( edge of antibody structure directs the...)Simple, Sensitive Isotyping of Mouse Monoclonal Antibodies

edge of antibody structure directs the strategies for purifying immunoglobulins. For example, protein A affinity purification of mouse IgG molecules is widely used for single-step affinity purification. However, mouse IgG₁ binds to protein A with only very low affinity. Therefore, purification of an IgG₁ antibody is best carried out using a protein G resin.¹

Isotyping Methods

Due to the importance of knowing the isotype of an immunoglobulin molecule, there are several methods currently available to determine the isotypes. The classic technique is the Ouchterlony (double diffusion) assay.² This method uses a thin layer of agarose in which several wells are punched, usually in a circular pattern around a centered well. A sample is added in the central well and isotyping antiserum is added in the peripheral wells. As the sample and isotyping antibodies in the wells diffuse, specific isotyping antibody will meet the immunoglobulin sample and form a precipitate. The precipitation line that occurs between the sample well and an antiserum well indicates the isotype. This assay is inexpensive and simple to use. However, the sensitivity can be low, and 12 to 24 hours may be required to obtain results.

An alternative to the Ouchterlony assay is the more sensitive enzyme-linked immunosorbent (ELISA) method.³ The ELISA method uses a 96-well microtiter plate as a solid support for anti-mouse immunoglobulin antibodies that are coated onto each well. Sample mouse immunoglobulins in solution are added and captured by the anti-mouse antibodies. Specific anti-mouse isotyping antibodies are then introduced and allowed to bind to the mouse-anti-mouse antibody complex. Finally, an enzyme-tagged antibody that reacts specifically with the anti-isotyping antibodies is added, which, together with a colorimetric substrate, indicate the immunoglobulin isotype of th
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