CN112763705A - Method for quantitatively detecting antigen - Google Patents

Method for quantitatively detecting antigen Download PDF

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Publication number
CN112763705A
CN112763705A CN202011460685.XA CN202011460685A CN112763705A CN 112763705 A CN112763705 A CN 112763705A CN 202011460685 A CN202011460685 A CN 202011460685A CN 112763705 A CN112763705 A CN 112763705A
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antibody
antigen
test strip
pad
solution
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王振新
徐凤琴
马立娜
朱群艳
张婳
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Changchun Institute of Applied Chemistry of CAS
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Changchun Institute of Applied Chemistry of CAS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7155—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/976—Trypsin; Chymotrypsin

Abstract

The invention relates to the technical field of biology, in particular to a method for quantitatively detecting an antigen. The method combines gold nanorod immunochromatographic test strip with photothermal detection to carry out quantitative detection on the antigen, and has the characteristics of simplicity, rapidness, less sample consumption, short detection time, universality and low cost. The lowest concentration of PSA and CEA detected by the method is 0.1ng/mL, and the standard curve range is 0.5-1000 ng/mL; the lowest concentration of sIL-2R detected by this method was 0.2ng/mL, with a standard curve ranging from 1 to 6250 ng/mL.

Description

Method for quantitatively detecting antigen
Technical Field
The invention relates to the technical field of biology, in particular to a method for quantitatively detecting an antigen.
Background
The colloidal gold immunochromatographic assay is a solid-phase membrane immunoassay method combining a nanogold immunoassay technology and a chromatographic chromatography technology, and can realize the purpose of rapid and accurate color development to detect an object to be detected. The colloidal gold test paper developed by the technology is widely applied to clinical diagnosis and drug detection, and the types of the test paper comprise: early pregnancy test paper, infectious disease test paper, drug residue test paper and the like. However, these tests are mainly colorimetric and only stay at a qualitative or semi-quantitative level. However, the test strip capable of realizing quantitative detection reported in the prior art has a problem that the linear range of detection is not wide enough. Therefore, it is of great practical significance to develop a method for quantitatively detecting an antigen that can broaden the linear range.
Disclosure of Invention
In view of the above, the present invention provides a method for quantitatively detecting an antigen. The method for quantitatively detecting the antigen has the characteristics of high sensitivity, linear range, short detection time, simple operation and low cost.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for quantitatively detecting an antigen, which comprises the steps of dripping a sample containing the antigen to be detected on a sample pad of an immunochromatography test strip, acting for 10-30 min, irradiating for 30-60 s by using 645-655 nm light, collecting a temperature signal by using a thermal imaging camera, and obtaining the content of the antigen in a sample to be detected according to a standard curve;
the immunochromatographic test strip consists of a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are arranged on the bottom plate and sequentially connected along the chromatography direction;
the NC membrane is sequentially marked with a detection line and a quality control line along the chromatography direction, and the detection line is coated with an antibody I; the combination pad is coated with an antibody II modified gold nanorod conjugate;
the antibodies I and II can be specifically combined with an analyte, and the antibodies I and II can identify different antigen epitopes on the analyte.
In some embodiments, the antigen to be tested comprises a protein, a small molecule compound, a polypeptide, a carbohydrate, a drug, or a nucleic acid.
In some embodiments, the antigen to be tested comprises that the antigen to be tested is a protein that is PSA, CEA, or sIL-2R.
In some embodiments, the wavelength of the irradiated light is 650nm and the irradiation time is 45 s.
In some embodiments, the quality control line of the immunochromatographic test strip is coated with a goat anti-mouse secondary antibody IgG.
In the method for quantitatively detecting the antigen, the preparation method of the immunochromatographic test strip comprises the following steps:
step 1: mixing a goat anti-mouse secondary antibody IgG and an antibody I with an antibody diluent respectively, and then sequentially coating the mixture on a nitrocellulose membrane along the chromatography direction to be used as a detection line and a quality control line respectively;
step 2: preparing an antibody II modified gold nanorod conjugate, and coating the antibody II modified gold nanorod conjugate on a bonding pad;
and step 3: and sticking the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad on the bottom plate to obtain the immune colloidal gold test strip.
In some embodiments, in step 1, the antibody dilution is 0.01mol/L phosphate buffer containing 3% sucrose.
In some embodiments, in step 1, the coating concentration of the goat anti-mouse secondary IgG and antibody I is 0.5-2 mg/mL, preferably 2 mg/mL.
In some embodiments, the antibody ii modified gold nanorod conjugate in step 2 is prepared by the following method:
and synthesizing the gold nanorods by adopting a seed growth method, and then carrying out covalent bonding on the polyacrylic acid sodium salt and the antibody II to obtain the antibody II modified gold nanorod conjugate.
In some embodiments, the antibody ii modified gold nanorod conjugate in step 2 is prepared by the following method:
mixing and stirring 1mL of the gold nanorod solution and 100 mu L of 1% sodium polyacrylate for 2h, centrifuging, dispersing in 1mL of 10mmol/LMES (pH is 7.0), adding 10 mu L of 100mmol/L EDC and 10 mu L of 100mmol/L NHS, stirring for a certain time, adding 10 mu g of PSA/CEA/sIL-2R monoclonal antibody II, and continuing to react for a preset time. And finally, adding 10 mu L of 10mg/mL BSA for blocking for 30min, centrifuging and dispersing in the diluent again to obtain the antibody II modified gold nanorod conjugate. The dilution was composed of pH 8.0, 10% BSA, 20% sucrose and 5% trehalose in 20 mmol/LTris-HCl. The dilution was composed of 20mmol/LTris-HCl at pH 8.0, 10% BSA, 20% sucrose and 5% trehalose.
In some embodiments, the antibody II modified gold nanorod conjugate is coated in an amount of 1-7 μ L. In some embodiments, the coating amount of the antibody II modified gold nanorod conjugate is 5 μ L.
In some embodiments, the synthesis of the gold nanorod solution by using a seed growth method comprises the following steps:
frozen 0.6mL NaBH4(0.01mol/L) solution was added to 5mL of HAuCl4The seed solution was obtained by mixing (0.5mmol/L) and 5mL of CTAB (0.2mol/L) with vigorous stirring and then allowing to stand for 2 hours. 50mL of HAuCl4(1mmol/L) solution, 50mL CTAB (0.2mol/L) solution, and 3mL of LGNO3(0.004mol/L) of the solution was mixed at room temperature, and 0.7mL of 0.1mol/L ascorbic acid was added thereto. And finally, adding 0.24mL of seed solution into the solution, standing the solution at 30 ℃ for 24 hours, centrifuging and washing the solution for 3 times, and dispersing the precipitate into deionized water to obtain the gold nanorod solution.
The invention relates to a method for quantitatively detecting an antigen, which has the detection principle that: the gold nanorods are used for marking an antibody II, a goat anti-mouse secondary antibody IgG and an antibody I are respectively coated on a nitrocellulose membrane, the antibody I and the antibody II identify different antigen epitopes on an object to be detected, the antibody I, the antigen and the antibody II form a double-antibody sandwich compound, and the test strip is prepared based on a double-antibody sandwich principle. When the sample solution is added to the sample pad of the test strip, the sample flows from the sample pad to the absorbent paper by capillary action. When the sample and the gold-labeled-antibody complex reach the region where the antibody is immobilized, an antigen-antibody specific binding reaction occurs, and when the reaction proceeds to a certain extent, a detection line visible to the naked eye appears (see fig. 1). Under the action of illumination, the gold nanorods show unique surface plasmon resonance characteristics, and can generate strong absorption effect on incident light through corresponding resonance effect. The absorbed energy can be dissipated through a series of micro relaxation processes and finally converted into heat, so that the effect of rapid temperature rise is achieved. Therefore, in this case, the detection line is irradiated with a 650nm laser for 45 seconds, and then a temperature signal is recorded by a thermal imager, so that photothermal detection of the antigen can be realized.
