CN115015543B - Multi-pathogen joint detection device and preparation method thereof - Google Patents

Multi-pathogen joint detection device and preparation method thereof Download PDF

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CN115015543B
CN115015543B CN202210402586.9A CN202210402586A CN115015543B CN 115015543 B CN115015543 B CN 115015543B CN 202210402586 A CN202210402586 A CN 202210402586A CN 115015543 B CN115015543 B CN 115015543B
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colloidal gold
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chitosan
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CN115015543A (en
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王洪波
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Jilin Xunzhun Biotechnology Co ltd
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    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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Abstract

The invention relates to a multinomial pathogen joint detection device and a preparation method thereof, belonging to the field of medical detection devices. The combined detection device comprises a sample pad, an immune colloidal gold antibody glass fiber membrane, an immune nitrocellulose membrane and an absorption pad, wherein the sample pad, the immune colloidal gold antibody glass fiber membrane, the immune nitrocellulose membrane and the absorption pad are respectively adhered to a plastic plate, two ends of the nitrocellulose membrane are respectively lapped with the absorption pad and the immune colloidal gold antibody glass fiber membrane, the other end of the immune colloidal gold antibody glass fiber membrane is lapped with the sample pad, and the IgM antibodies of human influenza virus A, influenza virus B, rhinovirus, parainfluenza virus, respiratory syncytial virus, adenovirus, mycoplasma pneumoniae and chlamydia pneumoniae in whole blood, serum and plasma specimens are prepared by utilizing a colloidal gold immunochromatography technology and a capture method principle. The method has the advantages of improving the sensitivity, accuracy and stability of the immunochromatography methodology, being simple and convenient to operate and high in practicability, and being capable of realizing sensitive, specific and comprehensive rapid detection of respiratory infectious diseases.

Description

Multi-pathogen joint detection device and preparation method thereof
Technical Field
The invention relates to the field of medical detection devices, in particular to an IgM (immunoglobulin M) antibody combined detection device for influenza viruses A and B, HRV, HPIV, RSV (respiratory syncytial virus), HAdV, MP (MP) and CP (CP) and a preparation method thereof.
Background
Influenza is an acute respiratory infectious disease caused by influenza virus infection, is rapidly transmitted through respiratory tracts, has short latency and obvious seasonality, is very easy to cause large-scale outbreak and has high control difficulty. Influenza viruses are classified into types A (A), B (B) and C (C) 3, wherein the influenza A virus and the influenza B virus are commonly infected, have strong infectivity and rapid spread and pose serious threats to human health. Because influenza viruses spread rapidly and viruses are subject to variation, epidemics of different sizes occur each year, even with global pandemics occurring every 10-15 years. The World Health Organization (WHO) speculates that about 65 million people worldwide die each year from influenza-related respiratory diseases. Influenza epidemics not only pose serious threats to the health of the population, but also cause huge social and economic losses. The clinical manifestations of influenza are fever, headache, myalgia and general malaise, and may be accompanied by respiratory system symptoms such as cough and expectoration, and may also be accompanied by digestive system symptoms such as vomiting and diarrhea. After children, pregnant and lying-in women, old people and people suffering from various chronic diseases or physical weakness suffer from influenza, serious complications or the original chronic diseases are easily developed, and even the corresponding organ failure is caused, so that the life is threatened. Therefore, understanding the characteristics of influenza facilitates early identification and timely treatment.
The variation of influenza virus, most important as type a, is often closely associated with a pandemic worldwide. Generally, antigenic variation of influenza virus refers to structural change of H and N antigens, and small variation (quantitative change) often occurs inside subtypes, which is called antigenic drift. Antigenic variation occurs only in type a viruses. It may be due to infection of 2 viruses of human and animal with the same cell, and genetic reassortment between the viruses. The produced virus hemagglutinin and neuraminidase are completely combined, so that the human group has no immunity. Antigenic shift is responsible for the global pandemic of influenza. Influenza a viruses have undergone 1 major variation approximately every decade, and since 1933 they have undergone 4 antigenic shifts: H0N1 (protoA, A0) in 1933-1946, H1N1 (methylene, A1) in 1946-1957, H2N2 (Asian A, A2) in 1957-1968, and H3N2 (hong Kong, A3) after 1968. Generally, there is a clear alternation between new and old subtypes, and after the new subtypes appear and become prevalent in a region, the old subtypes can no longer be separated. There are large and small variations between influenza b infections, but they are not classified into subtype changes. No antigenic variation has been found in influenza c viruses.
Although influenza b virus does not cause an influenza pandemic, it is circulating globally and annually like influenza a virus, causing local epidemics everywhere and can cause severe cases and death, and in some specific cases, the burden of disease is even greater than that of influenza a virus.
Acute and chronic respiratory infections are the most common human diseases, and the average annual respiratory infection rate of each human is more than 30% according to statistics, and the incidence rate and the fatality rate of children are higher. In addition to bacteria, viruses are one of the important pathogenic microorganisms causing respiratory tract infections. Rhinovirus pneumonia (HRV) is the main etiological agent of common cold in humans, and the main route of human rhinovirus invasion into the human body is the upper respiratory tract, after which the virus particles bind to ICAM-1 (CD 54 receptor) of epithelial cells. As the virus replicates and spreads, the infected cells release chemokines and cytokines, which signals can in turn activate inflammatory mediators. Infection occurs rapidly after 15 minutes after the virus enters the respiratory tract and binds to cell surface receptors. Only 50% of patients may experience less than two days of cold symptoms, 5% of cases may experience a latency of less than 20 hours, but still another 5% of cases may have a latency of more than half a day. Rhinovirus infection is intensified at temperatures of 33-35 ℃, which is also a major cause of its presence mainly in the nasal cavity.
After infection with Human Rhinovirus (HRV), symptoms of cold often appear, about 10-20% of adult colds are caused by HRV infection, and 15-30% of infant colds are caused by HRV. HRV is self-limiting in infection, but sometimes causes severe complications such as asthma, congestive heart failure, bronchiectasis, cystic fibrosis, and many infections are associated with other respiratory viruses, such as respiratory syncytial virus, adenovirus, parainfluenza virus, coronavirus, and enterovirus. It is worth mentioning that for children and susceptible groups with respiratory basic diseases, severe sequelae can also occur after HRV infection, which seriously affect the life quality of patients and bring heavy economic burden to families and society, so that the research on HRV is more and more paid attention by various circles.
Human parainfluenza virus (HPIV) is a virus that often causes lower respiratory tract infections in children, and is less pathogenic than Respiratory Syncytial Virus (RSV). Like RSV, human parainfluenza viruses can cause recurrent upper respiratory tract infections (e.g., colds and sore throats). It can also cause severe recurrent lower respiratory tract diseases (such as pneumonia, bronchitis and bronchiolitis), especially in the elderly and in people with immunodeficiency. The four subtypes of human parainfluenza virus have distinct clinical and epidemiological characteristics. The most typical clinical features of types I and II are the cause of laryngotracheobronchitis in children, with type I being the primary cause of laryngotracheobronchitis in children and type II being the next. Both type I and type II can cause other upper and lower respiratory tract diseases. Type III often leads to pneumonia and bronchiolitis. Type IV is difficult to detect, probably because it rarely causes severe disease. The incubation period of human parainfluenza virus is generally about 1 to 7 days. Epidemiological features are transmitted by close contact with infected persons or exposure to contaminants. However, infectious agents contact the mucous membranes of the human eyes, mouth or nose and cause infections, or infections by inhalation of droplets of respiratory secretions produced by sneezing and coughing. Human parainfluenza virus can survive in this suspended state for more than one hour. Human parainfluenza viruses are ubiquitous and most people have been infected in childhood. Serological monitoring indicated that 90% to 100% of children aged 5 years and older had antibodies against parainfluenza virus type III and approximately 75% had antibodies against parainfluenza virus types I and II.
Respiratory Syncytial Virus (RSV) is an RNA virus belonging to the family of the paramyxoviridae. The disease is spread by air droplets and close contact, is mostly seen in newborns and infants within 6 months, and has a latent period of 3-7 days; infants have severe symptoms, including hyperpyrexia, rhinitis, pharyngitis and laryngitis, and later manifested as bronchiolitis and pneumonia; the minority of sick children can be complicated by otitis media, pleuritis, myocarditis and the like. After infection in adults and older children, upper respiratory tract infection is mainly manifested. In the last decade, the syncytial virus pneumonia and the capillary bronchitis account for the first viral pneumonia of infants in China, the symptoms of the pneumonia and the pneumonia of parainfluenza virus, the pneumonia of mild influenza virus and the pneumonia of mild adenovirus can not be distinguished from each other in clinic, the treatment is mainly based on support and symptomatic treatment, and when secondary bacterial infection exists, the pneumonia and the pneumonia of mild adenovirus can be treated by antibacterial drugs. Preventing respiratory tract infection caused by other viruses.
