CN114113599A - Recombinant protein rP44-18ES kit for detecting anaplasma phagocytophilum antibody - Google Patents
Recombinant protein rP44-18ES kit for detecting anaplasma phagocytophilum antibody Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The application provides a recombinant protein rP44-18ES kit for detecting an anaplasma phagocytophila antibody, which comprises a double-antigen sandwich method gold-labeled detection strip, wherein the detection strip comprises a bottom plate, a sample pad, a gold-labeled pad, an analysis membrane and a water absorption pad are arranged on the bottom plate along the chromatography direction, the gold-labeled recombinant protein rP44-18ES and mouse IgG are arranged on the gold-labeled pad, and a detection line and a quality control line are arranged on the analysis membrane; the detection line is recombinant protein rP44-18ES, and the quality control line is an antibody combined with mouse IgG. The recombinant protein rP44-18ES is used as an antigen for detecting the HGA antibody, the method is more convenient and accurate, the sensitivity is high, the specificity is strong, the recombinant protein rP44-18ES has no cross reaction with other pathogens, the probability of missed detection in HGA serum detection is obviously reduced, large-scale instrument operation is not needed, and the method is widely applied to primary medical institutions.
Description
Technical Field
The application relates to the technical field of medical treatment, in particular to a recombinant protein rP44-18ES kit for detecting an anaplasma phagocytophila antibody.
Background
Anaplasma phagocytophila (anaplama phagocytophilum) belongs to obligate intracellular parasitic bacteria of anaplasmataceae of the order rickettsiae and can cause Human Granulocytic Anaplasmosis (HGA). The anaplasma phagocytophila is characterized in that it infects neutrophils directionally in the body and forms parasitic vacant cells (inclusion bodies) similar to mulberry (morula) in the cytoplasm to reproduce and proliferate. The P44 major outer membrane protein group with an associated antigenic variation on the surface of anaplasma phagocytosis is the major antigenic protein of the bacterium.
Since human granulocytic anaplasmosis has no specific clinical manifestations, the clinical symptoms are similar to those of certain viral diseases, and early detection is difficult. Currently, the more direct detection methods in clinical practice are blood smear staining methods, immunological methods such as Indirect fluorescence antibody assay (IFA) and Enzyme-Linked immunosorbent assay (ELISA), and in addition, the PCR (polymerase chain reaction) method is also a detection method commonly used for detecting anaplasma phagocytophilum, which is the most sensitive and high-specificity detection method for detecting anaplasma phagocytophilum.
However, the methods such as the immunofluorescence method, the enzyme-linked immunosorbent assay (ELISA), the Polymerase Chain Reaction (PCR) technology and the like require the use of designated instruments and equipment, have corresponding test conditions and skills, and are difficult to popularize at the basic level. The detection difficulty is increased due to the fact that antigenic variation is easy to generate, due to the fact that repeated sequences of a P44 multigene family exist in a genome of an anaplasma phagocytophila, exchange is easy to generate among homologous genes, different P44 outer membrane protein types are expressed in a body, so that the antigenic variation is caused, scientific basis is difficult to provide for quick and simple detection of human granulocytic anaplasmosis, and meanwhile, the detection risk of low sensitivity, low specificity and omission exists.
Disclosure of Invention
The application provides a recombinant protein rP44-18ES kit for detecting anaplasma phagocytophila antibody, sensitivity for solving existence in HGA antibody detection, the specificity is low and the rate of missed detection is high, easily produce the problem of antigenic variation, the recombinant protein rP44-18ES for detecting anaplasma phagocytophila antibody that this application provided, have sensitivity height, the strong advantage of specificity, do not have cross reaction with other pathogens simultaneously, and can show the probability of missed detection in reducing HGA serum detection, and the kit of making detects conveniently, high-efficiently, accurately, do not need large-scale instrument equipment, be convenient for extensively promote at basic medical institution.
The application provides a recombinant protein rP44-18ES kit for detecting an anaplasma phagocytophila antibody, which comprises a double-antigen sandwich gold-labeled detection strip, wherein the detection strip comprises a bottom plate, a sample pad, a gold-labeled pad, an analysis membrane and a water absorption pad are sequentially arranged on the bottom plate along a chromatography direction, a colloidal gold compound is arranged on the gold-labeled pad, and a detection line and a quality control line are arranged on the analysis membrane; the colloidal gold compound comprises a colloidal gold labeled recombinant protein rP44-18ES and a mouse IgG antibody, the detection line comprises the recombinant protein rP44-18ES, and the quality control line comprises an antibody combined with the mouse IgG antibody.
A double-antigen sandwich method is a convenient and rapid detection method, a sample pad in a kit is used for dropwise adding a sample to be detected, a gold label pad is soaked with recombinant protein rP44-18ES and mouse IgG antibody marked by nano gold particles and used for developing color during detection, a detection line and a quality control line are distributed on an analysis membrane, the detection line comprises recombinant protein rP44-18ES, whether the sample to be detected is infected with HGA virus or not is judged by the specific combination of the HGA antibody and the recombinant protein rP44-18ES existing in the sample to be detected, and the quality control line comprises the antibody combined with the mouse IgG antibody and used for detecting whether a gold label compound is qualified or not.
Through the scheme, the recombinant protein rP44-18ES is used for preparing the kit for detecting the anaplasma phagocytophila antibody by adopting a double-antigen sandwich method, the recombinant protein rP44-18ES is used as an antigen for detecting HGA, and whether a specific antibody exists in serum to be detected or not is specifically combined with the recombinant protein rP44-18ES, so that whether the animal is infected with the anaplasma phagocytophila or not is detected. The kit and the detection strip have the advantages of rapidness, simple and convenient operation and no need of special instruments and equipment, so that the detection is more convenient and faster.
Alternatively, the antibody that binds to a murine IgG antibody comprises one or more polyclonal antibodies of rabbit-, sheep-, horse-, camel-derived anti-murine IgG antibodies. The cross with the sample antibody is less, the specificity is good, the line signal of the quality control line is more stable, and the stability of the detection strip is improved.
