CN104880560B - PLA2R (phospholipase A2 receptor) antibody detection strip and preparation method and detection method thereof - Google Patents
PLA2R (phospholipase A2 receptor) antibody detection strip and preparation method and detection method thereof Download PDFInfo
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
- 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 relates to the biotechnology field and in particular relates to a PLA2R (phospholipase A2 receptor) antibody detection strip and a preparation method and a detection method of the PLA2R antibody detection strip. According to the PLA2R antibody detection strip, based on the double-antigen sandwiched antibody detection principle, PLA2R coats a nitrocellulose membrane, quantum dot coupled PLA2R is sprayed on a conjugate pad respectively, a sample pad, the conjugate pad, the nitrocellulose membrane and absorbent paper are sequentially lapped on a viscous PVC (polyvinyl chloride) bottom plate sequentially, the strip with certain width is cut out to form a detection card, the concentration value of the antibody is measured by a fluorescence analyzer, and the PLA2R antibody in the sample can be quantitatively detected; based on a quantum dot immunochromatographic method, the contents of PLA2R antibody in serum, plasma and whole blood can be quantitatively detected safely, accurately and rapidly in a noninvasive low-risk and low-cost manner, and the PLA2R antibody detection strip can provide assistance for preliminary screening of the idiopathic membranous nephropathy and illness monitoring.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of PLA2R antibody tests test strips and making, detection side
Method.
Background technology
Membranous nephropathy can be divided into idiopathic and Secondary cases membranous nephropathy by pathogenic factor, will rely on clinical manifestation in its diagnosis
And renal puncture art.Renal puncture art is a kind of diagnostic method of intrusive mood, has certain injury to patient.Foreign study finds
About 75% idiopathic membranous nephropathy patient can detect anti-PLA2R antibody by Serologic detection, and the country also has document to show
In compatriots, Positive rate is higher, reaches 82%, and the inspection of Secondary cases membranous nephropathy and other types Patients With Glomerular Diseases
Go out rate very low, healthy population is feminine gender.The detection by quantitative of serology PLA2R antibody can be the primary dcreening operation of idiopathic membranous nephropathy
Act on state of illness monitoring provided auxiliary.Therefore, non-wound, low-risk, safety, cheap, PLA2R accurately and quickly are developed quantitative
Determining test kit has extremely important meaning.
Immunochromatography technique is combined as fixing phase with various membrane materials, passes through capillary flow principle from sample-adding as the sample of liquid phase
One end swimming is flow in labeling pad, with the antigen or antibody labeling thing being fixed in labeling pad (such as gold colloidal, fluorescent rare earth unit
Element etc.) reaction forms immune complex, and this continues through to shove with markd immune complex and reaches fixed on reaction film
There is immunoreation so as to be trapped enrichment colour developing in the antigen or antibody in detection line, with detection line, reach the mesh of quick detection
's.The modal label of immunochromatography technique is gold colloidal, and application is widely, also extremely successful.Due to colloid gold label
It is the principle by Electrostatic Absorption, unstable in a fluid, the molecule of labelling easily comes off, and only colloid gold particle is enriched to
When certain macroscopic amount could interpretation, meanwhile, colloidal gold immunochromatographimethod technology is only to checking matter qualitative analyses.Mesh
Before, by colloidal gold immunochromatographimethod technology come quick detection phospholipase A2 receptor (PLA2R) antibody only in publication No. it is
Mention in CN102159951A, but as mentioned above, it is impossible to quantitative and gold colloidal stability is poor.
The content of the invention
It is an object of the invention to provide a kind of PLA2R antibody tests test strips and making, detection method, can accurately,
Quickly, the PLA2R antibody in sensitive, quantitative detection human blood.
The present invention solve its technical problem, the technical scheme for being adopted be to provide a kind of PLA2R antibody tests test strips and
Making, detection method.Specifically, including a detection kit, the detection kit includes PLA2R antibody tests card and sample
Buffer is savored, PLA2R antibody tests card is built with PLA2R antibody test test strips.Detection card can be measured by fluorescence analyser
PLA2R antibody concentration values.The test strips be included in overlapped in adhesive base successively sample pad, conjugate pad, cellulose nitrate
Plain film and absorbent paper；The quantum dot of PLA2R couplings is coated with described conjugate pad, is coated with described nitrocellulose filter
There are PLA2R antigens as detection line, PLA2R antibody is also coated with nitrocellulose filter as nature controlling line.Detection card is with double
Antigen sandwich method mode detection PLA2R antibody.
