SKU: 8464018572

Mouse IgG1 Detection Kit [Wide Range] (HICA)

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Description

Mouse IgG1 Detection Kit [Wide Range] (HICA)Product Specification Stability & Storage Store at 2~8C protected from light for 12 months; After reconstitution, the calibrator can be aliquoted and stored at 20C, avoiding repeated freeze thaw cycles. Background This product is used for the quantitative detection of mouse immunoglobulin G1 (mIgG1) concentration in buffer solutions, cell culture supernatants, or serum samples. Mouse immunoglobulin G1 is composed of two heavy chains and two light

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 product is used for the quantitative detection of mouse immunoglobulin G1 (mIgG1) concentration in buffer solutions, cell culture supernatants, or serum samples.

Mouse immunoglobulin G1 is composed of two heavy chains and two light chains and is one of the main subclasses of mIgG. Functionally, while mIgG1 has certain complement activation capabilities under specific conditions, its primary mechanism of action is through binding to Fc receptors to mediate opsonization, playing a significant role in immune regulation.

This kit employs a homogeneous luminescence competitive assay for the detection of mIgG1 concentration. Homogeneous luminescence is an immunoassay method based on energy transfer between donor and acceptor microspheres in close proximity to generate luminescence.

It uses acceptor microspheres conjugated with anti-mIgG1 antibodies (R1), biotin-labeled mIgG1 (R2), and donor microspheres conjugated with streptavidin (R3) to measure the concentration of mouse IgG1.

When the test sample contains no mIgG1, after mixing R2 and R1 with the sample, the biotin-labeled mIgG1 binds to the acceptor microspheres to form an immune complex, which then binds to the donor microspheres of R3 to form 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, and the acceptor microspheres produce corresponding emission light upon receiving the energy.

Conversely, when the test sample contains mIgG1, the mIgG1 in the sample competitively binds with R1 to form an immune complex, reducing the formation of luminescent complexes between donor and acceptor microspheres after adding R3. Upon laser excitation, the light signal produced by the acceptor microspheres decreases accordingly.

By collecting the light signal with the instrument's photosensitive elements and fitting a standard curve with calibrators, the concentration of mIgG1 in the sample can be calculated.

Components

Name

Active Ingredient

Content

mIgG1Detection ReagentR1

Receptor Microspheres Conjugated with Anti-Mouse IgG1 Antibody

2mL/vial×1

mIgG1Detection ReagentR2

Biotin-Labeled Mouse IgG1

2mL/vial×1

mIgG1Detection ReagentR3

Donor Microspheres Conjugated with Streptavidin

5mL/vial×1

mIgG1 Calibrator

Mouse IgG1

100μg,Lyophilized Powder

mIgG1 Calibrator Buffer

BSA

2mL/vial×1


Protocol

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 polymers.

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

[Assay Procedure for Reference]

Assay Procedure

Assay Procedure (37°C)

Assay Procedure (Room Temp.)

Step 1:

2 μL Calibrator/Sample + 4 μL R2* + 4 μL R1 + 10 μL R3,Protect from light/Green light

2 μL Calibrator/Sample + 4 μL R2* + 4 μL R1 + 10 μL R3,Protect from light/Green light

Step 2:

Incubate at 37°C for 30 minutes,Protect from light/Green light

Incubate at room temperature for 60 minutes,Protect from light/Green light

Reading

Read on instrument

Read on instrument


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

[Preparation of Calibrator Gradient Samples]

Reconstitute and prepare calibrator gradient samples using the same matrix as the test samples. For example, if the test samples are cell culture supernatants, use cell-free culture medium to reconstitute and prepare the calibrator gradient samples.

Reconstitute the lyophilized calibrator with 100 μL of matrix, then dilute the mIgG1 calibrator with the matrix. The recommended dilution scheme is shown in the table below:

Gradient

Concentration (μg/mL)

High Conc. mIgG1

Matrix (μL)

C8

1000

/

/

C7

300

21 μL C8

49

C6

100

7 μL C8

63

C5

30

7 μL C7

63

C4

10

7 μL C6

63

C3

3

7 μL C5

63

C2

1

7 μL C4

63

C1

0

63


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 represents the LOB.

Assay Procedure

Matrix

Limit of Blank (μg/mL)

Assay Procedure 1

Calibrator Diluent

0.61

Assay Procedure 2

Calibrator Diluent

0.48


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.

Assay Procedure

Matrix

Repeatability

Low Concentration

High Concentration

Assay Procedure 1

Calibrator Diluent

3.82%

1.62%

Assay Procedure 2

Calibrator Diluent

3.79%

4.33%


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

Assay Procedure

Matrix

Deviation

Low Concentration

High Concentration

Assay Procedure 1

Calibrator Diluent

3.45%

4.17%

Assay Procedure 2

Calibrator Diluent

-9.71%

-5.88%


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

Assay Procedure

Cross-reactant

Cross-reactivity Rate

Assay Procedure 1

Human IgG

0.17%

Rabbit IgG

0.18%

Porcine IgG

0.39%


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 test, with 2~3 replicates 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.

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