CN112858675A

CN112858675A - Novel coronavirus antigen and antibody combined intelligent detection device

Info

Publication number: CN112858675A
Application number: CN202110006935.0A
Authority: CN
Inventors: 陆维克; 陈金树; 张芳; 郭佳花; 高飞; 余立雁
Original assignee: Hangzhou Alltest Biotech Co ltd
Current assignee: Hangzhou Alltest Biotech Co ltd
Priority date: 2021-01-05
Filing date: 2021-01-05
Publication date: 2021-05-28
Anticipated expiration: 2041-01-05
Also published as: CN112858675B

Abstract

The invention relates to the technical field of immunodiagnosis, overcomes the problems of single detection form and low sensitivity of the existing new corona detection device, and discloses a novel coronavirus antigen and antibody combined intelligent detection device, wherein the detection device comprises an upper plastic shell and a lower plastic shell, the upper plastic shell is respectively provided with an antibody detection group and an antigen detection group, the antibody detection group comprises an antibody detection observation window and an antibody detection sample adding hole, and the antigen detection group comprises an antigen detection observation window and an antigen detection sample adding hole; an antibody detection reagent strip clamping groove and an antigen detection reagent strip clamping groove are formed in the inner side of the lower plastic shell; and an antibody detection reagent strip and an antigen detection reagent strip are respectively arranged in the antibody detection reagent strip clamping groove and the antigen detection reagent strip clamping groove. The invention has short detection period, adopts the mode of antigen-antibody combined detection, can directly shorten the detection window period of an antigen product, and has visual and accurate result display and strong specificity.

Description

Novel coronavirus antigen and antibody combined intelligent detection device

Technical Field

The invention relates to the technical field of immunodiagnosis, in particular to a novel coronavirus antigen and antibody combined intelligent detection device.

Background

The novel coronavirus pneumonia (new coronavirus pneumonia for short, COVID-19 for short) is an acute respiratory infectious disease caused by infection of the novel coronavirus (new coronavirus for short, SARS-CoV-2 for short). The novel coronavirus is a new strain of coronavirus that has not been previously discovered in humans. The initial symptoms of the patients are fever, hypodynamia and dry cough, and a few patients are accompanied with upper respiratory tract and digestive tract symptoms such as nasal obstruction, watery nasal discharge, diarrhea and the like. Severe cases often develop dyspnea after 1 week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis and hemorrhagic coagulation dysfunction, multiple organ failure, and the like. At present, effective antiviral drugs aiming at the pathogen are lacked, and mainly used for isolation treatment and symptomatic support treatment.

The current diagnostic methods for SARS-CoV-2 mainly include nucleic acid detection, antibody detection and antigen detection. The nucleic acid detection is currently used as a 'gold standard' for detecting the new coronavirus (SARS-CoV-2), primers and fluorescent probes are designed in a highly conserved region of a novel coronavirus ORF1ab/N gene sequence through fluorescence-PCR, and the virus RNA in a suspected novel coronavirus (SARS-CoV-2) infected patient specimen is amplified and detected through a full-automatic fluorescence PCR instrument, so that the qualitative detection of the novel coronavirus (SARS-CoV-2) RNA is realized, but the operation is complex, a special laboratory and professional training personnel are needed, the detection period is longer, and the labor cost is high. The reagent for detecting the neocorona antigen and the antibody is simple to operate, does not need special instruments and equipment, and can finish diagnosis in a short time. The combined diagnosis can be used as the differential diagnosis of the infection period, the infection is early, the patient does not have immune response, the antibody can not be detected in vivo or the detection rate of the antibody is low, but the antigen concentration is higher; in the middle and later stages after infection, the virus is possibly inhibited to reduce the concentration, and the in-vivo antibody (mainly IgG) is maintained at a certain titer and can be detected, so that the detection rate of the new coronary pneumonia is greatly improved.

The current common methods for detecting the new crown antibody comprise an enzyme-linked immunosorbent assay (ELISA), a chemiluminescence immunoassay and a colloidal gold immunochromatography; the detection of the new crown antigen is mainly focused on immunochromatography. ELISA is a high-sensitivity immunological experiment technology combining antigen, antibody specific reaction and high-efficiency catalytic action of enzyme on a substrate. The detection method has the advantages of high sensitivity, low carrier standardization difficulty, low detection speed, easy pollution and more complicated steps. The chemiluminescence immunoassay combines a high-sensitivity chemiluminescence assay technology with a high-specificity immunoreaction and is used for detecting various antigens, antibodies, hormones and the like. The method has sensitivity higher than that of ELISA, has the characteristics of strong specificity, wide linear range, stable result, simplified operation and the like, and is widely applied to detection of clinical specimens. However, the method has high requirements on equipment, operation and use environment, and a matched chemiluminescence instrument is required during use. The immunochromatography takes colloidal gold particles as a tracer marker, is a novel immunolabeling technology applied to antigen-antibody detection, can obtain a detection result only within 15min through visual observation, breaks through the limitation of the existing detection technology on personnel and places, shortens the detection time, is convenient and quick to operate, and is widely applied to basic medical units and field detection.

At present, the detection of new coronavirus is emphasized and cannot be replaced mutually, so that the development of differential diagnosis capable of simultaneously detecting antigen and antibody is crucial, the antigen and antibody combined detection can effectively shorten the detection window period, improve the positive detection rate and provide double guarantee for various possible risk groups.

The invention discloses a colloidal gold immunochromatographic assay test paper for combined diagnosis of COVID-19 and mycoplasma pneumoniae and a preparation method thereof, and relates to the technical field of immunodiagnosis, aiming at the problem of blank detection of COVID-19 and mycoplasma pneumoniae antibodies in the prior art, the invention discloses a colloidal gold immunochromatographic assay test paper for combined diagnosis of COVID-19 and mycoplasma pneumoniae, which specifically comprises the following preparation steps: 1) preparing a detection line T and a quality control line C; 2) preparing a colloidal gold conjugate treatment pad; 3) preparing detection test paper: and (3) preparing the water absorption pad, the sample pad and the colloidal gold conjugate treatment pad, and connecting and assembling the modified nitrocellulose membrane coated with the antibody and the PVC base plate in sequence to obtain a finished product. The prepared product can accurately detect whether the pneumonia of 2019-nCoV and mycoplasma is caused, the detection period is short, the detection process is simple, the equipment is portable, the antigen detection mode is adopted, the antigen has no incubation period, the result display is visual and accurate, the specificity is strong, the infection of new corona and pulmonary bronchi at different time can be diagnosed, and the detection efficiency is high.

The method has the disadvantages that only antibody detection is adopted, the detection mode is single, and the condition of missed detection is easy to occur.

Summary of the invention

The invention aims to overcome the problems of single detection form and low sensitivity of the existing new corona detection device, and provides a novel coronavirus antigen and antibody combined intelligent detection device, the combined diagnostic reagent has short detection period and simple detection process, the product adopts an antigen and antibody combined detection mode, an antigen product can directly shorten the detection window period, the result display is visual and accurate, the specificity is strong, the antibody product is helpful for identifying recent and past infections, the diagnosis of suspected patients is facilitated, the code information is scanned by APP on site, the information of the patients can be fed back to a related disease control big data platform in time, and the early discovery and the early control are realized.

In order to achieve the purpose, the invention adopts the following technical scheme:

a novel coronavirus antigen and antibody combined intelligent detection device comprises an upper plastic shell and a lower plastic shell, wherein the upper plastic shell is respectively provided with an antibody detection group and an antigen detection group, the antibody detection group comprises an antibody detection observation window and an antibody detection sample adding hole, and the antigen detection group comprises an antigen detection observation window and an antigen detection sample adding hole; an antibody detection reagent strip clamping groove and an antigen detection reagent strip clamping groove are formed in the inner side of the lower plastic shell; an antibody detection reagent strip and an antigen detection reagent strip are respectively arranged in the antibody detection reagent strip clamping groove and the antigen detection reagent strip clamping groove, and the antibody detection reagent strip sequentially consists of an antibody detection sample pad, an antibody detection processing pad, an antibody detection analysis pad and an absorption pad; the antigen detection reagent strip consists of an antigen detection sample pad, an antigen detection processing pad, an antigen detection analysis pad and an absorption pad in sequence.

