CN113702643B - Device for combined detection of novel coronavirus neutralizing antibody and nucleocapsid protein antibody - Google Patents
Device for combined detection of novel coronavirus neutralizing antibody and nucleocapsid protein antibody Download PDFInfo
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Abstract
The invention relates to the technical field of immunodiagnosis and discloses a device for combined detection of a novel coronavirus neutralizing antibody and a nucleocapsid protein antibody. The device comprises a reagent strip; the reagent strip comprises a bottom plate, and a sample pad, a marking pad and an antigen-coated nitrocellulose membrane which are arranged on the bottom plate; the labeling pad is coated with an anti-human IgG antibody-colloidal gold conjugate; the antigen-coated nitrocellulose membrane comprises a nitrocellulose membrane, and a T1 detection line and a T2 detection line which are arranged on the nitrocellulose membrane, wherein the T1 detection line is coated with an S-RBD antigen, and the T2 detection line is coated with a novel coronavirus nucleocapsid protein antigen. The device can realize the combined detection of the S-RBD antibody and the new coronavirus nucleocapsid protein antibody, can detect whether an individual has the capacity of preventing the new coronavirus or whether the individual is infected with the new coronavirus or not.
Description
Technical Field
The invention relates to the technical field of immunodiagnosis, in particular to a device for combined detection of a novel coronavirus neutralizing antibody and a nucleocapsid protein antibody.
Background
The novel coronavirus pneumonia (new coronavirus pneumonia for short, COVID-19 for short) is an acute respiratory infectious disease caused by infection of the novel coronavirus (new coronavirus for short, SARS-CoV-2 for short). At present, patients infected with the novel coronavirus are the main source of infection; asymptomatic infected persons may also be the source of infection. According to the current epidemiological investigation, the incubation period is 1 to 14 days, and is mostly 3 to 7 days. It is manifested as fever, asthenia, dry cough, nasal obstruction, nasal discharge, sore throat, myalgia and diarrhea in a few cases.
The genomic organization and expression of all coronaviruses are similar, with 16 nonstructural proteins (nsp1 to nsp16) encoded by the Open Reading Frame (ORF)1a/b at the 5' end, followed by the structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N). The 3' end is encoded by the other orf. The virus enters the host cell through binding of the S protein Receptor Binding Domain (RBD) to the target cell, particularly to the angiotensin converting enzyme 2(ACE2) receptor on the respiratory epithelial cells of the host. After SARS-CoV-2 infection, the host typically develops an immune response to the virus.
Neutralizing antibodies are antibodies that mimic pathogen invasion, and elicit an immune response after entering the body, promoting the immune system in the body to produce protection. If a new corona vaccine is inoculated, a new corona virus neutralizing antibody is generated in a human body, the neutralizing antibody is combined to an S-RBD epitope, the new corona virus is prevented from being combined with ACE2, so that a pathogen is prevented from infecting cells, and meanwhile, memory B cells and memory T cells are rapidly activated to start secondary reaction, and an infection source is eliminated before serious diseases are caused. In addition, when a pathogen first infects the body, unvaccinated people produce natural B and T cells that can interact with the pathogen, but in small numbers, and cause the person to become ill until the body itself can produce enough antibodies and effector T cells to clear the pathogen that proliferates in the body, a defense mechanism against the pathogen is established.
At present, in the field of in vitro diagnostic reagents, diagnosis aiming at a neutralizing antibody and a nucleocapsid protein antibody is emphasized, for example, patent CN202110119850.3 discloses a novel coronavirus neutralizing antibody detection test paper, and patent CN202010126261.3 discloses a novel coronavirus antigen colloidal gold rapid diagnostic kit and a preparation method thereof, which can be used for detecting the nucleocapsid protein of the novel coronavirus. In order to determine whether an individual has the ability to prevent infection with new coronavirus, whether the new coronavirus vaccine is successfully inoculated and whether the individual is infected with the new coronavirus needs to be comprehensively considered, so that combined detection of the neutralizing antibody and the nucleocapsid protein antibody of the new coronavirus is needed.
Disclosure of Invention
In order to solve the technical problems, the invention provides a device for combined detection of novel coronavirus neutralizing antibodies and nucleocapsid protein antibodies. The device can jointly detect the S-RBD antibody and the new coronavirus nucleocapsid protein antibody, can detect whether an individual has the capacity of preventing the new coronavirus or whether the individual is infected with the new coronavirus or not.
The specific technical scheme of the invention is as follows:
in a first aspect, the invention provides a device for combined detection of novel coronavirus neutralizing antibodies and nucleocapsid protein antibodies, which comprises a reagent strip; the reagent strip comprises a bottom plate, and a sample pad, a marking pad and an antigen-coated nitrocellulose membrane which are arranged on the bottom plate; the labeling pad is coated with an anti-human IgG antibody-colloidal gold conjugate; the antigen-coated nitrocellulose membrane comprises a nitrocellulose membrane, and a T1 detection line and a T2 detection line which are arranged on the nitrocellulose membrane, wherein the T1 detection line is coated with an S-RBD antigen, and the T2 detection line is coated with a novel coronavirus nucleocapsid protein antigen; the lower surface of the upstream end of the sample pad is connected with the upper surface of the bottom plate, the lower surface of the downstream end of the sample pad is connected with the upper surface of the upstream end of the marking pad, and the lower surface of the downstream end of the marking pad is connected with the upper surface of the upstream end of the nitrocellulose membrane.
