, Volume 66, Issue 1, pp 8–17 | Cite as

Evaluation of zero-length cross-linking procedure for immuno-magnetic separation of Leptospira

  • Stefan Schreier
  • Galayanee Doungchawee
  • Sudarat Chadsuthi
  • Darapond Triampo
  • Wannapong TriampoEmail author
Section Cellular and Molecular Biology


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.

Key words

Leptospira leptospirosis immuno-magnetic separation magnetic particles antibody immobilization zerolength cross-linker carbodiimide 



bovine serum albumin


capture efficiency


(1-3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride


Ellinghausen and McCullough liquid medium as modified by Johnson and Harris


isoelectric focusing


immuno-magnetic separation




sodium dodecyl sulphate polyacrylamide gel electrophoresis


room temperature


transmission electron microscopy


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 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.PubMedGoogle Scholar
  2. An Y. & Chen M. 2007. Preparation and self-assembly of carboxylic acid-functionalized silica. J. Colloid Interface Sci. 311: 507–513.CrossRefPubMedGoogle Scholar
  3. 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.CrossRefPubMedGoogle Scholar
  4. 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.CrossRefPubMedGoogle Scholar
  5. 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.CrossRefPubMedGoogle Scholar
  6. 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.CrossRefPubMedGoogle Scholar
  7. Brash J.L. & Wojciechowski P.W. 1996. Interfacial Phenomena and Bioproducts. Marcel Dekker, New York, 510 pp.Google Scholar
  8. 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.CrossRefGoogle Scholar
  9. Cabral J.M.S., Kennedy J.F. & Taylor R.F. 1991. Protein Immobilization: Fundamentals and Applications. Marcel Dekker, New York, 377 pp.Google Scholar
  10. 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.CrossRefGoogle Scholar
  11. Chen S., Liu L., Zhou J. & Jiang S. 2003. Controlling antibody orientation on charged self-assembled monolayers. Langmuir 19: 2859–2864.CrossRefGoogle Scholar
  12. 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.CrossRefPubMedGoogle Scholar
  13. 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.CrossRefPubMedGoogle Scholar
  14. 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.PubMedGoogle Scholar
  15. Faine S., Adler B., Bolin C. & Perolat P. 1994. Leptospira and leptospirosis. Medisci, Melbourne, 386 pp.Google Scholar
  16. 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.CrossRefPubMedGoogle Scholar
  17. 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.CrossRefGoogle Scholar
  18. 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.CrossRefGoogle Scholar
  19. 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.CrossRefPubMedGoogle Scholar
  20. 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.PubMedGoogle Scholar
  21. Grabarek Z., & Gergely J. 1990. Zero-length crosslinking procedure with the use of active esters. Anal. Biochem. 185: 131–135.CrossRefPubMedGoogle Scholar
  22. Henry R.A. & Johnson R.C. 1978. Distribution of the genus Leptospira in soil and water. Appl. Environ. Microbiol. 35: 492–499.PubMedGoogle Scholar
  23. Jönsson U., Malmqvist M. & Rönnberg I., 1985. Immobilization of immunoglobulins on silica surfaces. J. Biochem. 227: 373–378.Google Scholar
  24. Jung Y., Jeong J.Y. & Chung B.H. 2008. Recent advances in immobilization methods of antibodies on solid supports. Analyst 133: 697–701.CrossRefPubMedGoogle Scholar
  25. 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.CrossRefPubMedGoogle Scholar
  26. 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.CrossRefGoogle Scholar
  27. Laemmli U. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.CrossRefPubMedGoogle Scholar
  28. 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.Google Scholar
  29. Lund A., Hellemann A.L. & Vartdal F. 1988. Rapid isolation of K88+ Escherichia coli by using immunomagnetic particles. J. Clin. Microbiol. 26: 2572–2575.PubMedGoogle Scholar
  30. Meyerstein D. & Treinin A. 1962. The relation between lyotropic and spectroscopic properties of anions in solution. J. Chem. Soc. 66: 446–450.Google Scholar
  31. Ninham B.W. 2006. The present state of molecular forces. Progr. Colloid Polym. Sci. 133: 65–73.CrossRefGoogle Scholar
  32. 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.PubMedGoogle Scholar
  33. 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.CrossRefPubMedGoogle Scholar
  34. Skjerve E., Rorvik L.M. & Olsvik O. 1990. Detection of Listeria monocytogenes in foods by immunomagnetic separation. Appl. Environ. Microbiol. 56: 3478–3481.PubMedGoogle Scholar
  35. Smythe L.D. 1999. Leptospirosis worldwide. Wkly Epidemiol. Rec. 74: 237–242.Google Scholar
  36. 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.CrossRefGoogle Scholar
  37. 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.CrossRefGoogle Scholar
  38. 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.CrossRefPubMedGoogle Scholar
  39. 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.PubMedGoogle Scholar
  40. Visser H. 1992. Protein Interactions. VCH Verlag, Weinheim.Google Scholar
  41. 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.CrossRefPubMedGoogle Scholar
  42. Zhang Y. & Cremer P.S. 2006. Interactions between macromolecules and ions: the Hofmeister series. Curr. Opin. Chem. Biol. 10: 658–663.CrossRefPubMedGoogle Scholar

Copyright information

© © Versita Warsaw and Springer-Verlag Wien 2011

Authors and Affiliations

  • Stefan Schreier
    • 1
    • 2
    • 3
    • 4
  • Galayanee Doungchawee
    • 1
  • Sudarat Chadsuthi
    • 2
  • Darapond Triampo
    • 2
    • 5
  • Wannapong Triampo
    • 2
    • 3
    • 4
    Email author
  1. 1.Department of Pathobiology, Faculty of ScienceMahidol UniversityBangkokThailand
  2. 2.R&D Group of Biological and Environmental Physics, Department of Physics, Faculty of ScienceMahidol UniversityBangkokThailand
  3. 3.Center of Excellence for Vectors and Vector-Borne Diseases, Faculty of ScienceMahidol UniversityNakhon PathomThailand
  4. 4.ThEP CenterCHEBangkokThailand
  5. 5.Department of Chemistry (R3/1), Faculty of ScienceMahidol UniversityNakhon PathomThailand

Personalised recommendations