CN114034858A - 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 neutralizing antibodies generated after vaccination of Xinguan vaccine and a preparation method thereof, wherein the test strip comprises a PVC (polyvinyl chloride) bottom plate, and a sample pad (blood filtering membrane), a combining pad, an NC (numerical control) membrane and absorbent paper are sequentially overlapped on the PVC bottom plate from left to right; the binding pad is coated with a recombinant novel coronavirus RBD protein marked by colloidal gold; the NC film is coated with human angiotensin enzyme 2(ACE2) as a detection line T line; the NC membrane is coated with a chicken IgY antibody as a quality control line C line. The colloidal gold immunochromatographic test strip provided by the invention adopts a competition method to detect a neutralizing antibody generated in a human body after the new corona vaccine is inoculated, has the advantages of good specificity, sensitivity and stability, simple operation and short detection time, and provides a reliable choice for clinical rapid detection.
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 using an immune colloidal gold chromatography technology and a preparation method thereof.
Background
The novel coronavirus pneumonia is rolled around the world, and epidemic prevention and control become common tasks of governments and people of all countries. With the continuous marketing of new corona vaccines and the wide range of vaccination across the country and the world, the problem of evaluating the immune effect produced in individuals after vaccination has also received increasing attention.
Neutralizing antibodies induced in the convalescent patients with new coronary pneumonia and after vaccine injection are considered to be: when new coronavirus invades again, the spike protein on the virus can be recognized and further the virus is prevented from infecting cells through various mechanisms. Therefore, the detection of the neutralizing antibody can judge whether a sufficient amount of the novel coronavirus neutralizing antibody exists in human bodies, further indicate whether individuals have the capacity of preventing virus infection, and is also beneficial to the development and evaluation of vaccine products.
At present, the conventional detection technology of the neutralizing antibody is a neutralization test, live viruses or pseudo viruses are required to be used, all tests for evaluating the effect of related products by using the live viruses must be carried out in a biosafety level three (BSL-3) laboratory, and the conventional detection technology has very high requirements on personnel, equipment and operation and cannot meet the current clinical detection requirements.
The existing detection method aiming at the new crown neutralizing antibody comprises detection means such as enzyme-linked immunosorbent assay, chemiluminescence assay and the like, and corresponding kits are available on the market for the two methods. However, the detection operation of the kit is complicated and time-consuming, and is not suitable for the concomitant detection of the vaccine injection effect on a large scale.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an immunochromatographic test strip for detecting a novel coronavirus neutralizing antibody and a detection method.
The embodiment of the application aims to provide a novel coronavirus neutralizing antibody detection test paper, so as to solve the technical problems that the detection operation of the existing kit is complex, long in time consumption and not suitable for large-scale concomitant detection.
In order to achieve the purpose, the technical scheme adopted by the application is as follows: providing a novel coronavirus neutralizing antibody detection test paper, which comprises a PVC (polyvinyl chloride) bottom plate, a sample pad/blood filtering membrane, a combination pad, an NC (numerical control) membrane and absorbent paper, wherein the sample pad/blood filtering membrane, the combination pad, the NC membrane and the absorbent paper are arranged on the PVC bottom plate; wherein:
the sample pad/blood filtering membrane, the combining pad, the NC membrane and the absorbent paper are connected in sequence; the sample pad/blood filtering membrane, the combining pad, the NC membrane and the absorbent paper are sequentially overlapped in a staggered manner and then are adhered to the PVC bottom plate to form a test paper plate, and the test paper plate is cut to obtain the detection card;
the combination pad is provided with a novel coronavirus recombinant antigen RBD marked by colloidal gold and a chicken IgY antibody marked by the colloidal gold;
the detection line T line is angiotensin converting enzyme 2(ACE2) coated on the NC membrane, and the quality control line C line is goat anti-chicken antibody coated on the NC membrane;
the concentration of the colloidal gold labeled recombinant novel coronavirus RBD protein is 0.2-2mg/mL, and the coating amount of the colloidal gold labeled recombinant novel coronavirus RBD protein on the conjugate pad is 75 mu L;
the membrane scratching concentration of the angiotensin converting enzyme 2(ACE2) is 0.1-2mg/mL, and the coating amount is 30 mu L;
the membrane scratching concentration of the goat anti-chicken IgY is 0.5-2mg/mL, and the coating amount is 30 mu L;
the grain diameter of the colloidal gold particles marked with RBD on the colloidal gold pad is 20-30nm, and the grain diameter of the colloidal gold particles marked with chicken IgY on the colloidal gold pad is 15-20nm;
the detection lines T and the quality control lines C are arranged in parallel at intervals along the direction from the combined pad to the water absorption pad.
