CN112630426B - Colloidal gold test strip for detecting novel coronavirus COVID-19 - Google Patents
Colloidal gold test strip for detecting novel coronavirus COVID-19 Download PDFInfo
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Abstract
The invention provides a colloidal gold test strip for detecting novel coronavirus COVID-19, which is coated with: the novel coronavirus COVID-19 antigen is a recombinant antigen of S gene and partial fragment of N gene of the novel coronavirus COVID-19, and preferably, the sequence of the recombinant antigen is shown as SEQ ID NO. 6. The invention further provides a preparation method of the recombinant antigen. The test strip provided by the invention is convenient to operate, simple in sampling, rapid in result judgment, suitable for on-site rapid detection, and also suitable for non-professional operation.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a novel coronavirus COVID-19 colloidal gold detection test strip and a preparation method thereof.
Background
2019 novel coronavirus (2019-nCoV), belonging to the genus beta, has envelope, and the particles are round or elliptic, often polymorphic, and have diameters of 60-140 nm. The current research shows that the homology with bat SARS-like coronavirus (bat-SL-CoVZC 45) is more than 85%. At present, the preliminary judgment of spread of droplets, contact spread and faeces spread of coughing or sneezing of the COVID-19 is that mother and infant spread is possible, and spread can be carried out from person to person, and the crowd is generally susceptible because the crowd generally has no immunity to 2019-nCoV, and the aged or people with basic diseases are more easily infected. The COVID-19 mainly shows fever, hypodynamia, dry cough, the light patients only show low fever, slight hypodynamia and the like, no pneumonia shows, and partial patients have slight symptoms, can not have fever and recover after more than 1 week. Most patients have good prognosis, few patients have critical illness, severe patients have dyspnea and/or hypoxia after 1 week of onset, severe patients rapidly progress to acute respiratory distress syndrome, sepsis shock, metabolic acidosis and clotting dysfunction which are difficult to correct, and infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death.
Currently, detection of 2019 for the novel coronavirus disease covd-19 involves mainly one of the following methods.
Peripheral blood examination shows that peripheral blood leucocyte count is normal or reduced, lymphocyte count is reduced, C reactive protein and blood sedimentation are raised and procalcitonin is normal in most patients. It is believed that 2019-nCoV can destroy autoimmune functions of human bodies, bacterial infection causes increase of total number of neutrophil and leucocyte of peripheral blood neutral granulocyte, lymphocyte decrease, lymphocyte increase, and when mixed infection is carried out, total number of peripheral blood leucocyte is not increased after mutual neutralization, neutral granulocyte increase is caused by bacterial infection, and lymphocyte count is relatively reduced. The method is a preliminary screening of symptomatic patients.
Nucleic acid detection, mainly using pharyngeal swabs, nasal swabs, sputum, alveolar lavage, feces, blood or lung tissue, using real-time fluorescent quantitative PCR (RT-PCR) to detect 2019-nCoV RNA components extracted from the specimen, positive for the infected 2019-nCoV. The method is complex to operate, long in time and capable of being operated by a large instrument such as a fluorescent PCR instrument, but the method is high in detection accuracy and can be completed in a large hospital.
Virus isolation and culture 2019-nCoV from cells is difficult and needs to be performed in a biosafety laboratory of the designated three stages, which can be referred to WHO-related virus isolation and culture standard procedures. The method has the highest detection accuracy, but has extremely high requirements on experimental conditions, and cannot be completed in common laboratories and hospitals.
In the above methods, the peripheral blood screening is only preliminary screening, and the nucleic acid detection and virus isolation culture have higher accuracy, but all require corresponding equipment to complete, and the time is long, so that the requirements of on-site detection cannot be met.
Disclosure of Invention
In this regard, the invention provides a 2019-nCoV colloidal gold test strip for serodiagnosis and detection of 2019-nCoV specific IgM and IgG antibody levels. The test strip provided by the invention is convenient to operate, simple in sampling, rapid in result judgment, suitable for on-site rapid detection, and also suitable for non-professional operation.
Specifically, the invention provides a colloidal gold test strip for detecting novel coronavirus COVID-19, which is coated with: the anti-human mouse IgG, anti-human mouse IgM, anti-chicken IgY, and gold-labeled novel coronavirus COVID-19 antigen and gold-labeled chicken IgY, wherein the novel coronavirus COVID-19 antigen is a recombinant antigen of S gene and partial fragment of N gene of the novel coronavirus COVID-19.
