Protein A–Nanoluciferase Fusion Protein for Generalized, Sensitive Detection of Immunoglobulin G

Details

• Alternative Title:
  Anal Biochem
• Personal Author:
  Nandy, Suman ; Crum, Mary ; Wasden, Katherine ; Strych, Ulrich ; Goyal, Atul ; Maranholkar, Vijay ; Mo, William ; Vu, Binh ; Kourentzi, Katerina ; Willson, Richard C
• Description:
  Detection and quantification of antibodies, especially immunoglobulin G (IgG), is a cornerstone of ELISAs, many diagnostics, and the development of antibody-based drugs. Current state-of-the-art immunoassay techniques for antibody detection require species-specific secondary antibodies and carefully-controlled bioconjugations. Poor conjugation efficiency degrades assay performance and increases the risk of clinical false positives due to non-specific binding. We developed a generic, highly-sensitive platform for IgG quantification by fusing the IgG-Fc binding Z domain of Staphylococcal Protein A with the ultrabright bioluminescence reporter Nanoluc-luciferase (Nluc). We demonstrated the application of this fusion protein in a sandwich IgG detection immunoassay using surface-bound antigens to capture target IgG and protein A-Nanoluc fusion as the detector. We optimized the platform's sensitivity by incorporating multiple repeats of the Z domain into the fusion protein constructs. Using rabbit and mouse anti-SARS-CoV-2 Nucleoprotein IgGs as model analytes, we performed ELISAs in two different formats, either with SARS-CoV-2 Nucleoprotein as the capture antigen or with polyclonal chicken IgY as the capture antibody. Using standard laboratory equipment, the platform enabled the quantitation of antibody analytes at concentrations as low as 10 pg/mL (67 fM).
• Subjects:
  Animals Antibodies, Viral COVID-19* Immunoassay Immunoglobulin G Mice Nucleoproteins Rabbits SARS-CoV-2 Sensitivity And Specificity Staphylococcal Protein A

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• Source:
  Anal Biochem. 660:114929
• Pubmed ID:
  36270332
• Pubmed Central ID:
  PMC9826736
• Document Type:
  Journal Article
• Funding:
  R01 AR072742/AR/NIAMS NIH HHSUnited States/ ; U01 CK000512/CK/NCEZID CDC HHSUnited States/
• Volume:
  660
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:2e3d71038e490668a13a4bf402ed1b31f558747a5748d31ef16676fcc84e708f
• Download URL:
  https://stacks.cdc.gov/view/cdc/123513/cdc_123513_DS1.pdf
• File Type:
  [PDF - 1.35 MB ]