CN105988011A - ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for idiopathic membranous nephropathy and detection method thereof - Google Patents
ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for idiopathic membranous nephropathy and detection method thereof Download PDFInfo
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- CN105988011A CN105988011A CN201610201970.7A CN201610201970A CN105988011A CN 105988011 A CN105988011 A CN 105988011A CN 201610201970 A CN201610201970 A CN 201610201970A CN 105988011 A CN105988011 A CN 105988011A
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- 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
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- 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
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Abstract
The invention discloses an ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for idiopathic membranous nephropathy (IMN) and a detection method thereof. The ELISA detection kit comprises a coated ELISA plate, an enzyme-labeled secondary antibody, standard anti-PLA2R and anti-THSD7A blood serum, washing liquid, a substrate TMB (Tetramethylbenzidine) and a stopping solution, wherein the enzyme-labeled secondary antibody is an anti-IgG4 antibody; and the coated ELISA plate contains solid-phase antigens of PLA2R and THSD7A proteins. After the PLA2R and THSD7A proteins are prepared, a micro-pore plate is coated with the purified PLA2R and THSD7A proteins to prepare the coated ELISA plate; the standard anti-PLA2R and anti-THSD7A blood serum with known concentration and a sample to be detected with unknown concentration are added into the coated ELISA plate in sequence; and steps of incubating, adding the enzyme-labeled secondary antibody, washing, developing, determining and judging a result are carried out. According to the ELISA detection kit, the sensitivity to IMN diagnosis is up to 80%-92% and the specificity is up to 100%; and the obedience of patients can be improved, blood serum monitoring of IMN patients can be carried out in a non-invasive, specific, convenient, rapid, efficient and large-batch manner, and the ELISA detection kit is used for IMN diagnosis, disease condition monitoring, curative effect evaluation and prognosis evaluation.
Description
Technical field
The present invention relates to biological technical field, specifically a kind of idiopathic membranous nephropathy ELISA detection kit and detection method thereof.
Background technology
Idiopathic membranous nephropathy (idiopathic membranous nephropathy, IMN) being Urology Department commonly encountered diseases, frequently-occurring disease, in global range, sickness rate is about 1.2/100,000/ year, it is the modal cause of disease of white people's primary nephrotic syndrome, accounts for 20%.In China, IMN ratio in primary glomerulopathy is about 22.7%, is up to 39.6% more than 60 years old in patient.And day by day obvious along with Chinese society population structure ageing phenomenon, IMN occurred frequently in mid-aged population is the most in rising trend, threatens compatriots healthy.Despite 20%-35% patient can spontaneous remission in 2-3 initial after morbidity, but still have about 1/3 even more patient can gradually progress to end stagerenaldisease, need maintenance renal replacement therapies, serious harm human health.But at present the current treatment status of IMN is barely satisfactory, still depending on urine protein quantitation, renal function situation and rebiopsy for the state of illness monitoring of IMN, treatment choice etc., but urine protein quantitation, renal function lack specificity, the reflection state of an illness relatively lags behind；And rebiopsy is as a kind of invasive inspection, not only bring misery to patient, more having and perirenal hematoma, arteriovenous fistula occur, infect and damage the risk of other organs, patient is often difficult to accept repeated puncture, thus affects us and judge clinical efficacy and assess prognosis.Therefore clinical practice is needed serologic marker thing noninvasive, specific for IMN condition assessment and guiding treatment badly.
Anti-PLA2Autoantibody (the anti-PLA of R2And IMN R)
2009, Beck etc. found the M type phospholipase A being positioned on mankind's podocyte film first2Receptor is the target antigen of IMN: 1, has in 70% patients serum in 36 example IMN patients and detects anti-PLA2Autoantibody (the anti-PLA of R2And can be with PLA R),2R is incorporated under epithelial cell formation graininess immune complex.2、anti-PLA2R is IgG4 hypotype, and IgG4 is the mankind IMN main Types at glomerule Immune deposits.3, anti-PLA is confirmed by copolymerization Jiao's immuno-electron microscope detection renal biopsy2R and PLA2R co-deposits in upper subcutaneous, and can isolate this antibody from the immune complex of IMN patient's glomerule deposition, and secondary glomerulopathy rarely has detection, and this points out anti-PLA2R is antibody specific to IMN.The researcher Wei song Qin etc. of China also finds have the patient of 82% this antibody occur in IMN.Research finds anti-PLA simultaneously2The change of R with IMN patient's albuminuria is closely related, and can be as of assessment IMN renal function of patients Progressive symmetric erythrokeratodermia forfeiture risk early period parameters.
The autoantibody (anti-THSD7A) of anti-THSD7A and IMN
Although 70%-82%IMN has in the patient for PLA2The circulating antibody of R, but still have part IMN morbidity's mechanism the brightest.The pionerring research being published on NEJM magazine in November, 2014 confirms that 1 type thrombospondin 7A territory (THSD7A) being expressed in podocyte podocytic process is second antigen participating in IMN morbidity.Research finds 15 parts from PLA2Detection anti-THSD7A in IMN patient's (10%) serum of R negative antibody, and formation immune complex can be incorporated under podocyte with THSD7A.And 74 examples PLA2The IMN patient of R antibody positive, does not then detect this antigen in the serum specimen of 76 example difference Patients With Glomerular Diseases and 44 example normal healthy controls persons.This points out anti-THSD7A also antibody specific to IMN.And preliminary study finds anti-THSD7A level and Urinary protein quantification significant correlation.
Anti-PLA2The autoantibody of R and the autoantibody of anti-THSD7A
Although the antigen of this new identification of THSD7A and PLA2R is significantly different, but also has some and PLA2Structure that R is similar and biochemical characteristic, such as: be all expressed on podocyte film；The large-scale extracellular regions formed containing a N-glycosylation domains connected by disulfide bond；For THSD7A and PLA2The autoantibody of R is IgG4 hypotype, and IgG4 is the mankind IMN main Types at glomerule Immune deposits.
Summary of the invention
It is an object of the invention to provide a kind of sensitivity be up to 80-92%, specificity be up to 100% idiopathic membranous nephropathy ELISA detection kit and detection method thereof, with solve in above-mentioned background technology propose problem.
For achieving the above object, the present invention provides following technical scheme:
A kind of idiopathic membranous nephropathy ELISA detection kit, including coated elisa plate, ELIAS secondary antibody, standard anti-PLA2R and anti-THSD7A serum, cleaning mixture, substrate TMB and stop buffer, ELIAS secondary antibody body is anti-igg 4 antibody, and coated elisa plate contains PLA2The solid phase antigen of R and THSD7A albumen.
The detection method of described idiopathic membranous nephropathy ELISA detection kit, comprises the following steps:
(1) PLA is prepared2R and THSD7A albumen: extract glomerule total serum IgE, reverse transcription synthesis CDNA, separately design PLA2The primer of R and THSD7A gene carries out PCR amplification, then is connected with eucaryon plasmid carrier respectively, transfects HEK293T cell, and after cultivating 48h, centrifuging and taking supernatant extracts albumen, then purification PLA2R and THSD7A albumen；
(2) sample-adding: with the PLA of purification2R and THSD7A albumen is coated microwell plate, makes coated elisa plate, is sequentially added into standard anti-PLA of concentration known in coated elisa plate2R and anti-THSD7A serum and the measuring samples of unknown concentration；
(3) incubation, addition ELIAS secondary antibody: after incubation, add ELIAS secondary antibody, then be combined with Streptavidin-HRP, form immune complex；
(4) wash, develop the color: wash with cleaning mixture, add substrate TMB colour developing, add stop buffer and terminate reaction；
(5) measure and result judges.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention uses technique for gene engineering and ELISA experimental principle, use in conjunction serum anti-PLA2R and anti-THSD7A autoantibody prepares specific IMN detection kit, anti-PLA2The sensitivity that the use in conjunction of R and anti-THSD7A diagnoses for IMN is up to 80-92%, specificity is up to 100%, it it is the preferable Serologic markers of IMN, the present invention can improve patient compliance, noinvasive, special, convenient, fast, efficient, batch carry out IMN patients serum's monitoring, for IMN diagnosis, state of illness monitoring, therapeutic evaluation and prognosis evaluation.Thus promote IMN clinical practice.
