CN1975423A - Immuno magnetic bead and producing method, and method and test plate for detection - Google Patents

Abstract

An immunomagnetic bead and its production method, a method for detection and a test plate, the immunomagnetic bead is at least composed of magnetic carrier microspheres, and the magnetic carrier microspheres are combined with at least one immunoligand; the magnetic Carrier microspheres are composed of magnetic nanoparticles and polymer framework materials. The core is small metal particles, the outer core is polymer materials, and the outermost layer is a functional layer with various functional genes that can combine different immune ligands; immunomagnetic The production method of beads includes: pretreatment of magnetic beads; activation of magnetic beads; production of conjugated antibodies; blocking of conjugated antibodies with blocking solution; purification of immunomagnetic beads, etc. method and indirect method to detect the existence of different substances, and set up a control system on the test board: the test board is composed of coated test strips, coupling pads, sample pads, absorbent pads, covering films and test board outer cards, which have sensitivity High, accurate quantification, free from optical interference, stable magnetic signal, not easy to attenuate, simple, stable, low-cost reagents, fast detection, suitable for on-site detection, etc.

Description

Immunomagnetic beads and its production method and method for detection and test plate

technical field

The present invention relates to a marker for immunological detection, a method for making the marker, a method for detection and a test plate, in particular to an immunomagnetic bead, a method for making an immunomagnetic bead, and a method for detection and test board.

Background technique

Immunoassay is a method of analyzing and determining the corresponding antigen or antibody to be tested by using the property of antibody (Antibody) to specifically bind to corresponding antigen (Antigen) and hapten, by using specific antibody or antigen as a selective reagent. With the interpenetration of disciplines, the scope of immunology and its application continues to expand, and new immunological detection methods continue to emerge, playing an increasingly important role in the research and application of biology, medicine, agronomy and food science. role. In order to improve the sensitivity of antigen and antibody detection, the substances that are easily displayed on the known antibody or antigen label can be detected by detecting the marker to reflect whether there is an antigen-antibody reaction, so as to indirectly detect a small amount of antigen or antibody. Commonly used markers include enzymes, fluoresceins, radioactive isotopes, colloidal gold, and electron-dense substances. The specific reaction carried out by the display on the antigen or antibody label is called immunolabeling technology. Immunolabeling greatly increases assay sensitivity. At present, the immunolabeling techniques mainly include: radioimmunoassay, enzyme-linked immunoassay, chemiluminescent immunoassay, fluorescent immunoassay, and colloidal gold immunoassay, etc., each of which has advantages and disadvantages. Radioimmunity: high sensitivity, but the operator needs to be exposed to radioactive substances, which will cause environmental pollution after the measurement is completed; Fluorescence immunity: low sensitivity, expensive instruments, vulnerable to scattered light, background fluorescence from samples, and fluorescence quenching during analysis The interference of factors such as extinction; the sensitivity of chemiluminescence immunoassay is good, but the equipment is expensive, and the colloidal gold immune reaction is fast, but the sensitivity and specificity are poor; the most widely used at present is enzyme-linked immunoassay, its basic characteristics are in the middle of the above items, and the sensitivity Lower than radioimmunity, but the reaction time is long, the reaction is highly dependent on temperature, and cannot be quantified.

Contents of the invention

The object of the present invention is to overcome above-mentioned deficiency, and provide a kind of immune magnetic bead and this magnetic bead and preparation method that are used for detection with high sensitivity, short reaction time, cheap instrument and equipment, high safety;

Another object of the present invention is to provide a method for detecting the immunomagnetic beads and a special test plate.

The purpose of the present invention is achieved through the following technical scheme, which at least consists of magnetic carrier microspheres, and the magnetic carrier microspheres are combined with at least one immunoligand.

The magnetic carrier microspheres are composed of magnetic nanoparticles and a polymer framework material, the core of which is a small metal particle, the outer core is a polymer material, and the outermost layer is a functional gene that can combine with different immune ligands. layer.

The small metal particles are selected from Fe ₃ O ₄ or Fe ₂ O ₃ metal materials, and the outer polymer materials of the core are selected from polystyrene, polyvinyl chloride, polyacrylic acid, starch, dextran, gelatin, and ethyl cellulose. at least one.

A kind of preparation method of immunomagnetic beads, the steps are: (1) earlier pretreating magnetic beads with ethanesulfonic acid; (2) adding carbodiimide and N-hydroxysuccinimide of as small a volume as possible In the bead solution, shake and activate the magnetic beads on a vortex shaker; (3) Production of conjugated antibody; (4) Block the conjugated antibody with blocking solution; (5) Immunomagnetic bead purification

The pretreatment of the magnetic beads with ethanesulfonic acid (MES washing buffer) is to draw an appropriate amount of magnetic beads into an EP tube, place them in a magnetic field to separate the magnetic beads from the storage solution, and use 1-3 times the volume of ethanesulfonic acid (MES washing buffer) Washing buffer) to wash the magnetic beads at least once, and resuspend the magnetic beads in the original volume of ethanesulfonic acid (MES washing buffer);

The activation of magnetic beads is to add carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in the smallest possible volume to the magnetic bead solution, shake on a vortex shaker, and the temperature is 30-40 ℃, time 20-40 minutes, the molecular ratio of carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is 1:1-1:3, so that the number of molecules is 5 of the carboxyl groups on the surface of the magnetic beads -10 times, wash the magnetic beads with 1-3 times the volume of ethanesulfonic acid (MES Washing buffer) and the same volume of borate washing buffer.

The preparation of the conjugated antibody is to add an appropriate amount of antibody solution to the magnetic bead suspension, react at 25-37°C for 2-3hrs; the concentration of the antibody in the magnetic bead reaches >0.5mg/ml.

The blocking is to add 5-10 times the volume of antibody-magnetic beads-antibody blocking solution at 25-37° C. for 20-40 minutes.

The purification of the immunomagnetic beads is to wash the magnetic beads twice with 1 volume of borate washing buffer, and transfer the magnetic beads to a new tube.

A method for detection of immunomagnetic beads is: using immunological reaction sandwich method, competition method and indirect method to detect the existence of different substances, and setting a control system on the test plate:

(1) The antigen, antibody, or protein material related to the substance to be detected is prepared according to the above-mentioned preparation method of the immunomagnetic beads to produce corresponding immunomagnetic beads.

(2) Coat the antibody or antigen related to the substance to be detected on the test line position of the detection reagent plate, and set the corresponding antibody or antigen that can react with the immunomagnetic bead marker to coat the test line position.

(3) Add 200 microliters of the test sample into the sample hole on the test plate, and react at room temperature for 3 to 5 minutes, then put the test plate into the magnetic signal detector for detection.

This detection instrument can convert the biological response signals that have been converted into magnetic field signals, and quantitatively analyze the response properties of biological substances by measuring the changes in the voltage in the magnetic field.

A test plate used in a method based on immunomagnetic bead detection, which consists of a coated test strip, a coupling pad, a sample pad, a water-absorbing pad, a covering film and an outer card of the test plate, and the coated test strip includes a coated The test line formed by the target protein and the control line formed by the control antibody, the coupling pad is a marker strip added with immunomagnetic beads, and the sample pad is used to add the sample to be tested.

The making of described coating test paper strip is: (1) the purpose protein that will prepare to coat is loaded on the nitrocellulose membrane with the sample loading device of quantitative control, and loading is about 4 micrograms/centimeter (ug/cm) , dry at room temperature; (2) blocking treatment of nitrocellulose membrane: 1.5% bovine serum albumin-phosphate buffer (BSA-PBS) blocking 60 minutes, room temperature; (3) washing of membrane: 0.01% dodecyl sulfate Wash with sodium-phosphate buffer solution (BSA-PBS) for 15 minutes, a total of three times; (4) Preservation of the membrane: After drying at room temperature, store at 4-20 degrees with a humidity of less than 15%.

Compared with the prior art, the present invention has high sensitivity, accurate quantification, no interference from optical factors such as color, stable magnetic signal, not easy to attenuate, simple, stable and low-cost reagent, advanced method, simple and convenient instrument, fast detection, suitable for on-site detection Features.

Description of drawings

Fig. 1 is a schematic diagram of the structure of the test strip of the test board of the present invention.

Fig. 2 is a schematic diagram of the outer card structure of the test board of the present invention.

Fig. 3 is a comparison chart of test results of the present invention.

Fig. 4 is a comparison chart of another test result of the present invention.

Fig. 5 is a comparison chart of still another test result of the present invention.

Fig. 6 is a comparison chart of still another test result of the present invention.

Detailed ways

The magnetic nano-microsphere labeling technology of the present invention can be widely used in the detection of trace proteins, agricultural and veterinary drugs, hormones and other substances.

