CN108414748A - A kind of test strip and detection method of THSD7A antibody - Google Patents
A kind of test strip and detection method of THSD7A antibody Download PDFInfo
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- CN108414748A CN108414748A CN201810089697.2A CN201810089697A CN108414748A CN 108414748 A CN108414748 A CN 108414748A CN 201810089697 A CN201810089697 A CN 201810089697A CN 108414748 A CN108414748 A CN 108414748A
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- thsd7a
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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
- G01N33/58—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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- 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
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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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
- G01N33/58—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
- G01N33/587—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
Abstract
The invention belongs to technical field of biological, disclose a kind of test strip and detection method of THSD7A antibody.Test strips of the present invention include overlapping sample pad successively in adhesive base, bonding pad 1, bonding pad 2, NC films and blotting paper；THSD7A biotinylation conjugates are coated on the bonding pad 1, the bonding pad 2 is coated with SA fluorescent microspheres conjugate and DNP BSA fluorescent microsphere conjugates, and capture body is coated on the NC films as detection line.Detect the THSD7A antibody in sample, it is combined with biotinylated THSD7A, then it is combined with the fluorescent microsphere of SA, the captured body capture in capture body coated by NC films, in combination with the signal iodine of biotin and avidin, is developed the color by the signal of fluorescent microsphere, obtain signal reaction value result, high sensitivity, can easy, quick, accurate quantitative analysis the content for testing THSD7A antibody in detection sample.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of test strip of THSD7A antibody and detection side
Method.
Background technology
Membranous nephropathy (MN) is one of most common histological type of adult nephrotic syndrome, characteristic pathological change
It is the visible a large amount of immune complex deposits of glomerular capillary loop epithelial side.MN can be divided into idiopathic film property kidney by pathogenic factor
Sick (IMN) and secondary membranous nephropathy (SMN) two major classes.IMN is a kind of glomerulus chronic inflammation disease, mostly with anti-phosphatide
Enzyme A2 receptor antibodies are related, and anti-phospholipase A2 receptor antibody is combined with the corresponding antigens on sertoli cell, formed in situ immune compound
Object forms C5b-9 membrane attack complexes then by alternative pathway activating complement, damages sertoli cell, destroys glomerular filtration screen
Barrier, is typically characterised by albuminuria, with the increase of urine protein content, there is the development trend of kidney failure.And SMN is different from IMN,
It is secondary disease or concomitant disorders, drug therapy, drug abuse, infectious diseases, other autoimmune diseases and swollen
Tumor may all lead to the generation of SMN.Such as systemic loupus erythematosus, rheumatoid arthritis, hepatitis B virus infection and drug,
Poisonous substance, tumour or environmental factor etc..The drug of secondary membranous nephropathy is caused mainly to have some gold preparations, mercury, penicillamine, Bu Luo
Sweet smell, Diclofenac etc..Meanwhile SMN can improve with the treatment of basic disease.
The diagnosis of IMN and SMN relies primarily on clinical manifestation and renal puncture.Renal puncture, that is, Renal biospy, also referred to as renal fibroblast
Biopsy.It is a kind of wound diagnostics method of intrusive mood, there is certain injury to patient, and according to the diagnosis needs of clinical manifestation
Certain experience, and it is also required to the verification of histopathology.Research in recent years and document report, IMN are a kind of itself exempt from
Epidemic disease disease, it has been found that autoimmunity antigen phospholipase A2 receptors (PLA2R) be its main target antigen, the diagnosis to IMN
Positive rate does not detect the antibody up to 70%-82% in other diseases and normal human serum sample.Although 70%-
82%IMN patient's bodies have the circulating antibody for PLA2R, but still have part IMN patient not find that PLA2R antibody, IMN are suffered from
There is likely to be other autoantigens by person.THSD7A is that there are another new sertoli cells itself in IMN patient for discovered in recent years
Antigen has investigation to find that THSD7A antibody is present in the IMN of the anti-PLA2R negative antibodies of serum of Europe and North America 8%~14%
In patient, while proving that THSD7A and PLA2R has similar structure and biochemical characteristic and can induce based on IgG4
Humoral immune reaction generates cycle autoantibody.THSD7A and PLA2R is two antigens of spontaneous membranous nephropathy to spontaneity
The recall rate of membranous nephropathy is respectively 70%-75% and 5%-10%, and there is certain omission factor in when exclusive use, therefore develops
A kind of kit quantitatively detected to serology antibody THSD7A has the detection of the membranous nephropathy of idiopathic highly important
Meaning.
Application No. is 201610201970.7 Chinese patent disclose it is anti-using ELISA method test serum moderate resistance THSD7A
Body, but ELISA is cumbersome, the testing time is long.Application No. is 201710047530.5 Chinese patents to disclose using glue
Body gold method tests the THSD7A antibody in human serum, although colloidal gold method, which tests simple and quick this method, cannot be satisfied quantitative survey
Try the requirement of the THSD7A antibody in human serum.
Invention content
In view of this, it is an object of the invention to be directed in the prior art, operating process is cumbersome, detection time is long, without legal
The defect for measuring detection, provide it is a kind of can easy, the fast and accurately THSD7A antibody contents in quantitative test human serum detection
Test strips and detection method.
In order to achieve the object of the present invention, the present invention adopts the following technical scheme that：
A kind of test strip of THSD7A antibody, including overlap sample pad successively in adhesive base, bonding pad 1, knot
Close pad 2, nitrocellulose filter (NC) and blotting paper；THSD7A biotinylation conjugates, the knot are coated on the bonding pad 1
It closes pad 2 and is coated with Avidin (SA) fluorescent microsphere conjugate and DNP-BSA fluorescent microsphere conjugates, on the nitrocellulose filter
Capture body is coated with as detection line, anti-DNP-BSA antibody is also coated with as nature controlling line on the nitrocellulose filter.
Wherein, THSD7A described in THSD7A biotinylations conjugate described in the test strip includes but not limited to
For THSD7A molecules overall length or the albumen of Partial Fragment, or the variant of THSD7A albumen similar effects can be played, analog, replaced
For object.The THSD7A can be native purified THSD7A, can also be that technique for gene engineering recombination obtains.
Preferably, the grain size of fluorescent microsphere described in the test strip is 200nm.
It is anti-human IgG antibodies or can be combined with human IgG antibody preferably, captures body described in the test strip
Substance, such as albumin A or Protein G.More preferably mouse anti-human igg, such as mouse anti-human igg 4.
The present invention also provides the preparation methods of the test strip of the THSD7A antibody, include the following steps：
A, THSD7A protein biotinylations are obtained into THSD7A biotinylation conjugates, be sprayed on bonding pad 1；
B, pretreated Avidin and DNP-BSA and the fluorescent microsphere of activation are coupled respectively, it is micro- obtains Avidin fluorescence
Ball conjugate and DNP-BSA fluorescent microsphere conjugates are sprayed on bonding pad 2；
C, it will be used as detection line in capture body coating to nitrocellulose filter, by anti-DNP-BSA antibody coating to nitric acid fibre
It is used as nature controlling line on the plain film of dimension；
D, sample pad, the bonding pad 1 for being coated with THSD7A biotinylation conjugates, spraying are overlapped successively in adhesive base
There are the bonding pad 2, coating capture body and anti-DNP-BSA of Avidin fluorescent microsphere conjugate and DNP-BSA fluorescent microsphere conjugates
Nitrocellulose filter, the blotting paper of antibody.
The present invention does not limit the method for THSD7A protein biotinylations, may be used well known to a person skilled in the art
Method carries out.In some embodiments, the present invention gives birth to THSD7A albumen using common biotinylated kit
Object element marks.As using Thermo companiesNHS-LC-Biotin biotinylation kits.
It is to draw film instrument with metal spraying that THSD7A biotinylation conjugates, which are sprayed on the concrete operations on bonding pad 1, in step A
By THSD7A biotinylations conjugate with 1,37 DEG C of oven drying 2h of amount spraying bonding pad of 2 μ l/cm.
Coupling described in the preparation method step B of test strip of the present invention preferably centrifuges the fluorescent microsphere of activation
After removing supernatant, after being washed with the 50mM MES buffer solutions of pH7.0~8.0, it is affine that 0.1mg~0.2mg is added by every 100 μ l microballoons
Element or 0.2~0.4mg DNP-BSA, room temperature mixing react 1~2h.
Avidin described in the preparation method step B of test strip of the present invention and DNP-BSA with fluorescent microsphere
It is pre-processed before coupling.The 50mM MES buffer solutions of pH7.0~8.0 dialysis Avidin and DNP-BSA are preferably used respectively
Three times.
