CN111665353A - Compact immunoassay device and analysis method - Google Patents
Compact immunoassay device and analysis method Download PDFInfo
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- CN111665353A CN111665353A CN202010171362.2A CN202010171362A CN111665353A CN 111665353 A CN111665353 A CN 111665353A CN 202010171362 A CN202010171362 A CN 202010171362A CN 111665353 A CN111665353 A CN 111665353A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- 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
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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- G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus
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- G—PHYSICS
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- Immunology (AREA)
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- Molecular Biology (AREA)
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Abstract
The invention discloses a compact immunoassay device, which comprises a plurality of independent first cavities for storing liquid reagents and a second cavity for placing a solid-phase detection element, wherein the first cavities are separated from the second cavities, each first cavity comprises a first opening, each second cavity comprises a second opening, the first openings and the second openings can be sealed or opened, a plurality of labels are arranged on the upper surfaces of the second cavities, and the labels correspond to the first cavities respectively. The invention also discloses an immunoassay method, which can test one sample at a time without depending on an instrument to obtain the detection results of a plurality of indexes; the invention can be applied to the fields of medicine, veterinary medicine, agriculture, animal husbandry, food safety, environmental monitoring, biological safety and the like.
Description
Technical Field
The invention particularly relates to a compact immunoassay device and an immunoassay method.
Background
Immunological analysis methods are widely used in the detection of trace substances due to their unique specificity, high sensitivity, and operability. The method has wide application in the fields of medicine, veterinary medicine, agriculture, animal husbandry, food safety, environmental monitoring, biological safety and the like.
The analysis method can be classified into an instrument-dependent analysis method and an instrument-independent analysis method according to usage scenarios. The former includes radioimmunoassay, chemiluminescence, flow-through fluorescence, automated enzyme-linked immunoassay, turbidimetry, light-activated chemiluminescence and other methods which rely on large-scale analytical equipment, and also includes immunochromatography or diafiltration methods which rely on small-scale instruments for fluorescein, fluorescent microspheres, quantum dots, fluorescent proteins and the like. The latter include colloidal gold immunochromatography, colloidal gold percolation, and latex, quantum dots, fluorescent materials derived by observing changes in signals, and a variety of methods involving enzymes.
The analysis method can be divided into a common analysis method which is a single result and a chip analysis method which is a multi-result according to the number of the analyzed results. There are two current understandings for such chips, the first being the Lab on one chip. This method, which integrates analytical reagents into a single space, usually achieves a qualitative or quantitative detection result after each analysis. The design can be minimally instrument dependent, thereby obtaining relatively accurate detection results. The other is not to emphasize the integration and miniaturization of different functional components of the immunological reagent, but by proper design, a single sample can obtain qualitative or quantitative detection results of multiple indexes after passing through one analysis process. From the above analysis, when an enzyme and a corresponding substrate are used as a detectable signal in an immunoassay, the effect of obtaining a multi-index measurement result simultaneously without depending on an instrument can be achieved.
Currently, an immunoassay device and an immunoassay method are various, but the existing method has some disadvantages: firstly, mutual pollution is easy to occur in the detection process, and the accuracy of the detection result is influenced; second, the liquid reagent is pre-loaded in the device and the test element is not placed in the device but is otherwise stored, which increases the risk of loss due to separation, thereby failing the field application. Thirdly, the immunoassay device is large, occupies a large space, and is not convenient to carry. Fourthly, some devices carry more reagents, but the field detection does not need to carry incomplete reagents, and the redundant reagents cause troubles to the practical application.
Disclosure of Invention
In view of the above, the present invention provides a compact immunoassay device and method for analyzing an analyte, which overcome the drawbacks of the prior art.
In order to achieve the purpose, the invention provides the following technical scheme:
the utility model provides a compact immunoassay device, includes a plurality of independent first chambeies that are used for saving liquid reagent, is used for placing the second chamber of solid phase detection component, and first chamber is separated with the second chamber, and first chamber includes first opening, and the second chamber includes the second opening, and first opening and second opening homoenergetic are sealed or are opened, and second chamber upper surface is provided with a plurality of labels, and a plurality of labels correspond to a plurality of first chambeies respectively.
Further, the first opening is positioned on the upper surface of the device, and the second opening is positioned on the lower surface of the device; the first opening can be sealed by a first sealing element and the second opening can be sealed by a second sealing element.
Further, the liquid reagent comprises a sample diluent for diluting a sample to be tested; a substance labeled with a labeling substance; the cleaning solution is used for removing residual cleaning solution in the immune reaction process; substrate solution for amplifying reaction signal.
Further, the solid phase detection element includes a handle region and a detection region.
Further, the solid phase detection element refers to: solid phase materials with immobilized macromolecular materials including, but not limited to, polypeptides, proteins, polysaccharides, nucleic acids, deoxyribonucleic acids, polymeric compounds, and synthetic conjugates.
