CN113970637A - Canine distemper virus latex microsphere detection test strip and preparation method thereof - Google Patents
Canine distemper virus latex microsphere detection test strip and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, and discloses a canine distemper virus latex microsphere detection test strip and a preparation method thereof, wherein the canine distemper virus latex microsphere detection test strip comprises a shell and a sample diluent which is matched with the shell for use, the test strip is assembled in the shell and comprises a PVC (polyvinyl chloride) bottom plate, a sample pad, a marking pad, a coating film and a water absorption pad are adhered on the PVC bottom plate, the marking pad is a glass cellulose film, and a coupling marker of an anti-canine distemper virus F protein monoclonal antibody and latex microspheres is coated on the marking pad; the envelope film is a nitrocellulose film, and is coated with a detection line coated with a monoclonal antibody of the canine distemper virus H protein and a quality control line coated with a goat anti-mouse IgG antibody; the invention has the advantages of low preparation cost, convenient and quick detection, no need of professional equipment and instruments and no need of professional personnel.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a canine distemper virus latex microsphere detection test strip and a preparation method thereof.
Background
Canine distemper is an acute, virulent and highly contact infectious disease caused by canine distemper virus, has strong infectivity and mortality rate of 30-80 percent, and is widely popular in the world; in recent years, due to environmental changes, human intervention in animal habitats and virus evolution, the disease incidence rate of the disease in China is continuously increased, the host range of the disease is also continuously expanded, and the disease causes serious harm to dog breeding industry, economic animal breeding industry and wild animal protection industry, so the disease is particularly important to prevention, control and diagnosis.
The traditional detection method of the canine distemper virus comprises a serological detection method and a etiology detection method; the serological detection method mainly comprises the following steps: methods such as hemagglutination test, enzyme-linked immunosorbent assay, colloidal gold immunochromatography and the like have the problems of low sensitivity and low specificity, and the requirement on detection of pet disease antibodies is less than that of antigens.
The etiology detection method mainly comprises the following steps: virus separation, molecular biological detection, enzyme-linked immunosorbent assay and immunochromatography; although the virus isolation method can detect one infectious virion theoretically to confirm the infection of canine distemper virus, the virus isolation method is not suitable for clinical use due to the factors of complicated operation, high cost, slow confirmation and the like for isolating virus.
Molecular biological detection and enzyme-linked immunosorbent assay are not suitable for field detection in small clinics and fields due to the need of using a matched precise instrument, complicated detection steps, long time consumption, high price and high requirements on technical personnel and environment.
The immunochromatography method has the advantages of simple operation, convenience and quickness, no need of instruments and professionals, 3-10 minutes for result judgment, suitability for field use and the like, and is highly preferred by customers.
At present, most of the existing canine distemper virus antigen immunochromatographic assay test strips are marked by colloidal gold, but the phenomenon of uneven particles is easy to occur when the colloidal gold is fired, so that the product quality is unstable and the uniformity is poor; the existing canine distemper virus immunochromatographic test strip only adopts a monoclonal antibody resisting single protein to carry out antigen detection, and has relatively low sensitivity and specificity; in addition, there are also products for antigen detection by using virus particles to prepare monoclonal antibodies, but because the virus particles prepare monoclonal antibodies, the nonspecific epitope generates too many nonspecific antibodies, which brings great difficulty to screening positive clones, so the method has important significance for the process improvement and breakthrough of the immunochromatographic test strip.
Disclosure of Invention
The invention aims to solve the main technical problem of providing a canine distemper virus latex microsphere detection test strip and a preparation method thereof, which can solve the problems of unstable product quality and poor uniformity caused by uneven particles easily generated by firing of colloidal gold in the preparation process of the existing canine distemper virus antigen colloidal gold detection test strip; the test strip solves the problems that the existing test strip for detecting the canine distemper virus antigen only adopts a monoclonal antibody resisting single protein to detect the antigen, and the sensitivity and the specificity are relatively low; the test strip solves the problem that when the existing test strip for detecting the canine distemper virus antigen is marked by using monoclonal antibodies prepared from virus particles or used as a detection line to capture the antibodies, the screening of positive clones is very difficult due to too many nonspecific antibodies generated by nonspecific epitope.
In order to solve the technical problems, the invention provides the following technical scheme:
a canine distemper virus latex microsphere detection test strip comprises a shell and a sample diluent which is matched with the shell for use, wherein the test strip is assembled in the shell and comprises a PVC (polyvinyl chloride) bottom plate, a sample pad, a marking pad, a coating film and a water absorption pad are adhered to the PVC bottom plate, the marking pad is a glass cellulose film and is coated with a coupling marker of a canine distemper virus resistant F protein monoclonal antibody and a latex microsphere; the envelope film is a nitrocellulose film, and is coated with a detection line coated with a monoclonal antibody of the canine distemper virus H protein and a quality control line coated with a goat anti-mouse IgG antibody.
The following is a further optimization of the above technical solution of the present invention:
the sample diluent comprises a base solution, wherein 0.3 percent of sucrose and 0.05 percent of NaN are added into the base solution in percentage by mass3Tween-20 with the volume percentage of 0.4 percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.02mol/L, and the pH value of the PBS solution is 7.2.
