CN109239350A - Body fluid AGEs autoantibody is preparing the application in diabetogenous nephrosis disease early diagnosis reagent - Google Patents
Body fluid AGEs autoantibody is preparing the application in diabetogenous nephrosis disease early diagnosis reagent Download PDFInfo
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Abstract
The invention discloses body fluid AGEs autoantibodies to prepare the application in diabetogenous nephrosis disease early diagnosis reagent.AGEs autoantibody is preparing the application in diabetic nephropathy patients undergoing early diagnosis reagent as biomarker in body fluid.A kind of early diagnosis reagent of the diabetic nephropathy based on ELISA double antibody sandwich method, the anti-human IgG antibodies comprising AGEs antigen and peroxidase labelling.Provided by the present invention for the early diagnosis reagent of the diabetic nephropathy of double antibody sandwich method measurement body fluid AGEs autoantibody, it can be used on the detection devices such as semi- or fully automated spectrophotometer, and detection sensitivity is high, it is specific high, it is easy to operate, available practical popularization and use, will make it possible in time diagnose and tracking diabetic nephropathy generation and process, significantly improving at present can not prejudge diabetes to diabetic nephropathy, the awkward situation of early diagnosis and timing tracking has great economic results in society.
Description
Technical field
The invention belongs to biological medicine detection technique fields, are related to body fluid AGEs autoantibody and are preparing diabetic nephropathy morning
Application in phase diagnostic reagent, and in particular to one kind is using body fluid AGEs autoantibody as biomarker in diabetic nephropathy disease
Application in people's early diagnosis.
Background technique
Diabetic nephropathy (diabeticnephropathy, DN) is the main microvascular complication and terminal of diabetes
The most commonly encountered diseases of phase nephrosis because and one of the independent hazard factor of cardiovascular event.The case fatality rate of the concurrent DN of diabetic is not
30 times of concurrent DN person.The incidence of diabetic nephrosis and the course of disease of diabetes are related.Type 1 diabetes patient's course of disease < 5
Year person's DN incidence is lower, but 20~25 years person's incidences of the course of disease are 40%~50%.The diabetes B patient course of disease < 5 year,
DN illness rate be 7%~10%, course of disease 20-25, illness rate 20%~35%, the course of disease > 25 year, illness rate up to 50% with
On.In the U.S., the end stage renal failure as caused by DN (ESRD) has become the first cause of ESRD close to 50%, DN.And it is sub-
Continent end stage renal failure as caused by DN is also rising year by year.The situation of China is also pessimistic, and DN is the second master for causing ESRD
Cause is only second to ephritis, but the ratio is rising year by year, and is primarily due to China's diabetes prevalence and constantly rises, and
Cause diabetes complicated to the factor of diabetic nephropathy, mechanism is unclear, while lack can be used for early diagnosing it and in real time with
The specific biological marker of track, awareness are lower.
Kidney damage once occurs for diabetic, and continuous proteinuria occur, then the state of an illness is often irreversible, often develops
To end stage renal failure.Therefore, seeking early diagnosis and effective treatment is the problem of clinical and basic research is paid close attention to jointly.Root
According to the course of disease and pathologic, physiologic evolution process of DN, DN can be divided into 5 phases.The I phase: the filtration of glomerulus height and renal hypertrophy phase, kidney
Glomerular filtration rate (GFR) is higher than normal 25%~40%, and Kidney Volume increases about 20%, this phase is consistent with elevated blood glucose levels,
There is no micropathological damage, available part is alleviated after glycemic control.II phase: microalbuminuria phase after movement, Urine proteins row
Rate is let out normally or after movement to increase.Can there are basement membrane thickened and mesentery expansion.III phase: duration microalbuminuria phase, this phase
GFR remains to remain normal, and lesion is still invertibity, if not active intervention, the Most patients state of an illness is gradually in progress.IV phase: clinical
Diabetogenous nephrosis stadium or albuminuria phase, GFR decline more, there is typical DN pathological change.V phase: renal failure stage, pathology
For Nephrosclerosis.Since diabetic nephropathy early stage can be asymptomatic, most diabetic nephropathy early stage patients do not go to go to a doctor, when there is disease
Irreversible diabetic nephropathy middle and advanced stage is often had developed to when shape and loses the possibility of healing.