Further, a macromolecular material is immobilized on the detection region.
Further, the solid phase detecting element is a solid phase sheet, and the width of the handle region is larger than that of the detection region.
Further, the lower end of the detection area is provided with a tip; the handle area adopts an asymmetric structure.
An immunoassay method using the above-mentioned assay device, comprising the steps of:
(1) opening the second sealing element, taking out the solid phase sheet, and placing the immunoassay device forward;
(2) opening the first sealing element, injecting the sample into the first cavity B1 containing the sample diluent, and mixing the sample with the sample diluent in the first cavity B1;
(3) inserting the solid phase sheet into a first cavity B1, drawing up and down, and standing for 1-10 minutes;
(4) when the time is up, drawing out the solid phase sheet, scraping off the residual liquid, inserting the solid phase sheet into a first cavity B2 containing the cleaning liquid, drawing for about 10 times, and standing for 0-2 minutes;
(5) when the time is up, drawing out the solid phase sheet, scraping off the residual liquid, inserting the first cavity B3 containing the substance marked by the marker, drawing about 10 times, and standing for 1-10 minutes;
(6) when the time is up, drawing out the solid phase sheet, scraping off the residual liquid, inserting the solid phase sheet into a first cavity B4 containing the cleaning liquid, drawing for about 10 times, and standing for 0-2 minutes;
(7) when the time is up, drawing out the solid phase sheet, scraping off the residual liquid, inserting the solid phase sheet into a first cavity B5 containing the cleaning liquid, drawing for about 10 times, and standing for 0-2 minutes;
(8) when the time is up, drawing out the solid phase sheet, scraping off the residual liquid, inserting the solid phase sheet into a first cavity B6 containing substrate liquid, drawing for about 10 times, and standing for 1-10 minutes;
(9) when the time is up, drawing out the solid phase sheet, scraping off the residual liquid, inserting the solid phase sheet into a first cavity B7 containing the cleaning liquid, drawing for about 10 times, and standing for 0-2 minutes;
(10) when the time is up, the solid phase sheet is drawn out, the residual liquid is scraped off, the solid phase sheet is dried in the air, and the measurement result is observed.
Further, preparation of a solid phase sheet: spraying 0.2-2 μ L of macromolecular material with concentration of 0.5-5 μ g/mL and quality control of 0.01-0.1 μ g/mL on solid phase carrier, and fixing the protein sprayed on the solid phase carrier by physical adsorption or chemical covalent bonding; eliminating unfixed specific antigen, and drying to obtain a solid phase sheet. The macromolecular material comprises but is not limited to polypeptide, protein, polysaccharide, nucleic acid, deoxyribonucleic acid, macromolecular compound and artificially synthesized conjugate; for example, the macromolecular material may be a virus-specific antigen.
Further, any one of horseradish peroxidase, alkaline phosphatase and colloidal gold is used as the marker.
Further, the substance labeled with the labeling substance is an anti-animal species-specific antibody labeled with horseradish peroxidase, including but not limited to immunoglobulin (i.e., secondary antibody) of human, mammal, bird, fish, etc., and the source of the secondary antibody also includes but not limited to mammal and bird. The anti-animal species specific antibody can specifically recognize and combine with substances in the sample combined with the macromolecular material substance fixed on the detection area of the solid phase plate, including but not limited to various antibodies specifically recognizing and combining with the species of the detected sample.
Further, the quality control is a substance related to the detected object or related to the observed signal, an antibody specific to an animal species, including but not limited to immunoglobulin or analog of human, mammal, bird, fish, etc., and macromolecular materials, including but not limited to polypeptide, protein, polysaccharide, nucleic acid, deoxyribonucleic acid, macromolecular compound, and artificially synthesized conjugate.
The invention has the beneficial effects that:
(1) the invention can test one sample at a time without depending on an instrument to obtain the detection results of a plurality of indexes; the invention can be applied to the fields of medicine, veterinary medicine, agriculture, animal husbandry, food safety, environmental monitoring, biological safety and the like.
(2) The immunoassay device of the invention seals both the solid-phase detection element and the pre-loaded liquid reagents with different functions; the solid-phase detection element and the liquid reagent are respectively stored in different areas of the immunoassay device in a sealing way, and the different areas are isolated from each other and do not interfere with each other; the liquid reagents with different functions are also separated from each other and do not interfere with each other; the first opening is securely sealed after the first chamber is filled or injected with a reagent to ensure that any interference caused by leakage through the first opening is eliminated. And the solid-phase detection element and the pre-loaded liquid reagents with different functions are placed in the device, are matched for use and cannot be lost.
(3) According to the invention, the plurality of first cavities are arranged on the same straight line, the second cavity is positioned beside the first cavity, and the length direction of the second cavity is parallel to that of the first cavity, so that the space can be better utilized, and a solid phase sheet can be accommodated; the structure is compact, and the size of the immunoassay device is not too large. The length direction of second chamber is parallel with the length direction of first chamber, also is convenient for set up the label on second chamber surface, makes the label can correspond different first chambers better.
