Antagene Inc. offers free antigenicity analyses for the selection of antigenic peptides and provides assistance on designing peptides with respect to protein homology, antigenicity, hydrophilicity, surface probability, and synthetic suitability. Provide assistance on conjugation strategies.

We offer a low price on peptide synthesis at $12/AA for small scales (15–20 mg); these prices include HPLC and Mass spectrometry analyses. Post-synthesis peptide modifications such as phosphorylation, cyclization, terminal amidation, Farnesylation, sulfation, etc. are extra. Peptides of high purity (>80%) can be synthesized at extra cost.

Per Animal Minimum Recommended

Chickens 2.5mg - 5.0mg

Rabbits  2.5 - 5.0mg

Goats 5mg - 10mg

Sheep 5mg - 10mg

The amount of antigen required varies from a minimum to a maximum when additional injections might be needed to boost antibody production or to continue a maintenance program longer than the stated time. The ideal concentration is 1 mg/ml within PBS or other physical buffer, the purer, the better. Please ship it in dry ice.

Antagene Inc offers various conjugation options for individual investigators. A range of carrier proteins selection is available including KLH, Ovalbumin, BSA

We provide the options of antigen (peptide, protein, biologic, or small molecule) or preferred species (rabbit, chicken, mouse, or other big animal) in one of our 70-day conventional protocols for the production of high-quality antisera.

A Proven Protocol to give an anti-peptide response within ten weeks Antagene, Inc. guarantees a 1:10,000 ELISA titer in the last production bleed, or we will re-do the protocol for you for free.

Standard Protocol for 70 days with 4 immunizations, pre-immune bleeds (3-5 ml per rabbit), and final production bleeds (50 ml per rabbit).

Projects can be extended on a monthly basis for a nominal charge for booster injections and the care and handling of animals.

We can purify antibodies for various applications; some of the common procedures are Protein A, G, or L purification. 

Peptide affinity chromatography for specific anti-peptide antibodies. 

Selection of mono-specific polyclonal antibodies by successive batch elutions over multiple affinity matrices. 

Selection of phosphopeptide antibodies by selectively isolating phosphospecific antibodies from the phosphopeptide affinity column.

At Antagene Inc., we do anti-peptide ELISA by coating peptides coupled to a carrier protein other than that used for immunization using our optimized ELISA system. In this way, antibodies to carrier proteins are not detected. We perform ELISA on individual bleeds along with pre-immune serum at 3 dilutions (1:1K, 1:10 K, and 1:100K). We report ELISA results and titer values in a tabulated, easy-to-understand format.

Selection of phospho-peptide antibodies characterized by ELISA using phospho and dephospho peptides at 6 dilutions The Phospho-specific antibodies are later isolated by sequential affinity purification on dephospho and phosphopeptide affinity matrices.

The detailed methodology for ELISA is provided with each antibody progress report for easy reproduction of results in the investigator's laboratory if needed. All ELISA reagents are also available from Antagene, Inc.

Antagene Inc. can also characterize your antibodies by Western Blot analyses using antigens supplied by the investigator. The blots will be scanned on a high-resolution scanner. The digitized images will be sent electronically, and the digitized images will be saved on CD-ROM for permanent storage and shipment.

At Antagene Inc., We do all steps involved in custom antibody production in-house, which allows total control over project progress and allows investigators to have direct communication with our scientific staff.

Antibodies are designed to detect antigenic epitopes with high specificity on proteins in their native environment, and in order to do so, antibodies must have access to the part of the antigen against which they are raised. In a natural environment, proteins have a three-dimensional globular structure; some of the antigenic epitopes may be buried inside the protein and will be completely inaccessible to the fairly large antibody molecule. Due to its large size, antibody molecules may not be able to penetrate the protein matrix. In order for these antibodies to be useful in protocols where antigens have to be recognized in their native states, i.e., immunohistochemistry, flowcytometry, confocal microscopy, and native immunoprecipitation, the antibodies must be raised against an epitope that naturally lies exposed on the target proteins.