Embodiment one

Detection of Hepatitis B Virus Surface Antigen

Basic reaction principle: double antibody sandwich method:

Conjugated antibody: mouse anti-HBsAg monoclonal antibody 1

Coating antibody: mouse anti-HBsAg monoclonal antibody 2

Control coating: goat anti-mouse IgG secondary antibody

Reaction materials: absorbent pad, coupling pad, nitrocellulose membrane, sample pad, cover film

MES buffer pH 4.7 (0.1 mol/L MES, 0.9% Nacl)

10% Tween 20---MES buffer

MES wash buffer (MES buffer, pH 4.7, containing 0.05% Tween 20)

Boric acid buffer pH 8.5 (50 mmol/L)

10% Tween --- boric acid buffer

Boric acid wash buffer (boric acid buffer, pH 8.5, containing 0.05% Tween 20)

Magnetic bead storage solution (phosphate buffered saline, 0.05% thimerosal)

10% bovine serum albumin---boric acid buffer

Coupling buffer (pH7.0 phosphate buffer + 5% sucrose + 0.5% Tween 20 + 1% BSA)

Protein diluent: 25mM phosphate buffer (pH7.5)

Membrane blocking solution: 5mM phosphate buffer (pH7.5) + 1.5% BSA

Washing solution: 5mM phosphate buffer (pH7.5) + 0.01% SDS

The immunomagnetic beads are composed of magnetic carrier microspheres combined with mouse anti-HBsAg monoclonal antibody 1 immunoligand.

Preparation of immunomagnetic beads:

(1) Magnetic bead pretreatment: draw 20 microliters of magnetic beads into a small test tube, place them in a magnetic field to separate the magnetic beads from the storage solution, wash the magnetic beads once with 40 microliters of MES washing buffer, and resuspend the magnetic beads at 20 µl of MES wash buffer;

(2) Activation of magnetic beads: Add 70 micrograms of carbodiimide (EDC) and 80 micrograms of N-hydroxysuccinimide (NHS) to the magnetic bead solution, shake on a vortex shaker, and the temperature is 37°C , 30 minutes, then wash the magnetic beads with 2 times the volume of MES washing buffer and the same volume of boric acid washing buffer respectively;

(3) Production of the conjugate: Add 1 microliter of mouse anti-HBsAg monoclonal antibody 1 (5 mg/ml) to the magnetic bead suspension, react for 3 hours at 25-37°C; add 10% bovine Serum albumin blocking solution, 3 microliters, 25-37 ℃, time 30 minutes;

(4) The purification of the immunomagnetic beads is as follows: wash the magnetic beads twice with 1 volume of boric acid washing buffer, and transfer the magnetic beads to a new tube.

Test board and its preparation, as shown in Figure 1 and Figure 2, it consists of test strips containing coated test paper strips, coupling pads, sample pads, water-absorbent pads, covering film and test board outer card, coated test paper The strip includes a test line 3 formed by a coated target protein and a control line 2 formed by a control antibody, the immunomagnetic bead marking strip 4 is a coupling pad for adding immunomagnetic beads, and the sample pad 5 is used for adding the sample to be tested, 1 is an absorbent pad, and 6 is a sampling hole on the outer card of the test plate.

details as follows:

(1) Preparation of nitrocellulose membrane loaded with reactants

Load the target protein (mouse anti-HBsAg monoclonal antibody 2) to be coated on the nitrocellulose membrane with a quantitatively controlled sample loading device, with a load of about 4 micrograms/centimeter (ug/cm), as a test line; At the same time, add the control antibody and the second antibody of goat anti-mouse IgG to the lower edge of the test line, and dry at room temperature;

(2) Sealing treatment of nitrocellulose membrane: Add about 25 ml of 1.5% bovine serum albumin-phosphate buffer solution (BSA-PBS) into a petri dish to seal for 60 minutes at room temperature;

(3) Washing of the membrane: washing with 0.01% sodium dodecyl sulfate-phosphate buffer solution (SDS-PBS) for 15 minutes, three times in total;

(4) Preservation of the film: after drying at room temperature, store at 4-20° C. with a humidity of less than 15%. This finished product is a test paper strip;

(5) Preparation of magnetic bead labeling strips: load the immunomagnetic beads on the coupling pad with a quantitative loading device;

(6) The above-mentioned coated test paper strip, magnetic bead marker strip, sample pad, water-absorbing pad, cover film, test board outer card, etc. are formed into a test board;

(7) Test:

Add 200 microliters of the test sample into the sampling hole of the outer card of the test plate, and react at room temperature for 3-5 minutes, then put the test plate into the magnetic signal detector for detection.

