Time-resolved liquid phase immunoassay (TLIA) and combination screening, and hybrid screening

Abstract

To provide a method with which the specific interaction between molecules, such as an antibody and an antigen can be measured quickly and accurately with a simple operation.SOLUTION: An immunoassay (TLIA) method has been provided, which is simple, quick, and accurate, and includes bringing a target molecule and a molecule for detection into specific interaction in a solution without immobilization, and measuring the mass of an eluted nonbonded molecule for detection, thereby eliminating the need of a cleaning step. A "hybrid screening" method has been provided, which includes, from a large antibody library, after display screening in a colon bacillus, applying a production increasing method for an outer membrane vesicle (OMV) using a Tris buffer solution developed by the present inventors to continuously execute a colony assay method (combination screening) without crossing over vectors, and subsequently using the TLIA method to quickly and efficiently identify a desired antigen-specific monoclonal antibody.SELECTED DRAWING: Figure 12

Description

[Technical field]

[0001] The present invention relates to a novel "time-resolved liquid phase immunoassay (TLIA) method" for detecting and quantifying specific interactions between molecules such as antibodies and antigens quickly and easily with high sensitivity and accuracy, a novel "combination screening" method that enables seamless implementation of a liquid phase display method and a solid phase colony assay method, and a "hybrid screening" method that identifies antigen-specific monoclonal antibodies quickly and efficiently from an antibody library by successively implementing the "combination screening" method and the "TLIA" method. The TLIA method of the present invention is a homogeneous measurement method mainly in an aqueous solution, and since the unbound fraction is measured, a washing step is not required and high sensitivity is achieved. Not only the qualitative affinity of the target molecule but also the kinetic information of its binding and dissociation (k off , K D The present invention provides an immunoassay method capable of quantitatively measuring the above-mentioned markers, which is extremely useful in the fields of diagnosis, treatment, and research. That is, in the case of an antigen-antibody reaction as an example, the present invention is characterized in that at least one of the antigens (preferably both) is not immobilized, the antigen-antibody reaction is carried out in a solution, and the amount of unbound antibody eluted is measured, thereby eliminating the need for a washing process. In particular, the measurement after the antigen-antibody reaction in a solution focuses on the eluted unbound, non-immobilized antibody (antigen) rather than the bound antigen (antibody), which eliminates the need for a washing process and provides a simple, rapid, and accurate immunoassay method, which is revolutionary. Furthermore, when the TLIA method of the present invention is used in screening for the establishment of monoclonal antibodies, it is possible to select antibodies not only based on their high antigen affinity but also based on their binding mode (dissociation and association rates), and thus has the excellent property of enabling the efficient acquisition of desired monoclonal antibodies. Therefore, we developed a "combination screening" method that can seamlessly combine liquid-phase display and solid-phase colony assay methods as a screening method that can make the most of the excellent properties of the TLIA method. The "Hybrid Screening" method achieved by further continuously combining the "Combination Screening" method and the "TLIA" method is an epoch-making screening method that can surely and quickly identify target clones with an extremely low probability of existence from a huge antibody library, and it is a method that can identify positive clones surely without false positives.

Background Art

[0002] Specific interactions such as antibodies and antigens, receptors and signal molecules, and enzymes and substrates form the basis of life phenomena, and the detection of such interactions is very important in research, diagnosis, and treatment. In particular, immunoassays can quickly and simply detect and quantify target proteins with high sensitivity and high precision, and are very important detection methods in the diagnosis and research fields. Among immunoassays, Western blotting and ELISA that utilize the high specificity of antibodies are still widely used for the detection and quantification of target proteins, and are important basic technologies in research and diagnosis. In particular, ELISA has continued to be used as an indispensable tool for life science research and diagnosis for a long time.

[0003] <Problems of ELISA> ELISA is a method for detecting or quantifying a target antigen (or antibody) by immobilizing the target antigen (or antibody) on a microplate or beads, reacting it with an enzyme-labeled antibody (or antigen) or further with a secondary antibody, and detecting and measuring the change in the absorption spectrum caused by the enzymatic reaction between the enzyme-labeled antibody and the substrate. Depending on the combination of antigen-antibody reactions, "direct method", "indirect method", "sandwich method", "competitive method", etc. are known, but in any method, it is necessary to immobilize the target antigen or antibody on a solid phase, and therefore, multiple washing steps are always required after the enzymatic reaction.

[0004] That is, ELISA requires complicated procedures such as immobilization, washing, and blocking. Usually, the amount of either the antigen or the antibody is large, so that unbound antigen or antibody exists in the system, and a washing process is required for B / F (bound / free) separation. This makes the procedure complicated, laborious, and time-consuming, and causes data variability. In addition, automation is difficult due to the washing procedures and the large number of steps, which is a major problem, especially when there are many samples. Furthermore, even if the antigen-antibody is once bound, it is subjected to multiple washing steps and often dissociates quickly, especially when the affinity of the antigen-antibody is low. This makes it difficult to obtain accurate data, and the method is therefore only applicable to antigen-antibody combinations with high affinity. In the end, only qualitative affinity can be measured accurately using ELISA. The antigen (antibody) concentration can be estimated by comparing it with a calibration curve, but accurate measurement is not possible, especially in the low concentration range. In addition, information on the binding and dissociation kinetics of the antibody, which is necessary for quantitatively evaluating the affinity of the antibody, (k on :association rate constant [1 / Ms], k off : dissociation rate constant [1 / s]) and dissociation constant (K D However, there is a problem in that it is not possible to directly obtain

[0005] The standard method for measuring the kinetics of antibody binding and dissociation is the Biacore ELISA kit. (登録商標) This is an "SPR (surface plasmon resonance) method" that uses a device called "SPR Imaging Spectroscopy" (Non-Patent Documents 1, 2). Of the two molecules (antigen and antibody) whose interaction you wish to measure, one of the molecules, the antibody (ligand), is immobilized on the surface of a sensor chip, and a solution containing the other molecule, the antigen (analyte), is passed over the surface of the sensor chip. The increase in mass that occurs as a result of the interaction between the antigen and antibody is measured as a change in the refractive index of the solvent on the surface of the sensor chip. It is essential to fix the antibody to the sensor chip surface, and problems such as denaturation (changes in three-dimensional structure, etc.) due to solid-phase formation occur. If a sufficient amount of antigen is not supplied to the immobilized antibody per unit time, the accuracy of the measurement results cannot be guaranteed (=Mass Transport Limitation), there is a limit to the molecular size of the antigen that can be measured, and in order to measure the kinetics accurately, it may be necessary to align the orientation. In addition, since this method measures binding and dissociation in real time, it is difficult to accurately quantify the affinity for antibodies that dissociate slowly. In theory, it is possible to immobilize an antigen as a ligand on the sensor chip surface and flow a solution containing an antibody as an analyte over the chip surface. However, immobilizing an antigen is highly likely to cause denaturation of the antigen, and binding to an immobilized antigen is different from binding to an antigen in a natural aqueous solution, so the original K between the antigen and antibody may be lost. D and k on , k off It is not possible to measure the amount of (登録商標) However, since the exact concentration of the analyte (antigen) is required, K D It is impossible to analyze.

[0006] Similar to SPR, there is another method for analyzing molecular interactions in real time called "BLI (Bio-Layer Interferometry)" (Non-Patent Document 3). (登録商標) It is sold under the product name ". Specifically, an antibody is immobilized on the underside of the biosensor chip, white light is irradiated from above the biosensor, and the light reflected from the optical layer and the tip is observed. When an antigen in the solution binds to the tip, a delay in the reflected light from the liquid surface occurs, causing a shift in response that can be detected. Basically, it has the same problems as SPR. In particular, since it is necessary to fix the antibody to the sensor chip surface, there is a problem of loss of biological activity, and accurate kinetic information of binding and dissociation of natural antigen-antibody reaction in solution cannot be obtained. In addition, both methods require dedicated expensive measurement equipment.

[0007] As a method capable of measuring the kinetics of antibody binding and dissociation, a "kinetic exclusion assay (KinExA)" has been developed to quantify protein binding interactions in homogeneous solutions (Non-Patent Document 4), but this method also requires expensive dedicated measurement equipment. In the KinExA method, a ligand (antibody) and an analyte (antigen) are reacted in a solution, and a sample in equilibrium (or in the process of transitioning to equilibrium) is added to the antigen-bound beads, causing the unbound antibody to bind to the antigen on the surface of the beads. A fluorescently labeled secondary antibody is reacted with the unbound antibody bound to the beads, and the amount of the antibody is detected as a signal. Since the antigen-antibody reaction, which is the interaction between two molecules, occurs in an aqueous solution, problems caused by solidification such as mass transport limitation do not occur. In addition, since the sample is measured in equilibrium, the affinity of even ligands that dissociate slowly can be accurately quantified. Furthermore, it is possible to measure the molecular interactions of objects of various sizes, from biomolecules to cells. However, since it is necessary to repeat the washing process of binding unbound antibodies to antigen beads, washing them, reacting the unbound antibodies bound to the antigen beads with a secondary antibody, and then quantifying the amount of binding after washing, only antibodies with high affinity can be measured. Furthermore, when an antibody binds to an antigen on a bead, a dissociation reaction of the bound antigen and antibody occurs simultaneously in the aqueous solution, so the affinity must be so high that dissociation does not occur at that time. This is difficult to implement with the affinity of normal antibodies.

[0008] As a way to avoid the step of separating antigens that are bound to antibodies from those that are not (B / F separation), several "homogeneous immunoassay methods" have been developed that allow the reaction to occur in solution and do not require a washing step. For example, the Lumit (商標) In "Technology," NanoLuc (登録商標) Two antibodies are labeled with LgBiT and SmBiT fragments, which are fragments of a luminescent enzyme, and are allowed to act on a target antigen protein in a solution together with a luminescent substrate. By designing each antibody to recognize a different epitope on the target antigen protein, after both antibodies bind to the target antigen, the LgBiT and SmBiT fragments bound to each antibody approach each other and emit light as NanoLuc luminescent enzyme. No washing step is required, and the measurement can be performed accurately in a short time with simple operations (Non-Patent Document 5). However, the antibody that can be used in this method must be a specially designed antibody. In other words, the two antibodies used must recognize different epitopes on the same antigen, and after each antibody binds to the antigen, it is essential to design the two luminescent enzyme fragments bound to the antibodies to have an optimal distance, and it is not easy to prepare such an antibody. "Lumit (商標) The "AlphaLISA" method is known as a technology similar to the "AlphaLISA Technology" (Non-Patent Document 6). Two antibodies against a target antigen are prepared (antibody 1 and antibody 2), antibody 1 is bound to a donor bead, and antibody 2 is bound to an acceptor bead. When antibodies 1 and 2 bind to the antigen, the two beads approach each other, and excited singlet oxygen produced by the donor bead induces a chemiluminescence reaction in the nearby acceptor bead, making a washing step unnecessary. However, since the efficiency of acceptor bead excitation is not high, a high concentration of antibody on the bead is required to form an antigen-bead complex, and there is a drawback in that a high antibody concentration also increases the background. This "AlphaLISA" method also has the advantage that the K D It is possible to measure quantitative binding constants such as those above, but this is only possible in limited situations, such as when the affinity is very high, and it is difficult to apply this to general antigen-antibody reactions. It is not possible to measure the kinetics of binding.

[0009] In addition, a "Digital ELISA" has been developed as an improved ELISA method that does not require a washing step. In this method, the target antigen is captured by an antibody immobilized on a magnetic nanoparticle, which is magnetically collected and injected into a small reactor. The target antigen molecule on the nanoparticle reacts with the antibody immobilized on the reactor surface via a PEG linker. This method acquires bright-field images of the particles, classifies the patterns of particle movement, and digitally counts the particles that show binding patterns (Non-Patent Document 7). However, this method also requires complicated procedures and expensive specialized equipment, and has the same problems as SPR in that it requires immobilizing antibodies on nanoparticles.

