CN117554613A - Colloidal gold detection test strip for bovine coronavirus antigen, and preparation method and application thereof - Google Patents
Colloidal gold detection test strip for bovine coronavirus antigen, and preparation method and application thereof Download PDFInfo
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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
- G01N33/53—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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- 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
- G01N33/54391—Immunochromatographic test strips based on vertical flow
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of biological detection, and discloses a colloidal gold detection test strip for detecting bovine coronavirus antigen, which comprises a bottom plate, and a sample pad, a gold mark pad, a reaction membrane and a water absorption pad which are arranged on the bottom plate, wherein the sample pad, the gold mark pad, the reaction membrane and the water absorption pad are sequentially erected; the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with a BCoV monoclonal antibody 4B8, the quality control line is coated with a goat anti-mouse IgG antibody, and the gold mark pad is provided with a colloidal gold marker of a BCoV monoclonal antibody 6H 9. The colloidal gold detection test strip for detecting the bovine coronavirus antigen can be used for rapidly detecting bovine coronavirus in diseased cow dung samples, nose swabs and anus swabs, has high detection sensitivity and good specificity, is simple and convenient to use, is suitable for detecting clinical samples and environments, and is also suitable for identifying the antigenicity of bovine coronavirus vaccine development.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a colloidal gold detection test strip for bovine coronavirus antigen, a preparation method and application thereof.
Background
Bovine coronavirus (bovine coronavirus, BCoV) belongs to the order of the niporoviridae, family of coronaviridae, genus coronaviridae. BCoV belongs to the beta coronavirus genus 2b subclass. The virus is the main pathogen causing diarrhea in newborn calves, winter diarrhea in adult cattle and respiratory diseases in cattle of all ages. At present, BCoV is an important pathogen causing diarrhea of beef cattle and dairy cows and calves, the morbidity is 50-100%, and huge economic losses are caused for the cattle industry, and the important pathogens mainly comprise death of calves, reduction of growth performance of cattle in a feeding farm, reduction of milk yield of adult cows and the like. And infected susceptible animals carry BCoV for long periods of time, although BCoV is occasionally cleared by the immune system of the body, there is also a prolonged, recessive infection. No effective prevention and control method for the virus is reported at home and abroad, and no detection method and vaccine capable of effectively preventing BCoV infection are available.
Epidemiological studies have shown that BCoV has a high serum epidemiological positive rate in cattle groups in China, but the pathogenic epidemiological situation of BCoV in China is not clear; in addition, the calf diarrhea caused by BCoV requires differential diagnosis of other pathogens (BVDV, BRV) and the like. In addition, at present, no BCoV diagnosis method with independent intellectual property rights exists in China, foreign detection kits need to be purchased, and imported kits are long in shelf life and high in price and are not specially specific to BCoV. It is therefore more desirable to establish highly sensitive and specific detection methods to accurately detect BCoV. Thus, early rapid diagnosis of BCoV is important for preventing and controlling the spread of the disease.
The accurate diagnosis is an extremely important link for preventing and controlling the disease. The diagnosis of BCoV is mainly laboratory diagnosis, and the laboratory detection method mainly comprises the combination of etiology diagnosis, molecular biological diagnosis, immunological diagnosis and other technologies for diagnosis. At present, the etiology diagnosis method mainly comprises virus separation, electron microscope observation and the like, and the etiology diagnosis method is a gold standard for identifying various viruses all the time, but the electron microscope technology needs precise equipment, has low sensitivity and higher requirements on professional talents, takes at least 3 hours, and seriously hinders the popularization and application of the etiology diagnosis method. Molecular biological diagnostics mainly include reverse transcription polymerase chain reaction (RT-PCR), real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-qPCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), reverse transcription recombinase polymerase amplification (RT-RPA), and the like. Molecular biological diagnosis has the advantages of high accuracy, high speed, high sensitivity, high specificity and high repeatability, but requires special precise and expensive equipment and mature technical staff, and can also bear the risk of false positive results due to laboratory pollution. Immunological diagnosis mainly includes virus neutralization test, hemagglutination (HA) and Hemagglutination Inhibition (HI) test, indirect immunofluorescence technique (IFA), enzyme-linked immunosorbent assay (ELISA) and the like. In contrast, the ELISA method is one of the most widely applied large-scale rapid diagnosis methods in the current basic level, but ELISA experiments have high requirements on the accuracy of an enzyme-labeled instrument, have complicated operation, are long in time consumption, are not easy to be applied on a large scale on site, are easily interfered by cattle diseases caused by other viruses or bacteria, and are not suitable for being widely used by local farmers. Therefore, the development of the on-site screening method for the BCoV disease is simple, convenient, feasible, sensitive, specific and rapid, and has practical significance.
The colloidal gold immunochromatography technology is widely applied to the fields of chemical detection and immunological detection, and particularly has the advantages of simple operation, rapidness and sensitivity, clear result, easiness in judgment and preservation, no need of any instrument and equipment and the like, and is more suitable for large-scale application in clinical rapid diagnosis and epidemiological investigation in basic stations, rural areas and the like.
Disclosure of Invention
The invention aims to provide a colloidal gold detection test strip capable of specifically detecting bovine coronavirus antigen and a preparation method thereof.
The technical scheme for achieving the aim comprises the following steps.
The invention provides a colloidal gold detection test strip for detecting bovine coronavirus antigen, which comprises a bottom plate, and a sample pad, a gold label pad, a reaction membrane and a water absorption pad which are arranged on the bottom plate, wherein the sample pad, the gold label pad, the reaction membrane and the water absorption pad are sequentially arranged;
the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with a BCoV monoclonal antibody 4B8, the quality control line is coated with a goat anti-mouse IgG antibody, and the gold mark pad is provided with a colloidal gold marker of the BCoV monoclonal antibody 6H 9.
In some embodiments, the detection line is coated with 0.1-1.5 mg/mL of BCoV monoclonal antibody 4B8, wherein the coating amount is 0.5-1.5 mu L/cm; and/or the number of the groups of groups,
the gold mark pad is coated with 1 mu g/mL-100 mu g/mL of BCoV monoclonal antibody 6H9, and the coating amount is 1 mu L/cm-10 mu L/cm; and/or the number of the groups of groups,
the quality control line is coated with 0.8-1.5 mg/mL goat anti-mouse IgG antibody, and the coating amount is 0.6-1 mu L/cm.
In some embodiments, the detection line is coated with 1.1-1.3 mg/mL of BCoV monoclonal antibody 4B8, wherein the coating amount is 0.7-0.9 mu L/cm; and/or the number of the groups of groups,
the gold mark pad is coated with 9-11 mu g/mL of BCoV monoclonal antibody 6H9, and the coating amount is 1-3 mu L/cm; and/or the number of the groups of groups,
the quality control line is coated with goat anti-mouse IgG antibody of 1.1 mg/mL-1.3 mg/mL, and the coating amount is 0.7 mu L/cm-0.9 mu L/cm.
In some embodiments, the first end of the gold-labeled pad is disposed below the sample pad, the second end of the gold-labeled pad is disposed above the first end of the reaction membrane, and the second end of the reaction membrane is disposed below the water-absorbing pad.
In some embodiments, the gold mark pad is arranged on the sample pad and has a length of 1.5 mm-2.5 mm; and/or the gold mark pad is arranged on the reaction film, and the length of the gold mark pad is 1.5 mm-2.5 mm; and/or the water absorption pad is arranged on the reaction film, and the length of the water absorption pad is 1.5 mm-2.5 mm.
In some embodiments, the sample pad is treated with a sample pad treatment solution, wherein the sample pad treatment solution contains 0.3-1% bovine serum albumin, 0.3-1% S9, 0.5-1.5% PVP-40, 0.3 g/L-1 g/L PHA, 60 mg/mL-70 mg/mL RBC pH 7-8, and 0.1-0.5 mol/L phosphate buffer; preferably, the sample pad treatment solution contains 0.4-0.6% bovine serum albumin, 0.4-0.6% S9, 0.8-1.2% PVP-40, 0.4-0.6 g/L PHA, 65 mg/mL-67 mg/mL RBC pH 7.2-7.6 and 0.1 mol/L-0.3 mol/L phosphate buffer solution.
The second aspect of the invention provides an application of the colloidal gold test strip in detecting bovine coronavirus antigen in a sample.
In some of these embodiments, the sample is bovine feces, bovine anal secretion, or bovine nasal secretion.
The third aspect of the invention provides a method for preparing a colloidal gold test strip for detecting bovine coronavirus antigen, comprising the following steps:
adding a BCoV monoclonal antibody 6H9 into a colloidal gold solution to obtain a colloidal gold-antibody solution, adding bovine serum albumin into the colloidal gold-antibody solution, centrifuging to obtain a precipitate, and re-suspending the precipitate with a colloidal gold re-dissolving buffer solution to obtain a BCoV monoclonal antibody 6H9 colloidal gold marker solution;
spraying BCoV monoclonal antibody 6H9 colloidal gold marker solution on the gold mark pad;
preparing a detection line coated with a BCoV monoclonal antibody 4B8 and a quality control line coated with a goat anti-mouse IgG antibody on a reaction film;
assembling the sample pad, the gold mark pad, the reaction membrane, the water absorption pad and the bottom plate into a colloidal gold detection test strip;
wherein the colloidal gold reconstitution buffer solution contains 0.01M-0.03M Tris-HCl, 0.5-1.5% bovine serum albumin, 0.5-1.5% proclin-300 and 0.1-0.2% triton.
In some embodiments, the pH of the colloidal gold solution is 4.3-5.
In some embodiments, the pH of the colloidal gold solution is 4.4-4.6.
In some embodiments, the concentration of the BCoV monoclonal antibody 6H9 in the colloidal gold solution is 10-14 mug/mL.
In some embodiments, the concentration of the BCoV monoclonal antibody 6H9 in the colloidal gold solution is 10 [ mu ] g/mL-11 [ mu ] g/mL.
In some embodiments, the concentration of the bovine serum albumin in the colloidal gold-antibody solution is 55-65. Mu.L/mL.
In some embodiments, the concentration of the bovine serum albumin in the colloidal gold-antibody solution is 58-62. Mu.L/mL.
In some embodiments, the pH of the colloidal gold reconstitution buffer is 7-7.5.
In some of these embodiments, the colloidal gold reconstitution buffer has a pH of 7.4.