When a sample to be detected flows through the marking pad, the novel coronavirus neutralizing antibody (S-RBD IgG antibody) and the nucleocapsid protein antibody can be combined with the anti-human IgG antibody-colloidal gold conjugate to form a complex, and the complex formed by the neutralizing antibody can be specifically combined with the S-RBD antigen on the T1 detection line, so that a mauve strip is displayed on the T1 detection line; the complex formed by the nucleocapsid protein antibody can be specifically combined with the novel coronavirus nucleocapsid protein antigen on the T2 detection line, so that a purple red strip is displayed on the T2 detection line. Therefore, by observing the color development conditions of the T1 detection line and the T2 detection line, whether a sample to be detected contains a novel coronavirus neutralizing antibody and a nucleocapsid protein antibody or not can be judged, the combined detection of the two antibodies is realized, and whether an individual has the capacity of preventing a new coronavirus or not and whether the individual is infected with the new coronavirus or not can be judged.
In addition, the device provided by the invention is used for carrying out combined detection on the novel coronavirus neutralizing antibody and the nucleocapsid protein antibody, the detection result is relatively intuitive, the detection result can be judged and read through the depth of a purple red strip, the operation is simple, an additional instrument is not needed, the detection time is short, the efficiency is high, and the detection result can be obtained only by 15-20 min.
Preferably, the bottom plate is also provided with absorbent paper; the upper surface of the downstream end of the nitrocellulose membrane is connected with the lower surface of the upstream end of the absorbent paper, and the lower surface of the downstream end of the absorbent paper is connected with the bottom plate.
Preferably, a quality control line C is further arranged on the marking pad; the quality control line C is arranged at the downstream of the T1 detection line and the T2 detection line; the quality control line C is coated with a goat anti-mouse IgG antibody; the labeling pad is also coated with an anti-mouse IgG antibody-colloidal gold conjugate; mouse anti-erythrocyte antibodies are adsorbed on the sample pad.
The sample to be tested is mixed with a mouse anti-erythrocyte antibody (RBC) on a sample pad, after the sample pad is marked, the RBC and the anti-mouse IgG antibody-colloidal gold conjugate form a compound, and the compound is combined with the goat anti-mouse IgG antibody after reaching a C quality control line, so that a mauve strip is displayed on the C quality control line. By observing the color development condition of the C quality control line, whether enough samples reach the T1 and T2 detection lines or not can be judged, whether the chromatography process is normal or not can be judged, and meanwhile, the C quality control line can also be used as the internal control standard of the reagent.
Preferably, the nitrocellulose membrane (1.4.1) is a modified nitrocellulose membrane; modified polydopamine is crosslinked on a T1 detection line, a T2 detection line and a C quality control line of the modified cellulose nitrate membrane, and the modified polydopamine is polydopamine with a dimercaptosuccinic acid-glycerol copolymer grafted on hydroxyl groups.
The poly-dopamine contains a large amount of amino and hydroxyl, after the hydroxyl is grafted with dimercaptosuccinic acid-glycerol copolymer, the content of the hydroxyl and the carboxyl can be further improved, and the amino, the hydroxyl and the carboxyl can be utilized to improve the adsorption capacity of the nitrocellulose membrane to protein, so that the adsorption capacity of S-RBD antigen, novel coronavirus nucleocapsid protein antigen and goat anti-mouse IgG antibody on the membrane is improved, and the device disclosed by the invention has higher detection sensitivity.
In addition, the invention only modifies the T1 detection line, the T2 detection line and the C quality control line, and compared with the modification of the whole nitrocellulose membrane, the modification mode of the invention can avoid the phenomenon that the adsorption capacity of the membrane at the positions of the non-detection line and the quality control line to protein is improved, so that a large amount of antibody is adsorbed by the membrane when the antibody does not reach the detection line, and the detection accuracy and the sensitivity of the device are reduced.
Further, the preparation method of the modified nitrocellulose membrane is as follows:
(I) adding dopamine hydrochloride into a Tris buffer solution with the pH value of 8.0-8.5, and uniformly mixing to obtain a dopamine Tris buffer solution; spraying a dopamine Tris buffer solution on a T1 detection line, a T2 detection line and a C quality control line of an unmodified nitrocellulose membrane, standing for 4-6 hours at 20-30 ℃, and drying to obtain a polydopamine modified nitrocellulose membrane;
in the step (I), after the dopamine Tris buffer solution is sprayed on the nitrocellulose membrane, dopamine is subjected to polymerization reaction on the surface layer and the position close to the surface layer of the membrane and forms cross-linking with nitrocellulose, amino and hydroxyl in the polydopamine can improve the adsorption capacity of the membrane on protein, and meanwhile, the hydroxyl can provide a grafting site for the 2, 3-dimercaptosuccinic acid-glycerol copolymer.
(II) adding 2, 3-dimercaptosuccinic acid and glycerol into water, and uniformly mixing to obtain a modifier solution; spraying the modifier solution onto a T1 detection line, a T2 detection line and a C quality control line of the polydopamine modified nitrocellulose membrane, standing at 20-30 ℃ for reaction for 5-6 h, and drying to obtain a modified nitrocellulose membrane;
in the step (II), the 2, 3-dimercaptosuccinic acid-glycerol copolymer can be grafted to the hydroxyl of the polydopamine by utilizing the reaction between sulfydryl and hydroxyl, and a large amount of hydroxyl and carboxyl can be introduced on a T1 detection line, a T2 detection line and a C quality control line by utilizing the higher branching degree and the larger carboxyl and hydroxyl content of the copolymer, so that the adsorption capacity of the S-RBD antigen, the novel coronavirus nucleocapsid protein antigen and the goat anti-mouse IgG antibody is improved.
