title: Development of a custom pentaplex sandwich immunoassay using Protein-G coupled beads for the Luminex® xMAP® platform.
abstract: Multiplex bead-based assays have many advantages over ELISA, particularly for the analyses of large quantities of samples and/or precious samples of limited volume. Although many commercial arrays covering multitudes of biologically significant analytes are available, occasionally the development of custom arrays is necessary. Here, the development of a custom pentaplex sandwich immunoassay using Protein G-coupled beads, for analysis using the Luminex® xMAP® platform, is described. This array was required for the measurement of candidate biomarkers of vaccine safety in small volumes of mouse sera. Optimisation of this assay required a stepwise approach: testing cross-reactivity of the antibody pairs, the development of an in-house serum diluent buffer as well as heat-inactivation of serum samples to prevent interference from matrix effects. We then demonstrate the use of this array to analyse inflammatory mediators in mouse serum after immunisation. The work described here exemplifies how Protein G-coupled beads offer a flexible and robust approach to develop custom multiplex immunoassays, which can be applied to a range of analytes from multiple species.
authors.affiliation: Mucosal Infection & Immunity Group, Section of Virology, Imperial College London, St Mary's Campus, London W2 1PG, United Kingdom. Electronic address: jacqueline.mcdonald@imperial.ac.uk.
authors.forename: Jacqueline U
authors.initials: JU
authors.lastname: McDonald
authors.affiliation: Mucosal Infection & Immunity Group, Section of Virology, Imperial College London, St Mary's Campus, London W2 1PG, United Kingdom.
authors.forename: Julia
authors.initials: J
authors.lastname: Ekeruche-Makinde
authors.affiliation: Bacteriology Division, MHRA-NIBSC, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom.
authors.forename: Mei M
authors.initials: MM
authors.lastname: Ho
authors.affiliation: Mucosal Infection & Immunity Group, Section of Virology, Imperial College London, St Mary's Campus, London W2 1PG, United Kingdom.
authors.forename: John S
authors.initials: JS
authors.lastname: Tregoning
authors.affiliation: Bacteriology Division, MHRA-NIBSC, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom. Electronic address: Omodele.Ashiru@nibsc.org.
authors.forename: Omodele
authors.initials: O
authors.lastname: Ashiru
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis*; D001426:Bacterial Proteins / Q000737:chemistry*; D015415:Biomarkers; D002097:C-Reactive Protein / Q000276:immunology; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007118:Immunoassay / Q000379:methods*; D007252:Influenza Vaccines / Q000008:administration & dosage* / Q000009:adverse effects; D008562:Membrane Glycoproteins / Q000276:immunology; D051379:Mice; D008863:Microspheres; D009419:Nerve Tissue Proteins / Q000276:immunology; D011971:Receptors, Immunologic / Q000276:immunology; D000073871:Triggering Receptor Expressed on Myeloid Cells-1; D000099153:Pentraxins
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D015415:Biomarkers; D002097:C-Reactive Protein; D007252:Influenza Vaccines; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D000073871:Triggering Receptor Expressed on Myeloid Cells-1; D000099153:Pentraxins; C517262:TREM1 protein, mouse
keywords: Beads; Biomarkers; Protein G; Sandwich immunoassay; Serum; xMAP®