The gold nanorod immunochromatographic test strip is combined with photo-thermal detection for quantitative detection of the antigen for the first time, and the gold nanorod immunochromatographic test strip has the characteristics of simplicity, rapidness, low sample consumption, short detection time, universality and low cost. The lowest concentration of PSA and CEA detected by the method is 0.1ng/mL, and the standard curve range is 0.5-1000 ng/mL; the lowest concentration of sIL-2R detected by this method was 0.2ng/mL, with a standard curve ranging from 1 to 6250 ng/mL.
Drawings
FIG. 1 is a schematic diagram of photothermal detection using a colloidal gold immunochromatographic test strip according to the present invention;
FIG. 2 is a graph showing the photothermal signal of a PSA strip obtained by the method of the invention as a function of PSA concentration;
FIG. 3 shows a graph of the detection of PSA obtained with the method of the invention;
FIG. 4 is a graph showing the photothermal signal of the CEA test strip obtained by the method of the present invention as a function of the CEA concentration;
FIG. 5 shows a graph of the detection of CEA obtained by the method of the present invention;
FIG. 6 is a graph showing photothermal signals of sIL-2R test strips obtained by the method of the present invention as a function of sIL-2R concentration;
FIG. 7 shows a graph of the detection of sIL-2R obtained by the method of the present invention.
Detailed Description
The invention provides a method for quantitatively detecting an antigen. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1 method for quantitatively detecting CEA of the present invention
The goat anti-mouse anti-IgG and PSA monoclonal antibody I are respectively mixed with an antibody diluent, the concentration of each antibody diluent is 1mg/mL, wherein the antibody diluent is composed of 0.01mol/L phosphate buffer solution containing 3% sucrose. Sticking the nitrocellulose membrane on the corresponding area of the PVC base plate, and using an HM303 three-dimensional membrane scratching instrument to make the nitrocellulose membrane have a length of 1 muL cm-1The marking amount is respectively sprayed with goat anti-mouse anti-IgG and PSA monoclonal antibody I as a quality control line (C) and a detection line (T), and the mixture is dried at 37 ℃ overnight. A water absorbent pad was attached to one end of the nitrocellulose membrane, and a conjugate pad and a sample pad were attached to the other end, wherein the sample pad was completely soaked with a treatment solution (20mmol/LTris-HCl, pH 8.0, containing 0.1% Tween, 0.5% PVP) in advance, and then dried under vacuum at 37 ℃. And cutting the assembled PVC base plate into a preset width by using a ZQ2002 automatic cutting machine to obtain the primary test strip with the quality control line and the detection line.
PSA monoclonal antibody ii-modified gold nanorod conjugates were diluted with a diluent (20mmol/LTris-HCl, pH 8.0, 10% BSA, 20% sucrose, and 5% trehalose), and then applied to the conjugate pad of the primary test strip. The gold nanorods are synthesized by adopting a seed growth method, and then the polyacrylic acid sodium salt and the PSA monoclonal antibody II are covalently combined to form a gold nanorod conjugate, wherein the dosage of the gold nanorod solution is 1mL, and the dosage of the PSA monoclonal antibody II is 10 mu g, and the method specifically comprises the following steps:
frozen 0.6mL NaBH4(0.01mol/L) solution was added to 5mL of HAuCl4The seed solution was obtained by mixing (0.5mmol/L) and 5mL of CTAB (0.2mol/L) with vigorous stirring and then allowing to stand for 2 hours. 50mL of HAuCl4(1mmol/L) solution, 50mL CTAB (0.2mol/L) solution, and 3mL of LGNO3(0.004mol/L) of the solution was mixed at room temperature, and 0.7mL of 0.1mol/L ascorbic acid was added thereto. And finally, adding 0.24mL of seed solution into the solution, standing the solution at 30 ℃ for 24 hours, centrifuging and washing the solution for 3 times, and dispersing the precipitate in deionized water to obtain the gold nanorod solution.
