CN114814227A - Detection method and tool for novel coronavirus antibody - Google Patents

Detection method and tool for novel coronavirus antibody Download PDF

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Publication number
CN114814227A
CN114814227A CN202110118495.8A CN202110118495A CN114814227A CN 114814227 A CN114814227 A CN 114814227A CN 202110118495 A CN202110118495 A CN 202110118495A CN 114814227 A CN114814227 A CN 114814227A
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colloidal gold
antibody
protein
test strip
antigen
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张雁
刘大志
刘胤博
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Zhongke Meining Tianjin Pharmaceutical Technology Co ltd
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Zhongke Meining Tianjin Pharmaceutical Technology Co ltd
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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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

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Abstract

The application provides a colloidal gold test strip and a kit for detecting individual new coronavirus (COVID-19) antibodies, which can detect Receptor Binding Domain (RBD) antigens of S proteins of the new coronavirus and antibodies of N protein antigens, can detect IgG and IgM antibodies simultaneously, and have good detection performance and reliability.

Description

Detection method and tool for novel coronavirus antibody
Technical Field
The present application relates generally to the field of medical immunological testing; in particular, the present application provides methods and tools for the detection of antibodies elicited by a novel coronavirus (COVID-19) in an individual.
Background
Detection of infection by new coronaviruses is currently the most common nucleic acid detection method, but nucleic acid detection based on qPCR may show false negatives due to various factors such as differences in the number of virus particles in individual nasal swab samples, nucleic acid degradation or carbohydrate inhibition of PCR reactions during laboratory processing. Therefore, antibody testing is an important complementary means for the detection of new coronavirus infection.
Summary of The Invention
In a first aspect, the present application provides a colloidal gold test strip for detecting antibodies raised against infection with a new coronavirus (COVID-19) in an individual, comprising a loading zone, a colloidal gold labeled zone, a detection zone and a water uptake zone, wherein the colloidal gold labeled zone comprises a receptor binding domain antigen carrying a colloidal gold labeled new coronavirus S protein, a new coronavirus N protein antigen and an anti-chicken IgY antibody.
In some embodiments, the receptor binding domain antigen of the novel coronavirus S protein comprises the amino acid sequence set forth in SEQ ID NO. 1.
In some embodiments, the novel coronavirus N protein antigen comprises the amino acid sequence set forth in SEQ ID NO 2.
In some embodiments, the ratio of the receptor binding region antigen of the new coronavirus S protein to the new coronavirus N protein antigen is from 2:1 to 1:1, in terms of molar ratio.
In some embodiments, the anti-chicken IgY antibody is an anti-chicken IgY antibody of goat, sheep, mouse, rat, rabbit, human, guinea pig, horse, or cow origin.
In some embodiments, the ratio of the mass of the anti-chicken IgY antibody to the sum of the mass of the receptor binding region antigen of the neocoronavirus S protein and the mass of the antigen of the neocoronavirus N protein is 1:2 to 2: 1.
In some embodiments, the colloidal gold labeled region further comprises a blocking agent. In some embodiments, the blocking agent is Bovine Serum Albumin (BSA).
In some embodiments, the detection zone includes a first sub-detection zone coated with anti-human IgM antibody, a second sub-detection zone coated with anti-human IgG antibody, and a third sub-detection zone coated with chicken IgY antibody. In some embodiments, the amount of the anti-human IgM antibody, anti-human IgG antibody and chicken IgY antibody is substantially the same.
In some embodiments, the anti-human IgM antibody and the anti-human IgG antibody are independently selected from goat-, sheep-, mouse-, rat-, rabbit-, guinea-pig-, horse-, or bovine-derived antibodies. In some embodiments, the anti-human IgM antibody, the anti-human IgG antibody, and the anti-chicken IgY antibody are antibodies derived from the same species.
In a second aspect, the present application provides a kit for detecting or monitoring a neocoronavirus (COVID-19) infection in an individual, comprising:
the colloidal gold test strip of the first aspect, and
and the card box is matched with the colloidal gold test strip for use.
In a third aspect, the present application provides the use of the colloidal gold test strip of the first aspect in the manufacture of a kit for detecting or monitoring a neocoronavirus (COVID-19) infection in an individual.
In a fourth aspect, the present application provides a method for preparing the colloidal gold test strip of the first aspect, comprising the following steps:
(1) preparing or providing a colloidal gold solution;
(2) adding a protein to be marked (one or any combination of a receptor binding region antigen of a new coronavirus S protein, a new coronavirus N protein antigen and an anti-chicken IgY antibody) into the colloidal gold solution obtained in the step (1), and fully mixing to enable colloidal gold to fully coat the protein to be marked;
(3) adding a blocking agent (such as Bovine Serum Albumin (BSA)) into the product obtained in the step (2), uniformly mixing, standing, and removing a supernatant;
(4) adding a complex solution into the precipitate obtained in the step (3) to fully disperse the precipitate;
(5) applying the product of step (4) (if step (2) does not use a mixture of all the proteins to be labeled, mixing the products of step (4) of each protein to be labeled or a partial mixture) onto the colloidal gold-labeled zone material, and drying;
(6) marking the positions of the anti-human IgM antibody, the anti-human IgG antibody and the chicken IgY antibody, which correspond to the first sub-detection area, the second sub-detection area and the third sub-detection area, on the detection area material respectively, and drying; and
(7) and (4) assembling a sample loading area material, a water absorption area material, the colloidal gold labeled area material obtained in the step (5) and the detection area material obtained in the step (6).
In a fifth aspect, the present application provides a method of detecting or monitoring a new coronavirus (COVID-19) infection in an individual, comprising the steps of:
(1) providing the kit of the second aspect, and assembling the colloidal gold test strip and the cartridge together; and
(2) loading the individual biological sample from the loading area of the colloidal gold test strip and observing the results displayed in the detection area.
Brief description of the drawings
Fig. 1 shows a schematic structure of the colloidal gold kit of the present application.
Brief description of the sequences
SEQ ID NO. 1 is the amino acid sequence of the receptor binding region of the S protein of the novel coronavirus.
SEQ ID NO. 2 is the amino acid sequence of the N protein of the novel coronavirus.
Detailed Description
The development of an accurate and reliable new coronavirus infection detection tool has important medical and social significance. The colloidal gold test paper is a tool based on cross-flow immunochromatographic analysis, and is particularly suitable for detecting target protein in a biological sample of an individual. In the detection of new coronavirus infection, the colloidal gold test paper technology can be used for detecting antibodies, including IgG and IgM antibodies, caused by new coronavirus infection. The detection of the antibody of the new coronavirus has some similar applications to the detection of nucleic acid, such as the detection and discovery of asymptomatic infected persons, the examination before the cure and discharge of patients with new coronary pneumonia, the quarantine period monitoring of close contact persons or other people and the like, and therefore, can be used as an auxiliary tool for the detection of nucleic acid. Compared with nucleic acid detection, the equipment required by colloidal gold test paper detection is simpler, and even the detection can be carried out by the home of the object to be detected. In addition, due to the different properties of antibody detection and nucleic acid detection target molecules, antibody detection can bring some purposes that nucleic acid detection cannot achieve, such as community infection big data collection, immunity confirmation, identification of candidates that may become therapeutic antibody donors, vaccine effectiveness evaluation, and the like. Aiming at the aim, the inventor of the application carries out deep early-stage analysis and test, preparation process optimization and later-stage performance and reliability test, develops and obtains a new coronavirus infection detection tool and various inventions of the application. In addition, the detection of IgG and IgM antibodies is also of indicative significance for infection traceability and epidemiological studies.
While this application contains many specifics, these should not be construed as limitations on the scope of the invention or of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of particular inventions. Certain features that are described in the context of separate embodiments in this application can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.
Unless otherwise indicated, the terms herein have the same meaning as commonly understood by one of skill in the art, e.g., in reference to colloidal gold test strips, kits, and numerical units.
As used herein, the terms "comprises" and "comprising" mean either open or closed. For example, the terms "comprises" or "comprising" may mean that additional components or steps or other elements not listed may also be included or included, or that only the listed components or steps or other elements may be included or included.
Herein, the term "about" (e.g., in component amounts and reaction parameters) is to be interpreted in a sense that is generally understood by those skilled in the art. In general, the term "about" may be understood as any value within plus or minus 5% of a given value, for example, about X may represent any value in the range of 95% X to 105% X.
As used herein, the terms "substantially", "essentially" or the like represent the same/similar, coverage, completeness, etc. as can be determined by one of ordinary skill in the art based on the expertise. For example, the amounts of a and B are substantially the same, with the understanding that a and B are in the range of 95% to 105% of each other.
In a first aspect, the present application provides a colloidal gold test strip for detecting antibodies raised against infection with a new coronavirus (COVID-19) in an individual, comprising a loading zone, a colloidal gold labeled zone, a detection zone and a water uptake zone, wherein the colloidal gold labeled zone comprises a receptor binding domain antigen carrying a colloidal gold labeled new coronavirus S protein, a new coronavirus N protein antigen and an anti-chicken IgY antibody.
The colloidal gold test strip developed in the present application is similar to a conventional colloidal gold test strip in general working principle and structure, and generally includes a loading region (also referred to as a sample region, a sample pad, etc.), a colloidal gold labeling region (also referred to as a gold labeling region, a gold labeling pad, etc.), a detection region, and a water absorption region (also referred to as a water absorption pad, etc.). The structure schematic diagram is shown in figure 1. The term "subject" in this application generally refers to a human subject, but also includes other mammals.
The new coronavirus S protein refers to Spike protein of the new coronavirus, which is the most important pathogenic protein of the coronavirus, and a Receptor Binding Domain (RBD) contained in the Spike protein is responsible for recognizing a receptor of a cell, and the RBD is combined with transmembrane receptor protein ACE2 on a human cell membrane so as to help the cell to enter the interior of the cell, and an infected body can generate a corresponding RBD specific antibody.
The novel coronavirus N protein herein intertwines with viral genomic RNA to form a viral nucleocapsid, playing an important role in the synthesis of viral RNA. Meanwhile, the N protein is relatively conserved and accounts for the largest proportion in the structural proteins of the virus, so that the body can possibly generate high-level anti-N protein antibodies in the early infection stage. When viruses invade human bodies, the immune system of the human bodies automatically reacts under the stimulation of viral protein antigens to generate corresponding specific antibodies to resist the viruses, wherein the most common antibodies are IgG and IgM.
After research and molecular study, the inventor of the present application believes that antibody detection against the receptor binding region of the S protein and the N protein of the novel coronavirus can basically cover the infection of the novel coronavirus, and the infection caused by only the antibodies except the receptor binding region and the N protein is very little or negligible.
The anti-chicken IgY antibody contained in the colloidal gold labeled region is a quality control antibody, which ensures the completeness of the detection process together with the chicken IgY antibody coated in the quality control sub detection region (i.e., the third sub detection region) of the detection region. The anti-chicken IgY antibody may be an anti-antibody (i.e., a secondary antibody) of any species origin, such as goat, sheep, mouse, rat, rabbit, human, guinea pig, horse, or bovine origin. The application of the chicken IgY antibody/anti-chicken IgY antibody as a pair of quality control antibodies can improve the detection performance, and the possibility that the interference of quality control and detection presents false positive due to the anti-heterogeneous antibody in an individual human body is reduced as much as possible.
The structure of the new coronavirus is analyzed and sequenced, the RBD antigen of the S protein of the new coronavirus can comprise an amino acid sequence shown in SEQ ID NO. 1, and the N protein antigen can comprise an amino acid sequence shown in SEQ ID NO. 2. It will be appreciated by those skilled in the art that in the preparation of the colloidal gold strip, the RBD antigen of the S protein and the N protein are typically recombinantly expressed, and that recombinant expression inevitably introduces exogenous sequences such as purification tags (e.g., histidine tags), linker peptides, cleavage sites, etc., which would not be expected to affect the epitope, but would not have to be removed. Thus, the RBD antigen or N protein antigen of the novel coronavirus S protein of the present application may consist of the amino acid sequence shown in SEQ ID NO:1 or the amino acid sequence shown in SEQ ID NO:2, and optionally a purification tag (e.g., histidine tag), a linker peptide, and/or a cleavage site.
In the research and development process of the applicant, the proportion of the RBD antigen, the N protein antigen and the anti-chicken IgY antibody of the new coronavirus S protein in the colloidal gold labeled region has certain influence on the sensitivity and specificity of the detection result and is optimized. Thus, in some embodiments, the ratio of RBD antigen to N protein antigen of the S protein of the neocoronavirus to the N protein antigen is from 2:1 to 1: 1; the ratio of the mass of the anti-chicken IgY antibody to the sum of the mass of the RBD antigen of the new coronavirus S protein and the mass of the N protein antigen is 1:2 to 2: 1.
In some embodiments, the colloidal gold labeled region further comprises a blocking agent for reducing or eliminating non-specific binding of the protein. In some embodiments, the blocking agent is Bovine Serum Albumin (BSA).
The detection zone of the colloidal gold test paper strip of this application development can include the first sub-detection zone of peridium anti-human IgM antibody, peridium have anti-human IgG antibody the second sub-detection zone and peridium have chicken IgY antibody the third sub-detection zone (promptly, matter accuse sub-detection zone). Such an arrangement may allow for the simultaneous detection of both IgM and IgG antibodies, but may allow for the provision of only corresponding sub-detection zones if it is desired to detect only one of IgM and IgG antibodies. The location of the first, second and third sub-detection zones is not particularly required, but the third sub-detection zone, which is typically a quality control sub-detection zone, is located downstream in the direction of flow of the sample.
The amounts of anti-human IgM, anti-human IgG, and chicken IgY antibodies may be substantially the same, such a configuration may impart better contrast between the respective sub-detection zones.
Species sources of the anti-human IgM antibody and the anti-human IgG antibody are not particularly limited, and for example, they may be independently of goat, sheep, mouse, rat, rabbit, guinea pig, horse, or cow. However, from the viewpoint of improving the contrast, it is more desirable that the anti-human IgM antibody, the anti-human IgG antibody and the anti-chicken IgY antibody discussed above be derived from the same species.
For the results representative of the first, second, and third sub-detection zones, the third sub-detection zone must be developed to represent that the detection process is completed normally, so that the test in which the third sub-detection zone is not developed is regarded as an invalid test. In the case where the third sub-detection zone develops color, the first and second sub-detection zones develop color independently or simultaneously to represent detection of IgM antibody, IgG antibody, or both antibodies, respectively.
The colloidal gold test strip can also comprise a bottom plate, and the loading area, the colloidal gold marking area, the detection area and the water absorption area are supported by the bottom plate.
The material of the base plate and the respective regions may be a commonly used material. For example, the base plate may be made of polyvinyl chloride (PVC); the material of the sample loading area, the colloidal gold labeling area and the detection area can be a nitrocellulose membrane, a polyester fiber membrane or a glass fiber membrane carrier; the absorbent region may be made of an absorbent film or absorbent paper.
