CN112114140A - Novel coronavirus IgA antibody colloidal gold detection kit - Google Patents
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G01N33/532—Production of labelled immunochemicals
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Abstract
The invention provides a novel coronavirus IgA antibody colloidal gold detection kit, which comprises a detection card provided with a coating plate and a sample pad, wherein a gold mark area of the coating plate is coated with a colloidal gold mark anti-human IgA, a detection area is coated with a novel coronavirus recombinant antigen, and a quality control area is coated with goat anti-mouse IgG. The kit can be used for quickly detecting the novel coronavirus IgA antibody.
Description
Technical Field
The invention belongs to the technical field of immunoassay detection, and particularly relates to a novel coronavirus IgA antibody colloidal gold detection kit.
Background
The coronavirus belongs to single-stranded positive-strand RNA virus, and there are 6 kinds of coronavirus known to infect humans, namely HCoV-229E, HCoV-OC43, SARSr-CoV, HCoV-NL63, HCoV-HKU1 and MERSR-CoV. The novel coronavirus (2019-nCoV) belongs to the 7 th species.
The novel coronavirus pneumonia is an acute infectious pneumonia, and the pathogen of the novel coronavirus is a novel coronavirus which is not found in human before, namely 2019 novel coronavirus. Transmission via respiratory droplets and intimate contact is the primary transmission route, and there is the potential for transmission via aerosols during prolonged exposure to high concentrations of aerosols in a relatively closed environment. The initial symptoms of the patients are mostly fever, hypodynamia and dry cough, and the patients gradually show severe manifestations such as dyspnea and the like. The prognosis is good in most patients and acute respiratory distress syndrome or septic shock may occur in some severe cases and even death.
The novel coronavirus SARS-COV-2 is now known as a mucosa-targeted virus which stimulates the production of large amounts of secretory IgA, which in turn activates the mucosal immune response. However, no SARS-COV-2 specificity IgA antibody detection kit exists at present, and a method for rapidly detecting the novel coronavirus IgA antibody is provided.
Disclosure of Invention
In view of this, the invention aims to provide a novel coronavirus IgA antibody colloidal gold detection kit which can rapidly detect a novel coronavirus IgA antibody.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
the utility model provides a novel coronavirus IgA antibody colloidal gold detect reagent box, is equipped with the detection card that peridium board and sample pad, and the gold mark district of peridium board is wrapped there is anti human IgA of colloidal gold mark, and the detection zone parcel has novel coronavirus recombination antigen, and quality control district is wrapped goat anti mouse IgG.
Preferably, the novel coronavirus recombinant antigen is an S-RBD protein.
Preferably, the sample pad is coated with a sample treatment solution, and the sample treatment solution is tris buffer solution which contains BSA with the mass concentration of 0.5-1.5%, casein with the mass concentration of 0.5-1.2%, sucrose with the mass concentration of 0.3-1.2%, trehalose with the mass concentration of 0.1-1.2%, Procline with the volume concentration of 0.05-0.3%, and tween-20 with the volume concentration of 0.05-0.5% and has the pH value of 7.0-7.6; the preparation method of the sample pad comprises spraying or soaking the sample pad treating solution onto or into the glass fiber film or polyester film at 45-85 microliter per square centimeter, and oven drying at 37-45 deg.C for 8-16 hr.
Preferably, the preparation of the colloidal gold-labeled anti-human IgA comprises the step of adding K to a homogeneous colloidal gold solution2CO3Adjusting pH to 6.0-7.5, adding anti-human IgA into the solution with adjusted pH to make the protein content of the solution be 15-60 μ g/mL, coupling for 0.5-4 h, adding a stop solution to stop the coupling reaction, wherein the final concentration (mass-volume ratio) of the stop solution is 1-1.5%; centrifuging after 0.5-2 hours after the reaction is ended, centrifuging for 15-30min at the centrifugation parameter of 12000-18000RPM, discarding the supernatant, redissolving the precipitate with a redissolution, and storing at 2-8 ℃ for later use.
Preferably, the stop solution is an aqueous solution prepared from one or two of bovine serum albumin, skim milk powder and glycine, when the stop solution is one of the solutions, the stop solution needs to be prepared into a solution with a mass volume ratio of 5% -10%, and when the stop solution is two of the solutions, the mass volume ratio of the bovine serum albumin to the skim milk powder or the glycine is 1: 1-4: 1, the solid mass content and the liquid volume ratio are 10 percent; the compound solution is 0.01-0.05M Tris or PB buffer solution, the pH is 7.4-8.2, 0.5-1% of NaCl, 0.2-1% of trehalose and 0.1-3% of PEG20000 are added into the compound solution according to the mass-volume ratio; bovine serum albumin is 0.5-3% by mass volume, and Triton X-405 is 0.5-1% by volume.