The upper plastic shell and the lower plastic shell are combined through the fixing protrusions and the fixing boss grooves to fix the whole detection device, the reagent strip clamping grooves are used for placing reagent strips, the reagent strips are added into the reagent strips from the sample adding holes, the test results are observed through the observation windows, the detection device can accurately detect the results of the added reagent, the detection device is small in size, simple and convenient to drip, long in validity period and high in accuracy of the detection results. After the sample adding holes are used for adding samples, the samples are combined with the labeling pad in advance, the combined compound is migrated to the nitrocellulose membrane by the buffer solution for chromatography, and the sample and the raw materials on the labeling pad can be combined more fully by the two-window sample adding mode, so that the clinical detection rate is improved. And after the template is added with sample, the added sample cannot be influenced by the impact force of the buffer solution, so that the sample is impacted to the edge of the template, and the condition of missing detection is caused.

Preferably, the capture protein of the detection line IgM on the antibody detection and analysis pad is mouse anti-human IgM, the capture protein of the detection line IgG is mouse anti-human IgG, and the capture protein of the quality control line C is goat anti-mouse IgG antibody; the antibody detection processing pad is an antibody colloidal gold conjugate processing pad, and the colloidal gold conjugate is a composition of a novel coronavirus N protein colloidal gold conjugate, a novel coronavirus S protein colloidal gold conjugate and a goat anti-mouse IgG colloidal gold conjugate; or

The colloidal gold conjugate is a composition of a novel coronavirus N protein colloidal gold conjugate, a novel coronavirus RBD protein colloidal gold conjugate and a goat anti-mouse IgG colloidal gold conjugate.

Detection principle of the novel crown antibody: dual target detection based on novel coronavirus N and S proteins:

the reagent strip adopts a highly specific antibody antigen reaction and immunochromatography analysis technology, an analysis pad of the reagent strip contains a pre-coated anti-human IgG monoclonal antibody (IgG detection area) and an anti-human IgM monoclonal antibody (IgM detection area), and a treatment pad of the reagent strip is coated with a novel coronavirus N protein colloidal gold conjugate and a novel coronavirus S protein colloidal gold conjugate. During detection, if the sample contains new coronavirus IgG antibody and IgM antibody to be detected, the new coronavirus N protein colloidal gold conjugate and S protein colloidal gold conjugate on the treatment pad are combined with the substance to be detected to form an IgG-new coronavirus N protein colloidal gold conjugate complex, an IgG-new coronavirus S protein colloidal gold conjugate complex, an IgM-new coronavirus N protein colloidal gold conjugate complex and an IgM-new coronavirus N protein colloidal gold conjugate complex. The antigen-antibody complex is chromatographed upwards under capillary effect, and is respectively combined with an anti-human IgG monoclonal antibody of a detection area (IgG) and an anti-human IgM monoclonal antibody of a detection area (IgM). If the sample contains new coronavirus IgG antibodies, a purple red band appears in the detection area (IgG), if the sample contains new coronavirus IgM antibodies, a purple red band appears in the detection area (IgM), and if the sample does not contain the new coronavirus IgG antibodies and the IgM antibodies, the purple red band does not appear in the detection areas (IgG and IgM). A purple-red band appeared in the control region (C) regardless of the presence of the novel coronavirus antibody in the sample. The purple red band appearing in the quality control area (C) is a standard for judging whether enough samples exist or not and whether the chromatography process is normal or not, and is also used as an internal control standard of the reagent.

Double target detection based on novel coronavirus N protein and RBD protein:

the reagent strip adopts a highly specific antibody antigen reaction and immunochromatography analysis technology, an analysis pad of the reagent strip contains a pre-coated anti-human IgG monoclonal antibody (IgG detection area) and an anti-human IgM monoclonal antibody (IgM detection area), and a treatment pad of the reagent strip is coated with a novel coronavirus N protein colloidal gold conjugate and an RBD protein colloidal gold conjugate. During detection, if the sample contains new coronavirus IgG antibody and IgM antibody to be detected, the new coronavirus N protein colloidal gold conjugate and S protein colloidal gold conjugate on the treatment pad are combined with the substance to be detected to form an IgG-new coronavirus N protein colloidal gold conjugate complex, an IgG-new coronavirus RBD protein colloidal gold conjugate complex, an IgM-new coronavirus N protein colloidal gold conjugate complex and an IgM-new coronavirus RBD protein colloidal gold conjugate complex. The antigen-antibody complex is chromatographed upwards under capillary effect, and is respectively combined with an anti-human IgG monoclonal antibody of a detection area (IgG) and an anti-human IgM monoclonal antibody of a detection area (IgM). If the sample contains new coronavirus IgG antibodies, a purple red band appears in the detection area (IgG), if the sample contains new coronavirus IgM antibodies, a purple red band appears in the detection area (IgM), and if the sample does not contain the new coronavirus IgG antibodies and the IgM antibodies, the purple red band does not appear in the detection areas (IgG and IgM). A purple-red band appeared in the control region (C) regardless of the presence of the novel coronavirus antibody in the sample. The purple red band appearing in the quality control area (C) is a standard for judging whether enough samples exist or not and whether the chromatography process is normal or not, and is also used as an internal control standard of the reagent.

Preferably, the antigen detection processing pad is an antigen colloidal gold conjugate processing pad, and the colloidal gold conjugate is a composition of a novel coronavirus N protein antibody colloidal gold conjugate and a goat anti-mouse IgG colloidal gold conjugate; the capture protein of the corresponding antigen detection analysis pad detection line T is a novel coronavirus N protein antibody, and the capture protein of the quality control line C is a goat anti-mouse IgG antibody; or

The colloidal gold conjugate is a composition of a novel coronavirus N protein antibody colloidal gold conjugate, a novel coronavirus S protein antibody colloidal gold conjugate and a goat anti-mouse IgG colloidal gold conjugate; the capture protein of the corresponding antigen detection analysis pad detection line T is a composition of an anti-novel coronavirus N protein antibody and an anti-novel coronavirus S protein antibody, and the capture protein of the quality control line C is a goat anti-mouse IgG antibody.

The detection principle of the new corona antigen is as follows: novel coronavirus N protein-based assays;

the reagent strip adopts highly specific antibody antigen reaction and immunochromatography analysis technology, the analysis pad of the reagent strip contains a pre-coated novel coronavirus N protein antibody (T detection area), and the treatment pad of the reagent strip is coated with a novel coronavirus N protein antibody colloidal gold conjugate. During detection, if a sample contains the novel coronavirus antigen to be detected, the antigen (including the antigens such as N protein, S protein and the like) in the virus is firstly combined with the novel coronavirus N protein antibody colloidal gold conjugate on the treatment pad after being cracked and released by the cracking buffer solution to form a novel coronavirus antigen-novel coronavirus N protein antibody colloidal gold conjugate compound. The antigen-antibody complex is chromatographed upwards under capillary effect, and is combined with the novel coronavirus N protein antibody in the detection area (T), and a purple red strip appears in the detection area (T), if a sample does not contain the novel coronavirus antigen, no purple red strip exists in the detection area T. A purple-red band appears in the quality control region (C) regardless of whether the novel coronavirus antigen is present in the sample. The purple red band appearing in the quality control area (C) is a standard for judging whether enough samples exist or not and whether the chromatography process is normal or not, and is also used as an internal control standard of the reagent.

Based on the novel coronavirus N protein and S protein dual-target detection:

the reagent strip adopts a highly specific antibody antigen reaction and immunochromatography analysis technology, an analysis pad of the reagent strip contains a novel coronavirus N protein antibody and a novel coronavirus S protein antibody (T detection area) which are coated in advance, and a treatment pad of the reagent strip is coated with a novel coronavirus N protein antibody colloidal gold conjugate and a novel coronavirus S protein antibody colloidal gold conjugate. During detection, if a sample contains the novel coronavirus antigen, the antigen (including the antigens such as N protein, S protein and the like) in the virus is firstly combined with the novel coronavirus N protein antibody colloidal gold conjugate and the S protein antibody colloidal gold conjugate on the treatment pad after being cracked and released by the cracking buffer solution to form a novel coronavirus antigen (N protein) -novel coronavirus N protein antibody colloidal gold conjugate compound and a novel coronavirus antigen (S protein) -novel coronavirus S protein antibody colloidal gold conjugate compound. The antigen-antibody complex is chromatographed upwards under capillary effect, and is combined with the novel coronavirus N protein antibody in the detection area (T), and a purple red strip appears in the detection area (T), if a sample does not contain the novel coronavirus antigen, no purple red strip exists in the detection area T. A purple-red band appears in the quality control region (C) regardless of whether the novel coronavirus antigen is present in the sample. The purple red band appearing in the quality control area (C) is a standard for judging whether enough samples exist or not and whether the chromatography process is normal or not, and is also used as an internal control standard of the reagent.

