KR20080054647A - Real-time dual color assay for detecting protein-protein interaction and an apparatus therefor - Google Patents

Abstract

A dual color assay method is provided to detect real-time dynamics of temperature-dependent protein-protein interaction in solution in the single-molecule level by using a dual-wavelength laser-induced fluorescence, so that real-time fluorescence images of individual protein molecule labeled with different fluorescence dyes are monitored simultaneously without delay of time. A dual color assay method for detecting protein-protein interaction comprises the steps of: measuring the interaction between an analyte antigen protein and an antibody protein by using enzyme-linked immunosorbent assay; and interpreting the measured results by using the double-wavelength color of wavelength X and Y selected from 440 nm, 488 nm, 545 nm, 580 nm and 635 nm, wherein the enzyme-linked immunosorbent assay contains an analyte protein antigen A having a fluorescence label of wavelength X, a first antibody protein anti-A binding to the analyte protein antigen A, and a second antibody protein anti-B having a fluorescence label of wavelength Y, binding to the first antibody protein anti-A; and the analysis temperature is 12-32 deg. C.

Description

Real-time dual color assay for detecting protein-protein interaction and an apparatus therefor}

1 is a schematic diagram illustrating the labeling antigen, first antibody and second antibody complementary binding reaction steps for measuring protein-protein interaction of the present invention.

Figure 2 (a) is a photograph showing the instrument used to monitor single-protein molecules. Figure 2 (b) is a schematic diagram showing a capillary form of experimental instruments for monitoring single-protein molecules. In the figure, the MS is a mechanical shutter; CL is a cylindrical lens; OL is an objective lens; SC is a square capillary; TC is a thermal couple. Ar ⁺ laser (λ _ex = 488 nm) and He / Ne laser (λ _ex = 635 nm) are irradiated to the capillary center through the left and right sides of the capillary, respectively.

Figure 3 is a fluorescent video image (green) of the Alexa Fluor 488-labeled actin / anti-actin antibody complex (a) λ _ex = 488 nm in a buffered aqueous solution using a real-time dual color analysis system of the present invention (b) λ Fluorescent video image (red) of Alexa Fluor 633-labeled goat anti-rabbit IgG antibody at _ex = 635 nm and (c) Mixed protein specimen (Alexa Fluor 633-labeled goat anti-rabbit IgG antibody and Alexa Fluor 488-labeled actin / Fluorescence video image (anti-actin antibody complex). The ICCDN exposure time is 10 ms and the frame rate is 10 ms.

4 is a video image photograph comparing the sizes of actin protein molecules and 200-nm beads. At this time, the test apparatus used is to use the test apparatus of FIG.

5 is a video image photograph showing the effect of concentration on nonspecific protein-protein interactions using a real-time bi-color LIF detection system. Green, FITC-labeled protein kinase A; Red, Alexa Fluor 633-labeled goat anti-rabbit IgG antibody. Experimental conditions are the same as shown in FIG. 2. Dashed arrows indicate nonspecific binding of protein molecules, and straight arrows indicate aggregation of protein molecules.

6 is a video image photograph showing complementary binding of protein-protein interactions in solution using a real-time bi-color LIF detection system. Experimental conditions are the same as shown in FIG. 2. Green, Alexa Fluor 488-labeled actin / anti-actin antibody complex; Red, Alexa Fluor 633-labeled goat anti-rabbit IgG antibody.

7 shows Alexa Fluor 488-labeled actin / anti-actin antibody complex (green) and Alexa Fluor 633-labeled goat anti-rabbit IgG antibody (red) as a function of response time under capillary propulsion using a real-time bi-color LIF detection system. A video image photograph showing the interaction of. Experimental conditions are the same as shown in FIG. 2.

8 shows the number of binding molecules involved in the interaction between Alexa Fluor 488-labeled actin / anti-actin antibody complex (green) and Alexa Fluor 633-labeled goat anti-rabbit IgG antibody (red) at different temperatures as a function of reaction time ( Left y-axis) and relative molecular percentage (right y-axis). Experimental conditions are the same as shown in FIG. 2. Error bars represent standard deviation of five measurements. Red circle, 12 ° C .; Blue triangle, 20 ° C .; Yellow circle, 25 ° C .; Black square, 32 ° C.

