The Escherichia coli-derived Fab fragment of the IgM/kappa antibody IN-1 recognizes and neutralizes myelin-associated inhibitors of neurite growth.
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.
Institut für Hirnforschung, Universität Zürich, Switzerland.
C
C
Bandtlow
W
W
Schiweck
H H
HH
Tai
M E
ME
Schwab
A
A
Skerra
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007140:Immunoglobulin Fab Fragments / Q000235:genetics; D007075:Immunoglobulin M / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D009186:Myelin Sheath / Q000276:immunology*; D016501:Neurites / Q000276:immunology* / Q000648:ultrastructure; D009500:Neutralization Tests; D016716:PC12 Cells; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013116:Spinal Cord / Q000276:immunology / Q000378:metabolism
D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007075:Immunoglobulin M; D007145:Immunoglobulin kappa-Chains; D011994:Recombinant Proteins