CN108279309A - A kind of test strip and detection method of PLA2R antibody - Google Patents
A kind of test strip and detection method of PLA2R antibody Download PDFInfo
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Abstract
The invention belongs to technical field of biological, disclose a kind of test strip and detection method of PLA2R antibody.The test strip of PLA2R antibody of the present invention, including overlap sample pad successively in adhesive base, bonding pad 1, bonding pad 2, NC films and blotting paper；PLA2R 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, it is coated with capture body on the NC films as detection line, anti-DNP BSA antibody is also coated with as nature controlling line on the NC films.The present invention detects the test strips color speed of PLA2R antibody faster using prize law, from as long as sentence read result 7min is loaded onto, detection time is shorter, test strips color signal is stronger, weak sun is not susceptible to missing inspection, it is specific more preferable, it can quantitative, accurate, easily test the content of PLA2R antibody in human serum, blood plasma and whole blood.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of test strip of PLA2R 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.Serology PLA2R
Antibody, which quantitatively detects, to provide booster action for the primary dcreening operation and state of illness monitoring of the membranous nephropathy of idiopathic.Therefore non-wound is developed
The content of IMN related auto-antibodies in wound, low-risk, quick, safety, cheap, accurate quantitatively detection serum, blood plasma and whole blood
Method the detection of the membranous nephropathy of idiopathic is had a very important significance.
Pla2r antibody in the indirect immunofluorescence kit test serum of existing Ou Meng companies now, but it is indirectly immune
Fluorescence method operating process is cumbersome, and detection time is long.Application No. is 201510245291.5 Chinese patents to disclose using dual anti-
Former sandwich test serum PLA2R antibody test test strips, but this method sentence read result at least needs 15min, and color signal is weak, holds
The missing inspection of weakly positive easily occurs.Application No. is 201710047530.5 Chinese patents to disclose using colloidal gold immunochromatographimethod skill
Art tests serum pla2r antibody, but this method cannot be satisfied the requirement of quantitative test.
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, colour developing letter
Defect number weak, without standard measure detection, provide it is a kind of can it is quantitative, accurate, easily test in human serum, blood plasma and whole blood
The test strip and detection method of PLA2R antibody.
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 PLA2R antibody, including overlap sample pad successively in adhesive base, bonding pad 1, in conjunction with
Pad 2, nitrocellulose filter (NC) and blotting paper；PLA2R biotinylation conjugates, the combination are coated on the bonding pad 1
Pad 2 is coated with Avidin (SA) fluorescent microsphere conjugate and DNP-BSA fluorescent microsphere conjugates, is wrapped on the nitrocellulose filter
There is capture body as detection line, anti-DNP-BSA antibody is also coated with as nature controlling line on the nitrocellulose filter.
Wherein, PLA2R described in PLA2R biotinylations conjugate described in the test strip includes but not limited to be
The albumen of PLA2R molecules overall length or Partial Fragment, or the variant, analog, replacement of PLA2R albumen similar effects can be played
Object.The PLA2R can be native purified PLA2R, can also be that technique for gene engineering recombination obtains.
Preferably, the PLA2R molecular moieties segment is the molecule piece in the PLA2R combined with THSD7A protein sequences
Section.
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 PLA2R antibody, include the following steps：
A, PLA2R protein biotinylations are obtained into PLA2R 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 PLA2R 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 PLA2R protein biotinylations, may be used well known to a person skilled in the art
Method carries out.In some embodiments, the present invention carries out biology using common biotinylated kit to PLA2R albumen
Element label.As using Thermo companiesSulfo-NHS-LC-Biotin biotinylation kits
PLA2R biotinylation conjugates are sprayed on the concrete operations on bonding pad 1 in step A will to draw film instrument with metal spraying
PLA2R biotinylations conjugate is 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 0.2~0.4mgDNP-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 30~40min of 0.2-0.4 μ gEDC (1- (3- dimethylamino-propyls) -3- ethyl carbodiimides) room temperature mixing reaction.
