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

Sergei Y. Sokol,Andrew Hayhurst,Michael Klymkowsky,Keiji Itoh,Alice H. Reis

doi:10.1371/journal.pone.0216083

Abstract

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.

Highlights

For several decades, Xenopus laevis embryos have been a leading non-mammalian model for vertebrate embryology
We compared different approaches for the rapid isolation of multiple recombinant antibodies that react with Xenopus embryonic antigens
Embryos were injected at the 4-cell stage with 100 pg of Nuclear distribution element-like 1 (Ndel1) plasmid (25 pg per injection in each blastomere) and were cultured until stage 22 or 38 when they were lysed in 1% Triton X100, 50 mM NaCl, 1 mM EDTA and 10 mM Tris HCl

Summary

Introduction

Xenopus laevis embryos have been a leading non-mammalian model for vertebrate embryology. We describe the use of immune and non-immune phage display libraries for the isolation of several nanobodies specific for a variety of Xenopus antigens. Isolation of nanobodies against Xenopus antigens by phage display embryos were washed with PBS three times and homogenized with a pestle after adding 50 μl PBS in 1.5 ml Eppendorf tubes.

Results

Conclusion