[0010] Several other "one-step immunoassay methods without a washing step" have also been proposed. One is a one-step heterogeneous immunoassay that employs a "bound-free phase detection method" (Non-Patent Document 8). In this analysis method, an antibody is immobilized on magnetic beads, and a target antigen is immobilized on fluorescent beads. These two types of beads are mixed in a solution, and after the magnetic beads are collected by magnetic force, the amount of fluorescence from the free fluorescent beads is measured. Since no washing step is required and the method only involves supplying samples and reagents, incubation, separation, and detection, it can be said to be a method that can be easily miniaturized and automated. However, because it is necessary to immobilize the capture antibody on magnetic beads, there is a problem of loss of biological activity and it is not possible to obtain accurate kinetic information on the binding and dissociation of natural antigen-antibody reactions in solution. The other is a one-step immunoassay (Non-Patent Document 9) that uses two types of beads of different sizes and a filter. Antibodies are immobilized on the surfaces of both beads, and if there is an antigen in the reaction solution, it is captured by the antibodies on the surfaces of both beads. Since only small free beads can pass through the filter holes, the amount of antigen can be estimated by quantifying the unbound beads after filtration, but this method also has the same problems as the "non-bound phase detection method." In addition, in the above two methods, since both the antigen and antibody are immobilized on the beads, the binding rate in the aqueous solution is governed by the size of the beads, and there is also the problem that it takes a long time to achieve the equilibrium state of binding compared to when the antigen and antibody exist in the aqueous solution as free molecules.

[0011] As described above, the washing step, which was a drawback of ELISA, is not required, and the kinetics of antigen-antibody reactions in aqueous solutions (K D , k off Although many methods have been developed to enable this, they are still incomplete, as they require complicated operations, expensive measuring equipment, and are limited in scope. There is still a strong demand for the development of accurate, simple, and speedy assay methods for specific interactions such as antigens and antibodies.

[0012] <Antibody library screening method> Methods for identifying antigen-specific monoclonal antibodies from antibody gene libraries mainly include the "display method" and the "colony assay method." The advantages and disadvantages of each are discussed below.

[0013] (1) Display Law Display methods are essential for screening large libraries, Depending on the host, there are methods such as phage display, ribosome display, yeast display, and Escherichia coli display (Non-Patent Document 12).

[0014] (1-1) What is E. coli display? The E. coli display method is advantageous in that it is simple and quick (Non-Patent Documents 13, 14). Furthermore, efficient selection of positive clones is possible using MACS or FACS. Since many VHHs are displayed on the cell surface, antigen-specific VHHs can be efficiently selected (Non-Patent Document 15). The E. coli display method uses a technique in which an antibody fragment is fused to an outer membrane protein and the antibody fragment is displayed outside the cell. Examples of outer membrane proteins that are used include Lpp-OmpA and autotransporter (AIDA-I, Intimin) (Non-Patent Document 13). The method using Intimin as an outer membrane protein has attracted attention as an excellent method due to its high extracellular presentation ability and low toxicity (Non-Patent Document 14).

[0015] (1-2) Problems with the display method The advantage of the display method is that it can handle large libraries. This increases the probability of obtaining rare clones that have a low probability of existence. However, there is a problem that it is very difficult to identify positive clones when trying to identify antigen-binding clones using only the display method, because it requires a lot of effort and a long time. For example, 9 Library of 10 5 However, subsequent identification of the desired clone requires a great deal of effort and time.

[0016] (2) Colony assay method On the other hand, the "colony assay" method has the excellent feature that screening can be performed by observing actual binding, compared to the display system, and positive clones can be identified reliably without false positives. However, even the "expression automatic control colony assay method" (Patent Document 1), which the present inventors have developed through repeated improvements, can only handle a library size of 10 6 The degree of the display method is limited to 10 9 ~10 10 However, it has the disadvantage that it cannot handle huge libraries of this size.

[0017] As described above, the "display method" and the "colony assay method" each have their own advantages and problems, and both are insufficient as "antibody screening methods" that can reliably identify positive clones from a huge antibody library without false positives. There has been a strong demand for a revolutionary antibody screening technology that can solve these problems. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] Patent No. 7364242 [Non-patent literature]

[0019] [Non-Patent Document 1] Toshiharu Hase et al. (eds.): "Understanding the Functions of Proteins - Molecular Functions and Biological Effects" Kagaku Dojin (August 1, 2009) Chapter 2, 29-52 [Non-Patent Document 2] Tomoya Mitani: Japanese Society of Analytical Chemistry 2014 (1) 2-9 (2014.1) [Non-Patent Document 3] Takashi Saito: Japan Society for Analytical Chemistry 2020 (6) 202-203 (2020.6) [Non-Patent Document 4] Robert CB,et al.,Analytical Biochemistry 272,123-134 (1999) [Non-Patent Document 5] Rokhsareh Rozbeh et al.,Sci Rep 2021 Jun 15;11(1):12535. [Non-Patent Document 6] Christian D. Peters et al. Scand J Clin Lab Invest 2012 72(8): 583-92 [Non-Patent Document 7] Kenji Akama et al., ACS Nano 2019, 13, 13116-13126

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

[0020] The first objective of the present invention is to provide an immunoassay capable of detecting and measuring specific interactions between molecules, particularly specific interactions between proteins such as antigens and antibodies, receptors and signal molecules, and enzymes and substrates, with a simple operation similar to or even simpler than that of ELISA, quickly and with high accuracy. In addition, the ... simpler than that of ELISA, quickly and with high accuracy. In addition, the first objective of the present invention is to provide an immunoassay capable of detecting and measuring specific interactions between molecules, particularly specific interactions between proteins such as antigens and off , K D The object of the present invention is to provide a method for accurately and quickly measuring the following:

[0021] If such an immunoassay could be provided, rapid and accurate evaluation would be possible during screening when establishing monoclonal antibodies, making it possible to efficiently obtain monoclonal antibody-producing clones that are desired not only in terms of high antigen affinity but also in terms of the binding mode (dissociation and association rates). Therefore, another object of the present invention is to provide a screening method that can seamlessly perform liquid phase display method and solid phase colony assay method, as a screening method that can make the most of the excellent properties of the immunoassay of the present invention. The final objective is to provide a comprehensive screening method that can reliably and quickly identify desired monoclonal antibody-producing clones, the presence of which is extremely low, from a huge antibody library by further successively combining the screening method with the immunoassay of the present invention. [Means for solving the problem]

[0022] 1. About the assay method "TLIA" of the present invention: The present inventors first focused on the washing procedure, which is absolutely necessary for specific detection of target proteins in ELISA. The washing procedure is cumbersome, and insufficient or uneven washing will cause data variability, so it must be repeated. As a result, even if the detection itself is quick, the measurement will take a long time, and the measurement may become inaccurate. The present inventors have been trying to devise ways to specifically detect proteins and the like with high sensitivity without this washing procedure, and have noticed that in conventional techniques, high concentrations of antibodies and the like are used to detect trace amounts of target proteins fixed to a solid surface, and even if only a trace amount of the high concentration liquid remains, it will significantly change the data. This is because it is difficult to fix proteins to a solid surface, and only trace amounts can be fixed (for example, in the case of an IgG antibody, at most several hundred ng / cm can be fixed to a polystyrene surface). 2 This is due to the fact that the angle can only be fixed to a certain extent. Therefore, the inventors have invented a detection method that does not require washing procedures, but is rapid and highly sensitive, by causing an antigen-antibody reaction in solution and measuring the concentration of unbound antibody (or antigen) in the solution, rather than measuring the amount of target protein bound to a solid surface.

[0023] Specifically, after causing an antigen-antibody reaction between a biotin-labeled target antigen and an enzyme (fluorescence)-labeled antibody in an aqueous solution, the mixture is added to a streptavidin-labeled bead suspension to capture all of the target antigen, and then the unbound antibody is separated and its enzyme (fluorescence)-labeled signal is detected and measured, allowing the amount of unbound antibody to be calculated and the amount of bound antibody to be determined (Figure 1). At the same time, if a sample that may contain the target antigen is added to cause a competitive reaction, it becomes possible to estimate the presence or absence of the target antigen in the sample and calculate the amount of antigen. This method is a rapid and highly sensitive assay for detecting target antigens, and because it is a measurement with time resolution, it is possible to measure the binding and dissociation kinetics in the mid-binding and dissociation states, making it possible to determine the binding rate constant, dissociation rate constant, and dissociation constant of the antibody (Figures 2, 3, 4). Therefore, the immunoassay method of the present invention is also called "time-resolved liquid phase immunoassay (TLIA) method." In this specification, a typical antigen-antibody reaction is used to explain the intermolecular specific interaction, but the interaction is not limited to the antigen-antibody reaction.

[0024] 2. Screening method developed in the present invention (2-1) Development of a screening method that can maximize the characteristics of the TLIA method Another objective of the present invention is to provide a screening method that can make the most of the excellent properties of the TLIA method of the present invention, and to achieve this, we considered sequentially combining display screening and colony assay screening. Specifically, a large library is first concentrated by "screening by E. coli display", and then "expression automatic control colony assay (Patent Document 1)" (hereinafter simply referred to as "colony assay") is performed to identify positive clones reliably and quickly. In "screening by E. coli display", an antibody is fused with an outer membrane protein of E. coli, expressed on the outer membrane surface of E. coli, and E. coli expressing an antibody that binds to an antigen is selected. Next, by the "colony assay" method previously developed by the present inventors, E. coli expressing the antibody is seeded on a hydrophilic filter and an antigen-coated nitrocellulose membrane to form a colony. Then, expression of the antibody is induced, and the expressed antibody is released outside the bacterial cell, passes through the hydrophilic filter, and binds to the antigen on the antigen-coated nitrocellulose membrane. By detecting this antigen-bound antibody, a colony that produces an antibody that binds to the antigen is identified. In this way, if the two technologies can be combined, it becomes possible to identify a target clone from a huge library.

[0025] (2-2) Use of outer membrane vesicles (OMVs) However, the former is basically a display on the outer membrane in liquid-phase culture, while the latter is basically an expression in the periplasm in solid-phase culture, and because the expression patterns of the antibodies differ between the two, it is necessary to switch vectors, which is time-consuming and labor-intensive, and has the fatal drawback that the properties of the library are easily damaged during the switching process, resulting in the loss of the target clones. Therefore, the inventors focused on outer membrane vesicles (OMVs) released from the outer membrane of Gram-negative bacteria such as Escherichia coli, and came up with the idea that if an antibody protein is fused to an OMV protein and displayed on the surface of the OMV, it would be possible to proceed continuously to the colony assay process without the need for vector transfer.

[0026] (2-3) Culture method for increasing outer membrane vesicles (OMVs) First, we will explain outer membrane vesicles (OMVs). Membrane vesicles produced by bacteria are spherical structures composed of lipid bilayers of 20 to 400 nm, and are the outermost layer of microorganisms. They contain proteins, nucleic acids, signal molecules, etc., and are said to contribute to bacterial pathogenicity, resistance, and biological interactions (Non-Patent Document 17). In gram-negative bacteria such as Escherichia coli, the membrane vesicles are thought to be derived from the outer membrane and are called outer membrane vesicles (OMV). In recent years, it has become possible to produce outer membrane vesicles that present a target protein by fusing and expressing the target protein with the outer membrane protein to form outer membrane vesicles, and this technology is expected to have a promising future, as it can be used as a vaccine (Non-Patent Document 18).

