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December 2001
Vol. 4, No. 12, pp 28–32.
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Focus: Bioanalytical Methods
Feature Article
Antibodies and analysis


Immunology provides some of the most sensitive and precise bioanalytical techniques for drug and target discovery.

opening artOver evolutionary time, nature has designed what is perhaps the most specific and efficient analytical tool ever to be used in biomedical research—the antibody. Modern researchers have capitalized on this tool through a host of modification techniques, including antibody engineering using recombinant DNA methods. The use of antibodies has expanded from simple diagnostics to the detection of the fine structure of molecules, elucidation of gene function, localization of gene products, and rapid screening of biological effectors for drug discovery and testing. The use of these antibodies with fluorescent or enzymatic tags, in concert with advances in microscopy, improved enzyme-linked immunosorbent assay (ELISA) systems, and even microarrays, is already transforming disease research and the search for therapeutics. And not only do these newly designed and selected antibodies serve as powerful research tools, but many also show the potential to be used as drugs in their own right.

The “new” antibody
The growth in understanding of the molecular biology and genetics of the immune system is one of the greatest success stories in modern medical science. As part of this explosion of research, antibodies have become some of the best understood and manipulated protein molecules—and potentially the most valuable. The typical antibody, immunoglobulin G (IgG), is almost a stock character in modern biotechnology and is usually depicted as a capital letter Y. In nature, the antibody is a tetrameric molecule consisting of two copies of a heavy chain (H) polypeptide approximately 440 amino acids long and two copies of a light-chain (L) polypeptide some 220 amino acids long.

“Breeding” an antibody
As detailed in Using Antibodies: A Laboratory Manual, animals are estimated to produce 1012 different antigen-binding domains. How does the human body, for example, produce antibodies against this vast number of potential antigens with only 30,000 genes purportedly available, and where each different antibody must be secreted by a unique clone of B cells or their progeny plasma cells?

Four main sources of variation are involved. First, multiple coding sequences specify each different portion of the variable antigen-binding site; second, inaccurate homologous recombination realigns the various coding regions while also adding random nucleotides to the recombination “joints”; third, mutations are commonly generated in the coding regions; and finally, the various heavy and light chains (each of which has been undergoing these changes) can randomly associate.

These factors all lead to a diverse array of genetic recombination at the cellular level that transforms each antibody-producing cell into a unique entity, different at the DNA level from every other cell in the organism. When an antigen finds its preexisting cellular “match”, that cell begins division to produce clonal progeny that generate only a unique antibody to the particular antigen’s recognized site—hence the phenomenon of immunization. Further recombination can occur in the so-called constant region. This results in antibodies that can be differentially localized, causing them to appear either in serum or mucus secretions, for example.

Each H and L polypeptide contains a variable region and a constant region. At the tip of each arm of the Y-shaped antibody is a site consisting of the variable tips of the H and L subunits, which together bind to a specific, unique site on an antigen known as an epitope. The genetics of antibody variability and production is extraordinarily complex and would be a recipe for catastrophe for the production of any other protein that required maintaining structural fidelity (see box, “Breeding” an antibody).

Antibody technology has rapidly evolved from the original production and use of polyclonal antibody mixtures bled from rabbit ears or from horses in the first half of the 20th century, to the development of specific monoclonal antibodies through cell fusion techniques using mice spleens and cancers in the 1970s, to today’s engineering of uniquely designed mono- (one antigen attachment site) and divalent (two sites to different antigens) antibodies, and even to chimeric antibodies that partake of the nature of bacteriophages. The “new” antibody can find itself fused with a wide variety of other proteins that can modulate not only its activity but also its assayability and localization. Antibodies have even been isolated (and others deliberately designed) with unique catalytic activities that mimic several traditional enzyme behaviors and may interact significantly with compounds that play important roles in disease.

Major developments in current antibody research include the use of antibodies as carriers of drugs or prodrugs to directed sites, as well as the “humanization” of therapeutic antibodies to avoid the short- and long-term problems that can develop from using foreign immunologicals in human tissues. (For more on catalytic antibodies, visit Weapons of Tumor Mass Destruction.