The fourth aspect of the invention provides a kit for detecting bovine coronavirus antigen, which comprises a sample diluent and the colloidal gold detection test strip, wherein the sample diluent contains 5 g/L-10 g/L sodium chloride and 1 mL/L-5 mL/L proclin-300;
in the invention, a gold mark pad of a colloidal gold detection test strip is coated with a specific colloidal gold-labeled BCoV monoclonal antibody 6H9, a detection line of a reaction membrane is coated with a specific BCoV monoclonal antibody 4B8, a substance to be detected in a sample is firstly combined with a BCoV monoclonal antibody 6H9 colloidal gold mark on the gold mark pad to form a bovine coronavirus antigen-antibody 6H9 colloidal gold mark complex, and then is combined with a BCoV monoclonal antibody 4B8 on the detection line on the reaction membrane to form an antibody 6H9 colloidal gold mark-bovine coronavirus antigen-antibody 4B8 complex, so that the colloidal gold detection test strip can be used for detecting bovine coronavirus antigens through a double-antibody sandwich method formed by combining two BCoV monoclonal antibodies with different antigen epitopes, and can be used for rapidly detecting bovine coronaviruses in a sick cow dung sample, a nose swab and an anal swab, effectively eliminating bovine rotavirus, bovine viral diarrhea, bovine parainfluenza virus, bovine infectious rhinovirus and pestivirus to the detection of ruminant virus, and is also suitable for detecting the bovine coronavirus with high sensitivity and specificity, and is suitable for detecting the bovine coronavirus with a specific antigen, and a ruminant virus, and is suitable for the clinical detection of a vaccine, and the detection sample is also suitable for the clinical detection of a vaccine;
drawings
FIG. 1 is a graph showing the measurement results of colloidal gold-labeled BCoV monoclonal antibody 6H9 under different pH values.
FIG. 2 is a graph showing the measurement results of colloidal gold-labeled BCoV monoclonal antibody 6H9 under different monoclonal antibody 6H9 concentration conditions.
FIG. 3 is a graph showing the results of the detection of colloidal gold-labeled BCoV monoclonal antibody 6H9 under blocking conditions with BSA markers at different concentrations.
Fig. 4 is an assembly schematic of a BCoV antigen test paper card.
Fig. 5 is a schematic diagram showing the determination of the detection result of BCoV antigen detection card.
FIG. 6 is a graph of the detection result of the sensitivity of the BCoV antigen detection card; wherein, the reference numeral 1: BCoV dilution 2-fold; reference numeral 2: BCoV dilution 4-fold; reference numeral 3: BCoV dilution 8-fold; reference numeral 4: BCoV dilution 16-fold; reference numeral 5: BCoV dilution 32-fold; reference numeral 6: BCoV dilution 64-fold; reference numeral 7: negative control.
FIG. 7 is a graph of the results of BCoV antigen detection card specific assays; wherein, the reference numeral 1: bovine rotavirus; reference numeral 2: bovine viral diarrhea virus; reference numeral 3: bovine parainfluenza virus; reference numeral 4: infectious bovine rhinotracheitis virus; reference numeral 5: peste des petits ruminants virus; reference numeral 6: bovine coronavirus;
FIG. 8 is a graph of stability test results for a BCoV antigen test card;
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The embodiment of the invention provides a colloidal gold detection test strip for detecting bovine coronavirus antigen, which comprises a bottom plate, and a sample pad, a gold mark pad, a reaction membrane and a water absorption pad which are arranged on the bottom plate, wherein the sample pad, the gold mark pad, the reaction membrane and the water absorption pad are sequentially arranged;
the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with a BCoV monoclonal antibody 4B8, the quality control line is coated with a goat anti-mouse IgG antibody, and the gold mark pad is provided with a colloidal gold marker of the BCoV monoclonal antibody 6H 9.
In the above embodiment, the specific colloidal gold-labeled BCoV monoclonal antibody 6H9 is coated on the gold-labeled pad of the colloidal gold detection test strip, the specific BCoV monoclonal antibody 4B8 is coated on the detection line of the reaction membrane, and the substance to be detected in the sample is firstly combined with the BCoV monoclonal antibody 6H9 colloidal gold label on the gold-labeled pad to form a bovine coronavirus antigen-antibody 6H9 colloidal gold label complex, and then is combined with the BCoV monoclonal antibody 4B8 on the detection line on the reaction membrane to form an antibody 6H9 colloidal gold label-bovine coronavirus antigen-antibody 4B8 complex, so that the colloidal gold detection test strip can detect bovine coronavirus antigen through a double-antibody sandwich method by combining two BCoV monoclonal antibodies with different antigen epitopes, can rapidly detect bovine coronaviruses in a sickness cow dung sample, a nose swab and an anus swab, effectively eliminate bovine coronavirus, bovine parainfluenza virus, bovine infectious rhinovirus and rotavirus, and peste virus, has good sensitivity to detection of bovine coronavirus and ruminant virus, is also suitable for detecting the specific and testing of bovine coronavirus, and is easy and convenient for detecting the antigen of a clinical environment, and is suitable for the detection of a vaccine.
In some embodiments, the detection line is coated with 0.1-1.5 mg/mL of BCoV monoclonal antibody 4B8, wherein the coating amount is 0.5-1.5 mu L/cm; and/or the number of the groups of groups,
the gold mark pad is coated with 1 mu g/mL-100 mu g/mL of BCoV monoclonal antibody 6H9, and the coating amount is 1 mu L/cm-10 mu L/cm; and/or the number of the groups of groups,
the quality control line is coated with 0.8-1.5 mg/mL goat anti-mouse IgG antibody, and the coating amount is 0.6-1 mu L/cm.
In some embodiments, the detection line is coated with 1.1 mg/mL-1.3 mg/mL of BCoV monoclonal antibody 4B8, wherein the coating amount is 0.7 mu L/cm-0.9 mu L/cm; and/or the number of the groups of groups,
the gold mark pad is coated with 9-11 mu g/mL of BCoV monoclonal antibody 6H9, and the coating amount is 1-3 mu L/cm; and/or the number of the groups of groups,
the quality control line is coated with goat anti-mouse IgG antibody of 1.1 mg/mL-1.3 mg/mL, and the coating amount is 0.7 mu L/cm-0.9 mu L/cm.
In some embodiments, the first end of the gold-labeled pad is disposed below the sample pad, the second end of the gold-labeled pad is disposed above the first end of the reaction membrane, and the second end of the reaction membrane is disposed below the water-absorbing pad.
In some embodiments, the gold-labeled pad is arranged on the sample pad with a length of 1.5 mm-2.5 mm; and/or the gold mark pad is arranged on the reaction film, and the length of the gold mark pad is 1.5 mm-2.5 mm; and/or the water absorption pad is arranged on the reaction film, and the length of the water absorption pad is 1.5 mm-2.5 mm.
In some embodiments, the sample pad is treated with a sample pad treatment solution containing 0.3-1% bovine serum albumin, 0.3-1% S9, 0.5-1.5% PVP-40, 0.3 g/L-1 g/L PHA, 60 mg/mL-70 mg/mL RBC pH 7-8, 0.1-0.5 mol/L phosphate buffer; preferably, the sample pad treatment solution contains 0.4-0.6% bovine serum albumin, 0.4-0.6% S9, 0.8-1.2% PVP-40, 0.4-0.6 g/L PHA, 65 mg/mL-67 mg/mL RBC pH 7.2-7.6 and 0.1 mol/L-0.3 mol/L phosphate buffer solution.
The second aspect of the invention provides an application of the colloidal gold test strip in detecting bovine coronavirus antigen in a sample.
In some embodiments, the sample is bovine feces, bovine anal secretion, or bovine nasal secretion.
The third aspect of the invention provides a method for preparing a colloidal gold test strip for detecting bovine coronavirus antigen, comprising the following steps:
adding a BCoV monoclonal antibody 6H9 into a colloidal gold solution to obtain a colloidal gold-antibody solution, adding bovine serum albumin into the colloidal gold-antibody solution, centrifuging to obtain a precipitate, and re-suspending the precipitate with a colloidal gold re-dissolving buffer solution to obtain a BCoV monoclonal antibody 6H9 colloidal gold marker solution;
spraying BCoV monoclonal antibody 6H9 colloidal gold marker solution on the gold mark pad;
preparing a detection line coated with a BCoV monoclonal antibody 4B8 and a quality control line coated with a goat anti-mouse IgG antibody on a reaction film;
assembling the sample pad, the gold mark pad, the reaction membrane, the water absorption pad and the bottom plate into a colloidal gold detection test strip;
wherein the colloidal gold reconstitution buffer solution contains 0.01M-0.03M Tris-HCl, 0.5-1.5% bovine serum albumin, 0.5-1.5% proclin-300 and 0.1-0.2% triton.
In some embodiments, the pH of the colloidal gold solution is 4.3-5.
In some embodiments, the pH of the colloidal gold solution is 4.4-4.6.
In some embodiments, the concentration of the BCoV monoclonal antibody 6H9 in the colloidal gold solution is 10 [ mu ] g/mL-14 [ mu ] g/mL.
In some embodiments, the concentration of the BCoV monoclonal antibody 6H9 in the colloidal gold solution is 10 [ mu ] g/mL-11 [ mu ] g/mL.
In some embodiments, the concentration of the bovine serum albumin in the colloidal gold-antibody solution is 55. Mu.L/mL-65. Mu.L/mL.
In some embodiments, the concentration of the bovine serum albumin in the colloidal gold-antibody solution is 58-62. Mu.L/mL.
In the above steps, in the whole labeling process of the BCoV antibody colloidal gold label, the pH, the antibody labeling amount, the bovine serum albumin addition amount and the colloidal gold reconstitution buffer solution all have an influence on the stability and the sensitivity of the antibody 6H 9-colloidal gold label solution, and the finally obtained antibody 6H9 colloidal gold label solution can have excellent stability and sensitivity by optimizing the pH, the antibody labeling amount, the bovine serum albumin addition amount and the colloidal gold reconstitution buffer solution.
The fourth aspect of the invention provides a kit for detecting bovine coronavirus antigen, which comprises a sample diluent and the colloidal gold detection test strip, wherein the sample diluent contains 5 g/L-10 g/L sodium chloride and 1 mL/L-5 mL/L proclin-300.
The present invention will be described in further detail with reference to specific examples.
Example 1
The embodiment provides a preparation method of a colloidal gold detection test strip for detecting bovine coronavirus antigen, which comprises the following steps:
1) Preparing a gold-labeled pad sprayed with a BCoV monoclonal antibody 6H9 colloidal gold label;
2) Preparing a reaction membrane with a detection line and a quality control line respectively coated with a BCoV monoclonal antibody 4B8 and a goat anti-mouse IgG antibody;
3) And assembling the gold-labeled pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the previous two steps into the test strip.
The following is a stepwise detailed description:
1) Preparation of colloidal gold
1mL of 1% chloroauric acid (mass fraction) (purchased from Sigma company) is added into a conical flask containing 99ml of ultrapure water to prepare 0.01% chloroauric acid solution (volume fraction) (purchased from Sigma company), the mixture is uniformly shaken and heated to boiling by a constant-temperature electromagnetic stirrer, 1.6mL of 1% trisodium citrate (mass fraction) (purchased from Sigma company) is added under continuous high temperature and continuous stirring, the uniform stirring and heating are continued for about 10 minutes, the solution is firstly changed from yellow to colorless, then is changed to black and then is changed to transparent wine red slowly, the solution is reacted for 10 minutes after no more color changes, the solution is cooled to room temperature, and then the original volume is recovered by ultrapure water, and the solution is preserved in a dark place at 2-8 ℃. The prepared colloidal gold has pure appearance, is transparent, and has no sediment or floaters.
2) Preparation of BCoV monoclonal antibody 6H9 colloidal gold marker
The pH value of the colloidal gold solution was adjusted by using 0.2mol/L potassium carbonate solution (purchased from national pharmaceutical systems chemical reagent Co., ltd.) and 10. Mu.g of BCoV monoclonal antibody 6H9 (product number DWBN-102, shenzhen Lv Shi Yuan Biotechnology Co., ltd.) was added to each ml of the colloidal gold solution, and the mixture was left standing for 30 minutes after shaking and mixing, and 10% bovine serum albumin was added to give a final concentration of 0.5% (volume fraction) in the colloidal gold solution, and left standing for 30 minutes. Centrifuging at 8000r/min and 4deg.C for 15min, discarding supernatant, re-suspending precipitate with colloidal gold re-dissolving buffer solution with volume of 1/10 of that of colloidal gold, and standing at 4deg.C.
2.1 optimization of conditions for colloidal gold-labeled antibodies
In the whole labeling process of the BCoV antibody colloidal gold marker, optimizing the pH, the antibody labeling amount, the bovine serum albumin addition amount and the colloidal gold reconstitution buffer solution, wherein the optimization result is determined according to the stability and the sensitivity of the finally obtained antibody colloidal gold marker solution;
2.1.1 Screening of optimal pH value of colloidal gold labeled antibody:
the pH of the colloidal gold solution is regulated by using 0.2mol/L potassium carbonate solution, 1mL colloidal gold solution is added into a transparent centrifuge tube of 1.5 mL, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mu L potassium carbonate solution are respectively added, and after uniform mixing, the pH is respectively as follows: 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, colloidal gold solutions with different pH values are obtained. And respectively adding 10 mug of BCoV monoclonal antibody 6H9 with equal quantity, regulating the final concentration of the monoclonal antibody to 10 mug/mL, oscillating and uniformly mixing, standing and marking for 2 hours at room temperature, and obtaining different colloidal gold complexes. When the color of the colloidal gold is unchanged and the dosage of the potassium carbonate is the minimum, the optimal pH for combining the colloidal gold and the antibody 6H9 is obtained. As a result, as shown in FIG. 1, the colloidal gold-antibody complex obtained at an addition amount of 4.5uL of 0.2mol/L potassium carbonate solution had the best effect on the binding stability, i.e., the colloidal gold solution pH=4.5 was the optimal pH for binding of colloidal gold and antibody 6H 9.
2.1.2 screening of optimal concentration of colloidal gold-labeled mab 6H 9:
preparing a colloidal gold solution according to the determined optimal pH, taking 1mL, respectively adding the 1mL into 8 transparent centrifuge tubes of 1.5 mL, sequentially adding purified monoclonal antibody 6H9 with different concentrations to the final concentration of 0 mug/mL, 2 mug/mL, 4 mug/mL, 6 mug/mL, 8 mug/mL, 10 mug/mL, 12 mug/mL and 14 mug/mL, uniformly mixing, marking at room temperature, standing for 2 hours, and observing the color change of the colloidal gold. The most suitable amount of antibody labeling is when no further changes in colloidal gold in the tube occur and no precipitation occurs. The results of FIG. 2 show that the colloidal gold-labeled mab 6H9 was stable in pink color at greater than 10. Mu.g/mL, and that the concentration of monoclonal antibody at 10. Mu.g/mL was selected as the optimal concentration for colloidal gold labeling and used in subsequent operations.
2.1.3 optimization of bovine serum albumin content:
and (3) respectively adding 50, 60, 80 and 100 mu L of 10% bovine serum albumin into each milliliter of the colloidal gold-antibody solution to seal, uniformly mixing, marking at room temperature, standing for 2 hours, and observing the color change of the colloidal gold. The appropriate amount of bovine serum albumin added was determined based on the stability of the final antibody colloidal gold markers. The results showed that the colloidal gold binding stability with BCoV antibody was the best among the colloidal gold-antibody solutions having bovine serum albumin content of 60. Mu.L/mL.
2.1.4 optimization of colloidal gold reconstitution buffer:
three colloidal gold reconstitution buffers at pH7.4, 8.8, 9.6: preparation of 0.02M Tris-HCl (12% sucrose (from national pharmaceutical Co., ltd.), 1% bovine serum albumin (from Sigma Co.), 1% proclin-300 (from Sigma Co.), 0.15% triton (from Sigma Co.). And re-dissolving the centrifugal colloidal gold antibody markers after sealing by using the prepared three colloidal gold re-dissolving buffers with different pH values, and determining the proper colloidal gold re-dissolving buffer according to the stability and sensitivity of the markers. The results show that colloidal gold reconstitution buffer at pH7.4 works best.