Fluorescent microsphere described in step B needs preactivated processing.The specific method of the activation of the microballoon is preferably micro-
After ball is washed with 50mM pH6.0~6.5MES solution, 0.2~0.4 μ gNHS (N- maloyls are added by every 100 μ l microballoons
Imines) and 0.2~0.4 μ gEDC (1- (3- dimethylamino-propyls) -3- ethyl carbodiimides) room temperature mixing reaction, 30~40min.
It is further used as preferably, further includes after Avidin and DNP-BSA described in step B and the coupling of the fluorescent microsphere of activation
The step of closing.It is furthermore preferred that the closed concrete operations are that BSA to a concentration of 1%~5% (g/ml) is added, room temperature is mixed
30~40min of even reaction.BSA is added to a concentration of per 100ml 1g~5g, room temperature mixing reacts 30~40min.
Avidin fluorescent microsphere conjugate and DNP-BSA fluorescent microspheres conjugate can be with current existing systems, also can be previously prepared
After be stored in preserve liquid in.The store method is specially that Avidin fluorescent microsphere conjugate or DNP-BSA fluorescent microspheres is even
Preservation liquid is added to preserve again after joining object pH8.5 10mmol/L boric acid solution centrifuge washings.Preferably, the preservation liquid is containing 1%
~5%BSA, the pH8.510mmol/L boric acid solutions of 1%~2% trehalose.Wherein, the concentration of the BSA and trehalose is
Mass-volume concentration preserves liquid BSA containing 1g~5g, 1g~2g trehaloses based on g/ml per 100ml.
Avidin fluorescent microsphere conjugate and DNP-BSA fluorescent microsphere conjugates are sprayed on the tool on bonding pad 2 in step B
Gymnastics is used as draws film instrument respectively by Avidin fluorescent microsphere conjugate and DNP-BSA fluorescent microspheres conjugate with 2 μ l/cm with metal spraying
Amount spraying bonding pad 2,37 DEG C of oven drying 2h.
The preparation method step C of test strip of the present invention will be captured in body coating to nitrocellulose filter as inspection
Survey line will be used as nature controlling line in anti-DNP-BSA antibody coating to nitrocellulose filter.The step C is specially respectively with coating
Liquid dilution capture body and anti-DNP-BSA antibody by the capture body diluted and are resisted to 1~2mg/ml with metal spraying stroke film instrument respectively
DNP-BSA antibody is drawn by 1 μ l/cm on nitrocellulose filter, 37 DEG C of dry 16~22h；The coating buffer is containing 5%~10%
The PBS of trehalose.Wherein, a concentration of mass-volume concentration of the trehalose contains 5g based on g/ml per 100ml coating buffers
~10g trehaloses.The preparation method step D of test strip of the present invention overlaps sample pad, spray successively in adhesive base
It is coated with the bonding pad 1 of THSD7A biotinylation conjugates, is coated with Avidin fluorescent microsphere conjugate and DNP-BSA fluorescent microspheres
The bonding pad 2 of conjugate, the nitrocellulose filter of coating capture body and anti-DNP-BSA antibody, blotting paper assembling obtain test strips.
Wherein, preferably, the sample pad is in advance with the Tris- of the 0.5M pH=7.4 containing 5~10% trehalose
HCL buffer solutions impregnate 5~10min, 37 DEG C of dry 2h.Wherein, a concentration of mass-volume concentration of the trehalose, by g/ml
Meter, i.e., per Tris-HCL buffer solutions trehalose containing 5g~10g.
It will be appreciated by those skilled in the art that the present invention to the sample pad of the test strip of the THSD7A antibody, combine
Pad 1, bonding pad 2 material be not particularly limited, can be common material, such as polyester film, glass fibre.
The present invention also provides a kind of detection kit of THSD7A antibody, including above-mentioned test strip, sample are slow
The THSD7A antibody standard substances of fliud flushing and/or various concentration gradient.As the THSD7A antibody standard substances of 6~7 concentration gradients are used
In carrying out calibration test to test strips, according to statistical method, with detection line (T bands), the fluorescence intensity ratio of nature controlling line (C bands)
(T bands detected value/C bands detected value) is ordinate, and THSD7A concentration of standard solution is abscissa, establishes equation and is fitted to standard
Curve data is stored into instrument.