To raise an antibody that can bind native protein, the following criteria must be met for peptide selection:

1. The peptide sequence has to be unique and not conserved in any known protein.

2. The peptide must be selected from an accessible region of the protein if the resulting antibody is to be of use in immunohistochemistry, confocal microscopy, or native immunoprecipitation protocols. The most accessible areas on protein molecules are those that are hydrophilic and are exposed on the outside of the structure. As these regions are generally hydrophilic and are in contact with an aqueous environment.

3. The peptide should also adopt a conformation that mimics its shape when contained within the protein.

4. Finally, the peptide must be immunogenic. These parameters are generally selected with the aid of computer programs that can predict various functional domains based on the primary structure of the proteins.

The first step is to determine which parts of the protein are on the outside and thus available for antibody binding. Using hydrophilicity and hydropathy plots as a starting point, the protein can be mapped for its orientation in the natural environment. The hydrophilicity programs assign a "hydrophilic index" to each amino acid in a protein and then plot out a profile. The regions of hyrophilicity can then be seen. The hydropathy plots then determine the hydrophobic and hydrophilic regions. These programs will also predict the transmembrane domains, signal peptides, protein kinase sites, signal sequences, and proteolytic cleavage sites. There have also been attempts to produce algorithms to predict flexibility and secondary structure, parameters that may be important in antigenicity.

There is data available that suggests that longer peptides have a greater conformational similarity to the native protein and are therefore more likely to induce antibodies that recognize the natural protein.  There is also data to suggest that a single antigenic determinant (i.e. the smallest immunogenic peptide) is between 5 and 8 amino acids.  Consequently, a peptide length of 15-20 amino acids is preferable as it should contain at least one epitope and adopt a limited amount of conformation.  Other limitations that needs to be kept in mind while designing an antigenic peptide are:

1. Peptide selected must be synthesizeable, there are some sequences that are very difficult to

    put together (multiple hydrophobic amino acids in a cluster). 

2. The peptide should be readily soluble in an aqueous buffer for conjugation and use in

    biological assays.  If a hydrophilic region has been selected then peptide solubility should not

    be an issue.  However, even these regions may contain hydrophobic residues (e.g.

    tryptophan, valine, leucine, isoleucine and phenylalanine) and, if there is a choice, select a

    peptide with as few of these residues as possible.  Multiple glutamine is also avoided if

    possible since gutamine may cause insolubility due to its tendency to form inter molecular 

    hydrogen bonds.    

3. A cysteine in the selected sequence is useful for conjugation, however, if there are two  

    cysteines present, disulphide bonds may form inter- and intra-disulfide bonds.  We have seen

    that cyclic peptides have better antigenic response and longer half-life in animals compared to

    their linear counter part (Farooqui et al., J. Neurochemistry 57, 1363-1369, 1991).  However,  

    even for cyclic peptides conjugation to a carrier protein is necessary to render the peptide

    immunogenic and is covered in a later section.  A terminal cysteine is recommended for

    optimal conjugation of peptide with carrier proteins by a bifunctional cross linking agent such as

    MBS.

4. Tyrosine and proline are two important amino acids to consider for peptide selection criteria. 

    Proline can adopt a cis-amide bond structure (normally in peptides amide bonds are trans)

    consequently; it gives the peptide a bend that may mimic closely the shape of the peptide in

    the protein.  Normally, peptide chains tend to be random in structure and the introduction of a

    proline can induce structural motifs thereby enhancing its potential as an immunogen.  Tyrosine

    serves two purposes; firstly it is a large amino acid with a ring structure that again can induce

    structural motifs to enhance immunogenicity. Secondly, it can be used to couple the peptide to

    a carrier using bis-diazotised tolidine, or alternatively it could be labeled with iodine to monitor

    the coupling efficiency with carrier proteins (Farooqui et al., J. Neurochem. 57, 1363-1369,

    1991). 

5. The peptides should be amidated if it is not from the C-terminal region.  The C-terminal peptides should be used as carboxyl group to mimic natural existence.  If C-terminal peptide is a site for  

    lipid modifications (most of the G-proteins) then such lipid modifications should be made to the

    peptide before coupling to carrier protein.    

