CN111879925A - Colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis - Google Patents
Colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis Download PDFInfo
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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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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/23—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Brucella (G)
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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Abstract
The invention relates to the technical field of immunodiagnosis, and discloses a colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis aiming at the problem of inaccurate detection result caused by the detection of brucellosis antibodies by antigens in the prior art, and a preparation method of the colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis specifically comprises the following steps: 1) preparing a detection line T and a quality control line C; 2) preparing a gold label pad; 3) preparing detection test paper: and sequentially connecting and assembling the water absorption pad, the sample pad, the microsphere probe treatment pad, the modified nitrocellulose membrane coated with the antibody and the PVC bottom plate to obtain a finished product. The product can accurately detect whether the brucellosis exists or not, adopts a mode of detecting the antigen by the antibody, has no incubation period, is more accurate than a product for detecting the antibody, has higher accuracy of detecting the antigen than the product for detecting the antibody, has high detection efficiency and is simple in preparation process.
Description
Technical Field
The invention relates to the technical field of immunodiagnosis, in particular to colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis.
Background
Brucellosis, abbreviated as brucellosis, is an infectious allergic disease which is commonly suffered by human and livestock and has brucellosis. The disease is highly contagious, has been classified as a class B pathogen by the CDC, and is therefore considered a potential biological weapon. This disease exists in a number of countries and regions of the world 170. The spread of diseases among people and livestock is prevalent, about 1/5-1/6 of people in the world are threatened by the spread of diseases, about 500-600 thousands of people are spread of diseases in the world, and the economic loss caused by each year is up to billions of dollars. Moreover, the disease also poses serious threats to human health.
Infection by brucella in humans is due, on the one hand, to exposure to diseased animals and, on the other hand, to exposure to or consumption of various pathogen-contaminated animal products and their foodstuffs. The infection rate of the cloth disease is closely related to the occupation in which the veterinarian, herdsman, butcher, fur worker and meat food processor are high risk groups for the cloth disease. Dietary habits are important, and consumption of raw or unripe meat in milk, yogurt, or the like without high temperature sterilization increases the risk of infection. Cloth sickness can also be highly contagious via the respiratory tract.
At present, the diagnosis methods for the brucellosis mainly include tiger red plate agglutination experiments, complement fixation experiments (CFT) detection, enzyme-linked immunosorbent assay (ELISA), biomolecular diagnosis and the like, which are all suitable for laboratory research, have long detection time and are not suitable for basic detection, so that the research and development of a simpler and faster detection method is urgently needed.
The invention discloses a patent number CN201510477774.8 with the patent name of a Brucella colloidal gold antibody detection test strip, and relates to a rapid, efficient and accurate Brucella colloidal gold diagnosis method. The colloidal gold uses brucella S2 Lipopolysaccharide (LPS) as a test line coating antigen, so that the detection sensitivity is improved; the LPS purification and purity determination method is improved, so that the developed kit has good specificity; the invention utilizes the monoclonal antibody of the mouse anti-bovine IgG-Fc end to mark the colloidal gold to prepare the gold gel pad of the colloidal gold antibody detection test strip, thereby further improving the specificity of detection.
The method has the defects that the detection method adopts the antigen to detect the Brucella antibody, and the antibody has a window period, so that the latent period of pathogenic bacteria is long, and the detection result is not accurate enough.
Disclosure of Invention
The invention aims to overcome the problem that the detection result is inaccurate when brucellosis is detected by using an antigen in the prior art, and provides the colloidal gold immunochromatographic assay test paper for quickly diagnosing brucellosis.
In order to achieve the purpose, the invention adopts the following technical scheme:
a colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis comprises a PVC base plate, wherein the PVC base plate is sequentially provided with a sample pad, a gold-labeled probe treatment pad, a modified nitrocellulose membrane and a water absorption pad; the sample pad is arranged on the upper side of one end, connected with the PVC base plate, of the gold-labeled probe treatment pad, the bottom side of one end of the sample pad is connected with the upper side of the microsphere probe treatment pad, the bottom side of the other end of the sample pad is connected with the PVC base plate, and the non-connecting section of the modified nitrocellulose membrane is sequentially provided with a detection line T coated with a mouse anti-brucella antibody and a quality control line C coated with a goat anti-mouse IgG antibody from one side of the microsphere probe treatment pad.
The principle applied by the product is colloidal gold immunochromatography and a double-antibody sandwich method, when a sample contains brucella antigen, the antigen can be firstly combined with a mouse anti-brucella monoclonal antibody-gold-labeled probe in a labeling pad after sample addition to form an antigen-antibody compound, and the compound can be captured by the mouse anti-brucella monoclonal antibody coated on a nitrocellulose membrane along with the chromatography process to form the mouse anti-brucella monoclonal antibody-brucella antigen-mouse anti-brucella antibody-gold-labeled probe, and the color is purple red, clear and identifiable, no auxiliary equipment is needed, and the operation is convenient. Antibodies appear in patients 7-15 days after infection with brucella, and therefore detection of antigens eliminates brucella infection earlier than antibodies.
By using the test strip, a sample containing the Brucella antigen can be detected. Detecting the sample by using a test strip, wherein when red strips appear on the detection line T and the quality control line C, the sample is infected with brucella; if only the quality control line C has a red strip, indicating that the sample is not infected with the Brucella; and if the quality control line C does not have a red strip, the test strip is invalid.
Preferably, the gold-labeled probe treatment pad is a gold-labeled pad labeled with a brucella anti-brucella antibody-colloidal gold conjugate.
The colloidal gold probe is essentially a coating process in which a polymer such as a protein is adsorbed on the surface of a colloidal gold particle. The colloidal gold has negative charge in weak alkali environment, can form firm electrostatic combination with the positive charge group of protein molecules, and does not influence the biological characteristics of the protein. The colloidal gold probe comprises corresponding antibody and color developing particles, so that the colloidal gold probe plays a role in combination and color development in the whole process, and the better gold-labeled probe has the advantages of proper particle size, good uniformity and high color transparency.
Preferably, the concentration of the mouse anti-brucella antibody for coating coated on the detection line T is 0.1-1.0 mg/ml.
When the concentration of the coating antibody is lower than 0.1mg/ml, the brucella antigen in the sample is insufficiently captured by the mouse anti-brucella monoclonal antibody in the reaction process, and the condition of missing detection appears clinically. When the concentration of the coating antibody is higher than 1mg/ml, the foreign protein in the sample can be captured nonspecifically in the chromatography process, and the clinical risk of false positive can occur, so the optimal concentration range of the mouse anti-Brucella antibody for coating the detection line T is 0.1mg/ml-1 mg/ml.
Preferably, the concentration of the goat anti-mouse IgG antibody coated by the quality control line C is 1.0-3.0 mg/ml.
When the concentration of the quality control line coated antibody is lower than 1mg/ml, the combination of the mouse IgG monoclonal antibody-gold-labeled conjugate in the labeling pad and the goat anti-mouse polyclonal antibody is insufficient in the reaction process, and the problem of weak quality control line clinically occurs. When the concentration of the quality control line coated antibody is higher than 3mg/ml, the sheep anti-mouse polyclonal antibody treated on the nitrocellulose membrane can generate a diffusion phenomenon due to overflow, and the integral appearance is influenced. Therefore, the optimal concentration range of goat anti-mouse IgG polyclonal antibody coated with the quality control line C is 1mg/ml to 3 mg/ml.
Preferably, the diameter of the colloidal gold particles is 40-100nm, and the concentration of the labeled murine anti-Brucella monoclonal antibody-colloidal gold conjugate used herein should be OD1-OD 4. The chloroauric acid reacts with trisodium citrate serving as a reducing agent to form gold particles with a certain particle size, the performance of the product, the stability of the labeled conjugate and the color of the labeled conjugate are affected by the difference of the particle sizes, the color of the particles is reddish when the particle size is less than 40nm, and the sensitivity is weak; the particle size of more than 100nm is black, uneven in shape and easy to precipitate, so that the particle size of the colloidal gold particles is most suitable to be 40-100 nm. The mouse anti-brucella monoclonal antibody is marked into a mouse anti-brucella-colloidal gold conjugate by using 70nm gold particles, and is treated on glass fibers according to the absorbance values of OD0.5, OD1.0, OD2.0, OD3.0, OD4.0 and OD5.0, and is placed in a vacuum freeze-drying device for overnight (12-24 hours). With brucella positive reference 1:2000, 1:10000, 1:20000 and negative reference, the reading result of 10-20 minutes shows that the effect is best when the anti-Brucella murine monoclonal antibody-gold conjugate is used at the concentration of OD1-OD 4.
The preparation method of the colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis specifically comprises the following steps:
1) preparing a detection line T and a quality control line C: respectively diluting mouse anti-brucella antibody and goat anti-mouse IgG antibody solutions, respectively spraying the diluted solutions onto a nitrocellulose membrane at a speed of 1.0-1.2ul/cm to respectively serve as a detection line T and a quality control line C, and drying at 36-38 ℃ to obtain a detection line and a quality control line;
2) preparing a gold label pad: coating the purified gold-labeled antibody with the concentration of OD1-OD4 after dilution on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled pad;
3) preparing detection test paper: and sequentially connecting and assembling the water absorption pad, the sample pad, the microsphere probe treatment pad, the modified nitrocellulose membrane coated with the antibody and the PVC bottom plate to obtain a finished product.
The key process technology in the preparation process is the freeze drying treatment in the step 2), the freeze drying treatment is carried out under the vacuum condition, compared with the conventional air drying, the distribution of the gold-labeled conjugate on the carrier is more uniform, and the titer of the antigen or the antibody is less influenced. However, in the freeze-drying process, the temperature in the cabin body is likely to exceed the expected temperature due to the continuous temperature rise of the equipment, so that the product performance is affected, and in order to ensure that the temperature of the cabin body is maintained in the expected range, the temperature control equipment is installed on the freeze-drying machine, and the temperature of the cabin body is strictly controlled not to exceed 35 ℃ in the freeze-drying process, so that the performance of the product is not affected.
Preferably, the preparation process of the purified gold-labeled antibody comprises the following steps:
A. obtaining colloidal gold: adding 1.8-2.2ml of 0.8-1.2% trisodium citrate aqueous solution into 98-102ml of boiling 0.008-0.012% chloroauric acid aqueous solution to obtain colloidal gold with diameter of 65-75 nm;
B. labeling colloidal gold: with K of 0.18-0.22mol/L2CO3Adjusting the pH of the colloidal gold to 8.4-8.6 with the solution, adding the mouse anti-brucella antibody for marking into the colloidal gold with the adjusted pH, and marking for 20-25 min;
C. purifying the gold-labeled antibody: and D, adding 8-12% of BSA aqueous solution by mass into the labeled colloidal gold solution obtained in the step B until the final concentration of BSA is 0.8-1.2%, stopping adding, continuing to stir for 25-30min, continuing to add 8-12% of PEG20000 until the final concentration of PEG20000 is 0.18-0.22%, continuing to stir for 10-15min, centrifuging, and discarding the supernatant to obtain the purified gold-labeled antibody.
In the preparation process of the mouse anti-brucella monoclonal antibody-gold-labeled conjugate, the coupling process of colloidal gold and the antibody is particularly important, and because the particle size selected by the product is larger, the phenomena of gold floating and dead gold are easy to occur in the coupling process. In the reaction process, the phenomenon of gold bleaching and dead gold can be greatly reduced by adding the complex solution.
Preferably, the volume ratio of the mouse anti-brucella antibody used for marking in the step B to the colloidal gold after pH adjustment is 1: 1-1.2.
The mouse anti-brucella monoclonal antibody and the chloroauric acid are coupled according to the proportion of 1:0.5, 1:1, 1:1.2, 1:1.4,1:1.6 and 1:2.0, the final concentration values of the conjugates are all larger than OD100, the conjugates are diluted to OD2 by using gold-labeled diluent and are treated on glass fiber, the glass fiber is frozen and dried overnight (12 to 24 hours) in vacuum, and the Brucella positive reference product 1:2000, 1:10000, 1:20000 and a negative reference substance are tested, and the result of reading for 10-20 minutes shows that the result is optimal when the volume ratio is 1:1-1: 1.2.
Preferably, the centrifugation conditions in step C are: the centrifugation temperature is 4-6 ℃, the centrifugation rotation speed is 9900-10000rpm, and the centrifugation time is 30-40 min.
The protein is stable under the condition of 2-8 ℃, the centrifugation speed of the gold-labeled conjugate is generally set at 12000rpm, the centrifugation time is about 0.5 hour, and the product has the particle size of colloidal gold particles of about 70nm, so the centrifugation speed is controlled at 9900-10000rpm, the centrifugation time is 30-40 minutes, and the effect is optimal.
Therefore, the invention has the following beneficial effects:
(1) the colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis is provided, a product for detecting the antigen by using the antibody test paper is prepared, the detection time is short, only 10-20min is needed, and the requirement of field detection can be met;
(2) the accuracy of directly detecting the antigen is higher than that of detecting the antibody, the prepared antigen can accurately detect whether the brucellosis exists, the antigen has no incubation period, the antigen can be detected, the clinical missed detection condition caused by low antibody titer in a window period can be better avoided, and the product is more accurate than that of the antibody;
(3) the operation is simple and convenient, and other equipment and instruments are not needed; the detection result is displayed visually, can be judged by naked eyes and is suitable for personal use; the detection efficiency is high, the detection result is more direct, and the detection error caused by the antibody latency is avoided;
(4) the kit can simultaneously detect various animal brucella antigens, improves the clinical detection rate, and can obviously improve the product sensitivity due to the special mold design;
(5) the preparation process is simple, the test strip can be stored at normal temperature, special equipment and instruments are not needed, the test strip only needs to be kept dry, and the storage life can reach 2 years.