Preferably, the colloidal gold solution is prepared by preparing chloroauric acid into a solution with a mass-volume ratio of 1% -10%, adding the solution into boiled purified water until the mass-volume ratio of the chloroauric acid is one ten thousandth to four ten thousandth, continuing to boil for 2-5 minutes, adding 500-800 microliters of sodium citrate into 100mL chloroauric acid solution, and continuing to stir and heat for 10-20 minutes to prepare the colloidal gold solution with uniform particle size.
Preferably, the colloidal gold labeled anti-human IgA is spread on a labeling pad uniformly, and dried for 12 hours at 37 ℃ to obtain the labeling pad; preparing a solution with the protein content of 0.5-2.0mg/mL by using the novel coronavirus recombinant antigen and goat anti-mouse IgG, respectively coating the solution at the positions of a T line and a C line by using a film-cutting instrument, and drying at 37 ℃ to obtain a coated nitrocellulose membrane, wherein the coating parameter is 0.5-1.5 mu L/cm; and sequentially overlapping and sticking the absorbent paper, the nitrocellulose membrane, the marking pad, the blood filtering membrane and the sample pad on the PVC plate.
Preferably, the hemofiltration membrane is used after being treated with the anti-RBC antibody, the treatment concentration is 0.1mg/mL, the treatment amount is 60 microliters per square centimeter, and the hemofiltration membrane is dried for 12 hours at 37 ℃. Can be used for intercepting red blood cells in blood sample.
Preferably, the kit comprises a sample diluent and a 0.05M PB buffer solution, wherein the pH value is 8.0, the mass volume ratio of NaCl is 0.9-2%, and the volume ratio of tween 20 is 0.05-0.3%.
The invention also provides application of the kit in preparation of a novel coronavirus IgA detection reagent.
The conjugate pad is a module to which the labeled conjugate is attached, and the treatment solution for treating the conjugate pad is pH 8.0-8.5, 0.01-0.05M tris buffer, 0.1-0.3% tween-20.
The coating buffer solution for diluting the antigen-antibody raw material for coating is 0.01-0.05M buffer solution with pH of 7.4-8.5, 0.1-3% sucrose, 0.1-1% casein.
The principle of the invention is as follows:
the kit adopts immunochromatography, a blood sample to be detected is added on a detection card, a novel coronavirus IgA antibody and a non-novel coronavirus IgA antibody in the sample are combined with anti-human IgA marked by colloidal gold to form a novel coronavirus IgA antibody-anti-human IgA antibody compound, the non-novel coronavirus IgA antibody-anti-human IgA antibody compound, the immune compound moves forwards along the detection card under the capillary action, the novel coronavirus IgA antibody-anti-human IgA antibody compound can be captured by a novel coronavirus recombinant antigen coated on a detection area on a membrane to form a purple red (or pink) strip, and the non-novel coronavirus IgA antibody-anti-human IgA antibody compound continuously moves forwards, combined with a quality control region of a goat anti-mouse to form a purple (or pink) strip, and shows that the novel coronavirus IgA antibody is positive.
Compared with the prior art, the novel coronavirus IgA antibody colloidal gold detection kit disclosed by the invention has the following characteristics: when the probe is used as a single detection index, the probe has higher sensitivity and specificity.
Drawings
FIG. 1 shows the analysis of the condition of a test strip.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The present invention will be described in detail with reference to examples.
The utility model provides a novel coronavirus IgA colloidal gold detect reagent box, is including the detection card that is equipped with peridium board and sample pad, and the gold mark district of peridium board is wrapped there is anti human IgA of colloidal gold mark, and the detection zone parcel has novel coronavirus recombination antigen, and quality control district is wrapped goat anti mouse IgG. The novel coronavirus recombinant antigen is S-RBD protein.
The sample pad is coated with a sample treatment solution, and the sample treatment solution is a Tris buffer solution which contains BSA with the mass concentration of 1%, casein with the mass concentration of 0.5%, sucrose with the mass concentration of 0.5%, trehalose with the mass concentration of 1.0%, Proclin 300 with the volume concentration of 0.05%, tween-20 with the volume concentration of 0.08% and has the pH value of 7.4; the preparation method of the sample pad comprises spraying or soaking the sample pad treatment solution with 60 microliter per square centimeter on glass fiber film or polyester film, and oven drying at 37 deg.C for 12 hr.
