Biotechnology Letters

, Volume 29, Issue 11, pp 1735–1740 | Cite as

Production of polyclonal antibodies against peptide antigens using polystyrene beads as a carrier

  • Mihee Kim
  • Chul-Ho Yun
  • Seong-Ki Park
  • Jung-Hoon Seo
  • Taeho Ahn
Original Research Paper
First Online:
Received:
Revised:
Accepted:

Abstract

We describe a method for producing polyclonal antibody (PAb) against peptide antigen (the epitope region of cytochromes P450 1A2 and 3A4, HA tag, FLAG tag, and c-myc tag) using a polymer bead as a carrier. Carboxylated polystyrene beads (0.05, 0.5, 1, and 2 μm diam) were conjugated with the peptide, which is known as an epitope region, using a chemical cross-linker and injected into rabbits with adjuvant. By immunizing the animals intradermally twice at two week intervals, an immunoblot assay with anti-sera showed the efficient generation of the PAb.

Keywords

Carrier Conjugation Peptide antigen Polyclonal antibody Polystyrene bead 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgement

This work was supported by a Korea Research Foundation Grant KRF-2005-070-C00081.

References

  1. Ahn T, Yun C-H, Oh D-B (2005) Involvement of nonlamellar-prone lipids in the stability increase of human cytochrome P450 1A2 in reconstituted membranes. Biochemistry 44:9188–9196PubMedCrossRefGoogle Scholar
  2. Angeletti RH (1999) Design of useful peptide antigens. J Biomol Tech 10:2–10Google Scholar
  3. Castell JV, Cervera M, Marco R (1979) A convenient micromethod for the assay of primary amines and proteins with fluorescamine. A re-examination of the conditions of reaction. Anal Biochem 99:379–391PubMedCrossRefGoogle Scholar
  4. Edwards RJ, Singleton AM, Murray BP et al (1995) Short synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes. Biochem Pharmacol 49:39–47PubMedCrossRefGoogle Scholar
  5. Evan GI, Lewis GK, Ramsay G et al (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616PubMedGoogle Scholar
  6. Field J, Nikawa J, Broek D et al (1988) Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol 8:2159–2165PubMedGoogle Scholar
  7. Fifis T, Mottram P, Bogdanoska V et al (2004) Short peptide sequences containing MHC class I and/or class II epitopes linked to nano-beads induce strong immunity and inhibition of growth of antigen-specific tumor challenge in mice. Vaccine 23:258–266PubMedCrossRefGoogle Scholar
  8. Harlow E, Lane DP (1988) Antibodies: a laboratory manual. CSH Laboratory Press, Cold Spring Harbor, New York, p 128Google Scholar
  9. Kim K-H, Ahn T, Yun C-H (2003) Membrane properties induced by anionic phospholipids and phosphatidylethanolamine are critical for the membrane binding and catalytic activity of human cytochrome P450 3A4. Biochemistry 42:15377–15387PubMedCrossRefGoogle Scholar
  10. Knappik A, Pluckthun A (1994) An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments. BioTechniques 17:754–761PubMedGoogle Scholar
  11. Men Y, Gander B, Merkle HP et al (1996) Induction of sustained and elevated immune responses to weakly immunogenic synthetic malarial peptides by encapsulation in biodegradable polymer microspheres. Vaccine 14:1442–1450PubMedCrossRefGoogle Scholar
  12. Olivier V, Duval JL, Hindié M et al (2003) Comparative particle-induced cytotoxicity toward macrophages and fibroblasts. Cell Biol Toxicol 19:191–199CrossRefGoogle Scholar
  13. Scheerlinck JPY, Gloster S, Gamvrellis A et al (2006) Systemic immune responses in sheep, induced by a novel nano-bead adjuvant. Vaccine 24:1124–1131PubMedCrossRefGoogle Scholar
  14. Slobbe L, Medlicott N, Lockhart E et al (2003) A prolonged immune response to antigen delivered in poly (ε-caprolactone) microparticles. Immunol Cell Biol 81:185–191PubMedCrossRefGoogle Scholar
  15. Wang RW, Newton DJ, Liu NY et al (1999) Inhibitory anti-CYP3A4 peptide antibody: mapping of inhibitory epitope and specificity toward other CYP3A isoforms. Drug Metab Dispos 27:167–172PubMedGoogle Scholar
  16. Wilingsson LD, Sjöholm I (2002) Polyacryl starch microparticles as adjuvant in oral immunisation, inducing mucosal and systemic immune responses in mice. Vaccine 20:3355–3363CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Mihee Kim
    • 1
  • Chul-Ho Yun
    • 2
  • Seong-Ki Park
    • 3
  • Jung-Hoon Seo
    • 3
  • Taeho Ahn
    • 3
  1. 1.Department of Molecular EndocrinologyChonnam National UniversityGwangjuKorea
  2. 2.School of Biological Sciences and TechnologyChonnam National UniversityGwangjuKorea
  3. 3.Department of Biochemistry, College of Veterinary MedicineChonnam National UniversityGwangjuKorea

Personalised recommendations