High-Affinity Single-Domain Antibodies for Analyzing Human Apo- and Holo-Transferrin.

A highly efficient technology for generating new monoclonal single-domain recombinant antibodies (nanobodies) was used to obtain a panel of nanobodies recognizing human apo- and/or holo-transferrin. This article is devoted to the primary analysis of the properties of two different variants of the new nanobodies obtained by us, as well as to the demonstration of the unique potential of their application for diagnostic studies. The simultaneous use of immunosorbents based on these nanobodies apparently makes it possible to detect changes in the relative abundance of apo- and holo-transferrin in human biological fluids. Such changes could potentially be indicative of an increased risk or degree of development of pathological processes, such as malignant neoplasms in humans.

[Single-Domain Antibodies Used to Pretreat the Human Urinary Proteome in Cancer Biomarker Testing].

A number of single-domain antibodies (nanobodies) obtained previously to major marker blood proteins were tested as tools to preprocess urine samples from patients with bladder cancer. Nanobody-based tools demonstrated unique possibilities for noninvasive diagnostic studies along with other conventional methods, such as electrophoresis and, in prospect, mass spectrometric analysis. A testing of 22 samples from bladder cancer patients showed that the development of bladder cancer is accompanied by an increase in the urine contents of major blood proteins, including those known as potential bladder cancer biomarkers. New nanobody-based immunosorbents allow both specific enrichment and specific removal of particular antigenic proteins and subproteomes associated with them from a biological fluid. The isolation of immune complexes from the urine of a particular patient is of particular interest. An initial study of the complexes showed not only increased contents of IgA and IgG at advanced stages of the disease, but also many other components, which provide potential biomarkers of the pathological process in a particular patient. It is intended to use the approaches proposed in this work in a future larger-scale study of urine samples from patients with bladder cancer at different stages of the disease in order to identify new promising biomarkers of bladder cancer.

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

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.