A competitive alphascreen assay for detection of hyaluronan

Xiayun Huang, Tannin A. Schmidt, Claire Shortt, Shivani Arora, Akira Asari, Thorsten Kirsch, Mary Cowman

Research output: Contribution to journalArticle

Abstract

A method for specific quantification of hyaluronan (HA) concentration using AlphaScreen® (Amplified Luminescent Proximity Homogeneous Assay) technology is described. Two types of hydrogel-coated and chromophore-loaded latex nanobeads are employed. The proximity of the beads in solution is detected by excitation of the donor bead leading to the production of singlet oxygen, and chemiluminescence from the acceptor bead upon exposure to singlet oxygen. In the HA assay, the donor bead is modified with streptavidin, and binds biotin-labeled HA. The acceptor bead is modified with Ni(II), and is used to bind a specific recombinant HA-binding protein (such as HABP; aggrecan G1-IGD-G2) with a His-tag. Competitive inhibition of the HA-HABP interaction by free unlabeled HA in solution is used for quantification. The assay is specific for HA, and not dependent on HA molecular mass above the decasaccharide. HA can be quantified over a concentration range of approximately 30-1600 ng/mL using 2.5 μL of sample, for a detectable mass range of approximately 0.08-4 ng HA. This sensitivity of the AlphaScreen assay is greater than existing ELISA-like methods, due to the small volume requirements. HA can be detected in biological fluids using the AlphaScreen assay, after removal of bound proteins from HA and dilution or removal of other interfering proteins and lipids.

Original languageEnglish (US)
Pages (from-to)137-147
Number of pages11
JournalGlycobiology
Volume28
Issue number3
DOIs
StatePublished - Mar 1 2018

Fingerprint

Hyaluronic Acid
Assays
Singlet Oxygen
CD44 Antigens
Aggrecans
Chemiluminescence
Streptavidin
Hydrogel
Latex
Molecular mass
Chromophores
Biotin
Luminescence
Recombinant Proteins
Dilution
Proteins
Enzyme-Linked Immunosorbent Assay
Technology
Lipids
Fluids

Keywords

  • AlphaScreen
  • assay
  • hyaluronan
  • LOCI
  • quantification

ASJC Scopus subject areas

  • Biochemistry

Cite this

Huang, X., Schmidt, T. A., Shortt, C., Arora, S., Asari, A., Kirsch, T., & Cowman, M. (2018). A competitive alphascreen assay for detection of hyaluronan. Glycobiology, 28(3), 137-147. https://doi.org/10.1093/glycob/cwx109

A competitive alphascreen assay for detection of hyaluronan. / Huang, Xiayun; Schmidt, Tannin A.; Shortt, Claire; Arora, Shivani; Asari, Akira; Kirsch, Thorsten; Cowman, Mary.

In: Glycobiology, Vol. 28, No. 3, 01.03.2018, p. 137-147.

Research output: Contribution to journalArticle

Huang, X, Schmidt, TA, Shortt, C, Arora, S, Asari, A, Kirsch, T & Cowman, M 2018, 'A competitive alphascreen assay for detection of hyaluronan', Glycobiology, vol. 28, no. 3, pp. 137-147. https://doi.org/10.1093/glycob/cwx109
Huang X, Schmidt TA, Shortt C, Arora S, Asari A, Kirsch T et al. A competitive alphascreen assay for detection of hyaluronan. Glycobiology. 2018 Mar 1;28(3):137-147. https://doi.org/10.1093/glycob/cwx109
Huang, Xiayun ; Schmidt, Tannin A. ; Shortt, Claire ; Arora, Shivani ; Asari, Akira ; Kirsch, Thorsten ; Cowman, Mary. / A competitive alphascreen assay for detection of hyaluronan. In: Glycobiology. 2018 ; Vol. 28, No. 3. pp. 137-147.
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