Further optimization: the shell is provided with an observation window and a sample adding hole, after the test strip is assembled in the shell, the coating film is positioned at the position of the observation window, the sample pad is positioned at the position of the sample adding hole, the shell is provided with marks, namely C and T, the mark C corresponds to the quality control line, and the mark T corresponds to the detection line.
The invention also provides a preparation method of the test paper strip for detecting the canine distemper virus latex microspheres, which comprises the following steps:
s1, treatment of latex microspheres: adding 0.05% by mass of latex microspheres into a latex microsphere cleaning solution, centrifuging for 15 minutes at the temperature of 2-8 ℃, wherein the centrifugation speed is 13000 r/min, discarding the supernatant, adding a cleaning solution with the same volume as that of the latex microsphere cleaning solution into the precipitate for resuspension, and repeating the cleaning for 1 time, wherein an NHS solution with the concentration of 20mg/mL is firstly added into the suspended latex microsphere solution, an EDC solution with the concentration of 20mg/mL is then added, and the volume ratio of the NHS solution to the suspended latex microsphere solution is 1: 20; the volume ratio of the EDC solution to the suspended latex microsphere solution is 1: 100; then oscillating for 15 minutes at the oscillation speed of 140-150 r/min, centrifuging for 15 minutes at the temperature of 2-8 ℃ after oscillation is finished, wherein the centrifugation speed is 13000 r/min, discarding supernatant, and adding latex microsphere preservation solution with the volume of the original latex microsphere cleaning solution into the precipitate for resuspension;
s2, protein labeling: adding an anti-canine distemper virus F protein monoclonal antibody into the treated latex microspheres according to 1/10 of the mass of the latex microspheres, then oscillating for 1 hour at the oscillation speed of 140-150 r/min, then adding a sealing liquid with the volume of 1/10 of the original latex microsphere cleaning liquid, and oscillating for 30 minutes at the oscillation speed of 140-150 r/min; centrifuging for 15 minutes at the temperature of 2-8 ℃ after the oscillation is finished, wherein the centrifuging speed is 13000 r/min, discarding the supernatant after the centrifugation is finished, and adding a redissolution with the volume 2 times that of the original latex microsphere cleaning solution into the precipitate for redissolution;
s3, preparation of a marking pad: the labeling pad is a glass cellulose membrane, and the protein solution reconstituted in step S2 is spread on the glass cellulose membrane and then dried at room temperature of 37 ℃ for 2 hours to obtain the labeling pad.
The following is a further optimization of the above technical solution of the present invention:
the preparation method also comprises the following steps:
s4, preparation of coating film: selecting a nitrocellulose membrane as a coating membrane, diluting the anti-canine distemper virus H protein monoclonal antibody to the concentration of 1.0mg/mL by using a membrane scribing solution, and using the membrane scribing solution for detecting line scribing; diluting goat anti-mouse IgG antibody with the membrane scribing solution to the concentration of 1.2mg/mL for quality control line scribing; and (3) scribing a detection line and a quality control line on the nitrocellulose membrane by adopting a three-dimensional scribing and gold spraying instrument at the concentration of 1 mu L/cm in sequence, and then drying and coating for 2 hours in an environment at 37 ℃ to obtain a coating film.
Further optimization: the preparation method also comprises the following steps:
s5, preparing a sample diluent: the formula of the sample diluent comprises base liquid, wherein 0.3 percent of sucrose and 0.05 percent of NaN are added into the base liquid in percentage by mass3Tween-20 with the volume percentage of 0.4 percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.02mol/L, and the pH value of the PBS solution is 7.2.
S6, assembling the detection test strip: sequentially attaching the coating film, the marking pad, the sample pad and the water absorption pad to a PVC (polyvinyl chloride) base plate to prepare a test strip, and putting the test strip into a shell to obtain a canine distemper virus latex microsphere detection test strip;
s7, packaging: and (3) putting the assembled canine distemper virus latex microsphere detection test paper strip and the drying agent into an aluminum foil bag, sealing, labeling, and then putting the sample dilution tube into an outer packaging box.
Further optimization: the formula of the latex microsphere cleaning solution is as follows: MES solution with pH 6.1 and concentration 25 mmol/L.
Further optimization: the formula of the latex microsphere preservative fluid is as follows: MES solution with pH 7.2 and concentration 25 mmol/L.
Further optimization: the formula of the confining liquid comprises water, wherein 1.2 mass percent of BSA is added into the water;
the formula of the compound solution is as follows: the pH value is 7.2, the concentration is 0.02mol/L PBS buffer solution, and the PBS buffer solution is added with the following components by mass percent: 12% sucrose, 1.2% BSA, 0.05% PVP and 0.05% NaN3Tween-20 was also added to the PBS buffer in an amount of 0.5% by volume.
Further optimization: the formula of the film-scribing liquid comprises the following components: the pH value is 7.2, the concentration is 0.02mol/L PBS buffer solution, and the PBS buffer solution is added with the following components by mass percent: 1% sucrose, and 0.1% Tween-20 by volume in PBS buffer.
