Bacterial expression of a human monoclonal antibody-alkaline phosphatase conjugate specific for Entamoeba histolytica.
We previously produced human monoclonal antibody Fab fragments specific to Entamoeba histolytica in Escherichia coli. In order to use these Fab fragments for diagnostic purposes, an expression vector to produce a fusion protein of Fab and alkaline phosphatase (PhoA) in E. coli was designed and constructed. The E. coli PhoA gene was fused to the 3' terminus of the gene encoding the heavy-chain Fd region. The kappa and Fd genes from a previously prepared antibody clone, CP33, which is specific for the 260-kDa lectin of E. histolytica, were used as human antibody genes. When the fusion protein of CP33 and PhoA was incubated with paraformaldehyde-fixed trophozoites of E. histolytica and developed with a substrate, the trophozoites appeared to be stained. These results demonstrate the feasibility of bacterial expression of a human monoclonal antibody-PhoA conjugate specific for E. histolytica and that the antibody can be used to detect E. histolytica antigen without the use of chemically conjugated secondary antibodies.
Tachibana
Hiroshi
H
Departments of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan. htachiba@is.icc-u-tokai.ac.jp
Takekoshi
Masataka
M
Cheng
Xun-Jia
XJ
Nakata
Yuta
Y
Takeuchi
Tsutomu
T
Ihara
Seiji
S
D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D000913:Antibodies, Protozoan / Q000235:genetics*; D000953:Antigens, Protozoan; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004404:Dysentery, Amebic / Q000175:diagnosis; D004748:Entamoeba histolytica / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D004274:DNA, Recombinant; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase