CN113899901A - Preparation process and application of rod-shaped colloidal gold particles - Google Patents
Preparation process and application of rod-shaped colloidal gold particles Download PDFInfo
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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
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- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
The invention relates to the technical field of biology, in particular to a preparation process and application of colloidal gold particles, which comprises the following steps: (1) solution preparation; (2) mixing the feed liquid; (3) heating treatment; (4) and (6) cooling. By adopting the preparation process, 80-100nm rod-shaped colloidal gold particles can be obtained, the detection sensitivity of the antigen can be improved by utilizing the colloidal gold particles for marking, and particularly, when a weak positive sample is detected, the detection sensitivity is enhanced, and the false negative is reduced; compared with the conventional round colloidal gold particles, the round colloidal gold particles have the advantages of small dosage, fast color fading and difficult generation of gold spots.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation process and application of colloidal gold particles.
Background
The prior art has two general strategies for detecting pathogens, namely, the detection of the pathogens and the detection of antibodies generated by human bodies for resisting the pathogens, diseases caused by the pathogens invading the human bodies can be immediately detected, and the antibody detection needs human body adaptive immunity to generate antibodies with corresponding titer to be detected.
The existing kits for detecting antigens or antibodies on the market are various in types, wherein the colloidal gold immunochromatography antigen products generally adopt a mode of improving the particle size of colloidal gold to improve the sensitivity, the colloidal gold consumption is large, the chromatography speed is low, the hydrodynamic force is easy to be insufficient, the fading is slow, gold spots are easy to generate in an observation window, and the observation of the result is influenced. Research shows that the problems can be effectively solved by adopting rod-shaped colloidal gold particles.
Disclosure of Invention
The first object of the present invention is to: aiming at the defects in the prior art, the rod-shaped colloidal gold particles can be obtained by the preparation process, the sensitivity of antigen detection can be improved by using the colloidal gold particles to mark antibodies, the omission factor is effectively reduced, and the reagent bottom color fading speed is obviously improved.
In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:
a process for preparing rod-shaped colloidal gold particles, the process comprising the steps of:
(1) solution preparation: taking HAuCl4And trisodium citrate, each formulated to 0.01 wt% HAuCl4An aqueous solution and 0.5-1.2 wt% aqueous trisodium citrate solution;
(2) mixing the material liquid: 100ml of HAuCl obtained in step (1) was taken4Heating the aqueous solution to 80-88 ℃, adding 0.12-0.18ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, continuously heating to boil, adding 0.2-0.5ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, and uniformly stirring to obtain a boiling reaction liquid;
(3) heating treatment: taking the reaction liquid in the boiling state in the step (2), heating for 3-8min by using a heating sleeve under the voltage condition of 75-83V, continuing heating for 8-13min by using an electric heating sleeve under the voltage condition of 145-153V, observing the reaction liquid from light yellow to gray and from gray to black by naked eyes, and stopping heating when the reaction liquid from black to orange;
(4) cooling treatment: and (4) cooling the feed liquid heated by the heating jacket in the step (3) to room temperature to obtain 80-100nm rod-shaped colloidal gold particles.
As an improved technical scheme, the solution prepared in the step (1) is 0.01 weight percent of HAuCl4Aqueous solution and 0.8-1 wt% aqueous solution of trisodium citrate.
As an improved technical proposal, 100ml of HAuCl in the step (1) is taken when the feed liquid in the step (2) is mixed4And (2) heating the aqueous solution to 80-85 ℃, adding 0.12-0.15ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, continuously heating to boil, adding 0.3-0.45ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, and uniformly stirring to obtain a boiling reaction liquid.
As an improved technical scheme, during the heating treatment in the step (3), a heating sleeve is adopted to heat for 5-7min under the voltage condition of 75-80V, and then an electric heating sleeve is adopted to continue heating for 10-13min under the voltage condition of 145-150V.
The invention provides application of 80-100nm rod-shaped colloidal gold particles in a human immunodeficiency virus P24 antigen detection kit, a helicobacter pylori antigen detection kit and an influenza A virus detection kit.
