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.2024 Jul;10(4):e1532.
doi: 10.1002/vms3.1532.

Isolation of camel single domain antibodies against Yersinia pestis V270 antigen based on a semi-synthetic single domain antibody library and development of a VHH-based lateral flow assay

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Isolation of camel single domain antibodies against Yersinia pestis V270 antigen based on a semi-synthetic single domain antibody library and development of a VHH-based lateral flow assay

Bo Wang et al. Vet Med Sci.2024 Jul.

Abstract

Background: Antibodies have been proven effective as diagnostic agents for detecting zoonotic diseases. The variable domain of camel heavy chain antibody (VHH), as an antibody derivative, may be used as an alternative for traditional antibodies in existing immunodiagnostic reagents for detecting rapidly spreading infectious diseases.

Objectives: To expedite the isolation of specific antibodies for diagnostic purposes, we constructed a semi-synthetic camel single domain antibody library based on the phage display technique platform (PDT) and verified the validity of this study.

Methods: The semi-synthetic single domain antibody sequences consist of two parts: one is the FR1-FR3 region amplified by RT-PCR from healthy camel peripheral blood lymphocytes (PBLs), and the other part is the CDR3-FR4 region synthesised as an oligonucleotide containing CDR3 randomised region. The two parts were fused by overlapping PCR, resulting in the rearranged variable domain of heavy-chain antibodies (VHHs). Y. pestis low-calcium response V protein (LcrV) is an optional biomarker to detect the Y. pestis infection. The semi-synthetic library herein was screened using recombinant (LcrV) as a target antigen.

Results: After four cycles of panning the library, four VHH binders targeting 1-270 aa residues of LcrV were isolated. The four VHH genes with unique sequences were recloned into an expression vector and expressed as VHH-hFc chimeric antibodies. The purified antibodies were identified and used to develop a lateral flow immunoassay (LFA) test strip using latex microspheres (LM) for the rapid and visual detection of Y. pestis infection.

Conclusions: These data demonstrate the great potential of the semi-synthetic library for use in isolation of antigen-specific nanobodies and the isolated specific VHHs can be used in antigen-capture immunoassays.

Keywords: VHH; Y. pestis; diagnosis test; phage display.

© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Construction of the semi‐synthetic VHH library. (A) Schematic representation of steps involved in VHH antibody library construction. FR1 to FR4: framework 1 to 4; CH2: constant domain 2; CDR1 to 3: complementary determining region 1 to 3. The PCR products were analysed by electrophoresis on a 1.5% agarose gel prestained with ethidium bromide, including (B) first round PCR products amplified with primers CALL001 and CALL002; (C) second round PCR products (left) and asymmetric PCR products (right) amplified with primers VHH‐F and MCDR2; (D) fusion PCR products of FR1‐FR3 region and oligonucleotide CDR3M amplified with primers VHH‐F and VHH‐KAR.
FIGURE 2
FIGURE 2
Expression, purification, and identification of the V270 proteins ofY. pestis. (A) Lane M: the standard protein marker (Sangon Biotech, Shanghai, China). Lane 1 and 2: induction of rV270 expression at 42°C. Lane 3: uninduced at 37°C. (B) rV270 protein was purified by metal affinity chromatography using the AKTA system and analysed on a 12% SDS‐PAGE stained with coomassie blue. Lane 1–5: rV270 protein was eluted using 100, 150, 200, 250 and 300 mM of imidazole in PBS buffer. (C) Purified rV270 proteins were recognised by HRP‐anti‐HIS antibody using Western blot. Lane 1: control sample. Lane 2: rV270 protein.
FIGURE 3
FIGURE 3
Monoclonal phage‐ELISA characterisation of phages from the first round of selection binding to rV270 protein (A) and and fourth round of selection binding to rV270 protein (B). Irrelevant antigens, such as F1 protein ofY. pestis, heavy chain of botulinum neurotoxin type E (Ehc), fibre protein of human adenovirus type 55(F55), or heavy chain of botulinum neurotoxin type A(Ahc), were mixed and coated the plates parallelly as negative control.
FIGURE 4
FIGURE 4
Characterisation of the four anti‐rV270 VHH‐hFc antibodies. (A) The four anti‐rV270 antibodies were expressed as VHH‐hFc chimeric antibodies, purified and analysed on reducing (right) and nonreducing SDS‐PAGE gel (left). Lane 1–4: VHH1‐hFc∼VHH4‐hFc. (B) Specific binding of the four antibodies to the rV270 protein and other irrelevant proteins. (C) Binding activity of the four antibodies to rV270 protein. (D) Identification of the pairs useful for fabrication of a sensitive LFA test strip; the VHH1/VHH4 pair showed the highest signal scores among the 16 combinations.
FIGURE 5
FIGURE 5
Sensitivity, specificity, and stability of the plague test strips, the samples were analysed by applying 100 µL on the strips with PBS as a blank. (A) Lane 1–4: the sample isY. pestis EV76 strain, Brucella M5 strain,E.coli TG1, and PBS. (B) Lane 1–6: the rV270 protein at the concentration of 10, 5, 1, 0.5 and 0.25 ng/mL and PBS. (C) Lane 1–6: The same batch of latex microspheres conjugated strips stored at room temperature were tested once a month for a total of 6 months.
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