Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library

Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications

“…1,3 Similarly, the Ig isotype IgNAR (novel antigen receptor) discovered in the shark bloodstream is also a homodimeric structure of two heavy-chain polypeptides, each comprising a single variable domain and five constant domains (homodimeric structure, Figure 1G-I). 7 The percentage of HCAbs in the bloodstream of camelids varies greatly among species because of a variation in mutation rates.…”

“…Similar to the camelid VHHs, the V-NAR has abnormally long CDR3 loops that considerably reflect the greatest diversity of the V-NAR in both sequence and length. 7 Furthermore, highly frequent somatic mutation is also found in CDR1, at the equivalent site of the deleted CDR2, where the shorten loop forms a belt-like structure at the opposite side of the CDR1 and CDR3, and in a loop that resembles HV4 in T-cell receptors. 5,18 Similar to the camel VHHs, the shark V-NARs also evolved to generate equivalent disulfide bridges tethering the extended antigen-binding loops.…”

“…1,4,7,26,27 The technologies used for the preparation of nanobody library do not significantly differ from the ones otherwise used for recovering the Fab and scFv libraries. First of all, retrieval of nanobodies from an immune library is a priority consideration because somatic maturation in lymphocytes of immunized Camelidae will give antibody libraries more specific and higher affinity to antigens of interest.…”

“…27 Based on naturally occurring VHH or V-NAR sequences, the semisynthetic or synthetic nanobody libraries could be created by introducing length and sequence variations in CDR3 using randomized CDR3 primers, 18 or error-prone PCR combined with splice-overlap extension PCR method. 7,27 Using small blood samples (,10 mL), the complexity of diversity-enhanced semisynthetic or synthetic nanobody library is close to 10 9 , whereas the complexity of 10 6 of naive library could only be reached by using the same volume of blood samples. At least .1 L of blood samples might be consumed in order to obtain ~10 9 individual clones per library for the naive library.…”

“…1 Some cartilaginous fish, including nurse shark (Ginglymostoma cirratum), wobbegong (Orectolobus maculates), and dogfish (Squalus acanthias and Mustelus canis) sharks, also remarkably produce functional heavychain-only immunoglobulins (Igs), named IgNARs. 1,[5][6][7][8] Interestingly, some pathological and nonfunctional HCAbs were also discovered in sera of humans or in mouse hybridoma due to a partly genetic deletion of the variable heavychain (VH) and CH1 regions. 1 In camels or sharks, these HCAbs (or IgNARs) recognize the antigens via single variable domains, referred to as VHHs or V-NARs, respectively.…”