Antibodies are host proteins that are produced by the immune system in response to foreign molecules that enter the body. These foreign molecules are called antigens, and their molecular recognition by the immune system results in selective production of antibodies that are able to bind the specific antigen. Antibodies are made by B lymphocytes and circulate throughout the blood and lymph where they bind to their specific antigen, enabling it to be cleared from circulation.
This ability of animal immune systems to produce antibodies capable of binding specifically to antigens can be harnessed to manufacture probes for detection of molecules of interest in a variety of research and diagnostic applications. No other current technology allows researchers to design and manufacture such highly specific molecular recognition tools. In addition to their high specificity, several important features make antibodies particularly conducive to development as probes. For example, except in those portions that determine antigen binding, antibodies share a relatively uniform and well-characterized protein structure that enables them to be purified, labeled and detected predictably and reproducibly by generalized methods.
Procedures for generating, purifying and modifying antibodies for use as antigen-specific probes were developed during the 1970s and 1980s and have remained relatively unchanged since Harlow and Lane published their classic Antibodies: A Laboratory Manual in 1988 (Product # 15050). Antibody production involves preparation of antigen samples and their safe injection into laboratory or farm animals so as to evoke high-expression levels of antigen-specific antibodies in the serum, which may then be recovered from the animal. Alternatively, monoclonal hybridoma cell lines that produce one particular antigen-specific antibody can be prepared by fusion of individual antibody-secreting spleen cells from immunized mice with immortal myeloma cell lines.
Antibody purification involves isolation of antibody from serum (polyclonal antibody), ascites fluid or culture supernatant of a hybridoma cell line (monoclonal antibody). Purification methods range from very crude (precipitation of sample proteins including any antibodies present) to general (affinity purification of certain antibody classes without regard to antigen specificity) to specific (affinity purification of only those antibodies in a sample that bind to a particular antigen molecule). Which level of purification is necessary depends on the intended applications for the antibody.
Antibody characterization includes assessing antibody concentration and titer and determining the class and subclass of a purified antibody. Antibody concentration can be estimated by either a general protein assay or one of the species-specific Easy-Titer IgG Assay Kits. Antibody titer refers to the functional dilution of an antibody sample necessary for detection in a given assay, such as an enzyme-linked immunosorbent assay (ELISA). Determining the class (e.g., IgG vs. IgM) and subclass (e.g., IgG1 vs. IgG 2a ®) of an antibody is important for choosing an appropriate purification and modification method for the molecule. Class and subclass can be determined using an antibody isotyping kit (see page 22).
Purified antibodies may be modified for particular uses by several methods including fragmentation into smaller antigen-binding units, conjugation with enzyme or other detectable markers, and immobilization to solid supports. This handbook provides an overview of antibody structure and types, as well as of the procedures, reagents and tools used to produce, purify, fragment and label antibodies.