Bandtlow C, Schiweck W, Tai HH, Schwab ME, Skerra A
Institut fur Hirnforschung, Universitat Zurich, Switzerland.
A recombinant Fab fragment was prepared from the monoclonal IgM/kappa antibody
IN-1, which neutralizes
central nervous system myelin-associated neurite growth inhibitors both in vitro and in vivo. The variable domain
gene sequences were amplified and cloned after cDNA synthesis from the hybridoma RNA. After insertion into
the tet promoter vector pASK85, which provided the constant domains of class IgG1/kappa, equipped with a
His6 tag, large amounts of the Fab fragment were produced in Escherichia coli by medium cell density
fermentation. The Fab fragment was purified to homogeneity by immobilized metal-affinity chromatography and
its biochemical activity was compared with the original IN-1 antibody. In an assay for neurite outgrowth and
fibroblast spreading, the Fab fragment showed a similar neutralizing effect on inhibitory substrate properties of
central nervous system myelin as the unpurified IgM, although an approximately tenfold higher concentration was
necessary. Immunoprecipitation experiments revealed a more selective antigen-binding behaviour for the Fab
fragment. The Fab fragment was also successfully applied for antigen detection in immunohistochemical analyses.
Therefore, the recombinant Fab fragment of IN-1 shows full functionality in vitro and appears to be well suited
for replacing the monoclonal IgM in investigations on fiber tract regeneration in vivo.