CN108414478A - A kind of detection method of biological sample - Google Patents
A kind of detection method of biological sample Download PDFInfo
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- CN108414478A CN108414478A CN201810055545.0A CN201810055545A CN108414478A CN 108414478 A CN108414478 A CN 108414478A CN 201810055545 A CN201810055545 A CN 201810055545A CN 108414478 A CN108414478 A CN 108414478A
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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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 present invention provides a kind of detection methods of biological sample, include the following steps：(1) it is detached to acquiring from the biological sample of subject, obtains sample to be tested；(2) one or more in detection IgG4, PLA2R, Nephrin and NPHS2 in the sample to be tested, testing result is as the preliminary foundation for judging whether the subject needs progress Renal biospy.Detection method provided by the invention can carry out membranous nephropathy primary screening, and whether carrying out Renal biospy puncture for the later stage provides reference data；And the detection method is non-invasive, easy to operate and testing cost is relatively low.
Description
Technical field
The present invention relates to field of medical examination, and in particular to a kind of detection method of biological sample.
Background technology
China's chronic renal disease (CKD) illness rate is 10.8%, and membranous nephropathy (MN) is with glomerular basement membrane epithelium
One group of disease that immune complex deposit is characterized with basement membrane thickened under cell, is the common cause of adult nephrotic syndrome.
Membranous nephropathy accounts for about 6.0%-28.8% in primary glomerulopathy, becomes the primary glomerular disease after IgA nephrosis
Second cause of disease of disease, while being also the Important cause of disease for leading to end-stage renal disease (ESRD).Nanfang Hospital Hou Fanfan in 2016
One broad scale research of academician team shows that membranous nephropathy ratio is only second to IgA nephrosis (28.1%) up to 23.4%.
Whether clear according to pathogenic factor, membranous nephropathy is divided into spontaneous membranous nephropathy (IMN) and secondary membranous nephropathy
(SMN).IMN is the primary membranous nephropathy of the reasons such as removal system lupus erythematosus, infection, tumour and drug poisoning, is accounted for
75%-80%.Spontaneous membranous nephropathy (IMN) has with secondary membranous nephropathy (SMN) in the cause of disease, treatment and prognosis etc.
A great difference, thus it is particularly important to the early diagnosis and differential diagnosis of IMN and SMN.
Current clinically used kidney trouble detection method is direct progress biopsy, i.e. renal fibroblast inspection, film property kidney
The detect and diagnose of disease also relies on this inspection.However, renal fibroblast inspection is that one more complicated, technology content is higher
Inspection method is generally required in three-level to go to the hospital and its cooperation of coherent detection mechanism carries out, and popularization is poor.In addition, kidney is worn
Thorn is a kind of invasive detection means, detection there are risk, subject by while pain on body it is also possible to causing
Internal haemorrhage.Moreover, subject needs to be hospitalized overnight to complete to check, thus it is difficult to as clearly being treated during chronic disease therapy
Multiplicating detection methods needed for effect and prognosis.The expense for being additionally carried out renal fibroblast is also higher.In Chinese medical treatment money at present
Under the more deficient background in source, subject, which must not be not to wait for even longer time a couple of days, can just be scheduled for this inspection.
If in addition, biopsy the result is that patient and not suffering from nephrosis, patient is in the body brought in vain by puncture
Body pain and while undertake high costs, it is also possible to which the risk for undergoing internal haemorrhage causes secondary injury to body.This is to suffering from
Person, family members, hospital tripartite are potentially to bear.Therefore, to the doubtful patient with kidney trouble before carrying out renal fibroblast
Carry out that early period, noninvasive (Noninvasive), low expense, quickly reliable investigation is advantageous.
Have the study found that the basic cause of disease of spontaneous membranous nephropathy (IMN) is to be directed to M type phospholipase A2 receptors
(PLA2R) presence of autoantibody, the M type phospholipase A2 receptors are expressed in renal glomerulus.The blood of individual with IMN
This autoantibody containing detectable amount in clear.It, can be anti-using PLA2R according to the correlation of this autoantibody and IMN
Index of the Serologic detection of body as spontaneous membranous nephropathy diagnosis and prognosis.But clinical practice shows spontaneous film property
Nephrotic's blood-serum P LA2R antibody positive rates are only 68.5%, still there is 31.5% idiopathic membranous nephropathy patients serum PLA2R
Antibody is difficult to effectively detect for feminine gender.
