Preparation of peptide-protein immunogens using N-succinimidyl bromoacetate as a heterobifunctional crosslinking reagent

Anal Biochem. 1986 May 15;155(1):95-102. doi: 10.1016/0003-2697(86)90231-9.


Synthetic peptides derived from human fibrin were unidirectionally conjugated to three carrier proteins (bovine serum albumin, bovine alpha-lactalbumin, and keyhole limpet hemocyanin) by a method that employs N-succinimidyl bromoacetate. This heterobifunctional crosslinking reagent was prepared with a 79% yield in gram quantities from inexpensive starting materials. With this reagent, carrier proteins were first bromoacetylated, then reacted with the thiol groups of cysteine-containing peptides. The extent of peptide conjugation was assessed by amino acid analysis after acid hydrolysis, which liberated 1 mol of S-carboxymethylcysteine for each mole of thioether linkage between peptide and protein. The results of several conjugation experiments indicated that the efficiency of peptide incorporation ranged between 22 and 37% based on the recovery of S-carboxymethylcysteine relative to lysine. When the conjugates were used as immunogens, the S-carboxymethyl linkage was not antigenic in comparison with the S-maleimidobenzoyl linkage, even though their antipeptide immunoreactivities were similar.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antigens*
  • Cattle
  • Cross-Linking Reagents* / chemical synthesis
  • Hemocyanins / immunology
  • Immunochemistry
  • Lactalbumin / immunology
  • Peptides / immunology*
  • Proteins / immunology*
  • Serum Albumin, Bovine / immunology
  • Succinimides / chemical synthesis
  • Trinitrobenzenesulfonic Acid


  • Antigens
  • Cross-Linking Reagents
  • Peptides
  • Proteins
  • Succinimides
  • Serum Albumin, Bovine
  • N-succinimidyl bromoacetate
  • 3-maleimidobenzoyl N-hydroxysuccinimide
  • Trinitrobenzenesulfonic Acid
  • Hemocyanins
  • Lactalbumin
  • keyhole-limpet hemocyanin