CN112485436A - Colloidal gold immunochromatographic test strip for detecting in-vivo neutralizing antibody of neocorona vaccine after injection and preparation method thereof - Google Patents
Colloidal gold immunochromatographic test strip for detecting in-vivo neutralizing antibody of neocorona vaccine after injection and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a colloidal gold immunochromatographic test strip for detecting in vivo endocrine neutralizing antibodies after neocoronal vaccine injection and a preparation method thereof, wherein the test strip comprises a PVC (polyvinyl chloride) bottom plate, and a sample pad, a colloidal gold pad, a nitrocellulose membrane and water-absorbing filter paper are sequentially lapped on the PVC bottom plate from left to right; the colloidal gold pad is coated with a recombinant novel coronavirus RBD protein marked by colloidal gold; the cellulose nitrate membrane is coated with an angiotensin converting enzyme 2 (ACE 2) detection line and a mouse anti-chicken IgG antibody as a quality control line. The colloidal gold immunochromatographic test strip provided by the invention adopts a competition method to detect a neutralizing antibody in a human body, has good sensitivity, specificity, repeatability and stability, has a high recovery rate of a target compound, and has accurate and reliable test results. Realizes the rapid qualitative detection of the neutralizing antibody in human body after the injection of the new corona vaccine, has high sensitivity and small difference between batches, and provides great convenience for clinical use.
Description
Technical Field
The invention relates to a test strip for rapid qualitative detection of a blood sample and a preparation method thereof, in particular to a test strip for rapid qualitative detection of a neutralizing antibody of whole blood or serum after a human body is injected with a new corona vaccine by utilizing an immune colloidal gold chromatography technology and a preparation method thereof.
Background
At present, no specific medicine specially aiming at COVID-19 exists, and the new corona vaccine becomes an effective means for preventing COVID-19. Currently, research and development work of new coronary vaccines in China is generally in the leading position, vaccines entering clinical stages are arranged in each technical route, wherein 4 vaccines enter third-phase clinic in the inactivated vaccines and adenovirus vector vaccines, and the two technical routes. With the further success of subsequent vaccines, it is becoming a great need to verify the effectiveness of new corona vaccines. Therefore, it is feasible and necessary to establish a method for measuring neutralizing antibodies in human bodies after vaccine injection, which has the advantages of short detection time, simple operation, high sensitivity, high specificity and easy large-scale popularization, and provides early and accurate laboratory diagnosis for patients.
Disclosure of Invention
In order to detect the requirement of whether a human body generates a corresponding antibody after the injection of the new corona vaccine, the invention provides a rapid colloidal gold immunochromatographic test strip for rapidly and qualitatively detecting a neutralizing antibody in the human body after the injection of the new corona vaccine, and the colloidal gold immunochromatographic test strip effectively and qualitatively detects the problem of whether the human body contains the neutralizing antibody after the injection of the new corona vaccine by the competition of the neutralizing antibody in an actual sample and angiotensin converting enzyme 2 (ACE 2) coated on a nitrocellulose membrane. The test strip has good sensitivity, specificity, repeatability and stability, has high recovery rate to the target compound, and can provide more accurate and reliable test results.
The technical scheme for realizing the purpose of the invention is as follows:
the invention provides a colloidal gold immunochromatographic test strip for detecting in vivo endocrine neutralizing antibodies after injection of a new corona vaccine, which comprises a PVC (polyvinyl chloride) bottom plate, wherein a sample pad, a colloidal gold pad, a nitrocellulose membrane and water-absorbing filter paper are sequentially overlapped on the PVC bottom plate from left to right;
the colloidal gold pad is coated with a recombinant novel coronavirus RBD protein marked by colloidal gold;
the cellulose nitrate membrane is coated with an angiotensin converting enzyme 2 (ACE 2) detection line and a mouse anti-chicken IgG antibody as a quality control line.
