CN114088944A - Canine distemper virus antigen detection test strip based on nano antibody and preparation method thereof - Google Patents
Canine distemper virus antigen detection test strip based on nano antibody and preparation method thereof Download PDFInfo
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- CN114088944A CN114088944A CN202210057075.8A CN202210057075A CN114088944A CN 114088944 A CN114088944 A CN 114088944A CN 202210057075 A CN202210057075 A CN 202210057075A CN 114088944 A CN114088944 A CN 114088944A
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Abstract
The invention belongs to the technical field of biological detection, and discloses a canine distemper virus antigen detection test strip based on a nano antibody and a preparation method thereof, wherein the canine distemper virus antigen detection test strip based on the nano antibody 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 gold-labeled pad, a coating film and a water absorption pad are adhered on the PVC bottom plate, and the gold-labeled pad is a glass cellulose membrane and is coated with a coupling marker of a purified canine distemper H protein nano antibody and colloidal gold; the coated membrane is a nitrocellulose membrane, and a detection line coated with the purified anti-canine distemper virus H protein nano antibody and a quality control line coated with the goat anti-mouse IgG antibody are coated on the coated membrane.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a canine distemper virus antigen detection test strip based on a nano antibody and a preparation method thereof.
Background
Canine distemper is an acute, virulent and highly contagious infectious disease caused by canine distemper virus, has strong infectivity and mortality rate of 30-80 percent, and is widely prevalent worldwide; 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, but the methods have the problems of low sensitivity and low specificity; and pet disease antibody detection is in low demand relative to antigens.
The etiology detection method mainly comprises the following steps: virus separation, molecular biological detection, enzyme-linked immunosorbent assay and colloidal gold 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, high requirements on technical personnel and environment and the like.
The immunochromatography method adopts the canine distemper virus antigen immunochromatography detection test strip for detection, and is deeply favored by customers because of the advantages of simple operation, convenience and rapidness, no need of instruments and professionals, 3-10 minutes for result judgment, suitability for field use and the like.
At present, the existing canine distemper virus antigen immunochromatographic detection test paper strips all adopt a double-antibody sandwich method, wherein the double antibodies are monoclonal antibodies which are prepared by immunizing mice with purified proteins and adopting a hybridoma technology, and the monoclonal antibodies have the following defects: (1) the volume is relatively large, and the binding protein is unstable; (2) the development period is long, the production cost is high, and the yield is low; (3) difficult to mass produce; (4) easy degradation and high preservation cost; (5) easy to be polluted, high in maintenance cost, can be decomposed under the conditions of high temperature, strong acid and strong base, and must be stored at low temperature, otherwise, it can completely lose activity in several weeks.
In addition, monoclonal antibodies prepared by virus particles are available, but when the monoclonal antibodies are prepared by the virus particles, the nonspecific epitope generates too many nonspecific antibodies, which brings great difficulty to screening of positive clones, so that the method has important significance for improving and breaking through the technology of immunochromatography detection test strips from the aspect of raw materials.
Disclosure of Invention
The invention aims to solve the main technical problem of providing a canine distemper virus antigen detection test strip based on a nano antibody and having the advantages of stable binding protein, high detection sensitivity, low production cost and good thermal stability of antibody raw materials and a preparation method thereof.
In order to solve the technical problems, the invention provides the following technical scheme:
a canine distemper virus antigen detection test strip based on a nano antibody comprises a shell and a sample diluent which is matched with the shell, wherein the test strip is assembled in the shell and comprises a PVC (polyvinyl chloride) bottom plate, a sample pad, a gold-labeled pad, a coating film and a water absorption pad are adhered to the PVC bottom plate, and the gold-labeled pad is a glass cellulose membrane and is coated with a coupling marker of a purified canine distemper virus H protein nano antibody and colloidal gold; the envelope film is a nitrocellulose film, and is coated with a detection line coated with a purified anti-canine distemper virus H protein nano antibody and a quality control line coated with a goat anti-mouse IgG antibody;
the amino acid sequence of the canine distemper virus H protein nano antibody is shown as SEQ ID No. 1; the nucleotide sequence of the DNA molecule of the canine distemper virus H protein nano antibody is shown in SEQ ID No. 2.