The preparation of colloidal gold-labeled anti-human IgA comprises the step of adding K to a homogeneous colloidal gold solution2CO3Adjusting pH to 8.0, adding anti-human IgA to the pH adjusted solution to make its protein content 25 μ g/mL, coupling for 0.5 hrAdding a stop solution to the reaction mixture to stop the coupling reaction; centrifuging after 0.5 hour after reaction termination, wherein the centrifugation parameter is 12000RPM, centrifuging for 20min, discarding the supernatant, redissolving the precipitate with redissolving solution, and storing at 2-8 deg.C for later use; .
The stop solution is bovine serum albumin with the mass concentration of 10%; the complex solution is 0.02M PB buffer solution, the pH value is 7.4, and 0.9% of NaCl, 0.8% of trehalose, 1.5% of PEG 200001.5% bovine serum albumin and 1.32% of Triton X-1000.5% are added into the complex solution.
The colloidal gold solution is prepared by preparing 10% chloroauric acid solution by mass volume ratio, adding the solution into boiled purified water until the chloroauric acid content is two-fifths of a ten-thousandth, continuously boiling for 3 minutes, adding 580 microliters of sodium citrate into each 100mL chloroauric acid solution by 0.1M, continuously stirring and heating for 10 minutes, cooling to room temperature, thus preparing the colloidal gold solution with uniform particle size, and storing at 2-8 ℃ for later use.
Preparing the detection card, namely paving the colloidal gold-labeled anti-human IgA on a labeling pad uniformly, and drying for 12 hours at 37 ℃ to obtain the labeling pad; preparing the novel coronavirus recombinant antigen and goat anti-mouse IgG into 0.5-2.0mg/mL solutions respectively, coating the solutions at the positions of a T line and a C line respectively by using a film-cutting gold spraying instrument at 37 ℃, and drying to obtain coated nitrocellulose membranes, wherein the coating parameter is 0.5-1.5 mu L/cm; and sequentially overlapping and sticking the absorbent paper, the nitrocellulose membrane, the marking pad, the blood filtering membrane and the sample pad on the PVC plate.
The blood filter membrane is used after being treated by an anti-RBC antibody, the treatment concentration is 0.1mg/mL, the treatment capacity is 60 microliters per square centimeter, and the blood filter membrane is dried for 12 hours at the temperature of 37 ℃. Can be used for intercepting red blood cells in blood sample.
Also included was a sample diluent, 0.05PB buffer, pH 8.0, to which naci 0.9%, sucrose 2% was added.
The kit comprises the following reaction steps:
procedure of experiment
1 if the sample is stored in a refrigerated or frozen state, the sample to be tested and the required reagents are removed from the storage conditions and allowed to equilibrate to room temperature (20-30 ℃). Fully and uniformly mixing the sample to be detected after melting;
2 when in preparation for detection, opening the aluminum foil bag from the tear, taking out the detection card, and horizontally placing the detection card on a horizontal desktop;
marking a sample number on the detection card;
and 4, sucking 10 mu L of the sample to be detected from the sample tube by using a pipette or a dropper, and dripping 100 mu L of sample diluent to ensure that no bubbles exist in the operation process.
5, counting time for 15 minutes, judging the result, and after 20 minutes, the judging result is invalid.
Interpretation of test results
1, when only a quality control line C appears, the detection line T does not develop color, which indicates that the novel coronavirus antibody IgA is not detected, and the result is negative, as shown in (a) in the following figure 1;
2, a positive result, when the quality control line C and the detection line T both appear, indicating that the novel coronavirus antibody IgA is detected, as shown in (b) in fig. 1;
and 3, invalid result, namely when the quality control line C cannot be observed, whether T appears or not is an invalid result, and the result is detected again in (C/d) in FIG. 1.
Detecting 10 negative enterprise reference products, wherein the results are negative (-/-, 10/10); 5 positive business references with a positive result (+/+, 5/5); the sensitivity reference substance is not lower than L2, the precision reference substance is subjected to parallel detection for 10 times, the results are positive, and the color development is uniform;
the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A novel coronavirus IgA antibody colloidal gold detection kit is characterized in that: including being equipped with the detection card of peridium board and sample pad, the gold mark district of peridium board is wrapped up with anti human IgA of colloidal gold mark, and the detection zone is wrapped up with novel coronavirus recombinant antigen, and quality control district is wrapped up with goat anti mouse IgG.
2. The novel coronavirus IgA antibody colloidal gold assay kit according to claim 1, wherein: the novel coronavirus recombinant antigen is S-RBD protein.