By adopting the technical scheme, compared with the prior art, the invention has the following advantages:
by adopting the preparation process, 80-100nm rod-shaped colloidal gold particles can be obtained, the detection sensitivity of the antigen can be improved by utilizing the colloidal gold particles for marking, and particularly, when a weak positive sample is detected, the detection sensitivity is enhanced, and the false negative is reduced; compared with the conventional round colloidal gold particles, the round colloidal gold particles have the advantages of small dosage, fast color fading and difficult generation of gold spots.
Drawings
FIG. 1 is an electron microscope image of rod-like colloidal gold particles prepared by the preparation process of the present invention;
FIG. 2 is an electron micrograph of round colloidal gold particles.
Detailed Description
The present invention will be described in further detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A human immunodeficiency virus P24 antigen detection kit comprises a detection card, wherein a human immunodeficiency virus P24 antigen detection test strip is arranged in the detection card, and the test strip comprises a bottom plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad, wherein a colloidal gold marker is combined on the colloidal gold pad, and a detection line and a quality control line are arranged on the nitrocellulose membrane;
the preparation method of the test strip comprises the following steps:
1. preparation of colloidal gold pad
The preparation process of the rod-shaped colloidal gold particles comprises the following steps:
(1) solution preparation: taking HAuCl4And trisodium citrate, each formulated to 0.01 wt% HAuCl4An aqueous solution and 0.5 wt% aqueous trisodium citrate solution;
(2) mixing the material liquid: 100ml of HAuCl obtained in step (1) was taken4Heating the aqueous solution to 80 ℃, adding 0.12ml of trisodium citrate aqueous solution obtained in the step (1) under the stirring state, continuously heating to boiling,adding 0.2ml of trisodium citrate aqueous solution obtained in the step (1) under stirring, and uniformly stirring to obtain a boiling reaction liquid;
(3) heating treatment: heating the reaction liquid in the boiling state in the step (2) for 3min by using a heating sleeve under the voltage condition of 75V, then continuously heating for 8min by using an electric heating sleeve under the voltage condition of 145V, observing the reaction liquid from light yellow to gray, from gray to black by naked eyes, and stopping heating when the reaction liquid is from black to orange;
(4) cooling treatment: and (4) cooling the feed liquid heated by the heating jacket in the step (3) to room temperature to obtain rod-shaped colloidal gold particles of 100 nm.
Labeling of colloidal gold:
(1) adding 0.2M K into the above rod-shaped colloidal gold2CO3Adjusting pH to 5.8, stirring for 5 min; according to the concentration of one ten-thousandth colloidal gold solution per 1mL (the concentration of one ten-thousandth colloidal gold solution means that 1g HAuCl is contained in each 1 ten thousand mL of colloidal gold solution4) Adding 5ug of P24 monoclonal antibody 1 for reaction for 5min, adding 1mL of 10wt% PEG20000 solution into each 100mL of colloidal gold solution, adding 1mL of 10wt% BSA phosphate solution (0.1M, pH 7.4) into each 100mL of colloidal gold solution to block the stop marker, centrifuging at 4 ℃ at 7000rpm/min for 30min, and removing the supernatant to obtain a precipitate, namely a 100nm colloidal gold-antibody complex;
(2) re-dissolving the colloidal gold-antibody complex with a gold complex solution (1L of the gold complex solution contains pH7.4, 0.05M PBS, 10g glucose, 10g trehalose, 2mLTween-20, 5g BSA, 1g ProClin-300), uniformly spreading 40ml of the complex solution on glass fiber, and placing the glass fiber in a drying room to dry for 12 hours to form a colloidal gold pad;
2. coating of nitrocellulose membrane: respectively spraying 1.2mg/ml of P24 monoclonal antibody 2 and 2.5mg/ml of goat anti-mouse IgG antibody on a nitrocellulose membrane to form a detection line and a quality control line, and drying at 37 ℃ to obtain the nitrocellulose membrane;
3. sample pad: a sample treatment solution (1L of sample pad treatment solution prepared according to the following formulation: Tris-HCl, pH8.5, 15mM, 4g PEG20000, 5g casein, 2.5mL Tween-20, 10mg anti-erythrocyte membrane RBC monoclonal antibody; Perkin Elmer) was applied to glass fibers and dried for 8 hours to obtain a sample pad for use (sample pad specification: 30 cm. times.25 cm).