The concentration of the recombinant novel coronavirus RBD protein marked by the colloidal gold is 500 mu g/mL-5mg/mL, and the coating amount of the RBD protein on the colloidal gold pad is 400 uL;
the concentration of the angiotensin converting enzyme 2 (ACE 2) is 0.5-2.6mg/ml, and the coating amount is 30 uL;
the concentration of the mouse anti-chicken IgG antibody is 0.5-3mg/ml, and the coating amount is 30 uL.
The colloidal gold pad also comprises a separating agent, wherein the separating agent is any one or the combination of at least two of casein, BSA (bovine serum albumin) or PEG6000, and preferably PEG6000 with the concentration of 10%.
The inventor finds that the separating agent is added on the colloidal gold pad, so that the combination of the recombinant novel coronavirus RBD protein and the colloidal gold particles is more stable, and the detection limit of the colloidal gold immunochromatographic test strip is further reduced.
The cryptococcus antibody of the murine anti-human antibody of the neocoronavirus is a monoclonal antibody of an anti-cryptococcus capsular polysaccharide recombinant neocorona antigen and/or a polyclonal antibody of an anti-cryptococcus capsular polysaccharide recombinant neocorona antigen, and preferably a monoclonal antibody of a recombinant neocorona antigen anti-cryptococcus capsular polysaccharide antigen.
The grain diameter of the colloidal gold particles on the colloidal gold pad is 30-40 nm.
The distance between the detection line and the quality control line is 3-5cm, and the distance between the detection line and the quality control line is 3-5cm, so that the detection line and the quality control line are not interfered with each other.
The sample pad is a glass fiber film or a polyester fiber film; the colloidal gold pad is a glass fiber membrane.
In the invention, the colloidal gold immunochromatographic test strip adopts a competitive chromatography immunoassay, and a neutralizing antibody in an actual sample competes with angiotensin converting enzyme 2 (ACE 2) on a nitrocellulose membrane to mark a recombinant novel coronavirus RBD protein of colloidal gold in the lateral moving process. If the neutralizing antibody in the sample is enough, the result that the detection line has no color development and the quality control line has no color development is shown, which indicates that the antibody is generated after the vaccine is injected and the neutralizing antibody is generated by the vaccine; if the neutralizing antibody in the sample is insufficient, the results of detection line color development and quality control line color development are presented, which indicates that no antibody is generated after vaccine injection. The quality control line should have bands when detecting positive and negative samples, and the red bands are the standard for determining whether the chromatography process is normal, and are also used as the internal control standard of the reagent.
According to the invention, the virus antibody coated on the nitrocellulose membrane is angiotensin converting enzyme 2 (ACE 2), and the antibody used as the quality control line is a mouse anti-chicken IgG antibody. The murine antibody is the most common antibody form in the current commercial antibodies, and has the advantages of wide source, low cost, good specificity and stability. The virus antibody may be a polyclonal antibody or a monoclonal antibody, and methods for preparing the polyclonal antibody or the monoclonal antibody are well known to those skilled in the art, and for example, an animal, such as a mouse, may be immunized with the corresponding virus and the polyclonal antibody may be purified from the serum of the animal, but the polyclonal antibody may have a problem of poor specificity compared to the monoclonal antibody, and may have a false positive result. In the present invention, although polyclonal antibodies can be used as the viral antibodies, monoclonal antibodies have more significant advantages, and thus are preferred, and the monoclonal antibodies can be prepared, for example, by immunizing an animal such as a mouse with the corresponding virus, taking spleen B cells of the animal and fusing with mouse myeloma cells to form hybridoma cells, and obtaining monoclonal antibodies with higher specificity therefrom.