[0027] Outer membrane vesicles (OMVs) are approximately 200 nm in diameter and are formed outside the cell when the outer membrane is torn off. However, under normal culture conditions, their production frequency is not high, and the amount produced is too small to be used as antibody-producing OMVs for colony assays. As a result of extensive efforts, including the investigation of culture conditions, the present inventors have found that a large number of outer membrane vesicles can be produced by using a culture medium containing a Tris buffer during solid culture of E. coli. Using this phenomenon, it has become possible to produce a large number of outer membrane vesicles with antibodies produced on their surface in a culture medium containing a Tris buffer after display screening, and to seamlessly perform continuous screening by colony assay.

[0028] (2-4) Completion of the “combination screening” method This method allows for the production of more outer membrane vesicles and the performance of a "colony assay (Patent Document 1)," which allows direct screening without vector transfer, so there is no loss of positive clones due to transfer and no bias in the library. These outer membrane vesicles can pass through the hydrophilic filter (pore size 0.45 μm) used in the "colony assay (Patent Document 1)", and then bind to the antigen on the antigen-coated membrane, making them detectable. We succeeded in seamlessly and quickly screening both types of vesicles in succession. As a result, it became possible to rapidly and accurately identify positive clones that exhibit antigen binding from a large library. In the present invention, this method is called the "combination screening" method (Non-Patent Document 16).

[0029] (2-5) "Hybrid Screening" Method In the present invention, by continuously combining the above-mentioned "combination screening" method of the present invention with the "TLIA" method of the present invention, it is possible to continuously measure and evaluate the binding properties of antibodies, making it possible to simply and quickly identify extremely excellent antigen-specific monoclonal antibodies. In the present invention, a screening method that sequentially performs "combination screening" and "TLIA" methods is called "hybrid screening."

[0030] The "combination screening" method of the present invention is a screening method developed to take advantage of the excellent properties of the "TLIA" method. However, by combining various immunoassays, such as ELISA, and not limited to the "TLIA" method, it is possible to rapidly measure and evaluate the antigen recognition ability of the obtained antibodies, thereby identifying antibodies with more desirable binding properties.

[0031] That is, the present invention is as follows. [1] A method for detecting a target molecule in a sample or measuring the concentration thereof by using a molecule having a specific interaction with the target molecule as a detection molecule, comprising: (1) preparing an aqueous solution containing a sample possibly containing a target molecule and a quantity of a luminescently or fluorescently labeled detector molecule, comprising: (a) a fixed amount of biotin-labeled target molecule and streptavidin-labeled beads; (b) beads with a fixed amount of target molecule bound to their surface; (2) waiting for the specific interaction between the target molecule and the detector molecule in the aqueous solution to reach equilibrium, and separating and recovering the unbound luminescent or fluorescently labeled detector molecule from the beads; and (3) measuring the absorbance, luminescence or fluorescence intensity from the recovered detector molecule to determine the concentration of unbound detector molecule; 1. A method for detecting the presence of a target molecule or measuring the concentration of a target molecule in a sample, comprising: To be precise, the labeling of the detection molecule (antibody) can be expressed as "enzyme labeling, tag labeling, or fluorescent labeling." For example, in the case of luminescence using HiBit, a tag (11 amino acid residues) is attached to the detection molecule (antibody), and in the case of fluorescent labeling using GFP, a tag is also attached, so it can be called "tag labeling." In some cases, fluorescent dyes are chemically labeled. The measurement methods used at this time include measuring "absorbance, luminescence intensity, and fluorescence intensity." [2] Prior to step (1), a target molecule is reacted with a luminescent or fluorescently labeled detection molecule until equilibrium is reached in an aqueous solution in which the target molecule has been diluted to various concentrations, and the absorbance, luminescence or fluorescence intensity of each of the unbound detection molecules separated and recovered is measured, and a calibration curve of "signal intensities corresponding to the target molecule and the unbound detection molecule" is prepared. The method according to [1] above, further comprising a step of calculating the concentration of the target molecule in the sample from the deviation between the concentration of the unbound detection molecule obtained in step (3) and the corresponding target molecule concentration in the calibration curve. [3] A method for detecting a target molecule in a sample or measuring its concentration using a microfluidic device, comprising: The method according to claim 1, characterized in that the microchannel is composed of a main channel having a length sufficient to allow specific interactions between the target molecule and the detection molecule to occur until an equilibrium state is reached and a dam structure for blocking the beads, a sample introduction section, and a detection section. To be precise, the label of the detection molecule (antibody) can be expressed as an "enzyme label, tag label, or fluorescent label." [4] A method for measuring the concentration of a target molecule in a sample by using a molecule having a specific interaction with the target molecule as a detection molecule, comprising: (1) reacting a dilution series of unlabeled target molecules in an aqueous solution with a fixed concentration of luminescently or fluorescently labeled detector molecules and a fixed concentration of biotin-labeled target molecules; (2) After reaching equilibrium, the unbound luminescent or fluorescently labeled detection molecules are captured by streptavidin-labeled beads and then separated and recovered from the beads; (3) measuring the absorbance, or luminescence or fluorescence intensity from the recovered detection molecule, calculating the concentration of unbound detection molecule corresponding to the unlabeled target molecule in the dilution series, and creating a calibration curve for both; (4) reacting a sample containing an unknown concentration of target molecules with a constant concentration of luminescent or fluorescently labeled detection molecules and a constant concentration of biotin-labeled target molecules in an aqueous solution until equilibrium is reached, similar to steps (1) and (2) above, capturing the target molecules on streptavidin-labeled beads, and then separating and recovering unbound luminescent or fluorescently labeled detection molecules from the beads; and (5) measuring the absorbance, luminescence or fluorescence intensity from the recovered detection molecule and determining the concentration of the target molecule in the sample using the calibration curve prepared in (3); A method for measuring a target molecule concentration in a sample, comprising: To be precise, the label of the detection molecule (antibody) can be expressed as an "enzyme label, tag label, or fluorescent label." [5] The method for measuring a target molecule concentration in a sample according to any one of [1] to [4] above, characterized in that the target molecule is an antigen and the detection molecule is an antibody, or the target molecule is an antibody and the detection molecule is an antigen, and the specific interaction between the molecules is an antigen-antibody reaction. [6] By using a molecule that has a specific interaction with a target molecule as a detection molecule, the kinetics of binding of the specific interaction between molecules (k on A method for measuring (1) reacting a certain concentration of a luminescent or fluorescently labeled detection molecule with a certain concentration of a biotin-labeled target molecule in an aqueous solution containing streptavidin-labeled beads; (2) a step of collecting a portion of the solution at regular intervals before the specific interaction between the target molecule and the detection molecule in the aqueous solution reaches an equilibrium state, and separating and recovering the unbound detection molecule in the sample solution during the binding process; (3) measuring and recording the absorbance, or luminescence or fluorescence intensity, from unbound detector molecules for each sample solution; The method according to claim 1, further comprising: To be precise, the label of the detection molecule (antibody) can be expressed as an "enzyme label, tag label, or fluorescent label." [7] By using a molecule that has a specific interaction with a target molecule as a detection molecule, the kinetics (k off (1) reacting a constant concentration of a luminescent or fluorescently labeled detection molecule with a constant concentration of a biotin-labeled target molecule in an aqueous solution containing streptavidin-labeled beads; (2) After the two have sufficiently reached equilibrium, the beads are filtered to separate the supernatant, which is then suspended in a buffer, and a portion of the solution is sampled at regular intervals to separate and recover the unbound detection molecules in the sample solution during the dissociation process; (3) measuring and recording the absorbance, or luminescence or fluorescence intensity, from unbound detector molecules for each sample solution; The method according to claim 1, further comprising: To be precise, the label of the detection molecule (antibody) can be expressed as an "enzyme label, tag label, or fluorescent label." [8] By using a molecule that has a specific interaction with a target molecule as a detection molecule, the dissociation constant (K D A method for measuring (1) reacting a luminescent or fluorescently labeled detection molecule at a constant concentration with a biotin-labeled target molecule at multiple concentrations using a dilution series in an aqueous solution until equilibrium is reached; (2) After equilibration, the reaction solution of the target molecule and the detection molecule is applied to a filter column packed with streptavidin beads to capture the biotin-labeled target molecule on the streptavidin beads, the beads are filtered, and the supernatant is separated; (3) measuring the enzyme activity, fluorescence, or luminescence intensity in the supernatant to measure the amount of unbound antibody; (4) At each concentration of the target molecule, a Scatchard plot is made and the inverse of the slope of the graph is calculated to determine the dissociation constant (K D ) The method according to claim 1, further comprising: Here, a Scatchard plot can be created by, for example, in the case of an antigen-antibody reaction, calculating the value (X) obtained by dividing the bound antibody concentration (x) by the total antibody concentration (Abt) and the value (Y) obtained by dividing the bound antibody concentration (x) by the product of the total antibody concentration (Abt) and the free antigen concentration (Agf), i.e., "Y = x / (Abt × Agf)." To be precise, the label of the detection molecule (antibody) can be expressed as an "enzyme label, tag label, or fluorescent label." [9] The method for measuring the kinetics or dissociation constant of binding or dissociation between an antigen and an antibody according to any one of [6] to [8] above, wherein the target molecule is an antigen and the detection molecule is an antibody, or the target molecule is an antibody and the detection molecule is an antigen, and the specific interaction between the molecules is an antigen-antibody reaction.

[10] An antibody screening method in which the target molecule is a monoclonal antibody in an antibody library and the detection molecule is an antigen, comprising: A method for screening for an antibody with high antigen-binding ability, an antibody with rapid association, or an antibody with slow dissociation, using the method for measuring the kinetics of binding or dissociation between an antigen and an antibody, or the dissociation constant, described in any one of [6] to [8] above.

[11] A method for screening an antibody library for a monoclonal antibody with high antigen-binding ability, comprising the following steps (1) to (5): (1) A step of inserting an antibody gene library into an E. coli display library vector and inducing expression to produce an E. coli library displayed on the outer membrane surface of E. coli; (2) reacting the displayed antibody with a biotin-labeled antigen, then reacting it with anti-biotin antibody-labeled magnetic beads or streptavidin magnetic beads, and concentrating E. coli expressing the antigen-binding antibody using a magnetic column; (3) suspending a concentrated solution of E. coli expressing an antibody having antigen-binding ability in a culture medium containing a Tris buffer, and then forming colonies on a colony assay plate provided with an antigen-coated nitrocellulose membrane and a hydrophilic porous filter on an agar plate of the culture medium containing a Tris buffer; (4) separating the nitrocellulose membrane from the filter on which colonies are formed, and storing the filter on a new agar plate; (5) A process of detecting antibodies bound to the antigens on the nitrocellulose membrane, identifying colonies corresponding to the detected images on the filter in (4), and extracting the corresponding clones.

[12] The method according to

[11] , wherein the antibody gene library of (1) is a cDNA library of VHH antibody genes, and Intimin is used as an outer membrane protein for display on the outer membrane surface of Escherichia coli, and induced-expression Intimin-VHH is displayed on the outer membrane surface.