Further, in the step (I), the mass-to-volume ratio of the dopamine hydrochloride to the Tris buffer solution is 0.8-1.0 mg:1mL, and the spraying amount of the dopamine Tris buffer solution to a T1 detection line, a T2 detection line and a C quality control line is 4-5 muL/cm.
The spraying amount of the dopamine Tris buffer solution on the nitrocellulose membrane can influence the detection of the antibody, and when the spraying amount is too low, too little polydopamine is introduced on the nitrocellulose membrane, so that less amino, hydroxyl and carboxyl are introduced, and the improvement effect on the detection sensitivity is poor; when the spraying amount is too high, the polydopamine introduced on the nitrocellulose membrane is too much, so that the membrane aperture is reduced, more complexes of the nucleocapsid protein antibody and the anti-human IgG antibody-colloidal gold conjugate are retained at a T1 detection line, and the detection sensitivity of the nucleocapsid protein antibody at a T2 detection line is reduced.
Further, in the step (III), the mass-to-volume ratio of the 2, 3-dimercaptosuccinic acid to the glycerol to the water is 1mg: 0.7-1.3 mg: 0.8-1.2 mL, and the amount of the modifier solution sprayed on the T1 detection line, the T2 detection line and the C quality control line is 5.5-6.5 muL/cm.
Preferably, the preparation method of the antigen-coated nitrocellulose membrane is as follows: spraying an S-RBD antigen solution with the concentration of 0.50-0.55 mg/mL onto a T1 detection line in an amount of 1.0-1.3 muL/cm, spraying a novel coronavirus nucleocapsid protein antigen solution with the concentration of 0.20-0.25 mg/mL onto a T2 detection line in an amount of 1.0-1.3 muL/cm, spraying a goat anti-mouse IgG antibody solution with the concentration of 0.8-1.2 mg/mL onto a C quality control line in an amount of 1.0-1.3 muL/cm, and drying to obtain the antigen-coated nitrocellulose membrane.
In a second aspect, the present invention provides a method for performing combined detection of novel coronavirus neutralizing antibodies and nucleocapsid protein antibodies by using the device, comprising the following steps:
(1) dropwise adding a sample to be detected onto a sample pad, dropwise adding 75-85 mu L of buffer liquid onto the upstream of the sample dropwise adding position on the sample pad, and standing;
(2) observing the experimental result, and judging whether the sample to be detected contains the novel coronavirus neutralizing antibody and the nucleocapsid protein antibody according to the color development condition on the nitrocellulose membrane.
Preferably, in the step (1), the buffer solution contains Na with the concentration of 4.5-5.5 g/L, 2.8-3.3 g/L and 0.02-0.03 wt% respectively2HPO4NaCl, sodium caseinate (Casein-Na) and Proclin300, wherein the solvent is water, and the pH value is 7.3-7.5.
Preferably, in the step (1), the sample to be tested is a serum or plasma sample, and the volume is 10-13 μ L.
Preferably, in the step (1), the sample to be tested is a whole blood sample, and the volume of the whole blood sample is 18-23 μ L.
Compared with the prior art, the invention has the following advantages:
(1) by adopting the device, the combined detection of the novel coronavirus neutralizing antibody and the nucleocapsid protein antibody can be realized, so that whether an individual has the capacity of preventing the new coronavirus and whether the individual is infected with the new coronavirus is judged;
(2) the cellulose nitrate membrane is modified by the polydidopamine grafted and modified by the dimercaptosuccinic acid-glycerol copolymer, so that the adsorption capacity of S-RBD antigens, novel coronavirus nucleocapsid protein antigens and goat anti-mouse IgG antibodies on a T1 detection line, a T2 detection line and a C quality control line can be improved, and the detection sensitivity is improved.
Drawings
FIG. 1 is a schematic diagram of one configuration of a reagent strip in the apparatus of the present invention;
FIG. 2 is a top view of the apparatus of the present invention;
FIG. 3 is a schematic view of a process for testing a whole blood, serum or plasma sample using the device of the present invention;
FIG. 4 shows the results of measurement of the serum specimen in application example 1; FIGS. 4(A) to 4(D) are the results of detection of serum samples 1 to 4, respectively;
FIG. 5 shows the results of measurement of a plasma sample in application example 2; FIGS. 5(A) to 5(D) are the results of detection of plasma specimens 1 to 4, respectively;
FIG. 6 shows the results of measurement of a whole blood sample in application example 3; FIGS. 6(A) to 6(D) show the results of the detection of the whole blood samples 1 to 4, respectively.
The reference signs are: the kit comprises a reagent strip 1, a bottom plate 1.1, a sample pad 1.2, a label pad 1.3, an antigen-coated nitrocellulose membrane 1.4, a nitrocellulose membrane 1.4.1, a T1 detection line 1.4.2, a T2 detection line 1.4.3, a C quality control line 1.4, absorbent paper 1.5, a sample adding hole 2, a buffer adding hole 3 and a result observation window 4.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: device for combined detection of novel coronavirus neutralizing antibody and nucleocapsid protein antibody
A device for combined detection of novel coronavirus neutralizing antibodies and nucleocapsid protein antibodies comprises a reagent strip 1 and a shell.