Drawbacks described above based on the prior art, it is a kind of for the efficient, simple, non-intruding of membranous nephropathy there is an urgent need for developing
Detection method.
Invention content
The present inventor it has been investigated that, the autoantibody of the anti-PLA2R of membranous nephropathy patient's body is immunoglobulin man
Race (IgG).The present inventors have additionally discovered that in the blood testing of IMN patient, the Serological IgG level of IMN patient is compared with Healthy People
It is decreased obviously, about low IgG mass formed by blood stasis occurs for 65.75%IMN patient.And low IgG mass formed by blood stasis is not the specificity index of IMN, clinically
About 56.47% low IgG mass formed by blood stasis patient is diagnosed as IMN, and about 45.53% low IgG mass formed by blood stasis patient is diagnosed as minute lesion
(MCD).Therefore, the blood testing of current membranous nephropathy has limitation.
The present inventors have additionally discovered that PLA2R in the sertoli cell and cell fragment of the centrifugation sediment separation of membranous nephropathy Urine in Patients
And its antibody test is positive, and wherein the presence of IgG antibody and its content are related to membranous nephropathy.The present inventor sends out accordingly
It is bright go out a kind of non-invasive detection methods for membranous nephropathy Research of predicting markers, whether the testing result as judging the subject
Need the preliminary foundation of progress Renal biospy.Detection method provided by the invention can be as early period of existing Renal biospy method or auxiliary
Assistant section and/or provide reference data for Renal biospy.Certainly, under the premise of combining or not combining other factors, if clinical doctor
It is raw to think that the result of detection method is enough to determine that subject is based on the method or purposes with membranous nephropathy or subject
Result i.e. firmly believe its suffer from nephrosis, also directly can be treated or be remained to observe.
According to the present invention, a kind of detection method of biological sample is provided, is included the following steps：
(1) it is detached to acquiring from the biological sample of subject, obtains sample to be tested；With
(2) one or more in IgG4, PLA2R, Nephrin and NPHS2, detection knot is detected in the sample to be tested
Fruit is as the preliminary foundation for judging whether the subject needs progress Renal biospy.
Wherein, the biological sample is urine, and the sample to be tested is the urine precipitation that urine is obtained by centrifugal treating
Object, the sample to be tested include it is following in it is one or more：Cell, cell fragment and it is attached to cell or cell fragment
On immune complex.
Wherein, in step (2), detected in the sample to be tested by Immunofluorescence test IgG4, PLA2R,
It is one or more in Nephrin and NPHS2.
The Immunofluorescence test follows the steps below：
(A) it is contacted using immune composition as primary antibody with the biological sample, forms Antibody-antigen complex, washing removes
Remove unbonded primary antibody；
(B) secondary antibody that fluorescent marker is added is dyed, and washing removes unbonded secondary antibody；
(C) film-making, the position using fluorescence microscope fluorescent label signal and distribution,
Wherein, the immune composition in step (A) includes anti-IgG4 antibody, anti-PLA2R antibody, anti-Nephrin
It is one or more in antibody and anti-NPHS2 antibody.
The dyeing in above-mentioned steps (B) is independent dyeing, double staining, triple staining or its arbitrary combination.
Wherein, the independent dyeing includes individually carrying out immunofluorescence dyeing to IgG4 or PLA2R.
Wherein, the double staining includes carrying out immunofluorescence dyeing to two kinds of substances in following any combination：
Combine (1) IgG4 and PLA2R；
Combine (2) IgG4 and Nephrin；Or
Combine (3) IgG4 and NPHS2.
Wherein, the triple staining includes carrying out immunofluorescence dyeing to three kinds of substances in following any combination：
Combine (4) IgG4, PLA2R and Nephrin；Or
Combine (5) IgG4, PLA2R and NPHS2.
