Selection of large diversities of antiidiotypic antibody fragments by phage display.
Antiidiotypic antibodies (Ab2) are needed as tools for a better understanding of molecular mimicry and the immunological network, and for many potential applications in the biomedical and pharmaceutical field. Antiidiotypic antibodies mimicking carbohydrate or conformational epitopes (Ab2beta) are of considerable interest as surrogate immunogens for cancer vaccination. However, it has so far been difficult and tedious to produce Ab2s to a given antigen. Here we describe a fast and reliable technique for generating large diversities of antiidiotypic single chain antibody fragments from non-immunized phagemid libraries using phage display. Key elements are a specific elution with the original antigen followed by trypsin treatment of the eluted phages in combination with the protease sensitive helperphage KM13. This novel method was compared with various conventional selection and elution methods, including, specific elution with or without trypsin treatment, elution with glycine at pH 2.2 with or without trypsin treatment, and elution by trypsin treatment only. The results clearly show that specific elution in combination with trypsin treatment of the eluted phages is by far superior to the other conventional methods, enabling for the first time the generation of a large variety of Ab2s after only two to three rounds of selection, thereby maintaining maximum diversity. We obtained 28 to 88 antiidiotypes out of 96 tested clones after two to three rounds of selection with a diversity of 55-90 %. This was achieved for two carbohydrate (di-, and tetrasaccharides) and one conformational protein epitope using two large naïve libraries and their corresponding monoclonal Ab1. The antiidiotypic nature of the selected scFv-phages was verified by ELISA and immunocytochemistry inhibition experiments.
Goletz
Steffen
S
NEMOD GmbH, Robert-Rössle-Str. 10, Berlin-Buch, D-13125, FRG. Steffen.Goletz@Nemod.com
Christensen
Peter Astrup
PA
Kristensen
Peter
P
Blohm
Dietmar
D
Tomlinson
Ian
I
Winter
Greg
G
Karsten
Uwe
U
D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D000916:Antibody Diversity; D000941:Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D005455:Fluorescent Antibody Technique; D005998:Glycine / Q000378:metabolism; D006378:Helper Viruses / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D008280:Magnetics; D051379:Mice; D008863:Microspheres; D018716:Molecular Mimicry / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry / Q000276:immunology; D015202:Protein Engineering; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D000888:Antibodies, Anti-Idiotypic; D000941:Antigens; D000939:Epitopes; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011134:Polysaccharides; D014357:Trypsin; D005998:Glycine