CN112180083A - IgM antibody detection kit, detection card thereof and preparation method of detection card - Google Patents

IgM antibody detection kit, detection card thereof and preparation method of detection card Download PDF

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CN112180083A
CN112180083A CN202011058058.3A CN202011058058A CN112180083A CN 112180083 A CN112180083 A CN 112180083A CN 202011058058 A CN202011058058 A CN 202011058058A CN 112180083 A CN112180083 A CN 112180083A
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rare earth
igm antibody
detection
influenza
line
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张云
张肖
宋良
刘宁
林文娜
吴志滔
洪茂椿
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Xiamen Institute of Rare Earth Materials
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Abstract

The invention discloses an IgM antibody detection kit, a detection card thereof and a preparation method of the detection card, wherein the IgM antibody detection card comprises: a bottom lining, a coating film, a sample pad and absorbent paper; one end of the sample pad close to the coating film is sprayed with a microsphere line, and the microsphere line is an anti-human IgM antibody marked by a rare earth nano probe and a rabbit IgG antibody marked by the rare earth nano probe; the coating film is sequentially provided with a detection line and a quality control line, and the detection line is close to the sample pad; the detection line is coated with a novel coronavirus recombinant antigen, an influenza A virus recombinant antigen and an influenza B virus recombinant antigen, and the quality control line is coated with a goat anti-rabbit antibody. The nano probe is a rare earth fluoride nano material, has the advantages of low background, long luminescence life, strong fluorescence signal, high signal-to-noise ratio and the like, is stable in labeled product, has the characteristics of high sensitivity, high accuracy, rapidness, simplicity and convenience in detection and the like, can be used for screening and detecting early-stage new coronal suspected patients, and can be used for rapidly and specifically helping clinical diagnosis.

Description

IgM antibody detection kit, detection card thereof and preparation method of detection card
Technical Field
The invention relates to an IgM antibody detection kit, a detection card thereof and a preparation method of the detection card, in particular to a novel coronavirus/influenza A/influenza B virus IgM antibody detection kit, a detection card thereof and a preparation method of the detection card.
Background
The novel coronaviruses are based on current epidemiological investigation, and the incubation period is generally 3-7 days, and the longest day is not more than 14 days. The infected patients are mainly manifested as fever, fatigue and dry cough. The symptoms of upper respiratory tract such as nasal obstruction and nasal discharge are rare. Approximately half of patients develop dyspnea after one week, and severe patients rapidly progress to acute respiratory distress syndrome, septic shock, refractory metabolic acidosis, and procoagulant dysfunction. It is worth noting that the patients with severe or critical illness may have moderate or low fever, even without obvious fever.
The novel coronavirus, also known as Severe Acute Respiratory Syndrome (SARS) coronavirus-2, is defined as a β coronavirus, which is the 7 th member of the family of infecting human coronaviruses, and consists of RNA nucleic acid and Protein, etc., which are easily degraded, Protein capsid enclosing RNA therein, and an Envelope coat consisting of lipid and glycoprotein, so that RNA thereof is protected, viral RNA encoding four major structural proteins including Spike glycoprotein (S), small Envelope glycoprotein (E, Envelope Protein) and Membrane glycoprotein (M, Membrane Protein), and nucleocapsid (N) Protein, wherein S Protein plays a key role in virus recognition and invasion. The virus infects human respiratory epithelial cells through a molecular mechanism of interaction of S-protein and human ACE2, and after entering a human body, the virus mainly activates an immune system and causes lung injury through cytokines, inflammatory factors and the like. And replicating and propagating the cells using the host cells. Therefore, it is possible to confirm whether or not the body is infected by collecting a cell sample from a specific site of a human and detecting whether or not the cell sample contains viral RNA nucleic acid.
Nucleic acid detection is the gold standard for the current novel coronaviruse infection confirmed diagnosis, however, virus nucleic acid detection has a series of problems of long detection time and period, high environmental requirement, high technical force requirement, high hardware equipment requirement, high price and the like, can be only carried out in provincial/municipal disease control centers and large-scale hospitals, is not beneficial to rapid diagnosis and screening of pneumonia patients, and is not beneficial to prevention and control of epidemic situations.
The requirement of the diagnosis and treatment scheme for pneumonia infected by novel coronavirus is that the novel coronavirus pneumonia needs to be identified with other known virus pneumonia infections such as influenza virus and adenovirus. Virus gene sequencing or PCR technology for virus nucleic acid detection is used as the main identification method. However, the virus nucleic acid detection has the problems of long detection time, high requirements on environment, technology and equipment, and 'false positive' and 'false negative' caused by mutual interference of various viruses. At present, companies propose novel reagents for rapidly detecting coronavirus antibodies, but the reagents can only diagnose the infection of the novel coronavirus and cannot simultaneously diagnose the infection of the influenza A virus and the influenza B virus.
The invention develops a novel rapid triple combined detection kit for IgM antibodies infected by coronavirus, influenza A virus and influenza B virus based on a rare earth nano probe. By one-time detection, whether the infection is new coronavirus or influenza A or B virus can be accurately identified in early stage. Provides a field rapid high-throughput detection means for examining suspected patients and identifying new coronavirus and influenza virus infection, in particular to patients infected with new coronavirus and influenza simultaneously. The IgM antibody detection reagent can be used for joint detection with nucleic acid, and is beneficial to improving the detection rate of new coronavirus, avoiding false negative, improving the diagnosis rate and reducing the requirements on laboratories.
Novel IgM antibodies to coronary/influenza a/influenza b viruses are the first antibodies to appear in the human immune system during viral infection, appearing early after infection, peaking at the acute or convalescent phase. The early suspected patient can be screened and detected, and clinical screening and distinguishing can be rapidly and specifically assisted.
Therefore, the development of a novel rapid triple combined detection kit for the coronavirus, influenza A virus and influenza B virus infection IgM antibody with wide detection range and high sensitivity has important significance. The rare earth nano immunofluorescence method is to covalently bond an anti-human IgM antibody to a surface active group of a rare earth nano probe, and judges the result by judging whether a detection line generates fluorescence after excitation, so that the method is quick, convenient, accurate and quantitative, has the advantages of high sensitivity, stable marker and the like, screens and detects early suspected patients, and can quickly and specifically help clinical diagnosis.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides an IgM antibody detection kit which has wide detection range and high sensitivity and can quickly distinguish infection of novel coronavirus, influenza A virus and influenza B virus, a detection card thereof and a preparation method of the detection card.
