CN112630421A - Method, reagent strip and kit for labeling salmonella typhi recombinant antigen with colloidal gold - Google Patents
Method, reagent strip and kit for labeling salmonella typhi recombinant antigen with colloidal gold Download PDFInfo
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- CN112630421A CN112630421A CN202110242415.XA CN202110242415A CN112630421A CN 112630421 A CN112630421 A CN 112630421A CN 202110242415 A CN202110242415 A CN 202110242415A CN 112630421 A CN112630421 A CN 112630421A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
Abstract
The invention relates to a method for labeling a salmonella typhi recombinant antigen by colloidal gold, which comprises the following steps: (1) preparing a colloidal gold solution; (2) adjusting the pH value of the prepared colloidal gold solution to be 1.5-2.0 lower than the optimal pH value; (3) adding the marked salmonella typhi recombinant antigen once, wherein the adding amount is 70-80% lower than the most suitable amount of the marked salmonella typhi recombinant antigen; (4) adding a sealing agent and a stabilizing agent, and marking; the safety range is broken through, the product color development intensity and sensitivity can be obviously improved by placing the immunogold under a relatively unstable but controllable condition, the relatively unstable but controllable condition means that the pH value of the colloidal gold label is slightly lower than the isoelectric point of the labeled salmonella typhi recombinant antigen, the dosage of the labeled salmonella typhi recombinant antigen is unsaturated, and the sealer and the stabilizer are added simultaneously, so that the color development intensity on the product is obviously deepened finally, and the sensitivity is obviously improved.
Description
Technical Field
The invention belongs to the technical field of gold immunization, and particularly relates to a method, a reagent strip and a kit for labeling a salmonella typhi recombinant antigen by colloidal gold.
Background
The gold immunological technology is an immunological labeling technology using colloidal gold as a label, the colloidal gold is a hydrophobic colloidal solution with negative charge formed by chloroauric acid under the action of a reducing agent, the solution becomes a stable colloidal state due to electrostatic interaction, namely, the colloidal gold is obtained, the surface of the colloidal gold is negatively charged, positive charge groups of protein are coated on the surface of colloidal gold particles due to electrostatic adsorption force, and the process is the colloidal gold labeling and is also called gold immunization.
The characteristics of the immunogold are mainly influenced by gold particles, the dosage of the marked salmonella typhi recombinant antigen and the pH value, the dosage of the most suitable marked salmonella typhi recombinant antigen is generally the maximum quantity of the marked salmonella typhi recombinant antigen completely adsorbed on the colloidal gold, the most suitable pH value is generally close to or slightly higher than the isoelectric point of the salmonella typhi recombinant antigen to be marked, but the most suitable marking state can only ensure the stability of the immunogold, and the color development strength and the sensitivity of a product cannot be obviously improved in an immunochromatography.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for labeling a salmonella typhi recombinant antigen by colloidal gold, and the method can improve the color development intensity and sensitivity of a product.
The reagent strip for labeling the salmonella typhi recombinant antigen by the colloidal gold is high in detection sensitivity, high in color development speed and good in color development strength.
Also provides a kit for labeling the salmonella typhi recombinant antigen by colloidal gold, which is convenient to use and simple to operate.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for labeling a recombinant antigen of Salmonella typhi by colloidal gold comprises the following steps:
(1) preparing a colloidal gold solution;
(2) adjusting the pH value of the prepared colloidal gold solution to be 1.5-2.0 lower than the optimal pH value;
(3) adding the marked salmonella typhi recombinant antigen once, wherein the adding amount is 70-80% lower than the most suitable amount of the marked salmonella typhi recombinant antigen;
(4) adding a blocking agent and a stabilizing agent, and marking to end.
As an optimization scheme, the adding time of the marked salmonella typhi recombinant antigen in the step (3) is 2 s.
As an optimization scheme, the adding amount of the marked salmonella typhi recombinant antigen in the step (3) is as follows:
5.6-6.4 micrograms of salmonella typhi recombinant antigen is added into every 1mL of colloidal gold solution, and the reaction time is 10 min.
