CN1580773A - Urine neutral peptide chain endoprotease immunodetection method - Google Patents

Abstract

The present invention discloses an immuno-detection method for making quantitative detection of neutral endopeptidase in urine, and it is characterized by that said invention adopts ELISA method. Said method has good sensitivity, accuracy and repeatability, and the detected linear range can cover the clinical detection samples, and its method is simple and its cost is low.

Description

The immunologic detection method of urine neutral endopeptidase

Technical field

The present invention relates to urinate a kind of detection method of neutral endopeptidase (NEP), particularly the detection by quantitative immunization method of a kind of NEP.

Background technology

Neutral endopeptidase (international zymetology is numbered EC3.4.24.11 for neutral endopeptidase, NEP) is a kind of zinc-containing metal peptase of mammalian cell surface.It is a member in the NEP enzyme family, belongs to II type integral membrane proteins, plays an important role in regulating the metabolism of peptide class.This enzyme is at first come out from the brush border discovery and the purification of rabbit renal tubule in 1974 by Kerr and Kenny.NEP content in kidney and lung tissue is the abundantest, and the mRNA of three all type NEP (I type, IIa type, IIb type) all has expression at the near-end renal tubule, and wherein the IIb type is expressed the highest.This endopeptidase is positioned on the proximal tubule epithelium BBM in large quantities, enters urine following of physiological status slightly with teleblem segment form, handles appearing at the peptide class that the filtration in the TF is come out.(the γ-GT), NEP just is released into urine in large quantities when renal tubular cell is impaired as the amino peptase (AAP) of alanine, γ-paddy ammonia transpeptidase as the protein on other proximal tubule epithelial cell membrane surfaces.

The scholar has detected the activity of NEP respectively from several groups of kidney trouble patient urine, discovery this enzymatic activity in acute tubular toxic nephropathy patient (renal toxicity drug-induced) the highest (activity of NEP reaches 6.5 times of upper limits of normal in the urine), and after taking the renal toxicity medicine, promptly began to raise in second day.The NEP activity is increased to 2.5 times of upper limits of normal in acute tubular insufficiency (non-renal toxicity medicine causes) the patient urine, and in renal glomerular disease and the diabetic's urine activity of NEP in normal range.The inventor has studied the activity of the α 1-MG in the acute tubular insufficiency patient urine simultaneously, and after the activity of NEP had been reduced to normally in finding to urinate, the activity of α 1-MG was still increasing.

Scholars such as Nortier J have detected the NEP amount of accepting in the renal transplant recipients urine, find after transplanting the 1st, 2 day, and urine NEP significantly increases, and recovers normal after transplanting in 3～5 days.NEP discharges obviously increase in the early stage urine of transplanting, may be relevant to the damage of proximal tubule epithelium with ischemic and immunosuppressive drug.

J.F.Blaikley detected the patient's of row heart pass art urine NEP in the 1st, 4 days respectively at preceding 1 day of art, postoperative 3 hours, postoperative, find compared with the control, operation group postoperative urine NEP obviously increases, and NEP increases the most postoperatives of the most serious patient and enters acute renal failure and need hemodialysis.Because acute renal failure takes place after surgery is the main cause of death, so they advocate to detect urine NEP, so that early detection has the patient who develops into acute renal failure danger, and is in time treated.

Recently the ephrosis research that Chinese herbal medicine is caused is a lot of, because of the composition that wherein causes renal damage is mainly aristolochic acid, and the domestic Aristolochic acid nephropathy that is called.The renal damage that aristolochic acid causes also can show as acute tubular necrosis with the infringement of chronic renal Tubulointerstitial at most.Main pathological change is that renal tubule is badly damaged, and the renal tubule number reduces, kidney region fibrosis, atrophy, and cortical layer is got involved the heaviest in addition.People such as JOELLE L. measure Chinese herbs nephropathy (CHN) patient respectively, use Chinese herbal medicine but do not occur renal function affected individual, renal glomerular disease patient and normal healthy controls person as yet and urinate trace of albumin (comprising CC16, β 2-MG, α 1-MG) and urinate β-D-N-acetyl grape amine enzyme (NAG) and the amount of urine NEP, carry out the kidney pathological biopsy simultaneously, and these patients have been carried out following up a case by regular visits to of June～2 year.Found that in Chinese herbs nephropathy patient renal function, when severe is got involved, NEP concentration obviously descends in the urine, and trace of albumin is negative correlation and endogenous creatinine clearance rate is proportionate in its concentration level and the urine, and do not find that in the renal glomerular disease patient urine NEP and endogenous creatinine clearance rate have correlativity, and the degree that urine NEP descends is consistent with the degree of proximal tubule damage in the kidney biopsy.They think, the actual ratio that has reflected in the residual int brush border of proximal tubule of amount of urine NEP.

