Rapid detection reagent for yellow fever virus antibody and preparation method thereof
The combination of the ferromagnetic nanoparticles with the outer membrane E protein labeled by yellow heat viruses and rabbit anti-E protein polyclonal antibodies is solved by enriching magnetic nanoparticles, and the existing yellow heat virus detection methods are solved. Fast, sensitive and specific antibody detection is achieved, improving detection sensitivity and reducing costs.
Patent Information
- Application Number
- CN202411077348.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-07
- Publication Date
- 2024-12-27
AI Technical Summary
The existing yellow fever virus detection methods have a long detection time, complex process and are prone to infection, making it difficult to obtain test results quickly and effectively.
Magnetic nanoparticle capture reagent combined with immunochromatography test strips can enrich the triferromagnetic nanoparticles tetroxide and rabbit anti-E protein polyclonal antibodies marked by yellow heat virus outer membrane E protein through magnetic nanoparticles to achieve rapid and reliable antibodies Test.
It provides a fast, sensitive and specific yellow heat virus antibody detection method, which is low cost and does not require professional instruments. The results can be judged by naked eyes. The detection sensitivity is 10 times higher than the colloidal gold detection result.
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Figure CN119199127A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a detection reagent for yellow fever virus antibodies and a preparation method thereof, belonging to the technical field of biological detection. Background Art
[0002] Yellow fever virus belongs to the arbovirus group B in the togavirus family (togavirus), and is a member of the genus Flavivirus (Flavivirus) of the family Flaviridae. The clinical features of yellow fever include fever, severe headache, significant facial congestion, obvious relatively slow pulse, large amounts of black vomitus, large amounts of proteinuria, jaundice, etc. After infection with the yellow fever virus, clinical diseases appear, and the rest are latent infections. The incubation period is 7 days. Mild symptoms may only manifest as fever, headache, mild proteinuria, etc., without jaundice and bleeding, and will recover after a few days. Severe cases can generally be divided into infection period, poisoning period, and recovery period. The incubation period of this disease is 3 to 6 days and can be as long as 13 days. After infection, most patients are mild or subclinical infections. Only a few patients are seriously ill and eventually die (about 5% to 15%). Mild and latent infections are difficult to diagnose, and often rely on serum immunological tests including monoclonal antibodies, ELISA and other technologies to draw conclusions. PCR can also be used to detect viruses in blood samples, and blood can be inoculated into the brain of suckling mice to isolate the virus when necessary. If the patient has never been infected with the same virus before, positive results of hemagglutination inhibition test, neutralization test, complementation test, etc. can be used to make a diagnosis. However, these detection reagents take a long time to detect, are prone to infection during the process, are complicated to operate, and require detection instruments. How to develop yellow fever virus antibody detection reagents that can obtain test results with the naked eye and are fast and effective is imminent. Summary of the invention
[0003] 1. Technical issues to be resolved In order to solve the above problems in the prior art, the present invention provides a rapid detection reagent for yellow fever virus antibodies and a preparation method thereof.
[0004] (II) Technical solution In order to achieve the above object, the main technical solutions adopted by the present invention include: A rapid detection reagent for yellow fever virus antibodies comprises a magnetic nanoparticle capture reagent and an immunochromatographic test paper which is composed of a sample pad, a glass fiber pad, a nitrocellulose detection membrane and a water-absorbing pad which are overlapped in sequence and attached to a bottom liner card, wherein the nitrocellulose detection membrane is coated with a quality control line of a rabbit anti-E protein polyclonal antibody and a detection line of the outer membrane E protein of the yellow fever virus, and the magnetic nanoparticle capture reagent is ferroferric oxide magnetic nanoparticles marked with the outer membrane E protein of the yellow fever virus.
[0005] As described above, for the rapid detection reagent, preferably, the amino acid sequence of the yellow fever virus outer membrane E protein is as shown in SEQ ID NO.1.
[0006] As described above, the rapid detection reagent, preferably, the expression of the yellow fever virus outer membrane E protein is carried out using the gene sequence shown in SEQ ID NO.2.
[0007] As for the rapid detection reagent as described above, preferably, the sample pad is a glass fiber membrane that has been soaked in a treatment solution containing Tween-80.
[0008] In the rapid detection reagent as described above, preferably, the glass fiber mat is a glass fiber membrane soaked in a solution containing 0.1% by weight of Tris base, 2% by weight of BSA and 2% by weight of Triton X-100.
[0009] When the glass fiber pad in the immunochromatographic test paper is not treated in any way, a few test strips will show false positives of non-specific bands when the diluent of the sample to be tested is added. A large number of experiments have verified that when the glass fiber pad is soaked in a solution containing 0.1% Tris base, 2% BSA and 2% Triton X-100, the occurrence of false positives is effectively avoided.
[0010] A method for preparing a detection reagent for rapid detection of yellow fever virus antibodies, comprising the following steps: S1. After carboxylation of ferroferric oxide magnetic nanoparticles, yellow fever virus outer membrane E protein is added for labeling to obtain yellow fever virus outer membrane E protein labeled ferroferric oxide magnetic nanoparticle capture reagent; S2, soaking the glass fiber membrane in the heavy suspension and drying it to obtain a glass fiber mat; S3. A quality control line and a detection line are provided on the nitrocellulose membrane, the quality control line is coated with rabbit anti-E protein polyclonal antibody, and the detection line is coated with yellow fever virus outer membrane E protein, to obtain a nitrocellulose detection membrane; S4, immersing the glass fiber membrane in a treatment solution containing Tween-80, taking it out and drying it to obtain a sample pad; S5. Overlap the sample pad, glass fiber pad, nitrocellulose test membrane, and absorbent pad in sequence and stick them on the backing card. Cover the absorbent pad on one end of the nitrocellulose test membrane close to the quality control line, and cover the glass fiber pad on the other end close to the test line.
[0011] In the preparation method as described above, preferably, in step S1, the particle size of the ferroferric oxide magnetic nanoparticles is 15-40 nm, and the carboxylation is carried out using a glutaraldehyde solution.
[0012] According to the preparation method described above, preferably, in step S1, the concentration of the yellow fever virus outer membrane E protein label is 1.5 mg / mL, the reaction time during labeling is 2 h, the solution is discarded after magnetic adsorption, and then 0.8% BSA and 5% sucrose solution are added, and the mixture is shaken and blocked for 30 minutes; the solution is discarded after magnetic adsorption, and PBS containing 0.05% Tween-20 at a concentration of 0.01 mol / L is added for washing; and the resuspension is added to obtain an E protein-labeled ferroferric oxide magnetic nanoparticle capture reagent solution.
[0013] A large number of experiments have shown that if blocking is not performed with a solution containing 0.8% BSA and 5% sucrose, non-specific bands will appear, resulting in false positive results.
[0014] In the preparation method as described above, preferably, the yellow fever virus outer membrane E protein is obtained by expressing a gene sequence such as SEQ ID NO.2.
[0015] In the preparation method as described above, preferably, the resuspension solution contains 0.1% by weight of Tris base, 2% by weight of BSA and 2% by weight of Triton X-100 solution.
[0016] In the preparation method as described above, preferably, in step S3, the coating concentration of the rabbit anti-E protein polyclonal antibody is 2 mg / mL and the coating concentration of the yellow fever virus outer membrane E protein is 1.6 mg / mL.
[0017] The present invention obtains a reagent for rapid detection of yellow fever virus antibodies by wet immunochromatography through a large number of experimental verifications. A magnetic nanoparticle capture reagent is used, which is a magnetic nano-magnetic particle of ferroferric oxide labeled with the outer membrane E protein of the yellow fever virus. The adsorption of a magnet plays an enrichment role. The serum to be tested is added to the magnetic nano-particle capture reagent. If there are antibodies produced by the infected yellow fever virus in the serum to be tested, the antibodies will bind to the outer membrane E protein of the yellow fever virus, and the outer membrane E protein of the yellow fever virus is coated on the magnetic nano-magnetic particles of ferroferric oxide. Through the magnetic adsorption of the magnetic frame, the complex of the antibody-yellow fever virus outer membrane E protein-ferroferric oxide magnetic nano-magnetic particles will be adsorbed. At this time, the serum is discarded to achieve the purpose of enriching the antibody. The complex is then redissolved in 100 μL of buffer and added to the sample well of the immunochromatographic test paper prepared above. The detection sensitivity is effectively improved by magnet adsorption enrichment. The present invention also treats the sample pad, and through a large number of experiments, it is found that the sample pad treated with 0.05mol / L PBS containing 2.5% Tween-80 can effectively avoid the occurrence of false negatives. When the untreated sample pad is used for serum detection, the phenomenon of incomplete binding with the antigen on the strip will occur, resulting in the occurrence of false negatives. Through a large number of experimental studies, the present invention finds that when the magnetic nanoparticle capture reagent uses a resuspension solution with a weight percentage of 0.1% Tris base, 2% BSA and 2% Triton X-100, the optimal magnetic particle dispersion is obtained, which can effectively avoid the appearance of nonspecific strips and avoid false positives.