6. Protein regions that may be modified in natural proteins, such as glycosylation sites, protein

    kinases sites, cleavage sites should be avoided as any antibodies raised to these sequences

    may not recognize the modified native protein. 

7. Certain structural motifs with high mobility (high temperatures) in proteins are better antigenic   

    regions, however, not enough data is available to base selection of all peptides on this criteria. 

8. Finally, epitopes from transmembrane regions should be avoided as they may not be

    accessible and will have high sequence homology with proteins having similar structural motifs

    and orientation in the cell. 

    These selection processes will finally limit the antigenic epitopes to 2 or 3 peptides.  If

    possible at least 2 and preferably 3 peptides should be selected and synthesized.  This

    greatly increases the chance of peptide being successfully raising antibodies that will

    recognize native proteins.

    It is important to establish the integrity and purity of the peptides, and their amino-acid

    composition and molecular weight prior to use.  One of the most important aspects in raising

    good quality peptides antibodies is the purity of peptides.  The impurities in the peptides,

    incomplete synthesis and unprotected side groups will greatly influence the quality of the

    antibody response.

Normally, a good antigen is a large, complex molecule with a molecular weight greater than 10 kDa that, when injected into an animal, is able to promote a good immune response and induce high levels of specific antibodies. In contrast, peptides are small molecules, typically with a molecular weight ranging between 1000 and 2000 Daltons. Some peptides, when emulsified in adjuvant, are able to elicit a poor immune response. These molecules are called haptens. Immune response that results in a high level of antibody production and requires the stimulation of T cells to induce the B cells that recognize the antigen. 

Generally, haptens or emulsified peptides do not elicit such robust responses. One of the probable explanations is that at least two different "epitopes" are required within the antigen: one to stimulate the T cells, the other the B cells. A small peptide may not be large enough to contain two clear epitopes. In order to create multiple epitopes on the small peptides, peptides are coupled to a larger carrier molecule (e.g., keyhole limpet hemocyanin, bovine serum albumin, ovalbumin, etc.) that is inherently immunogenic. The T and B cells now have a whole range of "epitopes" to react to that result in the production of antibodies to both peptides and the carrier proteins. 

The immune system responds to the hapten-carrier conjugate as if it were a single molecule and makes antibodies against peptides as well. The proportion of antibodies made to the peptide is small compared to the overall response but is far higher than with the peptide alone. The drawback of this technique is that there will be high levels of anti-carrier antibodies produced, which may have to be removed to make the reagent usable. Using carrier proteins that are not found in the specimen to be analyzed with these antibodies generally solves such problems. An example of such a carrier protein is Keyhole limpet heamocyanin.

The next step is to covalently link the peptide to the carrier protein. This is not a random process and can be finely manipulated to ensure that the peptide is bound in a known orientation. 

The reagents used to link the peptide to the carrier are heterobifunctional, meaning that they have a reactive group at each end of the molecule that can cross-link proteins. These reagents can be used to link the peptide in a particular way to achieve an antibody that reacts with a particular part of the peptide. For example, a peptide common to two proteins can be used to generate two different antibodies depending on how the peptide is coupled to the carrier protein. If the C-terminus of the peptide is coupled to the carrier, the likelihood of cross-reactivity to this region is reduced simply because it is now "hidden" by the conjugating agent. where N-terminal conjugation will allow the C-terminal portion of the peptide to become more antigenic. 

The ratio of peptide to carrier has been the subject of much debate. Hapten carrier ratios of around 5:1 appear to give the best antibody response, which corresponds to about 5–25 molecules of hapten per 50,000 daltons of carrier protein. If feasible, a variety of carriers and/or coupling agents should be used so that the peptide is presented in a variety of ways to the immune system. This will increase the chances of generating an antibody with the desired characteristics. There are numerous reagents for cross-linking proteins; however, at Antagene Inc., there are four that are commonly used for the production of peptide antibodies.

