CN112180084A - Novel coronavirus enzyme-linked immunoassay kit based on microfluidics - Google Patents
Novel coronavirus enzyme-linked immunoassay kit based on microfluidics Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
Abstract
The invention provides a novel coronavirus enzyme-linked immunoassay kit based on microfluidics, which comprises: a microfluidic plate coated with a novel coronavirus S1 protein, an enzyme conjugate, a fluorescent substrate, and a sample diluent. The detection kit adopts the microfluidic plate coated with the novel coronavirus S1 protein to detect a sample to be detected, improves the working process of ELISA, and greatly improves the detection speed; meanwhile, the consumption of consumables such as antibodies, reagents and the like is obviously reduced, the dynamic range of the standard curve is improved, the sensitivity is good, and the rapid diagnosis and antibody quantification of a new coronavirus infected patient can be rapidly completed.
Description
Technical Field
The invention belongs to the technical field of immunoassay detection, and particularly relates to an enzyme-linked immunoassay kit, in particular to a novel coronavirus enzyme-linked immunoassay kit based on microfluidics.
Background
A novel coronavirus is a virus containing only ribonucleic acid (RNA), and the specific RNA sequence in the virus is a marker that distinguishes the virus from other pathogens. At present, a novel coronavirus detected by an RT-PCR method is used as a 'gold standard' for confirmed diagnosis, but the RT-PCR method has obvious defects, has a certain false negative rate, takes 4-6 hours for one-time complete nucleic acid detection, and easily causes medical extrusion in some areas with incomplete medical systems if all medical personnel only adopt nucleic acid detection, so that part of patients cannot be diagnosed after delay, treatment is delayed or epidemic situation is enlarged. For false negatives in nucleic acid detection, which are considered to be mainly affected by sampling, the rate of false negatives in samples obtained by outpatient collection mainly from nasopharyngeal swabs in the upper respiratory tract may change greatly with the change of sampling effectiveness.
At present, global prevention and control of epidemic situations enter a normalized stage, and considering that the virus has the characteristics of larger infectivity, more serious illness or a large number of asymptomatic infectors, in community infection, after the symptomatic infectors appear, the spreading chain is usually expanded in a larger range, so that the comprehensive spreading of the screening of the asymptomatic or mild infectors is particularly important for the final control and elimination of the epidemic situations, and therefore, besides a PCR test, antibody detection is also very important.
The IgM antibody is an antibody firstly stimulated to be generated after the new corona virus infects a human body, mostly begins to be positive after the disease occurs for 3-5 days, and part of patients show the positive blood IgM antibody when the nucleic acid detection is negative, and is one of the markers of the acute infection stage of the new corona virus. IgG generally appears in peripheral blood after one week of infection, IgG is generated and continuously exists, and the recovery period of IgG antibody titer is increased by 4 times or more than that of the acute period, and the IgG antibody titer can be used as an index of the previous infection.
Antibody detection can be determined by detecting blood samples by Enzyme-linked immunosorbent assay (ELISA). The enzyme-linked immunosorbent IgM/IgG antibody detection has low laboratory requirements, and is more suitable for primary hospitals and routine outpatients. In addition, compared with other existing immunodiagnostics for the new coronavirus, the immunodiagnosis method comprises the steps of carrying out immunodilation colloidal gold chromatography, carrying out nano magnetic bead chemiluminescence kit and the like. The colloidal gold method has a narrow detection range and cannot accurately quantify; some methods such as the nanobead chemiluminescence method require a specific detection instrument to be adapted.
However, conventional ELISA also has some significant drawbacks, such as: (1) each reaction step requires a reagent volume of at least 100. mu.L, whereas for some clinical serum samples that are difficult to obtain or expensive reagents, a 100. mu.L volume is large and difficult to obtain; (2) each incubation step requires 1-2 hours to achieve complete binding reaction, so that the total detection time can be completed within 5-6 hours; (3) the quantitative dynamic range of the obtained standard curve is narrow.
Therefore, aiming at the traditional ELISA detection kit, the development of a novel coronavirus ELISA detection kit with short detection time and less sample and reagent requirements is of great significance.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a novel coronavirus enzyme-linked immunoassay kit based on microfluidics, and a preparation method and a use method thereof. The detection kit can quickly, simply, sensitively and accurately detect the novel coronavirus antibody in a sample to be detected.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a novel coronavirus enzyme-linked immunoassay kit based on microfluidics, which comprises: a microfluidic plate coated with a novel coronavirus (SARS-CoV-2) S1 protein (Spike S1 protein), an enzyme conjugate, a fluorogenic substrate, and a sample diluent.
The kit provided by the invention adopts a microfluidic plate coated by the spike S1 protein of the novel coronavirus, and uses a blocking liquid to block blank sites, when the kit is used for a long time, serum or plasma with a certain dilution multiple of a patient to be detected is added, the antibody of the novel coronavirus in a positive sample is combined with the spike S1 protein in a microfluidic channel, and an enzyme conjugate is added to form an antigen-antibody-anti-antibody; adding a fluorescent substrate for color development, and comparing with a Spike IgG antibody standard curve with known content to quantitatively detect the concentration of the new coronavirus antibody in the serum; if the IgG antibody is detected, an antibody concentration change curve can be drawn through the content data of the Spike IgG antibody on different days, the disease course development of a patient can be judged, and meanwhile, the kit can also be applied to the quantitative detection of the specific IgG of the new crown Spike protein in the blood of a volunteer, and can effectively assist the subsequent vaccine development.
In a preferred embodiment of the present invention, the coating concentration of the novel coronavirus S1 protein on the microfluidic plate is 2-10. mu.g/mL, and may be, for example, 3. mu.g/mL, 4. mu.g/mL, 5. mu.g/mL, 6. mu.g/mL, 7. mu.g/mL, 8. mu.g/mL, or 9. mu.g/mL.
Preferably, the microfluidic plate comprises an optically transparent fluid detection channel comprising a liquid inlet and a liquid outlet within the fluid detection channel. For example: any of the array plates described in the patent (US20170097345a1) can be used as the microfluidic plate used in the present invention.
As a preferred technical scheme of the invention, the preparation method of the microfluidic plate coated with the novel coronavirus S1 protein comprises the following steps:
diluting the new coronavirus S1 protein, adding the diluted protein into a hole of the microfluidic plate, incubating for 40-80 min (for example, 50min, 55min, 60min, 65min, 70min and the like) at 20-28 ℃ (for example, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, or 27 ℃, and the like), transferring a sealing solution into the microfluidic plate, sealing for 20-40 min (for example, 22min, 25min, 30min, 35min, 38min and the like) at 20-28 ℃ (for example, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, or 27 ℃, and the like), washing, and drying to obtain the microfluidic plate coated with the new coronavirus S1 protein.
Illustratively, the microfluidic plate coated with the novel coronavirus S1 protein is prepared by the following method:
(1) coating: diluting a specific antigen, namely S1 protein to coating concentration, spotting the specific antigen into a sample adding funnel of the microfluidic plate at a rate of 20 mu L/hole to enable the S1 protein solution to fill the pore channel of the whole microfluidic plate, incubating at room temperature for 60min, and draining.
(2) And (3) sealing: spotting the blocking liquid into a sample-adding funnel by 20 mu L/hole to fill the microfluidic pore channel, combining with the vacant sites, sealing at room temperature for 30min, and draining to seal the redundant binding sites;
(3) washing: the microfluidic channels were washed 3 times with 20 μ L/well washing solution, i.e. PBST (phosphate tween buffer) was run through the microfluidic channels and then drained to remove excess blocking solution.
In a preferred embodiment of the present invention, the enzyme conjugate is an enzyme-labeled IgG antibody or an enzyme-labeled IgM antibody.
Preferably, the enzyme-labeled IgG antibody comprises a horseradish hydroxide enzyme-labeled IgG antibody.
Preferably, the enzyme-labeled IgM antibody comprises a horseradish hydroxide enzyme-labeled IgM antibody.
In a preferred embodiment of the present invention, the sample diluent comprises 0.008 to 0.012M (for example, 0.009M, 0.01M, 0.0105M, 0.011M, or 0.0115M) of Phosphate Buffered Saline (PBS).
