CN113092753A

CN113092753A - Colloidal gold duplex detection test strip for antibody of African swine fever virus and preparation method thereof

Info

Publication number: CN113092753A
Application number: CN202110364113.XA
Authority: CN
Inventors: 郑海学; �田宏; 万颖; 石正旺; 罗俊聪; 彭高闯; 王丽娟; 茹毅; 麻园; 周改静; 宋锐
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2021-04-03
Filing date: 2021-04-03
Publication date: 2021-07-09

Abstract

The invention discloses a colloidal gold duplex test strip for monitoring African swine fever virus antibodies in the whole process and a preparation method thereof, wherein the test strip comprises a PVC bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the PVC bottom plate from left to right, the nitrocellulose membrane is provided with a detection line and a quality control line, the combination pad is coated with ASFV p30 protein and ASFV p72 protein which are marked by colloidal gold, the detection line is coated with ASFV p72 and ASFV p30 protein, and the quality control line C is coated with an anti-ASFV p30 protein monoclonal antibody and/or an anti-ASFV p72 protein monoclonal antibody; the invention realizes the whole-process monitoring of the African swine fever virus antibody by optimizing the preparation of the binding pad, can more accurately learn the infection period of an organism compared with a single p30 or p72 test strip, is expected to become a technical means for on-site or laboratory detection of the African swine fever antibody by combining good specificity and higher sensitivity, and provides a new thought for accurate and rapid diagnosis of the African swine fever.

Description

Colloidal gold duplex detection test strip for antibody of African swine fever virus and preparation method thereof

Technical Field

The invention relates to the technical field of rapid immunochromatographic assay, in particular to a colloidal gold duplex test strip for detecting an antibody of African swine fever virus and a preparation method thereof.

Background

African Swine Fever (ASF) is an acute, virulent, highly contagious disease caused by African Swine Fever Virus (ASFV). The ASF is discovered in Kenya in the earliest 1920 years, and the African swine fever case appears for the first time in 8 months in 2018 in China and is rapidly spread to the whole country at present. ASFV particles have an icosahedral symmetrical structure and a diameter of about 200nm, and are large enveloped double-stranded DNA viruses that are not only the only members of Asfarviridae of the family Asfivirus, but also the only known DNA arboviruses. The genome length is about 170-193 kb, and codes more than 160 Open Reading Frames (ORFs). More than 50 proteins are packaged into virus particles and play a role in virus infection, such as pp220, pp62, p72, p54, p30, CD2v, p10, p12, p14.5 and p17, wherein the p30 protein is encoded by CP204L gene, is one of early virus proteins, has the relative molecular mass of 30kDa and is one of antigen structural proteins which are most involved in the process of infecting organisms by ASFV and have the strongest immunogenicity; the p72 protein is encoded by B646L (Vp72) gene, generally expressed in the late stage of virus infection, is the main structural protein of ASFV, has 73.2kDa of relative molecular mass, and has high antigen activity and immunogenicity because it is the main component of virus icosahedron. ASFV is generally susceptible to domestic pigs and wild pigs, and clinically it is usually manifested as acute, subacute and chronic infection according to the virulence of the strain. After acute infection, the symptoms of high fever, skin cyanosis, severe bleeding of lymph nodes and the like are usually presented, and the death rate is up to 100 percent; compared to the former, subacute infection is generally partially lethal and partially resistant; while chronic infection is not lethal, it develops recessive or persistent infection over a period of time. Because the virus has extremely high transmission speed, no effective therapeutic drug and effective vaccine are available at present, and the most effective measure for controlling the epidemic and transmission of the virus is killing when the epidemic situation occurs, the virus causes great loss to the pig industry all over the world. The world animal health Organization (OIE) ranks the animal epidemic disease as a legal report, and China ranks the animal epidemic disease as a type of animal epidemic disease for key prevention.

In the absence of current ASFV vaccination, a positive serological antibody test indicates that an infection is or has occurred. In epidemic areas, the antibody of the surviving animals can last for months or years after subacute infection, so the antibody detection can be applied to large-scale screening of animals with chronic or recessive infection. The ASF serology antibody detection method commonly used in the laboratory at present mainly comprises enzyme-linked immunosorbent assay (ELISA), fluorescent antibody assay (FAT), colloidal gold immunochromatography and the like. Although enzyme-linked immunosorbent assay (ELISA) is a serological diagnosis method recommended by OIE, which can effectively detect the ASFV specific antibody in the living pig body after ASFV infection, the method is time-consuming and can be completed by professional technicians and specific instruments, and the requirement of rapid, cheap and efficient monitoring of clinical antibody level is difficult to meet.

