CN114966011B - Test strip for detecting HIV-1 and HIV-2 antibodies in urine by colloidal gold method - Google Patents
Test strip for detecting HIV-1 and HIV-2 antibodies in urine by colloidal gold method Download PDFInfo
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- CN114966011B CN114966011B CN202210166846.7A CN202210166846A CN114966011B CN 114966011 B CN114966011 B CN 114966011B CN 202210166846 A CN202210166846 A CN 202210166846A CN 114966011 B CN114966011 B CN 114966011B
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- 208000031886 HIV Infections Diseases 0.000 title claims abstract description 26
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- 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 18
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- 239000000243 solution Substances 0.000 claims description 13
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 10
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 7
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- 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 claims description 5
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- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 4
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
- G01N33/569—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
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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
- G01N33/58—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
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- 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
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
- G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
- G01N2333/16—HIV-1, HIV-2
-
- 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
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
- G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
- G01N2333/16—HIV-1, HIV-2
- G01N2333/162—HIV-1, HIV-2 env, e.g. gp160, gp110/120, gp41, V3, peptid T, DC4-Binding site
-
- 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
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- 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
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- General Health & Medical Sciences (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- AIDS & HIV (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a test strip for detecting HIV-1 and HIV-2 antibodies in urine by a colloidal gold method, which comprises a gold-labeled pad, a nitrocellulose membrane coated with a detection line and a quality control line, a sample pad and filter paper. The invention reduces false positive, improves the accuracy of the product, simplifies the operation and reduces the cost. The invention does not need other equipment, has high sensitivity, is simple to operate, obtains a result in 15 minutes, uses urine as a sample, can be used for large-scale screening, can also be used for personal self-test, and has wide application range.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a test strip for detecting HIV antibodies in urine.
Background
AIDS self-detection is a process of independently collecting samples, detecting and reading results by individuals under the private alone or under the accompany of trusted people. The self-detection is an important supplement to the existing AIDS detection service, and is an acceptable, safe, accurate and effective method especially for high-risk groups. The HIV detection kit on the market mainly comprises blood sample detection, needs professional personnel to take blood in a professional laboratory for detection, has wounds in the detection process, and is easy to cross-infect due to the fact that HIV is transmitted through blood.
Current studies have demonstrated that HIV virus content in urine is very low and that HIV antibody IGg is present. However, the urine contains a low amount of antibodies, so that there are few products on the market. Staphylococcal protein a (SPA protein) binds to Fc fragments in human and various mammalian serum IgG molecules. IGg, which is capable of aggregating urine, is an ideal carrier.
The SPA protein is staphylococcus surface protein, has a simple structure, relatively small molecules, and is easy to fall off when being connected with colloid Jin Jingdian in an adsorption mode, so that the marking efficiency is low. The HIV antibody content in urine is reported to be only one thousandth of that in blood. The content is low, and the false positive rate of urine detection is high compared with blood products.
The nucleic acid similarity (nucleotide sequence identity) for both HIV-1 and HIV-2 subtypes is 55% and the amino sequence similarity (amino acid sequence identity) for several major proteins (gag, pol, env) is 33%. Is extremely easy to cross each other during detection. The rapid diagnosis test paper on the market at present is mainly based on non-parting detection.
Disclosure of Invention
The invention aims to overcome the defects that infection and wound are caused by blood test in the prior art, SPA protein is not easy to couple with colloidal gold and HIV-1 and HIV-2 can not be detected in a parting way, and provides a test strip for detecting HIV-1 and HIV-2 antibodies in urine by a colloidal gold method. In order to achieve the above purpose, the technical scheme of the invention is as follows:
a test strip for detecting HIV-1 and HIV-2 antibodies in urine by a colloidal gold method is characterized in that: the test paper strip comprises a gold mark pad, a nitrocellulose membrane coated with a detection line and a quality control line, a sample pad and filter paper; the gold mark pad is obtained by diluting SPA protein mark to working concentration, spraying the SPA protein mark on a polyester film soaked by Tris buffer solution, and drying.