Accompanying drawing explanation
Fig. 1 is idiopathic membranous nephropathy ELISA detection kit schematic diagram.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art are obtained under not making creative work premise, broadly fall into the scope of protection of the invention.
Embodiment 1
Referring to Fig. 1, in the embodiment of the present invention, idiopathic membranous nephropathy ELISA detection kit, including coated elisa plate, ELIAS secondary antibody, standard anti-PLA2R and anti-THSD7A serum, cleaning mixture, substrate TMB and stop buffer, ELIAS secondary antibody body is biotin labeled anti-igg 4 antibody.
The detection method of idiopathic membranous nephropathy ELISA detection kit, comprises the following steps:
(1) PLA is prepared2R and THSD7A albumen: extract glomerule total serum IgE, reverse transcription synthesis CDNA from people's nephridial tissue, separately design PLA2The primer of R and THSD7A gene carries out PCR amplification, then is connected with eucaryon plasmid carrier respectively, transfects HEK293T cell, cultivates after 48h, and centrifuging and taking supernatant extracts albumen, utilizes BCA method to measure protein concentration, then purifies PLA with affinity chromatography2R and THSD7A albumen.
(2) ELISA kit is prepared: with the PLA of purification2R and THSD7A albumen is coated microwell plate, makes coated elisa plate, is sequentially added into standard anti-PLA of concentration known in the micropore of envelope antigen2R and anti-THSD7A serum and the measuring samples of unknown concentration, with the rearmounted 37 DEG C of incubations of shrouding film shrouding；After incubation, add biotin labeled anti-igg 4 antibody, then be combined with Streptavidin-HRP, form immune complex.Adding substrate TMB colour developing, 37 DEG C of lucifuge colour developings after utilizing cleaning mixture thoroughly to wash, be eventually adding stop buffer and terminate reaction, now reagent is transferred to yellow by blueness.Anti-PLA in the depth of color and sample2R or anti-THSD7A content is proportionate.
Under 450nm wavelength, measure absorbance by microplate reader, calculate anti-PLA in sample by standard curve2R or anti-THSD7A content, thus carry out the diagnosis of IMN, state of illness monitoring, therapeutic evaluation and prognosis evaluation.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, and in the case of without departing from the spirit or essential attributes of the present invention, it is possible to realize the present invention in other specific forms.Therefore, no matter from the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is limited by claims rather than described above, it is intended that all changes fallen in the implication of equivalency and scope of claim included in the present invention.
In addition, it is to be understood that, although this specification is been described by according to embodiment, but the most each embodiment only comprises an independent technical scheme, this narrating mode of description is only for clarity sake, description should can also be formed, through appropriately combined, other embodiments that it will be appreciated by those skilled in the art that as an entirety, the technical scheme in each embodiment by those skilled in the art.
Claims (2)
1. an idiopathic membranous nephropathy ELISA detection kit, including coated elisa plate, ELIAS secondary antibody, standard anti-PLA2R and anti-THSD7A serum, cleaning mixture, substrate TMB and stop buffer, it is characterised in that ELIAS secondary antibody is anti-igg 4 antibody, and coated elisa plate contains PLA2The solid phase antigen of R and THSD7A albumen.
