Chitin-immobilized nanobodies for the detection of SARS-CoV-2


In a recent study published on bioRxiv*, researchers demonstrated a new strategy for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using chitin-bound nanobodies.

Study: Effective detection of SARS-CoV-2 using chitin-inactivated nanobodies manufactured in Ustilago maydis.  Image credit: CROCOTHERY / Shutterstock
Stady: Efficient detection of SARS-CoV-2 using chitin-assembled nanobodies synthesized in Ustilago maydis.. Image credit: CROCOTHERY / Shutterstock

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Shark and CamelidaeThe derived nanobodies are emerging alternatives to conventional antibodies. Nanobjects bind to ligands at the nanoscale and remain stable under heat/chemical stress conditions, making them promising candidates for large-scale dispersal. antigen Tests. Several anti-SARS-CoV-2 nanobodies have been designed via phage display or by immunizing llamas, sharks, and alpacas.

The microbial form of ustilago Maidis The maize fungi can be utilized for the synthesis of heterocyclic proteins such as nanobodies. Recently, the authors created a proof-of-principle for the synthesis of anti-SARS-CoV-2 nanobodies. Moreover, the unconventional secretion mechanism used by U Medics The export enzyme Cts1 chitinase can be utilized to secrete heterologous target proteins. Cts1 has chitin-binding activity and can therefore be exploited as an intrinsic stabilization and purification marker. Jps1, the stabilizing factor required for Cts1 secretion, could be an alternative vector.

Study and results

In this study, researchers have established a novel approach for detecting SARS-CoV-2 using chitin-immobilized nanobodies. First, they examined different nanobody-Cts1 fusion proteins for expression, unconventional secretion, and binding activity against the SARS-CoV-2 spike’s receptor-binding domain (RBD). Four nanobody-Cts1 fusion structures were synthesized using two llama-derived nanobody (VHH).e and VHHFifth) and two synthetic nanobodies (Sy15th And Lsi68).

In addition, a bivalent nanobody was generated by VHH couplingFifth with VHHe to produce VHHVE nano. Moreover, VHH is bivalentWhich The nanobody was produced to test the binding ability of the dimers. Published nanobody versions of Sy68/15-Jps1 and Sy68/15-Cts1 were controls. Secretion/excretion of the target fusion proteins was examined via western blots.

Culture supernatants showed sufficient secretion of Sy15th-Cts1, VHHFifth-Cts1, VHHe-Cts1, Sy68/15-jps1 and vhhWhichFusion proteins -Cts1. RBD-binding activity was evaluated using the enzyme-linked direct immunosorbent assay (ELISA) of cell extracts containing the nanobody-Cts1 fusion proteins. VHHWhich-Cts1 and Sy68/15-Jps1 showed the strongest binding to RBD, while VHH showed the strongest bindinge-Cts1 showed about half the signal intensity. The remaining fusion proteins lack apparent binding activity.

Furthermore, these three fusion proteins were purified and evaluated in a direct ELISA against the full-length SARS-CoV-2 S1 protein. All three fusion proteins showed significant binding activity; VHHWhich-Cts1 and Sy68/15-Jps1 had two-fold binding compared to VHHe-cts1. The researchers conducted modified neutralization assays to determine whether in the laboratory The activity has been translated into in vivo binding or neutralization.

VHHe-Cts1 lacks neutralizing activity, while VHHWhich-Cts1 and Sy68/15-Jps1 showed virus-inactivating activity. Since Cts1 can bind to chitin-coated surfaces such as magnetic chitin granules, this property can be taken advantage of to develop a new strategy for antigen testing. Chitin binding on chitin granules was recapitulated using purified recombinant Cts1. Chitin beads were mixed with recombinant Cts1, which confirmed the binding of the recombinant protein to chitin.

The β-glucuronidase (Gus)-Cts1 fusion protein was used to measure the results. The Gus-Jps1 fusion protein, which was predicted not to bind chitin, served as a negative control. Chitin beads were coated with Gus-Cts1 or Gus-Jps1 fusion proteins. Only the Gus-Cts1 fusion protein was bound to chitin granules, confirming the binding ability of the N-terminal Cts1 fusion proteins.

Signal intensity measurement revealed that 44% of the recombinant Cts1 protein and 68% of the Gus-Cts1 fusion protein were captured on the bead. The function of the fusion protein was evaluated after fixation (on beads). Specifically, Gus activity was detected on beads incubated with cell extracts containing Gus-Cts1, implying retention of functional (enzymatic) activity despite immobility on the beads.

Finally, sandwich immunosorbent assays were performed on ELISA plates and chitin beads to evaluate the ability of VHHWhich-Cts1 and VHHeFusion proteins -Cts1 nanobody. bad68/15-Jps1 was the control in both assays, since it should show activity in ELISA but not on chitin beads. Purified fusion proteins were coated on ELISA plates, incubated with serially diluted recombinant RBD, and detected by anti-RBD antibody and cognate horseradish peroxidase (HRP).

While all three nanobody fusion proteins could capture RBD in ELISA plates, only Sy68/15-jps1 and vhhWhich-Cts1 showed volumetric activity for serial dilution of RBD. VHHWhich-Cts1 showed the strongest binding event at the lowest concentration of RBD. The chitin beads were separately incubated with the three nanobody fusion proteins and mixed with RBD. Both are VHHWhich-Cts1 and VHHe-Cts1 retained binding activity, while Sy68/15-Jps1 lacks activity on chitin beads. Like the previous results, VHHWhich-Cts1 showed 2-fold stronger activity than VHHe-cts1.

The RBD capture ability of the chitin-based detection system was further characterized by quantitatively determining its binding activity using the most potent nanobody fusion protein (VHH).Which-cts1). Chitin beads were loaded with VHHWhich-Cts1 and incubated with RBD diluted with the recombinant sequence. Activity was detected using a sandwich of commercial antibody. The researchers observed a color reaction within two minutes, the intensity of which is proportional to RBD concentrations.

conclusions

To summarize, the study investigated Cts1-mediated secretion of mono- and divalent nanobodies against SARS-CoV-2. have provided a proof-of-principle for a SARS-CoV-2 antigen test based on chitin, which was facilitated by the unconventional secretion mechanism of Cts1 in U Medics. The authors investigated the applicability of the nanobody-Cts1 fusion proteins in detecting and neutralizing viruses in vivo. This confirmed that the nanobody could bind to the infectious virus, along with the RBD spike. The authors believe that this strategy could be turned into a lab-on-a-chip method for SARS-CoV-2 antigen testing.

*Important note

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, directing clinical practice/health-related behaviour, or treated as hard information.



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