[13] A method for screening an antibody library for a monoclonal antibody having high antigen-binding ability, rapid association, or slow dissociation, the method comprising the following steps (1) to (7) or further step (8): (1) A step of inserting a cDNA library of VHH antibody genes into an E. coli display library vector incorporating the N-terminus of Intimin so that Intimin-VHH can be expressed, and inducing expression to produce an E. coli library displayed on the outer membrane surface of E. coli; (2) reacting the displayed VHH with a biotin-labeled antigen, then reacting it with streptavidin magnetic beads, and concentrating E. coli expressing VHH with antigen-binding ability using a magnetic column; (3) suspending a concentrated solution of E. coli expressing antigen-binding VHH in a culture medium containing a Tris buffer, and then forming colonies on a colony assay plate having an antigen-coated nitrocellulose membrane and a hydrophilic porous filter on the surface of an agar plate in the culture medium containing a Tris buffer; (4) separating the nitrocellulose membrane from the filter on which colonies are formed, and storing the filter on a new agar plate; (5) detecting VHH bound to the antigen on the nitrocellulose membrane, identifying colonies corresponding to the detection image on the filter in (4), and extracting the corresponding clones; (6) collecting the obtained clones, culturing each in a liquid culture containing Tris buffer, inducing VHH expression, reacting with a biotin-labeled antigen, capturing the VHH on streptavidin beads, separating the streptavidin beads using a glass fiber filter, and quantifying unbound VHH; (7) selecting a monoclonal antibody that associates quickly with an antigen, or dissociates slowly from an antigen, or has high antigen-binding properties; (8) A process of expressing and purifying each VHH as a single molecule, and measuring the kinetics or dissociation constant of binding or dissociation between the antigen and the VHH using the measurement method described in any one of [6] to [8] above. Effect of the Invention

[0032] The "time-resolved liquid phase immunoassay (TLIA)" of the present invention provides a simple, rapid, sensitive and reliable alternative to conventional immunoassay methods including sandwich ELISA and Western blot. With a simple "add-separate-measure" protocol with no wash steps, the method is rapid and reliable, eliminating variability associated with repeated wash steps, and is easily adaptable to automation for large sample volume processing as a simple, highly sensitive assay. In addition, there is no need to immobilize the antigen or antibody on a solid surface, and the antigen-antibody binding reaction can be carried out in a natural state in an aqueous solution. A sample containing a target antigen is mixed with a biotin-labeled antigen, reacted with an antibody labeled with fluorescence or the like in the solution, and after trapping all the biotin-labeled antigen with streptavidin beads, the concentration of unbound antibody is measured by the amount of fluorescence or the like, and the antigen concentration in the sample can be determined by a competitive reaction method between the antigen in the sample and the biotin-labeled antigen. In this case, the antibody concentration does not need to be high, and it can be carried out even at a low concentration, so it has excellent S / N and dynamic range, can be measured in a wide range, and has the advantage of a low detection limit concentration of the target antigen. Furthermore, the antigen-antibody binding reaction can be carried out in a natural state in an aqueous solution, and the measurement has high time resolution, so it is possible to measure during the binding or dissociation process, and the kinetics (k on ,k off ) and the binding dissociation constant (K D ) can also be calculated.

[0033] The "combination screening" method of the present invention provides a simple and rapid method to replace the conventional display method, and it has become possible to rapidly identify desired clones continuously from a large library without vector transfer. In addition, by using the "hybrid screening" method, which combines the "combination screening" method with the "TLIA" method of the present invention, it has become possible to identify clones with a low probability of existence, which were previously very difficult to identify even if they were known to exist in a library. Since useful clones with high affinity or specificity to an antigen often have a low probability of existence, the "hybrid screening" method of the present invention has made it possible to establish more useful clones. [Brief description of the drawings]

[0034] [Figure 1] "Schematic diagram of time-resolved liquid immunoassay (TLIA)" [Diagram 2] Kinetics (kon): Schematic diagram of measurement of binding process [Diagram 3] Kinetics (koff): Schematic diagram of measurement of dissociation process [Figure 4] Conceptual diagram of KD measurement [Diagram 5] Signal intensity corresponding to antigen concentration and unbound antibody: After removing the SA beads, alkaline phosphatase (AP) activity in the supernatant was measured by measuring the absorbance at 405 nm (OD405). This absorbance corresponds to the amount of unbound antibody. [Figure 6]Antigen concentration, bound antibody concentration (x), and unbound antibody (Abf) concentration: the proportion of bound antibody can be calculated as "OD0-ODX / OD0". [Figure 7] Scatchard plot: For each concentration, the bound antibody concentration (x) divided by the total antibody concentration (Abt) (X) and the bound antibody concentration (x) divided by the product of the total antibody concentration (Abt) and the free antigen concentration (Agf) (Y) were calculated, i.e., "x / (Abt × Agf)," and a Scatchard plot was created. [Figure 8] Dissociation kinetics: The absorbance (OD405) from the dissociated antibody sampled at regular intervals was plotted, and koff was calculated from the slope of the fitted curve. [Figure 9] Calibration curve from a competitive experiment using the method of the present invention (TLIA): This is the measured value of absorbance (OD405) indicating AP activity corresponding to the amount of unbound antibody for unlabeled antigen in a dilution series (0 to 1000 nM), and corresponds to the "calibration curve" for unlabeled antigen (human IgG). [Figure 10] Conceptual diagram of microchannel experiment [Figure 11] Schematic diagram of the hybrid screening method [Figure 12] Procedure for carrying out the hybrid screening method DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] 1. About the "TLIA" method of the present invention (1) Overview of the "TLIA" method of the present invention: In this case, a typical antigen-antibody reaction is used as an example of a specific intermolecular interaction, but the present invention is not limited to the antigen-antibody reaction. In this case, a target antigen is used as a target molecule, and a target antibody is used as a detection molecule, but the same operation is possible even if the target antibody and detection antibody are used instead. Whether the antibody is monovalent or bivalent, the mechanism of the interaction is essentially the same and can be measured by this method.

[0036] The main embodiments of the "TLIA" method of the present invention include, as a first embodiment, a method for measuring the concentration of a target molecule (e.g., a target antigen), and as a second embodiment, a method for measuring the binding and dissociation kinetics of a target molecule. There is also a third embodiment, which is a simplified version of the first embodiment. (1-1) First aspect: concentration measurement A first aspect of the present invention is a method for quantitatively measuring the concentration of a target antigen (or antibody) in a sample. The basic technique is a competitive format, and is typically carried out using an antibody labeled with an enzyme or fluorescent substance and a biotin-labeled antigen. The feature of this method is that neither the antigen nor the antigen is immobilized (no immobilization), and the antigen-antibody reaction occurs in the liquid phase, and the amount of unbound antibody eluted is measured (unbound phase detection), so no washing process is required (no wash).As a result, this method is highly sensitive, rapid, and simple, and can be performed even with low antibody concentrations.Since the detection limit concentration of the target antigen is low, small amounts of sample are also possible.

[0037] The outline of the method is as follows. (1) A sample solution containing a target antigen and a biotin-labeled antigen are reacted with an antibody in the solution. The antibody is labeled with an enzyme or fluorescent substance. (2) After or during the antigen-antibody binding reaction, the reaction solution is added to a suspension of streptavidin-labeled beads to completely capture the biotin-labeled antigen. (3) Next, the beads are quickly collected by filtration, centrifugation, magnetic force, etc., and the unbound antibody is separated. The amount of unbound antibody is measured by measuring absorbance or fluorescence intensity, and the amount of antigen in the sample (antigen concentration) is calculated.

[0038] (1-2) Second aspect: kinetics, K D measurement The second aspect of the present invention is to measure the kinetics of antigen-antibody and DThis method requires that the antibody can be labeled 1:1 with an enzyme or fluorescent dye. The feature of this technique is that, like the first embodiment, it is performed in the liquid phase without immobilization and measures unbound antibodies in the solution (unbound phase detection), so no washing process is required (no wash). As a result, this method is capable of measuring kinetics by a quick and simple technique. This method is superior to conventional techniques such as SPR and BLI in that it can quantitatively measure kinetics for molecules (antibodies) freely dispersed in an aqueous solution.

[0039] The outline of the method is as follows. (1) Antigen and antibody are reacted in a solution. The antigen is labeled with biotin and the antibody is labeled with a fluorescent label or the like. (2) After or during the antigen-antibody binding reaction, the reaction solution is added to a suspension of streptavidin-labeled beads, and a fixed amount of the sample solution is sampled at regular intervals before equilibrium is reached. (3) Next, immediately after sampling, each sample is filtered, centrifuged, magnetically separated, or the like to instantly remove unbound antibodies. The amount of unbound antibodies in each sample is then measured to determine the change in the amount of fluorescent signal. The binding rate constant (k on ) and dissociation rate constant (k off ) is calculated. Conversely, after the equilibrium state is reached, the reaction solution is greatly diluted, and then samples are taken at regular intervals and measured to determine the dissociation rate constant (k off ) can be calculated. Also, by measuring the amount of binding at equilibrium, the dissociation constant (K D ) can be calculated. D value and the k obtained in the above experiment off From the value, k on The value can be calculated. Specifically, it can be calculated as follows: 1) Fit the kinetic curve (the time change of the binding process alone is acceptable, but it is more accurate if the time change of the dissociation process is included) to the theoretical curve, and calculate k on and k off K D =k off / k on From the relationship D Calculate. 2) From the amount of binding (unbound) at equilibrium at various concentrations, K D Calculate k from the dissociation process (e.g., large dilution after equilibrium). off Looking for K D =k off / k on From the relationship on Calculate.

[0040] (1-3) Third aspect: The present invention also includes a third aspect as a simplified version of the first aspect. That is, this is an embodiment that does not use a highly specific substance that forms a strong bond, such as biotin-streptavidin, as in the first embodiment. Specifically, this is a simple method in which, without using streptavidin-labeled beads or biotin-labeled antigens in which an antigen is bound to biotin, an antigen-antibody reaction is induced in a sample containing an antigen of unknown concentration using beads bound to an antibody labeled with a fluorescent substance or the like and the antigen, and after equilibrium is reached, the unbound antibody is recovered and the amount of fluorescence is measured.

[0041] Specifically, a fixed amount, preferably a large amount, of target antigen is bound to the surface of a bead, and an aqueous solution containing a fixed amount of antibody labeled with fluorescence or other labeling is added together with a sample containing an unknown concentration of target antigen, and the mixture is incubated. After the antigen-antibody reaction reaches equilibrium, unbound antibody is recovered by filtration or other means, and the amount of fluorescence is measured. This method also does not require a washing process (wash-free), and measures unbound antibody in solution (unbound phase detection), and is a highly sensitive, rapid, and simple method compared to conventional ELISA. Because a large amount of antigen is immobilized on the bead surface, competition can be observed even at low antibody concentrations. The present embodiment is superior to the first embodiment in terms of speed and simplicity, since it does not require the effort of binding the antigen in the sample with biotin to prepare a biotin-labeled antigen, or the effort of preparing streptavidin-labeled beads, as in the first embodiment. However, since it cannot be said that the antigen-antibody reaction occurs freely in an aqueous solution, the detection sensitivity and detection accuracy in the case of low-concentration antigen-antibody reactions are inferior to those of the first embodiment.

[0042] (2) Procedure for carrying out the "TLIA" method of the present invention (2-1) Measurement of concentration of target antigen in aqueous solution (first embodiment): (i) An antigen-antibody reaction is carried out in a solution without immobilizing the antigen or antibody. For example, a biotin-labeled antigen and a fluorescently labeled antibody are reacted in a solution. After or during the antigen-antibody binding reaction, the reaction solution is added to a suspension of streptavidin-labeled beads. Here, we will explain the case where the antigen is biotin-labeled, the antibody is enzyme (fluorescence)-labeled, and biotin-labeled beads are used, but the opposite case (biotin-labeled antibody, enzyme (fluorescence)-labeled antigen) can also be used.

