CN111983229A - Reagent strip for quantitatively detecting helicobacter pylori antibody by colloidal gold and detection method - Google Patents
Reagent strip for quantitatively detecting helicobacter pylori antibody by colloidal gold and detection method Download PDFInfo
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Classifications
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- G—PHYSICS
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- 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
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- 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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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
Abstract
The invention discloses a reagent strip for quantitatively detecting helicobacter pylori antibody by using colloidal gold, which comprises a bottom plate, and a sample pad, a colloidal gold pad, a nitrocellulose membrane and absorbent filter paper which are stuck on the bottom plate; the sample pad, the colloidal gold pad, the nitrocellulose membrane and the absorbent filter paper are sequentially overlapped end to end for a preset length; the nitrocellulose membrane is coated with a helicobacter pylori natural antigen by scribing; the colloidal gold pad is obtained by loading a helicobacter pylori recombinant protein antigen solution marked with colloidal gold on a glass cellulose membrane and drying. The reagent strip for quantitatively detecting the helicobacter pylori antibody can realize the quantitative detection of the helicobacter pylori antibody by a colloidal gold immunochromatography method. Correspondingly, the invention also provides a method for quantitatively detecting the helicobacter pylori antibody by using the reagent strip for quantitatively detecting the helicobacter pylori antibody.
Description
Technical Field
The invention relates to a detection technology of a helicobacter pylori antibody, in particular to a reagent strip for quantitatively detecting the helicobacter pylori antibody by using colloidal gold and a detection method for quantitatively detecting the helicobacter pylori antibody by using the reagent strip.
Background
Helicobacter pylori (H.p), was first discovered by Barry j. marshall and j. robin Warren, both of which thus received 2005 s reward for nobel physiology or medicine. Helicobacter pylori is a single-polar, multi-flagellar, blunt-ended, helically-curved bacterium, 2.5 to 4.0 μm long and 0.5 to 1.0 μm wide. The surface of the gastric mucosal epithelial cell is usually in a typical spiral shape or arc shape.
Helicobacter pylori infection is a major causative factor of chronic active gastritis, peptic gastric ulcer, and gastric mucosa-associated lymphoid tissue lymphoma, and has a close correlation with the occurrence of gastric cancer. Helicobacter pylori was identified as a class I carcinogen by the world health organization/international agency for research on cancer (WHO/IARC) in 1994. In Asian regions, the infection rates of helicobacter pylori in China continental, Vietnam, India and other juvenile are respectively 60%, 40% and 70%, the detection rate of helicobacter pylori in gastric mucosa biopsy specimens of chronic gastritis patients can reach 80% -90%, and digestive gastric ulcer patients are higher and can reach more than 95%, even approach 100%. Since the local epithelial cells of stomach cancer have been differentiated, the detection rate of stomach cancer is reported differently. Gastric infection with H.pylori has been a worldwide medical problem, and accurate diagnosis thereof has facilitated control H.p transmission and prevalence and monitoring of treatment for eradication H.p infections.
A number of methods have been developed for the diagnosis of H.pylori infection, including bacteriology, pathology, serology, isotopic tracing, molecular biology, etc. Generally, however, from the viewpoint of specimen collection, there are two main categories, invasive and non-invasive. Invasive methods mainly refer to methods that require biopsy specimens to be examined by gastroscopy, and are the current routine methods in the gastroenterology discipline. It includes the separation culture of bacteria, direct smear, fast urease test and drug sensitive test. The non-invasive method mainly refers to a method for diagnosing infection of helicobacter pylori specimens without taking biopsy specimens by gastroscopy. Such methods include antibody detection, antigen detection, urea 13C/14C breath assay, and the like. The existing methods for detecting the antibody comprise an enzyme-linked immunosorbent assay, an immunoblotting method, a colloidal gold method, a latex enhanced immunoturbidimetry method and the like.
Serological detection is a common non-invasive detection method, and is particularly suitable for areas with high helicobacter pylori infection rate. Helicobacter pylori causes chronic inflammation, and systemic immune response persists, producing IgG antibodies. The common serological tests comprise ELISA method, chemiluminescence, western blotting, colloidal gold immunochromatography and other methods, wherein the colloidal gold immunochromatography has the fastest detection speed, can obtain a result in only 10 minutes, and is particularly suitable for on-site rapid detection, but no quantitative helicobacter pylori antibody colloidal gold immunochromatography detection reagent is on the market at present.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a reagent strip for quantitatively detecting helicobacter pylori antibody by using colloidal gold. The reagent strip for quantitatively detecting the helicobacter pylori antibody can realize the quantitative detection of the helicobacter pylori antibody by a colloidal gold immunochromatography method. Correspondingly, the invention also provides a method for quantitatively detecting the helicobacter pylori antibody by using the reagent strip for quantitatively detecting the helicobacter pylori antibody.
For the reagent strip, the technical scheme of the invention is as follows:
the reagent strip for quantitatively detecting the helicobacter pylori antibody comprises a bottom plate, and a sample pad, a colloidal gold pad, a nitrocellulose membrane and absorbent filter paper which are stuck on the bottom plate; the sample pad, the colloidal gold pad, the nitrocellulose membrane and the absorbent filter paper are sequentially overlapped end to end for a preset length; the nitrocellulose membrane is coated with a helicobacter pylori natural antigen by scribing; the colloidal gold pad is obtained by loading a helicobacter pylori recombinant protein antigen solution marked with colloidal gold on a glass cellulose membrane and drying.
In an optimized embodiment of the present invention, in the reagent strip for quantitatively detecting helicobacter pylori antibody with colloidal gold, the colloidal gold pad is prepared by the following steps: step a, using 0.1-0.3 mol/L K2CO3The pH of the colloidal gold solution is adjusted to 7.0-9.0 by the solution; b, placing the colloidal gold solution in the step a into magnetic stirringStirring on a stirrer, dropwise adding the helicobacter pylori recombinant protein antigen into the colloidal gold solution according to the dosage of adding 0.5-2 mg of the helicobacter pylori recombinant protein antigen into every 100mL of the colloidal gold solution, and continuously stirring for 2-3 h; c, adding the stirred colloidal gold solution obtained in the step b into a mixed solution of BSA (bovine serum albumin) with the final concentration of 1% and casein with the final concentration of 1-2%, and carrying out closed labeling for 60 min; d, centrifuging the colloidal gold solution subjected to the closed labeling in the step c under the condition of 12000-15000 r/m, discarding supernatant, resuspending the precipitate by using 0.01mol/L PBS solution, and resuspending the precipitate into 1/10 of the volume of the original precipitate to obtain a resuspension solution; e, centrifuging the heavy suspension prepared in the step d for 30min under the condition of 2000-3000 r/min, taking supernatant liquid, adding the supernatant liquid into a sephadex chromatographic column for purification, and obtaining a helicobacter pylori recombinant protein solution of the labeled colloidal gold with the purity of more than 90%; and f, adding the helicobacter pylori recombinant protein antigen solution marked with the colloidal gold prepared in the step e onto a glass cellulose membrane, and drying for 4-6 hours at the temperature of 20-25 ℃ to prepare a colloidal gold pad for later use.
As an optimized scheme of the invention, in the reagent strip for quantitatively detecting helicobacter pylori antibody by using colloidal gold, the method for coating the helicobacter pylori natural antigen on the nitrocellulose membrane by scribing comprises the following steps: i, diluting the helicobacter pylori natural antigen to 0.2-0.5 mg/mL by using 0.01mol/L PBS; step ii, scribing and coating the helicobacter pylori natural antigen diluted in the step i on a nitrocellulose membrane according to 1 mu l/cm by using a membrane spotting instrument; and step iii, after the natural antigen coating of the helicobacter pylori is finished, drying the nitrocellulose membrane at room temperature in a drying room for later use.
As an optimized scheme of the invention, in the reagent strip for quantitatively detecting the helicobacter pylori antibody by the colloidal gold, the nitrocellulose membrane is coated with a mouse anti-helicobacter pylori monoclonal antibody by scribing and is used for quality control. Further, the method for coating the mouse anti-helicobacter pylori monoclonal antibody on the nitrocellulose membrane by streaking comprises the following steps: firstly, diluting a mouse anti-helicobacter pylori monoclonal antibody into 0.2-0.5 mg/mL by using 0.01mol/L PBS; step two, marking and coating the mouse anti-helicobacter pylori monoclonal antibody diluted in the step i on the nitrocellulose membrane according to 1 mul/cm by using a membrane spotting instrument; and step three, after the mouse anti-helicobacter pylori monoclonal antibody is coated, drying the nitrocellulose membrane at room temperature in a drying room for later use.
As an optimized scheme of the invention, in the reagent strip for quantitatively detecting helicobacter pylori antibody by using colloidal gold, the assembly of the reagent strip is completed in a drying room, and the specific steps are as follows: i, sticking a helicobacter pylori natural antigen nitrocellulose membrane coated in the center of a bottom plate; step II, pasting absorbent filter paper on the upper edge of the nitrocellulose membrane; step III, sticking a colloidal gold pad on the lower edge of the nitrocellulose membrane; and IV, sticking a sample pad on the lower edge of the colloidal gold pad, cutting the stuck test paper board into test paper strips by using a cutting machine after the sample pad is stuck, and sealing the test paper strips in an aluminum foil bag.
Preferably, the base plate may be made of PVC.
Compared with the prior art, the reagent strip for quantitatively detecting the helicobacter pylori antibody is prepared by adopting a helicobacter pylori recombinant protein antigen and natural antigen combined double-antigen sandwich method, when the reagent strip is used for detecting a clinical sample, a colloidal gold detector reads a T/C value and substitutes the T/C value into a standard curve, so that the content (AU/mL) of the helicobacter pylori antibody in the clinical sample can be obtained, and the rapid quantitative detection of the helicobacter pylori antibody (IgG antibody) is realized.
For the detection method, the technical scheme of the invention is as follows:
the method adopts the reagent strip for quantitatively detecting the helicobacter pylori antibody to carry out qualitative detection and quantitative detection on the helicobacter pylori antibody;
the qualitative detection process is as follows: horizontally placing the helicobacter pylori antibody detection strip, accurately dropwise adding 1 drop of serum or plasma sample on a sample pad of the test strip, reacting at room temperature for 10 minutes, wherein a red strip appears on a quality control line, a red strip appears on a detection line, and the detection result is positive helicobacter pylori IgG antibody; the quality control line has a red strip, the detection line has no red strip, and the detection result is helicobacter pylori IgG antibody negative; the quality control line does not have a red strip, or the quality control line and the detection line do not have a red strip, so that the detection of the reagent strip is invalid;
the quantitative detection process is as follows: horizontally placing the helicobacter pylori antibody reagent strip, accurately dropwise adding 1 drop of serum or plasma sample on a sample pad of the test strip, reacting at room temperature for 10 minutes, placing the helicobacter pylori antibody reagent strip into a colloidal gold quantitative reading instrument, reading the peak area ratio of a T line and a C line, namely a T/C value, substituting the T/C value into a helicobacter pylori antibody detection standard curve, and obtaining the content (AU/mL) of the helicobacter pylori antibody in the sample.
As an optimized scheme of the invention, in the detection method for quantitatively detecting the helicobacter pylori antibody by using the colloidal gold, the manufacturing method of the detection standard curve of the helicobacter pylori antibody comprises the following steps: diluting 1AU/mL helicobacter pylori antibody detection standard substance with helicobacter pylori antibody negative serum according to gradient, respectively taking 50 mu L of the helicobacter pylori antibody negative serum, adding the helicobacter pylori antibody negative serum into a sample pad of a reagent strip, reacting for 10min at room temperature, putting the reagent strip into a colloidal gold quantitative reading instrument, reading the peak area ratio of a T line and a C line, namely a T/C value, taking the T/C value as a horizontal coordinate and the AU value of the helicobacter pylori antibody detection standard substance as a vertical coordinate, and making a standard curve; the preparation method of the 1AU/mL helicobacter pylori antibody detection standard substance comprises the following steps: 20 parts of helicobacter pylori antibody positive serum are collected and mixed, and are subjected to affinity chromatography purification by saturated ammonium sulfate salting-out and Protein G column to obtain a purified helicobacter pylori antibody with the final concentration of 0.5mg/mL, which is determined as 1 AU/mL.
Compared with the prior art, the method of the invention has the following remarkable improvements: quantitative detection of helicobacter pylori is realized by using a colloidal gold immunochromatography.
Drawings
FIG. 1 is a schematic structural diagram of a reagent strip for quantitatively detecting helicobacter pylori antibody by using colloidal gold according to the present invention;
in the drawings, the reference numbers: 1-bottom plate, 2-sample pad, 3-colloidal gold pad, 4-absorbent filter paper, 5-nitrocellulose membrane, 501-detection line, 502-quality control line.
Detailed Description
The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.
Referring to fig. 1, the reagent strip for quantitatively detecting helicobacter pylori antibody of the present invention comprises a PVC-made base plate 1, and a sample pad 2, a colloidal gold pad 3, a nitrocellulose membrane 5 and absorbent filter paper 4 adhered to the PVC-made base plate 1; the sample pad 2, the colloidal gold pad 3, the nitrocellulose membrane 5 and the water-absorbing filter paper 4 are sequentially overlapped end to end for a preset length.
Wherein, the nitrocellulose membrane 5 is coated with helicobacter pylori natural antigen by scribing to form a detection line 501; the colloidal gold pad 3 is obtained by loading a helicobacter pylori recombinant protein antigen solution marked with colloidal gold on a glass cellulose membrane and drying.
When the invention is implemented, the nitrocellulose membrane 5 can be coated with the mouse anti-helicobacter pylori monoclonal antibody by scribing to form a quality control line 502 for quality control.
Example (b):
the reagent strip for quantitatively detecting helicobacter pylori antibody using colloidal gold of this example was prepared as follows.
Firstly, preparing a colloidal gold pad coated with a helicobacter pylori recombinant protein antigen: with 0.2mol/L K2CO3The pH of the colloidal gold solution was adjusted to 8. And then placing the solution on a magnetic stirrer to be slowly stirred, adding 1.25mg of helicobacter pylori recombinant protein antigen into every 100mL of colloidal gold solution to slowly drop the protein into the colloidal gold solution, continuously stirring for 2.5h, then dropping the protein into BSA (bovine serum albumin) and casein (1.5%) with final concentrations to carry out blocking marking for 60min, centrifuging at 13500r/m after the blocking marking is finished, removing supernatant, resuspending the precipitate with 0.01mol/L of PBS solution, resuspending the precipitate into 1/10 of the original volume, centrifuging for 30min at 2500r/min, taking the supernatant, adding the supernatant into a sephadex chromatographic column for purification, and obtaining the helicobacter pylori recombinant protein solution marked with the colloidal gold, wherein the purity of the helicobacter pylori recombinant protein solution is more than 90%. Then, the helicobacter pylori recombinant protein antigen solution marked with the colloidal gold is loaded on a glass cellulose membrane, and is dried for 5 hours at the temperature of 22 ℃ to prepare a colloidal gold pad 3, and is dried for standby.
Secondly, coating a nitrocellulose membrane by a helicobacter pylori natural antigen: diluting helicobacter pylori natural antigen to 0.3mg/mL by using 0.01mol/L PBS, and then marking and coating the helicobacter pylori natural antigen on the nitrocellulose membrane 5 by using a BIODOT membrane spotting instrument according to 1 mu L/cm to form a detection line 501; meanwhile, a mouse anti-helicobacter pylori monoclonal antibody is coated on the nitrocellulose membrane 5 according to 1 mul/cm and is used for a quality control line 502 of a product, and after the coating is finished, the nitrocellulose membrane 5 is dried for 8 hours at room temperature for later use.
Thirdly, assembling reagents: preparing a PVC-made bottom plate 1, a sample pad 2, a colloidal gold pad 3, a nitrocellulose membrane 5 and water-absorbing filter paper 4 in a drying room, sticking a helicobacter pylori natural antigen nitrocellulose membrane 5 coated on the center of the bottom plate 1, sticking the water-absorbing filter paper 4 on the upper edge of the nitrocellulose membrane 5, sticking a helicobacter pylori recombinant protein antigen colloidal gold pad 3 on the lower edge of the nitrocellulose membrane 5, sticking the sample pad 2 on the lower edge of the colloidal gold pad 3, and cutting the stuck test paper board into test paper strips with the width of 4mm by using a cutting machine after the completion. And sealing the test strip in an aluminum foil bag to complete the assembly of the reagent, thereby obtaining the product.
The invention is further described below in connection with the detection method and the necessary tests.
The method for rapidly and quantitatively detecting the helicobacter pylori antibody by using the reagent card comprises the following steps: the reagent strip is horizontally placed, 1 drop of serum or plasma sample is accurately dripped on a sample pad 2 of the test strip, and after reaction for 10 minutes at room temperature, quantitative detection is carried out according to the following method.
The quantitative detection process is as follows: horizontally placing the helicobacter pylori antibody reagent strip, accurately dropwise adding 1 drop of serum or plasma sample on a sample pad 2 of the test strip, reacting for 10 minutes at room temperature, placing the helicobacter pylori antibody reagent strip into a colloidal gold quantitative reading instrument, reading the peak area ratio of a T line and a C line, namely a T/C value, substituting into a helicobacter pylori antibody detection standard curve, and obtaining the content (AU/mL) of the helicobacter pylori antibody in the sample.
The method for establishing the helicobacter pylori antibody detection standard curve comprises the following steps: diluting 1AU/mL helicobacter pylori antibody detection standard substance with helicobacter pylori antibody negative serum according to gradient, adding 50 μ L into a sample pad 2 of a reagent strip, reacting at room temperature for 10min, putting the reagent strip into a colloidal gold quantitative reading instrument, reading the peak area ratio of a T line and a C line, namely a T/C value, taking the T/C value as a horizontal coordinate and the AU value of the helicobacter pylori antibody detection standard substance as a vertical coordinate, and making a standard curve. When the reagent is used for detecting clinical samples, a colloidal gold quantitative reading instrument reads a T/C value and substitutes the T/C value into a standard curve to obtain the content (AU/mL) of helicobacter pylori antibody in the samples.
The preparation method of the helicobacter pylori antibody detection standard substance comprises the following steps: 20 parts of helicobacter pylori antibody positive serum are collected and mixed, and are subjected to affinity chromatography purification by saturated ammonium sulfate salting-out and Protein G column to obtain a purified helicobacter pylori antibody with the final concentration of 0.5mg/mL, which is determined as 1 AU/mL.
Helicobacter pylori antibody test strip specificity test: helicobacter pylori IgG positive serum, hepatitis B virus IgG positive serum, cytomegalovirus IgG positive serum, herpes simplex virus IgG positive serum, adenovirus IgG positive serum, respiratory syncytial virus IgG positive serum and helicobacter pylori IgG negative serum are detected by a helicobacter pylori antibody immune colloidal gold detection test strip, only the detection result of the helicobacter pylori standard IgG positive serum is positive, and the others are negative, which indicates that the reagent strip has good specificity.
The test paper strip repeatability test of the helicobacter pylori antibody immune colloidal gold test: 20 parts of helicobacter pylori IgG positive serum and negative serum are randomly extracted and repeatedly detected at different time, the coefficient of variation is less than 5%, and the reagent strip is proved to have good stability and repeatability.
Clinical specimen detection contrast test:
comparison of the test strips with the ELISA test kit for helicobacter pylori IgG antibody, a BIOHIT kit in Finland:
the negative and positive results were compared for 200 specimens tested with two kits, as shown in table 1:
table 1 comparison with the kit of BIOHIT, Finland
A total of 195 cases (97.5%) and only 5 cases (2.5%) of the two kits were tested on 200 specimens, which also indicates that the reagents of the present invention have high sensitivity and specificity.
The above is a description of a specific embodiment of the present invention, and the object of the present invention can be achieved by other specific embodiments formed by adjusting the values of the relevant parameters in the above embodiments within the scope of the technical solution of the present invention, which are not listed here.
The reagent strip of the invention can also be used for qualitatively detecting helicobacter pylori antibody, and the specific method is as follows:
and horizontally placing the reagent strip, accurately dropwise adding 1 drop of serum or plasma sample on a sample pad 2 of the test strip, reacting at room temperature for 10 minutes, and then judging a qualitative result.
Positive: the quality control line 502 shows a red strip, and the detection line 501 shows a red strip, which is positive to the helicobacter pylori IgG antibody; negative: the quality control line 502 has a red strip, and the detection line 501 has no red strip, which is the H.pylori IgG antibody negative; and (3) failure: the red strip does not appear on the quality control line 502, and the reagent strip is judged to be invalid (when the sample is dripped, the quality control product is dripped at the same time).
The above general description of the invention and the description of the specific embodiments thereof referred to in this application should not be construed as limiting the technical solutions of the invention. Those skilled in the art can add, reduce or combine the technical features disclosed in the general description and/or the embodiments to form other technical solutions within the protection scope of the present application without departing from the present disclosure.
Claims (9)
1. The reagent strip for quantitatively detecting the helicobacter pylori antibody is characterized in that:
the reagent strip comprises a bottom plate, and a sample pad, a colloidal gold pad, a nitrocellulose membrane and absorbent filter paper which are stuck on the bottom plate; the sample pad, the colloidal gold pad, the nitrocellulose membrane and the absorbent filter paper are sequentially overlapped end to end for a preset length;
the nitrocellulose membrane is coated with a helicobacter pylori natural antigen by scribing; the colloidal gold pad is obtained by loading a helicobacter pylori recombinant protein antigen solution marked with colloidal gold on a glass cellulose membrane and drying.
2. The reagent strip for quantitatively detecting helicobacter pylori antibody according to claim 1, wherein the colloidal gold pad is prepared by the following method steps:
step a, using 0.1-0.3 mol/L K2CO3The pH of the colloidal gold solution is adjusted to 7.0-9.0 by the solution;
b, placing the colloidal gold solution obtained in the step a on a magnetic stirrer for stirring, dropwise adding the helicobacter pylori recombinant protein antigen into the colloidal gold solution according to the dosage of adding 0.5-2 mg of the helicobacter pylori recombinant protein antigen into every 100mL of the colloidal gold solution, and continuously stirring for 2-3 hours;
c, adding the stirred colloidal gold solution obtained in the step b into a mixed solution of BSA (bovine serum albumin) with the final concentration of 1% and casein with the final concentration of 1-2%, and carrying out closed labeling for 60 min;
d, centrifuging the colloidal gold solution subjected to the closed labeling in the step c under the condition of 12000-15000 r/m, discarding supernatant, resuspending the precipitate by using 0.01mol/L PBS solution, and resuspending the precipitate into 1/10 of the volume of the original precipitate to obtain a resuspension solution;
e, centrifuging the heavy suspension prepared in the step d for 30min under the condition of 2000-3000 r/min, taking supernatant liquid, adding the supernatant liquid into a sephadex chromatographic column for purification, and obtaining a helicobacter pylori recombinant protein solution of the labeled colloidal gold with the purity of more than 90%;
and f, adding the helicobacter pylori recombinant protein antigen solution marked with the colloidal gold prepared in the step e onto a glass cellulose membrane, and drying for 4-6 hours at the temperature of 20-25 ℃ to prepare a colloidal gold pad for later use.
3. The reagent strip for quantitatively detecting helicobacter pylori antibody according to claim 1, wherein the method for coating the helicobacter pylori natural antigen on the nitrocellulose membrane by streaking comprises the following steps:
i, diluting the helicobacter pylori natural antigen to 0.2-0.5 mg/mL by using 0.01mol/L PBS;
step ii, scribing and coating the helicobacter pylori natural antigen diluted in the step i on a nitrocellulose membrane according to 1 mu l/cm by using a membrane spotting instrument;
and step iii, after the natural antigen coating of the helicobacter pylori is finished, drying the nitrocellulose membrane at room temperature in a drying room for later use.
4. The reagent strip for quantitatively detecting helicobacter pylori antibody according to claim 1, wherein a mouse anti-helicobacter pylori monoclonal antibody is further coated on the nitrocellulose membrane by streaking for quality control.