The colloidal gold immunochromatographic assay detection technology is a novel diagnosis technology which takes colloidal gold as a tracer marker and combines antigen-antibody immunoreaction, has the advantages of simple and rapid operation, easy judgment, no need of any instrument, low cost and the like, and is suitable for field detection. Because the p30 protein and the p72 protein play important roles in the early infection stage and the late infection stage respectively, the protein has important application values in the aspects of immunological diagnosis and novel vaccine development. However, the existing colloidal gold test paper strips of p30 protein and/or p72 protein have the following problems: the existing African swine fever colloidal gold test strip can only detect p30 protein and/p 72 protein independently, and can not realize the whole-process monitoring of the African monkey swine fever antibody; even if the test strip for detecting the p30 protein and/or the p72 protein is prepared by a conventional method, the color development difference of the detection line and the quality control line of the positive sample and the negative sample is not obvious because the conventional gold-labeled protein protective agent and the gold-labeled protein confining liquid are adopted in the preparation process of the bonding pad, so that the result judgment is influenced; and thirdly, the conventional sample pad sealing liquid and sample diluent are adopted, so that the color development difference of the detection line and the quality control line of the positive sample and the negative sample is not obvious, and the result judgment is influenced.

Aiming at the problems, the invention provides the colloidal gold duplex immunochromatographic test strip capable of quickly detecting the antibody level after ASF infection, so that the ASF epidemic situation is effectively monitored to control the spread and prevalence of the ASF epidemic situation, and a technical support is provided for the prevention and control of African swine fever.

Disclosure of Invention

Aiming at the technical problems, the invention aims to provide a colloidal gold duplex test strip for monitoring the African swine fever virus antibody in the whole process and a preparation method thereof. The method specifically comprises the following steps:

the invention provides a colloidal gold duplex test strip for monitoring African swine fever virus antibodies in the whole process, which comprises a PVC (polyvinyl chloride) base plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped on the PVC base plate; the nitrocellulose membrane is provided with a detection line T and a quality control line C, the detection line T is arranged at one end close to the sample pad, and the quality control line C is arranged at one end close to the water absorption pad; the binding pad is coated with gold-labeled protein, and the gold-labeled protein comprises colloidal gold labeled ASFV p30 protein and colloidal gold labeled ASFV p72 protein; the detection line T comprises a detection line T1 coated with ASFV p72 protein and a detection line T2 coated with ASFV p30 protein; the quality control line C is coated with an anti-ASFV p30 protein monoclonal antibody and/or an anti-ASFV p72 protein monoclonal antibody.

Preferably, the preparation method of the bonding pad comprises the following steps:

adjusting the pH value of the colloidal gold solution to 8, adding ASFV p30 protein into the colloidal gold solution, reacting at 37 ℃, adding gold-labeled protein confining liquid for reacting, reacting at 37 ℃, and centrifuging; resuspending the precipitate with a gold-labeled protein protective agent to obtain an ASFV p30 gold-labeled protein solution;

adjusting the pH value of the colloidal gold solution to 7, adding ASFV p72 protein into the colloidal gold solution, reacting at 37 ℃, adding gold-labeled protein confining liquid for reacting, reacting at 37 ℃, and centrifuging; resuspending the precipitate with a gold-labeled protein protective agent to obtain an ASFV p72 gold-labeled protein solution;

spraying the prepared gold-labeled protein on a gold-labeled bonding pad to obtain a bonding pad;

the gold-labeled protein blocking solution is an ultrapure water solution containing 10% m/v BSA and 3% m/v casein in purity;

the gold-labeled protein protective agent is a PBS solution containing 3% m/v of sucrose, 1% m/v of PVP and 0.5% v/v of Tween-20.

Preferably, the gold-labeled protein solution of ASFV p30 consists of: each 1ml of the colloidal gold solution contains 60 mu g of ASFV p30 protein; the ASFV p72 gold-labeled protein solution comprises: 10. mu.g of ASFV p72 protein was added to 1ml of the colloidal gold solution.

Preferably, the detection line T1 is coated with 0.8mg/ml ASFV p72 protein; the detection line T2 is coated with 0.3mg/ml ASFV p30 protein.

Preferably, the sample pad is soaked in the sample pad sealing solution; the sample pad blocking solution is PBS solution containing 1% m/v PEG20000, 1% m/v PVP-40, 0.5% v/v Tween-20, 2% m/v BSA.

Preferably, the sequence number of the gene of the ASFV p30 protein is NC 044955.1; the gene sequence number of the ASFV p72 protein is NC 001659.2.

In a second aspect, the invention provides a method for preparing the colloidal gold duplex test strip for monitoring the african swine fever virus antibody in the first aspect, wherein the method comprises the following steps:

preparing a binding pad coated with gold-labeled protein;

preparing a nitrocellulose membrane containing a detection line and a quality control line;

and sequentially overlapping the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad on the PVC base plate to obtain the African swine fever virus antibody colloidal gold duplex detection test strip.

