CN114280296A - Novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit and preparation method thereof - Google Patents
Novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit, wherein the test strip comprises: the kit comprises a sample pad, a combination pad 1, a combination pad 2, a nitrocellulose membrane, a water absorption pad and a bottom liner; the labeling process of the N protein-tracer particle complex comprises the following steps: mixing the N protein and the casein sodium evenly, and then adding the mixture into the colloidal gold solution; the cellulose nitrate membrane is provided with a detection line T2, a detection line T1 and a quality control line C, a mouse anti-human IgM monoclonal antibody is coated on the detection line T2, a mouse anti-human IgG monoclonal antibody is coated on the detection line T1, and a goat anti-chicken IgY antibody is coated on the quality control line. In the process of marking the colloidal gold and the N protein, the N protein and the sodium caseinate are mixed uniformly in advance and then added into the colloidal gold solution, so that the problem that the phenomenon of gold coagulation and death is easy to occur when the N protein and the colloidal gold solution are marked is solved, and meanwhile, the detection performance of the test strip is remarkably improved.
Description
Technical Field
The invention relates to the field of medical inspection, in particular to a novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit and a preparation method thereof.
Background
The Gold Immunochromatography (GICA) is a solid-phase labeled immunoassay which organically combines Gold labeling, immunoassay, chromatography analysis, and the like. The principle is as follows: the strip-shaped fiber chromatography material is used as a solid phase, a substance to be detected in a sample and a receptor (antigen or antibody) aiming at the substance to be detected on the chromatography material generate immunoreaction with high specificity and high affinity through capillary action, an immune complex is enriched or trapped in a certain area (detection zone) of the chromatography material in the chromatography process, and a marker (colloidal gold) capable of being visually observed is used to obtain the experimental phenomenon (color development) of a straight tube. While the force label crosses the detection zone and automatically separates from the binding label. The analysis technique has the advantages of simple and quick operation, single measurement, no need of special instruments and the like, is suitable for all levels of medical units, and can also be used for diagnosis, health care, physical examination and the like of family members.
The colloidal gold can be combined with various biological macromolecules such as protein, and the colloidal gold particles have a layer of surface negative charges, and are adsorbed with the positive charges on the surface of the protein through electrostatic induction, so that the environmental pH and the ionic strength are main factors influencing adsorption, and other factors such as the size, the protein molecular weight, the protein concentration and the like of the colloidal gold particles also influence protein adsorption. Although most proteins can be easily combined and adsorbed with colloidal gold, the phenomenon that part of the proteins generate dead gold after being added into a colloidal gold solution (such as new coronavirus N protein) still exists. In general, this is the case when the pH of the colloidal gold is first adjusted and if it cannot be improved, the protein is subjected to simple treatments such as dilution, dialysis, etc. However, for some proteins, especially expensive materials, the protein treatment often results in large losses or the amount of protein is small and insufficient for a single treatment. An effective solution is needed at this time.
Disclosure of Invention
According to the invention, a proper amount of sodium caseinate is added in the process of marking the colloidal gold and the marked protein, so that abnormal phenomena such as gold coagulation and dead gold caused by charge action can be effectively solved, meanwhile, the protein has high marking efficiency by adjusting the pH value, and the detection performance (including stability, sensitivity and specificity) of the test strip is remarkably improved.
In order to achieve the purpose, the invention adopts the following technical means: a novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit is characterized by comprising a test strip and a diluent, wherein the test strip comprises: the test paper comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a bottom lining, wherein the sample pad, the combination pad 1, the combination pad 2, the nitrocellulose membrane and the water absorption pad are sequentially overlapped and stuck on the bottom lining; the labeling pad 1 is coated with RBD protein-tracer particle complexes, and the labeling pad 2 is coated with N protein-tracer particle complexes; and the cellulose nitrate membrane is provided with a detection line T2, a detection line T1 and a quality control line C.
Preferably, the tracer particles are colloidal gold.