As an optimized scheme, the solution used for adjusting the pH value in the step (2) is a potassium stannate solution, and the pH value of the colloidal gold solution is adjusted to 4.5-5.0 by using the potassium stannate solution.
The preparation method of the potassium stannate solution comprises the steps of weighing 2g of potassium stannate, dissolving the potassium stannate in 100mL of ultrapure water at room temperature, filtering the solution by using a 0.22 mu m filter membrane, and storing the solution at 4 ℃ for later use.
As an optimized scheme, the colloidal gold solution in the step (1) is two parts per million of nano-gold prepared by a trisodium citrate reduction method.
As an optimized scheme, the preparation method of the colloidal gold solution in the step (1) comprises the following steps:
chloroauric acid solution: 10g of chloroauric acid is dissolved in ultrapure water, the volume is determined to 1000mL after the chloroauric acid is completely dissolved, and the chloroauric acid is stored at 4 ℃ in a dark place for later use.
Trisodium citrate solution: 1g of trisodium citrate is weighed, dissolved in 100mL of ultrapure water at room temperature, filtered through a 0.22 μm filter membrane and stored at 4 ℃ for further use.
Adding 2mL of chloroauric acid solution and 98mL of ultrapure water into a three-neck flask, heating to boil, then adding 4.5mL of trisodium citrate solution, reacting for 20min when the liquid in the flask changes from bluish purple to wine red, and storing at room temperature in a dark place for later use after cooling.
As an optimization scheme, in the step (4), the blocking agent and the stabilizing agent are BSA solution and PEG20000 solution, the stabilizing agent PEG20000 solution is added firstly, 1mL PEG20000 solution is added to every 100mL of colloidal gold solution, then the blocking agent BSA solution is added, and 1mL LBSA solution is added to every 100mL of colloidal gold solution.
As an optimized scheme, the preparation method of the BSA solution comprises the steps of weighing 10g of BSA, dissolving in 100mL of ultrapure water at room temperature, storing at 4 ℃, and preparing at present; the PEG20000 solution is prepared by weighing 10g of PEG20000, dissolving in 100mL of ultrapure water at room temperature, storing at 4 deg.C, and preparing at present.
A reagent strip for colloidal gold labeling of a salmonella typhi recombinant antigen is prepared by the following steps:
(1) coating of film
The formula of the coating diluent is as follows: 0.01MPB, pH7.4 and trehalose with the mass percent concentration of 0.05 percent, and the detection line and the quality control line are sequentially coated on the NC membrane from bottom to top.
(2) Preparation of colloidal gold pad
Preparing a colloidal gold solution, and then respectively marking the salmonella typhi recombinant antigen according to a conventional method and the method; and after marking, centrifuging, concentrating by using a gold re-solution, uniformly spreading on glass fiber, and drying in a drying room for 24 hours to form a colloidal gold pad.
(3) Sample pad handling
Sample pad treatment fluid formulation: 0.1MPBS, pH7.4, sucrose with a mass percent concentration of 0.1%, BSA with a mass percent concentration of 0.1%, Tween-20 with a mass percent concentration of 0.5%, Triton X-100 with a mass percent concentration of 0.1% and NaN with a mass percent concentration of 0.5%3(ii) a The treatment solution was uniformly applied to glass fibers, and the resulting material was dried in a drying room for 24 hours to prepare a sample pad.
(4) Assembly of reagent strips
And assembling the absorbent paper, the NC membrane, the colloidal gold pad, the sample pad and the PVC base plate in sequence and then cutting the assembly into the test strip.
A kit for labeling a salmonella typhi recombinant antigen by colloidal gold comprises a detection card and sample diluent, wherein the detection card comprises a card body and a card cover which are mutually buckled, and the test strip is arranged between the card body and the card cover.