The following is a further optimization of the above technical solution of the present invention:
the sample diluent comprises a base liquid, wherein NaN with the mass percent of 0.05 percent is added into the base liquid30.05 percent of PEG20000, and 0.5 percent of Tween-20 by volume percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.01mol/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 discloses a preparation method for preparing the test strip for detecting the canine distemper virus antigen based on the nano antibody, which comprises the following steps:
s1, preparing a colloidal gold solution: heating a 0.01% chloroauric acid solution to boiling, quickly adding 1.5mL trisodium citrate with the concentration of 1% and continuously heating, wherein the solution is changed from light yellow to blue black and finally to wine red, the solution is continuously heated for 5 minutes after the color is stable, cooling is carried out at room temperature to obtain a colloidal gold solution, and the colloidal gold solution is stored at 4 ℃ for later use, and the diameter of the colloidal gold particles is 25 nm;
s2, protein labeling: with 1% strength K2CO3Adjusting the pH value of the colloidal gold solution to 7.2 by using the solution; then adding 10 mu g of anti-canine distemper virus H protein nano antibody into each 1mL of colloidal gold solution, and oscillating for 30 minutes at room temperature, wherein the oscillation speed is 130 r/min; then adding 20 microliter of bovine serum albumin with the concentration of 10 percent for sealing, and standing for 30 minutes; centrifuging for 40 minutes after standing is finished, wherein the centrifugation speed is 12000r/min, discarding the supernatant, adding a complex solution according to the proportion of the volume of the colloidal gold solution of 1/10 to dissolve and precipitate, and obtaining a labeled anti-canine distemper virus H protein nano antibody marker;
s3, preparing a gold-labeled pad: and selecting a glass cellulose membrane as a gold-labeled pad, adding 1mL of anti-canine distemper virus H protein nano antibody marker into each glass cellulose strip, and drying to obtain the gold-labeled 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 canine distemper virus H protein nano antibody with the membrane scribing solution to the concentration of 1mg/mL, and using the membrane scribing solution for detecting line scribing; diluting a goat anti-mouse IgG antibody with the membrane scribing liquid to the concentration of 1mg/mL for scribing a quality control line; scribing a detection line and a quality control line on a nitrocellulose membrane by adopting a three-dimensional scribing and gold spraying instrument at the concentration of 1 mu L/cm, and then drying to obtain a coating film;
s5, sample pad treatment: a glass cellulose film was selected as a sample pad, and the sample pad was immersed in the sample pad treatment solution for 30 minutes and then dried overnight in an environment at 37 ℃.
Further optimization: the preparation method also comprises the following steps: s6, preparing a sample diluent: the sample diluent comprisesBase liquid, wherein 0.05 percent of NaN is added into the base liquid in percentage by mass30.05 percent of PEG20000, and 0.5 percent of Tween-20 by volume percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.01mol/L, and the pH value of the PBS solution is 7.2.
Further optimization: the preparation method also comprises the following steps: s7, assembling the detection test strip: sequentially attaching the coating film, the gold label 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 the canine distemper virus antigen detection test strip based on the nano antibody;
s8, packaging: and (3) putting the assembled canine distemper virus antigen detection test strip based on the nano antibody 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 compound solution comprises the following components: a PBS buffer solution with the pH value of 8.0 and the concentration of 0.01mol/L, wherein sucrose with the mass percent of 5 percent and NaN with the mass percent of 0.05 percent are added into the PBS buffer solution3And 2% PEG20000, and 0.2% by volume of Tween-20 in PBS buffer.
Further optimization: the formula of the scribing liquid comprises the following components: PBS buffer solution with pH value of 7.2 and concentration of 0.01mol/L, wherein NaN with mass percent of 0.05 percent is added into the PBS buffer solution3Tween-20 was also added to the PBS buffer in an amount of 0.1% by volume.
Further optimization: the formula of the sample pad treatment solution comprises: the pH value is 7.2, the concentration is 0.01mol/L PBS solution, the PBS solution is added with the following components by mass percent: 1% BSA, 1% sucrose and 0.05% NaN3Tween-20 with a volume percentage of 0.5% was also added to the PBS solution.
By adopting the technical scheme, the invention has the advantages of ingenious conception, low cost, stable combination of the antibody and the protein, higher sensitivity and convenient and quick detection, does not need professional equipment and instruments and the technology of professionals, replaces the traditional monoclonal antibody with the nano antibody, not only reduces the cost and improves the detection efficiency, but also ensures the detection accuracy, and can be used for the on-site quick detection of pet communities, farms and animal hospitals.
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 is a graph showing the results of experiments performed in the embodiment of the present invention when selecting the optimal pH of the gold-labeled protein;
FIG. 9 is a graph showing the results of experiments performed in the present invention when the optimal amount of gold-labeled protein was selected;
FIG. 10 is a schematic diagram showing the titer of camel blood serum antibodies in the preparation process of the anti-canine distemper virus H protein nano antibody of the present invention;
FIG. 11 is a schematic diagram illustrating the construction and identification of a display library in the process for preparing the anti-canine distemper virus H protein nanobody of the present invention;
FIG. 12 is a schematic diagram of VHH titer in the process for preparing the anti-canine distemper virus H protein nanobody of the present invention;
FIG. 13 is a schematic diagram of the detection of VHH specificity in the preparation process of the anti-canine distemper virus H protein nanobody of the present invention;
FIG. 14 is a schematic diagram of Nb2 expression and purification in the preparation process of the anti-canine distemper virus H protein nanobody of the present invention.
In the figure: 1-sample pad; 2-PVC base plate; 3-gold label pad; 4-detection line; 5-quality control line; 6-coating film; 7-absorbent pad; 8-a housing; 81-observation window; 82-addition well.
