Conversion of a mouse Fab into a whole humanized IgG antibody for detecting botulinum toxin.
Antibodies serve as the gold standard in most immunodiagnostic assays. Recent advances in recombinant DNA technology have offered the production of antibody fragments or Fabs as promising alternatives. However, the lack of the Fc region of the antibody can be difficult in many standard diagnostic platforms. Therefore we sought to convert a murine Fab into a whole humanized IgG. The variable regions from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified humanized IgG demonstrated conversion to human IgG with no traces of mouse Fab as determined by Western blot analysis. In addition, the humanized IgG performed better as both a detection and capture reagent in an ELISA format and detected the botulinum toxoid at a lower concentration than the parental murine Fab. This technique offers the ability to convert various species of antibodies or antibody fragments into humanized antibodies with improved characteristics in immunodiagnostic assays, for use as human controls in serological assays, or for possible therapeutic benefit.
Palys
Thomas J
TJ
Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA.
Schmid
Kara E
KE
Scherer
John M
JM
Schoepp
Randal J
RJ
D000818:Animals; D001709:Biotechnology / Q000379:methods; D001905:Botulinum Toxins / Q000032:analysis* / Q000276:immunology*; D001906:Botulism / Q000175:diagnosis; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D014162:Transfection
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D001905:Botulinum Toxins