Assembly of test card
A water absorption pad is stuck above the position of the nitrocellulose membrane fixed on the bottom plate (PVC plate); sticking a colloidal gold pad and a sample pad below the position of the nitrocellulose membrane fixed on a bottom plate (PVC plate), pressing and flatly cutting into test strips; then the test strip is put into the card body, and the card cover is buckled to obtain a test card; and (4) putting the detection card and the sample diluent into a packaging box to obtain a kit product.
Example 2
A kit for detecting the human immunodeficiency virus P24 antigen, which is different from the kit in example 1 only in that rod-shaped colloidal gold particles are prepared:
(1) solution preparation: taking HAuCl4And trisodium citrate, each formulated to 0.01 wt% HAuCl4An aqueous solution and 0.8 wt% aqueous trisodium citrate solution;
(2) mixing the material liquid: 100ml of HAuCl obtained in step (1) was taken4Heating the aqueous solution to 83 ℃, adding 0.13ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, continuously heating to boiling, adding 0.3ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, and uniformly stirring to obtain a boiling reaction feed liquid;
(3) heating treatment: heating the reaction liquid in the boiling state in the step (2) for 7min by using a heating sleeve under the voltage condition of 78V, continuously heating for 13min by using an electric heating sleeve under the voltage condition of 148V, observing the reaction liquid from light yellow to gray, from gray to black by naked eyes, and stopping heating when the reaction liquid is from black to orange;
(4) cooling treatment: and (4) cooling the feed liquid heated by the heating jacket in the step (3) to room temperature to obtain the rod-shaped colloidal gold particles with the particle size of 90 nm.
Example 3
A kit for detecting the human immunodeficiency virus P24 antigen, which is different from the kit in example 1 only in that rod-shaped colloidal gold particles are prepared:
(1) solution preparation: taking HAuCl4And trisodium citrate, each formulated to 0.01 wt% HAuCl4An aqueous solution and a 1 wt% aqueous solution of trisodium citrate;
(2) mixing the material liquid: 100ml of HAuCl obtained in step (1) was taken4Heating the aqueous solution to 85 ℃, adding 0.15ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, continuously heating to boiling, adding 0.45ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, and uniformly stirring to obtain a boiling reaction feed liquid;
(3) heating treatment: heating the reaction liquid in the boiling state in the step (2) for 5min by using a heating sleeve under the voltage condition of 80V, then continuously heating for 10min by using an electric heating sleeve under the voltage condition of 150V, observing the reaction liquid from light yellow to gray, from gray to black by naked eyes, and stopping heating when the reaction liquid is from black to orange;
(4) cooling treatment: and (4) cooling the feed liquid heated by the heating jacket in the step (3) to room temperature to obtain 80nm rod-shaped colloidal gold particles.
In order to better prove that the rod-shaped colloidal gold particles obtained by the preparation process are used for marking the antibody, the sensitivity of the kit can be improved, the fading time is shortened, the specificity is improved, a comparative example is given, and specific detection results are shown in tables 1, 2 and 3.
Comparative example 1
The same procedure as in example 1 was repeated except that the antibody was labeled with a 100nm circular gold colloidal particle solution.
1. Sensitivity test
The test kits of example 1, example 2 and example 3 and the test kit of comparative example 1 were tested for the standard substances at concentrations of 100IU/mL, 10IU/mL, 5IU/mL, 2IU/mL, 1IU/mL, 0.5IU/mL and 0.25IU/mL, respectively, and the test results are shown in Table 1.
As can be seen from the data in Table 1 above, the sensitivity of the kits of examples 1-3 of the present invention in practical detection applications is greatly improved compared to comparative example 1.
2. Determination of the fading time
10 serum samples were selected and the time to white fading was observed using the two kits of example 1 and comparative example 1, respectively. The results are given in the following table:
the data in table 2 show that the time for fading of the kit prepared by using the antibody labeled with the rod-shaped colloidal gold is significantly better than that of the kit prepared by using the antibody labeled with the round colloidal gold particles, and the time for observing results can be shortened by using the kit prepared by using the antibody labeled with the rod-shaped colloidal gold particles.
3. Positive and negative coincidence assay
Serum samples of 20 HIV-infected patients and 1500 serum samples of healthy persons were collected and tested by comparing the reagents of example 1 with those of comparative example 1.
As shown in Table 3, the reagent kit prepared by using the rod-shaped colloidal gold particle labeled antibody in the colloidal gold particle preparation process improves the specificity of the reagent kit and reduces the omission factor.
Example 4
A helicobacter pylori antigen detection kit comprises a detection card and a sample diluent (each 1L of the sample diluent contains 10mM PBS (pH7.2), 3.5g casein, 2.5mLS9, 3g sodium carbonate and 1g ProClin-300), wherein a helicobacter pylori antigen detection test strip is arranged in the detection card and comprises a bottom plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad, wherein the colloidal gold pad is combined with a colloidal gold marker, and the nitrocellulose membrane is provided with a detection line and a quality control line;
the preparation method of the test strip comprises the following steps:
1. preparation of colloidal gold pad
The preparation process of the rod-shaped colloidal gold particles comprises the following steps:
(1) solution preparation: taking HAuCl4And trisodium citrate, each formulated to 0.01 wt% HAuCl4An aqueous solution and 0.5 wt% aqueous trisodium citrate solution;
(2) mixing the material liquid: 100ml of HAuCl obtained in step (1) was taken4Heating the aqueous solution to 80 ℃, adding 0.12ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, continuously heating to boiling, adding 0.2ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, and uniformly stirring to obtain a boiling reaction feed liquid;
(3) heating treatment: heating the reaction liquid in the boiling state in the step (2) for 3min by using a heating sleeve under the voltage condition of 75V, then continuously heating for 8min by using an electric heating sleeve under the voltage condition of 145V, observing the reaction liquid from light yellow to gray, from gray to black by naked eyes, and stopping heating when the reaction liquid is from black to orange;
(4) cooling treatment: and (4) cooling the feed liquid heated by the heating jacket in the step (3) to room temperature to obtain rod-shaped colloidal gold particles of 100 nm.
Labeling of colloidal gold:
adding 0.2M K into the above rod-shaped colloidal gold2CO3Adjusting pH to 6.4, stirring for 5 min; according to the concentration of one ten-thousandth colloidal gold solution per 1mL (the concentration of one ten-thousandth colloidal gold solution means that 1g HAuCl is contained in each 1 ten thousand mL of colloidal gold solution4) Adding 5 mu g of helicobacter pylori monoclonal antibody 1 for reaction for 5min, adding 1mL of 10wt% PEG20000 solution into each 100mL of colloidal gold solution, adding 1mL of 10wt% BSA phosphate (0.1M, pH 7.4) solution into each 100mL of colloidal gold solution to block the stop marker, centrifuging at 4 ℃ at 7000rpm/min for 30min, and removing the supernatant to obtain a precipitate, namely a 100nm colloidal gold-antibody complex;
(3) re-dissolving the colloidal gold-antibody complex with a gold complex solution (1L of the gold complex solution contains pH7.4, 0.05M PBS, 10g glucose, 10g trehalose, 2mLTween-20, 5g BSA, 1g ProClin-300), uniformly spreading 40ml of the complex solution on glass fiber, and placing the glass fiber in a drying room to dry for 12 hours to form a colloidal gold pad;
2. coating of nitrocellulose membrane: respectively spraying helicobacter pylori monoclonal antibody 2 of 0.5mg/ml and goat anti-mouse IgG antibody of 2.2mg/ml on a nitrocellulose membrane to form a detection line and a quality control line, and drying at 37 ℃ to obtain the nitrocellulose membrane;
3. sample pad: a sample pad (sample pad standard: 30 cm. times.25 cm) was prepared by coating a sample treatment solution (1L of sample pad treatment solution prepared according to the following formulation: 15mM Tris-HCl pH8.5, 4g gelatin, 5g casein, 2.5mL Tween-20, 3g sodium carboxymethylcellulose, 1.5g sodium chloride;) on glass fibers and drying for 8 hours.
Assembly of test card
A water absorption pad is stuck above the position of the nitrocellulose membrane fixed on the bottom plate (PVC plate); sticking a colloidal gold pad and a sample pad below the position of the nitrocellulose membrane fixed on a bottom plate (PVC plate), pressing and flatly cutting into test strips; then the test strip is put into the card body, and the card cover is buckled to obtain a test card; and (4) putting the detection card and the sample diluent into a packaging box to obtain a kit product.
