CN115283690A - Preparation method of colloidal gold solution for hepatitis B surface antibody detection kit, reagent strip and kit - Google Patents
Preparation method of colloidal gold solution for hepatitis B surface antibody detection kit, reagent strip and kit Download PDFInfo
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- B22F9/00—Making metallic powder or suspensions thereof
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Abstract
The invention relates to a preparation method of a colloidal gold solution for a hepatitis B surface antibody detection kit, a reagent strip and the kit, belonging to the technical field of colloidal gold preparation, wherein the preparation method of the colloidal gold solution for the hepatitis B surface antibody detection kit comprises the following steps: (1) Uniformly mixing a chloroauric acid solution and ultrapure water according to the volume ratio of 2; (2) After heating to 130-150 ℃, quickly adding trisodium citrate solution at one time, wherein the volume ratio of the trisodium citrate solution to the added chloroauric acid solution is 1:3.5, reacting for 1min; (3) adjusting the temperature to 100 ℃, and continuously reacting for 1min; (4) adjusting the temperature to 130-150 ℃, and continuously reacting for 3min; (5) adjusting the temperature to 90 ℃, and continuously reacting for 7min; the method is based on the traditional trisodium citrate reduction method, controls different reaction temperatures and further controls the nucleation process and the nucleation speed of the colloidal gold, finally prepares the colloidal gold solution with large and uniform particle size, and has the advantages of high detection sensitivity, good color development strength and good specificity of the presented product.
Description
Technical Field
The invention belongs to the technical field of colloidal gold preparation, and particularly relates to a preparation method of a colloidal gold solution for a hepatitis B surface antibody detection kit, a reagent strip and the kit.
Background
Hepatitis B virus, hepatitis B virus for short, is a DNA virus and belongs to the family of hepadnaviridae (hepadnaviridae).
The hepatitis B virus surface antibody generally appears after the body is infected with HBV or inoculated with hepatitis B vaccine, has protective immunity to hepatitis B virus, and is called a protective antibody.
At present, hepatitis B virus surface antibodies are usually detected by a hepatitis B surface antibody detection kit, a gold-labeled pad needs to be prepared in the preparation process of the hepatitis B surface antibody detection kit, colloidal gold is used in the preparation process of the gold-labeled pad, and the colloidal gold is gold particles with certain size formed by chloroauric acid under the action of a certain reducing agent; the gold particles are maintained in a stable colloidal state by electrostatic interaction to form a negatively charged hydrophobic colloidal solution, called colloidal gold.
The existing preparation method of colloidal gold generally adopts a reduction method, wherein a white phosphorus reduction method can synthesize gold particles with the particle size of about 3 nm; the ascorbic acid reduction method can synthesize gold particles with the particle size of about 10 nm; gold particles with the particle size of about 3-20nm can be synthesized by a sodium borohydride reduction method; the reduction method of sodium citrate can synthesize gold particles with the particle size of about 5-200 nm.
The colloidal gold with different particle diameters can directly influence the performances of the product such as color development intensity, sensitivity, specificity and the like. Under the same condition, the larger the grain size of the colloidal gold, the more irregular the shape, the higher the preparation difficulty and the larger the batch difference, but the stronger the color development intensity, the higher the sensitivity and the poorer the specificity. Therefore, the preparation of high-quality colloidal gold with regular shape and good performance is the foundation and the foundation of the gold immunization technology.
Disclosure of Invention
In order to solve the above problems, the invention provides a method for preparing a colloidal gold solution for a hepatitis B surface antibody detection kit, by which colloidal gold with large particle size and regular shape can be prepared, and simultaneously the color development strength and sensitivity of the product can be improved, and the specificity meets the requirements;
also provides a reagent strip for detecting the hepatitis B surface antibody, which has high detection sensitivity and good color development intensity;
also provides a hepatitis B surface antibody detection kit, which is convenient to use and simple to operate.
In order to solve the above problems, the technical scheme adopted by the invention is as follows: a method for preparing colloidal gold solution for a hepatitis B surface antibody detection kit comprises the following steps:
(1) Uniformly mixing a chloroauric acid solution and ultrapure water according to the volume ratio of 2;
(2) After heating to 130-150 ℃, quickly adding trisodium citrate solution at one time, wherein the volume ratio of the trisodium citrate solution to the added chloroauric acid solution is 1:3.5, reacting for 1min;
(3) Adjusting the temperature to 100 ℃, and continuously reacting for 1min;
(4) Adjusting the temperature to 130-150 ℃, and continuously reacting for 3min;
(5) Adjusting the temperature to 90 ℃, continuously reacting for 7min, and finishing the reaction process;
(6) Recovering to room temperature, and keeping at 2-8 deg.C in dark place;
in the step (1), the preparation method of the chloroauric acid solution comprises the following steps: dissolving 1g of chloroauric acid in ultrapure water, fixing the volume to 100mL, uniformly mixing, and storing at 2-8 ℃ in the dark;
in the step (2), the preparation method of the trisodium citrate solution comprises the following steps: dissolving 2g of trisodium citrate in ultrapure water, diluting to 100mL, mixing uniformly, and storing at 2-8 ℃.
In step (2), the mixture was heated to 140 ℃.
In step (4), the temperature is adjusted to 140 ℃.
A reagent strip for detecting hepatitis B surface antibody, the preparation method of which comprises the following steps:
(1) Scribing film
The liquid formula of the film scratching liquid is as follows: 0.2M Tris, trehalose with the mass percent concentration of 0.5 percent, pH8.5;
and coating the quality control line and the detection line on the nitrocellulose membrane in sequence.
(2) Preparation of gold label pad
The formula of the gold compound solution comprises: 0.2M Tris, casein with the mass percent concentration of 0.8%, tween 20 with the mass percent concentration of 0.5%, proclin 300 with the mass percent concentration of 0.1%, cane sugar with the mass percent concentration of 0.5% and pH8.0;
preparing a colloidal gold solution by using the preparation method of the colloidal gold solution for the hepatitis B surface antibody detection kit, then carrying out recombinant HBsAg labeling, carrying out centrifugal concentration on a gold redissolution, carrying out solid phase immobilization on glass fiber, and drying for 6h to finish the preparation of the gold labeled pad.
(3) Preparation of sample pad
Sample pad treatment fluid formulation: 0.5M Tris, 0.5% PVP10 by mass percentage concentration, 1% BSA by mass percentage concentration, 0.5% Tween 20 by mass percentage concentration, 0.1% Proclin 300 by mass percentage concentration, 0.5% sucrose by mass percentage concentration, pH8.0;
and (3) solidifying the sample pad treatment liquid on glass fiber, and drying for 6 hours to finish the preparation of the sample pad.
(4) Assembly of reagent strips
And assembling the absorbent paper, the nitrocellulose membrane, the gold label pad, the sample pad and the PVC base plate in sequence, and cutting into reagent strips.
A hepatitis B surface antibody detection kit comprises a detection card, wherein the detection card comprises a card cover and a card body which are mutually buckled, and the reagent strip is arranged between the card cover and the card body.
By adopting the technical scheme, the invention has the following advantages: the traditional trisodium citrate reduction method for preparing the colloidal gold is one-step nucleation, and has short reaction time and high nucleation speed; however, the bigger the colloidal gold particles are, the higher the preparation difficulty is, the more obvious the heterogeneity is, and the size and the uniformity of the particle size directly influence the product specificity, the color development strength and the sensitivity, so the patent of the invention is based on the traditional trisodium citrate reduction method, controls the nucleation process and the speed of the colloidal gold by controlling different reaction temperatures, and finally prepares the nano gold sol with large and uniform particle size, and the presented product has high detection sensitivity, good color development strength and good specificity; the reagent strip for detecting the hepatitis B surface antibody has high detection sensitivity and good color development intensity; a hepatitis B surface antibody detection kit, use convenient, easy to operate.
The present invention will be further described with reference to the following examples.
Detailed Description
Example 1: a method for preparing colloidal gold solution for a hepatitis B surface antibody detection kit comprises the following steps:
(1) Uniformly mixing a chloroauric acid solution and ultrapure water according to the volume ratio of 2;
(2) After heating to the first reaction temperature, rapidly adding a trisodium citrate solution in a volume ratio of 1:3.5, reacting for 1min;
(3) Adjusting the temperature to the second reaction temperature, and continuously reacting for 1min;
(4) Adjusting the temperature to the third reaction temperature, and continuously reacting for 3min;
(5) Adjusting the reaction temperature to the fourth reaction temperature, continuously reacting for 7min, and finishing the reaction process;
(6) After the temperature is recovered to the room temperature, the mixture is placed at 2-8 ℃ in a dark place for standby.