Drawings
FIG. 1 is a schematic diagram of the structure of the test strip of the present invention.
FIG. 2 is a schematic view of the structure of the test strip detection window of the present invention.
In the figure: 1. the kit comprises a sample pad, 2, a gold-labeled pad for marking brucella antibody-colloidal gold, 3, a detection line T for coating the brucella antibody, 4, a quality control line C for coating goat anti-mouse IgG antibody, 5, a nitrocellulose membrane, 6, a water absorption pad, 7 and a PVC bottom plate.
Detailed Description
The invention is further described with reference to specific embodiments.
The colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis comprises a PVC base plate 7, wherein the PVC base plate 7 is sequentially provided with a sample pad 1, a microsphere probe treatment pad 2, a modified nitrocellulose membrane 5 and a water absorption pad 6, the modified nitrocellulose membrane 5 is arranged in the middle of the PVC base plate 7, the two ends of the modified nitrocellulose membrane 5 are respectively provided with the microsphere probe treatment pad 2 and the water absorption pad 6, the bottom sides of one ends of the microsphere probe treatment pad 2 and the water absorption pad 6 are respectively connected with the two ends of the modified nitrocellulose membrane 5, and the bottom sides of the other ends of the microsphere probe treatment pad 2 and the water absorption pad 6 are respectively connected with the PVC; the upper side of one end of the microsphere probe treatment pad 2 connected with the PVC base plate 7 is provided with a sample pad 1, the bottom side of one end of the sample pad 1 is connected with the upper side of the microsphere probe treatment pad 2, the bottom side of the other end of the sample pad 1 is connected with the PVC base plate 7, and the non-connecting section of the modified nitrocellulose membrane 5 is sequentially provided with a detection line T3 coated with a mouse anti-brucella antibody and a quality control line C4 coated with a goat anti-mouse IgG antibody from one side of the microsphere probe treatment pad 2.
Preparation of murine anti-brucella antibody: (1) immunizing BALB/C mice with antigen for eight weeks, performing subcutaneous injection, injecting every three weeks for five times,measuring the antibody titer by ELISA (enzyme-linked immunosorbent assay) for blood collection, wherein the titer can be determined, and the injection is strengthened three days before the fusion; (2) cell fusion and culture: immunizing 3d mouse with superstrong immunity, removing neck to kill mouse, aseptically operating to take out spleen to prepare splenocyte, performing cell fusion with SP2/0 cell under the action of PEG, adding the fused cell suspension onto cell culture plate, standing at 37 deg.C and 5% CO2Selectively culturing in HAT and HT culture medium in an incubator; (3) and (3) cell screening: screening positive holes by using indirect competition E LISA, and carrying out three times of limiting dilution cloning on strong positive holes with vigorous cell growth; (4) production of ascites monoclonal antibody: six mice are taken, paraffin is injected into the abdominal cavity, each mouse is injected with 2.5ml, cloned hybridoma cell strains are injected after 10 days, ascites is extracted after 12 days, antibodies are purified by an ammonium caprylate-sulfate salting-out method, and the concentration of the antibodies is measured by an ultraviolet absorption method.
Example 1
The preparation method of the colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis specifically comprises the following steps:
1) preparing a detection line T and a quality control line C: respectively diluting mouse anti-brucella antibody and goat anti-mouse IgG antibody solutions, spraying the diluted solutions on a nitrocellulose membrane at a speed of 1.0ul/cm to serve as a detection line T and a quality control line C, and drying at 36 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-brucella antibody for coating of the detection line T is 0.1 mg/ml; the concentration of the goat anti-mouse IgG antibody coated by the quality control line C is 1.0 mg/ml;
2) preparing a gold label pad: coating the purified gold-labeled antibody with the concentration of OD1 after dilution on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled pad; the preparation process of the purified gold-labeled antibody comprises the following steps: A. obtaining colloidal gold: adding 1.8ml of 0.8% trisodium citrate aqueous solution into 98ml of boiling 0.008% chloroauric acid aqueous solution to obtain colloidal gold with the diameter of 65 nm; B. labeling colloidal gold: with 0.18mol/L of K2CO3Adjusting the pH of the colloidal gold to 8.4, adding a 1mg/ml labeling mouse anti-Brucella antibody into the pH-adjusted colloidal gold, wherein the volume ratio of the labeling mouse anti-Brucella antibody to the pH-adjusted colloidal gold is 1:1, and labeling for 20min to obtain a 40 nm-diameter mouse anti-Brucella antibodyA brucella antibody-colloidal gold conjugate; C. purifying the gold-labeled antibody: and C, adding BSA (bovine serum albumin) water solution with the mass fraction of 8% into the labeled colloidal gold solution in the step B until the final concentration of BSA is 0.8%, stopping adding, continuing to stir for 25min, continuing to add 8% PEG20000 to PEG20000 until the final concentration is 0.18%, continuing to stir for 10min, centrifuging at 4 ℃ and 9900rpm for 30min, and removing the supernatant to obtain the purified gold-labeled antibody.
3) Preparing detection test paper: and sequentially connecting and assembling the water absorption pad 6, the sample pad 1, the microsphere probe treatment pad 2, the modified nitrocellulose membrane 5 coated with the antibody and the PVC base plate 7 to obtain a finished product.
Example 2
The preparation method of the colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis specifically comprises the following steps:
1) preparing a detection line T and a quality control line C: respectively diluting mouse anti-brucella antibody and goat anti-mouse IgG antibody solutions, spraying the diluted solutions on a nitrocellulose membrane at a speed of 1.05ul/cm to serve as a detection line T and a quality control line C, and drying at 36.5 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-brucella antibody for coating of the detection line T is 0.3 mg/ml; the concentration of the goat anti-mouse IgG antibody coated by the quality control line C is 1.5 mg/ml;
2) preparing a gold label pad: coating the purified gold-labeled antibody with the concentration of OD2 after dilution on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled pad; the preparation process of the purified gold-labeled antibody comprises the following steps: A. obtaining colloidal gold: adding 1.9ml of 0.9% trisodium citrate aqueous solution into 99ml of boiling 0.009% chloroauric acid aqueous solution to obtain colloidal gold with a diameter of 68 nm; B. labeling colloidal gold: with 0.19mol/L of K2CO3Adjusting the pH value of the colloidal gold to 8.5 by using the solution, adding a mouse anti-brucella antibody for marking with the concentration of 2mg/ml into the colloidal gold after the pH value is adjusted, wherein the volume ratio of the mouse anti-brucella antibody for marking to the colloidal gold after the pH value is adjusted is 1:1.1, and marking for 21min to obtain a mouse anti-brucella antibody-colloidal gold conjugate with the diameter of 50 nm; C. purifying the gold-labeled antibody: adding BSA (bovine serum albumin) with the mass fraction of 9 percent into the labeled colloidal gold solution in the step BProtein) until the final concentration of BSA is 0.9%, stopping adding, continuing stirring for 26min, continuing adding 9% PEG20000 until the final concentration of PEG20000 is 0.19%, continuing stirring for 11min, centrifuging at 5 deg.C and 9920rpm for 32min, and discarding the supernatant to obtain the purified gold-labeled antibody.
Example 3
The preparation method of the colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis specifically comprises the following steps:
1) preparing a detection line T and a quality control line C: respectively diluting mouse anti-brucella antibody and goat anti-mouse IgG antibody solutions, spraying the diluted solutions on a nitrocellulose membrane at a speed of 1.1ul/cm to serve as a detection line T and a quality control line C, and drying at 37 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-brucella antibody for coating of the detection line T is 0.5 mg/ml; the concentration of the goat anti-mouse IgG antibody coated by the quality control line C is 2.0 mg/ml;
2) preparing a gold label pad: coating the purified gold-labeled antibody with the concentration of OD2 after dilution on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled pad; the preparation process of the purified gold-labeled antibody comprises the following steps: A. obtaining colloidal gold: adding 2.0ml of 1.0% trisodium citrate aqueous solution into 100ml of boiling 0.01% chloroauric acid aqueous solution to obtain colloidal gold with the diameter of 70 nm; B. labeling colloidal gold: with 0.2mol/L of K2CO3Adjusting the pH value of the colloidal gold to 8.5 by using the solution, adding a mouse anti-brucella antibody for marking with the concentration of 3mg/ml into the colloidal gold after the pH value is adjusted, wherein the volume ratio of the mouse anti-brucella antibody for marking to the colloidal gold after the pH value is adjusted is 1:1.1, and marking for 23min to obtain a mouse anti-brucella antibody-colloidal gold conjugate with the diameter of 60 nm; C. purifying the gold-labeled antibody: and D, adding a BSA (bovine serum albumin) aqueous solution with the mass fraction of 10% into the labeled colloidal gold solution obtained in the step B until the final concentration of the BSA is 1.0%, stopping adding, continuing to stir for 28min, continuing to add 10% PEG20000 to PEG20000 until the final concentration is 0.2%, continuing to stir for 12min, centrifuging at 9950rpm for 35min at 5 ℃, and removing the supernatant to obtain the purified gold-labeled antibody.
Example 4
The preparation method of the colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis specifically comprises the following steps:
1) preparing a detection line T and a quality control line C: respectively diluting mouse anti-brucella antibody and goat anti-mouse IgG antibody solutions, spraying the diluted solutions on a nitrocellulose membrane at a speed of 1.1ul/cm to serve as a detection line T and a quality control line C, and drying at 37.5 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-brucella antibody for coating of the detection line T is 0.8 mg/ml; the concentration of the goat anti-mouse IgG antibody coated by the quality control line C is 2.5 mg/ml;
2) preparing a gold label pad: coating the purified gold-labeled antibody with the concentration of OD1-OD4 after dilution on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled pad; the preparation process of the purified gold-labeled antibody comprises the following steps: A. obtaining colloidal gold: adding 2.1ml of 1.1% trisodium citrate aqueous solution into 101ml of boiling 0.011% chloroauric acid aqueous solution to obtain colloidal gold with the diameter of 72 nm; B. labeling colloidal gold: with 0.21mol/L of K2CO3Adjusting the pH value of the colloidal gold to 8.5 by using the solution, adding a mouse anti-brucella antibody for marking with the concentration of 4mg/ml into the colloidal gold after the pH value is adjusted, wherein the volume ratio of the mouse anti-brucella antibody for marking to the colloidal gold after the pH value is adjusted is 1:1.1, and marking for 24min to obtain a mouse anti-brucella antibody-colloidal gold conjugate with the diameter of 80 nm; C. purifying the gold-labeled antibody: and D, adding 11% by mass of BSA (bovine serum albumin) aqueous solution into the labeled colloidal gold solution obtained in the step B until the final concentration of BSA is 1.1%, stopping adding, continuing to stir for 29min, continuing to add 11% PEG20000 to PEG20000 until the final concentration is 0.21%, continuing to stir for 14min, centrifuging at 5.5 ℃ and 9980rpm for 38min, and removing the supernatant to obtain the purified gold-labeled antibody.
Example 5
The preparation method of the colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis specifically comprises the following steps:
1) preparing a detection line T and a quality control line C: respectively diluting mouse anti-brucella antibody and goat anti-mouse IgG antibody solutions, spraying the diluted solutions on a nitrocellulose membrane at a speed of 1.2ul/cm to serve as a detection line T and a quality control line C, and drying at 38 ℃ to obtain a detection line and a quality control line; the concentration of the mouse anti-brucella antibody for coating of the detection line T is 1.0 mg/ml; the concentration of the goat anti-mouse IgG antibody coated by the quality control line C is 3.0 mg/ml;
2) preparing a gold label pad: coating the purified gold-labeled antibody with the concentration of OD4 after dilution on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled pad; the preparation process of the purified gold-labeled antibody comprises the following steps: A. obtaining colloidal gold: adding 2.2ml of 1.2% trisodium citrate aqueous solution into 102ml of boiling 0.012% chloroauric acid aqueous solution to obtain colloidal gold with the diameter of 75 nm; B. labeling colloidal gold: with 0.22mol/L of K2CO3Adjusting the pH value of the colloidal gold to 8.6 by using the solution, adding a mouse anti-brucella antibody for marking with the concentration of 5mg/ml into the colloidal gold after the pH value is adjusted, wherein the volume ratio of the mouse anti-brucella antibody for marking to the colloidal gold after the pH value is adjusted is 1:1.2, and marking for 25min to obtain a mouse anti-brucella antibody-colloidal gold conjugate with the diameter of 100 nm; C. purifying the gold-labeled antibody: and C, adding BSA (bovine serum albumin) aqueous solution with the mass fraction of 12% into the labeled colloidal gold solution in the step B until the final concentration of BSA is 1.2%, stopping adding, continuing stirring for 30min, continuing adding 12% PEG20000 to the final concentration of PEG20000 of 0.22%, continuing stirring for 15min, centrifuging at 6 ℃ and 10000rpm for 40min, and discarding the supernatant to obtain the purified gold-labeled antibody.
Comparative example 1
The difference from example 3 is that the diameter of the murine anti-brucella antibody-colloidal gold conjugate is as small as 20 nm.
Comparative example 2
The difference from example 3 is that the step of purifying the gold-labeled antibody in step 2) is omitted.
Comparative example 3
The difference from example 3 is that the colloidal gold particle size in step A is too large to be 90 mm.