Detailed Description
Example 1: referring to fig. 1-2, the test strip for detecting canine distemper virus antigen based on nano-antibody 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, and a sample pad 1, a gold label 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 gold mark 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 purified anti-canine distemper virus H protein nano antibody, and the quality control line 5 is coated with an goat anti-mouse IgG antibody.
In this example, the goat anti-mouse IgG antibody is produced by Midean biosciences and can be purchased commercially.
The gold label pad 3 is a glass cellulose membrane coated with a coupling marker of the purified anti-canine distemper virus H protein nano antibody and colloidal gold.
In the present embodiment, the gold label pad 3 is made of a glass cellulose membrane model 8964, manufactured by shanghai jiening biotechnology limited, and can be purchased directly from the market. .
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.
And marks C and T are respectively arranged on one side of the shell 8, which is positioned on the observation window 81, 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 strip is assembled in the housing 8, the sample pad 1 is positioned at the location of the loading aperture 82.
The formula of the sample diluent comprises a base solution, wherein 0.05 percent of NaN by mass is added into the base solution30.05 percent of PEG20000, and 0.5 percent of Tween-20 by volume percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.01mol/L, and the pH value of the PBS solution is 7.2.
The invention also provides a preparation method of the canine distemper virus antigen detection based on the nano antibody, which comprises the following steps:
s1, preparing a colloidal gold solution: adding ultrapure water into a chloroauric acid solution with the volume percentage of 1% to prepare a chloroauric acid solution with the concentration of 0.01%, heating to boiling, quickly adding 1.5mL of trisodium citrate with the concentration of 1%, continuously heating, converting the solution from faint yellow to blue black, finally changing to wine red, continuously heating for 5 minutes after the color is stable, storing at room temperature after heating is finished, cooling to obtain a colloidal gold solution, and storing the colloidal gold solution at the temperature of 4 ℃ for later use, wherein the diameter of the colloidal gold particles is 25 nm.
S2, protein labeling: about 45 mu L of K is added into every 1mL of colloidal gold solution2CO3Solution (K)2CO3The concentration of the solution is 1%), and the pH value is adjusted to 7.2; then adding the anti-canine distemper virus H protein nano antibody, adding 10 mu g of the anti-canine distemper virus H protein nano antibody into every 1mL of colloidal gold solution, and then oscillating for 30 minutes at room temperature, wherein the oscillation speed is 130 r/min; then adding 20 μ L of 10% Bovine Serum Albumin (BSA) for blocking, and standing at room temperature for 30 min; and (3) centrifuging for 40 minutes after standing is finished, wherein the centrifugation speed is 12000r/min, discarding the supernatant, adding the complex solution according to the proportion of the volume of the colloidal gold solution of 1/10 to dissolve and precipitate, and thus obtaining the labeled anti-canine distemper virus H protein nano antibody marker.
The optimal amount of the marker protein in step S2 is 10 μ g of anti-canine distemper virus H protein nanobody added to 1mL of colloidal gold solution.
The pH value of the colloidal gold label is 7.2.
The formula of the compound solution in the step S2 comprises the following components: PBS buffer solution with pH value of 8.0 and concentration of 0.01mol/L, wherein sucrose with mass percent of 5 percent and NaN with mass percent of 0.05 percent are added into the PBS buffer solution3And 2% PEG20000, and 0.2% by volume of Tween-20 in PBS buffer.
S3, preparing a gold-labeled pad: selecting a glass cellulose membrane 8964 and preparing the glass cellulose membrane into glass cellulose membrane strips, wherein the specification of the glass cellulose membrane strips is 0.3cm multiplied by 30cm, the glass cellulose membrane strips are used as gold-labeled pads 3, 1mL of anti-canine distemper virus H protein nano antibody marker is added into each glass cellulose strip, then, a drying process is carried out, the drying temperature is 37 ℃, the drying time is 2 hours, the gold-labeled pads 3 are obtained, and the gold-labeled pads 3 are sealed in tin foil bags and stored at normal temperature for later use.
S4, preparation of coating film: selecting a nitrocellulose membrane as a coating membrane 6, diluting the anti-canine distemper virus H protein nano antibody with a membrane scribing solution to obtain an anti-canine distemper virus H protein nano antibody diluent, wherein the concentration of the anti-canine distemper virus H protein nano antibody diluent is 1mg/mL, and the anti-canine distemper virus H protein nano antibody diluent is used for scribing a detection line 4;
diluting the goat anti-mouse IgG antibody with the membrane scribing liquid to obtain a goat anti-mouse IgG antibody diluent, wherein the concentration of the goat anti-mouse IgG antibody diluent is 1mg/mL, and the goat anti-mouse IgG antibody diluent is used for scribing a quality control line 5;
and (3) sequentially scribing the anti-canine distemper virus H protein nano antibody diluent and the goat anti-mouse IgG antibody diluent on a nitrocellulose membrane at the concentration of 1 mu L/cm by using a three-dimensional scribing and gold spraying instrument to perform the scribing of a detection line 4 and a quality control line 5.
