CN111378018A - Test strip for detecting novel coronavirus antibody and preparation method and application thereof - Google Patents
Test strip for detecting novel coronavirus antibody and preparation method and application thereof Download PDFInfo
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention discloses a test strip for detecting a novel coronavirus antibody, a preparation method and application thereof, wherein the test strip disclosed by the invention comprises a sample pad, a combination pad, an analysis membrane and water-absorbing filter paper which are sequentially attached to a bottom plate, the analysis membrane is provided with a detection line T and a quality control line C, the detection line T is coated with an NP antigen shown as SEQ ID NO.1, the quality control line C is coated with an anti-IgG antibody, and the test strip can be used for detecting the antibody of the novel coronavirus or evaluating the inoculation effect after injection of a vaccine.
Description
Technical Field
The invention relates to a test strip for detecting a novel coronavirus antibody, and a preparation method and application thereof.
Background
A novel coronavirus (SARS-CoV-2) belonging to β genus, which has an envelope, a particle having a circular or elliptical shape, usually a polymorphism, and a diameter of 60-140nm, and has gene characteristics significantly different from those of SARSr-CoV and MERSR-CoV.
At present, the conventional detection method for the novel coronavirus infection is mainly real-time fluorescence RT-PCR, but the detection is long in time consumption and high in reagent cost, false negative detection results can be caused due to various reasons, and the detection method has high requirements on instruments and equipment, is only suitable for laboratory detection and is not suitable for rapid low-cost detection in basic medical institutions and fields. Meanwhile, various other detection methods are also in the research stage. Such as enzyme-linked immunoassay, chemiluminescence, time-resolved fluorescence, etc., but all of the above detection means require relatively precise laboratory instruments and rely to a considerable extent on the operating experience of laboratory personnel. In the seventh edition of the latest diagnosis and treatment scheme for novel coronavirus pneumonia, a novel serum-added specific IgM antibody is used as one of etiological diagnosis standards, and IgM is generally considered as a first defense line generated in a human body after virus infection, and is an important basis and means for judging the acute stage of a disease during the rapid and accurate detection of IgM. Especially, a great number of asymptomatic infectors exist in the explosive COVID-19, which greatly improves the difficulty of accurate diagnosis by methods such as PCR and the like, so that the development and research of a detection method aiming at the novel coronavirus IgM antibody can not only make up the deficiency of nucleic acid detection and improve the diagnosis efficiency of novel coronavirus infection, but also can greatly avoid the potential risks brought to medical staff and other related staff in the collection and transportation process of nasopharyngeal swab specimens of patients or suspected patients.
At present, the immunochromatography technology is gaining greater and greater attention in a plurality of fields, such as food safety, environmental monitoring, drug screening, medical detection and the like. Although there are many mature quantitative or semi-quantitative immunoassays with high specificity and sensitivity, such as fluorescence and electrochemical luminescence, their disadvantages (complicated operation and long time-consuming detection process) cannot be ignored. Therefore, a novel point-of-care (POCT) technique with simple operation, short time consumption and strong specificity is urgently needed.
The invention obtains the novel coronavirus nucleoprotein (NP protein), combines the specific binding property of an antigen antibody with the colloidal gold immunochromatographic technique, and prepares the colloidal gold immunochromatographic test strip for rapidly detecting the novel coronavirus IgM antibody on site by adopting an indirect method. The whole detection process does not need complex instruments and equipment with skillful operation and precision, and has low cost, convenient transportation and easy storage. Therefore, the invention provides a high-efficiency and practical field detection method for POCT rapid diagnosis of the novel coronavirus IgM antibody, and has very important practical significance and wide application prospect for comprehensive prevention and control of novel coronavirus pneumonia (COVID-19).
Disclosure of Invention
In order to make up the defects of the prior art, the invention aims to provide an indirect colloidal gold immunochromatographic test strip which is simple and rapid, has strong specificity and can meet the requirement of rapidly screening novel coronavirus IgM antibodies on site in a large scale, and a preparation method and related application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the present invention provides an antigen for detecting SARS-CoV-2 antibody, the amino acid sequence of the antigen is shown in SEQ ID NO.1, or the sequence is formed by replacing, deleting or adding one or more amino acids and has the same function.
Further, the antigen is obtained by designing a primer, using a nucleic acid positive case sample as a template, carrying out RT-PCR amplification on an N gene, inserting an expression vector, introducing the N gene into an escherichia coli system for soluble expression, and carrying out purification expression by using a Ni affinity chromatography column.
In a second aspect the invention provides a product for detecting SARS-CoV-2 virus infection/antibodies against SARS-CoV-2 virus, which product comprises an antigen as described in the first aspect of the invention.
Further, the product comprises a kit, a detection card or a test strip.
Further, the test strip is an immunochromatography test strip.
Further, the immunochromatographic test strip comprises a sample pad, a binding pad, an analysis membrane, water-absorbing filter paper and a bottom plate, wherein the binding pad is attached with a specific binding protein of SARS-CoV-2 virus antibody marked by a marker, the analysis membrane is provided with a detection line T and a quality control line C, and the detection line T is coated with the antigen of the first aspect of the invention; the quality control line C is coated with an anti-IgG antibody.
Further, the sample pad is a glass fiber film, a polyester fiber film or a non-woven fabric.
Further, the combination pad is a glass fiber film or a polyester fiber film.
Further, the pH for preparing the colloidal gold is 7 to 10, and as a preferable embodiment of the present invention, the pH is 8.
Further, the specific binding protein of SARS-CoV-2 virus antibody is an anti-IgM antibody which can bind to the IgM antibody of SARS-CoV-2 virus.
Further, the anti-IgM antibody is murine anti-human IgM.
Further, the anti-IgM antibody is a mixed antibody of different anti-IgM antibodies. In particular embodiments of the invention, the murine anti-human IgM is at two different specifications and concentrations.
Further, the mixing ratio of the different anti-IgM antibodies was 1:1 (mass ratio).
Further, the amount of the labeled mouse anti-human IgM per 200. mu.L of colloidal gold is 0.75. mu.g to 7.5. mu.g, and as a preferred embodiment of the present invention, the amount of the labeled mouse anti-human IgM per 200. mu.L of colloidal gold is 1.8. mu.g.
Further, the analysis membrane is a nitrocellulose membrane; the bottom plate is a PVC bottom plate.
Furthermore, the quality control line C is goat anti-mouse IgG.
Further, the coating concentration of the quality control line C is 1.0mg/mL to 2.0mg/mL, and as a preferred embodiment of the present invention, the coating concentration of the quality control line C is 2.0 mg/mL.
Further, the antigen coating concentration of the detection line T is 0.6-1.2 mg/mL, and as a preferred embodiment of the present invention, the antigen coating concentration is 1.2 mg/mL.
When the colloidal gold immunochromatographic test strip is used for detecting novel coronavirus IgM antibodies, the detection is carried out according to the following steps:
20 mu L of serum sample to be detected is absorbed in a sample hole on the detection card by a pipette or a disposable dropper, 2-3 drops (about 70-100 mu L) of sample diluent are immediately dropped in the sample hole, no bubble is generated in the process, and the result is interpreted after 10 minutes.
And (4) judging a result:
negative results: and only a quality control line C appears, the detection line T does not develop color, which indicates that the novel coronavirus IgM antibody is not detected, and the result is negative for the novel coronavirus IgM antibody.
Positive results: and the quality control line C and the detection line T are both present, which indicates that the novel coronavirus IgM antibody is detected, and the result is positive.
Invalid result: the quality control line C does not appear, and whether the detection line T appears or not is an invalid result.
The third aspect of the invention provides a preparation method of a colloidal gold immunochromatographic test strip for detecting SARS-CoV-2 virus infection/anti-SARS-CoV-2 virus antibody, which comprises the following steps:
(1) preparation of immune colloidal gold combined pad
Preparing a colloidal gold solution by using a gold chloride solution and a trisodium citrate solution, adjusting the pH of the colloidal gold solution by using potassium carbonate, sequentially adding a specific binding protein to be labeled and a BSA solution, centrifuging to obtain a precipitate, re-suspending and precipitating a gold colloidal complex solution to obtain an immune gold colloidal complex solution, and dripping the solution on a collection pad treated by a binding pad treatment solution;
(2) preparation of analytical membranes
Diluting the antigen scribing of the first aspect of the invention with a coating buffer solution to obtain a detection line T, diluting the anti-IgG antibody scribing with the coating buffer solution to serve as a quality control line C, and blocking with a phosphate buffer solution of BSA;
(3) assembly of test strips
And sequentially adhering a sample pad, a combination pad, an analysis membrane and absorbent paper on the viscous bottom plate, and cutting the sample pad, the combination pad, the analysis membrane and the absorbent paper into test strips with proper width.
Further, the pH in the step (1) is 7 to 10, and as a preferable embodiment of the present invention, the pH is 8.
Further, the specific binding protein is an anti-IgM antibody, which in a particular embodiment of the invention is a murine anti-human IgM antibody.
Further, the amount of the labeled mouse anti-human IgM per 200. mu.L of colloidal gold is 0.75. mu.g to 7.5. mu.g, and as a preferred embodiment of the present invention, the amount of the labeled mouse anti-human IgM per 200. mu.L of colloidal gold is 1.8. mu.g.
Further, the anti-IgM antibody is a mixed antibody of different anti-IgM antibodies.
Further, the mixing ratio of the different anti-IgM antibodies was 1: 1.
Further, the immunogold complex solution was a Tris solution containing 0.1% BSA (w/v), 2% sucrose (w/v), and 0.5% Tween-20 (v/v).
Further, the conjugate pad treatment solution was 0.1% Tween-20 (v/v), 2.5% sucrose (w/v), 0.1% PEG-20000(w/v), 0.5% BSA (w/v) in PBS.