The technical scheme for realizing the purpose of the invention is divided into three aspects:
the first technical scheme of the invention is as follows: an IgM antibody detection card comprising:
a substrate as a base for the IgM antibody detection card;
the coating film is arranged in the middle of the upper surface of the bottom liner;
a sample pad lapped and arranged at one end of the upper surface of the coating film;
a water absorbing paper lapped and arranged at the other end of the upper surface of the coating film;
one end of the sample pad, which is close to the coating film, is sprayed with a microsphere line, wherein the microsphere line is an anti-human IgM antibody marked by a rare earth nano probe and a rabbit IgG antibody marked by the rare earth nano probe; the coating film is sequentially provided with a detection line and a quality control line, and the detection line is close to the sample pad; the detection line is respectively coated with a novel coronavirus recombinant antigen, an influenza A virus recombinant antigen and an influenza B virus recombinant antigen, and the quality control line is coated with a goat anti-rabbit antibody.
Further, the rare earth nanometer probe is praseodymium-doped gadolinium lutetium fluoride-coated yttrium sodium fluoride with a core-shell structure, and comprises the following components: NaGdxLu1-y-xF4:yPr@NaYF4Wherein, NaGdxLu1-y-xF4As a matrix, the doping ion is Pr3+(ii) a The colon represents praseodymium doping; x and y are the rare earth ion doping mole percentage, the range of x is 20-90%, and the range of y is 1-10%; NaYF4As a shell layer, @ denotes NaYF4Coating with NaGdxLu1-y-xF4yPr.
Further, the particle size of the praseodymium-doped gadolinium lutetium fluoride coated yttrium sodium fluoride with the core-shell structure is 40-60 nm.
Furthermore, the rare earth nano probe is stable under a ground state, and emits fluorescence with the wavelength range of 450-700nm under the action of an excitation light source of 365 nm.
Further, the preparation method of the rare earth nanoprobe comprises the following steps:
the method comprises the following steps: synthesizing praseodymium-doped gadolinium lutetium fluoride: adding mixed solution of nitrate or acetate or chloride of oleic acid, 1-octadecene, gadolinium, nitrate or acetate or chloride of praseodymium, nitrate or acetate or chloride of lutetium, NaOH and ammonium fluoride in a container, and reacting; then washing with cyclohexane-ethanol mixed solution, dispersing in cyclohexane to obtain NaGdxLu1-y-xF4yPr nanoprobe cyclohexane solution;
step two: preparing a core-shell structure rare earth nanoprobe: adding oleic acid, 1-octadecene, yttrium acetate, NaOH and ammonium fluoride methanol mixed solution and NaGd into a containerxLu1-y-xF4yPr nanoprobe cyclohexane solution, and reaction is carried out byWashing the cyclohexane-ethanol mixed solution for 3-4 times, and dispersing in cyclohexane; transferring the probe to a water phase by an acid washing method, and modifying carboxyl on the surface of the probe to obtain water-soluble NaGdxLu1-y-xF4:yPr@NaYF4A rare earth nanoprobe;
step three: activating the rare earth nanoprobe: performing ultrasonic treatment and centrifugal treatment on the rare earth nanoprobe obtained in the step two, and washing the precipitate with 10-100 mM MES solution with pH of 5.0-7.0; adding carbodiimide and N-hydroxy thiosuccinimide, uniformly mixing, centrifuging at a high speed, and washing the precipitate with MES solution with the pH of 5.0-7.0 to obtain the activated rare earth nano probe.
Furthermore, the content of the anti-human IgM antibody marked by the rare earth nano probe and the content of the rabbit IgG antibody marked by the rare earth nano probe which are sprayed on the sample pad are respectively 50-200 mu g of antibody per 200 mu l of nano probe.
Furthermore, the coating concentration of the new coronavirus recombinant antigen, the influenza A virus recombinant antigen and the influenza B virus recombinant antigen in the detection line is 0.1-2 mg/ml, the dosage is 0.5-1.5 mul of coating liquid volume/cm of membrane, and the coating concentration of the goat anti-rabbit IgG antibody in the quality control line is 0.5-2 mg/ml, the dosage is 0.5-1.5 mul of coating liquid volume/cm of membrane.
The second technical scheme of the invention is as follows: the method for preparing the IgM antibody detection card comprises the following steps:
the method comprises the following steps: preparing an activated rare earth nanoprobe according to the preparation method of the rare earth nanoprobe;
step two: preparing a rare earth nano probe labeled anti-human IgM antibody and a rare earth nano probe labeled rabbit IgG antibody: adding a proper amount of anti-human IgM antibody and rabbit IgG antibody into the activated rare earth nano probe obtained in the first step, sealing with a sealing solution, then centrifuging at a high speed, washing with a preserving fluid, resuspending, and preserving at a temperature of 4 ℃ in a dark place;
step three: preparing a sample pad: soaking the combined pad in a sample pad treatment solution containing 0.5% NaCl, 1% sucrose, 0.5% Tween-20, 50mM of 0.5% BSA, and Tris-HCl with pH8.0, and drying at 37 ℃ for overnight; spraying an anti-human IgM antibody marked by a rare earth nano probe on one end of the sample pad close to the coating film for one line, and drying;
step four: preparation of a coating film: respectively using a novel coronavirus recombinant antigen, an influenza A virus recombinant antigen and an influenza B virus recombinant antigen as detection lines G, A and B and a goat anti-rabbit IgG antibody as quality control lines, parallel-scribing on a nitrocellulose membrane for coating, and drying;
step five: and (3) sequentially and mutually overlapping and sticking a sample pad, a coating film and absorbent paper on the bottom lining to obtain a test paper board, and cutting to obtain the IgM antibody detection card.
