Phospho-specific Antibodies Protocol: 

Phospho-Specific Antibody development requires careful selection and design of peptide that will be subsequently used to generate antibodies.  We provide complete proteomics analyses of your protein of interest for selection of antigenic peptides at no cost to you.  Our peptide selection criterion is based on theoretical modeling of protein for determination of application-specific antigenic determinants.  Antibodies raised against such peptides are able to detect antigenic epitopes with high specificity on proteins in their native environment.  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.  The large size of the antibody molecule will produce sterric hindrance that will not allow proper recognition of antigenic determinants that are not exposed and buried inside.  Due to its large size, antibody molecule may not be unable to penetrate the protein matrix.  For antibodies that will be used in applications that require detection of antigens in their native or semi native state (confocal microscopy, immunohistochemistry, flowcytometry, and native immunoprecipitation), the antibodies must be raised against an epitope that naturally lies out side the target protein.  By careful selection of antigenic peptides that are highly antigenic and naturally lie exposed on the surface of the protein allow us to make antibodies that are function specific. 

In order for the 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 found in any other 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.  Generally the exposed areas on the protein are hydrophilic and are generally in contact with an aqueous environment. 

(iii)               The peptide should not be more than 14 amino acid long as longer peptides provide additional epitopes that may not represent phospho peptides.  Such antigens will make pan antibodies to the native proteins that will be hard to remove by affinity purification. 

(iv)              The peptide should also have phosphorylation sites that lie in the middle of the peptide as this present antigenic determinants that are phosphorylated. 

(v)                Formation of cyclic peptides also enhances the quality of antibodies (Farooqui et. al., 1991).

(vi)              The final selection criteria also requires that these peptides should adopt a conformation that mimics its shape when contained within the protein (presence of proline in peptide suggest a kink in peptide structure). 

(vii)             Finally, the peptide must be immunogenic and should be devoid of any post-translational modifications (glycosylation, methylation, signal peptides etc.).

Sequence Selection:  We suggest that a peptide length of 12-15 amino acids is preferable as it should contain at least one epitope and adopt a limited amount of conformation. Although longer peptides have a greater conformational similarity to the native protein and are therefore more likely to induce antibodies that will recognize the natural protein but phosphospecific antibodies will then represent a very small fraction of the antibodies.  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. 

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 pu ty.  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 and Immunization:  Proteins with MW greater than 10 kDa are generally good immunogens to promote a robust immunogenic response.  However, peptides are small molecules (Haptens; MW range from 1000 to 2500 dalton) and are required to be conjugated to larger proteins () in order make them immunogenic.   Heptens when emulsified in adjuvents elecit poor immunogenic response. 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 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. 

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.

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.

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. 

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.  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.  

Selection of Phospho-specific antibodies:  In order to affinity purify phospho-specific antibodies from pan antibodies, multiple affinity matrices are required.  Imunization of rabbits with antigenic peptide produce three different type of antibodies, (i) Phospho-specific antibodies, (ii) non-phospho antibodies to the same epitope that generate phospo-specific antibodies.  This is mainly due to the fact that endogenous phosphatases some times dephosphorylates the phospho peptides, (iii) antibodies to other epitopes that do not involve phospho-peptides, generally termed as “pan antibodies”.   We isolate phospho-specific antibodies from non-phospho and pan antibodies via a sequential affinity chromatography over phospho and dephospho affinity matrices.  In the first pass, the phospho and pan antibodies are specifically eluted from the phospho-peptide matrix.  The flow through fraction continued other antibodies (irrelevant IgGs) and antibodies to non-phosphorylated region of the antigen.  The eluted fraction (containing phospho-and pan antibodies) are then applied on a non-phophorylated peptide column that provides the antigen for pan antibodies.  The phospho-antibodies are eluted in the flow through fraction.  The pan antibodies can also be separated from the column if they are present.  The Other methods for separation of phospho antibodies requires specific elution of phospho-antibodies that are bound to phosphorylated antigen immobilized on nitrocellulose membranes.  This technique, although give high purity antibodies, with yield is in ug quantities.