Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D.
A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively.
Dziegiel
M
M
Department of Clinical Immunology, University Hospital, Copenhagen, Denmark.
Nielsen
L K
LK
Andersen
P S
PS
Blancher
A
A
Dickmeiss
E
E
Engberg
J
J
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000032:analysis; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007518:Isoantibodies / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology; D012204:Rh-Hr Blood-Group System / Q000276:immunology*
D018076:DNA, Complementary; D007518:Isoantibodies; D011993:Recombinant Fusion Proteins; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen