Research paperA kinetic ELISA to determine the immunoreactive fraction of monoclonal antibodies
Introduction
Immunoglobulins are proteins that constitute the main protective component of animal innate and adaptive humoral immune systems. Their physicochemical properties such as heterogeneity, specificity, affinity, and polyreactivity make them useful reagents. From the early days of immunology, passive antibody therapy was used to combat bacterial toxins and microbial infections (Graham and Ambrosino, 2015). The advent of monoclonal antibody (mAb) technology prompted their extensive use in research, clinical diagnosis, infectious immunotherapy (Walker and Burton, 2018), as well as immunomodulation of cancer (Ribas and Wolchok, 2018;), autoimmune, and inflammatory disorders (Kumar et al., 2018).
In the course of immunoglobulin biosynthesis, purification, labeling, or conjugation to other molecules, antibodies lose activity, which can alter their original immune reactivity. Determination of the antibody immunoreactive fraction (IRF) is thus an important, necessary quality control for immunoglobulin-based reagents.
IRF determinations were initially based on cellular radioimmunoassay (RIA) to measure radiolabeled mAb bound to cell surface antigens in conditions of antigen excess (Lindmo et al., 1984; Lindmo and Bunn, 1986) or antibody excess (Dux et al., 1991; Fjeld and Skretting, 1992). IRF values were then obtained by analysis of binding data using graphic methods such as direct linear plot (Partridge et al., 1985), Scatchard plot (Abraham et al., 1991; Beaumier et al., 1986), mathematical curve fitting (Konishi et al., 2004) or Lindmo's linear modification of the double-inverse Lineweaver-Burk plot (Dux et al., 1991; Fjeld and Skretting, 1992; Lindmo et al., 1984).
The Lindmo method, with modifications, remains the canonical assay for IRF determination (Rhodes et al., 1990; Carpenet et al., 2015; Haisma et al., 1992; Ngai and Reilly, 1993; Zamora et al., 1994). In this assay, free and cell-bound antibody fractions at equilibrium are plotted as the percentage of [total]/[bound] antibody (on the y-axis) against the inverse of cell antigen concentration, that is, 1/[cells] (on the x-axis). At hypothetical infinite antigen concentration, 1/[cells] = 0, and the extrapolated line intersects the y-axis at y0 = 1/r, where r is the inverse of the IRF value. The reliability of IRF values obtained using this method rests in part on the assumptions that, at large antigen excess, total and free antigen concentrations are similar, and that all functional antibody molecules are bound to antigen. These requirements are met when [antigen] > > 1/Ka at very high antigen concentrations, or when working with high affinity mAb. In analyses using cells with low antigen expression, however, or with low affinity mAb (Ka ~107–108 M−1), the Lindmo method can generate less accurate IRF data (Mattes, 1995).
Traditional cellular RIA methods for IRF determination are now becoming outdated, particularly given the trend toward reduced radioligand use. To improve and facilitate IRF determination, we developed a kinetic ELISA in conditions of antigen excess to quantitate mAb IRF. Antibody IRF value was derived from the initial velocity gradient of the reactions between antigen-unbound mAb fractions and peroxidase-conjugated antiglobulin. This kinetic ELISA method is simple, inexpensive, highly repeatable, and allows determination of the IRF value of poly- and monoclonal antibodies.
Section snippets
Reagents and buffers
Materials used included Sepharose CL-4B (GE Healthcare-Life Sciences, Chicago, IL, USA), ProfinityTM epoxide resin (Bio-Rad, Madrid, Spain), ELISA 96-microplate wells (Maxi Sorp 442404; Nunc, Roskilde, DK), and Costar high binding plates (9018; Corning, NY, USA). Tween-20, ortho-phenylenediamine (OPD) and 2,4,6-trinitrobenzenesulfonic acid (TNBS), and Bovine serum albumin were from Sigma-Aldrich (St. Louis, MO, USA). High-trap Protein G HR was from GE Healthcare Life Sciences (Pittsburgh, PA,
Determination of antigen concentration range for plate adsorption
To establish the dynamic range of antigen concentration, we first studied the progress curve of antigen adsorption to plastic. Plate wells were sensitized with a series of increasing concentrations (0 to 600 ng/mL) of CT, DPT, BSA, and TNP-BSA. After adsorption, we determined the course of antigen binding to antigen-specific mAb. The binding curve varied depending on concentration and molecular characteristics of each antigen (Fig. 1). The working ranges used were CT (0–200 ng/mL), DPT
Discussion
Engineered mAbs are essential reagents for diagnosis, prevention, and treatment of human diseases (Chames et al., 2009; Marston et al., 2018; Saeed et al., 2017). Their correct use nonetheless requires previous knowledge of certain antibody parameters, including concentration, association (k +1) and dissociation (k-1) rate constants, equilibrium constant (Ka), and the immunoreactive fraction (IRF). Some of these parameters are interdependent; for instance, the rate constant (k +1) of
Conclusions
This study describes a two-step ELISA method to quantitate mAb IRF in antigen excess conditions. Antigen-unbound (free) mAb fractions were quantitated by kinetic ELISA, based on the slopes of their initial reaction velocity with peroxidase-conjugated antiglobulin. This kinetic method lacks the restrictions posed by other techniques, is simple, inexpensive, highly repeatable, and can be used to determine the IRF of poly- and monoclonal antibodies.
Acknowledgements
The authors thank Nela Díez for the development of SIM169-3.3.3 and SIM47-15.14.1 mAb, Vanessa Fernández for SIM253-19.1.1 and SIM283-9.5.1, Ana Belén Martín and Soledad Crespo for mAb culture supernatants, Sergio González and Francisco Toraño for help with artwork, and Catherine Mark for editorial assistance, critical reading, and sound advice on the manuscript.
This work was supported in part by grants from the Ministry of Economy, Industry and Competitiveness of Spain (FIS PI13/011446 and
Declaration of Competing Interest
The authors declare no conflict of interest.
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