3) Preparation of gold mark pad
The gold-labeled pad was soaked in a colloidal gold reconstitution buffer for 30min and then dried in an oven at 45 ℃, after which the reconstituted BCoV monoclonal antibody 6H9 colloidal gold-labeled solution (BCoV monoclonal antibody 6H9 concentration 10 μg/mL) was sprayed onto the treated gold-labeled pad with an AUTOKUN-scribing gold-spraying machine at a rate of 2.0 μl/cm per well, after which the sprayed gold-labeled pad was placed in an oven at 37 ℃ for 12H, and then sealed, added with a desiccant for drying and preservation.
4) Preparation of reaction film
BCoV monoclonal antibody 4B8 was coated onto nitrocellulose membrane (NC 140) to constitute a detection line, and goat anti-mouse IgG antibody (bailibo company (F020201)) was coated onto nitrocellulose membrane (NC 140) to constitute a quality control line.
The coating process comprises the following steps: diluting BCoV monoclonal antibody 4B8 (product number is DWBN-101 of Shenzhen green poem biotechnology Co., ltd.) to 1.2mg/mL with phosphate buffer, and coating the BCoV monoclonal antibody on a detection line (T line) on a nitrocellulose membrane with an AUTOKUN film-dividing metal-spraying mark machine, wherein the coating amount is 0.8 mu L/cm; the goat anti-mouse IgG antibody was diluted to 1.2mg/mL with phosphate buffer, and coated on a quality control line (C line) on a nitrocellulose membrane in an amount of 0.8. Mu.L/cm with an AUTOKUN film-dividing and metal-spraying machine. And (5) drying the coated reaction film for 12 hours at 37 ℃ for standby.
5) Preparation of sample absorbent pad
The sample absorbent pad was placed in an oven containing 0.5% bovine serum albumin (volume fraction), 0.5% S9 (volume fraction), 1% PVP-40 (volume fraction) (from Sigma Co.), 0.5g/L PHA (volume fraction), 66mg/mL RBC pH7.4, 0.2mol/L phosphate buffer, and baked for 12 hours to complete dryness at 37℃and kept in a sealed dry package for use.
6) Assembly of test strips
As shown in fig. 4, a reaction membrane (NC membrane in the figure), a gold-labeled pad (gold pad in the figure), a sample pad, and a water-absorbent pad (water-absorbent paper in the figure) are sequentially stuck on a base plate (PVC plate in the figure); the first end of the gold mark pad is arranged below the sample pad, the second end of the gold mark pad is arranged above the first end of the reaction membrane, the second end of the reaction membrane is arranged below the water absorption pad, the gold mark pad, the water absorption pad and the reaction membrane are overlapped by 2mm, and the sample pad and the gold mark pad are overlapped by 2 mm. The initial end of the sample pad is aligned with the initial end of the PVC bottom plate, and the tail end of the water absorbing pad is aligned with the tail end of the PVC bottom plate; the reaction film is provided with a detection line and a quality control line, and the detection line (T line) and the quality control line (C line) are strip-shaped strips which are perpendicular to the length of the test strip; the detection line is positioned at one side close to the tail end of the sample pad; the quality control line is positioned at one side close to the head end of the water absorption pad; cutting the test strip into strips with the width of 2.8mm by a machine, and filling the strips into a sealing bag with a drying agent, thus obtaining the colloidal gold test strip which can be stored for 12 months under the storage condition of 4 ℃.
Example 2 detection method of colloidal gold test strip for detecting BCoV in fecal sample to be detected
Preparing a sample diluent: weighing 9 g sodium chloride, dissolving in 800 mL ultrapure water, adding 1mL proclin-300, adding ultrapure water to constant volume of 1L, filtering, sterilizing, and quantitatively packaging.
Pretreatment of a sample to be detected: placing the throat test sample, the nose swab and the anus swab which are suspected to be infected by BCoV into 200ul of sample diluent, uniformly mixing and standing for 2min, and taking clear liquid for detection; the fecal treatment is vortexed with a suitable amount of sample diluent and centrifuged to extract the supernatant clear liquid for detection.
1. Detection was performed using the colloidal gold test strip for detection of bovine coronavirus antigen assembled in example 1
And vertically dripping the treated sample diluent onto a sample pad, starting timing when the liquid flows, reacting for 15min, and judging the result.
2. Detecting the determination result
The sample to be detected is detected by a colloidal gold test strip, and the read result within 15min is analyzed as follows:
positive (+): C. the T line is developed to show that BCoV exists in the sample to be detected (shown as A in figure 5);
negative (-). T-line does not develop, only C-line develops, indicating no BCoV in the sample to be tested (as shown in B in fig. 5);
invalidation: line C is not colored, indicating that the procedure is incorrect, or that the test card has failed, requiring retesting (as indicated by C in fig. 5, as indicated by D in fig. 5).
Example 3 colloidal gold test strip sensitivity and specificity test
Example 3 colloidal gold test strip sensitivity and specificity test
1. Sensitivity test
BCoV virus culture broth (10) was diluted with sample 5.0 TCID 50 Per mL) were serially diluted 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, while the sample dilutions were made negative controls. The sample was tested using the colloidal gold test strip for detection of bovine coronavirus antigen as in example 2, with the highest dilution of red precipitate line at T line being visible to the naked eye and the BCoV virus content as the sensitivity of the BCoV antigen test paper card. As shown in the result of FIG. 6, when the negative sample diluent is detected, the quality control line of the BCoV antigen detection test strip develops color, and the detection line does not develop color, so that the negative sample diluent is negative; when the BCoV is diluted to 1:64, the quality control line and the detection line both develop color test strips, and the test strips are positive, which shows that the sensitivity of the test strip card for detecting the BCoV is 10 3.9 TCID 50 and/mL, the test paper card prepared by the invention has higher sensitivity to BCoV antigen.