Optionally, the sample pad and the gold label pad are fiberglass film or polyester film. When in detection, the sample and the gold-labeled compound can be uniformly distributed and smoothly electrophoresed on the membrane, and the antibody in the sample and the gold-labeled compound are specifically combined, thereby being beneficial to improving the sensitivity and the accuracy of the detection strip.
Optionally, the analytical membrane is a nitrocellulose membrane. The nitrocellulose membrane is used as a bearing body of the detection line and the quality control line, and is also a place for immunoreaction, so that the antibody in the sample can be conveniently and specifically combined with the gold-labeled compound.
Optionally, the preparation method of the test strip comprises:
s1, preparing a gold-labeled pad: respectively preparing a recombinant protein rP44-18ES colloidal gold compound and a mouse IgG colloidal gold compound, redissolving the recombinant protein rP44-18ES colloidal gold compound and the mouse IgG colloidal gold compound, mixing, soaking a glass fiber membrane, drying the soaked glass fiber membrane at 37 ℃ for 3-8 hours, and preparing the gold-labeled pad.
The recombinant protein rP44-18ES and the mouse IgG antibody are respectively marked by using the colloidal gold solution to form a colloidal gold compound, which is substantially a coating process that protein macromolecules are adsorbed to the surfaces of colloidal gold particles, so that an immune compound can be obtained due to antigen-antibody reaction in the detection process, the immune compound is intercepted and developed on a detection line and/or a quality control line, and whether a sample to be detected is infected with HGA virus or not can be conveniently judged.
S2, preparation of an analysis membrane: respectively preparing a recombinant protein rP44-18ES diluted solution with the concentration of 1-3 mg/mL and an antibody diluted solution which is combined with a mouse IgG antibody and has the concentration of 0.3-3 mg/mL by using the diluted solution; scribing the two diluted solutions onto a nitrocellulose membrane, scribing the recombinant protein rP44-18ES as a detection line of a detection strip, scribing an antibody combined with a mouse IgG antibody as a quality control line of the detection strip, and drying for 14-24 hours at 37-45 ℃; the diluent is a mixture of 50mM Tris and 2% sucrose, and the pH of the diluent is 8.5-9.
And (3) scribing the recombinant protein rP44-18ES as a detection line of the detection strip, so that the HGA antibody in serum is specifically combined with the recombinant protein rP44-18ES, an antibody-antigen-gold-antigen complex is formed on the detection line and is retained on the detection line to form a red line, which represents a positive result. The antibody bound to the murine IgG antibody is scored as the control line of the test strip, preferably the antibody bound to the murine IgG antibody is rabbit anti-mouse IgG. And forming a mouse IgG antibody-gold-rabbit anti-mouse IgG compound on the quality control line, and reserving the compound on the quality control line to form a red line, wherein the appearance of the quality control line proves that the detection function of the detection strip is normal. The pH value of the diluent is 8.5-9, which is beneficial to maintaining the stability of the colloidal gold compound.
S3, assembling the sample pad, the prepared gold label pad, the analysis membrane and the water absorption pad on a bottom plate, and then cutting according to the specification of the test strip to obtain the test strip.
Optionally, the preparation method of the gold-labeled pad includes:
(1) preparation of recombinant protein rP44-18ES colloidal gold complex and mouse IgG colloidal gold complex: respectively putting 100mL of colloidal gold solution into a beaker, stirring and adding K with the molar concentration of 0.1-0.2M2CO3Adjusting the pH value of the colloidal gold solution to 6.5-9, respectively adding 2-3 mg of recombinant protein rP44-18ES or mouse IgG antibody, then adding 0.5-1.3 mg of triethanolamine and 0.3-0.6 mg of eudesmol, and stirring at room temperature for 15-30 min; then respectively adding 10-20 mL of gold-labeled confining liquid to seal the stirred product, stirring at room temperature for 15-20 min, centrifuging at 12000rpm for 10min, sucking out the supernatant, and removing to obtain precipitates, namely two colloidal gold compounds;
the pH value of the colloidal gold solution is adjusted to 6.5-9, the nano-gold particles and the protein are combined favorably, the combination of the nano-gold particles and the protein is more stable due to the addition of the triethanolamine and the eudesmol, the sensitivity and specificity of the recombinant protein rP44-18ES as a detection antigen are further improved, and the gold-labeled confining liquid can prevent the nonspecific combination of the antigen.
(2) Redissolving: respectively diluting the colloidal gold compound with a gold-labeled diluent to enable the colloidal gold compound to be respectively dissolved in the gold-labeled diluent, wherein the concentration of the colloidal gold compound is 10-250 mu g/mL, and respectively obtaining a recombinant protein rP44-18ES colloidal gold compound solution and a mouse IgG antibody colloidal gold compound solution;
(3) preparing a gold label pad: and mixing the recombinant protein rP44-18ES colloidal gold complex solution and the mouse IgG antibody colloidal gold complex solution, soaking the glass fiber membrane until liquid seeps out, taking out the glass fiber membrane, and drying at 37 ℃ for 3-8 hours to form the gold label pad.
Optionally, the gold-labeled confining liquid includes: the kit comprises a phosphate buffer solution, macromolecular protein with the mass concentration of 0.2-1.0% and Tween-20 with the mass concentration of 0.2-1.0%, wherein the phosphate buffer solution is a PBS solution with the pH of 7.4 and the mass concentration of 0.1M, and the macromolecular protein is any one of BSA, fetal calf serum and skimmed milk.
The phosphate buffer solution can maintain the pH of the colloidal gold compound, keep the stable state of the colloid, facilitate the protection of the gold-labeled protein due to the existence of macromolecular protein, and prevent the nonspecific combination of the antigen, and the Tween-20 serving as a surfactant is favorable for the release of the gold-labeled protein, thereby improving the sensitivity of the detection result.
