Created: 28th February 1999, last updated: 7th April 1999, © 1999 ABRF
Ruth Hogue Angeletti
Laboratory for Macromolecular Analysis,
Albert Einstein College of Medicine, Bronx,
NY 10461
Well-designed anti-peptide antibodies provide the specificity and control for everyday and cutting-edge applications. Pragmatic peptide antigen design principles can be used to help ensure production of successful antibodies. Issues of peptide chemistry, sequence selection, antigen preparation, antibody handling, and experimental needs must be taken into consideration in the planning stages. (J Biomol Tech 1999;10:2-10)
Key words: anti-peptide antibodies, peptide antigens, epitopes.
Address correspondence and reprint request to: Dr. Ruth Hogue Angeletti, Laboratory for Macromolecular Analysis, Albert Einstein College of Medicine, 405 Ullmann Building, 1300 Morris Park Avenue, Bronx, NY 10461 (email: angelett@aecom.yu.edu).
Selective antibodies are powerful tools of experimental biology. Relatively straightforward immunoblotting experiments can be used to identify a polypeptide antigen in a novel cell or tissue or to study cellular processing of a precursor protein that is required for biologic activity or targeting. Immunoprecipitation or immunoaffinity purification techniques, often coupled to radiolabeling procedures, permit tracking of protein maturation and identification of partners in macromolecular assemblies. The repertoire of immunohistochemistry has been expanded from more classic light and electron microscopy to include confocal microscopy, which can permit analysis of antigens in fixed and in live cells. Antibodies themselves can be injected into cells to alter biologic processes. A carefully prepared, specific antibody can provide experimental breakthroughs. Antibodies prepared, borrowed, or used uncritically or without appropriate controls can be time-wasting and useless, or worse, can produce misleading results. In the following detailed discussion, references will be limited to specific examples or procedures, because the literature on immunodetection
is so extensive.
Natural proteins are perhaps the ideal antigens, providing sequence-specific and surface structural epitopes. However, natural proteins are rarely available in completely pure form, and antibodies often develop against contaminating polypeptides. Antibodies against natural proteins, particularly monoclonal antibodies, sometimes have exquisite specificities, recognizing only subsets of the immunizing protein, when a more general reagent was desired. Important biologic insights can be gained once these specificities are understood.1 However, it may take longer than anticipated to determine how these antibodies focus on a region of the protein, such as one with posttranslational modifications.
Recombinant fusion proteins produced in bacteria are also used successfully to produce antibodies. Some may suggest that recombinant antigens better reflect the protein itself, providing antibodies against three-dimensional and sequence determinants. They also do not require coupling to carrier proteins, and the proteins bound to affinity resins can be used to purify the antibody from crude sera. However, these polypeptides are not necessarily correctly folded, or they may have incorrect disulfide bond pairing. Some types of proteins, such as membrane proteins, cannot easily be produced in bacterial or eukaryotic expression systems. Recombinant fusion proteins are often considered easier to produce than peptide antigens, requiring only relatively accessible molecular biologic techniques and kit-type purification procedures of the protein antigen. In our laboratory, the quality of mass spectra of recombinant antigens purified by only one pass over such affinity columns suggests a misplaced confidence in these purification techniques.
Synthetic peptides can be prepared in highly pure form and coupled to carrier proteins, making it possible to elicit antibodies against proteins that may never have been isolated, identified only by sequences of cDNA or EST clones, or to target specific regions of more well-understood proteins, even regions modified on their amino acid side chains by phosphate, for example. Structural features useful for quantitation, crosslinking, and eventual antibody purification can be designed into their amino acid sequences. Proper antigen design can avoid mistakes that might lead to generation of cross-reactive or misleading reagents.
There is a place in a laboratory's armamentarium of immunologic probes for antibodies produced to all of the previously described types of antigens. However, many scientists may exclude peptide antigens from their experimental designs because of apprehension about deciding which sequence to synthesize, because of concerns about preparing a good conjugate for immunization, or because of discomfort from a previous bad experience. There may also be some concerns about cost, but a high-quality synthetic peptide can be made and characterized more quickly and less expensively than a recombinant antigen, particularly when the cost of the time of laboratory personnel is taken into consideration.