The renal damage that heavy metal such as cadmium, lead can cause mainly involves proximal tubule in early days.There is the scholar to detect NEP, NAG and the amount of NAG-β and the content [albumin, β 2-MG, retinal binding protein (RBP), CC16] of some urine trace of albumin in workman's urine of the workman of 52 contact cadmiums and 54 non-professional property contact cadmiums.Found that: when cadmium was had only the low dosage contact, NEP promptly began rising (this moment, other proximal tubule mark raise as yet).Pointed out NEP can show the early stage infringement of proximal tubule function.

Scholars such as Huang H. have detected one-sided kidney stone respectively and have not accompanied uronephrosis, urinate value and the blood adhesion molecule value of NEP, NAG, β-GAL (beta galactose enzyme) in associated with acute ureterolithiasis homonymy uronephrosis and the control group.Found that have that urine NEP obviously reduces in the associated with acute ureterolithiasis uronephrosis patient group, and NAG, β-GAL compare all with control group and increase.Low-level urinary obstruction, the uronephrosis of having pointed out of urine NEP causes that renal tubular function is impaired, the proximal convoluted tubule atrophy, and remaining tubule number reduces.

When ischemic, poisoning etc. caused the acute tubular damage, the proximal tubule brush border came off in a large number, and NEP enters in the urine with the brush border that comes off, and NEP obviously increases in the urine.The heavy absorption function of part renal tubule this moment descends, and the heavy absorption function compensatory of part renal tubule increases, and therefore, the urine trace of albumin of the heavy absorption function of reflection near-end renal tubule does not still have significant change; Enter the renal tubule of chronic lesion phase, can not provide NEP to be released in the urine again, the NEP in its urine reduces.The degree of its renal tubule damage is heavy more simultaneously, and the heavy absorption function of renal tubule is just poor more, thereby the trace of albumin of discharging in the urine is just many more, occurs the negative correlation of varying degree between urine NEP and the urine trace of albumin.Relatively acute tubular damage group and chronic renal tubule damage group can be found, in the acute stage of renal tubule damage, trace of albumin does not still have obviously to increase or only have slightly and increases in some urine, urine NEP is existing obviously to be increased, and with progression of disease, in the chronic phase of renal tubule damage, after urine NEP had descended, the amount of urine α 1-MG and RBP was still increasing.Therefore, if the amount of NEP increases in the urine, and the amount of urine traces of albumin such as urine α 1-MG and RBP there is no obvious increase, and often pointing out the early stage acute lesion of renal tubule and weighing the absorption function compensatory with renal tubule increases.And urine NEP reduces, traces of albumin such as urine α 1-MG and RBP increase, can point out the chronic pathology of renal tubule, urinate NEP this moment because the atrophy of renal cells, the minimizing of renal tubule number and surperficial brush border and discharge minimizing, and because the heavy absorption function of renal tubule reduces gradually, the trace of albumin in the urine increases gradually.

In sum, in the acute tubular damage, urine NEP obviously increases, and urinate trace of albumin this moment does not obviously increase as yet, therefore detects urine NEP and can help us to find the acute injury of renal tubule more in early days; Urine NEP reduces in chronic renal tubule damage group, and is negative correlation with increasing of trace of albumin of urine; And renal glomerular disease is not when causing the renal tubule obvious damage as yet, and urine NEP compares no change with normal phase.Two-way variation appears in NEP in the different courses of disease just because of urine, when detecting in conjunction with other urine traces of albumin, it can help us to judge that the renal tubule damage is in acute stage or chronic phase, thereby helps to estimate to the selection of therapeutic scheme with to prognosis.

But at present domestic research to urine NEP still belongs to blank, and have only a few laboratory studying abroad, they adopt fluorescence method to detect (Joelle L, Nortier, Monique M, et al.Proximal tubular injury in Chinese herbs nephropathy:Monitoring by neutral endopeptidase enzymuria.Kidney International, 1997,51:288-293.), make substrate with succinyl-Ala-Ala-phenylalanine-aminomethyl cumarin (succinyl-alanyl-alanyl-phenylalanyl-AMC), NEP can succinyl-alanyl-alanyl-and-phenylalanyl-AMC between with this substrate hydrolysis, then further hatch with aminopeptidase M, disengage free aminomethyl cumarin (AMC), this material contains fluorophor, at λ ex=367nm, during λ em=440nm, can optionally measure free AMC.And do control tube simultaneously, add the specific inhibitor inhibitory enzyme activity of NEP before the reaction.The result who obtains deducts the data of the corresponding control group of inhibitor existence, with the error of avoiding non-specific substrate hydrolysis to cause.With the typical curve that people's kidney NEP of purifying sets up, can make the fluorescence of free aminomethyl cumarin be converted into the amount of enzyme.But because of this method needs special fluorescence radiation detector, about 100,000 dollars of this instrument costs an arm and a leg, and reactions steps is comparatively complicated, only is used for experimental study at present, can not promote on a large scale in clinical practice.

