KR20020032751A - A micro well plate protein chip using nitrocellulose membrane as a support and a method for the preparation thereof - Google Patents

Abstract

PURPOSE: A microwell plate protein chip using nitrocellulose membrane as a support and a producing method thereof are provided, therefore the protein can be easily fixed on the surface and a larger number of sample can be simultaneously analyzed. CONSTITUTION: The microwell plate protein chip contains the nitrocellulose membrane support fixing at least one kind of proteins, such as antigens or antibodies, capable of specifically binding with proteins to be analyzed, to its surface and of which rear side is coated with a polystyrene, polypropylene or polyethylene solution. The method for producing the microwell plate protein chip comprises the steps of: coating the rear side of the nitrocellulose membrane with hydrophobic polymer solution; cutting the nitrocellulose membrane to be inserted into each well of the microwell plate; sticking the nitrocellulose on the bottom of each well of the microwell plate; spraying 0.05 to 0.5 microliter of at least one kind of antigens or antibodies capable of specifically binding with a desired protein on the surface of the nitrocellulose membrane; and reacting them at 35 to 40 deg.C for 1 hour.

Description

A micro well plate protein chip using nitrocellulose membrane as a support and a method for the preparation

The present invention relates to a micro well plate protein chip using nitrocellulose membrane as a support and a method for producing the same.

In recent years, research on protein chips has been rapidly made as a state-of-the-art technology for analyzing proteins in samples in fields such as pathology and biotechnology, such as disease diagnosis and drug treatment efficacy evaluation, and interest in them is increasing day by day. In general, a protein chip is immobilized in a sample by immobilizing a protein such as an antigen or an antibody that specifically binds to a target protein on the surface of the support, and then analyzing the binding result using a reaction with a sample, mainly an immune reaction. Detect specific proteins.

Currently used protein chips use glass plates or substrates made of materials such as plastic or metal plates and support these proteins by using the physical properties of these substrates and the properties of proteins such as antigens and antibodies to be immobilized. It is fixed on the surface of. Specific methods include the following.

The first method is to use hydrophobicity between the support and the protein, and to fix the protein by using the property of attaching the hydrophobic portion of the protein to the surface of a plastic or metal plate which shows hydrophobicity. This method has the disadvantage that it takes a very long time to fix the protein on the surface of the plastic or metal, and the problem is that the degree of immobilization is very irregular and shows a different degree of immune response according to the degree. In addition, since the proteins to be immobilized exhibit different immobilization states according to their characteristics, it is difficult to fix various types of proteins together.

Secondly, EDC [1-Ethyl-3- (3-dimethylaminopropyl) -carbodiimide] is used to activate a carboxyl group on the surface of a support such as plastic, and then bind and react with an amino group of a synthetic protein. Techniques for attaching proteins on the surface of a support [see; Bioorg Khim. Ivanovskaia MG, Naryshkin NA, Shabarova ZA. 21 (6): 454-60 (1995, 6). However, even when a protein chip is prepared by permanently immobilizing a protein on a plastic surface by a covalent bond by this technique, there is a disadvantage that a considerable time is required for the reaction between the carboxyl group on the plastic surface and the amino term of the protein.

As such, there are a number of difficulties in immobilizing the protein on the surface of a conventional support such as a glass plate or a plastic or metal plate in manufacturing a protein chip, and because of the inflexibility of the support material itself, the microwell plate It was difficult to install a protein chip in a container or apparatus that can be automated, such as a problem.

Accordingly, the present invention recognizes problems in the manufacturing process of the conventional protein chip using a support such as a glass plate or a plastic or metal plate and the stability of the product, and to solve this problem, the protein can be easily and simply fixed on the surface. It is an object of the present invention to provide a protein chip that is efficient, responsive, and easily utilized in various automated devices.

1A is a photograph taken from above of a 96-well microplate, and FIG. 1B is a magnified view of each well of a 96-well microplate.

FIG. 2A is a schematic view of a nitrocellulose membrane immobilized with a protein (antigen or antibody) on its surface, and FIG. 2B is a microwell plate in which the nitrocellulose membrane of FIG. 2A is attached to the bottom surface inside the well of FIG. 1A. Schematic illustration of one well in a protein chip.

Figure 3 is a diagram for Example 1, Figure 3a is a sample of five hepatitis virus HCV antigens (1: immune response control, 2: core protein, 3: NS3 protein, 4: NS4 protein, 5: NS5 protein before sample reaction) Figure 1 is a schematic view showing one well in a microwell plate protein chip in a fixed state, Figure 3b is a diagram showing the results after the sample reaction.