The conjugate pad of the present invention may employ a carrier conventional in the art, including but not limited to a glass fiber membrane or a polyester fiber membrane. In a preferred embodiment of the present invention, the bonding pad is a glass fiber film.
Further, the quality control line C in the step (2) is goat anti-mouse IgG.
Further, the coating concentration of the quality control line C is 1.0 mg/mL-2.0 mg/mL, and as a preferred embodiment of the present invention, the coating concentration of goat anti-mouse IgG is 2.0 mg/mL.
Further, the antigen coating concentration of the detection line T is 0.6-1.2 mg/mL, and as a preferred embodiment of the present invention, the antigen coating concentration is 1.2 mg/mL.
Further, the BSA concentration of the BSA phosphate buffer is 0.5 to 2%, and as a preferred embodiment of the present invention, the BSA concentration is 1%.
The fourth aspect of the invention provides a method for preparing a test card for detecting SARS-CoV-2 virus infection/antibody against SARS-CoV-2 virus, wherein the test strip prepared by the preparation method of the third aspect of the invention is placed in a plastic housing, wherein the sample adding hole of the plastic housing is above the sample pad, and the observation window of the plastic housing is above the detection line and the quality control line.
The fifth aspect of the invention provides a preparation method of a kit for detecting SARS-CoV-2 virus infection/antibody against SARS-CoV-2 virus, the detection card prepared by the preparation method of the fourth aspect of the invention is put into an aluminum foil bag and is packed with a sampling tube, a sample diluent and an instruction book.
A sixth aspect of the invention provides the use of any one of:
1) the antigen of the first aspect of the invention is applied to the preparation of products for detecting SARS-CoV-2 virus infection/anti-SARS-CoV-2 virus antibody/evaluating vaccination effect;
2) the product of the second aspect of the invention is applied to the preparation of a tool for detecting SARS-CoV-2 virus infection/anti-SARS-CoV-2 virus antibody/evaluating the vaccination effect;
3) the preparation method of the third aspect of the invention is applied to the preparation of test paper for detecting SARS-CoV-2 virus infection/anti-SARS-CoV-2 virus antibody/evaluating vaccination effect;
4) the preparation method of the fourth aspect of the invention is applied to the preparation of the detection card for detecting SARS-CoV-2 virus infection/anti-SARS-CoV-2 virus antibody/evaluating vaccination effect;
5) the preparation method of the fifth aspect of the invention is applied to the preparation of a kit for detecting SARS-CoV-2 virus infection/anti-SARS-CoV-2 virus antibody/evaluating vaccination effect.
The invention has the advantages and beneficial effects that:
the colloidal gold immunochromatographic test strip prepared by the invention has good specificity and stability, is convenient to store, convenient to transport, simple to operate, long in quality guarantee period and high in detection speed, is suitable for large-scale rapid detection of SARS-CoV-2-IgM in all levels of medical institutions, and provides necessary assistance for diagnosis of COVID-19 better by matching with laboratories such as PCR, CT and the like and clinical diagnosis standards, thereby having good application prospect.
Drawings
FIG. 1 is a SDS-PAGE graph showing the result of SARS-CoV-2 virus NP protein expression;
FIG. 2 is a diagram showing the detection of different NP proteins as antigens;
FIG. 3 is a TEM topographic map of colloidal gold;
FIG. 4 is a graph showing the determination of the optimum pH of a colloidal gold-labeled antibody;
FIG. 5 is a graph showing the results of the optimal labeling amount of gold-labeled antibody;
FIG. 6 is a graph showing the results of dissolution and precipitation of different redissolutions;
FIG. 7 is a graph of the sealing results for different sealing fluids;
FIG. 8 is a graph of results of treatment of a conjugate pad with different conjugate pad treatment fluids;
FIG. 9 is a graph showing the detection of antibodies at different concentrations on the control line;
FIG. 10 is a graph showing the detection of different concentrations of antigen on a detection line;
FIG. 11 is an assembly diagram of a colloidal gold immunochromatographic test strip; wherein, a is a structural diagram of the colloidal gold immunochromatographic test strip, b is a chromatography schematic diagram of a positive sample, and c is a chromatography schematic diagram of a negative sample;
FIG. 12 is a graph showing the results of the test strip specificity test;
FIG. 13 is a graph showing the results of tests performed on test strips from lot to lot;
FIG. 14 is a graph showing the results of the test strips at different time points in the same batch;
FIG. 15 is a graph showing the results of detection of clinical specimens.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.
EXAMPLE 1 preparation and purification of SARS-CoV-2-NP
The method comprises the steps of preparing a virus RNA template from an inactivated SARS-CoV-2 strain, amplifying an SARS-CoV-2 protein open reading frame, inserting a bacterium expression plasmid pET-28(a) into the virus RNA template, obtaining a bacterium expression plasmid pET-28(a) -NP., conventionally transforming the plasmid into an E.CoLi BL21 strain, inducing protein expression in broth LB at 37 ℃ by using 50 mu g/ml kanamycin and 1mM isopropyl- β -D-thiogalactopyranoside, resuspending culture particles containing recombinant proteins in chromatographic binding buffer, carrying out ultrasonic treatment, centrifuging cell lysate, loading a nickel ion affinity column with supernatant, thoroughly washing the column by using the binding buffer, eluting the recombinant 6 His-labeled NP protein from the column by using the elution buffer, analyzing the purified protein by SDS-PAGE, and observing the protein by using Coomassie brilliant blue staining.
According to the structure prediction, the 2019-nCoV NP protein can be structurally divided into two domains (domains), which are respectively called NP-NT (amino acid sequence: SEQ ID NO.1) and NP-CT (amino acid sequence: SEQ ID NO.2), and the two domains and the NP full-length protein (amino acid sequence: SEQ ID NO.3) are respectively expressed. The SDS-PAGE results (FIG. 1) also showed that three proteins (1-recombinant SARS-CoV-2 virus NP, 2-recombinant NP-CT protein, 3-recombinant NP-NT protein) were purified
Example 2 ELISA test for Effect of recombinant expression proteins binding to Virus-specific IgM
Respectively using NP geneLong protein, NP-NT and NP-NC proteins coated ELISA plates at 100ng/well, 4C overnight. After washing, the cells were blocked with 5% skimmed milk powder and kept overnight at 4 ℃. Adding 100 μ L of sample diluent into each well, adding 1 μ L of serum to be detected, mixing, incubating at 37 deg.C for 30min, washing for 5 times, adding mouse anti-human IgM-HRP (μ chain specificity) for detecting virus specific IgM, incubating at 37 deg.C for 30min, washing for 5 times, adding TMB + H2O2Substrate, 100. mu.L/well, was left at room temperature for 5 minutes, and stop buffer was added at 50. mu.L/well. OD was measured at a wavelength of 450 nm.
As shown in FIG. 2, it was found that NP-NT was the most effective in detection, and thus NP-NT was used as the T-line envelope antigen in the present invention.
EXAMPLE 3 preparation of colloidal gold assay card for SARS-CoV-2
1. Materials and reagents
1.1 Immunochromatographic Material
The cellophane film, the nitrocellulose film, the absorbent paper, the viscous master and the rapid detection plastic card are all purchased from Shanghainej, a biotechnology limited company.
1.2 preparation of the principal agent
HAuCl4Solution: 1g of HAuCl4Dissolving in 100mL of ultrapure water, filtering with a 0.22 μm filter, wrapping with tin paper, and storing at 4 deg.C in dark place.
Trisodium citrate solution: 1g of trisodium citrate powder, adding ultrapure water to 100mL, dissolving, filtering by a 0.22 μm filter, and subpackaging at 4 ℃ for storage.
BSA solution: an appropriate amount of BSA powder was weighed in percentage, added to 100ml of phosphate buffer with ph 7.4, sufficiently dissolved, and left to stand. Stored at 4 ℃ for later use, but only for a short period of time. It is best to allocate the components in proper amount, and the components are allocated as they are used.
Immune gold colloid complex solution:
A. a 0.01M pH 7.4 PBS solution containing 1% BSA (w/v), 5 w/v% sucrose, 0.5% tween-20 (v/v);
B. tris 0.06M pH 8.5 solution containing 0.1% BSA (w/v), 2% sucrose (w/v), 0.5% Tween-20 (v/v).
Gold-glue combined pad treatment fluid:
A. 0.01M pH 7.4 PBS solution containing tween-20 (v/v) 0.2%, sucrose (w/v) 5%, PEG-20000(w/v) 0.2%, BSA (w/v) 1%;
B. 0.01M PBS pH 7.4 containing Tween 0.1% Tween-20 (v/v), sucrose 2.5% (w/v), PEG-20000(w/v) 0.1%, BSA 0.5% (w/v).
Preparation of murine anti-human IgM: mouse anti-human IgM: a (cat # JYAB35, Shanghai Jie-Biotechnology Ltd.) and murine anti-human IgM: b (cat # JYAB38, Shanghai Jie-Biotechnology Co., Ltd.) dialyzed overnight, mixed at a mass ratio of 1:1, adjusted to a concentration of 3mg/ml, and centrifuged to remove protein polymer. The preparation is prepared before each experiment, can not be stored for a long time and repeatedly frozen and thawed, and is placed in an ice water bath for storage in the experiment process.
Preparation of SARS-CoV-2-NP: after overnight dialysis of SARS-CoV-2-NP, the concentration was adjusted to 1.2mg/ml and the protein polymer was removed by centrifugation. The preparation is prepared before each experiment, can not be stored for a long time and frozen and melted repeatedly, and is stored in an ice-water bath during the experiment.