IgG4, PLA2R, Nephrin can also be detected in the sample to be tested by immune-blotting method in step (2)
With it is one or more in NPHS2.
The immune-blotting method be included in it is described acquisition from the biological sample of subject detect PLA2R presence and its
Molecular weight.
The detection method of biological sample provided by the invention has the advantages that：
(1) detection method is detected the immunological marker object in urine, obtains existing about immunological marker object, be distributed
Or the information of content, primary screening is carried out to membranous nephropathy using the above- mentioned information of immunological marker object, whether kidney is carried out for the later stage
Biopsy provides reference data.Certainly, this method does not directly obtain the diagnostic result whether subject suffers from membranous nephropathy,
The presence or absence of immunological marker object is to judge whether an intermediate result with membranous nephropathy, it is also necessary to carry out it is follow-up or its
He could make a definite diagnosis membranous nephropathy method；Certainly, under the premise of combining or not combining other factors, if clinician recognizes
For the method for the present invention or purposes credible result or assume patient adheres to being conclusion in this approach, also can directly carry out treatment or
It remains to observe.
(2) detection method is non-invasive, avoids causing unnecessary damage to subject, and sample it is easy,
Detection is quick and easy, expense is relatively low.
(3) detection method provided by the invention can carry out the normal of woundless testing as a kind of to idiopathic membranous nephropathy
Rule project receives renal needle biopsy for part refusal or has the patient of renal fibroblast contraindication or unconditional renal fibroblast of carrying out to live
The medical institutions of inspection provide a kind of reference detection method having much directive significance.
Description of the drawings
Fig. 1 shows the separation method flow chart according to the sample to be tested of one embodiment of the present invention.
Fig. 2 shows according to the separation of the sample to be tested of one embodiment of the present invention and detection method flow chart.
Fig. 3 shows the immunofluorescence test result of the membranous nephropathy patient according to one embodiment of the present invention, shows
The Immunofluorescent signals of IgG4 and NPHS2.
Fig. 4 shows the immunofluorescence test result of the membranous nephropathy patient according to one embodiment of the present invention, shows
The Immunofluorescent signals of PLA2R and Nephrin.
Fig. 5 shows the IgG4 Immunofluorescence test images of normal healthy subjects.
Fig. 6-7 shows the IgG4 Immunofluorescence test images of membranous nephropathy patient, wherein Fig. 6 is shown on intact cell
IgG4 signals, Fig. 7 show the IgG4 signals on cell fragment.
Fig. 8 shows the IgG4 Immunofluorescence test images of Focal segmental glomerulosclerosis (FSGS) patient.
Fig. 9 shows the IgG4 Immunofluorescence test images of diabetic nephropathy (DN) patient.
Figure 10 shows the IgG4 Immunofluorescence test images of minute nephropathy (MCD) patient.
Figure 11 shows the IgG4 Immunofluorescence test images of IgA nephrotics.
Figure 12 shows the Immunofluorescence test image of IgG4 and PLA2R double stainings in membranous nephropathy Urine in Patients.
Figure 13 shows the immunofluorescence of IgG4 and kidney sertoli cell marker NPHS2 double stainings in membranous nephropathy Urine in Patients
Detection image.
Figure 14 shows that PLA2R's and kidney sertoli cell marker Nephrin double stainings in membranous nephropathy Urine in Patients is immune
Fluoroscopic image.
Figure 15 shows the Western Blot detection knots of membranous nephropathy patient, normal subjects and other nephrotic's urines
Fruit.
Specific implementation mode
Below by the drawings and specific embodiments, the present invention is described in more detail.Pass through these explanations, the present invention
The characteristics of and advantage will become more apparent from it is clear.
Term used in the context of the invention " positive " refers to the detection signal that can be detected.
It may refer to " in one embodiment " and " in another embodiment " used in the context of the invention
Identical embodiment can also refer to different embodiments.
The present invention provides a kind of inspection for membranous nephropathy biomarker in subject's urine cell and cell fragment etc.