[0018] (III) Beneficial effects The beneficial effects of the present invention are: The present invention provides a yellow fever virus outer membrane E protein, which can be used as an antigen for the preparation of an immune reagent for detecting yellow fever virus antibodies. The magnetic nanoparticle immunochromatography reagent for rapid detection of yellow fever virus antibodies prepared using the yellow fever virus outer membrane E protein has high detection sensitivity and specificity, and can be effectively used for the detection of positive antibodies produced after infection with yellow fever virus.
[0019] The magnetic nanoparticle immunochromatography kit for rapid detection of yellow fever virus antibodies provided by the present invention is prepared by using the prepared yellow fever virus outer membrane E protein antigen. The immunochromatography reagent is used for detecting yellow fever virus antibodies.
[0020] The magnetic nanoparticle immunochromatographic reagent for detecting yellow fever virus antibodies provided by the present invention can capture more test objects in the system through magnetic adsorption enrichment, and improve the detection sensitivity through enrichment. The reagent cost is low, the detection reagent solution is stable, the specificity is strong, the sensitivity is high, and the sensitivity is 10 times higher than that of the colloidal gold detection result. The detection result is accurate and reliable, the operation is simple, and strong professional skills are not required for operation. The detection result can be judged by naked eye observation, and it can be judged without instruments, and it is easy to use. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The electrophoresis results of the uninduced bacterial solution and the induced bacterial solution are shown in Figure 2. Figure 2 This is the electrophoresis result of purified recombinant E antigen protein; Figure 3 The result of testing positive samples using the magnetic nanoparticle immunochromatographic rapid detection reagent for yellow fever virus antibodies prepared by the present invention; Figure 4 This is the result of testing positive samples using colloidal gold chromatography test paper. DETAILED DESCRIPTION
[0022] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings. If not particularly explained in the following embodiments, conventional technical methods in the art are adopted.
[0023] Example 1 Preparation of recombinant yellow fever virus outer membrane E protein antigen (1) Construction of recombinant plasmid The yellow fever virus outer membrane E protein was selected, and its nucleotide sequence (GNnBank sequence number: NP_740305.1) was modified according to the preferred codons of Escherichia coli O127:H6. After a large number of experimental studies, a higher expression efficiency was obtained, and the exogenous protein expression efficiency reached more than 73%. The secondary structure of the recombinant protein was screened by bioinformatics, and the optimized gene sequence was obtained as shown in SEQ ID NO.2, and its corresponding amino acid sequence was shown in SEQ ID NO.1. Shanghai Shenggong Company was commissioned to synthesize the optimized gene sequence (SEQ ID NO.2), and the gene was cloned into the expression vector PGEX-4T-2 to obtain the recombinant plasmid. Experimental studies have shown that if the unoptimized gene sequence is used for recombinant expression, the recombinant protein cannot be obtained.
[0024] SEQ ID NO.1: GTSYKICTDKMFFVKNPTDTGHGTVVMQVKVSKGAPCRIPVIVADDLTAAINKGILVTVNPIASTNDDEVLIEVNPPFGDSYIIVGRGDSRLTYQWHKEG SEQ ID NO.2: GGTACCTCTTACAAGATCTGTACCGACAAGATGTTCTTCGTTAAGAACCCTACCGACACCGGTCACGGTACCGTTGTTATGCAAGTTAAGGTTTCTAAGGGTGCTCCATGTCGTATCCCTGTTATCGTTGCTGACGACTTGACCGCTG CTATCAACAAGGGTATCCTGGTTACCGTTAACCCAATCGCTTCTACCAACGACGACGAAGTTTTGATCGAAGTTAACCCACCATTCGGTGACTCTTACATCATCGTTGGTCGTGGTGACTCTAGATTGACCTACCAATGGCACAAGGAAGGT.
[0025] (2) Expression and identification of recombinant proteins The recombinant plasmid with correct sequencing was transformed into Escherichia coli BL21, and the single colony with correct identification was inoculated into LB liquid culture medium containing ampicillin at a concentration of 10μg / mL, and cultured at 37℃ overnight. The next day, it was inoculated into a new LB liquid culture medium containing ampicillin at a ratio of 1:100 and cultured until OD600 reached 0.6~0.8, then the bacterial solution temperature was cooled to 22℃, and IPTG was added to make its final concentration 0.8 mmol / L, and induced expression was performed at 22℃; and 1mL of bacterial solution was aspirated after 1, 2, 3, 4, 5, and 6 hours, ultrasonic lysis, and bacterial solution was collected, boiled in a water bath for 10 minutes, centrifuged at 4℃ and 12000r / min for 5 minutes, placed on ice, and the supernatant and precipitate were aspirated for SDS-PAGN electrophoresis detection; after the electrophoresis, the gel was stained with Coomassie Brilliant Blue for 80 minutes, and then decolorized for 2 hours to observe the protein induction expression. The target protein with a molecular weight of about 38KD was expressed from 1 hour after induction, and the yield was the highest at 6 hours. The results are as follows Figure 1 As shown, from left to right in the figure, the first band M is Mark, the second band is the band of bacterial liquid precipitation after uninduction, and the third band is the band of bacterial liquid precipitation after uninduction.
[0026] (3) Purification of recombinant protein 2 mL of bacterial solution after 6 hours of induction expression was taken, and centrifuged at 4°C, 12000 r / min for 30 minutes. The cell pellet was collected and washed three times with 0.01M pH7.4 PBS, and suspended in pH8.0 lysis buffer (Tris-NaCl). After sonication and centrifugation, the pellet was suspended in 8 mol / L urea at pH8.0, and shaken on ice for 2 hours to dissolve the inclusion bodies. The supernatant was filtered through a 0.22 μm filter membrane and purified using a His Trap TMHP (histidine) affinity adsorption column purification system. The purity was identified by SDS-PAGN. Finally, the purified protein was dialyzed into pH7.4, 0.01 mol / L PBS and concentrated by PNG-20000 to obtain a purified recombinant protein with a molecular weight of about 38 KD. The results are shown in Figure 2. Figure 2 As shown, from left to right in the figure, the purified recombinant protein marked as E, M is Mark. 2 mg of the purified recombinant protein was taken to prepare polyclonal antibodies, and rabbits were immunized using conventional methods to obtain positive rabbit serum, which was purified to obtain rabbit anti-E protein polyclonal antibodies.
[0027] Example 2 Preparation of a reagent for rapid detection of yellow fever virus antibodies S1. Magnetic nanoparticle capture reagent: Take 200 μL of ferroferric oxide magnetic nanoparticle solution with a particle size of 20 nm and a concentration of 2 mg / mL, and use 200 μL of 22% glutaraldehyde solution to carboxylate the ferroferric oxide magnetic nanoparticles for 2 hours; then wash them with PBS solution for 3 times, discard the supernatant solution after magnetic adsorption, add 500 μL of yellow fever virus outer membrane E protein prepared in Example 1 with a concentration of 1.5 mg / mL to the ferroferric oxide magnetic nanoparticles, and shake and incubate for 2 hours; after magnetic adsorption, discard the solution, add 800 μL of 0.8% BSA and 5% sucrose solution, shake and incubate for 30 minutes, and block; after magnetic adsorption, discard the solution, add PBS containing 0.05% Tween-20 with a concentration of 0.01 mol / L 1mL, wash 3 times; add 1mL resuspension to obtain E protein labeled ferroferric oxide magnetic nanoparticle capture reagent solution, wherein the resuspension contains 0.1% Tris base, 2% BSA and 2% Triton X-100 solution by weight, mix well and store at 4°C. A large number of experimental studies have shown that the optimal magnetic particle dispersion is obtained by using this ratio of resuspension.
[0028] S2. Preparation of glass fiber mat: soak the glass fiber in a heavy suspension containing 0.1% by weight of Tris base, 2% by weight of BSA and 2% by weight of Triton X-100 for 30 minutes, take it out, dry it in an oven at 37°C for 3 hours, and then set it aside for use.
[0029] S3. Preparation of nitrocellulose test membrane: Using PBS (pH 7.4, 0.01 M) as coating buffer, dilute 2 mg / mL rabbit anti-E protein polyclonal antibody and 1.6 mg / mL yellow fever virus outer membrane E protein prepared in Example 1 to prepare quality control line coating solution and test line coating solution. Use a coating machine to streak the quality control line and test line working solutions onto the nitrocellulose membrane, dry in a drying oven at 37°C, and store dry.
