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Development of a solid-phase assay for measurement of sulfated glycosaminoglycan concentrations in equine synovial fluid

Stacey L. Oke DVM, MSc1,2, Mark B. Hurtig DVM, MVSc3, Robert A. Keates PhD4, and Jen R. Wright BSc5
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  • 1 Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1.
  • | 2 Present address is 20-7 Forest Hill Dr, Guelph, ON, Canada N1G 2E1.
  • | 3 Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1.
  • | 4 Department of Chemistry and Biochemistry, College of Physical and Engineering Science, University of Guelph, Guelph, ON, Canada N1G 2W1.
  • | 5 Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1.

Abstract

Objective—To develop a new 1,9-dimethylmethylene blue (DMMB) assay for measurement of sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF) by use of membrane technology and to compare the assay's ability to measure sGAG concentrations with that of 2 other established DMMB assays.

Sample Population—25 samples of SF collected from affected joints of 14 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses.

Procedure—A solid-phase DMMB assay was developed to measure sGAG concentrations in SF. Results for the assay were then compared with results obtained by use of the direct spectrophotometric method (ie, Farndale method) and microplate DMMB assay.

Results—The solid-phase assay and direct spectrophotometric assay measured the same sGAG concentrations in identical equine SF, but those concentrations differed significantly from results obtained by use of the microplate DMMB assay. All other aspects of the solidphase DMMB assay were comparable to both the direct spectrophotometric and microplate DMMB assays.

Conclusions and Clinical Relevance—The new solid-phase assay can be used interchangeably with the direct spectrophotometric method to measure sGAG concentrations in equine SF samples, but it cannot be interchanged with the microplate DMMB assay. Results can be rapidly obtained with the solidphase assay. Also, the solid-phase assay can detect nanogram quantities of sGAGs in SF, circumvent the problem of premature precipitation of sGAG-dye complexes, and provide quantitative or qualitative results. The solid-phase assay may replace other DMMB assays for measuring sGAG concentrations in SF obtained from horses. ( Am J Vet Res 2003; 64:894–899)

Abstract

Objective—To develop a new 1,9-dimethylmethylene blue (DMMB) assay for measurement of sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF) by use of membrane technology and to compare the assay's ability to measure sGAG concentrations with that of 2 other established DMMB assays.

Sample Population—25 samples of SF collected from affected joints of 14 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses.

Procedure—A solid-phase DMMB assay was developed to measure sGAG concentrations in SF. Results for the assay were then compared with results obtained by use of the direct spectrophotometric method (ie, Farndale method) and microplate DMMB assay.

Results—The solid-phase assay and direct spectrophotometric assay measured the same sGAG concentrations in identical equine SF, but those concentrations differed significantly from results obtained by use of the microplate DMMB assay. All other aspects of the solidphase DMMB assay were comparable to both the direct spectrophotometric and microplate DMMB assays.

Conclusions and Clinical Relevance—The new solid-phase assay can be used interchangeably with the direct spectrophotometric method to measure sGAG concentrations in equine SF samples, but it cannot be interchanged with the microplate DMMB assay. Results can be rapidly obtained with the solidphase assay. Also, the solid-phase assay can detect nanogram quantities of sGAGs in SF, circumvent the problem of premature precipitation of sGAG-dye complexes, and provide quantitative or qualitative results. The solid-phase assay may replace other DMMB assays for measuring sGAG concentrations in SF obtained from horses. ( Am J Vet Res 2003; 64:894–899)