KR20110092083A - Automatic enzyme immunoassay device and method using beads - Google Patents

Abstract

PURPOSE: An automatic ELISA apparatus using a bead is provided to detect plural pathogens using one bead and to diagnose various diseases. CONSTITUTION: An automatic ELISA apparatus using a bead comprises: a syringe pump for transferring a fluid(11) stored in a fluid storage tank(10); a multi valve(30) which selectively forms a flow path tube for moving a sample in a sample port(50) and antigen(BFP, GFP, RFP) in an antigen port(60); a plurality of reaction coils(70-75) for reaction by transferring the fluid, bead, a reagent, and the sample; a detector(80) for detecting three fluorescence protein reacted with three antigen; and a discharge port(90) for discharging the bead.

Description

Automatic enzyme-linked immunosorbent assay (ELISA) measuring device and method using the bead}

The present invention relates to an automated enzyme immunoassay apparatus and method using beads, more preferably, fine beads within a few tens ~ 200um, and the sample and the fluorescent protein are sequentially transferred to a plurality of reaction coils, respectively, to the antigen- After inducing the antibody response to enter different wavelengths, and using the emitted wavelength to measure the fluorescent protein bound to the bead surface to detect a number of diseases in an accurate and early time.

Enzyme immunoassay (ELISA), which is basically used for diagnosing diseases of humans and animals, has been used for a long time, and is currently used for diagnosing diseases in various fields based on the advantages of being inexpensive and easy to measure.

In general, an immunoassay refers to identifying one or both of an antigen and an antibody with a radioisotope or chemiluminescent materical, and confirming the presence of the antigen or antibody by an antigen antibody reaction. .

Methods using radioisotopes are sometimes referred to as radioimmunoassays, and methods using chemifluorescent materials are sometimes referred to as immunofluorescence. Recently, studies to perform such immunological assays using biochips, biosensors or microfluidic devices made by microfabrication techniques have been conducted. Among these, in order to perform a solid surface-based immunological assay using micron level beads in a microfluidic device, it is necessary to pack the beads into a predetermined space such as a channel or a chamber through which a sample solution flows.

Therefore, there is a need for a structure that can trap a large amount of beads in a microfluidic device. Recently, research is being conducted to perform various operations while moving fluids using centrifugal force in a compact disk-shaped microfluidic device. In order to carry out solid surface-based immunological assays using beads in such a compact disc type microfluidic device, an apparatus capable of effectively packing beads using centrifugal force is required.

On the other hand, when packing the beads in the microfluidic device, if the particle size of the beads is too small, as in the conventional commercially available magnetic beads, it is difficult to create a structure for confining the beads, as well as in the microfluidic device while passing the packed beads In this case, the pressure loss of the fluid is large. In particular, in the case of compact disc-type microfluidic devices, this pressure loss means that the rotation speed of the device must be very high for fluid transfer. In addition, if the particle diameter of the bead is too large, such a pressure loss can be reduced, but the gap between the packed beads also becomes large, which leads to a problem of low efficiency of the immunological response.

In addition, in the method of measuring enzyme immunity using beads, since only one antigen-antibody reaction is measured in beads transferred to one reaction coil, not only several diseases can be measured but also long time to measure. There was a problem that it takes time.

The present invention was created in order to solve the above problems, the microbeads, the sample and the antigen-antibodies are transferred to several reaction coils respectively to send the reacted beads to the detector, and then to measure the fluorescent protein by the detector The purpose of the present invention is to make it possible to diagnose more than one specific disease of an animal (eg, pig) at a time by judging the color development.

That is, the detector uses three types of filters to differently adjust the wavelength incident on the fluorescent beads and measure the emitted wavelength with a detector (CCD).

In addition, the present invention is to automate the existing enzyme immunoassay to enable faster and more economic diagnosis of the disease. The automation is a form of moving the fluid using a sequential injectioin analysis, and precisely diagnoses the disease by moving the microbeads in the fluid and inducing and detecting the antigen-antibody response on the surface of the microbeads .

Referring to the configuration of the present invention having the above object with reference to the accompanying drawings as follows.

As shown in FIG. 1, the syringe pump 20 for transferring the transfer fluid 11 stored in the transfer fluid storage tank 10 and the transfer fluid 11 correspond to the transfer operation of the syringe pump 20. ), To selectively form the flow path tube 31 to move the beads stored in the bit port 40, the sample stored in the sample port 50 and the antigen (BFP, GFP, RFP) stored in the antigen port 60 Protein A in the multi-valve 30, the plurality of reaction coils 70 to 75 for transporting and reacting the transfer fluid 11, the beads, the reagents and the samples, and the respective reaction coils 70 to 75 A detector 80 for detecting and diagnosing three fluorescent proteins reacted with three antigens to an antibody formed in response to the attached beads, and a discharge port for discharging the detected beads from the detector 80 Automatic using beads, characterized in that configured to include 90 It provides a small immunoassay device.

In addition, the present invention provides an automatic enzyme immunity measurement apparatus using beads, characterized in that the holding coil 13 for temporarily storing the beads between the transfer pipe 12 and the multi-valve 30.

In addition, the present invention provides an automatic enzyme immunity measurement apparatus using beads, characterized in that the reaction coil (70 ~ 75) and holding coil (30) is equipped with a connector is configured to discharge only the transfer fluid (11).

In addition, the present invention provides an automatic enzyme immunity measurement apparatus using beads, characterized in that configured to transport the wash water stored in the washing port 100 through the flow path tube 31 for washing the reaction coil (30).

In addition, the present invention, after reacting again with the BFP, GFP, RFP antigen to the antibody formed by reacting the beads with the protein A attached by the transfer fluid 11 and the sample into the plurality of reaction coils 30, respectively, It is characterized in that by using three kinds of filters 110 to adjust the wavelength incident to the reacted beads, respectively, and to measure the fluorescent protein bound to the bead surface with a detector (CCD) (80) using the emitted wavelength An automated enzyme immunoassay method using beads is provided.

According to the present invention, the bead having protein A attached to each of the plurality of reaction coils formed thereon is reacted with each sample (serum), and then reacted with three antigens with fluorescent protein attached to the detector. As a result of the diagnosis, a large number of pathogens can be diagnosed in one bead.

In other words, by first reacting the antibody to the beads (Agarose bead), the reaction of the three kinds of specific fluorescent proteins and the final detection has the advantage of diagnosing several diseases in one experiment.

In addition, since the beads are transferred to the reactor in which a plurality is formed and diagnosed by a detector, it is possible to accurately and quickly diagnose pathogens.

In addition, the present invention aims to shorten the time, reduce the manpower, and reduce the error of the experiment by automatically performing the washing in each experimental step.

1 is a view showing a fluid transfer process using a syringe pump and a multi-valve according to the present invention,
2 is a view showing a state in which beads react in the reaction coil of the present invention,
3 is a view showing a state of detecting the reacted beads of the present invention by a detector.

Hereinafter, with reference to the accompanying drawings will be described a preferred embodiment according to the present invention.

In the present invention, as shown in Figure 1, the storage tank 10 for storing the transfer fluid 11 to be transferred by the syringe pump 20 is formed, the syringe pump 20 to transfer the transfer fluid (11) Is configured to operate. At this time, the transfer fluid 11 uses a buffer solution.

In addition, the multi-valve 30, which can be mixed with the transfer fluid 11 conveyed by the syringe pump 20 and reacts, is connected to the transfer pipe 12 for transferring the transfer fluid 11.

The multi-valve 30 includes a bit port 40 for storing beads, a sample port 50 for storing a sample, an antigen port 60 for storing antigens of BFP, GFP, RFP, and a bead. After reacting the sample, a plurality of reaction coils (70 to 75) for reacting with the antigen attached with the fluorophore, a detector (80) for detecting and analyzing the beads reacted in the reaction coils (70 to 75) and In addition, it comprises a washing port 90 for washing the reaction coils 70 to 75 and a discharge port 100 for discharging the detected beads to the detector 80.

In this case, in the reaction coils 70 to 75, three kinds of filters 110 are used to react with the reacted beads, and the fluorescence coupled to the bead surface by the detector (CCD) 80 is adjusted by using the emitted wavelength. Configure to measure protein.

In addition, the beads use a fine bead of several tens to 200um, and in the center of the multi-valve 30 while rotating in the center of the bead, sample, BFP, GFP, RFP antigen, washing water, etc. 31) is formed.

In addition, the holding coil 13 is formed in the conveying pipe 12 and the multi-valve 30 for conveying the conveying fluid 11, and the beads are not discharged from the holding coil 13 and the reaction coil 13. A connector (not shown) is connected.

Hereinafter, an embodiment for measuring enzyme immunity using the beads of the present invention will be described based on the drawings.

As shown in FIG. 1, the transfer fluid (buffer solution) 11 stored in the transfer fluid storage tank 10 is transferred from the transfer fluid storage tank 10 by the operation of the syringe pump 20 to provide a plurality of ports 40, 50. , 60, 70, 80, 90 are conveyed to the multi-valve 30.

At this time, after the transfer fluid 11 is transferred to the multi-valve 30, the syringe pump 20 operates to suck the transfer fluid 11 and at the same time, the beads flow into the holding coil 13. At this time, the rotating body 31 formed inside the multi-valve 30 is rotated while the flow path tube 31 moves to the bead port 40 to introduce the bead into the holding coil 13.

