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Overview of Antibody Production Introduction Polyclonal antibodies are produced in vivo in response to immunization with different epitopes on an immunogen. Anti-serum can be raised in a wide range of animals with multiple injections of the antigen along with the adjuvant (a non-specific enhancer of the immune response). For many small molecules or haptens, a carrier protein (which provides determinants recognized by helper T-cells) is required for conjugation via various bi-functional coupling reagents. Upon repeated immunizations, the antibodies produced will be predominantly IgG with a reasonable high affinity. Monoclonal antibodies provide single epitope specificity and potentially limitless amounts of identical antibody. On the other hand, polyclonal antibodies provide multiple specificity and are limited in the amount of serum that can be obtained from the immunized animal. However, with medium-sized species, such as rabbits, enough serum can be obtained from a hyper-immunized animal to yield high titer antibodies which can be stored for several years. The specificity of polyclonal antibodies can also be improved by affinity chromatography using purified antigen.
Proteins, peptides, haptens, chemical compounds, can be used to generate antibodies. Peptides, haptens, and small compounds need to be conjugated to a carrier protein in order to elicit a good immune response. The antibody titer can be monitored by antigen-specific ELISA.
Synthetic Peptides as Antigens Synthetic short peptides have been used to generate protein-reactive antibodies. The advantage of immunizing with synthetic peptides is that unlimited quantity of pure stable antigen can be used. This approach involves synthesizing short peptide sequences, coupling them to a large carrier molecule, and immunizing the animal of choice with the peptide-carrier molecule. The properties of antibodies are dependent on the primary sequence information. A good response to the desired peptide usually can be generated with careful selection of the sequence and coupling method. Most peptides can elicit a good response. The advantage of anti-peptide antibodies is that they can be prepared immediately after determining the amino acid sequence of a protein and the particular regions of a protein can be targeted specifically for antibody production. The only disadvantage is that they might not recognize the native protein. The ability of anti-peptide antibodies to recognize the native protein, such as in immunoprecipitation or immunohistochemistry staining, depends on the peptide sequence displayed on the surface of the native protein in a conformation similar to that found in the peptide-carrier protein conjugate. Therefore, the successful production of anti-peptide antibodies is often determined by the prediction of the location of certain peptide sequences in the three-dimensional structure of the protein. Protein prediction programs are available for such analysis. Important factors to consider include protein hydrophilicity and flexibility (see Protein Prediction Data below).
The length of peptide is another important factor to consider. Approximately, a peptide of 10-15 residues is optimal for anti-peptide antibody production; longer peptides are better since the number of possible epitopes increases with peptide length. However, long peptides increase the difficulties in synthesis, purification, and coupling to carrier proteins. The peptide synthesis facility is very important. The quality of an antibody is dependent upon the quality of the peptide. Side products contained in peptide products can lead to low-quality antibodies. Peptide-carrier protein coupling is another factor involved in the production of high titer antibodies. Most coupling methods rely on the reactive functional groups in amino acids, such as -NH2, -COOH, -SH, and phenolic -OH. Site-directed coupling is the method of choice. Another peptide method used in anti-peptide antibody production is the Multiple Antigenic Peptide system (MAPs). The advantage of MAPs is that the conjugation method is not necessary. No carrier protein or linkage bond is introduced into the immunized host. One disadvantage is that the purity of the peptide is more difficult to control. In addition, MAPs can bypass the immune response system in some hosts. Horvath and his colleagues recently published a new system, Lipid Core Peptides (LCP). The antibody titer is higher (3200-fold) with the LCP system, than found with CFA. This lipid-based method is still in research stage.
Rabbits are commonly used for antibody production. Sheeps, goats, chicken, mice, rats, hamsters, and guinea pigs can also be used. A minimum of two rabbits should be used for each antigen; three to four is preferred.
After the third injection of the antigen, serum can be taken to check the production of specific antibodies. The test bleeds normally are assayed against the immunogen itself. ELISA is the most common and easiest method to check the titer. Usually, serum samples of 25 mL should be collected, approximately 7-10 days after each boost.
The lytic complement activity of serum can be abolished by heating at 56°C for 30 min. In order to avoid denaturation of the antibody, it is important not to exceed this temperature.
Antibodies can be stored in several different buffers at neutral pH, but are most commonly kept in 0.01M phosphate-buffered saline (PBS) at pH 7.4 containing 0.1% sodium azide to inhibit microbial growth. For long-term storage, antibodies should be kept at -20°C or -70°C, although very low temperature is usually not necessary. They should be stored at > 0.5 mg/mL or in the presence of a carrier protein (e.g., 1% bovine serum albumin (BSA)). If stored frozen, avoid repeated freezing and thawing by making a number of aliquots and keeping each individual aliquot at 4°C subsequent to thawing and prior to experiment. Antibodies kept at 4°C should either be sterile-filtered through a 0.22-mm pore size Millipore filter, or kept in the presence of 0.1% sodium azide. Antibody conjugates are usually stored at 4°C, but can also be stored at -20°C in 50% glycerol.
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