Further, in the third step, the anti-human IgM antibody marked by the rare earth nano probe and the rabbit IgG antibody marked by the rare earth nano probe are diluted by 8-30 times by using a microsphere diluent, and the using amount is 2-4 mu l of liquid per cm of the sample pad; the microsphere diluent is a 2mM boric acid buffer solution containing 0.5% BSA and 10% sucrose.
The third technical scheme of the invention is as follows: IgM antibody detection kit, including:
the IgM antibody detection card is adopted;
the method comprises the steps of determining calibrators with different antibody titers by an IgM antibody detection card, drawing a standard curve by taking the reciprocal of the antibody titer of the calibrator as a horizontal coordinate and the ratio of a fluorescence signal as a vertical coordinate, writing and generating corresponding two-dimensional code information and storing the two-dimensional code information in the ID card.
The principle of the invention is as follows: the reagent adopts the fluorescence immunochromatography principle to detect novel coronavirus/influenza A/influenza B virus IgM antibodies in human serum, plasma and whole blood. Carrying out chromatography on blood sample diluent containing the novel coronavirus IgM antibody/influenza A virus IgM antibody/influenza B virus IgM antibody to a sample pad microsphere line, forming a reaction complex with an anti-human IgM antibody marked by a rare earth nano probe, carrying out chromatography to a detection area, and respectively combining with a pre-coated novel coronavirus recombinant antigen/influenza A virus recombinant antigen/influenza B virus recombinant antigen to form a final reaction complex; and (3) scanning and detecting the detection area by using a light source (365nm), wherein the rare earth nanoprobes on the detection line and the quality control line emit fluorescence (610nm), and the autofluorescence of the organisms in the fluorescence range is weaker. And (3) delaying the measurement time, and measuring the specific fluorescence of the rare earth element after the naturally occurring short-life fluorescence (1-10 ns) in the sample matrix is completely decayed, so that the interference of the specific background fluorescence can be completely eliminated. The concentration of the substance to be detected in the sample can be analyzed through the strength and the ratio of the fluorescence intensity of the detection line and the fluorescence intensity of the quality control line.
After the technical scheme is adopted, the invention has the positive effects that: (1) the nano probe is a rare earth fluoride nano material, has the advantages of low background, long luminescence life, strong fluorescence signal, high signal to noise ratio and the like, is stable in labeled product, has the characteristics of wide detection range, high sensitivity (the dilution titer of a mixed sample of a sensitivity reference substance, namely a novel coronavirus positive sample, an influenza A virus positive sample and an influenza B virus positive sample is 1:256, and can be detected), high accuracy, quick, simple and convenient detection and the like, and can be used for quick detection.
(2) The rare earth doped nano material has the advantages of stable physical and chemical properties, narrow-band emission, wide Stokes displacement, long service life and the like, thereby eliminating the interference of exciting light, eliminating background fluorescence interference through time-resolved delay detection and being not easily influenced by the environment in the process of biological marking. The rare earth luminescent material, especially the rare earth fluoride, has low phonon energy and stable physical and chemical properties, is suitable for the matrix material of the rare earth luminescent material, and can reduce the non-radiative relaxation of the excited state of the active ion so as to improve the luminous efficiency of the active ion. Therefore, the rare earth fluoride nano particles are used as the markers, the luminescence service life is long, the excitation wavelength is 365nm, the emission wavelength is 610nm, the Stokes displacement is large, the interference of an excitation light source can be avoided, meanwhile, the time-resolved fluorescence immunoassay technology is combined, the signal resolution is carried out by detecting two parameters of wavelength and time, the interference of non-specific fluorescence can be effectively eliminated, the analysis sensitivity and accuracy are greatly improved, and the method has the characteristics of wide detection range, high sensitivity, high accuracy, quick, simple and convenient detection and the like, and can be used for quick detection.
(3) According to the invention, rare earth fluoride is used as a matrix, and the high-performance praseodymium-doped lutetium sodium fluoride nano probe is synthesized by doping different rare earth ions, the doping of lutetium and the doping proportion further reduce the phonon energy of the matrix, and the energy conversion efficiency is improved, and the praseodymium doping can utilize the light emission of praseodymium at about 610nm, so that the detection of the praseodymium is facilitated, and the rare earth nano probe with stable and strong photochemical property and long light emission life is prepared.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a schematic view showing the structure of an IgM antibody detection card of the present invention.
FIG. 2 shows NaGdxLu1-y-xF4:yPr@NaYF4Fluorescence spectrum of rare earth nanoprobe under 365nm excitation.
FIG. 3 shows NaGdxLu1-y-xF4:yPr@NaYF4The rare earth nanoprobe has a transmission electron microscope picture, and the particle size of the nanoprobe is 40-60 nm.
The reference numbers in the drawings are as follows:
bottom liner 1, sample pad 2, coating film 3, detection line G line 31, detection line A line 32, detection line B line 33, quality control line 34 and absorbent paper 4.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
(example 1)
The IgM antibody detection kit of the present embodiment comprises: IgM antibody detection cards and ID cards containing calibration curves.
The ID card containing the calibration curve is used for determining calibrators with different antibody titers by an IgM antibody detection card, drawing a standard curve by taking the reciprocal of the titer of the calibrator as the abscissa and the ratio of the fluorescence signal as the ordinate, writing and generating corresponding two-dimensional code information and storing the two-dimensional code information in the ID card. The corresponding two-dimensional code information on the reagent card can be read by a dry-type fluorescence immunoassay analyzer, and the corresponding antibody titer can be measured.
The IgM antibody detection card is shown in FIG. 1 and comprises:
a bottom lining 1; as a substrate for the IgM antibody detection card;
a coating film 3; the middle part of the upper surface of the bottom liner 1 is provided with a groove;
a sample pad 2; one end of the upper surface of the coating film 3 is lapped and arranged;
a water absorbent paper 4; the other end of the upper surface of the coating film 3 is overlapped;
one end of the sample pad 2, which is close to the coating film 3, is sprayed with a microsphere line, wherein the microsphere line is an anti-human IgM antibody marked by a rare earth nano probe and a rabbit IgG antibody marked by the rare earth nano probe; the coating film 3 is sequentially provided with a detection line G line 31, a detection line A line 32, a detection line B line 33 and a quality control line 34, the detection line 31 is close to the sample pad 2, the detection lines 31, 32 and 33 and the quality control line 34 are parallel to each other, and the spacing distance is 3-5 mm; the detection line G line 31 is coated with a novel coronavirus recombinant antigen, the line A is coated with an influenza A virus recombinant antigen, the line B is coated with an influenza B virus recombinant antigen, and the quality control line 34 is coated with a goat anti-rabbit antibody.