In some embodiments, the THSD7A antibody standard substances of the various concentration gradient specifically select 2RU/ml,
7 THSD7A antibody unit concentration of 10RU/ml, 20RU/ml, 50RU/ml, 100RU/ml, 200RU/ml, 500RU/ml carry out
Calibration test, with a batch of test strips, each point test 6 times.According to statistical method, with detection line (T bands), nature controlling line
The fluorescence intensity ratio (T bands detected value/C bands detected value) of (C bands) is ordinate, and concentration of standard solution is abscissa, foundation side
Journey is simultaneously fitted to standard curve, data storage to instrument.
The present invention also provides a kind of detection methods of THSD7A antibody, take sample to be tested that sample buffer, mixing is added
It 10 seconds afterwards, is added in the sample pad of above-mentioned test strip, test strip is inserted into the detection hole of fluorescence analyser, is put
It sets 3~4 minutes, the fluorescence intensity ratio (T bands detected value/C bands detected value) with detection line (T bands), nature controlling line (C bands) is vertical sit
Mark, THSD7A antibody concentration of standard solution are the concentration value that abscissa calculates THSD7A antibody.
The present invention is coated with the capture body of anti-human igg 4 (or IgG) on nitrocellulose filter, exists for capturing in sample
THSD7A antibody, while using biotin-avidin signal amplifying system by THSD7A antigens first with biotinylated examination
Agent carries out biotinylation, while Avidin (SA) being marked with fluorescent microsphere and is coupled.When detecting sample, in sample to be tested
The association reaction of specificity occurs with the biotinylated THSD7A on bonding pad 1 for THSD7A antibody, and biotinylated
THSD7A can be combined again with the fluorescent microsphere of Avidin on bonding pad 2, the quilt in capture body coated by nitrocellulose filter
Body capture is captured, in combination with the signal iodine of biotin and avidin, is developed the color, be can be obtained by the signal of fluorescent microsphere
The signal reaction value result that instrument is read.
Preferably, the sample to be tested is whole blood, serum, blood plasma, urine or saliva.Such as venous blood, peripheral blood etc..
As shown from the above technical solution, the present invention provides a kind of test strips and detection method of THSD7A antibody.
The test strip of THSD7A antibody of the present invention, including overlap sample pad successively in adhesive base, bonding pad 1, in conjunction with
Pad 2, nitrocellulose filter and blotting paper；THSD7A biotinylation conjugates, the bonding pad 2 are coated on the bonding pad 1
It is coated with Avidin fluorescent microsphere conjugate and DNP-BSA fluorescent microsphere conjugates, is coated with and catches on the nitrocellulose filter
Body is obtained as detection line, anti-DNP-BSA antibody is also coated with as nature controlling line on the nitrocellulose filter.The present invention is used and is caught
The THSD7A antibody in method detection sample is obtained, the knot of specificity occurs with biotinylated THSD7A for the THSD7A antibody in sample
Reaction is closed, and biotinylated THSD7A can be combined with the fluorescent microsphere of Avidin, coated by nitrocellulose filter
Captured body capture when body is captured, in combination with the signal iodine of biotin and avidin, passes through the signal of fluorescent microsphere
The signal reaction value result of instrument reading can be obtained in colour developing.The present invention is clever using the test strips of prize law detection THSD7A antibody
Sensitivity is high, and the content of THSD7A antibody, is idiopathic in energy simplicity, quick, accurate quantitative analysis test human serum, blood plasma and whole blood
Primary dcreening operation, diagnosis and the state of an illness and Prognosis scoveillance of membranous nephropathy provide good auxiliary direction effect.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 shows the test strip structure chart of THSD7A antibody of the present invention；
Fig. 2 shows the schematic diagram of the detection method of THSD7A antibody of the present invention.
Specific implementation mode
The invention discloses a kind of test strips and detection method of THSD7A antibody.Those skilled in the art can borrow
Reflect present disclosure, is suitably modified technological parameter realization.In particular, it should be pointed out that all similar substitutions and modifications are to this field
It is it will be apparent that they are considered as being included in the present invention for technical staff.The method and product of the present invention has passed through
Preferred embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to described herein
Method be modified or suitably change and combine, to realize and apply the technology of the present invention.
For a further understanding of the present invention, below in conjunction with the embodiment of the present invention, to the technical side in the embodiment of the present invention
Case is clearly and completely described, it is clear that and described embodiments are only a part of the embodiments of the present invention, rather than all
Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art institute without making creative work
The every other embodiment obtained, shall fall within the protection scope of the present invention.
