CN111879951A - Novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip - Google Patents
Novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip Download PDFInfo
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- CN111879951A CN111879951A CN202010827101.1A CN202010827101A CN111879951A CN 111879951 A CN111879951 A CN 111879951A CN 202010827101 A CN202010827101 A CN 202010827101A CN 111879951 A CN111879951 A CN 111879951A
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Abstract
The invention provides a novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip, which comprises: a colloidal gold adsorption pad and an antibody bearing film; the colloidal gold adsorption pad is coated with a colloidal gold-labeled novel coronavirus recombinant antigen and a colloidal gold-labeled mouse IgG; the antibody bearing film is provided with a detection line T1, a detection line T2 and a quality control line C which are arranged at intervals, and the detection line T1 is coated with an anti-human IgA antibody; detection line T2 was coated with anti-human IgG antibody. The immunochromatographic test strip can be used for simultaneously detecting novel coronavirus IgA and IgG antibodies, the detection time is short and is only 10min, the cost is low, the operation is easy, the technical requirement is low, the on-site sample collection (particularly oral mucosa exudate) is facilitated, and the immunochromatographic test strip is suitable for on-site, instant and rapid detection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a novel coronavirus IgA/IgG antibody detection kit.
Background
Coronavirus is an enveloped RNA virus widely distributed in humans, other mammals and birds, and causes respiratory, intestinal, liver and nervous system diseases. Seven coronaviruses are currently known to cause human disease. Four viruses-229E, OC43, NL63 and HKU 1-are ubiquitous and cause symptoms of the common cold in immunocompromised individuals. The other three strains, severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and 2019 novel coronavirus (covi-19), are zoonotic viruses, sometimes associated with a fatal disease. IgG and IgA antibodies were detected after 2-3 weeks of exposure of the novel coronavirus. Studies have shown that IgA performs particularly well in critically ill patients, but over time antibody levels decline and IgG remains positive.
Disclosure of Invention
In a first aspect, the invention provides a novel immunochromatographic test strip for combined detection of an IgA/IgG antibody of a coronavirus, which comprises: a colloidal gold adsorption pad and an antibody bearing film; the colloidal gold adsorption pad is coated with a colloidal gold-labeled novel coronavirus recombinant antigen and a colloidal gold-labeled mouse IgG; the antibody bearing film is provided with a detection line T1, a detection line T2 and a quality control line C which are arranged at intervals, and the detection line T1 is coated with an anti-human IgA antibody; detection line T2 was coated with anti-human IgG antibody.
Furthermore, the sequence of the novel coronavirus recombinant antigen is shown as SEQ ID NO. 1.
In a second aspect, the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip is applied to detection or auxiliary detection of whether a sample to be detected contains the novel coronavirus IgA/IgG antibody;
further, the sample candidate to be tested contains a novel coronavirus IgA/IgG antibody;
the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip is applied to preparation and detection or auxiliary detection of whether a sample to be detected contains a novel coronavirus IgA/IgG antibody product, wherein the sample to be detected is oral mucosa exudate.
Oral mucosal exudate is gently wiped along the upper and lower gingiva of the subject with the sampling tip of the oral sampler. The method specifically comprises the following steps: the mouth sampler is used for slowly wiping the mouth from one position of the mouth corner to the other position of the mouth corner and then continuously wiping the mouth along the supragingival line until the beginning, and the time is about 5 to 6 seconds. Turning over the oral cavity sampler, wiping the lower gum line by using the other side of the sampling end of the oral cavity sampler, starting from one end of a mouth corner, slowly wiping the other end of the mouth corner, and continuously wiping the mouth corner along the lower gum line until the beginning for 5-6 seconds; the oral cavity sampler is inserted into a sample extraction pipe containing a sample treatment solution, and the oral cavity sampler is extruded to scrape 6-8 times up and down on the inner wall of the sample extraction pipe.
Further, the sample should be oral mucosal exudate rather than saliva.
In a third aspect, a preparation method of the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip comprises the following steps:
coating the novel coronavirus recombinant antigen marked by the colloidal gold and the mouse IgG marked by the colloidal gold on a colloidal gold adsorption pad;
respectively coating the anti-human IgA monoclonal antibody, the anti-human IgG antibody and the anti-mouse IgG antibody on a detection line T1, a detection line T2 and a quality control line C of the antibody bearing membrane;
and the sample loading pad, the colloidal gold adsorption pad, the antibody bearing film and the water absorption pad are sequentially lapped on the substrate.
Further, the coating concentration of the anti-mouse IgG polyclonal antibody is (1.5-1.8) mg/mL;
the coating concentration of the anti-human IgG antibody is (0.3-0.5) mg/mL;
the coating concentration of the anti-human IgA antibody is (0.3-0.5) mg/mL;
the marker concentration of the novel coronavirus recombinant antigen is as follows: (1-2) mu g/mL;
the labeling concentration of the mouse IgG antibody was (3-5). mu.g/mL.
In a fourth aspect, the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic reagent card comprises the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip or the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip prepared by the preparation method.
Further, the kit comprises a card shell, wherein a novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip is arranged in the card shell;
the card shell comprises an upper cover and a lower cover;
the upper cover is provided with a sample adding hole and a window;
the sampling hole corresponds to a loading pad of the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip;
the window corresponds to an antibody bearing membrane of the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip.
In a fifth aspect, the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic kit comprises the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip, the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip prepared by the preparation method, or the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic reagent card.
And sixthly, the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip is prepared by using the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip, the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip prepared by the preparation method, or the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic reagent card.
The technical scheme of the invention has the following advantages:
1. the invention provides a novel immunochromatographic test strip for combined detection of an IgA/IgG antibody of a coronavirus, which comprises the following components: a colloidal gold adsorption pad and an antibody bearing film; the colloidal gold adsorption pad is coated with a colloidal gold-labeled novel coronavirus recombinant antigen and a colloidal gold-labeled mouse IgG; the antibody bearing membrane is provided with a detection line T1, a detection line T2 and a quality control line C which are arranged at intervals, the detection line T1 is coated with an anti-human IgA monoclonal antibody, the detection line T2 is coated with an anti-human IgG antibody, and the quality control line is coated with an anti-mouse IgG antibody; the immunochromatographic test strip can be used for simultaneously detecting novel coronavirus IgA and IgG antibodies, the detection time is short and is only 10min, the cost is low, the operation is easy, the technical requirement is low, the on-site sample collection (particularly oral mucosa exudate) is facilitated, and the immunochromatographic test strip is suitable for on-site, instant and rapid detection.
2. The novel coronavirus recombinant antigen has a sequence shown as SEQ ID NO.1, can be specifically combined with novel coronavirus IgA and IgG antibodies, and has good specificity, repeatability, stability and sensitivity.
3. The invention provides a novel immunochromatographic kit for combined inspection of an IgA antibody and an IgG antibody of a coronavirus, which is used for storing the novel immunochromatographic test strip for combined inspection of the IgA antibody and the IgG antibody of the coronavirus for a long time.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of a novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip in example 1 of the present invention;
FIG. 2 is a schematic diagram of an embodiment of the immunochromatographic reagent card for the combined detection of IgA and IgG antibodies of the novel coronavirus in example 3 of the present invention.
1-substrate, 2-sample loading pad, 3-colloidal gold adsorption pad, 4-antibody bearing membrane, 5-water absorption pad, 6-protective membrane, 7-clamping shell, 8-upper cover, 9-sample adding hole, 10-window, T1-detection line T1, T2-detection line T2 and C-quality control line C.
Detailed Description
Example 1
A novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip is shown in figure 1, wherein a sample loading pad 2, a colloidal gold adsorption pad 3, an antibody bearing film 4 and a water absorption pad 5 are sequentially overlapped and lapped on a substrate 1;
the colloidal gold adsorption pad 3 is coated with a colloidal gold-labeled novel coronavirus recombinant antigen and a colloidal gold-labeled mouse IgG; the sequence of the novel coronavirus recombinant antigen is shown as SEQ ID NO. 1;
the antibody bearing film 4 is provided with a detection line T1, a detection line T2 and a quality control line C which are arranged at intervals, the detection line is arranged at one side close to the colloidal gold adsorption pad 3, and the quality control line C is arranged at one side close to the water absorption pad 5; the detection line T1 is coated with an anti-human IgA monoclonal antibody, the detection line T2 is coated with an anti-human IgG monoclonal antibody, and the quality control line C is coated with an anti-mouse IgG polyclonal antibody. The distance between the detection line T1, the detection line T2, and the detection line T2 and the quality control line C is 4-6mm, and 4mm is selected in the present embodiment.
Further, the sample loading pad 2 and the absorbent pad 5 are both provided with a protective film 6.
Example 2
The present embodiment provides a method for preparing the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip of example 1, which comprises the following steps:
1. preparing an antibody bearing membrane:
(1) selecting a nitrocellulose membrane with the aperture of 3-10 mu m, and cutting the membrane into specifications with the width of 2.0cm and the length of 30.5cm according to requirements for later use.
(2) Preparing an anti-human IgG monoclonal antibody for coating the detection line by using 0.1M, pH 8.0.0 Tris-HCL buffer solution, wherein the antibody concentration is (0.3-0.5) mg/mL, and in the embodiment, the antibody concentration is 0.3 mg/mL; the anti-human IgA monoclonal antibody has an antibody concentration of (0.3-0.5) mg/mL, in this example 0.3mg/mL, and a sodium chloride buffer solution with a mass percentage content of 0.85% is used to prepare a goat anti-mouse IgG polyclonal antibody for quality control line coating, so that the antibody concentration is (1.5-1.8) mg/mL, in this example 1.5 mg/mL.
(3) Selecting an antibody coating surface of the nitrocellulose membrane and marking, coating the detection line to be coated and the antibody solution of the quality control line on the membrane in parallel and uniformly, wherein the distance between the detection line and the quality control line is as follows: C-IgG 4.0mm, IgG-IgA4.0mm, and nitrocellulose membrane drying at 2-30 deg.C.