As shown in figure 1, the reagent strip 1 comprises a bottom plate 1.1 made of PVC material, and a sample pad 1.2, a marking pad 1.3, an antigen-coated nitrocellulose membrane 1.4 and absorbent paper 1.5 which are arranged on the bottom plate. Mouse anti-erythrocyte antibodies are adsorbed on the sample pad 1.2. The label pad 1.3 is coated with an anti-human IgG antibody-colloidal gold conjugate and an anti-mouse IgG antibody-colloidal gold conjugate. The antigen-coated nitrocellulose membrane 1.4 comprises a nitrocellulose membrane 1.4.1, and a T1 detection line 1.4.2, a T2 detection line 1.4.3 and a C quality control line 1.4.4 which are arranged on the nitrocellulose membrane 1.4.1; the quality control line C1.4.4 is arranged at the downstream of the T1 detection line 1.4.2 and the T2 detection line 1.4.3; S-RBD antigen is coated on the T1 detection line 1.4.2, novel coronavirus nucleocapsid protein antigen is coated on the T2 detection line 1.4.3, and goat anti-mouse IgG antibody is coated on the C quality control line 1.4.4. The upper surface of sample pad 1.2's upstream end lower surface links to each other with bottom plate 1.1's upper surface, the lower surface of the downstream end of sample pad 1.2 links to each other with the upper surface of the upstream end of marker pad 1.3, the lower surface of the downstream end of marker pad 1.3 links to each other with nitrocellulose membrane 1.4.1's upstream end upper surface, nitrocellulose membrane 1.4.1's downstream end upper surface links to each other with the upper surface of the upstream end of absorbent paper 1.5, absorbent paper 1.5's downstream end lower surface links to each other with bottom plate 1.1.
The casing includes plastic casing and lower plastic casing, go up plastic casing and lower plastic casing and combine closely and fix through fixed arch and fixed recess, the reagent strip is located in the casing. As shown in fig. 2, a sample well 2, a buffer solution well 3 and a result observation window 4 are arranged on the upper plastic shell; the sample adding hole 2 and the buffer solution adding hole 3 are arranged above the sample pad 1.2, and the sample adding hole 2 is positioned at one side of the buffer solution adding hole 3 close to the marking pad 1.3.
The device was prepared by the following steps:
(1) preparation of antigen-coated nitrocellulose membrane: spraying an S-RBD antigen solution with the concentration of 0.50mg/mL onto a T1 detection line in an amount of 1.0 muL/cm, spraying a novel coronavirus nucleocapsid protein antigen solution with the concentration of 0.20mg/mL onto a T2 detection line in an amount of 1.0 muL/cm, spraying a goat anti-mouse IgG antibody solution with the concentration of 1.0mg/mL onto a C quality control line in an amount of 1.0 muL/cm, and drying to obtain an antigen-coated nitrocellulose membrane;
(2) preparing a marking pad: the whole process is carried out in a constant temperature heating system at 94 ℃, and trisodium citrate aqueous solution is added into chloroauric acid aqueous solution to obtain colloidal gold; by K2CO3After the pH value of the colloidal gold is adjusted to 10 by the solution, adding an anti-human IgG antibody, adding a BSA aqueous solution, continuously stirring, adding PEG20000, continuously stirring for 10min, centrifuging at 4 ℃ and 10000rpm for 15min, and removing the supernatant to obtain an anti-human IgG antibody-colloidal gold conjugate; replacing the anti-human IgG antibody with an anti-mouse IgG antibody, and obtaining an anti-mouse IgG antibody-colloidal gold conjugate by the same method; respectively diluting the anti-human IgG antibody-colloidal gold conjugate and the anti-mouse IgG antibody-colloidal gold conjugate to optical densities of OD60 and OD5, coating the diluted conjugates on a polyester film, and drying the conjugates for 24 hours to obtain a label pad;
(3) preparation of sample pad: preparing a mixed solution containing 6g/L Tris, 4.8g/L sodium caseinate, 9.5g/L polyvinylpyrrolidone, 3g/L Tween-20, 3g/L Tetronic, 0.1mg/mL blocking agent, 1/50 mouse-resistant RBC and 0.02 wt% of Proclin300 by using water as a solvent, adjusting the pH of the mixed solution to 8.4, spraying the mixed solution onto glass fibers, and drying to obtain a sample pad;
(4) preparing a reagent strip: and assembling the sample pad, the marking pad, the nitrocellulose membrane, the absorbent paper and the PVC bottom plate to obtain the device for jointly detecting the novel coronavirus neutralizing antibody and the nucleocapsid protein antibody.
Example 2: device for combined detection of novel coronavirus neutralizing antibody and nucleocapsid protein antibody
This example differs from example 1 only in that the device was prepared by the following steps:
(1) preparation of antigen-coated nitrocellulose membrane: spraying an S-RBD antigen solution with the concentration of 0.55mg/mL onto a T1 detection line in an amount of 1.3 muL/cm, spraying a novel coronavirus nucleocapsid protein antigen solution with the concentration of 0.25mg/mL onto a T2 detection line in an amount of 1.3 muL/cm, spraying a goat anti-mouse IgG antibody solution with the concentration of 1.5mg/mL onto a C quality control line in an amount of 1.0 muL/cm, and drying to obtain an antigen-coated nitrocellulose membrane;
(2) preparing a marking pad: the whole process is carried out in a constant temperature heating system at 96 ℃, trisodium citrate aqueous solution is added into chloroauric acid aqueous solution to obtain colloidal gold; by K2CO3Adjusting the pH value of the colloidal gold to 10.1 by using the solution, adding an anti-human IgG antibody, adding a BSA (bovine serum albumin) aqueous solution, continuously stirring, adding PEG20000, continuously stirring for 15min, centrifuging at 6 ℃ and 9500rpm for 10min, and removing the supernatant to obtain an anti-human IgG antibody-colloidal gold conjugate; replacing the anti-human IgG antibody with an anti-mouse IgG antibody, and obtaining an anti-mouse IgG antibody-colloidal gold conjugate by the same method; respectively diluting the anti-human IgG antibody-colloidal gold conjugate and the anti-mouse IgG antibody-colloidal gold conjugate to optical densities of OD60 and OD5, coating the diluted conjugates on a polyester film, and drying the conjugates for 20 hours to obtain a label pad;
(3) preparation of sample pad: preparing a mixed solution containing 6g/L Tris, 4.8g/L sodium caseinate, 9.5g/L polyvinylpyrrolidone, 3g/L Tween-20, 3g/L Tetronic, 0.1mg/mL blocking agent, 1/50 mouse-resistant RBC and 0.02 wt% of Proclin300 by using water as a solvent, adjusting the pH of the mixed solution to 8.6, spraying the mixed solution onto glass fibers, and drying to obtain a sample pad;
(4) preparing a reagent strip: and assembling the sample pad, the marking pad, the nitrocellulose membrane, the absorbent paper and the PVC bottom plate to obtain the device for jointly detecting the novel coronavirus neutralizing antibody and the nucleocapsid protein antibody.
Example 3: the device for combined detection of novel coronavirus neutralizing antibody and nucleocapsid protein antibody only differs from example 1 in that the nitrocellulose membrane is a modified nitrocellulose membrane and is prepared by the following steps: (I) dissolving trihydroxyaminomethane in water, wherein the mass-volume ratio of the trihydroxyaminomethane to the water is 1.2mg/mL, and adjusting the pH value to 8.0 to obtain a Tris buffer solution; adding dopamine hydrochloride into a Tris buffer solution, wherein the mass-volume ratio of the dopamine hydrochloride to the Tris buffer solution is 0.8mg:1mL, and uniformly mixing to obtain a dopamine Tris buffer solution;
(II) spraying a dopamine Tris buffer solution onto a T1 detection line, a T2 detection line and a C quality control line of the unmodified nitrocellulose membrane in an amount of 5 mu L/cm, standing for 4h at 25 ℃, and drying to obtain the polydopamine modified nitrocellulose membrane;
(III) adding 2, 3-dimercaptosuccinic acid and glycerol into water, wherein the mass-volume ratio of the 2, 3-dimercaptosuccinic acid to the glycerol to the water is 1mg:0.6mg:0.8mL, and uniformly mixing to obtain a modifier solution;
(IV) spraying the modifier solution onto a T1 detection line, a T2 detection line and a C quality control line of the polydopamine modified nitrocellulose membrane in an amount of 5.5 mu L/cm, standing and reacting for 5h at 25 ℃, and drying to obtain the modified nitrocellulose membrane.
Example 4: the device for combined detection of novel coronavirus neutralizing antibody and nucleocapsid protein antibody only differs from example 1 in that the nitrocellulose membrane is a modified nitrocellulose membrane and is prepared by the following steps: (I) dissolving trihydroxyaminomethane in water, wherein the mass-volume ratio of the trihydroxyaminomethane to the water is 1.6mg/mL, and adjusting the pH value to 8.5 to obtain a Tris buffer solution; adding dopamine hydrochloride into a Tris buffer solution, wherein the mass-volume ratio of the dopamine hydrochloride to the Tris buffer solution is 1.0mg:1mL, and uniformly mixing to obtain a dopamine Tris buffer solution;
(II) spraying a dopamine Tris buffer solution on a T1 detection line, a T2 detection line and a C quality control line of the unmodified nitrocellulose membrane in an amount of 4 mu L/cm, standing for 6h at 25 ℃, and drying to obtain the polydopamine modified nitrocellulose membrane;
(III) adding 2, 3-dimercaptosuccinic acid and glycerol into water, wherein the mass-volume ratio of the 2, 3-dimercaptosuccinic acid to the glycerol to the water is 1mg:0.8mg:1.2mL, and uniformly mixing to obtain a modifier solution;
(IV) spraying the modifier solution onto a T1 detection line, a T2 detection line and a C quality control line of the polydopamine modified nitrocellulose membrane in an amount of 6.5 mu L/cm, standing and reacting for 6h at 25 ℃, and drying to obtain the modified nitrocellulose membrane.
Comparative example 1
This comparative example differs from example 3 only in that steps (III) to (IV) are not carried out.
Comparative example 2
This comparative example differs from example 3 only in that the sprayed amount of dopamine Tris buffer in step (II) was 7. mu.L/cm.