In a third aspect, the invention provides an African swine fever virus antibody detection kit, which comprises the colloidal gold duplex detection test strip for monitoring the African swine fever virus antibody in the first aspect in the whole process.

Preferably, the kit further comprises a sample diluent which is a PBS solution containing 0.02M Tris-HCl, 0.85% M/v NaCl, 1% v/v Tween-20.

Preferably, the kit further comprises a sample pad sealing solution, a gold-labeled protein protective agent and a gold-labeled protein sealing solution; the sample pad blocking solution is PBS solution containing 1% m/v PEG20000, 1% m/v PVP-40, 0.5% v/v Tween-20, 2% m/v BSA; the gold-labeled protein protective agent is a PBS solution containing 3% m/v of sucrose, 1% m/v of PVP-40 and 0.5% v/v of Tween-20; the gold-labeled protein blocking solution is an ultra-pure water solution containing 10% m/v BSA and 3% m/v casein.

The invention has the beneficial effects that: the ASFV p30 and p72 duplex test strip provided by the invention can accurately know the infection period of an organism, and has the advantages of good specificity, good sensitivity, high accuracy and the like; compared with the prior ELISA detection method, the method has high accuracy and simplifies the detection steps and time; the method is expected to become a field or laboratory detection application technology of the African swine fever antibody, and provides a new idea for rapid diagnosis of African swine fever.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of p30 protein and p72 protein, wherein M is Marker; 1 is p30 protein; 2 is p72 protein;

FIG. 2 is a diagram showing the results of pH selection of optimum protein markers, wherein A is ASFV p30 protein, and B is ASFV p72 protein;

FIG. 3 is a graph showing the results of selection of optimum protein marker amounts, wherein A is ASFV p30 protein, and B is ASFV p72 protein;

fig. 4 is a schematic structural diagram of the test strip, wherein 1 is a water absorption pad, 2 is a quality control line C, 3 is a detection line T1, 4 is a detection line T2, 5 is a combination pad, 6 is a sample pad, 7 is a PVC base plate, and 8 is a nitrocellulose membrane (NC membrane);

FIG. 5 is a schematic diagram illustrating test strip result determination;

FIG. 6 shows the results of the colloidal gold test strips for the two antibodies p30 and p 72;

FIG. 7 shows the ELISA growth-eliminating results of two antibodies, p30 and p 72.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The attenuated strain of ASFV Δ 181/UK described in the following examples was derived from African swine fever regional reference laboratory (Lanzhou). Example 1 preparation of p30 protein and p72 protein

Preparation of p30 recombinant protein

Transforming the recombinant positive plasmid pET-32a-p30 into competent cells BL21(DE3), and culturing at 37 ℃ overnight; picking single colony, inoculating the single colony in LB culture medium containing ampicillin (100 mug/ml), and culturing at 37 ℃ for 12h to serve as a seed; inoculating the cultured seeds at a ratio of 1:100 into LB medium containing ampicillin (100. mu.g/ml), and culturing at 37 deg.C to OD₆₀₀When the nm is about 0.65, adding IPTG (isopropyl thiogalactoside) until the final concentration is 0.5mmol/L, and continuously culturing at 37 ℃ for 6-8 h; centrifuging to collect bacteria, resuspending the bacteria with an appropriate amount of buffer solution, ultrasonically crushing, centrifuging at 12000r/min for 10min, discarding the supernatant, washing with PBS for 4-6 times, dissolving the precipitate with an appropriate amount of 8mol of urea, gradually dialyzing into 8mol of urea, and purifying the p30 protein by using a nickel column, wherein the purification result is shown in figure 1. The result of the identification shows that,the p30 protein obtained has high purity.

Preparation of p72 truncated protein

Transforming the recombinant positive plasmid pET-28a-p72 into competent cells BL21(DE3), and culturing at 37 ℃ overnight; picking single colony to inoculate LB culture medium containing kanamycin (100 mug/ml), culturing 12h at 37 ℃ as seed; the cultured seeds were inoculated at 1:100 into LB medium containing kanamycin (100. mu.g/ml) and cultured at 37 ℃ to OD₆₀₀When the nm is about 0.65, adding IPTG (isopropyl thiogalactoside) until the final concentration is 0.5mmol/L, and continuously culturing at 37 ℃ for 6-8 h; centrifuging to collect bacteria, re-suspending the bacteria with an appropriate amount of buffer solution, ultrasonically crushing, centrifuging at 12000r/min for 10min, discarding the supernatant, washing with PBS for 4-6 times, dissolving the precipitate with an appropriate amount of 8mol of urea, gradually dialyzing into 8mol of urea, purifying p72 truncated protein by using a nickel column, wherein the purification result is shown in figure 2. The identification result shows that the p72 protein obtained has high purity.

Example 2 preparation of African swine fever virus antibody colloidal gold duplex test strip

1. Preparation of the conjugate pad

1.1 preparation of colloidal gold solution

Adding 1ml of 1% chloroauric acid into 100ml of ultrapure water, fully and uniformly mixing, heating until bubbles are obviously uniformly appeared and rise upwards, immediately adding 2.0ml of 1% trisodium citrate solution at one time, heating until the color is not changed any more, continuously heating for 2-3 min, carefully taking down a conical flask, cooling to room temperature, adding ultrapure water, and fixing the volume to 100 ml.

1.2 preparation of gold-labeled protein

(1) Optimum labeled pH value

0.1mg/ml K was used₂CO₃The pH of gold solution is respectively adjusted to 4-9, a proper amount of protein is added into the gold solution with different pH values, the gold solution is kept stand at room temperature for 2h, the gold solution is centrifuged at 12000r/min at 4 ℃ for 30min, a supernatant coated ELISA plate is collected, the reactivity of the gold solution with ASF positive serum is detected by adopting indirect ELISA, the pH is used as a horizontal coordinate, and OD is used as an abscissa₄₅₀The nm value was plotted as the ordinate on a scattergram to determine the optimum pH.

The results are shown in FIG. 2, and indicate that the optimum pH is 8 when p30 protein is labeled; the optimum pH for labeling p72 protein was 7.

(2) Optimum amount of marker

P30 protein was diluted to 0.00625mg/ml with PBS at 0.1mg/ml fold ratio; the p72 protein was diluted from 0.25mg/ml fold to 0.001953125mg/ml with PBS. And (3) sequentially adding 0.1ml of the proteins with different concentrations into the gold solution with the well-adjusted pH value, fully and uniformly mixing, standing at room temperature for 5-10 min, adding 0.1ml of 10% NaCl solution, observing color change after 2h, recording the minimum protein labeling amount, and adding 10-20% of the minimum protein labeling amount to obtain the optimal protein labeling amount.

The results are shown in FIG. 3, where 1-5 indicates that the p30 protein was diluted from 0.1mg/ml to 0.00625mg/ml (0.1 mg/ml, 0.05mg/ml, 0.025mg/ml, 0.0125mg/ml, 0.00625mg/ml, respectively) in PBS; 1-8 show that the p72 protein is diluted from 0.25mg/ml to 0.001953125mg/ml (0.25 mg/ml, 0.125mg/ml, 0.0625mg/ml, 0.03125mg/ml, 0.015625mg/ml, 0.0078125mg/ml, 0.00390625mg/ml and 0.001953125mg/ml) by PBS, respectively, and the result shows that the color is changed from wine red to grey red when the p30 protein concentration is 0.025mg/ml (A3), so that 0.05mg/ml (A2) is the minimum protein concentration, and the optimal protein concentration is 60 mug/ml after adding 20% of the minimum mark amount; when the p72 protein concentration was 0.00390625mg/ml (B7), the color changed from wine red to crimson, so that 0.0078125mg/ml (B2) was the minimum protein concentration, and 20% was added based on the minimum labeling amount to give the optimum protein concentration of 9.4. mu.g/ml, followed by 10. mu.g/ml.

(3) Preparing gold-labeled protein:

adjusting the pH value of the colloidal gold solution to 8, adding 60 mu g of ASFV p30 protein into 1ml of the colloidal gold solution, adding ASFV p30 protein, mixing uniformly, and reacting at 37 ℃ for 30 min; adding gold-labeled protein blocking solution, and reacting at 37 ℃ for 30 min; standing at 4 ℃ for 2h, centrifuging at 12000r/min at 4 ℃ for 30min, and resuspending the precipitate with a gold-labeled protein protective agent according to 1/20 of the total volume to obtain an ASFV p30 gold-labeled protein solution;

adjusting the pH value of the colloidal gold solution to 7, adding 9.4 mu g of ASFV p72 protein into 1ml of the colloidal gold solution, adding ASFV p72 protein, mixing uniformly, and reacting at 37 ℃ for 30 min; adding gold-labeled protein blocking solution, and reacting at 37 ℃ for 30 min; standing at 4 ℃ for 2h, centrifuging at 12000r/min at 4 ℃ for 30min, and resuspending the precipitate with a gold-labeled protein protective agent according to 1/20 of the total volume to obtain an ASFV p72 gold-labeled protein solution;

(4) gold-labeled protein confining liquid selection

The sealing effect of the sealing liquid is judged according to the dead gold amount after the gold is finished.