The water-soluble sulfo-SMCC is a heterobifunctional cross linker, allowing conjugation of peptides, proteins, and ligands that have free sulfhydral groups to the activated amino groups on the carrier proteins. This type of conjugation creates a flexible covalent bond that presents the antigenic peptide in its native form as it is present on the larger protein. We highly recommend this coupling for the generation of Phospho-specific antibodies.

This is the most widely used cross-linker for making peptide antibodies at Antagene Inc. The MBS will link peptides via the SH group on cysteine to the NH2 groups. This is a widely used reagent due to the fact that it unequivocally links the peptide through a specific cysteine residue. Cysteine can be included in the peptide chain either at the N or C terminus; both positions generally give similar antibodies to the peptide.

Carbodiimides make a covalent bond between free carboxyl and amino groups, whether C- or N-terminal or on side chains (i.e., lysine, aspartic acid, or glutamic acid), to form amide bonds. Amide bonds are extremely rigid. This can cause considerable steric hindrance as the peptide is tightly bound and unable to rotate.

Bdt will link peptides via the aromatic side chain of tyrosine and, to a lesser extent, histidine, to the same residues on the carrier proteins. This linker is a large molecule that provides an arm between peptide and carrier that may result in an enhanced antibody response due to the increased accessibility of the peptide.

Glutaraldehyde cross-links the primary amino groups on the peptide to those on the carrier protein. The primary amino groups are at the N-terminus of the peptide and/or the epsilon amino group of Lysine. So conjugation using glutaraldehyde will usually result in an N-terminally coupled peptide. The linkage formed by glutaraldehyde is such that there is a degree of flexibility between peptide and carrier. This will reduce the possibility of steric hindrance (interfering with access to the immune system) and result in a better response.

However, the agent is rather non-specific and can couple at various places in the peptide, resulting in several alternative conjugates that may give rise to a variety of antibody responses. This can be advantageous as it presents multiple alternatives to the immune system.

Alternative Systems

In addition to this classic approach to antibody production using peptide conjugates, there are now several novel systems currently being evaluated that do not require a carrier to be used. The MAP synthesis requires the use of poly-lysine resin, which was developed by using the two primary amine groups on lysine on a beta-alanine backbone. Normally, peptide synthesis occurs by coupling through the N-terminal amino group, but by using the side-chain group as well, a branching poly-lysine molecule can be built up. In brief, a single lysine is attached to the resin support. This has two sites for further reaction. Lysine has now been added to these two sites. As each lysine has two sites, there are now four sites available for coupling. Subsequent steps give 8 and finally 16 available sites for coupling.

The peptide is then synthesized at the ends of these branching lysines, and when it is cleaved from the resin, it results in a molecule containing 16 copies of the peptide. As its molecular weight may now be 20–30 kDa, it is immunogenic in its own right and does not require conjugation. Such an approach results in a higher proportion of anti-peptide responses as there are no contaminating antibodies to the carrier proteins.

It is important to have an assay system that will determine whether peptide antibodies will recognize the native proteins. Synthetic peptides may or may not represent the same structural configuration as they would exhibit in native proteins, and therefore a sensitive assay to test for anti-peptide antibody reactivity towards the protein will determine whether the antibodies will be useful for immunoassays. An ELISA system is the simplest method for determining antipeptide activity. Some problems may occur in the adsorption of some peptides to the plate. If the peptide binds to the matrix using the same amino acids that are recognized as antigenic epitopes by the antibody, then the antibody may not bind the immobilized peptide, resulting in a false-negative observation. Antagene Inc. has developed an extremely sensitive ELISA assay, especially for peptides that pose these issues.

After establishing antibody reactivity to a peptide, it should be screened for its ability to recognize the native protein. The choice of assays is generally dictated by the final use of this antibody in the investigator's laboratory. For example, if it is to be used in Western blotting experiments, then it should be screened by immunoblotting protocols. The final screening requires the ability of our antibodies to recognize antigen in native confirmation; such antibodies are useful for applications in immunohistochemistry, confocal microscopy, and immunoprecipitation experiments.