Preferably, the labeling process of the N protein-colloidal gold comprises: and uniformly mixing the N protein and the sodium caseinate, and then adding the mixture into the colloidal gold solution, wherein the mass ratio of the N protein to the sodium caseinate is 1.25-5.
Preferably, the pH of the colloidal gold solution is 7.0 to 9.0, preferably 8.0.
Preferably, the detection line T2 is coated with a mouse anti-human IgM monoclonal antibody, the detection line T1 is coated with a mouse anti-human IgG monoclonal antibody, and the quality control line is coated with a goat anti-chicken IgY antibody.
Preferably, the diluent comprises 0.2-2.0g/L buffer solution, 0.2-10g/L salt ion and 1-2g/L preservative; the buffer solution is selected from at least one of disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, MOPS and Tris buffer solution, and is preferably the combination of the disodium hydrogen phosphate dodecahydrate and the potassium dihydrogen phosphate; the salt ions are selected from at least one of sodium chloride or potassium chloride, and preferably the combination of sodium chloride and potassium chloride; the preservative is selected from at least one of sodium azide and Proclin-300, and is preferably Proclin-300.
A preparation method of a novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit is characterized by comprising the following steps:
(1) treatment of the bonding pad 1
Adding RBD protein into colloidal gold solution 1, labeling for 10-30min, adding 2-10% blocking agent, stirring for 10-30min, 10000rpm, centrifuging for 20 min, removing supernatant, precipitating with colloidal gold labeled compound solution to recover volume, and storing at 4 deg.C;
coating the RBD protein gold-labeled compound on the binding pad 1 at the concentration of OD20-OD50, and then carrying out freeze drying treatment to obtain the binding pad 1;
(2) treatment of the bonding pad 2
Uniformly mixing N protein and casein sodium, adding into the colloidal gold solution 2, marking for 10-30min, adding 2-10% of a sealing agent, stirring for 10-30min, centrifuging at 10000rpm for 20 min, removing supernatant, precipitating, and recovering volume of the colloidal gold-marked complex solution to obtain an N protein gold-marked complex, and storing at 4 ℃;
coating the N protein gold-labeled compound on a binding pad 2 at the concentration of OD10-OD40, and then carrying out freeze drying treatment to obtain the binding pad 2;
(3) treatment of nitrocellulose membrane:
coating a detection line T2, a detection line T1 and a quality control line C on a nitrocellulose membrane, and then drying:
a. preparation of detection line T2
Spraying the mouse anti-human IgM monoclonal antibody on the nitrocellulose membrane at a rate of 1.0-3.0ul/cm, which is close to the end of the sample pad;
b, preparing a T1 detection line:
coating the mouse anti-human IgG monoclonal antibody at the side of the T2 detection line by 1.0-3.0ul/cm, and keeping away from the sample pad end; (ii) a
d.C preparation of quality control line:
coating the goat anti-chicken IgY antibody solution at 1.0-3.0ul/cm on the side of the T1 detection line, and keeping away from the end of the sample pad;
(4) assembling:
sequentially adhering a sample pad, a nitrocellulose membrane and a water absorption pad on a bottom lining, and cutting into test strips with the width of 3-5 mm.
Preferably, in the step (1), the mass ratio of the N protein to the sodium caseinate is 1.25-5; in the step (1) and the step (2), the pH value of the colloidal gold solution 1 is 6.0-8.0, preferably 7.0; the pH value of the colloidal gold solution 2 is 7.0-9.0, preferably 8.0.
Preferably, the components of the colloidal gold labeled complex solution include: 20-50 mmol/buffer solution, 1-10 g/protein, 10-20g/L surfactant, 20-60g/L protective agent, 0.1-0.5g/L macromolecular substance and 1-2.0g/L preservative; the buffer solution is at least one selected from glycine, Tris buffer solution and MOPS buffer solution; the protein is selected from at least one of casein or BSA, preferably the combination of casein and BSA; the surfactant is selected from at least one of Tween-80, Tween-20, Triton X-100 or A90; the macromolecular substance is at least one of PVP, PEG or PVA; the preservative is selected from at least one of sodium azide or Proclin-300.