And (3) after the marking is finished, carrying out a drying process, wherein the drying temperature is 37 ℃, the drying time is 2 hours, obtaining a coating film 6 after the drying is finished, sealing the coating film 6 in a tin foil bag, and storing at normal temperature for later use.
In the step S4, the model of the three-dimensional film-scribing metal spraying instrument is HM 3030.
After the scribing is finished, the detection line 4 is coated with a purified nano antibody for resisting the canine distemper virus H protein; the quality control line 5 is coated with goat anti-mouse IgG antibody.
The formula of the membrane scribing liquid in the step S4 comprises the following components: PBS buffer solution with pH value of 7.2 and concentration of 0.01mol/L, wherein NaN with mass percent of 0.05 percent is added into the PBS buffer solution3Tween-20 was also added to the PBS buffer in an amount of 0.1% by volume.
S5, sample pad treatment: the glass cellulose membrane 8964 was selected as the sample pad 1, the sample pad 1 was immersed in the sample pad treatment solution for 30 minutes, and then dried at 37 ℃ overnight.
The formula of the sample pad treatment solution in the step S5 includes: the pH value is 7.2, the concentration is 0.01mol/L PBS solution, the PBS solution is added with the following components by mass percent: 1% BSA (bovine serum Albumin), 1% sucrose and 0.05% NaN3Tween-20 with a volume percentage of 0.5% was also added to the PBS solution.
In this embodiment, the specific formulation of the sample pad treatment solution includes: 100mL of PBS solution with pH value of 7.2 and concentration of 0.01mol/L, 1g of BSA, 1g of sucrose, 0.5mL of Tween-20 and 0.05g of NaN are added to the PBS solution3。
S6, preparing a sample diluent: the formula of the sample diluent comprises base liquid, wherein NaN with the mass percent of 0.05% is added into the base liquid30.05 percent of PEG20000, and 0.5 percent of Tween-20 by volume percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.01mol/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 pH of 7.2 and concentration of 0.01mol/L, 0.5mL of Tween-20, 0.05g of PEG20000 and 0.05g of NaN were added to the PBS solution3。
S7, assembling the detection test strip: the coated membrane 6, the gold label pad 3, the sample pad 1 and the water absorption pad 7 are sequentially attached to the PVC base plate 2, then a strip cutting machine is used for cutting strips to obtain a test strip, and the test strip is put into the shell 8 to obtain the canine distemper virus antigen detection test strip based on the nano antibody.
S8, packaging: and (3) putting the assembled canine distemper virus antigen detection test strip based on the nano antibody 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 discloses a use method of the test strip for detecting the canine distemper virus antigen based on the nano antibody, which comprises the following specific 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.
And if the detected sample is serum, dropwise adding 2-3 drops of serum into the diluent pipe, uniformly stirring, and taking the mixed solution for detection.
If the sample can not be detected immediately, the sample should be refrigerated in the dark, and the sample should be refrigerated for more than 24 hours; the sample was allowed to return to room temperature before re-testing.
(3) And (3) recovering the unopened canine distemper virus antigen detection test strip and the detection sample to room temperature.
(4) The canine distemper virus antigen detection test strip based on the nano antibody is horizontally placed, and 4-5 drops of detection liquid without air bubbles are slowly and dropwise added into a sample adding hole 82 of the canine distemper virus antigen detection test strip based on the nano antibody by 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 control line 5 does not show a red color band, and the test strip for detecting the canine distemper virus antigen based on the nano antibody is judged to be invalid no matter whether the detection line 4 shows the red color band or not.
In the embodiment, the amino acid sequence of the anti-canine distemper virus H protein nano antibody is shown as SEQ ID No. 1.
The anti-canine distemper virus H protein nano antibody is prepared by encoding DNA molecules, and the nucleotide sequence of the DNA molecules is shown as SEQ ID No. 2.