2. Specificity test
The colloidal gold test strip for detecting bovine coronavirus antigen was used to detect bovine rotavirus, bovine viral diarrhea virus, bovine parainfluenza virus, bovine infectious rhinotracheitis virus and peste des petits ruminants virus as in example 2. The result is shown in FIG. 7, and the result shows that the test strip quality control line develops color, the detection line does not develop color, and the result is negative. The test paper has no cross reaction to bovine rotavirus, bovine viral diarrhea, bovine parainfluenza virus, bovine infectious rhinotracheitis virus and peste des petits ruminants virus, and has better specificity.
3. Application of test paper strip
The colloidal gold test strip for detecting bovine coronavirus antigen was used to detect BCoV in 127 diarrhea stool samples collected from a farm in the manner of example 2, and the pathogen positive rate of BCoV was 53%. 15 positive samples are randomly selected for RT-PCR sequencing and sequence analysis, and the detection is confirmed to be correct, so that the accuracy of the detection result of the test strip is further verified.
Example 4 stability test
The test strips prepared in example 1 were packed in a sealed bag, filled with a desiccant, stored in an oven at 45℃and taken out for 7 days, 14 days, 21 days and 28 days, respectively, and positive and negative samples were tested for stability of the test strips, respectively. As shown in FIG. 8, the results of the test were not significantly changed after storage at 45℃for 7 days, 14 days, 21 days, and 28 days, and the reconstitution effect of the colloidal gold-labeled antibody on the gold conjugate pad was reduced, the T line and C line were light, and the sensitivity was slightly reduced. Storage at 45 ℃ for 28 days is equivalent to storage at normal temperature for 12 months.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention.
It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. The colloidal gold detection test strip for detecting bovine coronavirus antigen is characterized by comprising a bottom plate, and a sample pad, a gold label pad, a reaction membrane and a water absorption pad which are arranged on the bottom plate, wherein the sample pad, the gold label pad, the reaction membrane and the water absorption pad are sequentially arranged;
the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with a BCoV monoclonal antibody 4B8, the quality control line is coated with a goat anti-mouse IgG antibody, and the gold mark pad is provided with a colloidal gold marker of the BCoV monoclonal antibody 6H 9.
2. The colloidal gold test strip according to claim 1, wherein the test line is coated with 0.1mg/mL to 1.5mg/mL of BCoV monoclonal antibody 4B8 in an amount of 0.5 μl/cm to 1.5 μl/cm; and/or the number of the groups of groups,
the gold mark pad is coated with 1 mu g/mL-100 mu g/mL of BCoV monoclonal antibody 6H9, and the coating amount is 1 mu L/cm-10 mu L/cm; and/or the number of the groups of groups,
the quality control line is coated with 0.8-1.5 mg/mL goat anti-mouse IgG antibody, and the coating amount is 0.6-1 mu L/cm.
3. The colloidal gold test strip according to claim 2, wherein the detection line is coated with 1.1mg/mL to 1.3mg/mL of BCoV monoclonal antibody 4B8 in an amount of 0.7 μl/cm to 0.9 μl/cm; and/or the number of the groups of groups,
the gold mark pad is coated with 9-11 mu g/mL of BCoV monoclonal antibody 6H9, and the coating amount is 1-3 mu L/cm; and/or the number of the groups of groups,
the quality control line is coated with goat anti-mouse IgG antibody of 1.1 mg/mL-1.3 mg/mL, and the coating amount is 0.7 mu L/cm-0.9 mu L/cm.
4. The colloidal gold test strip according to claim 1, wherein the gold-labeled pad has a first end disposed below the sample pad and a second end disposed above the first end of the reaction membrane, and the reaction membrane has a second end disposed below the absorbent pad.
5. The colloidal gold test strip according to claim 4, wherein the gold-labeled pad is 1.5mm to 2.5mm long; and/or the gold mark pad is arranged on the reaction film, and the length of the gold mark pad is 1.5 mm-2.5 mm; and/or the water absorption pad is arranged on the reaction film, and the length of the water absorption pad is 1.5 mm-2.5 mm.
6. The colloidal gold test strip according to claim 1, wherein the sample pad is obtained by treating a sample pad treatment solution, and the sample pad treatment solution contains 0.3-1% bovine serum albumin, 0.3-1% S9, 0.5-1.5% PVP-40, 0.3 g/L-1 g/L PHA, 60 mg/mL-70 mg/mL RBC pH 7-8, and 0.1-0.5 mol/L phosphate buffer.
7. A preparation method of a colloidal gold detection test strip for detecting bovine coronavirus antigen is characterized by comprising the following steps:
adding a BCoV monoclonal antibody 6H9 into a colloidal gold solution to obtain a colloidal gold-antibody solution, adding bovine serum albumin into the colloidal gold-antibody solution, centrifuging to obtain a precipitate, and re-suspending the precipitate with a colloidal gold re-dissolving buffer solution to obtain a BCoV monoclonal antibody 6H9 colloidal gold marker solution;
spraying BCoV monoclonal antibody 6H9 colloidal gold marker solution on the gold mark pad;
preparing a detection line coated with a BCoV monoclonal antibody 4B8 and a quality control line coated with a goat anti-mouse IgG antibody on a reaction film;
assembling the sample pad, the gold mark pad, the reaction membrane, the water absorption pad and the bottom plate into a colloidal gold detection test strip;
wherein the colloidal gold reconstitution buffer solution contains 0.01M-0.03M Tris-HCl, 0.5-1.5% bovine serum albumin, 0.5-1.5% proclin-300 and 0.1-0.2% triton.