Preferably, the antibody detection sample pad is treated by a treatment solution I, and the treatment solution I comprises the following components: water, 4-8g/L Tris, 4-7g/L sodium caseinate, 6-15g/L polyvinylpyrrolidone, 0.1-10g/L Tween 20, 0.1-10g/L Tetronic, 0.05-0.5mg/mL blocking agent, 1/50 mouse anti-RBC and 0.02% Proclin300, wherein the pH is 7.5-8.5; and/or

The antigen detection sample pad is required to be treated by a treatment solution II, and the treatment solution II comprises the following components: water, 4-8g/L Tris, 4-7g/L sodium caseinate, 6-15g/L polyvinylpyrrolidone, 0.1-10g/L Tween 20, 0.1-10g/L Tetronic and 0.02% Proclin300, and the pH value is 7.5-8.5.

Preferably, the process of detecting the antibody detection reagent strip needs the assistance of a buffer solution, and the preparation process of the buffer solution comprises the following steps: mixing 8-15g/L sodium carbonate, 1-10g/L sodium chloride, 1-10g/L ethylenediamine tetraacetic acid and 0.02% liquid biological preservative in an aqueous solution, and adjusting the final pH of the mixed solution to 7.4 +/-0.1 to obtain a buffer solution.

The buffer solution mainly plays a role in filtering impurities in a sample and assisting the sample to run on a plate for chromatography, wherein the sodium chloride and the sodium carbonate are mainly used for providing a stable detection environment, and the ethylenediaminetetraacetic acid can eliminate part of interference combination in the buffer solution, so that the clinical false positive is reduced, and the clinical detection rate is improved.

Preferably, the process of detecting the antigen detection reagent strip needs the assistance of a lysate, and the preparation process of the lysate comprises the following steps: dissolving 1-10g/L sodium chloride, 0.5-5g/L Trizma-Base, 0.5-5g/L calf serum albumin, 1-10g/L LTriton X-504, 1-10g/L Tetronic and 0.02-0.04% proclin300 in water, and adjusting the final pH of the mixed solution to 8.5 +/-0.1 to obtain the lysate.

The function of the lysate is mainly to crack the envelope protein of the new coronavirus and expose the nucleocapsid protein in the virus envelope, so that the new coronavirus can be detected. Wherein sodium chloride and Trizma-Base provide a suitable environment for the cleavage, and bovine serum albumin is used to promote the cleavage of envelope proteins.

Preferably, the antibody detection processing pad or the antigen detection processing pad is provided with a silver ion pad, and the preparation of the silver ion pad comprises the following steps: dissolving silver nitrate in PBS at 0.1-1.0mg/ml, adjusting pH to 7-8, spraying on glass fiber membrane, and baking at 30-60 deg.C for 20-24 hr to obtain silver ion pad.

The silver ion pad is introduced to enhance signals, the colloidal gold and silver developing method is combined, the colloidal gold particles precipitated at antibody positions play a catalytic role, so that the silver ions in the developing solution are reduced into silver ions in the presence of a reducing agent (hydroquinone), a silver shell is formed around the gold particles, more silver ions are reduced due to the liquefaction of the gold particles, the silver shell is enlarged, and finally the antibody positions are amplified.

Preferably, the antibody detection reagent strip clamping groove comprises an end part flange, a side flange and a bottom support positioned between the end part flange and the side flange, and the end part flange is U-shaped.

Preferably, the inner side of the upper plastic shell is provided with a test paper compression column and a test paper compression block, the test paper compression column is located at two ends of the antibody detection observation window, and the test paper compression block is located between the antibody detection sampling hole and the antibody detection observation window.

The test paper pressing column is used for pressing the end part of the reagent strip to prevent the reagent strip from warping; the test paper compact heap is for compressing tightly the middle part of reagent strip, prevents the protruding perk of reagent strip interlude.

Preferably, the edges of the antibody detection observation window, the antibody detection sample adding hole, the antigen detection observation window and the antigen detection sample adding hole are all in an inverted horn mouth shape, and the surface of the upper plastic shell is provided with a two-dimensional code scanning area; the inner side of the upper plastic shell is provided with a fixing bulge which surrounds the edge of the inner side of the upper plastic shell; the inner side of the lower plastic shell is provided with a fixed boss groove which surrounds the inner side edge of the lower plastic shell, and the fixed protrusion is positioned in the fixed boss groove.

The dripping effect can be ensured when the sample liquid and the buffer liquid are dripped, so that the dripping liquid splashed around the hole can flow downwards along the edge of the inverted bell mouth, and smoothly reaches the detection test strip for relevant detection. The scene can adopt APP scanning two-dimensional code scanning region, can in time feed back patient's information to relevant big data platform of disease control, accomplishes early discovery, early control, and the operating efficiency is high, has promoted prevention and control efficiency.

Therefore, the invention has the following beneficial effects:

(1) the immunochromatography detection test paper for combined diagnosis of the COVID-19 antigen and antibody is provided, a product for detecting the new coronavirus by using the antigen and antibody detection test paper is prepared, the detection time is short, only 15-20min is needed, the requirement of field detection can be met, and the antigen and the antibody are mutually supplemented, so that the clinical omission risk can be reduced;

(2) the accuracy of directly detecting the antigen is higher than that of the detected antibody, whether the prepared antigen has the new coronavirus can be accurately detected, the antigen has no incubation period, and the detected antigen can better avoid the clinical missed detection condition caused by low antibody titer in the window period. The double detection can ensure the validity of the result;

(3) the operation is simple and convenient, and other equipment and instruments are not needed; the detection result is displayed visually, can be judged by naked eyes and is suitable for personal use; the detection efficiency is high, and the detection result is more direct; the code is swept to cooperation cell-phone APP, but on-the-spot real-time intelligent feedback testing result provides data for the epidemic prevention center, shortens the transmission time of information.

Drawings

Fig. 1 is a schematic top view of the outer structure of the upper plastic shell of the present invention.

Fig. 2 is a schematic view of the inner side structure of the upper plastic shell of the present invention.

Fig. 3 is a schematic view of the inner side of the lower plastic shell of the present invention.

FIG. 4 is a schematic diagram of the structure of a buffer-free window according to the present invention.

Fig. 5 is a top view of the present invention.

FIG. 6 is a diagram of the detection process of the present invention.

In the figure: 1. an upper plastic shell; 2. a lower plastic shell; 3. an antibody detection observation window; 4. antibody detection wells; 5. a buffer window; 6. an antigen detection observation window; 7. antigen detection wells; 8. an antibody detection reagent strip card slot; 8.1, end flanges; 8.2, side flanges; 8.21, connecting strips; 8.3, mounting; 9. an antigen detection reagent strip slot; 10. a fixed boss groove; 11. a fixed protrusion; 12. a two-dimensional code scanning area; 13. the test paper compresses the column; 14. test paper compact heap.

Detailed Description

The invention is further described with reference to the following detailed description and accompanying drawings.

General examples

In the embodiment shown in fig. 1-3, the novel coronavirus antigen and antibody combined intelligent detection device comprises an upper plastic shell 1 and a lower plastic shell 2, wherein the upper plastic shell 1 is respectively provided with an antibody detection group and an antigen detection group, the antibody detection group comprises an antibody detection observation window 3, an antibody detection sample adding hole 4 and a buffer solution window 5, and the antigen detection group comprises an antigen detection observation window 6 and an antigen detection sample adding hole 7. The window that detects novel coronavirus antibody is located the left side of surveying the board, separates sample window and buffer window, and the single window that detects the antigen with the right side easily distinguishes, is difficult to cause the wrong application of sample of experiment.

An antibody detection reagent strip clamping groove 8 and an antigen detection reagent strip clamping groove 9 are formed in the inner side of the lower plastic shell 2, the antibody detection reagent strip clamping groove 8 corresponds to the antibody detection observation window 3, and the antigen detection reagent strip clamping groove 9 corresponds to the antigen detection observation window 6.