FIG. 4 is a diagram for Example 2, and FIG. 4A shows five kinds of antibodies (1: immune response control group, 2: Mycobacterium tuberculosis antibody, 3: chlamydia antibody, 4: malaria antibody, 5: streptococcal antibody) before sample reaction. Figure 1 schematically shows one well in a fixed microwell plate protein chip, Figure 4b shows the results after the sample reaction.

In order to achieve the above object, the present invention provides a protein chip, that is, a micro well plate protein chip, to which a nitrocellulose membrane support is attached in a micro well plate. More specifically, at least one protein such as an antigen or an antibody that specifically binds to the protein to be analyzed is immobilized on the surface thereof, and a back surface of the nitrocellulose membrane support coated with a hydrophobic polymer has a bottom surface inside each well. It relates to a micro well plate protein chip attached to and a method for producing the same.

The protein chip of the present invention uses a nitrocellulose membrane as a support, on the surface of which one or more proteins, such as antigens or antibodies, which specifically bind to the protein to be analyzed are immobilized, and the back side is a hydrophobic polymer such as polystyrene, Characterized in that it has been treated with polypropylene or polyethylene.

The micro well plate protein chip of the present invention is prepared by a method comprising the following steps:

(1) coating the back side of the nitrocellulose membrane with a hydrophobic polymer solution,

(2) cutting the nitrocellulose membrane to a size sufficient to fit into a well of a microwell plate,

(3) applying an adhesive to the bottom surface of each well of the microwell plate, and then attaching the nitrocellulose membrane,

(4) spraying 0.05 to 0.5 [mu] l each of at least one antigen or antibody that specifically binds to the protein to be analyzed, at a predetermined position on the surface of the nitrocellulose membrane attached in the microwell plate, and

(5) reacting the sprayed antigen or antibody on the surface of the nitrocellulose membrane for 1 hour at 35-40 ° C. to immobilize the antigen or antibody.

The nitrocellulose membrane used as a support in the present invention sprays a protein such as an antigen or an antibody on the surface, and then reacts for about 1 hour at 35-40 ° C. so that the proteins are adsorbed and fixed on the surface. Alternatively, compared to plastic or metal plates, proteins can be fixed relatively easily and simply, and have excellent flexibility in terms of physical properties, so that they can be easily attached to various automation devices such as micro well plates.

At this time, the protein to be immobilized on the surface of the support using a variety of proteins that specifically bind to the protein to be analyzed, for example, proteins such as antigens or antibodies, each antigen or antibody to facilitate the result analysis It is preferable to label a protein such as using a conventional labeling substance, for example, enzymes and substrates by enzyme measurement, fluorescent dyes by fluorescence measurement, radioisotopes by radiometry. Alternatively, for analysis of the results, it is also possible to use secondary antibodies specific for the analyte protein to which the above-mentioned label is attached.

In the present invention, by coating the back of the nitrocellulose membrane with a hydrophobic polymer solution, water or oil introduced from the bottom of the membrane is treated so as not to affect the properties of the membrane. This process serves to prevent denaturation of the membrane due to adhesive components, especially in the process of attaching the membrane to various devices such as microwell plates.

Examples of the hydrophobic polymer coated on the back side of the nitrocellulose membrane as described above include polystyrene, polypropylene, polyethylene, and the like.

As the micro well plate used in the present invention, all micro well plates and similar test apparatuses used in the art can be used, and it is advantageous in many ways to use 96-well micro plates.

When the sample is analyzed using the microwell plate protein chip of the present invention, it is preferable to coat each well with BSA (bovine serum albumin) or the like to remove specific protein binding reactions before the sample reaction.

Compared with the use of individual protein chips, the micro well plate protein chip of the present invention has the advantage of being able to analyze many samples at the same time and using various automated devices.

1 shows a top shot of the 96-well microplate (FIG. 1A) and an enlarged photo of each well (FIG. 1B). In addition, a schematic representation of a nitrocellulose membrane immobilized with a protein (antigen or antibody) on its surface and a schematic representation of one well of a microwell plate protein chip having the nitrocellulose membrane attached to the bottom surface inside the well of FIG. The figures are provided as FIGS. 2A and 2B, respectively.

Referring to the process of manufacturing the micro-well plate protein chip of the present invention in detail as follows.

The back side of the nitrocellulose membrane is applied 2-3 times with a hydrophobic polymer solution, dried and coated, and then cut to size enough to fit into the wells of the micro well plate. An adhesive is applied to the bottom surface of the well of the micro well plate, and then the nitrocellulose membrane prepared above is attached to the adhesive surface. Care should be taken to ensure that the surface of the membrane does not contain any foreign matter that may affect the test results, and that the adhesive components used to attach the membrane do not penetrate the membrane. 0.5 μl each of the antigens (or antibodies) with different kinds of labeling substances are sprayed at different positions on the surface of the nitrocellulose membrane attached inside each well of the microwell plate, and then sprayed at 37 ° C. for 1 hour. The microwell plate protein chip of the present invention is prepared by using a nitrocellulose membrane as a support by allowing the antigen (or the antibody) to be immobilized by adsorption. Samples are injected into each well of the prepared micro well plate protein chip, an immune reaction is performed, and the labeling material is read to analyze the protein in the sample.