(4) Preparing a sealing treatment soaking solution, and adding purified water with actual production capacity into a liquid preparation tank; respectively weighing buffer solution, sugar, blocking protein and preservative, directly adding the above components into a liquid preparation tank, stirring until the components are completely dissolved, adding purified water to a desired volume, and fully and uniformly stirring for at least 10 minutes for later use; the prepared sealing treatment soaking solution contains 0.1Mol of buffer solution, 0.5 mass percent of sugar, 1 mass percent of sealing protein and 0.05 mass percent of preservative, wherein the buffer solution is phosphate buffer solution, the sugar is cane sugar, the preservative is thimerosal, and the sealing protein is bovine serum albumin.
(5) Placing the membranes coated with the detection lines and the quality control lines in a treatment tank, adding the prepared sealing treatment soaking liquid of the step (4), ensuring that each membrane is completely immersed in the sealing treatment soaking liquid of the step (4) for 30 minutes, ensuring that the membranes do not move and overlap, taking out the membranes from the treatment tank after 30 minutes, and pouring the sealing treatment soaking liquid of the step (4); and (3) placing the membrane on gauze by using tweezers, and airing to be slightly dry to obtain the antibody bearing membrane.
(6) Pasting a board and drying: tearing off white paper in the middle of a cutting line on double-sided adhesive of a rubber plate, putting an antibody bearing film subjected to sealing treatment right at the central blank position of the rubber plate by using tweezers, enabling the right side of the rubber plate to be flush with the right side of the film, avoiding errors in the production process, ensuring that the color development position is relatively accurate, pasting one end of all top quality control lines when pasting the plate, smearing the film surface through the double-sided adhesive paper after the film is pasted on the rubber plate, avoiding bubbles, controlling the temperature in a control room to be 18-28 ℃ and the relative humidity to be less than or equal to 40%, and ensuring that air can circulate in a drying room and air of a dehumidifier can not directly blow on the film surface. The drying time is more than or equal to 4 hours and is reserved.
2. Preparation of colloidal gold adsorption pad
(1) Preparing a colloidal gold complex solution: adding an actual production amount of purified water to the liquor preparation tank; trehalose, bovine serum albumin, trisodium citrate, polyethylene glycol and NaN were weighed using an electronic analytical balance3Directly adding into a liquid preparation tank, stirring until completely dissolving, adding purified water to desired volume, stirring thoroughly, and stirring for more than one timeThe obtained colloidal gold complex solution contains 5 percent by mass of trehalose, 2 percent by mass of bovine serum albumin, 0.5 percent by mass of trisodium citrate, 0.05 percent by mass of polyethylene glycol and 0.05 percent by mass of NaN3。
(2) Measuring colloidal gold with required labeling amount by a measuring cylinder, adjusting according to pH7.0-7.3, namely adding 0.2mol/L potassium carbonate solution according to the volume percentage content of 0.50%, stirring for 15 minutes on a magnetic stirrer, taking a novel coronavirus recombinant antigen, diluting the novel coronavirus recombinant antigen by double distilled water, labeling the colloidal gold according to 1ug/mL (namely, after adding the colloidal gold into the novel coronavirus recombinant antigen dilution, the labeling concentration of the novel coronavirus recombinant antigen is 1ug/mL), adding the novel coronavirus recombinant antigen into the colloidal gold, stirring for 30 minutes on the magnetic stirrer, adding 0.5 per mill stabilizer polyethylene glycol according to the volume percentage content, stirring for 30 minutes, centrifuging, collecting precipitates, re-dissolving the colloidal gold re-solution according to the volume percentage content of 3%, stirring on the magnetic stirrer until uniform mixing, obtaining the colloidal gold-novel coronavirus recombinant antigen conjugate compound solution with the volume percentage content of 3% for later use.
(3) Measuring the colloidal gold with the required labeling amount by using a measuring cylinder, adjusting according to the pH value of 7.0-7.3, namely adding 0.2mol/L potassium carbonate solution according to the volume percentage of 0.50%, stirring for 15 minutes on a magnetic stirrer, taking a mouse IgG antibody, diluting the mouse IgG antibody by using double distilled water, labeling the colloidal gold according to the volume percentage of 3ug/mL, adding the mouse IgG antibody into the colloidal gold, stirring for 30 minutes on the magnetic stirrer, adding 0.5 thousandth of stabilizer polyethylene glycol according to the volume percentage, stirring for 30 minutes, centrifuging, collecting precipitate, redissolving by using a colloidal gold complex solution according to the volume percentage of 3%, stirring on the magnetic stirrer until the mixture is uniformly mixed to obtain the colloidal gold-mouse IgG antibody conjugate complex solution with the volume percentage of 3%, and reserving for later use.