2. Preparation and assembly of colloidal gold immunochromatographic test strip
2.1 preparation of aqueous solution of colloidal gold nanoparticles
1) Glassware cleaning and siliconization
Firstly, the glassware is put in concentrated H2SO4Soaking for 24H, washing for 10 times to remove residual concentrated H2SO4Then, the glassware is brushed by using a detergent, the glassware is washed by tap water for 10 times, deionized water is repeatedly washed for 5 times, ultrapure water is washed for 3-4 times, and the glassware is silicified after being dried by an oven. Taking a proper amount of silicification liquid (chloroform solution containing 5% (w/v) dimethyl dichlorosilane) in a glass ware, and slowly rotating the glass ware to enable the inner wall of the glass ware to be soaked by the silicification liquid.
2) Preparation of colloidal gold nanoparticles
Gold nanoparticles having a particle size of 30nm were prepared using a trisodium citrate reduction method. The method was established by Frene in 1973 by adding 1mL of 1% HAuCl to 100mL of ultrapure water4Heating to boil, adding 1.5ml 1% trisodium citrate solution, and heatingAfter about 10 minutes until the solution turns wine red (mechanical stirring and heating are required during the whole process until the solution boils), the solution is cooled to room temperature with water after 10 minutes, and then the volume is increased to 100ml by using a volumetric flask. Filtering and sterilizing by using a 0.22 mu m filter, and storing at 4 ℃ in a dark place.
2.2 quality determination of colloidal gold nanoparticle aqueous solution
Diluting the colloidal gold solution by a proper multiple, lightly dipping the copper mesh in the diluted colloidal gold solution, placing the solution on filter paper for natural drying, and detecting the quality of the colloidal gold particles by using a transmission electron microscope.
The result is shown in fig. 3, the size and shape of the colloidal gold particles are uniform, the particle shape is round, and no impurity or gold particle aggregation phenomenon is observed under the transmission electron microscope, which indicates that the colloidal gold solution reaches the standard and can be used for labeling the antibody.
2.3 preparation and identification of colloidal gold immunocomplexes
1) Determination of optimum pH of immune gold gel
Respectively packaging 200 μ L of the above colloidal gold nanoparticle water solution in clean glass bottles, and respectively adding 0.1M K with different volumes2CO3And (4) dissolving, adjusting the pH value of the colloidal gold solution, and testing by using a precise pH test paper. A control group was also set up. Continuously adding 6 mu g of mouse anti-human IgM mu chain into each glass bottle tube, fully and uniformly mixing, and standing for 10min at room temperature. Add 45. mu.L of 10% NaCl solution into each tube, mix well, stand for 2h, observe the results.
The results are shown in FIG. 4, in which the pH of the control group (AuNPs) was 6.2 and the pH of the experimental group (AuNPs + K)2CO3+ rat anti-human IgM mu chain) No. 2-9 are respectively 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10, and as can be seen from the figure, the pH value of the reaction system has a great influence on the coupling efficiency of the colloidal gold and the antibody to be labeled, and generally, it is more appropriate to select a pH slightly higher than the isoelectric point of the antibody to be labeled. Under the condition, the immune gold gel is most stable and can bear higher ion concentration, such as NaCl and the like, and coagulation cannot occur. In conclusion, the pH value of the EP tube which still keeps red and has the lowest value is selected as the optimum pH value of the colloidal gold labeled antibody, namely the pH value is about 8.0.
2) Determination of optimum mouse anti-human IgM mu chain label quantity
Sub-packaging 200 μ L of the above colloidal gold nanoparticle aqueous solution in a clean glass bottle, and adding 0.1M K2CO3The solution was adjusted to the above optimum pH while a control group was set up. mu.L, 0.25. mu.L, 0.5. mu.L, 1. mu.L, 1.5. mu.L, 2. mu.L, 2.5. mu.L (corresponding to 0. mu.g, 0.75. mu.g, 1.5. mu.g, 3.0. mu.g, 4.5. mu.g, 6.0. mu.g, 7.5. mu.g of antibody) of mouse-anti-human IgM mu chain were continuously added to each glass bottle, mixed well and allowed to stand at room temperature for 10 min. Add 45. mu.L 10% NaCl solution into each tube, mix well, stand for 2h, observe the result. The result judgment standard is as follows: when the protein amount reaches or exceeds the minimum stable amount of the colloidal gold, the color of the EP tube keeps unchanged red; otherwise, the colloidal gold is coagulated in the environment of high concentration electrolyte, such as NaCl, due to too small amount of antibody, so that the solution is bluish purple.
As shown in FIG. 5, when the amount of murine anti-human IgM added was 1.5. mu.g, no significant color change was observed in the color of colloidal gold, and considering that the amount of the antibody required in the course of the experiment should be slightly larger than the amount of the most suitable labeled antibody, the amount of the antibody added in the course of the experiment was enlarged to 1.2 times the above-mentioned value, i.e., 1.8. mu.g.