In a second aspect, the present application provides a kit for detecting or monitoring a neocoronavirus (COVID-19) infection in an individual, comprising:
the colloidal gold test strip of the first aspect, and
and the card box is matched with the colloidal gold test strip for use.
In a third aspect, the present application provides the use of the colloidal gold test strip of the first aspect in the manufacture of a kit for detecting or monitoring a neocoronavirus (COVID-19) infection in an individual.
Generally, a colloidal gold assay kit is equipped with a cartridge that is used in conjunction with a test strip, and the test strip is loaded into the cartridge as described before the assay. The cartridge is typically provided with a sample window in communication with the sample loading zone for loading the sample to be tested, and a result viewing window in communication with the detection zone for exposing each of the sub-detection zones.
In some cases, the kit may further include a buffer for administration after loading of the sample to be tested to provide system fluidity.
In a fourth aspect, the present application provides a method for preparing the colloidal gold test strip of the first aspect, comprising the following steps:
(1) preparing or providing a colloidal gold solution;
(2) adding a protein to be marked (one or any combination of a receptor binding region antigen of a new coronavirus S protein, a new coronavirus N protein antigen and an anti-chicken IgY antibody) into the colloidal gold solution obtained in the step (1), and fully mixing to enable colloidal gold to fully coat the protein to be marked;
(3) adding a blocking agent (such as Bovine Serum Albumin (BSA)) into the product obtained in the step (2), uniformly mixing, standing, and removing a supernatant;
(4) adding a complex solution into the precipitate obtained in the step (3) to fully disperse the precipitate;
(5) applying the product of step (4) (if step (2) does not use a mixture of all the proteins to be labeled, mixing the products of step (4) of each protein to be labeled or a partial mixture) onto the colloidal gold-labeled zone material, and drying;
(6) marking the positions of the anti-human IgM antibody, the anti-human IgG antibody and the chicken IgY antibody on the detection area material, which correspond to the first sub detection area, the second sub detection area and the third sub detection area respectively, and drying; and
(7) and (4) assembling a sample loading area material, a water absorption area material, the colloidal gold labeled area material obtained in the step (5) and the detection area material obtained in the step (6).
The preparation method of the colloidal gold test strip can generally utilize a conventional preparation method of the colloidal gold test strip, and an inventor conducts repeated test and optimization in the research and development process and finds technological characteristics beneficial to improving test performance, and the preparation method comprises the following steps:
A. the mutual proportion of the RBD antigen, the N protein antigen and the anti-chicken IgY antibody of the new coronavirus S protein in the colloidal gold labeling area is shown above;
B. using the chicken IgY antibody/anti-chicken IgY antibody as a pair of quality control antibodies, see above;
C. the formula of the sealed redissolution is as follows:
sucrose: 1.5-2.5g/L,
polymer (b): one or more of PVP, BSA, PEG4000, PEG6000, PEG8000 and PEG20000 at a concentration of 20-50g/L,
surfactant (b): one or more of TRITON-X100, TWEEN-20, TWEEN-80, S9 and S19 at a concentration of 20-40g/L,
buffer solution: one or more of phosphate buffer, Tris-HCl buffer, PBS and borate buffer with concentration of 20-40mM and pH of 7.2-7.6,
NaCl：0.2-1g/L，
solvent: water;
D. the RBD antigen of the S protein and the N protein antigen are mixed to prepare colloidal gold labeled solution, the anti-chicken IgY antibody is separately prepared into the colloidal gold labeled solution, and the colloidal gold labeled solution is mixed before being applied to the material of the colloidal gold labeled area.
As a non-limiting example, the preparation method of the colloidal gold test strip of the present application may include the following processes:
preparation of colloidal gold
1) 1g of HAuCl was taken 4 Adding into 99g of ultrapure water to prepare 1% HAuCl 4 And (3) solution.
2) 1g of trisodium citrate is added to 99g of ultrapure water to prepare a 1% trisodium citrate solution.
3) 1g of 1% HAuCl was added to the flask 4 The solution was added with 99g of ultrapure water, and the flask was weighed and recorded.
4) Placing the triangular flask on a magnetic heating stirrer, stirring and heating, adding 1-2g of 1% trisodium citrate solution when the temperature reaches about 98 deg.C, changing the color from yellow to colorless to black, and finally changing into brown red, and continuing to heat for 5min when the color is unchanged.
5) Gradually cooling the solution obtained in the step 4) at room temperature, weighing the triangular flask, filling the triangular flask with ultrapure water to the mass recorded in the step 3), and scanning by an ultraviolet detector at 526nm to confirm that the particle size of the colloidal gold is about 20-30 nm.
6) The prepared colloidal gold is sealed by tin foil paper and stored at 4 ℃.
Preparation of composite solution
Preparing a re-solution according to a formula
Colloidal gold-labeled antigen
1) Adding 20-40 μ L of 0.1M K into 1mL of colloidal gold 2 CO 3 The pH is adjusted to be alkaline.
2) Adding RBD protein and N protein into the solution of 1) at a ratio of 2:1-1:1 and a total amount of 10-20 μ g, mixing vigorously to completely coat RBD protein and N protein with colloidal gold, and standing at room temperature for 5-10 min.
3) Adding 20 μ L of 20% BSA solution into the solution obtained in 2), blocking, turning up and down, mixing uniformly, and standing at room temperature for 5-10 min.
4) Centrifuging the solution obtained in the step 3) for 5-10min at 3000-8000rcf, removing the supernatant, adding 0.5-1mL of redissolution, and uniformly dispersing the precipitate by using ultrasonic waves.
5) Taking 1mL of colloidal gold, adding 10-30 mu L of 0.1M K 2 CO 3 。
6) Adding 10-20 μ g of goat anti-chicken IgY antibody into the solution obtained in step 5), violently mixing to completely coat the goat anti-chicken IgY antibody with colloidal gold, and standing at room temperature for 5-10 min.
7) Adding 20 μ L of 20% BSA solution into the solution obtained in 6), blocking, turning up and down, mixing well, and standing at room temperature for 5-10 min.
8) Centrifuging the solution obtained in the step 7) for 5-10min at 3000-8000rcf, removing the supernatant, adding 0.5-1mL of redissolution, and uniformly dispersing the precipitate by using ultrasonic waves.
9) Uniformly mixing the solution 4) and the solution 8) according to the volume ratio of 1:1, and uniformly spreading the mixture on a gold label pad by using a 20-200 mu L range pipetting gun.
10) And (3) placing the gold label pad in an oven for drying at 37-42 ℃ for 0.5-1h, and placing the dried gold label pad in a dry sealing place for storage.
Marking off
Respectively marking detection lines on a nitrocellulose membrane (NC membrane) by using 0.5-1.5mg/mL goat anti-human IgM antibody and goat anti-human IgG antibody, marking a quality control line by using 0.5-1.5mg/mL chicken IgY antibody, and marking the detection lines and the quality control line according to the speed of 0.6-1.0 mu L/cm. And (4) placing the marked NC film in an oven for drying for 1-3h at 37-42 ℃. And (5) placing the dried film in a dry place for sealing and storing.
Product manufacture
The water absorption pad, the NC membrane, the gold label pad and the sample pad are overlapped and stuck on a PVC base plate of 30cm from top to bottom in sequence, and the strip is cut into strips according to the required width (for example, 3-5mm) by a strip cutting machine to obtain the detection test strip. During detection, the test strip is loaded into the card box to start sampling.
In a fifth aspect, the present application provides a method of detecting or monitoring a new coronavirus (COVID-19) infection in an individual, comprising the steps of:
(1) providing the kit of the second aspect, and assembling the colloidal gold test strip and the cartridge together; and
(2) loading the individual biological sample from the loading area of the colloidal gold test strip and observing the results displayed in the detection area.
The colloidal gold kit provided by the application is substantially the same as the conventional colloidal gold kit in the field, and suitable detection samples comprise whole blood, serum, plasma and the like.
The invention has been described in detail with respect to a general description and specific embodiments thereof, but it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Examples
The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. Experimental procedures without specific conditions noted in the examples below are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Unless defined otherwise, all terms of art or science used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are exemplary only.
Example 1 preparation of colloidal gold test strip
Preparation of colloidal gold
1) 1g of HAuCl was taken 4 Adding into 99g of ultrapure water to prepare 1% HAuCl 4 And (3) solution.
2) 1g of trisodium citrate is added to 99g of ultrapure water to prepare a 1% trisodium citrate solution.
3) To the flask were added 1g of 1% HAuCl4 solution and 99g of ultrapure water, and the flask was weighed and recorded.
4) Placing the triangular flask on a magnetic heating stirrer, stirring and heating, adding 1-2g of 1% trisodium citrate solution when the temperature reaches about 98 deg.C, changing the color from yellow to colorless to black, and finally changing into brown red, and continuing to heat for 5min when the color is unchanged.
5) Gradually cooling the solution obtained in the step 4) at room temperature, weighing the triangular flask, filling the triangular flask with ultrapure water to the mass recorded in the step 3), and scanning by an ultraviolet detector at 526nm to confirm that the particle size of the colloidal gold is about 20-30 nm.
6) The prepared colloidal gold is sealed by tin foil paper and stored at 4 ℃.
Preparation of composite solution
Preparing a complex solution according to the following formula:
sucrose: 1.5-2.5g/L,
polymer (b): one or more of PVP, BSA, PEG4000, PEG6000, PEG8000 and PEG20000 at a concentration of 20-50g/L,
surfactant (b): one or more of TRITON-X100, TWEEN-20, TWEEN-80, S9 and S19 at a concentration of 20-40g/L,
buffer solution: one or more of phosphate buffer, Tris-HCl buffer, PBS and borate buffer with concentration of 20-40mM and pH of 7.2-7.6,
NaCl：0.2-1g/L，
solvent: and (3) water.
Colloidal gold-labeled antigen
1) 1mL of colloidal gold was added to 20-40. mu.L of 0.1M K2CO3 to adjust the pH to alkaline.