In a second aspect, the present invention provides a method for preparing the colloidal gold immunochromatographic test strip of the first aspect, comprising the following steps:
preparing a colloidal gold pad, and coating the colloidal gold pad with a recombinant novel coronavirus RBD protein marked by colloidal gold;
(2) preparing a nitrocellulose membrane, coating an angiotensin converting enzyme 2 (ACE 2) detection line on the nitrocellulose membrane, and coating a mouse anti-chicken IgG antibody as a quality control line;
(3) and sequentially overlapping the sample pad, the colloidal gold pad, the nitrocellulose membrane and the absorbent filter paper on the PVC base plate from left to right.
In the preparation method of the present invention, the antibody coating is a conventional technical means in the field, and is not particularly limited, and a person skilled in the art can select and prepare the antibody according to actual needs.
In the preparation method of the invention, the antibody coating buffer solution coated by the antibody is any one of or the combination of at least two of PBS buffer solution or PB buffer solution.
The pH value of the PBS buffer is 6-8.5, and preferably 7-8.
The pH value of the PB buffer solution is 7-8, and preferably 7.2-7.4.
In the preparation method, the preparation of the colloidal gold pad comprises the following steps:
(1) cleaning a vessel, adjusting the pH value of the colloidal gold particles to 8.5 by adopting potassium nitrate, adding the potassium nitrate into the vessel, adding a separating agent with the final concentration of 10 percent into the vessel, adding the recombinant novel coronavirus RBD protein to the final concentration of 30 mu g/ml, and stirring for 40min to obtain a colloidal gold solution;
(2) adding 10% of sealing liquid into the colloidal gold solution obtained in the step (1) until the final concentration reaches 1%, and sealing for 15 min;
the confining liquid is casein and/or BSA solution;
(3) centrifuging the colloidal gold solution obtained in the step (2) for 40min at 4 ℃ and 12000 rpm, removing a supernatant after centrifugation, and redissolving and precipitating by using a complex solution to obtain a recombinant novel coronavirus RBD protein labeling solution;
the complex solution is a mixture of Tween 20 and BSA;
(4) spraying the recombinant novel coronavirus RBD protein labeling solution obtained in the step (3) onto a colloidal gold pad by using a gold spraying and film scratching instrument according to the concentration of 0.1 mu L/mm;
(5) and (4) drying the colloidal gold pad in the step (4) in a drying oven at 37 ℃ for 240min, adding a drying agent after drying, and sealing and storing to obtain the colloidal gold pad.
In a third aspect, the invention provides a colloidal gold immunochromatographic kit for detecting in vivo neutralizing antibodies after injection of a new corona vaccine, which comprises the colloidal gold immunochromatographic test strip of the first aspect.
In a fourth aspect, the invention provides the colloidal gold immunochromatographic test strip of the first aspect and/or the detection kit of the third aspect for rapid qualitative detection of neutralizing antibodies secreted in a human body after injection of a new corona vaccine.
In the invention, the use method of the test strip specifically comprises the following steps:
(1) sucking 10 mu L of sample to be detected, and slowly dripping the sample to be detected into a sample adding hole of the test strip;
(2) timing, reading results within 10-15 minutes, and possibly generating abnormal results after 15 minutes.
And (4) analyzing results:
positive (+): the appearance of the line in the detection line and the quality control line indicates that the neutralizing antibody caused by the new corona vaccine exists in the sample.
Negative (-): only one red strip appears on the quality control line, and no red strip appears on the detection line, which indicates that the neutralizing antibody triggered by the new corona vaccine exists in the sample.
And (4) invalidation: the control line did not appear red, possibly due to improper handling or reagent failure. In any case, it should be retested. If the problem still exists, the use of the lot should be stopped immediately and the local supplier contacted.
Note that: the detection line and the red strip will appear differently dark and light in color due to the different concentrations of neutralizing antibodies in the sample. However, the test results of the reagent cannot be used as a basis for determining the level of the gradient of the neutralizing antibody in the sample.