The gold-labeled diluent comprises: 20mM Tris, 1 wt% BSA, 0.02 wt% NaN 31 wt% sucrose. The addition of Tris can stabilize the pH value of the colloidal gold complex in the detection, and is beneficial to the stabilization of the colloidal state. The sucrose as a drying protective agent can provide a relatively hydrophilic environment, and avoid the situation that the gold-labeled compound is not released or slowly released.
Optionally, the detection line and the quality control line are separated by 5-7 mm. The mutual influence of the detection line and the quality control line caused by a small distance is avoided, and the judgment of the detection result is further influenced.
Optionally, the assembling step of the test strip includes: installing a sample pad, a gold label pad, an analysis membrane and a water absorption pad on a bottom plate, wherein a part of the sample pad is superposed and pressed on the gold label pad, a part of the gold label pad is superposed and pressed on the analysis membrane, and a part of the water absorption pad is superposed and pressed on the other side of the analysis membrane; the analysis membrane is divided into a test area and a quality control area, the test area is provided with a detection line, the quality control area is provided with a quality control line, the detection line is close to the gold-labeled pad, and the quality control line is close to the water absorption pad.
Alternatively, the recombinant protein rP44-18ES consists of an amino acid sequence shown in SEQ ID NO. 1.
The recombinant protein rP44-18ES kit for detecting the anaplasma phagocytophilum antibody provided by the application realizes the effect of detecting the HGA virus in serum by a double-antigen sandwich method. Has the following beneficial effects:
(1) the recombinant protein rP44-18ES is used as a detection antigen for detecting an anaplasma phagocytophilum antibody, is more convenient and rapid, has high sensitivity and strong specificity, has no cross reaction with other pathogens, and can obviously reduce the probability of missed detection in HGA serum detection.
(2) By using a nanogold labeling method, the recombinant protein rP44-18ES and the mouse IgG antibody are labeled, which is equivalent to staining the recombinant protein rP44-18ES and the mouse IgG antibody, so that the color is more easily developed through the specific binding of antigen and antibody in the detection process, and the detection result is more visual.
(3) During the colloidal gold marking, triethanolamine and eudesmol are added simultaneously, and the synergistic effect of the triethanolamine and the eudesmol improves the combination of the gold nanoparticles and the protein, and improves the stability of the colloidal gold compound, so that the colloidal gold compound can be released smoothly in the detection process, and the sensitivity and the specificity of the kit can be improved when the reactivity of the recombinant protein rP44-18ES antigen is effectively reserved.
(4) The kit prepared by the application can preliminarily detect the condition whether people and livestock are infected with the HGA virus, provides a quick auxiliary means for screening and treating the anaplasma phagocytophila, does not need a large-scale instrument to operate, can be widely applied to detection of basic medical institutions, and has wide market value.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic structural diagram of a test strip according to an embodiment of the present disclosure;
FIG. 2 shows the overall results of the test performed on clinical serum samples using different kits according to the present application.
Description of reference numerals:
1: a base plate;
2: a sample pad;
3: a gold label pad;
4: an analytical membrane;
41: detecting lines;
42: a quality control line;
5: an absorbent pad.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application are clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present application, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Fig. 1 is a schematic structural diagram of a kit provided in an embodiment of the present application, and as shown in fig. 1, the present application provides a recombinant protein rP44-18ES kit for detecting an anaplasma phagocytophila antibody, the kit includes a double-antigen sandwich gold-labeled detection strip, the detection strip includes a bottom plate 1, a sample pad 2, a gold-labeled pad 3, an analysis membrane 4 and a water absorption pad 5 are sequentially disposed on the bottom plate along a chromatography direction, a colloidal gold compound is disposed on the gold-labeled pad, and the analysis membrane is provided with a detection line 41 and a quality control line 42; the colloidal gold compound comprises a colloidal gold labeled recombinant protein rP44-18ES and a mouse IgG antibody, the detection line comprises the recombinant protein rP44-18ES, and the quality control line comprises an antibody combined with the mouse IgG antibody.
Specifically, a sample pad in the kit is used for dropwise adding a sample to be detected, a gold-labeled pad is soaked with recombinant protein rP44-18ES and a mouse IgG antibody marked by nano gold particles and used for developing color during detection, detection lines and quality control lines are distributed on an analysis membrane, the detection lines comprise recombinant protein rP44-18ES, the HGA antibody in the sample to be detected and the recombinant protein rP44-18ES are subjected to specific binding, whether the sample to be detected is infected with HGA virus or not is judged, and the quality control lines comprise antibodies bound with the mouse IgG antibody and used for detecting whether a gold-labeled compound is qualified or not.
Through the scheme, the recombinant protein rP44-18ES is used for preparing the kit for detecting the anaplasma phagocytophila antibody, and the specific antibody in the serum to be detected is specifically combined with the recombinant protein rP44-18ES by using the recombinant protein rP44-18ES as the antigen for detecting the HGA through a double-antigen sandwich method so as to detect whether the animal is infected with the anaplasma phagocytophila. The detection strip has the advantages of rapidness, simple and convenient operation and no need of special instruments and equipment, so that the detection is more convenient and faster.
Alternatively, the antibody that binds to a murine IgG antibody comprises one or more polyclonal antibodies of rabbit-, sheep-, horse-, camel-derived anti-murine IgG antibodies.
Specifically, a mouse IgG antibody is used as a primary antibody, and an antibody combined with the mouse IgG antibody is used as a secondary antibody, and as a quality control line antibody, the primary antibody of a species different from the species of the sample is generally selected as much as possible, so as to prevent the secondary antibody from generating a cross reaction with the endogenous immunoglobulin of the sample. In the application, one of rabbit source, sheep source, horse source and camel source is selected as the second antibody, so that the cross with the sample antibody is less, the specificity is good, the line signal of a quality control line is more stable, and the stability of the detection strip is favorably improved.
Optionally, the sample pad and the gold label pad are fiberglass film or polyester film.
Specifically, the surfaces of the glass fiber film and the polyester film are flat, the fibers are uniformly and tidily distributed, the grains are fine, the components in the film are biologically inert, the normal reaction of bioactive substances is not influenced, the glass fiber film or the polyester film is used as a sample pad and a gold label pad, the sample and the gold label compound can be uniformly distributed and smoothly surged on the film during detection, and the antigen in the sample and the gold label compound are specifically combined, so that the sensitivity and the accuracy of the detection strip are improved.
Optionally, the analytical membrane is a nitrocellulose membrane.
Specifically, the nitrocellulose membrane is used as a bearing body of a detection line and a quality control line in the detection strip, and is also a generation part of immunoreaction, so that the antibody in the sample and the gold-labeled compound are conveniently subjected to specific binding.
Optionally, the preparation method of the test strip comprises:
s1, preparing a gold-labeled pad: respectively preparing a recombinant protein rP44-18ES colloidal gold compound and a mouse IgG colloidal gold compound, redissolving the recombinant protein rP44-18ES colloidal gold compound and the mouse IgG colloidal gold compound, mixing, soaking a glass fiber membrane, drying the soaked glass fiber membrane at 37 ℃ for 3-8 hours, and preparing the gold label pad.
Specifically, the adsorption of the colloidal gold label is that negative charges carried on the surface of the colloidal gold and positive charges carried by protein molecules are mutually attracted by electrostatic force, and a firm combination is formed by van der waals attraction. Since the labeling process is mainly through physical adsorption, it has no significant effect on the biological activity of the protein molecules. The method comprises the steps of respectively marking a recombinant protein rP44-18ES mouse IgG antibody by using a colloidal gold solution to form a colloidal gold compound, substantially performing a coating process of adsorbing protein macromolecules to the surfaces of colloidal gold particles, re-dissolving and mixing the recombinant protein rP44-18ES colloidal gold compound and the mouse IgG colloidal gold compound, soaking a glass fiber membrane in a colloidal gold compound mixed solution, and taking out the glass fiber membrane and drying the glass fiber membrane for 3-8 hours at 37 ℃ to obtain the gold label pad. The sample is electrophoresed to the gold-labeled pad through the sample pad, so that the HGA antibody in the sample is specifically combined with the recombinant protein rP44-18ES colloidal gold complex on the gold-labeled pad to form an HGA antibody-rP 44-18ES antigen-gold complex.
S2, preparation of an analysis membrane: respectively preparing a recombinant protein rP44-18ES diluted solution with the concentration of 1-3 mg/mL and an antibody diluted solution which is combined with a mouse IgG antibody and has the concentration of 0.3-3 mg/mL by using the diluted solution; scribing the two diluted solutions onto a nitrocellulose membrane, scribing the recombinant protein rP44-18ES as a detection line of a detection strip, scribing an antibody combined with a mouse IgG antibody as a quality control line of the detection strip, and drying for 14-24 hours at 37-45 ℃; the diluent is a mixture of 50mM Tris and 2% sucrose, and the pH of the diluent is 8.5-9.
Specifically, the analysis membrane material adopts a nitrocellulose membrane as a carrier of a detection line and a quality control line, the recombinant protein rP44-18ES and an antibody combined with a mouse IgG antibody are respectively diluted, scribing is carried out on the nitrocellulose membrane, and scribing of the recombinant protein rP44-18ES is taken as the detection line of the detection strip, so that the HGA antibody in serum is specifically combined with the recombinant protein rP44-18ES, an antibody-antigen-gold-antigen complex is formed on the detection line and is retained on the detection line to form a red line, which represents a positive result. The antibody bound to the murine IgG antibody is scored as the control line of the test strip, preferably the antibody bound to the murine IgG antibody is rabbit anti-mouse IgG. And forming a mouse IgG antibody-gold-rabbit anti-mouse IgG compound on the quality control line, and reserving the compound on the quality control line to form a red line, wherein the appearance of the quality control line proves that the detection function of the detection strip is normal.
S3, assembling the sample pad, the prepared gold label pad, the analysis membrane and the water absorption pad on a bottom plate, and then cutting according to the specification of the test strip to obtain the test strip.
Specifically, the sample pad, the gold label pad, the analysis membrane and the absorbent pad are assembled on a bottom plate, cut into test strips with the width of 4mm, packaged in a closed container containing a drying agent, and stored at room temperature.
Optionally, the preparation method of the gold-labeled pad includes:
(1) preparation of recombinant protein rP44-18ES colloidal gold complex and mouse IgG colloidal gold complex: respectively putting 100mL of colloidal gold solution into a beaker, stirring and adding K with the molar concentration of 0.1-0.2M2CO3Adjusting the pH of the colloidal gold solution to 6.5-9, and respectively addingAdding 2-3 mg of recombinant protein rP44-18ES or mouse IgG antibody, adding 0.5-1.3 mg of triethanolamine and 0.3-0.6 mg of eudesmol, and stirring at room temperature for 15-30 min; and then respectively adding 10-20 mL of gold-labeled confining liquid to seal the stirred product, stirring at room temperature for 15-20 min, centrifuging at 12000rpm for 10min, sucking out the supernatant, and removing, wherein the obtained precipitate is two colloidal gold compounds.
(2) The preparation of the colloidal gold compound is beneficial to the combination of the colloidal gold and the protein, so that the protein is easily adsorbed on the surface of the gold particles through electrostatic interaction to form a protein layer, and the mutual contact of the colloidal gold particles is prevented, so that the colloidal gold is in a stable state. Then triethanolamine and eudesmol are respectively added into the colloidal gold solution, so that the recombinant protein rP44-18ES and the mouse IgG are combined with the colloidal gold more firmly, and the colloid is in a stable state. The blocking is performed by a gold-labeled blocking solution, so that the nonspecific binding of the antigen is prevented, and the test result is influenced. Redissolving: and (3) respectively diluting the colloidal gold compound with a gold-labeled diluent to enable the colloidal gold compound to be respectively dissolved in the gold-labeled diluent, wherein the concentration of the colloidal gold compound is 10-250 mu g/mL, and respectively obtaining a recombinant protein rP44-18ES colloidal gold compound solution and a mouse IgG antibody colloidal gold compound solution.
Specifically, the protein needs to be stored in a certain diluent to ensure the stability of the protein and the gold particles, so the blocked colloidal gold complex is diluted by a gold-labeled diluent, and preferably, the concentration of the colloidal gold complex is 30-60 μ g/mL.
(3) Preparing a gold label pad: and mixing the recombinant protein rP44-18ES colloidal gold complex solution and the mouse IgG antibody colloidal gold complex solution, soaking the glass fiber membrane until liquid seeps out, taking out the glass fiber membrane, and drying at 37 ℃ for 3-8 hours to form the gold label pad.
Specifically, preparation of a colloidal gold solution: 100mL of double distilled water was measured and poured into a triangular flask, and 10% HAuCl was added4Adding 0.1mL of the solution, stirring, heating on a magnetic stirrer to boil, and quickly adding the prepared 1% Na3C6H5O72.5mL, continuously heating and stirring until boiling, changing the liquid from transparent to black and then to mauve transparent,adjusting the heating temperature to 400 ℃ to 200 ℃, reacting for 10min to obtain a colloidal gold particle solution with the diameter of 30-50 nanometers, and cooling to room temperature for later use.
Optionally, the gold-labeled confining liquid includes: the kit comprises a phosphate buffer solution, macromolecular protein with the mass concentration of 0.2-1.0% and Tween-20 with the mass concentration of 0.2-1.0%, wherein the phosphate buffer solution is a PBS solution with the pH value of 7.4 and the pH value of 0.1M, and the macromolecular protein is any one of BSA, fetal calf serum and skimmed milk;
the gold-labeled diluent comprises: 20mM Tris, 1 wt% BSA, 0.02 wt% NaN 31 wt% sucrose.
Specifically, the colloidal gold particles maintain colloidal stability by adsorbing surrounding ions and by electrostatic repulsion. When various conditions for maintaining the colloidal state are broken, aggregation of colloidal particles and formation of flocs occur, and therefore, it is necessary to maintain the stable state of the colloid. The phosphate in the gold-labeled confining liquid has a wide applicable pH value range, can be used for preparing buffer solutions with various pH values, has less influence of the pH value on the temperature, has strong buffering capacity, and has small pH change after being diluted. The existence of the macromolecular protein is favorable for protecting the gold-labeled protein and hindering the nonspecific combination of the antigen, and the Tween-20 serving as the surfactant is favorable for releasing the gold-labeled protein and improving the sensitivity of a detection result. The addition of Tris in the gold-labeled diluent can stabilize the pH value in electrophoresis, and is favorable for the stability of a gold-labeled compound. The sucrose as a drying protective agent can provide a relatively hydrophilic environment, and the situation that the gold-labeled compound is not released or slowly released due to the fact that the gold-labeled compound is firmly combined with the gold-labeled pad through hydrophobic effect is avoided. The gold-labeled diluent is beneficial to the stability of the gold-labeled compound, and the sensitivity of the detection strip is ensured.
Optionally, the detection line and the quality control line are separated by 5-7 mm.
Optionally, the assembling step of the test strip includes: installing a sample pad 2, a gold label pad 3, an analysis membrane 4 and a water absorption pad 5 on a bottom plate 1, wherein a part of the sample pad 2 is superposed and pressed on the gold label pad 3, a part of the gold label pad 3 is superposed and pressed on the analysis membrane 4, and a part of the water absorption pad 5 is superposed and pressed on the other side of the analysis membrane 4; the analysis membrane 4 is divided into a test area and a quality control area, the test area is provided with a detection line 41, the quality control area is provided with a quality control line 42, the detection line 41 is close to the gold-labeled pad 3, and the quality control line 42 is close to the absorbent pad 5.
Specifically, the sample pad 2 is partially superimposed and pressed on the gold label pad 3, the gold label pad 3 is partially superimposed and pressed on the analysis membrane 4, and the absorbent pad 5 is partially superimposed and pressed on the other side of the analysis membrane 4. So that the sample can be run from the sample pad 2 to the absorbent pad 5 on the detection strip by capillary effect, and the absorbent pad 5 adopts filter paper to absorb the residual sample and the colloidal gold compound.
The detection principle is as follows:
after a sample to be detected is loaded on the sample pad 2, an HGA antibody in the sample and a recombinant protein rP44-18ES colloidal gold complex on the gold label pad 3 form an HGA antibody-rP 44-18ES antigen-gold immune complex, the complex is electrophoresed towards the water absorption pad 5 due to capillary effect, the complex and the recombinant protein rP44-18ES antigen coated on the detection line 41 undergo an immune combination reaction to form an HGA antibody-rP 44-18ES antigen-gold-recombinant protein rP44-18ES antigen complex which is trapped on the detection line 41 and gradually enriched to form a red strip; the capillary action continues to move forwards, the colloidal gold labeled mouse IgG antibody and the rabbit anti-mouse IgG antibody coated on the quality control line 42 generate specific immunoreaction and are intercepted, a mouse IgG antibody-gold-rabbit anti-mouse IgG antibody compound is formed, the compound is gradually enriched on the quality control line 42 to form a red strip, and redundant non-combined substances continue to be chromatographed on the water absorption pad 5, so that the strip with color development on both the detection line 41 and the quality control line 42 is judged to be a positive result (the HGA antibody exists in a sample); if the sample to be detected does not contain the HGA antibody, the colloidal gold labeled recombinant protein rP44-18ES does not have specific binding reaction with the recombinant protein rP44-18ES on the detection line 41, so a chromogenic strip does not appear on the detection line 41, but the colloidal gold labeled mouse IgG antibody continues to swim forwards, and has specific immunoreaction with the rabbit anti-mouse IgG antibody coated on the quality control line 42, and the rabbit anti-mouse IgG antibody is intercepted and gradually enriched on the quality control line 42, so that the chromogenic strip appearing only on the quality control line 42 is judged to be a negative result (the HGA antibody does not exist in the sample).
