of addition!

 

【Calibrator Gradient Sample Preparation】

Use the same matrix as the test samples to reconstitute and prepare the calibrator gradient samples. For example, if the test sample is cell culture supernatant, use the cell-free culture medium to reconstitute and prepare the calibrator gradient samples.

Reconstitute the lyophilized calibrator with 100μL of matrix, then dilute the mIgG2a calibrator with the matrix. The recommended dilution scheme is as follows:

 

 

 

Gradient

Concentration (μg/mL)极>

mIgG2a high concentration

Matrix (μL)

C8

1000

/

/

C7

300

21μL C8

49

C6

100

7μL C8

63

C5

30

7μL C7

63

C4

10

7μL C6

63

C3

3

7μL C5

63

C2

1

7μL C4

63

C1

0

63


 

 

 


 

Example of a complete standard curve:

【Performance Parameters】

Limit of Blank (LoB): Repeat testing of calibrator C1 20 times, calculate the mean signal and SD. The concentration corresponding to the mean signal - 2×SD is the LoB.

Detection Procedure

Matrix

LoB (μg/mL)

Detection Procedure 1

Calibrator buffer

0.23

Detection Procedure 2

Calibrator buffer

0.54


 

Dynamic range: 0~1000 μg/mL.

Repeatability: Repeat testing of high and low concentration samples 10 times, calculate the concentration CV.

Detection Procedure

Matrix

Repeatability

Low concentration

High concentration

Detection Procedure 1

Calibrator buffer

1.25%

1.49%

Detection Procedure 2

Calibrator buffer

2.78%

2.40%


 

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

Detection Procedure

Matrix

Deviation

Low concentration

High concentration

Detection Procedure 1

Calibrator buffer

-0.65%

2.03%

Detection Procedure 2

Calibrator buffer

-0.29%

-1.31%


 

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

Detection Procedure

Cross-reactant

Cross-reactivity rate

Detection Procedure 1

Human IgG

13.14%

Rabbit IgG

1.00%

Pig IgG

0.38%


 

 

Guidelines

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.