Respectively adopting 4 kinds of confining liquids, which are respectively: 10% m/v BSA (purity: 90%); ② 10 percent m/v BSA (purity: 99.8 percent); ③ 10% m/v BSA (purity: 99.8%) + 3% m/v casein; fourthly, 3 percent m/v PEG 20000; and fifthly, not sealing.

The results are shown in table 1, and the results show that all dead gold is obtained when the gold-labeled protein is not sealed by the gold-labeled protein sealing liquid in the preparation process of the gold-labeled protein; when the confining liquid IV is used, dead gold is almost all generated; the amount of dead gold is less when the confining liquid is used; the blocking liquid (c) is used for minimizing the dead gold, which shows that the blocking effect of BSA is better after being assisted by inert protein. Therefore, the invention selects the confining liquid c as the gold-labeled protein confining liquid.

TABLE 1 blocking Effect of gold-labeled protein blocking solution

Numbering	Sealing liquid	Whether there is dead gold
			②	10% BSA (purity: 90%)	++
②	10% BSA (purity: 99.8%)	++
			③	10% BSA (purity: 99.8%) + 3% casein	+
④	3％PEG20000	+++
			⑤	Is not closed	++++

Note: -means no dead gold; + means less dead/almost no dead; + indicates more dead gold; + + + + indicates excessive dead gold; ++++

Indicates that all the gold is dead

(5) Gold-labeled protein protectant selection

The gold-labeled protein protective agent is selected so that the quality control line (C line) and the detection line (T line) are obvious in color development, and the T line contrast corresponding to the positive sample and the negative sample is obvious.

Respectively adopting 5 protein protective agents which are respectively: boric acid + 2% m/v PEG20000 in PBS; (iii) Tris-HC l + 2% m/v PEG20000 in PBS; ③ 0.02mol/L (pH9.0) of Tris-HCl + 0.85% m/v NaCl + 1% v/v of T ween-20 in PBS; tris (0.02mol/L) + 20% m/v BSA + 5% m/v sucrose + 0.04% m/v PEG20000+ 0.5% v/v Tween-20 in PBS solution; 3% m/v sucrose + 1% m/v PVP-40+ 0.5% v/v Tween-20 in PBS.

The results of the 5 gold-labeled protein protective agents on color development of the quality control line (C line) and the detection line (T line) are shown in Table 2. Under the action of the 5 different gold-labeled protein protective agents, the positive sample and the negative sample are respectively detected. The result shows that when the protein protectant I is adopted, the C line and the T line of the positive sample and the negative sample are weak in color development and the difference between the two T lines is not obvious; when the protein protective agent II is adopted, although the color development of the C line and the T line of the positive sample and the negative sample is slightly strong, the difference of the two T lines is not obvious; when the protein protective agents are adopted, the color development difference of the C line and the T line of the positive sample and the negative sample is obvious; when the protein protective agent is adopted, the color development difference of the C line and the T line of the positive sample and the negative sample is more obvious, so the protein protective agent is used as the gold-labeled protein protective agent.

TABLE 2 color development results of gold-labeled protein protectant on quality control line (C line) and detection line (T line)

Numbering	C line color development	T-line color development	Positive and negative T-line results comparison
				①	+	+	The difference is not obvious
②	++	++	The difference is not obvious
				③	++	++	The difference is obvious
④	+++	+++	The difference is obvious
				⑤	+++	+++	The difference is obvious

Note: the more "+" indicates that the color development month is obvious

1.3 preparation of conjugate pads

And uniformly spraying the prepared gold-labeled protein on a gold-labeled bonding pad by using a film spraying instrument, immediately placing the gold-labeled protein in a 37 ℃ oven for drying for 2-3 h, taking out the gold-labeled protein, storing the gold-labeled protein at 4 ℃, and sealing and storing the gold-labeled protein for later use.

2. Preparation of sample pad

And (3) soaking the sample pad in the sample pad sealing liquid at the temperature of 37-38 ℃ for 2h, taking out the sample pad, drying the sample pad in an oven for 12h, and drying the sample pad in an aluminum foil bag for later use.

Selection of sample pad blocking solution:

the invention adopts 5 sample pad sealing liquids, which are respectively as follows: firstly, 3% of m/v sucrose, 1% of m/v BSA, 1% of m/v PVP-40 and 0.5% of v/v Tween-20 ultra-pure water solution; (ii) 3% m/v sucrose + 1% m/v BSA + 1% m/v PVP-40+ 0.5% v/v Tween-20 in PBS; ③ 1 percent of m/v trehalose, 1 percent of m/v PEG20000, 2 percent of m/v BSA, 1 percent of m/v PVP-40 and 0.5 percent of v/v Tween-20 in PBS; (iv) 1% m/v trehalose + 1% m/v PEG20000+ 2% m/v BSA + 1% m/v PVP-40+ 0.5% v/v TtitonX-100 in PBS; 1% m/v PEG200000+ 1% m/v PVP-40+ 0.5% v/v Tween-20+ 2% m/v BSA in PBS.

