CN116643044A - Kit for detecting HIV-1 and HIV-2 antibodies in urine based on colloidal gold method, and preparation method and application thereof - Google Patents
Kit for detecting HIV-1 and HIV-2 antibodies in urine based on colloidal gold method, and preparation method and application thereof Download PDFInfo
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- CN116643044A CN116643044A CN202310716488.7A CN202310716488A CN116643044A CN 116643044 A CN116643044 A CN 116643044A CN 202310716488 A CN202310716488 A CN 202310716488A CN 116643044 A CN116643044 A CN 116643044A
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 171
- 238000000034 method Methods 0.000 title claims abstract description 33
- 241000713772 Human immunodeficiency virus 1 Species 0.000 title claims abstract description 28
- 208000031886 HIV Infections Diseases 0.000 title claims abstract description 26
- 241000713340 Human immunodeficiency virus 2 Species 0.000 title claims abstract description 26
- 210000002700 urine Anatomy 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 54
- 239000010931 gold Substances 0.000 claims abstract description 50
- 229910052737 gold Inorganic materials 0.000 claims abstract description 50
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 30
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 30
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 30
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 30
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- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 claims abstract description 27
- PYIDGJJWBIBVIA-UYTYNIKBSA-N lauryl glucoside Chemical compound CCCCCCCCCCCCO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O PYIDGJJWBIBVIA-UYTYNIKBSA-N 0.000 claims abstract description 24
- 239000007853 buffer solution Substances 0.000 claims abstract description 17
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- 102000036639 antigens Human genes 0.000 claims description 66
- 108091007433 antigens Proteins 0.000 claims description 66
- 239000002245 particle Substances 0.000 claims description 61
- 102000004169 proteins and genes Human genes 0.000 claims description 54
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- 239000007864 aqueous solution Substances 0.000 claims description 35
- 101800000385 Transmembrane protein Proteins 0.000 claims description 33
- 101800001690 Transmembrane protein gp41 Proteins 0.000 claims description 33
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- 241000283707 Capra Species 0.000 claims description 23
- 238000003908 quality control method Methods 0.000 claims description 22
- 238000002372 labelling Methods 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000003365 glass fiber Substances 0.000 claims description 17
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 15
- 102000011632 Caseins Human genes 0.000 claims description 15
- 108010076119 Caseins Proteins 0.000 claims description 15
- 239000002253 acid Substances 0.000 claims description 15
- 239000001509 sodium citrate Substances 0.000 claims description 15
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 15
- 229940038773 trisodium citrate Drugs 0.000 claims description 15
- 238000001035 drying Methods 0.000 claims description 14
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- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 10
- 229940098773 bovine serum albumin Drugs 0.000 claims description 10
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- 241000725303 Human immunodeficiency virus Species 0.000 claims description 7
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- 235000018102 proteins Nutrition 0.000 description 43
- 230000000052 comparative effect Effects 0.000 description 17
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 12
- -1 dodecyl glucoside compound Chemical class 0.000 description 12
- 239000005018 casein Substances 0.000 description 10
- 235000021240 caseins Nutrition 0.000 description 10
- 230000000903 blocking effect Effects 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 230000009871 nonspecific binding Effects 0.000 description 6
- 229910000029 sodium carbonate Inorganic materials 0.000 description 6
- 239000012266 salt solution Substances 0.000 description 5
- 208000030507 AIDS Diseases 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
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- 230000008569 process Effects 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241000713666 Lentivirus Species 0.000 description 2
- 229920000881 Modified starch Polymers 0.000 description 2
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000003317 immunochromatography Methods 0.000 description 2
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- 238000012986 modification Methods 0.000 description 2
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- 239000004094 surface-active agent Substances 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
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- 239000007983 Tris buffer Substances 0.000 description 1
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- 230000009471 action Effects 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
Classifications
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56988—HIV or HTLV
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
-
- 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
-
- 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
- G01N33/6854—Immunoglobulins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a kit for detecting HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method, and a preparation method and application thereof. The kit comprises a reagent strip, wherein the reagent strip comprises a PVC bottom plate, a sample pad, a gold label pad, an immune nitrocellulose membrane and absorbent paper. The buffer solution containing trehalose and polyethylene glycol can obviously increase the use stability of the kit affected by the environment. The invention uses the matching of the n-hexyl glucoside and the dodecyl glucoside to obviously increase the sensitivity of the kit and reduce the probability of false positive, so that the kit has more excellent detection efficiency.
Description
Technical Field
The invention relates to the field of colloidal gold immunochromatography, in particular to a kit for detecting HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method, and a preparation method and application thereof.
Background
AIDS belongs to the human lentivirus group in the genus lentivirus of the family retrovirus, and is classified into type 1 and type 2. HIV-1 is currently predominantly prevalent worldwide. Acquired immune deficiency syndrome, AIDS (AIDS), is an infectious disease caused by the Human Immunodeficiency Virus (HIV). HIV can continuously destroy the immune system of the body after entering the human body, resulting in impaired or even defective immune cell function, eventually accompanied by various serious opportunistic infections and tumors.
Because of the poor awareness and precaution of aids in many areas and high risk groups, hiv-infected people have spread rapidly in some areas. The spread of aids has spread from the special population to the general population, and cases spread via the sexually transmitted route are continuously increasing. Detection reagents such as urinary HIV-1 antibody enzyme-linked immunosorbent (E L I S A), western Blot (Western Blot) and the like are developed in succession at home and abroad, the sensitivity and the specificity of the detection reagents are equivalent to those of HIV blood detection reagents, but the detection cost is higher, special experimental conditions are required, and the detection reagents are not suitable for large-scale popularization.
