Comparative study of the three different fluorophore antibody conjugation strategies

Dilip Shrestha, Adrienn Bagosi, J. Szöllősi, A. Jenei

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1449-1463
Number of pages15
JournalAnalytical and Bioanalytical Chemistry
Volume404
Issue number5
DOIs
Publication statusPublished - Sep 2012

Fingerprint

Fluorophores
Antibodies
Amines
Antigens
Labeling
Carbohydrates
Cysteamine
Costs and Cost Analysis
Costs
Proteins
Derivatives
Oxidation
Research
Experiments

Keywords

  • Amine targeted
  • Carbohydrate targeted
  • Fluorophore antibody conjugation
  • Mercaptoethylamine
  • Sodium periodate
  • Sulfhydryl targeted

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry

Cite this

Comparative study of the three different fluorophore antibody conjugation strategies. / Shrestha, Dilip; Bagosi, Adrienn; Szöllősi, J.; Jenei, A.

In: Analytical and Bioanalytical Chemistry, Vol. 404, No. 5, 09.2012, p. 1449-1463.

Research output: Contribution to journalArticle

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