(4) Taking the colloidal gold-novel coronavirus recombinant antigen conjugate compound solution with the volume percentage of 3% and the colloidal gold-mouse IgG antibody conjugate compound solution with the volume percentage of 50% in the above (2) and (3), and re-dissolving the colloidal gold compound solution with the volume percentage of 50%Mixing uniformly on a magnetic stirrer according to the proportion of 50 mu L/cm2Pouring the colloidal gold onto a prepared colloidal gold adsorption pad, placing the colloidal gold adsorption pad in a drying chamber to be dried for more than or equal to 4 hours, controlling the temperature of the drying chamber to be 18-28 ℃, controlling the relative humidity to be less than or equal to 40%, ensuring smooth air and preventing airflow from directly blowing on the colloidal gold adsorption pad, placing the dried colloidal gold adsorption pad into an aluminum foil bag filled with a drying agent, sealing and storing, and making a labeled colloidal gold adsorption pad for later use.
Note: the colloidal gold is cast because the cast colloidal gold can be freely cut in width, thereby being convenient for adjusting the color development of the product.
3. Assembling and cutting:
(1) taking a semi-finished product of a transparent substrate adhered with an antibody bearing film, cutting a test strip colloidal gold adsorption pad into strips of 0.5cm multiplied by 30cm according to the width of 0.5cm, adhering the strips to the transparent substrate close to one side of a detection line, keeping the strips to be overlapped with the antibody bearing film for about 1mm, compounding a test strip water absorption pad on the transparent substrate close to one side of a quality control line and overlapped with the antibody bearing film for about 1mm, compounding a test strip sample loading pad on one end, far away from the antibody bearing film, of the test strip colloidal gold adsorption pad and overlapped with the antibody bearing film for about 1mm, and marking for later use;
(2) and cutting the assembled substrate into strip test paper for later use.
Example 3
The embodiment provides a novel immunochromatography reagent card for combined detection of an IgA/IgG antibody of a coronavirus, which comprises the novel immunochromatography test strip for combined detection of the IgA/IgG antibody of the coronavirus in the embodiment 1.
Further, as shown in fig. 2, the kit further comprises a card shell 7, and the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip in embodiment 1 is mounted inside the card shell.
The card shell 7 comprises an upper cover 8 and a lower cover;
the upper cover 8 is provided with a sample adding hole 9 and a window 10;
the sample adding hole 9 corresponds to a sample loading pad 2 of the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip;
the window 10 corresponds to the antibody bearing membrane 4 of the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip.
Example 4
Obtaining of samples to be tested
1A 3mL sample extraction tube containing 0.5mL of the sample treatment solution (0.01M PBS buffer, pH 7.0) was inverted 3 times to mix the solutions evenly.
1.2 sample Collection
1.2.1. Before collection, the person to be tested rinses the oral cavity with warm water.
1.2.2. The oral sampler is removed from the bag by tearing along a cut in the sealed package of the oral sampler (note: the end of the handle of the oral sampler is grasped during collection and the sampling end is not touched).
1.2.3. The test subject was gently wiped along the upper and lower gums with the sampling end of the oral sampler. The method specifically comprises the following steps: the patient is slowly wiped from one corner of the mouth to the other corner of the mouth, and then is continuously wiped along the supragingival line until the beginning, which takes about 5 to 6 seconds. And turning over the oral cavity sampler, wiping the lower gum line by using the other side of the sampling end of the oral cavity sampler, starting from one end of a mouth corner, slowly wiping the other end of the mouth corner, and continuously wiping the mouth corner along the lower gum line until the mouth corner begins, wherein the time is 5-6 seconds. And (3) injecting, wherein sampling is carried out according to strict operation requirements, and oral mucosa exudate is not saliva.
1.3 sample treatment
1.3.1 inserting the oral cavity sampler into a sample extraction tube containing a sample treatment solution, scraping the oral cavity sampler up and down on the inner wall of the sample extraction tube for 6-8 times by extruding the oral cavity sampler,
note that the samples were processed within 1 hour after collection at ambient temperature. If the treated sample cannot be detected in time, the treated sample is refrigerated for 2-8 ℃ and is detected within 12 hours after treatment. Samples were strictly prohibited from being subjected to freeze-thaw processing.
Note that the sample treatment solution should be covered immediately after use and placed in a cool place or a refrigerator.
Example 5
The embodiment provides a novel coronavirus IgA/IgG antibody joint inspection method, which comprises the step of using the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip prepared in the embodiment 1 or the embodiment 2 to specifically comprise the following steps:
during detection, the sample to be detected is returned to room temperature, the treated sample solution is taken by a plastic pipette, 2-3 drops (60-80 mu L) are vertically dropped into the sample adding hole 9 of the reagent card, the experimental result is read and recorded from the window 10 within 15 minutes, and the reading result is invalid within 20 minutes. (when a strong positive sample is detected, a positive result can be shown in 3-5 minutes).
And (4) interpretation of results:
when an IgA positive sample is detected, the novel coronavirus IgA antibody in oral mucosa exudate is combined with a coronavirus recombinant antigen marked by colloidal gold to form a compound, the compound moves forwards along a paper strip due to the chromatography effect, and is combined with a pre-coated anti-human IgA monoclonal antibody to form an Au-2019-nCoV Ag-2019-nCoV IgA Ab-anti-human IgA Ab compound when passing through a detection line T1, so that a purple red strip is displayed at the detection line T1, and free gold-marked mouse IgG is combined with an anti-mouse IgG antibody at a quality control line C, so that a purple red strip is displayed at the quality control line C. Negative specimens showed only a purple-red band at the control line C.