The invention adopts the combination of a binding pad of a recombinant novel coronavirus RBD protein marked with colloidal gold and a nitrocellulose membrane coated with angiotensin converting enzyme 2 (ACE 2) by spraying. By adopting the principle of competitive immunochromatography, the neutralizing antibody in the actual sample competes with angiotensin converting enzyme 2 (ACE 2) on the nitrocellulose membrane to label the recombinant novel coronavirus RBD protein of colloidal gold in the lateral moving process. If the neutralizing antibody in the sample is enough, the result that the detection line has no color development and the quality control line has no color development is shown, which indicates that the antibody is generated after the vaccine is injected and the neutralizing antibody is generated by the vaccine; if the neutralizing antibody in the sample is insufficient, the results of detection line color development and quality control line color development are presented, which indicates that no antibody is generated after vaccine injection. Realizes the rapid qualitative detection of the neutralizing antibody in human body after the injection of the new corona vaccine, has high sensitivity and small difference between batches, and provides great convenience for clinical use.
Compared with the prior art, the method has the following beneficial effects:
(1) the colloidal gold immunochromatographic test strip provided by the invention adopts a competition method to detect a neutralizing antibody in a human body, has good sensitivity, specificity, repeatability and stability, has a high recovery rate of a target compound, and has accurate and reliable test results;
(2) according to the colloidal gold immunochromatographic test strip, the separating agent is added into the colloidal gold pad, so that the combination of the recombinant novel coronavirus RBD protein and the colloidal gold particles is more stable, and the detection limit of the colloidal gold immunochromatographic test strip is further reduced;
(3) the colloidal gold immunochromatographic test strip provided by the invention has higher sensitivity and specificity for detection of neutralizing antibodies.
Drawings
FIG. 1 is a schematic structural diagram of a colloidal gold immunochromatographic test strip of the present invention;
in the figure, 1, a sample pad 2, a colloidal gold pad 3, a nitrocellulose membrane 4, a detection line 5, a quality control line 6, water absorption filter paper 7 and a PVC bottom plate.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited thereto.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were purchased from conventional biochemical manufacturers.
Sources of reagents and instrumentation used in the examples:
the novel recombinant coronavirus RBD protein, angiotensin converting enzyme 2 (ACE 2), and the mouse anti-chicken IgG antibody are purchased from Shenzhen power biotechnology Limited; nitrocellulose membranes were purchased from merck millipore; the three-dimensional plane film scribing instrument is purchased from Shanghai gold-labeled Biotech limited; the slitter is purchased from Shanghai gold-labeled Biotech Co., Ltd; PVC offset plate, absorbent paper, glass fiber membrane and card shell are purchased from Shanghai gold-labeled Biotech limited.
Example 1: preparation of colloidal gold immunochromatographic test strip
The colloidal gold immunochromatographic test strip is prepared according to the following method, and the structure of the colloidal gold immunochromatographic test strip is shown in figure 1:
a sample pad 1, a colloidal gold pad 2, a nitrocellulose membrane 3 and water-absorbing filter paper 6 are sequentially attached to a polyvinyl chloride (PVC) base plate 7 from left to right in a mutually overlapped manner, then the PVC base plate 7 and the attached material are cut into test strips with the width of 3mm, and the test strips are loaded into a card shell, and a sample adding area and a color developing area (an observation area) are arranged on the card shell.
Wherein, the sample pad 1 is a glass fiber film; the colloidal gold pad 2 is coated with a recombinant novel coronavirus RBD protein marked by colloidal gold; the cellulose nitrate film 8 is coated with an angiotensin converting enzyme 2 (ACE 2) detection line 4 and is also coated with a mouse anti-chicken IgG antibody as a quality control line 5; the distance between the detection line 4 and the quality control line 5 is 3 cm.