A fixing bulge 11 is arranged on the inner side of the upper plastic shell 1, and the fixing bulge 11 surrounds the edge of the inner side of the upper plastic shell 1; the inner side of the lower plastic shell 2 is provided with a fixed boss groove 10, the fixed boss groove 10 surrounds the inner side edge of the lower plastic shell 2, and the fixed protrusion 11 is positioned in the fixed boss groove 10. The antibody detection reagent strip clamping groove 8 and the antigen detection reagent strip clamping groove 9 are consistent in structure. The antibody detection reagent strip clamping groove 8 comprises an end part flange 8.1, a side flange 8.2 and a bottom support 8.3 positioned between the end part flange 8.1 and the side flange 8.2. The end part flanges 8.1 are U-shaped, and connecting strips 8.21 are arranged between the side flanges 8.2. The inner side of the upper plastic shell 1 is provided with a test paper compression column 13 and a test paper compression block 14, the test paper compression columns 13 are located at two ends of the antibody detection observation window 3, and the test paper compression block 14 is located between the antibody detection sampling hole 4 and the antibody detection observation window 3. A physical examination test reagent strip and an antigen detection reagent strip are respectively arranged in the antibody detection reagent strip clamping groove 8 and the antigen detection reagent strip clamping groove 9. The edges of the antibody detection observation window 3, the antibody detection sample adding hole 4, the buffer solution window 5, the antigen detection observation window 6 and the antigen detection sample adding hole 7 are all in an inverted horn mouth shape. The surface of the upper plastic shell 1 is provided with a two-dimensional code scanning area 12.

A B-shaped character is arranged beside the buffer liquid window 5; s-shaped samples are respectively arranged beside the antibody detection sample adding hole 4 and the antigen detection sample adding hole 7; the antibody detection observation window 3 is provided with IgM, IgG and C characters in sequence from one end close to the antibody detection sampling hole 4; the antigen detection observation window 6 is provided with the characters of Ag and C from one end close to the antigen detection sample adding hole 7. And a COVID-19 character is arranged between the observation window and the two-dimensional code scanning area 12.

The preparation steps of the antibody detection reagent strip are as follows:

(1) sample pad preparation: mixing and dissolving 4-8g/L Tris, 4-7g/L sodium caseinate, 6-15g/L polyvinylpyrrolidone, 0.1-10g/L Tween 20, 0.1-10g/L Tetronic, 0.05-0.5mg/ml blocking agent, 1/50 mouse anti-RBC and 0.02% Proclin300 in water, adjusting the pH value to 7.5-8.5, spraying the mixture on a glass fiber membrane, and drying;

(2) preparing a colloidal gold conjugate treatment pad: the whole process is carried out in a constant temperature heating system at 94-96 deg.C, adding trisodium citrate aqueous solution into chloroauric acid aqueous solution to obtain colloidal gold, and adding K₂CO₃Adjusting the pH value of the obtained colloidal gold to 6-10, sequentially adding the novel coronavirus antigen to be labeled, BSA and PEG20000, stirring for 10-20min, centrifuging at 9000-;

(3) preparing a silver ion treatment pad: dissolving silver nitrate in PBS at 0.1-1.0mg/ml, adjusting pH to 7-8, spraying on glass fiber membrane, and baking at 30-60 deg.C for 20-24 hr;

(4) preparation of an analysis pad: diluting mouse anti-human IgM, mouse anti-human IgG and goat anti-mouse IgG antibody solutions, spraying the diluted solutions onto a nitrocellulose membrane at a concentration of 1.0-1.2 μ L/cm to serve as a detection line IgM, a detection line IgG and a quality control line C, and drying at 30-60 ℃;

(5) preparing a detection reagent strip: and connecting and assembling the sample pad, the silver ion treatment pad, the colloidal gold conjugate treatment pad, the analysis pad, the water absorption pad and the PVC base plate in a certain sequence, and cutting to obtain the COVID-19IgG/IgM antibody detection reagent strip.

The process of detecting the antibody detection reagent strip needs the assistance of a buffer solution, and the preparation process of the buffer solution comprises the following steps: mixing 8-15g/L sodium carbonate, 1-10g/L sodium chloride, 1-10g/L ethylenediamine tetraacetic acid and 0.02% liquid biological preservative in an aqueous solution, and adjusting the final pH of the mixed solution to 7.4 +/-0.1 to obtain a buffer solution.

The preparation steps of the antigen detection reagent strip are as follows:

A. sample pad preparation: mixing and dissolving 4-8g/L Tris, 4-7g/L sodium caseinate, 6-15g/L polyvinylpyrrolidone, 0.1-10g/L Tween 20, 0.1-10g/L Tetronic and 0.02% Proclin300 in water, adjusting the pH of the mixed solution to 7.5-8.5, spraying the mixed solution on a glass fiber membrane, and drying;

B. preparing a colloidal gold conjugate treatment pad: the whole process is carried out in a constant temperature heating system at 94-96 deg.C, adding trisodium citrate aqueous solution into chloroauric acid aqueous solution to obtain colloidal gold, and adding K₂CO₃Adjusting the pH value of the obtained colloidal gold to 6-10, sequentially adding novel coronavirus antibody, BSA and PEG20000, stirring for 10-20min, centrifuging at 9000-;

C. preparing a silver ion treatment pad: dissolving silver nitrate in PBS at 0.1-1.0mg/ml, adjusting pH to 7-8, spraying on glass fiber membrane, and baking at 30-60 deg.C for 20-24 hr;

D. preparation of an analysis pad: respectively spraying the solutions of the novel coronavirus antibody (detection line T) and the goat anti-mouse IgG antibody (quality control line C) on a nitrocellulose membrane, and drying at 30-60 ℃;

E. preparation of detection test paper: and connecting and assembling the sample pad, the colloidal gold conjugate treatment pad, the silver ion treatment pad, the analysis pad, the water absorption pad and the PVC base plate in a certain sequence, and cutting to obtain the COVID-19 antigen detection reagent strip.

The process of detecting the antigen detection reagent strip needs the assistance of lysate, and the preparation process of the lysate comprises the following steps: dissolving 1-10g/L sodium chloride, 0.5-5g/L Trizma-Base, 0.5-5g/L calf serum albumin, 1-10g/L LTriton X-504, 1-10g/L Tetronic and 0.02-0.04% proclin300 in water, and adjusting the final pH of the mixed solution to 8.5 +/-0.1 to obtain the lysate.

As shown in fig. 4, the difference from example 1 is that the antibody detection group does not include a buffer window; the upper cover comprises a joint 1 connected with the lower plate, a plate surface 2, an antibody detection observation window 3, an antibody sample adding hole 4, an antigen detection observation window 6 and an antigen sample adding hole 7; the upper cover and the lower plate are mutually embedded and fixed to form a combined diagnosis novel coronavirus antigen and antibody detection reagent plate; the antibody detection observation window 3 and the antigen detection observation window 6 are arranged in parallel, so that the result interpretation is not influenced, and the result misjudgment is avoided; the parallel arrangement of the antibody well 4 and the antigen well 7 can avoid cross contamination when a sample is added.

The antibody sample addition hole is different from the former antibody sample addition hole in that the sample addition hole and the buffer solution hole are the same hole, so the antibody detection sample addition mode can also be realized by adding a sample into the buffer solution sample addition hole 4 in advance and then dripping the buffer solution.

Detection method (as shown in fig. 5-6):

detecting novel coronavirus antibody: the sample (10. mu.L serum plasma or 20. mu.L whole blood) was added in the sample window "S", and 2 drops of buffer were added in the buffer window "B". After 15min, the experimental results were observed.

And (4) judging a result: a mauve strip appears at any position of IgG in the second detection line or IgM in the third detection line, and the result is positive; IgG and IgM were negative with no magenta band. The C band was purple red regardless of whether the novel coronavirus antibody was detected.

Detecting novel coronavirus antigen: inserting the sterile swab into nostril of patient, reaching the back of nasopharynx, wiping the surface of nasopharynx, taking out the swab, putting the swab into a lysis solution bottle, fully stirring, squeezing out the cotton swab and discarding. Add 3 drops of solution dropwise to the sample window "S". After 15min, the experimental results were observed.

And (4) judging a result: if a purple red strip appears at the third detection line, the detection line is positive; if there is no purple-red band, it is negative. The C position was a purple red band regardless of whether the novel coronavirus antigen was detected.

Example 1

A novel coronavirus antigen and antibody combined intelligent detection device and application thereof comprise device plastic shell characteristics, embedded reagent strip preparation, device using methods and detection examples.

A novel coronavirus antigen and antibody combined intelligent detection device comprises an upper plastic shell 2 and a lower plastic shell 1, wherein the upper plastic shell is respectively provided with an antibody detection group and an antigen detection group, the antibody detection group comprises an antibody detection observation window 3, an antibody detection sample adding hole 4 and a buffer solution window 5, and the antigen detection group comprises an antigen detection observation window 6 and an antigen detection sample adding hole 7. The window that detects novel coronavirus antibody is located the left side of surveying the board, separates sample window and buffer window, and the single window that detects the antigen with the right side easily distinguishes, is difficult to cause the wrong application of sample of experiment.