When an IgG positive sample is detected, a new coronavirus IgG antibody in oral mucosa exudate is combined with a new coronavirus antigen marked by colloidal gold to form a compound, the compound moves forwards along a paper strip due to chromatography, and is combined with a pre-coated anti-human IgG antibody to form an Au-2019-nCoV Ag-2019-nCoV IgG Ab-anti-human IgG Ab sandwich when passing through a detection line T2, so that a mauve strip is displayed at the detection line T2, and free gold-labeled mouse IgG is combined with an anti-mouse IgG antibody at a quality control line C, so that a mauve strip is displayed at the quality control line C. Negative specimens showed only a purple-red band at the control line C.
Note that the test paper should be removed from the original package as soon as possible within 1 hour, especially at room temperature above 30 ℃ or in a high humidity environment. After the test is finished, the used detection card, dropper, extraction tube and oral cavity sampler are treated as biomedical waste.
Example 6
The embodiment provides a novel coronavirus IgA/IgG antibody combined detection immunochromatographic kit, which comprises the novel coronavirus IgA/IgG antibody combined detection immunochromatographic reagent card in the embodiment 3.
Examples of the experiments
First, the performance index detection related to the kit in the embodiment 6 of the invention
1. Overview
In the experiment, performance indexes related to the kit in embodiment 6 of the present invention are detected according to the requirements of "guidance principles (comments on comments) for evaluating analytical performance of in vitro diagnostic reagent and" evaluation points for registration technology of 2019 novel coronavirus antigen/antibody detection reagent ", and whether the performance indexes meet the requirements of design and research is evaluated.
2. Reagent information
2.1 batch number and Specification
The novel coronavirus (2019-nCoV) IgA/IgG antibody combined detection kit (colloidal gold method) (kit to be detected) is of a card type, and the packaging specifications are 10 persons/box, 20 persons/box, 25 persons/box and 40 persons/box. In the production process of the product, semi-finished test strips prepared by the same process are put into a card shell and then sealed into an aluminum foil bag, and the specifications of 10 parts/box, 20 parts/box, 25 parts/box and 40 parts/box are completely the same and are not different, so that the product performances of 10 parts/box, 20 parts/box, 25 parts/box and 40 parts/box packages are consistent. Performance evaluations were now made for 20 person/box card type products.
Card type: specification: 20 persons/box; batch number: 20200520.
2.2 Standard substance
Table 1 reference for performance evaluation
3. Performance evaluation process
3.1 appearance Properties
3.1.1 Experimental requirements
The experimenter should be familiar with the detection method and reagent operation; the appearance of the test kit product is tested.
3.1.2 Experimental materials and methods
And taking out the test paper, observing by using a visual inspection method under natural light, judging whether the appearance of the test paper card is flat or not, judging whether the fit of the plastic shell is firm or not, and judging whether the assembly of the test paper in the shell is flat or not according to rules.
3.1.3 results of the experiment
Table 2: test results of appearance Properties
3.1.4 conclusions of the experiment
As can be seen from the above table: the appearance meets the requirements.
3.2 film strip Width
3.2.1 requirements of the experiment
The experimenter should be familiar with the detection method and reagent operation; the width of the membrane strip was tested with a test kit pilot product.
3.2.2 Experimental materials and methods
The test strips were removed, the width of the membrane strip was measured with a vernier caliper (precision 0.02mm), 3 test strips were repeated, and the average was taken.
3.2.3 results of the experiment
Table 3: film strip width test results
3.2.4 conclusions of the experiment
As can be seen from the above table: the width of the membrane strip meets the requirement.
3.3 traveling speed
3.3.1 requirements of the experiment
The experimenter should be familiar with the detection method and reagent operation; the speed of travel is checked with the test kit product.
3.3.2 Experimental materials and methods
Measuring the length from the lower end of the reaction area to the upper end of the reaction area of the window by using a vernier caliper (the precision is 0.02mm), recording the length as (L), recording the time required by the liquid to be measured to move in the L area by using a stopwatch (the precision is 0.01s), recording the time as (t), calculating the L/t as the moving speed, repeatedly measuring for 3 times, taking an average value, and observing the chromatographic state, the background condition and the color development of a quality control line of the test paper.
3.3.3 results of the experiment
Table 4: test results of traveling speed
3.3.4 conclusions of the experiment
As can be seen from the above table: the migration speed, quality control line color development, chromatographic state and background condition all meet the requirements.
3.4 minimum detection Limit
3.4.1 requirements of the experiment
The experimenter should be familiar with the detection method and reagent operation; adopting a proper reference substance; the lowest detection limit is checked with the test kit product.
3.4.2 test materials and methods
The detection is carried out by using a novel coronavirus (2019-nCoV) IgA/IgG antibody enterprise reference, and an IgG sensitivity reference: s1, S2, S3; IgA sensitivity reference: s4, S5 and S6.
3.4.3 results of the experiment
Table 5: test results of minimum detection limit
3.4.4 conclusions of the experiment
As can be seen from the above table (G stands for IgG and A stands for IgA): s1 and S2 are IgG positive reactions, and S3 is IgG negative reactions; s4 and S5 are IgA positive reactions, and S6 is IgA negative reactions.