Survey method, testing result class can be as the reference indexs for judging whether subject needs further progress Renal biospy.This hair
It bright preliminary discriminating for membranous nephropathy and examines more afterwards and provides a kind of quick and noninvasive detection method, and is long for chronic kidney disease
Curative effect observation needed for phase treatment provides detectable means at any time, has significant clinical practical operation value.Testing result also has
There is high reference value.But it may be noted that the immunological marker object that the present invention detects is not or is not necessarily with membranous nephropathy
Ultimate diagnosis index, and only intermediate result.
According to the present invention, a kind of detection method of biological sample is provided, is included the following steps：
(1) it is detached to acquiring from the biological sample of subject, obtains sample to be tested；With
(2) one or more in IgG4, PLA2R, Nephrin and NPHS2, detection knot is detected in the sample to be tested
Fruit is as the preliminary foundation for judging whether the subject needs progress Renal biospy.
In a kind of embodiment according to the present invention, by Immunofluorescence test in the sample to be tested in step (2)
It is one or more in middle detection IgG4, PLA2R, Nephrin and NPHS2.
The Immunofluorescence test follows the steps below：
(A) it is contacted from the biological sample of subject using immune composition as primary antibody with the acquisition, forms antibody-antigene
Compound, washing remove unbonded primary antibody；
(B) secondary antibody that fluorescent marker is added is dyed, and washing removes unbonded secondary antibody；
(C) film-making, the position using fluorescence microscope fluorescent label signal and distribution.
Wherein, the immune composition in step (A) includes anti-IgG4 antibody, anti-PLA2R antibody, anti-Nephrin
It is one or more in antibody and anti-NPHS2 antibody.
According to the present invention, the immune composition can be with IgG4, PLA2R, kidney sertoli cell marker Nephrin and kidney foot
One or more specific bindings in cell sign object NPHS2 generate immune complex, to further pass through immunofluorescence
Detection or immune-blotting method detect IgG4, PLA2R, kidney sertoli cell marker Nephrin and kidney sertoli cell marker NPHS2
In one or more presence and distribution.
In one embodiment, the dyeing in above-mentioned steps (B) is independent dyeing, double staining, triple staining
Or its arbitrary combination.
Wherein, the independent dyeing includes individually carrying out immunofluorescence dyeing to IgG4 or PLA2R.
Wherein, the double staining includes carrying out immunofluorescence dyeing to two kinds of substances in following any combination：
Combine (1) IgG4 and PLA2R；
Combine (2) IgG4 and Nephrin；Or
Combine (3) IgG4 and NPHS2.
Wherein, the triple staining includes carrying out immunofluorescence dyeing to three kinds of substances in following any combination：
Combine (4) IgG4, PLA2R and Nephrin；Or
Combine (5) IgG4, PLA2R and NPHS2.
It, can also be by immune-blotting method described in step (2) in another embodiment according to the present invention
It is detected in sample to be tested one or more in IgG4, PLA2R, Nephrin and NPHS2.The immune-blotting method is included in
The acquisition detects presence and its molecular weight of PLA2R from the biological sample of subject.
The separation of sample to be tested
Acquisition of the present invention is preferably urine from the biological sample of subject, can pass through any side known in the art
Formula is acquired.
Subject of the present invention is mammal.In one embodiment, the subject is the mankind.In another kind
In embodiment, the subject is nephrotic.In another embodiment, the subject is to show as hypoproteinemia
The nephrotic of disease.In another embodiment, the subject is the doubtful human experimenter with membranous nephropathy.
The present invention provides a kind of method detached to cell in urine and cell fragment.This method is by being collected by centrifugation
Cell in urine and cell fragment, thus successfully by cell or the adhesion immune complex on cell fragment surface and generally
Dissolubility protein molecular is detached, and technical support is provided for subsequent detecting step.
This method can exclude to interfere sex chromosome mosaicism present in immune complex detection.This method has sample to be tested
Effect concentration also substantially increases the sensitivity of detection while removal soluble chaff interferent, and make to be difficult to detect originally low contains
Amount ingredient can detected easily.