The size and uniformity of the colloidal gold directly affect the stability and sensitivity of the product, and the inventor prepares the colloidal gold with the particle size of about 40-60nm through a large number of experiments.
To achieve the above colloidal gold, chloroauric acid, trisodium citrate, and glycine were used. The glycine can slow down the reaction speed of chloroauric acid and trisodium citrate, so that the formed gold particles are more uniform. Meanwhile, glycine helps SPA protein to be stably coupled to gold particles, and is not easy to fall off.
The SPA protein labeling method comprises the following steps: the pH of the colloidal gold is regulated to 7.0-9.0 by 0.01-1M potassium carbonate buffer solution, SPA protein is added in the proportion of 10-20 mu g/ml gold, after stirring reaction is carried out for 10-30 minutes, blocking is carried out by 0.1% -10% BSA, centrifugation is carried out for 10-30 minutes at 2-10 ℃ 10000rpm, and the supernatant is discarded. The precipitate is redissolved to 1-10% of original volume, and the redissolved solution is 50mM Tris-HCl (pH 7.0-8.5) buffer solution containing disodium hydrogen phosphate and BSA, casein, tween-20. Is used for protecting and stabilizing SPA marking liquid.
To assist in the curing of SPA colloidal gold conjugates on polyester films, the gold-labeled pads were used for gold-sprayed polyester films were soaked with 50mM Tris-HCl (pH 7.0-8.5) containing 0.01% -1% BSA and 0.1-2.5g/L dextran 40000 and dried.
The invention provides a three-line membrane dividing mode which is used for the purpose of HIV-1 and HIV-2 typing detection.
The detection line of the nitrocellulose membrane is coated with gp41 recombinant antigen capable of recognizing HIV-1 antibody and gp36 antigen capable of recognizing HIV-2 antibody, and the quality control line is coated with chicken anti-SPA protein.
The HIV-1 and the HIV-2 have 33 percent of amino acid sequences which are identical, so that the two are easy to cross, and the currently marketed products adopt a two-in-one detection mode. At present, HIV virus is mainly in HIV-1 type, in the invention, HIVgp36 is adopted in the middle, HIVgp41 is adopted at the lowest part, the dosage of the type II is reduced as much as possible in the use of the membrane concentration, and simultaneously, the non-specific combination of the two is reduced by using 50mM Tris-HCl (pH 7.0-8.5) buffer solution containing disodium hydrogen phosphate and BSA, casein, tween-20 as the membrane dilution. The control line used chicken anti-SPA protein and the membrane-dividing diluent was PBS.
Preferably, the nitrocellulose membrane detects gp41 recombinant antigen coated by the line, and the concentration is 0.8-2.5mg/ml; gp36 recombinant antigen coated on nitrocellulose membrane detection line with concentration of 0.4-2.5mg/ml; the nitrocellulose membrane control wire is coated with chicken anti-SPA protein, and the concentration is 0.8-2.5mg/ml.
The main components in urine are urea, salt and drug metabolites, wherein the salt is easy to destroy the colloidal gold chromatographic reagent, and the colloidal gold chromatographic reagent is mainly coupled with the colloidal gold, so that the colloidal gold running plate fails and stays on the NC membrane to form false positive. The drug metabolite also had the stability of the perineal colloidal gold and had run plate failure. The sample pad functions to process the sample in the test strip. The sample pad provided by the invention can well play an important role in regulating pH, filtering macromolecules and inorganic salts, and reducing false positive.
The sample pad treatment method comprises the following steps: glass fibers were prepared using a glass fiber containing BSA, tween-20,1% S9 and Na 2 CO 3 Is soaked in 50mM Tris-HCl (pH 7.0-8.5) buffer solution and dried.
The specific dosage is as follows: 100ml 10-50mM Tris-HCl buffer with pH of 8.0, tween-20 dosage of 1-10%, S9 dosage of 0.1-1g, BSA dosage of 1-10%,0.01-2M Na 2 CO 3 。
The sample pad is prepared by adopting the buffer solution system for treatment and adopting 45-degree drying for 12-24 hours.