The rare earth nanometer probe is made of praseodymium-doped gadolinium lutetium fluoride-coated yttrium sodium fluoride with a core-shell structure, the particle size is 40-60 nm, and the rare earth nanometer probe comprises the following components: NaGdxLu1-y-xF4:yPr@NaYF4Wherein, NaGdxLu1-y-xF4As a matrix, the doping ion is Pr3+(ii) a The colon represents praseodymium doping; x and y are the rare earth ion doping mole percentage, the range of x is 20-90%,the range of y is 1-10%; NaYF4As a shell layer, @ denotes NaYF4Coating with NaGdxLu1-y-xF4yPr. NaGdxLu1-y-xF4:yPr@NaYF4The transmission electron microscope image of the rare earth nanoprobe is shown in figure 3.
Furthermore, the rare earth nano probe is stable under a ground state, and emits fluorescence with the wavelength range of 450-700nm under the action of an excitation light source of 365 nm. NaGdxLu1-y-xF4:yPr@NaYF4The fluorescence spectrum of the rare earth nanoprobe under 365nm excitation is shown in figure 2.
Further, the preparation method of the rare earth nanoprobe comprises the following steps:
the method comprises the following steps: synthesizing praseodymium-doped gadolinium lutetium fluoride: adding a mixture of 3-6 by volume: 7-14 parts of oleic acid and 1-octadecene, and then adding x parts of nitrate or acetate or chloride of gadolinium, y parts of nitrate or acetate or chloride of praseodymium and 1-x-y parts of nitrate or acetate or chloride of lutetium, x and y being the rare earth ion doping mole percentage, x ranging from 20% to 70%, y ranging from 0% to 30%; mixing and stirring at room temperature, vacuumizing, heating to 100-120 ℃, reacting for 20-30 minutes, heating to 150-160 ℃, and reacting for 10-15 minutes to obtain a transparent solution; naturally cooling to 40-50 ℃, releasing vacuum, and adding a mixture of the components in a molar concentration ratio of 1-2: 1.6-3.4 of mixed solution of NaOH and ammonium fluoride methanol, and reacting for 20-30 min; heating to 90-100 ℃, exhausting air for 3-4 times, introducing nitrogen, heating to 280-300 ℃, reacting for 1-2 hours, centrifuging, washing for 3-4 times by using cyclohexane ethanol mixed solution, dispersing in cyclohexane to obtain NaGdxLu1-y-xF4: yPr nanoprobe cyclohexane solution;
step two: preparing a core-shell structure rare earth nanoprobe: adding a mixture of 3-6 by volume: adding 7-14 parts of oleic acid and 1-octadecene into yttrium acetate, mixing and stirring at room temperature, vacuumizing, heating to 120 ℃, reacting for 20 minutes, heating to 160 ℃, and reacting for 10 minutes to obtain a transparent solution; naturally cooling to 50 deg.C, and releasingAnd (3) adding a mixed solution of NaOH and ammonium fluoride methanol in vacuum, wherein the volume ratio of the mixed solution to oleic acid is 3-6: 2 to 4 of NaGdxLu1-y-xF4yPr nanometer probe cyclohexane solution, mixing and stirring, reacting for 20-30 min; heating to 90-100 ℃, exhausting air for 3-4 times, introducing nitrogen, heating to 280-290 ℃, reacting for 1-2 hours, centrifuging at 6000rpm, washing for 3-4 times by using a cyclohexane-ethanol mixed solution, and dispersing in cyclohexane; the weight ratio of the yttrium acetate, NaOH and ammonium fluoride is 0.2-0.4: 0.5-1: 0.8 to 1.6; transferring the probe to a water phase by an acid washing method, and modifying carboxyl on the surface of the probe to obtain water-soluble NaGd with good dispersibilityxLu1-y-xF4:yPr@NaYF4A rare earth nanoprobe;
step three: activating the rare earth nanoprobe: performing ultrasonic treatment and centrifugal treatment (12000-14000 rpm high speed) on the rare earth nanoprobe in the second step for 1-2 min, washing the precipitate with 10-100 mM MES solution with pH of 5.0-7.0, and performing ultrasonic treatment for 2-3 min; adding 20-100 mg/ml carbodiimide, uniformly mixing for 5-10 min, adding 20-100 mg/ml N-hydroxy thiosuccinimide, uniformly mixing for 10-20 min, then centrifuging at 12000-14000 rpm for 5-15 min at a high speed, and washing the precipitate with an MES solution with the pH of 5.0-7.0 to obtain the activated rare earth nano probe.
Furthermore, the content of the anti-human IgM antibody marked by the rare earth nano probe and the content of the rabbit IgG antibody marked by the rare earth nano probe sprayed on the sample pad 2 are respectively 50-200 mug antibody/200 mul nano probe.
Furthermore, the novel coronavirus recombinant antigen, influenza A virus recombinant antigen and influenza B virus recombinant antigen coated in the detection line G line 31, the detection line A line 32 and the detection line B line 33 respectively have the coating concentration of 0.1-2 mg/ml and the dosage of 0.5-1.5 mul of coating liquid amount/cm of membrane, and the goat anti-rabbit IgG antibody coated in the quality control line 34 has the coating concentration of 0.5-2 mg/ml and the dosage of 0.5-1.5 mul of coating liquid amount/cm of membrane.
After the preparation, fix it on the plastics end card that matches with detection card size, detect the card surface and compress tightly with the card face, and the card face reserves application of sample hole and observation window respectively in the part that corresponds sample pad 2 and envelope membrane 3.