2) Adding RBD protein and N protein (antigen amino acid sequences are respectively shown as SEQ ID NO:1 and SEQ ID NO:2, and obtained by in vitro recombinant expression mode) into the solution in the step 1) at a ratio of 2:1-1:1, and in a total amount of 10-20 μ g, violently mixing to completely coat the RBD protein and the N protein with colloidal gold, and standing at room temperature for 5-10 min.
3) Adding 20 μ L of 20% BSA solution into the solution obtained in 2), blocking, turning up and down, mixing uniformly, and standing at room temperature for 5-10 min.
4) Centrifuging the solution obtained in the step 3) for 5-10min at 3000-8000rcf, removing the supernatant, adding 0.5-1mL of redissolution, and uniformly dispersing the precipitate by using ultrasonic waves.
5) Take 1mL of colloidal gold, add 10-30. mu.L of 0.1M K2CO 3.
6) Adding 10-20 μ g of goat anti-chicken IgY antibody (commercially available) into the solution obtained in step 5), mixing vigorously to completely coat the goat anti-chicken IgY antibody with colloidal gold, and standing at room temperature for 5-10 min.
7) Adding 20 μ L of 20% BSA solution into the solution obtained in 6), blocking, turning up and down, mixing well, and standing at room temperature for 5-10 min.
8) Centrifuging the solution obtained in the step 7) for 5-10min at 3000-8000rcf, removing the supernatant, adding 0.5-1mL of redissolution, and uniformly dispersing the precipitate by using ultrasonic waves.
9) Uniformly mixing the solution 4) and the solution 8) according to the volume ratio of 1:1, and uniformly spreading the mixture on a gold label pad by using a 20-200 mu L range pipetting gun.
10) And (3) placing the gold label pad in an oven for drying at 37-42 ℃ for 0.5-1h, and placing the dried gold label pad in a dry sealing place for storage.
Marking off
Respectively marking detection lines on a nitrocellulose membrane (NC membrane) by using 0.5-1.5mg/mL goat anti-human IgM antibody (commercially available) and goat anti-human IgG antibody (commercially available), marking quality control lines by using 0.5-1.5mg/mL chicken IgY antibody (commercially available), and marking the detection lines and the quality control lines according to the speed of 0.6-1.0 mu L/cm. And (4) placing the marked NC film in an oven for drying for 1-3h at 37-42 ℃. And (5) placing the dried film in a dry place for sealing and storing.
Product manufacture
The water absorption pad, the NC membrane, the gold label pad and the sample pad are overlapped and stuck on a PVC base plate of 30cm from top to bottom in sequence, and the strip is cut into strips according to the required width (for example, 3-5mm) by a strip cutting machine to obtain the detection test strip. The test strip can be packaged in a sealed mode, and is packaged into a kit form together with a card box, a buffer solution and the like which are packaged in a sealed mode. During detection, the test strip is loaded into the card box to start sampling.
Example 2 detection of novel coronavirus antibodies
This example describes the general procedure for the detection of antibodies to the novel coronavirus using the kit of example 1, and the subsequent examples operate in accordance with this example.
Test sample
1. The kit is particularly suitable for detecting whole blood, serum or plasma samples.
2. If serum or plasma is used as the test sample, they are separated from the blood as quickly as possible to avoid hemolysis. Only clear, non-hemolyzed samples were used.
3. The test is best performed as soon as possible after the sample is collected. The samples were not allowed to stand at room temperature for a long time. Serum and plasma samples can be stored at 2-8 ℃ for up to 3 days. For long term storage, the samples should be kept below-20 ℃. If the test is performed within 2 days after collection, the whole blood collected by venipuncture should be stored at 2-8 ℃. Whole blood samples cannot be frozen. Whole blood collected from the fingertips is preferably tested immediately.
4. The samples were allowed to reach room temperature prior to testing. The frozen samples must be completely thawed and thoroughly mixed prior to testing. The sample is not suitable for transportation.
Test flow
Prior to testing, the kit, test sample, buffer (which may be included in the kit or may be additionally provided), and/or control sample (if any) are allowed to equilibrate to room temperature (15-30 ℃).
1. The kit is unpackaged and assembled (the strips and cartridges may also be assembled and hermetically sealed) and tested as soon as possible (preferably within 1 hour).
2. The test device was placed on a horizontal surface.
For serum or plasma samples:
mu.L of serum/plasma sample was loaded onto the sample zone, followed immediately by the addition of 2 drops (about 80. mu.L) of loading buffer.
For whole blood samples:
1 drop of whole blood (about 10. mu.L) was loaded onto the sample area, followed immediately by 2 drops (about 80. mu.L) of loading buffer.
3. Waiting for the appearance of a colored line. After 2 minutes, an additional 1 drop of loading buffer was added if the red color did not move through the test window or if blood was still present in the loading zone.
4. Results should be read within 5 minutes, with positive results seen as soon as possible in 2 minutes. The results after 15 minutes had no interpretation significance.