Example 2: antibody coating
Firstly, diluting a mouse anti-chicken IgG antibody and angiotensin converting enzyme 2 (ACE 2) by using 0.05mol/L PB (phosphate buffer) with the pH value of 7.2-7.4 to obtain a detection line 4 mouse anti-chicken IgG antibody coating solution and a quality control line 5 angiotensin converting enzyme 2 (ACE 2) diluent;
secondly, scratching the obtained diluent on a nitrocellulose membrane by using a gold spraying and film scratching instrument, and scratching according to the condition of 0.1 mu L/cm;
finally, putting the nitrocellulose membrane in an oven at 37 ℃ for drying for 3 hours, adding a drying agent after drying, and sealing and storing;
example 3: preparation of colloidal gold pad
(1) Cleaning a vessel, adjusting the pH value of the colloidal gold particles to 8.5 by adopting potassium nitrate, adding the potassium nitrate into the vessel, adding a PEG6000 separating agent with the final concentration of 10 percent into the vessel, adding the recombinant novel coronavirus RBD protein to the final concentration of 30 mu g/ml, and stirring the mixture for 40min to obtain a colloidal gold solution;
(2) adding 10% BSA blocking solution into the colloidal gold solution obtained in the step (1) until the final concentration reaches 1%, and blocking for 15 min;
(3) centrifuging the colloidal gold solution obtained in the step (2) for 40min at 4 ℃ and 12000 rpm, removing a supernatant after centrifugation, and redissolving and precipitating by using a complex solution to obtain a recombinant novel coronavirus RBD protein labeling solution;
the complex solution is a mixture of Tween 20 and BSA;
(4) spraying the recombinant novel coronavirus RBD protein labeling solution obtained in the step (3) onto a glass fiber membrane by using a gold spraying and membrane scratching instrument according to the volume of 0.1 mu L/mm;
(5) and (4) drying the glass fiber membrane obtained in the step (4) in an oven at 37 ℃ for 240min, adding a drying agent after drying, and sealing and storing to obtain the colloidal gold pad.
Example 4:
the assembly of the colloidal gold test strip is shown in fig. 1:
firstly, assembling a coated nitrocellulose membrane 3, a colloidal gold pad 2, water absorption filter paper 6 and a sample pad 1 on a PVC (polyvinyl chloride) bottom plate 7;
secondly, cutting the obtained assembled plate into 3mm colloidal gold test strips by using a strip cutting device;
and finally, placing the obtained test strip in a sealing bag, and adding a drying agent for preservation.
As shown in fig. 1, the detection principle of the colloidal gold test strip of the present invention is as follows:
after the sample to be tested is added to the sample addition zone (sample pad), the area of neutralizing antibodies is detected by capillary action: the conjugate of the neutralizing antibody-RBD-colloidal gold in the sample moves toward one end of the absorbent paper. If the detected sample contains the neutralizing antibody, when the sample moves to the detection line 4, namely angiotensin converting enzyme 2 (ACE 2), RBD in the conjugate of the neutralizing antibody-RBD-colloidal gold is not combined with angiotensin converting enzyme 2 (ACE 2) any more, and a red strip is not shown; the sample continues to flow, and when the sample flows to the quality control line 5, namely the mouse anti-chicken IgG antibody, a red strip appears, so that the effectiveness of the test strip is proved. If the neutralizing antibody-RBD-colloidal gold does not form immune complex in the absence of the neutralizing antibody in the actual sample to be tested, the RBD in the complex is combined with angiotensin converting enzyme 2 (ACE 2), a red band is shown at the test line 4, and then the color is shown at the quality control line 5. As long as the quality control line 5 does not develop color, the test strip is proved to be invalid, and the actual sample needs to be detected again.
Claims (10)
1. The utility model provides a detect colloidal gold immunochromatography test paper strip of internal neutralizing antibody of new coronary vaccine injection back, includes the PVC bottom plate, its characterized in that:
a sample pad, a colloidal gold pad, a nitrocellulose membrane and absorbent filter paper are sequentially lapped on the PVC base plate from left to right;
the colloidal gold pad is coated with a recombinant novel coronavirus RBD protein marked by colloidal gold;
the cellulose nitrate membrane is coated with an angiotensin converting enzyme 2 detection line and a mouse anti-chicken IgG antibody as a quality control line.