The preparation method of the IgM antibody detection card comprises the following steps:
the method comprises the following steps: preparing an activated rare earth nanoprobe according to the preparation method of the rare earth nanoprobe;
step two: preparing a rare earth nano probe labeled anti-human IgM antibody and a rabbit IgG antibody: carrying out ultrasonic treatment on the activated rare earth nanoprobe obtained in the first step for 1-2 min, respectively adding an anti-human IgM antibody and a rabbit IgG antibody according to 50-200 mug/200 mul, uniformly mixing for 1-3 h, sealing for 0.5-1 h by using 10-50 mM containing 0.5% BSA and 10-50.5 pH7.5-8.5 Tris-HCl sealing solution, then carrying out high-speed centrifugation for 5-15 min at 12000-14000 rpm, washing and resuspending by using 10-50 mM containing 1% NaCl, 0.5% BSA and 0.1% Tween-20 and 10-50 mM containing 7.5-8.5 Tris-HCl preservation solution, and storing in a dark place at 4 ℃;
step three: preparing a sample pad: soaking the combined pad in Tris-HCl (Tris-HCl) with the temperature of 37 ℃ and drying overnight, wherein the sample pad treatment solution contains 0.5% of NaCl, 1% of sucrose, 0.5% of Tween-20, 50mM of 0.5% of BSA (bovine serum albumin), and pH8.0, diluting the anti-human IgM antibody and rabbit IgG antibody marked by the rare earth nanoprobe on one side of the sample pad close to the coating film by 8-30 times of microsphere diluent, uniformly spraying the diluted solution on a line, wherein the dosage of the diluted solution is 2-4 microliter of solution per cm of the sample pad, placing the sample pad in a drying oven, and drying overnight at 37 ℃; the microsphere diluent is a 2mM boric acid buffer solution containing 0.5% BSA and 10% sucrose.
Step four: preparation of a coating film: respectively using the novel coronavirus recombinant antigen, the influenza A virus recombinant antigen and the influenza B virus recombinant antigen as detection lines, using a goat anti-rabbit antibody as a quality control line, parallel-scribing on a nitrocellulose membrane for coating, and drying; respectively adjusting the concentration of a novel coronavirus recombinant antigen, an influenza A virus recombinant antigen, an influenza B virus recombinant antigen and a goat anti-rabbit antibody to 0.5-1.5 mg/ml by using coating buffer solution, wherein the amount of the coating buffer solution is 0.5-1.5 microliter per centimeter of membrane, the coating buffer solution is respectively used as a detection line and a quality control line which are parallel to each other and marked on a nitrocellulose membrane for coating, the interval between the quality control line and the detection line is 3-7 mm, and the nitrocellulose membrane is placed in an oven and dried at 45 ℃ for overnight;
step five: and (3) sequentially and mutually overlapping and sticking a sample pad, a coating film and absorbent paper on the bottom lining to obtain a test paper board, and cutting to obtain the IgM antibody detection card.
The method for quantitatively detecting the novel coronavirus/influenza a/influenza b virus IgM antibody by the IgM antibody detection kit of the embodiment comprises the following steps:
the method comprises the following steps: taking a serum/plasma/whole blood sample as a detection sample;
step two: starting a dry type fluorescence immunoassay analyzer, preheating for 5min, and then inserting a corresponding ID card containing a calibration curve;
step three: using a pipette with a suitable range, 10. mu.l of the serum/plasma to be tested/15. mu.l of the whole blood sample is pipetted into the well, and two drops of the diluent (about 80. mu.l) are added into the well of the test card, taking care not to generate obvious bubbles during pipetting and loading.
Step four: the test card is inserted into the test slot, and the test key is pressed, so that the test card is automatically scanned by the test machine (please strictly control the time from sample loading to test for 10 min). And reading/printing detection results, wherein the new crown IgM is more than 0.8, the influenza A IgM is more than 0.7, and the influenza B IgM is more than 0.7 respectively indicate the possibility of infection of the new coronavirus, the influenza A virus and the influenza B virus.
Specific example tests were carried out and measured as follows.
Praseodymium-doped gadolinium lutetium fluoride yttrium sodium fluoride particles (NaGd) coated with core-shell structure praseodymium with specific excitation light (365 nm)/emission light (610nm) wavelength are arranged on a microsphere line on a sample pad 1 for IgM antibody detectionxLu1-y-xF4:yPr@NaYF4) The detection line is coated with a novel coronavirus recombinant antigen, an influenza A virus recombinant antigen and an influenza B virus recombinant antigen (0.5mg/ml), and the quality control line is coated with a goat anti-rabbit antibody with the concentration of 1mg/ml (wherein the anti-human IgM antibody is from the Feibo biological corporation, the novel coronavirus recombinant antigen is from the Beijing Qiaohanshizhou science and technology company Limited, the influenza A virus recombinant antigen and the influenza B virus recombinant antigen are from the Xiamen Wankelong biological science and technology company Limited, and the goat anti-rabbit antibody and the rabbit IgG antibody are purchased from the Luoyang Bai-Otong experimental material center). The dosage of the microsphere line is 4 muThe dosage of the coating liquid volume/cm of the sample pad, the detection line and the quality control line is 1 mul of the coating liquid volume/cm of the membrane.
The preparation of the IgM antibody detection kit comprises the following steps:
(1) synthesis of praseodymium-doped gadolinium lutetium fluoride:
adding 4.5mL of oleic acid and 12.5mL of 1-octadecene into a 100mL three-neck round-bottom flask, adding 1.6mmol of gadolinium acetate, 1.6mmol of lutetium acetate and 0.02mmol of praseodymium acetate according to a molar ratio, mixing and stirring at room temperature, vacuumizing, heating to 120 ℃, reacting for 20 minutes, heating to 160 ℃, and reacting for 10 minutes to obtain a transparent solution; naturally cooling to 50 ℃, releasing vacuum, adding a mixed solution of 1mmol of NaOH and 1.6mmol of ammonium fluoride methanol, and reacting for 30 min; heating to 100 deg.C, extracting air, ventilating for 3 times, introducing nitrogen, heating to 300 deg.C, reacting for 1.5 hr, centrifuging at 6000rpm, washing with cyclohexane-ethanol mixed solution for 3 times, and dispersing in cyclohexane.