Adenovirus (HAdV) is a non-enveloped particle with a diameter of 70-90 nm, consisting of 252 capsomeres arranged in a 20-hedron configuration. The diameter of each shell particle is 7-9 nm. Inside the capsid is a linear double-stranded DNA molecule of about 4.7kb with inverted repeats of about 100bp each at both ends. A circular structure of double stranded DNA may occur due to the 5' end of each DNA strand being covalently bound to a protein molecule of molecular mass 55X10 Da. Adenovirus infection is prevalent mainly in winter and spring, and is easily outbreak in kindergartens, schools and new military troops. Generally, adenoviruses are transmitted via the respiratory tract or contact, primarily via respiratory droplets, ocular secretions; intestinal infections are transmitted primarily through the digestive tract. The prevention measures are similar to the prevention of other respiratory tract and digestive tract infectious diseases, and mainly comprise the steps of washing hands and disinfecting the hands frequently, and avoiding contacting with the respiratory tract droplets of patients. Drinking water and eating vegetables and fruits at ordinary times, and paying attention to physical exercise; indoor ventilation is performed, and the indoor environment is kept clean; in the epidemic season of winter and spring, people can go to a public place with dense people as little as possible, and the mask is worn when people go out, so that the patients can be prevented from being contacted, and infection can be prevented. Once acute fever, sore throat and conjunctivitis occur, patients should be seen in hospital as soon as possible, and the disease should be isolated and treated as soon as possible. The disease condition of more than 5 people is reported to epidemic prevention departments in the area in time, and effective prevention and control measures are taken in time to avoid disease spreading. In the adenovirus epidemic season, the infant with upper respiratory tract infection in the nursery house should go home to separate and rest to avoid spreading and epidemic. The patients can see a doctor in a nearby hospital as much as possible after suffering from the diseases, and infusion in a large hospital observation room with more concentrated patients is avoided, so that cross infection is prevented. Severe cough and dyspnea are usually serious cases, and patients should be hospitalized in hospitals in time to avoid delaying the illness.
Pneumonia is a common respiratory medical disease. Pneumonia is classified into many types due to the difference in pathogens. Two common forms of pneumonia are mycoplasmal pneumonia and chlamydial pneumonia. Both mycoplasma and chlamydia pneumonia present similar clinical symptoms, but they differ. Two common forms of pneumonia are mycoplasmal pneumonia and chlamydial pneumonia. Although some of the same symptoms may appear in mycoplasma pneumonia and chlamydia pneumonia, they are distinct. Mycoplasma pneumonia and Chlamydia pneumonia all differ in pathogenic cause, clinical symptoms, therapeutic methods, and the like. Pneumonia must be treated in time. The pathogenic causes of mycoplasma pneumonia and chlamydial pneumonia are different. The main etiology of mycoplasma pneumonia is mycoplasma pneumoniae. And chlamydial pneumonia is caused by chlamydial infection. Both mycoplasmal pneumonia and chlamydial pneumonia are transmitted through respiratory secretions. The clinical symptoms of mycoplasma pneumonia and chlamydia pneumonia are also different. Common symptoms of mycoplasmal pneumonia include fever, anorexia, cough, chills, headache, pharyngalgia, substernal pain, and the like. Most patients cough heavily, with an initial dry cough followed by sputum. Infant patients develop wheezing and dyspnea. Serious patients may have complications such as pleural effusion, atelectasis, mediastinal pneumatosis, pneumothorax, necrotizing pneumonia, etc. While chlamydial pneumonia manifests as symptoms of upper respiratory tract infection in the early stages. The patient can have symptoms of fever, chills, myalgia, dry cough, non-pleura inflammatory chest pain, headache, discomfort, hypodynamia and the like. The symptoms of chlamydial pneumonia are minor compared to mycoplasmal pneumonia.
Influenza virus type a and B, HRV, HPIV, RSV, HAdV, MP, CP clinical treatment regimens differ, and therefore it is necessary to differentially diagnose which causative agent is and then treat it on a targeted basis; therefore, the IgM antibody combined qualitative detection project of influenza viruses of A type and B type, HRV, HPIV, RSV, HAdV, MP and CP can timely, conveniently, accurately and comprehensively detect which type of virus is infected, great benefit is brought to clinical diagnosis and treatment, and a powerful, comprehensive and accurate screening and diagnosis tool is provided for scenes of medical institutions of all levels, disease control centers and the like. Secondly, the detection sample is whole blood or serum or plasma, and the material is convenient and safe to obtain; the IgM antibodies of influenza virus A and B, HRV, HPIV, RSV, HAdV, MP and CP are the indexes of the infection of the current symptoms, and the false positive problem does not exist, so the method is more accurate and has great value for clinical diagnosis.
Generally, chick embryo or MDCK cell isolation culture and hemagglutination inhibition methods are adopted for virus confirmation, so that the operation is complicated, time and labor are wasted, and the requirement for rapid diagnosis is difficult to meet. With the development and application of rapid diagnosis technology, the types of influenza and 2019 rhinovirus products in the current market can be basically divided into 3 categories: nucleic acid diagnostics (PCR), enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography. The PCR method has the highest accuracy, is the first choice method for determining diagnosis, but has higher detection cost and longer detection time; the ELISA method is a classical method in hospital clinical laboratory and disease control system laboratory; the colloidal gold method has the advantages of quickest speed, mature and stable technology, simplicity, convenience and feasibility, and is suitable for universal screening and diagnosis of all levels of medical institutions, disease control centers and other occasions.
The Gold Immunochromatography (GICA) technique is a solid-phase membrane immunoassay technique using microporous membrane as carrier, which combines the gold immunochromatography technique with the protein chromatography technique. The colloidal gold immunochromatographic assay is a common immunochromatographic assay, is very suitable for field detection due to the characteristics of simple operation, time saving, low manufacturing cost, easy result interpretation and the like, and is widely applied to the fields of biology, medicines, foods and the like. Because the colloidal gold immunochromatographic assay is used for completing detection in one step, the interference factors in the detection process are more, the low sensitivity is a main factor for limiting the application range of the colloidal gold immunochromatographic assay, and the detection limit of the traditional colloidal gold immunochromatographic assay is higher than that of methods such as ELISA and the like.
In the colloidal gold immunochromatography assay, proteins are immobilized on nitrocellulose membranes (NC membranes) as capture reagents for a sample to be tested. Since the detection result completely depends on the good adsorption effect of the capture reagent on the membrane, the uniform and good adsorption of the protein on the membrane is very important for the detection result of the colloidal gold. If the amount of protein bound to the NC membrane is insufficient or the binding force of protein is not strong enough, a considerable problem occurs, and it is very obvious on the detection line of the detection result. If the amount of protein bound to the membrane is too low, the color development of the detection line is weak and the detection sensitivity is reduced in the result. If the protein is not firmly adsorbed to the NC membrane, the protein diffuses before adsorbing to the NC membrane, so that the detection line is wide, the color development is weak, the detection line is bright and clear, and the detection result is difficult to explain. Under extreme conditions, if the physical adsorption of the protein to the NC membrane is too weak, the protein assay and surfactant solution flowing through may wash the immobilized protein off the NC membrane, thereby revealing a wider or not clear detection line at all, making it difficult to interpret the detection results.
Disclosure of Invention
The invention provides a multinomial pathogen joint detection device and a preparation method thereof, aiming at solving the problems of insufficient protein adsorption amount and weak binding force of an NC membrane in the prior art.
The technical scheme adopted by the invention is as follows:
a multi-pathogen joint detection device comprises a sample pad, an immune colloidal gold antibody glass fiber membrane, an immune nitrocellulose membrane and an absorption pad which are respectively stuck on a plastic plate, wherein two ends of the nitrocellulose membrane are respectively lapped with the absorption pad and the immune colloidal gold antibody glass fiber membrane, and the other end of the immune colloidal gold antibody glass fiber membrane is lapped with the sample pad; the immune nitrocellulose membrane is provided with a first detection line T1, a second detection line T2, a third detection line T3, a fourth detection line T4, a fifth detection line T5, a sixth detection line T6 and a quality control line C, wherein an influenza virus A antigen is solid-phase on the first detection line T1, an influenza virus B antigen is solid-phase on the second detection line T2, a rhinovirus antigen is solid-phase on the third detection line T3, a parainfluenza virus antigen is solid-phase on the fourth detection line T4, a respiratory syncytial virus antigen is solid-phase on the fifth detection line T5, an adenovirus antigen is solid-phase on the sixth detection line T6, a goat anti-mouse IgG polyclonal antibody is sprayed on the quality control line C, and the first detection line T1, the second detection line T2, the third detection line T3, the fourth detection line T4, the fifth detection line T5 and the sixth detection line T6 which are arranged on the six lines respectively, and the quality control line C is arranged on each line.
The immunological nitrocellulose membrane of the invention is also provided with a seventh detection line T7 and an eighth detection line T8, wherein a mycoplasma pneumoniae antigen is solid phase on the seventh detection line T7, a chlamydia pneumoniae antigen is solid phase on the eighth detection line T8, and a quality control line C is respectively arranged on each of the two lines.
A preparation method of a multi-pathogen combined detection device comprises the following steps:
(a) Preparation of zinc oxide-chitosan-colloidal gold
1) Synthesis of N-carboxyethyl chitosan: adding 4.0g of chitosan into 200mL of water containing 2.80mL of acrylic acid, continuously stirring for 2 days at 50 ℃, after the reaction is finished, adding 1mol/L NaOH solution into the reaction mixed solution, adjusting the pH value of the solution to 10-12 to completely convert all carboxylic acids into sodium salts, pouring the mixed solution into acetone for re-precipitation, dialyzing in aqueous solution to remove unreacted acrylic acid, and freeze-drying after two days of dialysis to obtain N-carboxyethyl chitosan;
2) Preparing nano zinc oxide: weighing 1.3225gZnAc 2 ·2H 2 Dissolving O in 20mL of distilled water, slowly dropwise adding 9mL of 5mol/L NaOH solution, leading the solution to be white and turbid, continuously dropwise adding the solution until the turbidity is completely disappeared, then adding 11mL of distilled water, uniformly stirring, then pouring the mixture into a low-pressure reaction kettle, putting the reaction kettle into a drying oven, heating the mixture for 12 hours at 170 ℃, naturally cooling the mixture to room temperature to obtain precipitates, respectively washing the precipitates with water and ethanol, centrifuging the precipitates twice, and putting the precipitates into the drying oven to dry the precipitates for later use at 60 ℃;
3) Preparing a mixed solution of N-carboxyethyl chitosan and nano zinc oxide: adding 5.00g of N-carboxyethyl chitosan into 300mL of deionized water, violently stirring for 0.5h at room temperature, fully dissolving to obtain a water-soluble chitosan solution, mixing the water-soluble chitosan solution and nano zinc oxide according to a certain proportion, violently stirring the mixed solution for 3h, and uniformly mixing;
4) Preparing colloidal gold:
putting a round-bottom flask filled with 90-100ml of purified water into an electric heating jacket for heating, after heating for 10-12 minutes, starting a revolution control switch when water in the flask is boiled, regulating to 400rpm, slowly adding 1ml of 1% gold chloride solution along the edge of a vortex by using an injector, after the solution is boiled, quickly adding 1-2ml of 1% trisodium citrate solution, after the solution is cooled to room temperature, adding water, and weighing to complement the weight;
5) Preparing zinc oxide-chitosan-colloidal gold: crosslinking at normal temperature by using benzene dicarbaldehyde as a crosslinking agent; after crosslinking, carrying out vacuum drying on the obtained zinc oxide-chitosan-colloid Jin Xidi for 24h;
(b) Taking 1ml of zinc oxide-chitosan-colloidal gold cross-linked anti-human IgM antibody, ultrasonically dispersing uniformly, then dropwise adding the antibody while stirring, adding the antibody, reacting for about 1min, then ultrasonically treating the mixture on ultrasonic waves for 30 seconds, then reacting for 4 hours, adding BSA (bovine serum albumin) for sealing for 24 hours, centrifuging after sealing at the speed of 12000r/min for 20min, and reserving the centrifuged precipitate for later use;
(c) Respectively diluting the precipitated zinc oxide-chitosan-colloidal gold antibody centrifuged in the step (b) by using a colloidal gold antibody buffer solution to uniformly disperse the precipitated zinc oxide-chitosan-colloidal gold antibody, respectively obtaining immune colloid Jin Kangti solutions, and respectively spraying the immune colloid Jin Kangti solutions on a glass fiber pad by using an immune colloid Jin Kangti solution to prepare an immune colloidal gold antibody glass fiber membrane;
(d) Pretreating a nitrocellulose membrane by using a polydopamine treatment solution, and then spraying a detection line with an influenza A antigen, an influenza B antigen, a rhinovirus antigen, a parainfluenza virus antigen, a respiratory syncytial virus antigen and an adenovirus antigen, or spraying a detection line with an influenza A antigen, an influenza B antigen, a rhinovirus antigen, a parainfluenza virus antigen, a respiratory syncytial virus antigen, an adenovirus antigen, a mycoplasma pneumoniae antigen and a chlamydia pneumoniae antigen, and spraying a quality control line with a goat anti-mouse IgG antibody to prepare an immune nitrocellulose membrane;
(e) And (d) sequentially sticking the pretreated sample pad, the immune colloidal gold antibody glass fiber membrane prepared in the step (c), the immune nitrocellulose membrane prepared in the step (d) and absorbent paper on a plastic plate, cutting to obtain a detection reagent strip, and finally filling the detection reagent strip into a plastic shell.
In the step (a) of preparing the zinc oxide-chitosan-colloidal gold, 1) the synthesis of the N-carboxyethyl chitosan, when the N-carboxyethyl chitosan is dialyzed in aqueous solution, the intercepting molecular weight of the dialysis membrane adopted is 8000-12000g/mol.
In the step (a) of preparing the zinc oxide-chitosan-colloidal gold, 2) preparing nano zinc oxide particles, slowly dropwise adding 9mL 5mol/L NaOH solution, wherein the NaOH solution is newly prepared solution.
In the step (a) of preparing the zinc oxide-chitosan-colloidal gold, 3) preparing a mixed solution of N-carboxyethyl chitosan and nano zinc oxide, wherein the mixing ratio of the water-soluble chitosan solution and the nano zinc oxide is that the mass ratio of the N-carboxyethyl chitosan to the zinc oxide is 100/5, 100/8 or 100/10.
The colloidal gold antibody buffer solution of step (c) of the present invention comprises: tris-HCl solution at a concentration of 20mM, sucrose at 5%, trehalose at 1%, proclin at 1%, BSA at 1%, pH 8.5.
The method comprises the following steps of (d) pre-treating a nitrocellulose membrane with polydopamine treating solution: and (3) putting the nitrocellulose membrane into the polydopamine treatment solution, soaking for 1h, shaking at a low speed, taking out, washing for 3 times by using distilled water, and finally drying in a vacuum drying oven.
The polydopamine treating fluid has a polydopamine concentration of 1 percent and is filtered by a filter membrane of 0.22 mu m for later use.
The sample pad treatment solution adopted by the pre-treated sample pad in the step (e) of the invention comprises 0.1mol/LTris-HCL solution, 1% bovine serum albumin BSA, and 1% surfactant.
Dopamine (DA) has also been shown to have superior adhesion properties due to its combination of catechol functionality and lysine amino functionality. DA can be oxidized and polymerized in an alkaline solution to form a Polydopamine (PDA) layer with super-strong viscosity on the surface of the material, so that super-strong adhesion on the surface of various materials is realized. PDA is formed simply without organic solvent, and PDA layer can be formed on the surface by only immersing the material in alkaline Tris-HCl buffer solution containing DA or other alkaline solution, therefore, PDA is widely applied to surface modification of material. In conclusion, polydopamine can improve the coating efficiency of NC membrane protein in terms of both charge action and hydrophobic action.
Chitin is a renewable resource second to cellulose in the earth, chitosan is a deacetylated derivative thereof, and 2-amino-2-deoxy-beta-D-glucose is connected through a beta-1,4-glycosidic bond to form a polymer. The chitosan has the special functions of good biocompatibility, biodegradability, broad-spectrum antibacterial property, corrosion resistance, no antigenicity, hemostasis, wound healing promotion and the like, so that the chitosan becomes one of the biomacromolecules with the greatest application prospect in the tissue engineering scaffold material. Nano zinc oxide (ZnO), white hexagonal crystal or spherical particle with particle size less than 100nm, average particle size of 50nm, and specific surface area greater than 4m 2 (ii) in terms of/g. Has extremely high chemical activity and excellent catalytic and photocatalytic activity. The research discusses the influence of zinc oxide-chitosan-colloidal gold nanoparticles on NC membrane coated antibodies, firstly the antibodies for membrane scribing are combined with the zinc oxide-chitosan-colloidal gold nanoparticles, the mixture is sealed, centrifugally purified, unbound antibodies are removed, then the antibodies are redissolved to a certain proportion, and then the membrane scribing is carried out, so that the zinc oxide-chitosan-colloidal gold particles can be combined with a plurality of antibodies, the efficiency of coating the antibodies is increased, the coating efficiency of the antibodies is increased by means of the larger surface area of the zinc oxide-chitosan-colloidal gold particles, and the sensitivity of a colloidal gold immunochromatography experiment is improved.
In order to improve the sensitivity, stability and accuracy of a colloidal gold immunochromatography technique, a nitrocellulose membrane is pretreated by a polydopamine treatment solution, and an NC-coated antibody is combined with zinc oxide-chitosan-colloidal gold, so that the aim of improving various performance indexes of the test paper is fulfilled.