Drawings
FIG. 1 is a schematic view showing the construction of an immunoassay device of the present invention (wherein the internal structure of the device is drawn with a dotted line).
FIG. 2 is a schematic view of the construction of the immunoassay device of the present invention (showing the first opening and the label, with the first sealing member hidden).
FIG. 3 is a schematic longitudinal sectional view of the immunoassay device of the present invention (showing the internal structures of the first chamber and the second chamber).
FIG. 4 is a schematic view of the immunoassay device of the present invention, which is transversely cut.
FIG. 5 is a schematic view showing the structure of an immunoassay device in example 2, when a solid phase sheet reacts with a diluted sample.
FIG. 6 is a schematic view showing the configuration of an immunoassay device in example 2, when a solid phase sheet after reaction with a sample is washed.
FIG. 7 is a schematic view of an immunoassay device in example 2, when a solid-phase sheet was reacted with an anti-canine IgG antibody-HRP conjugate.
FIG. 8 is a schematic view showing the structure of an immunoassay device in example 2, when the solid phase sheet after the reaction with the anti-canine IgG antibody-HRP conjugate was washed for the first time.
FIG. 9 is a schematic view showing the structure of an immunoassay device in example 2, when the solid phase sheet after the reaction with the anti-canine IgG antibody-HRP conjugate was washed for the second time.
FIG. 10 is a schematic view showing the configuration of an immunoassay device in example 2, when a solid phase sheet is reacted with a substrate solution.
FIG. 11 is a schematic diagram showing the configuration of an immunoassay device in example 2, when a solid phase sheet reacted with a substrate solution is washed.
FIG. 12 is a schematic structural view of a solid phase sheet.
FIG. 13 is a graph showing the results of detection in example 2; the method comprises the following steps of (a) testing the overall appearance of a solid phase sheet after a negative sample is tested, (b) testing the overall appearance of a solid phase sheet after a positive sample is tested, (c) testing the result of a negative sample in a solid phase sheet testing area, and (d) testing the result of a positive sample in the solid phase sheet testing area.
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, and it should be noted that the detailed description is only for describing the present invention, and should not be construed as limiting the present invention.
In the following description, the concentration unit M represents mol/L. For example, a 0.01M phosphate buffer solution refers to: 0.01mol/L phosphoric acid buffer solution.
A compact immunoassay device, as shown in FIGS. 1 to 4, comprises a plurality of independent first cavities B for storing liquid reagents and a second cavity 2 for placing a solid phase detection element, wherein the plurality of first cavities B are separated from each other, the first cavities B are also separated from the second cavity 2, and the plurality of first cavities B and the second cavity 2 do not interfere with each other and do not influence each other; first chamber B includes first opening 3, and second chamber 2 includes the second opening, and first opening 3 and second opening homoenergetic are sealed or are opened, and 2 upper surfaces of second chamber are provided with a plurality of labels, and a plurality of labels correspond to a plurality of first chamber B respectively. The immunoassay device of the invention seals both the solid-phase detection element and the pre-loaded liquid reagents with different functions; the solid phase detection element and the liquid reagent are respectively stored in different areas of the immunoassay device in a sealing way, and the different areas are mutually isolated and do not mutually interfere; the liquid reagents with different functions are also separated from each other and do not interfere with each other; the first opening is securely sealed after the first chamber is filled or injected with a reagent to ensure that any interference caused by leakage through the first opening is eliminated.
In some preferred modes, the plurality of first cavities B are respectively perpendicular to the second cavities 2, that is, the length direction of the second cavities is perpendicular to the length direction of the first cavities, and the second cavities 2 are positioned beside one of the first cavities.
In other preferable modes, the plurality of first cavities B are respectively parallel to the second cavity 2, that is, the length direction of the second cavity is parallel to the length direction of the first cavity, as shown in fig. 1-2, the plurality of first cavities are in a straight line, which results in a longer length of the immunoassay device, the second cavity 2 is located beside the first cavity, and the length direction of the second cavity is parallel to the length direction of the first cavity, so that the space can be better utilized, the structure of the immunoassay device is more compact, the volume of the device is not too large, and the label can be conveniently arranged on the surface of the second cavity, so that the label can better correspond to different first cavities. If, the length direction of second chamber and the length direction in first chamber are on a straight line, will lead to immunoassay device longer like this, and is pleasing to the eye, also be convenient for carry and use, also be convenient for set up the label, can't correspond label and a plurality of first chambeies.
In some preferred forms, the labels may be numbers or letters, or other marks, and the labels may be used to distinguish different first chambers B, or to indicate the detection sequence, and in some preferred forms, as shown in fig. 2, the labels are located beside the first openings 3 of the first chambers B, and the labels correspond to the first chambers one by one.