The staphylococcus protein A (SPA protein) is a surface protein existing on the cell wall of staphylococcus, can be combined with human and various mammal IgG, and HIV antibodies in urine exist in the form of IgG, and can be specifically combined with the IgG. A colloidal gold conjugate pad is prepared by using SPA protein to be connected with colloid Jin Ou and using the SPA protein marked by colloidal gold as a chromogenic probe, and gp41 antigen and gp36 antigen are coated on a nitrocellulose membrane separately to be used as detection lines for detecting HIV 1 type antibodies and HIV 2 type antibodies. The HIV antibody in urine and SPA protein are combined and developed with gp41 or gp36 coated in NC membrane when passing through detection line, thus achieving the purpose of typing detection. Meanwhile, chicken anti-SPA is used as a control line film drawing raw material, and because SPA protein is fungus protein, the crossover and false positive caused by independent C lines are eliminated. By adopting a Tris-HCL buffer system, S9, tween-20 and Na are added 2 CO 3 The sample pad is prepared by treating glass fiber and drying, so that the problem of false positive caused by salt ions and drug metabolites in urine is well solved. The false positive is reduced, and the accuracy of the product is improved.
Compared with the prior art, the invention has the following advantages:
1. the invention creatively adopts urine as a sample, and detects HIV antibodies in the urine by a colloidal gold method, thereby avoiding infection and wound caused by blood detection.
2. The invention creatively adopts staphylococcus A protein as the specific recognition HIV antibody, can enrich rare HIV antibody IGg in urine, and improves the sensitivity.
Staphylococcus A is different from HIV coated antibody, so that specific binding is reduced, and the risk of false positive is reduced.
3. The gold conjugate pad was conjugated to colloid Jin Ou using staphylococcal protein a (SPA protein). The gold conjugate pad was conjugated to colloid Jin Ou using staphylococcal protein a (SPA protein). The invention creatively adopts chicken anti-SPA protein as a control line, directly captures SPA protein connected with colloid Jin Ou, 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.
4. In the prior art, the crossing of the HIV-1 and the HIV-2 is difficult to achieve, and the crossing of the HIV-1 and the HIV-2 is reduced by adjusting the membrane scribing diluent and the membrane scribing position.
5. The invention reduces false positive, improves the accuracy of the product, simplifies the operation and reduces the cost. The invention does not need other equipment, has high sensitivity, is simple to operate, obtains a result in 15 minutes, uses urine as a sample, can be used for large-scale screening, can also be used for personal self-test, and has wide application range.
6. The invention adopts four parts of sample pad, gold mark pad, NC film and filter paper to link, the sample pad has the function of processing sample, the gold mark pad carries marked Jin Biaoye, NC film coats the specific antigen GP41 for detecting HIV-1 and the specific antigen GP36 for detecting HIV-2 on the detection line, and coats chicken anti-SPA polyclonal antibody on the control line. The filter paper absorbs liquid to give the action of chromatographic power, and each part has clear functions and convenient composition. And, the sample pad is added with S9, tween-20 and Na by adopting a Tris-HCL buffer system 2 CO 3 The glass fiber is processed and dried for preparation, so that the problem of false positive caused by salt ions and drug metabolites in urine is well solved.
7. The invention adopts urine as a sample to detect HIV-1 and HIV-2 antibodies, completely adopts the nondestructive detection of HIV, and has the advantages of simple operation, simple interpretation, and no need of equipment and specialized training to complete self-detection. Improving the initiative of AIDS detection and enhancing the accessibility and convenience of AIDS detection. Can be used for large-scale primary screening of HIV.
Detailed Description
The invention will be further illustrated by the following detailed description
Example 1
1L of ultrapure water is taken in a beaker, placed on a heating magnetic stirrer and stirred at medium speed, 1ml of 10% chloroauric acid is added when the temperature is heated to 90 ℃, 1.45ml of 10% trisodium citrate is added after the solution is boiled, and after the solution is continuously heated for 15 minutes, the solution is cooled at room temperature to prepare the 60-80 nm colloidal gold 1. 1L of ultrapure water is taken in a beaker, placed on a heating magnetic stirrer and stirred at medium speed, when the temperature is heated to 90 ℃, 1ml of 10% chloroauric acid is added, then 0.008g of glycine is quickly added, after the solution is boiled, 1.45ml of 10% trisodium citrate is added, after the solution is heated for 15 minutes, the solution is cooled at room temperature, and then 60-80 nm of colloidal gold 2 is prepared.