3.5 reference compliance
3.5.1 requirements of the experiment
The experimenter should be familiar with the detection method and reagent operation; adopting a proper reference substance; and detecting the reference product coincidence rate by using a test kit product.
3.5.2 Experimental materials and methods
Negative reference product compliance rate: a batch of kits was tested with corporate reference (N1-N15).
Positive reference compliance rate: and detecting a batch of the kit by using enterprise reference products (P1-P8).
3.5.3 results of the experiment
Table 6: test result of reference product conformity rate
3.5.4 conclusion of the experiment
As can be seen from Table 6 (G stands for IgG and A stands for IgA):
negative reference product compliance rate: the detection is carried out by enterprise reference products (N1-N15).
All negative reference samples should be negative, and the negative coincidence rate (-/-) is 15/15.
Positive reference compliance rate: the detection is carried out by using enterprise reference products (P1-P8).
IgG antibody positive reference (P1-P3) coincidence rate: IgG has no negative reaction, and the positive coincidence rate (/ +) is 3/3;
IgA antibody positive reference (P4-P6) compliance rate: IgA has no negative reaction, and the positive coincidence rate (/ +) is 3/3;
the positive reference products (P7-P8) of the IgA/IgG antibody have the following coincidence rate: IgA/IgG showed no negative reaction, and the positive coincidence rate (+/+) was 2/2.
3.6 precision
3.6.1 requirements of the experiment
The experimenter should be familiar with the detection method and reagent operation; adopting a proper reference substance; tested with a test kit product.
3.6.2 Experimental materials and methods
A batch of test kits was tested in parallel 10 times each with a corporate precision reference (CV1, CV 2).
3.6.3 results of the experiment
Table 7: results of precision test
3.6.4 conclusion of the experiment
From the above table (G for IgG and A for IgA): the results of all batches are consistent, the color development is uniform, and the consistency of the kit to be detected is good.
Second, preparation method of enterprise reference of novel coronavirus (2019-nCoV) IgA/IgG antibody combined detection kit (colloidal gold method) in experimental example
1. The purpose of the test is as follows:
the method aims to determine enterprise reference products of a novel coronavirus (2019-nCoV) IgA/IgG antibody combined detection kit (colloidal gold method) through screening, verification and standardization of clinical samples.
2. And (3) test management:
2.1 sample sources: clinical cooperative units (20 negative: 5 of them specific antibodies, 10 positive).
2.2 Experimental materials:
(1) novel coronavirus (2019-nCoV) IgM/IgG antibody detection kit (colloidal gold method) (enokites (down mountain) biotechnology limited);
(2) a novel coronavirus (2019-nCoV) IgA/IgG antibody joint detection kit (colloidal gold method) (inventive example 6);
(3) an influenza A virus IgM antibody detection kit (Weifang City Kanghua biotechnology limited);
(4) a detection kit (enzyme-free percolation method) for influenza B virus IgM antibody (Qingdao Han Tang Biotech Co., Ltd.);
(5) a coxsackie group B virus IgM antibody detection kit (colloidal gold method) (Beijing emerging Tetraatlantoan biotechnology limited);
(6) adenovirus IgM antibody detection kit (colloidal gold method) (Beijing emerging atlantoandian biotechnology, Inc.);
(7) a respiratory syncytial virus IgM antibody detection kit (colloidal gold method) (Beijing emerging Tetraatlas biotechnology limited);
3. description of experimental design and protocol:
3.1 the collected clinical specimens (see example 4) were first subjected to inactivation treatment (56 ℃ water bath inactivation treatment for 60 minutes).
3.2 use third party kits: clinical samples were screened using a novel coronavirus (2019-nCoV) IgM/IgG antibody detection kit (colloidal gold method) from Ennote (Tangshan) Biotechnology Ltd.
3.3 evaluating the specificity of the screened negative reference substance.
3.4 carry out classification numbering to the sample, require: the 8 positive branches are marked as P1-P8, 15 negative branches are marked as N1-N15 (wherein N1 is positive to influenza A virus IgM antibody, N2 is positive to influenza B virus IgM antibody, N3 is positive to Coxsackie group B virus IgM antibody, N4 is positive to adenovirus IgM antibody, N5 is positive to respiratory syncytial virus IgM antibody), the lowest detectable amount is 6 branches are marked as S1-S6, and the 2 precision branches are marked as CV1 and CV 2.
3.5 validation of the reference (3 times total) was performed with a novel CoV IgA/IgG antibody combination assay kit (colloidal gold method) from Nantong Yishi Biotechnology Ltd.
3.6, subpackaging: 50 μ L/count, CV: 500 μ L/count.
Carrying out a stability test after accelerated destruction is carried out for 10 days at 3.737 ℃, and determining the validity period of a reference product; finally, subpackaging and warehousing, and freezing and storing at-18 ℃ to-25 ℃.
