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 k_on and k_off and the equilibrium dissociation constant K_d 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. 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 Δ _p − Δ _s across the microarray-covered surface is measured [14] . Here we define the OI-RD signal as Im {Δ _p − Δ _s }, 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 [16] have shown that Here θ_inc is the angle of incidence, and ε₀ , ε_d , and ε_s are respectively the optical dielectric constants of the surroundings, the molecular layer, and the substrate. When using OI-RD to monitor the immobilization of an antibody with surface-bound InlA targets, ε_d is the dielectric constant of the immobilized antibody, ε₀ is the dielectric constant of glass, and ε_s is the dielectric constant of the solutions in the flow cell. Monoclonal antibody against InlA was raised in mice. Briefly, six-week-old female mice were administered intra-peritoneally (i.p.) with approximately 1 × 10 ⁸ cells/ml of heat-killed L. monocytogenes serotype 4b. Two weeks later, a mixture of heat-killed L. monocytogenes and 50 μ g of rInlA prepared with incomplete Freund’s adjuvant was administered i.p. every week for 8 weeks. The splenocytes were harvested from the mouse and fused with murine Sp2/O-Ag14 myeloma cells. Selected hybridoma clones were administered to pristine-primed mice to produce ascetic fluid for antibody production. Monoclonal antibody was purified by affinity chromatography using a protein A-Sepharose 4B column (GE Healthcare), and the class of the monoclonal antibody was determined by ELISA experiments [8] . The experimental procedure consisted of several steps. First, the 80-kDa InlA target was printed on the epoxy-functionalized glass slide by a GMS 417 pin-and-ring contact-printing robot arrayer (Genetic Microsystems) at a concentration of 8.75 μ M in a solution of 1 × PBS (phosphate buffered saline, pH = 7.4). Bovine serum albumin (BSA) was also printed as the control. InlA and BSA bind covalently to epoxy groups [17] . The arrangement of printed spots is shown in Fig. 2 ; the center-to-center separation between spots is 400 μ m. For each row, six spots were printed as repeats, and the slide was washed with 1 × PBS solution to remove excess unbound and weakly bound proteins. Then the remaining epoxy groups on the surface were blocked with BSA (7.6 μ M in 1 × PBS) for 10 minutes, to prevent probe molecules from nonspecific binding. At last, the slide was reacted with Mab-2D12 antibody in 1×PBS at room temperature. We first filled the flow cell with antibody quickly at a rate of 30 mL/min, then slowed down the flow rate to 0.05 mL/min during observation of the association reaction. To observe the dissolution, we quickly replaced the probe solution with 1×PBS at a flow rate of 30 mL/min, then slowed down the flow rate to 0.05 mL/min during the subsequent dissociation. In Figure 2 we show an OI-RD image of the InlA microarray at each step of the process. It is a small 4×6 microarray. The circles indicate where the spots should be, as printed. At the blocking and association step, it is unclear whether the reaction has happened by simply looking at Fig. 2 , but from the difference image-the image after the association minus the image before-, it is clear that there were reactions between Mab-2D12 antibody and surface-bound InlA, while no reactions happened on the BSA spots. Here we rewrite Eq. (1) in terms of the surface mass density of the monolayer targets, given by Г_probe = d_probeρprobe [18] , ε_probe is the dielectric constant for the antibody, ρ _probe is the volume mass density of the antibody, and d _probe is the average thickness of the immobilized antibody, which increases with time during association. Eq. (2) indicates that Г_probe is proportional to Im {Δ _p − Δ _s }, which means the real-time OI-RD signal is proportional to the mass (quantity) of antibody molecules that have reacted with surface-bound InlA. The difference image in Fig. 2 shows positive signals at InlA spots, which proves that the reaction did take place. Figure 3 shows three real-time binding curves with antibody at concentrations of 200 nM, 300 nM, and 400 nM: association in the first hour, and dissociation in the following two hours. Each curve is normalized to the maximum value of the OI-RD signal during association with antibody at 400 nM, assuming that a monolayer of antibody was immobilized on the surface after the association process. It is shown in Fig. 3 that the rates to reach saturation for the three curves are different. Saturation only occurred when the antibody was at a concentration of 400 nM. We fit the association-dissociation curves using Langmuir-reaction kinetics [19 , 20] . In this model, the association rate is assumed to be proportional to the antibody concentration as k_on [ c ], while the dissociation rate k_off is independent of the antibody concentration. The equilibrium dissociation constant of the reaction is given by K_d = k_off / k_on . We fit the binding curves to a one-site Langmuir-reaction kinetic model that assumes one surface-bound InlA molecule reacts with one Mab-2D12 antibody molecule at a specific site. In the one-site model, let N and N ⁽⁰⁾ be respectively the reacted and initially available amounts of antibody. The total amount of reacted antibody during the association reaction is given by Here [ c ] is the concentration of antibody and t is time. After a time t₀ , the antibody solution is replaced with 1 × PBS. Subsequently, the total number of the reacted antibody decreases as There is no doubt that the surface mass density Г_probe is proportional to the amount of reacted antibody N ( t ). From Eq. (2), the optical signals shown in Fig. 3 are expected to vary as for 0 < t < t₀ , and for t > t⁰ . For the three association-dissociation curves in Fig. 3 , we performed a global fitting using Eqs. (5) and (6) with the same k_on and k_off as global fitting parameters. The three gray lines in the figure are the fits to the model. The kinetic constants for Mab-2D12 antibody reactions with InlA are k_on = 3.35×10 ⁻³ /(¼ μ M⋅s), k_off = 1.14×10 ⁻⁴ /s, and K_d = 3.40×10 ⁻² μ M = 3.40×10 ⁻⁸ M. The dissociation constant K_d obtained here falls within the range for biological molecules, from 1×10 ⁻⁴ to 1×10 ⁻¹¹ M [21] . The data indicate that the monoclonal antibody is specific to the InlA protein. This experiment proves that the Mab-2D12 antibody is capable of detecting InlA protein and also whole-cell L. monocytogenes . In summary, we performed real-time, label-free monitoring of the reaction between Mab-2D12 antibody and surfacebound InlA. The appropriate dissociation constant K_d = 3.40 × 10 ⁻⁸ M indicates that Mab-2D12 antibody as a useful monoclonal antibody for the detection of L. monocytogenes . The results presented here also demonstrate the versatility of the combined OI-RD scanner and microarray platform for real-time monitoring of protein reaction kinetics, which confirms that OI-RD can be used to determine affinity of a protein with an antibody in a label-free manner.