1. Magnetic bead based assays for complement component C5.
- Author
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DiScipio RG and Schraufstatter IU
- Subjects
- Complement C3-C5 Convertases metabolism, Complement C3b metabolism, Complement C5 metabolism, Complement C7 metabolism, Complement Hemolytic Activity Assay, Complement Inactivating Agents pharmacology, Dextran Sulfate pharmacology, Hemolysis, Humans, Iron chemistry, Polysaccharides pharmacology, Protein Binding, Sepharose chemistry, beta-Glucans pharmacology, Complement Activation drug effects, Complement C3b immunology, Complement C5 immunology, Complement C7 immunology, Immunologic Techniques, Magnetics
- Abstract
Two novel magnetic agarose bead based assays have been developed to measure complement component C5 interaction with C3b and the Factor I Modules (FIMs) of C7. One innovation was to couple C3b onto the magnetic agarose bead using the alternative pathway C3 convertase, which resulted in a linkage of the ligand by a covalent ester bond. A second innovation was to employ nickel ion charged N,N,N'-tris(carboxymethyl)ethylene-diamine-magnetic agarose to capture recombinantly prepared C7 FIMs that were expressed with an oligo-histidine linker followed by an acidic domain that provided a spacer enabling the C7 modules exposure to C5. Detection was brought about by peroxidase coupled to C5. Both assays exhibited adequate statistics suitable for screening. As examples of the utility of these new methods, we chose to examine influence of natural products on C5 interaction. Fucoidan and β-glucans were observed to inhibit C3b-C5 interaction, and dextran sulfate was similarly active; however, rosmarinic acid had no measurable effect. In contrast only β-glucans from two species of macrofungi were able to interfere with interaction of C5 with the FIMs of C7., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
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