Method of performing liquid immunoassay by using in vitro diagnostic cartridge having magnetic bead well, reaction well, washing well and measurement well

Abstract

The present invention relates to a method for performing a liquid immunoassay by using an in vitro diagnostic cartridge having a magnetic bead well, a reaction well, a washing well and a measurement well. The liquid immunoassay method of the present invention comprises the steps of: bringing a magnet close to a pipette tip so as to collect magnetic beads filled in the magnetic bead well, thereby moving same to the reaction well; providing a sample to the reaction well so as to perform an antigen-antibody reaction; bringing the magnet close to the pipette tip so as to collect a reactant in the reaction well, thereby moving the reactant to the washing well and washing same; and bringing the magnet close to the pipette tip, thereby moving the reactant washed in the washing well to the measurement well and optically analyzing same. According to the present invention, the collection yield of the magnetic beads can be enhanced.

Description

A method for performing a liquid immunoassay using an in vitro diagnostic cartridge having a magnetic bead well, a reaction well, a washing well, and a measurement well.

[0001] The present invention relates to a method for performing a liquid immunoassay using an in vitro diagnostic cartridge having a magnetic bead well, a reaction well, a washing well, and a measurement well, and particularly to a liquid immunoassay method for improving the capture yield of magnetic beads.

[0002] With the advancement of medicine, biotechnology, and related technologies, tests that detect various molecular markers, such as blood cells, genes, proteins, antigens, and pathogens, in specific biological samples like urine and blood are becoming widely used. The testing process typically involves collecting a sample, reacting it with a specific reagent appropriate for the target marker, and analyzing and observing the resulting changes. This allows for qualitative and / or quantitative analysis of various molecular markers contained in the sample, which can then be used to provide information regarding the diagnosis, progression, or prognosis of a disease.

[0003] One widely used technique in these testing processes is an immunoassay, also known as an Enzyme ImmunoAssay (EIA), which relies on specific antigen-antibody binding. These include chromogenic or colorimetric methods, which measure a color reaction based on absorbance, chemiluminescence, and fluorescence, depending on the type of substrate used to detect the analyte. Furthermore, depending on the analytical method, there are sandwich-based immunoassays, also known as enzyme-linked immunosorbent assays (ELISAs), and competitive-based immunoassays.

[0004] In these analyses, regardless of the method used, the removal of nonspecific reactants is desirable for high-specificity and high-sensitivity detection. In other words, after the reaction between the reagent and sample during the assay, purification or separation of the reaction product is necessary for accurate detection.

[0005] The most effective method for removing nonspecific reactants is physical washing or purification. Among these, washing using magnetic particles is preferred. This washing method using magnetic particles requires a separate separation and washing device, and nonspecific reactants can be removed by repeatedly dispersing and collecting the magnetic particles. However, this method of repeatedly dispersing and collecting magnetic particles requires a long washing process and suffers from the loss of a large amount of magnetic beads. Therefore, there is a pressing need for the development of testing devices and methods that can effectively remove nonspecific reactants in a short period of time without losing the magnetic beads.

[0006] Meanwhile, disposable cartridges filled with reagents are widely used in point-of-care (POC) diagnostic equipment for in vitro diagnostics. These disposable cartridges feature a plastic frame and are equipped with wells for filling reagents or performing various reactions / purifications. Depending on their purpose, these wells can be categorized into reagent wells, reaction wells, wash wells, measurement wells, and sample wells.

[0007] In POC assays using cartridges, liquid-phase immunoassay technology utilizing magnetic beads (MB) is becoming increasingly important for separating and purifying samples. Using antigen / antibody pairs or antibody / antibody pairs, one antibody or antigen is bound to the magnetic beads, which are then used to form antigen-antibody complexes. A permanent magnet is then used to isolate and purify the target component with high sensitivity for quantitative or qualitative analysis. In liquid-phase immunoassay technology, magnetic beads are a crucial material for high-purity purification of the target component, and in particular, techniques for increasing the effective capture yield of magnetic beads are crucial, as they are directly related to the overall sensitivity and specificity of the assay.

[0008] The purpose of the present invention is to provide a liquid immunoassay method for increasing the effective capture yield of magnetic beads.

[0009] In addition, another object of the present invention is to provide a liquid immunoassay method with improved analytical sensitivity and specificity.

[0010] In order to achieve the above-described object, the present invention provides a method for performing a liquid immunoassay using an in vitro diagnostic cartridge having a magnetic bead well, a reaction well, a wash well, and a measurement well, the method comprising: a step of bringing a magnet close to a pipette tip to capture magnetic beads filled in the magnetic bead well and moving them to the reaction well; a step of providing a sample to the reaction well to perform an antigen-antibody reaction; a step of bringing the magnet close to the pipette tip to capture reactants in the reaction well on the pipette tip and moving them to the wash well to wash them; and a step of bringing the magnet close to the pipette tip to move the reactants washed in the wash well to the measurement well to perform an optical analysis.

