Diagnosis of American cutaneous leishmaniasis by enzyme immunoassay using membrane antigens of Leishmania (Viannia) braziliensis.
This study evaluated the reactivity of membrane antigens of Leishmania (Viannia) braziliensis for the diagnosis of ACL by enzyme immunoassay (EIA). Promastigotes of L. (V.) braziliensis were grown in medium 199 and lysed in a sonicator. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that specific proteins of L. (V.) braziliensis (apparent molecular weights 36 kDa and 48-56 kDa) were recognized by sera from ACL patients. These proteins were eluted from the SDS-PAGE and tested in EIA-IgG with sera from ACL patients, healthy individuals, patients with toxoplasmosis, paracoccidioidomycosis, syphilis, tuberculosis, leprosy, and Chagas disease. The EIA-IgG with membrane antigens allowed us to distinguish patients with ACL from healthy individuals and patients with other diseases (P < 0.0001), and showed a sensitivity of 93.3% and specificity of 90.8%, not including Chagas disease patients. 2D-SDS-PAGE followed by Western blotting was performed to improve the characterization of the antigens, and showed a component with isoelectric points near the acid pH side and apparent molecular weights of 48-56 kDa. The results showed good sensitivity and specificity of EIA-IgG with membrane antigens, indicating their potential use for diagnosis of ACL, as well as seroepidemiological surveys and follow-up of clinically cured patients.
Postgraduate Program in Health Sciences, State University of Maringá, Maringá, Paraná, Brazil.
Cissiara Manetti
CM
Skraba
Postgraduate Program in Health Sciences, State University of Maringá, Maringá, Paraná, Brazil.
Raíssa Bocchi
RB
Pedroso
Postgraduate Program in Biosciences Applied to Pharmacy, State University of Maringá, Paraná, Brazil.
Adriana
A
Fiorini
State University of Maringá, Paraná, Brazil.
Fábio Rogério
FR
Rosado
Department of Clinical Analysis and Biomedicine, State University of Maringá, Maringá, Paraná, Brazil.
Sandra Mara Alessi
SM
Aristides
Department of Clinical Analysis and Biomedicine, State University of Maringá, Maringá, Paraná, Brazil.
Maria Valdrinez Campana
MV
Lonardoni
Department of Clinical Analysis and Biomedicine, State University of Maringá, Maringá, Paraná, Brazil.
Jorge Juarez Vieira
JJ
Teixeira
Department of Clinical Analysis and Biomedicine, State University of Maringá, Maringá, Paraná, Brazil. Electronic address: tgvsilveira@uem.br.
Thaís Gomes Verzignassi
TG
Silveira
D000913:Antibodies, Protozoan / Q000097:blood*; D000953:Antigens, Protozoan / Q000737:chemistry; D015153:Blotting, Western; D019411:Clinical Laboratory Techniques / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods; D007892:Leishmania braziliensis / Q000276:immunology*; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis* / Q000276:immunology; D008970:Molecular Weight; D012680:Sensitivity and Specificity
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan
American cutaneous leishmaniasis; Diagnosis; EIA; Western blotting