In an embodiment, the sample pad/blood filtration membrane, the conjugate pad, the NC membrane, and the absorbent paper are all adhered to the PVC base plate.
In an embodiment, the sample pad/hemofilter is pressed against the lower edge of the NC membrane, next the conjugate pad is pressed against the lower edge of the sample pad/hemofilter, and the bibulous paper is pressed against the upper edge of the NC membrane;
the conjugate pad contained 2% PEG 6000.
In an embodiment, the base plate is a PVC plate.
In an embodiment, the sample pad is a blood filtration membrane and the absorbent paper is absorbent filter paper.
The novel coronavirus neutralizing antibody detection test strip provided by the application adopts a competitive immunochromatography method, and utilizes the principle that the protein interaction between the RBD of SARS-CoV-2 spike protein and human angiotensin converting enzyme 2(ACE2) can be blocked by a neutralizing antibody specifically bound with the RBD, after sample addition, the neutralizing antibody existing in a sample is bound with the RBD marked by colloidal gold, and the binding between the RBD and ACE2 is prevented, and the RBD marked by the colloidal gold and not bound with the neutralizing antibody is captured by ACE2 protein on a detection line T line. With increasing concentration of neutralizing antibody, the detection line T line disappears when the neutralizing antibody concentration is sufficiently high. The colloidal gold-labeled chicken IgY antibody is combined with the goat anti-chicken IgY coated on the quality control line C, and the color band is the quality control line. The quality control line should be developed when detecting negative and positive samples, and the developed red strip can determine whether the chromatography process is normal, and is also the internal control standard of the test strip. In general, if the neutralizing antibody in the sample is insufficient, the color development of a detection line T line and the color development of a quality control line C line are presented; if sufficient neutralizing antibodies are present in the sample, the test line T is either shallow or completely absent.
The application provides a novel coronavirus neutralizing antibody test paper, its testing performance is stable, judges visual standard, and the specificity is strong, and does not need special equipment, can obtain the testing result in 15 min. The test paper can provide effective support for evaluation of the induced immune effect of the new coronary vaccine and detection of the level of the neutralizing antibody in the body of a new coronary pneumonia rehabilitation patient.
The preparation method of the colloidal gold immunochromatographic test strip provided by the application is as follows:
(1) after the optimal PH and the optimal antibody concentration of the colloidal gold particles are determined, K is utilized2CO3Adjusting the pH value of the colloidal gold particles to the optimal pH value, adding the recombinant novel coronavirus RBD with the optimal antibody concentration, and incubating at room temperature for 30min;
(2) adding 200 mu L of 5% BSA solution/1 mL of colloidal gold solution into the colloidal gold solution obtained in the step (1), sealing for 30min;
(3) centrifuging the colloidal gold solution obtained in the step (2) at 4 ℃ and 10000rpm for 20min, removing a supernatant after centrifugation, redissolving the redissolved solution for precipitation, and concentrating by 20% to obtain a recombinant novel coronavirus RBD protein labeling solution;
the complex solution is a mixture of BSA and sucrose;
(4) treatment of the bonding pad: spreading the bonding pad on a gold-plated plate, adding bonding pad treatment solution (containing 1% BSA, 1% sucrose, 1% S9, 0.02% casein, and 1% -10% PEG 6000) to wet completely, soaking for about 10min, and draining the treatment solution. Then the mixture is put into an oven and dried for 12 hours at 37 ℃. Drying, adding a drying agent, and sealing for storage;
(5) spraying the recombinant novel coronavirus RBD protein labeling solution obtained in the step (3) onto the colloidal gold pad obtained in the step (4) by using a gold spraying and film scratching instrument according to the volume of 0.1 mu L/mm; and (4) drying the colloidal gold pad obtained in the step (4) in an oven at 37 ℃ for 8h, adding a drying agent after drying, and sealing and storing.