Example 2
SPA protein is added in a proportion of 0.1M potassium carbonate buffer solution to adjust the pH of the colloidal gold 1 to 8.0 and 20 mu g/ml gold, after stirring and reacting for 30 minutes, blocking is carried out by 10% BSA, centrifugation is carried out at 2-10 ℃ 10000rpm for 10 minutes, the supernatant is discarded, the precipitate is redissolved to 1% of the original volume, and the redissolved solution is 50mM Tris-Hcl (pH 7.0-8.5) buffer solution containing disodium hydrogen phosphate and BSA, casein, tween-20.
Example 3
SPA protein is added in a proportion of 0.1M potassium carbonate buffer solution to adjust the pH of the colloidal gold 2 to 8.0 and 20 mu g/ml gold, after stirring and reacting for 30 minutes, blocking is carried out by 10% BSA, centrifugation is carried out at 2-10 ℃ 10000rpm for 10 minutes, the supernatant is discarded, the precipitate is redissolved to 1% of the original volume, and the redissolved solution is 50mM Tris-Hcl (pH 7.0-8.5) buffer solution containing disodium hydrogen phosphate and BSA, casein, tween-20.
Example 4
The polyester film was treated with 50mM Tris-HCl (pH 8.0) containing 0.01% BSA and 2g/L dextran 40000, and 1% (PVP-10) was added, and immersed in 0.1M sodium salt of complexing protein for 30min, and dried at 45℃for 18 hours.
Example 5
The polyester film was treated with 50mM Tris-HCl (pH 8.0) containing 1% (PVP-10) and 0.1M complex protein sodium salt, immersed for 30min and dried at 45℃for 18 hours.
Example 6
The membrane was scored using 0.5mg/ml HIVGP36 in the middle and 0.8mg/ml HIVGP41 in the bottom, with a 50mM Tris-HCl (pH 8.0) buffer containing 0.1M disodium hydrogen phosphate, 0.01% BSA, 0.1MCasein, 1% Tween-20. The control line used chicken anti-SPA protein and the membrane-dividing diluent was PBS.
Example 7
The membrane was scored with 0.5mg/ml HIVGP36 in the middle and 0.8mg/ml HIVGP41 in the lowest. The control line used chicken anti-SPA protein. The detection line and the control line are both PBS.
Example 8
The glass fibers in the sample pad were buffered with 100ml 10-50mM Tris-HCl buffer, pH8.0, 1% Tween-20,1%S9,1%BSA,0.01M Na 2 CO 3 Soaking in 1% PVP-10,1M complex protein sodium salt buffer solution for 30 min. Drying at 45 degrees for 18 hours.
Example 9
The glass fibers in the sample pad were soaked in 100ml of 10-50mM Tris-HCl buffer, pH8.0, 1% PVP-10,1M complex protein sodium salt buffer for 30 min. Drying at 45 degrees for 18 hours.
Example 10 comparison of different colloidal gold labelling effects
The test strip 1 prepared by assembling the embodiment 2.4.6.8 is used for testing the positive quality control and the negative urine of enterprises
Example 3.4.6.8 was assembled into test strip 2 using the Business cationic control and negative urine test
The test results are shown in Table 1:
| sample type | Test strip 1 chromaticity | Test strip 2 chromaticity |
| HIV-1 strong yang reference | G10*3 | G10*3 |
| Reference for HIV-1 | G4*1/G4.5*2 | G5*3 |
| Reference for HIV-1 weak yang | G2.5*2/G3*1 | G4*1/G3.5*2 |
| HIV-1 repetitive reference | G3.5*1/G3*1/G2*1 | G4*2/G4.5*1 |
| HIV-2 strong yang reference | G10*3 | G10*3 |
| Reference for HIV-2 | G3.5*1/G3*2 | G4*3 |
| Reference for HIV-2 weak yang | G1.5*2/G2*1 | G3*3 |
| HIV-2 repetitive reference | G3*2/G2*1 | G3*3 |
| N1 | G0*3 | G0*3 |
| N2 | G0*3 | G0*3 |
| N3 | G0*3 | G0*3 |
| N4 | G0*3 | G0*3 |
| N5 | G0*3 | G0*3 |
The larger the chromaticity number, the stronger the color development.