Example 5
A helicobacter pylori antigen detection kit, which is different from the kit in example 4 only in the preparation of rod-shaped colloidal gold particles:
(1) solution preparation: taking HAuCl4And trisodium citrate, each formulated to 0.01 wt% HAuCl4An aqueous solution and 0.8 wt% aqueous trisodium citrate solution;
(2) mixing the material liquid: 100ml of HAuCl obtained in step (1) was taken4Heating the aqueous solution to 83 ℃, adding 0.13ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, continuously heating to boiling, adding 0.3ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, and uniformly stirring to obtain a boiling reaction feed liquid;
(3) heating treatment: heating the reaction liquid in the boiling state in the step (2) for 7min by using a heating sleeve under the voltage condition of 78V, continuously heating for 13min by using an electric heating sleeve under the voltage condition of 148V, observing the reaction liquid from light yellow to gray, from gray to black by naked eyes, and stopping heating when the reaction liquid is from black to orange;
(4) cooling treatment: and (4) cooling the feed liquid heated by the heating jacket in the step (3) to room temperature to obtain the rod-shaped colloidal gold particles with the particle size of 90 nm.
Example 6
A helicobacter pylori antigen detection kit, which is different from the kit in example 4 only in the preparation of rod-shaped colloidal gold particles:
(1) solution preparation: taking HAuCl4And trisodium citrate, each formulated to 0.01 wt% HAuCl4An aqueous solution and a 1 wt% aqueous solution of trisodium citrate;
(2) mixing the material liquid: 100ml of HAuCl obtained in step (1) was taken4Heating the aqueous solution to 85 ℃, adding 0.15ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, continuously heating to boiling, adding 0.45ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, and uniformly stirring to obtain a boiling reaction feed liquid;
(3) heating treatment: heating the reaction liquid in the boiling state in the step (2) for 5min by using a heating sleeve under the voltage condition of 80V, then continuously heating for 10min by using an electric heating sleeve under the voltage condition of 150V, observing the reaction liquid from light yellow to gray, from gray to black by naked eyes, and stopping heating when the reaction liquid is from black to orange;
(4) cooling treatment: and (4) cooling the feed liquid heated by the heating jacket in the step (3) to room temperature to obtain 80nm rod-shaped colloidal gold particles.
In order to better prove that the rod-shaped colloidal gold particles obtained by the preparation process are used for marking the antibody, the sensitivity of the kit can be improved, the fading time is shortened, the specificity is improved, a comparative example is given, and specific detection results are shown in tables 4, 5 and 6.
Comparative example 1
The difference from example 5 is that the antibody was labeled with a 90nm circular gold colloidal particle solution, and the rest of the procedure was the same.
1. Sensitivity test
The test kit of example 5 and the test kit of comparative example 1 were each set to a concentration of 1X 106CFU/mL、1×105CFU/mL、1×104CFU/mL、1×103CFU/mL、1×102The CFU/mL and 10CFU/mL standards were tested, and the test results are shown in Table 4.
As can be seen from the data in Table 4, the sensitivity of the kit prepared by using the antibody labeled with the rod-shaped colloidal gold particles of the present invention is significantly improved as compared with the kit prepared by using the antibody labeled with the round colloidal gold particles in comparative example 1.
2. Fade time comparison
10 stool samples were selected, diluted and dissolved with the same sample diluent, and the whitening time was observed using the two kits of example 5 and comparative example 1, respectively.
As can be seen from the data in Table 5, the reagent kit prepared by using the rod-shaped colloidal gold particle labeled antibody of the invention and the reagent kit prepared by using the round colloidal gold particle labeled antibody in the comparative example 1 have the advantages that the whitening time of the reagent kit in the example 5 is obviously better than that of the comparative example 1, and the result observation time is shortened.
3. Positive and negative coincidence assay
150 stool samples of H.pylori-infected patients and 300 stool samples of healthy people were collected and tested by the kit of example 5 and comparative example 1, and the specific test results are shown in Table 6.