Mixing 1mL of the gold nanorod solution with 100. mu.L of 1% sodium polyacrylate, stirring for 2h, centrifuging, dispersing in 1mL of 10mmol/LMES (pH 7.0), adding 10. mu.L of 100mmol/L EDC and 10. mu.L of 100mmol/L NHS, stirring for a certain period, adding 10. mu.g of PSA monoclonal antibody II, and reacting for a predetermined period. And finally, adding 10 mu L of 10mg/mL BSA for blocking for 30min, centrifuging and then dispersing in a diluent again to obtain the PSA monoclonal antibody II modified gold nanorod conjugate.
And adding the sample solution of the PSA antigen to a sample pad of the colloidal gold immunochromatographic test strip for detecting the PSA antigen, and reacting for 10-20min to obtain the test strip for completing the PSA detection. The PSA sample solution is obtained by mixing a PSA reaction solution and PSA, wherein the PSA reaction solution consists of 0.01mol/L phosphoric acid buffer solution (pH is 7.4), and the PSA concentration is respectively 0.1ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 1000 ng/mL;
and irradiating the test strip for 45s by using a 650nm laser, and collecting temperature signals by using a thermal imager to obtain a photo-thermal signal for detecting the PSA antigen colloidal gold immunochromatographic test strip.
The specific process of the method is shown in figure 1, and figure 1 is a schematic process diagram of the method for carrying out photothermal detection by using the colloidal gold immunochromatographic test strip, wherein a is PSA antigen, b is a gold nanorod conjugate modified by PSA monoclonal antibody II, c is PSA monoclonal antibody I, and d is goat anti-mouse IgG; after the sample solution is added to the sample pad of the test strip, the liquid gradually flows through the combination pad and the nitrocellulose membrane under the capillary action. Due to the antigen-antibody interaction, the antibody binds to the biomolecules immobilized on the nitrocellulose membrane and finally reaches the absorbent pad. The nano gold rod can show unique surface plasma resonance characteristics under the stimulation of specific laser wavelength, and light energy is converted into heat energy through a series of micro relaxation processes, so that the temperature can be rapidly increased in a short time after the detection line is irradiated by a 650nm laser, and the purpose of photo-thermal detection by the gold nano rod is realized.
The results of the tests performed according to the above experimental procedure are shown in fig. 2 and 3.
FIG. 2 is a graph showing the photothermal signal of a PSA strip obtained by the method of the present invention as a function of the concentration of PSA; FIG. 3 is a graph of the detection of PSA obtained using the method of the present invention. They respectively show the photothermal signal image of the T/C line on the PSA test strip as the PSA concentration changes and the corresponding data extraction graph, wherein the abscissa is the PSA concentration and the ordinate is TT/TC(ratio of T line temperature to C line temperature), the concentrations of the goat anti-mouse secondary IgG and the PSA monoclonal antibody I in the experiment are both 1mg/mL, and the dosage of the gold nanorod conjugate modified by the PSA monoclonal antibody II is 5 muL. PSA concentrations were 0.1ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 1000ng/mL, respectively, and the detection range for PSA obtained using this method was 0.5-1000 ng/mL.