The Jin Biaofu solution of the prior art CN112816700A is prepared by adding 0.6g Tris,0.5g Casein Na,1g PVP,0.5g PEG20000,1g surfactant S9, 20g sucrose, 0.05g preservative Proclin300 into 80ml ultra-pure water, stirring until the solution is completely dissolved, adjusting the pH to 8.0 by using 1M hydrochloric acid, and adding ultra-pure water to a volume of 100 ml; the gold-labeled complex solution contains a surfactant S9 and a sucrose component, but the Jin Biaofu solution is deficient in improving the sensitivity and stability in use of the test paper. The prior art CN114966011a discloses a test strip for detecting HIV-1 and HIV-2 antibodies in urine by a colloidal gold method, although it can detect HIV-1 and HIV-2 antibodies simultaneously; however, the quality control line used by the method coats chicken anti-SPA protein, so that the detection rate is relatively low; the stability in use, accuracy and nonspecific binding effect of the product obtained by the scheme described in the patent are further improved.
The existing detection method for the HIV-1 and HIV-2 antibodies simultaneously usually takes blood as a detection object, and the problems of infection, low sensitivity, requirement of adopting independent C lines, non-specific binding, accuracy, detection rate and use stability caused by blood detection cannot be solved at the same time.
Disclosure of Invention
The invention provides a kit for detecting HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method and a preparation method and application thereof based on the solution of the technical problems.
In order to increase the sensitivity of the kit and reduce the probability of false positive, the invention creatively selects trehalose and polyethylene glycol 20000 as labeling buffer components; buffers comprising trehalose and polyethylene glycol can significantly increase the stability of the kit in use under environmental influences.
In order to increase the excellent detection sensitivity and accuracy of the kit, the invention uses n-hexyl glucoside and dodecyl glucoside, and the cooperation of the n-hexyl glucoside and the dodecyl glucoside can synergistically and remarkably increase the sensitivity of the kit and reduce the probability of false positive, so that the kit has more excellent detection efficiency.
Compared with other gold releasing agents such as Tween-20, the gold releasing agent obtained by the n-hexyl glucoside and dodecyl glucoside compound system has better effects, and can give the product of the invention a better detection effect. When the mass ratio of n-hexyl glucoside to dodecyl glucoside is 1: in the range of (0.4-0.8), the kit has more excellent detection performance.
The principle of the kit for detecting the HIV-1 and HIV-2 antibodies in urine based on the colloidal gold method is that the immunochromatography method using the colloidal gold as a marker is used or the detection method does not need an instrument, and the result can be directly interpreted by naked eyes.
The kit for detecting the HIV-1 and HIV-2 antibodies in urine based on the colloidal gold method provided by the invention comprises a test strip, the preparation of the kit is simple, the SPA protein colloidal gold conjugate is used as a chromogenic substance, casein sodium salt is added in sealing, non-specific binding is reduced, a gold seed releasing agent, trehalose and polyethylene glycol 20000 are added in a marking buffer, the influence of the environment on the test strip is reduced, and the detection rate and the detection accuracy are improved. The gp41 recombinant antigen capable of recognizing the HIV-1 antibody and the gp36 recombinant antigen capable of recognizing the HIV-2 antibody are mixed in the detection line, the goat anti-mouse IgG polyclonal antibody is adopted as a control line, SPA protein connected with colloid Jin Faou is directly captured, the quality control effect is achieved, an independent C line is not needed, the cross and false positive caused by the independent C line are eliminated, the specificity of a product is improved, meanwhile, the operation of preparing an independent C line system is reduced, and the characteristics of excellent performance and simplicity in manufacturing are shown.
In one aspect, the invention discloses a kit for detecting HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method, which comprises a test strip, wherein the test strip comprises a PVC bottom plate, a sample pad, a gold label pad, an immune nitrocellulose membrane and absorbent paper.
Preferably, the sample pad, the gold-labeled pad, the immune nitrocellulose membrane and the absorbent paper are sequentially connected end to end and fixed on the PVC bottom plate.
Preferably, the gold-labeled pad is coated with SPA protein labeled by colloidal gold particles.
Preferably, the preparation of the SPA protein marked by the colloidal gold particles comprises the steps of adding a trisodium citrate solution after boiling water and chloroauric acid solution, continuously boiling, and cooling to prepare a colloidal gold particle solution; adjusting pH to 5-6.9, adding SPA protein, mixing, adding bovine serum albumin solution, mixing, adding sodium caseinate aqueous solution, mixing, centrifuging, and collecting precipitate.
Preferably, the chloroauric acid solution is an aqueous solution of chloroauric acid with a mass concentration of 0.5-2.5%.
Preferably, the trisodium citrate solution is a trisodium citrate aqueous solution with a mass concentration of 0.5-4%.
Preferably, the volume ratio of the water to the chloroauric acid solution to the trisodium citrate solution is 100: (3-6): (2-4).
Preferably, the ratio of the volume usage of the colloidal gold particle solution to the mass usage of SPA protein is (0.5-1.5) mL: (6-15) μg.
Preferably, the bovine serum albumin solution is a bovine serum albumin aqueous solution with a mass concentration of 5-15%.
Preferably, the volume ratio of the bovine serum albumin solution to the colloidal gold particle solution is (5-15) μl:1mL.
Preferably, the casein sodium salt solution is 15-25% casein sodium salt solution by mass concentration.
Preferably, the volume ratio of the casein sodium salt solution to the colloidal gold particle solution is (15-25) μl:1mL.
Preferably, the preparation of the gold mark pad comprises the following steps: and (3) dissolving SPA protein marked by colloidal gold particles in a marking buffer solution to obtain a colloidal gold composite solution, spreading the colloidal gold composite solution on glass fibers, and drying to obtain the colloidal gold composite solution.