2. the detection method of an idiopathic membranous nephropathy ELISA detection kit, it is characterised in that comprise the following steps:
(1) PLA is prepared2R and THSD7A albumen: extract glomerule total serum IgE, reverse transcription synthesis cDNA, separately design PLA2The primer of R and THSD7A gene carries out PCR amplification, then is connected with eucaryon plasmid carrier respectively, transfects HEK293T cell, and after cultivating 48h, centrifuging and taking supernatant extracts albumen, then purification PLA2R and THSD7A albumen；
(2) sample-adding: with the PLA of purification2R and THSD7A albumen is coated microwell plate, makes coated elisa plate, is sequentially added into standard anti-PLA of concentration known in coated elisa plate2R and anti-THSD7A serum and the measuring samples of unknown concentration；
(3) incubation, addition ELIAS secondary antibody: after incubation, add ELIAS secondary antibody, then be combined with Streptavidin-HRP, form immune complex；
(4) wash, develop the color: wash with cleaning mixture, add substrate TMB colour developing, add stop buffer and terminate reaction；
(5) measure and result judges.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106885901A (en) * | 2017-01-22 | 2017-06-23 | 上海埃文生物科技有限公司 | Idiopathic membranous nephropathy duplex detection kit |
| CN107663235A (en) * | 2017-08-11 | 2018-02-06 | 南京诺唯赞医疗科技有限公司 | The related PLA2R recombinant proteins of idiopathic membranous nephropathy and its application |
| CN108414741A (en) * | 2018-01-19 | 2018-08-17 | 苏庆宁 | A kind of purposes of immune composition in preparing membranous nephropathy Non-invasive detection reagent |
| CN108414478A (en) * | 2018-01-19 | 2018-08-17 | 祝胜郎 | A kind of detection method of biological sample |
| CN109459572A (en) * | 2018-12-21 | 2019-03-12 | 江南大学 | A kind of primary membranous nephropathy diagnostic kit |
| CN111239417A (en) * | 2020-02-21 | 2020-06-05 | 深圳市伯劳特生物制品有限公司 | Coating buffer solution, kit and application |
| CN112557670A (en) * | 2020-12-07 | 2021-03-26 | 东南大学 | Method and kit for detecting IMN based on urine exosome PLA2R and application of method and kit |
| CN113447650A (en) * | 2021-07-01 | 2021-09-28 | 浙江大学医学院附属儿童医院 | Detection kit for anti-peptidyl prolyl cis-trans isomerase D-IgG antibody |
| CN118063606A (en) * | 2024-04-24 | 2024-05-24 | 江西赛基生物技术有限公司 | Anti-THSD 7A monoclonal antibody, and preparation method and application thereof |
-
2016
- 2016-03-31 CN CN201610201970.7A patent/CN105988011A/en active Pending
Non-Patent Citations (1)
| Title |
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| 李佳宁: "血清抗M型磷脂酶A2受体（PLA2R）抗体阴性的特发性膜性肾病患者的诊断研究", 《中国博士学位论文全文数据库（电子期刊）》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106885901A (en) * | 2017-01-22 | 2017-06-23 | 上海埃文生物科技有限公司 | Idiopathic membranous nephropathy duplex detection kit |
| CN107663235A (en) * | 2017-08-11 | 2018-02-06 | 南京诺唯赞医疗科技有限公司 | The related PLA2R recombinant proteins of idiopathic membranous nephropathy and its application |
| CN108414741A (en) * | 2018-01-19 | 2018-08-17 | 苏庆宁 | A kind of purposes of immune composition in preparing membranous nephropathy Non-invasive detection reagent |
| CN108414478A (en) * | 2018-01-19 | 2018-08-17 | 祝胜郎 | A kind of detection method of biological sample |
| CN109459572A (en) * | 2018-12-21 | 2019-03-12 | 江南大学 | A kind of primary membranous nephropathy diagnostic kit |
| CN111239417A (en) * | 2020-02-21 | 2020-06-05 | 深圳市伯劳特生物制品有限公司 | Coating buffer solution, kit and application |
| CN112557670A (en) * | 2020-12-07 | 2021-03-26 | 东南大学 | Method and kit for detecting IMN based on urine exosome PLA2R and application of method and kit |
| CN113447650A (en) * | 2021-07-01 | 2021-09-28 | 浙江大学医学院附属儿童医院 | Detection kit for anti-peptidyl prolyl cis-trans isomerase D-IgG antibody |
| CN113447650B (en) * | 2021-07-01 | 2022-05-13 | 浙江大学 | Detection kit for anti-peptidyl prolyl cis-trans isomerase D-IgG antibody |
| CN118063606A (en) * | 2024-04-24 | 2024-05-24 | 江西赛基生物技术有限公司 | Anti-THSD 7A monoclonal antibody, and preparation method and application thereof |
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