(2) Preparing a praseodymium-doped gadolinium lutetium fluoride coated yttrium sodium fluoride nanometer probe with a core-shell structure:
adding 3mL of oleic acid and 7mL of 1-octadecene into a 100mL three-neck round-bottom flask, adding 0.4mmol of yttrium acetate, mixing and stirring at room temperature, vacuumizing, heating to 120 ℃, reacting for 20 minutes, heating to 160 ℃, and reacting for 10 minutes to obtain a transparent solution; naturally cooling to 50 ℃, releasing vacuum, adding a mixed solution of 1mmol NaOH and 1.6mmol ammonium fluoride methanol and 4mL NaGdxLu1-y-xF4yPr nanometer probe cyclohexane solution, mixing and stirring, reacting for 30 min; heating to 100 ℃, exhausting and ventilating for 3 times, introducing nitrogen, heating to 290 ℃, reacting for 1 hour, centrifuging at 6000rpm, washing for 3 times by using a cyclohexane-ethanol mixed solution, and dispersing in cyclohexane; transferring the probe to a water phase by an acid washing method, and modifying carboxyl on the surface of the probe to obtain water-soluble NaGd with good dispersibilityxLu1-y-xF4:yPr@NaYF4Rare earth nanometer fluorescent probe. NaGdxLu1-y-xF4yPr nm probexThe mole percentage is preferably 40% and y is preferably 5%. The size of the material is about 60nm, the appearance is uniform, and the luminescence performance is good. The excitation wavelength of the nano-probe is 365nm,the emission wavelength was 610 nm.
(3) Activation of the rare earth nanoprobe:
after the rare earth nano probe is treated by ultrasonic for 2min, 200 mul of the nano probe is taken to be centrifuged for 15min at 14000rpm, and the precipitate is washed to 1ml by MES solution with 100mM and pH of 6.0 and treated by ultrasonic for 2 min; adding 50 μ l of 100mg/ml carbodiimide, mixing for 5min, adding 100 μ l of 100mg/ml N-hydroxy-thiosuccinimide, mixing for 15min, centrifuging at 14000rpm for 15min, and washing the precipitate with MES solution with pH of 6.0 to 1 ml.
(4) Preparing an anti-human IgM antibody marked by the rare earth nano probe and a rabbit IgG antibody marked by the rare earth nano probe:
after the activated fluorescent microspheres are subjected to ultrasonic treatment for 2min, anti-human IgM and rabbit IgG are respectively added according to 200 mug/200 ul, the mixture is mixed for 2 hours, the mixture is blocked by 50mM containing 0.5% BSA and pH8.0 Tris-HCl blocking solution for 1 hour, then the mixture is centrifuged at 14000rpm for 15min at a high speed, and the mixture is washed twice by buffer solution in 50mM containing 1% (w/w) NaCl, 0.5% (w/w) BSA and 0.1% (w/w) Tween-20 and pH8.0 Tris-HCl preservation solution and is subjected to ultrasonic treatment to be resuspended to 200 ul and preserved in a dark place at 4 ℃.
(5) Preparation of sample pad:
soaking the combined pad in a sample pad treatment solution containing 0.5% NaCl, 1% sucrose, 0.5% Tween-20, 50mM of 0.5% BSA, and Tris-HCl with pH8.0, and drying at 37 ℃ for overnight; the anti-human IgM antibody and rabbit IgG antibody marked by the rare earth nano probe are diluted by 20 times with a microsphere diluent (2 mM boric acid buffer solution containing 0.5% (w/w) BSA and 10% (w/w) sucrose) and uniformly sprayed on one line on one side of the sample pad close to the coating film, wherein the dosage is 4 mu l of liquid per cm of the sample pad. The mixture was placed in an oven and dried overnight at 37 ℃.
(6) Preparation of coating film:
respectively adjusting the concentration of a novel coronavirus recombinant antigen, an influenza A virus recombinant antigen and an influenza B virus recombinant antigen to 0.5mg/ml by using coating buffer solution (20 mM Tris-HCl buffer solution containing 2.5% (w/w) sucrose and having a pH value of 8.0), adjusting the concentration of a goat anti-rabbit IgG antibody to 1mg/ml, and using 1 microliter of coating liquid volume/cm membrane as a detection line G, a detection line A, a detection line B and a quality control line to be coated on a nitrocellulose membrane in parallel, wherein the interval between the quality control line G, the detection line A, the detection line B and the quality control line is 4mM, drying in an oven with the humidity of less than 30% and the temperature of 45 ℃ overnight, and sealing for later use;
(7) assembling the detection card:
a sample pad (30X 300mm in size, glass fiber cotton material), a coating film (25X 300mm in size, nitrocellulose material) and absorbent paper (20X 30cm in size) were sequentially stuck to a substrate (80X 300mm in size) in an overlapping manner to obtain a test paper sheet, which was cut into test strips of 4mm in width as required.
IgM antibody detects card in the plastic casing that the card formed by plastics epitheca and plastics inferior valve lock when using, and the plastics epitheca is equipped with two trompils, is adding the sample hole and observation window respectively, and the adding the sample hole is corresponding to sample pad 2, is provided with the bayonet socket of fixed detection test paper strip on the plastics inferior valve, and the result observation window is corresponding to detection line G31, detection line A32, detection line B33 and quality control line 34, and this IgM antibody detects the card and can follow and take out in this plastic casing.
In the IgM antibody detection kit, each kit contains an ID card (same batch of standard curves) of a standard curve, calibrators with different antibody titers are determined by rare earth nano fluorescent test strips, the reciprocal of the antibody titer of the calibrator is taken as the abscissa, the ratio of a fluorescent signal is taken as the ordinate, a standard curve is drawn, the ID card is written into the standard curve to generate a two-dimensional code, and the corresponding two-dimensional code information on the reagent card can be read by a dry-type fluorescence immunoassay analyzer and the corresponding concentration is measured.
The drawing of the standard curve is described in detail below
The titer is taken as 1:256 novel coronavirus IgM calibrator is diluted by 32, 64, 256 and 320 times by using negative clinical samples respectively, each concentration is repeatedly measured for three times, and a standard curve is established by taking the T/C value mean value and the calibrator concentration.