[0011] Preferably, the pipette tip is mounted on the in vitro diagnostic cartridge.

[0012] Preferably, the reactant is sucked into the pipette tip and the magnet is captured in the pipette tip by approaching the pipette tip from the outside of the pipette tip.

[0013] Preferably, the magnetic bead well is filled with a greater amount of magnetic beads than the amount required for the optical analysis.

[0014] The present invention, having the aforementioned configuration, provides a liquid-phase immunoassay method with a high effective capture yield of magnetic beads. The present invention also provides a liquid-phase immunoassay method with high analytical sensitivity and specificity.

[0015] Figure 1 illustrates the process of a sandwich immunoreaction using magnetic beads used in a device according to one embodiment of the present invention.

[0016] Figure 2 illustrates the process of a competitive immune reaction using magnetic beads used in a device according to one embodiment of the present invention.

[0017] Figure 3 is a configuration diagram of an in vitro diagnostic cartridge to which one embodiment of the present invention can be applied.

[0018] Figure 4 is a flow chart of a liquid immunoassay method according to one embodiment of the present invention.

[0019] Figure 5 illustrates a magnetic bead capture method according to one embodiment of the present invention.

[0020] FIG. 6 illustrates a case where a liquid immunoassay method according to one embodiment of the present invention is applied to the cartridge illustrated in FIG. 3.

[0021] To fully understand the present invention, preferred embodiments of the present invention will be described with reference to the accompanying drawings. The embodiments of the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the embodiments described in detail below. These embodiments are provided to more completely explain the present invention to those with average knowledge in the art. Therefore, the shapes of elements in the drawings may be exaggerated to emphasize a clearer description. It should be noted that in each drawing, the same parts are sometimes depicted with the same reference numerals. Detailed descriptions of well-known functions and configurations that may unnecessarily obscure the gist of the present invention are omitted.

[0022] Below, first, the terms used in this specification and the principles of chemical reactions used with this device are explained.

[0023] In order to determine the presence or amount of an analyte included in a sample, as described in the “detection” herein, after the reaction of a reagent and a sample, the reaction result or the reaction result is purified, and then the analyte included therein is quantitatively or qualitatively analyzed.

[0024] In this specification, “test” is used as a term encompassing detection, analysis and interpretation.

[0025] “Analyte” means the compound to be analyzed in a sample, also called a target or indicator, including but not limited to a protein component such as an antigen or a nucleic acid material such as a gene.

[0026] In this specification, “reagent” refers to a substance mixed with a sample for quantitative or qualitative analysis of an analyte contained in the sample, and varies depending on the type of specific analyte, and may include, for example, a reaction buffer or buffer, a dilution buffer, a detection buffer, a washing buffer, or a specific antibody, enzyme, or substrate that reacts with various substances in the sample, such as antigens, but is not limited thereto.

[0027] An immunoassay device according to one embodiment of the present invention is a method optimized for physical washing to separate unreacted substances from the reaction results using magnetic beads before detection of an analyte, as well as detection of a specific component or analyte contained in a biological sample or the like through an immunoassay (ELISA)-based reaction based on specific binding between antigens / antibodies, for example, a reaction such as that shown in FIGS. 1 and 2.

[0028] Figures 1 and 2 illustrate various ELISA analysis processes for analyzing analytes. A sandwich immunoassay is an immunoassay that sandwiches capture antibodies and detector antibodies, chemically binding an enzyme to the detector antibody to induce a quantitative reaction with the substrate. At this time, the capture antibody is chemically or physically bound to magnetic beads, and the detector antibody uses a conjugate that is bound to an enzyme. This sandwich reaction using magnetic beads can be broadly divided into two types, and depending on the number of washing steps, it is divided into a 1-step assay and a 2-step assay. A method in which the sample and capture antibody are reacted first, washed, and then reacted with the detector antibody is called a 2-step reaction, while a method in which the capture antibody and detector antibody are reacted simultaneously without distinction is called a 1-step reaction.

[0029] Competition assays, widely used to detect small amounts of protein molecules along with sandwich immunoreactions, are also divided into two types. Depending on whether a competing protein or antibody is conjugated to magnetic beads, they are classified as indirect or direct competitive reactions. Furthermore, they are further categorized as one-step or two-step reactions based on the specific steps of the immunoreaction. For example, Figure 2 illustrates one form of an indirect competitive reaction and one form of a direct competitive reaction.