[0030] S4. Sample pad treatment: The glass fiber was immersed in a 0.05 mol / L PBS treatment solution containing 2.5% Tween-80 for 1 hour, and then dried at 37°C for 3 hours to obtain a sample pad.
[0031] S5. Assemble into detection reagents: The sample pad, glass fiber pad, nitrocellulose test membrane, and absorbent pad are sequentially pasted on the PVC base plate, wherein the end of the nitrocellulose test membrane close to the quality control line is covered with the absorbent pad, and the other end close to the test line is covered with the glass fiber pad. The immunochromatographic test paper with a width of 4 mm is cut and finally the immunochromatographic test paper is loaded into the card shell to obtain the test reagent card. The absorbent pad is absorbent paper or pure cotton lint filter paper.
[0032] When testing for yellow fever virus antibodies, take 500 μL of the serum sample to be tested and add 20 μL of magnetic nanoparticle capture reagent, mix well, and place on a magnetic stand after 5 minutes. After 1 minute, the magnetic beads are enriched near the magnetic field, and the liquid is discarded. Then 100 μL of 0.01M PBS is added to resuspend the magnetic nanoparticle capture reagent, and then all are dripped on the sample pad. Then 100 μL of TMB application solution (containing a solution with a TMB content of 5 mg / ml and a molar concentration of hydrogen peroxide of 0.8 m mol / L) is dripped on the sample pad, and the result is judged after 10 minutes. If only the quality control band appears brown-yellow, the result is negative, indicating that the serum sample to be tested does not contain yellow fever virus antibodies; if both the quality control band and the test band appear brown-yellow, the result is positive, indicating that the serum sample to be tested contains yellow fever virus antibodies; if neither of the two bands is colored or only the test band is colored, it means that the detection reagent has expired and the result is invalid.
[0033] The invention discloses a magnetic ferroferric oxide nanoparticle capture reagent labeled with yellow fever virus outer membrane E protein, and a detection reagent strip with rabbit anti-E protein polyclonal antibody as a quality control line and yellow fever virus outer membrane E protein as a detection line for detecting yellow fever virus antibodies. Example 3
[0034] The yellow fever virus outer membrane E protein-labeled ferroferric oxide magnetic nanoparticle capture reagent prepared in Example 2, a detection strip with rabbit anti-E protein polyclonal antibody as the quality control line and yellow fever virus outer membrane E protein as the detection line, is used to detect a yellow fever virus antibody positive serum sample. The sample is diluted 10 times with PBS solution, and the sample volume is 100 μL. The ferroferric oxide magnetic nanoparticle capture reagent labeled with yellow fever virus outer membrane E protein is directly added to the sample for detection. The detection result shows that 10 -1 -10 -6 The results of the gradient dilution samples were all positive, 10 -7 The sample diluted 100 times is negative, see Figure 3 In the figure shown, 1-7 are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Serially dilute the samples and 8 blank PBS solutions.
[0035] At the same time, as a comparison: the colloidal gold chromatography test paper prepared by the yellow fever virus outer membrane E protein prepared in Example 1 was prepared by a conventional method, SPA was used to label the colloidal gold particles, the detection band on the nitrocellulose membrane was coated with the yellow fever virus outer membrane E protein antigen, and the quality control band was coated with goat anti-mouse IgG; the obtained colloidal gold immunochromatographic test paper was used to detect the positive samples of yellow fever virus antibodies after 10-fold dilution with PBS solution, and the sample volume was 50 μL. The test results showed that 10 -1 -10 -5 The results of the gradient dilution of the positive sample are shown in Figure 4 As shown, 1-7 in the figure are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 The gradient dilution samples and 8 are blank PBS solutions. Under the same conditions, the test results show that the magnetic nanoparticle immunochromatographic rapid detection of yellow fever virus antibodies of the present invention has a higher sensitivity than the colloidal gold chromatography test paper, and the sensitivity is improved by 10 times.