Preferably, the ratio of the mass of the SPA protein marked by the colloidal gold particles to the total mass of the SPA protein marked by the marking buffer and the colloidal gold particles is (10-20): 100.
preferably, the labeling buffer consists of: 5-10wt% of BSA, 0.3-0.5wt% of trehalose, 0.25-0.35wt% of polyethylene glycol 20000 and 2-5wt% of a catalyst, wherein the mass ratio is 1: (0.4-0.8) gold seed releasing agent composed of n-hexyl glucoside and dodecyl glucoside and the balance of aqueous solution with concentration of 30-60 millimoles/liter and pH of 7.0-8.5 Tris-HCl.
Preferably, when preparing the gold-labeled pad, the colloidal gold composite solution is spread on the glass fiber according to the proportion of spreading 0.5-20mL of the colloidal gold composite solution on the glass fiber per square decimeter, and the gold-labeled pad is prepared by drying.
Preferably, the immune nitrocellulose membrane is provided with a detection line for coating gp41 recombinant antigen and gp36 recombinant antigen and a quality control line for coating goat anti-mouse IgG polyclonal antibody.
On the other hand, the invention discloses a preparation method of a kit for detecting HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method, which comprises the following steps:
1) Preparing colloidal gold particle marked SPA protein: labeling SPA protein by using colloidal gold particles to obtain SPA protein labeled by the colloidal gold particles;
2) Preparation of a gold mark pad: preparing a gold-labeled pad by using the SPA protein marked by the colloidal gold particles obtained in the step 1);
3) Spraying gp41 recombinant antigen, gp36 recombinant antigen mixture and goat anti-mouse IgG polyclonal antibody on the positions of a nitrocellulose membrane detection line T and a quality control line C respectively, and drying to obtain an immune nitrocellulose membrane;
4) Sequentially sticking a sample pad, a gold mark pad, an immune nitrocellulose membrane and water absorbing paper on a PVC plate, and cutting to prepare a test strip; and packaging the test strip in a packaging box to obtain the kit for detecting the HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method.
Preferably, the preparation of the SPA protein marked by the colloidal gold particles in the step 1) comprises the following steps:
(1) Adding chloroauric acid solution into water, boiling, adding trisodium citrate solution, boiling, and cooling to obtain colloidal gold particle solution;
(2) Adding sodium carbonate solution to adjust the pH;
(3) Adding SPA protein and stirring;
(4) Adding the sealing liquid and stirring;
(5) Adding a terminator and stirring; centrifuging, and taking the precipitate to obtain the SPA protein marked by the colloidal gold particles.
Preferably, the chloroauric acid solution is an aqueous chloroauric acid solution. The mass concentration is preferably 0.5-2.5%, 0.5-2%, 0.8-1.5%, 0.9-1.2% and 1%.
Preferably, the trisodium citrate solution is a trisodium citrate aqueous solution. The mass concentration is preferably 0.5-4%, 1-3%, 1.5-2.5% and 2%.
Preferably, the volume ratio of water to chloroauric acid solution and trisodium citrate solution in the step (1) is 100: (3-6): (2-4), more preferably 100: (3.5-5.5): (2.5-3.5), 100: (3.5-5): (2.8-3.2), 100:4:3.
preferably, the cooling temperature is 15-35 ℃, more preferably 18-30 ℃, 18-28 ℃, 19-25 ℃ or room temperature.
Preferably, the sodium carbonate solution is an aqueous sodium carbonate solution. The concentration is preferably 0.05-0.5mol/L, 0.1-0.4mol/L, 0.15-0.3mol/L, 0.2mol/L.
Preferably, the pH value of the pH adjustment is 5-6.9, 5.2-6.8, 5.5-6.2, 6.
Preferably, the ratio of the volume of the colloidal gold particle solution to the mass of SPA protein is (0.5-1.5) mL: (6-15) μg; more preferably (0.8-1.2) mL: (8-12) μg, 1mL:10 mug.
Preferably, the blocking solution is a bovine serum albumin solution. More preferably an aqueous bovine serum albumin solution. The mass concentration is preferably 5-15%, 8-13%, 9-12% and 10%.
Preferably, the volume ratio of the blocking solution to the colloidal gold particle solution is (5-15) μl:1mL, (8-12) μL:1mL, 10. Mu.L: 1mL.
Preferably, the terminator is casein sodium salt solution. More preferably an aqueous casein sodium salt solution. The mass concentration is preferably 15-25%, 18-23% and 20%. The terminator can further reduce non-specific binding, and the obtained test strip has better specificity.
Preferably, the volume ratio of the terminator to the colloidal gold particle solution is (15-25) μl:1mL, (18-22) μL:1mL, 20. Mu.L: 1mL.
Preferably, the stirring speeds in steps (3) - (5) are each independently 50-1500rpm, 100-1000rpm, 150-800rpm, 200-600rpm. The stirring time in steps (3) - (5) is 1-120 minutes, 2-75 minutes, 3-60 minutes, 4-30 minutes each independently.
Preferably, the centrifugal speed is preferably 1000-10000rpm, 1500-8000rpm, 2000-5000rpm, 2500-4000rpm, 3000rpm. The centrifugation time is preferably 1 to 60 minutes, 2 to 50 minutes, 3 to 40 minutes, 4 to 30 minutes.
Preferably, the preparation of the SPA protein marked by the colloidal gold particles in the step 1) comprises the following steps:
(1) Adding chloroauric acid water solution with the mass concentration of 0.5-2.5% into water, boiling, adding trisodium citrate water solution with the mass concentration of 0.5-4%, boiling, and cooling to 15-35 ℃ to obtain colloidal gold particle solution; wherein the volume ratio of the water to the chloroauric acid solution to the trisodium citrate solution is 100: (3-6): (2-4);
(2) Adding 0.05-0.5mol/L sodium carbonate aqueous solution to adjust the pH to 5-6.9;
(3) Adding SPA protein and stirring; wherein the ratio of the volume usage of the colloidal gold particle solution to the mass usage of SPA protein is (0.5-1.5) mL: (6-15) μg;
(4) Adding a blocking solution of 5-15% bovine serum albumin aqueous solution, and stirring; wherein the volume ratio of the blocking solution to the colloidal gold particle solution is (5-15) mu L:1mL;
(5) Adding a terminator of 15-25% of sodium caseinate aqueous solution, and stirring; centrifuging, and taking a precipitate to obtain the SPA protein marked by the colloidal gold particles; wherein the volume ratio of the terminator to the colloidal gold particle solution is (15-25) mu L:1mL; the centrifugal rotating speed is 1000-10000rpm, and the centrifugal time is 1-60 minutes;
the stirring speeds in the steps (3) - (5) are respectively 50-1500rpm and the stirring time is respectively 1-120 minutes.