An antibody detection test reagent strip clamping groove 8 and an antigen detection reagent strip clamping groove 9 are formed in the inner side of the lower plastic shell, the antibody detection reagent strip clamping groove 8 corresponds to the antibody detection observation window 3, and the antigen detection reagent strip clamping groove 9 corresponds to the antigen detection observation window 6.

A fixing bulge 11 is arranged on the inner side of the upper plastic shell, and the fixing bulge 11 surrounds the edge of the inner side of the upper plastic shell; the inner side of the lower plastic shell is provided with a fixed boss groove 10, the fixed boss groove surrounds the inner side edge of the lower plastic shell, and the fixed protrusion 11 is positioned in the fixed boss groove 10. The antibody detection reagent strip clamping groove 8 and the antigen detection reagent strip clamping groove 9 are consistent in structure. The antibody detection reagent strip clamping groove 8 comprises an end part flange 8.1, a side flange 8.2 and a bottom support 8.3 positioned between the end part flange 8.1 and the side flange 8.2. The end part flanges 8.1 are U-shaped, and connecting strips 8.21 are arranged between the side flanges 8.2. The inner side of the upper plastic shell is provided with a test paper compression column 13 and a test paper compression block 14, the test paper compression columns 13 are located at two ends of the antibody detection observation window 3, and the test paper compression block 14 is located between the antibody detection sampling hole 4 and the antibody detection observation window 3. A physical examination test reagent strip and an antigen detection reagent strip are respectively arranged in the antibody detection reagent strip clamping groove 8 and the antigen detection reagent strip clamping groove 9. The edges of the antibody detection observation window 3, the antibody detection sample adding hole 4, the buffer solution window 5, the antigen detection observation window 6 and the antigen detection sample adding hole 7 are all in an inverted horn mouth shape. And a two-dimensional code scanning area 12 is arranged on the surface of the upper plastic shell.

An embedded antibody detection reagent strip and an antigen detection reagent strip of a novel coronavirus antigen and antibody combined intelligent detection device. Preparing the antibody detection reagent strip:

(1) sample pad preparation: mixing 6g/LTris, 5g/L sodium caseinate, 10g/L polyvinylpyrrolidone, 5g/L Tween 20, 5g/L Tetronic, 0.25mg/ml blocking agent, 1/50 mouse anti-RBC and 0.02% Proclin300, dissolving in water, adjusting pH to 8.0, spraying on glass fiber membrane, and oven drying;

(2) preparing a colloidal gold conjugate treatment pad: the whole process is carried out in a constant temperature heating system at 95 ℃, trisodium citrate aqueous solution is added into chloroauric acid aqueous solution to obtain colloidal gold, and K is used₂CO₃Adjusting the pH of the obtained colloidal gold to 8.0, sequentially adding the novel coronavirus S protein to be labeled and the novel coronavirus N protein, BSA and PEG20000, stirring for 10min, centrifuging at 5 deg.C and 11000rpm for 15min, collecting precipitate, diluting to a certain concentration, spraying onto polyester film, and drying for 22 h;

(3) preparing a silver ion treatment pad: dissolving silver nitrate in PBS at a concentration of 0.5mg/ml, adjusting pH to 7.5, spraying on glass fiber membrane, and baking at 45 deg.C overnight;

(4) preparation of an analysis pad: spraying mouse anti-human IgM, mouse anti-human IgG and goat anti-mouse IgG antibody solutions onto the nitrocellulose membrane at a concentration of 1.0 μ L/cm to serve as a detection line IgM, a detection line IgG and a quality control line C, and drying at 45 ℃;

(5) preparing a detection reagent strip: and connecting and assembling the sample pad, the colloidal gold conjugate treatment pad, the silver ion treatment pad, the analysis pad, the water absorption pad and the PVC base plate in a certain sequence, and cutting to obtain the COVID-19IgG/IgM antibody detection test paper.

Preparing the antigen detection reagent strip:

A. sample pad preparation: dissolving 6g/L Tris, 5g/L sodium caseinate, 10g/L polyvinylpyrrolidone, 5g/L Tween 20, 5g/L Tetronic and 0.02% Proclin300 in water, adjusting pH to 8.0, spraying on a glass fiber membrane, and drying;

B. preparing a colloidal gold conjugate treatment pad: the whole process is carried out in a constant temperature heating system at 95 ℃, trisodium citrate aqueous solution is added into chloroauric acid aqueous solution to obtain colloidal gold, and K is used₂CO₃Adjusting the pH of the obtained colloidal gold to 8.0, sequentially adding a novel coronavirus N protein antibody to be labeled and a novel coronavirus S antibody, BSA (bovine serum albumin) and PEG20000, continuously stirring for 15min, centrifuging at 5 ℃ and 11000rpm for 15min, taking a precipitate, diluting, spraying on a polyester film, and drying for 22 h;

C. preparing a silver ion treatment pad: dissolving silver nitrate in PBS at a concentration of 0.5mg/ml, adjusting pH to 7.5, spraying on glass fiber membrane, and baking at 45 deg.C overnight;

D. preparation of an analysis pad: respectively diluting the novel coronavirus N protein antibody, the novel coronavirus S protein antibody and a goat anti-mouse IgG antibody solution, spraying the diluted solutions on a nitrocellulose membrane at a concentration of 1.0 mu L/cm to serve as a detection line T and a quality control line C, and drying at 45 ℃;

A reagent matched with a novel coronavirus antigen and antibody combined intelligent detection device is disclosed:

preparing antigen detection lysate: 5g/L sodium chloride, 2.5g/L Trizma-Base, 2.5g/L calf serum albumin, 5g/L Triton X-504, 5g/L Tetronic and 0.02% proclin300 were mixed and dissolved in water, and the pH was adjusted to 8.5.

The preparation process of the antibody detection buffer solution comprises the following steps: mixing 11g/L sodium carbonate, 5g/L sodium chloride, 5g/L ethylene diamine tetraacetic acid and 0.02% liquid biological preservative, dissolving in water, and adjusting pH to 7.4.

A detection method of a novel coronavirus antigen and antibody combined intelligent detection device (figures 5-6):

Detecting a novel coronavirus antigen: inserting the sterile swab into nostril of patient, reaching the back of nasopharynx, wiping the surface of nasopharynx, taking out the swab, putting the swab into a lysis solution bottle, fully stirring, squeezing out the cotton swab and discarding. Add 3 drops of solution dropwise to the sample window "S". After 15min, the experimental results were observed.

The application example of the novel coronavirus antigen and antibody combined intelligent detection device takes a novel coronavirus recombinant antigen, a virus culture solution and a novel coronavirus antibody positive sample as comparison to verify the sensitivity and the accuracy of the detection device, and the results are shown in table 1.1; 50 PCR positive samples and 50 PCR negative samples of the novel coronavirus are collected, the sensitivity and the accuracy of the detection device are verified by taking the PCR detection result as reference, and the detection result is shown in table 1.2. The result shows that the detection device has higher accuracy and the detection rate of the novel coronavirus infection.

TABLE 1.1 detection results of novel coronavirus recombinant antigens, virus culture solutions and novel coronavirus antibody positive samples

". note: when the signal intensity is more than 3, a positive result can be considered; when the signal intensity is less than or equal to 3, a negative result can be considered.

TABLE 1.2 detection accuracy of novel coronavirus antigen and antibody combined intelligent detection device

	Positive match rate (%)	Negative match rate (%)	Total sample percent match (%)
				Antigen detection	96.0	98.0	97.0
Antibody detection	94.0	96.0	95.0
				Joint detection	100.0	96.0	98.0

Example 2

A novel coronavirus antigen and antibody combined intelligent detection device and application thereof comprise a device plastic shell, an embedded reagent strip preparation method, an embedded reagent strip application method and a detection example. The plastic housing of the device was the same as in example 1. The reagent strip for detecting the embedded antibody and the antigen of the device is prepared as follows:

preparing the antibody detection reagent strip:

(1) sample pad preparation: dissolving 4g/L of LTris, 4g/L of casein sodium salt, 6g/L of polyvinylpyrrolidone, 0.1g/L of Tween 20, 0.1g/L of Tetronic, 0.05mg/ml of blocking agent, 1/50 mouse-resistant RBC and 0.02% of Proclin300 in water, adjusting the pH value to 7.5, spraying the mixture on a glass fiber membrane, and drying;

(2) preparing a colloidal gold conjugate treatment pad: the whole process is carried out in a constant temperature heating system at 94 ℃, trisodium citrate aqueous solution is added into chloroauric acid aqueous solution to obtain colloidal gold, and K is used₂CO₃Adjusting the pH of the obtained colloidal gold to 6, sequentially adding the novel coronavirus S protein to be labeled and the novel coronavirus N protein, BSA, and PEG20000, continuously stirring for 10min, centrifuging at 9000rpm for 10min at 4 deg.C, collecting precipitate, diluting, spraying onto polyester film, and drying for 20 h;

(3) preparing a silver ion treatment pad: dissolving silver nitrate in PBS at 0.1mg/mL, adjusting pH to 7.0, spraying on glass fiber membrane, and baking at 30 deg.C overnight;

(4) preparation of an analysis pad: diluting mouse anti-human IgM, mouse anti-human IgG and goat anti-mouse IgG antibody solutions respectively, spraying the diluted solutions onto a nitrocellulose membrane at a concentration of 1.0 mu L/cm to serve as a detection line IgM, a detection line IgG and a quality control line C, and drying at 30 ℃;

Preparing the antigen detection reagent strip:

A. sample pad preparation: mixing 4g/L Tris, 4g/L sodium caseinate, 6g/L polyvinylpyrrolidone, 0.1g/L Tween 20, 0.1g/L Tetronic and 0.02% Proclin300 in an aqueous solution, adjusting the pH of the mixed solution to 7.5, spraying the prepared sample pad solution on glass fibers, and drying to obtain a sample pad;

B. preparing a colloidal gold conjugate treatment pad: the whole process is carried out in a constant temperature heating system at 94 ℃, trisodium citrate aqueous solution is added into chloroauric acid aqueous solution to obtain colloidal gold, the pH of the obtained colloidal gold is adjusted to 6 by K2CO3 solution, a novel coronavirus N protein antibody to be marked and a novel coronavirus S antibody, BSA and PEG20000 are sequentially added, stirring is carried out for 10min, centrifugation is carried out at 4 ℃ and 9000rpm for 10min, precipitates are taken, diluted and sprayed on a polyester film, and drying is carried out for 20 h;

C. preparing a silver ion treatment pad: dissolving silver nitrate in PBS at 0.1mg/mL, adjusting pH to 7.0, spraying on glass fiber membrane, and baking at 30 deg.C overnight;

D. preparation of an analysis pad: diluting the novel coronavirus N protein antibody, the novel coronavirus S protein antibody and the goat anti-mouse IgG antibody solution, spraying the diluted solutions on a nitrocellulose membrane at a concentration of 1.0 mu L/cm to serve as a detection line T and a quality control line C, and drying at 30 ℃;

E. preparing a test paper strip: and connecting and assembling the sample pad, the colloidal gold conjugate treatment pad, the silver ion treatment pad, the analysis pad, the water absorption pad and the PVC base plate in a certain sequence, and cutting to obtain the antigen detection reagent strip of COVID-19.

preparing antigen detection lysate: 1g/L sodium chloride, 0.5g/L Trizma-Base, 0.5g/L calf serum albumin, 1.0g/L Triton X-504, 1.0g/L Tetronic and 0.02% proclin300 were dissolved in water and the pH was adjusted to 8.5.

The preparation process of the antibody detection buffer solution comprises the following steps: 8g/L sodium carbonate, 1g/L sodium chloride, 1g/L ethylene diamine tetraacetic acid and 0.02% liquid biological preservative are mixed and dissolved in water, and the mixed pH is adjusted to 7.4.

The application example of the novel coronavirus antigen and antibody combined intelligent detection device comprises the following steps:

referring to the device using method of example 1, the sensitivity and accuracy of the detection device were verified by using the novel coronavirus recombinant antigen, the virus culture fluid and the novel coronavirus antibody positive sample as controls, and the results are shown in table 2.1. The result shows that the detection device has higher accuracy and the detection rate of the novel coronavirus infection.

TABLE 2.1 detection results of novel coronavirus recombinant antigen, virus culture fluid, and novel coronavirus antibody positive sample

Example 3

A novel coronavirus antigen and antibody combined intelligent detection device and application thereof comprise a device plastic shell, an embedded reagent strip preparation method, an embedded reagent strip application method and a detection example. The plastic housing of the device was the same as in example 1. The device embedded reagent strip is prepared as follows:

preparing the antibody detection reagent strip:

(1) sample pad preparation: dissolving 8g/L Tris, 7g/L sodium caseinate, 15g/L polyvinylpyrrolidone, 10g/L Tween 20, 10g/L Tetronic, 0.5mg/ml blocking agent, 1/50 rat anti-RBC and 0.02% Proclin300 in water, adjusting the pH to 8.5, spraying the prepared sample pad solution on glass fiber, and drying;

(2) preparing a colloidal gold conjugate treatment pad: the whole process is carried out in a constant temperature heating system at 96 ℃, trisodium citrate aqueous solution is added into chloroauric acid aqueous solution to obtain colloidal gold, and K is used₂CO₃Adjusting the pH of the obtained colloidal gold to 10, sequentially adding novel coronavirus S protein, novel coronavirus N protein, BSA, and PEG20000, and stirring for 15min, centrifuging at 14000rpm for 20min at 6 deg.C, collecting precipitate, diluting, spraying onto polyester film, and drying for 24 hr;

(3) preparing a silver ion treatment pad: dissolving silver nitrate in PBS at 0.1mg/mL, adjusting pH to 8.0, spraying on glass fiber membrane, and baking at 60 deg.C overnight;

(4) preparation of an analysis pad: respectively diluting mouse anti-human IgM, mouse anti-human IgG and goat anti-mouse IgG antibody solutions, spraying the diluted solutions on a nitrocellulose membrane at a spraying amount of 1.2 mu L/cm to serve as a detection line IgM, a detection line IgG and a quality control line C, and drying at 60 ℃ to obtain a detection line and a quality control line;

Preparing the antigen detection reagent strip:

A. sample pad preparation: mixing and dissolving 8g/L Tris, 7g/L sodium caseinate, 15g/L polyvinylpyrrolidone, 10g/L Tween 20, 10g/L Tetronic and 0.02% Proclin300 in water, adjusting the pH to 8.5, spraying the mixture on a glass fiber membrane, and drying;

B. preparing a colloidal gold conjugate treatment pad: the whole process is carried out in a constant temperature heating system at 96 ℃, trisodium citrate aqueous solution is added into chloroauric acid aqueous solution to obtain colloidal gold, and K is used₂CO₃Adjusting pH of the solution to 10, sequentially adding to-be-labeled anti-novel coronavirus N protein antibody and anti-novel coronavirus S antibody, BSA, and PEG20000, stirring for 20min, centrifuging at 6 deg.C and 14000rpm for 20min, collecting precipitate, diluting and spraying onto polyester film, and drying for 24 h;

C. preparing a silver ion treatment pad: dissolving silver nitrate in PBS at 0.6mg/ml, adjusting pH to 7.4, spraying on glass fiber membrane, and baking at 60 deg.C overnight;

D. preparation of an analysis pad: diluting the novel coronavirus N protein antibody, the novel coronavirus S protein antibody and the goat anti-mouse IgG antibody solution, spraying the diluted solutions on a nitrocellulose membrane at a concentration of 1.2 mu L/cm to serve as a detection line T and a quality control line C, and drying at 60 ℃;

E. preparation of detection test paper: and (3) sequentially adhering the sample pad, the colloidal gold conjugate treatment pad, the silver ion treatment pad, the analysis pad and the water absorption pad on a PVC (polyvinyl chloride) bottom plate, and cutting to obtain the COVID-19 antigen detection reagent strip.

preparing antigen detection lysate: 10g/L sodium chloride, 5.0g/L Trizma-Base, 5.0g/L calf serum albumin, 10g/L Triton X-504, 10g/L Tetronic and 0.02% proclin300 were dissolved in water and the pH was adjusted to 8.5.

The preparation process of the antibody detection buffer solution comprises the following steps: mixing 15g/L sodium carbonate, 10g/L sodium chloride, 10g/L ethylene diamine tetraacetic acid and 0.02% liquid biological preservative, dissolving in water, and adjusting pH to 7.4.

referring to the method of using the device of example 1, the accuracy of the test device was verified by using the novel coronavirus recombinant antigen, the virus culture medium and the novel coronavirus antibody positive sample as controls, and the results are shown in table 3.1. The result shows that the detection device has higher accuracy and the detection rate of the novel coronavirus infection.