5. The reagent strip for quantitatively detecting helicobacter pylori antibody according to claim 1, wherein the method for coating the mouse anti-helicobacter pylori monoclonal antibody on the nitrocellulose membrane by streaking comprises the following steps:
firstly, diluting a mouse anti-helicobacter pylori monoclonal antibody into 0.2-0.5 mg/mL by using 0.01mol/L PBS;
step two, marking and coating the mouse anti-helicobacter pylori monoclonal antibody diluted in the step i on the nitrocellulose membrane according to 1 mul/cm by using a membrane spotting instrument;
and step three, after the mouse anti-helicobacter pylori monoclonal antibody is coated, drying the nitrocellulose membrane at room temperature in a drying room for later use.
6. The reagent strip for quantitatively detecting helicobacter pylori antibody according to the colloidal gold of claim 1, wherein the assembly of the reagent strip is completed in a drying room by the following specific steps:
i, sticking a helicobacter pylori natural antigen nitrocellulose membrane coated in the center of a bottom plate;
step II, pasting absorbent filter paper on the upper edge of the nitrocellulose membrane;
step III, sticking a colloidal gold pad on the lower edge of the nitrocellulose membrane;
and IV, sticking a sample pad on the lower edge of the colloidal gold pad, cutting the stuck test paper board into test paper strips by using a cutting machine after the sample pad is stuck, and sealing the test paper strips in an aluminum foil bag.
7. The reagent strip for quantitatively detecting helicobacter pylori antibody according to the colloidal gold of any one of claims 1 to 6, wherein the bottom plate is made of PVC.
8. A method for quantitatively detecting a helicobacter pylori antibody by using colloidal gold, which comprises quantitatively detecting a helicobacter pylori antibody by using the reagent strip for quantitatively detecting a helicobacter pylori antibody according to any one of claims 1 to 7;
the quantitative detection process is as follows: horizontally placing the helicobacter pylori antibody reagent strip, accurately dropwise adding 1 drop of serum or plasma sample on a sample pad of the test strip, reacting at room temperature for 10 minutes, placing the helicobacter pylori antibody reagent strip into a colloidal gold quantitative reading instrument, reading the peak area ratio of a T line and a C line, namely a T/C value, substituting the T/C value into a helicobacter pylori antibody detection standard curve, and obtaining the content (AU/mL) of the helicobacter pylori antibody in the sample.
9. The method for quantitatively detecting helicobacter pylori antibody according to the colloidal gold of claim 8, wherein the method for preparing the standard curve for detecting helicobacter pylori antibody comprises the following steps:
diluting 1AU/mL helicobacter pylori antibody detection standard substance with helicobacter pylori antibody negative serum according to gradient, respectively taking 50 mu L of the helicobacter pylori antibody negative serum, adding the helicobacter pylori antibody negative serum into a sample pad of a reagent strip, reacting for 10min at room temperature, putting the reagent strip into a colloidal gold quantitative reading instrument, reading the peak area ratio of a T line and a C line, namely a T/C value, taking the T/C value as a horizontal coordinate and the AU value of the helicobacter pylori antibody detection standard substance as a vertical coordinate, and making a standard curve;
the preparation method of the 1AU/mL helicobacter pylori antibody detection standard substance comprises the following steps: 20 parts of helicobacter pylori antibody positive serum are collected and mixed, and are subjected to affinity chromatography purification by saturated ammonium sulfate salting-out and Protein G column to obtain a purified helicobacter pylori antibody with the final concentration of 0.5mg/mL, which is determined as 1 AU/mL.
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| CN202010925995.8A CN111983229A (en) | 2020-09-07 | 2020-09-07 | Reagent strip for quantitatively detecting helicobacter pylori antibody by colloidal gold and detection method |
| PCT/CN2021/123687 WO2022048693A1 (en) | 2020-09-07 | 2021-10-14 | Reagent strip for quantitatively detecting helicobacter pylori antibody by means of colloidal gold and detection method |
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| CN202010925995.8A CN111983229A (en) | 2020-09-07 | 2020-09-07 | Reagent strip for quantitatively detecting helicobacter pylori antibody by colloidal gold and detection method |
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| WO2022048693A1 (en) * | 2020-09-07 | 2022-03-10 | 三门县人民医院 | Reagent strip for quantitatively detecting helicobacter pylori antibody by means of colloidal gold and detection method |
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