Summary of the invention

The objective of the invention is to address the above problem, provide a kind of can be at the immunologic detection method of the urine neutral endopeptidase (NEP) of clinical application.

Purpose of the present invention realizes by following technical proposals: the immunologic detection method of a kind of urine neutral endopeptidase (NEP) is characterized in that this method adopts enzyme linked immunosorbent assay (ELISA) (ELISA) technology.

This method can be used for urinating the quantitative measurement of NEP.

The concentration determination scope of above-mentioned urine NEP is 20-200 μ g/L.

Above-mentioned ELISA method comprises the following steps:

1) urinate NEP antibody sandwich enzyme reaction plate with the anti-people of 5-15 μ g/ml animal, every hole 50-200 μ l, put 1-40 ℃ 12 hours;

2) discard liquid in the hole, washing dries;

3) 1-40 ℃ was sealed enzyme reaction plate 1-24 hour;

4) discard liquid in the hole, washing dries;

5) add standard items, urine sample, the damping fluid (negative control) that suitably dilutes, every part all adds multiple hole, and every hole 50-200 μ l was hatched 10 minutes-24 hours for 1-40 ℃;

6) discard liquid in the hole, washing dries;

7) add the monoclonal antibody that the anti-people of 2-10 μ g/ml animal urinates NEP, every hole 50-200 μ l was hatched 1-24 hour for 1-40 ℃;

8) discard liquid in the hole, washing dries;

9) antibody of the animal immune globulin in the contragradience rapid 7 of adding horseradish peroxidase (HRP) mark, every hole 50-200 μ l was hatched 1-24 hour for 1-40 ℃;

10) washing dries;

11) after the colour developing, cessation reaction;

12) microplate reader reads each hole absorbance OD value at λ=450nm place;

13) according to the absorbance of standard series concentration, on semilog coordinate, obtain typical curve;

14) regression equation of trying to achieve according to typical curve calculates corresponding absorbance the concentration of urinating NEP.

Wherein, the anti-people of the animal described in the above-mentioned steps 1 and 7 to urinate NEP antibody be that anti-people of rabbit or mouse-anti people urinate NEP antibody.

The antibody of the animal immune globulin in the contragradience rapid 7 of the HRP mark described in the above-mentioned steps 9 is 1: 1000 the goat-anti rabbit of HRP mark or the antibody of sheep anti mouse immunoglobulin (Ig).

Color condition in the above-mentioned steps 11 is for adding the substrate solution of now joining, and every hole 50-200 μ l is under the 1-40 ℃ of lucifuge environment.

Positive progressive effect of the present invention is: the ELISA method for immunologic detection method commonly used (the Shen Xia chief editor. Beijing in 2002, People's Medical Officer Press. clinical immunology and immunological test new technology. Shen Xia: chapter 2 enzyme immunoassay technique .P12-20.), but the present invention has adopted sandwich ELISA method to detect urine NEP, and this does not at home and abroad appear in the newspapers in the document as yet; The evaluation of methodology of carrying out simultaneously shows: this method has sensitivity preferably, accuracy and repeatability, and the range of linearity of detection can contain the clinical detection sample; And this law is easy and simple to handle, and method does not have wound, and required instrument is cheap, and the general medicine laboratory all can be purchased.

Because its experiment operation steps is simple, is easy to promote, cost is lower; And experimental result accurately and reliably, and clinical diagnosis and treatment is had bigger help, and therefore broad clinical application prospect is arranged.

Description of drawings

The NEP typical curve that Fig. 1 surveys for the inventive method.

Embodiment

A large amount of experiments such as selection of the selection of the selection of this research by the antagonist system, antibody working concentration, optimality criterion product/antibody action time sum up the ELISA method of urinating the NEP detection by quantitative.Several preferred embodiments of following act only are that the inventive method better is described, and are not limited.