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, capture body is coated with to conduct on nitrocellulose filter
Detection line will be used as nature controlling line in anti-DNP-BSA antibody coating to nitrocellulose filter.The step C is specially respectively with packet
Capture body and anti-DNP-BSA antibody are diluted to 1-2mg/ml by liquid, by the capture body diluted and resisted respectively with metal spraying stroke film instrument
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 the seas 5%-10%
The PBS of algae sugar.Wherein, a concentration of mass-volume concentration of the trehalose, based on g/ml, i.e., per 100ml coating buffers containing 5g~
10g trehaloses.
The preparation method step D of test strip of the present invention overlaps sample pad in adhesive base, is coated with successively
The bonding pad 1 of PLA2R biotinylation conjugates is coated with Avidin fluorescent microsphere conjugate and the coupling of DNP-BSA fluorescent microspheres
The bonding pad 2 of object, 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 of the trehalose containing 5-10%
HCL buffer solutions impregnate 5-10min, 37 DEG C of dry 2h.Wherein, a concentration of mass-volume concentration of the trehalose, based on g/ml,
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 PLA2R 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 kits of PLA2R antibody, including above-mentioned test strip, Sample Buffer
The PLA2R antibody standard substances of liquid and/or various concentration gradient.As the PLA2R antibody standard substances of 6-7 concentration gradient are used for examination
Paper slip carries out calibration test, according to statistical method, with detection line (T bands), fluorescence intensity ratio (the T band inspections of nature controlling line (C bands)
Measured value/C bands detected value) it is ordinate, PLA2R concentration of standard solution is abscissa, establishes equation and is fitted to standard curve number
According to being stored into instrument.
In some embodiments, the PLA2R antibody standard substances of the various concentration gradient specifically select 2RU/ml,
6 PLA2R antibody units concentration of 20RU/ml, 100RU/ml, 500RU/ml, 1000RU/ml, 1500RU/ml carry out calibration survey
Examination, with a batch of test strips, each point test 6 times.According to statistical method, with detection line (T bands), nature controlling line (C bands)
Fluorescence intensity ratio (T band detected value/C bands detected value) be ordinate, concentration of standard solution is abscissa, establishes equation and intends
Synthetic standards curve, data storage to instrument.
The present invention also provides a kind of detection methods of PLA2R antibody, take sample to be tested that sample buffer is added, after mixing
It 10 seconds, is added in the sample pad of above-mentioned test strip, test strip is inserted into the detection hole of fluorescence analyser, place
3-4 minutes, with detection line (T bands), nature controlling line (C bands) fluorescence intensity ratio (T band detected value/C bands detected value) for ordinate,
PLA2R antibody concentration of standard solution is the concentration value that abscissa calculates PLA2R 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
PLA2R antibody, while using biotin-avidin signal amplifying system by PLA2R antigens first with biotinylated reagent
Biotinylation is carried out, while Avidin (SA) being marked with fluorescent microsphere and is coupled.When detecting sample, the PLA2R in sample to be tested
The association reaction of specificity occurs for the biotinylated PLA2R on antibody and bonding pad 1, and biotinylated PLA2R can be with
The fluorescent microsphere of Avidin combines on bonding pad 2, the captured body capture in capture body coated by nitrocellulose filter, together
When in conjunction with biotin and avidin signal iodine, developed the color by the signal of fluorescent microsphere, the letter of instrument reading can be obtained
Number response value result (see Fig. 2).
