A NEW METHOD BASED ON THE USE OF IMMOBILIZED SINGLE-DOMAIN ANTIBODIES TO REMOVE CERTAIN MAJOR PROTEINS FROM BLOOD PLASMA HELPS TO REDUCE NONSPECIFIC SIGNAL IN AN IMMUNOASSAY

A generally underestimated problem of immunoassays is its susceptibility to various interferences, being the Achilles heel of these assays (interventions in the analysis). The presence of interfering substances in the patient’s specimen can cause erroneous test sample result, which may lead to incorrect diagnosis and catastrophic consequences for the patient. Hence, one should pay particular attention to identifying possible interferences in the test systems used and, when possible, develop and apply methods to overcome them. The issue of avoiding interference is particularly important for immunoassays of biomarkers in human plasma or serum. In order to reduce possible interferences, it would be desirable to have an opportunity of specific and adaptable pretreatment of blood samples for a specifically assayed marker protein, as applied to a specific test system. We assume that such pretreatment may be done by combining the immunosorbents based on the use of special single-domain antibodies (nanobodies).

The nanobodies are recombinant proteins, derivatives of single-domain antigen-recognizing variable fragments of specific antibodies, consisting of a dimer of truncated heavy chains in the complete absence of light chains. Such specific antibodies are detectable in the normal samples taken from members of the Camelidae family (Camelids), and in some species of cartilaginous fishes, along with the common antibodies. The special properties of nanobodies can provide certain advantages in their use, compared with antibodies of traditional structure and their derivatives.

In this paper, we have shown for the first time, that the immunosorbents based on certain combination of single-domain antibodies used as ligands able for specifically binding and removal of specific high-abundance human blood proteins, may be selected for a given marker blood antigen in such a way that they will be an effective tool for blood pretreatment, aiming to reduce possible effects of interference and increased sensitivity in the diagnostic “sandwich” enzyme immunoassay. A new method of plasma pretreatment is demonstrated with human plasma samples (at a dilution of 1:40) of two patients. A previously developed model system for detection of lactoferrin protein was used to analyze plasma samples. It is shown that a significantly increased ratio of total detected signal to the nonspecific background signal could be obtained after drastic reduction of this background by affinity removal of 3 major protein fractions, i.e., fibrinogen, IgG alpha-2-macroglobulin from blood plasma samples, using appropriate immobilized single domain antibodies. 

1. Goryainova O.S., Ivanova T.I., Rutovskaya M.V., Tillib S.V. A method for the parallel and sequential generation of single-domain antibodies for the proteomic analysis of human blood plasma. Molekulyarnaya biologiya = Molecular Biology, 2017, Vol. 51, no. 6, pp. 985-996.(In Russ.)

2. Tillib S.V., Vyatchanin A.S., Muyldermans S. Molecular analysis of heavy chain-only antibodies of Camelus bactrianus. Biokhimiya = Biochemistry, 2014, Vol. 79, no. 12, pp. 1687-1697. (In Russ.)

3. Boehm Т.К., Sojar Н., DeNardin E. Concentration-dependent effect of fibrinogen on IgG-specific antigen binding and phagocytosis. Cell. Immunol., 2010, Vol. 263, no. 1, pp. 41-48.

4. Bolstad N., Warren D.J., Nustad K. Heterophilic antibody interference in immunometric assays. Best Pract. Res. Clin. Endocrinol. Metab., 2013, Vol. 27, pp. 647-661.

5. Hamers-Casterman C., Atarhouch T., Muyldermans S., Naturally occurring antibodies devoid of light chains. Nature, 1993, Vol. 363, pp. 446-448.

6. Harmsen M.M., de Haard H.J. Properties, production, and applications of camelid single-domain antibody fragments. Appl. Microbiol. Biotechnol., 2007, Vol. 77, pp. 13-22.

7. Moreno-Expósito L., Illescas-Montes R., Melguizo-Rodrígueza L. Multifunctional capacity and therapeutic potential of lactoferrin. Life Sciences,2018, Vol. 195, pp. 61-64.

8. Muyldermans S., Baral T.N., Retamozzo V.C. Camelid immunoglobulins and nanobody technology. Vet . Immunol. Immunopathol., 2009, Vol. 128, pp. 178-183.

9. Muyldermans S. Nanobodies: natural single-domain antibodies. Annu. Rev. Biochem., 2013, Vol. 82, pp. 775-797.

10. Steeland S., Vandenbroucke R.E., Libert C. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discovery Today., 2016, Vol. 21, pp.1077-1113.

11. Tillib S.V., Privezentseva M.E., Ivanova T.I. Single-domain antibody-based ligands for immunoaffinity separation of recombinant human lactoferrin from the goat lactoferrin of trasgenic goat milk. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2014, pp. 48-57, 949-950.

12. Ward G., Simpson A., Boscato L., Hickman P. E. The investigation of interferences in immunoassay. Clin. Biochem., 2017, Vol. 50, pp. 1306-1311.

13. Wyatt A.R., Kumita J.R., Farrawell N/. Alpha-2-macroglobulin is acutely sensitive to freezing and lyophilization: Implications for structural and functional studies. PLoS ONE, 2015, Vol. 10, no. 6, e0130036. doi: 10.7868/S0026898417060106.