A Method for the Parallel and Sequential Generation of Single-Domain Antibodies for the Proteomic Analysis of Human Blood Plasma

Abstract

A new efficient method for the parallel and sequential stepwise generation of single-domain antibodies to various high-abundance human-plasma proteins has been described. Single-domain antibodies have a number of features that favorably distinguish them from classical antibodies. In particular, they are able to recognize unusual unique conformational epitopes of native target proteins, small in size, and relatively easily produced and modified; have enhanced stability; and rapidly renature after denaturation. As a consequence, the immunosorbents that utilize these antibodies can be reused without any significant loss of activity. The principal novelty and universality of the described method is that it enables the sequential generation of antibodies to a number of high-abundance and yet unknown antigens of a complex protein mixture without the need for purified antigens. The effectiveness of the method is demonstrated by the example of generation of single-domain antibodies to a number of high-abundance proteins of the human blood plasma. The produced antibodies are promising biotechnological tools that can be used to develop prototypes for new diagnostic and therapeutic agents, as well as appropriate immunoaffinity-based methods for removal, enrichment, analysis, and/or targeting of specified proteins and their complexes from (in) the human blood. As we show, the generated single-domain antibodies can be efficiently used in designing new immunosorbents. As a rule, commercially available analogous immunosorbents that utilize classical antibodies remove many major proteins from the blood plasma immediately, while immunosorbents for many individual proteins are difficult to find and rather expensive. Single-domain antibodies generated by our method are unique new materials that allow for the development of more efficient and delicate approaches to pretreatment of plasma and the analysis of various blood plasma biomarkers.