As shown in Table 6, the reagent kit prepared by using the rod-shaped colloidal gold particle labeled antibody in the colloidal gold particle preparation process of the invention improves the specificity of the reagent kit and reduces the omission factor.
Example 7
A type A influenza virus detection kit comprises a detection card and sample extract (each 1L of the sample extract is prepared from 1L of purified water, 10mM PBS (with the pH value of 7.2), 3.5g of casein, 2.5mL of Tween20, 3g of sodium carbonate and 1g of ProClin-300), wherein an A influenza virus antigen detection test strip is arranged in the detection card and comprises a bottom plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad, wherein the colloidal gold pad is combined with a colloidal gold marker, and the nitrocellulose membrane is provided with a detection line and a quality control line;
the preparation method of the test strip comprises the following steps:
1. preparation of colloidal gold pad
The preparation process of the rod-shaped colloidal gold particles comprises the following steps:
(1) solution preparation: taking HAuCl4And trisodium citrate, each formulated to 0.01 wt% HAuCl4An aqueous solution and 0.5 wt% aqueous trisodium citrate solution;
(2) mixing the material liquid: 100ml of HAuCl obtained in step (1) was taken4Heating the aqueous solution to 80 ℃, adding 0.12ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, continuously heating to boiling, adding 0.2ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, and uniformly stirring to obtain a boiling reaction feed liquid;
(3) heating treatment: heating the reaction liquid in the boiling state in the step (2) for 3min by using a heating sleeve under the voltage condition of 75V, then continuously heating for 8min by using an electric heating sleeve under the voltage condition of 145V, observing the reaction liquid from light yellow to gray, from gray to black by naked eyes, and stopping heating when the reaction liquid is from black to orange;
(4) cooling treatment: and (4) cooling the feed liquid heated by the heating jacket in the step (3) to room temperature to obtain rod-shaped colloidal gold particles of 100 nm.
Labeling of colloidal gold:
adding 0.2M K into the above rod-shaped colloidal gold2CO3Adjusting pH to 5.8, stirring for 5 min; according to one ten thousandth of colloidal gold solution per 1mL(the concentration of one-in-ten-thousandth colloidal gold solution means that 1g of HAuCl is contained in 1 ten thousand ml of colloidal gold solution4) Adding 5 mu g of influenza A monoclonal antibody 1 to react for 5min, adding 1mL of 10wt% PEG20000 solution into each 100mL of colloidal gold solution, adding 1mL of 10wt% BSA phosphate (0.1M, pH 7.4) solution into each 100mL of colloidal gold solution to seal the stop marker, centrifuging at 4 ℃ at 7000rpm/min for 30min, and removing the supernatant to obtain a precipitate, namely an 80nm colloidal gold-antibody complex;
(3) re-dissolving the colloidal gold-antibody complex with a gold complex solution (1L of the gold complex solution contains pH7.4, 0.05M PBS, 10g glucose, 10g trehalose, 2mLTween-20, 5g BSA, 1g ProClin-300), uniformly spreading 40ml of the complex solution on glass fiber, and placing the glass fiber in a drying room to dry for 12 hours to form a colloidal gold pad;
2. coating of nitrocellulose membrane: respectively spraying 0.5mg/ml FluA-11C6 (influenza A monoclonal antibody 2) and 2mg/ml goat anti-mouse IgG antibody on a nitrocellulose membrane to form a detection line and a quality control line, and drying at 37 ℃ to obtain the nitrocellulose membrane;
3. sample pad: a sample pad (sample pad standard: 30 cm. times.25 cm) was prepared by coating a sample treatment solution (1L sample pad treatment solution prepared according to the following formulation: 10mM Tris-HCl, pH8.5, 2g casein, 2.5mL Tween-20, 1g sodium carboxymethylcellulose, 1.5g sodium chloride, 5g sucrose, 2g EDTA) on glass fibers and drying for 8 hours.
Assembly of test card
A water absorption pad is stuck above the position of the nitrocellulose membrane fixed on the bottom plate (PVC plate); sticking a colloidal gold pad and a sample pad below the position of the nitrocellulose membrane fixed on a bottom plate (PVC plate), pressing and flatly cutting into test strips; then the test strip is put into the card body, and the card cover is buckled to obtain a test card; and (4) putting the detection card and the sample diluent into a packaging box to obtain a kit product.