In some preferred forms, as shown in fig. 2-3, the first opening 3 is located on the upper surface of the immunoassay device and the second opening is located on the lower surface of the immunoassay device; the first opening 3 can be sealed by a first sealing element 8 and the second opening can be sealed by a second sealing element 7. A solid phase detection element or other material (such as a desiccant 6) can be placed into the second chamber 2 through the second opening and then sealed, and similarly a liquid reagent can be placed into the first chamber B through the first opening 3 and then sealed; in this embodiment, the sealing is performed by a heat sealing method.
In some preferred modes, the liquid reagent includes, but is not limited to, a sample diluent for diluting a sample to be tested, a substance labeled with a labeling substance; the cleaning solution is used for removing residual cleaning solution in the immune reaction process; substrate solution for amplifying reaction signal. In some preferred embodiments, the sample diluent is a functional substance dissolved in the aqueous phase. As the dilution, the concentration of the substance to be detected in the sample can be adjusted to improve the signal-to-noise ratio between the specific binding between the antigen and the antibody and the signal, and meanwhile, the substance added in the sample dilution is also beneficial to the specific binding between the antigen and the antibody. The sample dilution contains balanced salt for buffering liquid pH, such as phosphate, trihydroxymethyl aminomethane (Tris) hydrochloric acid, N-2-hydroxyethyl piperazine-N' -2-ethanesulfonic acid (HEPES) and the like, and the pH value of the diluted sample can be controlled to be close to neutral (pH is 6-8), so that the binding between antigen and antibody is facilitated; the sample diluent can contain neutral salt ions, sodium chloride, potassium chloride and the like, so that the osmotic pressure of the solution can be adjusted, the release of the detected antigen or antibody is facilitated, the dissolution of substances is enhanced, the uniformity degree of the diluted sample is improved, and the combination of the antigen and the antibody is facilitated; the sample diluent can also contain a certain amount of surfactant, such as one or more of Tween series, NP series, Triton series and the like, so that the surface tension between solid and liquid phases is reduced, the binding microenvironment of antigen and antibody is improved, and part of non-specific binding is eliminated; the sample diluent may also contain a certain amount of high molecular polymer, such as Polyoxyethylene (PVA), polyvinylpyrrolidone (PVP), Dextran (Dextran), etc., which are confirmed not to affect the specific antigen-antibody binding, and are helpful for eliminating a part of non-specific binding and improving the dispersion of each substance in the liquid, thereby facilitating the antigen-antibody binding; the sample diluent can also contain a certain amount of biomacromolecules irrelevant to the antigen-antibody specific reaction, such as bovine serum albumin, gelatin, animal derived serum, a heterophile antibody inhibitor and the like, and is used for eliminating part of non-specific binding, thereby being beneficial to the correlation of a signal to be observed only with the specific binding of the antigen-antibody. The above substances are merely illustrative of the actual composition of the sample diluent, and the selection and use of each substance is closely related to the verification of the experimental results, and thus the composition of different sample diluents is not uniform. After the sample diluent is added, the subsequent immune reaction can be facilitated. The substance labeled with a label refers to a substance labeled with a label including, but not limited to, horseradish peroxidase (HRP), Alkaline Phosphatase (AP), colloidal gold, etc., and linked to an antibody or antigen in a relatively stable manner. The connection mode of the label can be covalent, such as the label of HRP and AP, and also can be physical adsorption, such as colloidal gold; for removing residual wash solution during immunoreaction, the wash solution includes but is not limited to PBST (0.01M phosphate buffer, 0.15M sodium chloride, 0.05% Tween20, pH 7.4), TBST (0.01M Tris, 0.15M sodium chloride, 0.05% Tween20, pH 7.4), etc.; the cleaning solution can remove residual liquid to ensure that the subsequent reaction is not interfered. The substrate fluid used to amplify the reaction signal may be a chromogen catalyzed by an enzyme as an observable signal for the enzyme. For example, when horseradish peroxidase (HRP) is used as the observed signal, a precipitation-type substrate solution may be used; in the case of Alkaline Phosphatase (AP) as a signal to be observed, a substrate solution which can cause precipitation or attachment may be used; for the use of suitable colloidal gold as the observed signal, silver enhancing agents may be used.
In some preferred forms, as shown in FIG. 12, the solid phase detection element includes a handle region 11 and a detection region 12.
In some preferred forms, solid phase sheet 5 refers to: solid phase materials with immobilized biological materials, macromolecular materials including but not limited to polypeptides, proteins, polysaccharides, nucleic acids or deoxyribonucleic acids, macromolecular compounds, synthetic conjugates.
In some preferred forms, the macromolecular material is immobilized in the detection zone 12.