3) Colloidal gold labeled mouse anti-human IgM mu chain
Adjusting the pH value of the colloidal gold nanoparticle aqueous solution to be the optimum, slowly and uniformly adding mouse anti-human IgM mu chain, shaking a table at 20 ℃ for 20min at 150r/min, and standing at room temperature for 10 min. A5% BSA (w/v) solution in Tris-HCl buffer was added to a final concentration of 0.5% BSA to achieve the effect of reducing non-specific aggregation. Continuing to stir at 20 ℃ for 150r/min, shaking the shaking table for 20min, and standing at room temperature for 10 min. Centrifuging the reaction mixture solution at 2000r/min and 4 deg.C for 10min, transferring the supernatant to a new centrifuge tube, discarding the precipitate, centrifuging at 13200r/min and 4 deg.C for 20min, discarding the supernatant, redissolving the precipitate with the above redissolution, and concentrating to 1/4 of the original volume. Storing at 4 ℃ in dark for later use.
4) Selection and optimization of different IgM antibody synergistic effect in different proportions
Mouse anti-human IgM alone: A. mouse anti-human IgM: and B, mixing the two in different proportions, coupling the mixture with colloidal gold, preparing a test strip, and comparing the test effect of the test strip.
The results are shown in Table 1, combining the color development effects of the T line and the C line, when IgM: a and IgM: at a ratio of 1:1 of B, the assay exhibits a synergy, and therefore IgM is selected: a and IgM: and B, preparing the test strip by using the mixed antibody.
TABLE 1 detection Effect of different IgM antibodies
5) Selection and optimization of immunogold complex solution
Preparing immune gold complex solutions A and B with two different components, and observing the dissolution condition of the colloidal gold coupled anti-human IgM reaction system after centrifuging and removing the supernatant.
As a result, as shown in FIG. 6, the reconstituted solution A was found to be wall-hung after dissolution, and it was difficult to sufficiently dissolve the solution again, and the reconstituted solution B containing Tris was selected because it was more effective in dissolving the precipitate again.
6) Selection and optimization of nitrocellulose membrane blocking solution
And (3) blocking the nitrocellulose membrane with the marked detection T line and the marked quality control C line for 2 hours by using phosphate buffer solutions of 0.5 percent, 1.0 percent and 2.0 percent BSA respectively, washing the nitrocellulose membrane for 5 times by using PBST buffer solution, and drying for later use. After the test strip is assembled, a positive sample is used for detection. And selecting the components of the optimal confining liquid according to indexes such as chromatography speed, uniformity of liquid crawling, cleanness of background of the nitrocellulose membrane after chromatography, clearness of a detection strip and the like.
As shown in FIG. 7, the chromatographic time of the sample was 8min, 10min and 15min for three different concentrations of BSA as blocking solutions, the blocking solution containing 1% BSA had stronger effect of eliminating nonspecific binding than 0.5% BSA, and the color of the developed band was darker than that of 2% BSA, so that a phosphate buffer containing 1% BSA was selected as the nitrocellulose membrane blocking solution.
7) Selection and optimization of gold-glue combined pad treatment fluid
Preparing gold-glue bonding pad treatment solution with different components, uniformly and flatly soaking the bonding pad in the treatment solution with different components, standing at room temperature for 30min, and then placing in an oven at 37 ℃ for 12 h. And after complete drying, uniformly dropwise adding the prepared immune gold colloid, continuing to place in an oven at 37 ℃ for 1.5h, and completely drying for later use. The binding pads before and after treatment were loaded with 100. mu.L of each sample, and the binding and release effects of the different binding pads on the immunogold gel were observed.
As a result, as shown in FIG. 8, the treatment solution B for conjugate pad was more effective, and the binding and release of the gold-labeled antibody were more uniform and complete.
8) Selection and optimization of detection T line and quality control C line envelope concentration
The colloidal gold immunochromatographic test strip quality control C line is coated with goat anti-mouse IgG, and in order to determine the optimal concentration, the colloidal gold immunochromatographic test strip is diluted in a gradient manner to be 2.0mg/mL, 1.5mg/mL, 1.0mg/mL and 0.5 mg/mL; t-line coating was detected with SARS-CoV-2-NP diluted to 1.2mg/mL, 0.6mg/mL and 0.3 mg/mL. And determining the coating concentration according to the coloring condition of the T line and the C line after sample application chromatography.
The coloring condition of the quality control C line is shown in FIG. 9, and with the increase of the concentration of the goat anti-mouse IgG, the strip of the quality control C line becomes deeper and deeper, and 2.0mg/mL is selected as the concentration of the goat anti-mouse IgG on the quality control C line by comprehensively considering the definition of the observation result, the long-term storage of the test strip, the interference of factors such as temperature environment and the like.
As shown in FIG. 10, the staining of the detection line T is more and more obvious as the concentration of SARS-CoV-2-NP increases, and 1.2mg/mL is selected as the coating concentration of the detection line T in consideration of various factors such as environment, sample, and storage time.
