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Solid-Phase Synthesis of a Biotin Derivative and its Application to the Development of Anti-Biotin Antibodies

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Abstract

A biotin derivative, namely biotin–aminocaproic acid–lysine (BAL), was synthesized with solid-phase chemistry, conjugated to a carrier-protein, and used for rabbit immunization. The aminocaproic acid–lysine “long-arm” was used in order to project the biotin-hapten above the carrier-protein surface. Lysine was selected due to its Nε-amino group, through which BAL was conjugated to the carrier-protein. BAL was synthesized on a commercially available resin with the Fmoc-solid-phase strategy; this has simplified the experimental procedure, overcome the need for intermediate purification steps, and led to a final product of high purity, with high yield. The anti-BAL antibodies recognized free biotin, as shown with an in-house-developed ELISA, in which biotin conjugated to a synthetic “lysine–dendrimer” was used to coat the ELISA microwells. In immunocytology and Western-blot experiments, the anti-BAL antibodies led to similar results with those obtained with streptavidin. Synthetic derivatives of hapten molecules that can be easily prepared with solid-phase chemistry, such as BAL, may be used for the development of specific antibodies for the corresponding hapten.

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Abbreviations

2-CTC:

2-Chloro-trityl-chloride

ABTS:

2,2′-Azino-bis-(3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt

Aca:

Aminocaproic acid

BCA:

Bicinchoninic acid

Boc:

Tert-butoxycarbonyl

Cy3:

Cyanide dye 3

DAB:

3,3′-Diaminobenzidine

DCM:

Dichloromethane

DIC:

N,N′-Diisopropylcarbodiimide

DMEM:

Dulbecco’s modified eagle medium

DMF:

N,N′-Dimethylformamide

DMSO:

Dimethyl-sulfoxide

DTT:

Dithiothreitol

EDC:

1-Ethyl-3-(3-dimethyl-amino-propyl) carbodiimide hydrochloride

EDTA:

Ethylenediaminetetra acetate

ELISA:

Enzyme-linked immunosorbent assay

FCS:

Fetal calf serum

FITC:

Fluorescein isothiocyanate

Fmoc:

9-Fluorenylmethoxycarbonyl

GA:

Glutaraldehyde

HOBt:

Hydroxybenzotriazole

HRP:

Horseradish peroxidase

MW:

Molecular weight

NHS-LC-Biotin:

N-Hydroxy-succinimidyl-long chain-biotin

NP-40:

Nonyl phenoxylpolyethoxyl-ethanol

OD:

Optical density

PBS:

Phosphate-buffered saline

PMSF:

Phenylmethylsulfonyl fluoride

PPD:

p-Phenylenediamine

r.t.:

Room temperature

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

THF:

Tetrahydrofuran

TFA:

Trifluoroacetic acid

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Authors and Affiliations

  1. Immunopeptide Chemistry Lab, Institute of Radioisotopes & Radiodiagnostic Products, National Centre for Scientific Research “Demokritos”, Patriarchou Grigoriou & Neapoleos, Aghia Paraskevi Attikis, Athens, 153 10, Greece

    Ilias Papasarantos, Persefoni Klimentzou, Vassiliki Koutrafouri, Christos Zikos, Maria Paravatou-Petsotas & Evangelia Livaniou

  2. Department of Neurology, Medical School, University of Athens, Eginition Hospital, Athens, Greece

    Maria Anagnostouli

Authors
  1. Ilias Papasarantos
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  2. Persefoni Klimentzou
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  3. Vassiliki Koutrafouri
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  4. Maria Anagnostouli
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  5. Christos Zikos
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  6. Maria Paravatou-Petsotas
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  7. Evangelia Livaniou
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Correspondence to Evangelia Livaniou.

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Papasarantos, I., Klimentzou, P., Koutrafouri, V. et al. Solid-Phase Synthesis of a Biotin Derivative and its Application to the Development of Anti-Biotin Antibodies. Appl Biochem Biotechnol 162, 221–232 (2010). https://doi.org/10.1007/s12010-009-8870-y

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  • DOI: https://doi.org/10.1007/s12010-009-8870-y

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