The PLA2R, can be PLA2R the or PLA2R pieces of native purified PLA2R, or gene recombinaton acquisition
Section；It, by the PLA2R antibody obtained by PLA2R immune animals, can be such as rabbit source, Yang Yuan or other sucklings that PLA2R antibody is
The polyclonal antibody, or Mus resource monoclonal antibody of animal origin.
The quantum dot is to include I B, II B, III A, IV A, V A, the two or more elementary composition single crystalline substance in VI A races
Nuclear structure, such as CdS, CdSe, CdTe, ZnSe, InP, InAs, HgCdTe etc., or nucleocapsid structure, such as CdSe/ZnS, ZnCdS/
ZnS, MnSe/ZnSe/ZnS, CuInZnS/ZnS etc..
Quantum dot is made up of above element outward, also with surface modification group, such as the base such as-COOH ,-NH2 ,-SH ,-CHO
Group, can by some chemical reagent such as glutaraldehyde, carbodiimide etc. coupling biomacromolecule, such as protein, nucleic acid and other
Organic molecule.
The particle size range of the quantum dot be 2～1000nm, excitation wavelength scope be 200～600nm, wavelength of transmitted light
For 400～800nm.
A kind of manufacture method of PLA2R antibody tests test strips, comprises the following steps：
A. quantum dot-PLA2R complex is obtained with quantum point coupling PLA2R, and be sprayed on thing pad；
B. PLA2R is coated with nitrocellulose filter as detection line respectively, by PLA2R antibody coating to cellulose nitrate
As nature controlling line on plain film, the distance between detection line and nature controlling line is 3～10mm.
C. overlap sample pad in adhesive base successively, be coated with the conjugate pad of quantum dot-PLA2R complex, arrange
There are nitrocellulose filter, the absorbent paper of detection line and nature controlling line, and cut into required width and be PLA2R antibody test reagent paper
Bar.
The preparation method of detection line and nature controlling line is：0.005-0.2M is used respectively, and the coating buffer of pH6.0-pH9.0 is dilute
PLA2R and PLA2R antibody is released to 0.1-5mg/ml, the coating buffer is phosphate buffer, borate buffer solution, carbonate
One kind in buffer.With film instrument or spray film instrument is drawn respectively by PLA2R and PLA2R antibody coating on nitrocellulose filter, wrap
It is 0.4-2 μ l/cm by concentration；After spraying is finished, ambient humidity is placed on for less than 30%, temperature is in 16 DEG C -45 DEG C of environment
In, more than 1h is dried, is sealed up for safekeeping.
The method that PLA2R is coupled quantum dot：PLA2R is added in quantum dot solution or is added to quantum dot
It is coupled in PLA2R solution and obtains quantum dot-PLA2R complex；The quantum dot can be activated in appropriate solution, activating reagent
Can be EDC, NHS or glutaraldehyde or other cross-linking agent etc., quantum dot is combined into quantum dot solution i.e. with activator.
On conjugate pad, the condition of spraying quantum dot-PLA2R complex is：Quantum dot-PLA2R complex is diluted to dilute
5-50 times being released, being sprayed on conjugate pad by the amount of 2-100 μ l/cm with spray film instrument, 30% is less than in ambient humidity, temperature exists
1-5h is toasted in the environment of 35-37 DEG C, is sealed up for safekeeping after being dried.
The PLA2R antibody tests card, including upper shell and lower house, are placed between the upper shell and lower house
PLA2R antibody test test strips, are provided with well, observation window and the information area, the well and PLA2R on the upper shell
Sample pad correspondence in antibody test test strips, observation window are corresponding with detection line and nature controlling line on nitrocellulose filter；It is described
It is coated with the information area for recognizing Quick Response Code, bar code or the magnetic card of patient ID；The PLA2R antibody tests card is slow with sample
Rush liquid to be mounted in test kit together, on test kit, be provided with demarcation chip.