9) Assembly of colloidal gold immunochromatographic test strip
The solid phase materials (adhesive substrate, nitrocellulose membrane (analytical membrane), sample pad, conjugate pad and absorbent pad) treated in the above steps were stuck together in an overlapping manner in the following order. The nitrocellulose membrane (analysis membrane) is fixed in the middle area of the viscous bottom lining support, the quality control line C is above, and the detection line T is below. The upper end of the treated conjugate pad was pressed against the lower end of the nitrocellulose membrane (analytical membrane) with an overlap of 1 mm. The upper end of the treated sample pad was pressed against the lower end of the conjugate pad, overlapping 2 mm. The water absorption pad is stuck on the viscous bottom lining, the upper end of the water absorption pad is flush with the viscous bottom lining, the lower end of the water absorption pad is pressed on the upper end of the nitrocellulose membrane (analysis membrane), and the water absorption pad is overlapped by 2 mm; the strip thus assembled was cut into test strips of 4mm width by a cutting slitter. The prepared and molded test strip is placed in a sealed bag, vacuumized and stored in a dry environment for later use. The assembly effect of the immunochromatographic test strip is shown in fig. 11.
10) Test strip test method and result judgment standard
20 mu L of serum sample to be detected is absorbed in a sample hole on the detection card by a pipette or a disposable dropper, 2-3 drops (about 70-100 mu L) of sample diluent are immediately dropped in the sample hole, and no bubble is generated in the process. And interpreting the result after 10 min.
Negative results: only the quality control line C appears, and the detection line T does not develop color, which indicates that SARS-CoV-2-IgM is not detected, and the result is SARS-CoV-2-IgM negative.
Positive results: the appearance of both the quality control line C and the detection line T indicates that SARS-CoV-2-IgM was detected, and the result is positive for SARS-CoV-2-IgM.
Invalid result: the quality control line C does not appear, and whether the detection line appears or not is an invalid result.
2.4 testing of colloidal gold immunochromatographic test strip Performance
1) Colloidal gold immunochromatographic test strip specificity test
Detection was performed with one new coronavirus (SARS-CoV-2) positive serum, two new bunyavirus (SFTSV) positive sera, two dengue virus (DFV) positive sera, and one healthy human serum, respectively.
The result is shown in figure 12, only SARS-CoV-2 positive serum is positive result, the rest is negative result, which shows that the colloidal gold immunochromatographic test strip has good specificity.
2) Repeatability test of colloidal gold immunochromatographic test strip
The test paper of different batches is used for carrying out batch-to-batch repeated detection on the simultaneous detection of SARS-CoV-2 positive serum and healthy human serum. SARS-CoV-2 positive serum and healthy human serum were tested repeatedly in batches at different time points using the same batch of test strips.
The results are shown in fig. 13 and 14, and the test strips in different batches and at different time points in the same batch have better repeatability.
3) Clinical serum sample testing
And collecting 19 parts of novel coronavirus positive and negative serum (the positive judgment standard is positive diagnosis of throat swab by PCR detection), and detecting by using the colloidal gold immunochromatographic test strip.
The results are shown in fig. 15 and table 2, which show that the test results of the colloidal gold immunochromatographic test strip have a sensitivity of 100%, a specificity of 93.3%, a Kappa coefficient of 0.872, a dawden coefficient of 0.933, a positive predictive value of 0.833, a negative predictive value of 100%, and a higher coincidence rate than the PCR results.
TABLE 2 comparison of test results of clinical samples
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
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Claims (10)
1. An antigen for detecting SARS-CoV-2 antibody, characterized in that, the amino acid sequence of the antigen is shown in SEQ ID NO.1, or the sequence is replaced, deleted or added with one or more amino acids to form an amino acid sequence with the same function.
2. A product for detecting SARS-CoV-2 virus infection/anti-SARS-CoV-2 virus antibodies, wherein the product comprises the antigen of claim 1, preferably the product comprises a kit, a test card or a test strip, preferably the test strip is an immunochromatographic test strip.
3. The product of claim 2, wherein the immunochromatographic test strip comprises a sample pad, a conjugate pad, an analytical membrane, a water-absorbing filter paper and a bottom plate, wherein a specific binding protein of SARS-CoV-2 virus antibody labeled with a marker is attached to the conjugate pad, a detection line T and a quality control line C are provided on the analytical membrane, and the detection line T is coated with the antigen of claim 1; the quality control line C is coated with anti-IgG antibody;
preferably, the sample pad is a glass fiber film, a polyester fiber film or a non-woven fabric;
preferably, the bonding pad is a glass fiber film or a polyester fiber film;
preferably, the marker is colloidal gold, and preferably, the diameter of the colloidal gold nanoparticle is 30 nm;
preferably, the pH for preparing the colloidal gold is 7-10, and preferably, the pH is 8;
preferably, the specific binding protein of the SARS-CoV-2 virus antibody is an anti-IgM antibody, preferably a mouse anti-human IgM, preferably, the labeling amount is 0.75 μ g to 7.5 μ g; the preferred amount of label is 1.8. mu.g;
preferably, the analytical membrane is a nitrocellulose membrane; the bottom plate is a PVC bottom plate;
preferably, the quality control line C is goat anti-mouse IgG, and preferably, the coating concentration of the quality control line C is 1.0 mg/mL-2.0 mg/mL, preferably 2.0 mg/mL;
preferably, the antigen coating concentration of the detection line T is 0.6-1.2 mg/mL, and preferably 1.2 mg/mL.
