Label-Free Real-Time Monitoring of Reactions Between Internalin A and Its Antibody by an Oblique-Incidence Reflectivity-Difference Method

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    Surface protein internalin (InlA) is a major virulence factor of the food-borne pathogen L. monocytogenes. It plays an important role in bacteria crossing the host's barrier by specific interaction with the cell adhesion molecule E-cadherin. Study of this protein will help to find better ways to prevent listeriosis. In this study, a monoclonal antibody against InlA was used to detect InlA. The reaction was label-free and monitored in real time with an oblique-incidence reflectivity-difference (OI-RD) technique. The kinetic constants kon and koff and the equilibrium dissociation constant Kd for this reaction were also obtained. These parameters indicate that the antibody is capable of detecting InlA. Additionally, the results also demonstrate the feasibility of using OI-RD for proteomics research and bacteria detection.


    Label-free , Reflectivity difference , Internalin


    L. monocytogenes is a human pathogen that causes listeriosis, a severe illness. Internalin A (InlA) is considered to be the most important surface protein of L. monocytogenes, because it promotes listeria uptake into intestinal epithelial cells by targeting the N-terminal domain of human E-cadherin, the dominant adhesion molecule of adherens junctions [1, 2]. Based on this fact, L. monocytogenes may cause illness in an intestinal, utero-placental, hepatic, or neurological phase of infection. Since the L. monocytogenes bacteria always hide in host cells, which makes it difficult for an antibiotic to work, a better way to prevent listeriosis is to block InlA from adhesion and invasion. Thus many researchers have turned their attention to InlA.

    Most anti-listeria antibodies available from research laboratories or commercial vendors are associated with the problems of low affinity [3], reaction to heterologous antigens [4, 5], or lack of reaction toward all serotypes of L. monocytogenes [6, 7]. Recently, a group of researchers announced a monoclonal antibody against InlA that reacts with both InlA and L. monocytogenes cells [8]. Known as Mab-2D12, it is reactive with all 13 serotypes of L. monocytogenes, but the affinity of this antibody to InlA has not been determined. To obtain information about the affinity of Mab-2D12, real-time measurements of the interaction between Mab-2D12 and InlA are desired. Further data analysis should provide the kinetic constants of the reaction.

    A common way to obtain affinity information is by using a surface plasmon resonance (SPR) biosensor [9-12]. Recently an optical oblique-incidence reflectivity difference (OI-RD) method has also shown great potential in high-throughput proteomics research [13, 14]. The OI-RD method detects a biomolecular microarray by measuring thickness and mass density of target spots. A small change in thickness or mass density in a molecular layer leads to changes in reflectivity. OI-RD is compatible with all flat substrates, which makes surface modification in OI-RD more flexible [15].

    In the present study, OI-RD was used for label-free, real-time monitoring of the binding reaction of Mab-2D12 antibody with surface-bound InlA targets. We will show how the kinetic parameters are obtained from the observed binding curves.


    A typical setup for OI-RD from a microarray-covered solid surface is sketched in Fig. 1. The microscope uses a He-Ne laser (λ = 633 nm) for illumination. The complex differential reflectivity change Δp − Δs across the microarray-covered surface is measured [14]. Here we define the OI-RD signal as Imp − Δs}, which can be obtained directly from experiments. Since the average thickness d of a biomolecular microarray is much less than the optical wavelength λ, Zhu and coworker