[0043] Biotin and enzyme labels can be chemically labeled using reactions with amino and thiol groups. Alternatively, biotin labeling can be performed using Avi-tag during protein expression. (商標) It is possible to add a sequence (SEQ ID NO:5) to the target protein and then enzymatically add biotin (Protein Science Society Archives, 13, e096 (2020)). For enzyme labeling such as alkaline phosphatase and peroxidase, the enzyme may be expressed as a fusion protein during protein expression (Hybody (商標)) Regarding fluorescent labeling, simply adding the C-terminal 16 amino acids of GFP (GFP11) during protein expression allows specific fluorescent labeling without impairing the function of the protein to be labeled, by subsequently associating it with the N-terminus of GFP (GFP1-10) (Non-Patent Document 11). Alternatively, fluorescent molecules may be labeled chemically. (商標) Technology" NanoLuc (登録商標) The small subunit of luciferase, SmBiT (Non-Patent Document 5), may be added to the end of the antibody, and LgBit and a luminescent substrate may be added to the unbound antibody to measure the amount of antibody by luminescence. Alternatively, fluorescent substances such as europium or chemiluminescent substances such as luminol may be used.

[0044] Addition of biotin causes only a very small change in motility in solution. Addition of a large enzyme to an antibody may cause some change in motility in aqueous solution, but the effect is also limited. When adding SmBiT to an antibody and measuring luminescence, or adding the C-terminal 13 amino acids of the above-mentioned GFP to measure fluorescence, the size of the added molecule is small, so the effect on antigen-antibody interaction can be ignored.

[0045] (ii) A sufficient amount of streptavidin-labeled beads is added to the sample to capture all the antigens onto the beads. The biotin-streptavidin bond is very strong (among the known non-covalent interactions, the dissociation constant is K D =10 -15 In M), biotin labeled polymers in solution bind quickly and almost irreversibly. Note that the object of the present invention can be achieved by utilizing highly specific interactions, not limited to biotin-streptavidin.

[0046] (iii) Wait until equilibrium is reached and separate the antibodies that are not bound to the antigen by filtering or centrifuging the beads. Magnetic separation using magnetic beads is also possible.

[0047] (iv) The absorbance of the fluorescent dye (enzyme) labeling the separated unbound antibody is measured to determine the amount of unbound antibody. Next, the amount of bound antibody is calculated by subtracting the amount of unbound antibody from the amount of antibody in the antibody solution before the reaction (Figure 1). (v) A sample solution containing an antigen of unknown concentration is added, and the antigen concentration is determined from the magnitude of the signal using the above-mentioned calibration curve (Figure 1) (competitive assay).

[0048] (2-2) Measurement of antibody binding / dissociation kinetics (second embodiment): (i) As in the above (2-1)(i), an antigen-antibody reaction is carried out in a solution without immobilizing the antigen or antibody. For example, a fluorescently labeled antibody and a biotinylated antigen are reacted in a solution. After or during the antigen-antibody binding reaction, the reaction solution is added to a suspension of streptavidin-labeled beads.

[0049] (ii) A sufficient amount of streptavidin-labeled beads is added to the sample to capture all the antigens onto the beads.

[0050] (iii) Before the binding between the antigen and the antibody reaches equilibrium, a fixed amount of the sample solution is sampled at regular intervals, and the antibodies that are not bound to the antigen on the beads in each sample are separated.

[0051] (iv) The fluorescent dye absorbance of the unbound antibody separated from each sample is measured, the amount of unbound antibody is calculated, and the binding kinetics (k on ) is measured (Figure 2). (v) Next, the amount of bound antibody is calculated by subtracting the amount of unbound antibody from the amount of antibody in the antibody solution before the reaction, and the dissociation constant K D It is possible to calculate (vi) After the binding reaches equilibrium, the mixture is suspended in a large volume of buffer, sampled at regular intervals, and the dissociation kinetics (k off) can be measured (Figure 3). This k on and K. D By this, k off The calculation of k off and K. D By this, k on It is possible to calculate the following.

[0052] 2. Application of the TLIA method of the present invention The "time-resolved liquid immunoassay (TLIA)" of the present invention can be used in the following ways. (1) Detection of target antigen or target antibody in a sample solution and measurement of its concentration in the solution: by competitive assay (1-1) Detection of target antigen: The presence of an antigen in a sample solution can be detected with high sensitivity by performing an antigen-antibody reaction in the solution without immobilizing the antigen or antibody, and simply measuring the amount of unbound antibody. In addition, antigens that can be used are not limited to proteins, but also include small molecules, peptides, viruses, cells, and the like. (1-2) Determination of antigen concentration of unknown antigen concentration sample: The antigen concentration can be determined with high accuracy by performing an antigen-antibody reaction in solution without immobilizing the antigen or antibody, adding unlabeled antigen to perform a competitive experiment, and measuring the concentration of unbound antibody.

[0053] (2) Kinetic measurement of antigen-antibody reaction and dissociation constant (K D ) Determination (2-1) The dissociation constant (K D ) can be determined (Figure 4: When a fluorescent signal is used). (2-2) The kinetics of the antigen-antibody reaction can be measured by performing an antigen-antibody reaction in a solution without immobilizing the antigen or antibody, and measuring the binding and dissociation processes during the binding process. The kinetic coefficient (k on ,k off) can be determined (Figures 2 and 3: when using fluorescent signals).

[0054] (3) Screening for the establishment of monoclonal antibodies The "TLIA" method of the present invention can be used to screen for monoclonal antibodies with high antigen specificity or monoclonal antibodies with slow or fast dissociation rates when producing various monoclonal antibodies using methods such as the hybridoma method and genetic recombination method. In particular, when used in a "hybrid screening" method in combination with the "combination screening" method of the present invention, the characteristics of "TLIA" can be maximized and it is extremely effective. The procedure for such a case is described in detail in 3. below.

[0055] In screening for the establishment of monoclonal antibodies, by measuring the binding and dissociation kinetics of the antibodies produced by the clones, it is possible to select antibodies with high affinity for the antigen as well as antibodies with different binding modes (dissociation and association rates). For example, k off This is particularly useful for screening of antibodies with small molecular weight or antibodies that dissociate slowly from the antigen.

[0056] The antibody library of interest here may be any antibody library, and may also be a protein library, not limited to antibodies. Preferred target antibodies include VHH antibodies from camelids such as alpacas, VNAR antibodies from sharks, single-chain antibodies such as scFv, Fab, etc., with VHH antibodies and VNAR antibodies being particularly preferred.

[0057] (4) Measurement using the "TLIA" method using microchannels In the "TLIA" method of the present invention, a mixed solution of the sample and fluorescently labeled antibody is added to the left of the microchannel in Figure 10, the sample is pumped through the microchannel, and passes through the streptavidin bead section, capturing all of the biotin antigen. Fluorescence measurement is then performed downstream, making it possible to measure the amount of unbound antibody. The diameter (D) of the streptavidin beads has a relationship of H>D with the height (H) of the channel in the figure, and if the relationship with the channel widths (W1, W2) is W1>D>W2, the beads will be stopped midway. The microchannel device can be easily fabricated by photolithography or the like using polydimethylpolysiloxane (PDMS), which is a material commonly used in microchannels.

[0058] 3. Experimental Procedures for "Combination Screening" and "Hybrid Screening" of the Present Invention (3-1) "Combination Screening" of the Present Invention In the "combination screening" of the present invention, a screening by the "E. coli display" method (Non-Patent Document 14) using Intimin as an outer membrane protein is combined with a screening by the "expression auto-control colony assay" method (Patent Document 1) previously developed by the present inventor (Figs. 11 and 12). In this case, the "culture method for increasing outer membrane vesicles" that we developed this time is used to increase the production of outer membrane vesicles.

[0059] (3-2) Library construction and screening by E. coli display method First, a VHH gene library made by amplifying alpaca-derived VHH genes is inserted into an E. coli display library vector to create an E. coli library. In this case, the outer membrane protein to which the target protein is fused and displayed extracellularly is not limited to Intimin, but the E. coli display method using Intimin is superior in terms of its high extracellular display ability and low toxicity (Non-Patent Document 14).

[0060] (3-3) Screening using E. coli display method with Intimin The screening procedure using the E. coli display method using Intimin is as follows. (1) A process for preparing an expression vector incorporating the N-terminus of Intimin. (2) A process of inserting VHH into the C-terminus of Intimin so that Intimin-VHH can be expressed, and preparing a VHH library. (3) Transforming the VHH library into E. coli to produce an E. coli library. (4) A process of inducing expression of Intimin-VHH and reacting the VHH displayed on the outer membrane surface of E. coli with a biotin-labeled antigen. (5) A step of reacting with anti-biotin antibody-labeled magnetic beads (or streptavidin magnetic beads). (6) A step of concentrating E. coli that binds to magnetic beads, i.e., E. coli that expresses antigen-binding VHHs, using a magnetic column.

[0061] (3-4) Screening by colony assay using a culture method to increase outer membrane vesicles (OMVs) Next, the E. coli displaying the Intimin-VHH selected in (3-3) is directly screened by "colony assay (expression automated control colony assay)" according to the following procedure. (1) To prepare a plate for the colony assay, an agar plate is prepared using culture medium containing Tris buffer, and an antigen-coated nitrocellulose membrane and a hydrophilic porous filter for colony formation are placed on top of the agar plate. (2) A concentrated solution of E. coli expressing the antigen-binding VHH obtained in (3-3)(6) above is suspended in a culture medium containing Tris buffer and seeded on the filter prepared in (1) above to form colonies. (3) An inducer such as an IPTG-containing solution is sprayed to induce the expression of Intimin-VHH. (4) OMVs carrying Intimin-VHH pass through the filter, and the antigen-affinity VHH bound to the OMV reacts with the antigen on the nitrocellulose membrane. (5) Separate the nitrocellulose membrane from the filter with the colonies, and store the filter by placing it on a new agar plate. (6) On the other hand, VHH bound to the antigen on the nitrocellulose membrane is detected using an anti-His antibody or the like, and the colonies on the filter in (5) corresponding to this detection image are identified and the corresponding clones are extracted.

[0062] (3-5) A culture method suitable for increasing “outer membrane vesicles” Here, the culture method for increasing "outer membrane vesicles" developed by the present inventors will be described below. The present inventors discovered that the production of outer membrane vesicles (OMVs) was increased by adding a Tris buffer to the culture medium during solid-state culture of E. coli, and established an OMV-increasing culture method. They found that colony assays were possible by using the OMVs increased using this method. This made it possible to perform E. coli display and colony assays consecutively without changing vectors. Whether the antibody was expressed in liquid phase or solid phase, it became possible to perform the same expression system by using the OMV-increasing culture method. By producing more outer membrane vesicles than before, it became possible to perform colony assays while the antibody gene was still in the display system vector. In addition, the subsequent TLIA assay could be performed using the vector as it was, making it possible to quickly and reliably identify clones with the desired binding properties.

[0063] (3-6) Selection (hybrid screening) procedure by TLIA method The multiple clones obtained in (3-4) above are collected and cultured in liquid medium to induce VHH expression, and outer membrane vesicles containing VHH are collected. Next, the VHH is reacted with a biotinylated antigen in a liquid, and the same measurement as in "Kinetics Measurement" of Example 2 above is performed. Specifically, the biotin-labeled antigen is captured by streptavidin beads, and then filtered through a glass fiber filter to separate unbound VHH, and the amount of unbound VHH is quantified by fluorescence measurement or the like. At this time, clones with a small amount of unbound VHH, i.e., clones with slow dissociation, are selected. Furthermore, by expressing them in E. coli and purifying each plasmid, the sequence of the VHH can be determined. If necessary, the selected clones can be cultured to express the VHH, and after purification, the dissociation constant, dissociation rate constant, etc. can be measured and evaluated by the TLIA method of the present invention.

