RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays

  1. Yan Ni
  2. Bas J.H.M. Rosier
  3. Eva A. van Aalen
  4. Eva T.L. Hanckmann
  5. Lieuwe Biewenga
  6. Anna-Maria Makri Pistikou
  7. Bart Timmermans
  8. Chris Vu
  9. Sophie Roos
  10. Remco Arts
  11. Wentao Li
  12. Tom F.A. de Greef
  13. Frank J. M. van Kuppeveld
  14. Berend-Jan Bosch
  15. Maarten Merkx

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Abstract

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into easy-to-use point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID ( Ra tiometric P lug-and- P lay Immuno d iagnostics), a “mix-and-measure” homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout that can be detected using a basic digital camera. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. We also introduce the use of a calibrator luciferase that provides a robust ratiometric signal, allowing direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. RAPPID combines ratiometric bioluminescent detection with antibody-based target recognition into an easy-to-implement standardized workflow, and therefore represents an attractive, fast, and low-cost alternative to traditional immunoassays, both in an academic setting and in clinical laboratories for point-of-care applications.

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  1. ScreenIT

    SciScore for 10.1101/2020.10.31.363044: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Photoconjugation: Cardiac Troponin I antibodies 19C7 (4T21) and 4C2 (4T21), and C-reactive protein antibodies C6 (4C28cc) and C135 (4C28) were obtained from Hytest.
    C-reactive protein antibodies C6
    suggested: None
    C135
    suggested: None
    Anti-adalimumab/TNFα monoclonal antibody (HCA207) and anti-infliximab (HCA213) were obtained from BioRad.
    Anti-adalimumab/TNFα
    suggested: None
    anti-infliximab (HCA213
    suggested: None
    Anti-SARS-COV-2 antibodies 47D11 and 49F1 were produced in HEK-293T cells and purified using Protein-A affinity chromatography as described previously44.
    Anti-SARS-COV-2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The SARS-COV-2 spike trimer protein was expressed in HEK-293T cells with a C-terminal trimerization motif and Strep-tag using the pCAGGS expression plasmid, and purified from the culture supernatant by Strep-Tactin chromatography as described previously44.
    HEK-293T
    suggested: None
    For lentiviral production of the two vectors, encoding either the RBD-LB or RBD-SB transgene, HEK293T cells (ATCC® CRL-3216™) were transiently co-transfected with the abovementioned transfer vectors, as well as the VSV-G envelope (pMD2.G) and packaging plasmids (pCMVR8.74), as previously described58.
    HEK293T
    suggested: None

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2020.10.31.363044v1 on bioRxiv