Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies.

Nobuyuki Kurosawa,Haruki Kitamura,Megumi Yoshioka,Shun Matsuzawa,Yoshihiro Kishi,Masaharu Isobe,Tomonao Inobe,Yuka Wakata

doi:10.1038/srep25174

Abstract

Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins.

Highlights

Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine
endoplasmic reticulum-based identification of antigen-specific antibody-producing cells (ERIAA) enables the generation of monoclonal antibodies (mAbs) against highly conserved antigens via the isolation of antigen-specific plasma cell (ASPC) from a variety of animals to break immune tolerance, the potential of this technology has been limited by the inability to isolate rare ASPCs with high purity
Attempting to increase the intensity of ASPC staining with fluorescently labeled antigens, we found that intracellular labeling of paraformaldehyde (PFA)-fixed cells with a fluorescently labeled antigen together with an antibody against the immunoglobulin results in very bright signals on ASPCs, far removing them from noise

Summary

Introduction

Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. In light of the current protein engineering technology, animal immunization might appear obsolete This approach is still commonly used to produce antibodies against a variety of antigens as complementary technologies for generating different types of mAbs. by taking advantage of the natural affinity maturation of the immune system, a variety of phosphorylation site-specific polyclonal antibodies have been generated by immunizing with phosphorylated peptides[10,11]. ERIAA enables the generation of mAbs against highly conserved antigens via the isolation of ASPCs from a variety of animals to break immune tolerance, the potential of this technology has been limited by the inability to isolate rare ASPCs with high purity This is because ASPC separation by ERIAA depends on weakly expressed cell surface immunoglobulins as a tag for a fluorescently labeled antigen, precluding a sufficiently strong signal on ASPCs to allow specific separation. We succeeded in amplifying full-length immunoglobulin variable (V) genes from individual fixed and intracellularly labeled cells

Methods

Results

Conclusion