A method for marking colloidal gold and a substance to be marked for an immunochromatography reagent is characterized by comprising the step of uniformly mixing the substance to be marked and sodium caseinate and then adding the mixture into a colloidal gold solution.
The invention has the beneficial effects that: according to the invention, by means of the mode of uniformly mixing the new coronavirus N protein and the casein sodium in advance and then adding the mixture into the colloidal gold solution, the problems that the new coronavirus N protein and the colloidal gold particles are difficult to mark and abnormal phenomena such as gold precipitation and the like are very easy to occur in the marking process are effectively solved, and meanwhile, by adjusting the pH value of the colloidal gold solution to a specific range and matching with the reagent preparation system disclosed by the invention, various detection performances of the reagent can be effectively improved, and the clinical detection requirements are fully met.
Drawings
FIG. 1 is a schematic structural diagram of the test strip of the present invention.
The reference signs are:
1-PVC base plate; 1-sample pad; 3-conjugate pad 1; 4-conjugate pad 2; 5-nitrocellulose membrane; 6-absorbent paper; 7-detection line T2; 8-detection line T1; 9-Mass control line C.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following examples are included to more clearly and clearly illustrate the technical solutions of the present invention by way of illustration. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The specific embodiments of the present invention are merely illustrative of the invention and are not intended to limit the invention in any way.
Example 1 preparation of novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit (colloidal gold method)
(1) Preparation of the Diluent
The preparation is carried out according to the following formula, and the mixture is fully stirred, evenly mixed and stored at the temperature of 2-8 ℃.
(2) Preparation of colloidal gold labeled compound solution
The preparation is carried out according to the following formula, and the mixture is fully stirred, evenly mixed and stored at the temperature of 2-8 ℃.
(3) Preparation of colloidal gold-antibody complexes
Taking 1ml of colloidal gold, and adding 0.2mol/L of K2CO3Adjusting the pH value of the colloidal gold to 7.4 by using the solution, adding 20ug of a labeling RBD antigen into the colloidal gold after the pH value is adjusted, labeling for 20 minutes, adding 30ul of 5% casein sodium, sealing and stirring for 20 minutes, then performing centrifugation at 10000rpm for 20 minutes, discarding supernatant, precipitating, labeling the volume with the colloidal gold, recovering the solution to obtain a RBD protein gold-labeled compound, and storing at 4 ℃.
Taking 1ml of colloidal gold, adjusting the pH of the colloidal gold to 8.0 by using 0.1mol/L NaOH solution, uniformly mixing 20ug of N protein antigen for marking and 1ul of 5% casein sodium, adding the mixture into the colloidal gold solution after the pH is adjusted, marking for 20 minutes, adding 30ul of 5% casein sodium, sealing and stirring for 20 minutes, then performing 10000rpm, centrifuging for 20 minutes, collecting supernatant, precipitating, and recovering the volume by using colloidal gold-marked compound solution to obtain an N protein gold-marked compound, and storing at 4 ℃.
(4) Preparation of bonding pad
Coating the purified RBD protein gold-labeled compound with the concentration of OD30 after dilution on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a bonding pad 1;
and (3) diluting the purified N protein gold-labeled compound, coating the diluted N protein gold-labeled compound on a glass fiber pad when the concentration is OD15, and treating the glass fiber pad in a freeze dryer to obtain the combined pad 2.
(5) Assembly of immunochromatographic test strip
a. Treatment of nitrocellulose membranes
Spraying 1.0mg/ml of mouse anti-human IgG monoclonal antibody on a nitrocellulose membrane at the speed of 1.0ul/cm to serve as a first detection line T1, spraying 1.0mg/ml of mouse anti-human IgM monoclonal antibody on the nitrocellulose membrane at the speed of 1.0ul/cm to serve as a second detection line T2, spraying 0.5mg/ml of goat anti-chicken IgY antibody solution on the nitrocellulose membrane at the speed of 1.0ul/cm to serve as a quality control line C, and placing the nitrocellulose membrane in a drying oven to be dried for 24 hours at the temperature of 45 ℃. The second detection line T2 is close to the gold-labeled pad end on the nitrocellulose membrane, the quality control line C is located close to the absorbent pad end, and the first detection line T1 is located between the second detection line and the quality control line.
b. Test strip assembly
Sequentially sticking the sample pad, the bonding pad 1, the bonding pad 2, the nitrocellulose membrane coated with the antibody and the water absorption pad on a PVC (shown in figure 1), cutting the stuck intermediate into strips with the width of 3.7mm by a cutting machine, and assembling the strips into a test strip card box to obtain the finished test strip.
