Leishmanin skin test in guinea pig with a single purified protein of Leishmania major

• Published in: Experimental parasitology Vol. 111; no. 4; pp. 239 - 243
• Main Authors: Khabiri, A.R., Bagheri, F., Alimohammadian, M.H., Assmar, M., Nadaf, S.R.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01-12-2005; Elsevier
• Subjects: Animals; Antibodies, Monoclonal - immunology; Antigens, Protozoan - immunology; Antigens, Protozoan - isolation & purification; Biological and medical sciences; Blotting, Western; Chromatography, Affinity; Chromatography, High Pressure Liquid; Delayed-type hypersensitivity; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Female; Fundamental and applied biological sciences. Psychology; Guinea Pigs; Hybridomas; Hypersensitivity, Delayed; Intradermal Tests - standards; Leishmania major; Leishmania major - immunology; Leishmaniasis - diagnosis; Leishmaniasis - immunology; Leishmanin; Life cycle. Host-agent relationship. Pathogenesis; Mammalia; Mice; Mice, Inbred BALB C; Monoclonal antibody; Protozoa; Protozoan Proteins - immunology; Protozoan Proteins - isolation & purification; Single antigen; Vertebrates: general zoology, morphology, phylogeny, systematics, cytogenetics, geographical distribution; Leishmanin; Single antigen; Monoclonal antibody; Delayed-type hypersensitivity; Kinetoplastida; Protozoa; Hypersensitivity; Rodentia; Parasite; Protein; Antigen; Vertebrata; Mammalia; Guinea pig; Skin; Leishmania major
• ISSN: 0014-4894; 1090-2449
• DOI: 10.1016/j.exppara.2005.08.011

A series of hybridomas was produced by fusion of SP2/0 myeloma cells with spleen cells of mice immunized with
Leishmania major (
L. major). The reactivity of secreted monoclonal antibodies (mAbs) was evaluated against available leishmanin antigen by enzyme linked immunosorbent assay. Only one hybridoma designated as 7F9 secreted IgG1 mAb which was shown to be reactive with leishmanin. This mAb was further tested against four species of
Leishmania (
L. donovani,
L. tropica,
L. infantum,
L. major) and a recombinant gp63. Among the four species tested it was shown to be only reactive with promastigotes of
L. major. The antigen recognized by this mAb was purified and analyzed from both sonicated and supernatant cultures of
L. major by immunoaffinity chromatography and reverse phase high performance liquid chromatography. The purified antigen, which gave a single band of 56
kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis elicited a strong delayed-type hypersensitivity (DTH) reaction in guinea pigs sensitized with
L. major. It was almost of the same degree as that produced by leishmanin. These results suggest that an
L. major—specific antigen is an alternative as a specific diagnostic skin test reagent, which could lead to a better understanding of the mechanism of DTH in
L. major.