CN111551746A - African swine fever virus N protein IgY antibody detection colloidal gold test paper and method - Google Patents
African swine fever virus N protein IgY antibody detection colloidal gold test paper and method Download PDFInfo
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Abstract
The invention relates to a colloidal gold test paper and a method for detecting an African swine fever virus N protein IgY antibody, belonging to the field of detection of African swine fever virus antibodies, wherein a combination pad of the test paper is a colloidal gold pad coated with a rabbit anti-chicken IgG-colloidal gold compound, and a nitrocellulose membrane of the test paper is provided with a quality control line coated by goat anti-rabbit IgG and a detection line coated by an ASFV recombinant antigen. Compared with the prior art, the test strip can rapidly detect the ASFV IgY antibody level, in order to improve the sensitivity and the linear range of detection, the invention specially prepares 2 purified ASFV recombinant antigens, and compared with other conventional ELISA antibody detection or colloidal gold detection methods, the invention is more convenient and rapid and has higher sensitivity.
Description
Technical Field
The invention relates to the technical field of rapid detection of African swine fever virus N protein IgY antibody, in particular to a colloidal gold test paper and a method for detecting the African swine fever virus N protein IgY antibody.
Background
African Swine Fever (ASF) is a highly contagious swine viral disease. High mortality rates approaching 100% can result in domestic pigs. ASF is caused by African Swine Fever Virus (ASFV), a large double-stranded DNA Virus that replicates primarily in the cytoplasm of macrophages, the ASFV being the only member of the genus Asfivirus of the family Asfarviridae. The natural hosts of ASFV include wild boars and arthropod vectors of the Ornithodoros genus. Infection of an ASFV in its reservoir host is usually asymptomatic and progresses to persistent infection. In contrast, infection of domestic pigs with ASFV can lead to fatal hemorrhagic fever.
The greatest characteristic of the african swine fever virus is "two high": high infection and high death rate. Once a pig farm is infected with African swine fever virus, the swine fever virus can spread to the periphery quickly, the death rate of infected live pigs reaches 100 percent, and none of the pigs survives. The African swine fever is generally discovered by a detection means, and the identification by naked eyes is not necessarily accurate. In pigs infected with the virus, the virus survives in the five viscera, blood and feces.
At present, the killing of the virus is difficult, no specific medicine can effectively kill the virus, and the corresponding vaccination vaccine is not mature and cannot be clinically applied.
The colloidal gold immunochromatography technology is increasingly widely applied to various fields of biomedicine as a new immunological method. The method is a mature and widely applied immune marking technology after the traditional three immune marking technologies. After a sample to be detected is added on a sample membrane, due to the capillary action of the microporous filter membrane, the antigen-antibody reaction is rapidly carried out on the solid phase membrane, the detection generally takes 5-10 min to obtain a result, and the detection time is greatly shortened compared with other methods (for example, ELISA needs 1-2 h, and a fluorescent quantitative PCR method needs 2 h). The test result is judged by a macroscopic color development strip, special instruments and equipment are not needed, only a test strip or a percolation kit is needed, and the sample is only subjected to very simple treatment or no pretreatment. The method has the advantages of low cost, simple operation, reagent stability, no influence of external factors such as temperature and the like, convenience, rapidness, specificity, sensitivity, strong stability, intuitive result judgment and the like, is particularly suitable for on-site rapid inspection, has huge development potential and wide application prospect, and represents the development direction of simple, rapid and convenient popularization of diagnostic reagents.
Chinese patent document CN201920618571.X discloses an African swine fever antibody colloidal gold detection card, the detection card includes test paper, and test paper includes the PVC bottom plate, and overlap joint sample pad, combination pad, holoderm, cellulose nitrate membrane and the pad that absorbs water on the PVC bottom plate in proper order, and the combination pad is wrapped up and is had the African swine fever recombinant antigen of colloidal gold mark, is equipped with detection line, half contrast line, contrast line and quality control line on the cellulose nitrate membrane in proper order, wrap up on detection line, half contrast line and the contrast line African swine fever virus recombinant antigen, the peridium rabbit anti-Staphylococcus aureus A polyclonal antibody on the quality control line.
