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Comparative study of the three different fluorophore antibody conjugation strategies

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The progression in bioconjugational chemistry has significantly contributed to the evolution and success of protein biology. Mainly, antibody chemistry has been a subject of intensive study owing to the expansion of research areas warranted by using various derivatives of conjugated antibodies. Three reactive moieties (amine, sulfhydryl and carbohydrate) in the antibodies are chiefly favored for the conjugational purpose. This feature is known for decades, nevertheless, amine based conjugation is still the most preferred strategy despite the appreciation the other two methods receive in conserving the antigen binding affinity (ABA). No single report has been published, according to our knowledge, where these three conjugation strategies were applied to the same fluorophore antibody systems. In this study, we evaluated conjugation yield, time demand and cost efficiency of these conjugation procedures. Our results showed that amine based conjugations was by far the best technique due to its simplicity, rapidity, ease of operation, higher conjugate yield, cheaper cost and potential for larger fluorophore/protein labeling ratio without having much effect in ABA. Furthermore, sulfhydryl labeling clearly excelled in terms of reduced non-specific binding and mild effect in ABA but was usually complicated by an asymmetric antibody reduction due to mercaptoethylamine while carbohydrate oxidation based strategy performed the worst during our experiment.

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Antigen binding affinity


Cluster of Differentiation 1d




Intercellular Adhesion Molecule 1


Major histocompatibility complex




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This work was supported, in whole or in part, by research grants from the Hungarian Scientific Research Fund (K68763), the European Commission grants (LSHC-CT-2005-018914) -ATTACK, MCRTN-CT-2006-036946-2 (IMMUNANOMAP), the New Hungary Development Plan co-financed by the European Social Fund and the European Regional Development Fund (TAMOP-4.2.2-08/1-2008-0019, TAMOP-4.2.1/B-09/1/KONV-2010-007 and TAMOP-4.2.2/B-10/1-2010-0024).We are thankful to the members of receptor tyrosine kinase group, University of Debrecen for their direct/indirect support during the study. We also appreciate the informal discussions that we had with György Vereb and Daniel Beyer.

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Correspondence to János Szöllősi.

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Shrestha, D., Bagosi, A., Szöllősi, J. et al. Comparative study of the three different fluorophore antibody conjugation strategies. Anal Bioanal Chem 404, 1449–1463 (2012).

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