Going through a phage
This month, the 12th International Antibody Engineering IBC Conference is scheduled to be held in San Diego ( One of the key technologies focused on is that of phage antibodies. Phage display libraries that can be used to produce human antibody fragments are constructed by fusing the genes coding for the variable region of the antibody isolated from human B cells with the coat protein gene of any of various bacteriophages, including M13. The antibody is “displayed” on the surface of the phage’s coat, and each phage produces its own unique antibody. Affinity capture can be used to isolate those phages that display an antibody recognizing a specific epitope or selection of epitopes. Subcloning of these phages provides an inexhaustible supply of highly specific antibodies for research use.

Another field of antibody engineering is the production of divalent (bispecific) antibodies. In these molecules, the binding sites for two different antigens are combined into one antibody. This was most recently accomplished by engineering the separate genes into a single bacterial clone for mass production. Such bispecific antibodies have been used to study and manipulate a wide variety of cellular processes by their ability to bind at one site to specific cells at the same time as they capture or deliver designated molecules—from cytotoxic factors to signaling triggers to simple labeling enzymes—on the other antigenic site ( The potential use of these antibodies for highly specific therapeutic delivery is being widely studied.

Research diagnostics
Although the key hallmark of antibody usefulness is in clinical practice—everyone is familiar with the use of antibodies to diagnose diseases—it is in the research laboratory that the modern antibody is proving most versatile. Highly specific and engineered antibodies hold the key to understanding the etiology and behavior of diseases, from infectious disorders to autoimmune conditions and even to cancers at the level of molecular targeting and signaling—the critical nexus for the development of inhibitory or preventive drugs. One of the most obvious problems in genomic analysis and the use of DNA microarrays for comparing different cell states is the fact that the old one gene/one protein model is a simplistic myth. Not surprisingly, the first real evidence of the phenomenal complexity of cellular proteins appeared from the routine use of that warhorse of modern antibody analysis—Western blotting.

The two-dimensional polyacrylamide gel electrophoresis used in Western blots showed clearly that the number of serologically related proteins routinely exceeded expectations and that cells somehow managed to develop unique proteins from an original template by highly sophisticated enzyme activities. Because the proteome is the product of complex posttranscriptional and posttranslational modifications (including RNA splicing and the glycosylation and variable folding of individual proteins), its study demands tools than can distinguish structure and identity far beyond the simplicities of mere sequence, be it at the nucleic or amino acid level. Because antibodies can reveal this complexity and be engineered to do even more, they are not only the ideal, but perhaps the only practical tool for the bioanalytical analysis of proteome complexities.

Epitope mapping
Today, structure is everything. Knowledge of the structure–activity relationship is some of the most sought-after information in molecular modeling for drug and target discovery. In proteins, the epitope can be considered a basic unit of functional analysis. An epitope is a distinguishable antigenic site, as defined by its ability to be recognized by an antibody. It can be the surface of a linear sequence of connected amino acids and accessory carbohydrates, or a nonlinear concatenation of folded molecules (such as often occurs at a receptor or enzyme site).

Imagine a nanomachine that could localize and differentiate specific amino acid or added carbohydrate sequences in individual proteins as they exist in nature, taking into account three-dimensional folding and posttranslational modifications. Imagine also that this submicroscopic machine could be used to locate these sites in individual cells or purify these proteins from complex cell mixtures. In concert, these nanomachines could be used to map the complex geography of individual proteins, providing sophisticated differentiation between related proteins with similar topographies in different cells or even different species. And to aid drug and target discovery, imagine that these nanomachines could perform this activity on membrane receptors or signaling proteins and could block specific binding locations of drug candidates or toxic disease-related molecules. Antibodies are such nanomachines—and the principal tool for epitope mapping.

Perhaps most significant for practical target discovery—once unique epitopes are discovered on known target molecules in enzyme active sites, specific binding regions, or unique conformational arrangements—is the fact that antibodies to these sites could be used as the basis of high-throughput screening (HTS) assays to discover related proteins that might also serve as potential targets.

Similarly, through the localization of unique epitopes that differentiate between closely related members of the same molecular family, sophisticated assays could be developed that would be able, for example, to detect the presence of one particular biological molecule (perhaps a cytokine) among a host of related ones. Antibodies to carefully selected epitopes can distinguish between antigenic molecules that are significantly different in their mode of action and effects but not easily distinguished by standard bioassays or less sophisticated antibody analysis. Such different antigens may even be products of the same gene, but differentially glycosylated or spliced in subtle ways. The ability to identify differences that may only be due to a change in molecular folding and not even the starting sequence is critical to analyzing the fine structure of cellular physiology, in which extremely similar molecules can have dramatically different metabolic and physiological consequences.

The therapeutic antibody
Modern antibody engineering has been specifically turned to the attempt to design ever-improved therapeutics. One critical requirement is the humanization of antibodies to prevent adverse reactions. Several companies have engineered mice to generate monoclonal antibodies from human gene inserts for the immunological response. Abgenix (Fremont, CA), for example, produces the XenoMouse, which can produce “fully human” monoclonal antibodies that have the “pharmacokinetics of normal human antibodies based on human clinical trials” (J. Immunol. Methods 1999, 10, 11–23).

The production and use of catalytic antibodies, otherwise known as abzymes, is also a burgeoning field of research into therapeutics. Hesed Biomed, Inc., for example, has turned to the use of peptidase catalytic antibodies to attack the HIV virus by cleaving a viral coat protein (gp20) at a conserved site. In laboratory experiments, HIV-CA rendered numerous strains of the virus noninfective and destroyed the ability of released gp120 protein to induce cell death.

A key way of using antibodies to discover new drug targets for diseases depends on their ability to block the epitopes to which they bind. Antibodies are screened to find those that are highly antagonistic to specific physiological or metabolic processes—including disease. Those which bind to and block surface membrane transport, signaling, or even toxin or virus receptor sites not only provide a perfect means of isolating and purifying such physiologically important structures, but also provide the means for testing the effectiveness of potential drugs through competitive binding assays. The discovery of narrowly defined and specific epitopes can be used to tailor competitive binding assays with potential drug candidates, eliminating experimental artifacts and dramatically improving the specificity of such assays. Even more important, such antibodies themselves may prove to be candidates for therapeutic use (see box, “The therapeutic antibody”).

Arrayed for screening
Most immunologically based HTS systems use some form of ELISA. Although ELISA has been the traditional system for the automated screening of antibodies and antigens, including drug targets (and the mainstay of many biotechnology companies whose profits derive from diagnostic kits), protein microarrays are becoming a promising alternative, with potentially higher speed and sensitivity.

Researchers at Stanford University, for example, used such arrays for “highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.” Using 115 antibody–antigen pairs spotted by printing microscopic spots of either antigens (to detect antibodies) or antibodies (to detect antigens) on a glass surface, Brian Haab and co-workers used fluorescence binding techniques to detect proteins at fraction-of-microgram quantities on average, and down to less than 1 ng/mL in some cases. Antigen microarrays have excellent potential for the monitoring and diagnosis of autoimmune diseases.

One company, Ciphergen, is using protein antibody arrays to do epitope mapping. Protein antigen is captured by the bound antibodies, and then enzymatic digestion with endoproteases removes all of the unattached antigen, leaving behind the epitope fragment bound to the antibody, which can then be released and characterized.

Synthetic peptides can be used to generate antibodies, even to the extent of creating the immunological equivalent of a combinatorial library that can be screened for huge numbers of unknown epitopes, which can then be easily characterized, especially with the use of easily clonable phage displays.

As the mainstay of a multibillion-dollar-a-year pharmaceutical support industry, companies are using antibodies as research tools, diagnostics, and drugs. Perhaps it is only fitting that the chemical system that evolved naturally to fight alien invaders is being so heavily enlisted by industry and public research institutions alike in the search for new drugs and the elucidation of new drug targets. From bioanalytical research tool, to clinical diagnostics, to hitherto unimaginably precise therapeutics, the future of modern antibody research seems rosy (or at least blood red).

Suggested reading

  • Harlow, E.; Lane, D. Using Antibodies: A Laboratory Manual; Cold Spring Laboratory Press: Cold Spring Harbor, NY, 1999.
  • The Antibody Gateway ( has extensive links to antibody sources and companies and is searchable by specific antibody or research area.
  • The Recombinant Antibody Pages, Link List.

Mark S. Lesney is a senior editor of Modern Drug Discovery. Send your comments or questions regarding this article to or the Editorial Office by fax at 202-776-8166 or by post at 1155 16th Street, NW; Washington, DC 20036.

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