Several examples of sequence-targeted antibodies may be helpful. In experiments studying depolarization-induced exocytosis in PC12 pheochromocytoma cells, antibodies to the carboxyl-terminal sequence of dopamine and beta-monooxygenase, a catecholamine biosynthetic enzyme, were used to visualize the inner faces of secretory vesicles transiently exposed on the cell surface.2,3 Despite its modest titer, the specificity of this antibody enabled this unique application.
In another example, the prohormone endoproteinase furin is present in low concentrations in cells, making it difficult to detect by immunohistochemical methods. Antibodies directed to its C-terminal cytosolic tail were able to localize endogenous furin to the trans-Golgi network.4 It had previously only been possible to localize epitope-tagged furin in cells transfected with the appropriate construct.5 In another example using fluorescence confocal microscopy,6 anti-peptide antibodies to the organic anion transport protein (OATP1), the first in a new family of membrane transporters, readily localize this polypeptide to the basolateral (blood) face of hepatocytes excluded from the surfaces of the bile canaliculi. These useful and highly specific antibodies were all planned, taking into consideration the pragmatic issues discussed in the subsequent sections.
Before preparing a set of sequence-specific antibodies, a laboratory must carefully evaluate its short-term and long-term experimental needs. A library of well-designed antibodies will be useful for many years, and efforts made at the outset to anticipate present and future needs and to prepare and characterize the antigen itself constitute an important investment in a research program. Structural features, intended use, and synthetic concerns must all be thought through.
A peptide must be soluble under the experimental conditions in which it will be used. Although it is possible to couple antigens to carrier proteins in the presence of solubilizing detergents, they must be removed before injection of the conjugate into an animal. Peptides cannot fold like longer polypeptides, in which stretches of hydrophobic residues are buried within a three-dimensional structure. Because peptides are short, even a small cluster of hydrophobic residues can render them insoluble. Extremely hydrophobic peptides have been known to form gels in solution. The contributions of the alpha-amino group and the terminal carboxyl moiety to the hydrophilicity and solubility are greater than in proteins. N-terminal acetylation and C-terminal amidation are often used to preserve biologic activity or, for antigens, to make the peptide more closely resemble an internal protein sequence. Acetylation or "capping" of the amino terminus assists in proper folding of peptide sequences.7 Because these modifications diminish the solubility of synthetic peptides, the choices between structural concerns and solubility must be balanced.
Some sequences are problematic in solid-phase peptide synthesis or cleavage, and they are best avoided; these include Asp-Pro sequences or stretches of amino acids that require bulky protecting groups on their side chains during synthesis.8-10 The latter includes residues such as Arg, Asn, Cys, Gln, and His. Mass spectral analysis of the products is essential to verify that the desired peptide has been produced.11-13 Mass spectrometry helps identify whether there may be truncation or deletion products or peptides with oxidized Met or Trp or with an uncleaved side chain protecting group remaining, but it cannot reveal their relative abundance in the peptide mixture. If these byproducts are apparent in the spectrum, high-performance liquid chromatography (HPLC) analysis can reveal whether they are minor or major components of the mixture.14-16 Amino acid analysis is essential for accurate peptide quantitation and helpful for evaluating the presence of modified, deleted, or mistaken amino acids.17 However, the expense and time required for this vital technique have caused many laboratories to discontinue providing this service.
The presence of truncation peptides is not necessarily troublesome, because they have the correct amino acid sequence but are shorter. However, peptides with specific residues deleted or with undeprotected side chains could potentially be more potent antigens than the desired natural sequence. Although Cys residues are often added to peptides to enable crosslinking to a carrier protein, a peptide synthesized with many Cys residues present can be difficult to handle and may not lead to a useful antibody. Multiple Cys residues may lead to the formation of covalently linked aggregates. The Cys-rich regions of proteins may have some disulfide bonds. The linear peptide with reduced Cys would therefore not represent the protein itself, which would be more structurally constrained.
