Use of recombinant fusion proteins for generation and rapid characterization of monoclonal antibodies. Application to the Kunitz domain of human beta amyloid precursor protein.
Production of peptides by recombinant DNA techniques is an efficient alternative to chemical synthesis of peptides. Proteins and peptides produced by recombinant DNA methods in E. coli are routinely used as antigens for the production of antibodies. However, most small peptides are rapidly degraded within the E. coli cell, and therefore, must initially be expressed as components of larger, more stable fusion proteins. The peptide of interest must be cleaved from the fusion protein, and purified prior to immunization to eliminate epitopes contributed by the fusion partner. We have now established methods for the production and characterization of monoclonal antibodies using partially purified, uncleaved fusion proteins. We have also described a method for efficient production and detection of the fusion protein, an EIA for rapid differential screening of hybridoma supernatants, and a strategy for epitope mapping of the antibodies. These methods have been applied to the production and characterization of monoclonal antibodies specific for a 75-amino-acid internal segment of the Alzheimer amyloid precursor protein, and should be applicable to a wide variety of other peptides and proteins.
Molecular Therapeutics, Inc., West Haven, CT.
D
D
Wunderlich
A
A
Lee
R P
RP
Fracasso
D V
DV
Mierz
R M
RM
Bayney
T V
TV
Ramabhadran
D016564:Amyloid beta-Protein Precursor / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007124:Immunoenzyme Techniques; D010446:Peptide Fragments / Q000276:immunology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology*
D016564:Amyloid beta-Protein Precursor; D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins