CN116298279A - Kit for detecting human immunodeficiency virus antibody in urine based on colloidal gold method - Google Patents
Kit for detecting human immunodeficiency virus antibody in urine based on colloidal gold method Download PDFInfo
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- CN116298279A CN116298279A CN202310585859.2A CN202310585859A CN116298279A CN 116298279 A CN116298279 A CN 116298279A CN 202310585859 A CN202310585859 A CN 202310585859A CN 116298279 A CN116298279 A CN 116298279A
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- OCKGFTQIICXDQW-ZEQRLZLVSA-N 5-[(1r)-1-hydroxy-2-[4-[(2r)-2-hydroxy-2-(4-methyl-1-oxo-3h-2-benzofuran-5-yl)ethyl]piperazin-1-yl]ethyl]-4-methyl-3h-2-benzofuran-1-one Chemical compound C1=C2C(=O)OCC2=C(C)C([C@@H](O)CN2CCN(CC2)C[C@H](O)C2=CC=C3C(=O)OCC3=C2C)=C1 OCKGFTQIICXDQW-ZEQRLZLVSA-N 0.000 description 1
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- AIDS & HIV (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The application relates to the technical field of biomedical detection, in particular to a kit for detecting human immunodeficiency virus antibodies in urine based on a colloidal gold method; the test strip comprises a bottom plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped and stuck on the bottom plate according to the chromatographic direction, a quality control line and a detection line are coated on the nitrocellulose membrane, a goat anti-chicken IgY is coated on the quality control line, SPA protein is coated on the detection line, and a colloidal gold-labeled GP160 antigen and GP41, GP36 and GP120 mixed antigen are coated on the colloidal gold pad; the invention uses colloidal gold as a carrier, is convenient and quick to detect, can complete result interpretation without the help of related instruments and equipment, and has higher sensitivity.
Description
Technical Field
The application relates to the technical field of biomedical detection, in particular to a kit for detecting human immunodeficiency virus antibodies in urine based on a colloidal gold method.
Background
AIDS is an acquired immunodeficiency syndrome caused by human immunodeficiency virus (human immune deficiency virus, HIV), a progressive immune deficiency characterized by a decrease in human CD4+ T lymphocytes, a complex disorder that can be secondary to various opportunistic infections, malignancies and central nervous system lesions in the later stages of the disease. So far, there is no effective therapeutic scheme and prevention and control vaccine for AIDS in medical field, so the prevention and control of AIDS depends on accurate identification and diagnosis in early infection stage.
The presence of antibodies specific for HIV in urine has been studied and demonstrated in large numbers, but the content of antibodies to HIV in urine is about 0.1 to 1% of the content in blood, which is slightly less than that in serum, and therefore the sensitivity requirements for the detection of antibodies in HIV urine are high.
At present, some manufacturers in China develop human immunodeficiency virus urine antibody detection kits, and an enzyme-linked immunosorbent assay and a colloidal gold method are adopted; the ELISA method has complex operation steps, long time consumption and the like, and an operator can be qualified through professional training, so that the use of common users is limited; the colloidal gold method is generally low in sensitivity due to the limitation of the size of the colloidal gold particles of the carrier.
Therefore, the human immunodeficiency virus urine antibody detection kit produced in the prior art cannot meet the requirement of rapid differential diagnosis.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a test strip and a kit for detecting human immunodeficiency virus antibodies based on a colloidal gold method, which have the advantages of convenient use, simple operation and convenient popularization, and the detection sample is urine, is subjected to noninvasive sampling, and avoids cross infection caused by using modes such as blood sampling and the like.
In a first aspect, the present application provides a test strip for detecting antibodies of human immunodeficiency virus in urine based on a colloidal gold method, which adopts the following technical scheme:
a test strip for detecting human immunodeficiency virus antibodies in urine based on a colloidal gold method comprises a bottom plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped and stuck on the bottom plate according to the chromatographic direction, a quality control line and a detection line are coated on the nitrocellulose membrane, a goat anti-chicken IgY is coated on the quality control line, SPA protein is coated on the detection line, and a colloidal gold-labeled GP160 antigen and a GP41, GP36 and GP120 mixed antigen are coated on the colloidal gold pad.
The invention adopts indirect principle to qualitatively detect the free HIV1/2 antibody in human urine.
When the HIV1/2 antibody exists in the sample during detection, the HIV1/2 antibody in the sample reacts with the mixed antigen of the colloidal gold marked GP41, GP36 and GP120 and the GP160 antigen on the colloidal gold pad to form a marked antigen-antibody complex, the complex is upwards chromatographed by capillary action, and is captured by a detection line (SPA protein) coated on a nitrocellulose membrane, and a red strip appears; the compound continues to be chromatographed upwards, is captured by a quality control line (C line) antibody (sheep anti-chicken IgY antibody) coated on a nitrocellulose membrane, and has red stripes; when the content of the to-be-detected object in the sample is lower than the lowest detection limit, the detection line does not develop color.
Preferably, the preparation process of the colloidal gold-labeled GP160 antigen and the mixed antigen of GP41, GP36 and GP120 is as follows:
(1) Adjusting the pH of the colloidal gold solution to 7.5, and adding HIV tag protein for mixing; adjusting the pH value of the solution to 8-9, adding the reactant, the GP41, the GP36 and the GP120 mixed antigen and mixing the GP160 antigens;
(2) Adding the blocking solution to mix, and obtaining the GP160 antigen marked by colloidal gold and the GP41, GP36 and GP120 mixed antigen.
Preferably, the addition amount of the colloidal gold solution in the step (1) is 8-12mL; the addition amount of HIV label protein is 150-200 mug; the addition amount of the reactant is 0.1-0.5mL; the addition amount of the GP41, GP36 and GP120 mixed antigen is 30-40 mug; the amount of GP160 antigen added is 30-40 μg.
Preferably, the blocking solution is added in the amount of 0.5-1mL in the step (2).
Preferably, the HIV label protein is added in the step (1) for reaction for 8-15min; adding the reactant, the GP41, the GP36 and the GP120 mixed antigen and the GP160 antigen for 8-15min; and (3) adding a sealing liquid in the step (2) for reaction for 10-20min.
Preferably, 0.2M potassium carbonate is added in the steps (1) and (2) to adjust the pH.
The added HIV tag protein is obtained by performing immune-purification-clone culture-purification-excision of the mixed antigen of GP41, GP36 and GP120 and the GP160 antigen, and the added HIV tag protein is only combined with the mixed antigen of GP41, GP36 and GP120 and the GP160 antigen in a specific way and is not combined with other proteins, so that the specificity is enhanced; colloid Jin Xian is coupled with tag proteins, and GP41, GP36 and GP120 mixed antigen and GP160 antigen are combined through the tag proteins, so that a reaction arm is prolonged, steric hindrance is reduced, specific combination of antigen and antibody is facilitated, and the sensitivity of a product is improved.
Before adding reactants, GP41, GP36 and GP120 mixed antigen and GP160 antigen for mixing, adjusting the pH value of the solution to 8-9, and adjusting the pH value of the solution to 8-9 by adding potassium carbonate, wherein the pH value of the solution is higher than isoelectric points of the GP41, GP36 and GP120 mixed antigen and the GP160 antigen, so that the GP41, GP36 and GP120 mixed antigen and the GP160 antigen are only combined with HIV tag proteins and are not combined with colloidal gold, namely, the combined sites of the colloid Jin Weijie are not combined with the GP41, GP36 and GP120 mixed antigen and the GP160 antigen; but also does not influence the GP41, GP36 and GP120 mixed antigen, GP160 antigen and HIV tag protein to improve the sensitivity of the product through specific combination.
Preferably, the reactant in the step (1) comprises the following raw materials in mass concentration: 50g/L-100g/L Tween-20, 50g/L-100g/L surfactant S22.
The tween-20 added in the method can elute nonspecific adsorption, reduce aggregation of colloidal gold conjugate particles and improve product specificity; the added surfactant S22 can increase the solubility of the protein in the solution, the protein in unit volume is more uniformly dispersed, the binding efficiency is higher, and the product sensitivity is improved.
Preferably, the mixed antigen of the colloidal gold-labeled GP160 antigen and GP41, GP36 and GP120 prepared in the step (2) is preserved by a preservation solution, wherein the preservation solution comprises the following raw materials in mass concentration: 2.4g/L-6g/L Tris, 5g/L-10g/L BSA, 100g/L-200g/L sucrose, 5g/L-10g/L MPC.
According to the preservation solution formula, the signal enhancer MPC is added, the MPC is an amphoteric copolymer, and has good biocompatibility and protein adsorption resistance, so that nonspecific adsorption can be reduced, and the sensitivity and specificity of a product are improved.
In a specific embodiment, a method for preparing a kit for detecting antibodies to human immunodeficiency virus in urine based on a colloidal gold method comprises the following steps:
step one, preparing a nitrocellulose membrane: diluting SPA protein and sheep anti-chicken IgY to 0.5-1.2mg/mL with coating liquid, sequentially coating on the corresponding position of nitrocellulose membrane with a film drawing instrument, and drying at 45deg.C for 2 hr;
wherein, the coating liquid comprises the following raw materials in mass concentration: 2.4g/L Tris (pH 8.0), 20g/L trehalose.
Step two, a preparation method of the colloidal gold-labeled GP160 antigen and the mixed antigen (colloidal gold conjugate) of GP41, GP36 and GP120 comprises the following steps: taking 8-12mL of colloidal gold solution in a beaker, regulating the pH value to 7.5 by using 0.2M potassium carbonate solution, adding 150-200 mu g of HIV tag protein, and stirring at room temperature for reacting for 8-15min to obtain solution A; continuously regulating the pH of the solution A to 8-9 by using 0.2M potassium carbonate solution, adding 0.1-0.5mL of reactant, 30-40 mug GP41, GP36 and GP120 mixed antigen and 30-40 mug GP160 antigen, and stirring and reacting for 8-15min at room temperature. Centrifuging (at 4deg.C, rotational speed 8000-10000rpm,15 min), removing supernatant, adding 0.5-1mL of blocking solution, sealing for 10-20min, centrifuging (at 4deg.C, rotational speed 8000-10000rpm,15 min), removing supernatant, adding 1mL of preserving solution, and redissolving to obtain colloidal gold labeled GP160 antigen and GP41, GP36, GP120 mixed antigen (colloidal gold conjugate).
Wherein, the sealing liquid comprises the following raw materials in mass concentration: 2.13g/L disodium hydrogen phosphate, 0.456g/L sodium dihydrogen phosphate, 10g/L BSA.
Step three, a preparation method of the colloidal gold pad comprises the following steps: and diluting the conjugate marked by the colloidal gold by 20 times, soaking the blank pad, taking out and drying at 37 ℃ to obtain the colloidal gold pad.
Step four, sample pad treatment: soaking the blank gasket in sample pad treatment liquid, taking out and drying at 37 ℃, wherein the formula of the sample pad treatment liquid is as follows: 12-24g/L Tris (pH 8.0), 5g/L Casein, 10g/L surfactant S9, 2.5g/L EDTA.
Step five, assembling a test strip: and sequentially overlapping and adhering the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad on the PVC bottom plate according to the chromatographic direction, and cutting according to the width requirement to obtain the test paper strip.
Preferably, the colloidal gold solution in the step (1) is a concentrated colloidal gold solution, and the preparation process thereof is as follows:
s1, mixing PEG with water;
s2, adding chloroauric acid solution, mixing, and heating to boil;
s3, adding a trisodium citrate solution to react to obtain a colloidal gold solution.
Preferably, the PEG in step S1 is added in an amount of 0.8-2g; the water addition amount is 50-150mL; the adding amount of chloroauric acid solution in the step S2 is 4-12mL; the adding amount of the trisodium citrate solution in the step S3 is 3.2-12mL.
Preferably, in step S1, the reaction time is 8-12 mm; in the step S2, the reaction is carried out for 1-2min after heating to boiling.
In a specific embodiment, the colloidal gold solution (concentrated colloidal gold solution) is prepared as follows:
s1, weighing 0.8-2g of PEG in a beaker, adding 50-150mL of purified water, stirring and uniformly mixing for 10min;
s2, transferring the solution of the S1 into a 250mL round-bottom flask, adding 4-12mL chloroauric acid solution with the concentration of 10g/L, placing the round-bottom flask in an oil bath pot, setting the oil temperature to 120 ℃, turning on a condensing device at the speed of 250rpm, heating to boil, and maintaining the boiling state for 2min;
s3, adding 3.2-12mL of trisodium citrate solution with the concentration of 20g/L, regulating the rotating speed to 150rpm after the solution changes color, closing a heating mode after the solution changes into purplish red, waiting for 15min, taking out the round bottom beaker, placing the round bottom beaker on a heat insulation sleeve for natural cooling, and collecting the solution in the round bottom flask in a brown reagent bottle to obtain the colloidal gold solution.
The concentration of gold particles in the concentrated colloidal gold solution is higher, the adding amount of chloroauric acid is more in the preparation process of the colloidal gold, the reaction process of the reducing agent (trisodium citrate) and the chloroauric acid is very uneven, the state of the whole system is very unstable, and gold particles with uniform and stable sizes are difficult to form.
In this application, PEG is added prior to the addition of chloroauric acid and a reducing agent (trisodium citrate). In the reaction process, PEG can be used as a protective agent to effectively prevent particle agglomeration and the prepared gold sol has good dispersibility. Au3 ＋ The gold particles and PEG form a coordination complex to generate electrostatic steric hindrance, so that aggregation in the gold particle forming process is reduced, hydrophobic acting force is formed between the formed gold particles and the PEG, the gold particles are well suspended in the solution, aggregation among the gold particles is avoided, and finally the gold particles with uniform and stable sizes are formed.
In addition, the PEG can prevent excessive trisodium citrate from changing the weak acidic environment of the system and maintain constant reaction efficiency.
In the method, PEG is added in advance before colloidal gold is prepared, so that gold particles with uniform and stable size can be obtained, and the prepared colloidal gold can be stored at 2-8 ℃ for a long time.
By adopting the method, the colloidal gold solution with higher multiple can be prepared, and the product prepared by adopting the colloidal gold solution (namely the concentrated colloidal gold solution) is superior to the prior art in sensitivity and specificity.
In a second aspect, the present application provides a kit, which adopts the following technical scheme:
a kit comprises the test strip.
In summary, the present application includes at least one of the following beneficial technical effects:
1. the kit for detecting the human immunodeficiency virus antibody provided by the invention can be used for directly detecting urine samples, performing noninvasive sampling, and avoiding cross infection caused by using modes such as blood sampling.
2. The invention uses colloidal gold as a carrier, is convenient and quick to detect, can complete result interpretation without the help of related instruments and equipment, and has high sensitivity and good specificity.
3. The MPC is added into the preservation solution, so that the nonspecific adsorption of the product can be reduced, and the sensitivity and specificity of the product can be improved.
4. The invention can prepare the colloidal gold solution with higher multiple through improving the colloidal gold preparation process, and the product prepared by adopting the colloidal gold solution has better sensitivity and specificity.
Detailed Description
All adopt the commercial product to the raw materials that this application relates to, wherein:
HIV tag proteins were purchased from: jiangsu Shenji Biotech Co., ltd., product model: t1.
Sheep anti-chicken IgY was purchased from: keqiao (Xiamen) Biotechnology Co.
GP41, GP36, GP120 mixed antigen was purchased from: jiangsu Shenji Biotech Co., ltd., product model: HIV recombinant antigen III (GP 41, GP36, GP120 mixed antigen).
Abbreviations and key term definitions referred to in this embodiment:
BSA: bovine serum albumin;
casein: casein;
tris: tris (hydroxymethyl) aminomethane;
CTAB: cetyl trimethyl ammonium bromide;
PVP40: polyvinylpyrrolidone 40000;
MPC: 2-methacryloyloxyethyl phosphate (acyl) choline;
SDS: sodium dodecyl sulfate;
PEG20000: polyethylene glycol 20000;
PVA: polyvinyl alcohol;
PEG: polyethylene glycol.
The present application is described in further detail below in connection with examples and comparative examples.
Example 1, kit for detecting human immunodeficiency Virus in urine based on colloidal gold method
1. Preparing a solution:
0.2M potassium carbonate: 2.76g of potassium carbonate is placed in a beaker, 100mL of purified water is added, and after stirring and mixing, the mixture is preserved at 4 ℃ for standby.
BSA preparation: 10g of BSA was weighed into a beaker, 100mL of purified water was added thereto, and after stirring and mixing, the mixture was kept at 4℃for further use.
Coating liquid preparation: 2.4g of Tris (pH 8.0) and 20g of trehalose are weighed, 1L of purified water is added into a beaker, and after stirring and mixing uniformly, the pH is adjusted to 8 by using an HCL solution, and the mixture is preserved at 4 ℃ for standby.
And (3) preparing a reactant: 5g of Tween-20 and 5g of surfactant S22 are weighed into a beaker, 100mL of purified water is added, and after stirring and mixing uniformly, the mixture is preserved at 4 ℃ for standby.
Preparing a sealing liquid: 0.213g of disodium hydrogen phosphate and 0.0456 g of sodium dihydrogen phosphate are weighed into a beaker, 1g of BSA is added with 100mL of purified water, and after being stirred uniformly, the mixture is preserved at 4 ℃ for standby.
Preparing a preservation solution: 2.4g of Tris (pH 8.0), 10g of BSA, 100g of sucrose and 5g of MPC were weighed into a beaker, 1L of purified water was added, and after stirring and mixing, the pH was adjusted to 8 with HCL solution and stored at 4℃for further use.
Sample pad treatment liquid: 24g of Tris (pH 8.0), 5g of casein, 10g of surfactant S9 and 2.5g of EDTA are weighed, 1L of purified water is added, and after stirring and mixing, the pH is adjusted to 8 by using an HCL solution, and the mixture is preserved at 4 ℃ for later use.
2. Preparation of colloidal gold solution (concentrated colloidal gold solution)
(1) Weighing 1g of PEG in a beaker, adding 100mL of purified water, stirring at room temperature, and mixing for 10min;
(2) Transferring the solution in the step (1) into a 250mL round-bottom flask, adding 10mL chloroauric acid solution with the concentration of 10g/L, placing the round-bottom flask in an oil bath, setting the oil temperature to 120 ℃, turning on the rotating speed to 250rpm, switching on a condensing device, heating to boiling, and keeping the boiling state for 2min;
(3) Adding 11mL of trisodium citrate solution with the concentration of 20g/L, regulating the rotating speed to 150rpm after the solution changes color, closing a heating mode after the solution changes into purplish red, waiting for 15min, taking out a round bottom beaker, placing the round bottom beaker on a heat insulation sleeve for natural cooling, and collecting the solution in the round bottom flask in a brown reagent bottle to obtain the colloidal gold solution.
3. Preparation of nitrocellulose membrane:
diluting SPA protein to 1mg/mL with coating liquid, and coating the SPA protein on the detection line position of the nitrocellulose membrane with a parameter of 1uL/cm by using a film drawing instrument;
diluting sheep anti-chicken IgY into 1mg/mL with coating liquid, and coating the IgY on a quality control line position on a nitrocellulose membrane with parameters of 1uL/cm by using a membrane drawing instrument;
the coated film was placed in a 45 ℃ oven and dried for 2 hours.
4. The preparation method of the colloidal gold conjugate comprises the following steps:
the preparation method of the colloidal gold conjugate comprises the following steps: taking 10mL of colloidal gold solution in a beaker, regulating the pH value to 7.5 by using 0.2M potassium carbonate solution, adding 150 mu g of HIV (human immunodeficiency Virus) tag protein, and stirring at room temperature for reacting for 10min to obtain solution A; continuously adjusting the pH value of the solution A to 8.5 by using 0.2M potassium carbonate solution, adding 0.2mL of reactant, uniformly mixing, adding 30 mug GP41, GP36 and GP120 mixed antigen and 30 mug GP160 antigen, and stirring and reacting for 10min at room temperature. Centrifuging (temperature 4 ℃ C., rotating speed 8500rpm,15 min), discarding supernatant, adding 1mL of sealing liquid, sealing for 20min, centrifuging (temperature 4 ℃ C., rotating speed 8500rpm,15 min), discarding supernatant, adding 1mL of preservation liquid, and redissolving to obtain the colloidal gold-labeled GP160 antigen and GP41, GP36 and GP120 mixed antigen (colloidal gold conjugate).
5. The preparation method of the colloidal gold pad comprises the following steps:
diluting the conjugate marked by the colloidal gold (GP 160 antigen marked by the colloidal gold and GP41, GP36 and GP120 mixed antigen) by 20 times with preservation solution, soaking the blank pad, taking out and drying at 37 ℃ to obtain the colloidal gold pad.
6. Sample pad treatment: the blank pad is soaked in sample pad treatment liquid, taken out and dried at 37 ℃.
7. Assembling a test strip: and sequentially overlapping and adhering the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad on the PVC bottom plate according to the chromatographic direction, and cutting into test strips with the width of 3 mm.
Example 2A kit for detecting human immunodeficiency Virus in urine based on colloidal gold method
1. Preparing a solution:
0.2M potassium carbonate: 2.76g of potassium carbonate is placed in a beaker, 100mL of purified water is added, and after stirring and mixing, the mixture is preserved at 4 ℃ for standby.
BSA preparation: 10g of BSA was weighed into a beaker, 100mL of purified water was added thereto, and after stirring and mixing, the mixture was kept at 4℃for further use.
Coating liquid preparation: 2.4g of Tris (pH 8.0) and 20g of trehalose are weighed, 1L of purified water is added into a beaker, and after stirring and mixing uniformly, the pH is adjusted to 8.0,4 ℃ by using an HCL solution and the mixture is preserved for standby.
And (3) preparing a reactant: 10g of Tween-20 and 10g of surfactant S22 are weighed into a beaker, 100mL of purified water is added, and after stirring and mixing uniformly, the mixture is preserved at 4 ℃ for standby.
Preparing a sealing liquid: 0.213g of disodium hydrogen phosphate and 0.0456 g of sodium dihydrogen phosphate are weighed into a beaker, 1g of BSA is added with 100mL of purified water, and after being stirred uniformly, the mixture is preserved at 4 ℃ for standby.
Preparing a preservation solution: 2.4g of Tris (pH 8.0), 10g of BSA, 100g of sucrose and 10g of MPC are weighed into a beaker, 1L of purified water is added, the mixture is stirred and mixed uniformly, the pH is adjusted to 8 by using an HCL solution, and the mixture is preserved at 4 ℃ for later use.
Sample pad treatment liquid: 12g of Tris (pH 8.0), 5g of casein, 10g of surfactant S9 and 2.5g of EDTA are weighed, 1L of purified water is added, and after stirring and mixing, the pH is adjusted to 8.0,4 ℃ by using an HCL solution and the mixture is preserved for later use.
2. Preparation of colloidal gold solution (concentrated colloidal gold solution)
(1) 1.2g of PEG is weighed into a beaker, and 100mL of purified water is added into the beaker to be stirred and mixed uniformly for 10min;
(2) Transferring the solution in the step (1) into a 250mL round-bottom flask, adding 10mL chloroauric acid solution with the concentration of 10g/L, placing the round-bottom flask in an oil bath, setting the oil temperature to 120 ℃, turning on the rotating speed to 250rpm, switching on a condensing device, heating to boil, and maintaining the boiling state for 2min;
(3) Adding 10mL of trisodium citrate solution with the concentration of 20g/L, regulating the rotating speed to 150rpm after the solution changes color, closing a heating mode after the solution changes into purplish red, waiting for 15min, taking out a round bottom beaker, placing the round bottom beaker on a heat insulation sleeve for natural cooling, and collecting the solution in the round bottom flask in a brown reagent bottle to obtain the colloidal gold solution.
3. Preparation of nitrocellulose membrane:
diluting SPA protein to 0.8mg/mL with coating liquid, and coating the SPA protein on the detection line position of the nitrocellulose membrane with a parameter of 1uL/cm by using a film drawing instrument;
diluting sheep anti-chicken IgY into 1mg/mL with coating liquid, and coating the IgY on a quality control line position on a nitrocellulose membrane with parameters of 1uL/cm by using a membrane drawing instrument;
the coated film was placed in a 45 ℃ oven and dried for 2 hours.
4. The preparation method of the colloidal gold conjugate comprises the following steps:
the preparation method of the colloidal gold conjugate comprises the following steps: taking 10mL of colloidal gold solution in a beaker, regulating the pH value to 7.5 by using 0.2M potassium carbonate solution, adding 200 mu g of HIV tag protein, and stirring at room temperature for reacting for 10min to obtain solution A; continuously adjusting the pH value of the solution A to 8.5 by using 0.2M potassium carbonate solution, adding 0.5mL of reactant, uniformly mixing, adding 40 mug GP41, GP36 and GP120 mixed antigen and 40 mug GP160 antigen, and stirring and reacting for 10min at room temperature. Centrifuging (temperature 4 ℃ C., rotating speed 8500rpm,15 min), discarding supernatant, adding 1mL of sealing liquid, sealing for 20min, centrifuging (temperature 4 ℃ C., rotating speed 8500rpm,15 min), discarding supernatant, adding 1mL of preservation liquid, and redissolving to obtain the colloidal gold-labeled GP160 antigen and GP41, GP36 and GP120 mixed antigen (colloidal gold conjugate).
5. The preparation method of the colloidal gold pad comprises the following steps:
diluting the conjugate marked by the colloidal gold (GP 160 antigen marked by the colloidal gold and GP41, GP36 and GP120 mixed antigen) by 20 times with preservation solution, soaking the blank pad, taking out and drying at 37 ℃ to obtain the colloidal gold pad.
6. Sample pad treatment: the blank pad is soaked in sample pad treatment liquid, taken out and dried at 37 ℃.
7. Assembling a test strip: and sequentially overlapping and adhering the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad on the PVC bottom plate according to the chromatographic direction, and cutting into test strips with the width of 3 mm.
Example 3A kit for detecting human immunodeficiency Virus in urine based on colloidal gold method
1. Preparing a solution:
0.2M potassium carbonate: 2.76g of potassium carbonate is placed in a beaker, 100mL of purified water is added, and after stirring and mixing, the mixture is preserved at 4 ℃ for standby.
BSA preparation: 10g of BSA was weighed into a beaker, 100mL of purified water was added thereto, and after stirring and mixing, the mixture was kept at 4℃for further use.
Coating liquid preparation: 2.4g of Tris (pH 8.0) and 20g of trehalose are weighed, 1L of purified water is added into a beaker, and after stirring and mixing uniformly, the pH is adjusted to 8 by using an HCL solution, and the mixture is preserved at 4 ℃ for standby.
And (3) preparing a reactant: 8g of Tween-20 and 8g of surfactant S22 are weighed into a beaker, 100mL of purified water is added, and after stirring and mixing uniformly, the mixture is preserved at 4 ℃ for standby.
Preparing a sealing liquid: 0.213g of disodium hydrogen phosphate and 0.0456 g of sodium dihydrogen phosphate are weighed into a beaker, 1g of BSA is added with 100mL of purified water, and after being stirred uniformly, the mixture is preserved at 4 ℃ for standby.
Preparing a preservation solution: 2.4g of Tris (pH 8.0), 10g of BSA, 100g of sucrose and 7.5g of MPC were weighed into a beaker, 1L of purified water was added, and after stirring and mixing, the pH was adjusted to 8 with HCL solution and stored at 4℃for further use.
Sample pad treatment liquid: 18g of Tris (pH 8.0), 5g of Casein, 10g of surfactant S9 and 2.5g of EDTA are weighed, 1L of purified water is added, and after stirring and mixing, the pH is adjusted to 8 by using an HCL solution, and the mixture is preserved at 4 ℃ for later use.
2. Preparation of colloidal gold solution (concentrated colloidal gold solution)
(1) 1.5g of PEG is weighed into a beaker, and 100mL of purified water is added into the beaker to be stirred and mixed uniformly for 10min;
(2) Transferring the solution in the step (1) into a 250mL round-bottom flask, adding 12mL chloroauric acid solution with the concentration of 10g/L, placing the round-bottom flask in an oil bath, setting the oil temperature to 120 ℃, turning on the rotating speed to 250rpm, switching on a condensing device, heating to boil, and maintaining the boiling state for 2min;
(3) Adding 12mL of trisodium citrate solution with the concentration of 20g/L, regulating the rotating speed to 150rpm after the solution changes color, closing a heating mode after the solution changes into purplish red, waiting for 15min, taking out a round bottom beaker, placing the round bottom beaker on a heat insulation sleeve for natural cooling, and collecting the solution in the round bottom flask in a brown reagent bottle to obtain the colloidal gold solution.
3. Preparation of nitrocellulose membrane:
diluting SPA protein to 1mg/mL with coating liquid, and coating the SPA protein on the detection line position of the nitrocellulose membrane with a parameter of 1uL/cm by using a film drawing instrument;
diluting sheep anti-chicken IgY into 1mg/mL with coating liquid, and coating the IgY on a quality control line position on a nitrocellulose membrane with parameters of 1uL/cm by using a membrane drawing instrument;
the coated film was placed in a 45 ℃ oven and dried for 2 hours.
4. The preparation method of the colloidal gold conjugate comprises the following steps:
the preparation method of the colloidal gold conjugate comprises the following steps: taking 10mL of colloidal gold solution in a beaker, regulating the pH value to 7.5 by using 0.2M potassium carbonate solution, adding 200 mu g of HIV tag protein, and stirring at room temperature for reacting for 10min to obtain solution A; the pH of the solution A is continuously regulated to 9 by 0.2M potassium carbonate solution, and after 0.3mL of reactant is added and evenly mixed, 40 mug of GP41, GP36 and GP120 mixed antigen and 30 mug of GP160 antigen are added and stirred at room temperature for reaction for 10min. Centrifuging (temperature 4 ℃ C., rotating speed 8500rpm,15 min), discarding supernatant, adding 1mL of sealing liquid, sealing for 20min, centrifuging (temperature 4 ℃ C., rotating speed 8500rpm,15 min), discarding supernatant, adding 1mL of preservation liquid, and redissolving to obtain the colloidal gold-labeled GP160 antigen and GP41, GP36 and GP120 mixed antigen (colloidal gold conjugate).
5. The preparation method of the colloidal gold pad comprises the following steps:
diluting the conjugate marked by the colloidal gold (GP 160 antigen marked by the colloidal gold and GP41, GP36 and GP120 mixed antigen) by 20 times with preservation solution, soaking the blank pad, taking out and drying at 37 ℃ to obtain the colloidal gold pad.
6. Sample pad treatment: the blank pad is soaked in sample pad treatment liquid, taken out and dried at 37 ℃.
7. Assembling a test strip: and sequentially overlapping and adhering the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad on the PVC bottom plate according to the chromatographic direction, and cutting into test strips with the width of 3 mm.
Example 4A kit for detecting human immunodeficiency Virus in urine based on colloidal gold method
1. Preparing a solution:
0.2M potassium carbonate: 2.76g of potassium carbonate is placed in a beaker, 100mL of purified water is added, and after stirring and mixing, the mixture is preserved at 4 ℃ for standby.
BSA preparation: 10g of BSA was weighed into a beaker, 100mL of purified water was added thereto, and after stirring and mixing, the mixture was kept at 4℃for further use.
Coating liquid preparation: 2.4g of Tris (pH 8.0) and 20g of trehalose are weighed, 1L of purified water is added into a beaker, and after stirring and mixing uniformly, the pH is adjusted to 8 by using an HCL solution, and the mixture is preserved at 4 ℃ for standby.
And (3) preparing a reactant: 8g of Tween-20 and 8g of surfactant S22 are weighed into a beaker, 100mL of purified water is added, and after stirring and mixing uniformly, the mixture is preserved at 4 ℃ for standby.
Preparing a sealing liquid: 0.213g of disodium hydrogen phosphate and 0.0456 g of sodium dihydrogen phosphate are weighed into a beaker, 1g of BSA is added with 100mL of purified water, and after being stirred uniformly, the mixture is preserved at 4 ℃ for standby.
Preparing a preservation solution: 6g of Tris (pH 8.0), 5g of BSA, 200g of sucrose and 7.5g of MPC were weighed into a beaker, 1L of purified water was added, and after stirring and mixing, the pH was adjusted to 8 with HCL solution and stored at 4℃for further use.
Sample pad treatment liquid: 18g of Tris (pH 8.0), 5g of Casein, 10g of surfactant S9 and 2.5g of EDTA are weighed, 1L of purified water is added, and after stirring and mixing, the pH is adjusted to 8 by using an HCL solution, and the mixture is preserved at 4 ℃ for later use.
2. Preparation of colloidal gold solution (concentrated colloidal gold solution)
(1) Weighing 2g of PEG in a beaker, adding 100mL of purified water, stirring at room temperature, and mixing for 10min;
(2) Transferring the solution in the step (1) into a 250mL round-bottom flask, adding 12mL chloroauric acid solution with the concentration of 10g/L, placing the round-bottom flask in an oil bath, setting the oil temperature to 120 ℃, turning on the rotating speed to 250rpm, switching on a condensing device, heating to boil, and maintaining the boiling state for 2min;
(3) Adding 9.6mL of trisodium citrate solution with the concentration of 20g/L, regulating the rotating speed to 150rpm after the solution changes color, closing a heating mode after the solution changes into purplish red, waiting for 15min, taking out a round bottom beaker, placing the round bottom beaker on a heat insulation sleeve, naturally cooling the round bottom beaker, and collecting the solution in the round bottom flask in a brown reagent bottle to obtain the colloidal gold solution.
3. Preparation of nitrocellulose membrane:
diluting SPA protein to 1mg/mL with coating liquid, and coating the SPA protein on the detection line position of the nitrocellulose membrane with a parameter of 1uL/cm by using a film drawing instrument;
diluting sheep anti-chicken IgY into 1mg/mL with coating liquid, and coating the quality control line position on the nitrocellulose membrane with a parameter of 1uL/cm by using a membrane drawing instrument;
the coated film was placed in a 45 ℃ oven and dried for 2 hours.
4. The preparation method of the colloidal gold conjugate comprises the following steps:
the preparation method of the colloidal gold conjugate comprises the following steps: taking 10mL of colloidal gold solution in a beaker, regulating the pH value to 7.5 by using 0.2M potassium carbonate solution, adding 200 mu g of HIV tag protein, and stirring at room temperature for reacting for 10min to obtain solution A; the pH of the solution A is continuously regulated to 9 by 0.2M potassium carbonate solution, and after 0.1mL of reactant is added and evenly mixed, 40 mug of GP41, GP36 and GP120 mixed antigen and 30 mug of GP160 antigen are added and stirred at room temperature for reaction for 10min. Centrifuging (temperature 4 ℃ C., rotating speed 8500rpm,15 min), discarding supernatant, adding 0.5mL of sealing liquid, sealing for 20min, centrifuging (temperature 4 ℃ C., rotating speed 8500rpm,15 min), discarding supernatant, adding 1mL of preservation liquid, and redissolving to obtain the colloidal gold-labeled GP160 antigen and GP41, GP36 and GP120 mixed antigen (colloidal gold conjugate).
5. The preparation method of the colloidal gold pad comprises the following steps:
diluting the conjugate marked by the colloidal gold (GP 160 antigen marked by the colloidal gold and GP41, GP36 and GP120 mixed antigen) by 20 times with preservation solution, soaking the blank pad, taking out and drying at 37 ℃ to obtain the colloidal gold pad.
6. Sample pad treatment: the blank pad is soaked in sample pad treatment liquid, taken out and dried at 37 ℃.
7. Assembling a test strip: and sequentially overlapping and adhering the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad on the PVC bottom plate according to the chromatographic direction, and cutting into test strips with the width of 3 mm.
Example 5: influence of different pH values on colloidal gold marking process
This example differs from example 1 in that the pH of solution a was different prior to the addition of the reactants, GP41, GP36, GP120 mix antigen, GP160 antigen.
The colloidal gold conjugates obtained by different pH value marks are prepared into corresponding kits for detecting HIV positive samples: and taking HIV positive samples for 10-time gradient dilution to obtain L1-L6 and 6 gradient samples, and detecting 6 samples, wherein the detection result is as follows.
TABLE 1 results of testing solution A at different pH values
As can be seen from the combination table 1, the sensitivity of the groups 2 to 3 is 10 times higher than that of the group 1, which indicates that before the HIV tag proteins are combined with the GP41, GP36, GP120 mixed antigen and GP160 antigen, the pH value is adjusted to be above the isoelectric point, so that the GP41, GP36, GP120 mixed antigen and GP160 antigen are combined with the HIV tag proteins only and are not combined with colloidal gold, thereby effectively improving the sensitivity of the product; and the sensitivity effect is optimal when the pH value of the solution A is 8-9.
Performance test 1:
the human immunodeficiency virus antibody urine national references were tested using the kits prepared in examples 1-4, example 5 (groups 2-3), and the results were as follows:
TABLE 2 detection results
The kits prepared in examples 1-4 and 5 (groups 2-3) meet the requirements of detection results of the human immunodeficiency virus antibody urine national reference.
Comparative example:
comparative example 1:
this comparative example differs from example 1 in that colloidal gold solution was prepared without the addition of PEG.
The preparation method of the colloidal gold solution comprises the following steps:
(1) Adding 100mL of purified water into a 250mL round-bottom flask, adding chloroauric acid solution with the concentration of 10g/L, placing the round-bottom flask into an oil bath, setting the oil temperature to 120 ℃, turning on the rotating speed to 250rpm, switching on a condensing device, heating to boiling, and maintaining the boiling state for 2min;
(2) Adding trisodium citrate solution with the concentration of 20g/L, regulating the rotating speed to 150rpm after the solution changes color, closing a heating mode after the solution changes into mauve, waiting for 15min, taking out the round bottom beaker, placing the round bottom beaker on a heat insulation sleeve for natural cooling, and collecting the solution in the round bottom flask in a brown reagent bottle to obtain the colloidal gold solution.
TABLE 3 raw materials proportioning table of colloidal gold solution
In the application, the gold solution with 0.01% chloroauric acid content is defined as 1 times gold, the gold solution with 0.02% chloroauric acid content is defined as 2 times gold, and the gold solution with 0.05% chloroauric acid content is defined as 5 times gold.