These discussions may make peptide antigen synthesis seem a daunting task, which is not the case. Thinking about these issues before starting the synthesis helps make the preparation of peptide antigens straightforward and seamless.
If there are no constraints on antigen choice for a cytosolic or soluble polypeptide or the rationale for choosing an epitope is ambiguous, a pragmatic approach to choosing a peptide sequence for immunization is to synthesize the N-terminal and C-terminal sequences of the protein. These sequences are often found to be solvent exposed and mobile in crystallographic structures of proteins. There may be a higher likelihood that an antibody prepared against these sequences will work well in immunoblot analysis and in analyses of native proteins by immunoprecipitation. When using an N-terminal peptide, conjugation to the carrier should be achieved through the carboxyl terminus, so that the peptide will mimic its position in the protein. Similarly, a C-terminal peptide is best conjugated at its amino terminus.
Computer algorithms are frequently used to select epitopes from predicted protein sequences. The most frequently used are those that predict predominantly hydrophilic and hydrophobic portions of polypeptides, helping identify solvent-exposed regions on the surface of the protein.18 Programs such as those provided by the Wisconsin Genetics Computer Group, commercial programs, and others available on the World Wide Web19 are helpful. However, protein folding is not entirely understood. Unless a prediction of surface residues is based on modeling of a known protein three-dimensional structure, these hydrophilicity plots are not experimental data but only useful guides. If possible, more than one of these sites should be chosen to produce antibodies specific to a desired protein. More information about which peptide sequences bind to class I and class II major histocompatibility complex molecules is also being discovered.20,21 This knowledge base may help future peptide antigen design.
It is possible that a desirable peptide antigen may share by chance a region of homology or identity with a sequence in a totally unrelated protein. This can lead to experimental findings that are misleading or that require much analytic work to understand.22 Searching several databases using Web-based protocols such as BLAST can help prevent the occurrence of this type of problem.23 Modifications to the standard search protocols are usually necessary when short amino acid sequences are used in the query and have been well described elsewhere.24
A major design concern about preparing sequence-specific antibodies is the choice of determinants common to a family of proteins or unique to one member of that family. This selection depends on the experiments a laboratory needs to perform. Specific and general reagents may be required. Programs located on the World Wide Web25,26 facilitate alignment of the amino acid sequences of multiple members of a protein family. Sequences or functional domains common to several related polypeptides can be highlighted. This alignment makes identification of sequences common to the same protein from multiple species straightforward, ensuring the general utility of the antibody reagent developed and enhancing its potential usefulness for discovering other related proteins. These same sequence alignments permit ready visualization of sequences that are found in only one member of a protein family or in one species of that family, aiding development of highly specific reagents.
Many common sequence motifs have been exploited during evolution to maintain specific protein functions. These can include helix-loop-helix sequences, GTP binding sites, RGD recognition motifs, SH2 domains, phosphorylation consensus sites, and others. Analyzing the predicted or known protein sequence by Web-based programs can exclude these regions as candidates for antigens.27 The programs that identify hydrophilic and hydrophobic regions within a polypeptide can help exclude the hydrophobic transmembrane regions of intrinsic membrane proteins. There are specific programs that predict these regions. Even if these sequences may elicit an antibody, the antibodies will not be as useful as antibodies to exposed portions of the membrane polypeptide.
Antibodies that discriminate between phosphorylated and unphosphorylated versions of a protein (or other modifications) can be powerful functional and positional markers in cell biology experiments.28 It is now relatively straightforward to synthesize phosphopeptides. Monoclonal antibodies directed against phosphorylated sequences can be readily prepared. Polyclonal antisera against phosphopeptides may contain antibodies to the unphosphorylated sequence, because some of the peptide becomes dephosphorylated after injection. However, use of peptide-affinity resins should permit purification of either or both antibodies to phosphorylated and unphosphorylated forms of the peptide or protein. Alternatively, monoclonal antibodies can be used to develop reagents specific for only one form of the peptide.28
If left with a set of peptide candidates, which ones should be chosen? If only one peptide will be made, the sequence that can provide the most straightforward synthesis and can be most soluble and easy to handle should be chosen. If funds for peptide synthesis, coupling, and immunization protocols are not limiting, at least two sequences, preferably from different regions of the polypeptide, should be chosen. Because epitope prediction programs are only partly based on experimental data, preparation of several antigens provides some assurance that the needs of a wide range of antibody applications can be met. Many proteins are processed proteolytically within the cell, often in a cell-specific manner. The availability of more than one sequence-specific antibody ensures that the protein can be universally detected and provides tools for studying in vivo processing of the polypeptide.
