Production of Antipeptide Antisera

John E. Coligan1, James P. Tam2, Jun Shao2

1 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, 2 Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.6
DOI:  10.1002/0471142301.ns0506s00
Online Posting Date:  May, 2001
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Abstract

This unit describes methods used for the chemical coupling of synthetic peptides to carrier proteins, required for the preparation of peptide immunogens. The carrier protein described here is keyhole limpet hemocyanin (KLH) because it is the one most commonly used. However, other proteins may be used in place of KLH, including bovine serum albumin (BSA) and ovalbumin. Coupling may be accomplished as described with MBS, which requires a Cys residue in the peptide, or with glutaraldehyde, EDCI, or BDB. Also included are an assay for detecting free sulfhydryl (SH) groups, a means of calculating coupling efficiency, an immunization schedule, an indirect ELISA, and a method for preparing a peptide affinity column. The final methodology described is the multiple antigen (MAP) system.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Coupling of Synthetic Peptide to Carrier Protein Using MBS
  • Alternate Protocol 1: Coupling of Synthetic of Peptide to Carrier Protein Using Glutaraldehyde
  • Alternate Protocol 2: Coupling of Synthetic Peptide to Carrier Protein Using EDCI
  • Alternate Protocol 3: Coupling of Synthetic Peptide to Carrier Protein Using BDB
  • Support Protocol 1: Detection of Free Sulfhydryl Groups in Peptides
  • Support Protocol 2: Calculation of the Molar Ratio of Peptide to Carrier Protein
  • Support Protocol 3: Immunization Schedule for Producing Antipeptide Sera in Rabbits
  • Support Protocol 4: Indirect ELISA to Determine Antipeptide Antibody Titer
  • Support Protocol 5: Preparation of Peptide Affinity Column
  • Basic Protocol 2: Use of Multiple Antigen Peptide(MAP) Systems
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Coupling of Synthetic Peptide to Carrier Protein Using MBS

  Materials
  • Keyhole limpet hemocyanin (KLH; Calbiochem)
  • 0.01 M potassium phosphate buffer, pH 7.0 ( appendix 2A)
  • 15 mg/ml m‐maleimidobenzoyl‐N‐hydroxysuccinimide ester (Pierce) in dimethylformamide (MBS/DMF), prepared within 1 hr of use
  • 0.05 M potassium phosphate buffer, pH 6.0 ( appendix 2A)
  • Cys‐containing synthetic peptide
  • PBS ( appendix 2A)
  • 0.1 M HCl and 0.1 M NaOH
  • Dialysis tubing (10,000 MWCO; Spectrapor; Fisher)
  • 10 × 75–mm (3‐ml) and 15‐ml glass test tubes
  • PD‐10 column, prepacked (Pharmacia Biotech)
NOTE: Tris buffers should not be used in this procedure.

Alternate Protocol 1: Coupling of Synthetic of Peptide to Carrier Protein Using Glutaraldehyde

  • Borate buffers, pH 10 and pH 8.5 (see recipe)
  • Synthetic peptide
  • 0.3% glutaraldehyde solution (see recipe)
  • l M glycine
NOTE: Tris buffers should not be used in this procedure.

Alternate Protocol 2: Coupling of Synthetic Peptide to Carrier Protein Using EDCI

  • 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide⋅HCl (EDCI), freshly prepared (Sigma or Pierce)
  • 12 × 75–mm glass test tubes (∼5‐ml capacity)
NOTE: Buffers containing phosphate, carboxylates, or amino groups (e.g., Tris) will interfere with this reaction and should be avoided.

Alternate Protocol 3: Coupling of Synthetic Peptide to Carrier Protein Using BDB

  • Benzidine⋅HCl (Sigma)
  • 0.2 M HCl
  • NaNO 2
  • Borate buffer, pH 9.0 (see recipe)
  • 15‐ml test tube
  • 50‐ml beaker
CAUTION:Benzedine and its salts and BDB are considered to be carcinogens. Use appropriate precautions when handling.

Support Protocol 1: Detection of Free Sulfhydryl Groups in Peptides

  Materials
  • 0.05 M potassium phosphate buffer, pH 8.0 ( appendix 2A)
  • DTNB solution (see recipe), freshly prepared
  • 3 mM 2‐mercaptoethanol (2‐ME), freshly prepared in H 2O
  • 5 mg/ml synthetic peptide in 0.05 M potassium phosphate buffer, pH 8.0
  • 10 × 75–mm glass test tubes

Support Protocol 2: Calculation of the Molar Ratio of Peptide to Carrier Protein

  Materials
  • Rabbits
  • Peptide/carrier conjugate (see protocol 1 and protocol 2Alternate Protocols 1, protocol 32, and protocol 43)
  • PBS ( appendix 2A)
  • Complete Freunds adjuvant (Sigma)
  • Incomplete Freunds adjuvant (Sigma)

Support Protocol 3: Immunization Schedule for Producing Antipeptide Sera in Rabbits

