What sample types can be tested with this kit?
Ascites fluid, hybridoma or myeloma culture supernatants can be tested. The kit is not intended for use with serum samples, which contain many IgG types, all of which will be detected by the assay.
How sensitive is the kit?
Antibodies are detected at concentrations greater than 3 ng/ml.
Do the kits include plates?
Yes. Five pre-coated ELISA plates in a strip-well format (8 x 12) are provided. The ready-to-use plates are blocked and contain eight pre-coated antibodies.
Which mouse-specific antibodies are identified in this kit?
Each 8-well strip of a plate tests for six mouse heavy-chain subclasses (IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) and two mouse light chain isotypes (kappa and lambda).
How long does it take to complete the isotyping protocol?
The assay can be completed in 1 hour. Optimizing for maximum sensitivity will extend the assay time to 1.5 hours.
I only need to test two samples. What can I do with the other sample strips in the plate frame so that they can be used later?
The supplied plates are strip-plates, meaning that individual 8-well strips (columns) can be removed from the plate frame. To test two samples, remove 10 strips, leaving two strips in the plate frame for testing. Place unused strips in the foil pouch with the provided desiccant. Store reserved strips at 2-8°C. Be sure the foil pouch is tightly sealed. After completing the assay, retain the plate frame for use with reserved strips for future assays.
How do I evaluate results?
The assay can be evaluated qualitatively by visual inspection or quantitatively by measuring absorbances in a spectrophotometer (plate reader) at 450 nm.
What if I see cross-reactivity for kappa light and IgG3 and IgG2?
Try diluting samples. Some antibodies have epitopes that cross-react with the IgG3 and IgG2 antibodies used in the kit; when the sample dilution is optimized, this cross-reaction will produce significantly less signal (color) compared to the true identity.
Will fetal bovine serum interfere?
No, the kit is compatible with FBS; the kit antibodies do not cross-react with bovine immunoglobulins.
How does this ELISA-based Isotyping Kit (#37503) differ from the Cassette-based Kits (#26178, 26179)?
The ELISA Kit procedure takes approximately 1.5 hours to complete and enables simultaneous testing of multiple samples (12 per plate). The Cassette-based Kits provide results in only 5 minutes but are single-use units that are less practical and more costly per sample than the ELISA Kit. The Cassette-based Kits are less sensitive (minimum antibody concentration is 10ng/ml) and provide only qualitative assessment.