Preferably, the specific steps of preparing the gold mark pad in the step 2) include:
A. dissolving SPA protein marked by colloidal gold particles by using a marking buffer solution to obtain a colloidal gold composite solution;
B. spreading the colloidal gold composite solution prepared in the step A on glass fiber, and drying to obtain the gold-labeled pad.
Preferably, the ratio of the mass of the SPA protein marked by the colloidal gold particles in the step A to the total mass of the SPA protein marked by the marking buffer and the colloidal gold particles is (10-20): 100. (12-18): 100. (13-17): 100. (14-16): 100.
preferably, the labeling buffer in the step a consists of: BSA, trehalose, polyethylene glycol 20000, a gold seed releasing agent and Tris-HCl aqueous solution.
Preferably, the amount of trehalose in the labeling buffer of step A is 0.3-0.5wt%.
Preferably, the amount of polyethylene glycol 20000 in the labeling buffer in step a is 0.25-0.35wt%.
Preferably, the amount of the gold seed releasing agent in the labeling buffer in the step A is 2-5wt%.
Preferably, the labeling buffer in the step a is composed of the following components: 5-10wt% of BSA, 0.3-0.5wt% of trehalose, 0.25-0.35wt% of polyethylene glycol 20000, 2-5wt% of gold seed releasing agent and the balance of Tris-HCl aqueous solution.
Preferably, the concentration of the Tris-HCl aqueous solution is 30-60 mmol/L, 35-55 mmol/L, 45-55 mmol/L, 50 mmol/L. Preferably, the pH of the aqueous Tris-HCl solution is 7.0-8.5, 7.1-8.3, 7.2-8.2, 7.3-8.0.
Preferably, the amount of BSA in the labelling buffer is 6.5-9.5wt%, 7-9wt% and 7.5-8.5wt%.
Preferably, the trehalose is used in an amount of 0.32-0.48wt%, 0.33-0.47wt%, 0.35-0.45wt% in the labeling buffer.
Preferably, the polyethylene glycol 20000 is used in an amount of 0.26-0.34wt%, 0.27-0.33wt%, 0.28-0.32wt% in the labeling buffer.
Preferably, the amount of the gold seed releasing agent in the labeling buffer is 2.2-4.8wt%, 2.3-4.6wt% and 2.5-4.5wt%.
Preferably, the gold seed releasing agent in the labeling buffer consists of n-hexyl glucoside and dodecyl glucoside.
Preferably, the mass ratio of the n-hexyl glucoside to the dodecyl glucoside is 1: (0.4-0.8), 1: (0.42-0.78), 1: (0.45-0.75), 1: (0.5-0.7).
Preferably, the labeling buffer in the step a is composed of the following components: 5-10wt% BSA, 0.3-0.5wt% trehalose, 0.25-0.35wt% polyethylene glycol 20000, 2-5wt% gold seed releasing agent and the balance Tris-HCl aqueous solution; the gold seed releasing agent in the marking buffer solution comprises the following components in percentage by mass: (0.4-0.8) n-hexyl glucoside and dodecyl glucoside; the concentration of the Tris-HCl aqueous solution is 30-60 mmol/L and the pH value is 7.0-8.5.
Preferably, the labeling buffer in the step a is formulated as follows: 7wt% of BSA, 0.4wt% of trehalose, 0.3wt% of polyethylene glycol 20000 and 3.5wt% of a modified starch comprising the following components in percentage by mass: (0.4-0.8) gold seed releasing agent composed of n-hexyl glucoside and dodecyl glucoside and the balance of 50 millimoles/liter Tris-HCl aqueous solution; the Tris-HCl aqueous solution had a pH of 7.5.
Preferably, in the step B, the colloidal gold solution is paved on glass fibers, 0.5-20mL of colloidal gold composite solution is paved on the glass fibers per square decimeter, and the gold standard pad is prepared by drying. More preferably, 1-15mL of the colloidal gold composite solution is spread on every square decimeter of the glass fiber, 2-10mL of the colloidal gold composite solution is spread on every square decimeter of the glass fiber, and 6mL of the colloidal gold composite solution is spread on every square decimeter of the glass fiber.
Preferably, the specific steps of preparing the gold mark pad in the step 2) include:
A. dissolving SPA protein marked by colloidal gold particles by using a marking buffer solution to obtain a colloidal gold composite solution; wherein, the ratio of the mass of the SPA protein marked by the colloidal gold particles to the total mass of the SPA protein marked by the marking buffer solution and the colloidal gold particles is (10-20): 100; the labeling buffer consists of: 5-10wt% of BSA, 0.3-0.5wt% of trehalose, 0.25-0.35wt% of polyethylene glycol 20000 and 2-5wt% of a catalyst, wherein the mass ratio is 1: (0.4-0.8) gold seed releasing agent composed of n-hexyl glucoside and dodecyl glucoside and the balance of aqueous solution with concentration of 30-60 millimoles/liter and pH of 7.0-8.5 Tris-HCl;
B. spreading the colloidal gold composite solution prepared in the step A on glass fibers, spreading 0.5-20mL of colloidal gold composite solution on each square decimeter of glass fibers, and drying to obtain the gold mark pad.