Material:

1.Elx800 type microplate reader (available from U.S. Bio-Tec company);

2.96 hole ELISA Plate (available from U.S. Labsystem company);

3. people NEP standard items (acquisition of Belgian laboratoire Pluridisciplinaire de RechercheExperimentale Biomedicale laboratory);

4. the anti-people NEP of rabbit polyclonal antibody (available from Shanghai genome company);

5. mouse anti human NEP monoclonal antibody (available from Shanghai genome company);

6. the goat-anti mouse of horseradish peroxidase (HRP) mark, goat anti-rabbit immunoglobulin antibody (acquisition of medical test key lab of Shanghai Second Emdical University);

7. wrap diluted liquid: pH9.6,0.05mol/L sodium carbonate-sodium bicarbonate buffer liquid;

8. confining liquid: the PB damping fluid that contains 0.5%BSA;

9. cleansing solution: 0.1%Tween-20;

10.NEP the PBS of and antibody diluent: pH7.4;

11. substrate solution: TMB (available from Sigma company);

The H of 12. stop buffer: 2mol/L ₂SO ₄

Embodiment 1

By enzyme reaction plate, every hole 100 μ l put 4 ℃, 12 hours to step 1. with the polyclonal antibody bag of the anti-people NEP of 10 μ g/ml rabbits;

Step 2. discards liquid in the hole, and cleansing solution washing 3 times each 3 minutes, dries;

Step 3. is sealed enzyme reaction plate with confining liquid, and every hole 200 μ l put 4 ℃, 18 hours;

Step 4. is with step 2;

Step 5. adds standard items, urine sample, the damping fluid (negative control) of suitably dilution, every hole 100 μ l, and every part of standard items, sample, contrast all add multiple hole, hatch 1 hour for 37 ℃;

Step 6. is with step 2;

Step 7. adds the monoclonal antibody of 6 μ g/ml mouse anti human NEP, and every hole 100 μ l were hatched 1 hour for 37 ℃;

Step 8. is with step 2;

Step 9. adds 1: the goat-anti mouse immuning ball protein antibody of 1000HRP mark, and every hole 100 μ l were hatched 1 hour for 37 ℃;

Step 10. cleansing solution is washed plate 4 times, dries in each 3 minutes;

Step 11. adds the substrate solution of now joining, every hole 100 μ l, and 37 ℃ of lucifuge environment colour developings down, after 15 minutes, every hole adds stop buffer 50 μ l cessation reactions;

Step 12. microplate reader reads each hole absorbance (OD) value at λ=450nm place;

Step 13. obtains typical curve according to the absorbance of standard series concentration on semilog coordinate;

The regression equation that step 14. is tried to achieve according to typical curve calculates corresponding absorbance the concentration of NEP.

Embodiment 2

By enzyme reaction plate, every hole 50 μ l put 40 ℃, 1 hour to step 1. with the polyclonal antibody bag of 15 μ g/ml mouse-anti people NEP;

Step 2. discards liquid in the hole, and cleansing solution washing 3 times each 3 minutes, dries;

Step 3. is sealed enzyme reaction plate with confining liquid, and every hole 100 μ l put 1 ℃, 24 hours;

Step 4. is with step 2;

Step 5. adds standard items, urine sample, the damping fluid (negative control) of suitably dilution, every hole 50 μ l, and every part of standard items, sample, contrast all add multiple hole, hatch 10 minutes for 40 ℃;

Step 6. is with step 2;

Step 7. adds the monoclonal antibody of the anti-people NEP of 2 μ g/ml rabbits, and every hole 50 μ l were hatched 24 hours for 1 ℃;

Step 8. is with step 2;

Step 9. adds 1: the goat anti-rabbit immunoglobulin antibody of 1000HRP mark, and every hole 50 μ l were hatched 4 hours for 40 ℃;

Step 10. cleansing solution is washed plate 4 times, dries in each 3 minutes;

Step 11. adds the substrate solution of now joining, every hole 200 μ l, and 1 ℃ of lucifuge environment colour developing down, after 30 minutes, every hole adds stop buffer 200 μ l cessation reactions;

Step 12. microplate reader reads each hole absorbance (OD) value at λ=450nm place;

Step 13. obtains typical curve according to the absorbance of standard series concentration on semilog coordinate;

The regression equation that step 14. is tried to achieve according to typical curve calculates corresponding absorbance the concentration of NEP.