In a kind of embodiment according to the present invention, the sediment that urine sample is obtained through centrifugal sedimentation carries out coverslip
Or glass slide smear, or carry out smear again after fixation, for detection, the detection includes but not limited to immunofluorescence
It is one or more in detection, immunochemistry detection, biochemistry detection.In one embodiment, the urine sample is 10-
40ml.In one embodiment, the urine sample preferably centrifuges 5-20 minutes by 1000-4000 revs/min of centrifugation,
More preferably centrifugation 10 minutes.
In one embodiment, centrifugal sedimentation can be substituted with the mode of multiple rejection tablet.
In one embodiment, the sediment passes through further centrifuge washing to remove the soluble egg in urine
In vain, then by the sediment after washing dissolving is resuspended for use in detection.
In one embodiment, the sediment is fixed by suspending, and preferably carries out suspension fixation with paraformaldehyde.
In a kind of embodiment, 30 minutes are fixed using the 4% paraformaldehyde suspension of 0.5ml.
In one embodiment, treated sample to be tested drop will be passed through on coverslip or glass slide, or
Sample to be tested is got rid of on the cover slip and dried with rejection tablet machine.
The sample to be tested include it is following in it is one or more：Cell, cell fragment and it is attached to cell or cell
Immune complex on fragment.
The detection of sample to be tested
In a kind of embodiment according to the present invention, the one or more of anti-PLA2R are detected in the sample to be tested
Autoantibody.One or more autoantibodies of the anti-PLA2R are one or more in immunoglobulin class.One
In kind embodiment, the immunoglobulin is IgG4.
In a kind of embodiment according to the present invention, the detection method further includes being detected in the sample to be tested
PLA2R receptors.
In a kind of embodiment according to the present invention, the detection method further includes being detected in the sample to be tested
IgG4-PLA2R immune complexs.
In a kind of embodiment according to the present invention, the detection method further includes detecting one in the sample to be tested
Kind or a variety of kidney sertoli cell markers.In one embodiment, one or more kidney sertoli cell markers are
Nephrin, NPHS2 or combination.In one embodiment, in the kidney sertoli cell in Nephrin and NPHS2 extremely
Few one is positive in the detection.
In a kind of embodiment according to the present invention, the common of following two or two or more substances exists described in instruction
Subject may suffer from membranous nephropathy, need to carry out Renal biospy further to make a definite diagnosis：PLA2R receptors, anti-PLA2R one kind or
A variety of autoantibodies, PLA2R receptor-antibodies immune complex, one or more kidney sertoli cell markers.
In one embodiment, the presence of the PLA2R receptors individually indicates that the subject may suffer from film property kidney
Disease needs to carry out Renal biospy further to make a definite diagnosis.In one embodiment, described and IgG4 common locations PLA2R receptors
Membranous nephropathy may be suffered from the presence of the instruction subject, need to carry out Renal biospy further to make a definite diagnosis.
In one embodiment, the presence of one or more autoantibodies of the anti-PLA2R individually instruction it is described by
Examination person may suffer from membranous nephropathy, need to carry out Renal biospy further to make a definite diagnosis.In one embodiment, IgG4 there are lists
It solely indicates that the subject may suffer from membranous nephropathy, needs to carry out Renal biospy further to make a definite diagnosis.
In one embodiment, the presence of the IgG4-PLA2R immune complexs individually indicates that the subject may
With membranous nephropathy, need to carry out Renal biospy further to make a definite diagnosis.
In one embodiment, the presence of one or more kidney sertoli cell markers individually indicates the subject
Membranous nephropathy may be suffered from, needs to carry out Renal biospy further to make a definite diagnosis.In one embodiment, described and IgG4 common locations
The presence of one or more kidney sertoli cell markers indicate that the subject may suffer from membranous nephropathy, need to carry out Renal biospy
Further to make a definite diagnosis.In one embodiment, the presence of at least one of Nephrin and NPHS2 individually refers in kidney sertoli cell
Show that the subject may suffer from membranous nephropathy, needs to carry out Renal biospy further to make a definite diagnosis.In one embodiment, kidney foot
The presence of at least one of Nephrin and NPHS2 and PLA2R receptors, IgG4 antibody, IgG4-PLA2R immune complexs in cell
At least one of presence indicate collectively the subject may suffer from membranous nephropathy, need carry out Renal biospy with further really
It examines.
