Which assay should you use?
We are committed to getting antibodies fit for your intended purpose, so please share with us your end use for the antibodies we will be producing. There are many different immunoassays and not all antibodies bind their antigens in all assays. For example, if your goal is to routinely assay vast numbers of clinical blood or plasma samples, you might require antibodies that bind native antigen in the presence of blood components, some of which could interfere with antigen-antibody binding. Likewise, a monoclonal antibody useful in Western blots will need to bind antigen that has been denatured with sodium dodecylsulfate and heat. By knowing your end use for the antibody you need made, we can consider alternative sample and assay conditions during the hybridoma selection phase of antibody production.
Below are several routine assays we use to select hybridoma antibodies:
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 is that some epitopes are highly dependent on the solution versus the surface properties of the antigen. This is an important consideration for small linear peptides which often require conjugation to a larger carrier molecule.
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). One format uses wells coated with rabbit or goat anti-murine immunoglobulin to capture mouse mAbs. Addition of biotin-labeled antigen followed by avidin peroxidase identifies murine antibodies that bind to the antigen. As with any solid-phase assay, critical parameters include the type of plate, the method of blocking nonspecific protein binding sites on the plate, incubation times and temperatures, the relative concentration of assay components, and the number of washes and composition of the wash buffer. This assay is often used for projects having less than 1 mg total antigen.
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. One use for this assay is the identification of anti-idiotypic mAbs. Binding of the mouse mAb in solution to the target antibody prevents binding of the target to its ligand on the surface of a well.
Immunoblotting (Western Blot)
Antibodies recognize both linear epitopes and nonlinear epitopes determined by the conformation of the antigen. Immunoblotting assays often detect a different set of antigenic determinants than other immunoassays because the antigen is denatured before electrophoresis. This assay is useful for detecting antigens in complex mixtures and for estimating antigen molecular weight.