2. The colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the concentration of the recombinant novel coronavirus RBD protein marked by the colloidal gold is 500 mu g/mL-5mg/mL, and the coating amount of the RBD protein on the colloidal gold pad is 400 uL;
the concentration of the angiotensin converting enzyme 2 is 0.5-2.6mg/ml, and the coating amount is 30 uL;
the concentration of the mouse anti-chicken IgG antibody is 0.5-3mg/ml, and the coating amount is 30 uL.
3. The colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the colloidal gold pad also comprises a separating agent, and the separating agent is any one or the combination of at least two of casein, BSA or PEG 6000.
4. The colloidal gold immunochromatographic test strip according to claim 3, characterized in that: the separating agent is PEG6000 with the concentration of 10%.
5. The colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the grain diameter of the colloidal gold particles on the colloidal gold pad is 30-40 nm.
6. The colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the distance between the detection line and the quality control line is 3-5cm, and the distance between the detection line and the quality control line is 3-5cm, so that the detection line and the quality control line are not interfered with each other.
7. The colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the sample pad is a glass fiber film or a polyester fiber film; the colloidal gold pad is a glass fiber membrane.
8. The method for preparing the colloidal gold immunochromatographic test strip according to any one of claims 1 to 7, which is characterized by comprising the following steps:
(1) preparing a colloidal gold pad, and coating the colloidal gold pad with a recombinant novel coronavirus RBD protein marked by colloidal gold;
(2) preparing a nitrocellulose membrane, coating an angiotensin converting enzyme 2 detection line on the nitrocellulose membrane, and coating a mouse anti-chicken IgG antibody as a quality control line;
(3) and sequentially overlapping the sample pad, the colloidal gold pad, the nitrocellulose membrane and the absorbent filter paper on the PVC base plate from left to right.
9. The method for preparing the colloidal gold immunochromatographic test strip according to claim 8, which is characterized in that: the preparation of the colloidal gold pad comprises the following steps:
(1) cleaning a vessel, adjusting the pH value of the colloidal gold particles to 8.5 by adopting potassium nitrate, adding the potassium nitrate into the vessel, adding a separating agent with the final concentration of 10 percent into the vessel, adding the recombinant novel coronavirus RBD protein to the final concentration of 30 mu g/ml, and stirring for 40min to obtain a colloidal gold solution;
(2) adding 10% of sealing liquid into the colloidal gold solution obtained in the step (1) until the final concentration reaches 1%, and sealing for 15 min;
the confining liquid is casein and/or BSA solution;
(3) centrifuging the colloidal gold solution obtained in the step (2) for 40min at 4 ℃ and 12000 rpm, removing a supernatant after centrifugation, and redissolving and precipitating by using a complex solution to obtain a recombinant novel coronavirus RBD protein labeling solution;
the complex solution is a mixture of Tween 20 and BSA;
(4) spraying the recombinant novel coronavirus RBD protein labeling solution obtained in the step (3) onto a colloidal gold pad by using a gold spraying and film scratching instrument according to the concentration of 0.1 mu L/mm;
(5) and (4) drying the colloidal gold pad in the step (4) in a drying oven at 37 ℃ for 240min, adding a drying agent after drying, and sealing and storing to obtain the colloidal gold pad.
10. A colloidal gold immunochromatographic kit for detecting in-vivo neutralizing antibodies after injection of a new corona vaccine is characterized in that: the kit comprises the colloidal gold immunochromatographic strip of any one of claims 1 to 7.
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| CN113203856A (en) * | 2021-04-30 | 2021-08-03 | 深圳迈瑞生物医疗电子股份有限公司 | Kit for detecting coronavirus antibody and detection method of coronavirus antibody |
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