CN113687070A - High-sensitivity semi-quantitative kit for detecting novel coronavirus neutralizing antibody - Google Patents
High-sensitivity semi-quantitative kit for detecting novel coronavirus neutralizing antibody Download PDFInfo
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention relates to the technical field of detection of neutralizing antibodies, in particular to a high-sensitivity semi-quantitative kit for detecting novel coronavirus neutralizing antibodies, which comprises a detection card, a sample diluent and a colorimetric card, wherein a gold pad is arranged in the detection card, and the gold pad contains a recombinant S-RBD antigen marked by colloidal gold; wherein the amount of the recombinant S-RBD antigen used per milliliter of colloidal gold is more than 5 micrograms. The product greatly improves the sensitivity of detecting the new crown neutralizing antibody, and can detect the neutralizing antibody with the concentration of 20ng/ml at least under the condition of direct observation by naked eyes. The kit for detecting the neutralizing antibody of the novel coronavirus in the blood sample has the advantages of rapidness, simplicity, convenience, easiness in popularization, semi-quantification, safety and reliability.
Description
Technical Field
The invention relates to the technical field of detection of neutralizing antibodies, in particular to a semi-quantitative kit for detecting novel coronavirus neutralizing antibodies.
Background
In order to prevent new serious infectious diseases, neutralizing antibody therapy is an important strategy for effective prevention and treatment. After the virus invades the human body, the target cells are invaded by binding to spike proteins on the surface of the virus. Neutralizing antibodies are soluble proteins secreted by adaptive immune response cells that recognize viral surface proteins and prevent their binding to cellular receptors.
With the global pandemic of new coronavirus pneumonia (COVID-19), the number of infections and deaths continues to rise, and scientists from various countries are always looking for treatment methods for COVID-19. Neutralizing antibodies are immunoglobulins directed against the receptor binding domain (S-RBD) of a novel coronavirus protein. The neutralizing antibody can inhibit the combination of the novel coronavirus surface protein and a human cell surface specific receptor ACE-2 through the combination of the novel coronavirus surface protein, thereby blocking the invasion of viruses. Therefore, it is expected that neutralizing antibodies will be an effective means against novel coronavirus diseases, and that neutralizing antibody detection can be used to monitor the presence or absence of neutralizing antibodies and the level of neutralizing antibody concentration in subjects vaccinated with the novel coronavirus vaccine or in people infected with the novel coronavirus, and can be used to evaluate the vaccination efficacy.
The virus neutralization test is the most traditional method for detecting the neutralizing antibody, but a cell culture facility and test conditions of using live viruses are needed, the test result needs at least 2-3 days, the risk is high, and the safety is low.
At present, the detection of the neutralizing antibody of the new coronavirus in the market mainly adopts a competitive binding principle, and the neutralizing antibody in blood is detected by combining with the RBD protein of the new coronavirus in a competitive way with a human cell surface specific receptor ACE-2. The commonly adopted technical methods comprise three methods, namely an enzyme-linked immunosorbent assay, a chemiluminescence assay and a colloidal gold assay. Among them, the chemiluminescence method and the enzyme-linked immunoassay method are capable of quantification, high sensitivity, high throughput, but require skilled laboratory personnel for operation and expensive detection equipment. The common colloidal gold method has the advantages of high detection speed and no need of depending on detection equipment, but also has the defects of low sensitivity and incapability of quantification.
The competitive colloidal gold immunochromatography technology adopted in the detection of the neutralizing antibody can inhibit the competitive combination of the RBD protein of the novel coronavirus surface protein and the ACE2 protein through the neutralizing antibody, thereby achieving the detection of the concentration of the neutralizing antibody in serum or plasma. The test zone (line T) of the novel coronavirus neutralizing antibody test kit was labeled with recombinant ACE-2 and the control line (line C) with polyclonal antibody against RBD. During the detection, the sample is dropped into the sample well, and the neutralizing antibody of the novel coronavirus is combined with the recombinant S-RBD antigen of the novel coronavirus labeled with the colloidal gold, so that the colloidal gold-labeled recombinant S-RBD antigen is prevented from being combined with ACE-2 on the T line. Sheep anti-S-RBD polyclonal antibody/novel coronavirus recombinant S-RBD antigen-colloidal gold complex is formed on C line. After detection, semi-quantitative detection of the neutralizing antibody of the novel coronavirus in the blood sample can be realized according to the colorimetric card.
However, the existing kit for detecting the neutralizing antibody by the competitive immunochromatography technology still has the following defects that the sensitivity is low, and the neutralizing antibody with the concentration of more than 200ng/ml can be detected at least; it is difficult to quantify and only qualitative detection of the presence of neutralizing antibodies in a sample is possible.
Therefore, the development of a high-sensitivity semi-quantitative kit for detecting a novel coronavirus neutralizing antibody is one of the technical problems which need to be solved at present.
Disclosure of Invention
The invention aims to provide a high-sensitivity semi-quantitative kit for detecting a novel coronavirus neutralizing antibody, which is improved by using a colloidal gold immunochromatography technology, optimizes the RBD colloidal gold amount, the size of gold nanoparticles, the ratio of RBD protein amount to ACE2 protein amount and optimizes the color development presentation mode, and achieves the purpose of improving the sensitivity of the neutralizing antibody detected by a competitive immunochromatography technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a high-sensitivity semi-quantitative kit for detecting a novel coronavirus neutralizing antibody comprises a detection card, a sample diluent and a colorimetric card, wherein a gold pad is arranged in the detection card, and a recombinant S-RBD antigen marked by colloidal gold is contained on the gold pad; wherein the amount of the recombinant S-RBD antigen used per mL of the colloidal gold is 5. mu.g or more.
Further, the particle size of the colloidal gold is 20-60 nm.
Further, the gold pad comprises a glass fiber membrane, wherein the glass fiber membrane is sprayed with recombinant S-RBD antigen containing colloidal gold markers, namely RBD colloidal gold complex, and the spraying amount is 0.5-3.0 muL/cm.
Furthermore, an NC membrane is arranged in the detection card, a detection line is arranged on the NC membrane, the detection line is coated with recombinant ACE-2 protein, and the concentration of the recombinant ACE-2 protein is 0.5-5.0 mg/mL.
Further, the recombinant ACE-2 protein is immobilized on an NC membrane in a lattice form.
The preparation method of the detection card comprises the steps of preparing a cellulose nitrate film, preparing a gold pad and preparing the detection card;
the preparation process of the coated nitrocellulose membrane comprises the following specific steps:
coating a quality control line and a detection line on a nitrocellulose membrane, and coating a recombinant ACE-2 protein on the detection line, wherein the use amount of the recombinant ACE-2 protein is 0.5-5.0 mg/mL; fixing the recombinant ACE-2 protein on a nitrocellulose membrane in a dot matrix form; then the coated nitrocellulose membrane is dried in an oven at 30 ℃ for standby.
The preparation process of the gold pad specifically comprises the following steps:
(1) preparation of colloidal gold
A.100ml distilled water is added with chloroauric acid with two ten-thousandth of final concentration, and the mixture is heated, stirred and boiled on a magnetic stirrer;
B. adding trisodium citrate with a final concentration of three ten-thousandth into the solution, and continuously heating and stirring for 10 minutes;
C. cooling at room temperature to obtain colloidal gold; the particle size of the colloidal gold is 20-60 nm;
(2) preparation of RBD colloidal gold complexes
A. Adding colloidal gold into a centrifuge tube, and adding 50 μ l of 1% K2CO3Mixing uniformly;
B. adding the recombinant S-RBD antigen into a centrifuge tube, uniformly mixing, and incubating for 30 minutes at room temperature; the amount of the recombinant S-RBD antigen used per mL of colloidal gold is more than 5 mug;
(3) preparation of colloidal gold bars
Treating and drying the glass fiber membrane by using a gold pad treatment solution, wherein the formula of the gold pad treatment solution is as follows: 80g sucrose, 2.42g Tris, 10g BSA, 4g Tween-20 dissolved in 1L distilled water, pH 8.0; the RBD colloidal gold complex was concentrated ten times using a metal spraying instrument and then treated with a 30cm by 1cm glass fiber membrane at a spray rate of 0.5-3.0. mu.L/cm.