Unless otherwise specified, reagent involved in the embodiment of the present invention is commercial product, can pass through business canal
Road purchase obtains.
The making of the fluorescent test paper strip of the detection THSD7A antibody of embodiment 1, the present invention
1. the preparation of Avidin (SA) fluorescent microsphere conjugate, DNP-BSA fluorescent microsphere conjugates
1.1 prepare 50mM pH7.0MES solution
1.2 prepare 50mM pH6.5MES solution
1.3 Avidin, the DNP-BSA that will be coupled use 50mM pH7.0MES buffer solutions to dialyse three times respectively, and 4 DEG C temporary
It is spare.
The activation of 1.4 microballoons：50mM pH6.5MES solution, 12000r/min centrifuge washings is added in 20 μ l of 200nm microballoons
After twice, 0.25 μ gNHS (N- hydroxysuccinimides) and 0.25 μ gEDC (1- (3- dimethylaminos is added by every 100 μ l microballoons
Propyl) -3- ethyl carbodiimides), room temperature mixing reacts 40min.
The coupling of 1.5 microballoons：After supernatant is removed in above-mentioned activated microballoon 12000r/min centrifugations, with the 50mM of pH7.0
MES buffer by centrifugation by every 100 μ l microballoons is separately added into the 0.2mg Avidins or 0.25mgDNP- dialysed after washing 2 times
BSA, room temperature mixing react 1h.
The closing of 1.6 microballoons:BSA is added in the good microballoon of above-mentioned coupling, makes BSA a concentration of 1% (1g/100ml), room temperature
Continue mixing and reacts 40min.
The preservation of 1.7 microballoons：The good SA microballoons of above-mentioned closing, DNP-BSA microballoons are used into pH=8.510mmol/L boron respectively
After acid solution 12000r/min is washed twice plus preserve 4 DEG C of preservations of liquid, the preservations liquid ingredient for containing 1%BSA (1g/100ml),
The pH8.5 10mmol/L boric acid solutions of 2% (2g/100ml) trehalose.
The preparation of 2.THSD7A biotinylation conjugates：Use Thermo-NHS-L C-
For Biotin biotinylation kits by THSD7A biotinylations, 4 DEG C temporary spare.
3. the preparation of bonding pad 1, bonding pad 2
The THSD7A biotinylations conjugate of above-mentioned preparation is drawn into film instrument with metal spraying, 1cm wide is sprayed at the volume of 2 μ l/cm
On glass fibre, bonding pad 1 is made, the SA fluorescent microspheres conjugate of above-mentioned preparation, DNP-BSA fluorescent microsphere conjugates are sprayed
Gold is drawn film instrument and is sprayed on 1cm wide glass fibres with the volume of 2 μ l/cm, and bonding pad 2 is made；Respectively by bonding pad 1 and bonding pad
2 are placed in 37 DEG C of oven drying 2h, and sealed storage is spare.
4. the coating of nitrocellulose filter (NC films)
It uses the 0.01M PBS containing 5% trehalose as coating buffer, mouse anti-human igg 4 is diluted to 1mg/ml, by rabbit-anti
DNP-BSA is diluted to 1mg/ml, using metal spraying draw film instrument the mouse anti-human igg 4 dilute, rabbit-anti DNP-BSA is by 1 μ l/cm strokes
On NC films, make T lines and C lines respectively, 37 DEG C of oven drying 22h, sealed storage is spare.
5. the assembling of test strips
Sample pad, bonding pad 1, bonding pad 2, nitrocellulose filter are overlapped successively on sticky PVC bottom plates (by above-mentioned steps
Be coated with), blotting paper, and the test strips for being cut into 4mm wide are assembled on card (see Fig. 1).
6. the calibration test of test strips
Select 2RU/ml, 10RU/ml, 20RU/ml, 50RU/ml, 100RU/ml, 200RU/ml, 500RU/ml, 7
THSD7A concentration gradient standard items carry out calibration test, with a batch of test strips, each point test 6 times.According to statistics
Method, with detection line (T bands), nature controlling line (C bands) fluorescence intensity ratio (T band detected value/C bands detected value) for ordinate, mark
Quasi- solution concentration is abscissa, establishes equation and is fitted to standard curve, data storage to instrument.