By adopting the technical scheme, the invention has the following beneficial effects:
1. the test strip for detecting the canine distemper virus latex microspheres has the advantages of low preparation cost, convenience and quickness in detection, no need of professional equipment and instruments, and no need of professional personnel.
2. According to the invention, the latex microspheres are adopted to replace colloidal gold for marking for the first time, so that the preparation process of the test strip has better repeatability, higher sensitivity and stronger stability.
3. The invention adopts the monoclonal antibody of the anti-canine distemper virus H protein and the monoclonal antibody of the anti-canine distemper virus F protein as the binding antibody and the capture antibody for the first time, and can improve the sensitivity and the specificity of the test strip compared with the monoclonal antibody of the anti-single protein.
4. The method can avoid the problem that the screening of the positive specific monoclonal antibody is difficult due to too many non-specific antibodies generated by adopting the virus particles to prepare the monoclonal antibody.
The invention is further illustrated with reference to the following figures and examples.
Drawings
FIG. 1 is a schematic diagram of the overall structure of an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a test strip in an embodiment of the present invention;
FIG. 3 is a diagram showing the determination of a strong positive result when the test strip is used in the embodiment of the present invention;
FIG. 4 is a diagram illustrating a weak positive result determination of the test strip in the embodiment of the present invention;
FIG. 5 is a diagram illustrating a negative result determination of the test strip in an embodiment of the present invention;
FIG. 6 is a graph showing the detection line only showing the invalid result when the test strip is used in the embodiment of the present invention;
FIG. 7 is a diagram illustrating the determination of the invalid result of the test strip in the embodiment of the present invention;
FIG. 8 shows the results of pH measurements of different labels in examples of the present invention;
FIG. 9 shows the results of experiments with different amounts of protein markers in the examples of the present invention;
FIG. 10 shows experimental results for different sealant formulations in accordance with an embodiment of the present invention;
FIG. 11 shows the results of experiments with different reconstituted solutions according to examples of the present invention;
FIG. 12 shows experimental results of various scribes in examples of the present invention;
FIG. 13 shows the result of the test for the optimal scribe line density of the test lines in the embodiment of the present invention;
FIG. 14 is a graph showing the results of an experiment for the optimal concentration of scribe lines in the quality control line according to an embodiment of the present invention;
FIG. 15 shows the results of detection of sensitivity in examples of the present invention;
FIG. 16 shows the results of specific detection in the examples of the present invention;
FIG. 17 is a graph showing experimental results of a first in-batch repeat test in an example of the present invention;
FIG. 18 is a graph showing the results of a second batch repeat test in accordance with an embodiment of the present invention;
FIG. 19 is a graph showing the results of a third batch repeat test conducted in accordance with an example of the present invention;
FIG. 20 is a graph showing experimental results of a first batch-to-batch repeat test in accordance with an embodiment of the present invention;
FIG. 21 is a graph showing the results of a second batch-to-batch repeat test in accordance with an embodiment of the present invention;
FIG. 22 is a graph showing the results of a third batch-to-batch repeat test conducted in accordance with an example of the present invention;
FIG. 23 is a graph showing the results of a fourth batch-to-batch repeat test in accordance with an example of the present invention.
In the figure: 1-sample pad; 2-PVC base plate; 3-a marker pad; 4-detection line (T-line); 5-quality control line (line C); 6-coating film; 7-absorbent pad; 8-a housing; 81-observation window; 82-addition well.
Detailed Description
Example (b): referring to fig. 1-2, a test strip for detecting canine distemper virus latex microspheres comprises a shell 8 and a sample diluent used therewith, wherein the test strip is assembled in the shell 8.
The test strip comprises a PVC base plate 2, wherein a sample pad 1, a marking pad 3, a coating film 6 and a water absorption pad 7 are sequentially adhered to the PVC base plate 2 from one side of the PVC base plate 2 to the other side.
The coating film 6 is a nitrocellulose film.
In this example, a nitrocellulose membrane model CN140 manufactured by Sartorius corporation was used as the coating film 6.
The coating film 6 is coated with a detection line 4 and a quality control line 5, the detection line 4 is coated at a position close to the marking pad 3, and the quality control line 5 is coated at a position close to the absorbent pad 7.
The detection line 4 is coated with a monoclonal antibody (CDV-Mab-H) of the anti-canine distemper virus H protein, and the quality control line 5 is coated with an anti-goat mouse IgG antibody.
The label pad is a glass cellulose membrane and is coated with a conjugate label of a monoclonal antibody (CDV-Mab-F) of anti-canine distemper virus F protein and a latex microsphere.
In the present embodiment, the marking pad 3 is made of a glass cellulose film model 8964, manufactured by shanghai jiening biotechnology limited, and is available on the market directly.
In this example, the anti-canine distemper virus H protein monoclonal antibody, the goat anti-mouse IgG antibody, and the anti-canine distemper virus F protein monoclonal antibody were all produced by madean life science, and were commercially available.
An observation window 81 is arranged on the shell 8 and close to the coating film 6 of the test strip.
After the strip is assembled in the housing 8, the envelope 6 is positioned at the position of the observation window 81.
The two sides of the observation window 81 on the shell 8 are respectively provided with marks C and T, the mark C corresponds to the quality control line 5, and the mark T corresponds to the detection line 4.
The shell 8 is provided with a sample adding hole 82 at a position close to the sample pad 1 of the test strip.
After the test strip is assembled in the housing 8, the sample pad 1 is positioned at the location of the loading hole 82.
The sample diluent comprises a base solution, wherein 1 percent of sucrose and 0.05 percent of NaN are added into the base solution in percentage by mass3Tween-20 with the volume percentage of 0.3 percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.02mol/L, and the pH value of the PBS solution is 7.2.
The invention also discloses a preparation method of the test paper strip for detecting the canine distemper virus latex microspheres, which comprises the following steps:
s1, treatment of latex microspheres: adding 0.05% of latex microspheres in percentage by mass into a latex microsphere cleaning solution, performing a centrifugation process by using a centrifuge at the ambient temperature of 2-8 ℃, wherein the centrifugation speed is 13000 r/min, the centrifugation time is 15 minutes, discarding the supernatant to obtain a precipitate, adding a cleaning solution with the same volume as that of the latex microsphere cleaning solution into the precipitate, and repeatedly cleaning for 1 time.
Adding NHS (N-hydroxysuccinimide) solution with the concentration of 20mg/mL into the suspended latex microsphere solution, and then adding EDC (1-ethyl- (3-dimethylaminopropyl) diimine hydrogen carbonate hydrochloride) solution with the concentration of 20mg/mL, wherein the volume ratio of the NHS solution to the suspended latex microsphere solution is 1: 20; the volume ratio of the EDC solution to the suspended latex microsphere solution is 1: 100.
And then oscillating in a room temperature environment, wherein the oscillation speed is 140-150 r/min, and the oscillation time is 15 minutes.
And after the oscillation is finished, performing a centrifugation process by using a centrifuge at the ambient temperature of 2-8 ℃, wherein the centrifugation speed is 13000 r/min, the centrifugation time is 15 minutes, discarding the supernatant after the centrifugation is finished to obtain a precipitate, and adding the latex microsphere preservation solution with the volume of the original latex microsphere cleaning solution into the precipitate for resuspension.
In step S1, the average diameter of the latex microspheres is 100 nm.
The latex microspheres can be obtained from commercial sources directly.
In step S1, the formula of the latex microsphere cleaning solution is as follows: MES solution (2- (N-morpholine) ethanesulfonic acid solution) with pH 6.1 and concentration of 25 mmol/L.
In step S1, the formula of the latex microsphere preservation solution is as follows: MES solution with pH 7.2 and concentration 25 mmol/L.
S2, protein labeling: adding a monoclonal antibody (CDV-Mab-F) against canine distemper virus F protein into the treated latex microspheres according to 1/10 of the mass of the latex microspheres, then oscillating at the room temperature, wherein the oscillation speed is 140-150 r/min, the oscillation time is 1 hour, then adding a sealing liquid with the volume of 1/10 of the original latex microsphere cleaning liquid, and continuing oscillating at the room temperature, wherein the oscillation speed is 140-150 r/min, and the oscillation time is 30 minutes.
And after the oscillation is finished, performing a centrifugation process by using a centrifuge at the ambient temperature of 2-8 ℃, wherein the centrifugation speed is 13000 r/min, the centrifugation time is 15 minutes, discarding the supernatant after the centrifugation is finished to obtain a precipitate, and adding a redissolution with the volume 5 times that of the original latex microsphere cleaning solution into the precipitate for redissolution.
In the step S2, the mass ratio of the latex microspheres to the labeled monoclonal antibody (CDV-Mab-F) against the canine distemper virus F protein is 10: 1.
In step S2, the formulation of the blocking solution includes water, and BSA (bovine serum albumin) is added to the water in a mass percentage of 1.2%.
In the step S2, the volume ratio of the latex microsphere cleaning solution to the confining liquid is 10: 1.
In step S2, the formula of the reconstituted solution is: the pH value is 7.2, the concentration is 0.02mol/L PBS buffer solution, and the PBS buffer solution is added with the following components by mass percent: 12% sucrose, 1.2% BSA, 0.05% PVP (polyvinylpyrrolidone) and 0.05% NaN3, and 0.5% Tween-20 by volume in PBS buffer.
In the step S2, the volume ratio of the latex microsphere cleaning solution to the composite solution is 1: 5.
S3, preparation of a marking pad: the marking pad 3 is a glass cellulose membrane, the protein solution reconstituted in the step S2 is spread on the glass cellulose membrane, and then the glass cellulose membrane is placed in a room temperature environment of 37 ℃ for drying for 2 hours to obtain the marking pad 3, and then the marking pad 3 is sealed in a tin foil bag and stored at normal temperature for standby.
After the preparation of the marking pad 6 is finished, a conjugate marker of a monoclonal antibody (CDV-Mab-F) of anti-canine distemper virus F protein and latex microspheres is coated.
S4, preparation of coating film: a nitrocellulose membrane is selected as a coating membrane 6, and a monoclonal antibody (CDV-Mab-H) for resisting the canine distemper virus H protein is diluted to the concentration of 1.0mg/mL by using a membrane scribing solution and is used for scribing a detection line 4 (T line).
Goat anti-mouse IgG antibody was diluted with the membrane at a concentration of 1.2mg/mL for control line 5 (line C) streaking.
And (3) scribing a detection line 4 and a quality control line 5 on the nitrocellulose membrane by adopting a three-dimensional scribing and metal spraying instrument at the concentration of 1 mu L/cm in sequence.
After the marking is finished, the coating is placed in an environment with the temperature of 37 ℃ to be dried and coated for 2 hours to obtain a coating film 6, and the coating film 6 is sealed in a tin foil bag and stored at normal temperature for standby.
The model of the three-dimensional film-scratching gold spraying instrument is HM 3030.
In the step S4, the formulation of the dope solution is: the pH value is 7.2, the concentration is 0.02mol/L PBS buffer solution, and the PBS buffer solution is added with the following components by mass percent: 1% sucrose, and 0.1% Tween-20 by volume in PBS buffer.
S5, preparing a sample diluent: the formula of the sample diluent comprises base liquid, wherein 0.3 percent of sucrose and 0.05 percent of NaN are added into the base liquid in percentage by mass3Tween-20 with the volume percentage of 0.4 percent is also added into the base fluid.
The base solution is PBS solution, the concentration of the PBS solution is 0.02mol/L, and the pH value of the PBS solution is 7.2.
After the diluent is prepared, the diluent is filled into a diluent tube.
In this embodiment, the specific formulation of the sample diluent is: 100mL of PBS solution with the pH value of 7.2 and the concentration of 0.02mol/L, 0.3g of cane sugar, 0.4mL of Tween-20 and 0.05g of NaN are added into the PBS solution3。
S6, assembling the detection test strip: the coated membrane 6, the marking pad 3, the sample pad 1 and the water absorption pad 7 are sequentially stuck on the PVC base plate 2, then a slitting machine is adopted for slitting to obtain a test strip, and the test strip is put into the shell 8 to obtain the canine distemper virus latex microsphere detection test strip.
S7, packaging: and (3) putting the assembled canine distemper virus latex microsphere detection test paper strip and the drying agent into an aluminum foil bag, sealing, labeling, and then putting the sample dilution tube into an outer packaging box.
The invention also provides a use method of the test paper strip for detecting the canine distemper virus latex microspheres, which comprises the following steps:
(1) taking appropriate amount of eye and conjunctival secretion, nasal fluid, and saliva with cotton swab, or taking serum for detection.
(2) Immersing a cotton swab in a sample dilution tube, fully stirring and uniformly mixing, standing for 5 minutes, and taking supernatant liquid as detection liquid by using a disposable dropper.
In the step (2), if the detected sample is serum, 2-3 drops of serum are dripped into the diluent pipe, and after the serum is uniformly stirred, the mixed solution is taken for detection.
In the step (2), if the sample cannot be detected immediately, the sample should be refrigerated in a dark place and stored for more than 24 hours, and the sample should be refrigerated and stored; the sample was allowed to return to room temperature before re-testing.
(3) And (3) returning the unopened canine distemper virus latex microsphere detection test strip and the detection sample to room temperature.
(4) Horizontally placing the canine distemper virus latex microsphere detection test strip, and slowly dropwise adding 4-5 drops of detection liquid without bubbles into a sample adding hole 82 of the canine distemper virus latex microsphere detection test strip by using a dropper.
(5) After the sample liquid is added, the red liquid flows out from the edge of the observation window 81 near the sample addition hole 82 and flows in the other direction.
(6) The result is judged in 5-10 minutes, and the result after 10 minutes is only used as reference.
And (5) judging a result: as shown in fig. 3-7, positive: the quality control line 5 shows a red color band, and the detection line 4 also shows a red color band, which is judged to be positive regardless of the color depth.
Negative: the quality control line 5 shows a red color band, and the detection line 4 shows no red color band, and the result is judged to be negative.
And (4) invalidation: the quality control line 5 does not display a red color band, and the test strip for detecting the canine distemper virus latex microspheres is judged to be invalid no matter whether the detection line 4 displays the red color band or not.
Example 2: in example 1 above, determination of the optimal protein labeling pH: respectively adopting MES solutions with pH values of 6.8, 7.2 and 7.6 to mark in the protein marking process of the latex microspheres, and comparing the influence of different pH values on the specificity and sensitivity of the detection test strip so as to determine the optimal protein marking pH value; the experimental structures are shown in the following table and fig. 8.
The following table: experimental results for different labeling pH values:
in the above table, "+" indicates positive; "-" indicates negative.
As can be seen from the above table and FIG. 8, the test strip test line has better color development at pH 7.2, and has poorer sensitivity at pH 6.8 and 7.2, and false negative appears, so the selection marker has a pH 7.2 according to the experimental results.
Example 3, selection of the optimal amount of protein marker in example 1 above: in the process of protein labeling of the latex microspheres, the ratio of the labeled protein CDV-Mab-F to the latex microspheres is 1:12, 1:10 and 1:8 respectively, and the experimental structure is shown in the following table and fig. 9.