In one embodiment, Immunofluorescence test, immunochemistry detection, biochemistry detection are carried out to the sample to be tested
In it is one or more.
The Immunofluorescence test is by one or more carry out immunofluorescence dyeings in following substance, to detect
The presence and distribution of these substances, the substance be PLA2R receptors, one or more autoantibodies of anti-PLA2R, PLA2R by
Body-antibody immune complex, one or more kidney sertoli cell markers.
The dyeing is independent dyeing, double staining, triple staining or its arbitrary combination.Combination includes but not limited to following
Mode：
Combination 1：
Individually dyeing：IgG4 or PLA2R
Double staining：IgG4 and NPHS2 (or Nephrin)
Combination 2：
Double staining：IgG4 and PLA2R
Double staining：IgG4 and NPHS2 (or Nephrin)
Combination 3：
Triple staining：IgG4, PLA2R and NPHS2 (or Nephrin)
In one embodiment, in the Immunofluorescence test of the sample to be tested, PLA2R receptors, IgG4 antibody,
The common location of at least one of IgG4-PLA2R immune complexs and kidney sertoli cell marker indicates that the subject may suffer from
There is membranous nephropathy, needs to carry out Renal biospy further to make a definite diagnosis.
In one embodiment, in the Immunofluorescence test of the sample to be tested, PLA2R receptors, IgG4 antibody,
The positive signal of at least one of IgG4-PLA2R immune complexs has weakened or has turned out cloudy instruction film property nephrotic disease
It improves or is completely recovered.
In one embodiment, the non-invasive detection methods can with blood testing use in conjunction, for making up film property
The deficiency of nephrosis blood testing.
Embodiment
Embodiment 1 detaches
As shown in Figure 1, detached to acquiring from the urine sample of subject, it is specific as follows：
The 40ml freshly voided urine samples for acquiring subject centrifuge 10 minutes through 1000-4000 revs/min, collect sediment
(containing cell and cell fragment).Sediment is resuspended with PBS, centrifuges 10 minutes, washes away through 1000-4000 revs/min again
Remaining soluble protein collects sediment.Sediment is resuspended with PBS and is distributed into three parts of samples to be tested I, II, III.
Wherein, sample to be tested I fixes 30 minutes with the suspension of 4% paraformaldehyde of 0.5ml, for immunohistochemistry, immune glimmering
Light.The observation property detection such as Electronic Speculum.Gel-runing buffer is added in sample to be tested II, is used for immune-blotting method (Western
Blot).Protease inhibitors is added in sample to be tested III, is placed in -80 DEG C of preservations, for detections such as protein science chips.
2 Immunofluorescence test of embodiment
As shown in Fig. 2, Immunofluorescence test is carried out to sample to be tested, it is specific as follows：
Using 4 antibody of anti-human igg, anti-human PLA2R antibody, anti-human NPHS2 antibody and anti-human IgA antibody as primary antibody, resist
Body serum origin is mouse, rabbit or sheep, monoclonal antibody or mostly anti-；
It is secondary antibody using the anti-mouse of the donkey of the fluorescent markers such as FITC, Cy3 and Cy5, donkey anti-rabbit or the anti-goat-anti body of donkey；
Detection mode is a kind of single dye (antibody primary antibody), double dyes (two kinds of primary antibodies) or three dyes (three kinds of primary antibodies)；
One antiantibody of immunofluorescence uses the phosphate buffer (PBS) containing 1% bovine serum albumin(BSA) (BSA) to dilute 100-200
Times, be incubated antibody before first with containing 0.2%Triton and 1%BSA PBS (PBST) be incubated 15-30 minute, primary antibody overnight incubation or
2-3 hours；
Two antiantibody of immunofluorescence also dilutes 100- with the phosphate buffer (PBS) containing 1% bovine serum albumin(BSA) (BSA)
200 times, incubation time 1-2 hours；
Primary antibody and secondary antibody are washed 3 times, every time 10 minutes with PBS after being incubated；
After secondary antibody has been incubated and has been washed with PBS, it is washed with water primary and then is contaminated 5 minutes with DAPI, mounting after washing 3 times；
Directly coupling, which can be used, in primary antibody has the corresponding antibodies of fluorescein or enzyme to carry out direct immuno-fluorescent antibody assay.