The preparation process of the canine distemper virus H protein nano antibody is the prior art, and specifically comprises the following steps:
s1, immunizing bactrian camel with inactivated seedlings: injecting 2mL of canine distemper inactivated vaccine into the neck of a bactrian camel in a subcutaneous injection mode, wherein the interval period of each immunization injection is two weeks, and collecting 100mL of peripheral blood at the neck vein of the bactrian camel by using an injector after the fifth immunization injection;
s2, cell separation: adding 2mL of blood and 2mL of PBS solution into a centrifuge tube, blowing and uniformly mixing for dilution, taking a new centrifuge tube, dropwise adding 3mL of ficoll separating medium into the centrifuge tube, dropwise adding the diluted blood into the centrifuge tube slightly to enable a mixed blood layer to be located on the upper layer of the ficoll separating medium, putting the centrifuge tube into a centrifuge, centrifuging for 25min at a centrifugal acceleration of 300g, wherein the centrifugal temperature is 18 ℃, after centrifugation, firstly absorbing a plasma layer on the upper layer in the centrifuge tube, then absorbing a middle white membrane layer, wherein the white membrane layer is lymphocytes, and the plasma layer is serum;
s3, amplification of the VHH gene and construction of a display carrier: putting collected 100mL of peripheral blood into an anticoagulation blood collection bag, adding diluted blood of a cell culture medium with the same volume, extracting RNA of peripheral blood lymphocytes by using an RNA extraction kit, taking the extracted RNA as a template, performing first round amplification by using a nested PCR method, recovering a strip near 700bp by using glue, as shown in A in figure 11, performing second-step amplification of a VHH gene by using cDNA as a template, putting a second round PCR product into an electrophoresis apparatus for electrophoresis, cutting off the strip near 400bp, as shown in B in figure 11, recovering a PCR product by using the glue recovery kit, selecting Pst I and Not I to perform enzyme digestion on an enzyme digestion site, performing enzyme digestion treatment on the VHH gene and a carrier gene, and connecting the VHH to a pCANTAB 5E carrier by using DNA ligase after performing enzyme digestion for 16 h;
s4, construction of a nano antibody library and library capacity determination: electrically transforming the ligation product obtained in the step S3 into TGI competent cells, adding 10mL of preheated SOC culture medium after transformation, culturing for 1h at 220rpm in a shaking table at 37 ℃, taking 50 μ L of thallus after culture, uniformly coating the thallus on an LB/Amp-GLU solid plate, putting the solid plate in an incubator upside down, culturing for 6h at 37 ℃, scraping off the lawn on a culture dish by using a cell scraper after culture, putting the lawn into a glycerol bottle with the concentration of 50%, hermetically storing the bacterial solution in a refrigeration device at-80 ℃, taking 1mL of bacterial solution, coating the bacterial solution on a fixed plate, putting the bacterial solution into the incubator for culturing for 12h, calculating a single colony with a regular shape to obtain the library capacity, and carrying out PCR identification on the bacterial solution, wherein the library capacity is shown as C in figure 11;
s5, recombinant phage titer determination: taking 1mL of library bacterial liquid out of a glycerol bottle, putting the library bacterial liquid into a 2 XTY/Amp-GLU culture medium, putting the culture medium into a shaking table for culturing to a logarithmic phase, adding M13K07 helper phage into 20 MOI, standing the culture medium for 30min at 37 ℃, centrifuging 400g for 15min, removing supernatant, adding 2 XTY/Amp-Kan culture medium to suspend the thalli, culturing for 14h at the rotating speed of 220r/min, centrifuging 12000g for 5min, taking out supernatant, adding 2mL of PEG solution, standing for 6h at 4 ℃, centrifuging 12000g for 5min, and adding 1mLPBS heavy suspension; uniformly smearing TG1 dipped bacterial liquid on an M9 flat plate, culturing for 40h at 37 ℃, selecting a single colony, culturing to a logarithmic phase, diluting recombinant phage by using a 2 xTY culture medium, adding 100 mu L of a sample with dilution of 10-9 and 10-10 into 200 mu L of TG1 bacterial liquid, standing for 30min at 37 ℃, uniformly smearing on an LB/Amp-GLU flat plate, and culturing for 8h at 37 ℃;
s6, panning H protein specific recombinant phage: ELISA plate each hole coated with 10 u gH protein, control hole coated with PBS solution, and added to 100 u L3% BSA solution, at room temperature conditions for 1h, then the PBS solution to plate washing 4 times, will dilute the phage to 5 x 1011pfu/ml, after completion of plate washingAdding 100 mu L of 0.1M triethylamine, placing the plate in a fume hood, standing for 10min, adding 100 mu L of 1M Tris-HCl for neutralization, wherein the pH value after neutralization is 7.4, measuring the titer of the eluted phage, taking 2mL of TG1 bacterial liquid in logarithmic growth phase, adding 100 mu L of phage solution, mixing uniformly, placing in an incubator at 37 ℃ for 30min, immediately adding 8mL of 2 XTY/Amp-GLU culture medium, and placing in a shaking table for culture until the logarithmic growth phase; adding 40 mu L of M13K07 helper phage into the culture medium for dip dyeing for 1h, then placing the liquid in the culture medium into a centrifuge for centrifugation for 5min at the rotating speed of 1500r/min, after centrifugation, using 2 XYT/AMP-KAN for heavy suspension, placing the conical flask into a shaking table for culture for 12h, repeating the steps twice, and completing three rounds of panning processes;
s7.H protein specificity recombinant phage enrichment: taking 100 mu L of phage eluate in the third round, smearing the phage eluate on a culture dish, culturing for 12H, selecting 48 bacterial colonies, placing the bacterial colonies in a TB culture medium for culturing, adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM to induce overnight expression when the logarithmic growth phase is reached, collecting thalli after 12000g of centrifugation for 10min, placing the bacterial colonies at the temperature of minus 80 ℃ for freezing for 30min, placing the bacterial colonies at the room temperature for cracking to release crude extracts, adding a 500 mu LPBS solution and centrifuging at the rotating speed of 12000g/min for 5min, collecting supernatant, coating 400ng of H protein in each hole of an ELISA plate, placing the ELISA plate at the temperature of 4 ℃ for 8H, and sealing 3% BSA for 1H; three washes with PBS' T solution, 100 μ L of ten-fold diluted supernatant per well, incubation for 1h, washing the plate three times again, and adding 1: 3000 diluted anti-M13 phage monoclonal antibody 100 u L, at 25 degrees C after 1h incubation, using PBS' T washing plate 3 times, adding TMB color solution and standing for 15min color development, to each hole adding 50 u L3M sulfuric acid termination, using the spectrophotometer reading absorbance.
