阅读说明：本技术 一种定性检测人血清中鲍曼不动杆菌特异性抗体的检测条 (Detection strip for qualitatively detecting specific antibody of acinetobacter baumannii in human serum ) 是由 杨波 胡征 王毅 于 2019-10-31 设计创作，主要内容包括：本发明涉及一种一种定性检测人血清中鲍曼不动杆菌特异性抗体的检测条。该检测条由样品垫、结合垫、硝酸纤维素膜、吸水垫、PVC板组成。结合垫上喷涂有彩色乳胶微球偶联的鼠抗人IgG单克隆抗体,硝酸纤维素膜上包被有鲍曼不动杆菌融合抗原的检测线和羊抗鼠IgG多克隆抗体的质控线。当加入的样品中含有鲍曼不动杆菌抗体时,鲍曼不动杆菌抗体首先与乳胶-鼠抗人IgG单克隆抗体形成复合物,在毛细作用下迁移至包被有鲍曼不动杆菌融合抗原的检测线时被捕捉,检测线呈相应的彩色,因而可检测样品中是否含有鲍曼不动杆菌抗体。该检测条具有快速、简捷、灵敏度高,特异性好,假阳性低的优点,能在十分钟之内得到明确的结果,有效用于鲍曼不动杆菌感染的辅助诊断。(The invention relates to a detection strip for qualitatively detecting specific antibodies of acinetobacter baumannii in human serum. The detection strip consists of a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC plate. The combination pad is sprayed with a mouse anti-human IgG monoclonal antibody coupled by colored latex microspheres, and the nitrocellulose membrane is coated with a detection line of acinetobacter baumannii fusion antigen and a quality control line of a goat anti-mouse IgG polyclonal antibody. When the added sample contains the acinetobacter baumannii antibody, the acinetobacter baumannii antibody firstly forms a compound with the latex-mouse anti-human IgG monoclonal antibody, the compound is captured when the compound migrates to a detection line coated with the acinetobacter baumannii fusion antigen under the capillary action, and the detection line is in corresponding color, so that whether the sample contains the acinetobacter baumannii antibody or not can be detected. The detection strip has the advantages of rapidness, simplicity, high sensitivity, good specificity and low false positive, can obtain a clear result within ten minutes, and is effectively used for auxiliary diagnosis of acinetobacter baumannii infection.)

1. An acinetobacter baumannii fusion antigen (Fhue + OmpA + Pilf), characterized in that: the protein sequence of the acinetobacter baumannii fusion antigen (Fhue + OmpA + Pilf) is as follows:

MFDGNYLDPVEGNSTEVGLKSAWFDGRLNGTLALYHIKQDNLAQEAGQVTRNGVKETYYRAAKGATSEGFEVEVSGQITPDWNITAGYSQFSAKDANDADVNTQLPRKMIQSFTTYKLPGGGSGGSGGSGGSLGYTFQDTQHNNGGKDGELTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGLAAGAEYKQKQINGNFGGSGGSGGSGGSAVKVRTQLAAEYIRSGDLDSAKRSLDQALSVDSRDATANMMMGILLQQEGSKSNLEKAEHYFKRAISSEPDNAQARNNYGTYLYQMERYN；

the complete sequence of the gene for the protein encoding the acinetobacter baumannii fusion antigen (Fhue + OmpA + Pilf) is:

2. a method for preparing the acinetobacter baumannii fusion antigen (Fhue + OmpA + Pilf) of claim 1, characterized in that: the method comprises the following steps:

respectively obtaining peptide segments with the most abundant antigenic epitopes in the extracellular domains of the acinetobacter baumannii surface protein Fhue, the surface protein OmpA and the surface protein Pilf, finding out the gene coding sequence of the peptide segment, optimizing the gene coding sequence of the peptide segment, and connecting the three segments of gene sequences by using the coding sequences of two segments of flexible connecting peptides to form a fusion gene;

the Acinetobacter baumannii surface protein Fhue, the surface protein OmpA and the surface protein Pilf respectively have access numbers of KMV27515, AJF83030 and AJF80497 in an NCBI protein database;

the amino acid sequence of the flexible connecting peptide is ggsggsggs;

simultaneously, enzyme cutting site NdeI is introduced into the 5 'end of the fusion gene, and termination signal TAA and enzyme cutting site BamHI are introduced into the 3' end of the fusion gene, and then a complete gene sequence is chemically synthesized and is marked as FhuOmpPil;

the complete gene sequence of the FhuOmpPil is as follows:

the protein sequence of the FhuOmpPil is:

