Evaluation of chemically modified carrier proteins for developing monoclonal antibodies against a clinically relevant mutation of cKIT

J Immunol Methods. 2012 Oct 31;384(1-2):171-6. doi: 10.1016/j.jim.2012.07.007. Epub 2012 Jul 23.

Abstract

In this report we show that combining double-chemically modified carrier proteins and hetero-functional cross-linkers allows preparing tailor-made hapten-protein carrier conjugates. Accordingly, a new carrier protein has been designed where carboxylic groups were transformed into highly reactive primary amino groups by reaction with ethylendiamine after activation with EDCI. The aminated protein carrier is then modified by different cross-linkers (hyper-activated proteins) at different conditions in order to control the conjugation ratio from 1 to >12 molecules of hapten per carrier protein. Finally, this novel strategy has been successfully used to develop antibodies against a short specific peptide corresponding to a point mutation (D816V) of cKIT, which is a clinically relevant mutation related to mastocytosis and gastrointestinal stroma tumor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / blood
  • Antibodies / immunology
  • Antibodies, Monoclonal / immunology*
  • Carrier Proteins / chemistry
  • Carrier Proteins / immunology*
  • Cattle
  • Cross-Linking Reagents / chemistry
  • Electrophoresis, Gel, Two-Dimensional
  • Enzyme-Linked Immunosorbent Assay
  • Ethylenediamines / chemistry
  • Gastrointestinal Stromal Tumors / genetics
  • Gastrointestinal Stromal Tumors / immunology
  • Immunoconjugates / chemistry
  • Immunoconjugates / immunology
  • Maleimides / chemistry
  • Mastocytosis / genetics
  • Mastocytosis / immunology
  • Mice
  • Models, Chemical
  • Models, Molecular
  • Molecular Structure
  • Mutation, Missense*
  • Peptides / genetics
  • Peptides / immunology
  • Protein Conformation
  • Proto-Oncogene Proteins c-kit / chemistry
  • Proto-Oncogene Proteins c-kit / genetics
  • Proto-Oncogene Proteins c-kit / immunology*
  • Serum Albumin, Bovine / chemistry
  • Serum Albumin, Bovine / immunology
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Antibodies
  • Antibodies, Monoclonal
  • Carrier Proteins
  • Cross-Linking Reagents
  • Ethylenediamines
  • Immunoconjugates
  • Maleimides
  • Peptides
  • maleimide
  • Serum Albumin, Bovine
  • Proto-Oncogene Proteins c-kit