The appearance of urinary albumin is clinically earliest detectable stage to DN, once there is clinical proteinuria, renal function
It will decline in progressive.Result of study shows that DN early stage receives the type 2 diabetic patient treated from the normal or microalbuminuria stage
The ratio for being transitioned into the albuminuria stage substantially reduces, therefore finds microalbuminuria as early as possible, is effectively treated, Ke Yiyan
Slow or reverse disease, avoids the occurrence and development of clinical DN.Microdose urine protein is currently used diagnosis early stage DN for 24 hours for measurement
Method, domestic and international numerous studies confirm that microdose urine protein for 24 hours can reflect the slight damage of glomerulus early stage, are the current country
A reliable index of sensitivity of generally acknowledged diagnosis early stage DN outside.But studies have found that, part of diabetes mellitus patient's urinary albumin
The change of renal function has occurred in normal range (NR) for level.Therefore Urine proteins are higher as marker rate of missed diagnosis.
In addition, finding in recent years, since diabetic is during renal function early lesion, there is the spy in some urine
Kind albumen, such as retinal binding protein (RBP), urine Uromucoid (THP), transferrins (TRF) are before
Toward in significantly increasing, although absolute content of these protein in urine is very low, and most (except urinary albumins) is i.e.
Make the advanced stage to disease that can not constitute significantly increasing for urine total protein, therefore measure special proteins in above-mentioned urine, to early stage glycosuria
The diagnosis of sick nephrosis has certain help, but these markers to the prediction sensitivity and specificity of diabetic nephropathy relatively
Difference, clinical degree of recognition and utilization rate are lower.
Therefore, for DN as diabetic's one of the most common type chronic complicating diseases, early stage correct diagnosis and therapeutic intervention are outstanding
It is important.Although there are many DN sensitive index, it is early that Linchuan still lacks effective, non-invasive, easy-to-use DN
Phase inspection method helps patient to early diagnose, and prevents the occurrence and development of DN, reduces the generation of end-stage renal disease.Therefore DN is found
Early diagnosis marker will not only mitigate the pain of patient and the financial burden of society, and more theories can be also provided for clinic
Foundation.
Advanced glycation end products (glycation end products, AGEs) are a kind of substances of typical isomerism,
Glucose can chemically be reacted by Schiff base under the conditions of non-enzymatic, be covalently attached to the amino to dissociate in protein
In group, stable ketoamine key conjugate, i.e. Amodori product are then formed through isomerization.The latter again through it is a series of dehydration, it is dense
Contracting, cracking, oxidation, cyclization, make protein that intramolecular and intermolecular crosslinking occur, form a kind of molecule that complexity is stable
Structure, i.e. AGEs.AGEs be in height heterogeneity, mainly include carboxymethyl-lysine (CML), pentosidine (Pentosidine),
Hydroxymethyl lysine (CEL) etc..Recently as AGEs, AGEs, mechanism of action is ground in the diabetic nephropathy Development process
Study carefully progress, discovery DN patient's Renal biospy mesangial region, Renal vascular wall, renal tubule wall, renal interstitial AGEs dyeing are obvious, and DN pathology damages
Evil is heavier, and dyeing is deeper, shows that AGEs may play an important role in the development process of diabetic nephropathy.Although existing
Research shows that AGEs is related to diabetic nephropathy, but its molecular mechanism not yet illustrates completely.In addition, there is hyperglycemia
In the case where, the diabetic why having progresses to diabetic nephropathy, and some diabetics do not progress to glycosuria all the life
The reason of sick nephrosis, is unclear.
In view of the foregoing, for inventor first from the pathogenic mechanism of AGEs, disclosing AGEs under certain condition can quilt
Antigen presenting cell phagocytosis, is handled and present antigen is to T cell and B cell, finally generates the autoantibody for being directed to AGEs.Due to
Autoantibody can be attacked with mediate complement system and T cell molecule and cell to expression antigen, for AGEs itself
Antibody identifies Renal Podocytes, the AGEs of basilar memebrane and mesangial cell surface leads to diabetic nephropathy so as to cause injury of kidney.
Our revealed AGEs pathogenesis are as shown in Figure 1.On this basis, the present invention, which is ground, shows Diabetic Nephropathy patients and model
An important factor for autoantibody in the serum of mouse for sertoli cell and basilar memebrane is not only diabetic nephropathy occurrence and development, together
When also can be used as early diagnosis diabetes de-velopment to diabetic nephropathy biomarker.
Summary of the invention
The object of the present invention is to provide a kind of body fluid AGEs autoantibodies in preparation early diagnosis diabetic nephropathy reagent
Application.
The purpose of the present invention can be achieved through the following technical solutions:
AGEs autoantibody is as biomarker in preparing diabetic nephropathy patients undergoing early diagnosis reagent in body fluid
Using.Body fluid AGEs autoantibody is preparing the application in diabetic nephropathy patients undergoing early diagnosis reagent as detection target spot.
The preferred serum of the body fluid, blood plasma or whole blood.
The diagnostic reagent is preferably based on the diagnostic reagent of ELISA double antibody sandwich method or based on immunochromatography double antibody
The diagnostic reagent of sandwich method.
A kind of early diagnosis reagent of the diabetic nephropathy based on ELISA double antibody sandwich method, it is characterised in that include
The anti-human IgG antibodies of AGEs antigen and peroxidase labelling.
A kind of diabetogenous nephrosis disease early diagnosis immune chromatography test paper of AGEs autoantibody in serum, it is characterised in that test paper
Detect the body fluid AGEs autoantibody with coating colloid gold label.
The utility model has the advantages that
DN is the common important complication of diabetes, is that diabetes are lethal, the main reason for disabling.Not in time such as diagnosis and treatment,
It, can only be using dialysis even kidney transplant when DN develops to end-stage of renal disease.The pathogenesis of DN is still unclear at present, but more next
More research evidences shows that AGEs plays an important role in the development process of DN.Present invention research shows first
There are AGEs and its principal mode CML (Fig. 2) for glomerular podocyte, mesentery and the basilar memebrane of diabetic nephropathy early stage patient.Body
Outside experiments have shown that glomerular podocyte has AGEs processing and submission function (Fig. 3).On this basis, inventor's discovery is in glycosuria
There is the autoantibody (Fig. 4) for being directed to AGEs in the serum of sick nephrosis early stage patient.Further, inventor takes Diabetic nephropathy
Early stage patient serum marker nephridial tissue finds there is the AGEs itself that specificity is directed to sertoli cell, mesentery and basilar memebrane in serum
Antibody (Fig. 5).In addition, the present invention is further found that this kind of autoantibody in serum can be used as biomarker to glycosuria
Sick nephrosis carries out early diagnosis and timing tracking (Fig. 6).
Therefore, provided by the present invention for the morning of the diabetic nephropathy of double antibody sandwich method measurement body fluid AGEs autoantibody
Phase diagnostic reagent can use on the detection devices such as semi- or fully automated spectrophotometer, and detection sensitivity is high,
It is specific high, easy to operate, available practical popularization and use, will make to diagnose in time and the generation of tracking diabetic nephropathy and
Process is possibly realized, and significantly improving at present can not prejudge diabetes to diabetic nephropathy, early diagnosis and timing
The awkward situation of tracking has great economic results in society.
Detailed description of the invention
Fig. 1 present invention discover that AGEs pathogenesis
Fig. 2 Immunofluorescence test AGEs and its be primarily present form A ML be present in diabetic nephropathy early stage patient kidney it is small
Ball sertoli cell, mesentery and basilar memebrane.
Fig. 3 Laser Scanning Confocal Microscope testing result.
There is the autoantibody for AGEs in the serum of Fig. 4 diabetic nephropathy early stage patient.
There is specificity during Fig. 5 Immunofluorescence test Diabetic Nephropathy Patients are clear and is directed to sertoli cell, mesentery and basilar memebrane
AGEs autoantibody.
Fig. 6 .ELISA detects the content of diabetic and Diabetic Nephropathy Patients AGEs antibody middle clearly.
