Patent Application
20230028665
The present invention relates to a cartridge for sandwich ELISA most commonly used among enzyme linked immunosorbent assays (ELISA) and a sandwich ELISA device using the cartridge.
Enzyme linked immunosorbent assay (ELISA), which is a method of confirming an antigen-antibody reaction by conjugating an enzyme to an antibody, is an immunoassay that shows high detection ability for a specific antibody or antigen. The ELISA is the most widely used antigen-antibody assay method because the ELISA has high detection ability, is simple and inexpensive, and can perform mass analysis.
Many modifications of the ELISA have been developed and used in different situations, but have the same four basic elements: A difference between these modifications comes from the difference between these basic elements.
{circle around (1)} Coating/Capture: direct or indirect immobilization of antigens to the surface of polystyrene microplate wells.
{circle around (2)} Plate Blocking: addition of irrelevant protein or other molecule to cover all unsaturated surface-binding sites of the microplate wells.
{circle around (3)} Probing/Detection: incubation with antigen-specific antibodies that affinity-bind to the antigens.
{circle around (4)} Signal Measurement: detection of the signal generated via the direct or secondary tag on the specific antibody.
More specifically, there are several types of ELISA as follows.
1) Direct ELISA
Direct ELISA is a method of directly conjugating an enzyme to an antibody that reacts with an antigen. In order to perform the direct ELISA, a method of demanding a labeled antibody for an antibody, immobilizing an antigen on a plate, and injecting a labeled antibody is used.
2) Indirect ELISA
Indirect ELISA is a method of using an enzyme that is not conjugated to an antibody (primary antibody) bound to an antigen, but is conjugated to an antibody (secondary antibody) bound to the primary antibody. In general, the indirect ELISA is commercialized and sold as an enzyme conjugated anti-isotype antibody type, and an experimenter purchases and uses an enzyme conjugated antibody that meets the isotype of the primary antibody that he/she uses.
3) Sandwich ELISA
The sandwich ELISA is a method in which an antibody against an antigen is first bound to a plate, and the antigen is bound to the antibody and then tested in a direct or indirect method in order to better immobilize the antigen on the plate. The sandwich ELISA is used when the amount of antigen is small or when the antigen contained in an unknown sample is tested, and requires two antigen-specific antibodies.
4) Competitive ELISA
The competitive ELISA is achieved by competition between two antigens for one identical binding site of an antibody. One of the two antigens is an antigen labeled by an isotope or an enzyme, and the other is unlabeled. The competitive ELISA requires a sample that contains a labeled antigen, a non-labeled antigen (that is, antigen to be quantified) to make a standard curve, a monoclonal antibody (or antiserum) specifically bound to the antigen, and an antigen. The antigen contained in the unknown sample is quantified by the standard curve using a weak label signal detected in a reaction binding substance by the reaction of the antibody with the unlabeled antigen contained in the sample. According to the competition principle, the free binding site of the antibody becomes a limit factor in the reaction solution.
Among them, the sandwich ELISA has high sensitivity, and therefore is considerably widely used. The sandwich ELISA process will be described in detail as follows.
{circle around (1)} Coating: Coat a capture antibody on a plate. In general, in the case of an experimental kit, the capture antibody is provided in a state already coated on the plate.
{circle around (2)} Plate Blocking: Block other surfaces for specific binding to a sample antigen. In general, in the case of the experimental kit, the capture antibody is coated on the plate and then provided in the blocked state.
{circle around (3)} Detection: In the case of the direct method, enzymes are conjugated to the detection antibody, and therefore it is possible to immediately perform the detection by adding only a substrate. In the case of the indirect method, it is possible to perform detection by adding the substrate after a process of binding an enzyme conjugated secondary antibody to the detection antibody is subjected.
{circle around (4)} Signal Measurement: Measure an optical density value using an ELISA reader.
The inference can be made from the above processes. In addition, as is well known from the actual process of the ELISA, in the ELISA, the processes of filling, emptying, and washing a variety of experimental reagents (detect antibody, washing buffer, HRP, TMB, and the like) are repeated on the plate coated with the capture antibody several times. It is necessary to repeatedly perform such a complicated process, and as a result, it is true that ELISA has a very high risk of human error.