When the amount of chloroauric acid added was higher, the number of the colloidal gold particles per unit volume was larger, and positive color development after labeling was stronger, and as shown in table 3, the preparation of the colloidal gold solution was performed without adding PEG, and when the chloroauric acid content exceeded 0.06%, the prepared colloidal gold solution was precipitated, so that the colloidal gold solution (5 times gold) having a chloroauric acid concentration of 0.05% was selected in this comparative example.
Comparative example 2:
the difference between this comparative example and example 2 is that no tag protein was added when the colloidal gold-labeled GP160 antigen was mixed with GP41, GP36, and GP120 antigens.
The preparation method of the colloidal gold conjugate comprises the following steps: 10mL of colloidal gold solution is taken in a beaker, the pH value is regulated to 7.5 by 0.2M potassium carbonate solution, 150 mug of GP41, GP36 and GP120 mixed antigen and 150 mug of GP160 antigen are added, and the mixture is stirred and reacted for 10min at room temperature. Centrifuging (at 4 ℃ C., 8500rpm,15 min), discarding the supernatant, adding 1mL of sealing liquid, sealing for 20min, centrifuging (at 4 ℃ C., 8500rpm,15 min), discarding the supernatant, adding 1mL of preservation liquid, and redissolving to obtain the colloidal gold conjugate.
Comparative example 3:
the difference between this comparative example and example 2 is that no reagent is added when the colloidal gold-labeled GP160 antigen is mixed with GP41, GP36, and GP120 antigens.
The preparation method of the colloidal gold conjugate comprises the following steps: taking 10mL of colloidal gold solution in a beaker, regulating the pH value to 7.5 by using 0.2M potassium carbonate solution, adding 200 mu g of HIV tag protein, and stirring at room temperature for reacting for 10min to obtain solution A; the pH of the solution A was adjusted to 8.5 with a 0.2M potassium carbonate solution, 40. Mu.g of GP41, GP36 and GP120 antigen mixture was added, and 40. Mu.g of GP160 antigen was stirred at room temperature for reaction for 10min. Centrifuging (at 4 ℃ C., 8500rpm,15 min), discarding the supernatant, adding 1mL of sealing liquid, sealing for 20min, centrifuging (at 4 ℃ C., 8500rpm,15 min), discarding the supernatant, adding 1mL of preservation liquid, and redissolving to obtain the colloidal gold conjugate.
Comparative example 4:
the difference between this comparative example and example 2 is that when the colloidal gold-labeled GP160 antigen and GP41, GP36, GP120 were mixed, tween-20 was not added to the reagent.
Comparative example 5:
the difference between this comparative example and example 2 is that the surfactant S22 is not added to the reagent when the colloidal gold-labeled GP160 antigen is mixed with the antigens GP41, GP36 and GP 120.
Comparative example 6:
this comparative example differs from example 3 in that no MPC was added to the preservation solution.
Performance test 2:
1. minimum detection limit test
Taking HIV positive samples for 10-time gradient dilution to obtain L1-L6 and 6 gradient samples, and detecting 6 samples, wherein the detection results are as follows:
table 4 lowest limit of detection results
Note that: "+" indicates positive and "-" indicates negative
As can be seen from Table 4, the lowest detection limit of examples 1-4 is higher than that of comparative examples 1-3 and comparative examples 5-6, which demonstrate that the product sensitivity is effectively improved by improving the preparation process of the kit in the present application.
The lowest detection limit of the embodiment 1 is lower, and the sensitivity is 100 times higher than that of the comparative embodiment 1, which shows that the concentrated colloidal gold solution prepared by adding PEG as a protective agent effectively improves the sensitivity of the product.
Example 2 is 100 times higher than comparative example 2, demonstrating that the method of combining HIV tagged proteins with GP41, GP36, GP120 mixed antigen and GP160 antigen can improve the sensitivity of the product.
Example 2 was 10 times more sensitive than comparative example 3 and comparative example 5 and was consistent with comparative example 4, demonstrating that the addition of surfactant S22 to the reactants used in this application increased product sensitivity.
Example 3 was 100 times more sensitive than comparative example 6, demonstrating that the addition of MPC to the preservation solution increased the product sensitivity.
2. Specific detection
400 confirmed negative urine samples are collected, 250 normal human physical examination urine samples, 100 pregnant woman urine samples and 50 drug-taking crowd urine samples are covered, and the samples are detected, wherein the detection results are as follows:
TABLE 5 preparation of reagent-specific assay results from examples 1-4
TABLE 6 preparation of reagent-specific assay results for comparative examples 1-3
TABLE 7 comparative examples 4-6 preparation of reagent-specific assay results
As can be seen from the comparison of tables 5-7, the specificity of examples 1-4 is better than that of comparative examples 1-4 and comparative example 6, which shows that the product specificity is effectively improved by improving the preparation process of the kit in the application.
The specificity of example 1 is better than that of comparative example 1, which shows that the concentrated colloidal gold solution prepared by adding PEG as a protective agent effectively improves the product specificity.
The specificity of example 2 is better than that of comparative example 2, demonstrating that the product specificity can be improved by the method of binding HIV tagged proteins to the GP41, GP36, GP120 mixed antigen, GP160 antigen.
The specificity of example 2 was better than that of comparative examples 3 and 4, consistent with comparative example 5, demonstrating that the addition of tween-20 to the reactants used in this application can increase product specificity.
Example 3 has better specificity than comparative example 6, demonstrating that MPC can reduce non-specific adsorption and thus can increase product specificity.
3. Influence of concentrated colloidal gold solution prepared by adding PEG on product performance
It can be seen from table 3 that when the colloidal gold solution was prepared without adding PEG, the colloidal gold solution prepared could be up to 5 times gold, and the colloidal gold solution prepared according to the methods of examples 1 to 4 in the present application (i.e., concentrated colloidal gold solution), when the colloidal gold solution was prepared with PEG (i.e., concentrated colloidal gold solution), the colloidal gold solution (i.e., concentrated colloidal gold solution) could be up to 10 times gold (i.e., examples 1 to 2, chloroauric acid content 0.1%) and 12 times gold (i.e., examples 3 to 4, chloroauric acid content 0.12%), and the kit prepared in examples 1 to 4 had better sensitivity and specificity than comparative example 1 in combination with the data of examples 4, 5 and 6, indicating that the higher multiple colloidal gold solution (i.e., concentrated colloidal gold solution) could be prepared using the solution of the present application, and the product prepared with the colloidal gold solution (i.e., concentrated colloidal gold solution) had better sensitivity and specificity than comparative example 1.
