The Use of Peptides As Antigens

AnaSpec, Inc.

Solid phase peptide synthesis (SPPS) method, pioneered by Bruce Merrifield (1) makes peptide synthesis relatively easier; especially when automated SPPS became available. A significant corollary development is the growing convenience of making antibodies towards whole proteins, yet using short synthetic peptides as antigens. Thousands of antibodies have been raised using synthetic peptides and even more are on the way.

This article presents a brief introduction to researchers who may be considering custom antibody production from synthetic peptides. It is not meant to be exhaustive in its scope. For this, an excellent review can be found in Ed Harlow and David Lane’s “Antibodies, A Laboratory Manual” (2).

Advantages
Raising antibodies from synthetic peptides is in general quicker and simpler than using a (recombinant) protein. Once the sequence is available, the antibody can be raised immediately according to the predicted amino acid sequence. It also gives the flexibility to target a certain region of a protein, resulting in specific epitope recognition (2). One major disadvantage is the possibility that it may not recognize the native protein (2).

How to Choose a Sequence?
In order to raise good antibodies from peptides, one has to choose a sequence that shows good antigenicity (for obvious reason), hydrophilicity (for ease of peptide synthesis, specifically peptide purification) and surface exposure (for antibody recognition and accessibility). Different computer programs, such as MacVector (Cary, NC), are available to assist one in choosing an antigenic peptide sequence based on these criteria. Generally, one epitope consists of 5-7 amino acids; and a 10-15 residue long peptide is adequate to raise good antibodies.

What Sequence to Avoid?
1. Some sequences to avoid include sequences with a stretch of consecutive hydrophobic amino acids such as Leu (L), Ala (A), Gly (G), Ile (I), Val (V), Phe (F), Trp (W) and Met (M). The number of hydrophobic amino acids should preferably not exceed 50% of the sequence, with 25% or less being ideal. A hydrophobic sequence makes the peptide insoluble, resulting in peptide purification difficulties.

2. Acidic peptides containing Cys will pose difficulty in purification, since the use of a basic solvent in purification will cause cysteines to dimerize.

3. Multiple cysteines-containing sequences have to be avoided as well, especially when the conjugation strategy is to use a single thiol group of the Cys for conjugation to carriers. One solution is to use Acm (Acetamidomethyl) -protected Cys for the internal cysteines that are not going to be used for conjugation.

4. N-Terminal glutamine (Gln) tends to cyclize to pyroglutamic acid (Pyr), substituting Gln (Q) with Pyr, removing Gln from the N-terminus, adding another amino acid at the N-terminus or acetylating the N-terminus are some of the viable alternatives.

5. Met tends to oxidize to methionine sulfoxide [Met(O)], norleucine (Nle) may be used instead.

After a sequence is selected, it is always a good idea to check if the sequence is unique for the protein of interest, i.e. it is not found in another protein. A website such as http://www.ncbi.nlm.nih.gov/blast/Blast.cgi is useful for this purpose.

Conjugation to Carriers Proteins
With the right conjugation strategies, a sequence with good antigenic index will induce a good immune response. Many peptides contain B-cell epitope, but not T-cell epitope, and may therefore not be immunogenic. Consequently, a peptide conjugated to carriers such as keyhole limpet hemocyanin (KLH) is commonly used in order to elicit an immune response. BSA can also be used as a carrier. However BSA is also a good immunogen (2) and since it has high nonspecific binding, it can be put to better use as a capture antigen when determining antibody titer in ELISA. For example, when using a KLH-conjugated peptide as an immunizing peptide, one can screen the antibodies using the same peptide conjugated to BSA. This way, the antibody titer obtained is from the immunizing peptide and not from KLH. Other carriers used include Ovalbumin and RSA (rabbit serum albumin).