The kit using method and the judgment standard are as follows:
when in use, a sample to be detected is dripped to the sample pad, the sample pad is placed at room temperature for 10min, then the detection result is judged,
(1) two color bands appear, wherein one color band is a detection line, the other color band is a quality control line and is positive, and the sample contains an HGA antibody;
(2) only one band appears on the quality control line, no band appears in the test area, the test area is negative, and the sample does not contain HGA antibody;
(3) no strip appears on the quality control line, which indicates that the detection strip is damaged, and whether the detection strip appears or not, the detection strip should be replaced with a new test strip for retesting.
Alternatively, the recombinant protein rP44-18ES consists of an amino acid sequence shown in SEQ ID NO. 1.
The present application will be described in further detail with reference to examples.
Example 1
A recombinant protein rP44-18ES kit for detecting phagocytophile anaplasma antibodies, comprising the steps of:
(1) preparing a colloidal gold solution: 100mL of double distilled water was measured and poured into a triangular flask, and 10% HAuCl was added4Adding 0.1mL of the solution, stirring, heating on a magnetic stirrer to boil, and quickly adding the prepared 1% Na3C6H5O72.5mL, continuously heating and stirring until boiling, changing the liquid from transparent to black and then to mauve transparent, adjusting the heating temperature to 400 ℃ to 200 ℃, reacting for 10min to obtain colloidal gold particle solution with the diameter of 3 nanometers, and cooling to room temperature for later use.
(2) Preparation of recombinant protein rP44-18ES colloidal gold complex and mouse IgG colloidal gold complex: respectively putting 100mL of colloidal gold solution into a beaker, and adding K with the molar concentration of 0.1M by stirring2CO3Adjusting the pH value of the colloidal gold solution to 7, respectively adding 2mg of recombinant protein rP44-18ES or mouse IgG antibody, then adding 0.5mg of triethanolamine and 0.5mg of eudesmol, and stirring at room temperature for 15 min; then respectively adding 10mL of gold-labeled confining liquid to seal the stirred product, stirring for 15min at room temperature, then centrifuging at 12000rpm for 10min, sucking out the supernatant, and discarding to obtain precipitates, namely two colloidal gold compounds. Respectively diluting the colloidal gold complex with gold-labeled diluentDiluting the solution to obtain a colloidal gold complex solution of the recombinant protein rP44-18ES and a colloidal gold complex solution of a mouse IgG antibody, wherein the concentration of the colloidal gold complex is 50 mu g/mL.
Wherein, the gold-labeled confining liquid comprises: phosphate buffer solution, macromolecular protein with the mass concentration of 0.2 percent and Tween-20 with the mass concentration of 0.2 percent, wherein the phosphate buffer solution is PBS solution with the pH value of 7.4 and 0.1M, and the macromolecular protein is BSA.
The gold-labeled diluent comprises: 20mM Tris, 1 wt% BSA, 0.02 wt% NaN 31 wt% sucrose.
(3) Preparing a gold label pad: and (3) mixing the two colloidal gold compound solutions prepared in the step (2), soaking the glass fiber membrane in the colloidal gold compound mixed solution until the glass fiber membrane has liquid exudation, taking out the glass fiber membrane, and drying at 37 ℃ for 4 hours to obtain the gold label pad.
(4) Preparation of analytical membranes: preparing a recombinant protein rP44-18ES diluted solution with the concentration of 1mg/mL and an antibody diluted solution with the concentration of 0.5mg/mL and combined with a mouse IgG antibody by using a diluent respectively, scribing the two diluted solutions onto a nitrocellulose membrane, scribing the recombinant protein rP44-18ES as a detection line of a detection strip, scribing a rabbit anti-mouse IgG antibody as a quality control line of the detection strip, and drying for 14 hours at 40 ℃; the diluent is a mixture of 50mM Tris and 2% sucrose, and the pH of the diluent is 8.5; the detection line and the quality control line are separated by 5 mm.
(5) Assembling: installing a sample pad, a gold label pad, an analysis membrane and a water absorption pad on a bottom plate, wherein a part of the sample pad is superposed and pressed on the gold label pad, a part of the gold label pad is superposed and pressed on the analysis membrane, and a part of the water absorption pad is superposed and pressed on the other side of the analysis membrane; the analysis membrane is divided into a test area and a quality control area, the test area is provided with a detection line, the quality control area is provided with a quality control line, the detection line is close to the gold-labeled pad, and the quality control line is close to the water absorption pad.
Comparative example 1:
a recombinant protein rP44-18ES kit for detecting antibodies to anaplasma phagocytophila, comprising the following steps, differing from example 1 in that:
0.5mg triethanolamine was added in step (2).
Comparative example 2:
a recombinant protein rP44-18ES kit for detecting antibodies to anaplasma phagocytophila, comprising the following steps, differing from example 1 in that:
in the step (2), 0.5mg of eudesmol is added.
Comparative example 3:
a recombinant protein rP44-18ES kit for detecting antibodies to anaplasma phagocytophila, comprising the following steps, differing from example 1 in that:
in the step (2), triethanolamine and eudesmol are not added.
Experimental example:
kit detection test
Taking the kits prepared in example 1 and comparative examples 1 to 3 as sample 1, sample 2, sample 3 and sample 4 respectively, taking recombinant protein P44-1 (amino acid sequence is shown in SEQ ID NO.2) which is conventionally used in the prior art as a control sample, and providing 15 parts of positive serum of the phagocytophilic anaplasma infection dog and 15 parts of negative serum of the dog; 40 parts of human serum positive to the anaplasma phagocytophila infection and 40 parts of human serum negative. The total of 110 serum samples were tested using 5 samples, and the results were counted and compared. And (5) counting the sensitivity and specificity of the kit, and calculating the overall coincidence rate. Each experimental group was set up with 3 replicates and the average was taken. The results are shown in FIG. 2. Both mouse IgG and rabbit anti-mouse IgG used in the experiment were purchased from Saimer Feishell science and technology (China) Co.
FIG. 2 shows the overall results of the test performed on clinical serum samples using different kits according to the present application. The results showed that 55 positive sera were detected in 55 positive sera and 0 missed test was performed using the sample 1 kit for recombinant protein rP44-18 ES; 1 part of false positive appears in 55 parts of negative serum; calculating to obtain: the sensitivity is 100%, the specificity is 98.2%, and the overall coincidence rate is 99.1%.