Specific embodiment
Embodiment 1
The nephridial tissue slice for collecting diabetic nephropathy early stage patient, passes through the anti-AGEs antibody and anti-CML bought
Antibody marks nephridial tissue to carry out Immunofluorescence test AGEs and its be primarily present form A ML in diabetic nephropathy early stage patient respectively
Glomerular podocyte, build up at mesentery and basilar memebrane.Experimental result (Fig. 2) display, in the kidney of diabetic nephropathy early stage patient
Bead sertoli cell, mesentery and basilar memebrane are there are AGEs and its are primarily present form A ML.
Embodiment 2
DQ-OVA is made of BODIPY FL and OVA height conjugation, this structure leads to Fluorescence self-quenching.In DQ-OVA egg
After plain boiled water solution, the dyestuff of peptide fragment is marked just to be able to observe green fluorescence.Sertoli cell is detected so that DQ-OVA is additional antigen
Antigen processing capabilities.After differentiation is cultivated 10 days in 37 DEG C of incubators by the sertoli cell system (HPC) of source of people, DQ-OVA is added to just
In normal culture medium or culture medium containing high sugared (40mM glucose).The HPC of this method processing, which is placed in 37 DEG C of incubators, to be continued
Skeleton dyeing is carried out with 594 phalloidine of Alexa Fluor after culture 4h, is then examined using laser confocal microscope
Green fluorescence is surveyed to react sertoli cell to the working process ability of OVA.It is shown as shown in figure 3, the high processed sertoli cell of sugar has
The antigen processing capabilities of work, after this shows high glucose induction Podocytes in Renal Tissue, can promote sertoli cell phagocytosis exotic antigen and to its into
Row processing, under the conditions of prompting diabetic nephropathy, sertoli cell may have the energy that processing, present antigen and induction autoantibody generate
Power.
Embodiment 3
It is the preparation of HSA-AGEs first, carries out by the following method:
(1) be formulated as follows reagent: the 1M sodium phosphate buffer (pH 7.4) of sterilizing, 10M NaOH solution and 30%HSA are molten
Liquid.
(2) it is molten in 20ml 1M sodium phosphate buffer vortex in sterile 50ml centrifuge tube, is added to weigh 4.51g glucose
Then solution is added the HSA solution (human serum albumin solution) of 8.3ml 30%, 21ml ddH is added after mild mixing2O。HSA
Control group is free of glucose, other compositions are as processing.
(3) two groups of solution of HSA-AGEs group and HSA group are respectively with the flask for going to sterilizing after 0.22 μm of filter filtering
In, it is put into 37 ° of incubators and is protected from light incubation 3 months or more.
(4) pH is adjusted with 10M NaOH solution weekly, stablizes pH 7.4, this pays attention to sterile in the process.
(5) it is incubated for the dialysis of each pipe solution terminated, to remove unreacted glucose and HAS.Dialysis process is as follows: choosing
The bag filter for filling solution is placed in the solution of the 0.01M PBS of its big 10 times of volumes, makes it not by the bag filter for selecting 3.5KD
Disconnected to carry out dialysis displacement (guaranteeing solution flowing), every 4 hours replacement dialyzates terminate dialysis after 48 hours.The HSA- to have dialysed
AGEs solution is used for subsequent experimental.
It is early with Western blotting technology detection diabetes, Diabetic nephropathy using the HSA-AGEs prepared as antigen
Whether there is or not the antibody for being directed to AGEs in phase and diabetic nephropathy middle and advanced stage patients serum, as a result, it has been found that diabetic nephropathy early stage (DN1)
With the diabetic that there is the antibody for being directed to HSA-AGEs in the serum of middle and advanced stage patient (DN2 and DN3) and be not suffering from nephrosis
(DM) there is no the antibody (Fig. 4) for HSA-AGEs in serum, the antibody for HSA-AGEs is prompted to can be used as diabetic nephropathy
Early diagnosis marker.
Embodiment 4
The nephridial tissue slice and serum for collecting diabetic nephropathy early stage patient, with Diabetic nephropathy early stage patient serum marker kidney
Tissue carries out the spontaneous antibody that whether there is specificity to be directed to kidney in Immunofluorescence test serum.As a result (Fig. 5) is shown, glycosuria
There is the AGEs autoantibody that specificity is directed to sertoli cell, mesentery and basilar memebrane in the serum of sick nephrosis early stage patient.