[0064] In this way, "hybrid screening," which combines "combination screening" and "TLIA" methods, makes it possible to obtain more desired antibodies from a huge antibody library. EXAMPLES

[0065] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to the following examples. Other terms and concepts in the present invention are based on the meanings of terms commonly used in the relevant field, and the techniques used to carry out the present invention, except for those whose sources are particularly specified, can be easily and reliably carried out by a person skilled in the art based on known literature, etc. Furthermore, various analyses, etc. were carried out mutatis mutandis according to the methods described in the instruction manuals, catalogs, etc. of the analytical instruments or reagents and kits used. The contents of the technical documents, patent publications, and patent application specifications cited in this specification are to be referred to as the contents of the present invention.

[0066] Example 1 Dissociation constant (K D )measurement (1-1) Preparation of enzyme (alkaline phosphatase: AP)-labeled antibodies In this example, the dissociation constant (K D The procedure for finding θ is as follows: Biotin-labeled hIgG (Human IgG: manufactured by Jackson) was used as the biotin-labeled antigen, and the enzyme-labeled antibody was a rabbit single-chain antibody against hIgG (anti-hIgG scFv (SEQ ID NO: 4)) described in the applicant's previous application (WO2020 / 071524). The dissociation constant (K D ) is measured. The "anti-hIgG scFv" is a single-chain antibody against human IgG screened from a rabbit scFv library in the above application (WO2020 / 071524). Next, a fusion protein (AP-scFv) with alkaline phosphatase (AP) was expressed and purified by the following method according to the method described in Example (9-2) of the same application.

[0067] (i) Expression vector and expression E. coli The expression vector pET-22b, into which the above-mentioned anti-hIgG scFv was inserted in the form of AP-scFv, was used to transform E. coli BL21DE3 strain (Novagen) for expression.

[0068] (ii) Expression and recovery of periplasmic fraction The clones were grown in 10 mL of LB medium containing ampicillin at 37°C at OD 600 The culture was then incubated until the affinity of the plasmid pK1 reached 0.6, and 0.5 mM IPTG was added to induce expression at 20° C. overnight. After cultivation, the E. coli were collected by centrifugation, and after expression, the culture solution was centrifuged at 5000g for 10 minutes to recover the E. coli. The amount of E. coli was measured, and 2 mL of PE Buffer (20% Sucrose, 1 mM EDTA, 100 mM Tris-HCl (pH 8.0)) was added to extract the periplasmic fraction from the E. coli, which was then treated on ice for 30 minutes and centrifuged at 1600G for 20 minutes. The supernatant was then collected and used as the periplasmic fraction.

[0069] (iii) Purification and Quantification The scFv was purified from the periplasmic fraction using its C-terminal His tag on a Ni column (Ni Sepharose 6 Fast Flow, Cytiva). After applying the sample to the column, it was washed with PBS containing 20 mM imidazole, and the bound scFv was eluted with PBS containing 200 mM imidazole and purified. After purification, the degree of purification was confirmed by SDS electrophoresis. All samples were purified to a degree of 95% or more. The weight of the purified scFv was determined using the Bradford method (Quick Start Protein Assay, BioRad).

[0070] (1-2) Antibody-antigen reaction A constant concentration (1 nM) of anti-hIgG scFv (AP enzyme-labeled antibody) is reacted with biotin-labeled antigen (hIgG) in a solution (1 mL) at room temperature until equilibration (about 30 minutes). A dilution series of concentrations of biotin-labeled antigen (hIgG) is used (50, 25, 12.5, 6.25, 3.13, 1.57, 0 nM). In order to enable accurate experiments, the antibody concentration was set at the K D It is necessary to set the value lower than the expected K D It was used at about 1 / 10 of the original amount.

[0071] (1-3) Capture of biotin-labeled antigens onto streptavidin beads After equilibration (approximately 30 minutes at room temperature), the antibody-antigen reaction solution is applied to a filter column packed with streptavidin beads (Micromod) to capture the biotin-labeled antigen on the streptavidin beads. The streptavidin (SA) beads used are silica beads with a diameter of 3 mm. The binding between streptavidin and biotin is strong (dissociation constant K D =10 -15 M), the binding rate is fast and the affinity is high, so it is almost irreversible. By using a large excess of SA beads, all of the biotin-labeled antigen in the reaction solution is instantly captured by the beads.

[0072] (1-4) Separation of the supernatant (unbound antibody) by filtration The beads are filtered by vacuum filtration using a glass fiber filter (pore size 0.7 mm, GF / F, Cytiva) to separate the supernatant. In this case, filtration is carried out within a few seconds for a solution of about 100 mL to 2 mL. It is also possible to use magnetic beads instead of silica beads as the SA beads, and to apply a method of recovering the beads by magnetic force in several tens of seconds.

[0073] (1-5) Quantification of unbound antibody 0.1 mL of the supernatant was taken, alkaline phosphatase substrate (p-nitrophenyl phosphate: pNPP, Merck) was added, and the enzyme activity was measured using a microplate reader (Thermo) as the absorbance at 405 nm (OD 405 ) The absorbance corresponds to the amount of unbound antibody. The results are shown in (Figure 5) as "Signal intensity corresponding to antigen concentration and unbound antibody." Here, it is important to ensure linearity between the amount of enzyme activity and the amount of unbound antibody, and care must be taken not to use an excessive amount of liquid from the supernatant.

[0074] (1-6) Calculation of the bound antibody ratio OD without antigen 405 (OD0) is the absorbance when all antibodies are unbound, so the absorbance OD when antigen is added is 405 (OD x ) and divide the result by OD0, i.e., "OD0-OD X " / OD0" is the proportion of bound antibody. By performing this calculation at each concentration, the proportion of bound and unbound antibody can be calculated, and the bound antibody concentration (x) and unbound (free) antibody concentration (Abf) can be obtained. The results are shown in Figure 6 as "antigen concentration and bound and unbound antibody concentrations."

[0075] (1-7) K from Scatchard plot D Calculation of A Scatchard plot, which plots the ratio of bound to unbound ligand concentrations against the bound ligand concentration, allows for a more accurate estimation of the K D can be sought. Specifically, for each concentration, the bound antibody concentration (x) divided by the total antibody concentration (Abt) was calculated as X, and the bound antibody concentration (x) divided by the product of the total antibody concentration (Abt) and the free antigen concentration (Agf) was calculated as Y = x / (Abt x Agf), and a Scatchard plot was created (Figure 7). The dissociation constant K D is 8.27 x 10 -9 I was able to find M.

[0076] Example 2 Kinetics measurement: dissociation time (k off )measurement (2-1) Kinetics measurement experimental procedure In this example, the kinetics (k) of antibody dissociation and binding were measured by the "TLIA" method of the present invention. off and k on In order to demonstrate that it is possible to measure dissociation kinetics, the procedure of FIG. 3 is followed. The above (Example 1) "K D The experiment was carried out following roughly the same procedure as in "Measurement". The antibody used was the same anti-hIgGscFv as used in (Example 1), purified AP fusion protein (AP-scFv), and the antigen used was biotin-labeled hIgG (Jackson). The biggest difference from Example 1 is that in the following (2-3), after the reaction reaches equilibrium, a small portion of the suspension is diluted with a large amount of buffer, sampled at regular intervals, filtered, and the degree of dissociation is measured. Also, in the antigen-antibody reaction in the following (2-2), a dilution series of biotin-labeled antigen (hIgG) is used in the above (1-2), whereas in this example, the reaction is carried out at a constant concentration.

[0077] (2-2) Antibody-antigen reaction A constant concentration (1 nM) of anti-hIgG scFv (enzyme-labeled antibody) was reacted with a constant concentration (100 nM) of biotin-labeled antigen (hIgG) in an aqueous solution (1 mL).

[0078] (2-3) Antigen capture on streptavidin beads After the antigen-antibody reaction reaches equilibrium, the antibody-antigen reaction solution is applied to a filter column packed with streptavidin beads (silica beads with a diameter of 3 mm) to capture the biotin-labeled antigen on the streptavidin beads.

[0079] (2-4) Separation of the supernatant (unbound antibody) and measurement of dissociation kinetics (2-4-1) Filtration After the antigen-antibody reaction reaches equilibrium, the beads are filtered by vacuum filtration using a glass fiber filter (pore size 0.7 mm, GF / F, Cytiva) and the supernatant is separated.

[0080] (2-4-2) Measurement of dissociation kinetics After filtration, the mixture is suspended in a buffer 30 times the volume of the reaction mixture. A small portion of the suspension is then sampled at regular intervals, filtered, and measured. In this case, the more the amount of buffer, the more ideal the dissociation process will be observed, but 30 times the amount of the reaction solution is sufficient. Even if the amount of buffer is increased, it will not cause any problems in the measurement because it will be filtered again in the next step. In addition, the amount of liquid sampled at regular intervals is small, so it does not affect the dissociation process.

[0081] (2-5) Quantification of unbound antibody 0.1 mL of the supernatant was taken, alkaline phosphatase substrate (pNPP, Merck) was added, and the enzyme activity was measured using a microplate reader (Thermo) as the absorbance at 405 nm (OD 405 ) This absorbance corresponds to the amount of unbound antibody.

[0082] (2-6) From the antigen-antibody dissociation kinetics, koff Calculate Sampling is performed at regular intervals, and the absorbance of the dissociated antibody is measured. The results are shown in (Figure 8) "Dissociation Kinetics." From the slope of the fitted curve of the experimental data, k off is 8.87 x 10 -4 s -1 It can be found that:

[0083] (2-7) Determination of dissociation and association rate constants From Example 1, the dissociation constant K D is 8.27 x 10 -9 Since it has been determined that M, k on is k on =k off / K D From the relationship, 1.07×10 5 M -1 s -1 It can be determined that: Here, we have shown how to measure and calculate the dissociation kinetics. However, the binding kinetics can also be measured in a similar manner following the procedure in Figure 2, and the k on As above, we can obtain K D From the value of k off It is possible to calculate the following.

[0084] Example 3 Determining the antigen concentration of unknown samples: Achieved by competitive assay (3-1) Experimental procedure In this example, it is shown that by using the "TLIA" method of the present invention, the antigen concentration in a sample with an unknown antigen concentration can be determined by a competitive reaction experiment quickly, simply, and accurately compared to the ELISA method. The antibody used was AP-scFv, which was obtained by fusing AP with the anti-hIgG scFv used above (Example 1), and purified, and biotin-labeled hIgG (Jackson) was used as the labeled antigen. And, K in the above (Example 1) DAs in the experimental procedure for "Measurement," the enzyme-labeled antibody is reacted with the biotin-labeled antigen and the target unlabeled antigen in the sample until equilibrium is reached, and then the amount of unbound antibody is measured. Specifically, the procedure is as follows:

[0085] (3-2) Antibody-antigen reaction A constant concentration (1 nM) of anti-hIgG scFv (enzyme-labeled antibody), a constant concentration (100 nM) of biotin-labeled antigen (human IgG), and a dilution series (0 to 100 nM) of unlabeled antigen (human IgG) are reacted in a solution (1 mL).

[0086] (3-3) Antigen capture on streptobeads After equilibration (30 minutes at room temperature), the antibody-antigen reaction solution is applied to a filter column packed with streptavidin beads (silica beads with a diameter of 3 mm) to capture the biotin-labeled antigen on the streptavidin beads.

[0087] The beads are then filtered by vacuum filtration using a glass fiber filter (pore size 0.7 mm, GF / F: Cytiva) to separate the supernatant (unbound antibody).