Example 3 method of Using test strips
1. Uniformly mixing a sample to be detected with the diluent for later use;
2. 2 drops (more than 80ul) of diluted sample are dropped into the sample adding hole of the detection card
3. The results were observed after 5-15 min.
Example 4 test strip Performance validation
In order to verify various performances of the test strip, two groups of kits are arranged for performance verification:
group A: the test strip of embodiment 1 of the invention;
group B: commercial kit I
Group C: commercial kit II
Wherein group a test strips were tested according to the method of use described in example 2, and group B and group C were tested according to their instructions.
(1) Occurrence of the phenomenon of gold accumulation and death
The test results show that in the process of labeling the colloidal gold and the protein of the test strips of the groups A and C, the test strips of the group A do not generate the colloidal gold coagulation phenomenon, and the test strips of the group C generate the obvious colloidal gold coagulation phenomenon (dark granular substances can be seen by naked eyes in the labeling liquid).
(2) Clinical evaluation
64 clinical positive samples and 93 clinical negative samples are selected, the three groups of test strips are used for respectively detecting the positive samples and the negative samples, and the detection results are shown in the following table:
TABLE 1 test strip clinical Properties
Injecting: clinical sensitivity (number of positive tests/number of positive samples)%; clinical specificity ═ (number of negative tests/number of negative samples)%; the total coincidence rate is (number of correct detections/total samples)%.
(3) Repeatability of
The three groups of test strips are used for respectively detecting 1 part of novel coronavirus antibody reference substance, each group of test strips is repeatedly detected for 10 times, wherein the detection results of the A group of test strips are positive after 10 times; the test result of the test strip B is positive for 9 times and negative for one time after 10 times; the test result of the test strip in the group C is 7 times positive and 3 times negative after 10 times of detection.
(4) Stability of
When the three groups of test strips are placed at 37 ℃ for 0, 5, 10, 15, 20, 25 and 30 days, the detection is carried out on the same sample, wherein the detection results of the test strips in the group A are the same within 30 days of thermal acceleration, the detection results of the test strips in the group B are deviated after 25 days of thermal acceleration, and the detection results of the test strips in the group C are deviated after 20 days of thermal acceleration.
According to the experimental results, the clinical sensitivity of the four groups of test strips is respectively 98.44%, 85.94% and 73.44%; clinical specificity was 100%, 99% and 97%, respectively; the total coincidence rates are 99.36%, 93.63% and 87.26% respectively; the experimental results show that the detection performance (including clinical compliance, repeatability and stability) of the test strip (group A) prepared in the embodiment 1 of the invention is obviously superior to that of the test strips of the groups B and C.
Example 5 Effect of sodium Casein addition on test strip Performance during labeling
In order to verify the influence of the sodium caseinate added in the process of marking the colloidal gold and the protein on the performance of the test strip, the following 4 groups of test strips are arranged:
group A: the test strip of embodiment 1 of the invention;
group B: the preparation method of the test strip is different from that of the test strip in the embodiment 1 only in that no sodium caseinate is added in the labeling process of the colloidal gold and the N protein;
group C: the preparation method of the test strip is different from that of the embodiment 1 in that sodium caseinate is not added in the labeling process of colloidal gold and N protein, and 1ul of 5% BSA is added;
group D: the preparation method of the test strip is different from that of the test strip in the embodiment 1 in that sodium caseinate is not added in the labeling process of the colloidal gold and the N protein, and 1ul 5% PEG20000 is added.