Present invention also offers a kind of detection method of PLA2R antibody：Comprise the following steps：
1) first fluorescence analyser is demarcated with the demarcation chip on test kit, preserve nominal data；
2) 10 microlitres of serum or plasma sample are taken, or 15 microlitres of whole blood samples adds sample buffer, 10 seconds after mixing,
Add detection card well；
3) by the detection hole of detection card insertion fluorescence analyser, place 3-4 minutes, fluorescence analyser can read PLA2R
The concentration value of antibody.
Beneficial effect：Quantum dot and immunochromatography technique are combined, test strips can be made both to have had the advantages that stability was high,
Meanwhile, detection by quantitative can be carried out again to PLA2R antibody, and detect that sensitivity is high again；The detection method also has detection time short,
The characteristics of efficiency high.Relative to traditional gold colloidal and organic fluorescent dye, quantum dot have the advantages that it is stable, can be quantitative, because
This, can be very good to detect the storage of PLA2R antibody.The present invention utilize quantum dot immunochromatographic method, non-wound, low-risk,
PLA2R antibody contents in safety, cheap, detection by quantitative serum accurately and quickly, blood plasma and whole blood, can be idiopathic film
Property nephropathy primary dcreening operation and state of illness monitoring provided auxiliary effect.
Specific embodiment
A kind of detection kit, the detection kit include PLA2R antibody tests card and sample buffer, and PLA2R resists
Health check-up surveys card built with PLA2R antibody test test strips.The test strips be included in overlapped in adhesive base successively sample pad,
Conjugate pad, nitrocellulose filter and absorbent paper.The quantum dot of PLA2R couplings is coated with described conjugate pad, it is described
PLA2R antigens are coated with nitrocellulose filter as detection line, PLA2R antibody is also coated with nitrocellulose filter as matter
Control line.Detection card is with dual-antigen sandwich method mode detection PLA2R antibody.
The PLA2R, can be PLA2R the or PLA2R pieces of native purified PLA2R, or gene recombinaton acquisition
Section, is replaced with PLA2R herein；It, by the PLA2R antibody obtained by PLA2R immune animals, can be all that PLA2R antibody is
Such as the polyclonal antibody, or Mus resource monoclonal antibody of rabbit source, Yang Yuan or other mammal sources.
The quantum dot is to include I B, II B, III A, IV A, V A, the two or more elementary composition single crystalline substance in VI A races
Nuclear structure, such as CdS, CdSe, CdTe, ZnSe, InP, InAs, HgCdTe etc., or nucleocapsid structure, such as CdSe/ZnS, ZnCdS/
ZnS, MnSe/ZnSe/ZnS, CuInZnS/ZnS etc..
Outside quantum dot is made up of above element, also with surface modification group, such as-COOH ,-NH2,-SH, the base such as-CHO
Group, can by some chemical reagent such as glutaraldehyde, carbodiimide etc. coupling biomacromolecule, such as protein, nucleic acid and other
Organic molecule.
The particle size range of the quantum dot be 2～1000nm, excitation wavelength scope be 200～600nm, wavelength of transmitted light
For 400～800nm.
The PLA2R antibody tests card, including upper shell and lower house, are placed between the upper shell and lower house
PLA2R antibody test test strips, are provided with well, observation window and the information area, the well and PLA2R on the upper shell
Sample pad correspondence in antibody test test strips, observation window are corresponding with detection line and nature controlling line on nitrocellulose filter；It is described
It is coated with the information area for recognizing Quick Response Code, bar code or the magnetic card of patient ID；The PLA2R antibody tests card is slow with sample
Rush liquid to be mounted in test kit together, on test kit, be provided with demarcation chip.
Present invention also offers a kind of detection method of PLA2R antibody：Comprise the following steps：
1) first fluorescence analyser is demarcated with the demarcation chip on test kit, preserve nominal data；
2) 10 microlitres of serum or plasma sample are taken, or 15 microlitres of whole blood samples adds sample buffer, 10 seconds after mixing,
Take 80 microlitres of mixed liquor and add detection card well；
3) by the detection hole of detection card insertion fluorescence analyser, place 3-4 minutes, fluorescence analyser can read PLA2R
The concentration value of antibody.
Embodiment 1：
A kind of manufacture method of PLA2R antibody tests test strips, comprises the following steps：
A. quantum dot-PLA2R complex is obtained with quantum point coupling PLA2R, and be sprayed on thing pad；
B. PLA2R is coated with nitrocellulose filter as detection line respectively, by PLA2R antibody coating to cellulose nitrate
As nature controlling line on plain film, the distance between detection line and nature controlling line is 5mm.
