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

Ricardo Jara-Acevedo; Maria Gonzalez-Gonzalez; María Jara-Acevedo; Jose Claros; Alfredo Conde; Ricardo López-Perez; Alberto Orfao; Manuel Fuentes

doi:10.1016/j.jim.2012.07.007

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.

Authors

Ricardo Jara-Acevedo¹, Maria Gonzalez-Gonzalez, María Jara-Acevedo, Jose Claros, Alfredo Conde, Ricardo López-Perez, Alberto Orfao, Manuel Fuentes

Affiliation

¹ ImmunoStep SL, Salamanca, Spain.

PMID: 22835433
DOI: 10.1016/j.jim.2012.07.007

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