A special set of sequence constraints should be considered for peptides with hormonal activity. Endocrine peptides are generated from longer precursor polypeptides after cleavage by a specific set of proteolytic enzymes in a defined order, perhaps in a unique subcellular compartment. Many endocrine precursors are multivalent, capable of generating several different sets of biologically active peptides in different cell types or different physiologic conditions. Peptides synthesized based on the predicted cleavage products can be most useful. These peptides often have unique posttranslational modifications important for their biologic activity. One of the most common is C-terminal amidation, suggested by the presence of a C-terminal glycine just before the basic proteolytic processing site. Peptide amides and N-terminally acetylated peptides are straightforward to prepare but must be made during synthesis, not after.8-19 Biologic activity and antibody specificity can depend on these modifications. Particularly for endocrine peptides, a Tyr residue is often incorporated into a portion of the peptide synthesis to provide a labeling site for later radioimmunoassay development.
Other concerns arise when posttranslational modifications are suspected. Unless these issues are the topic of the investigation, initial attempts at antibody preparation should avoid regions rich with probable disulfide bonds or modified residues.
As a final precaution before initiating synthesis of an antigen, the sequence selected should searched through available databases to ensure that the sequence is unique and that the antibody reagent developed will not later be found to cross-react with an unrelated protein with a chance identity in a small region of its sequence.22 Peptide antigen design issues related to conjugation to the carrier protein are discussed in the following sections.
The length of an antigenic peptide is a point of some dispute. Most peptide antigens requested range in length from 12 to 16 residues and are relatively easy to synthesize. Peptides that are 9 residues or shorter have been effective antigens, but peptides longer than 12 to 16 amino acids may contain several epitopes. Peptides in excess of 18 to 20 residues begin to present more synthetic challenges. Although these can usually be dealt with by modifying the strategy of the synthesis itself and by careful monitoring, this entails more cost in materials and in the work of expert personnel on the part of the resource laboratory.
There is some disagreement about whether high-quality peptide antigens require purification. An excellent peptide preparation with no apparent trace of truncation or deletion products or partially deprotected peptides should be able to be used to conjugate to carrier proteins with no further preparation, except
for neutralization where necessary to facilitate the crosslinking reactions. Any salts and small molecules present do not affect antigen conjugate preparation. However, it is conceivable that a small amount of
an undeprotected peptide would be more immunogenic than the designed peptide, if only because the protected peptide would not be as readily degraded in vivo.
If significant amounts of peptide byproducts are present in the preparation, two choices can be made. The peptide can be purified before conjugation and immunization, identifying the correct HPLC peak by mass spectrometry.12-16 Alternatively, the peptide can be purified to later prepare a peptide column for purification of the specific antibody. However, excellent anti-peptide antibodies with high titer have been prepared against unpurified antigens conjugated to carrier proteins.
Conjugation of the Peptide
Three methods are routinely used to crosslink a peptide to a carrier protein.29,30 These procedures take advantage of the sulfhydryl, amino, or carboxyl functional group. The most common method is to couple the peptide through the sulfhydryl group of a Cys residue designed into the peptide sequence. Depending on the chemistry chosen, this conjugation method usually produces a biologically stable thioether bond. Amino group functionalities are easily crosslinked by glutaraldehyde or other reagents. Because Cys residues are less abundant in protein sequences than Lys residues, insertion of Cys sequences for coupling purposes less often interferes with peptide design. To couple by means of an added Cys residue, the peptides must be in the reduced sulfhydryl form to crosslink. Although most Cys-containing peptide antigens appear to be in the sulfhydryl form after cleavage from the resin, the oxidation state can be verified by mass spectrometry or by reaction with Ellman's reagent.13
Alternatively, the peptide can be reduced in solution or by solid-state methods before proceeding. Carboxyl groups can also be crosslinked to the side chains of Lys residues of carrier proteins to produce stable pseudopeptide bonds. However, this strategy is not as routinely useful, because the acidic Glu and Asp residues are common in predicted antigenic or soluble portions of polypeptides. This chemistry is not as specific and can easily involve unintended crosslinking sites.