The invention has the beneficial effects that:
1. the detection device has a novel structure, coats the influenza A antigen, the influenza B antigen, the rhinovirus antigen, the parainfluenza virus antigen, the respiratory syncytial virus antigen, the adenovirus antigen, the mycoplasma pneumoniae antigen and the chlamydia pneumoniae antigen on the nitrocellulose membrane, has strong specificity, can simultaneously detect IgM antibodies of the influenza A virus, the influenza B virus, the rhinovirus, the parainfluenza virus, the respiratory syncytial virus and the adenovirus in a specimen, or IgM antibodies of the influenza A virus, the influenza B virus, the rhinovirus, the parainfluenza virus, the respiratory syncytial virus, the adenovirus, the mycoplasma pneumoniae and the chlamydia pneumoniae, and does not increase the complexity of production operation.
2. In the preparation step of the immune colloidal gold, the immune colloidal gold can be completely released by matching with a proper gold spraying buffer solution and a sample pad treatment solution, and simultaneously, the marker and the antibody can be firmly adsorbed together by the combination mode, so that the antibody or the antigen can not fall off from the surface of the marker, and the marking efficiency and the stability, sensitivity and precision of the immunochromatography methodology are improved; and under the same threshold value, the usage amount of the colloidal gold can be reduced, and the cost is saved.
3. The invention carries out polydopamine pretreatment on the nitrocellulose membrane, modifies the antibody coated on the nitrocellulose membrane, increases the coating efficiency of the antibody, improves the adsorption effect, and has more uniform protein distribution, thereby improving the sensitivity, the precision and the stability of the immunochromatography methodology.
4. The detection device does not need any special instrument and equipment, and has low detection cost.
5. The detection device is simple and convenient to operate, and does not need to be operated by professional staff. The practicability is strong.
Drawings
FIG. 1 is a schematic structural view of the present invention, in which detection lines T1, T2, T3, T4, T5 and T6 are respectively arranged on six lines in the transverse direction;
FIG. 2 isbase:Sub>A cross-sectional view A-A of FIG. 1;
FIG. 3 is a cross-sectional view B-B of FIG. 1;
FIG. 4 is a cross-sectional view C-C of FIG. 1;
FIG. 5 is a cross-sectional view D-D of FIG. 1;
FIG. 6 is a cross-sectional view E-E of FIG. 1;
FIG. 7 is a cross-sectional view F-F of FIG. 1;
FIG. 8 is a schematic view of the structure in which the detection lines T1, T2, T3, T4, T5, T6, T7 and T8 of the present invention are transversely arranged on eight lines, respectively;
FIG. 9 is a sectional view taken along line G-G of FIG. 8;
fig. 10 is a sectional view H-H of fig. 8.
Detailed Description
As shown in fig. 1 to 7, a sample pad 1, an immune colloidal gold antibody glass fiber membrane 2, an immune nitrocellulose membrane 3, and an absorption pad 4 are respectively adhered to a plastic plate 5, two ends of the nitrocellulose membrane 3 are respectively lapped with the absorption pad 4 and the immune colloidal gold antibody glass fiber membrane 2, and the other end of the immune colloidal gold antibody glass fiber membrane 2 is lapped with the sample pad 1; the immune nitrocellulose membrane is characterized in that a first detection line T1, a second detection line T2, a third detection line T3, a fourth detection line T4, a fifth detection line T5, a sixth detection line T6 and a quality control line C are arranged on the immune nitrocellulose membrane 3, an influenza virus A antigen is solid-phase on the first detection line T1, an influenza virus B antigen is solid-phase on the second detection line T2, a rhinovirus antigen is solid-phase on the third detection line T3, a parainfluenza virus antigen is solid-phase on the fourth detection line T4, a respiratory syncytial virus antigen is solid-phase on the fifth detection line T5, an adenovirus antigen is solid-phase on the sixth detection line T6, a goat anti-mouse IgG polyclonal antibody is sprayed on the quality control line C, and the first detection line T1, the second detection line T2, the third detection line T3, the fourth detection line T4, the fifth detection line T5 and the sixth detection line T6 which are arranged on the immune nitrocellulose membrane are respectively arranged on the six lines, and the quality control line C is respectively arranged on each line.
As shown in fig. 8 to 10, a seventh detection line T7 and an eighth detection line T8 are further disposed on the nitrocellulose membrane 3, the mycoplasma pneumoniae antigen is immobilized on the seventh detection line T7, the chlamydia pneumoniae antigen is immobilized on the eighth detection line T8, and a quality control line C is disposed on each of the two lines.
Example 1
Comprises the following steps:
(a) Preparation of zinc oxide-chitosan-colloidal gold
1) Synthesis of N-carboxyethyl chitosan: adding 4.0g of chitosan into 200mL of water containing 2.80mL of acrylic acid, continuously stirring for 2 days at 50 ℃, after the reaction is finished, adding 1mol/L NaOH solution into the reaction mixed solution, adjusting the pH value of the solution to 10 to convert all carboxylic acids into sodium salts, pouring the mixed solution into acetone for heavy precipitation, dialyzing in aqueous solution, intercepting the molecular weight of 8000g/mol by a dialysis membrane, removing unreacted acrylic acid, dialyzing for two days, and freeze-drying to obtain 3.6g of N-carboxyethyl chitosan with the yield of 90%;
2) Preparing nano zinc oxide: weighing 1.3225gZnAc 2 ·2H 2 Dissolving O in 20mL of distilled water, slowly dropwise adding 9mL of newly prepared 5mol/L NaOH solution, enabling the solution to be white and turbid first, enabling the turbidity to disappear completely if the solution is continuously dropwise added, then adding 11mL of distilled water, stirring uniformly, then pouring the mixture into a low-pressure reaction kettle, putting the reaction kettle into an oven, heating the reaction kettle at 170 ℃ for 12 hours, naturally cooling the reaction kettle to room temperature to obtain precipitates, respectively washing the precipitates with water and ethanol, centrifuging the precipitates twice, and putting the precipitates into the oven to dry the precipitates at 60 ℃ for later use;
3) Preparing a mixed solution of N-carboxyethyl chitosan and nano zinc oxide: adding 5.00g of N-carboxyethyl chitosan into 300mL of deionized water, violently stirring for 0.5h at room temperature, fully dissolving to obtain a water-soluble chitosan solution, mixing the water-soluble chitosan solution and nano zinc oxide according to a certain proportion to ensure that the mass ratio of the N-carboxyethyl chitosan to the zinc oxide is 100/5, and violently stirring the mixed solution for 3h to uniformly mix the mixed solution;
4) Preparing colloidal gold:
putting a round-bottom flask filled with 90ml of purified water into an electric heating jacket for heating, after heating for 10 minutes, starting a revolution control switch when the water in the flask is boiled, regulating the revolution control switch to 400rpm, slowly adding 1ml of 1% gold chloride solution along the edge of a vortex by using an injector, after the solution begins to boil, quickly adding 1ml of 1% trisodium citrate solution, and after the solution is cooled to the room temperature, adding water and weighing for complementing the weight;
5) Preparing zinc oxide-chitosan-colloidal gold: crosslinking at normal temperature by using benzene dicarbaldehyde as a crosslinking agent; after the crosslinking is finished, the obtained zinc oxide-chitosan-colloid Jin Xidi is dried in vacuum for 24 hours;
(b) Taking 1ml of zinc oxide-chitosan-colloidal gold suspension, ultrasonically dispersing the suspension uniformly, then dropwise adding the antibody while stirring, reacting for about 1min after adding the antibody, then performing ultrasonic treatment on the suspension for 30 seconds by ultrasonic waves, reacting for 4 hours again, adding BSA (bovine serum albumin) for sealing for 24 hours, centrifuging at the speed of 12000r/min for 20min after sealing, and reserving the centrifuged precipitate for later use;
(c) Respectively diluting the precipitated zinc oxide-chitosan-colloidal gold antibody centrifuged in the step (b) by using a colloidal gold antibody buffer solution to uniformly disperse the precipitated zinc oxide-chitosan-colloidal gold antibody to obtain immune colloid Jin Kangti solutions respectively, and spraying the immune colloid Jin Kangti solutions onto a glass fiber pad by using an immune colloid Jin Kangti solution respectively to prepare an immune colloidal gold antibody glass fiber membrane 2;
the colloidal gold antibody buffer solution comprises: tris-HCl solution with the concentration of 20mM, sucrose concentration of 5%, trehalose concentration of 1%, proclin concentration of 1%, BSA concentration of 1%, and pH of 8.5;
(d) Pretreating a nitrocellulose membrane by using a polydopamine treatment solution, and then spraying a detection line with an influenza A antigen, an influenza B antigen, a rhinovirus antigen, a parainfluenza virus antigen, a respiratory syncytial virus antigen and an adenovirus antigen, or spraying a detection line with an influenza A antigen, an influenza B antigen, a rhinovirus antigen, a parainfluenza virus antigen, a respiratory syncytial virus antigen, an adenovirus antigen, a mycoplasma pneumoniae antigen and a chlamydia pneumoniae antigen, and spraying a quality control line with a goat anti-mouse IgG antibody to prepare an immune nitrocellulose membrane 3; wherein the concentration of polydopamine in the polydopamine treatment solution is 1%, the polydopamine treatment solution is filtered by a 0.22-micrometer filter membrane for standby, the nitrocellulose membrane is put into the polydopamine treatment solution to be soaked for 1 hour, is shaken at a low speed, is taken out and then is washed for 3 times by distilled water, and finally is dried in a vacuum drying oven;
(e) Sequentially sticking the pretreated sample pad 1, the immune colloidal gold antibody glass fiber membrane 2 prepared in the step (c), the immune nitrocellulose membrane 3 prepared in the step (d) and absorbent paper 4 on a plastic plate 5, cutting to obtain a detection reagent strip, and finally filling the detection reagent strip into a plastic shell; the sample pad treatment fluid adopted by the pretreated sample pad comprises 0.1mol/LTris-HCL fluid, 1% bovine serum albumin BSA and 1% surfactant.