55 positive serums are detected by using a sample 2 kit of the recombinant protein rP44-18ES, and 0 part is missed; false positive 2 parts appear in 55 parts of negative serum; calculating to obtain: the sensitivity is 100%, the specificity is 96.4%, and the overall coincidence rate is 98.2%.
54 positive serums are detected by using a sample 3 kit of the recombinant protein rP44-18ES, and 1 negative serum is detected; 1 part of false positive appears in 55 parts of negative serum; calculating to obtain: the sensitivity is 98.2%, the specificity is 98.2%, and the overall coincidence rate is 98.2%.
53 positive samples are detected in 55 positive serums by using a sample 4 kit of the recombinant protein rP44-18ES, and 2 negative samples are detected; false positive 3 parts appear in 55 parts of negative serum; calculating to obtain: the sensitivity is 96.4%, the specificity is 94.5%, and the overall coincidence rate is 95.5%.
53 positive samples are detected in 55 positive serums by using a control sample kit of the recombinant protein P44-1, and 2 negative samples are detected; false positive of 4 parts appears in 55 parts of negative serum; calculating to obtain: the sensitivity is 96.4%, the specificity is 92.7%, and the overall coincidence rate is 94.5%.
As can be seen from FIG. 2, samples 1 to 4 have higher sensitivity and specificity and higher overall coincidence rate compared with the control, which indicates that the recombinant protein rP44-18ES of the present application can be used as an antigen protein for detecting anaplasma phagocytophilum antibodies, and can significantly reduce the probability of missed detection in HGA serum detection. And the triethanolamine and the eudesmol are added when the colloidal gold is marked, so that the binding rate of the gold particles with the recombinant protein rP44-18ES and the mouse IgG is improved, the stability of a colloidal gold compound is enhanced, and the sensitivity and the specificity of the prepared kit are further improved. By comparing samples 1 to 4, the sensitivity and specificity of the kit can be improved by adding triethanolamine and eudesmol at the same time, but the sensitivity of the kit can be improved when the triethanolamine is used alone, and the specificity of the kit can be enhanced when the eudesmol is added alone, so that the triethanolamine and the eudesmol act synergistically, and the kit containing the recombinant protein rP44-18ES has higher sensitivity and specificity. The kit prepared by the application is convenient, rapid, efficient and accurate to use, the detection result is easy to observe and distinguish, whether people and livestock are infected with the HGA virus or not can be preliminarily detected, a quick auxiliary detection means is provided for screening and treating anaplasma phagocytophila, a large instrument is not needed for operation, and the kit can be suitable for detection of basic medical institutions.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art; the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present application.
Sequence listing
<110> Hetao college
<120> recombinant protein rP44-18ES kit for detecting phagocytophilic anaplasma antibody
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 202
<212> PRT
<213> phagocytophilic cell anaplasmic (Anaplama phagocytophilum)
<400> 1
Met Trp Ser His Pro Gln Phe Glu Lys Tyr Asp Val Val Thr Gly Gln
1 5 10 15
Thr Asp Asn Leu Ala Ala Ala Leu Glu Ile Ser Asn Ser Gly Ile Gly
20 25 30
Lys Lys Val Cys Glu Thr Lys Arg Lys Asp Gly Asp Thr Thr Asn Arg
35 40 45
Phe Ala Lys Tyr Ile Val Gly Ala Gly Asp Ser Ser Asn Ala Gly Thr
50 55 60
Ser Leu Cys Gly Gly Lys Asn Gln Lys Ser Ser Asp Thr Asp Thr Gly
65 70 75 80
Val Glu Lys Ala Gln Ala Leu His Asp Phe Val Ser Asn Thr Leu Ser
85 90 95
Asp Gly Thr Lys Asn Trp Pro Thr Ser Ser Glu Thr Ser Lys Ser Asn
100 105 110
Asn Asp Asn Ala Lys Ala Val Ala Gly Asp Leu Thr Lys Lys Leu Thr
115 120 125
Pro Glu Glu Lys Thr Ile Val Ala Gly Leu Leu Ala Lys Thr Ile Glu
130 135 140
Gly Gly Glu Val Val Glu Ile Arg Ala Val Ser Ser Thr Ser Val Met
145 150 155 160
Val Asn Ala Cys Tyr Asp Leu Leu Ser Glu Gly Leu Gly Val Val Pro
165 170 175
Tyr Ala Cys Val Gly Leu Gly Gly Asn Phe Val Gly Val Val Asp Gly
180 185 190
His Ile Thr Pro Lys Leu Ala Tyr Arg Leu
195 200
<210> 2
<211> 211
<212> PRT
<213> phagocytophilic cell anaplasmic (Anaplama phagocytophilum)
<400> 2
Met Trp Ser His Pro Gln Phe Glu Lys Ala Gly Ser Asp Val Arg Ala
1 5 10 15
His Asp Asp Val Ser Ala Ser Pro Ala Phe Ser Lys Ile Arg Asp Phe
20 25 30
Ser Ile Arg Glu Ser Asn Gly Glu Thr Lys Ala Val Tyr Pro Tyr Leu
35 40 45
Lys Asp Gly Lys Ser Val Lys Leu Glu Ser His Lys Phe Asp Trp Asn
50 55 60
Thr Pro Asp Pro Arg Ile Gly Phe Lys Asp Asn Met Leu Val Ala Met
65 70 75 80
Glu Gly Ser Val Gly Tyr Gly Ile Gly Gly Ala Arg Val Glu Leu Glu
85 90 95
Ile Gly Tyr Glu Arg Phe Lys Thr Lys Gly Ile Arg Asp Ser Gly Ser
100 105 110
Lys Glu Asp Glu Ala Asp Thr Val Tyr Leu Leu Ala Lys Glu Leu Ala
115 120 125
Tyr Asp Val Val Thr Gly Gln Thr Asp Asn Leu Ala Ala Ala Leu Ala
130 135 140
Lys Thr Ser Gly Lys Asp Ile Val Gln Phe Ala Lys Ala Val Glu Ile
145 150 155 160
Ser Tyr Pro Ser Ile Asp Gly Lys Val Cys Ser Gly Lys His Ala Ala
165 170 175
Leu Ala Ala Asn Thr Asn Ala Glu Lys Lys Tyr Ala Val Glu Pro Ala
180 185 190
Asn Gly Gly Thr Asp Gly Ser Thr Ser Gln Cys Ser Gly Leu Ser Asn
195 200 205
Gly Ser Ala
210
Claims (10)
1. A recombinant protein rP44-18ES kit for detecting an anaplasma phagocytophila antibody is characterized by comprising a double-antigen sandwich gold-labeled detection strip, wherein the detection strip comprises a bottom plate, a sample pad, a gold-labeled pad, an analysis membrane and a water absorption pad are sequentially arranged on the bottom plate along a chromatography direction, a colloidal gold compound is arranged on the gold-labeled pad, and a detection line and a quality control line are arranged on the analysis membrane; the colloidal gold compound comprises a colloidal gold labeled recombinant protein rP44-18ES and a mouse IgG antibody, the detection line comprises the recombinant protein rP44-18ES, and the quality control line comprises an antibody combined with the mouse IgG antibody.
