CN111413506A - Application of detection test strip in preparation of kit for detecting P L A2R antibody - Google Patents
Application of detection test strip in preparation of kit for detecting P L A2R antibody Download PDFInfo
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- CN111413506A CN111413506A CN202010279263.6A CN202010279263A CN111413506A CN 111413506 A CN111413506 A CN 111413506A CN 202010279263 A CN202010279263 A CN 202010279263A CN 111413506 A CN111413506 A CN 111413506A
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
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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 for a P L A2R antibody and a detection method thereof, wherein the detection test strip for the P L A2R antibody comprises a sample pad, a combination pad 1, a combination pad 2, an NC membrane and absorbent paper which are sequentially overlapped on a viscous bottom plate, a P L A2R biotinylation conjugate is sprayed on the combination pad 1, an SA fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate are sprayed on the combination pad 2, a capture body is coated on the NC membrane to serve as a detection line, and an anti-DNP-BSA antibody is further coated on the NC membrane to serve as a whole blood quality control line.
Description
The application is a divisional application of the invention with the application date of 2018, 20/01 and 30, and the invention name of "test paper and a detection method for the P L A2R antibody" and the application number of 201810091384.0.
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 P L A2R 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, namely kidney biopsy, also called kidney puncture biopsy, is an invasive wound diagnosis method, has certain harm to patients, and the diagnosis according to the clinical manifestations requires certain experience and histopathological verification.
The method is characterized in that the indirect immunofluorescence kit of the European Mongolian company is used for testing the Pla2r antibody in serum, but the indirect immunofluorescence method is complicated in operation process and long in detection time, the Chinese patent with the application number of 201510245291.5 discloses a test strip for testing the serum P L A2R antibody by adopting a double-antigen sandwich, the method is used for judging the result for at least 15min, the chromogenic signal is weak, and the weak positive missed detection is easy to occur, and the Chinese patent with the application number of 201710047530.5 discloses a method for testing the serum Pla2r antibody by adopting a colloidal gold immunochromatography technology, but the method cannot meet the requirement of quantitative test.
Disclosure of Invention
In view of the above, the present invention aims to provide a test strip and a test method for quantitatively, accurately and conveniently testing the P L A2R antibody in human serum, plasma and whole blood, aiming at the defects of complicated operation process, long detection time, weak chromogenic signal 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:
an application of a detection test strip in preparation of a kit for detecting a P L A2R antibody is disclosed, 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 overlapped on a viscous bottom plate, a P L A2R biotinylation conjugate is sprayed on the combination pad 1, an avidin fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate are sprayed on the combination pad 2, 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.
A detection test strip for a P L A2R 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 a viscous bottom plate, wherein a P L A2R biotinylation conjugate is sprayed on the combination pad 1, an avidin (SA) fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate are sprayed on the combination pad 2, 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.
The P L A2R in the P L A2R biotinylated conjugate in the test strip includes but is not limited to protein which is full-length or partial fragment of P L A2R molecule, or variant, analogue and substitute which can play a similar role to P L A2R protein, the P L A2R can be natural purified P L A2R, or can be obtained by gene engineering technology recombination.
Preferably, the P L A2R molecular fragment is the molecular fragment in P L A2R combined with the THSD7A protein sequence.
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 P L A2R antibody, which comprises the following steps:
A. biotinylation of the P L A2R protein to obtain a P L A2R biotinylated conjugate, 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 P L A2R biotinylated 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 are sequentially overlapped on an adhesive bottom plate.
The method of biotinylation of the P L A2R protein is not limited and can be performed by methods well known to those skilled in the art
Sulfo-NHS-L C-Biotin biotinylation kit
A specific procedure for spraying the P L A2R biotinylated conjugate onto the conjugate pad 1 in step A was to spray the P L A2R biotinylated conjugate onto the conjugate 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 centrifugally remove the supernatant 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 into each 100 mul of microspheres, and uniformly mix the microspheres 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 to dilute the capture body and the anti-DNP-BSA antibody to 1-2mg/ml by using coating solution 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 film scratching instrument respectively, and dry the membrane for 16-22h 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 P L A2R 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 5-10min in advance, and is dried for 2h at 37 ℃, wherein the trehalose concentration is the mass volume concentration, namely, the concentration is 5 g-10 g of trehalose in g/ml of Tris-HC L buffer solution.
It will 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 P L A2R antibody in the present invention 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 P L A2R antibody, which comprises the detection test strip, a sample buffer solution and/or the P L A2R antibody standard products with different concentration gradients, for example, the P L A2R antibody standard products with 6-7 concentration gradients are used for carrying out calibration test on the test strip, 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 P L A2R standard solution as a horizontal coordinate.
In some embodiments, the P L A2R antibody standard with different concentration gradients is specifically selected from 2RU/ml, 20RU/ml, 100RU/ml, 500RU/ml, 1000RU/ml, 1500RU/ml 6P L A2R antibody unit concentrations for calibration test, each point is tested 6 times with the same batch of test strips, according to a statistical method, 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) is taken as an ordinate, the standard solution concentration is taken as an abscissa, an equation is established and fitted into a standard curve, and data is stored in an instrument.
The invention also provides a detection method of the P L A2R antibody, a sample to be detected is added into a sample buffer solution, the sample buffer solution is added to a sample pad of the detection test strip after being mixed for 10 seconds, the detection test strip is inserted into a detection hole of a fluorescence analyzer and is placed for 3 to 4 minutes, and the concentration value of the P L A2R antibody is calculated 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 an ordinate and taking the concentration of the P L A2R antibody standard solution as an abscissa.
The invention coats a capture body of anti-human IgG4 (or IgG) on a nitrocellulose membrane, is used for capturing P L A2R antibody existing in a sample, simultaneously utilizes a biotin-avidin signal amplification system to carry out biotinylation on the P L A2R antigen by using a biotinylation reagent, and simultaneously uses fluorescent microsphere label to couple avidin (SA). when the sample is detected, the P L A2R antibody in the sample to be detected and the biotinylated P L A2R on a binding pad 1 have specific binding reaction, while the biotinylated P L A2R can be combined with the fluorescent microsphere of avidin on the binding pad 2, and is captured by the capture body when passing through the capture body coated by the nitrocellulose membrane, and simultaneously, the signal amplification reaction of the biotin and the avidin is combined, and the signal of the fluorescent microsphere is developed, so that the signal reaction value result read by an instrument can be obtained (see figure 2).
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 test strip for the P L A2R antibody comprises a sample pad, a binding pad 1, a binding pad 2, a nitrocellulose membrane and water absorption paper which are sequentially overlapped on a viscous bottom plate, wherein a P L A2R biotinylated conjugate is sprayed on the binding pad 1, an avidin fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate are sprayed on the binding pad 2, 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.
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 is a diagram showing the structure of a test strip for detecting the P L A2R antibody according to the present invention;
FIG. 2 is a schematic diagram showing the method for detecting the P L A2R antibody according to the present invention.
Detailed Description
The invention has been described in terms of preferred embodiments, and it will be apparent to those skilled in the art that variations and modifications of the methods described herein, as well as other variations and modifications, are possible in light of the above teachings and are within the purview of those skilled in the art.
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 P L A2R antibody of 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 pH8.510 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 P L A2R biotinylated conjugates Using Thermo
The Sulfo-NHS-L C-Biotin biotinylation kit biotinylates P L A2R and temporarily stores it at 4 ℃ until use.
3. Preparation of bonding pad 1 and bonding pad 2
Spraying the prepared P L A2R biotinylated conjugate on 1cm wide glass fiber by a gold spraying and film scratching instrument in the volume of 2 mu l/cm to prepare a binding pad 1, spraying the prepared SA fluorescent microsphere conjugate and DNP-BSA fluorescent microsphere conjugate on 1cm wide glass fiber in the volume of 2 mu l/cm by the gold spraying and film scratching instrument to prepare a binding pad 2, respectively placing the binding pad 1 and the binding pad 2 in an oven for drying at 37 ℃ for 2h, and sealing and storing for later use.
4. Coating of nitrocellulose membranes (NC membranes)
Using 0.01M PBS containing 5% (5g/100ml) trehalose as a coating solution, mouse anti-human IgG4 was diluted to 1mg/ml, rabbit anti-DNP-BSA was diluted to 1mg/ml, diluted mouse anti-human IgG4 and rabbit anti-DNP-BSA were drawn on an NC membrane at 1. mu.l/cm using a gold-spraying membrane drawing instrument, and subjected to T-line and C-line, oven-dried at 37 ℃ for 22 hours, sealed and stored for later 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, 20RU/ml, 100RU/ml, 500RU/ml, 1000RU/ml, 1500RU/ml and 6P L A2R unit concentrations are selected for calibration test, each point is tested for 6 times by using the same batch of test strips, according to a statistical method, the fluorescence intensity ratio (T strip detection value/C strip detection value) of a detection line (T strip) and a quality control line (C strip) is taken as a vertical coordinate, the concentration of a standard solution is taken as a horizontal coordinate, an equation is established and fitted into a standard curve, and data are stored in an instrument.
Example 2 Performance testing of fluorescent test strips of the P L A2R antibody of the invention
Each sample was tested with 60. mu.l serum and the test results were read with a 7min instrument.
1. The test strip of the invention determines the critical value, the average value of the concentration of the anti-P L A2R antibody is 12.8RU/ml and the standard deviation is 1.17RU/ml by detecting 220 normal human serums, and the critical value is 16.31RU/ml by taking the sum of the average value and 3 times of standard deviation as the critical value.
2. The test strip disclosed by the invention is determined by selecting 3 parts of quality control serum with high, medium and low values (a high value control target value is 300RU/m L, a medium value control target value is 70RU/m L and a low value control target value is 30RU/ml), repeatedly measuring each part for 10 times in the same test, 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 (%) -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 portions of serum with 57 and 301RU/ml of anti-P L A2R antibody concentration were selected, the low-value serum and the high-value serum were mixed in a ratio of 1:4, 1:1 and 9:1 to obtain 3 portions of serum with different concentrations, the theoretical values were 252.2, 207.5 and 81.4RU/ml respectively, the consistency between the detection value and the theoretical value, i.e. the recovery rate, was calculated by using a kit, the calculation result of the recovery rate is shown in Table 2, and the calculation formula is that the recovery rate is × 100% of the test value/the theoretical value.
TABLE 2 results of recovery calculation
The results show that the recovery is between 91% and 109% and the average recovery is 99.3%. The accuracy meets the requirement.
4. Evaluation of test strip coincidence rate
Comparison with gold standard: 113 parts of serum of patients with idiopathic membranous nephropathy and 43 parts of serum of patients with non-idiopathic membranous nephropathy, which are confirmed by renal puncture, are selected, the renal puncture is used as a gold standard, and the statistical results are shown in the following table 3.
TABLE 3 compliance rate test
The result shows that the specificity of the test strip is 93.0%, and the sensitivity is 85.0%.
Example 3 comparison with the Chinese patent with application number 201510245291.5
1. 10 weak positive quality control sera were selected for testing, the test paper of the present invention and the test paper of Chinese patent application No. 201510245291.5 were used to test the concentration of P L A2R antibody in sera, the results were read at 7min and 15min, respectively, and the results are shown in Table 4.
TABLE 4 comparison of the test results of different reading times of weakly positive quality control sera
The result shows that the difference between the 7min interpretation result and the theoretical concentration of the test strip of the Chinese patent with the application number of 201510245291.5 is larger, the 7min and 15min judgment results of the test strip are consistent with the theoretical concentration, and the test result is more accurate.
2. 15 negative clinical samples are selected and are simultaneously tested and compared with the test strip of the Chinese patent with the application number of 201510245291.5 respectively, and the results are shown in table 5.
TABLE 5 comparison of negative clinical samples
Note: "-" indicates negative, "+ -" indicates weak positive
The result shows that 15 clinical negative serums are tested to be all negative by adopting the test strip disclosed by the invention, and 4 false positives appear in the test strip disclosed by the Chinese patent with the application number of 201510245291.5, which indicates that the test strip disclosed by the invention has better specificity.
The comparison data are combined to show that the test strip has shorter detection time and more accurate detection result.
Claims (10)
1. An application of a detection test strip in preparation of a kit for detecting a P L A2R antibody is disclosed, 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 overlapped on a viscous bottom plate, a P L A2R biotinylation conjugate is sprayed on the combination pad 1, an avidin fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate are sprayed on the combination pad 2, 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.
2. The use of claim 1, wherein the P L A2R of the biotinylated conjugate P L A2R is a full-length or partial-fragment of the molecule P L A2R, or a variant, analog or substitute capable of acting like the P L A2R 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. biotinylation of the P L A2R protein to obtain a P L A2R biotinylated conjugate, 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 P L A2R biotinylated 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 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 1-2mg/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 5-10min, 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 P L A2R antibody comprises a detection test strip for detecting the P L A2R antibody, a sample buffer solution and/or a P L A2R antibody standard substance 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 overlapped on a viscous bottom plate, a P L A2R biotinylation conjugate is sprayed on the combination pad 1, an avidin fluorescent microsphere conjugate and a DNP-BSA fluorescent microsphere conjugate are sprayed on the combination pad 2, 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.
9. A method for detecting the P L A2R 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 a sample pad of a detection test strip of the detection kit according to claim 8, inserting the detection test strip into a detection hole of a fluorescence analyzer, standing the detection test strip for 3 to 4 minutes, and calculating the concentration value of the P L A2R antibody by taking the fluorescence intensity ratio of a detection line (T strip) and a quality control line (C strip) as an ordinate and the concentration of a P L A2R 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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| CN115184620A (en) * | 2022-09-14 | 2022-10-14 | 山东子峰生物技术有限公司 | Quantum dot fluorescence detection test strip and kit for PLA2R antibody and application of quantum dot fluorescence detection test strip and kit |
| CN116183919A (en) * | 2023-02-02 | 2023-05-30 | 上海基宣科技有限公司 | Preparation method and application of fluorescent quantitative detection test strip for detecting pepsinogen I/II content in serum, plasma or whole blood |
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| CN108279309A (en) | 2018-07-13 |
| WO2019148754A1 (en) | 2019-08-08 |
| CN108279309B (en) | 2022-02-15 |
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