Comparative example 3
This comparative example differs from example 4 only in that in step (II), the sprayed amount of dopamine Tris buffer was 2. mu.L/cm.
Application example 1: the device in example 1 is used for combined detection of the novel coronavirus neutralizing antibody and the nucleocapsid protein antibody in the serum sample, as shown in fig. 3, the method specifically comprises the following steps:
(1) using water as solvent to prepare a solution containing 5g/L of Na2HPO4Adjusting the pH value to 7.4 to obtain a buffer solution by using a solution of 5g/L NaCl, 3g/L Casein-Na and 0.02 wt% of Proclin 300;
(2) sucking 10 mu L of serum specimen, and dripping the serum specimen from the sample adding hole onto the sample pad; sucking 80 mu L of buffer solution, dripping the buffer solution from the buffer solution adding hole onto the sample pad, and standing for 15 min;
(3) and observing the color development conditions of the T1 detection line, the T2 detection line and the C quality control line through a result observation window:
case 1: if a mauve strip appears at the T1 detection line and the C quality control line and no mauve strip appears at the T2 detection line, the serum specimen contains a new coronavirus neutralizing antibody and does not contain a new coronavirus nucleocapsid protein antibody;
case 2: if no mauve strip exists at the T1 detection line and mauve strips appear at the T2 detection line and the C quality control line, the serum specimen does not contain a new coronavirus neutralizing antibody and contains a new coronavirus nucleocapsid protein antibody;
case 3: if purple red bands appear at the detection lines of T1 and T2 and the quality control line of C, the serum sample contains a new coronavirus neutralizing antibody and a nucleocapsid protein antibody;
case 4: if no mauve strip exists at the detection lines of T1 and T2, and a mauve strip appears at the quality control line of C, the serum sample does not contain a new coronavirus neutralizing antibody and a nucleocapsid protein antibody;
case 5: if the mauve strip is not displayed on the C quality control line, the test strip is invalid, and the problems of insufficient sample amount or abnormal chromatography process and the like may exist.
The results of the detection of the serum samples 1 to 4 by the above-described method are shown in FIGS. 4(A) to (D), respectively. As can be seen from the figure: serum samples 1 and 4 do not contain neutralizing antibodies and nucleocapsid protein antibodies; serum sample 2 contains only nucleocapsid protein antibodies; serum sample 3 contains antibodies to nucleocapsid protein and neutralizing antibodies.
Application example 2: plasma specimen detection
The device in example 2 is used to perform combined detection on the novel coronavirus neutralizing antibody and the nucleocapsid protein antibody in the plasma sample, as shown in fig. 3, and specifically comprises the following steps:
(1) using water as solvent to prepare a solution containing 5g/L of Na2HPO4Adjusting the pH value to 7.4 to obtain a buffer solution by using a solution of 5g/L NaCl, 3g/L Casein-Na and 0.02 wt% of Proclin 300;
(2) sucking 10 mu L of plasma specimen, and dripping the plasma specimen from the sample adding hole onto the sample pad; sucking 80 mu L of buffer solution, dripping the buffer solution from the buffer solution adding hole onto the sample pad, and standing for 15 min;
(3) and observing the color development conditions of the T1 detection line, the T2 detection line and the C quality control line through a result observation window:
case 1: if a mauve strip appears at the T1 detection line and the C quality control line and no mauve strip appears at the T2 detection line, the plasma specimen contains a new coronavirus neutralizing antibody and does not contain a new coronavirus nucleocapsid protein antibody;
case 2: if no mauve strip exists at the T1 detection line and mauve strips appear at the T2 detection line and the C quality control line, the plasma specimen does not contain a new coronavirus neutralizing antibody and contains a new coronavirus nucleocapsid protein antibody;
case 3: if purple red bands appear at the detection lines of T1 and T2 and the quality control line of C, the plasma specimen contains a new coronavirus neutralizing antibody and a nucleocapsid protein antibody;
case 4: if no mauve strip exists at the detection lines of T1 and T2, and a mauve strip appears at the quality control line of C, the plasma specimen does not contain a new coronavirus neutralizing antibody and a nucleocapsid protein antibody;
case 5: if the mauve strip is not displayed on the C quality control line, the test strip is invalid, and the problems of insufficient sample amount or abnormal chromatography process and the like may exist.
The results of the detection of the plasma samples 1 to 4 by the above-described method are shown in FIGS. 5(A) to (D), respectively. As can be seen from the figure: plasma samples 1 and 4 do not contain neutralizing antibodies and nucleocapsid protein antibodies; plasma sample 2 contains only nucleocapsid protein antibodies; plasma sample 3 contains antibodies to nucleocapsid protein and neutralizing antibodies.