The titer is taken as 1:256 influenza A virus IgM calibrator is respectively diluted into titers of 32, 64, 256 and 366 times by using negative clinical samples, each concentration is repeatedly measured three times, and a standard curve is established by taking the T/C value mean value and the calibrator concentration.
The titer is taken as 1:256 influenza B virus IgM calibrator is respectively diluted into titers of 32, 64, 256 and 366 times by using negative clinical samples, each concentration is repeatedly measured three times, and a standard curve is established by taking the T/C value mean value and the calibrator concentration.
The results and analysis are shown in the following table:
TABLE 1 novel coronavirus IgM Standard Curve
Figure BDA0002711388870000111
TABLE 2 influenza A IgG Standard Curve
Figure BDA0002711388870000112
TABLE 3 Standard Curve of IgM for influenza B Virus
Figure BDA0002711388870000113
And (3) drawing a standard curve by using the reciprocal of the antibody titer and the average value of the T/C of the sample signal, wherein the curve data are shown in tables 1-3, and the new crown, the first class antibody titer and the second class antibody titer contained in the sample are quantitatively determined through the marked line.
The performance test was performed on the IgM antibody detection card as follows:
1. negative coincidence rate: and (5) detecting negative enterprise reference products (N1-N10), wherein 10 parts of the enterprise reference products are negative.
Wherein: the N series of enterprise reference products are derived from clinical samples of hospitals, 2 parts of serum of normal people (N1-N2) and 2 parts of plasma of normal people (N3-N4); 1 part of serum/plasma of metapneumovirus patient (N5), 1 part of serum/plasma of parainfluenza virus patient (N6), 1 part of serum/plasma of respiratory syncytial virus patient (N7), 1 part of serum/plasma of mycoplasma pneumoniae patient (N8), 1 part of serum/plasma of respiratory adenovirus patient (N9), and 1 part of serum/plasma of chlamydia pneumoniae patient (N10).
2. Positive compliance rate: detecting positive enterprise reference (P1-P15), wherein P1-P5 are positive for novel coronavirus, negative for influenza A virus and negative for influenza B virus; P6-P10 should be negative for novel coronavirus, positive for influenza A virus and negative for influenza B virus; P11-P15 should be negative for novel coronavirus, negative for influenza A virus and positive for influenza B virus.
Wherein: the P series enterprise reference products are derived from hospital clinical samples; P1-P5: clinically confirming a sample of a patient infected with the novel coronavirus; P6-P10: clinically confirming a specimen of an influenza a virus infected patient; P11-P15: clinically confirming a sample of a patient infected with influenza b virus;
3. minimum detection limit: detecting enterprise sensitivity reference products S1-S4 (formed by diluting enterprise sensitivity reference products S according to the ratio of 1:32, 1:64, 1:256 and 1: 512), wherein S1, S2 and S3 are positive for novel coronavirus, positive for influenza A virus and positive for influenza B virus, and S4 is negative for novel coronavirus, negative for influenza A virus and negative for influenza B virus.
The sensitivity reference substance S of the enterprise is prepared from clinically confirmed novel coronavirus positive, influenza A virus positive and influenza B virus positive samples, the sensitivity reference substance S is adjusted until the T/C fluorescence ratio of a reaction strip is consistent with the T/C fluorescence ratio when the same batch of reagent is used for detecting W, and the S is simultaneously diluted by 1:32, 1:64, 1:256 and 1:512 times to prepare sensitivity reference substances of S1-S4 series.
4. Repeatability: the method comprises the steps of respectively detecting an enterprise repetitive strong positive reference substance C1 and a repetitive weak positive reference substance C2, wherein each sample is parallelly detected for 10 times, and the Coefficient of Variation (CV) is not more than 15%. The detection of C1 can obtain new crown CV 10.29%, A flow CV 9.79%, B flow CV 8.63%, the detection of C2 can obtain new crown CV 12.79%, A flow CV 12.15%, B flow CV 12.21%.
The C series enterprise reference products are derived from hospital clinical samples, and C1 is prepared by mixing novel coronavirus positive, influenza A virus positive and influenza B virus positive samples; c2 is prepared by mixing the positive samples of the novel coronavirus, the positive samples of the influenza A virus and the positive samples of the influenza B virus, and diluting the samples until the T/C fluorescence ratio of the S3 sensitivity reference sample is consistent. The positive sample is collected by a positive patient which is confirmed by a clinician such as PCR diagnosis and clinical CT diagnosis.
The clinical test sample is a serum sample of 501 subjects, wherein the new crown IgM control group is a clinically confirmed case diagnosed according to a new crown nucleic acid result, and the A-flow and B-flow IgM detection control group is an influenza virus A-type IgM antibody, an influenza virus B-type IgM antibody and a parainfluenza virus IgM antibody joint detection kit (colloidal gold method) produced by Beijing Endotte biotechnology Limited.
The test results are summarized as follows:
1. according to the detection result of the test nuclear reagent in 121 cases of clinically confirmed positive samples of the new coronary nucleic acid test: 106 samples of the new crown IgM positive samples, 115 samples of the examination reagents in the control group A flow 118 positive samples, and 141 samples of the control group B flow 121 positive samples, which are confirmed by the examination reagent 118, wherein the examination results of the examination reagents are as follows: the new crown IgM negative samples were 140, the A-class IgM negative samples were 139, and the B-class IgM negative samples were 140.
2. The examination results of 501 clinical serum samples showed that:
Figure BDA0002711388870000131
therefore, the kit provided by the invention realizes high sensitivity, accurate quantification, simplicity and convenience in quantitative detection of the novel coronavirus/influenza A/influenza B virus IgM antibody by using the sensitivity of the rare earth nano fluorescence immunochromatography technology and combining with a dry immunofluorescence analyzer.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (9)

  1. An IgM antibody detection card characterized by comprising:
    a substrate as a base for the IgM antibody detection card;
    the coating film is arranged in the middle of the upper surface of the bottom liner;
    a sample pad lapped and arranged at one end of the upper surface of the coating film;
    a water absorbing paper lapped and arranged at the other end of the upper surface of the coating film;
    one end of the sample pad, which is close to the coating film, is sprayed with a microsphere line, wherein the microsphere line is an anti-human IgM antibody marked by a rare earth nano probe and a rabbit IgG antibody marked by the rare earth nano probe; the coating film is sequentially provided with a detection line and a quality control line, and the detection line is close to the sample pad; the detection line is coated with a novel coronavirus recombinant antigen, an influenza A virus recombinant antigen and an influenza B virus recombinant antigen, and the quality control line is coated with a goat anti-rabbit antibody.