2. The kit of claim 1, wherein the antibodies that bind to murine IgG antibodies comprise one or more polyclonal antibodies to murine IgG antibodies of rabbit, sheep, horse, camel origin.
3. The kit of claim 1, wherein the sample pad and gold label pad are fiberglass film or mylar film.
4. The kit of claim 1, wherein the analytical membrane is a nitrocellulose membrane.
5. The kit of claim 1, wherein the test strip is prepared by a method comprising:
s1, preparing a gold-labeled pad: respectively preparing a recombinant protein rP44-18ES colloidal gold compound and a mouse IgG colloidal gold compound, redissolving the recombinant protein rP44-18ES colloidal gold compound and the mouse IgG colloidal gold compound, mixing, soaking a glass fiber membrane, drying the soaked glass fiber membrane at 37 ℃ for 3-8 hours, and preparing a gold label pad;
s2, preparation of an analysis membrane: respectively preparing a recombinant protein rP44-18ES diluted solution with the concentration of 1-3 mg/mL and an antibody diluted solution which is combined with a mouse IgG antibody and has the concentration of 0.3-3 mg/mL by using the diluted solution;
scribing the two diluted solutions onto a nitrocellulose membrane, scribing the recombinant protein rP44-18ES as a detection line of the detection strip, scribing an antibody combined with a mouse IgG antibody as a quality control line of the detection strip, and drying for 14-24 hours at 37-45 ℃; the diluent is a mixture of 50mM Tris and 2% sucrose, and the pH of the diluent is 8.5-9;
and S3, assembling the sample pad, the prepared gold label pad, the analysis membrane and the water absorption pad on the bottom plate, and then cutting according to specifications to obtain the detection strip.
6. The kit according to claim 5, wherein the gold pad is prepared by a method comprising:
preparation of recombinant protein rP44-18ES colloidal gold complex and mouse IgG colloidal gold complex: respectively putting 100mL of colloidal gold solution into a beaker, stirring and adding K with the molar concentration of 0.1-0.2M2CO3Adjusting the pH value of a colloidal gold solution to 6.5-9, respectively adding 2-3 mg of the recombinant protein rP44-18ES or the mouse IgG antibody, then adding 0.5-1.3 mg of triethanolamine and 0.3-0.6 mg of eudesmol, and stirring at room temperature for 15-30 min; then respectively adding 10-20 mL of gold-labeled confining liquid to seal the stirred product, stirring at room temperature for 15-20 min, centrifuging at 12000rpm for 10min, sucking out the supernatant, and removing to obtain precipitates, namely two colloidal gold compounds;
redissolving: respectively diluting the colloidal gold compound with a gold-labeled diluent to enable the colloidal gold compound to be respectively dissolved in the gold-labeled diluent, wherein the concentration of the colloidal gold compound is 10-250 mu g/mL, and respectively obtaining a recombinant protein rP44-18ES colloidal gold compound solution and a mouse IgG antibody colloidal gold compound solution;
preparing a gold label pad: and mixing the recombinant protein rP44-18ES colloidal gold complex solution and the mouse IgG antibody colloidal gold complex solution, soaking the glass fiber membrane until liquid seeps out, taking out the glass fiber membrane, and drying at 37 ℃ for 3-8 hours to form the gold label pad.
7. The kit according to claim 6, wherein the gold-labeled blocking solution comprises: the kit comprises a phosphate buffer solution, macromolecular protein with the mass concentration of 0.2-1.0% and Tween-20 with the mass concentration of 0.2-1.0%, wherein the phosphate buffer solution is a PBS solution with the pH of 7.4 and the mass concentration of 0.1M, and the macromolecular protein is any one of BSA, fetal calf serum and skimmed milk;
the gold-labeled diluent comprises: 20mM Tris, 1 wt% BSA, 0.02 wt% NaN31 wt% sucrose.
8. The kit according to claim 5, wherein the detection line and the quality control line are separated by 5-7 mm.
9. The kit of claim 5, wherein the step of assembling the test strip comprises: installing the sample pad, the gold label pad, the analysis membrane and the water absorption pad on the bottom plate, wherein a part of the sample pad is superposed and pressed on the gold label pad, a part of the gold label pad is superposed and pressed on the analysis membrane, and a part of the water absorption pad is superposed and pressed on the other side of the analysis membrane; the analysis membrane is divided into a test area and a quality control area, the test area is provided with a detection line, the quality control area is provided with a quality control line, the detection line is close to the gold-labeled pad, and the quality control line is close to the water absorption pad.
10. The kit according to any one of claims 1 to 9, wherein the recombinant protein rP44-18ES consists of the amino acid sequence shown in SEQ ID No. 1.
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