Preferably, the fluorogenic substrate comprises: a fluorescent reaction substrate of horseradish peroxidase.
Preferably, the novel coronavirus enzyme-linked immunoassay kit further comprises a washing solution.
Preferably, the washing solution comprises: and a phosphate buffer containing tween 20 in a mass fraction of 0.03 to 0.06% (for example, 0.035%, 0.04%, 0.045%, 0.05%, or 0.055%).
In a second aspect, the present invention provides a method for preparing a novel coronavirus enzyme-linked immunoassay kit according to the first aspect, which specifically comprises the following steps:
preparing a microfluidic plate coated with a novel coronavirus S1 protein, then preparing an enzyme conjugate, a fluorogenic substrate and a sample diluent, and subpackaging to obtain the novel coronavirus enzyme-linked immunoassay kit;
preferably, the preparation method of the microfluidic plate coated with the novel coronavirus S1 protein comprises the following steps: and diluting the new coronavirus S1 protein, adding the diluted protein into a hole of the microfluidic plate, incubating at 20-28 ℃ for 40-80 min, transferring a confining liquid into the microfluidic plate, confining at 20-28 ℃ for 20-40 min, washing, and drying to obtain the microfluidic plate coated with the new coronavirus S1 protein.
Preferably, the sample loading volume is 15-25 μ L/well, for example, 16 μ L/well, 17 μ L/well, 18 μ L/well, 19 μ L/well, 20 μ L/well, 21 μ L/well, 22 μ L/well, 23 μ L/well or 24 μ L/well.
Preferably, the diluent used for diluting the new coronavirus S1 protein comprises 0.008-0.012M (for example, 0.009M, 0.01M, 0.0105M, 0.011M, or 0.0115M) phosphate buffer.
Preferably, the confining liquid comprises: a phosphate buffer containing 8 to 10% by mass (for example, 8.2%, 8.4%, 8.5%, 9%, 9.2%, 9.5%, 9.6%, or 9.8%) of bovine serum albumin.
In a third aspect, the present invention provides a method for using the novel coronavirus enzyme-linked immunoassay kit according to the first aspect, which specifically comprises the following steps:
(1) diluting a sample to be detected, a positive standard substance and a negative standard substance by using a sample diluent, respectively adding the diluted sample, the positive standard substance and the negative standard substance to a microfluidic plate coated with a novel coronavirus S1 protein, incubating and washing;
(2) adding an enzyme conjugate to the microfluidic plate, incubating again, and washing;
(3) and adding a fluorogenic substrate on the microfluidic plate, reacting, and detecting the fluorescence value of the microfluidic plate.
As a preferable technical scheme of the invention, the volume ratio of the sample to be measured and the sample diluent in the step (1) is 1 (5-1000), and may be, for example, 1:5, 1:8, 1:10, 1:20, 1:50, 1:80, 1:100, 1:120, 1:150, 1:200, 1:500, 1:800, 1:1000, or the like.
Preferably, the sample to be tested in step (1) comprises serum or plasma.
Preferably, the sample loading volume in step (1) is 15-25 μ L/well, for example, 16 μ L/well, 17 μ L/well, 18 μ L/well, 19 μ L/well, 20 μ L/well, 21 μ L/well, 22 μ L/well, 23 μ L/well or 24 μ L/well.
Preferably, the incubation time in step (1) is 13-20 min, such as 14min, 15min, 16min, 17min, 18min or 19 min.
Preferably, the incubation temperature in step (1) is 20-28 ℃, for example, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃ or 27 ℃.
As a preferred embodiment of the present invention, the volume of the enzyme conjugate in step (2) is 15 to 25. mu.L/well, and may be, for example, 16. mu.L/well, 17. mu.L/well, 18. mu.L/well, 19. mu.L/well, 20. mu.L/well, 21. mu.L/well, 22. mu.L/well, 23. mu.L/well or 24. mu.L/well.
Preferably, the incubation time in step (2) is 5-10 min, such as 6min, 7min, 8min or 9 min. Preferably, the reaction time in step (3) is 5-10 min, such as 6min, 7min, 8min or 9 min.
Preferably, the use method further comprises the operation of drawing a standard curve by using the positive control substance and the negative control substance.
As a preferable technical scheme of the invention, the using method comprises the following steps:
(1) diluting a sample to be detected, a positive control product and a negative control product respectively by using a sample diluent, adding samples to a microfluidic plate coated with a novel coronavirus S1 protein in a volume of 15-25 mu L/hole, incubating for 13-20 min at 20-28 ℃, and washing;
(2) adding an enzyme conjugate to the microfluidic plate, incubating for 5-10 min again, and washing;
(3) adding 13-20 mu L/hole of fluorogenic substrate on the microfluidic plate, reacting for 5-10 min, detecting the fluorescence value of the microfluidic plate, and drawing a standard curve by using the concentrations of the positive control substance and the negative control substance and the fluorescence value to obtain the concentration of the antibody in the sample to be detected.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) the detection kit provided by the invention adopts the microfluidic plate coated by the spike S1 protein of the novel coronavirus to detect a sample to be detected, improves the working process of ELISA, and greatly improves the detection speed; wherein the time including coating, sealing, sample incubation, enzyme-linked antibody incubation and substrate development is reduced from original about 20h to 2 h; the coating time is not included, the simple detection time is reduced to be within 30min from the original 4h, and the aim of quickly and quantitatively detecting and diagnosing the antibody of the novel coronavirus is fulfilled;
(2) the detection kit provided by the invention greatly reduces the consumption of consumables such as antibodies, reagents and the like, and reduces the consumption from the original 100 mu L to 20 mu L, and the reduction amount reaches 80%; meanwhile, the dynamic range of the standard curve is improved by 5-10 times by using fluorescent substrate for color development, and the kit detects the specific IgM/IgG antibody of the new coronavirus in the blood of an infected person by a microfluidic rapid ELISA method, so that rapid diagnosis and antibody quantification of a patient infected with the new coronavirus can be rapidly completed.
Drawings
FIG. 1 is a standard curve graph obtained by fitting according to the test results in example 4.
Detailed Description
The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.
Example 1
The embodiment provides an enzyme-linked immunoassay kit for detecting a novel coronavirus IgG antibody, which specifically comprises: a microfluidic plate coated with a novel coronavirus S1 protein, an enzyme conjugate, a fluorescent substrate, a sample diluent, a positive standard and a negative standard.
The enzyme conjugate is: goat Anti-Human IgG Fc Cross-adsorbed Secondary Antibody, HRP, Thermo, 31413; the fluorogenic substrates for the enzymatic reactions were: quantated Enhanced chemifluorcent HRP Substrate, Thermo, 15159; the sample diluent was 0.01M phosphate buffer.
The preparation method of the microfluidic plate coated with the novel coronavirus S1 protein comprises the following steps:
(1) coating: diluting a specific antigen, namely S1 protein, to a coating concentration of 5 mug/mL by using a phosphate buffer solution, spotting the specific antigen into a sample adding funnel of a microfluidic plate by using 20 mug/hole, filling the pore channel of the whole microfluidic plate with an S1 protein solution, incubating at room temperature for 60min, and draining;
wherein the microfluidic plate is prepared from Optofluidic Bioassay
OBK05, which is a microfluidic plate described in Patent US20170097345a1 (i.e. US Patent Number: 10,730,044);
(2) and (3) sealing: spotting the blocking liquid into a sample-adding funnel by 20 mu L/hole to fill the microfluidic pore channel, combining with the vacant sites, sealing at room temperature for 30min, and draining to seal the redundant binding sites;
(3) washing: the microfluidic channels were washed 3 times with 20 μ L/well washing solution, i.e. PBST (phosphate tween buffer) was run through the microfluidic channels and then drained to remove excess blocking solution.
Example 2
The embodiment provides an enzyme-linked immunoassay kit for detecting a novel coronavirus IgM antibody.
The difference from example 1 is that the enzyme conjugate is: horseradish hydroxide enzyme-labeled human IgM antibody.
