Mouse Monoclonal Antibody Isotyping ELISA

Mouse Monoclonal Antibody Isotyping ELISA:
For Research Use Only

Kit Sizes: 1 plate, 5 plate, contact Eagle for bulk pricing
Incubation Time: 1 hour
Sample Type: Tissue Culture and Hybridoma Supernatants
Sample Size: 100 µl
Samples / 96 wells: 12, 1 sample per strip
Isotypes Measured: anti-IgG₁, IgG_2a,IgG_2b,IgG_3,IgA, and IgM, kappa and lambda
Product Developed and Manufactured in the USA

Subtype Well Identifier Map

Intended Use
The Mouse Monoclonal Antibody Isotyping (mAb) ELISA kit is intended for the identification of mouse antibody isotypes (anti-IgG₁, IgG_2a,IgG_2b,IgG_3,IgA, and IgM, as well as light chain kappa and lambda) by a microplate enzyme immunoassay (ELISA). This kit employs a horseradish peroxidase labeled mouse anti-IgG isotype specific antibody system to provide a colorimetric change; identifying the isotype and light chain of the test antibody. Using the kit components provided tissue culture supernatant and purified antibodies can easily be screened for mouse antibody isotypes and light chain.

Summary and Explanation Of The Test
The Eagle Biosciences Mouse Monoclonal Antibody Isotyping (mAb) ELISA kit is designed to utilize a simple 96 strip-well (8x12) ELISA format. Up to 12 individual samples can be tested for anti-IgG₁, IgG_2a,IgG_2b,IgG_3,IgA, and IgM, as well as light chain kappa and lambda . One sample is used per strip.

In this ELISA, individual wells are pre-coated with anti-mouse subtype specific antibody or light chain specific antibody as the capture (Subtype specific anti-IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM, Light chain specific: kappa or lambda). Samples are added to the strip-wells with Goat Anti-Mouse IgG+IgA+IgM HRP Conjugate. After an hour incubation at room temperature, the wells are washed three times with Working Strength Wash Buffer before TMB is added to each well. After a short incubation, Stop Solution is added and the absorbance is measured at 450nm.

Assay Procedure

Equilibrate kit components to room temperature.
Add 100 µl of the appropriately diluted antibody sample to each well of the 8-well strip.
Add 100 µl of the Goat Anti-Mouse IgG+IgA+IgM Conjugate Buffer to each well of the 8-well strip.
Carefully cover wells with new adhesive film. Mix on plate shaker (300-900 rpm) for 1 hour at room temperature (18-25°C).
Carefully remove adhesive film, empty well contents and wash three times using 300µl per well of Working Strength Wash Buffer.
Add 100 µl of TMB Substrate to each well. Incubate 10 minutes (0.5 - 20 minutes depending upon intensity of signal) at room temperature (18–25°C). Positive results (blue color in designated well) may be observed in as little as 30 seconds depending upon intensity of the antibody concentration and isotype.
Add 100 µl of Stop Solution which changes blue color in wells to yellow.
Measure absorbance using plate reader at wavelength 450 nm. Some researchers may determine that visual reading is sufficient.

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