The present invention relates to a real-time dual color analysis method for measuring protein-protein interactions, and more particularly to an antigen having a fluorescent label, a first antibody bound to the antigen and a fluorescent label bound to the first antibody. The present invention relates to an assay method and apparatus for measuring protein-protein interaction using an enzyme immunoassay method consisting of a second antibody.

Protein-protein interactions play an important role in all cellular processes and functions. Several assays, including immunoassays, have been used to analyze protein-protein interactions. Common methods for early immune response assays were used for analysis by incubating the protein with an exogenous binding partner, usually at low temperatures of 2-8 ° C. overnight. However, accurate analysis is required for medical and commercial applications in the short time of the last 1-2 hours. When high-throughput protein-protein interaction assays are more widely used, the need to promote binding reactions and shorten assay times becomes even more important.

Various assays have been developed to measure protein-protein interactions in vivo. These include yeast two-hybrid system method, surface plasmon resonance, affinity column method, isothermal titration calorimetry, nuclear magnetic resonance spectroscopy or atomic force microscopy (AFM), equilibrium centrifugation method, labeled protein method And tandem affinity tablets.

PCT International Publication No. WO 2004/19967, Methods for Isolating Subpopulations Involving Protein-Protein Interactions, uses a chemically reactive support matrix to isolate proteins that are non-covalently bound to other proteins, thereby allowing calcium- or substrate-dependent protein interactions. A method for measuring is disclosed.

However, assays for measuring such protein-protein interactions are in need of further refinement and these assays are specialized, but require a large amount of protein and can confuse the labeling of interaction partners with spectroscopic probes. Problems often arise.

Fluorescent probes are now frequently used to measure protein-protein interactions and are versatile for studying the kinetics, affinity, and conformational changes of various protein interactions. However, protein-protein interactions and their kinetics are complex and heterogeneous. Conventional assays based on uniform hypotheses are not always able to analyze the kinetics of protein-protein interactions in real time. Thus, characterization of protein-protein interactions as well as complete interaction networks is essential for understanding these processes at the single-molecule level.

Single-molecule detection (SMD) technology offers powerful new technologies in the analytical chemistry and biophysics as well as in the life sciences. In particular, SMD not only visualizes protein interactions in vivo, but also performs on very limited samples in vitro. AFM and fluorescence techniques have become very common in the investigation of protein-protein interactions at the single-molecule level. However, AFM is not suitable for the real-time detection of power molecules for samples moving in solution in solution.

Some fluorescence detection methods can be used successfully to measure protein interactions with probes at the single-molecule level both in vitro and in living cells. Conventional fluorescence detection techniques use only single-wavelength light as an excitation energy source. Single-wavelength detection, however, cannot provide accurate images of protein-protein interactions as well as the kinetics of individual molecules in the mixture.

In the present invention, a dual-wavelength laser-induced fluorescence (LIF) detection system was applied to investigate the real-time kinetics of temperature-dependent protein-protein interactions in solution at the single-molecule level. The present invention has been accomplished by using a dual-wavelength single-molecule fluorescence detection system to allow real-time fluorescence images of individual protein molecules labeled with different fluorescent dyes to be simultaneously monitored in real time without delay.

The technical problem to be achieved in the present invention is to develop a double-wavelength laser-induced fluorescence detection method to investigate the real-time kinetics of temperature-dependent protein-protein interaction in solution at the single-molecule level. In the present invention, a dual-wavelength single-molecule fluorescence detection system is fabricated and a real-time dual color analysis method for real-time fluorescence imaging of individual protein molecules labeled with different fluorescent dyes can be simultaneously monitored without time delay. .

It is an object of the present invention to provide a sample protein antigen A with a fluorescent label of wavelength X; The first antibody protein anti-A that binds to sample protein antigen A; And a real-time dual color analysis method for measuring a protein-protein interaction consisting of a second antibody protein anti-B having a fluorescent label of wavelength Y bound to the first antibody protein anti-A, wherein the dual color analysis method It is to provide a real-time dual color analysis method characterized by measuring the interaction between the sample antigen protein and the antibody protein using an enzyme immune response, and reading the result through the double-wavelength color of wavelength X and Y. .

Meanwhile, the wavelengths X and Y are characterized in that the wavelength selected from 440 nm, 488 nm, 545 nm, 580 nm, 635 nm, respectively.

In addition, the real-time dual color analysis method, the concentration of the sample protein is characterized in that it can detect the interaction of the ultra-small amount of the sample protein to atomole (aM) to femtomol (fM) level.

In addition, the real-time dual color analysis method is characterized in that it is carried out at room temperature of 12 ~ 32 ℃.

On the other hand, another object of the present invention is iii) sample protein antigen A having a fluorescent label of wavelength X; The first antibody protein anti-A that binds to sample protein antigen A; And a capillary inserted with a sample protein consisting of a second antibody protein anti-B with a fluorescent label of wavelength Y bound to the first antibody protein anti-A; Ii) a laser light source for reading protein interactions in the capillary and an optical analyzer for reading its fluorescence wavelength; And iii) a real-time dual color analysis device for measuring protein-protein interactions of sample proteins consisting of computer equipment for deciphering optical analysis instruments.

Hereinafter, the present invention will be described in more detail.

Real-time dual color analysis instrument and measuring principle of the present invention

1 is a schematic diagram illustrating the labeling antigen, first antibody and second antibody complementary binding reaction steps for measuring protein-protein interaction of the present invention.

Protein kinase A-FITC conjugates were purchased from Promega (Madison, USA), and then the Alexa Fluor 488-labeled actin conjugate was prepared as a labeling antigen using an antigen-antibody binding procedure, and the anti-actin antibody at 4 ° C. Incubate for 60 minutes to generate the labeled antigen (Alexa Fluor 488-labeled actin) -first antibody (unlabeled anti-actin antibody) complex as the first antibody. Alexa Fluor 633-labeled goat anti-rabbit IgG antibody was added to the reaction mixture solution containing the labeled antigen (Alexa Fluor 488-labeled actin) -first antibody (unlabeled anti-actin antibody) complex as the second antibody.

Figure 2 (a) is a photograph showing the instrument used to monitor single-protein molecules. Figure 2 (b) is a schematic diagram showing a capillary form of experimental instruments for monitoring single-protein molecules. In the figure, the MS is a mechanical shutter; CL is a cylindrical lens; OL is an objective lens; SC is a square capillary; TC is a thermal couple. Ar ⁺ laser (λ _ex = 488 nm) and He / Ne laser (λ _ex = 635 nm) are irradiated to the capillary center through the left and right sides of the capillary, respectively.

As described above, FIG. 2 shows a schematic diagram and a photograph of a device used in a real-time dual color analysis method. Zeiss Axioskop2 microscopes (Zeiss, Germany) equipped with Dual-View ^™ (Optical Insight, LLC, Tucson, USA) are used for most of the irradiation. Zeiss 40 × / 0.75 NA Plan-Neofluar microscope objectives (Zeiss, Germany) are used.

Measurement of the action of single-protein molecules using real-time dual color analysis

Figure 3 is a fluorescent video image (green) of (a) λ _ex = 488 nm Alexa Fluor 488-labeled actin / anti-actin antibody complex in buffered aqueous solution using the real-time dual color analysis system of the present invention (green), (b) λ Fluorescent video image (red) of Alexa Fluor 633-labeled goat anti-rabbit IgG antibody at _ex = 635 nm and (c) Mixed protein specimen (Alexa Fluor 633-labeled goat anti-rabbit IgG antibody and Alexa Fluor 488-labeled actin / Fluorescence video image (anti-actin antibody complex). The ICCDN exposure time is 10 ms and the frame rate is 10 ms.

As shown in Figure 3 Alexa Fluor 488-labeled actin / 488 nm from the Ar ⁺ laser and 635 nm from the He / Ne laser are simultaneously excited and without any time loss when observed with Dual-View ^TM Fluorescence images of anti-actin antibody complexes and Alexa Fluor 633-labeled goat anti-rabbit IgG antibodies can be monitored in specific wavelength ranges. Individual protein molecules in the mixture sample are effectively concentrated and interact with complementary protein molecules and are measured by double-wavelength (excitation wavelength, λ _ex = 488 and 635 nm) LIF detection.

All mixed protein samples are co-excited by double-wavelength, but Alexa Fluor 488-labeled actin / anti-actin antibody complex molecules are clearly monitored in the 488 nm region (green in FIG. 3A) and Alexa Fluor 633-labeled goat Anti-rabbit IgG antibody molecules image only at 635 nm (red in FIG. 3B). Dual-wavelength LIF detection systems can provide accurate simultaneous imaging of individual proteins with different fluorophores without any time delay.

Two fluorescence emission images (520 and 650) of Alexa Fluor 488-labeled actin / anti-actin antibody complex and Alexa Fluor 633-labeled goat anti-rabbit IgG antibody in a single snapshot since there is no time delay between the images obtained nm) simultaneous acquisition is possible. In addition, when images at each specific wavelength overlap, two different protein molecules in the mixed sample can be measured.

4 is a video image photograph comparing the sizes of actin protein molecules and 200-nm beads. At this time, the test apparatus used is to use the test apparatus of FIG.

As shown in FIG. 4, the observed length of the individual Alexa Fluor 488-labeled actin was 96 ± 2.56% of the theoretical value of about 382 ± 9.79 (mean ± standard deviation) nm or 395 nm (43 kDa = 1161-bp DNA = 395). nm). The size of the actin protein molecule is almost similar to the 200-nm sulfur-green fluorescent FluoSphere beads. Individual protein molecules can be readily detected within the capillary if the capillary is dynamically coated with 0.5% PVP prior to injecting the sample with 10 mM CHES buffer.

Real-time binding kinetics of protein-protein interactions between Alexa Fluor 488-labeled actin / anti-actin antibodies and Alexa Fluor 633-labeled goat anti-rabbit IgG antibodies in solution using single-molecules using a real-time dual-color LIF detection system The level was investigated.

Even when protein molecules are not examples of complementary binding, high concentrations of protein samples (ie, at least 500 pM) resulted in aggregation and nonspecific binding of protein molecules. Protein concentrations of fM-aM levels are required to prevent nonspecific binding of non-complementary protein binding and allow accurate observation at the single-molecule level. The binding kinetics results between the 500aM Alexa Fluor 488-labeled actin / anti-actin antibody and Alexa Fluor 633-labeled goat anti-rabbit IgG antibody resulted in a more rapid interaction between individual protein molecules at lower temperatures according to collision and docking thermodynamic theory. Coupling and dissociation occur. The number of binding protein molecules decreased with increasing temperature in the range of 20-32 ° C.

This indicates that the characterization of protein-protein interactions as well as the overall interaction network essential for understanding the protein-protein interaction process is dependent on temperature and concentration at the single-molecule level. In the present invention, using the real-time dual color analysis system of the present invention, the real-time kinetics of protein-protein interaction at the single-molecule level without any time delay can be analyzed.

The present invention will be described in more detail with reference to the following examples. However, these examples do not limit the scope of the present invention.

Test Reagent

2- (cyclohexylamino) ethanesulfonic acid (CHES) was purchased from Sigma Chemcial Co. (St. Louis, MO, USA). 10 mM CHES was dissolved in ultrapure water (> 15 kPa) and the pH was titrated to 10 with 1 M NaOH (Showa Chemical Co. Ltd., Tokyo, Japan). Prior to use all buffers were filtered through a 0.2-μm membrane filter (MilliQ ™ / Milli-RO Water System, USA) and photo-bleached using a UV-B lamp (G15T8E, 280 315 nm, Philips, The Netherlands). To prevent protein adsorption to the capillary lining, 0.5% PVP (w / v) of 1 3000 000 Mr poly (vinylpyrrolidone) (PVP) purchased from Polyscience (Warrington, England, USA) was added to 10 mM CHES buffer. By dissolving a capillary power coating matrix was prepared.