To reduce this risk, attempts have been made to automate the ELISA process. However, since the conventional ELISA automation devices include a method of injecting necessary reagents step by step, there is a problem in that the device becomes large and expensive because it requires precise positioning and injection amount control.
The present invention has been devised to solve the problems of the related art as described above, and an object of the present invention is to provide a cartridge for sandwich ELISA pre-loaded with antigen customized detection reagent to more efficiently perform the sandwich ELISA and a sandwich ELISA device using the cartridge.
In one general aspect, a cartridge 100 for sandwich ELISA may include: a body 110 that has a plurality of wells 111 arranged in rows and columns in a width direction and a length direction, having reagents accommodated therein, and formed on a top surface thereof; a pin-assy 120 that includes a plurality of pins 121 extending in one direction and having a capture antibody coated on some areas of one end thereof and a single bar 122 extending in the width direction and coupled to the other end of the pins 121 so that the pins 121 are arranged to correspond to positions of the wells 111 included in the row; and a provider 130 that extends in the width direction to support the pin-assy 120 and is movably formed in the length direction from the top surface of the body 110 to provide the row composed of the pins 121 with the row composed of the wells 111.
In addition, according to the cartridge 100 for sandwich ELISA, the bar 122 may be rotatably coupled to the provider 130, and the one end of the pin 121 may be immersed in or taken out from the well 111 by the rotation of the bar 122.
In addition, according to the cartridge 100 for sandwich ELISA, the bar 122 may be rotated by the movement of the provider 130 in the length direction, the pins 121 may protrude in an opposite direction to a traveling direction of the provider 130 to be supported on the top surface of the body 110, when the provider 130 is arranged at a position where the wells 111 do not exist on the top surface of the body 110, and the one end of the pins 121 may be immersed in the wells 111 by the rotation of the bar 122, when the provider 130 is arranged at a position where the wells 111 exist on the top surface of the body 110.
In this case, according to the cartridge 100 for sandwich ELISA, the top surface of the membrane 112 may be covered with a membrane 112 to prevent the reagents accommodated in the wells 111 from being separated, and the one end of the pins 121 may be immersed in the wells 111 while crushing the membrane 112, when the bar 122 is rotated by the movement of the provider 130 in the length direction.
In addition, the pin 121 may include a tearer 123 whose one end surface is inclined so that a tip thereof is sharply formed.
In addition, the pin 121 may include sample-stations 124 recessed in a dish form that are formed in some areas on which the capture antibody is coated.
In this case, the sample-station 124 may be formed on an opposite side where the tearer 123 is formed.
In addition, the provider 130 may be provided with a pair of holes 131 that is formed on both ends of thereof in the width direction and grooves 132 that communicate with the pair of holes 131 and extend in the width direction so that the bar 122 may be accommodated in the groove 132 and both ends of the bar 122 may be fitted into the pair of holes 131 to replaceably couple the pin-assy 120 to the provider 130.
In addition, the provider 130 may be provided with a pair of guides 133 that is formed on a surface of an opposite direction to the traveling direction of the both ends of thereof in the width direction, the pair of guides 133 regulating the rotation of the pins 121, and a width between the pair of guides 133 may be formed to be the same as that of a row of any one of the wells 111 so that the guide 133 may prevent the pins 121 from being deviated from a fixed position corresponding to the positions of the wells 111 in the width direction.
In addition, according to the cartridge 100 for sandwich ELISA, a pair of rails 113 extending in the length direction may be formed at the both ends of the body 110 in the width direction and sliders 134 may be formed at lower ends of the both ends of the provider 130 in the width direction to engage with the rails 113 so that the rails 113 and the sliders 134 engage with each other to slidably move the provider 130 in the length direction on the body 110.
In another general aspect, a sandwich ELISA device according to the present invention may include the cartridge 100 for sandwich ELISA as described above.
