CN112782400A - Preparation method of reagent card for rapidly detecting pet virus colloidal gold - Google Patents
Preparation method of reagent card for rapidly detecting pet virus colloidal gold Download PDFInfo
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Abstract
The invention relates to a preparation method of a reagent card for rapidly detecting pet virus colloidal gold, which comprises the following steps: (1) preparing colloidal gold-labeled streptavidin; (2) preparing a biotinylated pet virus labeled antibody; (3) preparing a colloidal gold labeled streptomycin-biotinylated pet virus labeled antibody; (4) preparing a colloidal gold marker pad; (5) preparing a pet virus coated plate; (6) preparing a pet virus detection card: and (3) sticking the colloidal gold marker pad, the pet virus coated plate and the sample pad on the detection plate in sequence, cutting the colloidal gold marker pad, the pet virus coated plate and the sample pad into test strips, namely the pet virus detection test strips, and then putting the test strips into a card shell to obtain the pet virus detection reagent card. Compared with the prior art, the detection reagent card prepared by the invention not only retains the characteristics of simplicity and rapidness of the colloidal gold technology, but also improves the sensitivity of the colloidal gold, and is particularly suitable for the rapid diagnosis requirement of the basic level.
Description
Technical Field
The invention belongs to the technical field of preparation of detection kits, and relates to a preparation method of a reagent card for rapidly detecting pet virus colloidal gold.
Background
The colloidal gold immunity technology is originated from Faulk and Taylor in the early 70 s and is characterized in that colloidal gold is used as a labeled tracer, is applied to an immunity labeling technology of antigen-antibody reaction and is initially applied to an immune electron microscope technology. He is a relatively stable heterogeneous system centered on the gold core by electrostatic action, which has a characteristic color itself. Can be firmly combined with protein through charge action without influencing the activity of the protein, and the advantage enables the protein to be widely applied to the immunodiagnosis industry. To date, gold labeling has been used primarily in immunohistochemistry. In immunoassay, a gold-labeled conjugate is often matched with a membrane carrier to form a specific assay mode, such as an immunochromatography test and a dot immunodiafiltration test, which are simple, convenient and rapid detection methods widely applied at present.
For example, Canine Parvovirus (CPV) is a member of Parvovirus, a single-stranded small DNA virus. The virus particles are equiaxed and symmetrical icosahedron, are round or hexagonal in appearance and have no envelope, the virus particle size is 21-24 nm, and the virus particles are firstly separated from a sick dog suffering from enteritis in 1977. The sick dog is mainly characterized by vomit, hemorrhagic enteritis and leukopenia, the susceptibility of the puppy is high, the myocarditis of the puppy can be caused, the morbidity is 50-100%, the mortality is 0-50%, and the influence on the canine raising industry and the economic animal breeding industry is great, so a quick and effective detection method is established and the disease is effectively treated. The feline panleukopenia and feline infectious enteritis are acute high-contact infectious diseases of cats. The pathogenic feline panleukosis virus is a virus of parvoviridae and parvovirus. The clinical manifestations are mainly characterized by sudden high fever, intractable vomiting, diarrhea, dehydration, circulatory disturbance and acute reduction of leukocytes. The disease can occur all the year round, especially in winter and spring, and the diseased cat can discharge a large amount of virus from excrement, urine, vomit and various secretions, and the virus can survive for a long time in the environment. Besides the infection of domestic cats, the disease can also infect other felines (such as tigers and leopards), ferrets (minks) and raccoons of the family bear. Cats of all ages can be infected. Under most conditions, cats under 1 year of age are susceptible, the infection rate can reach 70%, the mortality rate is 50-60%, the mortality rate of young cats under 5 months of age can reach 80-90% at most, the morbidity rate is gradually reduced along with the increase of the age, and the cats in group culture can outbreak in whole group or in whole litter.
Currently, serological tests of antibodies, such as hemagglutination test, hemagglutination inhibition test, agar diffusion test and the like, exist in the relatively mature canine parvovirus detection methods, but the methods are often limited in practical application because cross serological reflection can occur among CPV, feline panleukopenia virus and mink enteritis virus. In addition, the ELISA method is relatively widely used, but the ELISA method is relatively complex to operate, requires a professional to perform the operation, requires a microplate reader to interpret the result, and has a long overall reaction time, so that the ELISA method is not suitable for the requirement of rapid diagnosis of the substrate. In the method for detecting the feline distemper virus, the fluorescence quantitative PCR method and the ELISA method are relatively widely applied, but the two methods are relatively complex to operate, require professional personnel to operate, need to be matched with professional instruments and equipment to interpret results, have long integral reaction time and are not suitable for the requirement of rapid diagnosis of a basic layer.
Disclosure of Invention
The invention aims to provide a preparation method of a reagent card for rapidly detecting pet virus colloidal gold, so as to realize rapid and accurate detection of canine parvovirus and/or feline distemper virus.
The purpose of the invention can be realized by the following technical scheme:
a preparation method of a reagent card for rapidly detecting pet virus colloidal gold comprises the following steps:
(1) preparing colloidal gold-labeled streptavidin:
adjusting the pH value of the colloidal gold solution to 9-10, adding a streptavidin solution, carrying out oscillation reaction, adding BSA, continuing oscillation, carrying out centrifugal separation to remove supernatant to obtain colloidal gold-labeled streptavidin, and re-dissolving the precipitate by using a gold-labeled diluent for later use;
(2) preparation of biotinylated pet virus labeled antibody:
diluting pet virus labeled antibody (5E2) with buffer solution, dialyzing to obtain pet virus labeled antibody solution, adding biotin solution, reacting at room temperature, and adding NH4Continuously stirring the Cl solution at room temperature for reaction, and then dialyzing and purifying by a column to obtain a biotinylated pet virus labeled antibody;
(3) preparing a colloidal gold labeled streptomycin-biotinylated pet virus labeled antibody:
mixing the colloidal gold-labeled streptavidin prepared in the step (1) and the biotinylated pet virus labeled antibody prepared in the step (2), carrying out oscillation reaction, centrifuging to remove supernatant to obtain a colloidal gold-labeled streptomycin-biotinylated pet virus labeled antibody, and then re-dissolving the precipitate with a gold-labeled diluent for later use;
(4) preparing a colloidal gold marker pad:
spraying the redissolved colloidal gold labeled streptomycin-biotinylated pet virus labeled antibody on a blank marker pad, and drying overnight to obtain the colloidal gold marker pad;
(5) preparation of a coated plate:
diluting goat anti-mouse IgG and pet virus coated antibody (5B10) with buffer solution, coating on blank coated plate stuck with nitrocellulose membrane and cotton pulp plate, and drying in oven overnight after coating;
(6) preparation of the detection card:
and (2) sticking the colloidal gold marker pad, the coated plate and the sample pad on a detection plate according to the sequence that the marker pad presses the coated plate for 1-2mm (namely, the two are overlapped in an area with the length of 1-2 mm) and the sample pad presses the marker pad for 1-2mm, cutting the sample pad into test strips, namely the pet virus detection test strips, and then putting the test strips into a card shell to obtain the pet virus detection reagent card.
Further, the pet virus is canine parvovirus or feline distemper virus, correspondingly, the pet virus labeled antibody is a canine parvomarker antibody or an feline distemper marker antibody, the biotinylated pet virus labeled antibody is a biotinylated canine parvomarker antibody or a biotinylated feline distemper marker antibody, and the pet virus coated antibody is a murine monoclonal antibody canine parvocoating antibody or a murine monoclonal antibody feline distemper coating antibody.
Further, in the step (1), the concentration of the colloidal gold solution is one ten thousandth (i.e. 0.01 wt%), the concentration of the streptavidin solution is 10mg/mL, and the addition amount of the streptavidin solution and the colloidal gold solution satisfies the following condition: after the two are mixed, the final concentration of the streptavidin solution is 20-50 mug/mL;
the adding amount of BSA meets the following requirements: the final mass concentration was 1%.
Further, in the step (1), adding a streptavidin solution, and oscillating for 20 min;
further, in step (1), after adding BSA, the shaking was continued for 20 min.