[0030] Figure 3 is a configuration diagram of an in vitro diagnostic cartridge (100) to which one embodiment of the present invention can be applied. As illustrated, the cartridge (100) includes a sample well (104), a magnetic bead well (106), a reagent well (108, 116, 118, 120), a reaction well (110), a washing well (112, 114), and a measurement well (122).

[0031] A sample is placed and filled into the sample well (104). A diagnostic reagent required for liquid-phase immunoassay is filled into the reagent wells (108, 116, 118, 120). A magnetic bead well (106) is filled with magnetic beads bound to a reagent. It is preferable to fill the magnetic bead well (106) with a greater amount of magnetic beads than the amount required for optical analysis.

[0032] In the reaction well (110), the magnetic beads with the reagents, the luminescent material, the target component in the sample, and other diagnostic reagents react to form an antigen-antibody complex. In the wash wells (112, 114), unreacted substances in the sample are removed. The measurement well (122) is connected to a signal detection device, and qualitative or quantitative analysis of the target component is performed by measuring the amount of luminescence in the measurement well (122). The pipette tip (102) can be mounted in the pipette tip well (124) formed in the cartridge (100).

[0033] FIG. 4 is a flowchart of a liquid immunoassay method according to one embodiment of the present invention, and FIG. 5 describes a magnetic bead capture method according to one embodiment of the present invention.

[0034] First, as shown in FIG. 5, by bringing the permanent magnet (302) close to the pipette tip (102), the magnetic beads (304) filled in the magnetic bead well (106) are captured using the magnetism of the permanent magnet (302) and moved to the reaction well (110) ((S202). That is, the magnetic beads (304) filled in the magnetic bead well (106) are selectively captured by the pipette tip (102) and the permanent magnet (302) and are introduced into the reaction well (110). Through this selective capture process, only good magnetic beads (304) among the magnetic beads (304) filled in the magnetic bead well (106) are moved to the reaction well (110). Next, the sample filled in the sample well (110) is introduced into the reaction well (110), and the reagent wells (108, 116, 118, The reagent filled in (120) is injected into the reaction well (110) to perform an antigen-antibody reaction ((S204).

[0035] Next, by bringing the permanent magnet (302) close to the pipette tip (102), the reactants bound to the magnetic beads in the reaction well (110) are captured in the pipette tip (102) and moved to the washing well (112, 114) for washing ((S206). The reactants are sucked into the pipette tip (102) and the permanent magnet (302) is brought close to the outside of the pipette tip (102), so that the reactants bound to the magnetic beads are captured inside the pipette tip (102). Next, by bringing the permanent magnet (302) close to the pipette tip (102), the washed reactants in the washing well (112, 114) are moved to the measurement well (122) for optical analysis ((S208).

[0036] If some of the magnetic beads (304) are lost during the well-to-well movement process, signal loss occurs. The reason why the magnetic beads (304) are lost during the capture and transport process is that some of the magnetic beads (304) react weakly to the permanent magnet (302) and are not attached to the permanent magnet (302) and remain in the solution. The reason why the magnetic beads (304) react weakly to the permanent magnet (302) is because some of the magnetic beads (302) are far from the permanent magnet (302) and are relatively strongly attached to the surface of the cartridge (100) and do not come off, so they are not captured. In addition, it is known that during the process of storing in an aqueous solution for several months or tens of months, a slow chemical reaction occurs by reacting with water, oxygen, and the components in the solution, and magnetite, which is the main material of the magnetic beads (304), changes into non-magnetic hematite.

[0037] In addition, differences in the capture yield of magnetic beads (304) may also occur due to differences in the liquid immunoassay equipment using magnetic beads (304). That is, differences in the capture yield may occur due to differences in the strength of the permanent magnets and differences in the distance between the permanent magnets and magnetic beads depending on the equipment.

[0038] A common method for increasing the capture yield of magnetic beads (304) is to increase the strength of the permanent magnet (or electromagnet) (302) used for capturing the magnetic beads (304) or to reduce the gap between the permanent magnet (302) and the magnetic beads (304). However, even these methods have limitations in capturing magnetic beads (304) that have been chemically modified and whose response to magnetic force has been significantly reduced.

[0039] In order to compensate for differences between equipment and improve capture yield, in this embodiment, a step of selectively capturing magnetic beads (304) is introduced prior to the step of performing an antigen-antibody reaction in a reaction well (110). In the conventional method, the antigen-antibody reaction was performed by simply introducing magnetic beads into the reaction well (110) without using a permanent magnet. In this embodiment, the magnetic beads are first captured in the magnetic bead well (106) using a permanent magnet (302), and then moved to the reaction well (110) where the antigen-antibody reaction occurs. Except for the addition of the step of selectively capturing magnetic beads, the remaining steps are the same as in the conventional method.