The application provides a colloidal gold immunochromatographic test strip for rapidly and qualitatively detecting a neutralizing antibody generated in a human body after a new corona vaccine is inoculated, and the using method is as follows:
(1) sucking 75 mu L of sample to be detected, slowly dripping the sample into the sample adding hole of the test strip, and then slowly dripping 75 mu L of diluent into the sample adding hole of the test strip;
(2) timing, reading the result after 10-15min, and reading after 15min, wherein the result may be abnormal;
(3) and (4) analyzing results:
negative (-): the T line of the detection line has consistent color development compared with the negative control, and the C line of the quality control line has color development, which indicates that a certain amount of novel coronavirus neutralizing antibody does not exist in the sample;
positive (+): the T line of the detection line is lighter than the negative control in color development and even completely disappears, and the C line of the quality control line is colored, which indicates that a certain amount of novel coronavirus neutralizing antibodies exist in the sample;
and (4) invalidation: the control line is not developed, possibly due to mishandling or reagent failure, in which case it should be retested or discontinued.
The coating shell comprises an outer shell with an opening and a cover body detachably connected to the outer shell, and the cover body can close or open the opening; the detection test paper is arranged in the shell, a sample dropping opening is formed in the shell or the cover body, and the sample dropping opening corresponds to a sample pad of the detection test paper.
In one embodiment, the casing is a cuboid structure with an upper opening, a placement groove extending along the length direction of the casing is formed in the casing, and the detection test strip is placed in the placement groove; the cover is provided with the sample dropping port.
In one embodiment, the cover body is further provided with a viewing port, and the viewing port corresponds to the NC film of the test strip.
The utility model provides a novel coronavirus neutralization antibody test paper strip still can combine with colloidal gold immunoassay reading instrument, accurate survey determinand content, sensitivity is high, detection range is wide, it is simple and convenient swift, the content of the novel coronavirus neutralization antibody in the short-term test human serum in 15min, blood plasma, the whole blood sample, the accessible filters the blood pad and handles whole blood sample, utilize colloidal gold immunochromatography technique, use the micro-sample, dropwise add sample buffer solution, insert colloidal gold immunoassay reading instrument, can show the result fast. The method can meet the requirement of rapidly detecting the content of the neutralizing antibody of the novel coronavirus in a single person, and has the advantages of less sample dosage, short detection time, simple and convenient operation and accurate and reliable detection result.
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, it is obvious that the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a side view of an immunochromatographic test strip for detecting neutralizing antibodies against a novel coronavirus according to the present invention;
FIG. 2 is a front view of an immunochromatographic test strip for detecting a novel coronavirus neutralizing antibody according to the present invention.
Reference numerals
1-PVC base plate, 2-sample pad (blood filtering pad), 3-combination pad, 4-NC membrane and 5-absorbent paper.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the technical solutions of the present invention is provided. But are not intended to limit the invention.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were commercially available from conventional biochemical manufacturers.
The above figures show the side and front views of an immunochromatographic test strip for detecting a novel coronavirus neutralizing antibody of the present invention.
Examples
An immunochromatographic test strip for detecting a novel coronavirus neutralizing antibody is shown in figures 1 and 2 and comprises a detection card and a sample diluent, wherein the detection card comprises a PVC (polyvinyl chloride) base plate 1, a sample pad (blood filtering membrane) 2, a combination pad 3, an NC (numerical control) membrane 4 and a piece of absorbent paper 5, the sample pad (blood filtering membrane) 2, the combination pad 3, the NC membrane 4 and the absorbent paper 5 are sequentially attached to the PVC base plate 1 in a mutually staggered and lapped mode, the size of the mutually staggered and lapped mode is 2mm, the size of the PVC base plate 1 is 80-300mm, a test paper plate is formed, the test paper plate is cut into test strips with the width of 3-4mm, and a drying agent is added for storage at normal temperature to obtain the detection card;
wherein, the sample pad (blood filtering membrane) 2 has the size of 18 x 300mm, and the absorbent paper 5 has the size of 18 x 300 mm;
the combination pad 3 is a glass fiber pad, and is sprayed with novel coronavirus RBD antigen labeled colloidal gold and chicken IgY labeled colloidal gold, wherein the complex solution of the RBD labeled colloidal gold is 1% BSA, 1% sucrose and 10mM PB buffer (PH = 8), and the complex solution of the chicken IgY labeled colloidal gold is 1% BSA, 1% sucrose and 10mM PB buffer (PH = 7.4)
The concrete preparation method of the test paper board is as follows:
the conjugate pad 3 was soaked in 10mM PBS conjugate pad treatment solution containing 1% BSA, 1% sucrose, 0.5S9%, 0.02% sodium caseinate, and 1% -10% PEG 6000. Oven dried at 37 ℃ overnight. The labeled colloidal gold was sprayed evenly onto the conjugate pad 3 and dried overnight at 37 ℃.
