Custom Antibody Production Services Summary

FabGennix Int. Inc. An Antibody Company

“Our services are flexible to the demands and needs of individual investigators”

Some of our custom peptide antibody related services are outlined below:

Antigenic Prediction

¨       FabGennix Inc. offers free antigenicity, BLAST, and hydropathy analyses for the selection of antigenic peptides.  The results of these analyses will be sent to you in an easy to understand graphical format.  FabGennix Inc. performs an exhaustive proteomics analyses on the selected sequences before starting the antibody protocol.  The details are outlined below.   

¨       On-line protein database access (We access a number of protein and nucleotide data bases available on the world wide web).

¨       Expert selection of peptides (See antigenic prediction criteria)

¨       Advice on conjugation strategy

Synthesis of peptides

¨       We offer fixed prices with no set up fee on peptide synthesis at 90% purity, these prices include HPLC purification, gradient elution, Mass Spec analyses and UV scans.  Post synthesis peptide modifications such as phosphorylation, cyclization, terminal amidation, Farnesylation, sulfation etc. are extra.  Peptides of higher purity <98% can be synthesized at extra cost.

Conjugation

¨       We offer various conjugation strategies, the chemistry for these coupling reactions have been optimized for best results.

¨       A range of carrier proteins selection is available including KLH, Ovalbumin, BSA)

Immunization

¨       Proven protocol to give an anti-peptide response in as little as ten weeks.  FabGennix Inc. guarantees a 1:1,000 ELISA titer in the last production bleed or we re-do the protocol for free. 

¨       Standard protocol for 65 days with 5 immunizations, 2 test bleeds (3-5 ml) and two production bleeds (17-20 ml).

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

Purification

¨       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 Phospho-peptide antibodies by selectively isolating phosphospecific antibodies from phosphopeptide affinity column.  

Screening

¨       At FabGennix Inc. we do anti-peptide ELISA by coating free peptides or peptides coupled to a carrier protein other than 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 5 dilutions ( 1:10 , 1:100, 1:1K, 1:10 K and 1:100K).  We report ELISA results and titer values in tabulated, easy to understand format.

¨       Selection of phospho-peptide antibody characterized by ELISA using phospho and dephospho peptides at 5 dilutions.  The Phospho-specific antibodies are later isolated by sequential affinity purification on dephospho and phospho peptide affinity matrices.   

¨       The detailed methodology for ELISA is provided with each antibody progress report for easy reproduction of results in investigators laboratory if needed.  All ELISA reagents are also available from FabGennix Inc.

¨       FabGennix can also characterize your antibodies by Western Blot analyses using antigens supplied be 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 FabGennix Inc. We do all steps involved in custom antibody production in “house” that allows total control on project progress and investigators can make direct communication with our scientific staff.  

Antigenic Prediction

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 natural environment proteins have 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 molecule may not be unable 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:

(i)                  The peptide sequence has to be unique and is not conserved in any known protein.

(ii)                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 for 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. 

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

(iv)              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 primary structure of the proteins. 

Computer Analysis:  The first step is to determine which parts of the protein are on the outside and thus available for antibody binding.  Using a hydrophilicity and hydropathy plots as a starting point the protein can be mapped for its orientation in 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. 

Sequence Selection:  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:

(i)                  Peptide selected must be synthesizeable, there are some sequences that are very difficult to put together (multiple hydrophobic amino acids in a cluster). 

(ii)                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.    

(iii)               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. . 

(iv)              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). 

(v)                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.    

(vi)              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. 

(vii)             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. 

(viii)           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.  FabGennix does not recommend material less than 85% pure to be used for antibody production (FabGennix Inc., uses peptides with 90% or more purity for antibody production).  The peptides must be purified on RP HPLC and eluted with salt gradient to achieve higher purity.  The HPLC chromatograms, Mas spec and UV spectra and other physical characteristics of the peptides will be provided to the investigator on first shipment along with preimmune and first test bleed.      

Conjugation

Normally, a good antigen is a large, complex molecule with a molecular weight greater than 10kDa and when injected in an animal is able to promote a good immune response and induce high levels of specific antibody.  In contrast, peptides are small molecules, typically with a molecular weight ranging between 1000-2000 Daltons.  Some peptides when emulsified in adjuvant are able to elicit poor immune response.  These molecules are called haptens.  Immune response that results in a high level of antibody production, that requires the stimulation of T cells to induce the B cells that recognize the antigen.  Generally haptens or emulsified peptide 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 haemocyanin, bovine serum albumin, ovalbumin etc.) that are inherently immunogenic. The T and B cells now have a whole range of "epitopes" to react to that result in production of antibodies to both peptides and the carrier proteins.  The immune system responds to the hapten-carrier conjugate as if it were as a single molecule and in so make antibodies against peptide as well.  The proportion of antibody made to the peptide is small compared to the overall response but is far higher than with peptide alone.  The draw back for this technique is that there will be high levels of anti-carrier antibody produced which may have to be removed to make the reagent useable.  Using carrier proteins that are not found in the specimen to be analyzed with these antibodies generally solves such problems.  An example of such 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 in two proteins can be used to generate two different antibodies depending upon 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 as 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 FabGennix Inc. there are four that are commonly used for the production of peptide antibodies. 

Sulfo-SMCC conjugation:
The water soluble sulfo-SMCC is a heterobifunctional cross linker allowing conjugation of peptides/proteins/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 the native form as present on the larger protein.  We highly recommend this coupling for generation of Phospho-specific antibodies.  

m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS):  This is the most widely used cross linker for making peptide antibodies at FabGennix Inc.  The MBS will link peptides via the - SH group on cysteine to - 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 position generally gives similar antibodies to the peptide. 

Carbodiimides (CDI):  Carbodiimides makes 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.

Bis-diazotised tolidine (Bdt):  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:  Glutaraldehyde cross-links 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 so result in a better response.

However, the agent is rather non-specific and so can couple at various places in the peptide that result in to several alternative conjugates which 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 prodution 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 that was developed by using the two primary amine groups on lysine on a beta alanine back bone. 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 is now added and couples to these two sites.  As each of the lysine has two sites, there are now 4 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, results in a molecule containing 16 copies of the peptide.  As its molecular weight may now be 20-30kDa, it is immunogenic in its own right and does not require conjugation.  Such an approach results in a higher proportion of anti-peptide response as there are no contaminating antibodies to a carrier proteins. 

Antibody Screening (titer and reactivity to native 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 to detect 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 anti peptide activity.  Some problems may occur in the adsorption of some peptides to the plate.  If the peptide binds to the matrix using same amino acids that are recognized as antigenic epitopes by the antibody, then the antibody may not bind the immobilized peptide resulting in to a false negative observation.  FabGennix Inc., has developed extremely sensitive ELISA assay specially for peptides that pose these issues.

The purity of the peptide is important for screening antibodies.  At FabGennix Inc., we use peptides that are 85% or better in purity and represent an insignificant contamination with incomplete or deleted sequences.  The presence of deleted sequences and other impurities in the peptide will result in false positive titer values.  In some cases during peptide synthesis there is incomplete removal of side chain protecting groups, which act as antigenic determinant.  The presence of such group also led to false positive observations in ELISA tests.   At FabGennix Inc. we use peptides that are full length and free from side chain protecting groups. 

After established antibody reactivity to 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 investigators 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.