Example 2
Comprises the following steps:
(a) Preparation of zinc oxide-chitosan-colloidal gold
1) Synthesis of N-carboxyethyl chitosan: adding 4.0g of chitosan into 200mL of water containing 2.80mL of acrylic acid, continuously stirring for 2 days at 50 ℃, after the reaction is finished, adding 1mol/L NaOH solution into the reaction mixed solution, adjusting the pH value of the solution to 11 to convert all carboxylic acids into sodium salts, pouring the mixed solution into acetone for re-precipitation, dialyzing in aqueous solution, intercepting the molecular weight of the mixed solution by a dialysis membrane to 10000g/mol, removing unreacted acrylic acid, dialyzing for two days, and freeze-drying to obtain N-carboxyethyl chitosan;
2) Preparing nano zinc oxide: weighing 1.3225gZnAc 2 ·2H 2 Dissolving O in 20mL of distilled water, slowly dropwise adding 9mL of newly prepared 5mol/L NaOH solution, enabling the solution to be white and turbid first, enabling the turbidity to disappear completely if the solution is continuously dropwise added, then adding 11mL of distilled water, stirring uniformly, then pouring the mixture into a low-pressure reaction kettle, putting the reaction kettle into an oven, heating the reaction kettle at 170 ℃ for 12 hours, naturally cooling the reaction kettle to room temperature to obtain precipitates, respectively washing the precipitates with water and ethanol, centrifuging the precipitates twice, and putting the precipitates into the oven to dry the precipitates at 60 ℃ for later use;
3) Preparing a mixed solution of N-carboxyethyl chitosan and nano zinc oxide: adding 5.00g of N-carboxyethyl chitosan into 300mL of deionized water, violently stirring for 0.5h at room temperature, fully dissolving to obtain a water-soluble chitosan solution, mixing the water-soluble chitosan solution and nano zinc oxide according to a certain proportion to ensure that the mass ratio of the N-carboxyethyl chitosan to the zinc oxide is 100/8, and violently stirring the mixed solution for 3h to uniformly mix the mixed solution;
4) Preparing colloidal gold:
putting a round-bottom flask filled with 95ml of purified water into an electric heating jacket for heating, after heating for 11 minutes, turning on a revolution control switch when water in the flask is boiled, adjusting to 400rpm, slowly adding 1ml of 1% gold chloride solution along the edge of a vortex by using an injector, after the solution begins to boil, quickly adding 1.5ml of 1% trisodium citrate solution, after the solution is cooled to room temperature, adding water and weighing to complement the weight;
5) Preparing zinc oxide-chitosan-colloidal gold: crosslinking at normal temperature by using benzene dicarbaldehyde as a crosslinking agent; after the crosslinking is finished, the obtained zinc oxide-chitosan-colloid Jin Xidi is dried in vacuum for 24 hours;
(b) Taking 1ml of zinc oxide-chitosan-colloidal gold cross-linked anti-human IgM antibody, ultrasonically dispersing uniformly, then dropwise adding the antibody while stirring, adding the antibody, reacting for about 1min, then ultrasonically treating the mixture on ultrasonic waves for 30 seconds, then reacting for 4 hours, adding BSA (bovine serum albumin) for sealing for 24 hours, centrifuging after sealing at the speed of 12000r/min for 20min, and reserving the centrifuged precipitate for later use;
(c) Respectively diluting the precipitated zinc oxide-chitosan-colloidal gold antibody centrifuged in the step (b) by using a colloidal gold antibody buffer solution to uniformly disperse the precipitated zinc oxide-chitosan-colloidal gold antibody to obtain immune colloid Jin Kangti solutions respectively, and spraying the immune colloid Jin Kangti solutions onto a glass fiber pad by using an immune colloid Jin Kangti solution respectively to prepare an immune colloidal gold antibody glass fiber membrane 2;
the colloidal gold antibody buffer solution comprises: tris-HCl solution with the concentration of 20mM, sucrose concentration of 5%, trehalose concentration of 1%, proclin concentration of 1%, BSA concentration of 1%, and pH of 8.5;
(d) Pretreating a nitrocellulose membrane by using a polydopamine treatment solution, and then spraying a detection line with an influenza A antigen, an influenza B antigen, a rhinovirus antigen, a parainfluenza virus antigen, a respiratory syncytial virus antigen and an adenovirus antigen, or spraying a detection line with an influenza A antigen, an influenza B antigen, a rhinovirus antigen, a parainfluenza virus antigen, a respiratory syncytial virus antigen, an adenovirus antigen, a mycoplasma pneumoniae antigen and a chlamydia pneumoniae antigen, and spraying a quality control line with a goat anti-mouse IgG antibody to prepare an immune nitrocellulose membrane 3; wherein the concentration of polydopamine in the polydopamine treatment solution is 1%, the polydopamine treatment solution is filtered by a 0.22-micrometer filter membrane for standby, the nitrocellulose membrane is put into the polydopamine treatment solution to be soaked for 1 hour, is shaken at a low speed, is taken out and then is washed for 3 times by distilled water, and finally is dried in a vacuum drying oven;
(e) Sequentially sticking the pretreated sample pad 1, the immune colloidal gold antibody glass fiber membrane 2 prepared in the step (c), the immune nitrocellulose membrane 3 prepared in the step (d) and absorbent paper 4 on a plastic plate 5, cutting to obtain a detection reagent strip, and finally filling the detection reagent strip into a plastic shell; the sample pad treatment fluid adopted by the pretreated sample pad comprises 0.1mol/LTris-HCL fluid, 1% bovine serum albumin BSA and 1% surfactant.
Example 3
Comprises the following steps:
(a) Preparation of zinc oxide-chitosan-colloidal gold
1) Synthesis of N-carboxyethyl chitosan: adding 4.0g of chitosan into 200mL of water containing 2.80mL of acrylic acid, continuously stirring for 2 days at 50 ℃, after the reaction is finished, adding 1mol/L NaOH solution into the reaction mixed solution, adjusting the pH value of the solution to 12 to convert all carboxylic acids into sodium salts, pouring the mixed solution into acetone for re-precipitation, dialyzing in aqueous solution, intercepting the molecular weight by a dialysis membrane to 12000g/mol, removing unreacted acrylic acid, dialyzing for two days, and freeze-drying to obtain N-carboxyethyl chitosan;
2) Preparing nano zinc oxide: weighing 1.3225gZnAc 2 ·2H 2 Dissolving O in 20mL of distilled water, slowly dropwise adding 9mL of newly prepared 5mol/L NaOH solution, enabling the solution to be white and turbid first, enabling the turbidity to disappear completely if the solution is continuously dropwise added, then adding 11mL of distilled water, stirring uniformly, then pouring the mixture into a low-pressure reaction kettle, putting the reaction kettle into an oven, heating the reaction kettle at 170 ℃ for 12 hours, naturally cooling the reaction kettle to room temperature to obtain precipitates, respectively washing the precipitates with water and ethanol, centrifuging the precipitates twice, and putting the precipitates into the oven to dry the precipitates at 60 ℃ for later use;
3) Preparing a mixed solution of N-carboxyethyl chitosan and nano zinc oxide: adding 5.00g of N-carboxyethyl chitosan into 300mL of deionized water, violently stirring for 0.5h at room temperature, fully dissolving to obtain a water-soluble chitosan solution, mixing the water-soluble chitosan solution and nano zinc oxide according to a certain proportion to ensure that the mass ratio of the N-carboxyethyl chitosan to the zinc oxide is 100/10, and violently stirring the mixed solution for 3h to uniformly mix the mixed solution;
4) Preparing colloidal gold:
putting a round-bottom flask filled with 100ml of purified water into an electric heating jacket for heating, after heating for 12 minutes, turning on a revolution control switch when water in the flask is boiled, adjusting to 400rpm, slowly adding 1ml of 1% gold chloride solution along the edge of a vortex by using an injector, after the solution begins to boil, quickly adding 2ml of 1% trisodium citrate solution, after the solution is cooled to room temperature, adding water and weighing to complement the weight;
5) Preparing zinc oxide-chitosan-colloidal gold: crosslinking at normal temperature by using benzene dicarbaldehyde as a crosslinking agent; after the crosslinking is finished, the obtained zinc oxide-chitosan-colloid Jin Xidi is dried in vacuum for 24 hours;
(b) Taking 1ml of zinc oxide-chitosan-colloidal gold cross-linked anti-human IgM antibody, ultrasonically dispersing uniformly, then dropwise adding the antibody while stirring, adding the antibody, reacting for about 1min, then ultrasonically treating the mixture on ultrasonic waves for 30 seconds, then reacting for 4 hours, adding BSA (bovine serum albumin) for sealing for 24 hours, centrifuging after sealing at the speed of 12000r/min for 20min, and reserving the centrifuged precipitate for later use;
(c) Respectively diluting the precipitated zinc oxide-chitosan-colloidal gold antibody centrifuged in the step (b) by using a colloidal gold antibody buffer solution to uniformly disperse the precipitated zinc oxide-chitosan-colloidal gold antibody to obtain immune colloid Jin Kangti solutions respectively, and spraying the immune colloid Jin Kangti solutions onto a glass fiber pad by using an immune colloid Jin Kangti solution respectively to prepare an immune colloidal gold antibody glass fiber membrane 2;
the colloidal gold antibody buffer solution comprises: tris-HCl solution with the concentration of 20mM, sucrose concentration of 5%, trehalose concentration of 1%, proclin concentration of 1%, BSA concentration of 1%, and pH of 8.5;
(d) Pretreating a nitrocellulose membrane by using a polydopamine treatment solution, and then spraying a detection line with an influenza A antigen, an influenza B antigen, a rhinovirus antigen, a parainfluenza virus antigen, a respiratory syncytial virus antigen and an adenovirus antigen, or spraying a detection line with an influenza A antigen, an influenza B antigen, a rhinovirus antigen, a parainfluenza virus antigen, a respiratory syncytial virus antigen, an adenovirus antigen, a mycoplasma pneumoniae antigen and a chlamydia pneumoniae antigen, and spraying a quality control line with a goat anti-mouse IgG antibody to prepare an immune nitrocellulose membrane 3; wherein the polydopamine treatment solution has a polydopamine concentration of 1%, is filtered through a 0.22-micron filter membrane for later use, and the nitrocellulose membrane is placed into the polydopamine treatment solution to be soaked for 1h, is shaken at a low speed, is washed for 3 times by distilled water after being taken out, and is finally dried in a vacuum drying oven;
(e) Sequentially sticking the pretreated sample pad 1, the immune colloidal gold antibody glass fiber membrane 2 prepared in the step (c), the immune nitrocellulose membrane 3 prepared in the step (d) and absorbent paper 4 on a plastic plate 5, cutting to obtain a detection reagent strip, and finally filling the detection reagent strip into a plastic shell; the sample pad treatment fluid adopted by the pretreated sample pad comprises 0.1mol/LTris-HCL fluid, 1% bovine serum albumin BSA and 1% surfactant.
The invention is further illustrated below by means of experimental examples.
Experimental example 1 comparison of adsorption capacities of Polydopamine treating solutions to nitrocellulose Membrane antibodies
1 materials and methods
1, material: nitrocellulose membrane, pore size 4.5um, available from general electric company of USA, polydopamine available from Sigma
2 nitrocellulose membrane treatment method
2.1 preparation of Polydopamine treating fluid
Preparing a polydopamine treatment fluid: the polydopamine is prepared into 1% concentration by distilled water, and is filtered by a 0.22 μm filter membrane for standby.
2.2 Nitrocellulose Membrane treatment method
And (3) putting the nitrocellulose membrane into the polydopamine treatment solution, soaking for 1h, shaking at a low speed, taking out, washing for 3 times by using distilled water, and finally drying in a vacuum drying oven.
3 Experimental methods
The untreated and treated nitrocellulose membranes were used to prepare IgM joint test paper for influenza a, influenza B, rhinovirus, parainfluenza, respiratory syncytial virus, adenovirus, mycoplasma pneumoniae, chlamydia pneumoniae according to the above procedures, and the differences in the adsorption and stability indexes of untreated and treated nitrocellulose membranes were compared with each other according to the test paper instructions.
4, results:
4.1 comparison of protein adsorption Capacity
The polydopamine-treated group test paper and the non-treated group test paper are respectively added into a sample to be detected, and the protein adsorption capacity of the treated membrane is judged by observing the color development condition, and the result is shown in table 1. The result shows that the treated nitrocellulose membrane is obviously better than the untreated membrane in the aspect of solution wettability, the color of the positive strip of the treated membrane is slightly dark, and particularly when the concentration is lower, the reaction sensitivity is improved, which shows that the protein adsorption capacity is obviously enhanced, and the reaction sensitivity is improved. The adsorption effect of the polydopamine group is obviously better than that of the untreated group.
TABLE 1 comparison of the adsorption Capacity of nitrocellulose membranes
Figure GDA0003752438980000151
4.2 comparison of stability of nitrocellulose membranes
The stability of the adsorbed protein on the nitrocellulose membrane after treatment was judged by taking the polydopamine-treated group and untreated group test paper and observing the color development through 37 ℃ accelerated experiments, and the results are shown in table 2. The results in Table 2 and the results in Table 1 show that the color change of the nitrocellulose membrane after the treatment is basically consistent with that before 10 days, and the stability is good.
TABLE 2 accelerated stability comparison of nitrocellulose membranes (10 days at 37 ℃ C.)
Figure GDA0003752438980000152
Experimental example 2 Zinc oxide-Chitosan-colloidal gold nanoparticles-modified anti-human IgM antibody
1 materials and methods
1.1 materials: nitrocellulose membrane, pore size 4.5um, available from general electric company of USA
1.2 Zinc oxide-Chitosan-colloidal gold nanoparticle modified anti-human IgM antibody
Zinc oxide-chitosan-colloidal gold is used as a carrier, 200 mu L of zinc oxide-chitosan-colloidal gold nanoparticle (1 mg/mL) solution and 25 mu of antihuman IgM antibody (40 mu mol/L) solution are added into ultrapure water, and the final reaction system is 1mL. And after the mixture is fully and uniformly mixed, setting the temperature of a shaking table to be 25 ℃ and the rotating speed to be 200r/m, and carrying out light-proof shake culture on the mixed solution in the shaking table for 2 hours. The mixed solution after the reaction was thoroughly centrifuged and washed 4 times with ultrapure water by an ultracentrifuge at a rotational speed of 13 000r/m, and the excess unreacted antibody in the supernatant was removed. The obtained precipitate is the zinc oxide-chitosan-colloidal gold-anti-human IgM antibody probe compound, and the volume of the compound is determined to be 1mL by ultrapure water and the compound is stored at 4 ℃.
2 method of experiment
The detection test paper is prepared by the zinc oxide-chitosan-colloidal gold nanoparticle modified anti-human IgM antibody and the unmodified anti-human IgM antibody according to the process flows of the above embodiments, the test flow refers to the test paper specification, and the difference of the protein adsorption force and the stability index of the zinc oxide-chitosan-colloidal gold nanoparticle treatment and the protein adsorption force of the non-treatment is compared.
3 results
3.1 comparison of protein adsorption Capacity
The test paper of the zinc oxide-chitosan-colloidal gold nanoparticle treatment group and the test paper of the untreated group are respectively added into a sample to be detected, and the protein adsorption capacity of the treated membrane is judged by observing the color development condition, and the result is shown in table 3. The result shows that the zinc oxide-chitosan-colloidal gold nanoparticle modified group membrane improves the reaction sensitivity particularly at lower concentration, which shows that the protein adsorption capacity is obviously enhanced and the reaction sensitivity is improved.
TABLE 3 comparison of protein adsorption Capacity of Zinc oxide-Chitosan-colloidal gold nanoparticle modifications
Figure GDA0003752438980000161
3.2 comparison of stability
Test paper of the zinc oxide-chitosan-colloidal gold nanoparticle treatment group and the test paper of the non-treatment group are taken, and the stability of the protein adsorbed on the nitrocellulose membrane modified by the zinc oxide-chitosan-colloidal gold is judged by observing the color development condition through an accelerated experiment at 37 ℃, and the result is shown in table 4. The results in Table 4 and the results in Table 3 show that the color change of the nitrocellulose membrane after the treatment is basically consistent with that before 10 days, and the stability is good.
TABLE 4 accelerated stability comparison (10 days at 37 ℃ C.)
