Preparation of recombinant human monoclonal antibody Fab fragments specific for Entamoeba histolytica.
Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis.
Tachibana
H
H
Departments of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan. htachiba@is.icc.u-tokai.ac.jp
Cheng
X J
XJ
Watanabe
K
K
Takekoshi
M
M
Maeda
F
F
Aotsuka
S
S
Kaneda
Y
Y
Takeuchi
T
T
Ihara
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D000913:Antibodies, Protozoan / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D004748:Entamoeba histolytica / Q000276:immunology* / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008101:Liver Abscess, Amebic / Q000469:parasitology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins