CN111413501A - Application of detection test strip in preparation of kit for detecting THSD7A antibody - Google Patents
Application of detection test strip in preparation of kit for detecting THSD7A antibody Download PDFInfo
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- CN111413501A CN111413501A CN202010279273.XA CN202010279273A CN111413501A CN 111413501 A CN111413501 A CN 111413501A CN 202010279273 A CN202010279273 A CN 202010279273A CN 111413501 A CN111413501 A CN 111413501A
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention belongs to the technical field of biological detection, and discloses a detection test strip and a detection method for a THSD7A antibody. The test strip comprises a sample pad, a combination pad 1, a combination pad 2, an NC membrane and absorbent paper which are sequentially lapped on a viscous bottom plate; the THSD7A biotinylated conjugate is sprayed on the binding pad 1, the SA fluorescent microsphere conjugate and the DNP-BSA fluorescent microsphere conjugate are sprayed on the binding pad 2, and a capture body is coated on the NC membrane to serve as a detection line. The THSD7A antibody in the detection sample is combined with biotinylated THSD7A, then combined with fluorescent microspheres of SA, captured by a capture body when passing through the capture body coated by an NC membrane, simultaneously combined with signal amplification reaction of biotin and avidin, and developed through signals of the fluorescent microspheres to obtain a signal reaction value result, so that the detection sample has high sensitivity, and the content of the THSD7A antibody in the detection sample can be simply, rapidly and accurately quantitatively tested.
Description
The application is a divisional application of the invention with the application date of 2018, month 01 and 30, and the invention name of the invention is 'test paper and a detection method for detecting THSD7A antibody', and the application number of 201810089697.2.
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a detection test strip and a detection method for a THSD7A antibody.
Background
Membranous Nephropathy (MN) is one of the most common types of pathology in adult nephrotic syndrome, characterized by the pathological changes seen on the epithelial side of the glomerular capillary loop with a large deposit of immune complexes. MN can be classified into two major categories, Idiopathic Membranous Nephropathy (IMN) and Secondary Membranous Nephropathy (SMN), depending on the cause of the disease. IMN is a chronic inflammatory disease of glomerulus, and is mostly associated with an anti-phospholipase a2 receptor antibody, and the anti-phospholipase a2 receptor antibody binds to a corresponding antigen on podocytes to form an in situ immune complex, and then activates complement through an alternative pathway to form a C5b-9 membrane attack complex, which damages podocytes and destroys the glomerular filtration barrier, and is typically characterized by proteinuria, with the increase of urine protein content, there is a trend of renal failure. Unlike IMN, SMN is a secondary or concurrent disease, and SMN may occur as a result of drug therapy, drug abuse, infectious diseases, other autoimmune diseases, and tumors. Such as systemic lupus erythematosus, rheumatoid arthritis, hepatitis B virus infection, and drugs, poisons, tumors or environmental factors. The medicines for secondary membranous nephropathy mainly comprise gold preparations, mercury, penicillamine, ibuprofen, diclofenac and the like. At the same time, SMN may improve with treatment of the underlying disease.
The diagnosis of IMN and SMN mainly depends on clinical manifestations and kidney puncture, kidney biopsy, also called kidney puncture biopsy, is an invasive wound diagnosis method, has certain harm to patients, and diagnosis according to clinical manifestations requires certain experience, and also requires histopathological verification, recent research and literature reports, IMN is an autoimmune disease, the discovered autoimmune antigen phospholipase A2 receptor (P L A2R) is the main target antigen, the positive rate for diagnosis of the IMN can reach 70% -82%, and the antibody is not detected in other disease and normal human serum samples, although 70% -82% of IMN patients have circulating antibody against P L A2R, some IMN patients still do not find P L A2R antibody, IMN patients may have other self antigen, THSD7A is another new podocyte autoantigen in IMN patients, there is found THSD7 and 34 antibody in European patients and kidney puncture, kidney biopsy kit also called kidney puncture biopsy, is also called kidney puncture biopsy, is an invasive wound diagnosis method, and has certain clinical diagnosis result that the antibody of spontaneous detection of spontaneous serological membrane abnormality of the antibody of the Western nephropathy is detected by spontaneous serological membrane-induced by the Western nephropathy induced by the antibody of the Western nephropathy, the antibody of the serum of the antibody of another new podocyte autoantibody, the antibody of.
The Chinese patent with application number of 201610201970.7 discloses that an E L ISA method is used for testing anti-THSD 7A antibodies in serum, but the E L ISA method is complex to operate and long in testing time, the Chinese patent with application number of 201710047530.5 discloses that a colloidal gold method is used for testing THSD7A antibodies in human serum, and although the colloidal gold method is simple and rapid in testing, the method cannot meet the requirement of quantitatively testing the THSD7A antibodies in the human serum.
Disclosure of Invention
In view of the above, the present invention aims to provide a test strip and a test method for quantitatively testing the THSD7A antibody content in human serum, which are simple, fast and accurate, aiming at the defects of complicated operation process, long detection time and incapability of quantitative detection in the prior art.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the application of the detection test strip in the preparation of the kit for detecting the THSD7A antibody is characterized in that the detection test strip comprises a sample pad, a combination pad 1, a combination pad 2, a nitrocellulose membrane and absorbent paper which are sequentially overlapped on an adhesive bottom plate; the detection kit comprises a binding pad 1, a nitrocellulose membrane, a capture body and an anti-DNP-BSA antibody, wherein the binding pad 1 is sprayed with a THSD7A biotinylation conjugate, the binding pad 2 is sprayed with an avidin fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate, the nitrocellulose membrane is coated with the capture body as a detection line, and the nitrocellulose membrane is further coated with the anti-DNP-BSA antibody as a quality control line.
A test strip for detecting a THSD7A antibody comprises a sample pad, a combination pad 1, a combination pad 2, a nitrocellulose membrane (NC) and absorbent paper which are sequentially overlapped on an adhesive bottom plate; the detection kit comprises a binding pad 1, a nitrocellulose membrane and a nitrocellulose membrane, wherein the binding pad 1 is sprayed with a THSD7A biotinylation conjugate, the binding pad 2 is sprayed with an avidin (SA) fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate, a capture body is coated on the nitrocellulose membrane to serve as a detection line, and an anti-DNP-BSA antibody is further coated on the nitrocellulose membrane to serve as a quality control line.
Wherein, the THSD7A in the THSD7A biotinylated conjugate in the detection test strip comprises but is not limited to a protein which is a full-length or partial fragment of a THSD7A molecule, or a variant, an analogue and a substitute which can play a similar role to the THSD7A protein. The THSD7A can be natural purified THSD7A, or obtained by gene engineering technology recombination.
Preferably, the particle size of the fluorescent microsphere in the test strip is 200 nm.
Preferably, the capture body in the test strip is an anti-human IgG antibody or a substance capable of binding to the human IgG antibody, such as protein A or protein G. More preferably a murine anti-human IgG, such as murine anti-human IgG 4.
The invention also provides a preparation method of the test strip for detecting the THSD7A antibody, which comprises the following steps:
A. the THSD7A protein is biotinylated to obtain a THSD7A biotinylated conjugate, which is sprayed on the binding pad 1;
B. coupling the pretreated avidin and DNP-BSA with the activated fluorescent microspheres respectively to obtain avidin fluorescent microsphere conjugates and DNP-BSA fluorescent microsphere conjugates, and spraying the avidin fluorescent microsphere conjugates and the DNP-BSA fluorescent microsphere conjugates on the binding pad 2;
C. coating a capture body on a nitrocellulose membrane as a detection line, and coating an anti-DNP-BSA antibody on the nitrocellulose membrane as a quality control line;
D. a sample pad, a binding pad 1 sprayed with a THSD7A biotinylated conjugate, a binding pad 2 sprayed with an avidin fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate, a nitrocellulose membrane coating a capture body and an anti-DNP-BSA antibody, and absorbent paper are sequentially overlapped on an adhesive bottom plate.
The method of biotinylation of the THSD7A protein according to the present invention is not limited, and may be performed by a method known to those skilled in the art. In some embodiments, the invention employs a commonly used biotinylation kit for biotin labeling of THSD7A protein. E.g. using Thermo corporation
Sulfo-NHS-L C-Biotin biotinylation kit.
The specific procedure for spraying the THSD7A biotinylated conjugate on the pad 1 in step A was to spray the THSD7A biotinylated conjugate on the pad 1 in an amount of 2. mu.l/cm using a gold spraying striping machine and oven dry at 37 ℃ for 2 h.
The coupling in the step B of the preparation method of the test strip is preferably to remove the supernatant by centrifugation of the activated fluorescent microspheres, wash the microspheres with MES buffer solution with the pH value of 7.0-8.050 mM, add 0.1-0.2 mg of avidin or 0.2-0.4 mg of DNP-BSA per 100 mul of microspheres, and mix the microspheres uniformly at room temperature for reaction for 1-2 hours.
In the preparation method of the test strip, the avidin and DNP-BSA in the step B need to be pretreated before being coupled with the fluorescent microspheres. Preferably, avidin and DNP-BSA are dialyzed three times against MES buffer of pH7.0 to 8.050 mM, respectively.
The fluorescent microspheres in the step B need to be activated in advance. The specific method for activating the microspheres is preferably that after the microspheres are washed by 50mM MES solution with pH of 6.0-6.5, 0.2-0.4 mu g of NHS (N-hydroxysuccinimide) and 0.2-0.4 mu g of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) are added into each 100 mu l of microspheres and mixed uniformly at room temperature for reaction for 30-40 min.
Further preferably, the coupling of avidin and DNP-BSA with activated fluorescent microspheres in step B further comprises a blocking step. More preferably, the blocking is carried out by adding BSA to a concentration of 1-5% (g/ml), and carrying out a uniform mixing reaction at room temperature for 30-40 min. Adding BSA to the solution until the concentration is 1 to 5g per 100ml, and uniformly mixing the solution at room temperature for reaction for 30 to 40 min.
The method comprises the steps of carrying out centrifugal washing on the avidin fluorescent microsphere conjugate or the DNP-BSA fluorescent microsphere conjugate by using a pH 8.510mmol/L boric acid solution, and then adding the preserving solution for preservation, wherein preferably, the preserving solution is a pH 8.510mmol/L boric acid solution containing 1-5% of BSA and 1-2% of trehalose, and the concentration of the BSA and the trehalose is the mass volume concentration, namely, each 100ml of the preserving solution contains 1-5 g of BSA and 1-2 g of trehalose.
In the step B, the specific operation of spraying the avidin fluorescent microsphere conjugate and the DNP-BSA fluorescent microsphere conjugate on the binding pad 2 is to respectively spray the avidin fluorescent microsphere conjugate and the DNP-BSA fluorescent microsphere conjugate on the binding pad 2 by using a gold spraying and membrane scribing instrument in an amount of 2 mu l/cm, and drying the avidin fluorescent microsphere conjugate and the DNP-BSA fluorescent microsphere conjugate in an oven at 37 ℃ for 2 hours.
And step C of the preparation method of the test strip, the capture body is coated on the nitrocellulose membrane to be used as a detection line, and the anti-DNP-BSA antibody is coated on the nitrocellulose membrane to be used as a quality control line. The step C is specifically to dilute the capture body and the anti-DNP-BSA antibody to 1-2 mg/ml by using coating solutions respectively, scratch the diluted capture body and the anti-DNP-BSA antibody on a nitrocellulose membrane according to 1 mul/cm by using a gold spraying and scratching instrument respectively, and dry the membrane for 16-22 h at 37 ℃; the coating solution is PBS containing 5-10% trehalose. Wherein the concentration of the trehalose is mass volume concentration, and the trehalose is measured by g/ml, namely, 5 g-10 g of trehalose is contained in each 100ml of coating liquid. And D, sequentially overlapping a sample pad, a binding pad 1 sprayed with a THSD7A biotinylation conjugate, a binding pad 2 sprayed with an avidin fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate, a nitrocellulose membrane coated with a capture body and an anti-DNP-BSA antibody, and absorbent paper on a viscous bottom plate to obtain the test strip.
Preferably, the sample pad is soaked in a Tris-HC L buffer solution with the pH value of 7.4 of 0.5M and containing 5-10% of trehalose for 5-10 min in advance, and is dried for 2h at 37 ℃, wherein the trehalose concentration is the mass volume concentration, namely, the trehalose is contained in a unit of g/ml of Tris-HC L buffer solution with the concentration of 5 g-10 g.
It can be understood by those skilled in the art that the materials of the sample pad, the bonding pad 1 and the bonding pad 2 of the test strip for detecting the THSD7A antibody are not particularly limited, and may be common materials, such as polyester film and glass fiber.
The invention also provides a detection kit of the THSD7A antibody, which comprises the detection test strip, a sample buffer solution and/or THSD7A antibody standard products with different concentration gradients. For example, the THSD7A antibody standard substance with 6-7 concentration gradients is used for carrying out calibration test on a test strip, and according to a statistical method, an equation is established and fitted into standard curve data to be stored in an instrument by taking the fluorescence intensity ratio (T band detection value/C band detection value) of a detection line (T band) and a quality control line (C band) as a vertical coordinate and the concentration of a THSD7A standard solution as a horizontal coordinate.
In some embodiments, the THSD7A antibody standard with different concentration gradients is specifically subjected to a calibration test using 7 THSD7A antibody unit concentrations of 2RU/ml, 10RU/ml, 20RU/ml, 50RU/ml, 100RU/ml, 200RU/ml, 500RU/ml, and each spot is tested 6 times using the same batch of test strips. According to a statistical method, an equation is established and fitted into a standard curve by taking the fluorescence intensity ratio (T band detection value/C band detection value) of a detection line (T band) and a quality control line (C band) as a vertical coordinate and the standard solution concentration as a horizontal coordinate, and data are stored in an instrument.
The invention also provides a detection method of the THSD7A antibody, which comprises the steps of adding a sample to be detected into a sample buffer solution, mixing the sample and the sample buffer solution for 10 seconds, adding the sample to the sample pad of the detection test strip, inserting the detection test strip into a detection hole of a fluorescence analyzer, standing for 3-4 minutes, and calculating the concentration value of the THSD7A antibody by taking the fluorescence intensity ratio (T-band detection value/C-band detection value) of a detection line (T-band) and a quality control line (C-band) as a vertical coordinate and the concentration of the THSD7A antibody standard solution as a horizontal coordinate.
The invention coats a capture body of anti-human IgG4 (or IgG) on a nitrocellulose membrane, is used for capturing THSD7A antibody existing in a sample, simultaneously biotinylates the THSD7A antibody by using a biotinylation reagent by using a biotin-avidin signal amplification system, and simultaneously labels and couples avidin (SA) by using fluorescent microspheres. When a sample is detected, the THSD7A antibody in the sample to be detected and biotinylated THSD7A on the binding pad 1 generate specific binding reaction, the biotinylated THSD7A can be bound with the fluorescent microspheres of the avidin on the binding pad 2, the fluorescent microspheres are captured by a capture body coated by the nitrocellulose membrane, meanwhile, the signal amplification reaction of the biotin and the avidin is bound, and the signal reaction value result read by an instrument can be obtained through the signal color development of the fluorescent microspheres.
Preferably, the sample to be tested is whole blood, serum, plasma, urine or saliva. Such as venous blood, peripheral blood, etc.
According to the technical scheme, the invention provides the test strip for detecting the THSD7A antibody and the detection method. The test strip for detecting the THSD7A antibody comprises a sample pad, a combination pad 1, a combination pad 2, a nitrocellulose membrane and absorbent paper which are sequentially lapped on an adhesive bottom plate; the detection kit comprises a binding pad 1, a nitrocellulose membrane, a capture body and an anti-DNP-BSA antibody, wherein the binding pad 1 is sprayed with a THSD7A biotinylation conjugate, the binding pad 2 is sprayed with an avidin fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate, the nitrocellulose membrane is coated with the capture body as a detection line, and the nitrocellulose membrane is further coated with the anti-DNP-BSA antibody as a quality control line. The THSD7A antibody in the sample is detected by a capture method, the THSD7A antibody in the sample and biotinylated THSD7A generate specific binding reaction, the biotinylated THSD7A can be bound with the fluorescent microsphere of avidin, the biotinylated THSD7A is captured by a capture body coated by a nitrocellulose membrane and simultaneously is bound with the signal amplification reaction of biotin and avidin, and the signal reaction value result read by an instrument can be obtained by the signal color development of the fluorescent microsphere. The test strip for detecting the THSD7A antibody by adopting the capture method has high sensitivity, can simply, conveniently, quickly and accurately quantitatively test the content of the THSD7A antibody in human serum, blood plasma and whole blood, and provides good auxiliary guidance for primary screening, diagnosis, disease condition and prognosis monitoring of idiopathic membranous nephropathy.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a structure diagram of a test strip for detecting THSD7A antibody according to the present invention;
FIG. 2 shows a schematic diagram of the detection method of THSD7A antibody according to the present invention.
Detailed Description
The invention discloses a test strip and a test method for detecting a THSD7A antibody. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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 invention.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.
Example 1 preparation of fluorescent test strip for detecting THSD7A antibody according to the present invention
1. Preparation of avidin (SA) fluorescent microsphere conjugate and DNP-BSA fluorescent microsphere conjugate
1.1 preparation of a 50mM solution of pH7.0MES
1.2 preparation of a 50mM solution of pH6.5 MES
1.3 avidin to be coupled, DNP-BSA were dialyzed against 50mM MES buffer pH7.0 for three times, and buffered at 4 ℃ for further use.
1.4 activation of microspheres: 20 mu.l of 200nm microspheres are added with 50mM MES solution with pH6.5, and after centrifugal washing is carried out twice at 12000r/min, 0.25 mu g of NHS (N-hydroxysuccinimide) and 0.25 mu g of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) are added into each 100 mu.l of microspheres, and the mixture is evenly mixed and reacted for 40min at room temperature.
1.5 coupling of microspheres: centrifuging the activated microspheres at 12000r/min to remove supernatant, washing with 50mM MES buffer solution of pH7.0 for 2 times, adding dialyzed 0.2mg avidin or 0.25mg DNP-BSA per 100 μ l microspheres, and mixing at room temperature for 1 hr.
1.6 blocking of microspheres, namely adding the coupled microspheres into BSA to ensure that the BSA concentration is 1% (1g/100ml), and continuously mixing uniformly at room temperature for reaction for 40 min.
1.7 preservation of microspheres, the above-mentioned SA microspheres and DNP-BSA microspheres which were blocked were washed twice with 12000r/min pH 8.510 mmol/L boric acid solution, and then stored at 4 ℃ in a preservation solution containing 1% BSA (1g/100ml) and 2% (2g/100ml) trehalose in pH 8.510mmol/L boric acid solution.
Preparation of THSD7A biotinylated conjugate: using Thermo
The Sulfo-NHS-L C-Biotin biotinylation kit biotinylates THSD7A and temporarily stores it at 4 ℃ until use.
3. Preparation of bonding pad 1 and bonding pad 2
Spraying the prepared THSD7A biotinylated conjugate on 1cm wide glass fiber by using a gold spraying and film scratching instrument in a volume of 2 mul/cm to prepare a binding pad 1, and spraying the prepared SA fluorescent microsphere conjugate and DNP-BSA fluorescent microsphere conjugate on 1cm wide glass fiber by using a gold spraying and film scratching instrument in a volume of 2 mul/cm to prepare a binding pad 2; the bonding pad 1 and the bonding pad 2 are respectively placed in an oven for drying at 37 ℃ for 2h, and are sealed and stored for standby.
4. Coating of nitrocellulose membranes (NC membranes)
The coating solution was 0.01M PBS containing 5% trehalose, mouse anti-human IgG4 was diluted to 1mg/ml, rabbit anti-DNP-BSA was diluted to 1mg/ml, the diluted mouse anti-human IgG4 and rabbit anti-DNP-BSA were drawn on the NC membrane at 1. mu.l/cm using a gold-jet membrane drawing instrument, and the NC membrane was subjected to T-line and C-line, oven-dried at 37 ℃ for 22 hours, and stored in a sealed state for future use.
5. Assembly of test strips
The sample pad, the conjugate pad 1, the conjugate pad 2, the nitrocellulose membrane (coated in the above-mentioned step), and the absorbent paper were sequentially overlapped on the adhesive PVC base plate, and cut into test strips of 4mm width and assembled on the card (see fig. 1).
6. Calibration test of test strips
2RU/ml, 10RU/ml, 20RU/ml, 50RU/ml, 100RU/ml, 200RU/ml, 500RU/ml, 7 THSD7A concentration gradient standards were selected for calibration testing, and each point was tested 6 times using the same test strip batch. According to a statistical method, an equation is established and fitted into a standard curve by taking the fluorescence intensity ratio (T band detection value/C band detection value) of a detection line (T band) and a quality control line (C band) as a vertical coordinate and the standard solution concentration as a horizontal coordinate, and data are stored in an instrument.
7. And (3) testing a clinical sample, determining a critical value of the test strip, and evaluating the related performances of the test strip, such as precision, accuracy, conformity rate and the like. And carrying out calibration test on unit concentration, establishing standard curve data by taking the concentration as an abscissa and the T/C as an ordinate, storing the standard curve data into a dry-type fluorescence immunoassay analyzer, and calculating the concentration of a test sample based on the curve.
Example 2 performance test of fluorescent test strip of THSD7A antibody of the invention
Each sample is tested with 60. mu.l serum and the test result is read by a 15min instrument.
1. The determination of the test strip critical value of the invention comprises the following steps: the mean value of the concentration of anti-THSD 7A antibody was 20.8RU/ml and the standard deviation was 1.1RU/ml by testing 220 normal human sera, and the cut-off value was 24.1RU/ml with the sum of the mean value plus 3 times the standard deviation as the cut-off value.
2. The test paper strip disclosed by the invention is determined by selecting 3 parts of quality control serum with high, medium and low values (a high value quality control target value is 290RU/ml, a medium value quality control target value is 75RU/m L, and a low value quality control target value is 30RU/m L), repeatedly measuring each part in the same test for 10 times, respectively calculating an average value and a standard deviation, calculating a variation coefficient CV (%) in the test, measuring for 1 time every day, continuously measuring for 10 days, and calculating a CV (%) value between tests, wherein the calculation formula is that CV (%) is standard deviation/average value × 100%, and the test data are shown in the following table 1.
TABLE 1 test paper strip precision results
Note: the intra-test means that in one test, the same test is repeatedly tested for multiple times; by inter-assay is meant that the same assay is tested repeatedly at different times.
The results in table 1 show that the test strip of the present invention meets the requirements for precision.
3. The test strip provided by the invention is used for detecting the accuracy (recovery rate experiment):
2 parts of serum with anti-THSD 7A antibody concentration of 60 and 300RU/ml are selected, the low value serum and the high value serum are mixed into 3 parts of serum with different concentrations according to the proportion of 1:4, 1:1 and 9:1 respectively, the theoretical value is 252.2, 207.5 and 81.4RU/ml respectively, the consistency of the detection value and the theoretical value, namely the recovery rate is calculated by the detection of a kit, the calculation result of the recovery rate is shown in a table 2, and the calculation formula is that the recovery rate is equal to the test value/the theoretical value of × 100%.
TABLE 2 results of recovery calculation
The results show that the recovery is between 97% and 102% and the average recovery is 99.0%. The accuracy meets the requirement.
4. Evaluation of test strip coincidence rate
Comparison with gold standard: selecting 115 parts of serum of patients with idiopathic membranous nephropathy and 45 parts of serum of patients with non-idiopathic membranous nephropathy, which are confirmed to be diagnosed by renal puncture, taking the renal puncture as a gold standard, and obtaining the statistical results shown in the following table 3.
TABLE 3 compliance rate test
The result shows that the specificity of the test strip is 92.0%, and the sensitivity is 84.0%.
Example 3 comparison with the Indirect immunofluorescence detection method (IIFT) developed by Euron corporation
Collecting 170 serum of patients with idiopathic membranous nephropathy and non-idiopathic membranous nephropathy, simultaneously testing with fluorescence test paper strip developed by the company and indirect immunofluorescence detection method (IIFT) developed by Euromeng company, and the statistical results are shown in Table 4
Table 4 comparison with IIFT test results
The results show that the positive coincidence rate is 88 percent and the negative coincidence rate is 92 percent compared with the indirect immunofluorescence detection method (IIFT) developed by the Euromeng company.
The data show that the detection of the THSD7A antibody can accurately test the content of the THSD7A antibody in serum, and can effectively perform preliminary screening and disease monitoring on idiopathic membranous nephropathy.
Claims (10)
1. The application of the detection test strip in the preparation of the kit for detecting the THSD7A antibody is characterized in that the detection test strip comprises a sample pad, a combination pad 1, a combination pad 2, a nitrocellulose membrane and absorbent paper which are sequentially overlapped on an adhesive bottom plate; the detection kit comprises a binding pad 1, a nitrocellulose membrane, a capture body and an anti-DNP-BSA antibody, wherein the binding pad 1 is sprayed with a THSD7A biotinylation conjugate, the binding pad 2 is sprayed with an avidin fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate, the nitrocellulose membrane is coated with the capture body as a detection line, and the nitrocellulose membrane is further coated with the anti-DNP-BSA antibody as a quality control line.
2. The use of claim 1, in the THSD7A biotinylated conjugate, the THSD7A is a protein which is a full-length or partial fragment of the THSD7A molecule, or a variant, analog or substitute which can play a similar role to the THSD7A protein.
3. The use of claim 1, wherein the fluorescent microspheres in the test strip have a particle size of 200 nm; the capture body is an anti-human IgG antibody or a substance capable of binding to the human IgG antibody.
4. The use of claim 1, the method for preparing the test strip, comprising the steps of:
A. the THSD7A protein is biotinylated to obtain a THSD7A biotinylated conjugate, which is sprayed on the binding pad 1;
B. coupling the pretreated avidin and DNP-BSA with the activated fluorescent microspheres respectively to obtain avidin fluorescent microsphere conjugates and DNP-BSA fluorescent microsphere conjugates, and spraying the avidin fluorescent microsphere conjugates and the DNP-BSA fluorescent microsphere conjugates on the binding pad 2;
C. coating a capture body on a nitrocellulose membrane as a detection line, and coating an anti-DNP-BSA antibody on the nitrocellulose membrane as a quality control line;
D. a sample pad, a binding pad 1 sprayed with a THSD7A biotinylated conjugate, a binding pad 2 sprayed with an avidin fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate, a nitrocellulose membrane coating a capture body and an anti-DNP-BSA antibody, and absorbent paper are sequentially overlapped on an adhesive bottom plate.
5. The use of claim 4, wherein the coupling in step B of the preparation method of the test strip is that after centrifugation is carried out on the activated fluorescent microspheres to remove supernatant, 0.1-0.2 mg of avidin or 0.2-0.4 mg of DNP-BSA is added to each 100 μ l of microspheres after washing with MES buffer solution with pH of 7.0-8.050 mM, and the mixture is mixed and reacted for 1-2 hours at room temperature.
6. The use according to claim 4, wherein the test strip is prepared by the method of step C, which comprises diluting the capture body and the anti-DNP-BSA antibody with a coating solution to 1-2 mg/ml, scratching the diluted capture body and the anti-DNP-BSA antibody with a gold-spraying scratching apparatus on a nitrocellulose membrane at 1 μ l/cm, and drying at 37 ℃ for 16-22 h; the coating solution is PBS containing 5-10% (g/ml) trehalose.
7. The application of claim 4, wherein the test strip is prepared by pre-soaking the sample pad in 5-10% (g/ml) trehalose in Tris-HC L buffer solution with pH of 0.5M and pH of 7.4 for 5-10 min, drying at 37 ℃ for 2h, pre-treating avidin and DNP-BSA by dialyzing with MES buffer solution with pH of 7.0-8.050 mM, activating the microspheres by washing the microspheres with 50mM MES solution with pH of 6.0-6.5, adding 0.2-0.4 μ g NHS (N-hydroxysuccinimide) and 0.2-0.4 μ g EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) per 100 μ l of microspheres, mixing at room temperature for 30-40 min, and coupling avidin and DNP-BSA with activated fluorescent microspheres, and then sealing, specifically adding BSA to a concentration of 1-5% (g/ml), and mixing at room temperature for 30-40 min.
8. A detection kit for a THSD7A antibody comprises a detection test strip for detecting the THSD7A antibody, a sample buffer solution and/or THSD7A antibody standard substances with different concentration gradients; wherein the detection test strip comprises a sample pad, a combination pad 1, a combination pad 2, a nitrocellulose membrane and absorbent paper which are sequentially lapped on an adhesive bottom plate; the detection kit comprises a binding pad 1, a nitrocellulose membrane, a capture body and an anti-DNP-BSA antibody, wherein the binding pad 1 is sprayed with a THSD7A biotinylation conjugate, the binding pad 2 is sprayed with an avidin fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate, the nitrocellulose membrane is coated with the capture body as a detection line, and the nitrocellulose membrane is further coated with the anti-DNP-BSA antibody as a quality control line.
9. A detection method of THSD7A antibody comprises adding a sample to be detected into a sample buffer solution, mixing for 10 seconds, adding the sample to a sample pad of a detection test strip of the detection kit of claim 9, inserting the detection test strip into a detection hole of a fluorescence analyzer, standing for 3-4 minutes, and calculating the concentration value of the THSD7A antibody by taking the fluorescence intensity ratio of a detection line (T band) and a quality control line (C band) as an ordinate and the concentration of a THSD7A antibody standard solution as an abscissa.
10. The method of claim 9, wherein the sample is whole blood, serum, plasma, urine or saliva.
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Also Published As
| Publication number | Publication date |
|---|---|
| WO2019148753A1 (en) | 2019-08-08 |
| CN108414748B (en) | 2022-02-15 |
| CN108414748A (en) | 2018-08-17 |
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