Preferably, the sequence of the recombinant antigen is shown as SEQ ID NO. 6.
The invention further provides a novel coronavirus COVID-19 antigen for preparing the test strip according to claim 1, wherein the sequence of the antigen is shown as SEQ ID NO. 6.
The invention further provides a nucleotide sequence encoding the antigen.
Preferably, the sequence is shown in SEQ ID NO. 5.
The invention further provides a plasmid vector containing the nucleotide.
The invention further provides a recombinant cell comprising said recombinant vector.
Preferably, the cell is a CHO cell.
The invention further provides a method for preparing the antigen, which is obtained by inducing expression of the recombinant cells and separating and purifying the recombinant cells.
The invention further claims the use of the antigen, the nucleotide, the plasmid vector and the recombinant cell in preparing a colloidal gold test strip for detecting novel coronavirus COVID-19.
The invention further discloses a preparation method of the colloidal gold test strip, which comprises the following steps: 1. the production process of the gold-labeled chicken IgY; 2. preparing a gold-labeled antigen; 3. preparing a gold mark pad; 4. preparing a coating film; 5. and (5) preparing a gold-labeled large plate.
Drawings
FIG. 1 shows the result of PCR detection electrophoresis of recombinant nucleotide sequences of novel coronaviruses
FIG. 2 is an electrophoretogram of novel coronavirus recombinant protein after gel chromatography purification
Fig. 3 is a graph showing a determination result of a detection result of a coronavirus disease covd-19 colloidal gold test strip, wherein the determination result is respectively from left to right: igG positive IgM positive; igG positive; positive IgM; negative.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1 novel coronavirus dominant epitope selection
From the structure of the novel coronavirus, the spike glycoprotein S is exposed on the outer surface of the whole virus, and the corresponding nucleocapsid protein N is also present in the viral nucleic acid released after cleavage of the virus. Therefore, the invention selects part of the S gene and N gene of the virus as dominant antigen epitope.
The hydrophilicity, antigenicity, and hydrophobicity of the novel coronavirus S and N fragment antigens were analyzed using the biological software EPIQUEST, while focusing on NO significant homology to other antigen sequences other than coronavirus antigens, the S antigen fragment (SEQ ID NO: 2) and the N antigen fragment (SEQ ID NO: 4) were obtained, respectively.
EXAMPLE 2 construction of recombinant antigen expression vectors
After the nucleotide sequences (SEQ ID NO: 1) for encoding the S antigen fragment and the nucleotide sequences (SEQ ID NO: 3) for encoding the N antigen fragment are connected in series, complete gene synthesis is carried out to obtain a recombinant nucleotide sequence (SEQ ID NO: 5) for encoding the fusion S antigen fragment and the N antigen fragment, wherein the recombinant protein sequence encoded by the recombinant nucleotide sequence (SEQ ID NO: 5) is a recombinant novel coronavirus S and N antigen fragment (SEQ ID NO: 6).
In the synthesis of the recombinant nucleotide sequence (SEQ ID NO: 5), bglI and BamHI cleavage sites were introduced at both ends of the sequence, respectively. Cloning the recombinant nucleotide sequence (SEQ ID NO: 5) into a high-efficiency eukaryotic expression vector phCMV1 by a conventional PCR and molecular cloning method to obtain a recombinant expression vector phCMV1-CoV, and carrying out PCR detection on the recombinant vector. The liposome method transfected CHO cells, and G418 (800 LgPml) was used to select resistant clones. The selection was maintained for 8 days with one change every 3 days. Positive cell clones were selected and maintained in culture with a medium containing G418 (300 LgPml). When the positive clone is 95% pooled, the culture medium is replaced by CHO serum-free medium. Serum-free culture solutions for 24, 48 and 72 hours were collected and cultured, 5000rP/min was centrifuged for 5 minutes, and the supernatant-20 e was stored for SDS to detect the expression level of the target protein in the serum-free medium.