[0036] Example 4 Specificity Detection The yellow fever virus outer membrane E protein-labeled ferroferric oxide magnetic nanoparticle capture reagent prepared by the method of Example 2, a detection reagent strip with rabbit anti-E protein polyclonal antibody as the quality control line and yellow fever virus outer membrane E protein as the detection line, and a commercial yellow fever antibody ELISA detection kit of NOVABIOS, USA, were used to simultaneously detect 42 normal human sera, and the detection results of the two methods were all negative (-) results; specific samples were also detected: plague positive serum, chikungunya fever positive serum, influenza A H1N1 virus positive serum, American eastern equine encephalitis virus positive serum, Japanese encephalitis positive serum and dengue virus positive serum (the above positive serum was provided by the Health Quarantine Laboratory of the Science and Technology Research Center of China Customs), and the detection results of the two methods were all negative (-) results, indicating that the magnetic nanoparticle immunochromatography rapid detection of yellow fever virus antibodies has accurate results and strong specificity.
[0037] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any person skilled in the art can use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.
Claims
1. A rapid detection reagent for yellow fever virus antibodies, characterized in that: The invention comprises a magnetic nanoparticle capture reagent and an immunochromatographic test paper which is composed of a sample pad, a glass fiber pad, a nitrocellulose detection membrane and a water-absorbing pad which are overlapped in sequence and attached to a bottom lining card, wherein the nitrocellulose detection membrane is coated with a quality control line of a rabbit anti-E protein polyclonal antibody and a detection line of the outer membrane E protein of the yellow fever virus, and the magnetic nanoparticle capture reagent is ferroferric oxide magnetic nanoparticles marked with the outer membrane E protein of the yellow fever virus.
2. The rapid detection reagent according to claim 1, characterized in that The amino acid sequence of the yellow fever virus outer membrane E protein is shown in SEQ ID NO.
1.
3. The rapid detection reagent according to claim 1, characterized in that The expression of the yellow fever virus outer membrane E protein is carried out using the gene sequence shown in SEQ ID NO.
2.
4. The rapid detection reagent according to claim 1, characterized in that The sample pad is a glass fiber membrane that has been soaked in a treatment solution containing Tween-80.
5. The rapid detection reagent according to claim 1, characterized in that The glass fiber mat is a glass fiber membrane soaked in a solution containing 0.1% by weight of Tris base, 2% by weight of BSA and 2% by weight of Triton X-100.
6. A method for preparing a detection reagent for rapid detection of yellow fever virus antibodies, characterized in that: The method comprises the following steps: S1. After carboxylation of ferroferric oxide magnetic nanoparticles, yellow fever virus outer membrane E protein is added for labeling to obtain yellow fever virus outer membrane E protein labeled ferroferric oxide magnetic nanoparticle capture reagent; S2, soaking the glass fiber membrane in the heavy suspension and drying it to obtain a glass fiber mat; S3. A quality control line and a detection line are provided on the nitrocellulose membrane, the quality control line is coated with rabbit anti-E protein polyclonal antibody, and the detection line is coated with yellow fever virus outer membrane E protein, to obtain a nitrocellulose detection membrane; S4, immersing the glass fiber membrane in a treatment solution containing Tween-80, taking it out and drying it to obtain a sample pad; S5. Overlap the sample pad, glass fiber pad, nitrocellulose test membrane, and absorbent pad in sequence and stick them on the backing card. Cover the absorbent pad on one end of the nitrocellulose test membrane close to the quality control line, and cover the glass fiber pad on the other end close to the test line.
7. The preparation method according to claim 6, characterized in that: In step S1, the particle size of the ferroferric oxide magnetic nanoparticles is 15-40 nm, and the carboxylation is carried out using a glutaraldehyde solution; The concentration of the yellow fever virus outer membrane E protein labeling is 1.5 mg / mL, the reaction time during labeling is 2 hours, the solution is discarded after magnetic adsorption, and then 0.8% BSA and 5% sucrose solution are added, and shaking is blocked for 30 minutes; after magnetic adsorption, the solution is discarded, and PBS containing 0.05% Tween-20 with a concentration of 0.01 mol / L is added for washing; and then a resuspension is added to obtain an E protein labeled ferroferric oxide magnetic nanoparticle capture reagent solution.
8. The preparation method according to claim 6, characterized in that: The yellow fever virus outer membrane E protein is obtained by expressing and preparing the gene sequence such as SEQ ID NO.
2.
9. The preparation method according to claim 6, characterized in that: The resuspension solution contains 0.1% by weight of Tris base, 2% by weight of BSA and 2% by weight of Triton X-100 solution.
10. The preparation method according to claim 6, characterized in that: In step S3, the coating concentration of the rabbit anti-E protein polyclonal antibody is 2 mg / mL and the coating concentration of the yellow fever virus outer membrane E protein is 1.6 mg / mL.