Sample preparation

Alexa Fluor 488-labeled actin conjugate and Alexa Fluor 633-labeled goat anti-rabbit IgG antibody were purchased from Molecular Probes (Eugene, OR, USA). Rabbit anti-actin antibodies were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Protein kinase A-FITC conjugate was purchased from Promega (Madison, USA). Alexa Fluor 488-labeled actin conjugate and anti-actin antibody were incubated at 4 ° C. for 60 minutes using an antigen-antibody binding procedure to antigen (Alexa Fluor 488-labeled actin) -antibody (unlabeled anti-actin antibody) The complex was created. Alexa Fluor 633-labeled goat anti-rabbit IgG antibody was added to the reaction mixture solution containing the antigen (Alexa Fluor 488-labeled actin) -antibody (unlabeled anti-actin antibody) complex (FIG. 1).

All protein samples were prepared at 1 μM concentration in 10 mM CHES buffer. For SMD experiments, protein samples were further diluted to 500 aM-5 pM using 10 mM CHES buffer just before the start of the experiment. Protein size was measured using 200 nm sulfur-green fluorescent FluoSpheres beads (Molecular Probes, Eugene, OR, USA) diluted to 500 aM concentration in 10 mM CHES buffer.

Real-time dual color analysis system and instrument

Figure 2 (a) is a photograph showing an analytical system used to monitor single-protein molecules. Figure 2 (b) is a schematic diagram showing a capillary form of experimental instruments for monitoring single-protein molecules. Zeiss Axioskop2 microscope (Zeiss, Germany) equipped with Dual-View ^™ (Optical Insight, LLC, Tucson, USA) was used for most of the investigation. Zeiss 40 × / 0.75 NA Plan-Neofluar microscope objectives (Zeiss, Germany) were used. Pentamax 512-EFT / 1EA enhanced CCD camera (ICCD, Princeton Instruments, Princeton, NJ, USA) was fixed on top of the microscope. Dual-View ^™ was fixed between the objective lens and the CCD camera. Dual-View ^TM filter box includes four mirrors, two of dichroic filters (565dcxr, Chroma technology Corp., Rockingham, USA), and two band-path filter (D680 / 35 and D535 / 40, Chroma Technology Corp., Rockingham, USA).

Two different lasers were used as excitation sources: wavelength adjustable argon ion laser (maximum output 150 mW at 488 nm; Melles Griot, Irvin, CA, USA) and 635 nm fiber coupled laser (maximum at 635 nm). Output 100 mW; B & W TEKINC, Newark, DE, USA) was used. The laser beam was focused along a 50-mm focal length 0.5-inch circular-cylinder-lens (Edmund Optic Inc., Barrington, NJ, USA) to focus the beam at the center of the capillary window. The digitization rate of the camera was 5 MHz (12 bits) with the software controller increase set to 3 and the hardware increase set to 70. The camera was operated in external co-occurrence mode.

The laser beam was transmitted through optical pinholes to eliminate extraneous light and plasma lines and to reduce laser diameter. Uniblitz mechanical shutters (model LS2Z2, Vincent Associates, Rochester, NY, USA) were used to block the laser beam when the camera was not working to reduce light bleaching. The shutter was controlled by the VMM-D1 shutter driver (Vincent Associates, Rochester, NY, USA). Experiment time was controlled using a Stanford Research System model DG-535 four channel digital / pulse generator (Stanford Research Systems Inc., Sunnyvale, CA, USA). The ICCD camera was operated at 0 ms time with a 10 ms duration TTL pulse.

Sampling frequency was 10 Hz with a shutter driver set to 10 ms exposure and 90 ms delay. Fluorescence from individual single-protein molecules was passed through a Dual-View ^™ filter box to remove unwanted light and to obtain images of all protein molecules. WinView / 32 ^™ (version 2.5.14.1, Downingtown, PA, USA) and MetaMorph 6.3 software (Universal Imaging Co., Downingtown, PA, USA) were used to collect images of individual single molecules and process the data.