Detection image is as shown in Figures 3 and 4, is in granular form the IgG4 positive signals of membranous nephropathy patient or small crumby more
Fragment, these fragments show slightly flat and unclear in microscope light field (DIC) image compared with cell and other cell fragments.
The urine IgG4 Immunofluorescence tests of the different nephrotics of embodiment 3 and normal subjects
According to present invention non-invasive detection methods described above, acquisition is suffered from from normal health volunteer, membranous nephropathy
Person, Focal segmental glomerulosclerosis (FSGS) patient, diabetic nephropathy (DN) patient, minute nephropathy (MCD) patient, IgA
The urine of nephrotic carries out IgG4 Immunofluorescence tests respectively, and detection image is shown in Fig. 5-11.
Fig. 5 is the Immunofluorescence test image of normal healthy subjects, as shown, normal human urine sediment IgG4 letters
Number for feminine gender.
Fig. 6-7 is the Immunofluorescence test image of membranous nephropathy patient, and Fig. 6 is the IgG4 signals on intact cell, and Fig. 7 is
IgG4 signals on cell fragment.As shown, IgG4 positive signals are mostly cell fragment, small part is on intact cell.
Fig. 8 is the Immunofluorescence test image of Focal segmental glomerulosclerosis (FSGS) patient, as shown, IgG4 signals
For feminine gender.
Fig. 9 is the Immunofluorescence test image of diabetic nephropathy (DN) patient, as shown, IgG4 signals are feminine gender.
Figure 10 is the Immunofluorescence test image of minute nephropathy (MCD) patient, as shown, IgG4 signals are the moon
Property.
Figure 11 is the Immunofluorescence test image of IgA nephrotics, as shown, minority IgA nephrotics are with faint
IgG4 signals.
The urine double staining Immunofluorescence test of 4 membranous nephropathy patient of embodiment
According to present invention non-invasive detection methods described above, to acquiring the dual dye of urine progress from membranous nephropathy patient
Color Immunofluorescence test, detection image are shown in Figure 12-14.
Figure 12 is the Immunofluorescence test image of IgG4 and PLA2R double stainings in urine, as shown, membranous nephropathy is suffered from
IgG4 and PLA2R distributions are consistent in person's urine.
Figure 13 is the Immunofluorescence test image of IgG4 and kidney sertoli cell marker NPHS2 double stainings in urine, is such as schemed
It is shown, IgG4 and NPHS2 common locations.
Figure 14 is the Immunofluorescence test image of PLA2R and kidney sertoli cell marker Nephrin double stainings in urine, such as
Shown in figure, PLA2R and Nephrin common locations.
Complete kidney sertoli cell quantity is few in membranous nephropathy urine sediment, IgG4 and kidney sertoli cell marker (NPHS2) weight
It closes, but this part sediment only accounts for seldom part of IgG4 positive signals；
Membranous nephropathy PLA2R positive signals are identical as IgG4 signal characteristics, the accidental PLA2R positive signals of other nephrosis, but
These signals are often weaker and IgG4 be it is negative or not with IgG4 common locations.
5 immune-blotting method of embodiment
To acquire from the urine of membranous nephropathy patient (3), normal subjects (1) and other nephrotics (3) into
Row Western Blot detections, the result is shown in Figure 15.
