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Updated: May 13, 2026

Identification of Protein Interacting Partners Using Tandem Affinity Purification
10:02

Identification of Protein Interacting Partners Using Tandem Affinity Purification

Published on: February 25, 2012

Use of T-2 toxin-immobilized amine-activated beads as an efficient affinity purification matrix for the isolation of

Soujanya Ratna Edupuganti1, Om Prakash Edupuganti, Richard O'Kennedy

  • 1Applied Biochemistry Group (ABG), School of Biotechnology, Dublin City University, Dublin 9, Ireland.

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
|March 19, 2013
PubMed

An affinity purification method that isolates T-2 toxin-specific IgY utilizing a T-2-toxin-immobilized column was developed. The T-2 toxin was covalently coupled via a carbonyldiimidazole-activated hydroxyl functional group to amine-activated sepharose beads. The affinity-purified IgY was characterized by gel electrophoresis, fast protein liquid chromatography, enzyme-linked immunosorbant assay, surface plasmon resonance and mass spectrometry. A competitive inhibition ELISA (CI-ELISA) was performed using affinity-purified IgY with a T-2 toxin detection sensitivity of 30 ng/mL, which falls within the maximum permissible limit of 100 ng/mL. The cross reactivity of IgY towards deoxynivalenol, zearalenone, fumonisin B1 and HT-2 was significantly reduced after affinity purification. A surface plasmon resonance (SPR)-based inhibition assay was also applied for quantitative determination of T-2 toxin in spiked wheat samples. The results obtained indicate the feasibility of utilizing this IgY-based assay for the detection of T-2 toxin in food samples.

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