Preparation of capillaries for real-time dual color analysis measurement

A25-cm length × 75-μm i.d. × 375-μm o.d. Square-form exposure fusion-silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) were used for all SMD experiments with a 0.5-cm window cleared at 12 cm from the end of the capillary. The capillary was fixed to the capillary base and both ends were immersed in a conventional 1.5 mL microcentrifuge polypropylene test tube used as a reservoir. To prevent any effects other than siphon action, holes were drilled into the reservoir using a 400-μm Pin Vise Drill (SUPLECO, PA, USA). Capillaries were washed successively with water for 5 minutes, with 0.1 M sodium hydroxide for 3 minutes, with water for 5 minutes, and with 10 mM CHES buffer for 5 minutes. Finally, the mixed protein sample was introduced into the capillary after dynamic coating with 0.5% PVP for 1 minute to prevent protein adsorption.

All reagents were introduced into the capillary by hydraulic injection into a 5 mL syringe. The height difference between the action buffer vials was controlled to activate or stop individual protein molecules in the capillary. The vial was placed at the same height as used for non-operating conditions and there was a height difference of 2 cm for the operating conditions.

Example 1 Direct Observation of Single-Protein Molecules Using Real-Time Bi-Color Analysis

As shown in FIG. 3, Alexa Fluor 488-labeled actin / anti-actin antibody was simultaneously excited at 488 nm from Ar ⁺ laser and 635 nm from He / Ne laser in real time and observed with Dual-View ^™ Fluorescence images of the complex and Alexa Fluor 633-labeled goat anti-rabbit IgG antibodies could be monitored in specific wavelength ranges. Individual protein molecules in the mixture sample were effectively concentrated and interacted with complementary protein molecules and detected by double-wavelength (excitation wavelength, λ _ex = 488 and 635 nm) LIF detection. All mixed protein samples were co-excited by double-wavelength, but Alexa Fluor 488-labeled actin / anti-actin antibody complex molecules were clearly monitored in the 488 nm region (green in FIG. 3A) and Alexa Fluor 633-labeled goat anti- Rabbit IgG antibody molecules imaged only at 635 nm (red in FIG. 3B). The dual-wavelength LIF detection system provided accurate simultaneous imaging of individual proteins with different fluorophores in real time.

Two fluorescence emission images (520 and 650) of Alexa Fluor 488-labeled actin / anti-actin antibody complex and Alexa Fluor 633-labeled goat anti-rabbit IgG antibody in a single snapshot since there was no time delay between the images obtained nm) simultaneous acquisition is possible. It was also possible to measure the position of each of two different protein molecules in a mixed sample when the images at each particular wavelength overlap.

The observed length of the individual Alexa Fluor 488-labeled actin was about 382 ± 9.79 (mean ± standard deviation) nm or 96 ± 2.56% of the theoretical value of 395 nm (43 kDa = 1161-bp DNA = 395 nm). The size of the actin protein molecule was nearly similar to the 200-nm sulfur-green fluorescent FluoSphere beads (FIG. 4). Individual protein molecules were readily detected within the capillary if the capillary was dynamically coated with 0.5% PVP prior to injecting the sample with 10 mM CHES buffer.

In addition, the real-time dual color analysis method was able to calculate 4.1 W 0.99 (mean W standard deviation) individual single molecules with 5 fM concentration. A concentration of 5 × 10 ⁻¹⁵ M means that only 4.5 single-protein molecules are present in the detection window provided in this system. This is theoretically 4244 protein molecules in each 1.41 μl volume or 75 μm wide × 75 μm deep × 25 cm long capillary region, and the detection window of Dual-View ^™ in a 40 ㅧ objective is 58.9 μm × 265 μm. Because. The accuracy of the number of protein molecules observed at this low detection volume and ultra-low concentration was about 91%. However, due to adsorption on the capillary injection region, no individual protein molecules could be observed without dynamic coating with 0.5% PVP. These results are consistent with the fact that PVP can prevent adsorption of protein molecules on fusion-silica surfaces and fusion-silica / water interfaces in capillary electrophoresis.