Figure GDA0003752438980000171
Experimental example 3 research on detection capability of multi-pathogen joint detection device in practical application to clinical specimens
1. General data of the study
In the clinical test, the serum of 1002 patients is selected in the first clinical hospital of the department of traditional Chinese medicine and science and technology of Jilin province and the people hospital of Jilin province, blood samples are detected, and the positive rate of any one detection line at least reaches 30%. Blood samples were tested in the clinical trial, and serum plasma samples of 334 patients were collected in the center. All the selected samples are tested, and the samples in the number have enough statistical significance to evaluate the consistency of the detection result of the serum of the test paper and the detection result of the control paper.
2. Consistency analysis of influenza A IgM assay results
In the qualitative detection process of influenza virus A IgM in serum, 186 cases exist in which the test reagent and the control reagent are positive; the test reagent is positive, and 0 cases exist when the control reagent is negative; 0 cases with negative test reagents and positive control reagents; 148 cases of test reagents and control reagents which are negative; p =1,P > 0.05, with no statistical significance for the differences between groups. The Kappa test coefficient was 1 and greater than 0.75, indicating better agreement of the test reagents with the control reagents.
The results are given in Table 5,6;
TABLE 5 qualitative determination results of influenza A IgM antibodies with two reagents
Figure GDA0003752438980000181
TABLE 6 compliance of the assessment agent with respect to the reference agent
Figure GDA0003752438980000182
3. Consistency analysis of influenza B IgM assay results
In the qualitative detection process of influenza virus B IgM in serum, 38 positive cases of test reagents and control reagents are available; test reagent positive, control reagent negative has 0 cases; 0 cases with negative test reagents and positive control reagents; 29 negative test reagents and control reagents are used; p =1,P > 0.05, with no statistical significance for the differences between groups. The Kappa test coefficient was 1 and greater than 0.75, indicating better agreement of the test reagents with the control reagents. The number of positive consistent cases of the test reagent is 38, the coincidence rate is 100%, the number of negative consistent cases is 29, the coincidence rate is 100%, the number of overall consistent cases of the test reagent is 67, and the overall coincidence rate is 100%. The result shows that the difference between the test reagent and the control reagent has no statistical significance and better consistency. The results are shown in tables 7 and 8;
TABLE 7 qualitative determination results of influenza B IgM antibodies with two reagents
Figure GDA0003752438980000191
TABLE 8 test agent compliance relative to reference
Figure GDA0003752438980000192
4. Consistency analysis of Human Rhinovirus (HRV) IgM assay results
In the qualitative detection process of Human Rhinovirus (HRV) IgM in serum, 173 cases exist in which the test reagent and the control reagent are positive; the test reagent is positive, and 4 cases of the control reagent are negative; 2 cases with negative test reagent and positive control reagent; 155 cases of test reagent and control reagent which are negative are provided; with McNemar test, P =0.6875, P > 0.05, no statistical significance for the differences between groups. The Kappa test coefficient was 1 and greater than 0.75, indicating better agreement of the test reagents with the control reagents. The number of positive concordant cases of the test reagent is 173, the concordant rate is 98.86%, the number of negative concordant cases is 155, the concordant rate is 97.48%, the number of overall concordant cases of the test reagent is 328, and the overall concordant rate is 98.20%. The result shows that the difference between the test reagent and the control reagent has no statistical significance and better consistency. The results are shown in tables 9 and 10;
TABLE 9 qualitative determination results of two reagents rhinovirus (HRV) IgM antibody
Figure GDA0003752438980000201
TABLE 10 compliance of the assessment agent with respect to the reference agent
Figure GDA0003752438980000202
5. Consistency analysis of human parainfluenza virus (HPIV) IgM assay results
In the qualitative detection process of human parainfluenza virus (HPIV) IgM antibody in serum, 50 cases exist in which the test reagent and the control reagent are positive; the test reagent is positive, and 0 cases exist when the control reagent is negative; 0 cases with negative test reagents and positive control reagents; the test reagent and the control reagent are negative, and 17 cases exist; p is more than 0.05, and the difference between groups has no statistical significance. The Kappa test coefficient was 1 and greater than 0.75, indicating better agreement of the test reagents with the control reagents. The number of positive consistent cases of the test reagent is 50, the coincidence rate is 100%, the number of negative consistent cases is 17, the coincidence rate is 100%, the number of overall consistent cases of the test reagent is 67, and the overall coincidence rate is 100%. The result shows that the difference between the test reagent and the control reagent has no statistical significance and better consistency. The results are shown in tables 11 and 12;
TABLE 11 qualitative determination of two reagents human parainfluenza virus (HPIV) IgM antibody
Figure GDA0003752438980000211
TABLE 12 compliance of the assessment agent with respect to the reference agent
Figure GDA0003752438980000212
6. Consistency analysis of human Respiratory Syncytial Virus (RSV) IgM assay results
In the process of qualitatively detecting the human Respiratory Syncytial Virus (RSV) IgM in the serum, 192 cases exist in which the test reagent and the control reagent are positive; the test reagent is positive, and 0 cases exist when the control reagent is negative; 0 cases with negative test reagents and positive control reagents; 142 cases exist where the test reagent and the control reagent are negative; using McNemar test, P =1,P > 0.05, the differences between groups were not statistically significant. The Kappa test coefficient was 1 and greater than 0.75, indicating better agreement of the test reagents with the control reagents. The number of positive consistent cases of the test reagent is 192, the coincidence rate is 100%, the number of negative consistent cases is 142, the coincidence rate is 100%, the overall consistent case number of the test reagent is 334, and the overall coincidence rate is 100%. The result shows that the difference between the test reagent and the control reagent has no statistical significance and better consistency. The results are shown in tables 13 and 14;
TABLE 13 qualitative determination of human Respiratory Syncytial Virus (RSV) IgM antibodies with two reagents
Figure GDA0003752438980000221
TABLE 14 test agent compliance relative to reference
Figure GDA0003752438980000222
7. Consistency analysis of results of human adenovirus (HAdV) IgM assay
In the qualitative detection process of human adenovirus (HAdV) IgM antibody in serum, 39 cases exist in which the test reagent and the control reagent are positive; test reagent positive, control reagent negative has 0 cases; 0 cases with negative test reagents and positive control reagents; 28 negative test reagents and 28 negative control reagents are available; p =1 > 0.05, with no statistical significance for the differences between groups. The Kappa test coefficient was 1 and greater than 0.75, indicating better agreement of the test reagents with the control reagents. The number of positive consistent cases of the test reagent is 39, the coincidence rate is 100%, the number of negative consistent cases is 28, the coincidence rate is 100%, the number of overall consistent cases of the test reagent is 67, and the overall coincidence rate is 100%. The result shows that the difference between the detection results of the test reagent and the control reagent has no statistical significance and has better consistency. The results are shown in tables 15 and 16;
TABLE 15 qualitative determination of two reagents human adenovirus (HAdV) IgM antibody
Figure GDA0003752438980000231
TABLE 16 test agent compliance relative to reference
Figure GDA0003752438980000232
8. Consistency analysis of human Mycoplasma Pneumoniae (MP) IgM assay results
In the qualitative detection process of the human Mycoplasma Pneumoniae (MP) IgM antibody in serum, 15 positive test reagents and 15 positive control reagents are provided; the test reagent is positive, and 0 cases exist when the control reagent is negative; 0 cases with negative test reagents and positive control reagents; the test reagent and the control reagent are negative, and 52 cases exist; with McNemar test, P =1,P > 0.05, no statistical difference between groups was significant. The Kappa test coefficient was 1 and greater than 0.75, indicating better agreement of the test reagents with the control reagents. The number of positive matching cases of the test reagents is 15, the matching rate is 100%, the number of negative matching cases is 52, the matching rate is 100%, the total number of matching cases of the test reagents is 67, and the total matching rate is 100%. The result shows that the difference between the test reagent and the control reagent has no statistical significance and has better consistency. The results are shown in tables 17 and 18;
TABLE 17 qualitative determination of two reagents Mycoplasma Pneumoniae (MP) IgM antibody
Figure GDA0003752438980000241
TABLE 18 compliance of the assessment agent with respect to the reference agent
Figure GDA0003752438980000242
9. Consistency analysis of human Chlamydia Pneumoniae (CP) IgM assay results