Compared with the prior art, the invention has the beneficial effects that:
the invention is innovatively improved on the basis of the original competitive immunochromatography technology, greatly improves the sensitivity of detecting the new crown neutralizing antibody, and can detect the neutralizing antibody with the concentration of 20ng/ml at least under the condition of direct visual observation. The kit has higher reference value for rapidly judging the existence of the neutralizing antibody in a subject inoculated with the novel coronavirus vaccine or a population infected with the novel coronavirus and evaluating the vaccination effect, and is suitable for popularization and application.
The kit for detecting the neutralizing antibody of the novel coronavirus in the blood sample has the advantages of rapidness, simplicity, convenience, easiness in popularization, semi-quantification, safety and reliability.
Drawings
FIG. 1 shows the results of experiments with different amounts of RBD protein.
FIG. 2 shows the results of different particle sizes.
Figure 3 is a 30cm by 1cm glass fiber membrane treated at different spray volumes.
Fig. 4-8 show the results of screening experiments for colloidal gold size, RBD and ACE2 usage.
Fig. 9 is a schematic diagram of a result interpretation method.
FIG. 10 shows the results of an experiment in the form of color development. Wherein, the dot matrix, the multiple lines and the single line are arranged from left to right in sequence.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The chemical reagents, experimental materials, and the like used in the following embodiments are all commercial products and are commercially available, unless otherwise specified.
A high sensitivity semi-quantitative kit for detecting neutralizing antibodies to a novel coronavirus comprising: a neutralizing antibody competition method detection card, a sample diluent and a colorimetric card for semi-quantitative detection.
The detection card for the neutralizing antibody competition method comprises a gold pad, a coated nitrocellulose membrane (NC membrane), absorbent paper and a bottom plate; the gold pad is marked with recombinant S-RBD antigen marked by colloidal gold tracer particles.
Wherein, the amount of the recombinant S-RBD antigen used per mL of the colloidal gold is more than 5 mug, and the particle size of the colloidal gold is 20-60 nm.
The gold pad comprises a glass fiber membrane, wherein the glass fiber membrane is sprayed with recombinant S-RBD antigen containing colloidal gold markers, namely RBD colloidal gold complex, and the spraying amount is 0.5-3.0 muL/cm.
And a detection line is arranged on the NC membrane, the detection line is coated with recombinant ACE-2 protein, and the concentration of the recombinant ACE-2 protein is 0.5-5.0 mg/mL. The recombinant ACE-2 protein is fixed on an NC membrane in a lattice form.
The preparation method of the detection card by the neutralizing antibody competition method comprises the preparation of a coated nitrocellulose membrane, the preparation of a gold pad and the preparation of the detection card;
the preparation process of the coated nitrocellulose membrane comprises the following specific steps:
and coating a quality control line and a detection line on the nitrocellulose membrane, wherein the quality control line is coated with a commercial goat anti-mouse IgG polyclonal antibody, and the coating concentration is 1.0 mg/mL. Coating a recombinant ACE-2 protein on the detection line, wherein the use amount of the recombinant ACE-2 protein is 0.5-5.0 mg/mL; fixing the recombinant ACE-2 protein on a nitrocellulose membrane in a dot matrix form; then the coated nitrocellulose membrane is dried in an oven at 30 ℃ for standby.