MFDGNYLDPVEGNSTEVGLKSAWFDGRLNGTLALYHIKQDNLAQEAGQVTRNGVKETYYRAAKGATSEGFEVEVSGQITPDWNITAGYSQFSAKDANDADVNTQLPRKMIQSFTTYKLPGGGSGGSGGSGGSLGYTFQDTQHNNGGKDGELTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGLAAGAEYKQKQINGNFGGSGGSGGSGGSAVKVRTQLAAEYIRSGDLDSAKRSLDQALSVDSRDATANMMMGILLQQEGSKSNLEKAEHYFKRAISSEPDNAQARNNYGTYLYQMERYN；

the protein sequence coded by the FhuOmpPil gene is 509-627aa of surface protein Fhue of acinetobacter baumannii, 31-104aa of surface protein OmpA and 28-117aa of surface protein Pilf; three protein sequences are connected by two flexible connecting peptides; cloning the gene fragment into prokaryotic expression vector pET-28a (+) according to conventional method, inducing recombinant Escherichia coli expression by IPTG, and using Ni²⁺Purifying the recombinant His-FhuOmpPil fusion protein by an affinity chromatography, and freeze-drying and storing for later use; the amino acid sequence of the flexible connecting peptide is ggsggsggs.

3. A test strip for qualitatively detecting specific antibodies of acinetobacter baumannii in human serum is characterized in that: the test strip for qualitatively detecting the acinetobacter baumannii specific antibody in human serum comprises a nitrocellulose membrane coated with an acinetobacter baumannii fusion antigen (Fhue + OmpA + Pilf) and a latex microsphere marker of a mouse anti-human IgG monoclonal antibody.

4. A method for preparing the test strip for qualitatively detecting acinetobacter baumannii specific antibodies in human serum according to claim 3, characterized in that: the method comprises the following steps:

1) preparing an acinetobacter baumannii fusion antigen (Fhue + OmpA + Pilf);

2) preparing a latex microsphere marker of a mouse anti-human IgG monoclonal antibody:

2.1) activation of latex microspheres:

taking 1mL of colored carboxylated polystyrene latex microsphere solution with the concentration of 10%, adding 9mLMES buffer solution, uniformly mixing, adding NHS and EDC until the final concentration of the two is 1mg/mL, slowly and uniformly mixing for 30 minutes at room temperature, centrifuging 19000g for 20 minutes after incubation is finished, removing supernatant, re-suspending the precipitate with 10mL of borax buffer solution, oscillating, and performing ultrasonic treatment to obtain activated latex microspheres; the MES buffer solution comprises the following components in percentage by weight: 0.1mol/LMES, pH of MES buffer is 8.5; the borax buffer solution comprises the following components in percentage by weight: 0.1mol/LNa₂B₄O₇The pH value of the borax buffer solution is 8.5; the grain size of the colored carboxylated polystyrene latex microspheres is 100 nm;

2.2) preparation of latex microsphere markers:

preparing a mouse anti-human IgG monoclonal antibody into 1mg/mL by using a borax buffer solution; adding 10mL of mouse anti-human IgG monoclonal antibody into 10mL of activated latex microspheres, slowly mixing uniformly for 30 minutes, centrifuging for 10 minutes at 19000g, and removing the supernatant; resuspending the precipitate with 10mL borax buffer solution containing 1% casein, repeating centrifugation for 1 time after ultrasonic pulverization, and removing supernatant; resuspending the precipitate by the same method, repeatedly centrifuging for 1 time after ultrasonic crushing, and removing supernatant; resuspending the precipitate with 10mL borax buffer solution containing 1% casein, namely the latex microsphere marker of the mouse anti-human IgG monoclonal antibody; the borax buffer solution comprisesThe amount is: 0.1mol/LNa₂B₄O₇The pH value of the borax buffer solution is 8.5;

3) preparation of the bonding pad:

spraying a latex microsphere marker of a mouse anti-human IgG monoclonal antibody on a bonding pad made of a polyester fiber material, wherein the spraying amount of a polyester fiber film per square centimeter is 20 mu L of the latex microsphere marker; drying at 37 deg.C in environment with relative humidity not more than 30%, cutting into required specification, sealing at 25 deg.C, drying and storing;

4) preparation of antigen solid-phase nitrocellulose membrane:

diluting the acinetobacter baumannii fusion antigen (Fhue + OmpA + Pilf) obtained in the step 1) to 1.5mg/mL by using a borax buffer solution, and then coating the diluted antigen to a detection line position on a nitrocellulose membrane by using a membrane spraying instrument as a detection line capture antigen, wherein the coating parameter is 1 mu L/cm; spraying goat anti-mouse IgG polyclonal antibody on a quality control line position on a nitrocellulose membrane as a control line to capture the antibody, wherein the concentration is 1mg/mL, and the coating parameter is 1 muL/cm; the distance between the detection line and the quality control line is 0.7cm, and the edge distances between the detection line and the quality control line and between the detection line and the nitrocellulose membrane are both 0.8 cm; after coating, putting the nitrocellulose membrane in an environment with the relative humidity not more than 30%, drying at 37 ℃, cutting into required specifications, sealing at 25 ℃, drying and storing; the borax buffer solution has the components and the content of 0.1mol/LNa₂B₄O₇The pH value of the borax buffer solution is 8.5;

5) preparation of sample pad:

taking a glass cellulose membrane, soaking the glass cellulose membrane in a sample pad treatment solution for at least 3h, placing the sample pad treatment solution in a biological safety cabinet for ventilation drying at 37 ℃, cutting the sample pad treatment solution into required specifications, and sealing, drying and storing the sample pad treatment solution at 25 ℃; thus, a sample pad was prepared; the sample pad treatment solution comprises the following components in percentage by weight: 0.01mol/LNa₂B₄O₇2g/L sodium chloride, 20g/L casein, 10ml/L Tween-20 and 10ml/L antifoaming agent S-17; the pH of the sample pad treatment solution was 8.5;

6) assembling the detection strip:

respectively sticking a water absorption pad, an antigen solid-phase nitrocellulose membrane, a combination pad and a sample pad which are made of water absorption filter paper materials on a PVC (polyvinyl chloride) base plate in sequence, wherein a quality control line on the nitrocellulose membrane is close to the end of the water absorption pad, a detection line is close to the end of the sample pad, and then cutting the nitrocellulose membrane into detection strips with certain width, sealing and packaging the detection strips, and drying the detection strips for low-temperature storage; thus, the test strip for qualitatively detecting the specific antibody of the acinetobacter baumannii in human serum is prepared.

Technical Field

The invention belongs to the fields of bioengineering and immunology, and particularly relates to a test strip for qualitatively detecting acinetobacter baumannii specific antibodies in human serum and a preparation method thereof.

Background

Acinetobacter baumannii (Ab) is a non-fermenting gram-negative bacillus. The bacterium is an important pathogenic bacterium of hospital infection, mainly causes respiratory tract infection, and also can cause bacteremia, urinary system infection, secondary meningitis, operation site infection, ventilator-associated pneumonia and the like. The resistance rate to commonly used antibiotics tends to increase year by year and is of serious concern to clinicians and microbiologists. Domestic data indicate that a. baumann ii accounts for approximately 70% or more of clinically isolated acinetobacter. The drug resistance rate of baumann ii to the third and fourth generation cephalosporins reaches 63.0-89.9%. The drug-resistant rate of the bacteria for the four aminoglycosides (amikacin, gentamicin, netilmicin and tobramycin) and ciprofloxacin reaches 96.3 percent. The vast majority of strains currently in China remain sensitive to imipenem, meropenem, cefperazone/sulbactam and polymyxin B, but have poor effects in the treatment of respiratory tract infections. In view of the recent trend toward further increase in the drug resistance of acinetobacter baumannii, this should be highly appreciated by clinicians and the microbiology. The clinician should pay attention to the acquired acinetobacter baumannii infection, and closely cooperate with the clinical microorganism laboratory to enhance the monitoring of the infection and effectively prevent and control the infection.