After the camel is immunized by the canine distemper inactivated vaccine, peripheral blood lymphocytes are separated, VHH genes of the bactrian camel are amplified by a nested PCR method and are connected with a pCANTAB 5E phage vector to construct a phage display library, 3 strains of H protein specific nano antibodies are screened by panning by using a phage display technology, tests prove that the nano antibodies can react with H protein specificity, wherein the H protein nano antibody has stronger binding force, the nano antibody prepared by the invention has the advantages of small molecular weight, good stability, high affinity, high specificity, easy genetic engineering modification and the like, the monoclonal antibody can be prepared by a prokaryotic expression system, still has biological activity, can specifically identify the canine distemper virus H protein, has wider application range compared with the conventional monoclonal antibody, has low cost, can be prepared in large quantities, and can avoid cross reaction with other virus proteins.
As shown in fig. 10, the H protein antibody titer was determined as follows: the serum separated in the step S2 is used for detecting the antibody titer aiming at the N protein, the antibody titer of the anti-H protein in the serum can reach 1:320000 by using the serum before immunization as a control and through indirect ELISA detection, the abscissa in figure 10 is the camel serum dilution ratio, and the ordinate is OD450nmAbsorbance of (b).
As shown in FIGS. 12-13, in the ELISA detection method of the H protein recombinant nano antibody, 400ng of each hole is coated with the H protein, PRRSV-N protein is used as a control protein, PBS 'T solution is used for washing for 3 times, 200 muL 3% BSA is added, blocking is performed at room temperature for 1H, then blocking solution is washed away, 100 muL of diluted nano antibody crude extract is added into the hole, incubation is performed at 25 ℃ for 1H, after PBS' T washing is finished, 100 muL of Mouse @ E-tag (1: 2000) antibody is added and incubated at room temperature for 1H, 100 muL of goat anti-Mouse IgG (1: 5000) marked by LHRP is added after three times of plate washing, and 100 muL of TMB is added into each hole after plate washing is finished and is subjected to light shading and color development for 15 min.
After the reaction, the reaction was terminated by adding 50. mu.L of 3M sulfuric acid, and OD was read450nmAnd (4) light absorption value.
And recording the number of the experimental wells more than three times higher than that of the control wells, sequencing corresponding positive clones, classifying sequences according to CDR regions of the positive clones, identifying the specificity and titer of the nano antibodies by ELISA (enzyme-linked immunosorbent assay), wherein the specificity and titer of the nano antibodies are shown in figures 12 and 13, and Nb1, Nb2 and Nb3 in figures 12 and 13 represent three screened positive nano antibodies.
As shown in FIG. 14, the construction and expression method of the Nb2 expression vector includes cloning Nb2 gene to pET25b, identifying the construction condition of recombinant plasmid through double enzyme digestion, gel recovery, ligation, transformation and PCR of bacterial liquid, purifying Ni column, performing SDS-PAGE, and observing blue staining, and the result is shown in FIG. 14, which shows that Nb2 is successfully expressed.
Example 2: in example 1 above, selection of the optimal pH of the colloidal gold-labeled protein: 1mL of the prepared colloidal gold solution was added to a 1.5mL centrifuge tube using K at a concentration of 0.1mol/L2CO3And 2mol/L HCl is used for adjusting the pH value of the colloidal gold solution to be: 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0.
And then adding 10 mu g of anti-canine distemper virus H protein nano antibody, shaking for 30 minutes at room temperature at a shaking speed of 130r/min, adding 100 mu L of 10% NaCL solution into each tube, shaking for 30 minutes at room temperature at a shaking speed of 130r/min, observing the color change of the solution, and recording the lowest pH (X) of the solution when the solution is kept red.
Then the pH values were adjusted to X-0.6, X-0.4, X-0.2, X, X +0.2, and X +0.4 (X is the basic pH value), and the optimum pH value was precisely determined, and the results were shown in FIG. 8.
According to the experimental results shown in FIG. 8, the optimal pH of the colloidal gold-labeled protein was determined to be 7.2.
Example 3: in example 1 above, selection of the optimal amount of colloidal gold-labeled protein marker: selecting 8 centrifuge tubes with the volume of 1.5 mL; and after 1mL of colloidal gold solution is added into 8 centrifuge tubes, the pH value of the colloidal gold solution is 7.2, and then anti-canine distemper virus H protein nano-antibodies are sequentially added into the 8 centrifuge tubes, so that the concentration of the marker protein is 0 mug/mL, 2 mug/mL, 4 mug/mL, 6 mug/mL, 8 mug/mL, 10 mug/mL, 12 mug/mL and 14 mug/mL.