Negative sample detection: There is no reaction at the test line, and there is an obvious peak change in the control line. Specifically as shown in Figure 3

Positive standard reference product detection: There are obvious peak changes in the test line and control line. Specifically as shown in Figure 4

Detection of samples to be tested: test with clear negative samples and positive samples of different concentrations, and the peak height of the test line has a certain correlation with the concentration of positive samples. Figure 5, Figure 6

Compared with the results of ELISA: dilute the positive control reference substances from several manufacturers in the market at different concentrations. After dilution to a certain extent, when the ELISA is negative, this method can still detect obvious peak changes. The reaction sensitivity can be at least 1-2 orders of magnitude higher than ELISA.

Embodiment two

Detection of hepatitis B virus surface antibody

Basic reaction principle: double antigen sandwich method:

Reaction raw material and basic reagent are with embodiment one

Coupling antigen: Hepatitis B virus surface antigen HBsAg

Coating antigen: Hepatitis B virus surface antigen HBsAg

Control coating: mouse anti-HBsAg monoclonal antibody

The immune magnetic beads are composed of magnetic carrier microspheres combined with hepatitis B virus surface antigen HBsAg immunoligand.

Preparation of immunomagnetic beads:

(1) Magnetic bead pretreatment: draw 20 microliters of magnetic beads into a small test tube, place them in a magnetic field to separate the magnetic beads from the storage solution, wash the magnetic beads once with 40 microliters of MES washing buffer, and resuspend the magnetic beads at 20 µl of MES wash buffer;

(2) Activation of magnetic beads: Add 70 micrograms of carbodiimide (EDC) and 80 micrograms of N-hydroxysuccinimide (NHS) to the magnetic bead solution, shake on a vortex shaker, and the temperature is 37°C , 30 minutes, then wash the magnetic beads with 2 times the volume of MES washing buffer and the same volume of boric acid washing buffer respectively;

(3) Production of the conjugate: add 1 microliter of hepatitis B virus surface antigen HBsAg (5 mg/ml) to the magnetic bead suspension, react for 3 hours at 25-37 ° C; add 10% bovine serum Albumin blocking solution, 3 microliters, 25-37°C, time 30 minutes;

(4) The purification of the immunomagnetic beads is as follows: wash the magnetic beads twice with 1 volume of boric acid washing buffer, and transfer the magnetic beads to a new tube.

The preparation of the test board is as follows:

(1) Preparation of nitrocellulose membrane loaded with reactants

Use a quantitatively controlled sample loading device to prepare the coated target protein hepatitis B virus surface antigen HBsAg on the nitrocellulose membrane, with a load of about 4 micrograms/centimeter (ug/cm), as a test line; Add control antibody mouse anti-HBsAg monoclonal antibody to the lower edge of the line, and dry at room temperature;

(2) Sealing treatment of nitrocellulose membrane: Add about 25 ml of 1.5% bovine serum albumin-phosphate buffer solution (BSA-PBS) into a petri dish to seal for 60 minutes at room temperature;

(3) Washing of the membrane: washing with 0.01% sodium dodecyl sulfate-phosphate buffer solution (SDS-PBS) for 15 minutes, three times in total;

(4) Preservation of the film: after drying at room temperature, store it at 4-20 degrees with a humidity of less than 15%. This finished product is a test paper strip;

(5) Preparation of magnetic bead labeling strips: load the immunomagnetic beads on the coupling pad with a quantitative loading device;

(6) The above-mentioned protein-coated nitrocellulose membrane, magnetic bead marker strip, sample pad, water-absorbing pad, cover film, test board outer card, etc. are used to form a test board;

(7) Test:

Add 200 microliters of the test sample into the sampling hole of the outer card of the test plate, and react at room temperature for 3-5 minutes, then put the test plate into the magnetic signal detector for detection.

Negative sample detection: There is no reaction at the test line, and there is an obvious peak change in the control line.

Positive standard reference product detection: There are obvious peak changes in the test line and control line.

Detection of samples to be tested: test with clear negative samples and positive samples of different concentrations, and the peak height of the test line has a certain correlation with the concentration of positive samples.