In some preferred embodiments, the solid phase detection element is a solid phase sheet, and as shown in FIG. 12, the width of the handle region 11 is greater than the width of the detection region 12. In some preferred forms, as shown in FIG. 12, solid phase sheet 5 has two regions different in width size, the region of small width being detection region 12 capable of being inserted into first cavity B and the region of large width being handle region 11 for taking solid phase sheet 5. In some preferred forms, the lower end of the detection region 12 has a pointed end that can be used to pierce the second sealing member, and in some preferred forms, the solid phase sheet has two different surfaces that are treated in a different manner, one smooth and one rough. In some detection items, the smooth surface is suitable, in some detection items, the rough surface is suitable, and in the embodiment, the virus specific antigen is fixed on the rough surface. In some preferred forms, the handle region is asymmetric. In some preferred embodiments, a protrusion 13 or other shape is provided on the upper end of the handle region 11 at a position other than the middle position, so that the handle region is asymmetric, which is advantageous in distinguishing and distinguishing between the two surfaces of the solid phase sheet, and determining which surface has the virus-specific antigen immobilized thereon.
In some preferred forms, as shown in fig. 3-4, a second chamber has solid phase sheet 5 and desiccant 6 stored therein.
An immunoassay method using the above-described assay device, comprising the steps of (as shown in fig. 5 to 11):
(1) opening second sealing member 7, taking out solid phase sheet 5, and then placing the immunoassay device in the forward direction;
(2) opening the first sealing member 8, injecting the sample into the first chamber B1 containing the sample diluent, and mixing the sample with the sample diluent in the first chamber B1;
(3) inserting solid phase sheet 5 into first cavity B1, twitching up and down, and standing for 5 minutes;
(4) when the time is up, drawing out the solid phase sheet 5, scraping off residual liquid, inserting into a first cavity B2 containing cleaning liquid, drawing for about 10 times, and standing for 1 minute;
(5) when the time is up, drawing out solid phase sheet 5, scraping off the residual liquid, inserting first chamber B3 containing the substance labeled with the labeling substance, twitching about 10 times, and standing for 5 minutes;
(6) when the time is up, drawing out the solid phase sheet 5, scraping off residual liquid, inserting into a first cavity B4 containing cleaning liquid, drawing for about 10 times, and standing for 1 minute;
(7) when the time is up, drawing out the solid phase sheet 5, scraping off residual liquid, inserting into a first cavity B5 containing cleaning liquid, drawing for about 10 times, and standing for 1 minute;
(8) when the time is up, drawing out the solid phase sheet 5, scraping off the residual liquid, inserting into the first cavity B6 containing the substrate liquid, drawing for about 10 times, and standing for 5 minutes;
(9) when the time is up, drawing out the solid phase sheet 5, scraping off residual liquid, inserting into a first cavity B7 containing cleaning liquid, drawing for about 10 times, and standing for 1 minute;
(10) when the time is up, the solid phase sheet 5 is drawn out, the residual liquid is scraped off, air-dried, and the measurement result is observed.
Example 1
In this embodiment, as shown in fig. 1-2, seven first cavities B and one second cavity 2 are included, the first cavities are B1, B2, B3, B4, B5, B6, and B7; the seven first cavities are parallel to the second cavity 2, independent from each other and do not influence each other; and seven first cavities B are in a straight line.
In this embodiment, the label is a number (1, 2, 3, 4, 5, 6, 7) and is marked on the upper surface of the second chamber 2, so that the different first chambers B can be distinguished simply and conveniently.
Canine Distemper Virus (CDV) detection reagent, Canine Parvovirus (CPV) and rabies virus (RABV) antibody detection reagent
The main component of the specific antigen is protein. There are two ways to obtain virus-specific antigens. One of them is to amplify the virus by using cells and then extract and purify the protein by using a biochemical method as a specific antigen of the virus. For example, CPV is amplified from F81 cells in large quantities and purified to obtain the relevant protein, which is used as specific antigen. The other is to find out the proper protein coding gene by analyzing the virus structure and comparing the immune response of the body. E.g., CDV F protein (fusion protein), N protein (nucleocapsid protein); VP2 protein of CPV (capsid protein which makes up about 90% of the virus surface); g protein (transmembrane glycoprotein) of RABV.
The sources of the various antigens as described above can be prepared on their own or purchased commercially.
