Isolation of neutralizing anti-C5a monoclonal antibodies from a filamentous phage monovalent Fab display library.
A panel of mAbs against the activated complement component C5a was obtained from a filamentous phage M13-Fab display library generated from mice immunized with human rC5a. Fabs isolated from the library after iterative selection vs rC5a bound to both rC5a and purified C5. To isolate Fabs specific for neoepitopes expressed on C5a but not on the native complement component C5 the library was rescreened in a competitive manner. The phage Fab library was first incubated with immobilized C5 to deplete C5 reactive Fabs. The C5 nonadherent phage were then incubated with immobilized rC5a in the presence of soluble C5. Bound phage were eluted and subjected to two additional cycles of subtraction with immobilized C5 and selection with immobilized rC5a in the presence of soluble C5. After three cycles of this competitive biopanning four Fabs reactive with rC5a were isolated. Two bound preferentially with and neutralized C5a. Competitive biopanning of phage display libraries may increase the probability of identification of Abs of the desired specificity.
Ames
R S
RS
Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Tornetta
M A
MA
Jones
C S
CS
Tsui
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000302:isolation & purification; D017104:Bacteriophage M13 / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D001667:Binding, Competitive; D015936:Complement C5a / Q000037:antagonists & inhibitors* / Q000276:immunology; D004247:DNA / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D007114:Immunization; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D015936:Complement C5a; D004247:DNA