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Isotype Determination of Antibodies
Publication Name:
Current Protocols in Immunology
Unit Number:
Unit 2.2
DOI:
10.1002/0471142735.im0202s01
Online Posting Date:
May, 2001 Abstract
Frequently it is necessary to know the amount and serological class of antibodies made by an immunized animal, produced by hybridomas, or present in the serum of patients with inflammatory or neoplastic conditions. The immunologist's approach to such a problem is to consider the antibody or immunoglobulin molecules themselves as antigens and to use anti-immunoglobulin antibodies as the specific and sensitive agents of detection. This unit describes two methods for measurement and classification of supernatant or serum immunoglobulins: an ELISA and a method employing electrophoresis and immunofixation.
Materials
Basic Protocol: Sandwich ELISA for Isotype Detection
Materials
- Capture anti-isotype antibodies: heavy-chain class-specific antibodies (anti-µ, -, -, -, -), heavy-chain subclass-specific antibodies (anti-1, -2a, -2b, -3, -4), or light-chain isotype-specific antibodies (anti-, -)
- PBS (appendix
3 (PBSN) - Test antibodies: hybridoma supernatants, ascites fluid, or antisera
- Blocking buffer (unit
- Standard isotype antibodies (i.e., purified antibodies of known isotypes)
- Developing reagent: anti-Ig antibody (specific for all heavy-chain classes)– alkaline phosphatase conjugate (see unit
- MUP or NPP substrate solution (unit
- Immulon 2 or 4 microtiter plates (or equivalent; unit
- Additional reagents and equipment for ELISA (unit
Basic Protocol: Detecting and Isotyping Antibodies by Electrophoresis and Immunofixation
Materials
- Serum (or other biological fluid)
- Normal saline
- 95% methanol/5% acetic acid
- 1% amido black (1 g in 100 ml of 2.5% acetic acid)
- 2.5% (v/v) acetic acid
- 2 × 2–cm cellulose acetate strip
- Monospecific anti-Ig, heavy chain–specific (, , µ, , or ) or light chain–specific ( or )
- 0.85% (w/v) NaCl
- Plexiglas electrophoresis cell with two agarose bridges
- Electrophoresis power supply (e.g., Pharmacia EPS 500/400)
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Figures
-
Figure 2.2.1Normal serum protein electrophoretic pattern obtained by agarose gel zone electrophoresis demonstrating the five protein zones. -
Figure 2.2.2Agarose gel zone electrophoresis of patient serum demonstrating a monoclonal band in the -globulin zone (A; arrow). Immunofixation electrophoresis with anti-µ (B) and anti- (C) demonstrate that the band is a µ- monoclonal immunoglobulin. There is no reactivity with the antisera to the other heavy (, , , ) and light () chains (data not shown).
Literature Cited
| Literature Cited | |
| Johnson, M.A. 1982. Immunofixation electrophoresis. Clin. Chem. 28:1797-1800. | |
| Johnson, M.A. 1986. Immunoprecipitation in gels. In Manual of Clinical Laboratory Immunology. (N.R. Rose, H. Friedman, and J.L. Fahey, eds.) pp. 14-24. Am. Soc. Microbiol., Washington, D.C. | |
| Papadopoulos, N.M., Elin, R.J., and Wilson, D.M. 1982. Incidence of banding in a healthy population by high-resolution immunofixation electrophoresis. Clin. Chem. 28:707-708. | |
| Tiselius, A. 1937. A new apparatus for electrophoretic analysis of colloidal mixtures. Trans. Faraday Soc. 33:524-526. | |
| Key References | |
| Johnson, 1986. See | |
| A concise discussion of the principles of immunoprecipitation with specific reference to immunofixation electrophoresis. | |
| Maggio, E.T. 1981. Enzyme Immunoassay. CRC Press, Boca Raton, Fla. | |
| A valuable reference describing parameters of ELISA technology. | |
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