  2. 2. The IgM antibody detection card of claim 1, characterized in that: the rare earth nanoprobe is made of praseodymium-doped gadolinium lutetium-coated yttrium sodium fluoride with a core-shell structure, and comprises the following components: NaGdxLu1-y-xF4:yPr@NaYF4Wherein, NaGdxLu1-y-xF4As a matrix, the doping ion is Pr3+(ii) a The colon represents praseodymium doping; x and y are the rare earth ion doping mole percentage, the range of x is 20-90%, and the range of y is 1-10%; NaYF4As a shell layer, @ denotes NaYF4Coating with NaGdxLu1-y-xF4yPr.
  3. 3. The IgM antibody detection card of claim 2, characterized in that: the rare earth nano probe is stable under the ground state and emits fluorescence with the wavelength range of 450-700nm under the action of an excitation light source of 365 nm.
  4. 4. The IgM antibody detection card according to claim 2 or 3, wherein the rare earth nanoprobe is prepared by a method comprising the steps of:
    the method comprises the following steps: synthesizing praseodymium-doped gadolinium lutetium fluoride: adding nitrate or acetate or chloride of oleic acid, 1-octadecene and gadolinium, nitrate or acetate or chloride of praseodymium, and,Nitrate or acetate or chloride of lutetium, NaOH and ammonium fluoride methanol mixed solution, and carrying out reaction; then washing with cyclohexane-ethanol mixed solution, dispersing in cyclohexane to obtain NaGdxLu1-y- xF4yPr nanoprobe cyclohexane solution;
    step two: preparing a core-shell structure rare earth nanoprobe: adding oleic acid, 1-octadecene, yttrium acetate, NaOH and ammonium fluoride methanol mixed solution and NaGd into a containerxLu1-y-xF4yPr nanometer probe cyclohexane solution, reacting, washing 3-4 times with cyclohexane-ethanol mixed solution, dispersing in cyclohexane; transferring the probe to a water phase by an acid washing method, and modifying carboxyl on the surface of the probe to obtain water-soluble NaGdxLu1-y-xF4:yPr@NaYF4A rare earth nanoprobe;
    step three: activating the rare earth nanoprobe: performing ultrasonic treatment and centrifugal treatment on the rare earth nanoprobe obtained in the step two, and washing the precipitate with 10-100 mM MES solution with pH of 5.0-7.0; adding carbodiimide and N-hydroxy thiosuccinimide, uniformly mixing, centrifuging at a high speed, and washing the precipitate with MES solution with the pH of 5.0-7.0 to obtain the activated rare earth nano probe.
  5. 5. The IgM antibody detection card of claim 4, characterized in that: the content of the anti-human IgM antibody marked by the rare earth nano probe and the content of the rabbit IgG antibody marked by the rare earth nano probe which are sprayed on the sample pad are respectively 50-200 mu g antibody/200 mu l nano probe.
  6. 6. The IgM antibody detection card of claim 5, characterized in that: the coating concentration of the novel coronavirus recombinant antigen, the influenza A virus recombinant antigen and the influenza B virus recombinant antigen in the detection line is 0.1-2 mg/ml, the dosage is 0.5-1.5 mu l of coating liquid amount/cm of membrane, and the coating concentration of the goat anti-rabbit IgG antibody in the quality control line is 0.5-2 mg/ml, and the dosage is 0.5-1.5 mu l of coating liquid amount/cm of membrane.
  7. 7. A method for producing the IgM antibody detection card according to claim 6, characterized by comprising the steps of:
    the method comprises the following steps: the method for preparing a rare earth nanoprobe according to claim 4, preparing an activated rare earth nanoprobe;
    step two: preparing a rare earth nano probe-labeled anti-human IgM antibody and a rare earth nano probe-labeled rabbit IgG antibody: respectively adding an anti-human IgM antibody and rabbit IgG to the activated rare earth nanoprobe obtained in the first step according to the concentration of 50-200 mug/200 mul, marking, sealing with a sealing solution, then centrifuging at a high speed, washing with a storage solution, resuspending, and storing at 4 ℃ in a dark place;
    step three: preparing a sample pad: soaking the sample pad in Tris-HCl solution containing 0.5% NaCl, 1% sucrose, 0.5% Tween-20, 50mM of 0.5% BSA and pH8.0, oven drying overnight at 37 deg.C, spraying anti-human IgM antibody marked with rare earth nanoprobe on the sample pad, and oven drying;
    step four: preparation of a coating film: respectively using a novel coronavirus recombinant antigen, an influenza A virus recombinant antigen and an influenza B virus recombinant antigen as a detection line G, A, B and a goat anti-rabbit antibody as a quality control line, parallel-scribing on a nitrocellulose membrane for coating, and drying;
    step five: and (3) sequentially and mutually overlapping and sticking a sample pad, a coating film and absorbent paper on the bottom lining to obtain a test paper board, and cutting to obtain the IgM antibody detection card.
  8. 8. The method for producing an IgM antibody detection card according to claim 7, characterized in that:
    in the third step, the anti-human IgM antibody and the rabbit IgG antibody marked by the rare earth nano probe are diluted by 8-30 times by using a microsphere diluent, and the using amount is 2-4 mu l of liquid per cm of sample pad; the microsphere dissociation solution is a 2mM boric acid buffer solution containing 0.5% BSA and 10% sucrose.
  9. An IgM antibody detection kit characterized by comprising:
    an IgM antibody detection card having a structure identical to that of the IgM antibody detection card of claim 1;
    the IgM antibody detection card is used for determining calibrators with different antibody titers, the reciprocal of the antibody titer of the calibrator is used as a horizontal coordinate, the ratio of the fluorescence signal is used as a vertical coordinate, a standard curve is drawn, and corresponding two-dimensional code information is written and generated and stored in the ID card.
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