Immunological characterization and expression in Escherichia coli and baculovirus systems of a Trypanosoma vivax antigen detected in the blood of infected animals.
A monoclonal antibody (MAb)Tv27 employed in an antigen-detection enzyme immunosorbent assay (Ag-ELISA) for diagnosis of Trypanosoma vivax infection was shown to react with a T. vivax-specific protein of an approximate molecular weight of 10 kDa. This protein is diffusely distributed throughout the cytosol and nucleus of metacyclic forms, bloodstream forms, and procyclic-like elongated trypomastigotes, but is not detectable in epimastigotes of T. vivax. The T. vivax-specific antigen prepared from parasite lysates appeared to be of lower molecular mass than the form expressed in either Escherichia coli or in baculovirus-infected silkworm insect cells. In the recombinant baculovirus-infected cells, the protein was expressed mostly as an 18-kDa peptide with less abundant forms of 13 and 12 kDa, while the protein expressed in E. coli was approximately 14 kDa. Both the low- and higher-molecular-weight proteins are recognized by the MAb Tv27 in Western blots and in Ag-ELISA. Although the crude preparations of the protein produced by the insect cells are labile when kept for more than 2 hr at 24 degrees C, they retained reactivity at temperatures below 4 degrees C for several weeks. The proteins expressed in both the insect cells and E. coli captured anti-T. vivax antibodies in sera prepared from trypanosome-infected animals. Since the recombinant protein expressed in the baculovirus-infected cells is available in large homogeneous quantities, it would serve as a positive control in Ag-ELISA and is also usable for antibody detection assays.
International Livestock Research Institute, (ILRI), Nairobi, Kenya.
R A
RA
Masake
O K
OK
ole-MoiYoi
T
T
Urakawa
H
H
Hirumi
P A
PA
Majiwa
C W
CW
Wells
S H
SH
Minja
J M
JM
Makau
V M
VM
Nantulya
D000818:Animals; D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000918:Antibody Specificity; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D012831:Bombyx / Q000166:cytology / Q000821:virology; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015151:Immunoblotting; D016780:Malaria, Vivax / Q000097:blood* / Q000175:diagnosis; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D016253:Microscopy, Immunoelectron; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000276:immunology; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D016689:Trypanosoma vivax / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D011993:Recombinant Fusion Proteins