Itoh K et al. (2019), Isolation of nanobodies against Xenopus embryon...

XB-ART-55927

PLoS One 2019 May 02;145:e0216083. doi: 10.1371/journal.pone.0216083.

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Isolation of nanobodies against Xenopus embryonic antigens using immune and non-immune phage display libraries.

Itoh K , Reis AH , Hayhurst A , Sokol SY .

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The use of Xenopus laevis as a model for vertebrate developmental biology is limited by a lack of antibodies specific for embryonic antigens. This study evaluated the use of immune and non-immune phage display libraries for the isolation of single domain antibodies, or nanobodies, with specificities for Xenopus embryonic antigens. The immune nanobody library was derived from peripheral blood lymphocyte RNA obtained from a llama immunized with Xenopus gastrula homogenates. Screening this library by immunostaining of embryonic tissues with pooled periplasmic material and sib-selection led to the isolation of several monoclonal phages reactive with the cytoplasm and nuclei of gastrula cells. One antigen recognized by a group of nanobodies was identified using a reverse proteomics approach as nucleoplasmin, an abundant histone chaperone. As an alternative strategy, a semi-synthetic non-immune llama nanobody phage display library was panned on highly purified Xenopus proteins. This proof-of-principle approach isolated monoclonal nanobodies that specifically bind Nuclear distribution element-like 1 (Ndel1) in multiple immunoassays. Our results suggest that immune and non-immune phage display screens on crude and purified embryonic antigens can efficiently identify nanobodies useful to the Xenopus developmental biology community.

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Species referenced: Xenopus laevis
Genes referenced: mapk1 mbp ndel1 npm1 npm2 pafah1b1

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	Fig 1. Distinct patterns are revealed by immunostaining of embryo sections with antibody pools.Phages expressing nanobody cDNA library were enriched by binding to immobilized antigens from total embryo homogenates. Periplasm was obtained from pools of 10–20 bacterial colonies infected with enriched phage population, except that pool 15N19 consisted of 3 colonies. Cryosections of stage 11 gastrula embryos were incubated with periplasm pools, followed by mouse anti-His antibodies and Alexa488-conjugated anti-mouse IgG antibodies. (A-A”) Pool G2 stains nuclei; B-B” pool G8 serves as a negative control. (C) Pool N21 reacts with yolk platelets and cytoplasmic puncta. (D) Pool 40D reacts with yolk platelets. (E) Pool 15N19 stains perinuclear Golgi-like structures of endodermal cells. DAPI staining visualizes nuclei (A, A”, B, B”, D, E). Ectoderm (ect), mesoderm (mes) and endoderm (endo) are indicated. Bar, 50 μm.
	Fig 2. Comparison of unique nanobody amino acid sequences.Nanobodies with different specificities were sequenced and the alignment of deduced amino acid sequences is shown for nanobodies staining nuclei (N), perinuclear Golgi-like structures (G), cytoplasmic puncta (P) and yolk platelets (Y). Complementarity determining regions (CDR1, 2 and 3) are marked by blue boxes, conserved cysteines are indicated as orange boxes.
	Fig 3. Immunoreactivity of purified nanobodies.Staining of gastrula cryosections with 1.6 μg/ml of NbG18 (A), 3 μg/ml of NbN19 (B) or 1 μg/ml of NbN21 (C) shows nuclear, perinuclear or punctate staining, respectively. DAPI stains nuclei (A’, B, C). Ectoderm (ect), mesoderm (mes), endoderm (endo), blastocoel and blastopore (bp) are indicated. Bars are 50 μm. D-E, immunoblot analysis of embryo lysates at different embryonic stages. Equal amounts of embryo lysates corresponding to the equivalent of 0.5 embryo were separated on 10% SDS-PAGE, transferred to PVDF membrane and probed with different nanobodies. D, NbG18 and NbN39 detect a single protein band of approx. molecular weight of 170 kDa; E, NbN19 detects a broad protein band of 80–95 kDa.
	Fig 4. Identification of Npm2 as an antigen recognized by NbG12.(A) Reverse proteomics approach to identify the antigen recognized by NbG12. Lysates from 50 gastrula embryos were precipitated with 7 μg of NbG12 or NbT15 and 25 μl of Ni ion resin and separated by SDS-PAGE. After Simply Blue staining, 170 kDa protein band that was precipitated with NbG12 and the corresponding gel area from NbT15 pulldown were excised for LC-MS/MS analysis. (B) Top hits from the LC-MS/MS analysis of the 170 kDa band. Npm2 is detected with high abundance in NbG12 but not in NbT15 pulldowns.
	Fig 5. Isolation of Ndel1-specific nanobodies.(A) amino acid sequence alignment of six nanobodies. Orange boxes indicate the two conserved cysteine residues, and blue boxes indicate the three complementarity-determining regions (CDRs). (B) wells coated with purified Ndel1-MBP protein (1 μg/ml) are positive in ELISA after probing with several Ndel1-specific nanobodies but not with negative controls (no Nb Co or NbN39). Control BSA coating confirms specificity. (C) immunoblotting of Xenopus Ndel1. Lysates from xNdel1-transfected or control (-) HEK293T cells were separated by SDS-PAGE and visualized with 2 μg/ml of purified AP-NbE7 and AP substrate. Purified Ndel1-MBP, but not LIS1-MBP is detected with NbE7. ERK1/2 antibody controls loading. (D, E) Ndel1 is precipitated by NbE9 from transfected cell lysates (D) and injected Xenopus embryos (E). (D) HEK293T cells were transfected with Xenopus Ndel1 plasmid as indicated. Cells were lysed after 24–48 h culture. Pulldowns were carried out with 2 μg of 6His-AP-NbE9 (asterisk) and 20 μl of Ni-agarose beads. (E) four-cell embryos were injected with 100 pg of xNdel1 DNA. The injected embryos were lysed at stage 38 and the pulldown was performed as in D. Arrows in D, E, indicate the 39 kD band corresponding to xNdel1. AP-NbE9 and AP substrate were used in immunoblotting for signal detection (D, E).

References [+] :

Asashima, Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor). 1990, Pubmed, Xenbase