Chinese patent document CN201310197035.4 discloses a preparation method of a colloidal gold immunochromatographic test strip for detecting antibodies of African swine fever viruses, and specifically discloses a preparation method of a colloidal gold immunochromatographic test strip by using colloidal gold particles to mark and purify a p54 recombinant antigen, using Staphylococcus aureus protein A as a detection line, and using p54 immune serum as a quality control line.
The invention can only qualitatively detect whether the antibody exists in the pig body, but cannot quantitatively detect whether the content of the antibody reaches the titer of the anti-virus, and cannot judge whether the antibody in the pig body is enough to resist the African swine fever virus. The 2 purified ASFV recombinant antigens ASFV-1 and ASFV-2 prepared by the invention can be mixed for use, not only can qualitatively detect the African swine fever virus, but also can improve the detection sensitivity and detection range, the antibody concentration is between 50ng/ml and 800ng/ml, and has a very high detection line, thereby obviously improving the detection linear range.
Disclosure of Invention
The invention aims to solve the problems and provide a novel colloidal gold test paper for detecting the African swine fever virus N protein IgY antibody and a preparation method thereof, so as to improve the sensitivity and the linear range of the detection of the African swine fever virus N protein IgY antibody.
The invention realizes the purpose through the following technical scheme:
the invention provides a colloidal gold test paper for detecting an African swine fever virus N protein IgY antibody, which comprises a PVC (polyvinyl chloride) base plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially lapped on the PVC base plate, wherein the combination pad is a colloidal gold pad coated with a rabbit anti-chicken IgG-colloidal gold compound, and the nitrocellulose membrane is provided with a quality control line coated by goat anti-rabbit IgG and a detection line coated by an ASFV recombinant antigen.
As a further optimization scheme of the invention, the detection line comprises 2 ASFV recombinant antigens.
The invention also provides a preparation method of the African swine fever virus N protein IgY antibody detection colloid test paper, which comprises the following steps:
(1) preparing a purified ASFV recombinant antigen;
(2) preparing a nitrocellulose membrane: respectively diluting goat anti-rabbit IgG and the purified ASFV recombinant antigen, and coating the diluted antigen on a quality control line and a detection line of a nitrocellulose membrane;
(3) preparing colloidal gold;
(4) colloidal gold labeled rabbit anti-chicken IgG: taking rabbit anti-chicken IgG, mixing the colloidal gold and the rabbit anti-chicken IgG, centrifuging, and carrying out heavy suspension precipitation by using a PBS (phosphate buffer solution) solution to obtain a rabbit anti-chicken IgG-colloidal gold compound;
(5) preparing a colloidal gold pad: uniformly spraying the rabbit anti-chicken IgG-colloidal gold compound on the bonding pad, and fully drying to obtain a colloidal gold pad;
(6) treatment of the sample pad: soaking the sample pad in 20mmol/L PBS buffer solution for 2-5 hr, shaking while mixing, adding activator into the buffer solution, wherein the activator is selected from one of PEG6000, PEG8000 and Triton 100, the final concentration is 1-2% by mass, and the dosage of the activator is 1-2% of one of PEG6000, PEG8000 and Triton 100. (ii) a After soaking, putting the sample pad into an oven for drying;
(7) assembling: and adhering the sample pad, the colloidal gold pad, the coated nitrocellulose membrane and the water absorption pad on a PVC plate in a dry environment, and cutting to obtain the test paper.
As a further preferable embodiment of the present invention, the step (1) comprises preparing 2 purified recombinant ASFV antigens ASFV-1 and ASFV-2, and comprises the steps of: preparing an escherichia coli recombinant strain of ASFV N protein according to known gene sequences of ASFV N-1 and N-2 proteins, fermenting and expressing the recombinant strain, centrifuging to obtain a thallus, carrying out ultrasonic disruption on the thallus, centrifuging, taking a supernatant, and passing the supernatant through an affinity chromatography column to obtain the purified ASFV-1 recombinant antigen.
As a further optimization scheme of the invention, in the step (2), the goat anti-rabbit IgG and the purified ASFV recombinant antigen are respectively diluted to a final concentration of 1mg/ml by 25-80mmol/L Tris buffer solution, and then sprayed and coated on the quality control line and the detection line by a spraying amount of 0.75 muL/cm, and fully dried.
