The Antibody Component

By virtue of their high levels of specificity and binding affinities, antibodies are the ideal choice of agent for drug detection. Antibodies are produced by the immune system as weapons to eliminate invading pathogens (1). Antibodies to a specific DOA can be produced by immunizing animals with the selected DOA conjugated to a carrier protein. A carrier protein is necessary because small chemicals (DOAs) by themselves are usually not immunogenic enough (as a hapten) to elicit an antibody response (2-6). Common protein carriers for this purpose include bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). However, this technique is not as simple as it sounds and is not for the amateur protein chemist. The type of linker used, the length of the linker used, the molar ratio of the drug to the carrier protein, and the type of carrier protein to be used are only some of the factors that must be carefully considered to achieve effective conjugation.

Once immunized, the host animal will then produce antibodies to areas of the whole complex antigen. It is likely that there will be antibodies to the drug, to areas formed by the drug and the carrier, and to the carrier. All of these antibodies in the antibody preparation will bind to the colloidal gold label and cause unwanted cross-reactions. This type of unwanted cross-reactivity is often observed in an inhibition assay as a failure to block a signal even when levels of free drug are taken above the normal working range. This does not always occur, but should be considered possible if cut-off is difficult to achieve. To circumvent this problem, the antibodies of unwanted specificity must be "absorbed out" by mixing the sera with the carrier protein and recovering the unbound antibodies. Alternatively, it is sensible to use one drug-carrier (e.g., KLH) to raise the antibody and to use a different drug-carrier (e.g., BSA) for the capture complex.

2.1.1. Polyclonal vs Monoclonal Antibodies

Antibodies can be produced polyclonally or monoclonally. Polyclonal antibodies are derived from blood sera of animals immunized with an antigen and, as the name implies, consist of a mixture of antibodies produced by different B-cell clones, each with a different specificity and binding affinity. Monoclonal antibodies, on the other hand, are derived by immortalization of a specific antibody-producing cell (a B-cell hybridoma) (7,8) such that its progeny produce antibodies of a single specificity. Production of monoclonal antibodies is not a trivial exercise. Laboratories not equipped to produce their own monoclonal antibodies are advised to subcontract the job to commercial sources. Polyclonal antibodies have the advantage of being simple to produce. Small animals such as mice and rats can be used to produce polyclonal antibodies for initial specificity testing. Larger animals such as rabbits, horses, and cows can yield large amounts of antisera. The fact that polyclonal antisera contain multiple antibody specificities may prove useful for some end users who have to detect drugs with related structures, e.g., methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA). A monoclonal antibody, on the other hand, is specific for a single epiotpe. The screening and isolation of a monoclonal antibody-secreting cell line is labor intensive. In addition, monoclonal antibodies can be made only in rodents (mice, rats, and hamsters) because fusion partners (myeloma cells) (7) from large animals are presently not available. Once a monoclonal-secreting line is identified, antibodies are produced by growing the cell line and antibodies purified from culture supernatants. Large quantities of monoclonal antibodies can also be produced by injecting hybridoma cell lines into the peritoneal cavity of pristine-primed mice and collecting ascite fluids for further purification.

2.1.2. Purification of Antibodies

All antibodies, whether monoclonal or polyclonal, should be purified before use. Besides antibodies, sera and ascitic fluids (7) all contain extraneous blood proteins, which may interfere with antibody binding. Monoclonal antibodies grown in culture usually will have similar proteins derived from culture supplements such as fetal calf serum. Purification has the added benefit of concentrating the antibody preparation, which aids in the stability of the antibody protein.

Simple methods of antibody purification include DEAE or Sephadex column separation, or precipitation with a solution of saturated ammonium sulfate. In addition to immunoglobulins, the purified preparation will contain a mass of proteins. All of these proteins will compete for binding sites on the surface of the labeled gold colloid. Those with the greatest level of the three residues controlling protein binding to colloid—cysteine, tryptophan, and lysine—will conjugate most readily to the naked negatively charged colloid (discussed later), and these may not be the proteins required to drive the assay. This will result in a low degree of sensitivity of the detector reagent in the assay. More specific purification of antibodies can be achieved with protein A or protein G derivatives (9-10). These bacterial cell-wall proteins have high binding affinity for the Fc region of immunoglobulins, making them the ligands of choice for antibody isolation. Protein A or protein G can be coupled to sepharose beads and antibody-containing preparations (sera, ascite fluids, culture supernatants) can be passed through a chromatographic column. Bound antibodies are then eluted with acidic buffers. Protein A and G can bind immunoglobulins from many animal species, including large animals commonly used for polyclonal antibody preparation (e.g., sheep, goat). It should be noted that protein A and protein G do not bind all immunoglobulin isotypes and subtypes equally. For example, protein A does not bind immunoglobulin G (IgG) or IgA subclasses efficiently. For these isotypes, protein G and protein L are appropriate choices, respectively. Furthermore, because the binding specificities of protein A and protein G are the Fc region of immunoglobulins, all immunoglobulins, irrespective of their antigen specificities, will be bound to the column and eluted with the DOA-specific antibodies. Monoclonal antibody culture supernatants, while containing the majority of specific antibodies, also contain fetal calf serum as a culture supplement. Naturally occurring calf immunoglobulins will co-purify with the DOA-specific antibodies and, as pointed out above, will bind to the gold colloid and lower the level of sensitivity of the DOA assay.

The highest degree of purity comes from affinity purification. In this case, the DOA of choice is coupled to a specific matrix. Antibody-containing preparations are passed over the drug-matrix column, which binds only those antibodies specific for the DOA. All other immunoglobulins and nonspecific proteins are washed through the column. The specific antibodies are then eluted by altering the salt or pH of the column buffer. Affinity purification may be costly in terms of time, money, and serum, but produces a quality conjugate, which should be considered as a key raw material for any assay.

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