Example 2 Concentration Effect on Protein-Protein Interaction

Several techniques have been developed, applied, and further refined over the years to characterize the interaction of purified proteins in vitro. These techniques are specialized and require a relatively large amount of protein. In the present invention, equilibrium and interaction kinetics could be analyzed because the interactions could be detected in real time and provide experimentally robust parameters important for characterizing various protein interactions. Investigations on systems with binding kinetics are possible with rate constants ranging from 1 s ⁻¹ to 1 × 10 ⁻⁴ s ⁻¹ and equilibrium dissociation constants ranging from 100 pM to 100 μM. Thus, real-time dual-color fluorescence video images of nonspecific protein-protein interactions dependent on the concentration of proteins in the sample at room temperature to single-molecule levels (ie, FITC-labeled protein kinase A and Alexa Fluor 633-labeled goat anti-rabbit IgG) Antibody) could be obtained (FIG. 5).

Images of individual protein molecules were directly monitored at a 10 Hz frame rate 10 minutes after hydrodynamic injection of the protein specimens. The operation of the protein molecules by hydraulic injection and gravity flow was accomplished by positioning the ends of the capillaries into the sample and then moving the sample container and the capillary ends by a 2 cm height difference. Real-time dual color analysis systems were able to obtain images of protein-protein interactions where fast photobleaching was a problem.

However, if the protein sample is not an example of complementary interactions (ie, antigen-antibody interactions) then nonspecific protein-protein interactions and aggregation of FITC-labeled protein kinase A and Alexa Fluor 633-labeled goat anti-rabbit IgG antibodies> It occurred at a 500 pM concentration (Figures 5c and d). This indicates that high protein concentrations can induce nonspecific protein-protein interactions even though protein molecules do not normally exhibit complementary binding. Thus, protein-protein interactions could be observed at 500 aM or less under non-propulsion conditions for 60 minutes with non-complementary binding samples and complementary binding tables of antigen-antibody interactions. Protein-protein interactions appeared only after a few seconds of observation of complementary antigen-antibody interactions, Alexa Fluor 488-labeled actin / anti-actin antibody complex and Alexa Fluor 633-labeled goat anti-rabbit IgG antibody (FIG. 6).

Example 3 Temperature Effect on Protein-Protein Interaction

The binding affinity of the complex is dependent on Gibbs binding free energy ΔG, and temperature T, as defined by the correlation KA = e ^{− (ΔG / RT)} . The same affinity can reflect different combinations of many different rate constants. Similarly, Gibbs free energy can reflect many different combinations of enthalpy and entropy change, depending on the specific chemical nature of the non-covalent force that is in effect during complex formation. Moreover, the nature of the temperature response can indicate the chemical nature of these forces.

Antigen-antibody interactions generally follow the following basic thermodynamic principles: higher temperatures result in higher binding / dissociation rate constants. It was therefore possible to observe the kinetics of real-time protein-protein interactions as a function of temperature and concentration below 50 fM, which did not indicate nonspecific binding of individual protein molecules (FIG. 7). Since the molecules were eluted through capillaries, monitoring of individual protein molecules showed repeated appearance and disappearance of bi-color fluorescence emission, but real-time dynamic images of the protein molecules were obtained without difficulty. There was no evidence of protein-protein binding immediately after two different protein samples were mixed under capillary propulsion (FIG. 7A). Complementary binding of individual protein molecules occurred a few seconds after the protein samples were mixed.

Most Alexa Fluor 633-labeled goat anti-rabbit IgG antibody molecules were continuously bound with Alexa Fluor 488-labeled actin / anti-actin antibody complex molecules within 40-80 minutes at room temperature (FIGS. 7B and C). However, after 80 minutes at room temperature, dissociation between Alexa Fluor 633-labeled goat anti-rabbit IgG antibody and Alexa Fluor 488-labeled actin / anti-actin antibody molecule began to occur (FIG. 7D).

The kinetics of protein-protein interactions in solution were investigated as temperature functions (12, 20, 25 and 32 ° C.) at the single-molecule level using a real-time dual color analysis system. As shown in FIG. 8, the number of molecules as a function of reaction time for the binding of Alexa Fluor 488-labeled actin / anti-actin antibody complex (green) and Alexa Fluor 633-labeled goat anti-rabbit IgG antibody molecule (red) (FIG. 8) Left y-axis) and relative percentage of molecules (right y-axis of FIG. 8) varied with temperature (FIG. 8).