TABLE 3.1 detection results of novel coronavirus recombinant antigen, virus culture fluid, and novel coronavirus antibody positive sample

Example 4

A novel coronavirus antigen and antibody combined intelligent detection device and application thereof comprise device plastic shell characteristics, embedded reagent strip preparation, device using methods and detection examples. The plastic housing of the device was the same as in example 1. The reagent strip embedded in the device is prepared by the following steps:

(2) preparing a colloidal gold conjugate treatment pad: the whole process is carried out in a constant temperature heating system at 95 ℃, trisodium citrate aqueous solution is added into chloroauric acid aqueous solution to obtain colloidal gold, and K is used₂CO₃Adjusting the pH of the obtained colloidal gold to 8.0, sequentially adding the novel coronavirus RBD to be labeled, the novel coronavirus N protein, BSA and PEG20000, stirring for 10min, centrifuging at 5 deg.C and 11000rpm for 15min, collecting precipitate, diluting to a certain concentration, spraying onto polyester film, and drying for 22 h;

Preparing the antigen detection reagent strip:

B. preparing a colloidal gold conjugate treatment pad: the whole process is carried out in a constant temperature heating system at 95 ℃, trisodium citrate aqueous solution is added into chloroauric acid aqueous solution to obtain colloidal gold, and K is used₂CO₃Adjusting the pH value of the obtained colloidal gold to 8.0, and sequentially adding the colloidal gold to be treatedLabeled anti-novel coronavirus N protein antibodies and anti-novel coronavirus S antibodies, BSA. PEG20000, stirring for 15min, centrifuging at 5 deg.C and 11000rpm for 15min, collecting precipitate, diluting, spraying onto polyester film, and drying for 22 h;

the sensitivity and accuracy of the detection device of the present invention were verified by using the novel coronavirus recombinant antigen, the virus culture solution, and the novel coronavirus antibody positive sample as controls with reference to the method of using the device of example 1, and the results are shown in table 4.1. The result shows that the detection device has higher accuracy and the detection rate of the novel coronavirus infection.

TABLE 4.1 detection results of novel coronavirus recombinant antigens, virus culture solutions and novel coronavirus antibody positive samples

Example 5 (different from example 1, the antigen detection reagent strip only adopts the novel coronavirus N protein antibody) a novel coronavirus antigen and antibody combined intelligent detection device and application, comprising device plastic shell characteristics, embedded reagent strip preparation, device using method and detection example.

Preparing the antigen detection reagent strip:

B. preparing a colloidal gold conjugate treatment pad: the whole process is carried out in a constant temperature heating system at 95 ℃, trisodium citrate aqueous solution is added into chloroauric acid aqueous solution to obtain colloidal gold, and K is used₂CO₃Adjusting the pH of the obtained colloidal gold to 8.0, sequentially adding a novel coronavirus N protein antibody to be labeled, BSA and PEG20000, continuously stirring for 15min, centrifuging at 5 ℃ and 11000rpm for 15min, taking precipitate, diluting, spraying onto a polyester film, and drying for 22 h;

D. preparation of an analysis pad: respectively diluting the novel coronavirus N protein antibody, the novel coronavirus S protein antibody and a goat anti-mouse IgG antibody solution, spraying the diluted solutions on a nitrocellulose membrane at a concentration of 1.0 mu L/cm to serve as a detection line T and a quality control line C, and drying at 45 ℃; E. preparation of detection test paper: and connecting and assembling the sample pad, the colloidal gold conjugate treatment pad, the silver ion treatment pad, the analysis pad, the water absorption pad and the PVC base plate in a certain sequence, and cutting to obtain the COVID-19 antigen detection reagent strip.

Referring to the plastic shell of the device, the detection matching reagent and the using method of the embodiment 1, the accuracy of the detection device is verified by taking the novel coronavirus recombinant antigen, the virus culture solution and the novel coronavirus antibody positive sample as comparison results, the results are shown in table 5.1, and the comparison result with the embodiment 1 (table 1.1) shows that the novel coronavirus antigen-antibody combined detection device provided by the invention has higher accuracy and the detection rate of novel coronavirus infection.

TABLE 5.1 detection results of novel coronavirus recombinant antigen, virus culture fluid, and novel coronavirus antibody positive sample

Comparative example 1 (different from example 1 in that no silver ion treatment pad was added to the test strip.)

The comparative example device plastic shell, the matching reagent and the using method are the same as the example 1. The difference is that the embedded reagent strip of the device is not added with a silver ion processing pad, and other structures and preparation methods of the reagent strip are the same as those of the embodiment 1.

The accuracy of the test device was verified by using the novel coronavirus recombinant antigen, virus culture medium, and novel coronavirus antibody positive sample as controls, according to the method of using the device of example 1, and the results are shown in Table 6.1. The color development values of the positive antigen detection samples without using the silver ion treatment pad (table 6.1) were: 7. 5.5, 4.5 and 3.5, after the silver ion pad is introduced (Table 1.1), the color rendering values of the antigen detection positive samples are 8, 6.5, 5 and 7.5, and the comparison result shows that the colorimetric values of the antigen detection results can be improved by 0.5-1 by introducing the silver ion treatment pad; the silver ion treatment pad (table 6.1) is not used, the color development values of the detection results of the antibody detection positive samples are respectively 3.5, 3.5 and 3.5, and the color development values of the detection results of the antibody detection positive samples are respectively 4, 4 and 4 after the silver ion treatment pad is introduced (table 1.1), and the comparison result shows that the colorimetric values of the antibody detection results can be improved by 1 by introducing the silver ion treatment pad. In summary, the comparison with example 1 shows that the incorporation of a silver treated pad into the device improves the detection sensitivity.

TABLE 6.1 detection results of novel coronavirus recombinant antigens, virus culture solutions and novel coronavirus antibody positive samples

Comparative example 2 (different from example 1 in that different lysis solution was used in the antigen detection process)

The comparative example device was a plastic housing, embedded reagent strip, and used in the same manner as example 1. The difference is that one of the reagents used in the device of this embodiment, antigen detection lysate, is different from that used in embodiment 1. The preparation method of the antigen lysate comprises the following steps: 3g/L of tris (hydroxymethyl) aminomethane, 10g/L of sodium chloride, 3g/L of Chemal LA-9, 5g/L of Triton X-100 and 0.02 percent of liquid biological preservative.

Referring to the method of using the device of example 1, the accuracy of the detection device was verified by using the novel coronavirus recombinant antigen, the virus culture solution and the novel coronavirus antibody positive sample as controls, and the results are shown in table 7.1, and the color rendering values of the detection results of the antigen positive sample (virus culture solution) of the comparative example are as follows: 5.5, the color values of the detection results of the antigen-positive samples (virus culture solutions) using the lysate of the present invention and those of examples 1 to 4 (tables 1.1 to 4.1) were: 8. 6.5; 7.5 and 6; 8. 6.5; 8. 6.5; the contrast result shows that the color rendering value of the virus culture solution using the lysis solution provided by the invention is higher than that of the lysis solution not provided by the invention, so that the detection sensitivity and accuracy of the device can be reduced by the lysis solution not provided by the invention.

TABLE 7.1 detection results of novel coronavirus recombinant antigen, virus culture fluid, and novel coronavirus antibody positive sample

Comparative example 3 (different from example 1 in that the buffer solution used in the antibody detection process was different)

The comparative example device was a plastic housing, embedded reagent strip, and used in the same manner as example 1. The difference is that one of the reagents used in the kit of the comparative example, antibody detection buffer, was different from that used in example 1. The preparation method of the antibody detection buffer solution comprises the following steps: 5g/L of sodium chloride, 3g/L of Tris, 6g/L of polyvinylpyrrolidone and 0.02% of liquid biological preservative are dissolved in water, and the pH value is adjusted to 7.4.

Referring to the device using method of example 1, the sensitivity and accuracy of the detection device were verified by using the novel coronavirus recombinant antigen, the virus culture solution, and the novel coronavirus antibody positive sample as controls, the results are shown in table 8.1, and the color rendering values of the detection results of the antibody positive sample of the comparative example are 3, 3.5, and 3, respectively; the color values of the detection results of the antibody positive samples obtained by the lysis method of the present invention and the detection results of the antibody positive samples of examples 1 to 4 (tables 1.1 to 4.1) are respectively as follows: 4. 4, 4; 4. 4, 3.5 and 4; 4.5, 4; 4.5, 4; the comparison result shows that the color rendering value of the detection result of the positive sample is higher than that of the buffer solution which is not used, so that the detection sensitivity and accuracy can be reduced by the buffer solution which is not in the formula concentration range provided by the invention.