In the step (1), the preparation method of the chloroauric acid solution comprises the following steps: dissolving 1g of chloroauric acid in ultrapure water, fixing the volume to 100mL, uniformly mixing, and storing at 2-8 ℃ in the dark;
in the step (2), the preparation method of the trisodium citrate solution comprises the following steps: dissolving 2g of trisodium citrate in ultrapure water, diluting to 100mL, uniformly mixing, and storing at 2-8 ℃;
in the step (2), the first reaction temperature is 130 ℃;
in the step (3), the second reaction temperature is 100 ℃;
in the step (4), the third reaction temperature is 130 ℃;
in the step (5), the fourth reaction temperature is 90 ℃.
Example 2: a preparation method of a colloidal gold solution for a hepatitis B surface antibody detection kit comprises the following steps:
(1) Uniformly mixing a chloroauric acid solution and ultrapure water according to the volume ratio of 2;
(2) After heating to the first reaction temperature, rapidly adding a trisodium citrate solution in one step, wherein the volume ratio of the trisodium citrate solution to the added chloroauric acid solution is 1:3.5, reacting for 1min;
(3) Adjusting the temperature to the second reaction temperature, and continuously reacting for 1min;
(4) Adjusting the temperature to the third reaction temperature, and continuously reacting for 3min;
(5) Adjusting the reaction temperature to the fourth reaction temperature, continuously reacting for 7min, and finishing the reaction process;
(6) After the temperature is recovered to the room temperature, the mixture is placed at 2-8 ℃ in a dark place for standby.
In the step (1), the preparation method of the chloroauric acid solution comprises the following steps: dissolving 1g of chloroauric acid in ultrapure water, fixing the volume to 100mL, uniformly mixing, and storing at 2-8 ℃ in the dark;
in the step (2), the preparation method of the trisodium citrate solution comprises the following steps: dissolving 2g of trisodium citrate in ultrapure water, diluting to 100mL, uniformly mixing, and storing at 2-8 ℃;
in the step (2), the first reaction temperature is 140 ℃;
in the step (3), the second reaction temperature is 100 ℃;
in the step (4), the third reaction temperature is 140 ℃;
in the step (5), the fourth reaction temperature is 90 ℃.
Example 3: a preparation method of a colloidal gold solution for a hepatitis B surface antibody detection kit comprises the following steps:
(1) Uniformly mixing a chloroauric acid solution and ultrapure water according to a volume ratio of 2;
(2) After heating to the first reaction temperature, rapidly adding a trisodium citrate solution in a volume ratio of 1:3.5, reacting for 1min;
(3) Adjusting the temperature to the second reaction temperature, and continuously reacting for 1min;
(4) Adjusting the temperature to the third reaction temperature, and continuously reacting for 3min;
(5) Adjusting the reaction temperature to the fourth reaction temperature, continuously reacting for 7min, and finishing the reaction process;
(6) Returning to room temperature, and keeping away from light and at 2-8 deg.C for use.
In the step (1), the preparation method of the chloroauric acid solution comprises the following steps: dissolving 1g of chloroauric acid in ultrapure water, fixing the volume to 100mL, uniformly mixing, and storing at 2-8 ℃ in the dark;
in the step (2), the preparation method of the trisodium citrate solution comprises the following steps: dissolving 2g of trisodium citrate in ultrapure water, diluting to 100mL, uniformly mixing, and storing at 2-8 ℃;
in the step (2), the first reaction temperature is 150 ℃;
in the step (3), the second reaction temperature is 100 ℃;
in the step (4), the third reaction temperature is 150 ℃;
in the step (5), the fourth reaction temperature is 90 ℃.
Example 4: a reagent strip for detecting hepatitis B surface antibody by a colloidal gold method is prepared by the following steps:
(1) Scribing film
The liquid formula of the scribing liquid is as follows: 0.2M Tris, trehalose with the mass percent concentration of 0.5%, pH8.5;
and coating the quality control line and the detection line on the nitrocellulose membrane from top to bottom in sequence.
(2) Preparation of gold-labeled pad
The formula of the gold compound solution comprises: 0.2M Tris, casein with the mass percent concentration of 0.8%, tween 20 with the mass percent concentration of 0.5%, proclin 300 with the mass percent concentration of 0.1%, cane sugar with the mass percent concentration of 0.5%, and pH8.0;
preparing a colloidal gold solution by using the detection kit for the hepatitis B surface antibody in one of the embodiments 1 to 3 and a preparation method of the colloidal gold solution, then labeling the recombinant HBsAg, performing centrifugal concentration on a gold redissolution, performing solid phase polymerization on glass fiber, and drying for 6 hours to complete preparation of the gold labeled pad.
(3) Preparation of sample pad
Sample pad treatment fluid formulation: 0.5M Tris, PVP10 with the mass percent concentration of 0.5%; BSA with the mass percent concentration of 1 percent; tween 20 with the mass percent concentration of 0.5 percent; proclin 300 with the mass percent concentration of 0.1 percent, sucrose with the mass percent concentration of 0.5 percent and pH8.0;
and (3) solidifying the sample pad treatment liquid on the glass fiber, and drying for 6h to finish the preparation of the sample pad.
(4) Reagent strip assembly
And assembling the absorbent paper, the nitrocellulose membrane, the gold label pad, the sample pad and the PVC base plate in sequence, and cutting into reagent strips.
Example 5: a reagent kit for detecting hepatitis B surface antibody by a colloidal gold method comprises a detection card, wherein the detection card comprises a card cover and a card body which are mutually buckled, and a reagent strip described in embodiment 4 is arranged between the card cover and the card body.
The experimental data of the hepatitis B surface antibody detection are as follows:
to better show the technical advantages of the present patent, colloidal gold solutions were prepared according to examples 1-3 and the conventional trisodium citrate method, respectively, and then a reagent strip for detecting antibody on hepatitis B surface was prepared for comparison, illustration and description.
(1) Scribing film
The liquid formula of the film scratching liquid is as follows: 0.2M Tris, trehalose with the mass percent concentration of 0.5 percent, pH8.5;
raw materials for quality control line: goat anti-rabbit IgG polyclonal body with a membrane scratching concentration of 1.0mg/mL;
raw materials for detection lines: blood-derived HBsAg with the membrane scratching concentration of 2.0mg/mL;
and coating the raw materials for the quality control line and the raw materials for the detection line on the nitrocellulose membrane from top to bottom in sequence.
(2) Preparation of gold label pad
The formula of the gold compound solution comprises: 0.2M Tris, casein with the mass percent concentration of 0.8%, tween 20 with the mass percent concentration of 0.5%, proclin 300 with the mass percent concentration of 0.1%, cane sugar with the mass percent concentration of 0.5%, and pH8.0;
meanwhile, colloidal gold solutions are respectively prepared according to examples 1-4 and a conventional trisodium citrate method, then the colloidal gold labeling is carried out to recombine HBsAg and rabbit IgG polyclonal antibody, the gold re-solution is used for centrifugal concentration, solid phase is carried out on glass fiber, and the gold labeled pad is placed in a drying room to be dried for 6 hours, so that the preparation of the gold labeled pad is completed.
Conventional trisodium citrate preparation colloidal gold method: uniformly mixing a chloroauric acid solution with the mass percentage concentration of 1% and ultrapure water according to the volume ratio of 2; after the mixture is heated to boiling by adopting a constant-temperature electric heating jacket, quickly adding a trisodium citrate solution with the mass percentage of 2% at one time, wherein the volume ratio of the trisodium citrate solution to the added chloroauric acid solution is 1:3.5, after the color of the liquid in the flask is changed into dark purple, continuously heating for 10min, and finishing the reaction;
the method for labeling the recombinant HBsAg by the colloidal gold comprises the following steps: adding 9uL 0.2M potassium stannate solution into each mL of colloidal gold solution, uniformly mixing, quickly adding 5ug of recombinant HBsAg at one time, reacting for 15min, adding 50uL 10% BSA solution by mass percentage, and finishing the reaction after 30 min;
the method for labeling the rabbit IgG polyclonal antibody by using the colloidal gold comprises the following steps: adding 11uL 0.2M potassium stannate solution into each mL of colloidal gold solution, uniformly mixing, quickly adding 7ug of rabbit IgG polyclonal antibody at one time, reacting for 15min, adding 50uL 10% BSA solution in percentage by mass concentration, and ending the reaction after 30 min.
(3) Preparation of sample pad
Sample pad treatment fluid formulation: 0.5M Tris, PVP10 with the mass percent concentration of 0.5%; BSA with the mass percentage concentration of 1 percent; tween 20 with the mass percent concentration of 0.5 percent; proclin 300 with the mass percent concentration of 0.1 percent, sucrose with the mass percent concentration of 0.5 percent and pH8.0;
and (3) solidifying the sample pad treatment liquid on the glass fiber, and drying in a drying room for 6h to finish the preparation of the sample pad.
(4) Assembled reagent strip
The absorbent paper, the nitrocellulose membrane, the gold label pad, the sample pad and the PVC base plate are assembled in sequence and then cut into reagent strips.
The differences in the color development intensity, sensitivity and specificity of the hepatitis B surface antibody detection product produced by different colloidal gold preparation methods are compared through experiments.
The experimental data are as follows:
"-" indicates negative, "+" indicates positive, and more "+" indicates stronger positive development.
Color strength results:
sensitivity results:
specific results:
according to the results, the method for preparing the colloidal gold is used in the hepatitis B surface antibody detection kit, and the color development intensity, the sensitivity and the specificity of the product are obviously improved.
Claims (5)
1. A method for preparing colloidal gold solution for a hepatitis B surface antibody detection kit is characterized by comprising the following steps: the method comprises the following steps:
(1) Uniformly mixing a chloroauric acid solution and ultrapure water according to the volume ratio of 2;
(2) After heating to 130-150 ℃, rapidly adding a trisodium citrate solution at one time, wherein the volume ratio of the trisodium citrate solution to the added chloroauric acid solution is 1:3.5, reacting for 1min;
(3) Adjusting the temperature to 100 ℃, and continuously reacting for 1min;
(4) Adjusting the temperature to 130-150 ℃, and continuously reacting for 3min;
(5) Adjusting the temperature to 90 ℃, continuously reacting for 7min, and ending the reaction process;
(6) Recovering to room temperature, and keeping away from light at 2-8 deg.C;
in the step (1), the preparation method of the chloroauric acid solution comprises the following steps: dissolving 1g of chloroauric acid in ultrapure water, fixing the volume to 100mL, uniformly mixing, and storing at 2-8 ℃ in a dark place;
in the step (2), the preparation method of the trisodium citrate solution comprises the following steps: dissolving 2g of trisodium citrate in ultrapure water, diluting to a constant volume of 100mL, mixing uniformly, and storing at 2-8 ℃.
2. The method for preparing the colloidal gold solution for the hepatitis B surface antibody detection kit according to claim 1, which is characterized in that: in step (2), the mixture was heated to 140 ℃.
3. The method for preparing the colloidal gold solution for the hepatitis B surface antibody detection kit according to claim 2, which is characterized in that: in step (4), the temperature is adjusted to 140 ℃.
4. A reagent strip for detecting hepatitis B surface antibody is characterized in that: the preparation method of the reagent strip comprises the following steps:
(1) Scribing film
The liquid formula of the film scratching liquid is as follows: 0.2M Tris, trehalose with the mass percent concentration of 0.5 percent, pH8.5;
coating the quality control line and the detection line on the nitrocellulose membrane in sequence;
(2) Preparation of gold label pad
The formula of the gold compound solution comprises: 0.2M Tris, casein with the mass percent concentration of 0.8%, tween 20 with the mass percent concentration of 0.5%, proclin 300 with the mass percent concentration of 0.1%, cane sugar with the mass percent concentration of 0.5%, and pH8.0;
preparing a colloidal gold solution by using the preparation method of the colloidal gold solution for the hepatitis B surface antibody detection kit according to claim 1, then carrying out recombinant HBsAg labeling, carrying out centrifugal concentration on a gold re-solution, carrying out solid phase immobilization on glass fibers, and drying for 6h to finish the preparation of a gold label pad;
(3) Preparation of sample pad
Sample pad treatment fluid formulation: 0.5M Tris, 0.5% PVP10 by mass, 1% BSA by mass, 0.5% Tween 20 by mass, 0.1% Proclin 300 by mass, 0.5% sucrose by mass and pH8.0;
solidifying the sample pad treatment liquid on glass fiber, and drying for 6h to finish the preparation of the sample pad;
(4) Assembly of reagent strips
The absorbent paper, the nitrocellulose membrane, the gold label pad, the sample pad and the PVC base plate are assembled in sequence and then cut into reagent strips.
5. A hepatitis B surface antibody detection kit, characterized by: the kit comprises a detection card, wherein the detection card comprises a card cover and a card body which are buckled with each other, and the reagent strip of claim 4 is arranged between the card cover and the card body.
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| CN117538520A (en) * | 2024-01-05 | 2024-02-09 | 山东康华生物医疗科技股份有限公司 | HBc-IgM antibody detection kit and preparation method thereof |
| CN117538520B (en) * | 2024-01-05 | 2024-04-02 | 山东康华生物医疗科技股份有限公司 | HBc-IgM antibody detection kit and preparation method thereof |
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