Preparing a T line marking liquid: diluted with coated antibody dilutions in 10 mpbs buffer containing 1% sucrose (PH = 7.4). Dilution of human angiotensin converting enzyme 2: diluting ACE2 protein to 0.1-2 mg/mL; c, preparing a line marking liquid: diluted with coated antibody dilutions in 10 mpbs buffer containing 1% sucrose (PH = 7.4). And diluting the goat anti-chicken IgY to 0.5-2mg/mL by using a coating antibody diluent.
Adopting a metal spraying membrane scribing instrument, coating a C line antibody and a T line antibody on an NC membrane fixed on a bottom plate respectively, and scribing the liquid: 1 mu L/cm; film scratching speed: 100 mm/s;
and (3) placing the drawn test paper board into a drying box, drying the test paper board at 37 ℃ overnight, adding a drying agent, and sealing for later use.
The detection card also comprises a shell, the cut test paper strip is assembled in the plastic shell formed by buckling an upper shell made of plastic and a lower shell made of plastic, a sample adding hole and an observation window are arranged on the plastic upper shell, the sample adding hole corresponds to the sample pad 1 (blood filtering film), and the observation window corresponds to the detection line T line and the quality control line C line.
The sample diluent was phosphate buffered saline at pH7.0 containing 0.9% NaCl, 0.5% BSA.
As shown in fig. 1, the detection principle of the colloidal gold test strip of the present invention is as follows:
after a sample to be detected is added into a sample pad, a neutralizing antibody-RBD-colloidal gold conjugate on the conjugate pad is chromatographed towards one end of absorbent paper through capillary action, if a certain amount of novel coronavirus neutralizing antibody is contained in a detected sample and can be completely combined with RBD on the conjugate pad, the detected sample moves to a detection line T line, namely angiotensin converting enzyme 2(ACE2), no residual gold-labeled RBD or a small amount of residual gold-labeled RBD can be combined with T angiotensin converting enzyme 2(ACE2), and the T line is slightly light in color or does not appear; when the sample solution is chromatographed to the position of the quality control line C, the chicken IgY marked by the colloidal gold is combined with the goat anti-chicken IgY on the C line, and the C line of the quality control line is developed, so that the effectiveness of the test strip is proved. If a certain amount of neutralizing antibodies do not exist in the detected sample, the redundant novel coronavirus recombinant protein RBD on the combination pad is combined with the human angiotensin converting enzyme 2(ACE2 protein) on the T line of the detection line, and the T line is developed; the C line then also developed normally. If the C line of the quality control line does not develop color, the test strip is invalid, and the actual sample needs to be detected again.
Claims (10)
1. A colloidal gold immunochromatographic test strip for detecting a neutralizing antibody generated in vivo after inoculation of a novel coronavirus vaccine is characterized in that:
sequentially overlapping a sample pad/blood filtering membrane, a combination pad, a nitrocellulose membrane and absorbent paper on the PVC base plate from left to right, and sequentially overlapping the sample pad/blood filtering membrane, the combination pad, the nitrocellulose membrane and the absorbent paper and then pasting the sample pad/blood filtering membrane, the combination pad, the nitrocellulose membrane and the absorbent paper on the PVC base plate to form a test paper large plate, and cutting the test paper large plate to obtain the colloidal gold immunity test paper strip;
the colloidal gold pad is coated with a recombinant novel coronavirus RBD protein marked by colloidal gold;
the cellulose nitrate film is coated with angiotensin converting enzyme 2(ACE2) protein as a detection line T line, and a sheep anti-chicken IgY antibody as a quality control line C line;
the treatment solution of the conjugate pad is 1% BSA, 1% sucrose, 0.5% S9, 0.02% sodium caseinate, 1% -10% PEG 6000;
spraying 10% concentrated 20-30nm colloidal gold labeled novel coronavirus recombinant protein RBD on the binding pad; 5% of concentrated 15-20nm colloidal gold labeled goat anti-chicken IgY antibody is sprayed on the combined pad; drying in a constant temperature drying oven at 37 deg.C overnight;
the nitrocellulose membrane is sequentially marked with a detection line T line and a quality control line C line which are parallel to each other and have a spacing distance of 3-8 mm; the detection line T line is close to the sample pad/blood filter membrane, and the quality control line C line is close to the absorbent paper.