EXAMPLE 3 purification of antigen of interest
And (3) separating and purifying the recombinant novel coronavirus S and N antigen fragments secreted and expressed in the serum-free culture medium by adopting a gel filtration method. Accurately weighing 15g of Sephadex G-100 gel, soaking and swelling with deionized water for more than 20h, pouring the supernatant, pouring into a chromatographic column, and eluting with PB buffer solution (pH 8.8) until the pH value of the effluent is close to that of the PB buffer solution. Serum-free culture medium containing target protein is slowly injected into the gel column, and the flow rate is controlled to be 1mL/min, so that the sample and the gel have full action. When the sample was sufficiently immersed in the gel, PB buffer was slowly injected for elution, with a constant flow rate of 3 mL/tube. The 20 tubes were collected, concentrated by polyethylene glycol dialysis, and the recombinant protein was approximately 56KD in size as detected by SDS-PAGE.
Example 4 preparation of COVID-19 colloidal gold test strip for coronavirus disease
Materials: 10% chloroauric acid, 12% trisodium citrate and purified water. Taking 200ml as an example, heating to boiling for 5 minutes, adding chloroauric acid, stirring and heating for 5 minutes, and adding trisodium citrate and heating for 10 minutes. After cooling, the volume was restored to 200ml. Rabbit anti-chicken IgY-quality control region: diluted to 1mg/ml with 0.02 MPB. Can be adjusted according to the properties of the raw materials. Murine anti-human IgG-detection line G region: diluted to 1mg/ml with 0.02M PB. Can be adjusted according to the properties of the raw materials. Murine anti-human IgM-detection line M region: diluted to 1mg/ml with 0.02M PB. Can be adjusted according to the properties of the raw materials.
1. The gold-labeled chicken IgY production process includes re-dissolving the working solution, 0.02M phosphate buffer solution, 0.5% BSA, 5% sucrose and 0.2% Tween-20.PH 7.4, and is prepared at present. 10% BSA, pH 7.0. Colorless or yellowish clear solution, without impurity. Is prepared in the prior art. 0.2M PB (pH 7.4) was added to 50. Mu.l/ml colloidal gold to adjust the pH. Antigen ratio: typically 10ug chicken IgY/ml colloidal gold. Can be adjusted according to the properties of the raw materials. Marking time: 5 minutes. Closed condition: 0.5% BSA for 10 min. The separation method comprises the following steps: low temperature and high speed centrifugation. 10000rpm for 15 minutes, the upper light solution was discarded and the lower dark precipitate was retained. The solution was reconstituted with 10% by volume of working fluid.
2. Preparation of gold-labeled antigen
1) Taking 0.2M PB (pH 7.4), 10% BSA and neocrown antigen (prepared in example 3) according to production requirements, taking colloidal gold from chicken IgY by using a glass graduated cylinder, and adding the colloidal gold into a container; taking 0.2M PB by using a pipette according to the number of instruction sheets, and adding the PB into a container; the new crown antigen was taken using a pipette and added to the container. Mixing for 5 minutes; 10% BSA was pipetted into the container. Mixing for 10 min; the solution was placed in a centrifuge tube. Centrifuging at 2-8deg.C and 10000rpm for 15 min, discarding upper light solution, and retaining lower dark precipitate; and taking gold-labeled diluent by using a pipette, adding the diluent into each centrifuge tube, and redissolving and precipitating. 2) Taking colloidal gold by using a glass measuring cylinder, and adding the colloidal gold into a container; pipette 0.2M PB into the vessel; according to the number of instruction sheets, the chicken IgY is taken by using a liquid transfer device and added into a container. Mixing for 5 minutes; 10% BSA was pipetted into the container. Mixing for 10 min; the solution was placed in a centrifuge tube. Centrifuging at 10000rpm for 15 min at 2-8deg.C; discarding the upper light color solution, and reserving the lower dark color sediment; and taking gold-labeled diluent by using a pipette, adding the diluent into each centrifuge tube, and redissolving and precipitating. 3) Mixing the gold-labeled COVID-19 antigen prepared in the step 1) and the step 2) with the gold-labeled chicken IgY in equal volume.
3. Preparing a gold mark pad, wherein a glass fiber single layer is obliquely arranged on a screen window; slowly and uniformly spreading gold-labeled antigen on glass fiber by a pipette; and (3) keeping the indoor air humidity at not more than 30%, and placing the laid gold-labeled pad in a drying oven to be dried for 20-28 hours at 37 ℃. The gold-labeled pad was cut into small strips at a width of 5 mm. The edge portion was discarded.
4. The preparation of the coating film comprises the steps of taking a film plate, a coating liquid (T-G) -mouse anti-human IgG (COVID-19), a coating liquid (T-M) -mouse anti-human IgM (IgM), and a coating liquid (C) -rabbit anti-chicken IgY (C) -according to production requirements. The film drawing machine is cleaned before use according to the requirements. The scribing distance of the scribing machine is set to be 0.5cm, and the coating volume is set to be 1ul/cm. The film plate is punched with water absorbing paper. The film dividing position is in the middle of the nitric acid film, and the coating is started after the film dividing position is positioned by a magnetic stripe.
5. Preparing a gold-labeled large plate, namely taking a coating film, a gold-labeled pad and a sample pad according to production requirements; sticking gold label pad: the upper edge of the gold paper is covered with the lower edge of the nitrocellulose membrane by about 1.0mm (0.5-2 mm); sticking a sample pad: the upper edge of the sample pad should be 1/2 of the gold mark pad, the highest must not exceed the gold mark pad, and the lowest must not leave the lower edge of the gold mark pad. And (5) loading the assembled large plate into a large sealing pocket, adding a drying agent, and sealing by placing an aluminum foil pocket.
Example 5 determination of the detection accuracy of coronavirus COVID-19 colloidal gold test strip
774 patients were diagnosed with the disease by PCR and CT methods, and the detection results are shown in Table 1 below.
Table 1: clinical sample conditions
The colloidal gold method of the present invention was used to recheck for each of the above identified, suspected patients and healthy individuals, with the results shown in tables 2-4 below.
Table 2: accuracy protocol for colloidal gold detection of diagnosed patients
The relative accuracy of the detection is: 216/243=88.9%
Table 3: detection of suspicious samples
Antibody detection rate=43/125=34.4%
Table 4: detection of healthy people
Relative detection rate=401/406=98.8%
Accuracy = (216+401)/(243+406) =95.1%
Example 6 detection sensitivity determination of coronavirus disease COVID-19 colloidal gold test strip
Samples of two cases of the diagnosed coronavirus disease COVID-19 were diluted 1:2,1:4,1:8,1:16,1:64,1:126,1:256,1:1024,1:2048, respectively, and then assayed using the colloidal gold kit of the invention, with the following results:
table 5: colloidal gold sensitivity detection result
++ Strong positive+positive.+ -. Weak positive-negative
Example 7 detection stability assay of coronavirus disease COVID-19 colloidal gold test strip
Taking 3 batches of colloidal gold test strips produced at different dates, and respectively placing the colloidal gold test strips in an environment with the relative humidity of 60% at 45 ℃ for 0,7,14 days. And then the samples are respectively measured. The measurement results were as follows:
table 6: colloidal gold test strip stability detection result
3-means that the negative sample is negative after 3 repeated tests
3+ indicates that the positive sample is positive after 3 repeated detection
The detection result shows that the colloidal gold test strips produced in different batches are stored for 0,7,14 days in an environment with the relative humidity of 60% at 45 ℃, and higher detection accuracy can still be ensured, so that the stability of the test strips is higher.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
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cctgctaaca atgctgcaat cgtgctacaa cttcctcaag gaacaacatt gccaaaaggc 1380
ttctacgcag aagggagcag aggcggcagt caagcctctt ctcgttcctc atcacgtagt 1440
cgcaacagtt caagaaattc aactccaggc agcagtaggg gaacttctcc tgctagaatg 1500
gctggcaatg gcggtgatgc tgctcttgct ttgctgctgc ttgacagatt gaaccagctt 1560
gagagcaaa 1569
<210> 6
<211> 523
<212> PRT
<213> person ()
<400> 6
Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu
1 5 10 15
Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg
20 25 30
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
35 40 45
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
50 55 60
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
65 70 75 80
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
85 90 95
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
100 105 110
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
115 120 125
Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
130 135 140
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
145 150 155 160
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
165 170 175
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro
180 185 190
Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
195 200 205
Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
210 215 220
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
225 230 235 240
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe
245 250 255
Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe
260 265 270
Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala
275 280 285
Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser
290 295 300
Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln
305 310 315 320
Val Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala
325 330 335
Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly
340 345 350
Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His
355 360 365
Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Thr Arg Arg Ile
370 375 380
Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe
385 390 395 400
Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn
405 410 415
Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro
420 425 430
Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val
435 440 445
Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu
450 455 460
Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser
465 470 475 480
Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser
485 490 495
Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu
500 505 510
Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys
515 520
Claims (7)
1. A colloidal gold test strip for detecting novel coronavirus covd-19, characterized in that the test strip is coated with: the novel coronavirus COVID-19 antigen is a recombinant antigen of a partial fragment of an S gene and a partial fragment of an N gene of the novel coronavirus COVID-19, and the sequence of the recombinant antigen is shown as SEQ ID NO: shown at 6.
2. A novel coronavirus covd-19 antigen for use in the preparation of the test strip of claim 1, wherein the antigen has the sequence set forth in SEQ ID NO: shown at 6.
3. A nucleotide sequence according to SEQ ID NO: shown at 5.
4. A plasmid vector comprising the nucleotide sequence of claim 3.
5. A recombinant cell comprising the plasmid vector of claim 4, wherein the cell is a CHO cell.
6. A method for producing the antigen according to claim 2, characterized in that the recombinant cell according to claim 5 is subjected to induction expression, and isolated and purified.
7. Use of the antigen of claim 2, the nucleotide of claim 3, the plasmid vector of claim 4 and the recombinant cell of claim 5 for preparing a colloidal gold test strip for detecting novel coronavirus covd-19.
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Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2004245896A1 (en) * | 2003-06-10 | 2004-12-16 | Agency For Science, Technology And Research | Method of diagnosing SARS corona virus infection |
| WO2005042579A1 (en) * | 2003-10-31 | 2005-05-12 | Fujirebio Inc. | Anti-sars virus antibody, hybridoma producing the antibody and immunoassay reagent using the antibody |
| CN106053785A (en) * | 2016-05-18 | 2016-10-26 | 北京北方生物技术研究所有限公司 | Solid antibody pre-coating method for competitive chemiluminescent immunoreactions |
| CN111187354A (en) * | 2020-02-20 | 2020-05-22 | 北京新创生物工程有限公司 | Novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection kit |
| CN111239392A (en) * | 2020-02-26 | 2020-06-05 | 浙江诺迦生物科技有限公司 | Novel coronavirus pneumonia (COVID-19) serological diagnosis kit |
| CN111426842A (en) * | 2020-02-21 | 2020-07-17 | 南京岚煜生物科技有限公司 | Novel coronavirus IgM/IgG detection reagent, reagent card, kit and preparation method thereof |
| CN111505314A (en) * | 2020-05-07 | 2020-08-07 | 厦门万渤生物技术有限公司 | Novel combined detection kit for coronavirus antibody and application thereof |
| CN111537720A (en) * | 2020-05-25 | 2020-08-14 | 浙江峻山生物科技有限公司 | Preparation method of novel coronavirus 2019-nCoV IgM/IgG antibody combined detection kit |
| CN111638332A (en) * | 2020-07-13 | 2020-09-08 | 浙江省人民医院 | Novel coronavirus IgA/IgM/IgG antibody ELISA detection kit |
| CN111735962A (en) * | 2020-06-08 | 2020-10-02 | 南通伊仕生物技术股份有限公司 | Novel coronavirus IgM/IgG antibody joint detection immunochromatography test strip |
| CN111796096A (en) * | 2020-04-30 | 2020-10-20 | 江苏达伯药业有限公司 | Preparation method of novel coronavirus IgGIgM antibody combined detection reagent |
| CN111856003A (en) * | 2020-02-04 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Novel coronavirus (2019-nCoV) IgM and IgG antibody detection test strip, kit and method |
| CN111848751A (en) * | 2020-06-18 | 2020-10-30 | 河南省生物工程技术研究中心 | Dominant epitope antigen of new coronavirus S protein and application thereof |
| CN111848752A (en) * | 2020-06-18 | 2020-10-30 | 河南省生物工程技术研究中心 | N protein dominant epitope antigen of new coronavirus and application thereof |
| CN111879951A (en) * | 2020-08-17 | 2020-11-03 | 南通伊仕生物技术股份有限公司 | Novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip |
| CN111896747A (en) * | 2020-08-02 | 2020-11-06 | 广州德成生物科技有限公司 | Immunochromatography test paper for detecting novel coronavirus IgM and IgG |
-
2020
- 2020-11-17 CN CN202011283294.5A patent/CN112630426B/en active Active
