Native llama Nanobody Library Panning Performed by Phage and Yeast Display Provides Binders Suitable for C-Reactive Protein Detection

C-reactive protein (CRP) is an inflammation biomarker that should be quantified accurately during infections and healing processes. Nanobodies are good candidates to replace conventional antibodies in immunodiagnostics due to their inexpensive production, simple engineering, and the possibility to obtain higher binder density on capture surfaces. Starting from the same pre-immune library, we compared the selection output resulting from two independent panning strategies, one exclusively exploiting the phage display and another in which a first round of phage display was followed by a second round of yeast display. There was a partial output convergence between the two methods, since two clones were identified using both panning protocols but the first provided several further different sequences, whereas the second favored the recovery of many copies of few clones. The isolated anti-CRP nanobodies had affinity in the low nanomolar range and were suitable for ELISA and immunoprecipitation. One of them was fused to SpyTag and exploited in combination with SpyCatcher as the immunocapture element to quantify CRP using electrochemical impedance spectroscopy. The sensitivity of the biosensor was calculated as low as 0.21 μg/mL.

Intrabody Targeting HIF-1α Mediates Transcriptional Downregulation of Target Genes Related to Solid Tumors

Uncontrolled growth of solid tumors will result in a hallmark hypoxic condition, whereby the key transcriptional regulator of hypoxia inducible factor-1α (HIF-1α) will be stabilized to activate the transcription of target genes that are responsible for the metabolism, proliferation, and metastasis of tumor cells. Targeting and inhibiting the transcriptional activity of HIF-1 may provide an interesting strategy for cancer therapy. In the present study, an immune library and a synthetic library were constructed for the phage display selection of Nbs against recombinant PAS B domain protein (rPasB) of HIF-1α. After panning and screening, seven different nanobodies (Nbs) were selected, of which five were confirmed via immunoprecipitation to target the native HIF-1α subunit. The inhibitory effect of the selected Nbs on HIF-1 induced activation of target genes has been evaluated after intracellular expression of these Nbs in HeLa cells. The dramatic inhibition of both intrabody formats on the expression of HIF-1-related target genes has been confirmed, which indicated the inhibitory efficacy of selected Nbs on the transcriptional activity of HIF-1.

Construction of scFv-antibodies to the active center of the Sunn bug (Eurygaster integriceps Put) gluten-hydrolyzing protease GHP3

This work describes the preparation of immune library and selection of recombinant single-chain (scFv) antibodies to the active center of the gluten-destroying proteinase GHP3 of the Eurygaster integriceps Put. bug.