Determination of IgG and IgA Antibodies by Fluorescence Polarization Using Fluorescently Labeled Recombinant Nanobodies

Objective: Quantitative, rapid and high-throughput analysis of IgG and IgA immunoglobulins is necessary to determine the content of these proteins and their associated molecules in the patient’s physiological fluids. The analysis of these proteins is necessary in the diagnosis of specific antibody deficiency as an auxiliary test for the detection of general variable immunodeficiency, as well as for risk stratification of patients with low IgA levels. IgG content determination can help in prescribing revaccination to patients and supporting their treatment strategy, can be used to monitor the patient's humoral immune system, as well as in the development and subsequent production of most therapeutic antibodies in biopharmaceuticals. Methods: Miniature recombinant single-domain antibodies (nanobodies) have a number of advantages over classical antibodies, such as their relative simplicity of operation, high stability over a wide range of temperature and pH values, the ability to recognize highly specific conformational epitopes of the target protein, as well as the possibility of using them as probes for detecting larger target antigen proteins in the fluorescence polarization method. Results and Discussion: Fluorescently labeled FITC-anti-IgG and FITC-anti-IgA nanobodies to human IgG and IgA were obtained and characterized. The KD values of the FITC-anti-IgG*IgG and FITC-anti-IgA*IgA complexes were determined; they confirmed the high affinity of the immunoreagents. The possibility of specifically determining IgG and IgA levels in human serum in the range of 35– 120 μg/mL (for IgA) and 75–260 μg/mL (for IgG) was demonstrated. Eighteen human sera were tested for IgG and IgA levels, and the content of antibodies in the samples was confirmed using commercial enzyme immunoassay kits. FITC-anti-IgG and FITC-anti-IgA did not interact with other human proteins: albumin, plasminogen, fibrinogen, lactoferrin, and transferrin. Testing of human and animal sera by FITC-anti-IgG and FITC-anti-IgA demonstrated specific binding to human and monkey antisera, but not to animal sera: bovine, canine, feline, rabbit, and sheep. Conclusions: Thus, the FPIA method can be used for the rapid and specific determination of human IgG and IgA.