Further, in the step (2), the pet virus labeled antibody is firstly diluted to 1mg/mL by using a buffer solution, the concentration of the biotin solution is 1mg/mL, and NH is added4The concentration of the Cl solution is 1 mol/L;
biotin solution, pet virus labeled antibody solution and NH4The addition ratio of the Cl solution is as follows: adding 120uL biotin solution into each milliliter of pet virus labeled antibody solution, and adding 1uL NH into each 25ug of biotin4Cl。
Further, in the step (2), after the biotin solution is added, the reaction time at room temperature is 2-4 h.
Further, in the step (2), NH is added4After the Cl solution, the reaction was stirred at room temperature for 10 min.
Further, in the step (3), the molar ratio of the streptavidin to the biotinylated antibody is 1: (5-10).
Further, in the step (3), the reaction time is 15min under shaking and the temperature is room temperature.
Further, the detection plate is a polyethylene plate.
The invention is a detection technology which takes colloidal gold as a tracer and is applied to antigen-antibody reaction by combining a biotin-streptavidin system. Firstly, the surface of colloidal gold particles in a colloidal gold solution is provided with a large amount of negative charges, and the negative charges and streptavidin are combined into a colloidal gold-streptavidin compound through electrostatic adsorption; then, coupling biotin and a labeled antibody to form a biotin-labeled antibody complex; and finally, reacting the colloidal gold-streptavidin complex with a biotinylated antibody complex to finally obtain the colloidal gold-streptavidin-biotin-labeled antibody complex.
The addition amount of various raw materials in the reaction process of the invention is the optimal usage amount after the research and optimization of the production process, and if the addition amount is not within the usage amount of the process condition range defined by the invention, the preparation efficiency of the colloidal gold-streptavidin-biotin-labeled antibody compound is reduced and the stability of the conjugate is deteriorated.
The invention utilizes the multistage amplification effect of streptavidin-avidin and combines the colloidal gold labeling technology. Not only retains the characteristics of simplicity and rapidness of the colloidal gold technology, but also improves the sensitivity of the colloidal gold. Compared with the prior art, the invention has the following advantages:
1. the marking efficiency is improved;
2. the labeled antibody is saved, and the cost is reduced;
3. the sensitivity of the product is improved;
4. the precision of the product is improved.
Drawings
FIG. 1 is a graph showing the effects of the test cards obtained in example 1 and comparative example 1.
FIG. 2 is a graph showing the comparison of the test results of the test cards obtained in example 1 and comparative example 2.
FIG. 3 is a graph showing the effects of the test cards obtained in example 2 and comparative example 3.
Detailed Description
The present invention will be described in detail with reference to specific examples. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.
In the following examples, for example, the murine monoclonal antibody canine parvo-labeled antibody (5E2), the murine monoclonal antibody canine parvo-coated antibody (5B10), the murine monoclonal antibody feline panleukopenia-labeled antibody (7D5), and the murine monoclonal antibody feline pansy-coated antibody (2F7) were commercially available raw materials in the art, and the quality control line (C-line) coated antibody goat anti-mouse IgG was commercially available monoclonal antibody, and purchased from shanghai jejg biotechnology limited. Otherwise, unless otherwise specified, all the conventional commercial raw materials or conventional processing techniques are used in the art.
In addition, the preparation process of the used colloidal gold solution is specifically as follows: dissolving chloroauric acid into 0.01% solution with purified water, and heating 100mL to boil; adding 1mL of trisodium citrate solution with the mass concentration of 1%, continuously heating, and observing that a light yellow chloroauric acid aqueous solution changes into gray after sodium citrate is added, then changes into black, then gradually stabilizes into red, continuously heating and boiling until the color is stable, and continuously heating for 15 minutes; the fired colloidal gold solution was cooled to room temperature and returned to the original volume with purified water. According to different addition amounts of sodium citrate, colloidal gold particles with different particle sizes can be prepared, and the basic rule is that the smaller the consumption amount of trisodium citrate is, the larger the particle size of the colloidal gold particles is. The relationship between the addition amount of trisodium citrate and the particle size of colloidal gold is shown in the following table:
| 0.01% chloroauric acid (mL) | 1% trisodium citrate (mL) | Average particle size (nm) of colloidal gold |
| 250 | 7.5 | ±15 |
| 250 | 3.75 | ±30 |
| 250 | 2.50 | ±50 |
| 250 | 1.90 | ±60 |
Example 1:
a preparation method of a reagent card for rapidly detecting pet virus colloidal gold comprises the following steps:
(1) preparing colloidal gold-labeled streptavidin:
the pH of the colloidal gold solution was adjusted to 9-10, and streptavidin was dissolved to 10 mg/mL. And (3) taking 10mL of colloidal gold solution, adding streptavidin to the solution until the final concentration is 50ug/mL, oscillating the solution for 20 minutes, adding 20% BSA (bovine serum albumin) by mass concentration to enable the final concentration of the BSA to be 1%, and continuously oscillating the solution for 20 minutes. The pellet was then centrifuged at 10000rpm for 10 minutes, the supernatant removed and finally the pellet reconstituted with 1mL of gold-labelled dilution.
(2) Preparation of biotinylated Canine Small labeled antibody (5E2)
A. Diluting canine fine labeled antibody (5E2) to be biotinylated to 1mg/ml with 0.1mol/L sodium bicarbonate buffer (pH8.0) or 0.5mol/L boric acid buffer (pH 8.6), and the biotinylation volume generally used in the laboratory is 1-2.5 ml;
B. the interaction was extensively dialyzed against canine parvomarker antibody with 0.1mol/L sodium bicarbonate buffer (pH8.0) or 0.5mol/L boric acid buffer (pH 8.6);
C. dissolve NHSB (i.e., biotin) 1mg with 1ml DMSO;
D. to 1ml of canine parvo-labeled antibody solution (i.e., containing 1mg of protein) was added 120. mu.l of NHSB solution (i.e., containing 120. mu.g of NHSB);
E. continuously stirring at room temperature, and keeping the temperature for 2-4 hours;
F. 9.6 μ L of 1mol/L NH was added4Cl (1. mu.l per 25. mu.g NHSB), stirred at room temperature for 10 min;
G. dialyzing the PBS thoroughly at 4 ℃ to remove free biotin;
H. loading the sample on a 1ml molecular sieve column, slowly eluting with PBS, collecting 1 ml/tube, and washing off the canine tiny marker antibody between 1ml and 3 ml;
I. finally, sodium azide (final concentration 0.5g/L) and 1.0g/L BSA were added to the sample. Storing the combined product at 4 deg.C in dark, and at-20 deg.C if long-term storage is required.
(3) Preparing a colloidal gold labeled streptomycin-biotinylated dog fine labeled antibody:
mixing the prepared colloidal gold-labeled streptavidin and biotinylated antibody, and enabling the mass ratio of the streptavidin to the biotinylated antibody to be 1: 5. after 15 minutes of reaction with shaking, the supernatant was removed by centrifugation. If the pad is manually coated, gold-labeled diluent is used for re-dissolving streptomycin in the same volume according to the labeling of colloidal gold; if the pad is sprayed by an instrument, gold-labeled diluent is used for redissolving according to one quarter volume of the colloidal gold-labeled streptomycin.
(4) Preparing a colloidal gold marker pad:
spraying the re-dissolved colloidal gold labeled streptomycin-biological dog fine labeled antibody on a blank marker pad, and drying overnight to obtain the product. Manual pad coating: cutting the blank label pad into 5mm × 30cm, and coating the colloidal gold label binder according to 25 uL/cm; spraying a cushion by an instrument: blank marker pads were cut to 8.5cm by 30cm and the instrument set to 4uL/cm for spraying.
(5) Preparation of canine parvo coated plate:
goat anti-mouse IgG was diluted to 0.5mg/mL with the coating solution, and canine parvo-coated antibody (5B10) was diluted to 1 mg/mL. Coating on a blank coated plate stuck with the nitrocellulose membrane and the cotton pulp plate according to the spraying amount of 0.6ul/cm, and after coating is finished, putting the blank coated plate into an oven and drying the blank coated plate overnight to obtain the nitrocellulose filter.
(6) Preparation of sample pad: and (3) pretreating the cut glass cellulose membrane by using the sample pad treatment solution, and after the treatment is finished, putting the glass cellulose membrane into an oven for drying.
(7) Preparation of a dog small detection card:
and (3) sequentially sticking the colloidal gold marker pad, the coated plate after coating and the processed sample pad on a polyethylene plate according to the sequence to form the large card. And longitudinally cutting the assembled large card into test strips with the length of 4mm, namely the test strips for detecting the canine parvovirus. And then, putting the test strip into a card shell to obtain the canine parvovirus detection reagent card.
Comparative example 1:
compared with example 1, most of them are the same, except that the introduction of streptavidin and biotin is omitted, i.e. the labeled antibody is directly connected with the colloidal gold particles, thereby obtaining the colloidal gold-labeled antibody conjugate.
The same canine parvovirus positive sample was simultaneously detected using the reagent cards obtained in example 1 and comparative example 1, and the results are shown in fig. 1, and it can be seen that the color development intensity (i.e., the circled portion in the figure) of the product prepared in example 1 is significantly better than that of the general labeling method.
Therefore, the invention obtains the colloidal gold-streptavidin-biotin-labeled antibody by combining the colloidal gold labeling technology through the multi-stage amplification effect of the streptavidin-biotin (one streptavidin can be combined with four biotinylated labeled antibodies), thereby greatly improving the combination efficiency of the labeled antibody and the colloidal gold, saving the labeled antibody and improving the sensitivity of the product.
Comparative example 2:
the principle of specific reaction of streptavidin and biotin is adopted, so that a signal amplification and detection sensitivity improvement mode is realized, and the labeling can be realized through the following embodiment modes:
(1) preparation of streptavidin-colloidal gold-labeled antibody conjugate pad
Adjusting the pH of the colloidal gold solution to 9-10, taking 10mL of the colloidal gold solution, then adding streptavidin and a canine small labeled antibody (5E2) to enable the final concentration of the streptavidin to be 50ug/mL and the final concentration of the canine small labeled antibody (5E2) to be 10ug/mL, carrying out oscillation reaction for 20 minutes at room temperature, then adding BSA with the mass concentration of 20% to enable the final concentration of the BSA to be 1%, and continuing oscillation and sealing for 20 minutes. The pellet was then centrifuged at 10000rpm for 10 minutes, the supernatant removed and finally the pellet reconstituted with 1mL of gold-labelled dilution. And spraying the re-dissolved streptavidin-colloidal gold-labeled antibody conjugate on a blank label pad, and drying overnight to obtain the streptavidin-colloidal gold-labeled antibody conjugate. Manual pad coating: cutting the blank label pad into 5mm × 30cm, and coating the colloidal gold label binder according to 25 uL/cm; spraying a cushion by an instrument: blank marker pads were cut to 8.5cm by 30cm and the instrument set to 4uL/cm for spraying.
(2) Preparation of colloidal gold-biotin conjugate pad
Adjusting the pH value of the colloidal gold solution to 7 +/-0.5, taking 10mL of the colloidal gold solution, then adding biotin to enable the final concentration to be 50ug/mL, carrying out oscillation reaction for 20 minutes at room temperature, then adding BSA with the mass concentration of 20% to enable the final concentration of the BSA to be 1%, and continuing the oscillation reaction for 20 minutes. The pellet was then centrifuged at 10000rpm for 10 minutes, the supernatant removed and finally the pellet reconstituted with 1mL of gold-labelled dilution. And spraying the redissolved colloidal gold-biotin conjugate on a blank marker pad, and drying overnight to obtain the product. Manual pad coating: cutting the blank label pad into 5mm × 30cm, and coating the colloidal gold label binder according to 25 uL/cm; spraying a cushion by an instrument: blank marker pads were cut to 8.5cm by 30cm and the instrument set to 4uL/cm for spraying.
(3) Preparation of canine parvo coated plate:
goat anti-mouse IgG was diluted to 0.5mg/mL with the coating solution, and canine parvo-coated antibody (5B10) was diluted to 1 mg/mL. Coating on a blank coated plate stuck with the nitrocellulose membrane and the cotton pulp plate according to the spraying amount of 0.6ul/cm, and after coating is finished, putting the blank coated plate into an oven and drying the blank coated plate overnight to obtain the nitrocellulose filter.
(4) Preparation of sample pad: cutting the glass cellulose membrane into 1.8cm by 30cm, pretreating with the sample pad treatment solution, and drying in an oven overnight after the treatment.
(5) Preparation of a dog small detection card:
and adhering the streptavidin-colloidal gold-labeled antibody conjugate pad, the colloidal gold-biotin conjugate pad, the dog fine coated plate and the treated sample pad to a detection plate according to the sequence that the sample pad presses the colloidal gold-biotin conjugate pad for 1-1.5mm, the colloidal gold-biotin conjugate pad presses the streptavidin-colloidal gold-labeled antibody conjugate pad for 1-1.5mm, and the streptavidin-colloidal gold-labeled antibody conjugate pad presses the dog nitrocellulose membrane for 1-1.5mm to form the large card. And longitudinally cutting the assembled large card into test strips with the length of 4mm, namely the test strips for detecting the canine parvovirus. And then, putting the test strip into a card shell to obtain the canine parvovirus detection reagent card.
In comparison with example 1, in example 2, two marker pads, namely a streptavidin-colloidal gold-labeled antibody conjugate pad and a colloidal gold-biotin conjugate pad, were prepared, and after the two marker pads were assembled into a canine parvovirus detection reagent card, another identical canine parvovirus positive sample was examined at the same time, and the results are shown in fig. 2.
As can be seen, the color development intensity (i.e., the circled portion in the figure) of the test card prepared by the labeling method of example 1 is better than that of the labeling method of example 2 as a whole. However, after the same sample was tested 5 times at the same time, the results showed that the detection card prepared by the labeling method of example 1 had a uniform line color development, and the precision of the product was significantly better than the detection card prepared by the labeling method of example 2.
In example 2, after the sample is added, the conjugate in the colloidal gold-biotin conjugate pad and the streptavidin-colloidal gold-labeled antibody conjugate pad is released, streptavidin binds to biotin, and the labeled antibody binds to canine parvovirus in the sample, forming a complex of canine parvovirus-canine parvolabeled antibody-streptavidin-biotin-colloidal gold, which mainly comprises two reactions: 1. combining the canine parvovirus with the canine parvovirus labeled antibody; 2. binding of streptavidin and biotin. Moreover, the combination of streptavidin and biotin is influenced by the release effect of the colloidal gold-biotin conjugate pad, so that the precision of the product is influenced to a certain extent.
Example 3:
a preparation method of a reagent card for rapidly detecting feline pestivirus colloidal gold comprises the following steps:
(1) preparation of biotinylated feline panleukopenia marker antibody (7D5)
A. The feline panleukopenia marker antibody to be biotinylated (7D5) was diluted to 1mg/mL with 0.1mol/L sodium bicarbonate buffer (pH8.0) or 0.5mol/L boric acid buffer (pH 8.6), typically with biotinylation volumes of 1-2.5mL for laboratory use;
B. the interaction was extensively dialyzed against the feline panleukopenia marker antibody with 0.1mol/L sodium bicarbonate buffer (pH8.0) or 0.5mol/L boric acid buffer (pH 8.6);
C. dissolve NHSB (i.e., biotin) 1mg with 1ml DMSO;
D. to 1ml of cat plague labeled antibody solution (i.e., containing 1mg of protein) was added 120. mu.l of NHSB solution (i.e., containing 120. mu.g of NHSB);
E. continuously stirring at room temperature, and keeping the temperature for 2-4 hours;
F. 9.6 μ L of 1mol/L NH was added4Cl (1. mu.l per 25. mu.g NHSB), stirred at room temperature for 10 min;
G. dialyzing the PBS thoroughly at 4 ℃ to remove free biotin;
H. loading the sample on a 1ml molecular sieve column, slowly eluting with PBS, collecting 1 ml/tube, and washing the feline panleukopenia marker antibody between 1 and 3 ml;
I. finally, sodium azide (final concentration 0.5g/L) and 1.0g/L BSA were added to the sample. The binding product is stored at 4 ℃ in the dark, if long-term storage is needed, and stored at-20 ℃.