Preparation of solid phase sheet 5: spraying 0.2-2 μ L of CDV, CPV, RABV virus specific antigen with concentration of 0.5-5 μ g/mL, and quality control (such as canine IgG) with concentration of 0.01-0.1 μ g/mL onto solid phase carrier, wherein the protein sprayed onto the solid phase carrier can be fixed on the solid phase carrier by physical adsorption or chemical covalent binding; removing unfixed protein, and drying to fix the above antigen or quality control on solid phase carrier to obtain solid phase sheet. The solid phase carrier includes, but is not limited to, PS (polystyrene), PVC (polyvinyl chloride), PP (polypropylene), glass, PMMA (polymethyl methacrylate), and high molecular polymer; the solid support may be a mixture of one or more of the above materials. For the convenience of observation, titanium dioxide and related auxiliary agents can be added to the solid phase carrier material which can be injection molded or blow molded as above, and the background of the formed solid material is modulated to be opaque white. The CDV, CPV, RABV virus specific antigens are mainly immobilized in the detection zone 12. Relatively speaking, the quality control is positioned at the top, the RABV, CPV and CDV virus specific antigens are downwards, the three antigens are mutually independent, the positions of the three antigens are not overlapped, and particularly, the position of the three antigens can be any position below the quality control point. The order of these three antigens may be freely arranged without particular limitation. In this example, the quality control is located at the top, and RABV, CPV and CDV virus specific antigens are arranged downwards in sequence. Of course, other virus-specific antigens may be selected, and are not limited to the several virus-specific antigens listed in this example, and the number of antigens may be set to 1 or 2 or 3 or other numbers as needed.
Putting the solid phase sheet 5 into the second cavity 2 through the second opening, and adding the drying agent 6; as shown in fig. 3-4.
50-300. mu.L of sample diluent, 50-300. mu.L of washing solution, 50-300. mu.L of anti-canine IgG antibody-HRP conjugate at a concentration of 0.1-10. mu.g/mL, and 50-300. mu.L of substrate solution were injected into the first chamber B through the first opening 3, respectively. In this embodiment, the sample diluent is: 0.01M tris buffer containing 10mg/mL BSA, 0.1% Triton X100, 0.15M sodium chloride, 0.05% Proclin 300 by mass fraction, pH 7.4; the cleaning solution is as follows: 0.01M phosphate buffer containing 0.05% Tween20 by volume fraction, 0.15M sodium chloride by mass fraction of 0.05% Proclin 300, pH 7.4; 50-300 μ L of anti-canine IgG antibody-HRP conjugate at a concentration of 0.1-10 μ g/mL: the diluent is 0.05M MES,0.15M sodium chloride, 1mg/mLBSA and 1mg/mL protein protective agent, the mass fraction is 0.05 percent of Proclin 300, and the pH value is 6.0; the substrate solution was a conventional commercially available TMB substrate solution.
The second chamber 2 containing the solid phase sheet 5 and the above-described liquid reagent, respectively, is sealed with the first chamber B by heat sealing in this example, using a heat seal film as the first sealing member 8 and a heat seal bar as the second sealing member 7.
Example 2. a compact immunoassay method using the assay device described above (as shown in FIGS. 5-11):
(1) a second sealing member 7 of solid phase sheet 5 is scratched with an instrument having a certain hardness and a sharp angle, and solid phase sheet 5 is taken out; placing the immunoassay device in a forward direction;
(2) sucking 5 mu L of dog serum or 10 mu L of dog whole blood sample by using a pipette, puncturing the heat-sealing film on the first cavity B1 by using a pipette tip in a puncturing mode, injecting the sample, and fully and uniformly mixing;
(3) as shown in FIG. 5, by holding solid phase sheet handle region 11 with hand and inserting solid phase sheet 5 into first chamber B1 into which a specimen has been added, the liquid in first chamber B1 rises and submerges the quality control point; the surface of the solid phase sheet is sufficiently wetted by the upward and downward twitching several times, so that the antigen specific to the surface of the solid phase sheet is sufficiently brought into contact with the diluted specimen, for example, 10 times of upward and downward twitching and left standing for 5 minutes, during which the antigen on the surface of the solid phase sheet can undergo an antigen-antibody binding reaction with the antibody in the specimen. The standing time can be adjusted according to the concentration of the immobilized antigen on the surface of the solid phase sheet, the sample dilution multiple and the performance of the sample diluent, and can be selected from 1-10 minutes for the purpose of rapidness of operation.
(4) When the time is up, holding solid phase sheet handle region 11 with hand, drawing out solid phase sheet 5, scraping off the residual liquid droplet at the first opening edge, keeping the residual liquid as much as possible to infiltrate the surface part of the solid phase sheet, usually not more than 5 μ L, piercing the tip of solid phase sheet 5 through first sealing element 8, inserting into first cavity B2 containing cleaning liquid, the liquid in first cavity B2 will rise and submerge the quality control point; pumping for several times to fully soak the surface of the solid phase sheet, standing for 1 minute, adjusting the standing time according to the components of the washing solution, and selecting 0-2 minutes for the purpose of rapidness and convenience in operation. The following settings regarding time are basically for the sake of quickness and convenience.
(5) When the time is up, the handle area of the solid phase sheet is held by hand, the solid phase sheet 5 is drawn out, residual liquid is scraped, the sharp end of the solid phase sheet 5 pierces the first sealing element 8 and is inserted into the first cavity B3 containing the anti-canine IgG antibody-HRP conjugate (the anti-canine IgG antibody-HRP conjugate is the rabbit secondary antibody labeled by horseradish peroxidase), and the liquid in the first cavity B3 rises and submerges the quality control point; twitching for about 10 times and standing for 5 minutes;
since the amount of anti-canine IgG antibody-HRP conjugate used was much larger than the amount of enzyme that could generate an observable signal in the substrate solution, in order to minimize the amount of enzyme remaining on solid phase sheet 5, the following (6) and (7) were used for two washes.