The flow path tube 31 is moved to the reaction coil so as to transfer the specialized beads introduced into the holding coil 13 to the reaction coils 70 to 75 formed in the multi-valve 30 by the operation of the syringe pump 20. 70-75 by rotating it, the beads are conveyed together with the conveying fluid (11). At this time, the specialized beads are attached to Protein A.

After the transfer of the sample stored in the sample port 50 using the transfer fluid 11 to the reaction coil (70 ~ 75) formed in a plurality of multi-valve 30 in the same manner, the washing water port (90) The flow path tube 31 is rotated and the sample transferred into the reaction coils 70 to 75 is transported and washed with a solution of PBS-Tween. Then, the flow path tube 31 is rotated and connected to the sample port 50 to transfer the sample to the reaction coils 70 to 75. In this case, the sample 1 is transferred to the reaction coil 1 (70), the sample 2 is transferred to the reaction coil 2 (71), the sample 3 is transferred to the reaction coil 3 (73), and the sample 4 is the reaction coil 4 (74). Sample 5 is transferred to reaction coil 5 (75), and sample 6 is transferred to reaction coil (76). That is, different samples are transferred to the respective reaction coils 70 to 75 and reacted for a predetermined time. After the reaction is performed for a predetermined time, as described above, using the wash water stored in the wash water port 90 to wash the reacted beads in the reaction coil (70 ~ 75).

Then, the flow path tube 31 rotates to connect the antigen port 60 to transfer antigens of BFP, GFP, and RFP, which are fluorescent proteins combined with specific proteins of a specific disease, to respective reaction coils 70 to 75. Let reaction.

At this time, as shown in Figure 2, in the reaction coil (70 to 75), the microbead attached with Protein A reacts with the antibodies A, B, and C in the sample, and the reacted beads are again divided into three fluorescent proteins (BFP, GFP). , And react with an antigen having RFP) to form fluorescent beads.

The reacted fluorescent beads are transferred to the detector again, and as shown in FIG. 3, the respective reaction coils 70 to 75 through the detector 80 using wavelengths emitted through the filters 110 having different wavelengths. Fluorescent protein bound in response to the surface of the beads in the) to detect the disease.

That is, when the wavelength is incident to the reacted light emitting beads is adjusted to 380nm, 488nm, 560nm, the emitted wavelength is emitted to BFP 446nm, GFP 509nm, RFP 583nm, respectively. By using the differently emitted wavelengths, the diseased reaction in the beads may be measured by the detector 70.

 The detected beads may be discharged by connecting to the discharge port 100 while the flow pipe 31 rotates, and then measures the enzyme immunity using the beads while repeating the above operation.

10. Transfer fluid reservoir 11. Transfer fluid
12. Transfer pipe 13. Reaction coil
20. Syringe pump 30. Multi-valve
31. Euro pipe 40. Bead port
50. Sample port 60. Antigen port
70 to 76. Reaction coil 80. Detector
90. Washing water port 100. Draining port

Claims (5)

 A syringe pump 20 for transferring the transfer fluid 11 stored in the transfer fluid storage tank 10, and beads stored in the transfer fluid 11 and the bit port 40 to correspond to the transfer operation of the syringe pump 20. And a multi-valve 30 for selectively forming a flow path tube 31 to move the sample stored in the sample port 50 and the antigen (BFP, GFP, RFP) stored in the antigen port 60, and the transfer fluid. (11) an antibody formed in response to a plurality of reaction coils (70 to 75) for transporting and reacting beads, reagents and samples, and beads having protein A attached thereto in the respective reaction coils (70 to 75). And a detector 80 for detecting and diagnosing three fluorescent proteins reacted with the three antigens, and a discharge port 90 for discharging the detected beads from the detector 80. Automated enzyme immunoassay device using beads. The method of claim 1,
An automatic enzyme immunity measurement device using beads, characterized in that the holding coil for temporarily storing the beads between the transfer pipe and the multi-valve. The method of claim 1,
The reaction coil and the holding coil is equipped with a connector for automatic enzyme immunity measurement device using a bead, characterized in that configured to discharge only the transport fluid. The method of claim 1,
Automatic enzyme immunity measurement apparatus using beads, characterized in that configured to transport the wash water through the flow path for washing the reaction coil. After reacting the BFP, GFP and RFP antigens to the antibody formed by reacting the beads with Protein A attached by the transfer fluid and the sample inside the plurality of reaction coils, the beads were reacted with three kinds of filters. A method for measuring automatic enzyme immunity using beads, characterized in that for adjusting the incident wavelengths and measuring the fluorescent protein bound to the bead surface by a detector (CCD) using the emitted wavelength.

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