Pierce™ Rapid Antibody Isotyping Kit - Mouse - FAQs

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 10 ng/mL) and provide only qualitative assessment.

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The Pro-Detect Rapid Antibody Isotyping Kit is a lateral-flow assay with high sensitivity for rapid determination of the immunoglobulin class, subclass, and light chain type of monoclonal antibodies. Two different formats of rapid isotyping kits are available. These include dipstick format and cassette-based format. One can use these kits to identify the isotype of a mouse, rat, or human antibody. Make sure to follow the recommended dilution guidelines.

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1) It is likely that there could be a substance interfering with the assay. Check the compatibility table on this page (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/pro-detect-rapid-assays.html), under “Detergent and salt compatibility limits”.
2) The expression level of the actual tagged protein is below the detectable range of the assay.

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Yes, first, make sure that the protein of interest is tagged and that we offer a Pro-Detect assay kit that can detect that tag. Secondly, make sure that you follow the guidelines to detect proteins in crude lysate and if necessary increase or decrease the lysate concentration to bring the protein of interest within the detectable range of the assay.

Note that the guidelines for the recommended detection ranges reflect the concentration of the pure tagged protein in a given sample mixture.

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As the concentration of the tag in the protein sample increases, the number of lines will decrease until they all disappear. The concentration of the tagged proteins is inversely related to the number of lines appearing on the strip.

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Yes. Western blotting is a widely used and accepted tool to identify a specific protein in a complex mixture. However, it is a time-consuming and labor-intensive process, with many preparative steps and time intervals to manage. The Pro-Detect rapid assays are able to provide comparable accuracy but with much shorter processing time (10-15 min) and less hands-on steps. For further information, please see the data shown on this page (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/pro-detect-rapid-assays.html) under “Results are comparable to western blotting”.

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Except for MBP Pro-Detect strips which cross-react with bacterial proteins, no other cross-reactivity of Pro-Detect strips has been observed. An example is shown in the figure on this page (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/pro-detect-rapid-assays.html) under “No cross-reactivity with other targets” where there was no cross-reactivity of Pro-Detect rapid GST assay strips with proteins endogenous to mammalian, bacterial, Sf9 cells (lanes 1-5) or with any other non-specific tag proteins (lanes 7-19). In addition, even when using 100% of Minimum Essential Medium with 10% FBS, there was no cross-reactivity detected.

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We examined the performance of the Pro-Detect rapid assay in the presence of various concentrations of detergents and salts that are commonly used in protein expression and isolation. Reagents tested include NaCl, Urea, Triton-X 100, SDS, NP40, EDTA, Glycerol, KCl, CHAPS, RIPA, BPER, and MPER. For estimates of each assay tolerance level for the tested detergents and salts, please refer to the table under “Detergent and salt compatibility limits” at https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/pro-detect-rapid-assays.html

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For sandwich assays, the range is 0.1-10 µg/mL of pure tagged protein.
Note: The lateral flow assays can detect outside of this range; however, intensity of the bands will be lower at concentrations higher and lower than the working range. It is not recommended to use at concentrations less than 0.01 µg/mL. In a mixture of proteins such as lysates, the target protein has to be in the detectable range (0.1-10 µg/mL) for the assay to be successful.

For competition assays, the range is 4-20 µg/ml.
Note: The competitive lateral flow assays can detect down to 1 µg/mL, but loss of band intensity may be less distinguished. As there is no upper limit, the lateral flow strip will continue to show positive results at concentrations greater than 20 µg/mL, visualized by complete loss of test lines (test lines closest to the sample will disappear first). It is not recommended to use at concentrations less than 1 µg/mL.

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