The existing method for detecting the pathogen in the respiratory tract mainly adopts the traditional method, namely a separation identification method, the method needs long time, generally takes 2-3 days, and the requirement of quick identification is difficult to meet; the PCR technology developed in recent years is a quick, sensitive and specific technology, but at present, the technology still relies on the previous enrichment step of the traditional method, PCR inhibitors are often contained in the enrichment liquid, so that the amplification effect is influenced, and false positive is also a prominent defect of the method. Meanwhile, the technology also needs professional detection equipment, and is not suitable for bedside detection. Immunological detection using antibodies as diagnostic targets has become an indispensable important technical means for detecting infection of pathogenic microorganisms in humans. Various specific immunoassay techniques, such as Radioimmunoassay (RIA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), Chemiluminescence Immunoassay (CIA), immunoprecipitation, immunoagglutination, ELISA detection kit, immune colloidal gold test strip, immune latex detection reagent, and the like, have been developed. The immune latex test strip and other immunological detection technologies based on antigen and antibody have become indispensable important means for detecting pathogenic microorganisms due to the characteristics of simplicity, rapidness, sensitivity, accuracy and practicability. Therefore, research and development of marker antigens of pathogenic microorganisms with proprietary intellectual property rights are the basis for development of methods for detecting pathogen antibodies such as ELISA and latex microsphere immunochromatography with proprietary intellectual property rights.

The preparation of antigen is the key to the specificity of antibody detection. In the research, surface proteins fhuE receptor, OmpA, PilF and the like with interspecies specificity are selected as antigen targets to prepare fusion antigens with good specificity, and the fusion antigens are applied to preparation of acinetobacter baumannii antibody latex microsphere immunochromatography detection strips.

Disclosure of Invention

The invention aims to develop a detection strip for qualitatively detecting the acinetobacter baumannii specific antibody in human serum, which is simple to operate, low in cost, quick and rapid by using an immune latex labeling technology on the basis of fusion antigen.

The purpose of the invention is realized by the following technical scheme:

a preparation method of a test strip for qualitatively detecting specific antibodies of acinetobacter baumannii in human serum is characterized in that: the preparation method of the detection strip for qualitatively detecting the acinetobacter baumannii specific antibody in human serum comprises the following steps:

1) preparation of acinetobacter baumannii fusion antigen (Fhue + OmpA + Pilf):

respectively obtaining peptide segments with the most abundant antigenic epitopes in the extracellular domains of the acinetobacter baumannii surface protein Fhue, the surface protein OmpA and the surface protein Pilf, finding out the gene coding sequence of the peptide segment, optimizing the gene coding sequence of the peptide segment, and connecting the three segments of gene sequences by using the coding sequences of two segments of flexible connecting peptides to form a fusion gene;

the Acinetobacter baumannii surface protein Fhue, the surface protein OmpA and the surface protein Pilf respectively have access numbers of KMV27515, AJF83030 and AJF80497 in an NCBI protein database;

the amino acid sequence of the flexible connecting peptide is ggsggsggs;

simultaneously, enzyme cutting site NdeI is introduced into the 5 'end of the fusion gene, and termination signal TAA and enzyme cutting site BamHI are introduced into the 3' end of the fusion gene, and then a complete gene sequence is chemically synthesized and is marked as FhuOmpPil;

the complete sequence of the FhuOmpPil gene is

The encoding amino acid sequence of the FhuOmpPil is as follows:

MFDGNYLDPVEGNSTEVGLKSAWFDGRLNGTLALYHIKQDNLAQEAGQVTRNGVKETYYRAAKGATSEGFEVEVSGQITPDWNITAGYSQFSAKDANDADVNTQLPRKMIQSFTTYKLPGGGSGGSGGSGGSLGYTFQDTQHNNGGKDGELTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGLAAGAEYKQKQINGNFGGSGGSGGSGGSAVKVRTQLAAEYIRSGDLDSAKRSLDQALSVDSRDATANMMMGILLQQEGSKSNLEKAEHYFKRAISSEPDNAQARNNYGTYLYQMERYN；

the protein sequence coded by the FhuOmpPil gene is 509-627aa of surface protein Fhue of acinetobacter baumannii, 31-104aa of surface protein OmpA and 28-117aa of surface protein Pilf; three protein sequences are connected by two flexible connecting peptides; cloning the gene fragment into prokaryotic expression vector pET-28a (+) according to conventional method, inducing recombinant Escherichia coli expression by IPTG, and using Ni²⁺Purifying the recombinant His-FhuOmpPil fusion protein by an affinity chromatography, and freeze-drying and storing for later use; the amino acid sequence of the flexible connecting peptide is ggsggsggs;

2) preparing a latex microsphere marker of a mouse anti-human IgG monoclonal antibody:

2.1) activation of the latex microspheres

Taking 1mL of colored carboxylated polystyrene latex microsphere solution with the concentration of 10%, adding 9mL of LMES (2- (N-morpholinyl) ethanesulfonic acid) buffer solution, uniformly mixing, adding NHS (N-hydroxysuccinimide) and EDC (1- (3-dimethylaminopropyl) -3-ethyl 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) until the final concentration of the NHS and the EDC is 1mg/mL, slowly and uniformly mixing for 30 minutes at room temperature, centrifuging for 20 minutes at 19000g after the incubation is finished, removing supernatant, re-suspending the precipitate with 10mL of borax buffer solution, oscillating, and performing ultrasonic treatment to obtain activated latex microspheres; the MES buffer solution comprises the following components in percentage by weight: 0.1mol/LMES, pH of MES buffer is 8.5; the size of the colored carboxylated polystyrene latex microspheres is 100 nm; the borax buffer solution comprises the following components in percentage by weight: 0.1mol/LNa₂B₄O₇The pH value of the borax buffer solution is 8.5;

2.2) preparation of latex microsphere markers

Preparing a mouse anti-human IgG monoclonal antibody into 1mg/mL by using a borax buffer solution; adding 10mL of mouse anti-human IgG monoclonal antibody into 10mL of activated latex microspheres, slowly mixing uniformly for 30 minutes, centrifuging for 10 minutes at 19000g, and removing the supernatant; resuspending the precipitate with 10mL borax buffer solution containing 1% casein, repeating centrifugation for 1 time after ultrasonic pulverization, and removing supernatant; resuspending the precipitate by the same method, repeatedly centrifuging for 1 time after ultrasonic crushing, and removing supernatant; resuspending the precipitate with 10mL borax buffer solution containing 1% casein, namely the latex microsphere marker of the mouse anti-human IgG monoclonal antibody; the borax buffer solution comprises the following components in percentage by weight: 0.1mol/LNa₂B₄O₇The pH value of the borax buffer solution is 8.5;

3) preparation of the bonding pad:

spraying a latex microsphere marker of a mouse anti-human IgG monoclonal antibody on a bonding pad made of a polyester fiber material, wherein the spraying amount of a polyester fiber film per square centimeter is 20 mu L of the latex microsphere marker; drying at 37 deg.C in environment with relative humidity not more than 30%, cutting into required specification, sealing at 25 deg.C, drying and storing;

4) preparation of antigen solid-phase nitrocellulose membrane:

diluting the acinetobacter baumannii fusion antigen (Fhue + OmpA + Pilf) obtained in the step 1) to 1.5mg/mL by using a borax buffer solution, and then coating the diluted antigen to a detection line position on a nitrocellulose membrane by using a membrane spraying instrument as a detection line capture antigen, wherein the coating parameter is 1 mu L/cm; spraying goat anti-mouse IgG polyclonal antibody on a quality control line position on a nitrocellulose membrane as a control line to capture the antibody, wherein the concentration is 1mg/mL, and the coating parameter is 1 muL/cm; the distance between the detection line and the quality control line is 0.7cm, and the edge distances between the detection line and the quality control line and between the detection line and the nitrocellulose membrane are both 0.8 cm; after coating, putting the nitrocellulose membrane in an environment with the relative humidity not more than 30%, drying at 37 ℃, cutting into required specifications, sealing at 25 ℃, drying and storing; the borax buffer solution has the components and the content of 0.1mol/LNa₂B₄O₇The pH value of the borax buffer solution is 8.5;

5) preparation of sample pad

Taking a glass cellulose membrane, soaking the glass cellulose membrane in a sample pad treatment solution for at least 3h, placing the sample pad treatment solution in a biological safety cabinet for ventilation drying at 37 ℃, cutting the sample pad treatment solution into required specifications, and sealing, drying and storing the sample pad treatment solution at 25 ℃; thus, a sample pad was prepared; the sample pad treatment solution comprises the following components in percentage by weight: 0.01mol/LNa₂B₄O₇2g/L sodium chloride, 20g/L casein, 10ml/L Tween-20 and 10ml/L antifoaming agent S-17; the pH of the sample pad treatment solution was 8.5;

6) assembly of test strips

Respectively sticking a water absorption pad, an antigen solid-phase nitrocellulose membrane, a combination pad and a sample pad which are made of water absorption filter paper materials on a PVC (polyvinyl chloride) base plate in sequence, wherein a quality control line on the nitrocellulose membrane is close to the end of the water absorption pad, a detection line is close to the end of the sample pad, and then cutting the nitrocellulose membrane into detection strips with certain width, sealing and packaging the detection strips, and drying the detection strips for low-temperature storage; thus, the test strip for qualitatively detecting the specific antibody of the acinetobacter baumannii in human serum is prepared.