The following table: experimental results for different amounts of protein labeling:
in the above table, "+" indicates positive; "-" indicates negative.
As can be seen from the above table and FIG. 9, when the mass ratio of CDV-Mab-F to latex microspheres is 1:12, the sensitivity is poor; the sensitivity and the specificity are better when the mass ratio is 1:10, and the color development effect of the test strip is not obviously changed when the mass ratio is 1:8 compared with 1:10, so that the mass ratio of CDV-Mab-F to the latex microspheres is 1:10 finally selected according to the experimental result.
Example 4: in example 1 above, the selection of the optimal sealant formulation: the final concentration of 0.7%, 1.2% and 1.7% BSA solution was used as blocking solution to block the labeled protein latex microspheres, and the experimental structure is shown in the following table and FIG. 10.
The following table: experimental results for different sealant formulations:
in the above table: "+" indicates positive; "-" indicates negative.
As can be seen from the above table and FIG. 10, the specificity was poor at a final concentration of 0.7% BSA solution; the sensitivity and specificity are better when the final concentration of the BSA solution is 1.2 percent; the sensitivity is relatively poor when the final concentration of the BSA solution is 1.7%, and the detection line is relatively weak in color development, so that the final concentration of the BSA solution is 1.2% according to the experimental result.
Example 5: in example 1 above, selection of the optimal reconstituted solution: different redissolving solutions are adopted for redissolving, the formula of the redissolving solution is shown in the following table, and the optimal formula of the redissolving solution is determined according to the influence of the different formulas of the redissolving solutions on the specificity and sensitivity of the test strip.
The following table: different redissolution formulas:
the experimental structures of the effects of different redissolving formulations on the specificity and sensitivity of the test strips are shown in the following table and fig. 11.
The following table: experimental results for different double solutions:
in the above table, "+" indicates positive; "-" indicates negative.
As can be seen from the above table and fig. 11, no false positive occurred in any of the 4 groups of reconstituted solutions from the aspect of specificity; from the aspect of sensitivity, the redissolved solution 3 has the highest sensitivity, and the detection line has the best color development, so the redissolved solution 3 is finally selected as the redissolved solution of the product.
Example 6: in example 1 above, the selection of the optimal formulation of the dope solution: adopting different formulas of the membrane scribing solution (shown in the following table), diluting CDV-Mab-H to 1.0mg/mL for scribing a detection line 4 (T line), and diluting goat anti-mouse IgG antibody to 1.2mg/mL for scribing a quality control line 5 (C line); and determining the optimal formula of the scribing liquid according to the influence of different formulas of the scribing liquid on the specificity and sensitivity of detection.
The following table shows the formulations of 3 different streaking solutions
The effect of different formulation of the streaking solutions on the specificity and sensitivity of the assay, the experimental structure is shown in the following table and FIG. 12.
The following table: experimental results of 3 different solutions for scratching
In the above table: "+" indicates positive; "-" indicates negative.
According to the table and the figure 12, the sensitivity and the specificity of the PBS buffer system in the 3 buffer systems to be selected are better than those of the detection cards of the PBST buffer system and the TRIS buffer system, wherein the TRIS buffer system has poorer sensitivity, and the detection line has weaker color development; the specificity of the PBST buffer system is poor, and false positive appears, so the PBS buffer system with the pH value of 7.2 and the concentration of 0.02mol/L is finally selected as the optimal formula of the scribing liquid.
Example 7: in example 1 above, determination of the optimal scribe line density of the detection line: the concentrations of CDV-Mab-H were set to 0.6mg/mL, 0.8mg/mL, 1.0mg/mL, and 1.2mg/mL, respectively, for streaking on a test line (T line), and the specificity and sensitivity of the test strip of the present invention were measured to determine the optimal streaking concentration on the test line, and the experimental structure is shown in Table 13 below.
The following table: test results of the detection line for the optimal scribing concentration:
in the above table: "+" indicates positive; "-" indicates negative.
As can be seen from the above table and FIG. 13, the sensitivity was poor and false negative was observed when the CDV-Mab-H concentration was 0.6 mg/mL; the detection line color is weak at 0.8 mg/mL; when the concentration is 1.0mg/mL, the sensitivity is higher, and the color development of the detection line is darker; since the color of the detection line at 1.2mg/mL did not change significantly from that at 1.0mg/mL, 1.0mg/mL was finally selected as the streaking concentration of the detection line.
Example 8: in example 1 above, determination of the optimal scribe line concentration of the quality control line: the concentrations of goat anti-mouse IgG antibody were 0.8mg/mL, 1.0mg/mL, 1.2mg/mL, and 1.4mg/mL, respectively, for streaking of the control line 5 (line C).
The detection line is scribed by CDV-Mab-H, the concentration of the CDV-Mab-H is set to be 1.0mg/mL, scribing amounts are all 1 mu L/cm, a 37 ℃ air blast drying box is arranged after scribing for 2 hours, the experimental condition of the quality control line 5 is observed, the optimal scribing concentration of the quality control line 5 is determined, and the experimental structure is shown in the following table and figure 14.