Interpretation of results
Regardless of the test line results, the quality control line must be developed for effective testing. And observing test lines representing the IgM antibody and the IgG antibody under the condition of the color development of the quality control line, wherein one or both of the test lines represent corresponding positive results. The kit may furthermore be equipped with a colorimetric card for semiquantifying positive results, for example in a strong/medium/weak text rating mode or in a numerical rating mode such as 3/2/1 (the latter being used for the kits of the examples of the present application).
Example 3 assay Performance of antibodies against New coronavirus
Serum samples were obtained from 45 new coronavirus nucleic acid detection positive individuals and 20 uninfected healthy individuals under informed consent. The inventors tested the above 65 samples using the kits of the examples of the present application, respectively. Tests using the kit of the present application gave negative results for all 20 healthy serum samples. For 45 nucleic acid detection positive serum samples, the positive results of antibody detection obtained by the test using the kit of the present application are shown in table 1.
Table 1.
Figure BDA0002921185130000091
Figure BDA0002921185130000101
Further expanding the sample amount experiment, the statistical data obtained by detecting more than 200 nucleic acid detection positive samples shows that the sensitivity of the kit of the application is 96% for IgG antibody detection and 82% for IgM antibody detection.
Example 4 reliability/validity test
4.1 comparison with chemiluminescence
Sample and method
Serum samples were provided by Davita for a total of 85 individuals including both new coronavirus infected and uninfected individuals. The concentrations of IgG and IgM antibodies against the new coronaviruses were determined beforehand by the Diazyme DZ-Lite New coronaviruses IgM and IgG CLIA test (CLIA test) which was run on a Diazyme Maglumi 2000Plus chemiluminescence analyzer with a cut-off set at 1 AU/mL. The colloidal gold kit is used for testing the serum sample, a reaction zone on the test strip is shot, the reaction zone is digitized by ImageJ, the density cut-off value is set to be 1, and three independent observers visually detect and verify the consistency through eyeballs.
Results
Table 3 shows the percent agreement of the two test results.
TABLE 3
Figure BDA0002921185130000102
From the above results, the colloidal gold kit of the present application has high sensitivity, the detection limit thereof is equivalent to the detection limit of 1AU/mL in CLIA chemiluminescence test, and the consistency of the results is also high.
4.2 negative sample testing
Sample and method
Serum samples from 91 subjects were obtained from a study collected in 2017 evaluating Brain Natriuretic Peptide (BNP) as a biomarker for cardiovascular disease. Prior to this test, these serum samples were stored at-80 ℃ and frozen/thawed no more than 3 times in previous BNP studies. The collection date of these serum samples was far earlier than the date when the first new instance of coronavirus pneumonia was reported and was considered a negative sample. The 91 serum samples were tested using the kits of the present application.
Results
Table 4 shows the test results.
TABLE 4
Figure BDA0002921185130000103
Figure BDA0002921185130000111
As shown in the results of Table 4, the colloidal gold kit of the present application has a low false positive rate and a good specificity, and the serum samples stored at-80 ℃ for a long time have little influence on the negative results.
4.3 fingertip blood test
Sample and method
As negative serum samples, serum samples of a total of 20 individuals in the study were selected for evaluation of anti-CCP 1 (cyclic citrullinated peptide 1) antibody collected in 2017 as a biomarker for Rheumatoid Arthritis (RA). Prior to this test, these serum samples were stored at-80 ℃ and frozen/thawed no more than 3 times in previous RA studies. The collection date of these serum samples was far earlier than the date when the first new instance of coronavirus pneumonia was reported and was considered a negative sample.
The positive serum sample was 20 serum samples selected from example 4.1 identified by the use of chemiluminescence and the kit of the present application.
Fingertip whole blood was collected from healthy individuals and the above positive and negative serum samples were spiked into fingertip whole blood at a ratio of 1: 5.
The above 40 serum samples were tested using the kit of the present application.
Results
Table 5 shows the test results.
TABLE 5
Figure BDA0002921185130000112
As shown in the results of table 5, the colloidal gold kit of the present application can be used for fingertip blood detection, but IgM may appear to be false negative because IgM in serum is estimated to be low in some cases.
4.4 Cross-reactivity test
Sample and method
The positive serum sample was 6 serum samples selected from example 4.1 identified by the use of chemiluminescence and the kit of the present application.
And (3) treating the serum sample:
(1) pre-diluted to weak positive with PBS (1% BSA) buffer (20-400X);
(2) pre-diluted and mixed with commercial heat-inactivated fresh coronavirus (isolate: USAWA1/2020) (1:5 vol.);
(3) pre-diluted and mixed with respiratory pathogen combination a from ZeptoMetrix;
(4) pre-diluted and mixed with respiratory pathogen combination B from ZeptoMetrix;
Figure BDA0002921185130000113
Figure BDA0002921185130000121
the serum samples treated as described above were tested using the kits of the present application.
Results
Table 6 shows the test results.
TABLE 6
Figure BDA0002921185130000122
As shown in the results of table 6, when the IgG/IgM antibody positive sample of the new coronavirus was mixed with the inactivated new coronavirus, the antibody/antigen bound so that the antibody was no longer detected, and the various respiratory pathogens in respiratory pathogen combinations a and B had no effect on the detection results, indicating that the kit of the present application has good pathogen specificity, low or no cross-reactivity.