As shown in figure 15, three membranous nephropathy Urine in Patients samples are in Immunofluorescence test and Western blot detections
PLA2R signals are the positive, but PLA2R signals are feminine gender in the renal fibroblast immunofluorescence of No. 3 patients, this shows membranous nephropathy
Renal fibroblast detection has deficiency.Western Blot are the result shows that the molecular weight of PLA2R differs markedly from 1 in No. 3 Urine in Patients
Number and No. 2 patients, this may be caused by the difference, genetic mutation or protein degradation of PLA2R gene expression products, this is also not
The reason of Different Results being obtained with the PLA2R antibody (epitope different) in source.
A kind of example of the present invention has shown and described in above description, but as previously described, it should be understood that the present invention is not office
Be limited to form disclosed herein, be not to be taken as excluding other embodiments, and can be used for various other combinations, modification and
Environment, and can be changed by the above teachings or related fields of technology or knowledge in the scope of the invention is set forth herein
It is dynamic.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, then it all should be appended by the present invention
Add in scope of the claims.
It is described the invention in detail above in association with preferred embodiment and exemplary example.But needs are stated
It is that these specific implementation modes are only the illustrative explanations to the present invention, do not constitute any limit to protection scope of the present invention
System.Without departing from spirit of that invention and protection domain, the technology of the present invention content and embodiments thereof can be carried out
Various improvement, equivalencing or modification, these each fall in protection scope of the present invention.Protection scope of the present invention is with appended power
Subject to profit requires.
Claims (10)
1. a kind of detection method of biological sample, includes the following steps：
(1) it is detached to acquiring from the biological sample of subject, obtains sample to be tested；With
(2) one or more in IgG4, PLA2R, Nephrin and NPHS2, testing result work is detected in the sample to be tested
To judge whether the subject needs the preliminary foundation of progress Renal biospy.
2. according to the method described in claim 1, it is characterized in that, the biological sample is urine, the sample to be tested is urine
Liquid passes through the obtained urine deposits of centrifugal treating, the sample to be tested include it is following in it is one or more：Cell, cell are broken
Piece and the immune complex being attached on the cell or the cell fragment.
3. according to the method described in claim 1, it is characterized in that, in the step (2), by Immunofluorescence test come
It is detected in the sample to be tested one or more in the IgG4, the PLA2R, the Nephrin and the NPHS2.
4. according to the method described in claim 3, it is characterized in that, the Immunofluorescence test follows the steps below：
(A) it is contacted using immune composition as primary antibody with the biological sample, forms Antibody-antigen complex, washing removes not
In conjunction with the primary antibody；
(B) secondary antibody that fluorescent marker is added is dyed, and washing removes the unbonded secondary antibody；
(C) film-making, the position using fluorescence microscope fluorescent label signal and distribution,
Wherein, the immune composition in the step (A) includes anti-IgG4 antibody, anti-PLA2R antibody, anti-Nephrin
It is one or more in antibody and anti-NPHS2 antibody.
5. according to the method described in claim 4, it is characterized in that, the dyeing is independent dyeing, double staining, triple staining
Or its arbitrary combination.
6. according to the method described in claim 5, it is characterized in that, the independent dyeing includes to the IgG4 or described
PLA2R individually carries out immunofluorescence dyeing.
7. according to the method described in claim 5, it is characterized in that, the double staining includes to two in following any combination
Kind substance carries out immunofluorescence dyeing：
It combines (1)：The IgG4 and PLA2R；
It combines (2)：The IgG4 and Nephrin；Or
It combines (3)：The IgG4 and NPHS2.
8. according to the method described in claim 5, it is characterized in that, the triple staining includes to three in following any combination
Kind substance carries out immunofluorescence dyeing：
It combines (4)：The IgG4, the PLA2R and the Nephrin；Or
It combines (5)：The IgG4, the PLA2R and the NPHS2.
9. according to the method described in claim 1, it is characterized in that, in the step (2), by immune-blotting method come
It is detected in the sample to be tested one or more in the IgG4, the PLA2R, the Nephrin and the NPHS2.
10. according to the method described in claim 9, it is characterized in that, the immune-blotting method is included in the biological sample
The presence of the middle detection PLA2R and its molecular weight.
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