(6) When the time is up, holding the handle area of the solid phase sheet, pulling out the solid phase sheet 5, scraping off the residual liquid, piercing the tip of the solid phase sheet 5 through the first sealing member 8, inserting the solid phase sheet into the first chamber B4 containing the cleaning liquid, and allowing the liquid in the first chamber B4 to rise and submerge the quality control point; pumping up and down for about 10 times, and standing for 1 minute;
(7) when the time is up, holding the handle area of the solid phase sheet, pulling out the solid phase sheet 5, scraping off the residual liquid, piercing the tip of the solid phase sheet 5 through the first sealing member 8, inserting the solid phase sheet into the first chamber B5 containing the cleaning liquid, and allowing the liquid in the first chamber B5 to rise and submerge the quality control point; twitching for about 10 times and standing for 1 minute;
(8) when the time is up, holding the handle area of the solid phase sheet, drawing out the solid phase sheet 5, scraping off the residual liquid, piercing the tip of the solid phase sheet 5 through the first sealing member 8, inserting the solid phase sheet into the first chamber substrate liquid B6 containing the substrate liquid, and allowing the liquid in the first chamber B6 to rise and submerge the quality control point; twitching for about 10 times and standing for 5 minutes;
(9) when the time is up, holding the handle area of the solid phase sheet, pulling out the solid phase sheet 5, scraping off the residual liquid, piercing the tip of the solid phase sheet 5 through the first sealing member 8, inserting the solid phase sheet into the first chamber B7 containing the cleaning liquid, and allowing the liquid in the first chamber B7 to rise and submerge the quality control point; twitching for about 10 times and standing for 1 minute;
(10) when the time is up, holding the handle area of the solid phase sheet, drawing out the solid phase sheet 5, scraping off residual liquid, drying in the air, and observing;
the process does not exceed 20 minutes, and after airing, the determination results of CDV, CPV and RABV can be obtained simultaneously. The immunoassay method of the invention can obtain qualitative results and semi-quantitative results; a semi-quantitative result can be given based on the grey scale of the spot color. In this example, the results of negative detection are shown in FIGS. 13(a) and (c), and the results of positive detection are shown in FIGS. 13(b) and (d). In FIG. 13(d), from top to bottom, 4 spots are the quality control, RABV, CPV, CDV virus specific antigens, and the color depth in the figure can be used to obtain the detection results: antibodies to RABV and CDV were weakly positive and antibodies to CPV were strongly positive.
It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. The utility model provides a compact immunoassay device, characterized by includes a plurality of independent first chambeies that are used for saving liquid reagent, is used for placing the second chamber of solid phase detection component, and first chamber is separated with the second chamber, and first chamber includes first opening, and the second chamber includes the second opening, and first opening and second opening homoenergetic are sealed or are opened, and second chamber upper surface is provided with a plurality of labels, and a plurality of labels correspond to a plurality of first chambeies respectively.
2. The compact immunoassay device of claim 1, wherein the first opening is located on an upper surface of the device and the second opening is located on a lower surface of the device; the first opening can be sealed by a first sealing element and the second opening can be sealed by a second sealing element.
3. The compact immunoassay device of claim 1, wherein the liquid reagent comprises a sample diluent for diluting a sample to be tested; a substance labeled with a labeling substance; the cleaning solution is used for removing residual cleaning solution in the immune reaction process; substrate solution for amplifying reaction signal.
4. The compact immunoassay device of claim 1, wherein the solid phase detection element comprises a handle region and a detection region.
5. The compact immunoassay device of claim 4, wherein the solid phase detection element is: the solid phase material is fixed with macromolecular material, the macromolecular material adopts at least one of polypeptide, protein, polysaccharide, nucleic acid, deoxyribonucleic acid, macromolecular compound and artificially synthesized conjugate; the macromolecular material is immobilized on the detection zone.
6. The compact immunoassay device of claim 4, wherein the solid phase detecting element is a solid phase sheet, and the width of the handle region is larger than the width of the detection region; the lower end of the detection area is provided with a tip; the handle area adopts an asymmetric structure.
