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Production of antibodies to the α7-subunit of human acetylcholine receptor with the use of immunoactive synthetic peptides

Abstract

Potential B epitopes and T-helper epitopes in the N-terminal extracellular domain of the α7-subunit of human acetylchloline receptor (AChR) were theoretically calculated in order to reveal peptides that can induce the formation of specific antibodies to this domain. Four peptides structurally corresponding to four α7-subunit regions containing 16–23 aa and three of their truncated analogues were synthesized. Rabbits were immunized with both free peptides and protein conjugates of their truncated analogues, and a panel of antibodies to various exposed regions of the N-terminal extracellular domain of the AChR α7-subunit was obtained. All of the four predicted peptides were shown to induce the production of antipeptide antibodies in free form, without conjugation with any protein carrier. The free peptides and the protein conjugates of truncated analogues induced the formation of almost equal levels of antibodies. Most of the obtained antisera contained antibodies that bind to the recombinant extracellular N-terminal domain of the rat AChR α7-subunit and do not react with the analogous domain of the α1-subunit of the ray Torpedo californica AChR.

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Abbreviations

AChR:

acetylcholine receptor

AChBP:

acetylcholine-binding protein

DIEA:

N-ethyldiisopropylamine

DIPC:

N,N′-diisopropylcarbodiimide

DMAP:

4-dimethylaminopyridine

FIA:

Freund’s incomplete adjuvant

FCA:

Freund’s complete adjuvant

Fmoc:

9-fluorenylmethoxycarbonyl

KLH:

keyhole limpet hemocyanin

PBS:

phosphate-buffered saline (0.15 N NaCl in 0.01 N NaH2PO4, pH 7.4)

Pbf:

2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl

TBTU:

O-(benzotriazole-1-yl)-N,N,N′,N′-tetramethylurea tetrafluoroborate

TIS:

triisopropylsilane

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Correspondence to O. M. Volpina.

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Original Russian Text © O.M. Volpina, M.A. Titova, D.O. Koroev, T.D. Volkova, M.B. Oboznaya, M.N. Zhmak, T.A. Aleekseev, V.I. Tsetlin, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 2, pp. 169–175.

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Volpina, O.M., Titova, M.A., Koroev, D.O. et al. Production of antibodies to the α7-subunit of human acetylcholine receptor with the use of immunoactive synthetic peptides. Russ J Bioorg Chem 32, 154–159 (2006). https://doi.org/10.1134/S1068162006020075

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  • DOI: https://doi.org/10.1134/S1068162006020075

Key words

  • acetylcholine receptor
  • antibodies
  • α7-subunit
  • B epitopes
  • T-helper epitopes
  • synthetic peptides