Development of SPC-ELISA: a new assay principle for the study of sulfated polysaccharide-protein interactions.
A sulfated polysaccharide-coating enzyme-linked immunosorbent assay (SPC-ELISA), a new screening assay for the study of interactions between sulfated polysaccharides and proteins, has been developed. Fibrinogen was used as representative for the protein. A microplate is coated with the sulfated polysaccharide to be tested and then incubated with various concentrations of fibrinogen. The bound fibrinogen is quantified by ELISA technique. The assay has been optimized with respect to coating procedure, incubation times, antibody concentrations, and detection conditions. Its capacity was demonstrated using three different sulfated polysaccharides: heparin, a sulfated glucuronogalactan extracted from a red algae, and a semisynthetic xanthan sulfate. Furthermore, the fibrinogen binding of semisynthetic laminarin sulfates with different degrees of sulfation showed good correlation with their anticoagulant effect as measured by fibrinogen clotting time. The intraassay as well as the interassay variations were lower than 8%. The binding properties observed in the SPC-ELISA correlated well with those found utilizing conventional gel permeation chromatography and fibrinogen affinity gel electrophoresis. Compared to these methods, the SPC-ELISA has several advantages: It is more rapid and far easier to perform, allows high throughput screening, and is suitable for automation. Furthermore, it is inexpensive, highly sensitive, and reproducible and has no special equipment requirements. Finally, the method represents the basis for multiple variations with regard to the target proteins as well as the detection methods.
Alban
S
S
Institute of Pharmacy, University of Regensburg, Regensburg, Germany. Susanne.Alban@chemie.uni-regensburg.de
Gastpar
R
R
D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D015203:Reproducibility of Results; D013464:Sulfuric Acids / Q000737:chemistry*
D011134:Polysaccharides; D013464:Sulfuric Acids; D005340:Fibrinogen