4. The product according to claim 3, wherein the anti-IgM antibodies are a mixture of different anti-IgM antibodies, preferably in a ratio of 1: 1.
5. A preparation method of a colloidal gold immunochromatographic test strip for detecting SARS-CoV-2 virus infection/antibody of SARS-CoV-2 virus is characterized by comprising the following steps:
(1) preparation of immune colloidal gold combined pad
Preparing a colloidal gold solution by using a gold chloride solution and a trisodium citrate solution, adjusting the pH of the colloidal gold solution by using potassium carbonate, sequentially adding a specific binding protein to be labeled and a BSA solution, centrifuging to obtain a precipitate, re-suspending the precipitate in a gold colloid re-solution to obtain an immune gold colloid compound solution, and dripping the solution on a collection pad treated by a binding pad treatment solution;
(2) preparation of analytical membranes
Diluting the antigen of claim 1 with a coating buffer to obtain a detection line T, diluting anti-IgG antibody with a coating buffer to obtain a line as a quality control line C, and blocking with a phosphate buffer solution of BSA;
(3) assembly of test strips
And sequentially adhering a sample pad, a combination pad, an analysis membrane and absorbent paper on the viscous bottom plate, and cutting the sample pad, the combination pad, the analysis membrane and the absorbent paper into test strips with proper width.
6. The method according to claim 5, wherein the pH in the step (1) is 7 to 10, preferably 8;
preferably, the specific binding protein is an anti-IgM antibody, preferably a mouse anti-human IgM, preferably, the labeling amount is 0.75 μ g to 7.5 μ g; preferably, the amount of label is 1.8 μ g; preferably, the anti-IgM antibodies are mixed antibodies of different anti-IgM antibodies, preferably, the mixing ratio is 1: 1;
preferably, the immunogold complexing solution is a Tris solution of 0.1% BSA (w/v), 2% sucrose (w/v), 0.5% Tween-20 (v/v);
preferably, the conjugate pad treatment solution is a PBS solution of 0.1% Tween-20 (v/v), 2.5% sucrose (w/v), 0.1% PEG-20000(w/v), 0.5% BSA (w/v);
preferably, the conjugate pad is a glass fiber film or a polyester fiber film, and preferably, the conjugate pad is a glass fiber film.
7. The method according to claim 5, wherein the quality control line C in step (2) is goat anti-mouse IgG, preferably, the coating concentration of the quality control line C is 1.0 mg/mL-2.0 mg/mL, preferably 2.0 mg/mL;
preferably, the antigen coating concentration of the detection line T is 0.6-1.2 mg/mL, and preferably 1.2 mg/mL.
Preferably, the BSA concentration of the BSA phosphate buffer is 0.5-2%, and preferably, the BSA concentration is 1%.
8. A method for preparing a test card for detecting SARS-CoV-2 virus infection/anti-SARS-CoV-2 virus antibodies, comprising placing a test strip prepared by the method of any one of claims 5 to 7 in a plastic housing, wherein the sample application hole of the plastic housing is above the sample pad, and the observation window of the plastic housing is above the detection line and the quality control line.
9. A method for preparing a kit for detecting SARS-CoV-2 virus infection/antibody against SARS-CoV-2 virus, which comprises placing the detection card prepared by the method of claim 8 into an aluminum foil bag, and packaging with a sampling tube, a sample diluent, and an instruction sheet.
10. Use according to any one of the following:
1) use of the antigen of claim 1 in the preparation of a product for detecting SARS-CoV-2 virus infection/anti-SARS-CoV-2 virus antibody/evaluating vaccination effect;
2) use of the product of any of claims 2-4 for the preparation of a means for detecting infection by/antibodies against SARS-CoV-2 virus/evaluating the effectiveness of vaccination;
3) use of the preparation method of any one of claims 5 to 7 for the preparation of a test strip for detecting SARS-CoV-2 virus infection/antibodies against SARS-CoV-2 virus/evaluation of vaccination effect;
4) the use of the preparation method of claim 8 for preparing a detection card for detecting SARS-CoV-2 virus infection/anti-SARS-CoV-2 virus antibody/evaluating vaccination effect;
5) the use of the method of claim 9 in the preparation of a kit for detecting SARS-CoV-2 virus infection/anti-SARS-CoV-2 virus antibody/evaluating vaccination effect.
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| CN111378018B (en) | 2021-01-19 |
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