The position of the reactive moiety should be considered. If it is desired to crosslink by the N-terminal alpha-amino group or the epsilon-amino group of an added Lys, there should be no Lys residues internal to the designed peptide sequence. The crosslinking reaction may favor the epsilon-amino group on the Lys side chain because of the differences in pK, and the peptide will be coupled to the carrier in the middle of its sequence. The amino acid residues near the Lys will be very close to the carrier and may be sterically hindered and not truly available as antigen. If Lys residues are located within the peptide sequence, it is wise to design a Cys residue at either end of the peptide. Similar arguments could be made for positioning the Cys residue for conjugation to the carrier. No Cys residues should be internal to the peptide sequence. After termination of the reaction, the conjugate must be dialyzed or otherwise desalted before use to remove EDTA, which is harmful to the animals.
Many laboratories successfully prepare anti-peptide antibodies using standardized procedures without characterizing the final peptide conjugate. However, other laboratories prefer to estimate or quantitate
the number of peptides conjugated to each carrier polypeptide. The methods available cover a range of technical difficulty and accuracy. A classic method uses a Tyr residue incorporated into the peptide sequence. A known amount of peptide can be trace labeled by radioiodination before coupling, and the calculated specific activity is used to estimate the amount of peptide crosslinked to a known amount of carrier protein. Alternatively, if the carrier protein has a different retention time by reverse-phase HPLC than the peptide, an estimate of the percent of crosslinking can be obtained by analytic HPLC separations. An aliquot of the crosslinking mixture is analyzed at zero time and at the end of the incubation period.
The reduction in the area integrated under the peptide peak at these two times provides an estimate of the percentage of peptide crosslinked. Certain crosslinking reagents release colored compounds as the reaction proceeds. A simple reading with the spectrophotometer permits quantitation of the amount of crosslink used and therefore of peptide coupled. The most accurate method for quantitation is amino acid analysis.17 If this technique is used, a norleucine, beta-alanine, or other quantifiable nonnatural amino acid residue should be incorporated into the peptide design in proximity to the coupling site. Amino acid analysis can identify the quantity of peptide chains by this signature amino acid. Each amino acid analysis system may require a different novel amino acid.
Carrier polypeptides range from natural proteins to synthetic polymers. The most commonly used carrier protein is keyhole limpet hemocyanin (KLH), a respiratory heme protein from a sea snail. This protein is commonly used because it is a large protein complex with no homology with vertebrate proteins. A rabbit immunized with KLH produces antibodies against it. However, this reactivity will not be revealed in immunologic examination of vertebrate tissue. Antibodies to be used in experiments with invertebrate species should be prepared with carrier proteins such as serum albumin or ovalbumin, which have no correlates in species from these phyla. Synthetic polymers provide a more immunologically neutral carrier, preferred by some laboratories. Vendors can provide carrier proteins treated to maintain solubility and preactivated for use in coupling to the sulfhydryls of cysteine residues.
The ability to purify a sequence-specific antibody provides extra assurance of the specificity of these precious biologic reagents. Competition studies with solutions of free peptide can be used to verify the specificity of the antibody in immunoblot or immunohistochemical experiments. However, some useful antibodies may have a high titer but an unexceptional affinity for the unconjugated peptide antigen. These peptide antigens cannot be used for competition at workable peptide concentrations. Antibodies with a high affinity but low titer can be more useful if enriched. A solution to both problems is to purify the antibody using a peptide affinity column. Affinity purification removes antibodies to the carrier protein.31 Purification also removes antibodies to bacterial or invertebrate pathogens, which may interfere with immunologic applications in invertebrate species, such as Drosophila melanogaster, Dictyostellium discoideum, and Caenorhabditis elegans. Peptide affinity columns can be prepared using cyanogen bromide-activated Sepharose-type matrices or specialized, commercially available media that are preactivated to react with amino or sulfhydryl groups.30,31 When using these purification procedures, it is important to test all fractions for biologic activity.
