Abstract
The first step in determining whether a fluorescent dye can be used for antibody labeling consists in collecting data on its physical interaction with the latter. In the present study, the interaction between the 2-(2-hydroxy-5-nitrobenzylidene)-1,3-indanedione (HNBID) dye and the IgG1 monoclonal mouse antibody anti-human heart fatty acid binding protein (anti-hFABP) has been investigated by fluorescence and circular dichroism spectroscopies and complementary structural results were obtained by molecular modeling. We have determined the parameters characterizing this interaction, namely the quenching and binding constants, classes of binding sites, and excited state lifetimes, and we have predicted the localization of HNBID within the Fc region of anti-hFABP. The key glycosidic and amino acid residues in anti-hFABP interacting with HNBID have also been identified. A similar systematic study was undertaken for the well-known fluorescein isothiocyanate fluorophore, for comparison purposes. Our results recommend HNBID as a valuable alternative to fluorescein isothiocyanate for use as a fluorescent probe for IgG1 antibodies.
Highlights
Recent studies have demonstrated that heart-type fatty acid binding protein can be used as an early marker for diagnosis of acute myocardial infarction and prognosis [1,2], and other myocardial events [3]
In the present study we investigated the interaction of the fluorescent dye, 2-(2-hydroxy-5-nitrobenzylidene)-1,3-indanedione (HNBID, Scheme 1), with the immunoglobulin G1 (IgG1) isotype of monoclonal mouse anti-heart-type fatty acid binding protein (hFABP) antibody of the 9F3 family, by combining two experimental spectroscopic methods with molecular modeling computations
Before performing a comparative study of the effects of HNBID and fluorescein isothiocyanate (FITC) on the fluorescence characteristics of anti-hFABP, the photophysics of the isolated ligands has been investigated in dimethyl sulfoxide (DMSO) and phosphate buffer of pH 7.4, the media most frequently used in vitro to mimic in vivo systems [16], and in anti-hFABP solution
Summary
Recent studies have demonstrated that heart-type fatty acid binding protein (hFABP) can be used as an early marker for diagnosis of acute myocardial infarction and prognosis [1,2], and other myocardial events [3]. Antibodies, labeled by chemical conjugation with fluorescent dyes, bind to the antigen of interest, allowing antigen detection by means of fluorescence techniques. The main drawback to this labeling strategy is that, usually, it results in a significant decrease of the antigen-binding activity of the labeled antibody because the amino groups distributed in the Fab region can react with FITC [6]. This covalent interaction, combined with FITC physical adsorption in this region, may significantly affect the availability of the binding sites on the antibody. Many other fluorescent molecules have been synthesized and described in the literature, only a handful came to being used as labeling agents due to drawbacks such as low fluorescence quantum yield and insufficient photostability
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