C. overlap sample pad in adhesive base successively, be coated with the conjugate pad of quantum dot-PLA2 complex, be provided with
The nitrocellulose filter of detection line and nature controlling line, absorbent paper, overlapped 1mm between each membrane material, cut into 4mm's after gluing
The test strips of width are PLA2R antibody test test strips.
PLA2R is coupled the method that quantum dot prepares quantum dot-PLA2R complex：
0.1ml is taken by CdSe, surface group is quantum dot (excitation wavelength 365nm, the launch wavelength of-COOH compositions
620nm) solution is placed in the MES buffer that 1ml concentration is 0.1mol/L PH5.0, while adding 0.02ml concentration 50mg/ml
The N- hydroxies succinyl of 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides (EDC) and 0.08ml concentration 20mg/ml is sub-
Amine (sulfo-NHS), after incubation at room temperature 20min, is centrifuged (23000rpm, 30min), abandons supernatant, wash to obtain quantum dot solvent, phase
It is centrifuged with the conditions of, abandons supernatant, obtain final quantum dot solvent, addition 0.1mg's is natural in final quantum dot solvent
The PLA2R of purification, is incubated at room temperature 2h, adds 1% glycine, is incubated at room temperature 0.5h, is centrifuged under the same terms, adds after abandoning supernatant
0.1ml preserves liquid (phosphate buffer of 0.02mol/L, containing 1%BSA, 0.04%Proclin300), and the quantum dot of gained-
PLA2R complex preserves stand-by in being positioned over 4 DEG C of environment.
The concrete preparation method of quantum dot conjugate pad：
Conjugate pad pretreatment：The glass fibre element film of a model 8975 is taken, 0.02mol/L phosphate buffers are used
Take out after (including 1% bovine serum albumin, 0.5% polysorbas20) immersion 30min, dry under conditions of 37 DEG C.Will be above-mentioned
Good quantum dot-PLA2R the complex of labelling with quantum dot working buffer solution (contain 1% bovine serum albumin, 0.05% polysorbas20,
The 0.01mol/L phosphate buffers of 0.05%Proclin300) by 1：After 200 dilutions, sprayed by the amount of 20 μ l/cm with spray film instrument
Be coated onto on the glass fibre membrane of above-mentioned pretreatment, 30% be less than in ambient humidity, 5h is toasted in the environment of 37 DEG C, be dried, obtain final product
Quantum dot conjugate pad, is put into desiccant and seals up for safekeeping.
The concrete preparation method of detection line and nature controlling line：
Take the nitrocellulose filter of model M180, in room temperature, relative humidity be 55% environmental condition under balance
After 30min, diluted with the phosphate buffer of 0.02M, pH7.0 and rule on nitrocellulose filter after PLA2R to 0.5mg/ml,
It is 2 μ l/cm to draw film parameters, used as detection line；PLA2R antibody is diluted to 0.5mg/ with the phosphate buffer of 0.02M, pH7.0
Ml rules on nitrocellulose filter, and it is 2 μ l/cm to draw film concentration, used as nature controlling line；At a distance of 5mm between detection line and nature controlling line.
In ambient humidity below 30%, temperature is dried more than 2h, is sealed up for safekeeping at 37 DEG C.The PLA2R antibody is polyclonal antibody.
Carry out elaboration detection, accuracy detection to above-mentioned gained test kit respectively and contrast with goldstandard, judge this
The precision and accuracy of test kit.Concrete grammar is：
The determination of marginal value
Whether marginal value is medical science decision level, is some limit values, be higher or lower than these limit values by Observe and measure value,
Can play a part of to exclude in medical diagnosis on disease or confirm, or some diseases are classified or are classified, or prognosis is made an estimate,
To point out which kind of processing mode doctor should clinically take, inspection in a certain respect is such as further carried out, or determine to take certain
Plant remedy measures etc..
By detecting 220 portions of normal human serums, the meansigma methodss of anti-PLA2R antibody concentration are 12.8RU/ml and standard deviation is
1.17RU/ml, using meansigma methodss plus 3 times of standard deviation sums as marginal value, then marginal value is 16.31RU/ml.