Example 2 method for quantitatively detecting CEA of the present invention
The goat anti-mouse anti-IgG and CEA monoclonal antibody I is respectively mixed with an antibody diluent, the concentration of the antibody diluent is 1mg/mL, wherein the composition of the antibody diluent is 0.01mol/L phosphate buffer solution containing 3% sucrose. Sticking the nitrocellulose membrane on the corresponding area of the PVC base plate, and using an HM303 three-dimensional membrane scratching instrument to make the nitrocellulose membrane have a length of 1 muL cm-1The marking amount is respectively sprayed with goat anti-mouse secondary antibody IgG and CEA monoclonal antibody I as a quality control line (C) and a detection line (T), and the mixture is dried at 37 ℃ for overnight. A water absorbent pad was attached to one end of the nitrocellulose membrane, and a conjugate pad and a sample pad were attached to the other end, wherein the sample pad was completely soaked with a treatment solution (20mmol/LTris-HCl, pH 8.0, containing 0.1% Tween, 0.5% PVP) in advance, and then dried under vacuum at 37 ℃. Cutting the assembled PVC base plate into blocks by using a ZQ2002 automatic cutting machineAnd (5) determining the width to obtain the primary test strip with the quality control line and the detection line.
The CEA monoclonal antibody ii-modified gold nanorod conjugate was diluted with a diluent (20mmol/LTris-HCl, pH 8.0, containing 10% BSA, 20% sucrose and 5% trehalose), and then applied to the binding pad of the primary test strip. The gold nanorods are synthesized by adopting a seed growth method, and then the polyacrylic acid sodium salt and the CEA monoclonal antibody II are covalently combined to form a gold nanorod conjugate, wherein the dosage of the gold nanorod solution is 1mL, and the dosage of the CEA monoclonal antibody II is 10 mu g, and the method specifically comprises the following steps:
frozen 0.6mL NaBH4(0.01mol/L) solution was added to 5mL of HAuCl4The seed solution was obtained by mixing (0.5mmol/L) and 5mL of CTAB (0.2mol/L) with vigorous stirring and then allowing to stand for 2 hours. 50mL of HAuCl4(1mmol/L) solution, 50mL CTAB (0.2mol/L) solution, and 3mL of LGNO3(0.004mol/L) of the solution was mixed at room temperature, and 0.7mL of 0.1mol/L ascorbic acid was added thereto. And finally, adding 0.24mL of seed solution into the solution, standing the solution at 30 ℃ for 24 hours, centrifuging and washing the solution for 3 times, and dispersing the precipitate in deionized water to obtain the gold nanorod solution.
Mixing 1mL of the gold nanorod solution with 100 μ L of 1% sodium polyacrylate, stirring for 2h, centrifuging, dispersing in 1mL of 10mmol/LMES (pH 7.0), adding 10 μ L of 100mmol/L EDC and 10 μ L of 100mmol/L NHS, stirring for a certain period, adding 10 μ g of CEA monoclonal antibody II, and reacting for a predetermined period. And finally, adding 10 mu L of 10mg/mL BSA for blocking for 30min, centrifuging and then dispersing in the diluent again to obtain the CEA monoclonal antibody II modified gold nanorod conjugate.
And adding the sample solution of the CEA antigen to a sample pad of the colloidal gold immunochromatographic test strip for detecting the CEA antigen, and reacting for 10-20min to obtain the test strip for completing the CEA detection. The CEA sample solution is obtained by mixing a CEA reaction solution and CEA, wherein the CEA reaction solution consists of a 0.01mol/L phosphoric acid buffer solution (pH is 7.4), and the CEA concentrations are respectively 0.1ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 1000 ng/mL;
and irradiating the test strip for 45s by using a 650nm laser, and collecting a temperature signal by using a thermal imaging instrument to obtain a photo-thermal signal for detecting the CEA antigen colloidal gold immunochromatographic test strip.
The specific process is shown in figure 1, and figure 1 is a schematic diagram of the process of the method for performing photothermal detection by using the colloidal gold immunochromatographic test strip, wherein a is a CEA antigen, b is a gold nanorod conjugate modified by a CEA monoclonal antibody II, c is a CEA monoclonal antibody I, and d is goat anti-mouse IgG; after the sample solution is added to the sample pad of the test strip, the liquid gradually flows through the combination pad and the nitrocellulose membrane under the capillary action. Due to the antigen-antibody interaction, the antibody binds to the biomolecules immobilized on the nitrocellulose membrane and finally reaches the absorbent pad. The nano gold rod can show unique surface plasma resonance characteristics under the stimulation of specific laser wavelength, and light energy is converted into heat energy through a series of micro relaxation processes, so that the temperature can be rapidly increased in a short time after the detection line is irradiated by a 650nm laser, and the purpose of photo-thermal detection by the gold nano rod is realized.