The preparation process of the gold pad comprises the following specific steps:
(1) preparation of colloidal gold
A.100ml distilled water is added with chloroauric acid with two ten-thousandth of final concentration, and the mixture is heated, stirred and boiled on a magnetic stirrer;
B. adding trisodium citrate with a final concentration of three ten-thousandth into the solution, and continuously heating and stirring for 10 minutes;
C. cooling at room temperature to obtain colloidal gold; the particle size of the colloidal gold is 20-60 nm;
(2) preparation of RBD colloidal gold complexes
A. Adding colloidal gold into a centrifuge tube, and adding 50 μ l of 1% K2CO3Mixing uniformly;
B. adding the recombinant S-RBD antigen into a centrifuge tube, uniformly mixing, and incubating for 30 minutes at room temperature; the amount of the recombinant S-RBD antigen used per mL of colloidal gold is more than 5 mug;
and (3) uniformly mixing, standing at room temperature for 30min, centrifuging at 15000rpm for 30min, removing supernatant, washing and precipitating for 3 times by using a labeled cleaning solution with the same volume of chloroauric acid solution, resuspending labeled colloidal gold particles by using a colloidal gold preserving solution with the same volume, and preserving at 2-8 ℃ for later use.
(3) Preparation of colloidal gold bars
Treating and drying the glass fiber membrane by using a gold pad treatment solution, wherein the formula of the gold pad treatment solution is as follows: 80g sucrose, 2.42g Tris, 10g BSA, 4g Tween-20 dissolved in 1L distilled water, pH 8.0; the RBD colloidal gold complex was concentrated ten times using a metal spraying instrument and then treated with a 30cm by 1cm glass fiber membrane at a spray rate of 0.5-3.0. mu.L/cm. And drying and storing the gold pad at room temperature for later use after freeze-drying.
The preparation of the detection card comprises the following steps:
cutting the gold pad into strips with the width of about 0.5cm, sequentially sticking the cut gold pad, the coated nitrocellulose membrane, the absorbent paper and the blank glass fiber on a bottom plate to assemble a large colloidal gold detection card, cutting according to the width of 3-4 mm, filling the large colloidal gold detection card into a card shell, filling the card shell and a drying agent into an aluminum foil bag, and sealing to prepare the detection card.
The sample diluent comprises 0.2-0.3 g/L of monopotassium phosphate, 1.4-0.15 g/L of disodium hydrogen phosphate, 7-9 g/L of sodium chloride, 0.1-0.3 g/L of potassium chloride and the addition amount of Tween-20 is 0.08-0.16% of the weight of the sample diluent; the preferable addition amount of the sample diluent is 0.24g/L of monopotassium phosphate, 1.44g/L of disodium phosphate, 8g/L of sodium chloride, 0.2g/L of potassium chloride and Tween-20, and the addition amount is 0.1 percent of the weight of the sample diluent.
The colorimetric card for semi-quantitative detection comprises a color development strip with a neutralizing antibody titer of 1:8 and a color development strip with a neutralizing antibody titer of 1: 64.
The specific optimization experiment process of the detection card is as follows:
determination of the amount of RBD protein
(1) Preparing colloidal gold:
a.100ml of distilled water was added with chloroauric acid at a final concentration of two parts per million, and heated, stirred and boiled on a magnetic stirrer.
B. Three parts per million of trisodium citrate is added to the solution, and heating and stirring are continued for 10 minutes.
C. And cooling at room temperature to obtain the colloidal gold.
(2) Preparing an RBD colloidal gold compound, determining the use amount of RBD protein:
A. 1ml of colloidal gold is respectively put into different centrifuge tubes, and 50 mul of 1% K is added into each centrifuge tube2CO3And (4) uniformly mixing.
D. Mu.l of 10% NaCl solution was added to each tube, and mixed well to observe color change.
As a result: 5 mug of RBD labeled colloidal gold is extremely unstable at high salt concentration, while more than 10 mug of RBD can ensure the stability of gold particles, so that at least 10 mug of RBD protein is required to label each ml of colloidal gold (as shown in figure 1).