(1) Gold precipitation during marking
The test results show that in the process of labeling the colloidal gold and the N protein, except for the test strips in the groups A and C, the test strips in the groups B and D have the phenomenon of colloidal gold coagulation (dark granular substances can be seen by naked eyes in the labeling liquid).
(2) Clinical evaluation
64 clinical positive samples and 93 clinical negative samples are selected, the four groups of test strips are used for respectively detecting the positive samples and the negative samples, and the detection results are shown in the following table:
TABLE 3 clinical Properties of test strips
Injecting: clinical sensitivity (number of positive tests/number of positive samples)%; clinical specificity ═ (number of negative tests/number of negative samples)%; the total coincidence rate is (number of correct detections/total samples)%.
According to the experimental results, the clinical sensitivity of the four groups of test strips is 100%, 45.31%, 59.38% and 76.56% respectively; clinical specificities were 100.00%, 100%, 97.00% and 98.00%, respectively; the total percent of compliance was 100%, 77.07%, 81.53% and 89.17%, respectively. The experimental results show that the clinical compliance of the test strip (group A) prepared in the embodiment 1 of the invention is significantly better than that of the test strips of the groups B, C and D. According to the experimental result, sodium caseinate is presumed to have the function of interfering static electricity as a protein, and sodium caseinate is in a dispersion state in a solution and has a repulsive effect on a gold-labeled compound, so that the dispersion degree of the gold-labeled compound is increased, the colloidal stability of colloidal gold is maintained, and therefore, sodium caseinate can be used for treating abnormal phenomena such as gold coagulation death and the like caused by the charge effect in the labeling process, particularly, for a to-be-labeled object which is very easy to generate gold coagulation death, such as new coronavirus N protein, a proper amount of sodium caseinate is added in the labeling process of the to-be-labeled object and the colloidal gold, so that the technical problem of gold coagulation can be effectively solved, the detection performance of the kit is remarkably improved, and the clinical requirements are fully met.
Example 5 Effect of sodium Casein addition on test strip Performance during labeling
In order to verify the influence of the addition of sodium caseinate on the performance of subsequent test strips in the process of marking colloidal gold and N protein, the following 4 groups of test strips are arranged:
group A: the preparation method of the test strip is different from that of the test strip in the embodiment 1 only in that 0.1ul of 5% sodium caseinate is added in the labeling process of the colloidal gold and the N protein;
group B: the preparation method of the test strip is different from that of the test strip in the embodiment 1 only in that 0.5ul of 5% casein sodium is added in the labeling process of the colloidal gold and the N protein;
group C: the preparation method of the test strip is different from that of the test strip in the embodiment 1 only in that 2ul of 5% sodium caseinate is added in the labeling process of the colloidal gold and the N protein;
group D: the preparation method of the test strip is different from that of the test strip in the embodiment 1 only in that 3ul of 5% sodium caseinate is added in the labeling process of the colloidal gold and the N protein;
(1) gold precipitation during marking
The test results show that in the process of labeling the colloidal gold and the N protein, the 4 groups of test strips do not generate the colloidal gold coagulation phenomenon except that the A group of test strips generate the trace colloidal gold coagulation phenomenon (dark granular substances can be seen by naked eyes in the labeling liquid), and the B group, the C group and the D group of test strips do not generate the colloidal gold coagulation phenomenon.
(2) Clinical evaluation
64 clinical positive samples and 93 clinical negative samples are selected, the four groups of test strips are used for respectively detecting the positive samples and the negative samples, and the detection results are shown in the following table:
TABLE 3 clinical Properties of test strips
Injecting: clinical sensitivity (number of positive tests/number of positive samples)%; clinical specificity ═ (number of negative tests/number of negative samples)%; the total coincidence rate is (number of correct detections/total samples)%.
According to the experimental results, the clinical sensitivity of the four groups of test strips is 67%, 98%, 100% and 83% respectively; clinical specificities were 97%, 100% and 99%, respectively; the total percent of compliance was 85%, 99%, 100% and 92%, respectively. The experimental result shows that the test strip has the best detection performance when the mass ratio of the sodium caseinate to the new coronavirus N protein is within the range of 1.25-5 in the process of marking the colloidal gold and the new coronavirus N protein.