4. And (3) test results:
table 8: the reference samples consisted of 31 samples in total and consisted of:
| categories | Numbering | Specification of |
| Negative reference substance | N1～N15 | 50 μ L/count |
| IgG antibody positive reference substance | P1～P3 | 50 μ L/count |
| IgA antibody positive reference substance | P4～P6 | 50 μ L/count |
| IgA/IgG antibody positive reference substance | P7～P8 | 50 μ L/count |
| IgG sensitivity reference | S1～S3 | 50 μ L/count |
| IgA sensitivity reference substance | S4～S6 | 50 μ L/count |
| Precision reference product | CV1、CV2 | 500 μ L/count |
Remarking: in the negative reference product, N1 is positive for influenza A virus IgM antibody, N2 is positive for influenza B virus IgM antibody, N3 is positive for coxsackie group B virus IgM antibody, N4 is positive for adenovirus IgM antibody, and N5 is positive for respiratory syncytial virus IgM antibody.
4.2 the positive samples were diluted by using No. 01 of the negative samples as a diluent.
4.3 negative reference: the negative samples were measured to be diluted with sample treatment solutions No. 02, 10, 08, 14, 15, 03, 04, 05, 06, 07, 11, 12, 13, 17, 18 at a ratio of 1:100, and defined as N1-N15.
4.4IgG positive reference: the test is carried out, and the test sample is diluted by No. 01 negative sample, No. 23 negative sample is diluted by No. 01 negative sample at a ratio of 1: 160 and then diluted by sample treatment fluid at a ratio of 1:100, and the test sample is defined as P1; 26 diluted 1: 60 with No. 01 negative sample and then diluted 1:100 with sample treatment solution, defined as P2; no. 28 was diluted 1: 20 with No. 01 negative sample and then 1:100 with sample treatment solution, which was defined as P3.
4.5IgA positive reference: through determination, diluting each IgA positive reference substance by using a No. 01 negative sample, and diluting the No. 24 negative sample by using a No. 01 negative sample at a ratio of 1: 160, and then diluting the sample by using a sample treatment solution at a ratio of 1:100, wherein the sample is defined as P4; no. 25 was diluted 1: 80 with No. 01 negative sample and then 1:100 with sample treatment solution, defined as P5; no. 29 was diluted 1: 30 with No. 01 negative sample and then 1:100 with sample treatment solution, which was defined as P6.
4.6IgG/IgA positive references: the samples for positive Nos. 21 and 22 were defined as P7 and P8 by dilution with the sample treatment solution 1: 100.
4.7IgG sensitivity reference: IgG positive P1 was selected, diluted 1:50, 1:100, 1: 200 with No. 01 negative sample, and then diluted 1:100 with sample treatment solution, which was defined as S1, S2, and S3, respectively.
4.8IgA sensitivity reference: IgA positive P4 was selected, and the samples were diluted with No. 01 negative sample at 1: 40, 1: 80, and 1: 160 and then diluted with sample treatment solution at 1:100, which were defined as S4, S5, and S6, respectively.
4.9 precision reference: through determination, after S1 and S41: 1 are mixed uniformly, the mixture is diluted by sample treatment fluid 1:100, and the CV1 is defined; after S2 and S51: 1 were mixed well, the mixture was diluted with 1:50 sample treatment solution, defined as CV 2.
4.10 accelerated destruction at 37 ℃ for 10 days, the result is almost unchanged, so the validity period is temporarily determined to be one year (-18 ℃ to-25 ℃).
5. Quality standard:
5.1 lowest detection limit:
respectively detecting with enterprise sensitivity reference products S1, S2, S3 and S4, S5 and S6, and judging results in 15 minutes, wherein the detection results of S1 and S2 are IgG positive, and the detection results of S3 are IgG negative; the results of S4 and S5 show that the IgA detection is positive, and the result of S6 shows that the IgA detection is negative.
5.2 Positive compliance:
8 parts of enterprise positive reference P1-P8 are used for detection respectively, and the results are judged in 15 minutes, wherein the results require that P1-P3 is IgG positive 3/3(+/+), P4-P6 is IgA positive 3/3(+/+), and P7-P8 IgG and IgA are positive 2/2 (+/+).
5.3 negative match rate:
15 parts of enterprise negative reference products N1-N15 are used for detection respectively, and the result is read in 15 minutes, so that the detection is only negative 15/15 (-/-).
5.4 precision:
and (3) respectively carrying out parallel detection on 10 parts by using 2 parts of enterprise precision reference CV, judging results in 15 minutes, wherein 110 parts of CV are required to be positive and have consistent color development intensity, and 210 parts of CV are required to be positive and have consistent color development intensity.
6. Storage conditions and effective period:
storage conditions were as follows: freezing at-18 deg.C to-25 deg.C
The validity period is as follows: temporarily for one year
The experiment summary:
1. no. 01-20 can be used as a negative sample.
2. No. 21-30 can be used as positive samples.
Three, specific cross test
1. Clinical samples collected: positive influenza A virus IgM antibody, positive influenza B virus IgM antibody, positive coxsackie B virus IgG antibody, positive adenovirus IgM antibody, positive adenovirus IgG antibody, positive respiratory syncytial virus IgM antibody, positive enterovirus 71 antibody IgG, positive EB virus, positive mumps virus IgG antibody, positive varicella-zoster virus IgM antibody, positive varicella-zoster virus IgG antibody, positive mycoplasma pneumoniae IgM antibody and positive mycoplasma pneumoniae IgG antibody. And none of the above clinical samples was diagnosed as positive for the novel coronavirus.