The positive and negative samples were tested separately under the influence of 5 different sample pad blocking solutions. As shown in Table 3, when the blocking solutions (i) and (iv) were used, the C-line and T-line of the positive sample and the negative sample were both weak in color development, and the difference in color development between the corresponding T-lines was not significant; when the sealing liquid II and III is adopted, the T lines of the positive sample and the negative sample are obviously different, but the C line and the T line are weak in color; when the sealing liquid is adopted, the C line and the T line of the positive sample and the negative sample are obvious in color development and the corresponding T lines are obvious in difference. Therefore, (. sup.1% m/v PEG200000+ 1% m/v PVP-40+ 0.5% v/v Tween-20+ 2% m/v BSA in PBS) was selected as the sample pad blocking solution.

TABLE 3 color development results under the action of the sample pad blocking solution

Numbering	C line color development	T-line color development	Positive and negative T-line results comparison
				①	++	++	Almost indistinguishable
②	+	+	The difference is obvious
				③	+	+	The difference is obvious
④	++	++	The difference is not obvious
				⑤	+++	+++	The difference is obvious

Note: more "+" indicates more obvious color development

3. Preparation of nitrocellulose membranes

Marking ASFV p72 protein with the film spraying concentration of 0.8mg/ml and ASFV p30 protein with the concentration of 0.3mg/ml on a nitrocellulose membrane to form detection lines T1 and T2;

drawing a line on the nitrocellulose membrane by using an equal proportion mixture containing 1mg/ml of the anti-p 72 protein monoclonal antibody and 1mg/ml of the anti-p 30 protein monoclonal antibody to form a quality control line C;

the interval between the two detection lines is 50 mm; the shortest distance between the quality control line and the detection line is 60 mm; and after the three lines are drawn, drying the three lines at 37-38 ℃ for 2-3 h.

The monoclonal antibodies are selected as follows:

and (3) monoclonal antibody selection: p30 monoclonal antibodies, 2H3, 4E6 and 3H8 are obtained by construction of a company; constructing to obtain p72 monoclonal antibodies which are 3G7, 3H5 and 6B6 respectively;

and (3) monoclonal antibody subtype identification: the subtype of the monoclonal antibody is identified by using a subtype identification test strip kit, and the identification result shows that the monoclonal antibodies 2H3, 4E6 and 3H8 of the p30 monoclonal antibodies belong to IgG2a, IgG2a and IgG1 types respectively, and the monoclonal antibodies 3G7, 3H5 and 6B6 of the p72 monoclonal antibodies belong to IgG2B, IgG1 and IgG2a types respectively.

And (3) specific identification:

the p30 protein is selected as a positive control, the p72 protein, the p54 protein, the 177L protein and the 17-22 protein are selected as negative controls, meanwhile, the coating ELISA plate carries out specificity detection on 3 monoclonal antibodies 2H3, 4E6 and 3H8 corresponding to the p30 protein, the detection result shows that both 4E6 and 3H8 have cross reaction with the p72 protein, the p54 protein and the 177L protein and are ineffective monoclonal antibodies, and the 2H3 is selected as an effective monoclonal antibody against the p30 protein.

The p72 protein is selected as a positive control, the p30 protein, the p54 protein, 177L and 17-22 protein are selected as negative controls, the coating ELISA plate is used for carrying out specific detection on 3 monoclonal antibodies 3G7, 3H5 and 6B6 corresponding to the p72 protein, the detection result shows that 3G7 and 3H5 have cross reaction with the p30 protein and the p54 protein, and the 6B6 is selected as an effective monoclonal antibody against the p72 protein.

And (3) indirect ELISA (enzyme-Linked immunosorbent assay) for measuring titer: coating an ELISA plate with p30 protein, and extracting p30 monoclonal antibody 2H3 from 10²Diluting to 10⁷The detection result shows that the 2H3 titer reaches 10⁶(ii) a Coating an ELISA plate with p72 protein, and extracting p72 monoclonal antibody 6B6 from 10²Diluting to 10⁷The detection result shows that the 2H3 titer reaches 10⁶。

4. Preparation of test paper strip

The quality control line coated antibody is exemplified by p30 monoclonal antibody 2H3 and p72 monoclonal antibody 6B 6.