(2) Preparing colloidal gold-labeled streptavidin:
the pH of the colloidal gold solution was adjusted to 9-10, and streptavidin was dissolved to 10 mg/mL. And (3) taking 10mL of colloidal gold solution, adding streptavidin to the solution until the final concentration is 50ug/mL, oscillating the solution for 20 minutes, adding 20% BSA (bovine serum albumin) by mass concentration to enable the final concentration of the BSA to be 1%, and continuously oscillating the solution for 20 minutes. The pellet was then centrifuged at 10000rpm for 10 minutes, the supernatant removed and finally the pellet reconstituted with 1mL of gold-labelled dilution.
(3) Preparing a colloidal gold labeled streptomycin-biotinylated feline panpest labeled antibody:
mixing the prepared colloidal gold-labeled streptavidin and biotinylated antibody, and enabling the mass ratio of the streptavidin to the biotinylated antibody to be 1: 5. after 15 minutes of reaction with shaking, the supernatant was removed by centrifugation. If the pad is manually coated, gold-labeled diluent is used for re-dissolving streptomycin in the same volume according to the labeling of colloidal gold; if the pad is sprayed by an instrument, gold-labeled diluent is used for redissolving according to one quarter volume of the colloidal gold-labeled streptomycin.
(4) Preparing a colloidal gold marker pad:
spraying the re-dissolved colloidal gold labeled streptomycin-biological feline panleukosis labeled antibody on a blank marker pad, and drying overnight to obtain the product. Manual pad coating: cutting the blank label pad into 5mm × 30cm, and coating the colloidal gold label binder according to 25 uL/cm; spraying a cushion by an instrument: blank marker pads were cut to 5cm by 30cm and the instrument set to 2.8uL/cm for spraying.
(5) Preparation of the cat plague coated plate:
goat anti-mouse IgG was diluted to 0.5mg/mL with coating solution and feline panleukopenia coated antibody (2F7) was diluted to 1 mg/mL. Coating on a blank coated plate stuck with the nitrocellulose membrane and the cotton pulp plate according to the spraying amount of 0.6ul/cm, and after coating is finished, putting the blank coated plate into an oven and drying the blank coated plate overnight to obtain the nitrocellulose filter.
(6) Preparation of sample pad: and (3) pretreating the cut glass cellulose membrane by using the sample pad treatment solution, and after the treatment is finished, putting the glass cellulose membrane into an oven for drying.
(7) Preparation of the feline panleukopenia detection card:
and (3) sequentially sticking the colloidal gold marker pad, the coated plate after coating and the processed sample pad on a polyethylene plate according to the sequence to form the large card. And longitudinally cutting the assembled large card into test strips with the length of 4mm, namely the test strips for detecting the feline pestivirus. And then, putting the test strip into a card shell to obtain the reagent card for detecting the feline pestivirus.
Comparative example 3:
compared with example 2, most of them are the same, except that the introduction of streptavidin and biotin is omitted, namely, the labeled antibody is directly connected with the colloidal gold particles, thereby obtaining the colloidal gold-labeled antibody conjugate.