2. The above samples were tested with the kit of example 6 in triplicate and the results are shown in Table 9.
The experimental results are as follows:
table 9: detection sample result of novel coronavirus IgA/IgG antibody oral mucosa detection kit
3. Experimental conclusions and improvements
From the results of the above table, it can be seen that:
positive influenza A virus IgM antibody, positive influenza B virus IgM antibody, positive Coxsackie B virus IgG antibody, positive adenovirus IgM antibody, positive adenovirus IgG antibody, positive respiratory syncytial virus IgM antibody, positive enterovirus 71 antibody IgG, positive EB virus, positive mumps virus IgG antibody, positive varicella-zoster virus IgM antibody, positive varicella-zoster virus IgG antibody, positive mycoplasma pneumoniae IgM antibody, positive mycoplasma pneumoniae IgG antibody, no cross reaction with the novel coronavirus IgA/IgG antibody detection kit (colloidal gold method) in example 1.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Sequence listing
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Claims (9)
1. A novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip is characterized by comprising: a colloidal gold adsorption pad and an antibody bearing film; the colloidal gold adsorption pad is coated with a colloidal gold-labeled novel coronavirus recombinant antigen and a colloidal gold-labeled mouse IgG; the antibody bearing film is provided with a detection line T1, a detection line T2 and a quality control line C which are arranged at intervals, and the detection line T1 is coated with an anti-human IgA antibody; detecting line T2 coated with anti-human IgG antibody; the control line was coated with anti-mouse IgG antibody.
2. The novel coronavirus IgA/IgG antibody combined detection immunochromatographic test strip according to claim 1, wherein the sequence of the novel coronavirus recombinant antigen is shown in SEQ ID No. 1.
3. Use of the novel coronavirus IgA/IgG antibody combined detection immunochromatographic test strip of claim 1 or 2 for detecting or assisting in detecting whether a sample to be detected contains the novel coronavirus IgA/IgG antibody; and/or, the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip of claim 1 or 2 is used for preparing a product for detecting or assisting to detect whether a sample to be detected contains the novel coronavirus IgA/IgG antibody, wherein the sample to be detected is an oral mucosa exudate.
4. A preparation method of a novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip is characterized by comprising the following steps:
coating the novel coronavirus recombinant antigen marked by the colloidal gold and the mouse IgG marked by the colloidal gold on a colloidal gold adsorption pad;
respectively coating the anti-human IgA monoclonal antibody, the anti-human IgG antibody and the anti-mouse IgG antibody on a detection line T1, a detection line T2 and a quality control line C of the antibody bearing membrane;
and the sample loading pad, the colloidal gold adsorption pad, the antibody bearing film and the water absorption pad are sequentially lapped on the substrate.
5. The method according to claim 4, wherein the coating concentration of the anti-mouse IgG polyclonal antibody is (1.5-1.8) mg/mL when the antibody carrier film and the colloidal gold adsorption pad are prepared;
the coating concentration of the anti-human IgG antibody is (0.3-0.5) mg/mL;
the coating concentration of the anti-human IgA antibody is (0.3-0.5) mg/mL;
the marker concentration of the novel coronavirus recombinant antigen is as follows: (1-2) mu g/mL;
the labeling concentration of the mouse IgG antibody was (3-5). mu.g/mL.
6. A novel coronavirus IgA/IgG antibody joint inspection immunochromatography reagent card, which comprises the novel coronavirus IgA/IgG antibody joint inspection immunochromatography test strip of claim 1 or 2 or the novel coronavirus IgA/IgG antibody joint inspection immunochromatography test strip prepared by the preparation method of claim 4 or 5.
7. The novel immunochromatography reagent card for combined IgA/IgG antibody detection of coronavirus according to claim 6, which comprises a card shell, wherein the card shell is internally provided with a novel immunochromatography test strip for combined IgA/IgG antibody detection of coronavirus;
the card shell comprises an upper cover and a lower cover;
the upper cover is provided with a sample adding hole and a window;
the sampling hole corresponds to a loading pad of the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip;
the window corresponds to an antibody bearing membrane of the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip.
8. A novel coronavirus IgA/IgG antibody combined detection immunochromatographic kit comprising the novel coronavirus IgA/IgG antibody combined detection immunochromatographic test strip of claim 1 or 2, the novel coronavirus IgA/IgG antibody combined detection immunochromatographic test strip prepared by the preparation method of claim 4 or 5, or the novel coronavirus IgA/IgG antibody combined detection immunochromatographic reagent card of claim 6 or 7.
9. A novel coronavirus IgA/IgG antibody joint inspection method, characterized in that the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip of claim 1 or 2, the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip prepared by the preparation method of claim 4 or 5, or the novel coronavirus IgA/IgG antibody joint inspection immunochromatographic reagent card of claim 6 or 7 are used.
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