7. the test of clinical sample determines test strips critical value, to test strips precision, accuracy, the correlations such as coincidence rate
Performance is assessed.Unit concentration carries out calibration test, and with a concentration of abscissa, T/C is that ordinate establishes standard curve data
It is stored into dry type fluorescence immunity analyzer, test sample concentration is calculated based on the curve.
The performance test of the fluorescent test paper strip of the THSD7A antibody of embodiment 2, the present invention
Testing each sample below adds 60 μ l Virus monitories, 15min instruments to read testing result.
1. the determination of test strips critical value of the present invention：By detecting 220 portions of normal human serums, anti-THSD7A antibody is dense
The average value of degree is 20.8RU/ml and standard deviation is 1.1RU/ml, using the sum of average value plus 3 times of standard deviations as critical value, then
Critical value is 24.1RU/ml.
2. test strips precision of the present invention determines：Choose high, medium and low value 3 parts of quality controlled serums (high level Quality Control target value：
290RU/ml, intermediate value Quality Control target value 75RU/mL, low value Quality Control target value 30RU/mL), every part of replication 10 in homogeneous is tested
It is secondary, average value, standard deviation are calculated separately, coefficient of variation CV (%) in experiment is calculated；It measures 1 time daily, METHOD FOR CONTINUOUS DETERMINATION 10 days, meter
Calculate test bay CV (%) value.Calculation formula is：CV (%)=standard deviation/average value × 100%.The experimental data are shown in the following table 1.
1 test strips precision result of table
Note：Refer to being tested repeatedly identical test repeatedly in primary experiment in experiment；Test bay refers to identical test not
With retest in the time.
Table 1 is the results show that test strips accuracy of the present invention meets the requirements.
3. the detection (rate of recovery experiment) of test strips accuracy of the present invention：
It is respectively 2 parts of serum of 60 and 300RU/ml, this low value serum and high level serum to select anti-THSD7A antibody concentrations
Respectively with 1:4、1:1 and 9:1 ratio is mixed into the serum of 3 parts of various concentrations, and theoretical value is respectively 252.2,207.5 and
81.4RU/ml.It is detected by kit, calculates the consistency of detected value and theoretical value, the i.e. rate of recovery.Rate of recovery result of calculation is shown in
Table 2.Calculation formula is:The rate of recovery=test value/theoretical value × 100%.
2 rate of recovery result of calculation of table
The results show that the rate of recovery is between 97%-102%, average recovery rate 99.0%.Accuracy meets the requirements.
4. test strips coincidence rate evaluation of the present invention
It is compared with goldstandard：It chooses and 115 parts of idiopathic membranous nephropathy patients serum, non-spy is diagnosed as by renal puncture art
45 parts of hair property membranous nephropathy patients serum, using renal puncture art as goldstandard, statistical result such as the following table 3.
3 coincidence rate of table is tested
The results show that the specificity of test strips of the present invention is 92.0%, sensibility 84.0%.
Embodiment 3, with Ou Meng companies exploitation indirect immunofluorescene assay method (IIFT) compared with
Collection is diagnosed as totally 170 parts of idiopathic membranous nephropathy patients serum, non-idiopathic membranous nephropathy patients serum, simultaneously
Indirect immunofluorescene assay method (IIFT) test of the fluorescent test paper strip developed with our company and the exploitation of Ou Meng companies, statistics knot
Fruit is shown in Table 4
Table 4 is compared with IIFT testing results
The results show that being compared with the indirect immunofluorescene assay method (IIFT) that Ou Meng companies develop, positive coincidence rate
88%, negative match-rate 92%
In summary data are it is found that the detection of THSD7A antibody of the present invention accurately can test THSD7A in serum by geodetic
The content of antibody effectively can carry out primary dcreening operation and state of illness monitoring to the membranous nephropathy of idiopathic.
Claims (11)
1. a kind of test strip of THSD7A antibody, including overlap sample pad successively in adhesive base, bonding pad 1, in conjunction with
Pad 2, nitrocellulose filter and blotting paper；THSD7A biotinylation conjugates, the bonding pad 2 are coated on the bonding pad 1
It is coated with Avidin fluorescent microsphere conjugate and DNP-BSA fluorescent microsphere conjugates, is coated with and catches on the nitrocellulose filter
Body is obtained as detection line, anti-DNP-BSA antibody is also coated with as nature controlling line on the nitrocellulose filter.
