![]() IGF control injections clearly show no signal response of mAb anti–IGF-1(74–90) with IGF-2, and, conversely, of mAb anti–IGF-2(53–65) with IGF-1. The mAb anti–IGF-2(53–65) was quantified with KD 0.7 n m IGF-2 affinity with ka 1.2E+06 m −1 s −1 and kd 7.7E-04 s −1 and t ½ diss 15 min. 5) with KD 6 p m IGF-1 affinity with ka 3.0E+06 m −1 s −1 association rate constant and dissociation rate constant kd 1.7E-05 s −1 and t ½ diss 680 min. The mAb anti–IGF-1(74–90) was quantified by a 120-min dissociation time experiment ( Fig. Sensorgrams, exemplifying affinity and specificity measurements, are depicted in Fig. Kinetic data of the finally selected mAb IGF-1(74–90) and mAb IGF-2(53–65) are listed in Table 2. We tested several commercially available anti-IGF mAbs, but all were disqualified because of considerable fast-on/-off kinetics versus the respective counterpart IGF isoform (data not shown). Generation of monoclonal antibodies (mAbs) The high thermal stability of the WT scaffold proteins was generally somewhat lowered by the grafted insert but was sufficient for the robust production of stable immunogens and screening reagents. It is noteworthy that, after thermally induced partial unfolding up to 90 ☌, the TgSlyD-IGF-2(53–65) graft variant was able to fold back reversibly when the protein solution was chilled from 90 to 20 ☌. The graft-bearing variant TgSlyD-IGF-2(53–65) was stable up to 78 ☌ ( Fig. TgSlyD-wt maintained its native baseline up to 76 ☌, whereas TgSlyD-ΔIF retained its native conformation up to 80 ☌ (data not shown). TtSlyD-IGF-1(74–90) was stable up to 60 ☌, unfolds at temperatures between 61–65 ☌, and seemed to aggregate at higher temperatures. It showed a midpoint of the unfolding transition at ∼70 ☌ and was completely unfolded at ∼85 ☌. The baseline of the denatured protein could not be reached in the accessible temperature range of the unfolding experiment ( Fig. TtSlyD-wt remained in a stable folded conformation up to 80 ☌, and partially unfolded until 90 ☌. ![]() REDLIVPVPIEQFTSAGLEPVEGMYVMTDAGIAKILKVEEKTVRLDFNHPL MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMP MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPHĪGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHHHHHHH MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPHĭPEGVQVVPLSAFPEDAEVVPGAQFYAQDMEGNPMPLTVVAVEGEEVTVDFNHPL Table 1 SlyD FKBP scaffold amino acid sequences SlyD type In conclusion, FKBP domains from thermostable SlyD proteins are highly suitable as a generic scaffold platform for epitope grafting. The respective SlyD scaffolds display favorable engineering properties in that they are small, monomeric, and cysteine-free and can be produced in high yields in a prokaryotic host, such as Escherichia coli. The selected antibodies bound with high affinity to the distinct IGF epitopes displayed on the protein scaffolds, as well as on the mature human IGF isoforms. Chimeric SlyD-IGF proteins allowed for the development of exceptionally specific IGF-1 and IGF-2 monoclonal antibodies. ![]() We found Thermus thermophilus sensitive to lysis D (SlyD) and Thermococcus gammatolerans SlyD FK-506–binding protein (FKBP) domains suitable for presentation of the predefined epitopes, namely the IGF-1 and IGF-2 loops. We sought to address this unmet demand by investigating novel chimeric immunogens as carriers for recombinant peptide motif grafting. the IGF loop, provides enough difference in sequence to discriminate between the two molecules. There is an urgent need for highly specific IGF-1 and IGF-2 antibodies, yet only a short sequence element, i.e. High sequence and structural homology between mature human insulin-like growth factors IGF-1 and IGF-2 makes serological discrimination by immunodiagnostic IGF tests a challenging task.
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