7. An immunoassay method using the analysis device according to any one of claims 1 to 6, comprising the steps of:
(1) opening the first sealing element, injecting the sample into the first cavity B1 containing the sample diluent, and mixing the sample with the sample diluent in the first cavity B1;
(2) inserting the solid phase sheet into a first cavity B1, drawing up and down, and standing for 1-10 minutes;
(3) when the time is up, drawing out the solid phase sheet, scraping off the residual liquid, inserting the solid phase sheet into a first cavity B2 containing the cleaning liquid, drawing for about 10 times, and standing for 0-2 minutes;
(4) when the time is up, drawing out the solid phase sheet, scraping off the residual liquid, inserting the first cavity B3 containing the substance marked by the marker, drawing about 10 times, and standing for 1-10 minutes;
(5) when the time is up, drawing out the solid phase sheet, scraping off the residual liquid, inserting the solid phase sheet into a first cavity B4 containing the cleaning liquid, drawing for about 10 times, and standing for 0-2 minutes;
(6) when the time is up, drawing out the solid phase sheet, scraping off the residual liquid, inserting the solid phase sheet into a first cavity B5 containing the cleaning liquid, drawing for about 10 times, and standing for 0-2 minutes;
(7) when the time is up, drawing out the solid phase sheet, scraping off the residual liquid, inserting the solid phase sheet into a first cavity B6 containing substrate liquid, drawing for about 10 times, and standing for 1-10 minutes;
(8) when the time is up, drawing out the solid phase sheet, scraping off the residual liquid, inserting the solid phase sheet into a first cavity B7 containing the cleaning liquid, drawing for about 10 times, and standing for 0-2 minutes;
(9) when the time is up, the solid phase sheet is drawn out, the residual liquid is scraped off, the solid phase sheet is dried in the air, and the measurement result is observed.
8. The immunoassay method according to claim 7, wherein the preparation of the solid phase sheet comprises: spraying 0.2-2 μ L of macromolecular material with concentration of 0.5-5 μ g/mL and quality control material with concentration of 0.01-0.1 μ g/mL onto solid phase carrier, and fixing the protein sprayed onto the solid phase carrier by physical adsorption or chemical covalent bonding; the unfixed protein was removed and dried to obtain a solid phase sheet.
9. An immunoassay according to claim 7, wherein the labeling substance is any one of horseradish peroxidase, alkaline phosphatase and colloidal gold.
10. The immunoassay method according to claim 7, wherein the substance labeled with the labeling substance is an antibody specific to an animal species and labeled with horseradish peroxidase.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999056130A1 (en) * | 1998-04-29 | 1999-11-04 | Queen Mary & Westfield College | Method for assaying a plurality of analytes |
| WO2003050537A1 (en) * | 2001-12-12 | 2003-06-19 | Proteome Systems Intellectual Property Pty Ltd | Diagnostic testing process |
| CN102147408A (en) * | 2010-12-31 | 2011-08-10 | 深圳市亚辉龙生物科技有限公司 | Method for testing anti-SmD1 antibody IgG and reagent device |
| CN104502586A (en) * | 2014-06-11 | 2015-04-08 | 陈岩松 | Immunochromatography detection method and test paper |
| JP2018132423A (en) * | 2017-02-15 | 2018-08-23 | 国立大学法人京都工芸繊維大学 | Immuno chromatography apparatus, method for detecting existence of object to be detected in sample solution using immuno chromatography apparatus, method for manufacturing immuno chromatography apparatus, and kit |
| RU2686490C1 (en) * | 2018-06-01 | 2019-04-29 | Федеральное бюджетное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФБУН ГНЦ ВБ "Вектор" Роспотребнадзора) | Kit for multiplex immunochemical analysis of antibodies and antigens in blood preparations |
| CN212622620U (en) * | 2020-03-12 | 2021-02-26 | 杭州布封科技有限公司 | Compact immunoassay device |
-
2020
- 2020-03-12 CN CN202010171362.2A patent/CN111665353A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999056130A1 (en) * | 1998-04-29 | 1999-11-04 | Queen Mary & Westfield College | Method for assaying a plurality of analytes |
| WO2003050537A1 (en) * | 2001-12-12 | 2003-06-19 | Proteome Systems Intellectual Property Pty Ltd | Diagnostic testing process |
| CN102147408A (en) * | 2010-12-31 | 2011-08-10 | 深圳市亚辉龙生物科技有限公司 | Method for testing anti-SmD1 antibody IgG and reagent device |
| CN104502586A (en) * | 2014-06-11 | 2015-04-08 | 陈岩松 | Immunochromatography detection method and test paper |
| JP2018132423A (en) * | 2017-02-15 | 2018-08-23 | 国立大学法人京都工芸繊維大学 | Immuno chromatography apparatus, method for detecting existence of object to be detected in sample solution using immuno chromatography apparatus, method for manufacturing immuno chromatography apparatus, and kit |
| RU2686490C1 (en) * | 2018-06-01 | 2019-04-29 | Федеральное бюджетное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФБУН ГНЦ ВБ "Вектор" Роспотребнадзора) | Kit for multiplex immunochemical analysis of antibodies and antigens in blood preparations |
| CN212622620U (en) * | 2020-03-12 | 2021-02-26 | 杭州布封科技有限公司 | Compact immunoassay device |
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