Patent Citations (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1639374A1 (en) * | 2003-06-10 | 2006-03-29 | Agency for Science, Technology and Research | Method of diagnosing sars corona virus infection |
| AU2004245896A1 (en) * | 2003-06-10 | 2004-12-16 | Agency For Science, Technology And Research | Method of diagnosing SARS corona virus infection |
| WO2005042579A1 (en) * | 2003-10-31 | 2005-05-12 | Fujirebio Inc. | Anti-sars virus antibody, hybridoma producing the antibody and immunoassay reagent using the antibody |
| CN106053785A (en) * | 2016-05-18 | 2016-10-26 | 北京北方生物技术研究所有限公司 | Solid antibody pre-coating method for competitive chemiluminescent immunoreactions |
| CN111856003A (en) * | 2020-02-04 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Novel coronavirus (2019-nCoV) IgM and IgG antibody detection test strip, kit and method |
| CN111187354A (en) * | 2020-02-20 | 2020-05-22 | 北京新创生物工程有限公司 | Novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection kit |
| CN111426842A (en) * | 2020-02-21 | 2020-07-17 | 南京岚煜生物科技有限公司 | Novel coronavirus IgM/IgG detection reagent, reagent card, kit and preparation method thereof |
| CN111239392A (en) * | 2020-02-26 | 2020-06-05 | 浙江诺迦生物科技有限公司 | Novel coronavirus pneumonia (COVID-19) serological diagnosis kit |
| CN111796096A (en) * | 2020-04-30 | 2020-10-20 | 江苏达伯药业有限公司 | Preparation method of novel coronavirus IgGIgM antibody combined detection reagent |
| CN111505314A (en) * | 2020-05-07 | 2020-08-07 | 厦门万渤生物技术有限公司 | Novel combined detection kit for coronavirus antibody and application thereof |
| CN111537720A (en) * | 2020-05-25 | 2020-08-14 | 浙江峻山生物科技有限公司 | Preparation method of novel coronavirus 2019-nCoV IgM/IgG antibody combined detection kit |
| CN111735962A (en) * | 2020-06-08 | 2020-10-02 | 南通伊仕生物技术股份有限公司 | Novel coronavirus IgM/IgG antibody joint detection immunochromatography test strip |
| CN111848751A (en) * | 2020-06-18 | 2020-10-30 | 河南省生物工程技术研究中心 | Dominant epitope antigen of new coronavirus S protein and application thereof |
| CN111848752A (en) * | 2020-06-18 | 2020-10-30 | 河南省生物工程技术研究中心 | N protein dominant epitope antigen of new coronavirus and application thereof |
| CN111638332A (en) * | 2020-07-13 | 2020-09-08 | 浙江省人民医院 | Novel coronavirus IgA/IgM/IgG antibody ELISA detection kit |
| CN111896747A (en) * | 2020-08-02 | 2020-11-06 | 广州德成生物科技有限公司 | Immunochromatography test paper for detecting novel coronavirus IgM and IgG |
| CN111879951A (en) * | 2020-08-17 | 2020-11-03 | 南通伊仕生物技术股份有限公司 | Novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip |
Non-Patent Citations (5)
| Title |
|---|
| Development of a Sensitive Immunochromatographic Method Using Lanthanide Fluorescent Microsphere for Rapid Serodiagnosis of COVID-19;Meng Feng 等;ACS Sens;第5卷(第8期);全文 * |
| Evaluation of Nucleocapsid and Spike Protein-Based Enzyme-Linked Immunosorbent Assays for Detecting Antibodies against SARS-CoV-2;Wanbing Liu 等;J Clin Microbiol;第58卷(第6期);全文 * |
| Evaluations of the serological test in the diagnosis of 2019 novel coronavirus (SARS-CoV-2) infections during the COVID-19 outbreak;Dachuan Lin 等;Eur J Clin Microbiol Infect Dis;第39卷(第12期);全文 * |
| 丙型肝炎病毒抗体胶体金免疫层析法的建立;郑嘉庚, 程缤雁;标记免疫分析与临床;第25卷(第5期);全文 * |
| 用于新型冠状病毒肺炎防治的生物制品研究策略;晏彩霞;李佳;沈鑫;罗丽;李燕;李明远;;四川大学学报(医学版)(第2期);全文 * |
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