A novel approach to enhance antibody sensitivity and specificity by peptide cross-linking

Anal Biochem. 2008 Dec 15;383(2):265-9. doi: 10.1016/j.ab.2008.08.024. Epub 2008 Aug 30.


Most current techniques employed to improve antigen-antibody signals in Western blotting and in immunohistochemistry rely on sample processing prior to staining (e.g., microwaving) or using a more robust reporter (e.g., a secondary antibody with biotin-streptavidin). We have developed and optimized a new approach intended to stabilize the complexes formed between antigens and their respective primary antibodies by cupric ions at high pH. This technique improves the affinity and lowers cross-reactivity with nonspecific bands of approximately 20% of antibodies tested (5/25). Here we report that this method can enhance antigen-antibody specificity and can improve the utility of some poorly reactive primary antibodies.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Antibody Affinity / drug effects
  • Antibody Specificity* / drug effects
  • Antigen-Antibody Reactions / drug effects
  • Biuret Reaction
  • Copper / chemistry*
  • Copper / pharmacology
  • Cross Reactions / drug effects
  • Cross-Linking Reagents / chemistry*
  • Cross-Linking Reagents / pharmacology
  • Humans
  • Mice
  • Peptides / chemistry*
  • Peptides / metabolism*


  • Cross-Linking Reagents
  • Peptides
  • Copper