[0040] The pipette tip (102) can be cylindrical or conical. The magnetic bead solution contained in the magnetic bead well (106) is sucked into the pipette tip (102), a permanent magnet with high magnetic force is brought near from the outside of the pipette tip (102) to attach the magnetic beads to the inner wall of the pipette tip (102), and then the remaining solution is discharged, thereby collecting the magnetic beads. This is repeated 1 to 20 times to concentrate the magnetic beads. After discharging the remaining solution, the magnetic beads are attached to the inner wall of the pipette tip (102), and then the magnet is removed by using another well, and the suction and discharge process is repeated with the solution of the corresponding well, thereby discharging the magnetic beads to the corresponding well. The magnetic beads are captured and transferred in this manner.

[0041] By using a method for selectively collecting magnetic beads, the differences between diagnostic equipment can be narrowed. Even if some of the magnetic beads have denatured due to long-term storage and their magnetism has weakened, by collecting the magnetic beads in a preliminary step and transferring them to the reaction well (110) rather than directly applying them to the antigen-antibody reaction, only those magnetic beads with sufficient magnetism can be selected and used by the equipment, thereby narrowing the loss rate during the well-to-well distribution and collection process of the magnetic beads.

[0042] During the screening capture process, chemically denatured magnetic beads are not captured, so the amount of magnetic beads captured may decrease. However, since the amount of magnetic beads used for antigen-antibody reactions is usually excessive rather than quantitative, even if some magnetic beads are lost during the screening capture process, it does not affect the analysis.

[0043] Figure 6 illustrates a case where a liquid immunoassay method according to one embodiment of the present invention is applied to the cartridge illustrated in Figure 3. In this embodiment, magnetic beads are moved a total of four times to a reaction well (110), two wash wells (112, 114), and a measurement well (122).

[0044] Table 1 shows the case where magnetic beads in the magnetic bead well (106) were introduced into the reaction well (110) without a selection collection process of the magnetic beads (Comparative Example 1). The absorbance in the reaction well (110) of the first cartridge was 0.3058, and the absorbance in the measurement well (122) was 0.2517. That is, the transfer rate of the magnetic beads in the first cartridge was 82.3%. The transfer rate of the magnetic beads in the second cartridge was 100.7%, and the transfer rate of the magnetic beads in the third cartridge was 83.8%. Since the transfer rate cannot be greater than 100%, the transfer rate in the second cartridge is due to a signal measurement error.

[0045]

[0046] Table 2 shows the case where the magnetic beads in the magnetic bead well (106) were selectively collected and introduced into the reaction well (110) according to the present embodiment (Example 1). The absorbance in the reaction well (110) was 0.248433. That is, during the selective collection process, 85.28% of the magnetic beads were transferred from the magnetic bead well (106) to the reaction well (110). In this case, the transfer rate of the magnetic beads was 97.7% in the first cartridge, 89.4% in the second cartridge, and 97.1% in the third cartridge.

[0047]

[0048] Table 3 shows the mean (Mean), standard deviation (SD), and coefficient of variation (CV) of the transfer rate in Comparative Example 1 and Example 1. From Table 3, it can be confirmed that the reproducibility in Example 1 is much higher than that in Comparative Example 1.

[0049]

[0050] The embodiments of the present invention described above are merely exemplary, and those skilled in the art will readily appreciate that various modifications and equivalent other embodiments are possible. Therefore, it will be readily understood that the present invention is not limited to the forms mentioned in the detailed description above. Accordingly, the true technical protection scope of the present invention should be defined by the technical spirit of the appended claims. Furthermore, the present invention should be understood to include all modifications, equivalents, and alternatives within the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A method for performing a liquid immunoassay using an in vitro diagnostic cartridge having a magnetic bead well, a reaction well, a washing well, and a measurement well, A step of bringing a magnet close to a pipette tip to capture magnetic beads filled in the magnetic bead well and moving them to the reaction well; A step of performing an antigen-antibody reaction by providing a sample to the above reaction well, A step of bringing the magnet close to the pipette tip to capture the reactant in the reaction well in the pipette tip and moving it to the washing well to wash it, A step of moving the washed reactant from the washing well to the measurement well by bringing the magnet close to the pipette tip to perform optical analysis. A liquid immunoassay method characterized by comprising:

2. In paragraph 1, A liquid immunoassay method, characterized in that the above pipette tip is mounted on the above in vitro diagnostic cartridge.

3. In paragraph 1, A liquid-phase immunoassay method, characterized in that the reactant is sucked into the pipette tip and the magnet is captured in the pipette tip by approaching the pipette tip from the outside of the pipette tip.

4. In paragraph 1, A liquid immunoassay method, characterized in that the magnetic bead well is filled with a greater amount of magnetic beads than the amount required for the optical analysis.

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