The number of individual protein molecules was measured by counting the number of binding proteins on 200 frames of the video image. The number of individual single-protein molecules over 200 frames was 23 ± 2 (mean ± standard deviation n = 5) for the Alexa Fluor 488-labeled actin / anti-actin antibody complex and the Alexa Fluor 633-labeled goat anti-rabbit IgG antibody. The case was 20 ± 1.

The highest relative binding (or number of binding molecules) between protein molecules at temperatures ranging from 12 to 32 ° C. occurred 40-50 minutes after two different protein molecules were mixed. The number and persistence of binding molecules increased with decreasing reaction temperature, while the dissociation rate decreased. On average they were mixed at 12 ° C., 20 ° C. and 25 ° C. and within 40-50 minutes 90% of Alexa Fluor 488-labeled actin / anti-actin antibody complexes bound to Alexa Fluor 633-labeled goat anti-rabbit IgG antibody. However, at 32 ° C., only 50% of Alexa Fluor 488-labeled actin / anti-actin antibody complex was bound.

The number of binding molecules by protein-protein interactions increased as they were mixed and decreased in temperature from 32 ° C. to 12 ° C. by 50 minutes. In general, the relative percentage of bond is expected to increase with increasing temperature because the increase in temperature increases both the binding rate constant and the dissociation rate constant.

However, these results at the single-molecule level were in contrast to general predictions. These results can be interpreted using the theory of collisions and docking thermodynamics: temperature has a differential effect on the collision and docking steps of these antigen-antibody complexes. According to this theory, temperature affects affinity by changing the distribution of free energy changes between the collision and docking steps. The docking step is more temperature-sensitive and less energetic at higher temperatures than the crashing step. Longer time is required for docking at higher temperatures and the apparent increase in off-rate reflects the higher proportion of molecules present in the impulse state.

Thus, the nature and scale of the temperature effect can be a characteristic of the binding kinetics of protein-protein interactions: binding between Alexa Fluor 488-labeled actin / anti-actin antibody complex and Alexa Fluor 633-labeled goat anti-rabbit IgG antibody The kinetics were shown to be more sensitive at lower temperatures (12-25 ° C) than at higher temperatures (32 ° C).

It is an effect of the present invention to provide a dual-wavelength laser-induced fluorescence detection method and apparatus for investigating the real-time kinetics of temperature-dependent protein-protein interactions in solution at the single-molecule level. The present invention provides a real-time dual color analysis method and apparatus for real-time fluorescence image monitoring of individual protein molecules labeled with different fluorescent dyes using a double-wavelength single-molecule fluorescence detection system simultaneously without time delay. .

Claims (5)

Sample protein antigen A with a fluorescent label of wavelength X; The first antibody protein anti-A that binds to sample protein antigen A; And In the real-time dual color analysis method for measuring the protein-protein interaction consisting of the second antibody protein anti-B having a fluorescent label of wavelength Y bound to the first antibody protein anti-A, Real-time dual color analysis characterized by measuring the interaction between the sample antigen protein and the antibody protein using the enzyme immunoreaction by the dual color analysis method, and the result is read through the double-wavelength color of the wavelength X and Y Way The method of claim 1, wherein the wavelengths X and Y are wavelengths selected from 440 nm, 488 nm, 545 nm, 580 nm, and 635 nm, respectively. The method according to claim 1, wherein the real-time dual color analysis method The concentration of the sample protein is a real-time double, characterized in that the detection of the ultra-small amount of the sample protein low to the level of Atomol (aM) to femtomol (fM) Color analysis method The method of claim 1, wherein the real time dual color analysis method is performed at a room temperature of 12 to 32 ℃. V) sample protein antigen A with a fluorescent label of wavelength X; The first antibody protein anti-A that binds to sample protein antigen A; And a capillary inserted with a sample protein consisting of a second antibody protein anti-B with a fluorescent label of wavelength Y bound to the first antibody protein anti-A; Ii) a laser light source for reading protein interactions in the capillary and an optical analyzer for reading its fluorescence wavelength; And Iii) consisting of computer equipment for deciphering the optical analyzer Real-time dual color analyzer for measuring protein-protein interactions of sample proteins

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