Example 3
Provided in this example is a method for detecting IgG antibodies using the detection kit as described in example 1. The method comprises the following specific steps:
(1) sample incubation: diluting the sample and the standard substance with a sample diluent, spotting the sample and the standard substance into a sample-adding funnel by the volume of 20 mu L/hole to fill the microfluidic pore channel and combine with the specific antigen, incubating for 15min at room temperature to remove the redundant liquid after the sample and the standard substance are captured by the coated antigen.
(2) Washing: washing with the washing solution for 3 times, allowing the washing solution with appropriate concentration to flow through the micro-flow pore channel, and pumping to remove excessive sample.
(3) Incubation with enzyme-linked antibody: and (3) spotting the enzyme-linked antibody diluent into a sample adding funnel to fill the microfluidic pore channel with the enzyme-linked antibody diluent, and removing the redundant liquid after incubating for 5min at room temperature.
(4) Washing: washing for 4 times, flowing PBST washing solution through the microfluidic channel, draining, and repeating for 4 times.
(5) Spotting 13 mu L/hole fluorogenic substrate solution into a sample adding funnel, filling a microfluidic pore channel with the fluorogenic substrate solution, and incubating for 5min at room temperature.
(6) The excitation wavelength was set at 550nm and the emission wavelength was set at 605nm on a microplate reader, and the fluorescence value was measured.
Comparing the time required for the detection method of the present invention with the time required for the conventional ELISA method, as shown in table 1 below, the washing time is neglected in the table below for the longer time-consuming steps of coating and the like:
TABLE 1
As can be seen from the above table, the detection kit provided by the invention can obviously reduce the detection time, and the microfluidic plate is adopted, so that the coating time, the sample incubation time and the enzyme-linked antibody incubation time are obviously reduced.
Example 4
This example uses the method described in example 3 to detect IgG antibody concentrations in positive and negative standards. The method specifically comprises the following steps:
the positive standard was serum, and was diluted in a gradient of 400ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 20ng/mL, 10ng/mL and 5 ng/mL. The positive standard substance and the negative standard substance with the 7 concentrations are detected, and the detection results are shown in the following table 2:
TABLE 2
As can be seen from the above table, the accuracy of the retest at each concentration point of 5-400 ng/mL is 100% +/-10%, the accuracy requirement is met, and the lowest limit of quantitation (LOQ) is 5 ng/mL;
and a standard curve is drawn according to the method described in embodiment 3, the standard curve being fitted with four parameters, as shown in fig. 1, the standard curve being:
Y＝(4670.557-93559.003)/(1+(X/55.359)1.595)+93559.003，R2＝1。
therefore, the detection kit provided by the invention has the lowest limit of quantitation of 5ng/mL and the limit of detection (LOD) of 1 ng/mL.
Example 5
This example performs a matrix interference assay on a blank human serum using the method provided in example 3.
Human serum-containing samples were diluted and scaled at 1:1, 1:2, 1:5, 1:10 and 1:20, respectively, to a concentration of 100.000 ng/mL.
The results of the measurements are shown in Table 3 below:
TABLE 3
As can be seen from the above table, the spiked recoveries at 5-, 10-and 20-fold dilutions corresponded to 100% ± 25%, indicating that the minimal dilution of the interference-depleted serum was 5-fold. Since the lowest limit of quantitation was 5 ng/mL. Therefore, the lowest concentration of IgG antibody of serum spike protein S1 that can be quantitatively detected by using the method is 25 ng/mL.
In addition, the detection kit provided by the invention has the advantages that the detection time is obviously reduced, and the advantages are shown in the following table 4:
TABLE 4
| Comparing items | General ELISA method | ELISA method provided in the invention |
| Coating conditions | Coating overnight at 2-8 DEG C | Room temperature for 1 hour |
| Reagent/sample consumption | 100μL | 20μL |
| Lower limit of detection | 1ng/mL | 1ng/mL |
| Dynamic range of standard curve | 1～100 | 1～400 |
As can be seen from the above table, the detection kit provided by the invention has the advantages that the use conditions and the use scenes are relatively common, the detection can be carried out at room temperature, the sample consumption is relatively low, and the dynamic range of the standard curve is obviously large.
