One of the most important items is to show that the claimed antibody is an alternative to known antibodies. To avoid competition, but also to prevent a threat from subsequently detected deviations from the patented sequences, as was the case for N14, patent claim rules usually permit changes in the CDR sequences provided that 90 or 95% sequence identity is retained (van der Hoff, 2014 ). With the decreasing cost of genomic sequencing, the sequences of the V H and V L domains (or of the set of six CDRs) started to dominate. In order to successfully patent an antibody, various claims are stated, with the most common being the sequence (Holliday, 2009 ). Additional instances of variable-chain glycosylations of largely unknown function detected in PDB models are compiled in Supplementary Table S1 (see § 3.3).ġ.3. We report crystallographic evidence for a rare glycosylation at Asn26L at the onset of the variable light-chain L1 loop (V L1) of the complementarity-determining region (CDR). Glycosylations in the variable regions that are functionally relevant to antigen (Ag) binding, for example, have been described at Asn58H, Asn60H and Asn54H (Gala & Morrison, 2004 ). Genomic cDNA analysis reveals that about 15–25% of Fabs are expected to be glycosylated overall (Anumula, 2012 ), while only ∼9% of the variable regions are glycosylated based on genomic cDNA analysis (Arnold et al., 2007 ). In addition to the frequent and in part conserved glycans of antibody Fc (fragment, crystallizable) domains (Arnold et al., 2007 ), glycosylations in the Fab regions of IgG antibodies emerging primarily through somatic hypermutation have gained increasing interest owing to their influence on IgG function and immune regulation (van de Bovenkamp et al., 2016 ). The N14 Fab displays a number of interesting structural features and its crystallization in two crystal forms with non-apparent isomorphism also allows an extended analysis of its structural flexibility and of the practice and effects of extensive solvent model building. A potential role of afamin in the glucose metabolism in papillary thyroid carcinoma has been reported (Shen et al., 2016 ), and the N14 Fab (fragment, antigen binding) can serve as a scaffolding partner in AFM crystallization. Strong interest in the AFM crystal structure results from the fact that it seems to be, at least in vitro, a carrier for Wnt signalling proteins (which are relevant in cell proliferation pathways), which are otherwise very hard to solubilize and to purify (Mihara et al., 2016 ). Afamin (AFM) is a biomarker for metabolic syndrome and related cardiovascular disease as well as for ovarian cancer (Dieplinger et al., 2009 Kronenberg et al., 2014 Seeber et al., 2014 ). Horseradish peroxidase-conjugated murine N14 IgG1 κ monoclonal antibody (mAB) is the detecting antibody in a novel sandwich ELISA used for quantification of the human glycoprotein afamin (Dieplinger et al., 2013 Dieplinger & Dieplinger, 2015 ), a plasma vitamin E-binding glycoprotein of the albumin gene family (Voegele et al., 2002 ). The N14 monoclonal antibody: function and unique features of its antibody fragment Improvements to the PDB validation reports affecting ligands, clashscore and buried surface calculations are suggested.ġ.1. Finally, a conservatively refined parsimonious model is presented and its statistics are compared with those from a less conservatively built model that has been modelled more enthusiastically. In addition, the map quality at 1.9 Å resolution was sufficient to crystallographically re-sequence the variable V L and V H domains and to detect discrepancies in the hybridoma-derived sequence. The crystal structures of two non-apparent (pseudo) isomorphous crystals of the N14 Fab were analyzed, which differ significantly in the elbow angles, thereby cautioning against the overinterpretation of domain movements upon antigen binding. As a rare occurrence, the N14 Fab is N-glycosylated at Asn26L at the onset of the V L1 antigen-binding loop, with the α-1–6 core fucosylated complex glycan facing out of the L1 complementarity-determining region. The monoclonal antibody N14 is used as a detection antibody in ELISA kits for the human glycoprotein afamin, a member of the albumin family, which has recently gained interest in the capture and stabilization of Wnt signalling proteins, and for its role in metabolic syndrome and papillary thyroid carcinoma.
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