Example 6 Effect of pH on test strip Performance during labeling
In order to verify the influence of the pH value on the performance of a subsequent test strip in the process of marking colloidal gold and N protein, the following 5 groups of test strips are arranged:
group A: the preparation method of the test strip is different from that of the example 1 only in that the pH value of the colloidal gold solution is 6.0 in the marking process;
group B: the preparation method of the test strip is different from that of the example 1 only in that the pH value of the colloidal gold solution is 7.0 in the marking process;
group C: the preparation method of the test strip is different from that of the test strip in the embodiment 1 only in that the pH value of colloidal gold solution is 9.0 in the marking process;
group D: the preparation method of the test strip is different from that of the example 1 only in that the pH value of the colloidal gold solution is 10.0 in the marking process;
(1) gold precipitation during marking
The test result shows that in the process of marking the colloidal gold and the protein, no colloidal gold coagulation phenomenon occurs in all the four groups of test strips.
(2) Clinical evaluation
Selecting 53 positive samples for completing injection of the new corona vaccine and 119 negative samples for not inoculating the new corona vaccine, and detecting the positive samples and the negative samples by using the three groups of test strips respectively, wherein the detection results are shown in the following table:
TABLE 3 clinical Properties of test strips
Injecting: the positive sample is a sample for completing vaccination; the negative sample is a sample without vaccination; clinical sensitivity (number of positive tests/number of positive samples)%; clinical specificity ═ (number of negative tests/number of negative samples)%; the total coincidence rate is (number of correct detections/total samples)%.
According to the experimental results, the clinical sensitivity of the four groups of test strips is 83%, 97%, 98% and 77% respectively; clinical specificities of 98%, 100% and 96%, respectively; the total percent of compliance was 92%, 99% and 88%, respectively. The experimental result shows that the detection performance is optimal when the pH value is within 7.0-9.0 in the process of labeling the N protein and the colloidal gold solution.
Although embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments without departing from the principles and spirit of the present invention.
Claims (10)
1. A novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit is characterized by comprising a test strip and a diluent, wherein the test strip comprises: the test paper comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a bottom lining, wherein the sample pad, the combination pad 1, the combination pad 2, the nitrocellulose membrane and the water absorption pad are sequentially overlapped and stuck on the bottom lining; the labeling pad 1 is coated with RBD protein-tracer particle complexes, and the labeling pad 2 is coated with N protein-tracer particle complexes; and the cellulose nitrate membrane is provided with a detection line T2, a detection line T1 and a quality control line C.
2. The novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit of claim 1, wherein the tracer particle is colloidal gold.
3. The novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit of claim 2, wherein the labeling process of N protein-colloidal gold comprises: and uniformly mixing the N protein and the sodium caseinate, and then adding the mixture into the colloidal gold solution, wherein the mass ratio of the N protein to the sodium caseinate is 1.25-5.
4. The novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit according to claim 3, wherein the colloidal gold solution has a pH of 7.0-9.0, preferably 8.0.
5. The novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit according to claim 4, wherein a mouse anti-human IgM monoclonal antibody is coated on the detection line T2, a mouse anti-human IgG monoclonal antibody is coated on the detection line T1, and a goat anti-chicken IgY antibody is coated on the quality control line.
6. The novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit according to claim 5, wherein the diluent comprises 0.2-2.0g/L buffer solution, 0.2-10g/L salt ions, 1-2g/L preservative; the buffer solution is selected from at least one of disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, MOPS and Tris buffer solution, and is preferably the combination of the disodium hydrogen phosphate dodecahydrate and the potassium dihydrogen phosphate; the salt ions are selected from at least one of sodium chloride or potassium chloride, and preferably the combination of sodium chloride and potassium chloride; the preservative is selected from at least one of sodium azide and Proclin-300, and is preferably Proclin-300.