The enzyme-linked immunoassay kit for detecting the novel coronavirus IgG antibody provided in example 1 is verified in the above examples, and the enzyme-linked immunoassay kit for detecting the novel coronavirus IgM antibody provided in example 2 is also verified by the same method, and the obtained result is compared with a common ELISA method, so that the enzyme-linked immunoassay kit has the advantages of short time consumption, less sample consumption, high sensitivity and obviously large dynamic range of a standard curve.
In conclusion, the invention adopts the micro-fluidic plate technology, can rapidly detect the novel coronavirus IgG/IgM antibody in human serum or plasma, performs small-volume quantification on a sample to be detected, saves economic cost, simultaneously has better adaptability of the instrument, and can perform reading and data processing on a common enzyme-labeling instrument; the antibody detection method provided by the invention is complementary with the nucleic acid detection method, the false negative rate of the detection method is reduced by an antigen-antibody pairing detection mode, and the requirement on a laboratory is reduced.
The novel coronavirus IgM and IgG antibody detection kit adopts serum or plasma as a detection sample type, blood samples are convenient to collect, the general blood samples are low in toxicity or do not contain viruses, the infection risk of medical workers can be greatly reduced, complex processing procedures of the samples during laboratory detection are omitted, the detection result can be quickly obtained, the operation is simple, the safety of the medical workers can be protected, and meanwhile, the current huge clinical diagnosis and treatment pressure can be greatly relieved.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
Claims (10)
1. A novel coronavirus enzyme-linked immunoassay kit based on microfluidics, which is characterized by comprising:
a microfluidic plate coated with a novel coronavirus S1 protein, an enzyme conjugate, a fluorescent substrate, and a sample diluent.
2. The novel coronavirus enzyme-linked immunoassay kit according to claim 1, wherein the coating concentration of the novel coronavirus S1 protein on the microfluidic plate is 2-10 μ g/mL;
preferably, the microfluidic plate comprises an optically transparent fluid detection channel comprising a liquid inlet and a liquid outlet within the fluid detection channel.
3. The novel coronavirus ELISA kit of claim 1 or 2, wherein the microfluidic plate coated with the novel coronavirus S1 protein is prepared by a method comprising:
and diluting the new coronavirus S1 protein, adding the diluted protein into a hole of the microfluidic plate, incubating at 20-28 ℃ for 40-80 min, transferring a confining liquid into the microfluidic plate, confining at 20-28 ℃ for 20-40 min, washing, and drying to obtain the microfluidic plate coated with the new coronavirus S1 protein.
4. The novel coronavirus ELISA detection kit according to any one of claims 1 to 3, wherein the enzyme conjugate is an enzyme-labeled IgG antibody or an enzyme-labeled IgM antibody;
preferably, the enzyme-labeled IgG antibody comprises a horseradish hydroxide enzyme-labeled human IgG antibody;
preferably, the enzyme-labeled IgM antibody comprises a horseradish hydroxide enzyme-labeled human IgM antibody.
5. The novel coronavirus ELISA test kit according to any one of claims 1 to 4, wherein the sample diluent comprises 0.008 to 0.012M phosphate buffer;
preferably, the fluorogenic substrate comprises a fluorogenic reaction substrate for horseradish peroxidase;
preferably, the novel coronavirus enzyme-linked immunoassay kit further comprises a washing solution;
preferably, the washing solution comprises: phosphate buffer solution containing 0.03-0.06% of Tween 20 by mass;
preferably, the novel coronavirus enzyme-linked immunoassay kit further comprises a positive standard substance and/or a negative standard substance.