The result test strip 2 is prepared by using a colloidal gold containing glycine, and has higher color rendering degree and higher sensitivity when a weak positive reference sample is tested. Test strip 2 performed better in uniformity when testing for a repetitive reference. The poor uniformity is probably due to uneven SPA protein on the surface of the gold particles, and the possibility of falling off, and the colloidal gold containing glycine ensures that the SPA protein is combined with the gold particles more stably and the running plate is more uniform.
Example 11 comparison of the effects of different polyester film treatments
Example 3.4.6.8 was assembled into test strip 3 using the Business cationic control and negative urine test
Example 3.5.6.8 was assembled into test strip 4 using the Business cationic control and negative urine test
The test results are shown in Table 2:
the larger the chromaticity number, the stronger the color development.
As a result, test strip 4 showed color development of HIV-2 when tested as a positive reference for HIV-1, and crossed the two. Test strip 3 showed no cross-bars when tested for positive reference. Description the polyester film treating solution of example 4 can eliminate the crossover phenomenon between the two subtypes.
Example 12 comparison of the effects of different marking fluids
Example 3.4.6.8 was assembled into test strip 5 using the Business cationic control and negative urine test
Example 3.4.7.8 was assembled into test strip 6 using the Business cationic control and negative urine test
The test results are shown in Table 3:
the larger the chromaticity number, the stronger the color development.
As a result, test strip 6 showed color development of HIV-2 when tested as a positive reference for HIV-1, and crossed the two. HIV-2 presents false positive, and test strip 5 does not present a cross line when testing positive reference. Illustration the cross-over and false positive between the two subtypes was eliminated by the streak dilution in example 6.
Example 13 comparison of the effects of different sample pad treatments
Example 3.4.6.8 was assembled into test strip 7 using the Business cationic control and negative urine test
Example 3.4.6.9 was assembled into test strip 8 using the Business cationic control and negative urine test
The test results are shown in Table 4:
the larger the chromaticity number, the stronger the color development.
As a result, test strip 8 showed color development of HIV-2 when tested as a positive reference for HIV-1, and crossed the two. Both HIV-1 and HIV-2 showed false positive, and test strip 7 showed no cross-bars when tested as a positive reference. Illustration the sample pad treated with the glass fiber treatment fluid of example 8 eliminates crossover and false positive between the two subtypes. And the sensitivity of the test strip is not affected.
Effect confirmation
1. National reference test
The test strip 7 prepared by the invention is used for testing human immunodeficiency virus antibody urine from national food and drug verification institute batch number 220020-2005041502 in national reference, and the result is shown in table 5:
the result shows that the sensitivity of the test strip 7 for detecting the human immunodeficiency virus antibody urine national reference is 100%, the specificity is 100%, and the test strip meets the detection standard of Chinese food and drug detection institute.