4. Positive sample detection
100 urine samples of patients positive for periodic re-detection of HIV, 50 urine samples of patients with early-stage HIV infection are collected, 150 urine samples are detected, and the detection results are as follows:
TABLE 8 preparation of test results for reagent positive samples examples 1-4
TABLE 9 comparative examples 1-3 preparation of reagent positive sample test results
Table 10 comparative examples 4-6 preparation of test results for reagent positive samples
As can be seen from tables 8-10, the positive detection rate of examples 1-4 of the present application is higher than that of comparative examples 1-6, which shows that the human immunodeficiency virus (HIV-1) urine antibody detection reagent prepared by the colloidal gold method of the present application can meet the clinical demands.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Claims (10)
1. A test strip for detecting human immunodeficiency virus antibodies in urine based on a colloidal gold method is characterized in that: the test strip comprises a bottom plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped and stuck on the bottom plate according to the chromatographic direction, a quality control line and a detection line are coated on the nitrocellulose membrane, a goat anti-chicken IgY is coated on the quality control line, an SPA protein is coated on the detection line, and a colloidal gold-marked GP160 antigen and a GP41, GP36 and GP120 mixed antigen are coated on the colloidal gold pad.
2. The test strip for detecting human immunodeficiency virus antibodies in urine based on the colloidal gold method according to claim 1, wherein the preparation process of the colloidal gold-labeled GP160 antigen and the mixed antigen of GP41, GP36 and GP120 is as follows:
(1) Adjusting the pH of the colloidal gold solution to 7.5, and adding HIV tag protein for mixing; adjusting the pH value of the solution to 8-9, adding the reactant, the GP41, the GP36 and the GP120 mixed antigen and mixing the GP160 antigens;
(2) Adding the blocking solution to mix, and obtaining the GP160 antigen marked by colloidal gold and the GP41, GP36 and GP120 mixed antigen.
3. The test strip for detecting human immunodeficiency virus antibodies in urine based on the colloidal gold method according to claim 2, wherein the test strip is characterized in that: the addition amount of the colloidal gold solution in the step (1) is 8-12mL; the addition amount of HIV label protein is 150-200 mug; the addition amount of the reactant is 0.1-0.5mL; the addition amount of the GP41, GP36 and GP120 mixed antigen is 30-40 mug; the addition amount of GP160 antigen is 30-40 mug; the addition amount of the sealing liquid in the step (2) is 0.5-1mL.
4. The test strip for detecting human immunodeficiency virus antibodies in urine based on the colloidal gold method according to claim 2, wherein the test strip is characterized in that: the HIV label protein is added in the step (1) for reaction for 8-15min; adding the reactant, the GP41, the GP36 and the GP120 mixed antigen and the GP160 antigen for 8-15min; and (3) adding a sealing liquid in the step (2) for reaction for 10-20min.
5. The test strip for detecting human immunodeficiency virus antibodies in urine based on the colloidal gold method according to claim 2, wherein the reagent in the step (1) comprises the following raw materials in mass concentration: 50g/L-100g/L Tween-20, 50g/L-100g/L surfactant S22.
6. The test strip for detecting human immunodeficiency virus antibodies in urine based on the colloidal gold method according to claim 2, wherein the mixed antigen of the colloidal gold-labeled GP160 antigen and GP41, GP36 and GP120 prepared in the step (2) is preserved by using a preservation solution, wherein the preservation solution comprises the following raw materials in mass concentration: 2.4g/L-6g/L Tris, 5g/L-10g/L BSA, 100g/L-200g/L sucrose, 5g/L-10g/L MPC.
7. The test strip for detecting human immunodeficiency virus antibodies in urine based on the colloidal gold method according to claim 2, wherein the colloidal gold solution in the step (1) is a concentrated colloidal gold solution, and the preparation process is as follows:
s1, mixing PEG with water;
s2, adding chloroauric acid solution, mixing, and heating to boil;
s3, adding a trisodium citrate solution to react to obtain a colloidal gold solution.
8. The test strip for detecting human immunodeficiency virus antibodies in urine based on the colloidal gold method according to claim 7, wherein the test strip is characterized in that: the addition amount of PEG in the step S1 is 0.8-2g; the water addition amount is 50-150mL; the adding amount of chloroauric acid solution in the step S2 is 4-12mL; the adding amount of the trisodium citrate solution in the step S3 is 3.2-12mL.
9. The test strip for detecting human immunodeficiency virus antibodies in urine based on the colloidal gold method according to claim 7, wherein in step S1, the reaction time is 8-12 mm; in the step S2, the reaction is carried out for 1-2min after heating to boiling.
10. A kit, characterized in that: a test strip comprising the test strip of any one of claims 1-9.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040016035A1 (en) * | 2002-07-15 | 2004-01-22 | Floyd Alton David | Quality control of assays |
| CN101581720A (en) * | 2008-05-14 | 2009-11-18 | 上海英旻泰生物技术有限公司 | Kit for confirming human immunodeficiency virus antibody |
| CN111273004A (en) * | 2020-03-09 | 2020-06-12 | 北京华晟源医疗科技有限公司 | Reagent strip for detecting HIV (l +2) antibody in urine based on colloidal gold method and preparation method thereof |
| CN115541882A (en) * | 2022-12-02 | 2022-12-30 | 南京申基医药科技有限公司 | Preparation method and kit for improving detection test strip of human immunodeficiency virus antibody |
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- 2023-05-23 CN CN202310585859.2A patent/CN116298279B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040016035A1 (en) * | 2002-07-15 | 2004-01-22 | Floyd Alton David | Quality control of assays |
| CN101581720A (en) * | 2008-05-14 | 2009-11-18 | 上海英旻泰生物技术有限公司 | Kit for confirming human immunodeficiency virus antibody |
| CN111273004A (en) * | 2020-03-09 | 2020-06-12 | 北京华晟源医疗科技有限公司 | Reagent strip for detecting HIV (l +2) antibody in urine based on colloidal gold method and preparation method thereof |
| CN115541882A (en) * | 2022-12-02 | 2022-12-30 | 南京申基医药科技有限公司 | Preparation method and kit for improving detection test strip of human immunodeficiency virus antibody |
Non-Patent Citations (1)
| Title |
|---|
| 张文通;魏凤;王金良;肖跃强;沈志强;: "猪圆环病毒2型胶体金抗体检测方法的建立", 中国预防兽医学报, no. 09 * |
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