The results obtained according to the above experimental procedure are shown in fig. 4 and 5. FIG. 4 is a graph of photothermal signals of CEA test strips obtained by the method of the present invention as a function of CEA concentration; FIG. 5 is a graph of the detection of CEA obtained by the method of the present invention. They respectively represent photothermal signal images of T/C lines on the CEA test strip, which change with the CEA concentration, and corresponding data extraction graphs, wherein the abscissa is the CEA concentration, and the ordinate is TT/TC(the ratio of the T-line temperature to the C-line temperature), the concentrations of the goat anti-mouse secondary IgG and the CEA monoclonal antibody I in the experiment are both 1mg/mL, and the dosage of the gold nanorod conjugate modified by the CEA monoclonal antibody II is 5 mu L. The CEA concentrations are respectively 0.1ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 1000ng/mL, and the detection range of the CEA obtained by the method is 0.5-1000 ng/mL.
EXAMPLE 3 method for quantitatively detecting sIL-2R of the present invention
The goat anti-mouse anti-IgG and the sIL-2R monoclonal antibody I were mixed with the antibody diluent at concentrations of 1mg/mL and 0.8mg/mL, respectively. Wherein the antibody is dilutedThe composition of the release solution is 0.01mol/L phosphate buffer solution containing 3% of sucrose. Sticking the nitrocellulose membrane on the corresponding area of the PVC base plate, and using an HM303 three-dimensional membrane scratching instrument to make the nitrocellulose membrane have a length of 1 muL cm-1The marking amount is respectively sprayed with goat anti-mouse secondary IgG and sIL-2R monoclonal antibody I as a quality control line (C) and a detection line (T), and the mixture is dried at 37 ℃ for overnight. A water absorbent pad was attached to one end of the nitrocellulose membrane, and a conjugate pad and a sample pad were attached to the other end, wherein the sample pad was completely soaked with a treatment solution (20mmol/LTris-HCl, pH 8.0, containing 0.1% Tween, 0.5% PVP) in advance, and then dried under vacuum at 37 ℃. And cutting the assembled PVC base plate into a preset width by using a ZQ2002 automatic cutting machine to obtain the primary test strip with the quality control line and the detection line.
The sIL-2R mab ii modified gold nanorod conjugates were diluted with diluent (20mmol/l tris-HCl, pH 8.0, 10% BSA, 20% sucrose and 5% trehalose) and applied to the conjugate pad of the primary test strip. The gold nanorods are synthesized by adopting a seed growth method, and then a gold nanorod conjugate is formed by covalent bonding of polyacrylic acid sodium salt and an sIL-2R monoclonal antibody II, wherein the dosage of a gold nanorod solution is 1mL, and the dosage of the sIL-2R monoclonal antibody II is 10 mu g, and the method specifically comprises the following steps:
frozen 0.6mL NaBH4(0.01mol/L) solution was added to 5mL of HAuCl4The seed solution was obtained by mixing (0.5mmol/L) and 5mL of CTAB (0.2mol/L) with vigorous stirring and then allowing to stand for 2 hours. 50mL of HAuCl4(1mmol/L) solution, 50mL CTAB (0.2mol/L) solution, and 3mL of LGNO3(0.004mol/L) of the solution was mixed at room temperature, and 0.7mL of 0.1mol/L ascorbic acid was added thereto. And finally, adding 0.24mL of seed solution into the solution, standing the solution at 30 ℃ for 24 hours, centrifuging and washing the solution for 3 times, and dispersing the precipitate in deionized water to obtain the gold nanorod solution.
