Identification of surface antigens of <i>Trichomonas vaginalis</i>

, Woo, N S; , Min, D Y; , Leem, M H; , Choi, Y K

doi:1993.31.1.37

Korean J Parasito > Volume 31(1):1993 > Article

Original Article


Korean J Parasitol. 1993 Mar;31(1):37-42. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1993.31.1.37
Copyright © 1993 by The Korean Society for Parasitology


Identification of surface antigens of Trichomonas vaginalis
Nam-Sick Woo,¹Duk-Young Min,^*¹Mi-Hyea Leem,¹ and Yong-Keel Choi²
¹Department of Parasitology, College of Medicine, Hanyang University, Seoul 133-791, Korea.
²Department of Biology, College of Natural Science, Hanyang University, Seoul 133-791, Korea.

Received December 28, 1992; Accepted January 26, 1993.
Abstract
Surface proteins of Trichomonas vaginalis (T. vaginalis) were analyzed to study the antigenic variation. The surface proteins of protozoa were labelled by N-hydroxysuccinimide-biotin (NHS-biotin), the NHS-biotin-labelled proteins were immunoprecipitated with rabbit antiserum to purify the antigenic fractions and analysed by SDS-PAGE plus electroblotting. The results obtained in this study were as follows; Biotinylated T. vaginalis-proteins obtained from intact cell and cells disrupted prior to labelling were detected by anti-biotin-peroxidase in Western blots. Labelled proteins were immunoprecipitated by T. vaginalis-immunized rabbit serum and the six bands with the molecular weights of 46, 60, 68, 90, 130 and 220 kDa were identified as having antigenicity. T. vaginalis HY-1, HY-15 and ATCC 50148 were immunoprecipitated by immune rabbit serum after biotinylation and there were no difference from antigenic bands among these strains by this technique. In conclusion with the results obtained in the present study, it was assumed that surface proteins of T. vaginalis were labelled by biotinylation and the six labelled bands at 46, 60, 68, 90, 130 and 220 kDa in their molecular weight were identified as having antigenicity by immunoprecipitation (IP) and this biotinylation-IP technique may be used for further study of surface antigen of T. vaginalis.

Figures

Fig. 1
Immunoprecipitation of biotinylated T. vaginalis. 120 µl aliquots of trichomonal proteins labelled at concentrations of 1 mM (A) or 10 mM (B) NHS-biotin were incubated with 50 µl of normal (lane 5) and immune rabbit serum in the presence of 0 (lane 1), 125 (lane 2), 450 (lane 3) or 1075 µl (lane 4) lysis buffer respectively. Immunoprecipitated complexes were separated by SDS-PAGE and electroblotting and labelled proteins were detected by antibiotin-HRP.


	Fig. 2 Western blots of biotin-labelled proteins of T. vaginalis HY-1 obtained from intact cells (lane 1) and from control cells disrupted prior to labelling (lane 2). A: Amidio black staining. B: Anti-biotin detection of biotinylated proteins. C: immuno-precipitated proteins labelled by NHS-biotin and detected by anti-biotin-HRP.


	Fig. 3 Western blots of biotin-labelled proteins of T. vaginalis HY-1 (lane-1), HY-15 (lane 2) and ATCC 50148 (lane 3). A: Amido black staining of T. vaginalis proteins. B: Anti-biotin detection of biotinylated proteins obtained from intact cells. C: Immunoprecipitation of biotinylated proteins obtained from intact cells.

References


1.	Min DY, Leem MH, Kim JM, Choi YK. [Comparative antigen analysis of Trichomonas vaginalis by enzyme-linked immunoelectrotransfer blot technique]. Korean J Parasitol 1992;30(4):323–328.

2.	Alderete JF. Antigen analysis of several pathogenic strains of Trichomonas vaginalis. Infect Immun 1983;39(3):1041–1047.

3.	Alderete JF. Identification of immunogenic and antibody-binding membrane proteins of pathogenic Trichomonas vaginalis. Infect Immun 1983;40(1):284–291.

4.	Alderete JF, Demes P, Gombosova A, Valent M, Fabusova M, Janoska A, Stefanovic J, Arroyo R. Specific parasitism of purified vaginal epithelial cells by Trichomonas vaginalis. Infect Immun 1988;56(10):2558–2562.

5.	Alderete JF, Suprun-Brown L, Kasmala L, Smith J, Spence M. Heterogeneity of Trichomonas vaginalis and discrimination among trichomonal isolates and subpopulations with sera of patients and experimentally infected mice. Infect Immun 1985;49(3):463–468.

6.	Anderson DJ, Blobel G. Immunoprecipitation of proteins from cell-free translations. Methods Enzymol 1983;96:111–120.

7.	Andrews BJ, Bjorvatn B. Immunoprecipitation studies with biotinylated Entamoeba histolytica antigens. Parasite Immunol 1991;13(1):95–103.

8.	Cole SR, Ashman LK, Ey PL. Biotinylation: an alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates. Mol Immunol 1987;24(7):699–705.

9.	Diamond LS. Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae. J Parasitol 1968;54(5):1047–1056.

*10.*	Finn FM, Titus G, Horstman D, Hofmann K. Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor. Proc Natl Acad Sci U S A 1984;81(23):7328–7332.

*11.*	Hurley WL, Finkelstein E, Holst BD. Identification of surface proteins on bovine leukocytes by a biotin-avidin protein blotting technique. J Immunol Methods 1985;85(1):195–202.

*12.*	Kendall C, Ionescu-Matiu I, Dreesman GR. Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA). J Immunol Methods 1983;56(3):329–339.

*13.*	Krieger JN, Ravdin JI, Rein MF. Contact-dependent cytopathogenic mechanisms of Trichomonas vaginalis. Infect Immun 1985;50(3):778–786.

*14.*	Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680–685.

*15.*	Lima MF, Villalta F. Trypanosoma cruzi trypomastigote clones differentially express a parasite cell adhesion molecule. Mol Biochem Parasitol 1989;33(2):159–170.

*16.*	Scott MT, Snary D. Protective immunisation of mice using cell surface glycoprotein from Trypanosoma cruzi. Nature 1979;282(5734):73–74.

*17.*	Slot JW, Geuze HJ. A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol 1985;38(1):87–93.

*18.*	Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150(1):76–85.

*19.*	Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76(9):4350–4354.

*20.*	Tsang VC, Peralta JM, Simons AR. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol 1983;92:377–391.