Compared with the results of ELISA: dilute the positive control reference substances of several manufacturers in the market at different concentrations. After dilution to a certain extent, when the ELISA shows a negative reaction, this method can still detect obvious peak changes, and the reaction sensitivity can be higher than that of ELISA. Out at least 1-2 orders of magnitude.

Embodiment three

Detection of Small Organic Molecules in the Environment and Food—Taking 2,4-D as an Example

Fundamental Response Principles: Competition Law

Reaction raw material and basic reagent are the same as embodiment one and two

Conjugated antigen: whole antigen conjugated to chloramphenicol and ovalbumin (OVA)

Coating antibody: mouse anti-chloramphenicol-bovine serum albumin monoclonal antibody

Control coating: goat anti-OVA polyclonal antibody

The immunomagnetic beads are composed of magnetic carrier microspheres combined with whole antigen immunoligands coupled with chloramphenicol and ovalbumin (OVA).

Preparation of immunomagnetic beads:

(1) Magnetic bead pretreatment: draw 20 microliters of magnetic beads into a small test tube, place them in a magnetic field to separate the magnetic beads from the storage solution, wash the magnetic beads once with 40 microliters of MES washing buffer, and resuspend the magnetic beads at 20 µl of MES wash buffer;

(2) Activation of magnetic beads: Add 70 micrograms of carbodiimide (EDC) and 80 micrograms of N-hydroxysuccinimide (NHS) to the magnetic bead solution, shake on a vortex shaker, and the temperature is 37°C , 30 minutes, then wash the magnetic beads with 2 times the volume of MES washing buffer and the same volume of boric acid washing buffer respectively;

(3) Production of the conjugate: add 1 microliter of chloramphenicol and ovalbumin (OVA)-coupled whole antigen (5 mg/ml) to the magnetic bead suspension, 25-37 ° C, reaction 3 Hours; add 10% bovine serum albumin blocking solution, 3 microliters, 25-37 ° C, time 30 minutes;

(4) The purification of the immunomagnetic beads is as follows: wash the magnetic beads twice with 1 volume of boric acid washing buffer, and transfer the magnetic beads to a new tube.

The preparation of the test board is as follows:

(1) Preparation of nitrocellulose membrane loaded with reactants

The target protein mouse anti-chloramphenicol-bovine serum albumin monoclonal antibody prepared to be coated is loaded on the nitrocellulose membrane with a quantitatively controlled sample loading device, and the load is about 4 micrograms/centimeter (ug/cm), as Test line; at the same time, add the control antigen goat anti-OVA polyclonal antibody to the lower edge of the test line, and dry at room temperature;

(2) Sealing treatment of nitrocellulose membrane: Add about 25 ml of 1.5% bovine serum albumin-phosphate buffer solution (BSA-PBS) into a petri dish to seal for 60 minutes at room temperature;

(3) Washing of the membrane: washing with 0.01% sodium dodecyl sulfate-phosphate buffer solution (SDS-PBS) for 15 minutes, three times in total;

(4) Preservation of the film: after drying at room temperature, store it at 4-20 degrees with a humidity of less than 15%. This finished product is a test paper strip;

(5) Preparation of magnetic bead labeling strips: load the immunomagnetic beads on the coupling pad with a quantitative loading device;

(6) The above-mentioned protein-coated nitrocellulose membrane, magnetic bead marker strip, sample pad, water-absorbing pad, cover film, test board outer card, etc. are used to form a test board;

(7) Test:

Add 200 microliters of the test sample into the sampling hole of the outer card of the test plate, and react at room temperature for 3-5 minutes, then put the test plate into the magnetic signal detector for detection.

Negative sample detection: There are obvious peak changes in the test line and control line.

Positive standard reference product detection: According to the concentration of the positive standard reference product, the test line has different peak changes.

Detection of samples to be tested: use clear negative samples and positive samples of different concentrations for testing, and the peak height of the test line has a certain negative correlation with the concentration of positive samples.

Basic results: According to the concentration of the reference product, the detection concentration lower than the current national standard can be detected.

Claims (8)

1, a kind of immunomagnetic beads, it is made up of the magnetic carrier microballoon at least, it is characterized in that this magnetic carrier microballoon is combined with at least a immune aglucon.