As a further optimized scheme of the present invention, in the step (3), the method for preparing the colloidal gold is a trisodium citrate reduction method, and the steps include:
taking HAuCl with the mass fraction of 0.005-0.015 percent4Heating and boiling 100ml of the aqueous solution;
and (II) quickly adding 1ml of trisodium citrate aqueous solution with the mass fraction of 0.5-5%, continuously boiling, changing the solution from light yellow to gray, then changing to black, and then gradually stabilizing to wine red to prepare the colloidal gold solution.
As a further optimized scheme of the present invention, in the step (4), the step of labeling rabbit anti-chicken IgG with colloidal gold comprises:
(i) dissolving rabbit anti-chicken IgG in 0.1-0.2ml 10mM buffer solution, and adjusting the pH value of the colloidal gold solution to 7.2;
(ii) adding 10ml of colloidal gold solution into 20-50mg of rabbit anti-chicken IgG;
(iii) mixing for 3-5min, adding BSA with mass fraction of 0.05%, and acting at room temperature for 15 min;
(iv) centrifuging at low speed of 2000r/min for 15min, and centrifuging at high speed of 12000r/min for 25 min;
(v) the precipitate was resuspended in 100mmol/L pH7.8 buffer containing 0.2% of Proclin300 by mass fraction to obtain rabbit anti-chicken IgG-colloidal gold complex.
As a further optimization scheme of the invention, the buffer solution is Tris buffer solution, PBS buffer solution or Hepes buffer solution.
As a further optimization scheme of the invention, the spraying amount of the rabbit anti-chicken IgG-colloidal gold compound on the glass fiber membrane is 10 mu L/cm.
The invention also provides a method for detecting the ASFVIgY antibody by using the African swine fever virus N protein IgY antibody detection colloidal test paper, which comprises the following steps:
firstly, horizontally placing the test paper, taking 50-100 mu L of a serum sample to be detected, and adding the serum sample into a sample pad;
step two, standing at room temperature for 15min, and judging: if the quality control line has a purple red line and the detection line has no color line, the result is negative; if the quality control line and the detection line both have purple red lines, the result is positive; the color intensity of the detection line is directly proportional to the antibody titer.
The invention has the beneficial effects that: the invention provides African swine fever virus N protein IgY antibody detection colloidal test paper and a preparation method and application thereof. In order to improve the sensitivity and the linear range of detection, 2 purified ASFV recombinant antigens are specially prepared, and compared with other conventional ELISA antibody detection or colloidal gold detection methods, the method is more convenient and rapid and has higher sensitivity.
Drawings
FIG. 1 is a schematic diagram of the overall structure of the African swine fever virus N protein IgY antibody detection colloid test paper of the present invention.
Detailed Description
The present application will now be described in further detail with reference to the drawings, it should be noted that the following detailed description is given for illustrative purposes only and is not to be construed as limiting the scope of the present application, as those skilled in the art will be able to make numerous insubstantial modifications and adaptations to the present application based on the above disclosure.
Example 1
The preparation method of the African swine fever virus N protein IgY antibody detection colloid test paper comprises the following steps:
(1) preparation of purified ASFV recombinant antigen
ASFV N-1 and N-2 protein gene sequences (ASFV N-1 and ASFV N-2 are obtained from GENBANK (https: i/www.ncbi.nlm.nih.gov) by intercepting from GENBANK gene accession number: MK128995.1, wherein the gene sequence corresponding to 60 amino acids after the signal peptide of MK128995.1 gene coding protein is used as ASFV N-1, and the gene sequence corresponding to the remaining amino acids after 60 amino acids is used as ASFV N-2), sending to Huada gene company to prepare Escherichia coli recombinant bacteria of ASFV protein, fermenting and expressing the recombinant bacteria and centrifuging to obtain bacteria, crushing the bacteria by using a high-pressure homogenizer, secondarily crushing by using an ultrasonic crusher, centrifuging to obtain supernatant, passing the supernatant through an affinity chromatography column to obtain purified ASFV, and respectively naming two purified antigens as ASFV-1 and ASFV-2.
(2) Preparation of nitrocellulose membranes
The goat anti-rabbit IgG and the purified ASFV recombinant antigen (ASFV-1, ASFV-2) are respectively diluted by 25-80mmol/L Tris buffer solution (the buffer solution contains 0.9% NaCl, Tween20 and pc300) to the final concentration of 1mg/ml, and then are respectively and uniformly sprayed on a quality control line C and a detection line T on a nitrocellulose membrane, wherein the spraying amount is 0.75 mu L/cm, and the two are fully dried.