Preferably, in the step 3), the goat anti-mouse IgG polyclonal antibody sprayed on the nitrocellulose membrane quality control line C is added in the form of a goat anti-mouse IgG polyclonal antibody solution, and the concentration is 0.5.0-4.0mg/ml, 0.8-3.5mg/ml, 0.9-2.5mg/ml, and 1.0-2.0mg/ml.
Preferably, 3-10ml of goat anti-mouse IgG polyclonal antibody solution is sprayed on each 50m long nitrocellulose membrane, more preferably 4-9ml, 5-8ml, and 6ml.
Preferably, in the step 3), the gp41 recombinant antigen and the gp36 recombinant antigen mixture sprayed on the nitrocellulose membrane detection line T is added in the form of a solution containing both the gp41 recombinant antigen and the gp36 recombinant antigen.
Preferably, the concentration of gp41 recombinant antigen in the solution containing gp41 recombinant antigen and gp36 recombinant antigen is 0.2-5.0mg/ml, 0.3-4.0mg/ml, 0.6-3mg/ml and 1.0-2.0mg/ml.
Preferably, the concentration of gp36 recombinant antigen in the solution containing gp41 recombinant antigen and gp36 recombinant antigen is 0.2-5.0mg/ml, 0.5-3.0mg/ml, 0.8-2.5mg/ml and 1.0-1.5mg/ml.
Preferably, 3-10ml of solution containing gp41 recombinant antigen and gp36 recombinant antigen are sprayed on each 50m long nitrocellulose membrane, more preferably 4-9ml, 5-8ml and 6ml
Preferably, the spacing of the quality control lines is 1.0-10.0mm, 2.0-8.0mm, 3.0-7.0mm, 4.0-6.0mm.
Preferably, the step 3) includes: spraying gp41 recombinant antigen, gp36 recombinant antigen mixture and goat anti-mouse IgG polyclonal antibody on the positions of a nitrocellulose membrane detection line T and a quality control line C respectively, and drying to obtain an immune nitrocellulose membrane; wherein, the goat anti-mouse IgG polyclonal antibody sprayed on the nitrocellulose membrane quality control line C is added in the form of a goat anti-mouse IgG polyclonal antibody solution, and the concentration is 0.5.0-4.0mg/ml; 3-10ml of goat anti-mouse IgG polyclonal antibody solution is sprayed on each 50m long nitrocellulose membrane; the gp41 recombinant antigen and gp36 recombinant antigen mixture sprayed on the nitrocellulose membrane detection line T are added in the form of a solution containing gp41 recombinant antigen and gp36 recombinant antigen at the same time; the concentration of gp41 recombinant antigen in the solution containing gp41 recombinant antigen and gp36 recombinant antigen is 0.2-5.0mg/ml, and the concentration of gp36 recombinant antigen is 0.2-5.0mg/ml; 3-10ml of solution simultaneously containing gp41 recombinant antigen and gp36 recombinant antigen is sprayed on each 50m long nitrocellulose membrane; the distance between the detection line T line and the quality control line C line is 1.0-10.0mm.
On the other hand, the invention discloses an application of the kit for detecting the HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method in detecting the HIV-1 and HIV-2 antibodies.
On the other hand, the invention discloses a method for using a colloidal gold method kit for detecting HIV-1 and HIV-2 antibodies in urine, which comprises the following steps:
1. and (3) detecting a sample: sucking 2-3 drops of sample liquid by a suction pipe, adding the sample liquid into a sample hole, carrying out chromatography on a sample to be detected to a water absorption paper end under the capillary action, and sequentially passing through a sample pad, a gold mark pad and an immune nitrocellulose membrane; after reaching the gold-labeled pad, the antibodies marked by colloidal gold are fully identified and combined, and the antibodies are continuously chromatographed on the nitrocellulose membrane, and immunoreact with gp41 recombinant antigen or gp36 recombinant antigen coated on the nitrocellulose membrane, so that corresponding red lines are displayed;
2. interpretation of results: interpreting the result 15 minutes after dripping the sample;
positive: a red line appears at the positions of a detection line and a quality control line of the detection test strip respectively, which indicates that the HIV antibody exists in the sample;
negative: only a red line appears at the position of the quality control line, which indicates that no HIV antibody exists in the sample;
invalidation: the quality control line position has no red line, which indicates that the result is invalid and the retry should be performed.
The beneficial effects are that:
the invention uses urine as a detection carrier, is convenient to detect, and avoids organism damage to a detected human body. The invention innovatively adopts the goat anti-mouse IgG polyclonal antibody as a control line, directly captures SPA protein connected with colloid Jin Faou, plays a role in quality control, and discards an independent C line system. The cross and false positive caused by independent C lines are reduced, the operation is reduced, and the cost is lowered.
The gp41 recombinant antigen capable of recognizing the HIV-1 antibody and the gp36 recombinant antigen capable of recognizing the HIV-2 antibody are mixed in the detection line of the invention, so that the HIV-1 and the HIV-2 can be detected simultaneously.
The detection kit has high detection sensitivity, accurate detection result and high use stability influenced by environment.
According to the invention, casein sodium salt is added in the sealing process, so that non-specific binding is reduced, the accuracy of the product is improved, the operation is simplified, and the cost is reduced.
The invention uses trehalose and polyethylene glycol to increase the sensitivity of the kit and reduce the probability of false positive. Trehalose and polyethylene glycol 20000 have the synergistic effect of compounding on increasing the sensitivity of the kit and reducing the false positive effect. The trehalose and the polyethylene glycol can obviously increase the use stability of the kit affected by the environment.
The invention uses the n-hexyl glucoside and the dodecyl glucoside to obviously increase the sensitivity of the kit and reduce the probability of false positive; the combination of the two can endow the kit with excellent detection sensitivity and accuracy. The mass ratio of the n-hexyl glucoside to the dodecyl glucoside is 1: in the range of (0.4-0.8), the kit has more excellent detection performance.