[0088] (3-4) Quantification of unbound antibody 0.1 mL of the supernatant was taken, alkaline phosphatase substrate (pNPP, Merck) was added, and the enzyme activity was measured by absorbance at 405 nm using a microplate reader (Thermo). The absorbance corresponds to the amount of unbound antibody. The results are shown in (Figure 9).

[0089] (3-5) Determination of concentration of unknown samples (Figure 9) shows the absorbance (OD ) that indicates the AP activity corresponding to the amount of unbound antibody for unlabeled antigen (human IgG) in a dilution series (0 to 100 nM). 405) and corresponds to the "standard curve" corresponding to the unlabeled antigen concentration, but it can be seen that competition occurs over a wide range of antigen concentrations. If a sample containing an unknown concentration of antigen is similarly measured and the absorbance is obtained, the concentration can be determined using the standard curve in (Figure 9).

[0090] In competitive experiments using ELISA, the amount of immobilized antigen is low, so the concentration of the enzyme-labeled antibody for detection must be high, and a competitive reaction occurs in the high antigen concentration range, making it impossible to measure at low concentrations. If the concentration of the enzyme-labeled antibody for detection is low, a competitive reaction occurs in the low antigen concentration range, but the signal becomes very weak, and measurement is often impossible due to the influence of measurement errors. The technique of the present invention has higher sensitivity and a wider measurable dynamic range than ELISA. The method of the present invention requires fewer procedures and is simpler than ELISA, and the time required for measurement is less than one-fourth of that required for ELISA.

[0091] Example 4 Screening of monoclonal antibodies by binding and dissociation patterns (4-1) Experimental procedure In this example, it is shown that the "TLIA" method of the present invention makes it possible to screen monoclonal antibodies using dissociation / association rates as indicators. Furthermore, during the primary screening, antibodies screened using the "primary screening using a display system" and the colony assay method previously developed by the inventors were evaluated using the "TLIA" method of the present invention, demonstrating that screening for antibodies with high recognition ability can be easily performed.

[0092] (4-2) Generation of alpaca VHH naive library After blood was collected from the alpaca, peripheral blood lymphocytes (PBMCs) were separated and mRNA was purified. After cDNA was synthesized from the mRNA, VHH genes were amplified using VHH-specific primers. The VHH library was treated with a restriction enzyme (XbaI) and inserted into a library vector for E. coli display. The library vector for E. coli display is a vector in which Intimin is inserted into a pUC18 vector so that it can be expressed on the extracellular membrane. It is designed so that VHH is inserted into the C-terminus of Intimin using XbaI (Non-Patent Document 10). At the C-terminus of VHH, there is a His tag and 16 residues (215-230) of the C-terminus of GFP (GFP11: SEQ ID NO: 6).

[0093] (4-3) Primary screening using a display system The E. coli display library was transformed into the JM109 strain to produce an E. coli library. Expression of Intimin-VHH was induced by adding IPTG, and the mixture was mixed with 50 nM of biotinylated antigen (mouse IgG), washed, and then mixed with streptavidin magnetic beads (Miltenyi). By passing the mixture through a magnetic bead column, only E. coli displaying VHH reactive with the antigen (mouse IgG) could be isolated, and the eluate was spread on a solid-phase surface, solid-phase cultured, and a colony assay was performed. Here, this method can be used as an "off-rate screening" to select clones that have high affinity with the antigen and are unlikely to lose their binding even after dilution.

[0094] (4-4) Secondary screening using colony assay Hereinafter, the colony assay was performed according to the procedure of the "colony assay method (Patent Document 1)" developed by the present inventors. (4-4-1) Preparing plates for colony assay An agar plate was prepared using 2xYT culture medium containing 50 mM Tris, and the antigen-coated nitrocellulose membrane and a hydrophilic porous filter for colony formation were placed on top of the plate.

[0095] (4-4-2) Library colony formation The E. coli obtained in (4-3) above was suspended in 2xYT culture medium containing 50 mM Tris, seeded on the filter prepared in (4-4-1) above, and incubated overnight at 30° C. to form colonies.

[0096] (4-4-3) Induction of VHH expression, outer membrane vesicle formation, and colony assay using outer membrane vesicles 400 μL of 0.1 mM IPTG was sprayed onto the cells, and the cells were incubated at 25° C. for 5 hours to induce the expression of Intimin-VHH. OMVs carrying Intimin-VHHs pass through the filter, and the VHHs on the OMVs can react with the antigens present on the nitrocellulose membrane. VHHs with affinity for the antigens bind to the antigens on the nitrocellulose membrane. The nitrocellulose membrane and the filter containing the colonies were separated, and the filter was placed on a new agar plate and stored at 4°C. On the other hand, VHHs bound to the antigen on the nitrocellulose membrane were detected using an anti-His antibody (Wako). Colonies on the filter corresponding to this detection image were identified, and the corresponding clones were extracted. In this way, multiple clones producing VHHs that specifically bind to the antigen were identified. In this way, the addition of Tris buffer to the medium allowed the production of more outer membrane vesicles (OMVs) displaying VHH, enabling colony assays to be performed using the display vector as is (combinatorial screening).

[0097] (4-5) Positive clones were cultured and expression was induced Below, we will further show a procedure for selecting clones with slow dissociation (off-rate screening) by applying kinetic measurement of the TLIA method while keeping the same display vector. First, the VHH-producing clone obtained in (4-4-3) above is collected and cultured in liquid to express "outer membrane vesicles (OMV)" containing the VHH. After preculture, the clone is cultured in LB medium at 25°C until OD 600 When the ΔΨ reaches 0.6, VHH expression is induced by administration of 0.1 mM IPTG and 150 mM Tris.

[0098] (4-6) Assay off-rate screening The E. coli cells after expression induction are pelleted by centrifugation, and the outer membrane vesicles containing the VHH in the supernatant are collected. The cells are reacted with biotinylated antigen in the liquid, and the same measurement as in the "Kinetics Measurement" in Example 2 above is performed. After equilibration, 30 volumes of buffer are added, and the mixture is applied to a filter column filled with streptavidin (silica) beads (Micromod) at 0 and 30 minutes after dilution, and the biotin-labeled antigen is captured by the streptavidin beads. The mixture is then filtered through a glass fiber filter (pore size 0.7 mm, GF / F, Cytiva) to separate unbound VHH. GFP1-10 (SEQ ID NO: 7: 1-214 residues) is added, and the amount of unbound VHH is quantified by measuring the fluorescence. At this time, clones that had a small amount of unbound VHH even 30 minutes after dilution with buffer, i.e., clones with a slow dissociation rate, were selected. GFP1-10 (SEQ ID NO: 7) was inserted into a pET30 vector (Novagen), expressed in E. coli, and purified using a GFP-specific antibody (Abcam).

[0099] (4-7) Establishment and Evaluation Plasmids were purified from each of the selected clones 1 to 3 with slow dissociation rates, and the VHH sequences were determined (SEQ ID NOs: 1 to 3). The purified plasmids were used to amplify the VHH regions by PCR. These were then inserted into an expression vector (with AP label) and transformed into the JM109 strain. Each clone was cultured in liquid culture, and VHH expression was induced by administration of IPTG.

[0100] ·evaluation Meanwhile, the selected clones 1 to 3 were each cultured, and VHH was expressed and purified by His tag in the same manner as described above (Example 1) (1-1) as follows.

[0101] Specifically, clones 1 to 3 were cultured in 10 mL of LB medium containing ampicillin at 37°C until OD 600The culture was grown until the affinity of the plasmid pK1 reached 0.6, and then 0.5 mM IPTG was added to induce expression at 20°C overnight. After cultivation, the E. coli were collected by centrifugation, and after expression, the culture solution was centrifuged at 5000g for 10 minutes to recover the E. coli. The amount of E. coli was measured, and 2 mL of PE Buffer (20% Sucrose, 1 mM EDTA, 100 mM Tris-HCl (pH 8.0)) was added to extract the periplasmic fraction from the E. coli, which was then treated on ice for 30 minutes and centrifuged at 1600G for 20 minutes. The supernatant was then collected and used as the periplasmic fraction.

[0102] Next, VHH was purified from the periplasmic fraction using its C-terminal His tag on a Ni column (Cytiva). After applying the sample to the column, it was washed with PBS containing 20 mM imidazole, and the bound VHH was eluted with PBS containing 200 mM imidazole and purified. After purification, the degree of purification was confirmed by SDS electrophoresis. All samples were purified to a degree of 95% or more. The weight of the purified VHH was determined using the Bradford method (BioRad).

[0103] For each VHH antibody, the TLIA method of the present invention was applied using biotin-labeled human IgG (manufactured by Jackson), streptavidin beads (manufactured by Micromod), and a glass fiber filter (pore size 0.7 mm, GF / F: Cytiva) in the same manner as in (1-2) to (1-7) above (Example 1), and the dissociation constant, dissociation rate constant, etc. were measured and verified as shown below (Table 1).

[0104] [Table 1]

[0105] In this way, it was verified that by applying the "TLIA" method to multiple clones obtained by the "combination screening" method of the present invention, clones with small dissociation rate constants were obtained.

[0106] Example 5 A culture method for producing more outer membrane vesicles (OMVs) during solid culture We verified that the culture method using Tris buffer, which was used in the "combination screening" of the present invention, indeed produces more "outer membrane vesicles (OMVs)." (5-1) Colony formation and recovery of E. coli Agar plates were prepared from 2xYT culture medium containing 50 mM Tris buffer and 2xYT culture medium (referred to as Tris-2xYT plate and 2xYT plate, respectively). 0.1 mL of E. coli (DH10b, NEB) was inoculated onto each plate and cultured overnight at 37°C to allow colonies to form. All colonies were collected with a scraper and suspended in 2xYT culture medium (referred to as Tris-2xYT suspension and 2xYT suspension, respectively).

[0107] (5-2) Isolation of outer membrane vesicles (OMVs) from E. coli suspension Using a commercially available OMV detection kit ("ExoBacteria OMV Isolation Kit (System Biosciences)"), outer membrane vesicles (OMVs) were isolated from each of the 2xYT suspensions and 2xYT suspensions in (5-1) above. The suspensions were centrifuged twice at 5,000 x g, 20 minutes, and 4°C to remove bacteria and debris, and then filtered through a 0.22 μm suction filter. The column was added to the "ExoBacteria OMV Isolation Kit" and washed three times with 15 ml of binding buffer. 1.5 ml of elution buffer was added to the column and incubated at room temperature for 2 minutes, after which the OMVs were eluted.

[0108] (5-3) Quantification of outer membrane vesicles (OMVs) The outer membrane vesicles (OMV) isolated in (5-2) above were quantified using the "ExoELISA-ULTRA Complete Kit (System Biosciences)". The outer membrane vesicles (OMV) suspension was added to an ELISA plate and incubated at 37°C for 2 hours to immobilize. A capture (primary) antibody and an HRP-labeled secondary antibody were added in sequence and incubated at 37°C for 1 hour. The antibody solution was drained, washed, and TMB ELISA substrate was added. After the reaction was stopped, the absorbance at 450 nm was measured using a plate reader (model 680, Bio-Rad). The amount of OMV from the Tris-2xYT plate and the 2xYT plate is shown in Table 2.

[0109] [Table 2]

[0110] As described above, it was demonstrated that by applying a culture method using TRIS buffer when culturing E. coli on agar plates used in colony assays, a greater amount of "outer membrane vesicles (OMVs)" was indeed produced.