The most common linker used for conjugating carrier proteins to peptides is sulfo-SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Sulfo-SMCC links the carrier protein to the peptide by forming a bond between the maleimide group and the peptide Cys group. Cys is usually introduced on either the N- or the C-terminus. If a sequence is derived from the N-terminus, then Cys goes on the C-terminus, and vice-versa. If the peptide sequence is derived from an internal sequence, Cys can go on either end. For sequences with multiple cysteines, all except one Cys (towards the N or C terminus preferably) must be protected (usually with Acm). Other conjugation methods include the use of EDC (for conjugation to free carboxyl groups) and glutaraldehyde (for conjugation to free amino groups).

Applications
Antibodies are used in different applications such as western blots, immunoprecipitations, immunohistochemistry and flow cytometry. Below are some examples of how different antibodies were used in research studies: Western Blots and IHCs of Sarcoglycan Subunits Antibodies.

In a paper (3) reported by Dr. Yiu-mo Michael Chan, antibodies each specific to the different subunits of the sarcoglycan (SG) protein were raised. “Some of the SG subunits share significant sequence homology,” AnaSpec, the custom antibody provider “did a good job in selecting the right sequences for generating peptides.” To confirm specificities of the antibodies, he performed an experiment where COS-1 cells were transfected with different SG subunits (, , , , and ). Total cell lysate (20ug) was then analyzed by Western blots using antibodies raised against each of the different subunits (See Figure 1, panel A). Immunohistochemistry (IHC) results are shown in panel B of Figure 1 (western blot and IHC data courtesy of Dr. Yiu-mo Michael Chan, Carolinas Medical Center, Charlotte, NC).

Figure 1. Specificity of anti-sarcoglycan antibodies (Panel A). COS-1 cells were transfected with -SG (lane 1), -SG (lane 2), -SG (lane 3), -SG (lane 4), -SG (lane 5), and -SG (lane 6). Total cell lysate (20ug) was analyzed by Western blots using antibodies (from AnaSpec) against -SG (ANA-), -SG (ANA-), -SG (ANA-), -SG (ANA-), -SG (ANA-), and -SG (ANA-). Note that the ANA- and ANA- antibodies recognized the corresponding recombinant sarcoglycans and did not cross-react with other sarcoglycans. Actin was used as a loading control. Localization of sarcoglycans in peripheral nerve (Panel B). Cryosections of adult rat sciatic nerves were stained with sarcoglycan antibodies (green) [-SG, -SG, -SG polyclonal antibodies from AnaSpec, -SG, -SG, -SG monoclonal antibodies from Novocastra]. Sections were also co-stained with anti-neurofilament antibody (NF-H) to reveal neurofilaments in the axons (red). Bar, 12mm (courtesy of Dr. Yiu-mo Michael Chan, Carolinas Medical Center, Charlotte, NC).

Western Blots of p53 Phosphospecific Antibodies
In normal, undamaged cells, p53, an important mammalian cell cycle checkpoint protein, is rapidly degraded; but when cells are treated with DNA damage-inducing agents, there is a transient accumulation of this tumor suppressor protein and it is activated as a transcription factor. In several types of human cancers, p53 is mutated (4, 5). Phosphorylation at serines 6, 9, 15, 20, 33, 37 occurs after cells are exposed either to ionizing radiation or to UV light (6, 7). Serines 6 and 15 were demonstrated to be among the strongest and earliest phosphorylated sites in response to DNA damage-induced posttranslational modifications (8, 9). As shown in western blots (Figure 2), Cos-7 cells treated with hydroxyurea, a known inhibitor of DNA synthesis, express an increasing amount of phosphorylated serine p53’s (pSER 9 and 15), while no signal increase is seen in non-phosphospecific p53.

Since reversible protein phosphorylation/dephosphorylation has been shown to have a principal role in the regulation of essentially all cellular functions and most aspects of cell life, more and more researchers are looking into raising antibodies specific for the phosphorylated version of the proteins and not just the non-phosphorylated protein. AnaSpec, a leader in the production of kinase and phosphatase peptide substrates also offers custom production of phosphospecific antibodies. Our experience has shown that the phosphopeptide immunogen should at least be 90% pure in order to raise good antibodies.