The following table: experimental results of the optimal concentration of scribed lines of the quality control line:
as can be seen from the above table and FIG. 14, the color of the control line is weak when the concentration of goat anti-mouse IgG antibody is 0.8mg/mL and 1.0 mg/mL; when the concentration is 1.2mg/mL, the sensitivity is higher, and the color development of the detection line is darker; since the color of the detection line at 1.4mg/mL did not change significantly from that at 1.2mg/mL, 1.2mg/mL was finally selected as the streaking concentration of the detection line.
Example 9: and (3) sensitivity detection: sequentially diluting canine distemper virus positive samples to 1000TCID in multiple proportion50500TCID50、250TCID50、125TCID50、62.5TCID50、31.3TCID50、15.6TCID50、7.8TCID50And 3.9TCID50Then, the test strip of the present invention is used for detection, and the detection structure is shown in the following table and fig. 15.
The following table: sensitivity detection results:
note: "+" indicates canine distemper virus positive, and "-" indicates canine distemper virus negative.
As can be seen from the above table and FIG. 15, the serum samples were diluted to 15.6TCID50, and the test results were all positive.
Example 10: and (3) specificity test: the detection test strip provided by the invention is used for detecting canine distemper virus negative samples (N1 and N2), canine parvovirus (N3), canine parainfluenza virus (N4), canine adenovirus type (N5) and canine coronavirus (N6), and the detection structures are shown in the following table and FIG. 16.
The following table: and (3) specific detection results:
in the above table: "-" indicates canine distemper virus negative.
As can be seen from the table and fig. 16, the detection structure of the test strip of the present invention for canine distemper virus negative samples (N1 and N2), canine parvovirus (N3), canine parainfluenza virus (N4), canine adenovirus type (N5) and canine coronavirus (N6) is negative, so that the test strip of the present invention can satisfy the requirement of specificity.
Example 11: and (3) repeatability test: in-batch repeat testing:
the detection test paper strip of the invention is adopted to randomly draw 5 boxes in the same batch, 30 known canine distemper virus positive clinical samples and negative clinical samples are detected, each sample is repeatedly detected for 4 times, and the detection structure is shown in figures 17-19.
In said FIGS. 17-19, "+" indicates positive and "-" indicates negative; as can be seen from fig. 17 to 19, 30 known positive samples were all positive, and 30 known negative samples were all negative.
Batch-to-batch repeat test: 3 batches of the test strip are adopted, 5 boxes are randomly extracted from each batch, 30 known canine distemper virus positive clinical samples and 30 known canine distemper virus negative clinical samples are detected, each sample is repeated for 4 times, and the detection structure is shown in figures 20-23.
In FIGS. 20 to 23, "+" indicates positive and "-" indicates negative. As can be seen from fig. 20 to 23, 30 known positive samples were all positive, and 30 known negative samples were all negative.
It will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in the embodiments described above without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims.
Claims (10)
1. The utility model provides a canine distemper virus latex microsphere detection test paper strip, includes shell (8) and the supporting sample diluent that uses, is equipped with the test paper strip in shell (8), and the test paper strip includes PVC bottom plate (2), pastes on PVC bottom plate (2) and has sample pad (1), mark pad (3), envelope membrane (6) and pad (7) that absorb water, its characterized in that: the marking pad (3) is a glass cellulose membrane and is coated with a coupling marker of the anti-canine distemper virus F protein monoclonal antibody and the latex microspheres; the coating film (6) is a nitrocellulose film, and a detection line (4) coated with a monoclonal antibody of the anti-canine distemper virus H protein and a quality control line (5) coated with a goat anti-mouse IgG antibody are coated on the coating film (6).
2. The test strip for detecting the canine distemper virus latex microspheres of claim 1, which is characterized in that: the sample diluent comprises a base solution, wherein 0.3 percent of sucrose and 0.05 percent of NaN are added into the base solution in percentage by mass3Tween-20 with the volume percentage of 0.4 percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.02mol/L, and the pH value of the PBS solution is 7.2.
3. The test paper strip for detecting the canine distemper virus latex microspheres of claim 2, which is characterized in that: an observation window (81) and a sample adding hole (82) are formed in the shell (8), after the test strip is assembled in the shell (8), the coating film (6) is located at the position of the observation window (81), the sample pad (1) is located at the position of the sample adding hole (82), marks C and T are arranged on the shell (8), the mark C corresponds to the quality control line (5), and the mark T corresponds to the detection line (4).