6. The preparation method of the novel coronavirus enzyme-linked immunoassay kit according to any one of claims 1 to 5, wherein the preparation method comprises the following steps:
preparing a microfluidic plate coated with a novel coronavirus S1 protein, then preparing an enzyme conjugate, a fluorogenic substrate and a sample diluent, and subpackaging to obtain the novel coronavirus enzyme-linked immunoassay kit;
preferably, the preparation method of the microfluidic plate coated with the novel coronavirus S1 protein comprises the following steps: diluting the new coronavirus S1 protein, adding the diluted protein into a hole of the microfluidic plate, incubating at 20-28 ℃ for 40-80 min, transferring a confining liquid into the microfluidic plate, confining at 20-28 ℃ for 20-40 min, washing, and drying to obtain the microfluidic plate coated with the new coronavirus S1 protein;
preferably, the volume of the sample adding is 15-25 muL/hole;
preferably, the diluent used for diluting the new coronavirus S1 protein comprises 0.008-0.012M phosphate buffer solution;
preferably, the confining liquid comprises: and phosphate buffer solution containing 8-10% of bovine serum by mass.
7. The use method of the novel coronavirus enzyme-linked immunoassay kit according to any one of claims 1 to 6, wherein the use method comprises the following steps:
(1) diluting a sample to be detected by using a sample diluent, adding the sample to a microfluidic plate coated with a novel coronavirus S1 protein, incubating and washing;
(2) adding an enzyme conjugate to the microfluidic plate, incubating again, and washing;
(3) and adding a fluorogenic substrate on the microfluidic plate, reacting, and detecting the fluorescence value of the microfluidic plate.
8. The use method of claim 7, wherein the volume ratio of the sample to be tested to the sample diluent in the step (1) is 1 (5-1000);
preferably, the sample to be tested in step (1) comprises serum or plasma;
preferably, the volume of the sample adding in the step (1) is 15-25 muL/hole;
preferably, the incubation time in the step (1) is 13-20 min;
preferably, the incubation temperature in the step (1) is 20-28 ℃.
9. The use of claim 7 or 8, wherein the volume of the enzyme conjugate in step (2) is 15 to 25 μ L/well;
preferably, the incubation time in the step (2) is 5-10 min;
preferably, the reaction time in the step (3) is 5-10 min;
preferably, the use method further comprises the operation of drawing a standard curve by using the positive control substance and the negative control substance.
10. Use according to any one of claims 7 to 9, comprising the following steps:
(1) diluting a sample to be detected, a positive control product and a negative control product respectively by using a sample diluent, adding samples to a microfluidic plate coated with a novel coronavirus S1 protein in a volume of 15-25 mu L/hole, incubating for 13-20 min at 20-28 ℃, and washing;
(2) adding an enzyme conjugate to the microfluidic plate, incubating for 5-10 min again, and washing;
(3) adding 13-20 mu L/hole of fluorogenic substrate on the microfluidic plate, reacting for 5-10 min, detecting the fluorescence value of the microfluidic plate, and drawing a standard curve by using the concentrations of the positive control substance and the negative control substance and the fluorescence value to obtain the concentration of the antibody in the sample to be detected.
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| CN115290877A (en) * | 2022-08-22 | 2022-11-04 | 深圳市科瑞达生物技术有限公司 | Microfluidic chip for multi-item joint detection of whole blood sample, and whole blood detection method and system |
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| US20170097345A1 (en) * | 2015-10-01 | 2017-04-06 | The Regents Of The University Of Michigan | Assay plate and uses thereof |
| CN111505316A (en) * | 2020-05-22 | 2020-08-07 | 无锡市孚维尔生物医疗科技有限公司 | Production method of novel coronavirus 2019-nCoV antibody spectrum detection kit |
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| US20170097345A1 (en) * | 2015-10-01 | 2017-04-06 | The Regents Of The University Of Michigan | Assay plate and uses thereof |
| CN111505316A (en) * | 2020-05-22 | 2020-08-07 | 无锡市孚维尔生物医疗科技有限公司 | Production method of novel coronavirus 2019-nCoV antibody spectrum detection kit |
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| CN115290877A (en) * | 2022-08-22 | 2022-11-04 | 深圳市科瑞达生物技术有限公司 | Microfluidic chip for multi-item joint detection of whole blood sample, and whole blood detection method and system |
| CN115290877B (en) * | 2022-08-22 | 2023-10-03 | 深圳市科瑞达生物技术有限公司 | Microfluidic chip for multi-item joint inspection of whole blood sample and whole blood detection method and system |
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