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

Abstract

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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Abbreviations

BSA:

bovine serum albumin

CE:

capture efficiency

EDC:

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

EMJH:

Ellinghausen and McCullough liquid medium as modified by Johnson and Harris

IEF:

isoelectric focusing

IMS:

immuno-magnetic separation

NHS:

N-hydroxysuccinimide

SDS-PAGE:

sodium dodecyl sulphate polyacrylamide gel electrophoresis

RT:

room temperature

TEM:

transmission electron microscopy

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Correspondence to Wannapong Triampo.

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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

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Key words

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