By adopting the technical scheme, the invention has the following advantages:
the process of labeling salmonella typhi recombinant antigen by colloidal gold is a process of mutual adsorption through electrostatic interaction of positive and negative charges, the more salmonella typhi recombinant antigen adsorbed on colloidal gold particles is in a stable safety range of the immunogold, the higher the color development intensity and sensitivity of the product is, but if the safety range is broken, the color development intensity and sensitivity of the product can be obviously improved by placing the immunogold under a relatively unstable and controllable condition, wherein the relatively unstable and controllable condition means that the pH value of the colloidal gold label is slightly lower than the isoelectric point of the labeled salmonella typhi recombinant antigen, the dosage of the labeled salmonella typhi recombinant antigen is unsaturated, and a sealing agent and a stabilizing agent are added simultaneously, so that the condition can cause the part of the immunogold to be agglomerated but stably controlled, the color development intensity of the product is obviously deepened at the end, and the sensitivity is obviously improved.
The present invention will be further described with reference to the following examples.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, in which specific conditions are not specified, and which are performed according to conventional conditions or conditions recommended by manufacturers. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.
The embodiments described are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without any inventive work belong to the protection scope of the present invention.
Example 1: a method for labeling a recombinant antigen of Salmonella typhi by colloidal gold comprises the following steps:
(1) liquid formulation
Chloroauric acid solution: 10g of chloroauric acid is dissolved in ultrapure water, the volume is determined to 1000mL after the chloroauric acid is completely dissolved, and the chloroauric acid is stored at 4 ℃ in a dark place for later use.
Trisodium citrate solution: 1g of trisodium citrate is weighed, dissolved in 100mL of ultrapure water at room temperature, filtered through a 0.22 μm filter membrane and stored at 4 ℃ for further use.
Potassium stannate solution: 2g of potassium stannate was weighed, dissolved in 100mL of ultrapure water at room temperature, filtered through a 0.22 μm filter membrane, and stored at 4 ℃ for further use.
BSA solution: 10g BSA was weighed, dissolved in 100mL ultrapure water at room temperature, stored at 4 ℃ and ready to use.
PEG20000 solution: 10g of PEG20000 is weighed, dissolved in 100mL of ultrapure water at room temperature, stored at 4 ℃ and prepared as-is.
(2) Preparation of colloidal gold solution
Adding 2mL of chloroauric acid solution and 98mL of ultrapure water into a three-neck flask, heating to boil, then adding 4.5mL of trisodium citrate solution, reacting for 20min after the liquid in the flask changes from bluish purple to wine red, cooling, and storing at room temperature in a dark place for later use.
(3) Colloidal gold labeling
And adjusting the pH value of the colloidal gold solution to 5 by using a potassium stannate solution, then adding 5.6 micrograms of salmonella typhi recombinant antigen into every 1mL of the colloidal gold solution, after reacting for 10min, firstly adding a stabilizer PEG20000 solution, adding 1mL of PEG20000 solution into every 100mL of the colloidal gold solution, then adding a sealant BSA solution, adding 1mL of LBSA solution into every 100mL of the colloidal gold solution, and ending the marking.
Wherein the saturated addition amount of the salmonella typhi recombinant antigen is 8 mug added to every 1mL of colloidal gold solution, namely 8 mug is the optimal marked salmonella typhi recombinant antigen usage amount, the adjusted pH value is 1.5 lower than the optimal pH value, namely the optimal pH value is 6.5, the adding time of the marked salmonella typhi recombinant antigen is 2s, and the addition amount is 70% of the optimal marked salmonella typhi recombinant antigen usage amount.
Example 2: a method for labeling a recombinant antigen of Salmonella typhi by colloidal gold comprises the following steps:
(1) liquid formulation
Chloroauric acid solution: 10g of chloroauric acid is dissolved in ultrapure water, the volume is determined to 1000mL after the chloroauric acid is completely dissolved, and the chloroauric acid is stored at 4 ℃ in a dark place for later use.
Trisodium citrate solution: 1g of trisodium citrate is weighed, dissolved in 100mL of ultrapure water at room temperature, filtered through a 0.22 μm filter membrane and stored at 4 ℃ for further use.
Potassium stannate solution: 2g of potassium stannate was weighed, dissolved in 100mL of ultrapure water at room temperature, filtered through a 0.22 μm filter membrane, and stored at 4 ℃ for further use.
BSA solution: 10g BSA was weighed, dissolved in 100mL ultrapure water at room temperature, stored at 4 ℃ and ready to use.
PEG20000 solution: 10g of PEG20000 is weighed, dissolved in 100mL of ultrapure water at room temperature, stored at 4 ℃ and prepared as-is.
(2) Preparation of colloidal gold solution
Adding 2mL of chloroauric acid solution and 98mL of ultrapure water into a three-neck flask, heating to boil, then adding 4.5mL of trisodium citrate solution, reacting for 20min after the liquid in the flask changes from bluish purple to wine red, cooling, and storing at room temperature in a dark place for later use.
(3) Colloidal gold labeling
Adjusting the pH value of the colloidal gold solution to 4.5 by using a potassium stannate solution, then adding 6.4 micrograms of salmonella typhi recombinant antigen into every 1mL of the colloidal gold solution, after reacting for 10min, firstly adding a stabilizer PEG20000 solution, adding 1mL of PEG20000 solution into every 100mL of the colloidal gold solution, then adding a sealant BSA solution, adding 1mL of LBSA solution into every 100mL of the colloidal gold solution, and finishing the marking.
Wherein the saturated addition amount of the salmonella typhi recombinant antigen is 8 mug added to every 1mL of colloidal gold solution, namely 8 mug is the optimal marked salmonella typhi recombinant antigen usage amount, the adjusted pH value is 2.0 lower than the optimal pH value, namely the optimal pH value is 6.5, the adding time of the marked salmonella typhi recombinant antigen is 2s, and the addition amount is 80% of the optimal marked salmonella typhi recombinant antigen usage amount.
Example 3: a method for labeling a recombinant antigen of Salmonella typhi by colloidal gold comprises the following steps:
(1) liquid formulation
Chloroauric acid solution: 10g of chloroauric acid is dissolved in ultrapure water, the volume is determined to 1000mL after the chloroauric acid is completely dissolved, and the chloroauric acid is stored at 4 ℃ in a dark place for later use.
Trisodium citrate solution: 1g of trisodium citrate is weighed, dissolved in 100mL of ultrapure water at room temperature, filtered through a 0.22 μm filter membrane and stored at 4 ℃ for further use.
Potassium stannate solution: 2g of potassium stannate was weighed, dissolved in 100mL of ultrapure water at room temperature, filtered through a 0.22 μm filter membrane, and stored at 4 ℃ for further use.
BSA solution: 10g BSA was weighed, dissolved in 100mL ultrapure water at room temperature, stored at 4 ℃ and ready to use.
PEG20000 solution: 10g of PEG20000 is weighed, dissolved in 100mL of ultrapure water at room temperature, stored at 4 ℃ and prepared as-is.
(2) Preparation of colloidal gold solution
Adding 2mL of chloroauric acid solution and 98mL of ultrapure water into a three-neck flask, heating to boil, then adding 4.5mL of trisodium citrate solution, reacting for 20min after the liquid in the flask changes from bluish purple to wine red, cooling, and storing at room temperature in a dark place for later use.
(3) Colloidal gold labeling
And adjusting the pH value of the colloidal gold solution to 4.8 by using a potassium stannate solution, then adding 6 micrograms of salmonella typhi recombinant antigen into every 1mL of the colloidal gold solution, after reacting for 10min, firstly adding a stabilizer PEG20000 solution, adding 1mL of PEG20000 solution into every 100mL of the colloidal gold solution, then adding a sealant BSA solution, adding 1mL of LBSA solution into every 100mL of the colloidal gold solution, and ending the marking.
Wherein the saturated addition amount of the salmonella typhi recombinant antigen is 8 mug added to every 1mL of colloidal gold solution, namely 8 mug is the optimal marked salmonella typhi recombinant antigen usage amount, the adjusted pH value is 1.7 lower than the optimal pH value, namely the optimal pH value is 6.5, the adding time of the marked salmonella typhi recombinant antigen is 2s, and the addition amount is 75% of the optimal marked salmonella typhi recombinant antigen usage amount.
Example 4: a reagent strip for colloidal gold labeling of a salmonella typhi recombinant antigen is prepared by the following steps:
(1) coating of film
The formula of the coating diluent is as follows: 0.01M PB, pH7.4, the mass percent concentration is trehalose of 0.05%, will detect line and matter control line from bottom to top wrap in proper order on the NC membrane.
(2) Preparation of colloidal gold pad
Preparing a colloidal gold solution, marking the salmonella typhi recombinant antigen by a conventional method and the method of any one of the embodiments 1-3, centrifuging after marking, concentrating by using a gold re-solution, uniformly paving on glass fiber, and drying in a drying room for 24 hours to form a colloidal gold pad.
The formula of the gold compound solution comprises: 0.05M PBS, pH7.4, 0.01% glucose by mass, 0.01% trehalose by mass, 0.05% Tween-20 by mass, 0.01% BSA by mass, 0.01% NaN by mass3。
(3) Sample pad handling
Sample pad treatment fluid formulation: 0.1M PBS, pH7.4, sucrose with a mass percent concentration of 0.1%, BSA with a mass percent concentration of 0.1%, Tween-20 with a mass percent concentration of 0.5%, Triton X-100 with a mass percent concentration of 0.1% NaN3(ii) a The treatment solution was uniformly applied to glass fibers, and the resulting material was dried in a drying room for 24 hours to prepare a sample pad.
(4) Assembly of reagent strips
And assembling the absorbent paper, the NC membrane, the colloidal gold pad, the sample pad and the PVC base plate in sequence and then cutting the assembly into the test strip.
Example 5: a kit for labeling a salmonella typhi recombinant antigen by colloidal gold comprises a detection card and sample diluent, wherein the detection card comprises a card body and a card cover which are mutually buckled, and the test strip of embodiment 4 is arranged between the card body and the card cover.
The experimental data of the salmonella typhi recombinant antigen are as follows:
in order to illustrate the advantages of the technical scheme adopted by the patent, the reagent is used in an immunochromatography typhoid three-line detection reagent and is compared with a conventional labeling method.
The preparation method of the colloidal gold test strip comprises the following steps:
(1) coating of film
The formula of the coating diluent is as follows: 0.01M PB, pH7.4, mass percent concentration of 0.05% trehalose.
And sequentially coating the detection line 1 mouse anti-human IgM monoclonal antibody, the detection line 2 mouse anti-human IgG monoclonal antibody and the quality control line sheep anti-chicken IgY polyclonal antibody on the NC membrane from bottom to top, wherein the coating concentrations are 0.8mg/mL, 1.0mg/mL and 1.0mg/mL respectively.
(2) Preparation of colloidal gold pad
Preparing a colloidal gold solution, then marking chicken immunoglobulin Y and the salmonella typhi recombinant antigen according to a conventional method, and then marking the salmonella typhi recombinant antigen by using the method of any one of embodiments 1-3; and after marking, centrifuging, concentrating by using a gold re-solution, uniformly spreading on glass fiber, and drying in a drying room for 24 hours to form a colloidal gold pad.
The conventional marking method comprises the following steps: firstly, determining the optimal pH value and the optimal protein amount of the colloidal gold marker by using a ratio, then adjusting the colloidal gold to the optimal pH value by using a potassium stannate solution, adding the salmonella typhi recombinant antigen to be marked according to the optimal protein amount, reacting for 10min, adding a BSA solution, adding 1mL of BSA solution with 10% mass percentage concentration into every 100mL of colloidal gold solution, and finishing marking.
The formula of the gold compound solution comprises: 0.05M PBS, pH7.4, 0.01% glucose by mass, 0.01% trehalose by mass, 0.05% Tween-20 by mass, 0.01% BSA by mass, 0.01% NaN by mass3。
(3) Sample pad handling
Sample pad treatment fluid formulation: 0.1M PBS, pH7.4, sucrose concentration of 0.1% by mass, BSA concentration of 0.1% by mass, Tween-20 concentration of 0.5% by mass, Triton X-100 concentration of 0.5% by mass, NaN concentration of 0.1% by mass3(ii) a The treatment solution was uniformly applied to glass fibers, and the resulting material was dried in a drying room for 24 hours to prepare a sample pad.
(4) Assembling and testing reagent strips
And assembling the absorbent paper, the NC membrane, the colloidal gold pad, the sample pad and the PVC base plate in sequence and then cutting the assembly into the test strip.
The differences of titer, sensitivity, specificity and stability of typhoid detection products are made by comparing different labeling methods through tests.
The specific experimental data are as follows:
remarking: "-" indicates negative result, "+" indicates positive result, and more "+" indicates darker color development, higher titer, and stronger positive.
And (3) titer comparison:
| positive sample 1 | Positive sample 2 | Positive sample 3 | Positive sample 4 | Positive sample 5 | Positive sample 6 | |
| Unsaturated labelling method | ++ | ++ | +++ | ++++ | +++++ | ++++ |
| Conventional marking method | + | + | ++ | +++ | ++++ | ++++ |
And (3) sensitivity comparison:
| positive sample 7 | Positive sample 8 | Positive sample 9 | Positive sample 10 | Positive sample 11 | |
| Unsaturated labelling method | + | + | + | ++ | + |
| Conventional marking method | - | - | - | + | + |
And (3) specific comparison:
| negative sample 1 | Negative sample 2 | Negative sample 3 | Negative sample 4 | Negative sample 5 | Negative sample 6 | |
| Unsaturated labelling method | - | - | - | - | - | - |
| Conventional marking method | - | - | - | - | - | - |
And (3) stability comparison:
from the data, compared with the conventional labeling method, the labeling of the salmonella typhi recombinant antigen by the unsaturated labeling method has the advantages that the titer, the sensitivity and the stability are obviously improved.
Finally, it should be noted that: the above are only preferred embodiments of the present invention, and are not intended to limit the present invention, and the embodiments are examples, wherein the details that are not described are all the common general knowledge of those skilled in the art, and the technical solutions described in the foregoing embodiments can be modified or some technical features can be equivalently replaced by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for colloidal gold labeling of a salmonella typhi recombinant antigen is characterized by comprising the following steps: the marking method comprises the following steps:
(1) preparing a colloidal gold solution;
(2) adjusting the pH value of the prepared colloidal gold solution to be 1.5-2.0 lower than the optimal pH value;
(3) adding the marked salmonella typhi recombinant antigen once, wherein the adding amount is 70-80% lower than the most suitable amount of the marked salmonella typhi recombinant antigen;
(4) adding a blocking agent and a stabilizing agent, and marking to end.
2. The method of labeling the recombinant antigen of salmonella typhi with colloidal gold according to claim 1, wherein: and (4) adding the marked salmonella typhi recombinant antigen in the step (3) for 2 s.
3. The method of labeling the recombinant antigen of salmonella typhi with colloidal gold according to claim 1, wherein: the adding amount of the marked salmonella typhi recombinant antigen in the step (3) is as follows:
5.6-6.4 micrograms of salmonella typhi recombinant antigen is added into every 1mL of colloidal gold solution, and the reaction time is 10 min.
4. The method of labeling the recombinant antigen of salmonella typhi with colloidal gold according to any one of claims 1 to 3, characterized in that: adjusting the pH value of the colloidal gold solution to 4.5-5.0 by using a potassium stannate solution which is a solution used for adjusting the pH value in the step (2);
the preparation method of the potassium stannate solution comprises the steps of weighing 2g of potassium stannate, dissolving the potassium stannate in 100mL of ultrapure water at room temperature, filtering the solution by using a 0.22 mu m filter membrane, and storing the solution at 4 ℃ for later use.
5. The method of labeling the recombinant antigen of salmonella typhi with colloidal gold according to any one of claims 1 to 3, characterized in that: the colloidal gold solution in the step (1) is two parts per million of nano gold prepared by a trisodium citrate reduction method.
6. The method of labeling the recombinant antigen of salmonella typhi with colloidal gold according to any one of claims 1 to 3, characterized in that: the preparation method of the colloidal gold solution in the step (1) comprises the following steps:
chloroauric acid solution: dissolving 10g of chloroauric acid in ultrapure water, after complete dissolution, fixing the volume to 1000mL, and storing at 4 ℃ in a dark place for later use;
trisodium citrate solution: weighing 1g of trisodium citrate, dissolving in 100mL of ultrapure water at room temperature, filtering with a 0.22 mu m filter membrane, and storing at 4 ℃ for later use;
adding 2mL of chloroauric acid solution and 98mL of ultrapure water into a three-neck flask, heating to boil, then adding 4.5mL of trisodium citrate solution, reacting for 20min when the liquid in the flask changes from bluish purple to wine red, and storing at room temperature in a dark place for later use after cooling.
7. The method of labeling the recombinant antigen of salmonella typhi with colloidal gold according to any one of claims 1 to 3, characterized in that: in the step (4), the blocking agent and the stabilizing agent are BSA solution and PEG20000 solution, the stabilizing agent PEG20000 solution is firstly added, 1mL PEG20000 solution is added into each 100mL of colloidal gold solution, then the blocking agent BSA solution is added, and 1mL LBSA solution is added into each 100mL of colloidal gold solution.
8. The method of labeling the recombinant antigen of salmonella typhi with colloidal gold according to claim 7, wherein: the preparation method of the BSA solution comprises the steps of weighing 10g of BSA, dissolving in 100mL of ultrapure water at room temperature, storing at 4 ℃, and preparing at present; the PEG20000 solution is prepared by weighing 10g of PEG20000, dissolving in 100mL of ultrapure water at room temperature, storing at 4 deg.C, and preparing at present.
9. A reagent strip for colloidal gold labeling of a salmonella typhi recombinant antigen is characterized in that: the preparation method of the reagent strip comprises the following steps:
(1) coating of film
The formula of the coating diluent is as follows: 0.01MPB, pH7.4, trehalose with the mass percent concentration of 0.05%, and sequentially coating the detection line and the quality control line on an NC membrane from bottom to top;
(2) preparation of colloidal gold pad
Preparing a colloidal gold solution, and then labeling the salmonella typhi recombinant antigen according to a conventional method and the method of any one of claims 1 to 8, respectively; centrifuging after marking, concentrating with gold re-solution, uniformly spreading on glass fiber, and drying in a drying room for 24 hours to form colloidal gold pad;
(3) sample pad handling
Sample pad treatment fluid formulation: 0.1MPBS, pH7.4, sucrose with a mass percent concentration of 0.1%, BSA with a mass percent concentration of 0.1%, Tween-20 with a mass percent concentration of 0.5%, Triton X-100 with a mass percent concentration of 0.1% and NaN with a mass percent concentration of 0.5%3(ii) a Uniformly coating the treatment solution on glass fiber, and drying for 24 hours in a drying room to prepare a sample pad;
(4) assembly of reagent strips
And assembling the absorbent paper, the NC membrane, the colloidal gold pad, the sample pad and the PVC base plate in sequence and then cutting the assembly into the test strip.
10. A kit for colloidal gold labeling of a salmonella typhi recombinant antigen is characterized in that: the kit comprises a detection card and a sample diluent, wherein the detection card comprises a card body and a card cover which are mutually buckled, and the test strip of claim 9 is arranged between the card body and the card cover.
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