The 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.

Nearly all medical or cell biology researchers doing any kind of molecular analysis make use of antibody technology in one form or another. Depending upon their specific research needs, scientists will differ in the extent to which they concern themselves with antibody production and purification.

Introduction

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. Certainly, no other current technology allows researchers to design and manufacture such highly specific molecular recognition tools. Several important features besides their high specificity 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.

Antibody production and purification guide

The updated Antibody Production and Purification Technical Handbook is an essential resource for any laboratory working with antibodies. The handbook provides an overview of antibody structure and types, as well as technical information on the procedures, reagents and tools used to produce, purify, fragment and label antibodies.


Antibody production

The term "antibody production" has both general and specific meanings. In the broad sense, it refers to the entire process of creating a usable specific antibody, including steps of immunogen preparation, immunization, hybridoma creation, collection, screening, isotyping, purification, and labeling for direct use in a particular method. In the more restricted sense, antibody production refers to the steps leading up to antibody generation but does not include various forms of purifying and labeling the antibody for particular uses.

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 can then be recovered from the animal. Polyclonal antibodies are recovered directly from serum (bleeds). Monoclonal antibodies are produced by fusing antibody-secreting spleen cells from immunized mice with immortal myeloma cell to create monoclonal hybridoma cell lines that express the specific antibody in cell culture supernatant.

Successful antibody production depends upon careful planning and implementation with respect to several important steps and considerations:

  • Synthesize or purify the target antigen (e.g., peptide or hapten)
  • Choose an appropriate immunogenic carrier protein
  • Conjugate the antigen and carrier protein to create the immunogen
  • Immunize animals using appropriate schedule and adjuvant formula
  • Screen serum (or hybridoma) for antibody titer and isotype (also called antibody characterization; see below)

Antibody purification

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 to highly specific:

  • Crude—precipitation of a subset of total serum proteins that includes immunoglobulins
  • General—affinity purification of certain antibody classes (e.g., IgG) without regard to antigen specificity
  • Specific—affinity purification of only those antibodies in a sample that bind to a particular antigen molecule

Which level of purification is necessary to obtain usable antibody depends upon the intended application(s) for the antibody.


Antibody characterization

Antibody characterization involves three kinds of activities that are usually performed at various stages throughout an entire antibody production and purification project

  • Screening—identifying antibody samples having antigen-binding specificity
  • Titering—measuring antibody concentration and functional assay titer
  • Isotyping—determining a monoclonal antibody class and subclass identity

Screening is first required during production to identify which animals and hybridoma clones are producing a high level of antigen-specific antibody. This is usually accomplished using ELISA techniques.

Antibody concentration can be estimated using either a general protein assay or a species- and immunoglobulin-specific method, such as with specialized microagglutination assay kits. Antibody titer is related to concentration but refers more specifically to the effective potency of a given antibody sample. Measuring titer usually means determining the functional dilution of an antibody sample necessary for detection in a given assay, such as ELISA.

Isotyping involves determining the class (e.g., IgG vs. IgM) and subclass (e.g., IgG1 vs. IgG2a) of a monoclonal antibody. This is a critical step in antibody production, as it is necessary for choosing an appropriate purification and modification method for the molecule. Isotyping is most easily accomplished with commercial, ready-to-use antibody isotyping kits.


Antibody fragmentation

Purified antibodies can 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. Most often antibodies are used in whole-molecule form. However, the performance of some techniques and experiments can be improved by using antibodies whose nonessential portions have been removed.

Antibody Fragmentation refers to procedures for cleaving apart whole antibody molecules and removing portions that are not necessary for binding antigen. Fab and F(ab)'2 are antibody fragments of IgG that are most frequently created and utilized by researchers.


Antibody labeling and immobilization

Antibodies are produced and purified for use as antigen-specific probes. However, their utility in any given technique (ELISA, Western blotting, cellular imaging, immunohistochemistry) depends upon having a mechanism to secondarily detect the antibody.

Techniques that utilize antibodies for immunoprecipitation or other form of affinity purification depend upon mechanisms for attaching or immobilizing them to chromatography media (e.g., beaded agarose resin). Strategies for accomplishing this involve the same considerations and chemical methods as antibody labeling.


References

  1. Alberts, B., et al. (1983). Molecular Biology of the Cell. Garland Publishing, Inc., New York, NY.
  2. Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  3. Sites, D.P., et al. (1976). Basic & Clinical Immunology. Lange Medical Publication, Los Altos, CA.