Application example 3: whole blood sample testing
The device in example 1 is used to perform combined detection of the novel coronavirus neutralizing antibody and the nucleocapsid protein antibody in the whole blood sample, as shown in fig. 3, and specifically comprises the following steps:
(1) using water as solvent to prepare a solution containing 5g/L of Na2HPO4Adjusting the pH value to 7.4 to obtain a buffer solution by using a solution of 5g/L NaCl, 3g/L Casein-Na and 0.02 wt% of Proclin 300;
(2) sucking 20 mu L of whole blood sample, and dripping the whole blood sample onto the sample pad from the sample adding hole; sucking 80 mu L of buffer solution, dripping the buffer solution from the buffer solution adding hole onto the sample pad, and standing for 15 min;
(3) and observing the color development conditions of the T1 detection line, the T2 detection line and the C quality control line through a result observation window:
case 1: if a mauve strip appears at the T1 detection line and the C quality control line and no mauve strip exists at the T2 detection line, the whole blood sample contains a new coronavirus neutralizing antibody and does not contain a new coronavirus nucleocapsid protein antibody;
case 2: if no mauve strip exists at the T1 detection line and mauve strips appear at the T2 detection line and the C quality control line, the whole blood sample does not contain a new coronavirus neutralizing antibody and contains a new coronavirus nucleocapsid protein antibody;
case 3: if purple red bands appear at the detection lines of T1 and T2 and the quality control line of C, the whole blood sample contains a new coronavirus neutralizing antibody and a nucleocapsid protein antibody;
case 4: if no mauve strip exists at the detection lines of T1 and T2, and a mauve strip appears at the quality control line of C, the whole blood sample does not contain a new coronavirus neutralizing antibody and a nucleocapsid protein antibody;
case 5: if the mauve strip is not displayed on the C quality control line, the test strip is invalid, and the problems of insufficient sample amount or abnormal chromatography process and the like may exist.
The results of the detection of the whole blood samples 1 to 4 by the above-described method are shown in FIGS. 6(A) to (D), respectively. As can be seen from the figure: whole blood samples 1 and 4 do not contain neutralizing antibodies and nucleocapsid protein antibodies; whole blood sample 2 contains only nucleocapsid protein antibodies; the whole blood sample 3 contains antibodies to the nucleocapsid protein and neutralizing antibodies.
Test example: sensitivity detection
Adding the neutralizing antibody of the new coronavirus and the nucleocapsid protein antibody into PBS buffer solution, preparing antibody standard solutions with the concentrations of 0, 5, 10, 15, 20, 50 and 100BAU/mL (the antibody standard solution with the concentration of c represents that the concentrations of the neutralizing antibody and the nucleocapsid protein antibody in the solutions are both c), using the devices of examples 1, 3 and 4 and comparative examples 1-3, changing the serum sample in the step (2) into the antibody standard solution according to the steps in the application example 1, observing the color development of the nitrocellulose membrane, and obtaining the detection limits of the devices on the two antibodies, wherein the results are shown in Table 1.
TABLE 1
As can be seen from table 1:
(1) compared with example 1 using an unmodified NC membrane, examples 3 and 4 have significantly improved detection sensitivity for both antibodies after the NC membrane is modified by the method of the invention. The reason is that: the polydopamine contains a large amount of amino and hydroxyl, after the dimercaptosuccinic acid-glycerol copolymer is grafted on the hydroxyl, the content of the hydroxyl and the carboxyl can be further improved, and the amino, the hydroxyl and the carboxyl can be utilized to improve the adsorption capacity of an NC membrane to protein, so that the adsorption capacity of S-RBD antigen and novel coronavirus nucleocapsid protein antigen on the membrane is improved.
(2) Compared with comparative example 1 without grafting dimercaptosuccinic acid-glycerol copolymer, in example 3, after the copolymer is grafted in polydopamine, the detection sensitivity of two antigens is obviously improved. The reason is that: after the 2, 3-dimercaptosuccinic acid-glycerol copolymer is grafted to the hydroxyl of the polydopamine, a large amount of hydroxyl and carboxyl can be introduced on a T1 detection line and a T2 detection line by utilizing the higher branching degree and the larger carboxyl and hydroxyl content of the copolymer, so that the adsorption quantity of S-RBD antigens and novel coronavirus nucleocapsid protein antigens is improved.
(3) In comparative example 2 and example 3, the spraying amount of the dopamine Tris buffer is 7 μ L/cm and 5 μ L/cm, respectively, and the detection sensitivity of comparative example 2 to the nucleocapsid protein antibody is reduced compared with example 3. The reason is that: when the spraying amount of the dopamine Tris buffer solution is too high, the pore diameter of the membrane is reduced due to excessive polydopamine introduced on the nitrocellulose membrane, and more complexes of the nucleocapsid protein antibody and the anti-human IgG antibody-colloidal gold conjugate are retained at a T1 detection line, so that the detection sensitivity of the nucleocapsid protein antibody at a T2 detection line is reduced.
(4) In comparative example 3 and example 4, the spraying amount of the dopamine Tris buffer is 2 μ L/cm and 4 μ L/cm, respectively, and the detection sensitivity of comparative example 3 to the two antibodies is reduced compared with example 4. The reason is that: when the spraying amount of the dopamine Tris buffer solution is too low, too little polydopamine is introduced into the nitrocellulose membrane, so that less amino groups, hydroxyl groups and carboxyl groups are introduced, and the effect of improving the detection sensitivity is poor.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (9)
1. A device for combined detection of novel coronavirus neutralizing antibody and nucleocapsid protein antibody is characterized by comprising a reagent strip (1); the reagent strip (1) comprises a bottom plate (1.1), and a sample pad (1.2), a marking pad (1.3) and an antigen-coated nitrocellulose membrane (1.4) which are arranged on the bottom plate; the anti-human IgG antibody-colloidal gold conjugate is coated on the label pad (1.3); the antigen-coated nitrocellulose membrane (1.4) comprises a nitrocellulose membrane (1.4.1), and a T1 detection line (1.4.2), a T2 detection line (1.4.3) and a C quality control line (1.4.4) which are arranged on the nitrocellulose membrane (1.4.1), wherein the T1 detection line (1.4.2) is coated with an S-RBD antigen, and the T2 detection line (1.4.3) is coated with a novel coronavirus nucleocapsid protein antigen; the lower surface of the upstream end of the sample pad (1.2) is connected with the upper surface of the bottom plate (1.1), the lower surface of the downstream end of the sample pad (1.2) is connected with the upper surface of the upstream end of the marking pad (1.3), and the lower surface of the downstream end of the marking pad (1.3) is connected with the upper surface of the upstream end of the nitrocellulose membrane (1.4.1); the nitrocellulose membrane (1.4.1) is a modified nitrocellulose membrane; modified polydopamine is crosslinked on a T1 detection line, a T2 detection line and a C quality control line of the modified cellulose nitrate membrane, and the modified polydopamine is polydopamine with a dimercaptosuccinic acid-glycerol copolymer grafted on hydroxyl groups.
