Direct production of the Fab fragment derived from the sperm immobilizing antibody using polymerase chain reaction and cDNA expression vectors.
OBJECTIVE
Sperm immobilizing antibodies cause infertility mainly through complement dependent sperm immobilization. To analyze any effect of sperm immobilizing antibody on fertilization, we had already established cell lines that secrete IgM monoclonal antibody (MAb H6-3C4) and IgG monoclonal antibody (MAb EnBCMGS). The latter was a class-switched recombinant IgG antibody that shares the same variable region as MAb H6-3C4. The biological effects of the IgG antibody were also reported previously to eliminate sperm immobilizing or sperm agglutinating activities. However, the method of chemical digestion of IgG had some disadvantage to prepare the purified Fab fragment stably and in large quantities. This time we report a unique method to obtain the recombinant Fab fragments (Fab EnBCMGS) using polymerase chain reaction (PCR) and cDNA expression vectors.


METHODS
Two kinds of PCR primers were designed to make a truncated heavy chain (Fd) gene of MAb EnBCMGS. The amplified Fd gene and light chain gene were ligated into cDNA expression vectors and then transfected into mammalian cells.


RESULTS
Expression of the Fd gene and light chain gene were confirmed by Northern blotting. Secretion of the recombinant Fab fragment from mammalian cells was also confirmed by Western blotting. The Fab fragment showed biological activity as is expected by FACS analysis.


CONCLUSIONS
This method enables the stable production of genuine Fab fragments of IgG in mammalian cells without any chemical treatment that may be time consuming and affect the quality of the Fab fragments.
Sawai
H
H
Department of Obstetrics and Gynecology, Hyogo College of Medicine, Nishinomiya, Japan.
Yamasaki
N
N
Shigeta
M
M
Komori
S
S
Karasuyama
H
H
Koyama
K
K
Isojima
S
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D005803:Genes, Immunoglobulin / Q000276:immunology; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013078:Sperm Immobilizing Agents / Q000276:immunology*
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D013078:Sperm Immobilizing Agents