And then oscillating for 30 minutes at room temperature with the oscillation speed of 130r/min, adding 100 muL of 10% NaCL solution into each tube, oscillating for 30 minutes at room temperature with the oscillation speed of 130r/min, observing the color change of the colloidal gold solution, recording the amount of the lowest anti-canine distemper virus H protein nano antibody keeping red, namely the optimal protein labeling amount, and detecting the result as shown in figure 9.
According to the experimental result shown in FIG. 9, the marker amount of the selected anti-canine distemper virus H protein nano antibody is finally determined to be 10 mug/mL.
Example 4: and (3) sensitivity test: canine distemper virus antibody based on nano antibody and prepared by 3 batches of methodsThe original test paper strip is used for detecting and diluting canine distemper virus cell culture solution samples with different diluted concentrations, and the diluted concentrations are as follows in sequence: 400TCID50、200TCID50、100TCID50、50TCID50、25TCID50、12.5TCID50(ii) a The results of the measurements are shown in the following table.
The following table: results of sensitivity measurement:
in the above table, "+" indicates that the canine distemper virus is positive, and the results show that red bands can appear on both the detection line and the quality control line when the concentration of the virus diluent is greater than or equal to 50TCID50, namely, the red bands are positive.
Example 5: and (3) specificity test: the test paper strip for detecting canine distemper virus antigen based on the nano antibody is used for detecting canine parvovirus, canine coronavirus, canine parainfluenza virus, peste des petits ruminants virus, pseudorabies virus and newcastle disease virus in 3 batches, and the detection results are shown in the following table.
The following table: and (3) specific detection results:
in the above table, "-" indicates negative, and it can be seen from the above table that the test strip for detecting canine distemper virus antigen based on the nano-antibody is negative when used for detecting canine parvovirus, canine coronavirus, canine parainfluenza virus, peste des petits ruminants virus, pseudorabies virus and newcastle disease virus.
Therefore, the test strip for detecting the canine distemper virus antigen based on the nano antibody can not be used for detecting canine parvovirus, canine coronavirus, canine parainfluenza virus, peste des petits ruminants virus, pseudorabies virus and newcastle disease virus.
Example 6: and (3) repeatability test: in-batch repeat testing: the same batch of test paper for detecting the canine distemper virus antigen based on the nano antibody is randomly extracted for 5 boxes, 30 known canine distemper virus positive samples and 30 known canine distemper virus negative samples are detected, each sample is repeatedly detected for 4 times, and the detection results are shown in the following tables 1 to 3.
Table 1:
table 2:
table 3:
in tables 1 to 3, "+" indicates positive canine distemper virus and "-" indicates negative canine distemper virus.
As can be seen from tables 1 to 3, 30 known positive samples are all positive, and 30 known negative samples are all negative, so that the test strip for detecting the canine distemper virus antigen based on the nano-antibody has high detection precision and accurate detection data.
Batch-to-batch repeat test: 3 batches of the test strip for detecting the canine distemper virus antigen based on the nano antibody are adopted, 5 boxes are randomly extracted from each batch, 30 known canine distemper virus positive samples and 30 known canine distemper virus negative samples are detected, each sample is repeatedly detected for 4 times, and the experimental results are shown in the following tables 4-7.
Table 4:
table 5:
table 6:
table 7:
table 4-table 7: "+" indicates canine distemper virus positive, and "-" indicates canine distemper virus negative.
As can be seen from tables 4 to 7, 30 known positive samples are all positive, and 30 known negative samples are all negative, so that the test strip for detecting the canine distemper virus antigen based on the nano-antibody has high detection precision and accurate detection data.
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.
Sequence listing
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Claims (9)
1. Canine distemper virus antigen detection test paper strip based on nanometer antibody, including shell (8) and the supporting sample diluent that uses, be equipped with the test paper strip in shell (8), the test paper strip includes PVC bottom plate (2), pastes on PVC bottom plate (2) and has sample pad (1), gold mark pad (3), envelope membrane (6) and pad (7) that absorb water, its characterized in that: the gold label pad (3) is a glass cellulose membrane coated with a coupling marker of the purified anti-canine distemper virus H protein nano antibody and colloidal gold; the coating film (6) is a nitrocellulose film, and a detection line (4) coated with a purified anti-canine distemper virus H protein nano antibody and a quality control line (5) coated with a goat anti-mouse IgG antibody are coated on the coating film (6);
the amino acid sequence of the canine distemper virus H protein nano antibody is shown as SEQ ID No. 1; the nucleotide sequence of the DNA molecule of the canine distemper virus H protein nano antibody is shown in SEQ ID No. 2.
2. The test strip for detecting canine distemper virus antigen based on nano-antibody according to claim 1, characterized in that: the sample diluent comprises a base liquid, wherein NaN with the mass percent of 0.05 percent is added into the base liquid30.05 percent of PEG20000, and 0.5 percent of Tween-20 by volume percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.01mol/L, and the pH value of the PBS solution is 7.2.