Compared with other gold releasing agents such as Tween-20, the gold releasing agent obtained by the n-hexyl glucoside and dodecyl glucoside compound system has better effects, and can give the product of the invention a better detection effect.
Detailed Description
The invention will be described in connection with specific embodiments. It will be appreciated by those skilled in the art that these embodiments are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Example 1
A preparation method of a kit for detecting HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method comprises the following steps:
1) Preparing SPA protein marked by colloidal gold particles: (1) Adding chloroauric acid water solution with the mass concentration of 1% into water, boiling, adding trisodium citrate water solution with the mass concentration of 2%, boiling, and cooling to room temperature to obtain colloidal gold particle solution; wherein the volume ratio of the water to the chloroauric acid solution to the trisodium citrate solution is 100:4:3, a step of;
(2) Adding 0.2mol/L sodium carbonate aqueous solution to adjust the pH to 6;
(3) Adding SPA protein and stirring; wherein, the ratio of the volume consumption of the colloidal gold particle solution to the mass consumption of SPA protein is 1mL:10 μg;
(4) Adding a blocking solution of a bovine serum albumin aqueous solution with the mass concentration of 10% and stirring; wherein, the volume ratio of the blocking solution to the colloidal gold particle solution is 10 mu L:1mL;
(5) Adding a terminator of a sodium caseinate aqueous solution with the mass concentration of 20% and stirring; centrifuging, and taking a precipitate to obtain the SPA protein marked by the colloidal gold particles; wherein, the volume ratio of the terminator to the colloidal gold particle solution is 20 mu L:1mL; the centrifugal rotating speed is 6000rpm, and the centrifugal time is 15 minutes;
the stirring speeds in the steps (3) - (5) are respectively 600rpm and the stirring times are respectively 10 minutes;
2) Preparing a gold mark pad: A. dissolving SPA protein marked by colloidal gold particles by using a marking buffer solution to obtain a colloidal gold composite solution; wherein, the ratio of the mass of the SPA protein marked by the colloidal gold particles to the total mass of the SPA protein marked by the marking buffer solution and the colloidal gold particles is 15:100; the labeling buffer consists of: 7wt% of BSA, 0.4wt% of trehalose, 0.3wt% of polyethylene glycol 20000 and 3.5wt% of a modified starch comprising the following components in percentage by mass: 0.6 of n-hexyl glucoside and dodecyl glucoside, and the balance of 50 millimoles/liter of aqueous solution of Tris-HCl with the pH value of 7.5;
B. spreading the colloidal gold composite solution prepared in the step A on glass fibers, spreading 6mL of colloidal gold composite solution on each square decimeter of glass fibers, and drying to obtain a gold mark pad;
3) Spraying gp41 recombinant antigen, gp36 recombinant antigen mixture and goat anti-mouse IgG polyclonal antibody on the positions of a nitrocellulose membrane detection line T and a quality control line C respectively, and drying to obtain an immune nitrocellulose membrane; wherein, the goat anti-mouse IgG polyclonal antibody sprayed on the nitrocellulose membrane quality control line C is added in the form of a goat anti-mouse IgG polyclonal antibody solution, and the concentration is 1.2mg/ml; 6ml of goat anti-mouse IgG polyclonal antibody solution is sprayed on each 50m long nitrocellulose membrane; the gp41 recombinant antigen and gp36 recombinant antigen mixture sprayed on the nitrocellulose membrane detection line T are added in the form of a solution containing gp41 recombinant antigen and gp36 recombinant antigen at the same time; the concentration of gp41 recombinant antigen in the solution containing gp41 recombinant antigen and gp36 recombinant antigen is 1.5mg/ml, and the concentration of gp36 recombinant antigen is 1.2mg/ml; 6ml of solution simultaneously containing gp41 recombinant antigen and gp36 recombinant antigen is sprayed on each 50m long nitrocellulose membrane; the distance between the detection line T line and the quality control line C line is 5mm;
4) Sequentially sticking the sample pad, the gold mark pad, the immune nitrocellulose membrane and the absorbent paper on a PVC plate, and cutting to prepare a test strip; and packaging the test strip in a packaging box to obtain the kit for detecting the HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method.