Hereinafter, the case where the micro well plate protein chip of the present invention is used for a sample analysis test will be described in detail with reference to specific examples. The following examples are provided to illustrate the invention and should not be understood as limiting the invention.

Example 1 HCV Antibody Identification Test in Sample Using Method of the Present Invention

According to the method of the present invention, among the antigen proteins of HCV, core protein, NS3 protein (non-structural 3 protein), NS4 protein (non-structural 4 protein), NS5 protein (non-structural 5 protein), together with a control group, 0.5 μl each was sprayed in a row on the membrane surface of the prepared microwell plate protein chip and incubated at 37 ° C. for about 1 hour to fix each antigenic protein on the membrane surface. Patient serum samples were injected into each well and an immune response was performed at room temperature for 30 minutes to allow HCV specific antibodies present in the serum to bind antigen on the protein chip. Serum samples were removed from the microwells and washed three times with wash solution (PBS-T; 10 mM phosphate buffer pH7.4, 150 mM NaCl, 0.05% Tween20) to remove unbound antibodies. Secondary antibodies with fluorescent dyes that can specifically bind to human antibodies were injected into each well to react for specific antibody-antibody binding reactions. The microwells were washed three times with the washing solution to remove unbound antibody. The microwells were analyzed with a fluorescent ccd camera to determine antigen-antibody binding.

The results are shown in FIG. According to Figure 3b, it was found that the antibody to HCV in the test blood.

Example 2 Test for Confirming the Presence of Pathogenic Microorganisms in a Sample Using the Method of the Present Invention

According to the process of the present invention, the Mycobacterium tuberculosis (M.TB), chlamydia (Chlamydia), malaria (Malaria), Streptococcus with a specific antibody for (Streptococus) and the control group, respectively on the membrane surface of a microwell plate protein chip prepared 0.5 μl of each line was sprayed and incubated at 37 ° C. for about 1 hour to immobilize each antibody protein on the membrane surface. The patient's sample was injected into each well and an immune response was performed at room temperature for 30 minutes to allow pathogens present in the sample to bind to antibodies on the protein chip. Serum samples were removed from the microwells and washed three times with wash (PBS-T; 10 mM phosphate buffer pH7.4, 150 mM NaCl, 0.05% Tween20). Secondary antibodies with fluorescent dyes that can specifically bind to each pathogen were injected into each well and reacted to achieve a specific antigen-antibody binding reaction. The microwells were washed three times with the washing solution to remove unbound antibody. The microwells were analyzed with a fluorescent ccd camera to determine antigen-antibody binding.

The results are shown in FIG. According to FIG. 4B, it was found that Mycobacterium tuberculosis was present in the test sample.

Since the microwell plate protein chip of the present invention uses a nitrocellulose membrane as a support, the protein can be immobilized on the surface relatively easily and simply compared to a glass plate or a plastic or metal plate that has been used in the past, and individual protein chips are used. Compared to the analysis, many samples can be analyzed at the same time, and it is advantageous to use various automated devices. It provides excellent efficiency and more accurate analysis results when testing.

Claims (4)

A microwell plate protein chip in which a nitrocellulose membrane support on which at least one protein that specifically binds a desired protein is immobilized on a surface is attached to each well of a microwell plate. The method of claim 1, The back of the nitrocellulose membrane is a protein chip, characterized in that the coating with a polystyrene, polypropylene or polyethylene solution. The method of claim 2, Protein chip, characterized in that the protein is an antigen or antibody specific for the protein of interest. (1) coating the back side of the nitrocellulose membrane with a hydrophobic polymer solution, (2) cutting the nitrocellulose membrane to a size sufficient to fit into a well of a microwell plate, (3) applying an adhesive to the bottom surface of each well of the microwell plate, and then attaching the nitrocellulose membrane, (4) spraying 0.05 to 0.5 [mu] l each of at least one antigen or antibody that specifically binds to the protein to be analyzed, at a predetermined position on the surface of the nitrocellulose membrane attached in the microwell plate; (5) A method of making a microwell plate protein chip comprising the step of reacting the sprayed antigen or antibody on the surface of the nitrocellulose membrane for 1 hour at 35-40 ° C. to immobilize the antigen or antibodies.