According to the present invention, the pins coated with the capture antibody are stepwise inserted into the wells pre-loaded with the reagents required for the sandwich ELISA. Accordingly, according to the present invention, it is possible to very easily simplify and automate the processes of adding, reacting, emptying, and washing the reagents, and adding other reagents again as in the related art. In particular, in the related art, the capture antibody was coated on the plate, and as a result, there is a high risk of human error in the process of sequentially adding several reagents to the plate and washing the reagents, but according to the present invention, required reagents are stepwise pre-loaded into the wells in accordance with the pre-designed experimental stage, and as a result, the risk of human error is basically eliminated by pre-loading the reagents. That is, according to the present invention, there is an effect of maximizing the accuracy, promptness, and efficiency of the sandwich ELISA.
In addition, according to the present invention, appropriate reagents can be pre-loaded according to the antigen to be detected. Accordingly, the customized cartridge for the detection antigen can be easily manufactured. In addition, the cartridge according to the present invention has a simple configuration and is easy to manufacture. As such, as the cartridge in which the required reagent is pre-loaded, the analytical device can also be configured simply and compactly to have only basic functions, such that the sandwich ELISA device itself can be reduced in size and cost.
Hereinafter, a cartridge for a sandwich ELISA pre-loaded with an antigen customized detection reagent according to the present invention having the above-described configuration and a sandwich ELISA device using the cartridge will be described in detail with reference to the accompanying drawings.
The body 110 has a plurality of wells 111 that are arranged in rows and columns in a width direction and a length direction, and formed on a top surface thereof. Reagents are accommodated in the wells 111, in which the reagents accommodated in the wells 111 can be any reagents except for a sample to be tested among various reagents required for a sandwich ELISA process. In addition, as shown in
As shown, the pin-assy 120 includes a plurality of pins 121 and a bar 122 that is coupled to the pins 121 to fixedly support the pins 121. The pins 121 are formed to extend in one direction, and some areas of one ends thereof are coated with a capture antibody. In this case, as shown in
The provider 130 extends in the width direction to support the pin-assy 120 and is movably formed in the length direction from the top surface of the body 110. As described above, since the plurality of pins 121 included in the pin-assy 120 are arranged to correspond to the positions of the wells 111 included in any one row, when the positions of the pins 121 overlap the positions of the wells 111 forming any one row while the provider 130 moves the pin-assy 120, the pins 121 may be immersed in the wells 111. That is, the provider 130 serves to provide a column composed of the wells 111 with a column composed of the pins 121.
In summary, the cartridge 100 for sandwich ELISA according to the present invention is arranged in rows and columns, and includes the body 110 in which the wells 111 accommodating reagents are formed, the pin-assy 120 including the pins 121 that have the capture antibody coated on one ends thereof, and the provider 130 that moves the pin-assy 120 to provide the well 111 with the pin 121 to allow the reagents and the capture antibody to be into contact and react with each other. In the related art, the ELISA was progressed by repeatedly performing processes of immobilizing the capture antibody on the plate, performing the reaction with the reagent by filling the plate with the reagent, emptying the reagent after the reaction for the next step, and washing the plate. However, in the present invention, the reagent is pre-loaded into the wells 111 on the body 110, and the capture antibody is coated on the pin 121. In this state, when the provider 130 moves the pin-assy 120 in the length direction of the body 110, the rows composed of the pins 121 may be sequentially immersed in or taken out from the rows composed of the wells 111. In other words, in the conventional process, a complex process of filling and discarding several reagents in and from the plate on which the capture antibody is immobilized is replaced with a simple process of sequentially containing or taking out the pins 121 on which the capture antibody is immobilized in or from the wells 111 pre-loaded with several reagents. There is a problem in that in the conventional complex process, the risk of human error and the like is very high, and when the process is automated as it is, a motion of a person should be replaced with a machine, and as a result, the device is very complicated. However, the present invention fundamentally prevents these problems because the process itself is much simplified, and as a result, the accuracy, promptness, and efficiency of the ELISA are maximized. In addition, the sandwich ELISA device including the cartridge 100 for sandwich ELISA according to the present invention can be made in a much simpler configuration than the conventional automation device by simply configuring the analytical device in a very compact form so as to have only basic functions, such that the sandwich ELISA device itself can be compact and inexpensive.
Hereinafter, embodiments of the pin-assy 120 and the provider 130 will be described in more detail.