The reagent card obtained in example 2 and the reagent card obtained in comparative example 3 are used for detecting the same positive sample of feline pestivirus, and the result is shown in fig. 1, and it can be seen that the product prepared in example 2 has a color development intensity (i.e. the circle part in the figure) which is obviously better than that of the general labeling method.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A preparation method of a reagent card for rapidly detecting pet virus colloidal gold is characterized by comprising the following steps:
(1) preparing colloidal gold-labeled streptavidin:
adjusting the pH value of the colloidal gold solution to 9-10, adding a streptavidin solution, carrying out oscillation reaction, adding BSA, continuing oscillation, carrying out centrifugal separation to remove supernatant to obtain colloidal gold-labeled streptavidin, and re-dissolving the precipitate by using a gold-labeled diluent for later use;
(2) preparation of biotinylated pet virus labeled antibody:
diluting the pet virus labeled antibody with buffer solution, dialyzing to obtain pet virus labeled antibody solution, adding biotin solution, reacting at room temperature, and adding NH4Continuously stirring the Cl solution at room temperature for reaction, and then dialyzing and purifying by a column to obtain a biotinylated pet virus labeled antibody;
(3) preparing a colloidal gold labeled streptomycin-biotinylated pet virus labeled antibody:
mixing the colloidal gold-labeled streptavidin prepared in the step (1) and the biotinylated pet virus labeled antibody prepared in the step (2), carrying out oscillation reaction, centrifuging to remove supernatant to obtain a colloidal gold-labeled streptomycin-biotinylated pet virus labeled antibody, and then re-dissolving the precipitate with a gold-labeled diluent for later use;
(4) preparing a colloidal gold marker pad:
spraying the redissolved colloidal gold labeled streptomycin-biotinylated pet virus labeled antibody on a blank marker pad, and drying overnight to obtain the colloidal gold marker pad;
(5) preparation of a coated plate:
diluting goat anti-mouse IgG and pet virus coated antibody with buffer solution, coating on a blank coated plate stuck with a nitrocellulose membrane and a cotton pulp plate, and after coating is finished, putting the blank coated plate into an oven to dry overnight;
(6) preparation of the detection card:
and (3) sticking the colloidal gold marker pad, the coated plate and the sample pad on the detection plate in sequence, cutting the colloidal gold marker pad, the coated plate and the sample pad into test strips, namely the pet virus detection test strips, and then putting the test strips into a card shell to obtain the pet virus detection reagent card.
2. The method for preparing the reagent card for rapidly detecting the pet virus colloidal gold according to claim 1, wherein the pet virus is canine parvovirus or feline distemper virus, correspondingly, the pet virus labeled antibody is a canine parvomarker antibody or a feline distemper marker antibody, the biotinylated pet virus labeled antibody is a biotinylated canine parvomarker antibody or a biotinylated feline distemper marker antibody, and the pet virus coated antibody is a murine monoclonal antibody canine parvocoated antibody or a murine monoclonal antibody feline distemper coated antibody.
3. The method for preparing the reagent card for rapidly detecting the pet virus colloidal gold according to claim 1, wherein in the step (1), the concentration of the colloidal gold solution is 0.01 wt%, the concentration of the streptavidin solution is 10mg/mL, and the addition amounts of the streptavidin solution and the colloidal gold solution satisfy the following requirements: after the two are mixed, the final concentration of the streptavidin solution is 20-50 mug/mL;
the adding amount of BSA meets the following requirements: the final mass concentration was 1%.
4. The method for preparing the reagent card for the rapid colloidal gold detection of pet viruses as claimed in claim 1, wherein in the step (1), a streptavidin solution is added, and the reaction time is 20min with shaking.
5. The method for preparing a reagent card for colloidal gold rapid assay of pet virus according to claim 1, wherein in the step (1), after adding BSA, the shaking is continued for 20 min.
6. The method for preparing pet virus colloidal gold rapid detection reagent card according to claim 1, wherein in the step (2), the pet virus labeled antibody is firstly diluted to 1mg/mL by buffer solution, the concentration of biotin solution is 1mg/mL, and NH is added4The concentration of the Cl solution is 1 mol/L;
biotin solution, pet virus labeled antibody solution and NH4The addition ratio of the Cl solution is as follows: adding 120 mu L biotin solution into each ml of pet virus labeled antibody solution, and adding 1 mu L NH into each 25 mu g of biotin4Cl。
7. The method for preparing the reagent card for the rapid colloidal gold assay of pet viruses as claimed in claim 1, wherein in the step (2), the reaction time at room temperature is 2-4h after the biotin solution is added;
addition of NH4After the Cl solution, the reaction was stirred at room temperature for 10 min.
8. The method for preparing the reagent card for rapidly detecting the pet virus colloidal gold according to claim 1, wherein in the step (3), the molar ratio of the streptavidin to the biotinylated antibody is 1: (5-10).
9. The method for preparing a reagent card for colloidal gold rapid assay of pet virus according to claim 1, wherein in the step (3), the shaking reaction time is 15min and the temperature is room temperature.
10. The method for preparing a reagent card for colloidal gold rapid assay of pet virus according to claim 1, wherein the assay plate is a polyethylene plate.
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