Elaboration is detected
High, medium and low 3 parts of quality controlled serums of value are chosen, every part of replication 10 times, is calculated averagely respectively in the homogeneous test
Value, standard deviation, calculate coefficient of variation CV (%) in test；Determine 1 time daily, METHOD FOR CONTINUOUS DETERMINATION 10 days, calculate test bay CV (%)
Value.Computing formula is：CV (%)=standard deviation/meansigma methodss × 100%
Table 1. tests the interior CV value results with test bay
Note：Refer in test in once testing, identical test is tested repeatedly repeatedly；Test bay refers to identical test not
With retest in the time；
Accuracy is detected：
Anti- PLA2R antibody concentration is selected to be respectively 57 and 301RU/ml 2 parts of serum, this low value serum and high level serum point
Not with 1：4、1：1 and 9：1 ratio is mixed into the serum of 3 parts of variable concentrations, and theoretical value is respectively 252.2,207.5 and 81.4RU/
ml.Detected by test kit, calculate the concordance of detected value and theoretical value, the i.e. response rate.Computing formula is:The response rate=test
Value/theoretical value × 100%
2. response rate result of calculation of table
Between 91%-109%, average recovery rate is 99.3% to the response rate.
Contrast with goldstandard：
Selection is diagnosed as 113 parts of idiopathic membranous nephropathy patients serum, non-idiopathic membranous nephropathy by renal puncture art
43 parts of patients serum, with renal puncture art as goldstandard, counts as follows with four fold table：
Specificity=true negative/feminine gender sum × 100%
Sensitivity=true positives/positive sum × 100%
Positive predictive value=true positives/(true positives+false positive) × 100%
Negative predictive value=true negative/(true negative+false negative) × 100%
3. four fold table statistical result of table
Therefore the specificity of this test kit is 93.0%, and sensitivity is 85.0%, and positive predictive value is 97.0%, negative pre-
Measured value is 70.2%.
Embodiment 2：
A kind of manufacture method of PLA2R antibody tests test strips, comprises the following steps：
A. quantum dot-PLA2R complex is obtained with quantum point coupling PLA2R, and be sprayed on thing pad；
B. PLA2R is coated with nitrocellulose filter as detection line respectively, by PLA2R antibody coating to cellulose nitrate
As nature controlling line on plain film, the distance between detection line and nature controlling line is 3mm.
C. overlap sample pad in adhesive base successively, be coated with the conjugate pad of quantum dot-PLA2 complex, be provided with
The nitrocellulose filter of detection line and nature controlling line, absorbent paper, overlapped 1mm between each membrane material, cut into 4mm's after gluing
The test strips of width are PLA2R antibody test test strips.
PLA2R is coupled the method that quantum dot prepares quantum dot-PLA2R complex：
0.1ml is taken by CdSe/ZnS, surface group is quantum dot (excitation wavelength 365nm, the launch wavelength of-COOH compositions
620nm) solution is placed in the MES buffer that 1ml concentration is 0.1mol/L PH5.0, while adding 0.02ml concentration 50mg/ml
The N- hydroxies succinyl of 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides (EDC) and 0.08ml concentration 20mg/ml is sub-
Amine (sulfo-NHS), after incubation at room temperature 20min, is centrifuged (18000rpm, 30min), abandons supernatant, wash to obtain quantum dot solvent, phase
It is centrifuged with the conditions of, abandons supernatant, obtain final quantum dot solvent, addition 0.1mg's is natural in final quantum dot solvent
The PLA2R of purification, is incubated at room temperature 2h, adds 1% glycine, is incubated at room temperature 0.5h, is centrifuged under the same terms, adds after abandoning supernatant
0.1ml preserves liquid (phosphate buffer of 0.02mol/L, containing 1%BSA, 0.04%Proclin300), and the quantum dot of gained-
PLA2R complex preserves stand-by in being positioned over 4 DEG C of environment.
The concrete preparation method of quantum dot conjugate pad：
Conjugate pad pretreatment：The glass fibre element film of a model 8975 is taken, 0.02mol/L phosphate buffers are used
Take out after (including 1% bovine serum albumin, 0.5% polysorbas20) immersion 30min, dry under conditions of 37 DEG C.Will be above-mentioned
Good quantum dot-PLA2R the complex of labelling with quantum dot working buffer solution (contain 1% bovine serum albumin, 0.05% polysorbas20,
The 0.01mol/L phosphate buffers of 0.05%Proclin300) by 1：After 200 dilutions, sprayed by the amount of 20 μ l/cm with spray film instrument
Be coated onto on the glass fibre membrane of above-mentioned pretreatment, 30% be less than in ambient humidity, 5h is toasted in the environment of 37 DEG C, be dried, obtain final product
Quantum dot conjugate pad, is put into desiccant and seals up for safekeeping.