(3) Preparation of colloidal gold solution
The 10-40nm colloidal gold is prepared by a trisodium citrate reduction method, and the specific operation method is as follows:
taking HAuCl with the mass fraction of 0.005-0.015 percent4The aqueous solution (100 ml) was boiled under heating.
(II) rapidly adding 1ml of trisodium citrate aqueous solution with the mass fraction of 1% -5%, and continuously boiling for about 5 min;
(III) after gold particles with the particle size of 10-40nm are prepared, the light yellow chloroauric acid aqueous solution can be observed to quickly turn into gray after the trisodium citrate is added, and then turn into black, and then gradually stabilize to be wine red;
(IV) cooling to room temperature, and storing at 4 ℃ in a dark place.
(4) Preparation of colloidal gold-labeled Rabbit anti-Chicken IgG
(i) Dissolving high-purity rabbit anti-chicken IgG in 0.1-0.2ml of 10mM Tris buffer solution, and adjusting the pH value of the colloidal gold solution to 7.2;
(ii) rabbit anti-chicken IgG was mixed with colloidal gold in the following combinations: adding 10ml of 40nm colloidal gold solution into every 20-50mg of rabbit anti-chicken IgG;
(iii) mixing for 3-5min, adding BSA with mass fraction of 0.05%, and acting at room temperature for 15 min;
(iv) centrifuging at low speed of 2000r/min for 15min, and centrifuging at high speed of 12000r/min for 25 min;
(v) the precipitate was resuspended in 100mmol/L Tris buffer pH7.8 containing 0.2% Proclin300 by mass fraction to obtain rabbit anti-chicken IgG-colloidal gold complex.
(5) Preparation of the colloidal gold pad 3
The rabbit anti-chicken IgG-colloidal gold complex prepared above was uniformly sprayed on a glass cellulose membrane at a spray rate of 10. mu.L/cm, and sufficiently dried to obtain a colloidal gold pad 3.
(6) Treatment of sample pad 2
In order to improve the accuracy and stability of the detection and make the colors of the C line and the T line more uniform when detecting the sample, the sample pad 2 is specially processed. The treatment method comprises the following steps: the sample pad 2 was soaked in 20mmol/L PBS buffer for 2-5 hours and mixed well with shaking, wherein 1% PEG8000 was added to the buffer. After the soaking, the sample pad 2 is placed in an oven for drying, in this embodiment, the drying temperature is 37 ℃ for 14 hours.
(7) Assembly of test strips
Preparing a water absorption pad 5, a sample pad 2 and a PVC base plate 1 in a drying room, pasting a nitrocellulose membrane 4 coated in the step 2.2 on the center of the PVC base plate 1, pasting the water absorption pad 5 on the upper edge of the nitrocellulose membrane 4, pasting a colloidal gold pad 3 prepared in the step 2.5 on the lower edge of the nitrocellulose membrane 4, pasting the sample pad 2 treated in the step 2.6 on the lower edge of the colloidal gold pad 3, wherein the mutual superposition part of each component is 1-2mm, and cutting the pasted test paper plate into test paper strips with the width of 4.1mm by using a cutting machine after the completion, wherein the structure is shown in figure 1. And (3) putting the cut test strip into a card shell, and sealing the test strip and the drying agent in an aluminum foil bag to complete the assembly of the product.
Example 2
Obtaining an ASFV N-1 protein gene sequence from GENBANK (https:// www.ncbi.nlm.nih.gov), sending the sequence to Huada gene company to prepare an Escherichia coli recombinant strain of ASFV protein, fermenting and expressing the recombinant strain and centrifuging to obtain a strain, crushing the strain by using a high-pressure homogenizer, crushing the strain by using an ultrasonic crusher for the second time, centrifuging to obtain a supernatant, and passing the supernatant through an affinity chromatography column to obtain the purified ASFV-1 recombinant antigen.
The goat anti-rabbit IgG and the purified ASFV-1 recombinant antigen are respectively diluted by 25-80mmol/L Tris buffer solution (containing 0.9% NaCl, Tween20 and pc300) to a final concentration of 1mg/ml, and then are respectively and uniformly sprayed on a quality control line C and a detection line T on a nitrocellulose membrane, wherein the spraying amount is 0.75 mu L/cm, and the test line T is fully dried.
Preparing 10-40nm colloidal gold by a trisodium citrate reduction method.
Preparing the colloidal gold-labeled rabbit anti-chicken IgG, uniformly spraying the colloidal gold-labeled rabbit anti-chicken IgG on a glass cellulose membrane, and fully drying to obtain the colloidal gold pad.
And (3) soaking the sample pad in PBS buffer solution containing an activating agent and an anti-interference substance, and drying.
And assembling the water absorption pad, the sample pad, the PVC bottom plate, the nitrocellulose membrane coated with the quality control line and the detection line and the colloidal gold pad in a drying room, and cutting to obtain the test paper.
Example 3
Obtaining an ASFV N-2 protein gene sequence from GENBANK (https:// www.ncbi.nlm.nih.gov), sending the sequence to Huada gene company to prepare an Escherichia coli recombinant strain of ASFV protein, fermenting and expressing the recombinant strain and centrifuging to obtain a strain, crushing the strain by using a high-pressure homogenizer, crushing the strain by using an ultrasonic crusher for the second time, centrifuging to obtain a supernatant, and passing the supernatant through an affinity chromatography column to obtain the purified ASFV-2 recombinant antigen.
The goat anti-rabbit IgG and the purified ASFV-2 recombinant antigen are respectively diluted by 25-80mmol/L Tris buffer solution (containing 0.9% NaCl, Tween20 and pc300) to a final concentration of 1mg/ml, and then are respectively and uniformly sprayed on a quality control line C and a detection line T on a nitrocellulose membrane, wherein the spraying amount is 0.75 mu L/cm, and the test line T is fully dried.
Preparing 10-40nm colloidal gold by a trisodium citrate reduction method.
Preparing the colloidal gold-labeled rabbit anti-chicken IgG, uniformly spraying the colloidal gold-labeled rabbit anti-chicken IgG on a glass cellulose membrane, and fully drying to obtain the colloidal gold pad.
And (3) soaking the sample pad in PBS buffer solution containing an activating agent and an anti-interference substance, and drying.
And assembling the water absorption pad, the sample pad, the PVC bottom plate, the nitrocellulose membrane coated with the quality control line and the detection line and the colloidal gold pad in a drying room, and cutting to obtain the test paper.
Test example 1
Test paper was prepared according to the method of example 1, except that the test paper was soaked in a buffer solution containing no activator and no anti-interference substance.
Test example 2
Acquiring ASFV N-1 and N-2 protein gene sequences from GENBANK (https:// www.ncbi.nlm.nih.gov), sending the sequences to Huada gene company to prepare Escherichia coli recombinant bacteria of ASFV protein, fermenting and expressing the recombinant bacteria and centrifuging to obtain bacteria, crushing the bacteria by using a high-pressure homogenizer, secondarily crushing the bacteria by using an ultrasonic crusher, centrifuging to obtain supernatant, passing the supernatant through an affinity chromatography column to obtain purified ASFV recombinant antigen, and respectively naming the two purified antigens as ASFV-1 and ASFV-2.
Rabbit anti-staphylococcus aureus A polyclonal antibody is uniformly sprayed on a quality control line C on a nitrocellulose membrane instead of goat anti-rabbit IgG in example 1, and purified ASFV recombinant antigens (ASFV-1, ASFV-2) are uniformly sprayed on a detection line T of the nitrocellulose membrane.
Preparing 10-40nm colloidal gold by a trisodium citrate reduction method.
Preparing the colloidal gold-labeled rabbit anti-chicken IgG, uniformly spraying the colloidal gold-labeled rabbit anti-chicken IgG on a glass cellulose membrane, and fully drying to obtain the colloidal gold pad.
And (3) soaking the sample pad in PBS buffer solution containing an activating agent and an anti-interference substance, and drying.
And assembling the water absorption pad, the sample pad, the PVC bottom plate, the nitrocellulose membrane coated with the quality control line and the detection line and the colloidal gold pad in a drying room, and cutting to obtain the test paper.
Test example 3
In this test example, referring to chinese patent document CN201310197035.4, a recombinant p54 antigen is labeled with a colloidal gold particle, which is sprayed on a glass cellulose membrane to prepare a colloidal gold pad, and a test paper is prepared using staphylococcus aureus protein a as a detection line and p54 immune serum as a quality control line.