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 PLA2R antibody.This
The test strip of the PLA2R antibody is invented, including overlaps sample pad successively in adhesive base, bonding pad 1, bonding pad 2,
Nitrocellulose filter and blotting paper；PLA2R biotinylation conjugates are coated on the bonding pad 1, the bonding pad 2 is coated with
Avidin fluorescent microsphere conjugate and DNP-BSA fluorescent microsphere conjugates are coated with capture body on the nitrocellulose filter and make
For detection line, anti-DNP-BSA antibody is also coated with as nature controlling line on the nitrocellulose filter.The present invention is examined using prize law
PLA2R antibody in test sample sheet, coating capture body, biotinylated antigen PLA2R on the nitrocellulose filter of test strip
The fluorescent microsphere that the PLA2R antibody in sample to be tested is coupled in combination with Avidin is combined to be moved on nitrocellulose filter
It is coated the detection line position capture of capture body, the fluorescent microsphere of retention generates signal.The present invention is detected using prize law
The test strips color speed of PLA2R antibody faster, if from sentence read result 7min is loaded onto, with application No. is
When the disclosed method using double antigens sandwich test serum PLA2R antibody of 201510245291.5 Chinese patent is compared to detection
Between shorter (dual-antigen sandwich method sentence read result at least wants 15min), and the present invention using prize law detection PLA2R antibody examination
Paper slip color signal is stronger, the weak more difficult generation missing inspection of sun, and specificity is more preferable, can quantitative, accurate, easily tester
The content of PLA2R antibody in serum, blood plasma and whole blood.
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 PLA2R antibody of the present invention；
Fig. 2 shows the schematic diagram of the detection method of PLA2R antibody of the present invention.
Specific implementation mode
The invention discloses a kind of test strips and detection method of PLA2R 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 PLA2R 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.
2.PLA2R the preparation of biotinylation conjugate：Use ThermoSulfo-NHS-LC-Biotin gives birth to
For object element kit by PLA2R biotinylations, 4 DEG C temporary spare.
3. the preparation of bonding pad 1, bonding pad 2
The PLA2R 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)
Use the 0.01M PBS containing 5% (5g/100ml) trehalose that mouse anti-human igg 4 is diluted to 1mg/ as coating buffer
Rabbit-anti DNP-BSA is diluted to 1mg/ml by ml, draws film instrument using metal spraying and the mouse anti-human igg 4 that has diluted, rabbit-anti DNP-BSA are pressed
1 μ l/cm are drawn on NC films, make T lines and C lines respectively, 37 DEG C of oven drying 22h, and 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, 20RU/ml, 100RU/ml, 500RU/ml, 1000RU/ml, 1500RU/ml, 6 PLA2R units
Concentration carries out calibration test, with a batch of test strips, each point test 6 times.According to statistical method, with detection line (T
Band), the fluorescence intensity ratio of nature controlling line (C bands) (T bands detected value/C bands detected value) be ordinate, concentration of standard solution is horizontal seat
Mark, establishes equation and is fitted to standard curve, data storage to instrument.
The performance test of the fluorescent test paper strip of the PLA2R antibody of embodiment 2, the present invention
Testing each sample below adds 60 μ l Virus monitories, 7min 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-PLA2R antibody is dense
The average value of degree is 12.8RU/ml and standard deviation is 1.17RU/ml, using the sum of average value plus 3 times of standard deviations as critical value, then
Critical value is 16.31RU/ml.
2. test strips precision of the present invention determines：Choose high, medium and low value 3 parts of quality controlled serums (high level control target value：
300RU/mL, middle Quality Control target value：70RU/mL, low value control target value：30RU/ml), interior every part of replication is tested 10 times in homogeneous,
Average value, standard deviation are calculated separately, coefficient of variation CV (%) in experiment is calculated；It measures 1 time, METHOD FOR CONTINUOUS DETERMINATION 10 days, calculates daily
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 57 and 301RU/ml, this low value serum and high level serum point to select anti-PLA2R antibody concentrations
Not 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.Meter
Calculating 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 91%-109%, average recovery rate 99.3%.Accuracy meets the requirements.
4. test strips coincidence rate evaluation of the present invention
It is compared with goldstandard：It chooses and 113 parts of idiopathic membranous nephropathy patients serum, non-spy is diagnosed as by renal puncture art
43 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 93.0%, sensibility 85.0%.
Embodiment 3, compared with application No. is 201510245291.5 Chinese patents
1. choose 10 weakly positive quality controlled serums tested, be respectively adopted test strips of the present invention and application No. is
The test strips of 201510245291.5 Chinese patents carry out the detection of PLA2R antibody concentrations in serum, respectively at 7min and 15min
It reads as a result, the results are shown in Table 4.