Example 2
A preparation method of a kit for detecting HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method comprises the following steps:
1) Preparing SPA protein marked by colloidal gold particles: (1) Adding chloroauric acid aqueous solution with the mass concentration of 1.2% into water, boiling, adding trisodium citrate aqueous solution with the mass concentration of 1.7%, boiling, and cooling to room temperature to obtain colloidal gold particle solution; wherein the volume ratio of the water to the chloroauric acid solution to the trisodium citrate solution is 100:3.8:3.1;
(2) Adding 0.2mol/L sodium carbonate aqueous solution to adjust the pH value to 5.9;
(3) Adding SPA protein and stirring; wherein, the ratio of the volume consumption of the colloidal gold particle solution to the mass consumption of SPA protein is 1mL:10 μg;
(4) Adding a blocking solution of a bovine serum albumin aqueous solution with the mass concentration of 10% and stirring; wherein, the volume ratio of the blocking solution to the colloidal gold particle solution is 10 mu L:1mL;
(5) Adding a terminator of 18% casein sodium salt aqueous solution, and stirring; centrifuging, and taking a precipitate to obtain the SPA protein marked by the colloidal gold particles; wherein, the volume ratio of the terminator to the colloidal gold particle solution is 20 mu L:1mL; the centrifugal rotating speed is 5000rpm, and the centrifugal time is 14 minutes;
the stirring speeds in the steps (3) - (5) are respectively 800rpm and the stirring times are respectively 10 minutes;
2) Preparing a gold mark pad: A. dissolving SPA protein marked by colloidal gold particles by using a marking buffer solution to obtain a colloidal gold composite solution; wherein, the ratio of the mass of the SPA protein marked by the colloidal gold particles to the total mass of the SPA protein marked by the marking buffer solution and the colloidal gold particles is 16:100; the labeling buffer consists of: 6wt% BSA, 0.3wt% trehalose, 0.25wt% polyethylene glycol 20000, 3.2wt% of the total mass ratio of 1:0.4 of n-hexyl glucoside and dodecyl glucoside, and the balance of 50 millimoles/liter of aqueous solution of Tris-HCl with the pH value of 7.5;
B. spreading the colloidal gold composite solution prepared in the step A on glass fibers, spreading 6mL of colloidal gold composite solution on each square decimeter of glass fibers, and drying to obtain a gold mark pad;
3) Spraying gp41 recombinant antigen, gp36 recombinant antigen mixture and goat anti-mouse IgG polyclonal antibody on the positions of a nitrocellulose membrane detection line T and a quality control line C respectively, and drying to obtain an immune nitrocellulose membrane; wherein, the goat anti-mouse IgG polyclonal antibody sprayed on the nitrocellulose membrane quality control line C is added in the form of a goat anti-mouse IgG polyclonal antibody solution, and the concentration is 1.2mg/ml; 6ml of goat anti-mouse IgG polyclonal antibody solution is sprayed on each 50m long nitrocellulose membrane; the gp41 recombinant antigen and gp36 recombinant antigen mixture sprayed on the nitrocellulose membrane detection line T are added in the form of a solution containing gp41 recombinant antigen and gp36 recombinant antigen at the same time; the concentration of gp41 recombinant antigen in the solution containing gp41 recombinant antigen and gp36 recombinant antigen is 1.5mg/ml, and the concentration of gp36 recombinant antigen is 1.2mg/ml; 6ml of solution simultaneously containing gp41 recombinant antigen and gp36 recombinant antigen is sprayed on each 50m long nitrocellulose membrane; the distance between the detection line T line and the quality control line C line is 5mm;
4) Sequentially sticking the sample pad, the gold mark pad, the immune nitrocellulose membrane and the absorbent paper on a PVC plate, and cutting to prepare a test strip; and packaging the test strip in a packaging box to obtain the kit for detecting the HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method.
Comparative example 1: the difference from example 1 is that: the aqueous solution of sodium caseinate with the mass concentration of 20% in the step (5) of the step (1) is replaced by the same amount of water; the other steps are the same as in example 1.
Comparative example 2: the difference from example 1 is that: the trehalose of step A in step 2) is replaced with the same amount of polyethylene glycol 20000; the other steps are the same as in example 1.
Comparative example 3: the difference from example 1 is that: the polyethylene glycol 20000 of step a in step 2) is replaced with the same amount of trehalose; the other steps are the same as in example 1.
Comparative example 4: the difference from example 1 is that: replacing the gold seed releasing agent in the step 2) by using the same amount of water; the other steps are the same as in example 1.
Comparative example 5: the difference from example 1 is that: the gold seed releasing agent in step 2) consists of only n-hexyl glucoside; the other steps are the same as in example 1.
Comparative example 6: the difference from example 1 is that: the gold seed releasing agent in the step 2) consists of only dodecyl glucoside; the other steps are the same as in example 1.
Comparative example 7: the difference from example 1 is that: the mass ratio of the n-hexyl glucoside to the dodecyl glucoside in the gold seed releasing agent in the step 2) is 1:0.1; the other steps are the same as in example 1.
Comparative example 8: the difference from example 1 is that: the mass ratio of the n-hexyl glucoside to the dodecyl glucoside in the gold seed releasing agent in the step 2) is 1:1.5; the other steps are the same as in example 1.
Comparative example 9: the difference from example 1 is that: the gold seed releasing agent in the step 2) is Tween-20; the other steps are the same as in example 1.
Comparative example 10: the difference from example 1 is that: the polyethylene glycol 20000 and the trehalose of the step A in the step 2) are replaced by using the same amount of water; the other steps are the same as in example 1.
Performance test:
in order to verify the sensitivity, specificity and stability of use affected by the environment of the product of the invention. 500 HIV-1 positive specimens, 500 HIV-2 positive specimens and 5000 negative specimens were selected for detection. The stability in use affected by the environment is to disassemble the packaging box, place the test strip for detection in the environment with the relative humidity of 45+/-2% and the temperature of 30 ℃ for 3 hours, and then test. The results of the detection are shown in tables 1 and 2.
Table 1: sensitivity and specificity test results
Table 2: environmental impact stability in use test results
As can be seen from the test results in Table 1 and Table 2, the test box has high detection sensitivity, accurate detection result and good use stability under the influence of environment.
As can be seen from a comparison of example 1 and comparative example 1, the addition of casein sodium salt in the blocking of the present invention reduces non-specific binding and increases the detection performance of the product.
As can be seen from a comparison of example 1 and comparative examples 2-3, the use of trehalose and polyethylene glycol according to the present invention increases the sensitivity of the kit and reduces the probability of false positives. One of trehalose or polyethylene glycol 20000 is omitted in comparative examples 2-3 and the total dosage of the trehalose and polyethylene glycol 20000 is kept the same as that in example 1, and the sensitivity and the false positive effect of the obtained kit are lower than those in example 1, which means that the trehalose and the polyethylene glycol 20000 have a compound synergistic effect in increasing the sensitivity and reducing the false positive effect of the kit. As can be seen from a comparison of example 1 and comparative examples 2-3, 10 in tables 1-2, trehalose and polyethylene glycol according to the invention significantly increase the stability of the kit in use under environmental influences.
As can be seen from a comparison of example 1 and comparative examples 4-5, the use of n-hexyl glucoside and dodecyl glucoside according to the invention can significantly increase the sensitivity of the kit and reduce the probability of false positives; the combination of the two can endow the kit with excellent detection sensitivity and accuracy. As is clear from comparison of comparative examples 7 to 8 with example 1, the mass ratio of n-hexyl glucoside and dodecyl glucoside of the present invention is 1: in the range of (0.4-0.8), the kit has more excellent detection performance.