An alternative procedure for preparing a peptide affinity resin for antibody purification is to take advantage of the high affinity of avidin for the biotin molecule.31 If a portion of the peptide antigen is synthesized with an N-terminal biotin instead of N-terminal Cys or Lys, that peptide can be bound to an avidin or streptavidin matrix at high affinity. After washing, the peptide column is ready for use. When using this method, the biotin should be placed near or at the site of crosslinking of the peptide to carrier to mimic the conjugate used for immunization. If background contamination or stringent specificity is such a serious concern that it may not be sufficiently addressed using purified polyclonal antibodies, production of monoclonal antibodies should be considered as an alternative.
Coupling chemistry can also take into consideration future needs for preparing peptide surfaces for quantitative bioassays. Enzyme-linked immunosorbent assays have been routine for many years.32 Although simple adsorption of peptides to multiwell plates has often been used, the variability of peptide chemistry makes other approaches attractive. Because proteins usually bind better to the plastic surfaces, coating plates with a protein to which the peptide can be covalently crosslinked or can bind at high affinity presents an advantage. If one maleimide-activated carrier protein is used for antigen preparation (eg, KLH), another protein such as ovalbumin should be used to prepare conjugates for these assays, so that antibodies to the carrier protein do not contribute to the signal. Streptavidin can be bound to plate surfaces. Biotinylated peptides then bind with high affinity to the streptavidin-coated surface. The latter is a popular method for linking peptides to surfaces in biosensor instruments, which are sensitive measures of the kinetics of antigen-antibody and other binding reactions. Covalent crosslinking of peptides through amino, carboxyl, and sulfhydryl moieties is also possible for biosensor technology.33
Multiple Antigen Peptide System (MAPS) peptides provide an alternate approach to preparation of sequence-specific antibodies.34,35 MAPS peptides are synthesized or purchased as four or eight identical peptides on a core structure that can be dialyzed before immunization. Antisera produced to MAPS peptides do not contain antibodies to the commonly used carrier proteins, such as KLH, although they may contain antibodies specific to the MAPS core structure. However, these antisera should not cross-react with natural antigens.
The ease in use for the investigator requiring sequence-specific antibody places all responsibility for antigen preparation on the synthetic chemist in the resource laboratory. Although some laboratories have not found MAPS antigens to be as effective as conjugated linear peptides,36,37 others have found MAPS peptides to be an excellent alternative when no other antigen or immunization approach has produced an effective antibody.38-40 Few extensive comparisons have been carried out, probably because of the cost involved. Few laboratories report detailed analytic data for their peptides and conjugates, making comparisons difficult. The structures of MAPS peptides are a challenge to verify by mass spectrometry and HPLC, even in the most experienced laboratories.41 The goal of having a well-characterized antigen may sometimes be elusive. Nonetheless, MAPS antigens provide an important alternative to more conventional peptide-carrier conjugates.
When an antibody does not function as predicted, peptide design should be carefully reevaluated, as should immunization protocols and the original working hypothesis for the final experiment. Having the ability to purify the antibody or perform effective controls permits a laboratory to distinguish between unanticipated but meaningful reactivity and mere artifact. In our experience, most anti-peptide antibodies work well in immunoblot experiments, although not necessarily in immunohistochemical or immunoprecipitation experiments, confocal microscopy, or many other applications. Sometimes, an antiserum may have extensive nonspecific cross-reactivity or a low titer. When the problem can be identified, it may result from a peptide chemistry or design issue or from the biologic component of antibody production.
If the antibody was prepared against a sequence identified only from direct Edman sequencing, it is possible that the sequence represented a contaminating polypeptide and that the protein of interest had a blocked amino terminus. Alternatively, the cDNA sequence may be incorrect. In either case, the antibody would be directed against an irrelevant sequence. Another potential reason for lack of immunoreactivity is that this sequence in the cell has a posttranslational modification that is not identified by the antibody directed against the unmodified peptide. If the posttranslational modification involves proteolytic cleavage, it is possible that this sequence does not exist in the finished polypeptide. An antibody may also have been directed against a partially protected peptide or truncation peptide sequence that is more stable biologically but not present in the natural protein.