The testing result of 4 weakly positive quality controlled serum difference read access time of table compares
The results show that application No. is the test strips of 201510245291.5 Chinese patents test 7min sentence read results and theory
Concentration difference is larger, tests 7min using test strips of the present invention and 15min judging results are consistent with theoretical concentration, and test
As a result it wants more acurrate.
2. choosing 15 negative clinical samples, test strips of the present invention are used respectively and application No. is 201510245291.5
The test strips of Chinese patent are carried out at the same time test and comparison, the results are shown in Table 5.
The negative clinical sample comparison result of table 5
Note："-" indicates negative, and " ± " indicates weak sun
The results show that 15 clinical negative serums test total negative using test strips of the present invention, and use application
Number there are 4 false sun for the test strips test of 201510245291.5 Chinese patents, illustrates test strips specificity of the present invention
More preferably.
In summary for correction data it is found that the ELISA test strip time of the present invention is shorter, testing result is more acurrate.
Claims (11)
1. a kind of test strip of PLA2R antibody, including overlap sample pad successively in adhesive base, bonding pad 1, bonding pad
2, nitrocellulose filter and blotting paper；PLA2R biotinylation conjugates are coated on the bonding pad 1, the bonding pad 2 sprays
There are Avidin fluorescent microsphere conjugate and DNP-BSA fluorescent microsphere conjugates, capture body is coated on the nitrocellulose filter
Anti- DNP-BSA antibody is also coated with as detection line, on the nitrocellulose filter as nature controlling line.
2. test strip according to claim 1, PLA2R is PLA2R molecules in the PLA2R biotinylations conjugate
The albumen of overall length or Partial Fragment, or the variant, analog, substitute of PLA2R 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 claim 1-3 any one, the capture body is anti-human IgG antibodies or energy and people
The substance that IgG antibody combines.
5. the preparation method of the test strip described in claim 1-4 any one, includes the following steps：
A, PLA2R protein biotinylations are obtained into PLA2R 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 PLA2R 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 used in advance containing 5-10% (g/ml)
The Tris-HCL buffer solutions of the 0.5M pH=7.4 of trehalose impregnate 5-10min, 37 DEG C of dry 2h；The Avidin and DNP-
The pretreatment of 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 used for microballoon
After the washing of 50mM pH6.0~6.5MES solution, 0.2~0.4 μ gNHS (N- hydroxysuccinimides) are added by every 100 μ l microballoons
30~40min is reacted with 0.2-0.4 μ gEDC (1- (3- dimethylamino-propyls) -3- ethyl carbodiimides) room temperature mixing；The parent
Further include the steps that closing after being coupled with element and DNP-BSA and the fluorescent microsphere of activation, BSA to a concentration of 1% is specially added
~5% (g/ml), room temperature mixing react 30~40min.
9. a kind of detection kit of PLA2R antibody, including the test strip described in claim 1-4 any one, sample
The PLA2R antibody standard substances of buffer solution and/or various concentration gradient.
10. a kind of detection method of PLA2R antibody takes sample to be tested that sample buffer is added and is added to above-mentioned 10 seconds after mixing
Test strip sample pad on, by test strip be inserted into fluorescence analyser detection hole, place 3~4 minutes, with detection
Line (T bands), nature controlling line (C bands) fluorescence intensity ratio be ordinate, PLA2R antibody concentration of standard solution be abscissa calculating
The concentration value of PLA2R antibody.
11. detection method according to claim 10, the sample to be tested is whole blood, serum, blood plasma, urine or saliva.
Priority Applications (3)
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| CN202010279263.6A CN111413506A (en) | 2018-01-30 | 2018-01-30 | Application of detection test strip in preparation of kit for detecting P L A2R antibody |
| CN201810091384.0A CN108279309B (en) | 2018-01-30 | 2018-01-30 | Detection test strip and detection method for PLA2R antibody |
| PCT/CN2018/092846 WO2019148754A1 (en) | 2018-01-30 | 2018-06-26 | Test strip and testing method for pla2r antibody |
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| CN108279309B (en) | 2022-02-15 |
| CN111413506A (en) | 2020-07-14 |
| WO2019148754A1 (en) | 2019-08-08 |
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