And (3) test strip detection effect verification:
the test paper strip and an enzyme-linked immunosorbent assay (ELISA) are respectively used for detecting the antibody concentration, and the steps comprise: the test strip is horizontally placed on a desktop, according to the antibody level required by the ASFV IgY antibody when the ASFV IgY antibody leaves a factory, the ASFV IgY antibody is taken and diluted to 50ng/ml, 100ng/ml, 200ng/ml, 400ng/ml and 800ng/ml by using 50mM Tris solution as a detection sample, and a blank control is set at the same time. Respectively taking 50-100 μ L of detection sample and blank control, adding sample pad 2, standing at room temperature for 15min, reading results according to the color of quality control line and detection line: if the quality control line has a purple red line and the detection line has no color line, the result is negative; if the quality control line and the detection line both have purple red lines, the result is positive; the color intensity of the detection line is directly proportional to the antibody titer. The concentration of the antibody is detected by enzyme-linked immunosorbent assay (ELISA) according to the conventional ELISA method.
The results are shown in table 1 below:
table 1: results of different methods for detecting ASFV IgG antibody concentration
| Concentration of sample | 50ng/ml | 100ng/ml | 200ng/ml | 400ng/ml | 800ng/ml | Blank space |
| Example 1 | Positive (light color) | Positive (light color) | Positive (normal) | Positive (normal) | Positive (deep color) | Negative of |
| Example 2 | Negatives (Wireless) | Positive (light color) | Positive (light color) | Positive (normal) | Positive (deep color) | Negative of |
| Example 3 | Negatives (Wireless) | Positive (light color) | Positive (light color) | Positive (normal) | Positive (deep color) | Negative of |
| Test example 1 | Negatives (Wireless) | Negatives (Wireless) | Positive (light color) | Positive (light color) | Positive (normal) | Negative of |
| Test example 2 | Negatives (Wireless) | Positive (light color) | Positive (light color) | Positive (normal) | Positive (deep color) | Negative of |
| Test example 3 | Negatives (Wireless) | Positive (light color) | Positive (light color) | Positive (normal) | Positive (deep color) | Negative of |
| ELISA | Positive for | Positive for | Positive for | Positive for | Positive for | Negative of |
Description of the drawings: example 1, the present invention uses the results of 2 ASFV recombinant antigens; examples 2 to 3 are the results of detection using 1 ASFV recombinant antigen, respectively; test example 1 was the sample pad untreated; test example 2 is a test result of the cn201920618571.x patent method; test example 3 is the test result of the method of patent CN 201310197035.4;
table 1 shows that the detection result of the colloidal gold test strip of the present invention is highly consistent with the ELISA detection result, which indicates that the accuracy of the colloidal gold test strip of the present invention is 100%, and it is feasible to detect ASFV IgY antibody and its content using the colloidal gold test strip of the present invention. The depth of the color line is in direct proportion to the antibody titer, the higher the antibody titer is, the deeper the color line is, whether the commercial ASFV IgY antibody product reaches the factory antibody level can be detected, so as to judge the quality of the purchased product, furthermore, the embodiment 1 of the invention uses 2 ASFV recombinant antigens which are obviously superior to the following embodiments, and the concentration of 50ng/ml antibody can be detected, so that a light color line can be seen.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. The colloidal gold test paper for detecting the African swine fever virus N protein IgY antibody comprises a PVC base plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially lapped on the PVC base plate, and is characterized in that the combination pad is a colloidal gold pad coated with a rabbit anti-chicken IgG-colloidal gold compound, and the nitrocellulose membrane is provided with a quality control line coated by goat anti-rabbit IgG and a detection line coated by an ASFV recombinant antigen.
2. The African swine fever virus N protein IgY antibody detection colloidal gold test paper of claim 1, wherein the detection line comprises 2 ASFV recombinant antigens.
