Leptospirosis constitutes a major health problem in tropical and subtropical countries and is caused by pathogenic Leptospira. Immuno-magnetic separation (IMS) is considered to be an effective pre-enrichment method to isolate Leptospira from liquid specimen. We applied an inexpensive and simple IMS protocol using zero-length cross-linkers to immobilize polyclonal anti-leptospiral antibodies onto magnetic particles. The IMS-system has been optimized and evaluated by the assessment of the capture efficiency (CE). Main parameters that influence the conjugation procedure were optimized, including the amount of protein per milligram of magnetic particles, the pH and ionic strength of the conjugation buffer. The bead-bound leptospiral fraction was identified by using acridine orange fluorescence dye. The highest value for CE occurred when using high molar phosphate saline buffer at a pH around the isoelectric point of the antibodies. Finally, up to 3×108 leptospiral cells per mL could have been captured with approximately 50 μg of antibody-labelled particles. Strong particle agglutination could be observed during incubation for leptospiral concentrations in the range of 107–108 cells per mL. Despite covalent binding, we show that the physical adsorption parameters pH and ionic strength of the conjugation buffer greatly affect the entire immobilization process with regard to the CE, thus being able to increase the reactivity of the particles. We therefore conclude that a well-adjusted conjugation buffer for the used chemistry could possibly replace expensive and more complicated antibody immobilization methods.
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bovine serum albumin
Ellinghausen and McCullough liquid medium as modified by Johnson and Harris
sodium dodecyl sulphate polyacrylamide gel electrophoresis
transmission electron microscopy
Alexander A.D., Evans L.B., Baker M.F., Baker H.J., Ellison D. & Marriapan M. 1975. Pathogenic leptospiras isolated from Malaysian surface waters. Appl. Microbiol. 29: 30–33.
An Y. & Chen M. 2007. Preparation and self-assembly of carboxylic acid-functionalized silica. J. Colloid Interface Sci. 311: 507–513.
Arakawa T. & Timasheff S.N. 1984. Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding. Biochemistry 23: 5912–5923.
Batchelor J.D., Olteanu A., Tripathy A. & Pielak G.J. 2004. Impact of protein denaturants and stabilizers on water structure. J. Am. Chem. Soc. 126: 1958–1961.
Bharti A.R., Nally J.E., Ricaldi J.N., Matthias M.A., Diaz M.M., Lovett M.A., Levett P.N., Gilman R.H., Willig M.R. & Gotuzzo E. 2003. Leptospirosis: a zoonotic disease of global importance. Lancet Infect. Dis. 3: 757–771.
Bradford M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.
Brash J.L. & Wojciechowski P.W. 1996. Interfacial Phenomena and Bioproducts. Marcel Dekker, New York, 510 pp.
Buijs J., White D.D. & Norde W. 1997. The effect of adsorption on the antigen binding by IgG and its F(ab′)2 fragments. Colloids Surfaces B: Biointerfaces 8: 239–249.
Cabral J.M.S., Kennedy J.F. & Taylor R.F. 1991. Protein Immobilization: Fundamentals and Applications. Marcel Dekker, New York, 377 pp.
Chang I.N., Lin J.N., Andrade J.D. & Herron J.N. 1995. Adsorption mechanism of acid pretreated antibodies on dichlorodimethylsilane-treated silica surfaces. J. Colloid Interface Sci. 174: 10–23.
Chen S., Liu L., Zhou J. & Jiang S. 2003. Controlling antibody orientation on charged self-assembled monolayers. Langmuir 19: 2859–2864.
Danczyk R., Krieder B., North A., Webster T., HogenEsch H. & Rundell A. 2003. Comparison of antibody functionality using different immobilization methods. Biotechnol. Bioeng. 84: 215–223.
Deponte S., Steingroewer J., Löser C., Boschke E. & Bley T. 2004. Biomagnetic separation of Escherichia coli by use of anion-exchange beads: measurement and modeling of the kinetics of cell-bead interactions. Anal. Bioanal. Chem. 379: 419–426.
Doungchawee G., Phulsuksombat D., Naigowit P., Khoaprasert Y., Sangjun N., Kongtim S. & Smythe L. 2005. Survey of leptospirosis of small mammals in Thailand. Southeast Asian J. Trop. Med. Public Health 36: 1516–1522.
Faine S., Adler B., Bolin C. & Perolat P. 1994. Leptospira and leptospirosis. Medisci, Melbourne, 386 pp.
Fernandes C.P.H., Seixas F.K., Coutinho M.L., Vasconcellos F.A., Moreira Â.N., Conceiçăo F.R., Dellagostin O.A. & Aleixo J.A.G. 2008. An immuno-magnetic separation-PCR method for detection of pathogenic Leptospira in biological fluids. Hybridoma 27: 381–386.