As described above, in the present invention, the rows (hereinafter referred to as “pin-row”) composed of the pins 121 are provided to the row (hereinafter referred to as “well-row”) composed of the wells 111, and are sequentially provided to the well 111-rows arranged in the length direction. Of course, even if the device is configured to move each pin 121 by a separate actuator, the above operation can be realized. In this case, however, the device may be unnecessarily excessively complicated. In realizing the above-described operation, it is preferable that the pins 121 forming one row are interlocked with each other to make the same movement. In the present invention, as the simplest structure for realizing such an operation, the same structure as the pin-assy 120 as shown in
Meanwhile, the provider 130 operates to sequentially provide the pin-rows to the well-rows arranged in the length direction. In realizing the provider 130, for example, the structure and the like in which the motor is directly attached to the bar 122 and rotated may be adopted to move the pin-row. However, in the same manner as in the pin-assy 120 design, it is preferable to realize the above-described operation while having the simplest structure as much as possible in order to prevent the device from being unnecessarily excessively complicated.
As described above, it is preferred that the membrane 112 is covered on the top surface of the body 110 to prevent the reagents immersed in the wells 111 from being separated. In the case where the membrane 112 exists, when the bar 122 is rotated by the movement of the provider 130 in the length direction, one end of the pins 121 is immersed in the wells 111 while crushing the membrane 112. In this case, however, when one end of the pins 121 is bluntly formed, there is a possibility that one end of the pins 121 is supported by the membrane 112 to disturb the rotation of the bar 122. In order to avoid this problem, as shown in
In this case, when the sample-stations 124 are formed together on a surface on which the tearer 123 is formed, the positions of the sample-stations 124 are raised too much, and therefore there is a possibility that the sample-stations 124 may not be sufficiently immersed in the reagent. To avoid this problem, it is preferable that the sample-station 124 is formed on the opposite side where the tearer 123 is formed.
In view of the above operation process, it may be seen that the provider 130 may be formed to be movable only in one direction with respect to the length direction of the body 110. However, the provider 130 is preferably formed to be movable in both directions with respect to the length direction of the body 110, which will be described in detail as follows. When the provider 130 moves in the same direction as the arrow in the top view of
The structures of the pin-assy 120 and the provider 130 of the present invention designed in a simplified structure while being able to realize all of these operations will be described again in more detail with reference to
As described above, the pin-assy 120 is formed in a form in which a plurality of the pins 121 are fixedly arranged in a single bar 122 in a row. At this time, as shown in
In this way, the pin-assy (120) is free to move in the width direction in the process of allowing both ends of the bar 122 to fit into the holes 131 at both ends of the provider 130, and therefore is likely to be arranged at a position deviating from a fixed position. At this time, since the plurality of pins 121 are fixedly arranged in line with the rows of the wells 111, when the pin-assy 120 itself deviates from the fixed position, the pins 121 are not correctly immersed in the wells 111. In order to prevent such a problem, the provider 130 may have a pair of guides 133, that regulates the rotation of the pins 121, provided on the opposite side of the traveling direction of both ends in the width direction. A width between the pair of the guides 133 is formed to be the same as that of a row of any one of the wells 111. Therefore, when the pins 121 deviate from the fixed positions corresponding to the positions of the wells 111 in the width direction, the rotational movement of the pins 121 is regulated by the guide 133. Therefore, as a result, it is possible to prevent the pins 121 from deviating from the fixed positions corresponding to the positions of the wells 111 in the width direction by the guide 133.
Meanwhile, as the structure in which the provider 130 is movably coupled in the length direction with respect to the body 110, the provider 130 may employ a rail structure as shown in
The present invention is not limited to the abovementioned exemplary embodiments, but may be variously applied. In addition, the present invention may be variously modified by those skilled in the art to which the present invention pertains without departing from the gist of the present invention claimed in the claims.
The present invention uses a cartridge for sandwich ELISA to prevent human errors by sequential and stepwise contact and reaction of antibodies and reagents with high accuracy. Accordingly, according to the present invention, since the accuracy, promptness, and efficiency of the sandwich ELISA are maximized, the utilization in medical and bio fields is very high.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/004538 | 4/3/2020 | WO |