[0111] Example 6 Measurement of "TLIA" method using microchannels This example shows that the use of a microfluidic system enables measurements to be performed using a smaller amount of sample in a shorter time. (6-1) Design of microfluidic devices We designed a flow channel structure in which the antigen-antibody reaction solution passes through silica beads (or streptavidin beads) on which antigens are immobilized in the tiny space of a microfluidic device, reliably capturing the antigens in the reaction solution (Figure 10). The microchannel is composed of a sample introduction section, a main channel, and a detection section. The sample introduction section is a site for introducing a sample containing an antigen of unknown concentration, silica beads or streptavidin beads with immobilized antigens, and an aqueous solution (buffer solution) containing luminescent or fluorescently labeled antibodies. The main channel has a length that ensures that the antigen-antibody reaction occurs until an equilibrium state is reached, and has a dam structure that can hold back the beads. In the detection section, the absorbance or fluorescence intensity of the luminescent or fluorescent substance labeled to the unbound antibody is measured. For example, if the diameter (D) of the streptavidin beads is in the relationship of H>D with the height (H) of the flow channel in the figure, and the width (W1, W2) of the flow channel is in the relationship of W1>D>W2, the beads will be blocked midway. The dimensions of the flow channel are width: 1000mm and 30mm, height: 200mm, and the streptavidin beads are silica beads with a diameter of 50mm.

[0112] (6-2) Fabrication of microfluidic device The microfluidic device is fabricated using polydimethylsiloxane as the material, a mold made by microfabrication technology, and photolithography.

[0113] (6-3) Liquid Delivery The antigen-antibody reaction solution is pumped through the microchannel device by opening and closing valves driven by an electroosmotic pump (Nanofusion, Inc.).

[0114] (6-4) Reaction of antigen with antigen solution Biotin-labeled human IgG (Jackson) is used as the antigen, and FITC-labeled anti-human IgG antibody (Jackson) is used as the antibody. The concentration of biotin-labeled human IgG is 100nM, the antibody concentration is 10nM, and human IgG (Jackson) at a concentration of 1-100nM is added, and the experiment is performed with an antigen-antibody reaction solution volume of 10-200mL. Streptavidin beads are 50mm diameter silica beads manufactured by Micromod.

[0115] (6-5) Measurement of unbound antibody amount The amount of unbound antibody in the antigen-antibody reaction solution can be quantified in the measurement section by exciting the fluorescent dye (FITC) labeled on the antibody with excitation light of 492 nm wavelength and measuring the amount of emitted fluorescence (wavelength 520 nm).

Claims

1. A method for detecting a target molecule in a sample or measuring a concentration thereof by using a molecule having a specific interaction with the target molecule as a detection molecule, comprising the steps of: (1) preparing an aqueous solution containing a sample possibly containing a target molecule and a certain amount of a luminescently or fluorescently labeled detector molecule, comprising: (a) a fixed amount of biotin-labeled target molecule and streptavidin-labeled beads; (b) beads having a fixed amount of target molecules bound to their surface; (2) waiting for the specific interaction between the target molecule and the detector molecule in the aqueous solution to reach equilibrium, and separating and recovering the unbound luminescent or fluorescently labeled detector molecule from the beads; and (3) measuring the absorbance, luminescence or fluorescence intensity from the recovered detector molecule to determine the concentration of unbound detector molecule; 1. A method for detecting the presence of a target molecule or measuring the concentration of a target molecule in a sample, comprising:

2. Prior to step (1), a target molecule is reacted with a luminescent or fluorescently labeled detection molecule until equilibrium is reached in an aqueous solution in which the target molecule has been diluted to various concentrations, and the absorbance, luminescence or fluorescence intensity of each of the unbound detection molecules separated and recovered is measured to create a calibration curve of "signal intensities corresponding to the target molecule and the unbound detection molecule." The method according to claim 1, further comprising a step of calculating the concentration of the target molecule in the sample from the deviation between the concentration of the unbound detector molecule obtained in step (3) and the corresponding target molecule concentration in the calibration curve.

3. A method for detecting or measuring a concentration of a target molecule in a sample using a microfluidic device, comprising: The method according to claim 1, characterized in that the microchannel is composed of a main channel having a length sufficient to allow specific interaction between the target molecule and the detection molecule to occur until an equilibrium state is reached and a dam structure for blocking the beads, a sample introduction section, and a detection section.

4. A method for measuring the concentration of a target molecule in a sample by using a molecule having a specific interaction with the target molecule as a detection molecule, comprising the steps of: (1) reacting a dilution series of unlabeled target molecules with a constant concentration of luminescently or fluorescently labeled detection molecules and a constant concentration of biotin-labeled target molecules in an aqueous solution; (2) After reaching equilibrium, the unbound luminescent or fluorescently labeled detection molecules are captured by streptavidin-labeled beads and then separated and recovered from the beads; (3) measuring the absorbance, or luminescence or fluorescence intensity from the recovered detection molecule, calculating the concentration of unbound detection molecule corresponding to the unlabeled target molecule in the dilution series, and creating a calibration curve for both; (4) reacting a sample containing an unknown concentration of target molecules with a constant concentration of luminescent or fluorescently labeled detection molecules and a constant concentration of biotin-labeled target molecules in an aqueous solution until equilibrium is reached, similar to steps (1) and (2), capturing the target molecules on streptavidin-labeled beads, and then separating and recovering unbound luminescent or fluorescently labeled detection molecules from the beads; and (5) measuring the absorbance, luminescence or fluorescence intensity from the recovered detection molecule and determining the target molecule concentration in the sample using the calibration curve prepared in (3); A method for measuring a target molecule concentration in a sample, comprising:

5. The method for measuring a target molecule concentration in a sample according to any one of claims 1 to 4, characterized in that the target molecule is an antigen and the detection molecule is an antibody, or the target molecule is an antibody and the detection molecule is an antigen, and the specific interaction between the molecules is an antigen-antibody reaction.

6. By using a molecule that has a specific interaction with a target molecule as a detection molecule, the kinetics of binding of the specific interaction between molecules (k ｏｎ ) a method for measuring (1) reacting a certain concentration of a luminescent or fluorescently labeled detection molecule with a certain concentration of a biotin-labeled target molecule in an aqueous solution containing streptavidin-labeled beads; (2) A step of collecting a portion of the solution at regular intervals before the specific interaction between the target molecule and the detection molecule in the aqueous solution reaches an equilibrium state, and separating and recovering the unbound detection molecule in the sample solution during the binding process; (3) measuring and recording the absorbance, or luminescence or fluorescence intensity from unbound detector molecules for each sample solution; The method according to claim 1, further comprising:

7. By using a molecule that has a specific interaction with a target molecule as a detection molecule, the kinetics of dissociation of the specific interaction between molecules (k ｏｆｆ ) a method for measuring (1) reacting a certain concentration of a luminescent or fluorescently labeled detection molecule with a certain concentration of a biotin-labeled target molecule in an aqueous solution containing streptavidin-labeled beads; (2) After the two have sufficiently reached equilibrium, the beads are filtered to separate the supernatant, which is then suspended in a buffer, and a portion of the solution is sampled at regular intervals to separate and recover unbound detection molecules in the sample solution during the dissociation process; (3) measuring and recording the absorbance, or luminescence or fluorescence intensity from unbound detector molecules for each sample solution; The method according to claim 1, further comprising:

8. By using a molecule that has a specific interaction with a target molecule as a detection molecule, the dissociation constant (K Ｄ ) a method for measuring (1) A step of reacting a luminescent or fluorescently labeled detection molecule at a constant concentration with a biotin-labeled target molecule adjusted to a plurality of concentrations using a dilution series in an aqueous solution until equilibrium is reached; (2) After reaching equilibrium, the reaction solution of the target molecule and the detection molecule is applied to a filter column packed with streptavidin beads to capture the biotin-labeled target molecule on the streptavidin beads, the beads are filtered, and the supernatant is separated; (3) measuring the enzyme activity, or the fluorescence or luminescence intensity in the supernatant to measure the amount of unbound antibody; (4) At each concentration of the target molecule, a Scatchard plot was prepared and the inverse of the slope of the graph was calculated to determine the dissociation constant (K Ｄ ) The method according to claim 1, further comprising:

9. The method for measuring the kinetics or dissociation constant upon binding or dissociation between an antigen and an antibody according to any one of claims 6 to 8, characterized in that the target molecule is an antigen and the detection molecule is an antibody, or the target molecule is an antibody and the detection molecule is an antigen, and the specific interaction between the molecules is an antigen-antibody reaction.

10. In an antibody screening method in which the target molecule is a monoclonal antibody in an antibody library and the detection molecule is an antigen, A method for screening for an antibody having high antigen-binding ability, rapid association, or slow dissociation, using the method for measuring the kinetics or dissociation constant of binding or dissociation between an antigen and an antibody according to any one of claims 6 to 8.

11. A method for screening an antibody library for a monoclonal antibody with high antigen-binding ability, comprising the following steps (1) to (5): (1) inserting an antibody gene library into an E. coli display library vector and inducing expression to prepare an E. coli library displayed on the outer membrane surface of E. coli; (2) reacting the displayed antibody with a biotin-labeled antigen, then reacting it with anti-biotin antibody-labeled magnetic beads or streptavidin magnetic beads, and concentrating E. coli expressing the antibody having antigen-binding ability using a magnetic column; (3) suspending a concentrated solution of E. coli expressing an antibody having antigen-binding ability in a culture medium containing a Tris buffer, and then forming colonies on a colony assay plate provided with an antigen-coated nitrocellulose membrane and a hydrophilic porous filter on an agar plate of the culture medium containing a Tris buffer; (4) separating the nitrocellulose membrane from the filter on which colonies have been formed, and storing the filter on a new agar plate; (5) A step of detecting the antibody bound to the antigen on the nitrocellulose membrane, identifying the colony corresponding to the detected image on the filter in (4), and extracting the corresponding clone.

12. The method according to claim 11, characterized in that the antibody gene library (1) is a cDNA library of VHH antibody genes, in which Intimin is used as an outer membrane protein for display on the outer membrane surface of Escherichia coli, and Intimin-VHH induced by expression is displayed on the outer membrane surface.

13. A method for screening an antibody library for a monoclonal antibody having high antigen-binding ability, rapid association, or slow dissociation, comprising the following steps (1) to (7) or further step (8): (1) inserting a cDNA library of VHH antibody genes into an E. coli display library vector incorporating the N-terminus of Intimin so that Intimin-VHH can be expressed, and inducing expression to prepare an E. coli library displayed on the outer membrane surface of E. coli; (2) reacting the displayed VHH with a biotin-labeled antigen, then reacting it with streptavidin magnetic beads, and concentrating E. coli expressing the VHH with antigen-binding ability using a magnetic column; (3) suspending a concentrated solution of E. coli expressing antigen-binding VHH in a culture medium containing a Tris buffer, and then forming colonies on a colony assay plate having an antigen-coated nitrocellulose membrane and a hydrophilic porous filter on the surface of an agar plate of the culture medium containing a Tris buffer; (4) separating the nitrocellulose membrane from the filter on which colonies have been formed, and storing the filter on a new agar plate; (5) detecting VHH bound to the antigen on the nitrocellulose membrane, identifying colonies corresponding to the detection image on the filter in (4), and extracting the corresponding clones; (6) collecting the obtained clones, culturing each in a liquid culture containing Tris buffer to induce VHH expression, reacting with a biotin-labeled antigen, capturing the VHH on streptavidin beads, separating the streptavidin beads with a glass fiber filter, and quantifying unbound VHH; (7) selecting a monoclonal antibody that associates quickly with an antigen, or dissociates slowly from an antigen, or has high antigen-binding properties; (8) A step of expressing and purifying each VHH as a single molecule, and measuring the kinetics or dissociation constant of binding or dissociation between an antigen and a VHH using the measurement method described in any one of claims 6 to 8.

Images