7. The method for preparing a novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit according to any one of claims 1 to 5, comprising the steps of:
(1) treatment of the bonding pad 1
Adding RBD protein into colloidal gold solution 1, labeling for 10-30min, adding 2-10% blocking agent, stirring for 10-30min, 10000rpm, centrifuging for 20 min, removing supernatant, precipitating with colloidal gold labeled compound solution to recover volume, and storing at 4 deg.C;
coating the RBD protein gold-labeled compound on the binding pad 1 at the concentration of OD20-OD50, and then carrying out freeze drying treatment to obtain the binding pad 1;
(2) treatment of the bonding pad 2
Uniformly mixing N protein and casein sodium, adding into the colloidal gold solution 2, marking for 10-30min, adding 2-10% of a sealing agent, stirring for 10-30min, centrifuging at 10000rpm for 20 min, removing supernatant, precipitating, and recovering volume of the colloidal gold-marked complex solution to obtain an N protein gold-marked complex, and storing at 4 ℃;
coating the N protein gold-labeled compound on a binding pad 2 at the concentration of OD10-OD40, and then carrying out freeze drying treatment to obtain the binding pad 2;
(3) treatment of nitrocellulose membrane:
coating a detection line T2, a detection line T1 and a quality control line C on a nitrocellulose membrane, and then drying:
a. preparation of detection line T2
Spraying the mouse anti-human IgM monoclonal antibody on the nitrocellulose membrane at a rate of 1.0-3.0ul/cm, which is close to the end of the sample pad;
b, preparing a T1 detection line:
coating the mouse anti-human IgG monoclonal antibody at the side of the T2 detection line by 1.0-3.0ul/cm, and keeping away from the sample pad end; (ii) a
d.C preparation of quality control line:
coating the goat anti-chicken IgY antibody solution at 1.0-3.0ul/cm on the side of the T1 detection line, and keeping away from the end of the sample pad;
(4) assembling:
sequentially adhering a sample pad, a nitrocellulose membrane and a water absorption pad on a bottom lining, and cutting into test strips with the width of 3-5 mm.
8. The method for preparing the novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit according to claim 7, wherein in the step (1), the mass ratio of the N protein to the sodium caseinate is 1.25-5; in the step (1) and the step (2), the pH value of the colloidal gold solution 1 is 6.0-8.0, preferably 7.0; the pH value of the colloidal gold solution 2 is 7.0-9.0, preferably 8.0.
9. The method for preparing the novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit according to claim 8, wherein the colloidal gold labeled complex solution comprises the following components: 20-50 mmol/buffer solution, 1-10 g/protein, 10-20g/L surfactant, 20-60g/L protective agent, 0.1-0.5g/L macromolecular substance and 1-2.0g/L preservative; the buffer solution is at least one selected from glycine, Tris buffer solution and MOPS buffer solution; the protein is selected from at least one of casein or BSA, preferably the combination of casein and BSA; the surfactant is selected from at least one of Tween-80, Tween-20, Triton X-100 or A90; the macromolecular substance is at least one of PVP, PEG or PVA; the preservative is selected from at least one of sodium azide or Proclin-300.
10. A method for marking colloidal gold and a substance to be marked for an immunochromatography reagent is characterized by comprising the step of uniformly mixing the substance to be marked and sodium caseinate and then adding the mixture into a colloidal gold solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115201474A (en) * | 2022-08-12 | 2022-10-18 | 万华普曼生物工程有限公司 | Novel rapid detection kit for coronavirus antibody and preparation method thereof |
| CN117554613A (en) * | 2024-01-10 | 2024-02-13 | 深圳市绿诗源生物技术有限公司 | Colloidal gold detection test strip for bovine coronavirus antigen, and preparation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115201474A (en) * | 2022-08-12 | 2022-10-18 | 万华普曼生物工程有限公司 | Novel rapid detection kit for coronavirus antibody and preparation method thereof |
| CN117554613A (en) * | 2024-01-10 | 2024-02-13 | 深圳市绿诗源生物技术有限公司 | Colloidal gold detection test strip for bovine coronavirus antigen, and preparation method and application thereof |
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