99-1000 - FAQs

To estimate the contribution of the non-specific interaction and Fc receptor binding, staining protocols using an antibody directed to an irrelevant antigen (for example, DNP) having the same isotype as the antibody of interest may be analyzed in parallel with the antibody of interest. The antibody directed to the irrelevant antigen is known as the isotype control.

Antibodies are useful for identifying and localizing proteins and other antigens both on the surface of cells as wells as inside cells. The utility of antibodies lies in their specificity and avidity for their antigens, an interaction which is mediated by the antigen binding sites. Antibodies can also react with components other than their antigens in several ways. They can associate with cellular components by non-specific protein-protein interactions. They can associate with fatty components by hydrophobic interaction. Antibodies can also bind to antibody receptors expressed on the surface of some cell types. For example, antibodies of the IgG class can bind to the cell surface Fc receptors known as CD16, CD32, and CD64. Under ideal conditions, various types of non-specific binding will be prevented by including blocking proteins (BSA, milk, or animal serum products) in antibody incubation reaction mixtures. Antibody binding to Fc receptors can be prevented by preincubation of cells with serum preparations containing immunoglobulins.

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No. A customer notified us early in 2014 that Cat. No. 99-1000 did not detect IgG subclasses from cynomolgus monkeys. As far as other nonhuman primates are concerned, we do not know if this kit will work with samples from these animals.

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In the IgG Subclass Human ELISA Kit (Cat. No. 99-1000), the human IgG Subclass Standard is Part Number 50287HK. Each batch of 50287HK is calibrated against a WHO reference standard designated 67/97. More details about this standard are in the abstract from Klein F et al. (1985) Clin Chem Acta 150 (2):119-127.

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In the IgG Subclass Human ELISA Kit (Cat. No. 99-1000), all of the wells of the 8-well strips provided are coated with a goat polyclonal anti-FITC antibody, which serves as a general capture reagent. When you add the individual FITC-labeled, subclass-specific monoclonals to the wells (step 2 in the assay procedure on page 2 of the manual - https://tools.thermofisher.com/content/sfs/manuals/PI99-1000_Human%20IgG%20ELISA%20Kit%20Rev%201208.pdf), they bind to the goat capture antibody. This second layer of the ELISA sandwich now performs the subclass-specific capture function. When you add samples (i.e., serum, standards, and controls) to the wells, any human IgG present in the samples will bind to the subclass-specific antibodies that are captured on the plate via their FITC labels. The subclass-specific detection is enabled by each of these subclass-specific mouse monoclonal anti-human IgG antibodies provided in the kit. The actual subclass detection occurs after you add the HRP-labeled anti-human IgG antibody and the TMB chromogen solution to the wells. The amount of each IgG subclass is determined separately in its own set of wells.

An example of one of these subclass-specific sets is shown under step 1 in the assay procedure on page 2 of the manual - https://tools.thermofisher.com/content/sfs/manuals/PI99-1000_Human%20IgG%20ELISA%20Kit%20Rev%201208.pdf. In this case, the setup shown is for IgG1, but if you wanted to measure only IgG4, for example, you would follow the same setup. However, instead of loading these wells with mouse anti-human IgG1, you would use the mouse anti-human IgG4 instead. If all you want to detect is IgG4, the rest of the wells in the plate can be used for other samples instead of additional standard curves for the other subclasses and their respective samples. However, we suggest running a standard curve for the IgG subclass of interest on each plate that you prepare, and each time you run the assay.

The control in the kit consists of lyophilized human serum, and the human IgG standard provided contains all four subclasses at the concentrations indicated on the lot-specific manual. Even though you may want to measure only 1 or 2 subclasses, you'll be using a standard that contains all of them. The other 3 subclasses don't interfere with detection of IgG3, for example, because the capture antibody is IgG3-specific. The detection antibody in the kit is an HRP conjugate of an anti-human IgG that detects all of the subclasses equally effectively and detects all of the human IgG captured in the wells.

Note that the antibody-coated plates in the kit come as 8-well strips that you snap into the frame provided. You do not have to run an entire plate or both plates at one time. Store any unused 8-well strips at 2-8 degrees C and keep them dry. Unused wells in individual strips should be sealed securely to prevent the entry of moisture while running the assay.

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