Comparative example 4
The difference from example 3 is that the final centrifugation speed is reduced to 6000r/min and centrifugation is carried out for 0.5 hours.
Comparative example 5
The difference from example 3 is that the marking mat treated in step 1) is dried overnight (12-24 hours) in a forced air drying apparatus at 40 ℃.
And (3) performing a sensitivity test, namely dripping the brucella standard substance with the low concentration, the middle concentration and the high concentration and the negative sample into a sample adding hole of the test strip by using the sample adding amount of 2 drops of a 10ul dropper, adding 2 drops of buffer at the sample adding hole of the buffer solution for detection, repeating the setting of each concentration for three times, and ensuring that the detection results are completely consistent, thereby proving that the test strip has stable and reliable detection results.
The stability test adopts an accelerated stability test, the test strips of the same batch are respectively placed in ovens at 45 ℃ and 55 ℃, and the Brucella standard substances are respectively detected within the short time of the following table.
The results of the tests on the finished products obtained in examples 1 to 5 and comparative examples 1 to 5 are shown in Table 1.
Table 1 shows the performance evaluation indexes of the items related to the colloidal gold immunochromatographic test paper
Conclusion analysis: the colloidal gold immunochromatographic test paper with high detection sensitivity, accurate detection result and long service cycle can be prepared in the embodiments 1 to 5.
Comparative examples 1-5 the diameter of the murine anti-brucella antibody-colloidal gold conjugate in comparative example 1 was as small as 20nm, compared to example 3. The coupling mode of the antibody and the colloidal gold is physical adsorption, and although the colloidal gold with undersize grain diameter is stable, the number of the mouse anti-brucella monoclonal antibodies adsorbed on the surface of the colloidal gold is limited, thereby influencing the sensitivity of the product.
In comparative example 2, the step of purifying the gold-labeled antibody in step 2) is omitted, and the risk of clinical false positive may occur due to interference of the hybrid protein in the labeling process.
The particle size of the colloidal gold in step A of comparative example 3 was too large to be 90 mm. The colloidal gold used in the product has overlarge particle size, gold bleaching and dead gold are easy to occur in the marking process, and the marked gold-labeled conjugate is black in color and easy to generate false positive clinically.
In comparative example 4, the final centrifugation speed was reduced to 6000r/min and the centrifugation was carried out for 0.5 hour. The centrifugation speed is slow, the time is short, the centrifugation is insufficient, the concentration value of the gold-labeled conjugate is low, part of effective components are not collected, and the sensitivity is weak.
The marking pad treated in step 1) of comparative example 5 was dried overnight (12-24 hours) in a 40 c forced air drying apparatus. The temperature of the blast drying equipment is high, molecules are easy to diffuse to the edge due to the blast effect, the dried marking pad is not uniform, and individual differences can occur clinically.
As shown in fig. 2, the test line of the test strip was colored after the dropping of the positive standard, and the test line of the test strip was not colored after the dropping of the negative standard. The brucella antigen detection kit adopts a double-hole sample adding mode, a detection sample and a dilution buffer solution are added in two different sample adding holes, the detection sample is firstly dripped in an S sample hole to be combined and reacted with a mouse anti-brucella antibody in a marking pad, the dilution buffer solution is added in a B sample hole, and the brucella antigen-antibody compound is assisted to flow to a detection line position T on a nitrocellulose membrane to be combined and reacted with the mouse anti-brucella antibody in a detection line T.
Clinical sample validation
The inventor collects 50 samples infected with the Brucella and detects the Brucella by using the colloidal gold test strip prepared by the invention, and the detection result shows that 50 samples are positive and completely consistent with the detection result of a latex agglutination experiment. The test strip provided by the invention is reliable in result, strong in specificity, simple and rapid to operate, can be used for detecting clinical samples without any equipment, and is intuitive and accurate in detection result display.