2. test strip according to claim 1, THSD7A is THSD7A in the THSD7A biotinylations conjugate
The albumen of molecule overall length or Partial Fragment, or the variant, analog, substitute of THSD7A albumen similar effects can be played.
3. the grain size of test strip according to claim 1 or 2, the fluorescent microsphere is 200nm.
4. according to the test strip described in claims 1 to 3 any one, the capture body is anti-human IgG antibodies or can be with
The substance that human IgG antibody combines.
5. the preparation method of the test strip described in Claims 1 to 4 any one, includes the following steps：
A, THSD7A protein biotinylations are obtained into THSD7A biotinylation conjugates, be sprayed on bonding pad 1；
B, pretreated Avidin and DNP-BSA and the fluorescent microsphere of activation are coupled respectively, it is even obtains Avidin fluorescent microsphere
Connection object and DNP-BSA fluorescent microsphere conjugates are sprayed on bonding pad 2；
C, it will be used as detection line in capture body coating to nitrocellulose filter, by anti-DNP-BSA antibody coating to nitrocellulose
Nature controlling line is used as on film；
D, it overlaps sample pad successively in adhesive base, the bonding pad 1 for being coated with THSD7A biotinylation conjugates, be coated with parent
With the bonding pad 2 of plain fluorescent microsphere conjugate and DNP-BSA fluorescent microsphere conjugates, coating capture body and anti-DNP-BSA antibody
Nitrocellulose filter, blotting paper.
6. preparation method according to claim 5, the centrifugation of the fluorescent microsphere of activation is is gone by coupling described in the step B
After clear, after wash with the 50mM MES buffer solutions of pH7.0~8.0, by every 100 μ l microballoons addition 0.1mg~0.2mg Avidins or
0.2~0.4mg DNP-BSA, room temperature mixing react 1~2h.
7. preparation method according to claim 5 or 6, the step C be specially respectively with coating buffer dilute capture body and
Anti- DNP-BSA antibody draws film instrument respectively by the capture body diluted and anti-DNP-BSA antibody by 1 μ to 1~2mg/ml, with metal spraying
L/cm is drawn on nitrocellulose filter, 37 DEG C of dry 16~22h；The coating buffer is containing 5%-10% (g/ml) trehalose
PBS。
8. according to the preparation method described in claim 5~7 any one, the sample pad is in advance with containing 5~10% (g/ml)
Trehalose 0.5M pH=7.4 Tris-HCL buffer solutions impregnate 5~10min, 37 DEG C of dry 2h；The Avidin and
The pretreatment of DNP-BSA is to be dialysed with the 50mM MES buffer solutions of pH7.0~8.0；The specific method of the activation of the microballoon is micro-
After ball is washed with 50mM pH6.0~6.5MES solution, 0.2~0.4 μ gNHS (N- maloyls are added by every 100 μ l microballoons
Imines) and 0.2~0.4 μ gEDC (1- (3- dimethylamino-propyls) -3- ethyl carbodiimides) room temperature mixing reaction, 30~40min；
Further include the steps that closing after the Avidin and DNP-BSA and the coupling of the fluorescent microsphere of activation, BSA is specially added to concentration
For 1%~5% (g/ml), room temperature mixing reacts 30~40min.
9. a kind of detection kit of THSD7A antibody, including the test strip described in Claims 1 to 4 any one, sample
Savor the THSD7A antibody standard substances of buffer solution and/or various concentration gradient.
10. a kind of detection method of THSD7A antibody takes sample to be tested that sample buffer is added and is added to 10 seconds after mixing
In the sample pad for the test strip stated, test strip is inserted into the detection hole of fluorescence analyser, is placed 3-4 minutes, with inspection
Survey line (T bands), nature controlling line (C bands) fluorescence intensity ratio be ordinate, THSD7A antibody concentration of standard solution be abscissa meter
Calculate the concentration value of THSD7A antibody.
11. detection method according to claim 10, the sample to be tested is whole blood, serum, blood plasma, urine or saliva.
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| CN202010279273.XA CN111413501A (en) | 2018-01-30 | 2018-01-30 | Application of detection test strip in preparation of kit for detecting THSD7A antibody |
| PCT/CN2018/092769 WO2019148753A1 (en) | 2018-01-30 | 2018-06-26 | Test strip and testing method for thsd7a antibody |
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| WO2019148753A1 (en) | 2019-08-08 |
| CN108414748B (en) | 2022-02-15 |
| CN111413501A (en) | 2020-07-14 |
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