Embodiment 5
Using the AGEs antibody design double antibodies sandwich in AGE and Diabetes Mellitus, ELISA detect diabetic and
The content of Diabetic Nephropathy Patients AGEs antibody middle clearly.As a result, it has been found that the content of Diabetic Nephropathy Patients AGEs antibody middle clearly
Higher than the content (Fig. 6) of AGEs antibody in the non-nephrotic's serum of patient of diabetes, the AGEs autoantibody in this prompt serum can
To carry out early diagnosis and timing tracking to diabetic nephropathy as biomarker.
Embodiment 6
Using immunochromatography double antibody sandwich method, it is developed based on the diabetic nephropathy early stage of AGEs autoantibody in serum
Diagnose test paper.Test paper AGEs of the detection with coating colloid gold label, control band are the coated control antibodies of colloidal gold.We pass through
The non-nephrotic of diabetes and Diabetic Nephropathy Patients are detected clearly, discovery due to Diabetic Nephropathy Patients it is clear in contain
There is the autoantibody for AGEs, when serum sample siphon passes through colloid gold label AGEs, makees for the autoantibody of AGEs
Antigen-antibody colloidal gold composite can be formed with the antibody on test paper for antigen, compound continues to creep, and passes through coated second
When antibody, double-antibody sandwich colloidal gold composite is formed, red is presented at coating line, excessive colloidal gold antibody continues to climb
Capable and control line forms colloid gold immune compound, and control band presents red;And due to not in the non-nephrotic's serum of diabetes
Containing the autoantibody for being directed to AGEs, red is not presented when serum sample detects band by test paper, only control band is presented red
Color.
Claims (5)
1. body fluid AGEs autoantibody is preparing the application in diabetic nephropathy patients undergoing early diagnosis reagent as biomarker.
2. application according to claim 1, it is characterised in that the body fluid is serum or blood plasma.
3. application according to claim 1, it is characterised in that the diagnostic reagent is based on ELISA double antibody sandwich method
Diagnostic reagent or diagnostic reagent based on immunochromatography double antibody sandwich method.
4. a kind of diabetogenous nephrosis disease early diagnosis reagent based on ELISA double antibody sandwich method, it is characterised in that anti-comprising AGEs
Former and peroxidase labelling anti-human IgG antibodies.
5. the diabetogenous nephrosis disease early diagnosis immune chromatography test paper of AGEs autoantibody in a kind of serum, it is characterised in that test paper inspection
The body fluid AGEs autoantibody of measuring tape coating colloid gold label.
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Citations (3)
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JP2001004627A (en) * | 1999-04-19 | 2001-01-12 | Nisshin Flour Milling Co Ltd | Immunological detection method for autoantibody against maillard reaction latter-period product |
CN1327159A (en) * | 2000-06-05 | 2001-12-19 | 刘永详 | Immune analysis method, reagent and device for detecting saccharified protein |
CN105189778A (en) * | 2012-10-30 | 2015-12-23 | 香港中文大学 | Novel biomarkers for diabetic kidney diseases and use thereof |
-
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- 2018-08-06 CN CN201810885927.6A patent/CN109239350A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001004627A (en) * | 1999-04-19 | 2001-01-12 | Nisshin Flour Milling Co Ltd | Immunological detection method for autoantibody against maillard reaction latter-period product |
CN1327159A (en) * | 2000-06-05 | 2001-12-19 | 刘永详 | Immune analysis method, reagent and device for detecting saccharified protein |
CN105189778A (en) * | 2012-10-30 | 2015-12-23 | 香港中文大学 | Novel biomarkers for diabetic kidney diseases and use thereof |
Non-Patent Citations (3)
Title |
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JALALUDDIN MOHD.ASHRAF, ET AL.: "Recent Advances in Detection of AGEs: Immunochemical, Bioanalytical and Biochemical Approaches.", 《IUBMB LIFE》 * |
NORIE ARAKI, ET AL.: "Study of autoantibodies against advanced glycation endproducts of the Maillard reaction.", 《INTERNATIONAL CONGRESS SERIES》 * |
RIE SHIBAYAMA, ET AL.: "Autoantibody Against Nε-(Carboxymethyl) lysine:An Advanced Glycation End Product of the Maillard Reaction.", 《DIABETES》 * |
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