( Monoclonal antibody isotyping in only...)Simple, Sensitive Isotyping of Mouse Monoclonal Antibodies

Monoclonal antibody isotyping in only 5 minutes

Weiping Yang Matt C. Petre Darrel Holmes Peter L. Pingerelli
Stratagene Cloning Systems, Inc.

Stratagenes IsoDetect mouse monoclonal antibody isotyping kit is a simple, rapid and sensitive method for identifying mouse monoclonal antibody isotypes in a single step. Each IsoDetect test card is a self-contained assay, using colloidal gold-conjugated anti-mouse antibodies for isotype determination of 0.1 g/ml of antibody in 5 minutes.

For decades, antibodies have been used as key reagents for biological research. The development of hybridoma technology, which generates monoclonal antibodies, has resulted in the development of a significant number of immunoglobulins for research and diagnostics. As knowledge concerning the production of monoclonal antibodies has grown, these powerful tools have become more commonplace, and thousands of new monoclonal antibodies are made each year.

Mouse immunoglobulins have been classified into five primary classes (IgG, IgM, IgA, IgD and IgE) according to differences in their heavy chain polypeptides (the immunoglobulin classes have g, , a, d and e heavy chains, respectively). The antigenic differences among the five classes are located primarily on the Fc fragments of the heavy chains. These differences allow immunoglobulins to function at particular stages in a variety of immune responses. IgG molecules are further subclassed based on the antigenicity of the four isotypes. Mouse IgG isotypes are designated IgG₁, IgG_2a, IgG_2b and IgG₃.

Because the biological functions and biochemical characteristics of classes and isotypes differ, distinguishing the classes and isotypes of an immunoglobulin molecule is critical. Knowledge 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 the sample. This assay is highly sensitive and relatively fast (2 to 4 hours), but the required multistep operations of the assay are labor-intensive. A plate reader, which is best for quantitating ELISA results, may be an investment that is prohibitive for some laboratories.

A rapid method of isotyping uses anti-mouse antibody that is conjugated to an indicator molecule, such as colloidal gold or latex, and isotyping antibodies coated on a membrane in separate, discreet lines. The antibody in a sample forms an antigen-antibody complex with the indicator conjugate and is exposed to the isotyping membrane. When the sample complex meets the specific isotyping antibody, a colored line appears. Results are judged by reading the line at a specific location. This format allows multiple samples to be analyzed in a simple one-step operation. Fast results (5 minutes) and high sensitivity (as low as 1.0 g/ml of antibody, according to manufacturers) characterize this newest immunoglobulin isotyping method. Because this method is easy to use and gives rapid results, it is likely to replace the ELISA method for mouse immunoglobulin isotyping.

Single-Step Isotyping

figure 1

Stratagenes IsoDetect mouse monoclonal antibody isotyping kit is a rapid method for determining the isotypes of mouse monoclonal antibodies. This kit uses a colloidal-gold conjugation technique to label rabbit anti-mouse l and k light chain antibodies as indicating reagents, and rabbit anti-mouse isotyping immunoglobulin coated on a membrane. When a specific mouse immunoglobulin is present in a sample, a complex containing mouse immunoglobulin and rabbit anti-mouse l or k gold-conjugated antibody will be formed. This complex will bind to a specific anti-mouse isotype antibody that is immobilized on the membrane. The appearance of a pink band on the IsoDetect isotyping membrane at a defined location indicates the specific isotype of the mouse immunoglobulin tested. The sensitivity of the test is 0.1 g/ml of mouse immunoglobulin, and test results are determined visually within 5 minutes (figure 1). The IsoDetect kit is sensitive, simple, highly specific and requires no equipment. Unlike other isotyping products, the IsoDetect kit requires only a simple dilution of ascites or culture media with the provided phosphate buffered saline before applying the sample to the IsoDetect card. No agitation, mixing of reagents or long incubation periods are necessary. The IsoDetect card is completely self-contained and is ideally formatted for archiving permanent records. For added convenience, Stratagene is also offering the IsoDetect isotyping control kit, which contains purified mouse immunoglobulin isotype standards. The IsoDetect control kit can be used as an adjunct to the IsoDetect kit or can be used for other applications, such as dot blot assays.

Conclusions

Stratagenes IsoDetect mouse monoclonal antibody isotyping kit is a rapid method for determining isotypes of mouse monoclonal antibodies from tissue culture and ascites samples. The method uses a colloidal-gold conjugate technique to achieve sensitivities of 0.1 g/ml in 5 minutes. In addition, Stratagene is offering the IsoDetect isotyping control kit, which is a series of standards that can be used with the IsoDetect kit or for other isotyping applications.

REFERENCES

1. Harlowe, E., and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

2. Ouchterlony, (1949) Ark. Mineral Geol. 26: 1.

3. Coutler, A.R., et al. (1989) Med. Lab. Sci. 46: 54-58.