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Highly Sensitive Immuno-CRISPR Assay for CXCL9 Detection.

Inseon Lee1, Seok-Joon Kwon1, Mirco Sorci1

  • 1Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, United States.

Analytical Chemistry
|December 6, 2021
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Summary

A novel immuno-CRISPR assay detects CXCL9, a biomarker for kidney transplant rejection, with high sensitivity and accuracy. This CRISPR-based method offers a promising noninvasive tool for early detection of transplant complications.

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Area of Science:

  • Biotechnology
  • Molecular Diagnostics
  • Immunology

Background:

  • CRISPR-based detection systems leverage target recognition and collateral cleavage for signal amplification.
  • CXCL9 protein is a key biomarker elevated in urine during kidney transplant rejection episodes.
  • Current diagnostic methods like ELISA have limitations in sensitivity and speed for early rejection detection.

Purpose of the Study:

  • To develop and validate a modified immunoassay format, termed "immuno-CRISPR", for sensitive detection of the CXCL9 protein.
  • To enhance the sensitivity and efficiency of CRISPR-based detection for a protein biomarker in a clinical context.
  • To establish CXCL9 as a noninvasive point-of-care biomarker for kidney transplant rejection.

Main Methods:

  • Developed an immuno-CRISPR assay utilizing anti-CXCL9 antibody-DNA barcode conjugates for target capture.
  • Employed Cas12a enzyme for sequence-specific trans-cleavage of FQ reporter substrates, generating a fluorescent signal.
  • Engineered DNA barcodes with multiple Cas12a sites and utilized biotin-streptavidin complex formation to amplify signal generation.

Main Results:

  • The immuno-CRISPR assay achieved an 8-fold improvement in CXCL9 detection rate compared to monomeric DNA barcodes.
  • Demonstrated a limit of detection (LOD) of 14 pg/mL for CXCL9, representing a 7-fold improvement over traditional ELISA.
  • Successfully detected CXCL9 in urine samples from kidney transplant recipients with 100% accuracy, showing high specificity against CXCL1.

Conclusions:

  • The developed immuno-CRISPR assay provides a highly sensitive and specific method for detecting CXCL9 protein.
  • This assay shows significant potential as a noninvasive, point-of-care diagnostic tool for early kidney transplant rejection monitoring.
  • The engineered DNA barcode system enhances CRISPR-based detection capabilities for protein biomarkers.