3. The method for preparing the test strip for detecting the canine distemper virus antigen based on the nano-antibody according to any one of claims 1-2, which is characterized by comprising the following steps: the method comprises the following steps:
s1, preparing a colloidal gold solution: heating a 0.01% chloroauric acid solution to boiling, quickly adding 1.5mL trisodium citrate with the concentration of 1% and continuously heating, wherein the solution is changed from light yellow to blue black and finally to wine red, the solution is continuously heated for 5 minutes after the color is stable, cooling is carried out at room temperature to obtain a colloidal gold solution, and the colloidal gold solution is stored at 4 ℃ for later use, and the diameter of the colloidal gold particles is 25 nm;
s2, protein labeling: with 1% strength K2CO3Adjusting the pH value of the colloidal gold solution to 7.2 by using the solution; then adding 10 mu g of anti-canine distemper virus H protein nano antibody into each 1mL of colloidal gold solution, and oscillating for 30 minutes at room temperature, wherein the oscillation speed is 130 r/min; then adding 20 microliter of bovine serum albumin with the concentration of 10 percent for sealing, and standing for 30 minutes; centrifuging for 40 minutes after standing is finished, wherein the centrifugation speed is 12000r/min, discarding the supernatant, adding a complex solution according to the proportion of the volume of the colloidal gold solution of 1/10 to dissolve and precipitate, and obtaining a labeled anti-canine distemper virus H protein nano antibody marker;
s3, preparing a gold-labeled pad: and selecting a glass cellulose membrane as a gold-labeled pad (3), adding 1mL of anti-canine distemper virus H protein nano antibody marker into each glass cellulose strip, and drying to obtain the gold-labeled pad (3).
4. The method for preparing the test strip for detecting the canine distemper virus antigen based on the nano-antibody according to claim 3, which is characterized in that: the preparation method also comprises the following steps:
s4, preparation of coating film: selecting a nitrocellulose membrane as a coating membrane (6), diluting the canine distemper virus H protein resistant nano antibody with a membrane scribing solution to a concentration of 1mg/mL, and scribing for a detection line (4); diluting the goat anti-mouse IgG antibody with the membrane scribing solution to the concentration of 1mg/mL for scribing a quality control line (5); scribing a detection line (4) and a quality control line (5) on a nitrocellulose membrane by adopting a three-dimensional scribing and gold spraying instrument at the concentration of 1 mu L/cm, and then drying to obtain a coating film (6);
s5, sample pad treatment: a glass cellulose film was selected as the sample pad (1), and the sample pad (1) was immersed in the sample pad treatment solution for 30 minutes and then dried overnight at 37 ℃.
5. The method for preparing the test strip for detecting the canine distemper virus antigen based on the nano-antibody according to claim 4, wherein the method comprises the following steps: the preparation method also comprises the following steps: s6 preparation of sample diluentPreparing: the formula of the sample diluent comprises base liquid, wherein NaN with the mass percent of 0.05% is added into the base liquid30.05 percent of PEG20000, and 0.5 percent of Tween-20 by volume percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.01mol/L, and the pH value of the PBS solution is 7.2.
6. The method for preparing the test strip for detecting the canine distemper virus antigen based on the nano-antibody according to claim 5, which is characterized in that: the preparation method also comprises the following steps: s7, assembling the detection test strip: sequentially sticking the coating film (6), the gold label 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 the canine distemper virus antigen detection test strip based on the nano antibody;
s8, packaging: and (3) putting the assembled canine distemper virus antigen detection test strip based on the nano antibody 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 strip for detecting canine distemper virus antigen based on the nano-antibody according to claim 6, which is characterized in that: the compound solution comprises the following components: PBS buffer solution with pH value of 8.0 and concentration of 0.01mol/L, wherein sucrose with mass percent of 5 percent and NaN with mass percent of 0.05 percent are added into the PBS buffer solution3And 2% PEG20000, and 0.2% by volume of Tween-20 in PBS buffer.
8. The method for preparing the test strip for detecting the canine distemper virus antigen based on the nano-antibody according to claim 7, which is characterized in that: the formula of the scribing liquid comprises the following components: PBS buffer solution with pH value of 7.2 and concentration of 0.01mol/L, wherein NaN with mass percent of 0.05 percent is added into the PBS buffer solution3Tween-20 was also added to the PBS buffer in an amount of 0.1% by volume.
9. Nanobody-based canine distemper virus antigen of claim 8The preparation method of the test strip is characterized in that: the formula of the sample pad treatment solution comprises: the pH value is 7.2, the concentration is 0.01mol/L PBS solution, the PBS solution is added with the following components by mass percent: 1% BSA, 1% sucrose and 0.05% NaN3Tween-20 with a volume percentage of 0.5% was also added to the PBS solution.
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| CN115963278A (en) * | 2023-03-15 | 2023-04-14 | 山东康华生物医疗科技股份有限公司 | Detection kit for dog virus antibody triple-joint detection and preparation method thereof |
| 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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