4. The method for preparing the test paper strip for detecting the canine distemper virus latex microspheres as claimed in any one of claims 1 to 3, which is characterized in that: the method comprises the following steps:
s1, treatment of latex microspheres: adding 0.05% by mass of latex microspheres into a latex microsphere cleaning solution, centrifuging for 15 minutes at the temperature of 2-8 ℃, wherein the centrifugation speed is 13000 r/min, discarding the supernatant, adding a cleaning solution with the same volume as that of the latex microsphere cleaning solution into the precipitate for resuspension, and repeating the cleaning for 1 time, wherein an NHS solution with the concentration of 20mg/mL is firstly added into the suspended latex microsphere solution, an EDC solution with the concentration of 20mg/mL is then added, and the volume ratio of the NHS solution to the suspended latex microsphere solution is 1: 20; the volume ratio of the EDC solution to the suspended latex microsphere solution is 1: 100; then oscillating for 15 minutes at the oscillation speed of 140-150 r/min, centrifuging for 15 minutes at the temperature of 2-8 ℃ after oscillation is finished, wherein the centrifugation speed is 13000 r/min, discarding supernatant, and adding latex microsphere preservation solution with the volume of the original latex microsphere cleaning solution into the precipitate for resuspension;
s2, protein labeling: adding an anti-canine distemper virus F protein monoclonal antibody into the treated latex microspheres according to 1/10 of the mass of the latex microspheres, then oscillating for 1 hour at the oscillation speed of 140-150 r/min, then adding a sealing liquid with the volume of 1/10 of the original latex microsphere cleaning liquid, and oscillating for 30 minutes at the oscillation speed of 140-150 r/min; centrifuging for 15 minutes at the temperature of 2-8 ℃ after the oscillation is finished, wherein the centrifuging speed is 13000 r/min, discarding the supernatant after the centrifugation is finished, and adding a redissolution with the volume 2 times that of the original latex microsphere cleaning solution into the precipitate for redissolution;
s3, preparation of a marking pad: the labeling pad (3) is a glass cellulose membrane, and the protein solution reconstituted in step S2 is spread on the glass cellulose membrane and then dried at room temperature of 37 ℃ for 2 hours to obtain the labeling pad (3).
5. The preparation method of the test paper strip for detecting the canine distemper virus latex microspheres, which is characterized by comprising the following steps: the preparation method also comprises the following steps:
s4, preparation of coating film: selecting a nitrocellulose membrane as a coating membrane (6), diluting the anti-canine distemper virus H protein monoclonal antibody to the concentration of 1.0mg/mL by using a membrane scribing solution, and scribing by using a detection line (4); diluting the goat anti-mouse IgG antibody with the membrane scribing solution to the concentration of 1.2mg/mL for scribing a quality control line (5); and (3) scribing a detection line (4) and a quality control line (5) on the nitrocellulose membrane by adopting a three-dimensional scribing and gold spraying instrument at the concentration of 1 mu L/cm in sequence, and then drying and coating for 2 hours in an environment at 37 ℃ to obtain a coating film (6).
6. The preparation method of the test paper strip for detecting the canine distemper virus latex microspheres of claim 5, which is characterized by comprising the following steps: the preparation method also comprises the following steps:
s5, preparing a sample diluent: the formula of the sample diluent comprises base liquid, wherein 0.3 percent of sucrose and 0.05 percent of NaN are added into the base liquid in percentage by mass3Tween-20 with the volume percentage of 0.4 percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.02mol/L, and the pH value of the PBS solution is 7.2;
s6, assembling the detection test strip: sequentially sticking the coating film (6), the marking pad (3), the sample pad (1) and the water absorption pad (7) on the PVC base plate (2) to prepare a test strip, and putting the test strip into a shell (8) to obtain a canine distemper virus latex microsphere detection test strip;
s7, packaging: and (3) putting the assembled canine distemper virus latex microsphere detection test paper strip and the drying agent into an aluminum foil bag, sealing, labeling, and then putting the sample dilution tube into an outer packaging box.
7. The preparation method of the test paper strip for detecting the canine distemper virus latex microspheres of claim 6, which is characterized by comprising the following steps: the formula of the latex microsphere cleaning solution is as follows: MES solution with pH 6.1 and concentration 25 mmol/L.
8. The method for preparing the test paper strip for detecting the canine distemper virus latex microspheres according to claim 7, which is characterized in that: the formula of the latex microsphere preservative fluid is as follows: MES solution with pH 7.2 and concentration 25 mmol/L.
9. The method for preparing the test paper strip for detecting the canine distemper virus latex microspheres according to claim 8, which is characterized in that: the formula of the confining liquid comprises water, wherein 1.2 mass percent of BSA is added into the water;
the formula of the compound solution is as follows: the pH value is 7.2, the concentration is 0.02mol/L PBS buffer solution, and the PBS buffer solution is added with the following components by mass percent: 12% sucrose, 1.2% BSA, 0.05% PVP and 0.05% NaN3Tween-20 was also added to the PBS buffer in an amount of 0.5% by volume.
10. The method for preparing the test paper strip for detecting the canine distemper virus latex microspheres according to claim 9, which is characterized in that: the formula of the film-scribing liquid comprises the following components: the pH value is 7.2, the concentration is 0.02mol/L PBS buffer solution, and the PBS buffer solution is added with the following components by mass percent: 1% sucrose, and 0.1% Tween-20 by volume in PBS buffer.
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| CN202111303322.XA Pending CN113970637A (en) | 2021-11-05 | 2021-11-05 | Canine distemper virus latex microsphere detection test strip and preparation method thereof |
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Cited By (1)
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| CN116008543A (en) * | 2022-10-31 | 2023-04-25 | 北京天之泰生物科技有限公司 | Lateral chromatography system for detecting canine distemper virus antibody and preparation method thereof |
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