The concrete preparation method of detection line and nature controlling line：
Take the nitrocellulose filter of model M180, in room temperature, relative humidity be 55% environmental condition under balance
After 30min, diluted with the phosphate buffer of 0.02M, pH7.0 and rule on nitrocellulose filter after PLA2R to 0.5mg/ml,
It is 2 μ l/cm to draw film parameters, used as detection line；PLA2R antibody is diluted to 0.5mg/ with the phosphate buffer of 0.02M, pH7.0
Ml rules on nitrocellulose filter, and it is 2 μ l/cm to draw film parameters, used as nature controlling line；At a distance of 3mm between detection line and nature controlling line.
In ambient humidity below 30%, temperature is dried more than 2h, is sealed up for safekeeping at 37 DEG C.The PLA2R antibody is polyclonal antibody.
Carry out elaboration detection, accuracy detection to above-mentioned gained test kit respectively and contrast with goldstandard, judge this
The precision and accuracy of test kit.Concrete grammar is：
The determination of marginal value
By detecting 220 portions of normal human serums, the meansigma methodss of anti-PLA2R antibody concentration are 12.8RU/ml and standard deviation is
1.17RU/ml, using meansigma methodss plus 3 times of standard deviation sums as marginal value, then marginal value is 16.31RU/ml.
Elaboration is detected
High, medium and low 3 parts of quality controlled serums of value are chosen, every part of replication 10 times, is calculated averagely respectively in the homogeneous test
Value, standard deviation, calculate coefficient of variation CV (%) in test；Determine 1 time daily, METHOD FOR CONTINUOUS DETERMINATION 10 days, calculate test bay CV (%)
Value.Computing formula is：CV (%)=standard deviation/meansigma methodss × 100%
Table 1. tests the interior CV value results with test bay
Accuracy is detected：
Anti- PLA2R antibody concentration is selected to be respectively 57 and 301RU/ml 2 parts of serum, this low value serum and high level serum point
Not with 1：4、1：1 and 9：1 ratio is mixed into the serum of 3 parts of variable concentrations, and theoretical value is respectively 252.2,207.5 and 81.4RU/
ml.Detected by test kit, calculate the concordance of detected value and theoretical value, the i.e. response rate.Computing formula is:The response rate=test
Value/theoretical value × 100%
2. response rate result of calculation of table
Between 92%-101%, average recovery rate is 97.4% to the response rate.
Contrast with goldstandard：
Selection is diagnosed as 113 parts of idiopathic membranous nephropathy patients serum, non-idiopathic membranous nephropathy by renal puncture art
43 parts of patients serum, with renal puncture art as goldstandard, counts as follows with four fold table：
Specificity=true negative/feminine gender sum × 100%
Sensitivity=true positives/positive sum × 100%
Positive predictive value=true positives/(true positives+false positive) × 100%
Negative predictive value=true negative/(true negative+false negative) × 100%
3. four fold table statistical result of table
Therefore the specificity of this test kit is 97.7%, and sensitivity is 83.0%, and positive predictive value is 99.0%, negative pre-
Measured value is 68.9%.
Embodiment 3：
A kind of manufacture method of PLA2R antibody tests test strips, comprises the following steps：
A. quantum dot-PLA2R complex is obtained with quantum point coupling PLA2R, and be sprayed on thing pad；
B. PLA2R is coated with nitrocellulose filter as detection line respectively, by PLA2R antibody coating to cellulose nitrate
As nature controlling line on plain film, the distance between detection line and nature controlling line is 10mm.
C. overlap sample pad in adhesive base successively, be coated with the conjugate pad of quantum dot-PLA2 complex, be provided with
The nitrocellulose filter of detection line and nature controlling line, absorbent paper, overlapped 1mm between each membrane material, cut into 4mm's after gluing
The test strips of width are PLA2R antibody test test strips.