8. The method according to claim 7, wherein the pH of the colloidal gold solution is 4.3 to 5; and/or the number of the groups of groups,
the concentration of the BCoV monoclonal antibody 6H9 in the colloidal gold solution is 10-14 mug/mL; and/or the number of the groups of groups,
the concentration of the bovine serum albumin in the colloidal gold-antibody solution is 55 mu L/mL-65 mu L/mL;
and/or the pH value of the colloidal gold re-dissolution buffer solution is 7-7.5.
9. The preparation method of claim 8, wherein the pH of the colloidal gold solution is 4.4-4.6; and/or the number of the groups of groups,
the concentration of the BCoV monoclonal antibody 6H9 in the colloidal gold solution is 10 mu g/mL-11 mu g/mL; and/or the number of the groups of groups,
the concentration of the bovine serum albumin in the colloidal gold-antibody solution is 58-62 mu L/mL.
10. A kit for detecting bovine coronavirus antigen, which is characterized by comprising a sample diluent and the colloidal gold test strip according to any one of claims 1 to 6, wherein the sample diluent contains 5g/L to 10g/L sodium chloride and 1 mL/L to 5mL/L proclin-300.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105486859A (en) * | 2015-11-20 | 2016-04-13 | 润和生物医药科技(汕头)有限公司 | Novel improved immunochromatographic test strip, and preparation and application thereof |
| CN106970219A (en) * | 2017-04-28 | 2017-07-21 | 北京金豪制药股份有限公司 | One kind is based on HIV in colloidal gold method detection urine（1+2）Antibody reagent |
| CN111856003A (en) * | 2020-02-04 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Novel coronavirus (2019-nCoV) IgM and IgG antibody detection test strip, kit and method |
| CN112326968A (en) * | 2021-01-07 | 2021-02-05 | 山东康华生物医疗科技股份有限公司 | Kit for detecting malignant malaria and vivax malaria |
| CN113533736A (en) * | 2021-07-15 | 2021-10-22 | 上海伯杰医疗科技有限公司北京分公司 | Mycoplasma pneumoniae IgM antibody colloidal gold immunochromatographic assay detection kit and preparation method thereof |
| CN114113597A (en) * | 2021-12-08 | 2022-03-01 | 江苏晶红生物医药科技股份有限公司 | Novel coronavirus antigen detection kit and preparation method thereof |
| CN114280296A (en) * | 2021-12-29 | 2022-04-05 | 中元汇吉生物技术股份有限公司 | Novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit and preparation method thereof |
| CN116449002A (en) * | 2022-12-30 | 2023-07-18 | 杭州新脉生物科技有限公司 | Colloidal gold chromatographic test strip for screening vaccine immunity and novel coronavirus infection and application thereof |
| CN116559439A (en) * | 2023-04-24 | 2023-08-08 | 江苏省农业科学院 | Bovine coronavirus indirect ELISA antibody detection kit and application |
-
2024
- 2024-01-10 CN CN202410034869.1A patent/CN117554613A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105486859A (en) * | 2015-11-20 | 2016-04-13 | 润和生物医药科技(汕头)有限公司 | Novel improved immunochromatographic test strip, and preparation and application thereof |
| CN106970219A (en) * | 2017-04-28 | 2017-07-21 | 北京金豪制药股份有限公司 | One kind is based on HIV in colloidal gold method detection urine（1+2）Antibody reagent |
| CN111856003A (en) * | 2020-02-04 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Novel coronavirus (2019-nCoV) IgM and IgG antibody detection test strip, kit and method |
| CN112326968A (en) * | 2021-01-07 | 2021-02-05 | 山东康华生物医疗科技股份有限公司 | Kit for detecting malignant malaria and vivax malaria |
| CN113533736A (en) * | 2021-07-15 | 2021-10-22 | 上海伯杰医疗科技有限公司北京分公司 | Mycoplasma pneumoniae IgM antibody colloidal gold immunochromatographic assay detection kit and preparation method thereof |
| CN114113597A (en) * | 2021-12-08 | 2022-03-01 | 江苏晶红生物医药科技股份有限公司 | Novel coronavirus antigen detection kit and preparation method thereof |
| CN114280296A (en) * | 2021-12-29 | 2022-04-05 | 中元汇吉生物技术股份有限公司 | Novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit and preparation method thereof |
| CN116449002A (en) * | 2022-12-30 | 2023-07-18 | 杭州新脉生物科技有限公司 | Colloidal gold chromatographic test strip for screening vaccine immunity and novel coronavirus infection and application thereof |
| CN116559439A (en) * | 2023-04-24 | 2023-08-08 | 江苏省农业科学院 | Bovine coronavirus indirect ELISA antibody detection kit and application |
Non-Patent Citations (1)
| Title |
|---|
| 梁彤等: "牛冠状病毒 N 蛋白的原核表达及其单克隆抗体的制备", 中国动物传染病学报, 25 November 2022 (2022-11-25), pages 1 - 12 * |
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