As shown by the test results of the example 1 and the comparative example 9, the gold releasing agent obtained by the n-hexyl glucoside and dodecyl glucoside compound system has better effect than other gold releasing agents such as Tween-20 and the like, and can give better detection effect to the product.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. A kit for detecting HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method comprises a reagent strip, and is characterized in that: the reagent strip comprises a PVC bottom plate, a sample pad, a gold mark pad, an immune nitrocellulose membrane and absorbent paper; the gold mark pad is coated with SPA protein marked by colloidal gold particles;
the preparation of the gold mark pad comprises the following steps: dissolving SPA protein marked by colloidal gold particles in a marking buffer solution to obtain a colloidal gold composite solution, spreading the colloidal gold composite solution on glass fibers, and drying to obtain the colloidal gold composite solution;
the marking buffer solution consists of aqueous solution of BSA, trehalose, polyethylene glycol 20000, gold seed releasing agent and Tris-HCl;
the trehalose dosage in the marking buffer solution is 0.3-0.5wt%;
the dosage of polyethylene glycol 20000 in the marking buffer is 0.25-0.35wt%;
the gold seed releasing agent consists of n-hexyl glucoside and dodecyl glucoside;
the mass ratio of the n-hexyl glucoside to the dodecyl glucoside is 1: (0.4-0.8);
the sample pad, the gold mark pad, the immune nitrocellulose membrane and the absorbent paper are sequentially connected end to end and fixed on the PVC bottom plate;
the immune nitrocellulose membrane is provided with a detection line for coating gp41 recombinant antigen and gp36 recombinant antigen and a quality control line for coating goat anti-mouse IgG polyclonal antibody.
2. The kit of claim 1, wherein: the trehalose dosage in the marking buffer solution is 0.32-0.48wt%; and/or the dosage of polyethylene glycol 20000 in the labeling buffer is 0.26-0.34wt%.
3. The kit of claim 1, wherein: the dosage of the gold seed releasing agent in the marking buffer solution is 2-5wt%.
4. The kit of claim 1, wherein: the mass ratio of the n-hexyl glucoside to the dodecyl glucoside is 1: (0.42-0.78).
5. A method of preparing a kit according to any one of claims 1 to 4, comprising the steps of:
1) Preparing SPA protein marked by colloidal gold particles: labeling SPA protein by using colloidal gold particles to obtain SPA protein labeled by the colloidal gold particles;
2) Preparing a gold mark pad: preparing a gold-labeled pad by using the SPA protein marked by the colloidal gold particles obtained in the step 1);
3) Spraying gp41 recombinant antigen, gp36 recombinant antigen mixture and goat anti-mouse IgG polyclonal antibody on the positions of a nitrocellulose membrane detection line T and a quality control line C respectively, and drying to obtain an immune nitrocellulose membrane;
4) Sequentially sticking the sample pad, the gold mark pad, the immune nitrocellulose membrane and the absorbent paper on a PVC plate, and cutting to prepare a test strip; and packaging the test strip in a packaging box to obtain the kit for detecting the HIV-1 and HIV-2 antibodies in urine based on a colloidal gold method.
6. The method of claim 5, wherein the preparing of the SPA protein labeled with the colloidal gold particles in step 1) comprises:
(1) Adding chloroauric acid aqueous solution into water, boiling, adding trisodium citrate aqueous solution, boiling, and cooling to obtain colloidal gold particle solution;
(2) Regulating the pH value to 5-6.9;
(3) Adding SPA protein and stirring; wherein the ratio of the volume usage of the colloidal gold particle solution to the mass usage of SPA protein is (0.5-1.5) mL: (6-15) μg;
(4) Adding the sealing liquid of the bovine serum albumin aqueous solution and stirring;
(5) Adding a terminator of 15-25% of sodium caseinate aqueous solution, and stirring; centrifuging, and taking a precipitate to obtain the SPA protein marked by the colloidal gold particles; wherein the volume ratio of the terminator to the colloidal gold particle solution is (15-25) mu L:1mL.
7. The method according to claim 5, wherein the preparing step of the gold mark pad in the step 2) comprises:
A. dissolving SPA protein marked by colloidal gold particles by using a marking buffer solution to obtain a colloidal gold composite solution; the marking buffer solution consists of a gold seed releasing agent consisting of BSA, trehalose, polyethylene glycol 20000, n-hexyl glucoside and dodecyl glucoside and an aqueous solution of Tris-HCl;
B. spreading the colloidal gold composite solution prepared in the step A on glass fiber, and drying to obtain the gold-labeled pad.
8. The method of claim 7, wherein the labeling buffer comprises the following composition: 5-10wt% of BSA, 0.3-0.5wt% of trehalose, 0.25-0.35wt% of polyethylene glycol 20000 and 2-5wt% of a catalyst, wherein the mass ratio is 1: (0.4-0.8) gold seed releasing agent composed of n-hexyl glucoside and dodecyl glucoside and the balance of aqueous solution with concentration of 30-60 millimoles/liter and pH of 7.0-8.5 Tris-HCl.
9. The method of claim 5, wherein step 3) comprises: the goat anti-mouse IgG polyclonal antibody sprayed on the nitrocellulose membrane quality control line C is added in the form of a goat anti-mouse IgG polyclonal antibody solution;
and/or, the gp41 recombinant antigen and gp36 recombinant antigen mixture sprayed on the nitrocellulose membrane detection line T is added in the form of a solution containing the gp41 recombinant antigen and the gp36 recombinant antigen at the same time.
10. Use of a kit according to any one of claims 1 to 4 or obtainable by a method according to any one of claims 5 to 9, wherein said use comprises use in the detection of HIV.
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