Part:BBa_K5466004

AntiAFB1- Nb28-S102D

Single-domain antibody (sdAb) with improved binding activity to AFB1, without a start codon or stop codon, prepared for yeast surface display.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Nanobody

We chose a single-domain antibody (sdAb), also known as a nanobody (Nb), to capture AFB1 due to its high solubility, exceptional stability, and high affinity. There are two types of single-domain antibodies: VHH and VNAR. Immunoglobulins without light chains were first discovered in camelids, such as bactrian camels, dromedaries, and llamas. This discovery marked a significant breakthrough because the single N-terminal domains of these antibodies can bind to antigens without the need for domain pairing. VHHs are derived from the heavy chain and represent the N-terminal domain of these antibodies.

Later, single-domain antibodies were identified in certain cartilaginous fish, known as VNARs. VNARs are derived from the immunoglobulin new antigen receptor (IgNAR), a heavy-chain-only antibody. This receptor is a homodimeric protein, featuring antigen-binding regions at the ends of each heavy chain, which allows for effective binding to antigens.

While both VHH and VNAR share similar characteristics, most research on the biotechnological applications of single-domain antibodies has focused on camelids due to their ease of handling and immunization.

Like conventional VH domains, VHHs consist of four framework regions (FRs) that create the core structure of the immunoglobulin domain, along with three complementarity-determining regions (CDRs) responsible for antigen binding. In contrast, shark single-domain antibodies possess a vestigial CDR2 that does not participate in antigen binding.

Nb are significantly smaller than conventional antibodies and fragments such as Fab and scFv (~15 kDa). Despite this, they maintain a broad reactivity with diverse epitopes, aided by the extended flexible CDR3 loop that allows recognition of antigenic sites that are normally not recognized by conventional antibodies such as enzyme active sites and hidden or cryptic epitopes. Recent studies indicate that Nbs can form concave-shaped binding sites for small molecules, similar to conventional antibodies. Given these advantageous properties, Nbs are emerging as promising alternatives to traditional antibodies.

Nb28

Nb28 is a VHH against aflatoxin, produced in Vicugna pacos. To assess its sensitivity and binding capacity, the ratio of nanobody without ligand to nanobody with ligand was measured at different aflatoxin concentrations, resulting in an IC50 of 3.84 ng/mL.

The half-maximal inhibitory concentration (IC50) is a metric used to assess the potency of a substance in inhibiting a biological or biochemical function. It represents the concentration of an inhibitory agent (such as a drug) required to reduce a specific biological process or target by 50% in vitro. This target could be an enzyme, a cell, or a cell receptor. In this case, the lower the IC50, the less AFB1 is required to bind the 50% of the nanobodies, indicating a higher binding of Nb28.

He et al., (2021) introduce the S102D mutation by mimicking Nb26, which exhibits a novel binding mode distinct from other anti-hapten nanobodies by accommodating AFB1 in a cavity formed by the CDR3 and FR2 regions. By comparing the structure and sequence of Nb26 and Nb28, they discovered that Nb26 forms two hydrogen bonds with AFB1 through residues D102 and G103, and observed that no intermolecular hydrogen bond in the docking model of AFB1 binding to Nb28. In Nb28, S102 and G103 at the corresponding positions did not even participate in the formation of the binding pocket. The Nb28 mutants were constructed based on these findings, and the mutant showed increased sensitivity to AFB1 compared to the wild type, by introducing mutations into the FR2 and CDR3 regions.

Nb 28-S102D has an IC50 of 1.18 ng/mL, 3.3-fold sensitivity enhancement over the wild type and by about 1.5-fold when compared to the Nb26.

References

English, H., Hong, J., & Ho, M. (2020). Ancient species offers contemporary therapeutics: an update on shark VNAR single domain antibody sequences, phage libraries and potential clinical applications. Antibody Therapeutics, 3(1), 1–9. https://doi.org/10.1093/abt/tbaa001

Harmsen, M. M., & De Haard, H. J. (2007). Properties, production, and applications of camelid single-domain antibody fragments. Applied Microbiology and Biotechnology, 77(1), 13–22. https://doi.org/10.1007/s00253-007-1142-2

He, T., Wang, Y., Li, P., Zhang, Q., Lei, J., Zhang, Z., Ding, X., Zhou, H., & Zhang, W. (2014). Nanobody-Based Enzyme Immunoassay for Aflatoxin in Agro-Products with High Tolerance to Cosolvent Methanol. Analytical Chemistry, 86(17), 8873-8880. https://doi.org/10.1021/ac502390c

He, T., Nie, Y., Yan, T., Zhu, J., He, X., Li, Y., Zhang, Q., Tang, X., Hu, R., Yang, Y., & Liu, M. (2021). Enhancing the detection sensitivity of nanobody against aflatoxin B1 through structure-guided modification. International Journal Of Biological Macromolecules, 194, 188-197. https://doi.org/10.1016/j.ijbiomac.2021.11.182