Figure 2. Western blots of hydroxyurea-treated COS-7 cells showing an increasing amount of phosphorylated p53 at phosphoryated SER 9 and SER 15, while no signal increase is seen in non-phosphospecific p53.

Immunohistochemistry, IHC
An example of antibodies used in indirect IHC is shown in Figure 3 in which secondary antibodies labeled with AnaSpec’s patented HiLyte Fluorsup 488 and HiLyte Fluor^TM 555 were used in visualizing the co-localization of beta-amyloid and tau (10). Antibodies labeling kits, such as the AnaTag^™ HiLyte Fluor^™ 555 Protein Labeling Kit, can be used for directly labeling primary antibodies. AnaSpec’s AnaTag^TM series of protein labeling kits provides researchers a range of dyes from blue to near infra-red wavelengths with which to label their antibodies.

Figure 3. Coexpression of Aβ and tau in neurofibrillary tangles. (A–C) Tau [(A0024 labeled with Hilyte Fluor^TM 488-labeled goat anti-rabbit IgG (H+L)] exhibiting a green fluorescence (A) and Aβ [4G8 labeled with Hilyte Fluor^TM 555-labeled goat anti-mouse IgG (H+L)] showing red fluorescence (B) are coexpressed in tangle bearing neurons of the entorhinal cortex in a case of AD as shown by the yellow fluorescence in the merged image (C). (D) A lower-power merged image showing a mixture of tangled neurons coexpressing Aβ and tau (yellow color) and tau only (green color) as well as extracellular Aβ deposits (red color). (Scale bars, 50 µm A and 100 µm in D.) (Courtesy of Dr. Patrick L. McGeer, Kinsmen Laboratory of Neurological Research, University of British Columbia, Vancouver, Canada).

Multiple Antigenic Peptide System, MAPS
Multiple antigenic peptide system (MAPS) has been used successfully in producing high-titer anti-peptide antibodies (11, 12), as well as synthetic peptide vaccines (13). This kind of peptide antigen consists of several copies of the same peptide sequence attached to an inert lysine core (see Figure 3). Tetrameric or octameric constructs synthesized from 4-branched or 8-branched MAPS are the most commonly used. The molecular weights of MAPS peptides are difficult to be detected by mass spectrometry as is routinely done for linear peptides. It can only be characterized by amino acid analysis. HPLC profile of MAPS peptides exhibit broad peaks. Since MAPS constructs are big molecules, conjugations to carrier proteins are not necessary. A recent paper by Fukuda et al. (14) describes the use of an 8-branched MAPS peptide consisting of 4 amino acids (GWRQ) which was used in raising polyclonal antibodies.

Figure 4. Structure of an 8-branched MAPS.

Conclusion
Raising successful antibodies start with choosing an appropriate peptide sequence, having the peptide synthesized correctly, choosing the right host, getting a reasonable antibody titer and lastly, getting the antibodies to work in the desired applications.

References:
1. Merrifield RB. Fed. Proc. Amer. Soc. Exp. Biol. 1962 21, 412
2. Harlow E. and D. Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Press: New York 1988
3. Cai H. et al. Exp. Neurol. 2007 doi:10.1016/j.expneurol.2007.02.015
4. Brown, JM. et al. Cancer Res. 1999 59, 1391
5. Albrechtsen, N. et al. Oncogene 1999 18, 7706
6. Burns, TF. and El-Deiry, WS. J. Cell Physiol. 1999 181, 231
7. Oren, M. et al. J. Biol. Chem. 1999 274, 36031
8. Lakin, ND. et al Oncogene 1999 18, 7644
9. Higashimoto, Y. et al. J. Biol. Chem. 2000 275, 23199
10. Guo, J-P. et al. Proc. Natl. Acad. Sci. USA 2006 103, 1953
11. Wang, CY. et al. Science 1991 254, 285
12. Posnett, D. et al. J. Biol. Chem. 1988 263, 1719
13. Tam JP. PNAS USA 1988 85, 5409
14. Sugihara, K. et al. Proc. Natl. Acad. Sci. 2007 104, 3799

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