Example 8
An influenza a virus detection kit, which is different from the kit of example 7 only in the preparation of rod-shaped colloidal gold particles:
(1) solution preparation: taking HAuCl4Hening lemonTrisodium citrate, 0.01 wt% HAuCl4An aqueous solution and 0.8 wt% aqueous trisodium citrate solution;
(2) mixing the material liquid: taking 95ml of HAuCl in the step (1)4Heating the aqueous solution to 83 ℃, adding 0.13ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, continuously heating to boiling, adding 0.3ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, and uniformly stirring to obtain a boiling reaction feed liquid;
(3) heating treatment: heating the reaction liquid in the boiling state in the step (2) for 7min by using a heating sleeve under the voltage condition of 78V, continuously heating for 13min by using an electric heating sleeve under the voltage condition of 148V, observing the reaction liquid from light yellow to gray, from gray to black by naked eyes, and stopping heating when the reaction liquid is from black to orange;
(4) cooling treatment: and (4) cooling the feed liquid heated by the heating jacket in the step (3) to room temperature to obtain the rod-shaped colloidal gold particles with the particle size of 90 nm.
Example 9
An influenza a virus detection kit, which is different from the kit of example 7 only in the preparation of rod-shaped colloidal gold particles:
(1) solution preparation: taking HAuCl4And trisodium citrate, each formulated to 0.01 wt% HAuCl4An aqueous solution and a 1 wt% aqueous solution of trisodium citrate;
(2) mixing the material liquid: 100ml of HAuCl obtained in step (1) was taken4Heating the aqueous solution to 85 ℃, adding 0.15ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, continuously heating to boiling, adding 0.45ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, and uniformly stirring to obtain a boiling reaction feed liquid;
(3) heating treatment: heating the reaction liquid in the boiling state in the step (2) for 5min by using a heating sleeve under the voltage condition of 80V, then continuously heating for 10min by using an electric heating sleeve under the voltage condition of 150V, observing the reaction liquid from light yellow to gray, from gray to black by naked eyes, and stopping heating when the reaction liquid is from black to orange;
(4) cooling treatment: and (4) cooling the feed liquid heated by the heating jacket in the step (3) to room temperature to obtain 80nm rod-shaped colloidal gold particles.
In order to better prove that the rod-shaped colloidal gold particles obtained by the preparation process are used for marking the antibody, the sensitivity of the kit can be improved, the fading time is shortened, the specificity is improved, a comparative example is given, and specific detection results are shown in tables 7, 8 and 9.
Comparative example 1
The same procedure as in example 9 was repeated except that the antibody was labeled with a solution of 80nm round colloidal gold particles.
1. Sensitivity test
The test kit of example 9 and the test kit of comparative example 1 were tested against the standards at concentrations of 500TCID50/ml, 200TCID50/ml, 100TCID50/ml, 50TCID50/ml, 20TCID50/ml and 10TCID50/ml, respectively, and the test results are shown in Table 7.
The data in Table 7 show that the sensitivity of the kit prepared by using the rod-shaped colloidal gold particle labeled antibody is greatly improved, and the kit is obviously superior to the kit prepared by using the round colloidal gold particle labeled antibody in the comparative example 1.
2. Fade time comparison
10 nasopharyngeal swab samples were selected, diluted and dissolved with the same sample diluent, and the time for whitening was observed using the kit of example 7 and the kit of comparative example 1, respectively.
The data in table 8 show that the time for fading of the kit prepared by using the rod-shaped colloidal gold particle labeled antibody is obviously superior to that of the kit prepared by using the round colloidal gold particle labeled antibody, and the time for observing results is shortened.
3. Positive and negative coincidence assay
Collecting nasopharyngeal swabs or oropharyngeal swabs infected with influenza A virus for 30 cases; 100 nasopharyngeal swabs or oral swabs of healthy people are collected and detected by an influenza A virus antigen detection reagent.
As can be seen from Table 9, the kit prepared by using the rod-shaped colloidal gold labeled antibody is used for detecting the influenza A virus antigen, and compared with the kit prepared by using the round colloidal gold labeled antibody, the specificity of the kit product is greatly improved, and the omission factor is reduced.
The present patent is not limited to the above-mentioned embodiments, and those skilled in the art can make various changes without creative efforts from the above-mentioned conception, and fall within the protection scope of the present patent.
Claims (5)
1. A preparation process of rod-shaped colloidal gold particles is characterized by comprising the following steps:
(1) solution preparation: taking HAuCl4And trisodium citrate, each formulated to 0.01 wt% HAuCl4An aqueous solution and 0.5-1.2 wt% aqueous trisodium citrate solution;
(2) mixing the material liquid: 100ml of HAuCl obtained in step (1) was taken4Heating the aqueous solution to 80-88 ℃, adding 0.12-0.18ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, continuously heating to boil, adding 0.2-0.5ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, and uniformly stirring to obtain a boiling reaction liquid;
(3) heating treatment: taking the reaction liquid in the boiling state in the step (2), heating for 3-8min by using a heating sleeve under the voltage condition of 75-83V, continuing heating for 8-13min by using an electric heating sleeve under the voltage condition of 145-153V, observing the reaction liquid from light yellow to gray and from gray to black by naked eyes, and stopping heating when the reaction liquid from black to orange;
(4) cooling treatment: and (4) cooling the feed liquid heated by the heating jacket in the step (3) to room temperature to obtain 80-100nm rod-shaped colloidal gold particles.
2. The process according to claim 1, wherein the gold rod particles are prepared by the following steps: the solution prepared in the step (1) is 0.01 wt% of HAuCl4Aqueous solution and 0.8-1 wt% aqueous solution of trisodium citrate.
3. The process according to claim 1, wherein the gold rod particles are prepared by the following steps: when the feed liquid in the step (2) is mixed, 100ml of HAuCl in the step (1) is taken4And (2) heating the aqueous solution to 80-85 ℃, adding 0.12-0.15ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, continuously heating to boil, adding 0.3-0.45ml of trisodium citrate aqueous solution obtained in the step (1) under a stirring state, and uniformly stirring to obtain a boiling reaction liquid.
4. The process according to claim 1, wherein the gold rod particles are prepared by the following steps: during the heating treatment in the step (3), the heating sleeve is adopted to heat for 5-7min under the voltage condition of 75-80V, and then the electric heating sleeve is adopted to continue heating for 10-13min under the voltage condition of 145-150V.
5. Use of the gold rod-shaped colloidal particles in the process for producing gold rod-shaped colloidal particles according to claim 1 in a human immunodeficiency virus P24 antigen detection kit, a helicobacter pylori antigen detection kit, and an influenza A virus detection kit.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115060904A (en) * | 2022-08-16 | 2022-09-16 | 山东康华生物医疗科技股份有限公司 | Preparation method of colloidal gold solution for hepatitis B surface antigen detection kit, reagent strip and kit |
| CN115283690A (en) * | 2022-08-16 | 2022-11-04 | 山东康华生物医疗科技股份有限公司 | Preparation method of colloidal gold solution for hepatitis B surface antibody detection kit, reagent strip and kit |
| CN116660517A (en) * | 2023-07-31 | 2023-08-29 | 山东康华生物医疗科技股份有限公司 | Colloidal gold preparation method for gastric helicobacter pylori IgG antibody detection test strip and test strip preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115060904A (en) * | 2022-08-16 | 2022-09-16 | 山东康华生物医疗科技股份有限公司 | Preparation method of colloidal gold solution for hepatitis B surface antigen detection kit, reagent strip and kit |
| CN115283690A (en) * | 2022-08-16 | 2022-11-04 | 山东康华生物医疗科技股份有限公司 | Preparation method of colloidal gold solution for hepatitis B surface antibody detection kit, reagent strip and kit |
| CN116660517A (en) * | 2023-07-31 | 2023-08-29 | 山东康华生物医疗科技股份有限公司 | Colloidal gold preparation method for gastric helicobacter pylori IgG antibody detection test strip and test strip preparation method |
| CN116660517B (en) * | 2023-07-31 | 2023-10-20 | 山东康华生物医疗科技股份有限公司 | Colloidal gold preparation method for gastric helicobacter pylori IgG antibody detection test strip and test strip preparation method |
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