TABLE 8.1 detection results of novel coronavirus recombinant antigen, virus culture fluid, and novel coronavirus antibody positive sample

Comparative example 4 (different from example 1 in that only antigen detection is used)

The comparative example device comprises a plastic shell, an embedded reagent strip, a matched reagent and a using method, and is the same as example 1. The difference is that the embedded reagent strip of the device of the embodiment only has an antigen detection reagent strip. Referring to the using method of the device in example 1, the results of comparing the sensitivity and specificity of single antigen detection with those of example 1 are shown in table 9.1, the positive coincidence rate and the negative coincidence rate of the single antigen detection in the comparative example are respectively 96% and 98%, and the positive coincidence rate and the negative coincidence rate of the combined detection in example 1 are respectively 100% and 96%, and the comparison results show that the combined detection provided by the invention can obtain higher detection sensitivity and accuracy.

TABLE 9.1 accuracy of detection of novel coronaviruses

	Positive match rate (%)	Negative match rate (%)	Total sample percent match (%)
				Antigen detection	96.0	98.0	97.0
Antibody detection	/	/	/
				Combined assay (example 1)	100.0	96.0	98.0

Comparative example 5 (different from example 1 in that only antibody detection was used)

The device of the embodiment comprises a plastic shell, an embedded reagent strip, a matched reagent and a using method, which are the same as those in the embodiment 1. The difference is that the embedded reagent strip of the device of the embodiment is only the antibody detection reagent strip. Referring to the using method of the device in example 1, the results of comparing the sensitivity and specificity of single antibody detection with those of example 1 are shown in table 10.1, the positive coincidence rate and the negative coincidence rate of the single antibody detection in the comparative example are respectively 94% and 96%, and the positive coincidence rate and the negative coincidence rate of the combined detection in example 1 are respectively 100% and 96%, and the comparison results show that the combined detection provided by the invention can obtain higher detection sensitivity and accuracy.

TABLE 10.1 accuracy of detection of novel coronaviruses

	Positive match rate (%)	Negative match rate (%)	Total sample percent match (%)
				Antigen detection	/	/	/
Antibody detection	94.0	96.0	95.0
				Combined assay (example 1)	100.0	96.0	98.0

As can be seen from the data of examples 1 to 5 and comparative examples 1 to 5, the above requirements can be satisfied in all aspects and the most accurate detection result can be obtained only by the scheme within the scope of the claims of the present invention. The change of the mixture ratio, the replacement/addition/subtraction of raw materials or the change of the feeding sequence can bring corresponding negative effects.

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A novel coronavirus antigen and antibody joint intelligent detection device comprises an upper plastic shell (1) and a lower plastic shell (2), and is characterized in that the upper plastic shell (1) is respectively provided with an antibody detection group and an antigen detection group, the antibody detection group comprises an antibody detection observation window (3) and an antibody detection sample adding hole (4), and the antigen detection group comprises an antigen detection observation window (6) and an antigen detection sample adding hole (7); an antibody detection reagent strip clamping groove (8) and an antigen detection reagent strip clamping groove (9) are formed in the inner side of the lower plastic shell (2); an antibody detection reagent strip and an antigen detection reagent strip are respectively arranged in the antibody detection reagent strip clamping groove (8) and the antigen detection reagent strip clamping groove (9), and the antibody detection reagent strip sequentially consists of an antibody detection sample pad, an antibody detection processing pad, an antibody detection analysis pad and an absorption pad; the antigen detection reagent strip consists of an antigen detection sample pad, an antigen detection processing pad, an antigen detection analysis pad and an absorption pad in sequence.

2. The novel combined intelligent detection device for the coronavirus antigens and the antibodies as claimed in claim 1, wherein the capture protein of the detection line IgM on the antibody detection and analysis pad is mouse anti-human IgM, the capture protein of the detection line IgG is mouse anti-human IgG, and the capture protein of the quality control line C is goat anti-mouse IgG antibody; the antibody detection processing pad is an antibody colloidal gold conjugate processing pad, and the colloidal gold conjugate is a composition of a novel coronavirus N protein colloidal gold conjugate, a novel coronavirus S protein colloidal gold conjugate and a goat anti-mouse IgG colloidal gold conjugate; or

3. The device for the combined intelligent detection of the novel coronavirus antigen and antibody according to claim 1, wherein the antigen detection treatment pad is an antigen colloidal gold conjugate treatment pad, and the colloidal gold conjugate is a composition of a novel coronavirus N protein antibody colloidal gold conjugate and a goat anti-mouse IgG colloidal gold conjugate; the capture protein of the corresponding antigen detection analysis pad detection line T is a novel coronavirus N protein antibody, and the capture protein of the quality control line C is a goat anti-mouse IgG antibody; or

4. The novel coronavirus antigen-antibody combined intelligent detection device as claimed in claim 1, wherein the antibody detection sample pad is treated by a treatment solution I, and the treatment solution I comprises the following components: water, 4-8g/L Tris, 4-7g/L sodium caseinate, 6-15g/L polyvinylpyrrolidone, 0.1-10g/L Tween 20, 0.1-10g/L Tetronic, 0.05-0.5mg/mL blocking agent, 1/50 mouse anti-RBC and 0.02% Proclin300, wherein the pH is 7.5-8.5; and/or

5. The novel coronavirus antigen-antibody combined intelligent detection device as claimed in claim 1, wherein the detection process of the antibody detection reagent strip needs the assistance of a buffer solution, and the preparation process of the buffer solution comprises the following steps: mixing 8-15g/L sodium carbonate, 1-10g/L sodium chloride, 1-10g/L ethylene diamine tetraacetic acid and 0.02% of liquid biological preservative in an aqueous solution, and adjusting the final pH =7.4 +/-0.1 of the mixed solution to obtain the buffer solution.

6. The novel coronavirus antigen and antibody combined intelligent detection device as claimed in claim 1 or 4, wherein the antigen detection reagent strip detection process needs the assistance of a lysis solution, and the preparation process of the lysis solution comprises the following steps: dissolving 1-10g/L sodium chloride, 0.5-5g/L Trizma-Base, 0.5-5g/L calf serum albumin, 1-10g/L LTriton X-504, 1-10g/L Tetronic and 0.02-0.04% proclin300 in water, and adjusting the final pH of the mixed solution to be 8.5 +/-0.1 to obtain the lysate.

7. The novel coronavirus antigen-antibody combined intelligent detection device as claimed in claim 2 or 3, wherein a silver ion pad is arranged on the antibody detection treatment pad or the antigen detection treatment pad, and the preparation of the silver ion pad comprises the following steps: dissolving silver nitrate in PBS at 0.1-1.0mg/ml, adjusting pH to 7-8, spraying on glass fiber membrane, and baking at 30-60 deg.C for 20-24 hr to obtain silver ion pad.

8. The novel coronavirus antigen-antibody joint intelligent detection device according to claim 1, wherein the antibody detection reagent strip clamping groove (8) comprises an end rib (8.1), a side rib (8.2) and a bottom support (8.3) positioned between the end rib (8.1) and the side rib (8.2), and the end rib (8.1) is U-shaped.

9. The novel coronavirus antigen and antibody joint intelligent detection device as claimed in claim 1, wherein a test paper compression column (13) and a test paper compression block (14) are arranged on the inner side of the upper plastic shell (1), the test paper compression columns (13) are positioned at two ends of the antibody detection observation window (3), and the test paper compression block (14) is positioned between the antibody detection sample adding hole (4) and the antibody detection observation window (3).

10. The novel coronavirus antigen and antibody combined intelligent detection device as claimed in claim 1, wherein the edges of the antibody detection observation window (4), the antibody detection sample adding hole (4), the antigen detection observation window (6) and the antigen detection sample adding hole (7) are all in an inverted bell mouth shape, and a two-dimensional code scanning area (12) is arranged on the surface of the upper plastic shell (1); a fixing bulge (11) is arranged on the inner side of the upper plastic shell (1), and the fixing bulge (11) surrounds the edge of the inner side of the upper plastic shell (1); the inner side of the lower plastic shell (2) is provided with a fixed boss groove (10), the fixed boss groove (10) surrounds the inner side edge of the lower plastic shell (2), and the fixed protrusion (11) is positioned in the fixed boss groove (10).