And sequentially connecting the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad on the PVC base plate to obtain the test strip, and slitting the test strip according to the size of the test strip card shell. The structure schematic diagram of the test strip is shown in fig. 4, wherein 1 is a water absorption pad, 2 is a quality control line C, 3 is a detection line T1, 4 is a detection line T2, 5 is a combination pad, 6 is a sample pad, 7 is a PVC bottom plate, and 8 is a nitrocellulose membrane (NC membrane).

Example 3 African swine fever virus antibody colloidal gold duplex test paper strip use and result determination

Diluting a sample to be detected by using a sample diluent at a ratio of 1:10, and dripping 100 mu l of sample on a sample pad at room temperature for 5-10 min to observe the result; the determination result is shown in fig. 5, and if the detection line (T line) and the quality control line (C line) are both red, the result is positive; if the detection line (T line) does not develop color and the quality control line (C line) develops color, the detection line is negative; if the quality control line (C line) does not develop color, the test strip can be judged to be invalid.

Detection and decision principle: when a sample to be detected is added on the sample pad, forward chromatography is carried out through capillary action, when the sample to be detected reaches the binding pad, if p30 and/or p72 antibodies are contained in the sample to be detected, protein on the binding pad can perform antigen-antibody binding reaction with corresponding antibodies, so that corresponding complexes are formed, forward chromatography is carried out continuously until a red strip which is visible to the naked eye is formed at a detection line T2(p30) or T1(p72) or red strips are formed at T2(p30) and T1(p 72). And no matter whether the sample to be detected contains p30 and p72 antibodies or not, the gold-labeled protein can continuously carry out chromatography forwards to reach the quality control line and react with the monoclonal antibody compound of the anti-p 30 and the anti-p 72 at the quality control line, so that a red band which can be seen by naked eyes is formed. Thus, if the control line does not show a red band, it indicates that the strip has failed.

Selection of sample diluent:

this study adopted 6 sample diluents, which were:

firstly, boric acid solution; Tris-HCl solution; ③ PBS solution; Tris-HCl + PBS solution with different concentrations of BSA, 0.2% m/v, 0.5% m/v and 1% m/v; Tris-HCl + 0.2% m/v, 0.5% m/v, 1% m/v BSA + 0.8% v/v NaCl PBS solution; sixthly, 0.02M (pH9.0) of a PBS solution of Tris-HCl + 0.85% M/v NaCl + 1% v/v Tween-20.

The positive and negative samples were tested separately under the action of 6 different dilutions of the sample, and the results of color development are shown in Table 4 below. The result shows that when the confining liquid is (I) -III, the C line and the T line of the positive sample and the negative sample are weak in color development and the color development difference of the corresponding T lines is not obvious; when the diluent is IV, the C line and the T line of the positive sample and the negative sample are obvious in color development but the corresponding T lines are not obvious in difference; when the diluent is sixth, the C line and the T line of the positive sample and the negative sample are obvious in color development and the corresponding T lines are obvious in difference. Therefore, PBS solution containing 0.02M (pH9.0) Tris-HCl + 0.85% M/v NaCl + 1% v/v Tween-20 is used as sample diluent.

TABLE 4 color development results after sample dilution treatment

Numbering	C line color development	T-line color development	Positive and negative T-line results comparison
				①	+	+	The difference is not obvious
②	+	+	The difference is not obvious
				③	+	+	The difference is not obvious
④	++	++	The difference is not obvious under the condition of 3 concentrations
				⑤	++	++	The difference is not obvious under the condition of 3 concentrations
⑥	+++	+++	The difference is obvious

Note: more "+" indicates more obvious color development

Example 4 clinical application of African swine fever virus antibody colloidal gold duplex test strip

The swine Nos. 1, 2, 3, 4 and 5 are immunized by using ASFV delta 181/UK attenuated strain, immune sera of 0d, 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d, 21d, 23d, 25d, 27d and 29d are respectively adopted, the test strip prepared by the invention is used for detecting antibody growth, and the test strip is compared with a Swedish SVANOVA p30 indirect ELISA kit and a Spanish INGENASA p72 blocking ELISA kit to illustrate the growth law of the two antibodies of p30 and p 72.

The results of the two antibodies showed that: p30 appeared at about day 7 earliest and at least about day 11 latest, whereas p72 was later compared to p30, generally at 9 th and 15 th latest (fig. 6). Almost completely accords with the two imported kits (figure 7), so the invention achieves the aim of dynamically monitoring the ASF by observing the growth and decay rules of the two antibodies.

The ASFV antibody colloidal gold duplex immunochromatographic test strip prepared by the invention, an ASFV p30 antibody detection kit prepared by Swedish SVANOVA company and an ASFV p72 ELISA antibody detection kit prepared by Spanish INGENASA company are used for simultaneously detecting 84 clinical serum samples (40 ASFV delta 181/UK attenuated strain immune serum, 14 infected inactivated serum and 30 ASFV negative serum), and the coincidence rate of the ASFV antibody colloidal gold test strip and two commercial ELISA kits is compared so as to explain the specificity and sensitivity of the test strip. The results of the measurements are shown in Table 5 below.