2, immunomagnetic beads according to claim 1, it is characterized in that described magnetic carrier microballoon is made up of magnetic nano particle and high-molecular bone frame material, its core is a small metal particles, core is outward a macromolecular material, outermost layer be have various can be in conjunction with the functional layer of the immune aglucon functional gene of difference.

3, immunomagnetic beads according to claim 2 is characterized in that described small metal particles selects Fe for use ₃O ₄Or Fe ₂O ₃Metal material, the outer macromolecular material of core are selected at least a in polystyrene, Polyvinylchloride, polyacrylic acid, starch, glucosan, gelatin, the ethyl cellulose for use.

4, a kind of method for making as claim 1 or 2 or 3 described immunomagnetic beadses is characterized in that: (1) earlier with ethyl sulfonic acid to the magnetic bead pre-service; (2) carbodiimides of volume that will be as far as possible little and N-hydroxy-succinamide add in the magnetic bead solution, concussion activation magnetic bead on the vortex oscillator; (3) making of coupling antibody; (4) the antithesis len antibody seals with confining liquid; (5) immunomagnetic beads purifying.

5, the method for making of immunomagnetic beads according to claim 4, it is characterized in that: described is to draw an amount of magnetic bead to the EP pipe with ethyl sulfonic acid to the magnetic bead pre-service, place magnetic field that magnetic bead is separated with stock solution, ethyl sulfonic acid with 1-3 times of volume cleans magnetic bead at least once, and resuspended magnetic bead is in the ethyl sulfonic acid of original volume; Described activation magnetic bead adds in the magnetic bead solution for carbodiimides and the N-hydroxy-succinamide with as far as possible little volume, on the vortex oscillator, shake, temperature 30-40 ℃, time 20-40 minute, the molecular proportion that makes carbodiimides and N-hydroxy-succinamide is 1: 1-1: 3, the 5-10 that makes it molecular amounts and be the magnetic bead surfaces carboxyl doubly, ethyl sulfonic acid and the equal volume boric acid with 1-3 times of volume cleans magnetic bead respectively.

Being made as of described coupling antibody is added to an amount of antibody liquid in the magnetic bead suspension, 25-37 ℃, reacts 2-3 hour; The concentration of antibody in magnetic bead reaches＞0.5mg/ml.

Described sealing is for adding the magnetic bead-antibody confining liquid of 5-10 times of antibody volume, 25-37 ℃, time 20-40 minute.

Described immunomagnetic beads purifying cleans magnetic bead twice for the boric acid with 1 times of volume, and magnetic bead is transferred in the new pipe.

6, a kind of method that is used to detect as claim 1 or 2 or 3 described immunomagnetic beadses, it is characterized in that: utilize the immunological response sandwich method, competition law and indirect method detect the existence of different material, and control systems is set on test board: the antigen that (1) will be relevant with material to be detected, antibody or relative protein-based raw material, make corresponding immunomagnetic beads according to the method for making of above-mentioned immunomagnetic beads; (2) antibody that material to be detected is correlated with or antigen coated in the p-wire position of detectable plate, and antibody or antigen coated position at detection line corresponding and that the immunomagnetic beads label can react are set; (3) test sample 200 microlitres are added sample hole on the test board, after reacting 3 ~ 5 minutes under the room temperature, test board is put into the magnetic signal detector, detect.

7, a kind of used test board of method that is used to detect based on the described immunomagnetic beads of claim 6, it is characterized in that this test board by the bag formed by test strips, coupling pad, sample pad (5), adsorptive pads (1), coverlay and test board wild card, bag is comprised by wrapping p-wire (3) that is formed by destination protein and the control line (2) that is formed by control antibodies by test strips, the coupling pad is for adding the slug (4) of immunomagnetic beads, and sample pad (5) is used to add sample to be tested.

8, the immunomagnetic beads according to claim 7 used test board of method that is used to detect, it is characterized in that described bag by test strips is: (1) with quantitative control the sample adding device destination protein that will prepare to wrap quilt be stated from the nitrocellulose filter, about 4 micrograms of carrying capacity/centimetre, dry under the room temperature; (2) sealing treatment of nitric acid cellulose membrane: 1.5% bovine serum albumin(BSA)-phosphate buffer (BSA-PBS) sealing 60 minutes, room temperature; (3) washing of film: 0.01% lauryl sodium sulfate-phosphate buffer (BSA-PBS) washing 15 minutes, totally three times, the preservation of (4) film: after the drying at room temperature, be stored in 4 ~ 20 degree, humidity is less than 15%.

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