Fu Z., Rogelj S. & Kieft T.L. 2005. Rapid detection of Escherichia coli O157: H7 by immunomagnetic separation and real-time PCR. Food Microbiol. 99: 47–57.
Fuentes M., Mateo C., Guisan J.M. & Fernández-Lafuente R. 2005. Preparation of inert magnetic nano-particles for the directed immobilization of antibodies. Bionsens. Bioelectron. 20: 1380–1387.
Ganoza C.A., Matthias M.A., Collins-Richards D., Brouwer K.C., Cunningham C.B., Segura E.R., Gilman R.H., Gotuzzo E. & Vinetz J.M. 2006. Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira. PLoS Med. 3: e308.
Goodridge L., Chen J. & Griffiths M. 1999. Development and characterization of a fluorescent-bacteriophage assay for detection of Escherichia coli O157: H7. Appl. Environ. Microbiol. 65: 1397–1404.
Grabarek Z., & Gergely J. 1990. Zero-length crosslinking procedure with the use of active esters. Anal. Biochem. 185: 131–135.
Henry R.A. & Johnson R.C. 1978. Distribution of the genus Leptospira in soil and water. Appl. Environ. Microbiol. 35: 492–499.
Jönsson U., Malmqvist M. & Rönnberg I., 1985. Immobilization of immunoglobulins on silica surfaces. J. Biochem. 227: 373–378.
Jung Y., Jeong J.Y. & Chung B.H. 2008. Recent advances in immobilization methods of antibodies on solid supports. Analyst 133: 697–701.
Klose J. & Kobalz U. 1995. Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electrophoresis 16: 1034–1059.
Ko A.I., Goarant C. & Picardeau M. 2009. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nature Rev. Microbiol. 7: 736–747.
Laemmli U. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
LoNostro P., Lopes J.R., Ninham B.W. & Baglioni P. 2002. Effects of cations and anions on the formation of polypseudorotaxanes. J. Phys. Chem. 106: 2166–2174.
Lund A., Hellemann A.L. & Vartdal F. 1988. Rapid isolation of K88+ Escherichia coli by using immunomagnetic particles. J. Clin. Microbiol. 26: 2572–2575.
Meyerstein D. & Treinin A. 1962. The relation between lyotropic and spectroscopic properties of anions in solution. J. Chem. Soc. 66: 446–450.
Ninham B.W. 2006. The present state of molecular forces. Progr. Colloid Polym. Sci. 133: 65–73.
Pugh B.F. & Cox M.M. 1988. High salt activation of recA protein ATPase in the absence of DNA. J. Biol. Chem. 263: 76–83.
Schreier S., Triampo W., Doungchawee G., Triampo D. & Chadsuthi S. 2009. Leptospirosis research: fast, easy and reliable enumeration of mobile leptospires. Biol. Res. 42: 5–12.
Skjerve E., Rorvik L.M. & Olsvik O. 1990. Detection of Listeria monocytogenes in foods by immunomagnetic separation. Appl. Environ. Microbiol. 56: 3478–3481.
Smythe L.D. 1999. Leptospirosis worldwide. Wkly Epidemiol. Rec. 74: 237–242.
Taylor M.J., Ellis W.A., Montgomery J.M., Yan K.T., McDowell S.W.J. & Mackie D.P. 1996. Magnetic immuno capture PCR assay (MIPA): detection of Leptospira borgpetersenii serovar hardjo. Vet. Microbiol. 56: 135–145.
Timasheff S.N., Lee J.C., Pittz E.P. & Tweedy N. 1976. The interaction of tubulin and other proteins with structure-stabilizing solvents. J. Colloid Interface Sci. 55: 658–663.
Van Erp R., Linders Y.E., van Sommeren A.P. & Gribnau T.C. 1992. Characterization of monoclonal antibodies physically adsorbed onto polystyrene latex particles. J. Immunol. Methods 152: 191–199.
Varshney M., Yang L., Su X.L. & Li Y. 2005. Magnetic nanoparticle-antibody conjugates for the separation of Escherichia coli O157: H7 in ground beef. J. Food Prot. 68: 1804–1811.
Visser H. 1992. Protein Interactions. VCH Verlag, Weinheim.
Yan K.T., Ellis W.A., Montgomery J.M., Taylor M.J., Mackie D.P. & McDowell W.J. 1998. Development of an immunomagnetic antigen capture system for detecting leptospires in bovine urine. Res. Vet. Sci. 64: 119–124.
Zhang Y. & Cremer P.S. 2006. Interactions between macromolecules and ions: the Hofmeister series. Curr. Opin. Chem. Biol. 10: 658–663.
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Schreier, S., Doungchawee, G., Chadsuthi, S. et al. Evaluation of zero-length cross-linking procedure for immuno-magnetic separation of Leptospira . Biologia 66, 8–17 (2011). https://doi.org/10.2478/s11756-010-0143-2
- immuno-magnetic separation
- magnetic particles
- antibody immobilization
- zerolength cross-linker