title: Evaluation of truncated LipL32 expressed by Escherichia coli and Pichia pastoris for serodiagnosis of Leptospira infection in rodents.
abstract: The applicability of the recombinant LipL32 for serodiagnosis of leptospiral infection in field rodents was assessed in this study. An immunodominant region of LipL32 was determined by monoclonal antibodies, and then, truncated LipL32 (tLipL32) was designed to contain the region (87-188th amino acid). The tLipL32 was compared between two recombinant expression hosts Escherichia coli and Pichia pastoris in ELISA. With field rat sera, tLipL32 expressed by P. pastoris (tLipL32p) had high antigenicity without background reactions, while tLipL32 expressed by E. coli (tLipL32e) showed high background reactions, which were reduced by pre-adsorption of sera with E. coli. To evaluate tLipL32-ELISA, field rat sera were tentatively divided into a Leptospira infection positive (12 sera) and a negative group (12 sera) based on the results from flaB gene PCR of kidney samples and WB with whole Leptospira cell. Consequently, the sensitivity of tLipL32p-ELISA for field rat sera was 83% . A similar result was obtained from tLipL32e-ELISA with adsorbed sera, (92%). However, sensitivity of tLipL32e-ELISA using sera without an adsorption treatment was 50%. Regardless of the expression host, tLipL32-ELISA had 100% specificity and sensitivity in experimentally infected laboratory rats. These results suggest that recombinant LipL32 expressed by P. pastoris is more applicable for serodiagnosis in field rats due to a lack of background reaction.
authors.affiliation: Department of Microbiology, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan.
authors.forename: Kanae
authors.initials: K
authors.lastname: Shiokawa
authors.forename: Chandika D
authors.initials: CD
authors.lastname: Gamage
authors.forename: Nobuo
authors.initials: N
authors.lastname: Koizumi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sakoda
authors.forename: Kenta
authors.initials: K
authors.lastname: Shimizu
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Tsuda
authors.forename: Kumiko
authors.initials: K
authors.lastname: Yoshimatsu
authors.forename: Jiro
authors.initials: J
authors.lastname: Arikawa
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D007919:Leptospira / Q000276:immunology; D007922:Leptospirosis / Q000175:diagnosis*; D008074:Lipoproteins / Q000235:genetics / Q000276:immunology; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D010843:Pichia / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012698:Serologic Tests
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; C406589:LipL32 protein, Leptospira; D008074:Lipoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins