CN117347639A - Emulsion immunoassay kit for determining helicobacter pylori parting antibody, and preparation method and application thereof - Google Patents
Emulsion immunoassay kit for determining helicobacter pylori parting antibody, and preparation method and application thereof Download PDFInfo
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- CN117347639A CN117347639A CN202311231837.2A CN202311231837A CN117347639A CN 117347639 A CN117347639 A CN 117347639A CN 202311231837 A CN202311231837 A CN 202311231837A CN 117347639 A CN117347639 A CN 117347639A
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- helicobacter pylori
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- 239000000839 emulsion Substances 0.000 title abstract description 5
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- 229930006000 Sucrose Natural products 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
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- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 claims description 5
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- 229920000742 Cotton Polymers 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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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
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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
-
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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- G—PHYSICS
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- 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
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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Abstract
The invention relates to the technical field of biological detection, in particular to a latex immunoassay kit for determining helicobacter pylori parting antibodies, a preparation method and application thereof, wherein the kit comprises a detection reagent card, the detection reagent card comprises a sample pad, a release pad, a nitrocellulose membrane, absorbent paper and a bottom plate, the release pad comprises antibody microspheres marked with mouse anti-human IgG antibodies, a first detection line, a second detection line and a quality control line are sequentially arranged on the nitrocellulose membrane, a Urease antigen is coated on the first detection line, a CagA antigen and VacA antigen complex is coated on the second detection line, and goat anti-mouse IgG is coated on the quality control line. The emulsion immunoassay kit for determining helicobacter pylori parting antibodies can automatically detect helicobacter pylori parting antibodies, does not need any special instrument and equipment, and has low detection cost; the kit is simple and convenient to operate, and does not need professional operation.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a latex immunoassay kit for determining helicobacter pylori parting antibodies, a preparation method and application thereof.
Background
Gastric cancer is the fifth most common cancer worldwide, and is the third most leading cancer in mortality. Gastric cancer occurs as a result of a combination of factors including infection, environmental factors, nutritional status, diet, genetics, and the like. Helicobacter pylori (Helicobacter pylori, h.pyrri, abbreviated HP) infection is a major causative agent of chronic active gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer. About 50% of the population worldwide is infected with HP, and the rate of infection in china is high, eradicating helicobacter pylori is a viable measure for the treatment of peptic ulcers and prevention of gastric cancer.
According to the toxin secreted by helicobacter pylori, the toxin is classified into two types, namely type I and type II, of a virulent strain and a nontoxic strain. Type I: the cytotoxin-producing HP strain, namely: the Cap A or Vac A antibodies are expressed positively, and infection of the strain can cause cavitation, deformation and damage of gastric epithelial cells, cause ulcers and induce canceration. Type II: the HP strain which does not produce cytotoxin, namely: the Cap A and Vac A antibodies are negative in expression, and the HP strain is relatively low in toxicity, and generally causes only chronic superficial gastritis without clinical symptoms after infection. Type i strains are known to cause more severe gastric mucosal damage and inflammation and a higher probability of gastric cancer than type ii strains. Helicobacter pylori infection is huge in population, and there is a need for self-detection, particularly for individual detection for efficacy judgment after eradication therapy. And a detection technology based on individuals and families is established, so that accessibility and convenience of related diagnosis can be improved, and medical cost is effectively reduced. At present, methods for detecting helicobacter pylori typing antibodies capable of home self-test are lacking in the market.
In view of the above, there is a need for a helicobacter pylori typing antibody detection reagent that is simple to operate, accurate in results, and capable of home detection.
Disclosure of Invention
In view of the above, the invention aims to provide a latex immunoassay kit for determining helicobacter pylori parting antibodies, and a preparation method and application thereof, and the kit has the characteristics of convenience and rapidness in operation, accurate results and capability of self-detection.
The invention solves the technical problems by the following technical means:
in a first aspect, the invention provides a latex immunoassay kit for determining helicobacter pylori parting antibodies, the kit comprises a detection reagent card, the detection reagent card comprises a sample pad, a release pad, a nitrocellulose membrane, absorbent paper and a bottom plate, the release pad comprises antibody microspheres marked with mouse anti-human IgG antibodies, the nitrocellulose membrane is sequentially provided with a first detection line, a second detection line and a quality control line, the first detection line is coated with a Urease antigen, the second detection line is coated with a CagA antigen and VacA antigen complex, and the quality control line is coated with goat anti-mouse IgG.
With reference to the first aspect, in some embodiments, the sample pad, the release pad, the nitrocellulose membrane, and the absorbent paper are sequentially mounted on the base plate, one end of the sample pad is adhered to the base plate, and the other end is stacked on one end of the release pad; the other end of the release pad is stacked on one end of the nitrocellulose membrane, one end of the water absorbing paper is stacked on the other end of the nitrocellulose membrane, the other end of the water absorbing paper is stuck on the bottom plate, and the first detection line, the second detection line and the quality control line are all free from shielding.
In a second aspect, the invention provides a method for preparing a latex immunoassay kit for determining helicobacter pylori typing antibodies, comprising the steps of:
adding activated microspheres into an activation buffer solution, carrying out ultrasonic resuspension and dispersion, then adding 0.5-1 mg of mouse anti-human IgG, fully and uniformly mixing by a vortex mixer, carrying out mixed rotation incubation for 2.5-3 h at 37 ℃ and 250rpm in a shaking incubator, centrifuging for 10-20 min at 3-5 ℃ and 12500-13500 xg, removing the supernatant, then adding a microsphere sealing solution for microsphere sealing, thus obtaining antibody microspheres, and adding the antibody microspheres into a microsphere preservation solution for preservation;
preparing a release pad, namely spraying antibody microspheres on the pretreated latex bonding pad according to the amount of 0.8-1.2 mu L/cm, and drying to obtain the release pad coated with the antibody microspheres marked with the mouse anti-human IgG antibody;
preparing a nitrocellulose membrane, adhering the nitrocellulose membrane to a bottom plate, respectively diluting a complex of a Urease antigen, a CagA antigen and a VacA antigen to be 0.5-1 mg/m L by using a membrane buffer solution, diluting a goat anti-mouse IgG to be 1.5-2 mg/m L by using a membrane buffer solution, respectively spraying the diluted solution on the nitrocellulose membrane in sequence according to the amount of 1.5-2 mu L/cm, and drying to prepare a first detection line, a second detection line and a quality control line;
and (3) assembling the detection reagent card, namely installing the sample pad, the release pad and the absorbent paper on the bottom plate to obtain the detection reagent card.
With reference to the second aspect, in some embodiments, the activation of the microsphere comprises the steps of:
adding the microspheres into an activation buffer solution, centrifuging for 10-20 min at the temperature of 3-5 ℃ and under the condition of 12500-13500 xg, and removing the supernatant;
adding an activation buffer solution, using an ultrasonic crusher to ultrasonically resuspend the microspheres, then adding the activation buffer solution containing 10mg/mL of NHS, fully and uniformly mixing by a vortex mixer, then adding the activation buffer solution containing 10mg/mL of EDC, fully and uniformly mixing by the vortex mixer, carrying out mixed rotation incubation for 20-30min at 37 ℃ and 250rpm in a shaking incubator, and finally centrifuging for 10-20 min at 3-5 ℃ and 12500-13500 xg, and removing the supernatant to obtain the activated microspheres.
With reference to the second aspect, in some embodiments, the composition of the activation buffer is as follows: 45-55 mmol/L of morpholinoethanesulfonic acid and purified water.
With reference to the second aspect, in some embodiments, the microsphere blocking fluid comprises the following composition: 45-55 mmol/L of 4-hydroxyethyl piperazine ethane sulfonic acid, 0.8-1.2% of bovine serum albumin with mass percentage concentration, 0.08-0.12% of proclin300 with mass percentage concentration and the balance of purified water;
alternatively, the microsphere preservation solution comprises the following components: 45-55 mmol/L of morpholinoethanesulfonic acid, 0.8-1.2% of bovine serum albumin with mass percentage concentration, 1.8-2.2% of trehalose with mass percentage concentration, 0.08-0.12% of proclin300 with mass percentage concentration and the balance of purified water.
With reference to the second aspect, in some embodiments, the pretreatment of the latex binding pad is as follows: the latex bonding pad is immersed in a latex bonding pad optimizing buffer solution, and then the humidity is dried and controlled below 30%, wherein the latex bonding pad optimizing buffer solution comprises 45-55 mmol/L of tris, 0.8-1.2% of bovine serum albumin, 0.4-0.6% of Tween 20, 1.8-2.2% of sucrose, 0.08-0.12% of proclin300 and the balance of purified water.
With reference to the second aspect, in some embodiments, the printing film buffer solution includes PBS buffer solution with a concentration of 0.01-0.03 mol/L, pH of 7-7.4, trehalose with a mass percentage concentration of 0.8-1.2%, ethylene diamine tetraacetic acid with a concentration of 8-12 mmol/L, and proclin300 with a mass percentage concentration of 0.08-0.12%.
In a third aspect, the invention provides an application of the latex immunoassay kit for determining helicobacter pylori parting antibodies or the latex immunoassay kit prepared by the preparation method in preparation of products for determining helicobacter pylori parting antibodies.
With reference to the third aspect, in some embodiments, the product further comprises a sample diluent comprising PBS buffer at a concentration of 0.02mol/L, pH of 7.2, 2% glucose by mass, and 0.1% proclin300 by mass.
The latex immunoassay kit for determining helicobacter pylori parting antibodies adopts a latex immunoassay method to detect the Urea antibody, the CagA antibody and the VacA antibody in whole blood, and can judge the infection type of helicobacter pylori according to the detection result, thereby providing auxiliary diagnosis for related clinical diseases. The latex immunoassay kit has the advantages of convenience and quickness in operation and accuracy in result. Experiments prove that in the application of the latex immunoassay kit for judging the infection type of helicobacter pylori, the detected negative coincidence rate is 100%, and the positive coincidence rate is 100%.
The emulsion immunoassay kit for determining helicobacter pylori parting antibodies can automatically detect helicobacter pylori parting antibodies, does not need any special instrument and equipment, and has low detection cost; the kit is simple and convenient to operate, and does not need professional operation.
Drawings
FIG. 1 is a schematic diagram of the structure of a detection reagent card of the present invention;
the device comprises a sample pad 1, a release pad 2, a nitrocellulose membrane 3, absorbent paper 4, a bottom plate 5, a first detection line 6, a second detection line and a quality control line 8.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The following examples were conducted under conventional conditions or conditions recommended by the manufacturer, without specifying the specific conditions. The raw materials, equipment or instruments used are conventional products commercially available without identifying the manufacturer.
According to the toxin secreted by helicobacter pylori, the toxin is classified into two types, namely type I and type II, of a virulent strain and a nontoxic strain. Type I: the cytotoxin-producing HP strain, namely: the Cap A or Vac A antibodies are expressed positively, and infection of the strain can cause cavitation, deformation and damage of gastric epithelial cells, cause ulcers and induce canceration. Type II: the HP strain which does not produce cytotoxin, namely: the Cap A and Vac A antibodies are negative in expression, and the HP strain is relatively low in toxicity, and generally causes only chronic superficial gastritis without clinical symptoms after infection. Type i strains are known to cause more severe gastric mucosal damage and inflammation and a higher probability of gastric cancer than type ii strains. Helicobacter pylori infection is huge in population, and there is a need for self-detection, particularly for individual detection for efficacy judgment after eradication therapy. And a detection technology based on individuals and families is established, so that accessibility and convenience of related diagnosis can be improved, and medical cost is effectively reduced. At present, methods for detecting helicobacter pylori typing antibodies capable of home self-test are lacking in the market.
Therefore, the invention provides a latex immunoassay kit for determining helicobacter pylori parting antibodies, please refer to fig. 1, the latex immunoassay kit for determining helicobacter pylori parting antibodies comprises a detection reagent card, the detection reagent card comprises a sample pad 1, a release pad 2, a nitrocellulose membrane 3, absorbent paper 4 and a bottom plate 5, the release pad 2 comprises antibody microspheres marked with mouse anti-human IgG antibodies, a first detection line 6, a second detection line 7 and a quality control line 8 are sequentially arranged on the nitrocellulose membrane 3, a Urease antigen is coated on the first detection line 6, a CagA antigen and VacA antigen complex are coated on the second detection line 7, goat anti-mouse IgG is coated on the quality control line 8, further, the sample pad 1, the release pad 2, the nitrocellulose membrane 3 and absorbent paper 4 are sequentially arranged on the bottom plate 5, one end of the sample pad 1 is adhered on the bottom plate 5, and the other end is stacked on one end of the release pad 2; the other end of the release liner 2 is stacked on one end of the nitrocellulose membrane 3, one end of the water absorbing paper 4 is stacked on the other end of the nitrocellulose membrane 3, the other end of the water absorbing paper 4 is stuck on the bottom plate 5, and the first detection line 6, the second detection line 7 and the quality control line 8 are all free from shielding.
The preparation method of the emulsion immunoassay kit for determining helicobacter pylori parting antibody comprises the following steps:
adding activated microspheres into an activation buffer solution, carrying out ultrasonic resuspension and dispersion, then adding 0.5-1 mg of mouse anti-human IgG, fully and uniformly mixing by a vortex mixer, carrying out mixed rotation incubation for 2.5-3 h at 37 ℃ and 250rpm in a shaking incubator, centrifuging for 10-20 min at 4 ℃ and 13000xg, removing the supernatant, then adding a microsphere sealing solution for microsphere sealing, obtaining antibody microspheres, and adding the antibody microspheres into a microsphere preservation solution for preservation;
preparing a release pad, namely spraying antibody microspheres on the pretreated latex bonding pad according to the amount of 0.8-1.2 mu L/cm, and drying to obtain the release pad coated with the antibody microspheres marked with the mouse anti-human IgG antibody;
preparing a nitrocellulose membrane, adhering the nitrocellulose membrane to a bottom plate, respectively diluting a complex of a Urease antigen, a CagA antigen and a VacA antigen to be 0.5-1 mg/m L by using a membrane buffer solution, diluting a goat anti-mouse IgG to be 1.5-2 mg/m L by using a membrane buffer solution, respectively spraying the diluted solution on the nitrocellulose membrane in sequence according to the amount of 1.5-2 mu L/cm, and drying to prepare a first detection line, a second detection line and a quality control line;
and (3) assembling the detection reagent card, namely installing the sample pad, the release pad and the absorbent paper on the bottom plate to obtain the detection reagent card.
Specifically, the preparation method of the latex immunoassay kit for determining helicobacter pylori parting antibodies comprises the following steps:
step 1, microsphere labelling
(1) Microsphere activation
The microspheres are fully and evenly mixed by a vortex mixer, 1-4 mg of the color microspheres are added into 0.975-3.9 mL of activation buffer solution, and the mixture is centrifuged for 10-20 min under the condition of 13000xg at the temperature of 4 ℃ to remove the supernatant.
Adding 0.975-3.9 mL of activation buffer solution into a centrifuge tube, resuspending the dispersed microsphere precipitate, carrying out ultrasonic resuspension on the microsphere by using an integrated ultrasonic crusher to uniformly disperse the microsphere, firstly adding 0.02-0.08 mL of activation buffer reagent a (10 mg/mL of NHS, the activation buffer solution is dissolved and is used for preparation at present), fully and uniformly mixing by using a vortex mixer, then adding 0.01-0.04 mL of activation buffer b (10 mg/mL of EDC, the activation buffer solution is dissolved and is used for preparation at present), fully and uniformly mixing by using a vortex mixer, carrying out mixed spinning incubation at 250rpm for 20-30min at 37 ℃ in a shaking incubator, centrifuging for 10-20 min at 4 ℃ and 13000xg, and removing supernatant to obtain the activated microsphere.
The composition of the activation buffer was as follows: 45-55 mmol/L of morpholinoethanesulfonic acid and purified water.
(2) Coupling of microspheres to antibodies
Adding 1-4 mL of the activated microsphere into an activation buffer solution, carrying out ultrasonic resuspension and dispersion, adding 0.5-1 mg of mouse anti-human IgG into a centrifuge tube, fully and uniformly mixing by a vortex mixer, and carrying out mixed rotation incubation for 2.5-3 h at 37 ℃ and 250rpm in a vibration incubator; centrifuging at 13000Xg for 10-20 min at 4 ℃ to remove the supernatant.
(3) Closure and preservation of microspheres
Adding 1-4 mL of microsphere sealing liquid into a centrifuge tube, carrying out ultrasonic resuspension and dispersion, and carrying out mixed rotation incubation for 2-3h at 37 ℃ and 250rpm in a shake incubator; centrifuging for 10-20 min at 4 ℃ and 13000xg, and removing the supernatant; adding 1-4 mL of microsphere sealing liquid into a centrifuge tube, carrying out ultrasonic resuspension and dispersion, centrifuging for 10-20 min at 4 ℃ and 13000xg, and removing the supernatant; adding 0.2-0.8 mL of microsphere preservation solution into a centrifuge tube, carrying out ultrasonic resuspension and dispersion to obtain antibody microspheres, and then storing at 4 ℃ for later use in a dark place.
The microsphere sealing liquid comprises the following components: 45-55 mmol/L of 4-hydroxyethyl piperazine ethane sulfonic acid, 0.8-1.2% of bovine serum albumin with mass percentage concentration, 0.08-0.12% of proclin300 with mass percentage concentration and purified water.
The composition of the microsphere preservation solution is as follows: 45-55 mmol/L of morpholinoethanesulfonic acid, 0.8-1.2% of bovine serum albumin with mass percentage concentration, 1.8-2.2% of trehalose with mass percentage concentration, 0.08-0.12% of proclin300 with mass percentage concentration and purified water.
Step 2, preparation of Release pad
The antibody microsphere prepared in the step 1 is sprayed on the pretreated latex bonding pad in an amount of 0.8-1.2 mu L/cm, for example, 1mg microsphere is taken, 0.2mL microsphere preservation solution is added, and the final concentration of the antibody microsphere is 5mg/mL. The latex conjugate pads were 0.35cm in length and 0.8cm in width and were uniformly sprayed at a spray rate of 1. Mu.L/cm, each containing 0.35. Mu.L of antibody microspheres, i.e., each latex conjugate pad (0.28 square cm) contained 0.00175mg of antibody microspheres. Drying to obtain release pad coated with microsphere marked mouse anti-human IgG antibody.
The pretreatment method of the latex bonding pad is as follows: the latex bonding pad is immersed in a latex bonding pad optimizing buffer solution, and then the humidity is dried and controlled below 30%, wherein the latex bonding pad optimizing buffer solution comprises 45-55 mmol/L of tris, 0.8-1.2% of bovine serum albumin, 0.4-0.6% of Tween 20, 1.8-2.2% of sucrose, 0.08-0.12% of proclin300 and the balance of purified water.
Step 3, preparation of nitrocellulose Membrane
The method comprises the steps of adhering a nitrocellulose membrane to a base plate, respectively diluting a complex of a Urease antigen, a CagA antigen and a VacA antigen to 0.5-1 mg/mL by using a membrane buffer solution, respectively diluting a goat anti-mouse IgG to 1.5-2 mg/mL by using a membrane buffer solution, respectively spraying the diluted solution on the nitrocellulose membrane by 1.5-2 mu L/cm, and drying to obtain a first detection line, a second detection line and a quality control line, wherein the membrane buffer solution comprises a PBS buffer solution with the concentration of 0.01-0.03 mol/L, pH of 7-7.4, trehalose with the mass percentage concentration of 0.8-1.2%, ethylenediamine tetraacetic acid with the concentration of 8-12 mmol/L and proclin300 with the mass percentage concentration of 0.08-0.12%.
Step 4, assembling the detection reagent card
And installing the sample pad, the release pad and the absorbent paper on the bottom plate to obtain the detection reagent card.
The latex immunoassay kit for determination of helicobacter pylori-parting antibody and the preparation method thereof of the present invention are described below by way of example 1:
example 1
The preparation method of the latex immunoassay kit for determining helicobacter pylori parting antibodies of the embodiment is as follows:
(1) Optimization of sample pad
Preparing sample pad optimization buffer solution: 50mmol/L, pH is 8.2 tris, tween 20 at a mass percent concentration of 0.5%, proclin300 at a mass percent concentration of 0.1%, and the balance purified water.
Immersing the sample pad in the sample pad optimized buffer solution for 30-45 min, and then drying for more than 5h in a drying room, wherein the humidity is controlled below 30%.
(2) Pretreatment of latex bond pads
Preparing an optimized buffer solution of the latex binding pad: 50mmol/L, pH is tris 8.2, bovine Serum Albumin (BSA) at a mass percentage concentration of 1%, tween (Tween) 20 at a mass percentage concentration of 0.5%, sucrose at a mass percentage concentration of 2%, proclin300 at a mass percentage concentration of 0.1%, and the balance purified water.
Immersing the release pad in release pad optimized buffer solution for 30-45 min, and then drying for more than 5h in a drying room, wherein the humidity is controlled below 30%.
(3) Preparation of antibody microspheres
(1) The following solutions were prepared separately:
activation buffer: 50mmol/L of morpholinoethanesulfonic acid, and the balance purified water, pH 6.0.
Microsphere sealing liquid: 50mmol/L of 4-hydroxyethyl piperazine ethane sulfonic acid, 1% bovine serum albumin by mass percentage concentration, 0.1% proclin300 by mass percentage concentration, and the balance of purified water, wherein the pH value is 8.0.
Microsphere preservation solution: 50mmol/L of morpholinoethanesulfonic acid, 1% by mass of bovine serum albumin, 2% by mass of trehalose, 0.1% by mass of proclin300 and the balance of purified water.
(2) Activation of microspheres
The microspheres were thoroughly mixed by vortex mixer, 1mg of microspheres was added to 1mL of activation buffer, centrifuged at 13000Xg for 15min at 4℃and the supernatant removed.
Adding 1mL of an activation buffer solution into a centrifuge tube, re-suspending dispersed microsphere precipitation, carrying out ultrasonic re-suspension on the microsphere by using an integrated ultrasonic crusher to uniformly disperse the microsphere, firstly adding 0.04mL of an activation buffer reagent a (10 mg/mL of NHS, dissolved in the activation buffer solution for preparation at present), fully and uniformly mixing by using a vortex mixer, then adding 0.02mL of an activation buffer b (10 mg/mL of EDC, dissolved in the activation buffer solution for preparation at present), fully and uniformly mixing by using the vortex mixer, and carrying out mixed rotation incubation for 20min at 37 ℃ and 250rpm in a vibration incubator; the supernatant was removed by centrifugation at 13000Xg at 4℃for 15 min.
(3) Coupling of microspheres to antibodies
Adding 1mL of the activated microsphere into an activation buffer solution, carrying out ultrasonic resuspension and dispersion, adding 0.8mg of mouse anti-human IgG into a centrifuge tube, fully and uniformly mixing by a vortex mixer, carrying out mixed rotation incubation for 3h at 37 ℃ and 250rpm in a shaking incubator, centrifuging for 20min at 4 ℃ and 13000xg, and removing the supernatant.
(4) Closure and preservation of microspheres
Adding 1mL of microsphere sealing liquid into a centrifuge tube, carrying out ultrasonic resuspension and dispersion, carrying out mixed rotation incubation for 2 hours at 37 ℃ and 250rpm in a shake incubator, centrifuging for 15 minutes at 4 ℃ and 13000xg, and removing the supernatant; adding 1mL of microsphere sealing liquid into a centrifuge tube, carrying out ultrasonic resuspension and dispersion, centrifuging for 15min at 4 ℃ and 13000xg, and removing the supernatant; adding 0.25mL of microsphere preservation solution into the centrifuge tube, carrying out ultrasonic resuspension and dispersion to obtain microsphere markers, and then storing at 4 ℃ for later use in a dark place.
(4) The preparation of the release liner is carried out,
spraying the microsphere marker prepared in the step (3) on a release pad in an amount of 1 mu L/cm, and drying to prepare the release pad coated with the microsphere-labeled mouse anti-human IgG antibody;
(5) Preparation of nitrocellulose membranes
Sticking a nitrocellulose membrane onto a bottom plate, respectively diluting a Urease antigen, a CagA antigen and a VacA antigen complex to 0.8mg/mL by using a membrane buffer, diluting a goat anti-mouse IgG by using a membrane buffer to 1.5mg/m L, respectively spraying the diluted goat anti-mouse IgG by using an amount of 1.6 mu L/cm on the nitrocellulose membrane, and drying to obtain a first detection line, a second detection line and a quality control line, wherein the membrane buffer comprises PBS buffer with the concentration of 0.02mol/L and pH7.2, trehalose with the mass percentage concentration of 1.2%, ethylenediamine tetraacetic acid with the concentration of 10mmol/L and proclin300 with the mass percentage concentration of 0.1%;
(6) And (3) assembling the base plate, the sample pad, the release pad and the absorbent paper which are treated by the step (4) and are stuck with the nitrocellulose membrane, so as to obtain the detection reagent card.
Example 2
The embodiment discloses a method for using the kit of the embodiment 1, which comprises the following specific steps:
(1) Keeping the fingers clean, and sterilizing the fingers by using alcohol;
(2) Taking out the sampling needle to puncture the finger, removing the first drop of blood by using the cotton swab, and sucking 20 mu L of fingertip blood by using the blood collection tube;
(3) And (3) inserting the blood collection tube into the sample diluent, stirring and mixing uniformly, adding 2-3 drops of sample on a sample pad of the reagent card, reacting for 10-15 minutes, and reading the result. Wherein the sample diluent comprises PBS buffer with the concentration of 0.02mol/L and pH of 7.2, 2% glucose with the mass percentage concentration and 0.1% proclin300 with the mass percentage concentration.
(4) And (3) reading a detection result:
when the quality control line is provided with a strip, the first detection line and the second detection line are not provided with a strip, and the detection result is negative, namely helicobacter pylori is not infected;
when the quality control line and the first detection line are provided with a strip, the second detection line is not provided with a strip, and the detection result is helicobacter pylori infection II;
when the quality control line, the first detection line and the second detection line are all provided with strips, the detection result is helicobacter pylori infection type I.
(5) Negative-positive coincidence rate comparison
20 cases of homologous serum and whole blood (namely, the Urase antibody, the CagA antibody and the VacA antibody are all negative) which are clinically detected as negative are taken, and the detection results are shown in Table 1 by using the kit of the example 1.
Table 1:
| name of the name | Urea antibody | CagA/VacA antibodies |
| Negative homologous Whole blood 1 | - | - |
| Negative homologous Whole blood 2 | - | - |
| Negative homologous Whole blood 3 | - | - |
| Negative homologous Whole blood 4 | - | - |
| Negative homologous Whole blood 5 | - | - |
| Negative homologous Whole blood 6 | - | - |
| Negative homologous Whole blood 7 | - | - |
| Negative homologous Whole blood 8 | - | - |
| Negative homologous Whole blood 9 | - | - |
| Negative homologous Whole blood 10 | - | - |
| Negative homologous whole blood 11 | - | - |
| Negative homologous whole blood 12 | - | - |
| Negative homologous Whole blood 13 | - | - |
| Negative homologous Whole blood 14 | - | - |
| Negative homologous Whole blood 15 | - | - |
| Negative homologous Whole blood 16 | - | - |
| Negative homologous Whole blood 17 | - | - |
| Negative homologous Whole blood 18 | - | - |
| Negative homologous Whole blood 19 | - | - |
| Negative homologous Whole blood 20 | - | - |
The data in Table 1 shows that the negative compliance rate for detecting H.pylori-typed antibodies using the kit prepared in example 1 is 100%.
20 cases of homologous serum and whole blood (namely, urease antibody positive, cagA antibody positive and VacA antibody positive) which are detected as positive clinically are respectively taken, and the detection results are shown in tables 2-4 by using the kit of the example 1.
Table 2:
table 3:
| name of the name | Urea antibody | CagA/VacA antibodies |
| CagA antibody positive homologous whole blood 1 | + | + |
| CagA antibody positive homologous Whole blood 2 | + | + |
| CagA antibody positive homologous Whole blood 3 | + | + |
| CagA antibody positive homologous Whole blood 4 | + | + |
| CagA antibody positive homologous whole blood 5 | + | + |
| CagA antibody positive homologous Whole blood 6 | + | + |
| CagA antibody positive homologous Whole blood 7 | + | + |
| CagA antibody positive homologous Whole blood 8 | + | + |
| CagA antibody positive homologous whole blood 9 | + | + |
| CagA antibody positive homologous Whole blood 10 | + | + |
| CagA antibody positive homologous whole blood 11 | + | + |
| CagA antibody positive homologous whole blood 12 | + | + |
| CagA antibody positive homologous whole blood 13 | + | + |
| CagA antibody positive homologous Whole blood 14 | + | + |
| CagA antibody positive homologous Whole blood 15 | + | + |
| CagA antibody positive homologous whole blood 16 | + | + |
| CagA antibody positive homologous whole blood 17 | + | + |
| CagA antibody positive homologous whole blood 18 | + | + |
| CagA antibody positive homologous Whole blood 19 | + | + |
| CagA antibody positive homologous Whole blood 20 | + | + |
Table 4:
the data in tables 2-4 show that the positive compliance rate for detecting H.pylori-typed antibodies using the kit prepared in example 1 is 100%.
The results of the comparison of the kit of example 1 with the existing methods for detecting helicobacter pylori typing antibodies are shown in Table 5.
Table 5:
the data in Table 5 shows that the results of the kit of example 1 in determining the helicobacter pylori-typing antibodies are consistent with the results of the detection of the marketed products.
Example 3
The preparation method of the latex immunoassay kit for determining helicobacter pylori parting antibodies of the embodiment is as follows:
(1) Optimization of sample pad
Preparing sample pad optimization buffer solution: 50mmol/L, pH is 8.2 tris, tween 20 at a mass percent concentration of 0.5%, proclin300 at a mass percent concentration of 0.1%, and the balance purified water.
Immersing the sample pad in sample pad optimized buffer solution for 30min, and drying in a drying room for more than 5h, wherein the humidity is controlled below 30%.
(2) Pretreatment of latex bond pads
Preparing an optimized buffer solution of the latex binding pad: 45mmol/L, pH is tris 8.2, bovine Serum Albumin (BSA) at a mass percentage concentration of 0.8%, tween (Tween) 20 at a mass percentage concentration of 0.4%, sucrose at a mass percentage concentration of 1.8%, proclin300 at a mass percentage concentration of 0.08%, and the balance purified water.
Immersing the release pad in release pad optimized buffer solution for 30min, and drying in a drying room for more than 5h, wherein the humidity is controlled below 30%.
(3) Preparation of antibody microspheres
(1) The following solutions were prepared separately:
activation buffer: 45mmol/L of morpholinoethanesulfonic acid, and the balance purified water, pH 6.0.
Microsphere sealing liquid: 45 mmol/L4-hydroxyethyl piperazine ethane sulfonic acid, 0.8% bovine serum albumin, 0.08% proclin300, and the balance purified water, pH 8.0.
Microsphere preservation solution: 45mmol/L of morpholinoethanesulfonic acid, 0.8% by mass of bovine serum albumin, 1.8% by mass of trehalose, 0.08% by mass of proclin300, and the balance purified water.
(2) Activation of microspheres
The microspheres were thoroughly mixed by vortex mixer, 1mg of microspheres was added to 1mL of activation buffer, centrifuged at 3℃and 12500Xg for 10min, and the supernatant was removed.
Adding 2mL of activation buffer solution into a centrifuge tube, re-suspending dispersed microsphere sediment, carrying out ultrasonic re-suspension on the microsphere by using an integrated ultrasonic crusher to uniformly disperse the microsphere, firstly adding 0.04mL of activation buffer reagent a (10 mg/mL of NHS, dissolved by the activation buffer solution for preparation at present), fully and uniformly mixing by using a vortex mixer, then adding 0.02mL of activation buffer b (10 mg/mL of EDC, dissolved by the activation buffer solution for preparation at present), fully and uniformly mixing by using the vortex mixer, and carrying out mixed spin incubation at 37 ℃ and 250rpm in a vibration incubator for 25min; the supernatant was removed by centrifugation at 12500Xg for 10min at 3 ℃.
(3) Coupling of microspheres to antibodies
Adding 1mL of the activated microsphere into an activation buffer solution, carrying out ultrasonic resuspension and dispersion, adding 0.5mg of mouse anti-human IgG into a centrifuge tube, fully and uniformly mixing by a vortex mixer, carrying out mixed rotation incubation for 2.5h at 37 ℃ and 250rpm in a shaking incubator, centrifuging for 20min at 3 ℃ and 12500xg, and removing the supernatant.
(4) Closure and preservation of microspheres
Adding 2mL of microsphere sealing liquid into a centrifuge tube, carrying out ultrasonic resuspension and dispersion, carrying out mixed rotation incubation for 2h at 37 ℃ and 250rpm in a shake incubator, centrifuging for 15min at 3 ℃ and 12500xg, and removing the supernatant; adding 1mL of microsphere sealing liquid into a centrifuge tube, carrying out ultrasonic resuspension and dispersion, centrifuging at 3 ℃ and 12500xg for 15min, and removing the supernatant; adding 0.25mL of microsphere preservation solution into the centrifuge tube, carrying out ultrasonic resuspension and dispersion to obtain microsphere markers, and then storing at 4 ℃ for later use in a dark place.
(4) The preparation of the release liner is carried out,
spraying the microsphere marker prepared in the step (3) on a release pad in an amount of 0.8 mu L/cm, and drying to prepare the release pad coated with the microsphere-labeled mouse anti-human IgG antibody;
(5) Preparation of nitrocellulose membranes
Sticking a nitrocellulose membrane onto a bottom plate, respectively diluting a Urease antigen, a CagA antigen and a VacA antigen complex to 0.5mg/mL by using a membrane buffer, diluting a goat anti-mouse IgG by using a membrane buffer to 1.8mg/m L, respectively spraying the diluted goat anti-mouse IgG by using an amount of 1.5 mu L/cm on the nitrocellulose membrane, and drying to obtain a first detection line, a second detection line and a quality control line, wherein the membrane buffer comprises a PBS buffer with the concentration of 0.01mol/L, pH of 7.0, trehalose with the concentration of 0.8% by mass, ethylenediamine tetraacetic acid with the concentration of 8mmol/L and proclin300 with the concentration of 0.08% by mass;
(6) And (3) assembling the base plate, the sample pad, the release pad and the absorbent paper which are treated by the step (4) and are stuck with the nitrocellulose membrane, so as to obtain the detection reagent card.
Example 4
The preparation method of the latex immunoassay kit for determining helicobacter pylori parting antibodies of the embodiment is as follows:
(1) Optimization of sample pad
Preparing sample pad optimization buffer solution: 50mmol/L, pH is 8.2 tris, tween 20 at a mass percent concentration of 0.5%, proclin300 at a mass percent concentration of 0.1%, and the balance purified water.
Immersing the sample pad in sample pad optimized buffer solution for 45min, and drying in a drying room for more than 5h, wherein the humidity is controlled below 30%.
(2) Pretreatment of latex bond pads
Preparing an optimized buffer solution of the latex binding pad: 55mmol/L, pH is tris 8.2, bovine Serum Albumin (BSA) at a mass percentage concentration of 1.2%, tween (Tween) 20 at a mass percentage concentration of 0.6%, sucrose at a mass percentage concentration of 2.2%, proclin300 at a mass percentage concentration of 0.12%, and the balance purified water.
Immersing the release pad in release pad optimized buffer solution for 45min, and drying in a drying room for more than 5h, wherein the humidity is controlled below 30%.
(3) Preparation of antibody microspheres
(1) The following solutions were prepared separately:
activation buffer: 55mmol/L of morpholinoethanesulfonic acid, and the balance purified water, pH 6.0.
Microsphere sealing liquid: 55mmol/L of 4-hydroxyethyl piperazine ethane sulfonic acid, 1.2% bovine serum albumin with a mass percentage concentration, 0.12% proclin300 with a mass percentage concentration, and the balance of purified water, wherein the pH value is 8.0.
Microsphere preservation solution: 55mmol/L of morpholinoethanesulfonic acid, 1.2% by mass of bovine serum albumin, 2.2% by mass of trehalose, 0.12% by mass of proclin300 and the balance purified water.
(2) Activation of microspheres
The microspheres were thoroughly mixed by vortex mixer, 1mg of microspheres was added to 1mL of activation buffer, centrifuged at 13500Xg for 15min at 5℃and the supernatant was removed.
Adding 4mL of an activation buffer solution into a centrifuge tube, re-suspending dispersed microsphere precipitation, carrying out ultrasonic re-suspension on the microsphere by using an integrated ultrasonic crusher to uniformly disperse the microsphere, firstly adding 0.04mL of an activation buffer reagent a (10 mg/mL of NHS, dissolved in the activation buffer solution for preparation at present), fully and uniformly mixing by using a vortex mixer, then adding 0.02mL of an activation buffer b (10 mg/mL of EDC, dissolved in the activation buffer solution for preparation at present), fully and uniformly mixing by using the vortex mixer, and carrying out mixed rotation incubation for 30min at 37 ℃ and 250rpm in a vibration incubator; the supernatant was removed by centrifugation at 13500Xg at 5℃for 20 min.
(3) Coupling of microspheres to antibodies
Adding 4mL of the activated microsphere into an activation buffer solution, carrying out ultrasonic resuspension and dispersion, adding 0.8mg of mouse anti-human IgG into a centrifuge tube, fully and uniformly mixing by a vortex mixer, carrying out mixed rotation incubation for 3h at 37 ℃ and 250rpm in a shaking incubator, centrifuging for 20min at 5 ℃ and 13500xg, and removing the supernatant.
(4) Closure and preservation of microspheres
Adding 1mL of microsphere sealing liquid into a centrifuge tube, carrying out ultrasonic resuspension and dispersion, carrying out mixed rotation incubation for 2h at 37 ℃ and 250rpm in a shake incubator, centrifuging for 20min at 5 ℃ and 13500xg, and removing the supernatant; adding 1mL of microsphere sealing liquid into a centrifuge tube, carrying out ultrasonic resuspension and dispersion, centrifuging at 5 ℃ and 13500xg for 15min, and removing the supernatant; adding 0.25mL of microsphere preservation solution into the centrifuge tube, carrying out ultrasonic resuspension and dispersion to obtain microsphere markers, and then storing at 4 ℃ for later use in a dark place.
(4) The preparation of the release liner is carried out,
spraying the microsphere marker prepared in the step (3) on a release pad in an amount of 1 mu L/cm, and drying to prepare the release pad coated with the microsphere-labeled mouse anti-human IgG antibody;
(5) Preparation of nitrocellulose membranes
Sticking a nitrocellulose membrane onto a bottom plate, respectively diluting a Urease antigen, a CagA antigen and a VacA antigen complex to 0.5mg/mL by using a membrane buffer, diluting a goat anti-mouse IgG by using a membrane buffer to 1.6mg/m L, respectively spraying the diluted goat anti-mouse IgG by using an amount of 1.6 mu L/cm on the nitrocellulose membrane, and drying to obtain a first detection line, a second detection line and a quality control line, wherein the membrane buffer comprises a PBS buffer with the concentration of 0.01mol/L, pH of 7.0, trehalose with the concentration of 0.8% by mass, ethylenediamine tetraacetic acid with the concentration of 8mmol/L and proclin300 with the concentration of 0.08% by mass;
(6) And (3) assembling the base plate, the sample pad, the release pad and the absorbent paper which are treated by the step (4) and are stuck with the nitrocellulose membrane, so as to obtain the detection reagent card.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention. The technology, shape, and construction parts of the present invention, which are not described in detail, are known in the art.
Claims (10)
1. The latex immunoassay kit for determining helicobacter pylori parting antibodies is characterized by comprising a detection reagent card, wherein the detection reagent card comprises a sample pad, a release pad, a nitrocellulose membrane, absorbent paper and a bottom plate, the release pad comprises antibody microspheres marked with mouse anti-human IgG antibodies, a first detection line, a second detection line and a quality control line are sequentially arranged on the nitrocellulose membrane, a Urease antigen is coated on the first detection line, a CagA antigen and VacA antigen complex is coated on the second detection line, and goat anti-mouse IgG is coated on the quality control line.
2. The latex immunoassay kit for determining a helicobacter pylori parting antibody according to claim 1, wherein the sample pad, the release pad, the nitrocellulose membrane, and the absorbent paper are sequentially mounted on a base plate, one end of the sample pad is stuck to the base plate, and the other end is stacked on one end of the release pad; the other end of the release pad is stacked on one end of the nitrocellulose membrane, one end of the water absorbing paper is stacked on the other end of the nitrocellulose membrane, the other end of the water absorbing paper is stuck on the bottom plate, and the first detection line, the second detection line and the quality control line are all free from shielding.
3. The preparation method of the latex immunoassay kit for determining helicobacter pylori parting antibodies is characterized by comprising the following steps:
adding activated microspheres into an activation buffer solution, carrying out ultrasonic resuspension and dispersion, then adding 0.5-1 mg of mouse anti-human IgG, fully and uniformly mixing by a vortex mixer, carrying out mixed rotation incubation for 2.5-3 h at 37 ℃ and 250rpm in a shaking incubator, centrifuging for 10-20 min at 3-5 ℃ and 12500-13500 xg, removing the supernatant, then adding a microsphere sealing solution for microsphere sealing, thus obtaining antibody microspheres, and adding the antibody microspheres into a microsphere preservation solution for preservation;
preparing a release pad, namely spraying antibody microspheres on the pretreated latex bonding pad according to the amount of 0.8-1.2 mu L/cm, and drying to obtain the release pad coated with the antibody microspheres marked with the mouse anti-human IgG antibody;
preparing a nitrocellulose membrane, adhering the nitrocellulose membrane to a bottom plate, respectively diluting a complex of a Urease antigen, a CagA antigen and a VacA antigen to be 0.5-1 mg/m L by using a membrane buffer solution, diluting a goat anti-mouse IgG to be 1.5-2 mg/m L by using a membrane buffer solution, respectively spraying the diluted solution on the nitrocellulose membrane in sequence according to the amount of 1.5-2 mu L/cm, and drying to prepare a first detection line, a second detection line and a quality control line;
and (3) assembling the detection reagent card, namely installing the sample pad, the release pad and the absorbent paper on the bottom plate to obtain the detection reagent card.
4. A method of preparing a latex immunoassay kit for determining a helicobacter pylori-typing antibody according to claim 3, wherein the activation of the microsphere comprises the steps of:
adding the microspheres into an activation buffer solution, centrifuging for 10-20 min at the temperature of 3-5 ℃ and under the condition of 12500-13500 xg, and removing the supernatant;
adding an activation buffer solution, using an ultrasonic crusher to ultrasonically resuspend the microspheres, then adding the activation buffer solution containing 10mg/mL of NHS, fully and uniformly mixing by a vortex mixer, then adding the activation buffer solution containing 10mg/mL of EDC, fully and uniformly mixing by the vortex mixer, carrying out mixed rotation incubation for 20-30min at 37 ℃ and 250rpm in a shaking incubator, and finally centrifuging for 10-20 min at 3-5 ℃ and 12500-13500 xg, and removing the supernatant to obtain the activated microspheres.
5. The method for preparing a latex immunoassay kit for determining a helicobacter pylori parting antibody according to claim 4, wherein the composition of the activation buffer is as follows: 45-55 mmol/L of morpholinoethanesulfonic acid and purified water.
6. The method for preparing a latex immunoassay kit for determining a helicobacter pylori parting antibody according to claim 3, wherein the microsphere blocking solution has the following composition: 45-55 mmol/L of 4-hydroxyethyl piperazine ethane sulfonic acid, 0.8-1.2% of bovine serum albumin with mass percentage concentration, 0.08-0.12% of proclin300 with mass percentage concentration and the balance of purified water;
alternatively, the microsphere preservation solution comprises the following components: 45-55 mmol/L of morpholinoethanesulfonic acid, 0.8-1.2% of bovine serum albumin with mass percentage concentration, 1.8-2.2% of trehalose with mass percentage concentration, 0.08-0.12% of proclin300 with mass percentage concentration and the balance of purified water.
7. The method for preparing a latex immunoassay kit for determining a helicobacter pylori typing antibody according to claim 3, wherein the pretreatment of the latex binding pad is as follows: the latex bonding pad is immersed in a latex bonding pad optimizing buffer solution, and then the humidity is dried and controlled below 30%, wherein the latex bonding pad optimizing buffer solution comprises 45-55 mmol/L of tris, 0.8-1.2% of bovine serum albumin, 0.4-0.6% of Tween 20, 1.8-2.2% of sucrose, 0.08-0.12% of proclin300 and the balance of purified water.
8. The method for preparing a latex immunoassay kit for determining a helicobacter pylori parting antibody according to claim 3, wherein the printing film buffer solution comprises PBS buffer solution with the concentration of 0.01-0.03 mol/L, pH of 7-7.4, trehalose with the mass percentage concentration of 0.8-1.2%, ethylenediamine tetraacetic acid with the concentration of 8-12 mmol/L and proclin300 with the mass percentage concentration of 0.08-0.12%.
9. Use of the latex immunoassay kit for determination of helicobacter pylori parting antibodies according to claim 1 or 2 or the latex immunoassay kit prepared by the preparation method of any one of claims 3 to 8 in preparation of products for determination of helicobacter pylori parting antibodies.
10. The use of the latex immunoassay kit according to claim 9, wherein the product further comprises a sample diluent comprising PBS buffer with a concentration of 0.02mol/L, pH of 7.2, 2% glucose by mass, and 0.1% proclin300 by mass.
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