In the qualitative detection process of human Chlamydia Pneumoniae (CP) IgM antibody in serum, 34 positive cases exist in the test reagent and the control reagent; the test reagent is positive, and the control reagent is negative, wherein the number of the test reagent is 2; 1 case with negative test reagent and positive control reagent; 297 cases exist when the test reagent and the control reagent are negative; using McNemar test, P =1,P > 0.05, the differences between groups were not statistically significant. The Kappa test coefficient was 0.95 and greater than 0.75, indicating better consistency. The number of positive matching cases of the test reagent was 34, the matching rate was 97.14%, the number of negative matching cases was 297, the matching rate was 99.33%, the number of overall matching cases of the test reagent was 331, and the overall matching rate was 99.10%. The difference between groups of detection results of the test reagent and the contrast reagent has no statistical significance and has better consistency. The results are shown in tables 19 and 20;
TABLE 19 qualitative determination of Chlamydia Pneumoniae (CP) IgM antibodies with two reagents
Figure GDA0003752438980000251
TABLE 20 compliance of the assessment agent with respect to the reference agent
Figure GDA0003752438980000252
The above description is only a preferred example of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A preparation method of a multi-pathogen combined detection device is characterized by comprising the following steps:
(a) Preparation of zinc oxide-chitosan-colloidal gold
1) Synthesis of N-carboxyethyl chitosan: adding 4.0g of chitosan into 200mL of water containing 2.80mL of acrylic acid, continuously stirring for 2 days at 50 ℃, after the reaction is finished, adding 1mol/L NaOH solution into the reaction mixed solution, adjusting the pH value of the solution to 10-12 to completely convert all carboxylic acids into sodium salts, pouring the mixed solution into acetone for re-precipitation, dialyzing in aqueous solution to remove unreacted acrylic acid, and freeze-drying after two days of dialysis to obtain N-carboxyethyl chitosan;
2) Preparing nano zinc oxide: weighing 1.3225gZnAc 2 ·2H 2 Dissolving O in 20mL of distilled water, slowly dropwise adding 9mL of 5mol/L NaOH solution, leading the solution to be white turbid first, continuously dropwise adding the solution until the turbidity is completely disappeared, then adding 11mL of distilled water, stirring uniformly, then pouring the solution into a low-pressure reaction kettle, putting the reaction kettle into an oven, heating the reaction kettle for 12 hours at 170 ℃, naturally cooling the reaction kettle to room temperature, and finally obtaining the productAfter obtaining the precipitate, respectively washing with water and ethanol, centrifuging twice, and drying the precipitate in an oven at 60 ℃ for later use;
3) Preparing a mixed solution of N-carboxyethyl chitosan and nano zinc oxide: adding 5.00g of N-carboxyethyl chitosan into 300mL of deionized water, violently stirring for 0.5h at room temperature, fully dissolving to obtain a water-soluble chitosan solution, mixing the water-soluble chitosan solution and nano zinc oxide according to a certain proportion, violently stirring the mixed solution for 3h, and uniformly mixing;
4) Preparing colloidal gold:
putting a round-bottom flask filled with 90-100ml of purified water into an electric heating jacket for heating, after heating for 10-12 minutes, turning on a revolution control switch when water in the flask is boiled, adjusting to 400rpm, slowly adding 1ml of 1% gold chloride solution along the edge of a vortex by using an injector, after the solution begins to boil, quickly adding 1-2ml of 1% trisodium citrate solution, after the solution is cooled to room temperature, adding water, weighing and complementing the weight;
5) Preparing zinc oxide-chitosan-colloidal gold: crosslinking at normal temperature by using benzene dicarbaldehyde as a crosslinking agent; after the crosslinking is finished, the obtained zinc oxide-chitosan-colloid Jin Xidi is dried in vacuum for 24 hours;
(b) Taking 1ml of zinc oxide-chitosan-colloidal gold cross-linked anti-human IgM antibody, ultrasonically dispersing uniformly, then dropwise adding the antibody while stirring, adding the antibody, reacting for 1min, then ultrasonically treating the mixture on ultrasonic waves for 30 seconds, then reacting for 4 hours, adding BSA (bovine serum albumin) for sealing for 24 hours, centrifuging after sealing at the speed of 12000r/min for 20min, and reserving the centrifuged precipitate for later use;
(c) Respectively diluting the precipitated zinc oxide-chitosan-colloidal gold antibody centrifuged in the step (b) by using a colloidal gold antibody buffer solution to uniformly disperse the precipitated zinc oxide-chitosan-colloidal gold antibody, respectively obtaining immune colloid Jin Kangti solutions, and respectively spraying the immune colloid Jin Kangti solutions on a glass fiber pad by using an immune colloid Jin Kangti solution to prepare an immune colloidal gold antibody glass fiber membrane;
(d) Pretreating a nitrocellulose membrane by using a polydopamine treatment solution, and then spraying a detection line with an influenza A antigen, an influenza B antigen, a rhinovirus antigen, a parainfluenza virus antigen, a respiratory syncytial virus antigen and an adenovirus antigen, or spraying a detection line with an influenza A antigen, an influenza B antigen, a rhinovirus antigen, a parainfluenza virus antigen, a respiratory syncytial virus antigen, an adenovirus antigen, a mycoplasma pneumoniae antigen and a chlamydia pneumoniae antigen, and spraying a quality control line with a goat anti-mouse IgG antibody to prepare an immune nitrocellulose membrane;
(e) And (3) sequentially sticking the pretreated sample pad, the immune colloidal gold antibody glass fiber membrane prepared in the step (c), the immune nitrocellulose membrane prepared in the step (d) and absorbent paper on a plastic plate, cutting to obtain a detection reagent strip, and finally filling the detection reagent strip into a plastic shell.
2. The method for preparing a multi-pathogen combined detection device according to claim 1, wherein the method comprises the following steps: in the step (a) of preparing the zinc oxide-chitosan-colloidal gold, 1) the synthesis of the N-carboxyethyl chitosan, and the intercepting molecular weight of the adopted dialysis membrane is 8000-12000g/mol when the dialysis is carried out in aqueous solution.
3. The method for preparing a multi-pathogen joint detection device according to claim 1, wherein the method comprises the following steps: in the step (a) of preparing the zinc oxide-chitosan-colloidal gold, 2) preparing nano zinc oxide particles, and slowly dropwise adding 9mL 5mol/L NaOH solution, wherein the NaOH solution is newly prepared solution.
4. The method for preparing a multi-pathogen combined detection device according to claim 1, wherein the method comprises the following steps: in the step (a), during the preparation of the zinc oxide-chitosan-colloidal gold, 3) preparing a mixed solution of N-carboxyethyl chitosan and nano zinc oxide, wherein the mixing ratio of the water-soluble chitosan solution and the nano zinc oxide is that the mass ratio of the N-carboxyethyl chitosan to the zinc oxide is 100/5, 100/8 or 100/10.
5. The method for preparing a multi-pathogen joint detection device according to claim 1, wherein the method comprises the following steps: the colloidal gold antibody buffer of step (c) comprises: tris-HCl solution at a concentration of 20mM, sucrose at 5%, trehalose at 1%, proclin at 1%, BSA at 1%, pH 8.5.
6. The method for preparing a multi-pathogen combined detection device according to claim 1, wherein the method comprises the following steps: pretreating the cellulose nitrate membrane by the polydopamine treating solution in the step (d): and (3) putting the nitrocellulose membrane into the polydopamine treatment solution, soaking for 1h, shaking at a low speed, taking out, washing for 3 times by using distilled water, and finally drying in a vacuum drying oven.
7. The method for preparing a multi-pathogen combined detection device according to claim 6, wherein the method comprises the following steps: the polydopamine treatment solution is prepared by filtering a polydopamine 1% filter membrane of 0.22 mu m for later use.
8. The method for preparing a multi-pathogen joint detection device according to claim 1, wherein the method comprises the following steps: the sample pad treatment solution adopted by the pretreated sample pad of the step (e) comprises 0.1mol/LTris-HCL solution, 1% bovine serum albumin BSA (bovine serum Albumin) and 1% surfactant.
9. A multi-pathogen joint detection device made by the method of any of claims 1~8 comprising: the sample pad, the immune colloidal gold antibody glass fiber membrane, the immune nitrocellulose membrane and the absorption pad are respectively stuck on the plastic plate, two ends of the nitrocellulose membrane are respectively lapped with the absorption pad and the immune colloidal gold antibody glass fiber membrane, and the other end of the immune colloidal gold antibody glass fiber membrane is lapped with the sample pad; the immune nitrocellulose membrane is provided with a first detection line (T1), a second detection line (T2), a third detection line (T3), a fourth detection line (T4), a fifth detection line (T5), a sixth detection line (T6) and a quality control line (C), wherein an influenza A antigen is solid-phase on the first detection line (T1), an influenza B antigen is solid-phase on the second detection line (T2), a rhinovirus antigen is solid-phase on the third detection line (T3), a parainfluenza virus antigen is solid-phase on the fourth detection line (T4), a respiratory syncytial virus antigen is solid-phase on the fifth detection line (T5), an adenovirus antigen is solid-phase on the sixth detection line (T6), goat anti-mouse IgG polyclonal antibodies are spotted on the quality control line (C), the first detection line (T1), the second detection line (T2), the third detection line (T3), the fourth detection line (T4), the fifth detection line (T5) and the sixth detection line (T6) are respectively arranged on the six detection lines, and each quality control line (C) is respectively arranged on the six detection lines.
10. The combined detection device for multiple pathogens according to claim 9, wherein: the immune nitrocellulose membrane is also provided with a seventh detection line (T7) and an eighth detection line (T8), the solid phase on the seventh detection line (T7) is provided with mycoplasma pneumoniae antigen, the solid phase on the eighth detection line (T8) is provided with chlamydia pneumoniae antigen, and each line of the two lines is respectively provided with a quality control line (C).
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