2. The strip of claim 1, wherein the concentration of the RBD protein of the novel coronavirus is 0.2-2mg/mL, and the coating amount on the gold pad is 75 μ L.
3. The colloidal gold immunochromatographic test strip for detecting a novel coronavirus neutralizing antibody according to claim 1, which is characterized in that the detection line T line has a concentration of angiotensin converting enzyme 2, ACE2 protein of 0.1-2mg/mL and a coating amount of 30 μ L; the concentration of the goat anti-chicken IgY antibody is 0.5mg-2mg/mL, and the coating amount is 30 mu L.
4. The colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the colloidal gold pad also comprises a separating agent, wherein the separating agent is BSA and PEG 6000.
5. The colloidal gold test strip of claim 1, wherein: the grain diameter of the colloidal gold particles marked with RBD on the colloidal gold pad is 20-30nm, and the grain diameter of the colloidal gold particles marked with chicken IgY on the colloidal gold pad is 15-20 nm.
6. The colloidal gold immune test strip of claim 1, which is characterized in that: the distance between the detection line and the quality control line is 3-5cm, and the detection line and the quality control line are not interfered with each other.
7. The colloidal gold immune test strip of claim 1, which is characterized in that: the sample pad is a blood filtering membrane, and the combining pad is a glass cellulose 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 bonding pad, and coating the bonding pad with the recombinant novel coronavirus RBD protein marked by colloidal gold;
(2) preparing a nitrocellulose membrane, and coating the cellulose membrane with angiotensin converting enzyme 2: an ACE2 protein detection line and a goat anti-chicken IgY antibody coated with the ACE2 protein detection line are used as quality control lines;
(3) and sequentially overlapping the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper on the PVC base plate from left to right to obtain the colloidal gold immune test strip.
9. The method for preparing the colloidal gold immunochromatographic test strip according to claim 7, which is characterized in that: the preparation of the combined pad comprises the following steps:
(1) after the optimal PH and the optimal antibody concentration of the colloidal gold particles are determined, adjusting the PH of the colloidal gold particles to the optimal PH value by adopting K2CO3, adding a recombinant novel coronavirus RBD, and incubating for 30min at room temperature;
(2) adding 200 mu L of 5% BSA solution/1 mL of colloidal gold solution into the colloidal gold solution obtained in the step (1), sealing for 30min;
(3) centrifuging the colloidal gold solution obtained in the step (2) at 4 ℃ and 10000rpm for 10min, removing supernatant after centrifugation, and redissolving the precipitation in redissolved solution to obtain recombinant novel coronavirus RBD protein labeling solution;
the complex solution is a mixture of Tween 20 and BSA;
(4) treatment of the bonding pad: spreading the bonding pad on a gold-plating plate, adding a bonding pad treatment solution (containing 1% BSA, 1% sucrose, 1% S9, 0.02% casein, and 1% -10% PEG 6000) to completely wet, soaking for about 10min, and drying the treatment solution; then putting the mixture into a drying oven, drying the mixture for 12 hours at 37 ℃, adding a drying agent after drying, and sealing and storing the mixture;
(5) spraying the recombinant novel coronavirus RBD protein labeling solution obtained in the step (3) onto the colloidal gold pad obtained in the step (4) by using a gold spraying and film scratching instrument according to the volume of 0.1 mu L/mm;
(6) and (4) drying the colloidal gold pad obtained in the step (4) in an oven at 37 ℃ for 240min, adding a drying agent after drying, and sealing and storing.
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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| CN202111381088.2A CN114034858A (en) | 2021-11-20 | 2021-11-20 | Colloidal gold immunochromatographic test strip for detecting in-vivo neutralizing antibody of neocorona vaccine after injection and preparation method thereof |
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| CN202111381088.2A CN114034858A (en) | 2021-11-20 | 2021-11-20 | Colloidal gold immunochromatographic test strip for detecting in-vivo neutralizing antibody of neocorona vaccine after injection and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116027035A (en) * | 2023-03-30 | 2023-04-28 | 济南玖方生物科技有限公司 | Kit for improving detection accuracy of HIV1/2 urine colloidal gold immunochromatography and preparation method thereof |
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Cited By (1)
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| CN116027035A (en) * | 2023-03-30 | 2023-04-28 | 济南玖方生物科技有限公司 | Kit for improving detection accuracy of HIV1/2 urine colloidal gold immunochromatography and preparation method thereof |
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