TABLE 5 test results

The results show that:

the total coincidence rate of the test strip and the Swedish ELISA is 98.8% ((39+14+30)/(40+14+ 30));

the positive coincidence rate of the test strip and the Swedish ELISA is 98% ((39+14)/(40+ 14));

③ the test strip has 100 percent of negative coincidence rate with the Swedish ELISA (30/30);

the total coincidence rate of the test strip and Spanish ELISA is 98.6% ((39+30)/(40+ 30));

the positive coincidence rate of the test strip and Spanish ELISA is 97.5% (39/40);

sixthly, the negative coincidence rate of the test strip and Spanish ELISA is 100 percent (30/30);

in conclusion, the test strip prepared by the invention has high coincidence rate with a reference method, and can realize the joint detection of the African swine fever virus p30 and p72 antibodies in one step, realize the whole-process monitoring of the African swine fever virus antibodies and greatly simplify the detection steps and time.

Claims

1. A colloidal gold duplex test strip for monitoring African swine fever virus antibodies in the whole process comprises a PVC base plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped on the PVC base plate; the nitrocellulose membrane is provided with a detection line T and a quality control line C, the detection line T is arranged at one end close to the sample pad, and the quality control line C is arranged at one end close to the water absorption pad; it is characterized in that the preparation method is characterized in that,

the binding pad is coated with gold-labeled protein, and the gold-labeled protein comprises colloidal gold labeled ASFV p30 protein and colloidal gold labeled ASFV p72 protein;

the detection line T comprises a detection line T1 coated with ASFV p72 protein and a detection line T2 coated with ASFV p30 protein;

the quality control line C is coated with an anti-ASFV p30 protein monoclonal antibody and/or an anti-ASFV p72 protein monoclonal antibody 1:1 mixture.

2. The colloidal gold duplex test strip of claim 1, wherein the preparation method of the conjugate pad comprises:

mixing the prepared ASFV p30 gold-labeled protein and ASFV p72 gold-labeled protein in equal proportion, and spraying the mixture on a gold-labeled bonding pad to obtain a bonding pad;

the gold-labeled protein blocking solution is an ultrapure water solution containing 10% m/v BSA and 3% m/v casein;

3. The colloidal gold duplex test strip of claim 2, wherein the ASFV p30 gold-labeled protein solution consists of: each 1ml of the colloidal gold solution contains 60 mu g of ASFV p30 protein; the ASFV p72 gold-labeled protein solution comprises: 10. mu.g of ASFV p72 protein was added to 1ml of the colloidal gold solution.

4. The colloidal gold duplex test strip of claim 1, wherein the test line T1 is coated with 0.8mg/ml of ASFV p72 protein; the detection line T2 is coated with 0.3mg/ml ASFV p30 protein.

5. The colloidal gold duplex test strip of claim 1, wherein the sample pad is soaked in a sample pad blocking solution; the sample pad blocking solution is PBS solution containing 1% m/v PEG20000, 1% m/v PVP-40, 0.5% v/v Tween-20, 2% m/v BSA.

6. The colloidal gold duplex test strip of claim 1, wherein the gene sequence number of the ASFV p30 protein is NC 044955.1; the gene sequence number of the ASFV p72 protein is NC 001659.2.

7. The method for preparing a colloidal gold duplex test strip according to any one of claims 1 to 6, wherein the method comprises the following steps:

preparing a binding pad coated with gold-labeled protein;

8. An African swine fever virus antibody detection kit, which is characterized in that the kit comprises the colloidal gold duplex detection test strip of any one of claims 1-6.

9. The African swine fever virus antibody detection kit of claim 8, wherein the kit further comprises a sample diluent which is a PBS solution containing 0.02M Tris-HCl, 0.85% M/v NaCl, 1% v/v Tween-20.

10. The African swine fever virus antibody detection kit of claim 8 or 9, wherein the kit further comprises a sample pad blocking solution, a gold-labeled protein protective agent, a gold-labeled protein blocking solution; the sample pad blocking solution is PBS solution containing 1% m/v PEG20000, 1% m/v PVP-40, 0.5% v/v Tween-20, 2% m/v BSA; the gold-labeled protein protective agent is a PBS solution containing 3% m/v of sucrose, 1% m/v of PVP-40 and 0.5% v/v of Tween-20; the gold-labeled protein blocking solution is an ultra-pure water solution containing 10% m/v BSA and 3% m/v casein.