  Materials
  • 0.2 to 2.5 µM synthetic peptide in carbonate buffer (see recipe for buffer)
  • PBS( appendix 2A) containing 0.05% Tween 20 (PBS/Tween; Sigma)
  • 10 mg/ml BSA in PBS/Tween
  • Antipeptide antiserum (see protocol 6)
  • Goat anti‐rabbit globulin conjugated to alkaline phosphatase (Sigma)
  • Enzyme substrate: 1 mg/ml p‐nitrophenyl/phosphate (NPP; Sigma) in 0.1 M diethanolamine buffer, pH 9.8 (see recipe)
  • 96‐well microtiter plate
  • Microtiter plate reader: spectrophotometer with 405‐nm filter or spectrofluorometer (Dynatech) with 365‐nm excitation filter and 450‐nm emission filter

Support Protocol 4: Indirect ELISA to Determine Antipeptide Antibody Titer

  Materials
  • CNBr‐activated Sepharose‐4B (Pharmacia)
  • 1 × 10–cm column (e.g., Bio‐Rad Econo Column)
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Figures

Videos

Literature Cited

Literature Cited
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   Dyson, H.J., Cross, K.J., Houghten, R.A., Wilson, I.A., Wright, P.E., and Lerner, R.A. 1985. The immunodominant site of a synthetic immunogen has a conformational preference in water for a type‐II reverse turn. Nature 318:480‐483.
   Francis, M.J., Hastings, G.Z., Brown, F., McDermed, J., Lu, Y.A., and Tam, J.P. 1991. Immunological evaluation of the multiple antigen peptide (MAP) system using the major immunogenic site of foot‐and‐mouth disease virus. Immunology 73:249‐254.
   Geerligs, H.J., Weijer, W.J., Bloemhoff, W., Welling, G.W., and Welling‐Wester, S. 1988. The influence of pH and ionic strength on the coating of peptides of herpes simplex virus type 1 in an enzyme‐linked immunosorbent assay. J. Immunol. Methods 106:239‐244.
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   Nardelli, B., Lu, Y.A., Shim, D.R., Delpierre‐Defoort, C., Profy, A.T., and Tam, J.P. 1992. A chemically defined synthetic vaccine model for HIV‐1. J. Immunol. 148:914‐920.
   Pessi, A., Valmori, D., Migliorni, P., Tougne, C., Bianchi, E., Lambert, P.‐H., Corradin, G., and Giudice, del G. 1991. Lack of 2 restriction of plasmodium falcoparum (NANP) sequence as multiple antigen peptide. Eur. J. Immunol. 21:2273‐2276.
   Posnett, D., McGrath, H., and Tam, J.P. 1988. A novel method for producing anti‐peptide antibodies. J. Biol. Chem. 263:1719‐1725.
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   Tam, J.P. 1988. Synthetic peptide vaccine design: Synthesis and properties of a high‐density multiple antigenic peptide system. Proc. Natl. Acad. Sci. U.S.A. 85:5409‐5413.
   Tam, J.P. and Lu, Y.A. 1989. Vaccine engineering: Enhancement of immunogenicity of synthetic peptide vaccine related to hepatitis in chemically defined models consisting of T and B cell epitopes. Proc. Natl. Acad. Sci. U.S.A. 86:9084‐9088.
   Tam, J.P. and Shao, J. 1993. Synthesis and use of multiple antigen peptide (MAP) systems. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober,) pp.9.6.1‐9.6.19. John Wiley & Sons, New York.
   Tam, J.P., Clavijo, P., Lu, Y.A., Nussenzweig, R.S., Nussenzweig, V., and Zavala, F. 1990. Incorporation of T and B epitopes of the circumsporozoite protein in a chemically defined synthetic vaccine against malaria. J. Exp. Med. 171:299‐306.
   Tam, J.P. and Shao, J. 1993. Synthesis and use of multiple antigen peptide (MAP) systems. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 9.6.1‐9.6.19. John Wiley & Sons, New York.
   Van Regenmortel, M.H.V., Briand, J.P., Muller, S., and Plaué, S. 1988. Synthetic polypeptides as antigens. In Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 19 (R.H. Burdon and P.H. VonKnippenberg, eds.). Elsevier, Amsterdam and New York.
   von Heijne, G. 1986. A new method for predicting signal sequence cleavage sites. Nucl. Acids Res. 14:4683‐4690.
   Walter, G., Scheidtmann, K.‐H., Carbone, A., Laudano, A.P., and Doolittle, R.F. 1980. Antibodies specific for the carboxy‐ and amino‐terminal regions of simian virus 40 large tumor antigen. Proc.Natl. Acad. Sci. U.S.A. 77:5197‐5200.
   Westhof, E., Altschuh, D., Moras, D., Bloomer, A.C., Mondragon, A., Klug, A., and Van Regenmortel, M.H.V. 1984. Correlation between segmental mobility and the location of antigenic determinants in proteins. Nature 311:123‐126.
Key References
   Harlow, E. and Lane, D. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  Discusses preparation of peptide/carrier conjugates and immunization of rabbits.
   Van Regenmortel et al., 1988. See above.
  Presents detailed discussion of selection of peptides for preparing antipeptide sera reactive with native proteins.
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