If the peptide antigen sequence was not searched through the available sequence databases, it may be possible that cross-reactivity or loss of specificity may result from a common sequence motif or an unfortunate random occurrence.22 The computer algorithms for predicting protein structure are not 100% accurate. A predicted solvent-exposed site may not be readily accessible. Use of more than one peptide antigen provides a better chance of obtaining antibody useful in all types of experiments.
Unanticipated cross-reactivity may be real and important. If so, a well-planned antibody production as described previously can help generate the proper controls or purify the antibody so that new hypotheses can be tested.
Antibody production involves a biologic component, usually a rabbit or mouse, which may have genetic variability among strains in the ability to respond to the peptide sequence.39,42 Anecdotally, it has frequently been found that the antiserum from only one rabbit works in immunohistochemical experiments, whereas the antiserum from another rabbit may function better in immunoprecipitation analysis. If possible, multiple animals should be immunized. Occasionally, for highly conserved antigen sequences, it may be helpful to use a different species. Use of several species is often helpful when several antibodies must be used in one experiment or when large amounts of antiserum are required. Secondary antibodies or reagents can distinguish rabbit, rat, goat, and chicken immunoglobulins.
Unsatisfactory antibodies can result from mishandling in a laboratory animal facility, where workers from many laboratories enter to inject and bleed the animals, typically rabbits. Even a veteran laboratory, well known for its meticulous work and useful antibody probes, may assign an educated but inexperienced laboratory member to physically handle the production of a new antibody. If this scientist is unfamiliar with animal laboratory procedures, such as tattooing and cage labeling and changing, his or her uninformed judgment can lead to the injection of another laboratory's animal. Such errors can go undiscovered, providing an antibody of mystifying reactivity. Animal facilities in which only professional workers familiar with all procedures inject and bleed the animals are far less susceptible to these problems.
Antibody production requires blood flow and trafficking through immune tissues, such as spleen and lymph nodes. In years past, immunization was initiated with a foot pad injection, which provided slow antigen release to a nearby set of lymph nodes. For humane reasons, this procedure is now rarely used. In this laboratory's experience of many years, inclusion of a subscapular injection in the immunization protocol in addition to subcutaneous and intradermal injections improves the likelihood of obtaining an antibody of high titer and good specificity. This may improve access to nearby lymph nodes and to the circulation.
Anti-peptide antibodies are excellent tools for biologic research and discovery. Amino acid sequences selected should provide soluble peptides, with determinants of required specificity, and structural features that facilitate peptide conjugation, antibody purification, control experiments, and various immunochemical applications. Advanced planning with the help of a checklist of properties (Table 1) can eliminate many of the uncertainties in preparation of these valuable reagents.
TABLE 1.
CHECKLIST FOR PEPTIDE ANTIGEN PLANNING
|
|
|||
| Peptide chemistry | Crosslink | ||
| Solubility | Amino | ||
| Length | Sulfhydryl | ||
| Free carboxyl or amide | Other | ||
| Free amino or acetyl | Quantitation | ||
| Challenging chemistry | Carrier | ||
| Multiple Antigen Peptide | |||
| System (MAPS) | |||
| Sequence selection | Assays and purification | ||
| N-terminal | Tyrosine | ||
| C-terminal | Biotin | ||
| Membrane | Crosslink | ||
| Motifs | |||
| Modifications | |||
| Alignment, common | |||
| Alignment, unique | |||
| Database search | |||
|
|
|||
Work was supported by the Albert Einstein College of Medicine, by National Institutes of Health program grants to the Albert Einstein Cancer Research Center (P30-CA13330), by the Diabetes Research and Training Center (P60-DK20541), and by a grant from the March of Dimes Foundation (FY98-0195). The author thanks Dr. Barbara Birshstein for helpful discussions.
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