Mixing 1mL of the gold nanorod solution with 100. mu.L of 1% sodium polyacrylate, stirring for 2h, centrifuging, dispersing in 1mL of 10mmol/LMES (pH 7.0), adding 10. mu.L of 100mmol/L EDC and 10. mu.L of 100mmol/L NHS, stirring for a certain period, adding 10. mu.g of sIL-2R monoclonal antibody II, and reacting for a predetermined period. And finally, adding 10 mu L of 10mg/mL BSA for blocking for 30min, centrifuging and then dispersing in a diluent again to obtain the sIL-2R monoclonal antibody II modified gold nanorod conjugate.
And adding the sample solution of the sIL-2R antigen to a sample pad of the colloidal gold immunochromatographic test strip for detecting the sIL-2R antigen, and reacting for 10-20min to obtain the test strip for completing the detection of the sIL-2R. The sIL-2R sample solution is obtained by mixing an sIL-2R reaction solution and an sIL-2R, wherein the composition of the sIL-2R reaction solution is 0.01mol/L phosphate buffer solution (pH is 7.4), and the concentrations of the sIL-2R are respectively 0.2ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL and 6250 ng/mL;
and irradiating the test strip for 45s by using a 650nm laser, and collecting temperature signals by using a thermal imaging instrument to obtain a photo-thermal signal for detecting the sIL-2R antigen colloidal gold immunochromatographic test strip.
The specific process is shown in figure 1, and figure 1 is a schematic process diagram of the method for carrying out photothermal detection by using the colloidal gold immunochromatographic test strip, wherein a is sIL-2R antigen, b is a gold nanorod conjugate modified by an sIL-2R monoclonal antibody II, c is an sIL-2R monoclonal antibody I, and d is goat anti-mouse IgG; after the sample solution is added to the sample pad of the test strip, the liquid gradually flows through the combination pad and the nitrocellulose membrane under the capillary action. Due to the antigen-antibody interaction, the antibody binds to the biomolecules immobilized on the nitrocellulose membrane and finally reaches the absorbent pad. The nano gold rod can show unique surface plasma resonance characteristics under the stimulation of specific laser wavelength, and light energy is converted into heat energy through a series of micro relaxation processes, so that the temperature can be rapidly increased in a short time after the detection line is irradiated by a 650nm laser, and the purpose of photo-thermal detection by the gold nano rod is realized.
The results obtained according to the above experimental procedure are shown in fig. 6 and 7. FIG. 6 is a graph of photothermal signals of sIL-2R test strips obtained by the method of the invention as a function of sIL-2R concentration; FIG. 7 is a graph of CEA detection obtained by the method of the present invention. They respectively show the photothermal signal images of T/C lines on the sIL-2R test strip along with the change of the concentration of sIL-2R and corresponding data extractionGraph with the abscissa of the concentration of sIL-2R and the ordinate of TT/TC(ratio of T line temperature to C line temperature), the concentrations of the goat anti-mouse secondary IgG and the sIL-2R monoclonal antibody I in the experiment are both 1mg/mL, and the dosage of the gold nanorod conjugate modified by the sIL-2R monoclonal antibody II is 3 mu L. The concentrations of sIL-2R were 0.2ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL and 6250ng/mL, respectively, and the detection range of sIL-2R obtained by this method was 1-6250 ng/mL.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (10)

1. A method for quantitatively detecting an antigen is characterized in that a sample containing the antigen to be detected is dripped on a sample pad of an immunochromatography test strip and acts for 10-30 min, light of 645-655 nm is used for irradiating for 30-60 s, a thermal imaging instrument is used for collecting temperature signals, and the content of the antigen in a sample to be detected is obtained according to a standard curve;
the immunochromatographic test strip consists of a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are arranged on the bottom plate and sequentially connected along the chromatography direction;
the NC membrane is sequentially marked with a detection line and a quality control line along the chromatography direction, and the detection line is coated with an antibody I; the combination pad is coated with an antibody II modified gold nanorod conjugate;
the antibodies I and II can be specifically combined with an analyte, and the antibodies I and II can identify different antigen epitopes on the analyte.
