Assays

Which assay should you use?

When choosing an immunoassay, consider the type of antibody (monoclonal or polyclonal), number of different antibodies, and amount of antibodies available; the purity and amount of the available antigen; the nature of the sample (media, plasma, tissue extract); the number of anticipated samples (tens, hundreds, or thousands); and special equipment required for the assay (washers, spectrophotometers, gamma counters, etc.).

Solid-phase ELISA (with antigen coated plates)
Our standard solid-phase ELISA uses antigen-coated microtiter plates, a secondary antibody (for example, goat anti-mouse IgG conjugated with horseradish peroxidase), and a chromogenic substrate. The output signal (absorbance of the chromogenic substrate following oxidation) depends on the formation of primary antibody-antigen complexes. Under certain conditions, the assay can be linear with respect to antibody concentration and, therefore, useful for comparing the relative concentrations of antibodies in different samples. One factor to consider with this assay is that antigen bound to a surface can be different than antigen in solution. Some epitopes are highly dependent on the solution versus the surface properties of the antigen. This is particularly true for small linear peptides.

Antigen Capture ELISA
This solid-phase assay uses antibody-coated microtiter plates (with antibody either directly bound or bound via another protein, such as protein A). We first capture antigen from solution, then detect antigen-antibody complexes using either biotin-labeled antigen with horseradish peroxidase-conjugated streptavidin, or a second antibody positioned at a site distinct from the first antibody. As with any solid-phase assay, critical parameters include the type of plate, the method of blocking nonspecific protein binding sites on the plate, the relative concentration of assay components, and the number of washes and composition of the wash buffer. We generally use biotinylated-antigen in this format.

Competitive ELISA for antigen in solution
This assay measures the amount of free antibody remaining in an equilibrium mixture of antibody and antigen in solution.

Immunoblotting (Western Blot)
Antibodies recognize both linear epitopes and nonlinear epitopes determined by the conformation of the antigen. For this reason, immunoblotting assays often detect a different set of antigenic determinants than other immunoassays, and antibodies or antigens screened in this method can behave differently from other assay methods. This assay is useful for detecting antigens in complex mixtures and for estimating molecular weight.