As can be seen from the data of examples 1-5 and comparative examples 1-5, only the protocol within the scope of the claims of the present invention can satisfy the above requirements in all aspects, and an optimized protocol can be obtained, and a colloidal gold immunochromatographic test strip with optimal performance can be obtained. The change of the mixture ratio, the replacement/addition/subtraction of raw materials or the change of the feeding sequence can bring corresponding negative effects.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (9)
1. The colloidal gold immunochromatographic assay test paper for rapidly diagnosing brucellosis is characterized by comprising a PVC base plate (7), wherein the PVC base plate (7) is sequentially provided with a sample pad (1), a microsphere probe treatment pad (2), a modified nitrocellulose membrane (5) and a water absorption pad (6), the modified nitrocellulose membrane (5) is arranged in the middle of the PVC base plate (7), the two ends of the modified nitrocellulose membrane (5) are respectively provided with the microsphere probe treatment pad (2) and the water absorption pad (6), the bottom sides of one ends of the microsphere probe treatment pad (2) and the water absorption pad (6) are respectively connected to the two ends of the modified nitrocellulose membrane (5), and the bottom sides of the other ends of the microsphere probe treatment pad and the water absorption pad are respectively connected with the PVC base plate (; the upper side of one end of the microsphere probe treatment pad (2) connected with the PVC base plate (7) is provided with a sample pad (1), the bottom side of one end of the sample pad (1) is connected with the upper side of the microsphere probe treatment pad (2), the bottom side of the other end of the sample pad is connected with the PVC base plate (7), and the non-connecting section of the modified nitrocellulose membrane (5) is sequentially provided with a detection line T (3) coated with a Brucella antibody and a quality control line C (4) coated with a goat anti-mouse IgG antibody from one side of the microsphere probe treatment pad (2).
2. The test paper for colloidal gold immunochromatography for rapid diagnosis of brucellosis according to claim 1, wherein said microsphere probe treatment pad (2) is a gold-labeled pad labeled with a brucella anti-antibody-colloidal gold conjugate.
3. The test paper for colloidal gold immunochromatography for rapid diagnosis of brucellosis according to claim 1, wherein the concentration of the mouse anti-brucellosis antibody for coating coated by the detection line T (3) is 0.1-1.0 mg/ml.
4. The test paper for rapidly diagnosing brucellosis according to claim 1, wherein the concentration of goat anti-mouse IgG antibody coated by the quality control line C (4) is 1.0-3.0 mg/ml.
5. The test strip for rapidly diagnosing brucellosis according to claim 2, wherein the diameter of the colloidal gold particle is 40-100nm, and the concentration of the labeled mouse anti-brucellosis monoclonal antibody-colloidal gold conjugate is OD1-OD 4.
6. The method for preparing the colloidal gold immunochromatographic test strip for rapidly diagnosing brucellosis according to any one of claims 1 to 5, which is characterized by comprising the following steps:
1) preparing a detection line T and a quality control line C: respectively diluting mouse anti-brucella antibody and goat anti-mouse IgG antibody solutions, respectively spraying the diluted solutions onto a nitrocellulose membrane at a speed of 1.0-1.2ul/cm to respectively serve as a detection line T and a quality control line C, and drying at 36-38 ℃ to obtain a detection line and a quality control line;
2) preparing a gold label pad: coating the purified gold-labeled antibody with the concentration of OD1-OD4 after dilution on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled pad;
3) preparing detection test paper: and sequentially connecting and assembling the water absorption pad (6), the sample pad (1), the microsphere probe treatment pad (2), the modified nitrocellulose membrane (5) coated with the antibody and the PVC bottom plate (7) to obtain a finished product.
7. The method for preparing the test paper for rapidly diagnosing the brucellosis according to claim 6, wherein the preparation process of the purified gold-labeled antibody comprises the following steps:
A. obtaining colloidal gold: adding 1.8-2.2ml of 0.8-1.2% trisodium citrate aqueous solution into 98-102ml of boiling 0.008-0.012% chloroauric acid aqueous solution to obtain colloidal gold with diameter of 65-75 nm;
B. labeling colloidal gold: with K of 0.18-0.22mol/L2CO3Adjusting the pH of the colloidal gold to 8.4-8.6 with the solution, adding the mouse anti-brucella antibody for marking into the colloidal gold with the adjusted pH, and marking for 20-25 min;
C. purifying the gold-labeled antibody: and D, adding 8-12% of BSA aqueous solution by mass into the labeled colloidal gold solution obtained in the step B until the final concentration of BSA is 0.8-1.2%, stopping adding, continuing to stir for 25-30min, continuing to add 8-12% of PEG20000 until the final concentration of PEG20000 is 0.18-0.22%, continuing to stir for 10-15min, centrifuging, and discarding the supernatant to obtain the purified gold-labeled antibody.
8. The method for preparing the test paper for the colloidal gold immunochromatographic assay for the rapid diagnosis of brucellosis according to claim 7, wherein the volume ratio of the mouse anti-brucellosis antibody used for labeling in step B to the colloidal gold after pH adjustment is 1: 1-1.2.
9. The method for preparing the test paper for rapidly diagnosing the brucellosis by using the colloidal gold immunochromatography according to claim 7, wherein the centrifugation conditions in the step C are as follows: the centrifugation temperature is 4-6 ℃, the centrifugation rotation speed is 9900-10000rpm, and the centrifugation time is 30-40 min.
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Application publication date: 20201103 |