3. The novel coronavirus IgA antibody colloidal gold assay kit according to claim 1, wherein: the sample pad is coated with sample treatment liquid, and the sample treatment liquid is tris buffer solution which contains BSA with the mass concentration of 0.5-1.5%, casein with the mass concentration of 0.5-1%, sucrose with the mass concentration of 0.3-1.2%, trehalose with the mass concentration of 0.1-1.2%, Procline with the volume concentration of 0.05-0.3%, tween-20 with the volume concentration of 0.05-0.5% and has the pH value of 7.0-7.6; the preparation method of the sample pad comprises spraying or soaking the sample pad treating solution onto or into the glass fiber film or polyester film at 45-85 microliter per square centimeter, and oven drying at 37-45 deg.C for 8-16 hr.
4. The novel coronavirus IgA antibody colloidal gold assay kit according to claim 1, wherein: the preparation of colloidal gold-labeled anti-human IgA comprises the step of adding K to a homogeneous colloidal gold solution2CO3Adjusting pH to 6.0-7.5, adding anti-human IgA into the solution with adjusted pH to make the protein content of the solution be 15-60 μ g/mL, coupling for 0.5-4 h, adding a stop solution to stop the coupling reaction, wherein the final concentration (mass-volume ratio) of the stop solution is 1-1.5%; centrifuging after 0.5-2 hours after the reaction is ended, centrifuging for 15-30min at the centrifugation parameter of 12000-18000RPM, discarding the supernatant, redissolving the precipitate with a redissolution, and storing at 2-8 ℃ for later use.
5. The novel coronavirus IgA antibody colloidal gold assay kit according to claim 4, wherein: the stop solution is an aqueous solution prepared from one or two of bovine serum albumin, skim milk powder or glycine, when the stop solution is one of the solutions, the stop solution needs to be prepared into a solution with a mass-volume ratio of 5% -10%, and when the stop solution is two of the solutions, the mass-volume ratio of the bovine serum albumin to the skim milk powder or the glycine is 1: 1-4: 1, the solid mass content and the liquid volume ratio are 10 percent; the compound solution is 0.01-0.05M Tris or PB buffer solution, the pH is 7.4-8.2, 0.5-1% of NaCl, 0.2-1% of trehalose and 0.1-3% of PEG20000 are added into the compound solution according to the mass-volume ratio; bovine serum albumin is 0.5-3% by mass volume, and Triton X-405 is 0.5-1% by volume.
6. The novel coronavirus IgA antibody colloidal gold assay kit according to claim 4, wherein: the colloidal gold solution is prepared by preparing chloroauric acid into a solution with the mass volume ratio of 1% -10%, adding the solution into boiled purified water until the mass volume ratio of the chloroauric acid is one ten thousandth to four ten thousandth, continuously boiling for 2-5 minutes, adding 800 microliters of 500-one sodium citrate into each 100mL chloroauric acid solution, and continuously stirring and heating for 10-20 minutes to prepare the colloidal gold solution with uniform particle size.
7. The novel coronavirus IgA antibody colloidal gold assay kit according to claim 1, wherein: uniformly spreading the colloidal gold labeled anti-human IgA on a labeling pad, and drying for 12 hours at 37 ℃ to obtain the labeling pad; preparing a solution with the protein content of 0.5-2.0mg/mL by using the novel coronavirus recombinant antigen and goat anti-mouse IgG, respectively coating the solution at the positions of a T line and a C line by using a film-cutting instrument, and drying at 37 ℃ to obtain a coated nitrocellulose membrane, wherein the coating parameter is 0.5-1.5 mu L/cm; and sequentially overlapping and sticking the absorbent paper, the nitrocellulose membrane, the marking pad, the blood filtering membrane and the sample pad on the PVC plate.
8. The novel coronavirus IgA antibody colloidal gold assay kit according to claim 1, wherein: the blood filter membrane is used after being treated by an anti-RBC antibody, the treatment concentration is 0.1mg/mL, the treatment capacity is 60 microliters per square centimeter, and the blood filter membrane is dried for 12 hours at the temperature of 37 ℃.
9. The novel coronavirus IgA antibody colloidal gold assay kit according to claim 1, wherein: the kit also comprises a sample diluent and a 0.05M PB buffer solution, wherein the pH is 8.0, the mass volume ratio of NaCl is 0.9-2%, and the volume ratio of tween 20 is 0.05-0.3%.
10. Use of a kit according to claims 1-9 for the preparation of a novel reagent for the detection of coronavirus IgA.
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| CN113834934A (en) * | 2021-11-29 | 2021-12-24 | 北京百普赛斯生物科技股份有限公司 | Immunochromatography test strip and preparation method thereof |
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