2. The device according to claim 1, characterized in that the bottom plate (1.1) is further provided with a water-absorbent paper (1.5); the upper surface of the downstream end of the nitrocellulose membrane (1.4.1) is connected with the lower surface of the upstream end of the absorbent paper (1.5), and the lower surface of the downstream end of the absorbent paper (1.5) is connected with the bottom plate (1.1).
3. The apparatus of claim 1, wherein the C quality control line (1.4.4) is provided downstream of the T1 detection line (1.4.2) and the T2 detection line (1.4.3); the quality control line C (1.4.4) is coated with a goat anti-mouse IgG antibody; the labeling pad (1.3) is also coated with an anti-mouse IgG antibody-colloidal gold conjugate; mouse anti-erythrocyte antibodies are adsorbed on the sample pad (1.2).
4. The device of claim 3, wherein the modified nitrocellulose membrane is prepared by the following method:
(I) adding dopamine hydrochloride into a Tris buffer solution with the pH value of 8.0-8.5, and uniformly mixing to obtain a dopamine Tris buffer solution; spraying a dopamine Tris buffer solution on a T1 detection line, a T2 detection line and a C quality control line of an unmodified nitrocellulose membrane, standing for 4-6 hours at 20-30 ℃, and drying to obtain a polydopamine modified nitrocellulose membrane;
(II) adding 2, 3-dimercaptosuccinic acid and glycerol into water, and uniformly mixing to obtain a modifier solution; and spraying the modifier solution onto a T1 detection line, a T2 detection line and a C quality control line of the polydopamine modified nitrocellulose membrane, standing at 20-30 ℃ for reaction for 5-6 h, and drying to obtain the modified nitrocellulose membrane.
5. The apparatus of claim 4, wherein:
in the step (I), the mass-to-volume ratio of the dopamine hydrochloride to the Tris buffer solution is 0.8-1.0 mg:1mL, and the amount of the dopamine Tris buffer solution sprayed on a T1 detection line, a T2 detection line and a C quality control line is 4-5 muL/cm; and/or
In the step (III), the mass-to-volume ratio of the 2, 3-dimercaptosuccinic acid to the glycerol to the water is 1mg: 0.7-1.3 mg: 0.8-1.2 mL, and the amount of the modifier solution sprayed on the T1 detection line, the T2 detection line and the C quality control line is 5.5-6.5 muL/cm.
6. The device according to claim 1 or 3, wherein the antigen-coated nitrocellulose membrane (1.4) is prepared by the following method: spraying an S-RBD antigen solution with the concentration of 0.50-0.55 mg/mL onto a T1 detection line in an amount of 1.0-1.3 muL/cm, spraying a novel coronavirus nucleocapsid protein antigen solution with the concentration of 0.20-0.25 mg/mL onto a T2 detection line in an amount of 1.0-1.3 muL/cm, spraying a goat anti-mouse IgG antibody solution with the concentration of 0.8-1.2 mg/mL onto a C quality control line in an amount of 1.0-1.3 muL/cm, and drying to obtain the antigen-coated nitrocellulose membrane.
7. A method for the combined detection of novel coronavirus neutralizing antibodies and nucleocapsid protein antibodies using the device according to any one of claims 1 to 6, comprising the steps of:
(1) dropwise adding a sample to be detected onto a sample pad, dropwise adding 75-85 mu L of buffer liquid onto the upstream of the sample dropwise adding position on the sample pad, and standing;
(2) observing the experimental result, and judging whether the sample to be detected contains the novel coronavirus neutralizing antibody and the nucleocapsid protein antibody according to the color development condition on the nitrocellulose membrane.
8. The method according to claim 7, wherein in the step (1), the buffer solution contains Na in concentrations of 4.5 to 5.5g/L, 2.8 to 3.3g/L and 0.02 to 0.03wt%, respectively2HPO4NaCl, sodium caseinate and Proclin300, wherein the solvent is water, and the pH value is 7.3-7.5.
9. The method of claim 7, wherein:
in the step (1), the sample to be detected is a serum or plasma sample, and the volume of the sample to be detected is 10-13 mu L;
or the sample to be detected is a whole blood sample, and the volume of the sample to be detected is 18-23 muL.
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| CN115015563B (en) * | 2022-04-17 | 2023-02-14 | 吉林迅准生物技术有限公司 | MPO, cTnI or cTnT, IMA combined detection device and preparation method |
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