PLA2R is coupled the method that quantum dot prepares quantum dot-PLA2R complex：
0.1ml is taken by CdSe/ZnS, surface group is-NH2Quantum dot (excitation wavelength 365nm, the launch wavelength of composition
620nm) solution is placed in the carbonate buffer solution of 0.05mol/L PH9.0 of the 1ml containing 5% glutaraldehyde, is put in water bath, 37 DEG C
Water-bath is centrifuged (18000rpm, 30min), abandons supernatant, wash to obtain quantum dot solvent with reference to after 2h, is centrifuged, abandons under the same terms
Clearly, final quantum dot solvent is obtained, in final quantum dot solvent, adds the native purified PLA2R of 0.1mg, room temperature to incubate
2h is educated, 1%BSA is added, 0.5h is incubated at room temperature, is centrifuged under the same terms, after abandoning supernatant, add 0.1ml to preserve liquid (0.02mol/L
Phosphate buffer, containing 1%BSA, 0.04%Proclin300), the quantum dot-PLA2R complex of gained is positioned over 4 DEG C
Preserve in environment stand-by.
The concrete preparation method of quantum dot conjugate pad：
Conjugate pad pretreatment：The glass fibre element film of a model 8975 is taken, 0.02mol/L phosphate buffers are used
Take out after (including 1% bovine serum albumin, 0.5% polysorbas20) immersion 30min, dry under conditions of 37 DEG C.Will be above-mentioned
Good quantum dot-PLA2R complex quantum dot the working buffer solutions of labelling (include 1% bovine serum albumin, 0.05% tween
The 0.01mol/L phosphate buffers of 20,0.05%Proclin300) by 1：After 200 dilutions, with spray film instrument by 20 μ l/cm's
Amount is sprayed on the glass fibre membrane of above-mentioned pretreatment, is less than 30% in ambient humidity, 5h is toasted in the environment of 37 DEG C, is dried,
Quantum dot conjugate pad is obtained final product, desiccant is put into and is sealed up for safekeeping.
The concrete preparation method of detection line and nature controlling line：
Take the nitrocellulose filter of model M180, in room temperature, relative humidity be 55% environmental condition under balance
After 30min, diluted with the phosphate buffer of 0.02M, pH7.0 and rule on nitrocellulose filter after PLA2R to 0.5mg/ml,
It is 2 μ l/cm to draw film parameters, used as detection line；PLA2R antibody is diluted to 0.5mg/ with the phosphate buffer of 0.02M, pH7.0
Ml rules on nitrocellulose filter, and it is 2 μ l/cm to draw film concentration, used as nature controlling line；Between detection line and nature controlling line apart
10mm.In ambient humidity below 30%, temperature is dried more than 2h, is sealed up for safekeeping at 37 DEG C.The PLA2R antibody is Anti-TNF-α
Body.
Carry out elaboration detection, accuracy detection to above-mentioned gained test kit respectively and contrast with goldstandard, judge this
The precision and accuracy of test kit.Concrete grammar is：
The determination of marginal value
By detecting 220 portions of normal human serums, the meansigma methodss of anti-PLA2R antibody concentration are 12.8RU/ml and standard deviation is
1.17RU/ml, using meansigma methodss plus 3 times of standard deviation sums as marginal value, then marginal value is 16.31RU/ml.
Elaboration is detected
High, medium and low 3 parts of quality controlled serums of value are chosen, every part of replication 10 times, is calculated averagely respectively in the homogeneous test
Value, standard deviation, calculate coefficient of variation CV (%) in test；Determine 1 time daily, METHOD FOR CONTINUOUS DETERMINATION 10 days, calculate test bay CV (%)
Value.Computing formula is：CV (%)=standard deviation/meansigma methodss × 100%
Table 1. tests the interior CV value results with test bay
Accuracy is detected：
Anti- PLA2R antibody concentration is selected to be respectively 57 and 301RU/ml 2 parts of serum, this low value serum and high level serum point
Not with 1：4、1：1 and 9：1 ratio is mixed into the serum of 3 parts of variable concentrations, and theoretical value is respectively 252.2,207.5 and 81.4RU/
ml.Detected by test kit, calculate the concordance of detected value and theoretical value, the i.e. response rate.Computing formula is:The response rate=test
Value/theoretical value × 100%
2. response rate result of calculation of table
Between 94%-103%, average recovery rate is 97.7% to the response rate.