2. The method of claim 1, wherein the test antigen comprises a protein, a small molecule compound, a polypeptide, a carbohydrate, a drug, or a nucleic acid.
3. The method of claim 1, wherein the antigen to be tested comprises the antigen to be tested being a protein, and wherein the protein is PSA, CEA, or sIL-2R.
4. The method of claim 1, wherein the irradiated light has a wavelength of 650nm and an irradiation time of 45 s.
5. The method of claim 1, wherein the quality control line of the immunochromatographic test strip is coated with a goat anti-mouse secondary IgG.
6. The method of any one of claims 1 to 5, wherein the preparation method of the immunochromatographic test strip comprises the following steps:
step 1: mixing a goat anti-mouse secondary antibody IgG and an antibody I with an antibody diluent respectively, and then sequentially coating the mixture on a nitrocellulose membrane along the chromatography direction to be used as a detection line and a quality control line respectively;
step 2: preparing an antibody II modified gold nanorod conjugate, and coating the antibody II modified gold nanorod conjugate on a bonding pad;
and step 3: and sticking the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad on the bottom plate to obtain the immune colloidal gold test strip.
7. The method of claim 6, wherein in step 1, the antibody diluent is 0.01mol/L phosphate buffer containing 3% sucrose.
8. The method of claim 6, wherein in step 1, the coating concentration of the goat anti-mouse secondary IgG and antibody I is 0.5-2 mg/mL.
9. The method according to claim 6, wherein the antibody II modified gold nanorod conjugate in the step 2 is prepared by the following method:
and synthesizing the gold nanorods by adopting a seed growth method, and then carrying out covalent bonding on the polyacrylic acid sodium salt and the antibody II to obtain the antibody II modified gold nanorod conjugate.
10. The method according to claim 6, wherein the antibody II modified gold nanorod conjugate is coated in an amount of 1-7 μ L.
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CN113376372A (en) * 2021-06-16 2021-09-10 郑州凌思生物科技有限责任公司 Immunofluorescence colloidal gold nano microsphere material
CN113504377A (en) * 2021-07-15 2021-10-15 中国科学院长春应用化学研究所 Test strip for bimodal detection of CRP (C-reactive protein), preparation method and detection method
CN115097130A (en) * 2022-05-17 2022-09-23 中国石油大学(华东) Capillary glass tube detection method for rapidly qualitatively-quantitatively detecting coronavirus

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CN107478642A (en) * 2017-07-27 2017-12-15 山东师范大学 A kind of colloidal gold strip quantitative testing device and method based on photo-thermal effect detection
US20180128827A1 (en) * 2012-01-31 2018-05-10 Regents Of The University Of Minnesota Lateral flow assays with thermal contrast readers

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US20180128827A1 (en) * 2012-01-31 2018-05-10 Regents Of The University Of Minnesota Lateral flow assays with thermal contrast readers
CN107478642A (en) * 2017-07-27 2017-12-15 山东师范大学 A kind of colloidal gold strip quantitative testing device and method based on photo-thermal effect detection

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Publication number Priority date Publication date Assignee Title
CN113376372A (en) * 2021-06-16 2021-09-10 郑州凌思生物科技有限责任公司 Immunofluorescence colloidal gold nano microsphere material
CN113504377A (en) * 2021-07-15 2021-10-15 中国科学院长春应用化学研究所 Test strip for bimodal detection of CRP (C-reactive protein), preparation method and detection method
CN115097130A (en) * 2022-05-17 2022-09-23 中国石油大学(华东) Capillary glass tube detection method for rapidly qualitatively-quantitatively detecting coronavirus
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