2. Clinical sample test of the test strip 7 manufactured by the invention
20 HIV samples confirmed by immunoblotting are obtained from a certain HIV fixed-point hospital (test results are shown in Table 6), and 100 negative urine samples are collected from the inside of the company (physical examination report shows that HIV negative population including non-AIDS population samples such as immune diseases and common cold) are confirmed (test results are shown in Table 7)
Table 6:
| positive sample | HIV-1 | HIV-2 | Positive sample | HIV-1 | HIV-2 |
| 1 | G10 | G0 | 11 | G6 | G0 |
| 2 | G6 | G0 | 12 | G5 | G0 |
| 3 | G6 | G0 | 13 | G10 | G0 |
| 4 | G7 | G0 | 14 | G10 | G0 |
| 5 | G7 | G0 | 15 | G8 | G0 |
| 6 | G5 | G0 | 16 | G8 | G0 |
| 7 | G8 | G0 | 17 | G9 | G0 |
| 8 | G8 | G0 | 18 | G7 | G0 |
| 9 | G7 | G0 | 19 | G10 | G0 |
| 10 | G8 | G0 | 20 | G10 | G0 |
Table 7:
the summary of the clinical results is shown in Table 8:
the results show that the test of small batches of clinical samples shows that the positive coincidence rate is 100 percent, the negative coincidence rate is 100 percent, and the expected requirement is met. Also illustrates that the invention can effectively eliminate and reduce false positive and improve the accuracy of the product
The invention reduces false positive, improves the accuracy of the product, simplifies the operation and reduces the cost. The invention does not need other equipment, has high sensitivity, is simple to operate, obtains a result in 15 minutes, uses urine as a sample, can be used for large-scale screening, can also be used for personal self-test, and has wide application range.
Claims (1)
1. A test strip for detecting HIV-1 and HIV-2 antibodies in urine by a colloidal gold method is characterized in that: the preparation method comprises the following steps:
adjusting the pH of colloidal gold to 8.0 by using 0.1M potassium carbonate buffer solution, adding SPA protein in the proportion of 20 mug/ml colloidal gold, stirring for reacting for 30 minutes, blocking by using 10% BSA, centrifuging at 2-10 ℃ 10000rpm for 10 minutes, discarding supernatant, and redissolving the precipitate to 1% of original volume, wherein the redissolved solution is 50mM Tris-Hcl buffer solution containing disodium hydrogen phosphate and BSA, casein, tween-20, and the pH is 7.0-8.5;
the polyester film was treated with 50mM Tris-HCl containing 0.01% BSA and 2g/L dextran 40000, pH=8.0, and immersed in 1% PVP-10,0.1M complex protein sodium salt for 30min, oven dried at 45℃for 18 hours;
in the membrane separation, 0.5mg/ml of HIVgp36 is arranged in the middle, 0.8mg/ml of HIVgp41 is arranged at the bottom, and the membrane separation diluent is prepared into 50mM Tris-HCl buffer solution containing 0.1M disodium hydrogen phosphate, 0.01% BSA, 0.1MCasein and 1% Tween-20, wherein the pH=8.0; the control line adopts chicken anti-SPA protein, and the membrane drawing diluent is PBS;
the glass fibers in the sample pad were buffered with 100ml 10-50mM Tris-HCl buffer, pH8.0, 1% Tween-20,1%S9,1%BSA,0.01M Na 2 CO 3 1% PVP-10,1M complex protein sodium salt buffer solution is soaked for 30 min; drying at 45 ℃ for 18 hours;
the preparation method of the colloidal gold comprises the steps of taking 1L of ultrapure water in a beaker, placing the ultrapure water on a heating magnetic stirrer, stirring at medium speed, heating to 90 ℃, adding 1ml of 10% chloroauric acid, then rapidly adding 0.008g glycine, adding 1.45ml of 10% trisodium citrate after the solution is boiled, continuously heating for 15 minutes, and cooling at room temperature to prepare the 60-80 nm colloidal gold.
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| CN116500260A (en) * | 2022-01-18 | 2023-07-28 | 广州达安基因股份有限公司 | Test strip, test card and test method for rapid qualitative detection of immunodeficiency virus antigen and antibody |
| CN116539876A (en) * | 2023-04-08 | 2023-08-04 | 南京申基医药科技有限公司 | Kit for detecting human immunodeficiency virus P24 antigen and antibody based on latex method and preparation method thereof |
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| BRPI0520182A2 (en) * | 2005-05-20 | 2011-04-05 | Calypte Biomedical Corp | rapid immunochromatographic test of oral fluid |
| CN101266247A (en) * | 2008-04-30 | 2008-09-17 | 天津中新科炬生物制药有限公司 | HIV antibody immune magnetic beads rapid detection reagent kit |
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| CN111562366A (en) * | 2020-06-15 | 2020-08-21 | 郑州方欣生物科技有限责任公司 | Urine HIV antibody rapid detection kit, preparation method and use method |
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