Secondly, determining the size of the colloidal gold particles, the spraying amount of RBD colloidal gold and the using amount of ACE-2 protein
(1) Preparing colloidal gold particles of different sizes
Referring to the first step of the first experiment, the gold particle size was adjusted by controlling the ratio of chloroauric acid to trisodium citrate, and the gold particle diameter was measured by an instrument to prepare gold particles having diameters of 30nm, 35nm, and 40 nm. The ratio of chloroauric acid to trisodium citrate used for 30nm, 35nm and 40nm gold particles was 1:1.75, 1:1.5 and 1:1.35, respectively.
As a result: the results of particle size detection are shown in FIG. 2, and the colloidal gold of 30nm, 35nm and 40nm was successfully prepared.
(2) Preparing colloidal gold strips with different RBD spraying amounts
The RBD colloidal gold complex was concentrated ten-fold using a metal sprayer and then treated with 30cm by 1cm glass fiber membranes (FIG. 3) at a spray rate of 1.0. mu.L/cm, 1.5. mu.L/cm, and 2.0. mu.L/cm. The glass fiber membrane is treated and dried by gold pad treatment liquid in advance. The formula of the gold pad treating fluid is as follows: 80g of sucrose, 2.42g of Tris, 10g of BSA, 4g of Tween-20 were dissolved in 1L of distilled water at a pH of 8.0.
(3) Preparation of different concentrations of ACE-2 protein treated NC membranes
NC membranes were treated with 1.0mg/mL, 1.5mg/mL, and 2.0mg/mL of ACE-2, respectively, and were dried in an oven at 30 ℃ until ready for use.
(4) The detection cards shown in example 1 were prepared by matching NC films of different gold nanoparticle sizes, different RBD colloidal gold spraying amounts, and different ACE2 protein usage amounts (the kit and the rest of the detection card were kept unchanged), as shown in table 1. And detecting different detection cards in the table 1 by using the negative sample and the neutralizing antibody sample with the concentration of 20ng/ml, and screening the optimal collocation.
TABLE 1
The results of the tests of Nos. 1 to 27 are shown in FIGS. 4 to 8.
The result interpretation method refers to fig. 9. The method specifically comprises the following steps:
positive: the presence of a red or orange-red dot at the position of the mass control line (line C) and a reddish or no dot at the position of the detection line (line T) in the observation window indicates that the detection of the novel coronavirus in the sample was positive.
Negative: the presence of two colored bands in the viewing window, i.e., a red or magenta color spot at the location of the mass control line (line C) and the test line (line T), but at a color depth between the colors of the test results for standards at concentrations of 0ng/ml and 20ng/ml on the colorimetric card, indicates that the neutralizing antibodies against the novel coronavirus in the sample are negative or below the test value.
And (4) invalidation: no red dot appears on the test area or control line, or only a red dot appears on the detection line, but no red dot appears on the control line.
As can be seen from the results of fig. 4 to 8, the collocation No. 13 (35nm colloidal gold +1.5 μ L/cm RBD spray amount +1.0mg/mL ACE-2 dot film amount) is the optimal collocation, and the color development difference can be judged by naked eyes, and the other collocation differences are small. Therefore, the 13 th collocation is the optimal choice, and can be used for detecting samples with the concentration of the neutralizing antibody of 20ng/mL under the condition.
Thirdly, determining the color rendering form
(1) Immobilization of detection zone ACE-2
The same concentration of ACE-2(1.0mg/ml) was immobilized on NC membranes in three different forms, single line, multiple line and dot matrix.
(2) Matching and assembling of detection card
And combining the optimized gold-labeled pad and sample pad under the same conditions with NC films in different forms to form the detection card.
(3) Color development Effect test
And testing the assembled neutralizing antibody detection card by using a neutralizing antibody quality control product with a concentration gradient respectively to optimize an optimal color development scheme.
