Phage display-mediated immuno-polymerase chain reaction (PD-IPCR) is an ultrasensitive detection technology that combines the advantages of immuno-PCR and phage display. The phage particle, which displayed antibody fragments including single-chain fragment variable (scFv), variable domain of heavy-chain antibodies (VHH), and antigen-binding fragment (Fab) on the surface can be directly used in IPCR, supplying both the detection antibody and deoxyribonucleic acid (DNA) template. In this work, we used ochratoxin A (OTA) as a model system to study the capacity of PD-IPCR in the detection of toxic small molecular weight compounds, especially mycotoxins. An alpaca-derived VHH library was constructed and subjected to four cycles of panning. In total, 16 clones with four unique sequences were selected by competitive binding with OTA. The clone VHH-28 resulted in the lowest 50% inhibitory concentration of 0.31 ng/mL in the phage enzyme-linked immunosorbent assay (ELISA) and was selected to develop the VHH phage-based real-time immuno-PCR (RT-IPCR). The detection limit of the VHH phage-based RT-IPCR was 3.7 pg/L, with a linear range of 0.01–1000 pg/mL. This method was compared with conventional ELISA, and validation results indicated the reliability of VHH phage-based RT-IPCR in the detection of OTA in cereal samples. This study provides a new idea for the ultrasensitive detection of mycotoxins and other toxic small molecular weight compounds.

Summary: Heavy‐chain‐only antibodies (HCAbs), which are devoid of light chains, have been found naturally occurring in various species including camelids and cartilaginous fish. Because of their high thermostability, refoldability and capacity for cell permeation, the variable regions of the heavy chain of HCAbs (VHHs) have been widely used in diagnosis, bio‐imaging, food safety and therapeutics. Most immunogenetic and functional studies of HCAbs are based on case studies or a limited number of low‐throughput sequencing data. A complete picture derived from more abundant high‐throughput sequencing (HTS) data can help us gain deeper insights. We cloned and sequenced the full‐length coding region of VHHs in Alpaca (Vicugna pacos) via HTS in this study. A new pipeline was developed to conduct an in‐depth analysis of the HCAb repertoires. Various critical features, including the length distribution of complementarity‐determining region 3 (CDR3), V(D)J usage, VJ pairing, germline‐specific mutation rate and germline‐specific scoring profiles (GSSPs), were systematically characterized. The quantitative data show that V(D)J usage and VHH recombination are highly biased. Interestingly, we found that the average CDR3 length of classical VHHs is longer than that of non‐classical ones, whereas the mutation rates are similar in both kinds of VHHs. Finally, GSSPs were built to quantitatively describe and compare sequences that originate from each VJ pair. Overall, this study presents a comprehensive landscape of the HCAb repertoire, which can provide useful guidance for the modeling of somatic hypermutation and the design of novel functional VHHs or VHH repertoires via evolutionary profiles.

Deoxynivalenol (DON), or vomitoxin, is a mycotoxin of the trichothecene group produced by Fusarium species. The present work described construction of a phage display library based on variable domain of heavy chain of HCAbs (VHHs) and panning for binders of DON. Two pair PCR primers designed based on the VHHs conserved domains were used for cloning coding sequences of complementary DNA (cDNA). After total RNA extraction, semi-nested PCR, subcloning and electro transformation, the primary library was generated containing 1.9×10⁷ independent clones. Phage display library was rescued by infection of the primary library with helper phage KM13, and the titre of the library was 5.6×10¹² CFU/ml. Three clones with specific DON-binding affinity were obtained after three rounds of panning. Since the library was derived from non-immune animal, it has potential to be used for panning binders of other mycotoxins or low molecular weight haptens. The binders with specific affinity provided new recourses for developing detection methods or tools of mycotoxins in foodstuffs.