3. The method for preparing the African swine fever virus N protein IgY antibody detection colloid test paper of any one of claims 1-2, which is characterized by comprising the following steps:
(1) preparing a purified ASFV recombinant antigen;
(2) preparing a nitrocellulose membrane: respectively diluting goat anti-rabbit IgG and the purified ASFV recombinant antigen, and coating the diluted antigen on a quality control line and a detection line of a nitrocellulose membrane;
(3) preparing colloidal gold;
(4) colloidal gold labeled rabbit anti-chicken IgG: taking rabbit anti-chicken IgG, mixing the colloidal gold and the rabbit anti-chicken IgG, centrifuging, and carrying out heavy suspension precipitation by using a PBS (phosphate buffer solution) solution to obtain a rabbit anti-chicken IgG-colloidal gold compound;
(5) preparing a colloidal gold pad: uniformly spraying the rabbit anti-chicken IgG-colloidal gold compound on the bonding pad, and fully drying to obtain a colloidal gold pad;
(6) treatment of the sample pad: soaking the sample pad in 20mmol/L PBS buffer solution for 2-5 hours, and shaking and uniformly mixing, wherein an activating agent is added into the buffer solution; after soaking, putting the sample pad into an oven for drying;
(7) assembling: and adhering the sample pad, the colloidal gold pad, the coated nitrocellulose membrane and the water absorption pad on a PVC plate in a dry environment, and cutting to obtain the test paper.
4. The method for preparing African swine fever virus N protein IgY antibody detection colloid test paper according to claim 3, wherein the step (1) comprises preparing 2 purified ASFV recombinant antigens ASFV-1 and ASFV-2, and the steps comprise: preparing an escherichia coli recombinant strain of ASFV N protein according to known gene sequences of ASFV N-1 and N-2 proteins, fermenting and expressing the recombinant strain, centrifuging to obtain a thallus, carrying out ultrasonic disruption on the thallus, centrifuging, taking a supernatant, and passing the supernatant through an affinity chromatography column to obtain the purified ASFV-1 recombinant antigen.
5. The method for preparing African swine fever virus N protein IgY antibody detection colloid test paper according to claim 3, characterized in that in the step (2), goat anti-rabbit IgG and the purified ASFV recombinant antigen are respectively diluted with 25-80mmol/L Tris buffer solution to a final concentration of 1mg/ml, and then sprayed and coated on a quality control line and a detection line with a spraying amount of 0.75 μ L/cm, and fully dried.
6. The method for preparing African swine fever virus N protein IgY antibody detection colloid test paper according to claim 3, wherein in the step (3), the method for preparing the colloidal gold is trisodium citrate reduction method, and the steps comprise:
taking HAuCl with the mass fraction of 0.005-0.015 percent4Heating and boiling 100ml of the aqueous solution;
and (II) quickly adding 1ml of trisodium citrate aqueous solution with the mass fraction of 0.5-5%, continuously boiling, changing the solution from light yellow to gray, then changing to black, and then gradually stabilizing to wine red to prepare the colloidal gold solution.
7. The method for preparing African swine fever virus N protein IgY antibody detection colloid test paper according to claim 3, wherein in the step (4), the step of labeling rabbit anti-chicken IgG with colloid gold comprises:
(i) dissolving rabbit anti-chicken IgG in 0.1-0.2ml 10mM buffer solution, and adjusting the pH value of the colloidal gold solution to 7.2;
(ii) adding 10ml of colloidal gold solution into 20-50mg of rabbit anti-chicken IgG;
(iii) mixing for 3-5min, adding BSA with mass fraction of 0.05%, and acting at room temperature for 15 min;
(iv) centrifuging at low speed of 2000r/min for 15min, and centrifuging at high speed of 12000r/min for 25 min;
(v) the precipitate was resuspended in 100mmol/L pH7.8 buffer containing 0.2% of Proclin300 by mass fraction to obtain rabbit anti-chicken IgG-colloidal gold complex.
8. The method for preparing African swine fever virus N protein IgY antibody detection colloid test paper according to claim 3, characterized in that the buffer solution is Tris buffer solution, PBS buffer solution or Hepes buffer solution.
9. The method for preparing African swine fever virus N protein IgY antibody detection colloid test paper according to claim 3, wherein the spraying amount of the rabbit anti-chicken IgG-colloidal gold compound on the glass fiber membrane is 10 μ L/cm.
10. A method for detecting ASFV IgY antibody using the african swine fever virus N protein IgY antibody detection colloidal test strip of any one of claims 1-2, comprising the steps of:
firstly, horizontally placing the test paper, taking 50-100 mu L of a serum sample to be detected, and adding the serum sample into a sample pad;
step two, standing at room temperature for 15min, and judging: if the quality control line has a purple red line and the detection line has no color line, the result is negative; if the quality control line and the detection line both have purple red lines, the result is positive; the color intensity of the detection line is directly proportional to the antibody titer.
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