The invention has the advantages that:

1) the invention adopts the modes of antigen structure analysis, gene optimization and the like to construct a brand new fusion antigen gene, and successfully obtains the soluble recombinant Fhue/OmpA/Pilf fusion protein for the first time through soluble over-expression. The gene engineering fusion protein has high expression amount, high protein solubility, high antigenicity and low preparation cost.

2) The recombinant antigen prepared by using the extracellular exposure epitopes of the three structural proteins on the surface of the acinetobacter baumannii for the first time has high recognition capability on serum antibodies and strong capture capability, greatly reduces the possibility of missed detection, has high sensitivity of a detection strip, and has the advantages of rapidness, high efficiency and the like.

3) The specificity of the detection strip is good, and the result of a specificity experiment carried out by using 78 serum samples of Acinetobacter baumannii infected persons confirmed by etiological identification and 100 serum samples of non-Acinetobacter baumannii infected persons shows that the detection strip has good specificity and sensitivity, can detect the serum of all tested Acinetobacter baumannii infected persons, has no cross reaction with the serum samples of the non-Acinetobacter baumannii infected persons, and is very suitable for clinical non-diagnostic application.

4) The detection strip can be preserved for two years at normal temperature, effectively prolongs the shelf life and reduces the storage condition; non-professional persons can finish the whole-process detection by using the detection test paper, the operation is simple, and the popularization of the method is facilitated; the whole detection process can be finished within 10min at the fastest speed, and is more suitable for bedside detection.

Drawings

FIG. 1 is a schematic diagram of an explosion structure of a test strip for qualitatively detecting Acinetobacter baumannii specific antibodies in human serum provided by the invention;

FIG. 2 is a schematic diagram of an assembly structure of the test strip for qualitatively detecting Acinetobacter baumannii specific antibody in human serum provided by the present invention;

wherein:

1-sample pad; 2-a conjugate pad; 3-NC film; 4-absorbent pad; 5-PVC sheet.

Detailed Description

The present invention is further specifically described by the following examples.

Sources of the various materials used or employed in the present invention

1. Latex microspheres: the latex microspheres used in the invention are carboxylated modified polystyrene latex microspheres, are products of Shanghai Yanghi Biotech Co., Ltd, have the particle size of 100nm and the color of red, have the tolerance of the average diameter of the products within 10 percent, are in the form of 10 percent solid aqueous suspension, and have the product code of MSI-CAR100 NM.

2. Glass cellulose membrane: the thickness is 0.45-0.55mm, the water absorption capacity is 800mg/m²The glass fiber has a diameter of 0.6-3 μm and good hydrophilicity, and is available from Shanghai gold-labeled Biotech Co., Ltd (model number BT 50).

3. Polyester fiber film: has a thickness of 0.25-0.35mm, a climbing speed of 15-40mm/60s, excellent hydrophilicity, and is used for preparing a bonding pad, and is available from Shanghai gold-labeled Biotech Co., Ltd (model number VL 98).

4. Cellulose nitrate membrane: model number Millipore Corp SHF135, with liner plates, was purchased from Millipore corporation.

5. Water-absorbing filter paper: the thickness is 0.95mm, the water absorption speed is 60s/4cm, and the water absorption capacity is 700mg/cm²Has good water absorption and is used as a material for manufacturing the water absorption pad. Purchased from Shanghai gold Biotech, Inc. (model CH 37K).

6. A bottom plate: is made of high-whiteness PVC material, and is coated with a single-layer high-polymer pressure-sensitive adhesive SM31 purchased from Shanghai gold-labeled Biotech Co.

7. The microorganism samples used in the present invention were purchased from the American Type Culture Collection (ATCC).

8. pET28a (+): e.coli expression vectors, introduced from Novagen, USA.

9. Escherichia coli (e. coli) BL21(DE 3): purchased from northern Biotechnology, Inc., Shanghai.

10. Goat anti-mouse IgG polyclonal antibody: product No. BA1038, product of bosch de bioengineering limited.

11. Mouse anti-human IgG monoclonal antibody: product of Abcam, USA, cat # ab771, which is a monoclonal antibody against human IgG Fab segments secreted by hybridoma cells [ 4A11 ].

The methods used in the following examples are conventional methods unless otherwise specified.