As can be clearly seen from the results of fig. 10, the relationship between the sensitivity of sample detection is: dot matrix > multiple lines > single line. Therefore, the invention selects a dot matrix form as a color display mode.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. The utility model provides a detect high sensitivity semi-quantitative kit of novel coronavirus neutralizing antibody, includes detection card, sample diluent and colour comparison card, its characterized in that: a gold pad is arranged in the detection card, and the gold pad contains a recombinant S-RBD antigen marked by colloidal gold; wherein the amount of the recombinant S-RBD antigen used per mL of the colloidal gold is 5. mu.g or more.
2. The high-sensitivity semi-quantitative kit for detecting neutralizing antibodies against novel coronaviruses according to claim 1, characterized in that: the particle size of the colloidal gold is 20-60 nm.
3. The high-sensitivity semi-quantitative kit for detecting neutralizing antibodies against novel coronaviruses according to claim 1, characterized in that: the gold pad comprises a glass fiber membrane, wherein the glass fiber membrane is sprayed with recombinant S-RBD antigen containing colloidal gold markers, namely RBD colloidal gold compound, and the spraying amount of the RBD colloidal gold compound is 0.5-3.0 muL/cm.
4. The high-sensitivity semi-quantitative kit for detecting neutralizing antibodies against novel coronaviruses according to claim 1, characterized in that: the detection card is characterized in that an NC membrane is further arranged in the detection card, a detection line is arranged on the NC membrane, recombinant ACE-2 protein is coated on the detection line, and the use amount of the recombinant ACE-2 protein is 0.5-5.0 mg/mL.
5. The high-sensitivity semi-quantitative kit for detecting neutralizing antibodies against novel coronaviruses according to claim 4, characterized in that: the recombinant ACE-2 protein is fixed on an NC membrane in a lattice form.
6. The high-sensitivity semi-quantitative kit for detecting neutralizing antibodies against novel coronaviruses of claims 1 to 5, characterized in that: the preparation method of the detection card comprises the steps of preparing a cellulose nitrate film, preparing a gold pad and preparing the detection card;
the preparation process of the coated nitrocellulose membrane comprises the following specific steps:
coating a quality control line and a detection line on a nitrocellulose membrane, and coating a recombinant ACE-2 protein on the detection line, wherein the use amount of the recombinant ACE-2 protein is 0.5-5.0 mg/mL; fixing the recombinant ACE-2 protein on a nitrocellulose membrane in a dot matrix form; then the coated nitrocellulose membrane is dried in an oven at 30 ℃ for standby.
7. The high-sensitivity semi-quantitative kit for detecting neutralizing antibodies against a novel coronavirus as set forth in claim 6, wherein: the preparation process of the gold pad specifically comprises the following steps:
(1) preparation of colloidal gold
A.100ml distilled water is added with chloroauric acid with two ten-thousandth of final concentration, and the mixture is heated, stirred and boiled on a magnetic stirrer;
B. adding trisodium citrate with a final concentration of three ten-thousandth into the solution, and continuously heating and stirring for 10 minutes;
C. cooling at room temperature to obtain colloidal gold; the particle size of the colloidal gold is 20-60 nm;
(2) preparation of RBD colloidal gold complexes
A. Adding colloidal gold into a centrifuge tube, and adding 50 μ l of 1% K2CO3Mixing uniformly;
B. adding the recombinant S-RBD antigen into a centrifuge tube, uniformly mixing, and incubating for 30 minutes at room temperature; the amount of the recombinant S-RBD antigen used per mL of colloidal gold is more than 5 mug;
(3) preparation of colloidal gold bars
Treating and drying the glass fiber membrane by using a gold pad treatment solution, wherein the formula of the gold pad treatment solution is as follows: 80g sucrose, 2.42g Tris, 10g BSA, 4g Tween-20 dissolved in 1L distilled water, pH 8.0; the RBD colloidal gold complex was concentrated ten times using a metal spraying instrument and then treated with a 30cm by 1cm glass fiber membrane at a spray rate of 0.5-3.0. mu.L/cm.
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