4.5 model analysis
Sample and method
Serum samples from 15 individuals with negative, weak positive and strong positive results (5 each) in the new coronavirus IgG/IgM antibody test were selected.
mu.L of the above serum samples were spiked into 8. mu.L of serum, plasma, venipuncture whole blood and fingertip whole blood from the same donor (confirmed to be antibody negative by serum testing prior to the study), respectively, and then the above pooled blood samples were tested using the kit of the present application.
As a result, the
The results of this study (specific data not shown) show that the kit of the present application has consistent results in different models of serum, plasma, venipuncture whole blood and fingertip whole blood samples, and has strong applicability.
4.6 Distinguishing of IgG and IgM antibodies
Sample and method
This example determines the discriminatory properties of the kits of the present application for IgG and IgM antibodies by the different neutralizing properties of DTT for IgG and IgM antibodies.
The test samples were serum samples from 5 individuals who gave positive results in the new coronavirus IgG/IgM antibody test.
Control samples were recombinant anti-Spike humanized IgG and IgM antibodies.
The test and control samples were pretreated with 5mM DTT for 15 minutes at 37 ℃ and then dialyzed against 500mL PBS buffer (pH 7.5) overnight at 4 ℃ in a button dialysis bag to remove DTT. Meanwhile, in the absence of DTT, the same operation was repeated for the test sample and the control sample as a control.
The obtained samples were tested using the kit of the present application.
Results
Table 7 shows the test results.
TABLE 7
Figure BDA0002921185130000131
As shown in the results of table 7, the kit of the present application enables good discrimination between IgG and IgM antibodies.
Finally, it should be understood that while the various aspects of the present specification describe specific embodiments, those skilled in the art will readily appreciate that the disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Accordingly, it is to be understood that, unless explicitly stated otherwise, the disclosed subject matter is not limited to the particular compositions, methods, and/or formulations, etc., described herein. Moreover, those of ordinary skill in the art will recognize that certain changes, modifications, permutations, variations, additions, subtractions and sub-combinations may be made in accordance with the teachings herein without departing from the spirit of the present specification. It is therefore intended that the following appended claims be interpreted as including all such alterations, modifications, permutations, variations, additions, subtractions and sub-combinations as fall within the true spirit and scope thereof.
Sequence listing
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Claims (10)

1. A colloidal gold test strip for detecting antibodies caused by infection of a new coronavirus (COVID-19) in an individual comprises a loading area, a colloidal gold labeling area, a detection area and a water absorption area, wherein the colloidal gold labeling area comprises a receptor binding area antigen with a colloidal gold labeled new coronavirus S protein, a new coronavirus N protein antigen and an anti-chicken IgY antibody.
2. The colloidal gold test strip of claim 1, wherein the antigen of the receptor binding region of the new coronavirus S protein comprises the amino acid sequence shown in SEQ ID NO. 1 and/or the antigen of the new coronavirus N protein comprises the amino acid sequence shown in SEQ ID NO. 2.
3. The colloidal gold test strip of claim 1 or 2, wherein the ratio of the receptor binding domain antigen of the neocoronavirus S protein to the neocoronavirus N protein antigen is from 2:1 to 1:1 in terms of molar ratio.
4. The colloidal gold test strip of any one of claims 1-3, wherein the anti-chicken IgY antibody is of goat, sheep, mouse, rat, rabbit, human, guinea pig, horse or cow origin.
5. The colloidal gold test strip of any one of claims 1-4, wherein the ratio of the mass of the anti-chicken IgY antibody to the sum of the mass of the receptor binding region antigen of the neocoronavirus S protein and the mass of the antigen of the neocoronavirus N protein is 1:2 to 2: 1.
6. The colloidal gold test strip of any one of claims 1-5, wherein the colloidal gold labeled region further comprises a blocking agent, such as Bovine Serum Albumin (BSA).
7. The colloidal gold test strip of any one of claims 1-6, wherein the detection zone comprises a first sub-detection zone coated with anti-human IgM antibody, a second sub-detection zone coated with anti-human IgG antibody, and a third sub-detection zone coated with chicken IgY antibody, preferably, the amounts of the anti-human IgM antibody, the anti-human IgG antibody, and the chicken IgY antibody are substantially the same.
8. The colloidal gold test strip of claim 7, wherein the anti-human IgM antibody and the anti-human IgG antibody are independently selected from antibodies of goat, sheep, mouse, rat, rabbit, guinea pig, horse or cow origin, preferably the anti-human IgM antibody, the anti-human IgG antibody and the anti-chicken IgY antibody are antibodies of the same species origin.
9. A kit for detecting or monitoring a neocoronavirus (COVID-19) infection in an individual, comprising:
the colloidal gold test strip of any one of claims 1-8, and
and the card box is matched with the colloidal gold test strip for use.
10. Use of the colloidal gold test strip of any one of claims 1-8 in the manufacture of a kit for detecting or monitoring a neocoronavirus (COVID-19) infection in an individual.
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