A CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples

The COVID-19 pandemic highlighted the urgent need for sensitive and specific assays to detect SARS-CoV-2 infections and the resulting immune responses. While CRISPR-based diagnostics have shown promise in detecting viral RNA, their application in detecting clinically relevant proteins, such as antibodies, has been limited. Existing immunoassays like ELISA and CLIA, while commercially available, often lack the sensitivity needed for early diagnosis or monitoring antibody levels in immunocompromised populations. These limitations motivated the development of a new assay with sensitivity comparable to CRISPR-based nucleic acid tests (NATs). This study aimed to develop a simple, highly sensitive assay capable of early COVID-19 diagnosis and providing insights into humoral immune responses in both infected individuals and vaccinated populations, particularly immunocompromised individuals.

The introduction section extensively discusses the limitations of existing methods for detecting anti-SARS-CoV-2 antibodies. It mentions the success of CRISPR-Cas13 (SHERLOCK) and Cas12 (DETECTR) systems in detecting viral RNA but notes the lack of application in sensitive antibody detection. The limitations of conventional immunoassays like ELISA and CLIA, such as insufficient sensitivity for early infection detection and monitoring in immunocompromised populations, are highlighted. The need for a simple, ultra-sensitive assay with a limit-of-detection (LOD) comparable to CRISPR-based NATs is established, emphasizing the importance of early diagnosis and monitoring humoral immune responses.

The UCAD assay converts antibody detection into CRISPR-based nucleic acid detection. It uses a dsDNA barcode containing a 20-nucleotide binding domain in the CRISPR guide RNA (crRNA). This barcode is truncated to prevent Cas12a activation in the absence of the target antibody. The truncated strands are conjugated with SARS-CoV-2 spike protein RBD and an anti-human IgG (or IgM) antibody. In the presence of anti-RBD antibodies, the binding of the conjugated antibodies brings the DNA probes into proximity, forming a stable duplex that activates Cas12a. The assay proceeds in three steps: (1) antibody-specific primer extension to produce dsDNA barcodes; (2) RPA amplification; and (3) Cas12a-mediated cleavage of fluorophore-quencher (FQ) labeled ssDNA reporters. The entire process is performed homogeneously at 37°C in a single tube. Optimization of assay conditions enabled detection of as low as 10 anti-RBD human monoclonal antibodies. The assay's specificity and modularity were demonstrated by its ability to distinguish between antibodies against SARS-CoV-2 and other coronaviruses, and its adaptability to detect antibodies against different SARS-CoV-2 variants by simply changing the RBD input. A lateral flow readout was also developed by replacing the FQ-labeled reporter with FAM and Dig-labeled reporters. Clinical samples were analyzed using both UCAD and standard ELISA or CLIA, with a focus on kidney transplant recipients (KTRs). Standard ELISA and CLIA were performed according to manufacturer's instructions. Flow cytometry was used for blood cell analysis in KTRs.

UCAD demonstrated significantly higher sensitivity (10,000-fold) compared to commercial ELISA kits. Clinical validation using 197 serum samples showed 100% sensitivity and 98.5% specificity. UCAD successfully detected anti-SARS-CoV-2 antibodies in vaccinated KTRs, where standard assays yielded undetectable results. The modular design allowed for the specific detection of antibodies against different SARS-CoV-2 variants (WT, Delta, Omicron) by switching the RBD input. The assay's high sensitivity was confirmed using PAGE analysis. A lateral flow readout was developed, demonstrating adaptability to resource-limited settings. In a cohort of vaccinated KTRs, UCAD revealed significantly lower levels of anti-RBD IgG and IgM compared to vaccinated healthy individuals. Following a third vaccine dose, a significant increase in anti-RBD IgG levels was observed in most KTRs.

UCAD represents a significant advance in CRISPR diagnostics, expanding its applications beyond nucleic acid detection to ultrasensitive protein analysis. Its high sensitivity, modularity, and simplicity make it suitable for various diagnostic settings. The ability to detect low antibody levels in immunocompromised individuals is particularly valuable. The assay's compatibility with existing CRISPR platforms and adaptability to different detection methods (fluorescence, lateral flow) further enhance its versatility. Compared to other ultrasensitive protein detection techniques, UCAD offers significant advantages in terms of portability and ease of use. Future research will focus on tracking antibody level changes over time and developing UCAD as a point-of-care test (POCT) for detecting other clinically important proteins.

UCAD offers a highly sensitive, specific, and adaptable method for detecting anti-SARS-CoV-2 antibodies. Its superior performance, particularly in detecting low antibody levels in immunocompromised individuals, makes it a promising tool for early diagnosis, monitoring of immune responses, and potential development as a POCT. Future studies should focus on broader clinical validation and adapting UCAD for detecting other clinically relevant proteins.

While the study demonstrated high sensitivity and specificity, a larger-scale clinical validation with diverse populations is needed to confirm its generalizability. Further investigation is required to fully understand the clinical significance of detecting low levels of anti-SARS-CoV-2 antibodies in immunocompromised individuals. The study primarily focused on anti-RBD antibodies; further research might explore the applicability of UCAD to other SARS-CoV-2 antigens.