The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning.

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A new single-chain variable fragment (scFv) antibody library was generated and human serum albumin (HSA)-specific clones were characterized to investigate the usefulness of porcine antibodies. Phage libraries were developed from pigs immunized with the model antigen HSA. The library size was 1.5 × 10(7) for kappa (VL) and 1.4 × 10(7) for lambda fragments. Eight HSA-specific clones from the kappa library and one clone from the lambda library were isolated using affinity selection. The binding specificity of these clones was confirmed using a phage enzyme-linked immunosorbent assay (ELISA). The scFvs were expressed in Escherichia coli and purified from the periplasm fraction for further investigation. Based on the results of ELISA and Western blot analysis, four scFv clones with high activity and high yield were selected and purified. The purified scFvs from four of the nine clones exhibited an approximate KD of 10(-8) M. This is the first report describing isolation of HSA-specific porcine scFv antibodies from an antibody phage library and characterization of their binding properties.

It is difficult to link antibody repertoire expansion with changes in the physical characteristics of antigen-responding antibodies as correct light-chain and heavy-chain matching and conventional antibody production are laborious. Utilization of single-domain antibody solved these problems. A blood of immunized alpaca was collected weekly for three months for antibody repertoire analysis. The sequences processed to cluster and recombinant antibodies were generated to determine whether the clusters respond to antigens. The repertoire expansion in most of the antigen-responding clusters exhibited distinct patterns as compared to that of antigen irrelevant clusters. In addition, the sequences at the tips and root in the molecular phylogenetic cluster tree have strong and weak antigen affinity, respectively. These features may be utilized to predict clusters of antigen-binding antibodies and physical characteristics of antibodies.

VHH antibodies or nanobodies, which are antigen-binding domains of heavy chain antibodies from camelid species, have several advantageous characteristics, including compact molecular size, high productibility in bacteria, and easy engineering for functional improvement. Focusing on these advantages of VHHs, we attempted to establish an immunoassay system for detection of Legionella, the causative pathogen of Legionnaires’ disease. A VHH phage display library was constructed using cDNA from B cells of alpacas immunized with Legionella pneumophila serogroup1 (LpSG1). Through biopanning, two specific VHH clones were isolated and used to construct a Legionella detection system based on the latex agglutination assay. After engineering the VHHs and improving the assay system, the sensitive detection system was successfully established for the LpSG1 antigen. The immunoassay developed in this study should be useful in easy and sensitive detection of Legionella, the causative agent of Legionnaires’ disease, which is a potentially fatal pneumonia.