Contrast with goldstandard：
Selection is diagnosed as 113 parts of idiopathic membranous nephropathy patients serum, non-idiopathic membranous nephropathy by renal puncture art
43 parts of patients serum, with renal puncture art as goldstandard, counts as follows with four fold table：
Specificity=true negative/feminine gender sum × 100%
Sensitivity=true positives/positive sum × 100%
Positive predictive value=true positives/(true positives+false positive) × 100%
Negative predictive value=true negative/(true negative+false negative) × 100%
3. four fold table statistical result of table
Therefore the specificity of this test kit is 88.4%, and sensitivity is 87.6%, and positive predictive value is 95.2%, negative pre-
Measured value is 73.1%.
To sum up test data analyzer can be obtained, this test strips and detection method high precision, good stability, and can be with detection by quantitative
The concentration of PLA2R antibody.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (1)
1. a kind of manufacture method of PLA2R antibody tests test strips, it is characterised in that：Comprise the following steps：
A. quantum dot-PLA2R complex is obtained with quantum point coupling PLA2R, and be sprayed on thing pad；
B. PLA2R is coated with nitrocellulose filter as detection line respectively, by PLA2R antibody coating to nitrocellulose filter
On as nature controlling line, the distance between detection line and nature controlling line is 3～10mm；
C. overlap sample pad in adhesive base successively, be coated with the conjugate pad of quantum dot-PLA2R complex, be provided with inspection
The nitrocellulose filter of survey line and nature controlling line, absorbent paper, and cut into required width, as PLA2R antibody tests test strips；
The concrete manufacture method of step b is：The coating buffer of 0.005-0.2M, pH6.0-pH9.0 is used to dilute respectively
To 0.1-5mg/ml, the coating buffer is phosphate buffer to PLA2R and PLA2R antibody, borate buffer solution, carbonate delay
The one kind rushed in liquid；With film instrument or spray film instrument is drawn respectively by PLA2R and PLA2R antibody coating on nitrocellulose filter, it is coated with
Concentration is 0.4-2 μ l/cm；After spraying is finished, be placed on ambient humidity for less than 30%, temperature in 16 DEG C -45 DEG C of environment,
Drying more than 1h, seals up for safekeeping；
The concrete manufacture method of step a is：PLA2R is added in quantum dot solution or is added to quantum dot
It is coupled in PLA2R solution and obtains quantum dot-PLA2R complex；
Quantum dot-PLA2R complex is diluted into 5-200 times, is sprayed on conjugate pad by the amount of 2-100 μ l/cm with spray film instrument,
30% is less than in ambient humidity, temperature toasts 1-5h in the environment of 35-37 DEG C, is sealed up for safekeeping after being dried.
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| CN105548556A (en) * | 2015-12-04 | 2016-05-04 | 深圳市伯劳特生物制品有限公司 | PGI rapid quantitative detection kit and making method and detection method thereof |
| CN105606796A (en) * | 2016-02-01 | 2016-05-25 | 苏州东尼生物技术有限公司 | Nitrocellulose membrane pretreatment method applied to gold immunochromatography assay |
| CN111413506A (en) * | 2018-01-30 | 2020-07-14 | 深圳市伯劳特生物制品有限公司 | Application of detection test strip in preparation of kit for detecting P L A2R antibody |
| CN108982828A (en) * | 2018-06-28 | 2018-12-11 | 长春万成生物电子工程有限公司 | A kind of quantum dot examines the preparation and application of test paper fastly |
| CN109459572A (en) * | 2018-12-21 | 2019-03-12 | 江南大学 | A kind of primary membranous nephropathy diagnostic kit |
| CN115184620B (en) * | 2022-09-14 | 2023-01-24 | 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 |
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| CN101339196A (en) * | 2008-07-29 | 2009-01-07 | 上海师范大学 | Rapid checking method for bladder cancer by quantum dot mark immunity-chromatography test paper |
| WO2014176753A1 (en) * | 2013-04-30 | 2014-11-06 | 成都领御生物技术有限公司 | Quantum-dot immunochromatographic test strip detection system and use thereof |
| WO2015004603A1 (en) * | 2013-07-09 | 2015-01-15 | Institut National De La Sante Et De La Recherche Medicale | Anti-pla2r antibody and uses thereof |
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