Embodiment 3

By enzyme reaction plate, every hole 200 μ l put 1 ℃, 24 hours to step 1. with the polyclonal antibody bag of the anti-people NEP of 5 μ g/ml rabbits;

Step 2. discards liquid in the hole, and cleansing solution washing 3 times each 3 minutes, dries;

Step 3. is sealed enzyme reaction plate with confining liquid, and every hole 50 μ l put 40 ℃, 1 hour;

Step 4. is with step 2;

Step 5. adds standard items, urine sample, the damping fluid (negative control) of suitably dilution, every hole 200 μ l, and every part of standard items, sample, contrast all add multiple hole, hatch 24 hours for 1 ℃;

Step 6. is with step 2;

Step 7. adds the monoclonal antibody of 10 μ g/ml mouse anti human NEP, and every hole 200 μ l were hatched 3 hours for 40 ℃;

Step 8. is with step 2;

Step 9. adds 1: the goat-anti mouse immuning ball protein antibody of 1000HRP mark, and every hole 200 μ l were hatched 24 hours for 1 ℃;

Step 10. cleansing solution is washed plate 4 times, dries in each 3 minutes;

Step 11. adds the substrate solution of now joining, every hole 50 μ l, and 40 ℃ of lucifuge environment colour developings down, after 5 minutes, every hole adds stop buffer 10 μ l cessation reactions;

Step 12. microplate reader reads each hole absorbance (OD) value at λ=450nm place;

Step 13. obtains typical curve according to the absorbance of standard series concentration on semilog coordinate;

The regression equation that step 14. is tried to achieve according to typical curve calculates corresponding absorbance the concentration of NEP.

Statistical method:

Carry out correlation regression analysis and descriptive statistic with SAS6.12 and EXCEL software.

The result:

(1) range of linearity

It is 400 μ g/L, 200 μ g/L, 100 μ g/L, 50 μ g/L, 20 μ g/L, 10 μ g/L, 5 μ g/L, 2.5 μ g/L that the pure product of NEP that people's nephridial tissue is extracted are diluted to concentration with the PBS damping fluid, blank well adds the PBS damping fluid, logarithm with concentration is mapped to corresponding absorbance, when NEP concentration during at 20～200 μ g/L, above-mentioned data are carried out the correlation regression analysis, finding has good linear relationship (Y=A*LgX+B) between the logarithm of its concentration and absorbance, shows as Fig. 1.

At different time, in the line taking scope, the repeatability of typical curve is detected.

Get the typical curve in the different time points range of linearity, the CV of each normal concentration is 2.063～4.170%, average 2.987%.

All typical curve r＞0.99 (p＜0.001), repeatability is good.

(2) accuracy detects

Reclaim experiment and be used for the accuracy of test experience, i.e. the degree of closeness of measured value and true value, the recovery commonly used is represented.In five parts of urine samples, add the NEP of variable concentrations, calculate its recovery separately.The recovery 93.6～107.3%, average 101.7%, good accuracy is arranged.

(3) degree of accuracy detects

Get the different urine sample of two parts of concentration in respectively surveying in a collection of 10 times, calculate variation within batch coefficient (CV); Respectively two parts of urine samples are measured 10 times CV between calculating is criticized 10 difference detection days.

Variation within batch coefficient (CV) 4.152～5.469%, average 4.811%, interassay coefficient of variation (CV) 6.241～6.395%, average 6.318%, good accuracy is arranged.

(4) detect limit value

Detect the minimum detected value that limit value is a certain experiment, as detecting limit value, drawing as calculated and detecting limit value is 3.20 μ g/L with the pairing concentration value of the average X+2S of blank absorbency.

Application Example

One, material and method

One) study subject

1, grouping situation

It is five groups that patient group is divided into, and comprises the CRF group (specifically grouping situation sees Table 16) due to acute tubular damage group, chronic renal tubule damage group, Aristolochic acid nephropathy group, simple renal glomerular disease group, the renal glomerular disease.All cases all are at the Ruijin Hospital Attached to Shanghai Medical Univ No.2 inpatient between year October in January, 2002 to 2002.Be pathological diagnosis or the clinical diagnosis person of making a definite diagnosis.Clinical diagnosis is according to (except that other routine tests, detecting patient's routine urinalysis, urine α simultaneously by medical history, health check-up, laboratory examination ₁-microglobulin (α ₁-MG), urine transferrins (TRU), urine RBP, urine NAG, urine immunoglobulin (Ig) (IgG), UCr, blood urine osmotic pressure, blood Scr, twenty-four-hour urine electrolyte, urine titratable acid and urine ammonium, blood β ₂-MG, blood α ₁-MG, urine β ₂-MG, urine α ₁-MG, urine amino acid mensuration, urinary glucose determination, urine protein electrophoresis, the experiment of urine concentration and dilution etc.).The normal control group all is the healthy student at school of the court's health and medical staff and two medical universities.

68 customary kidney biopsy centesis and do pathological section in patient group.The male sex's 36 examples wherein, women's 32 examples.The all capable light microscopic of all pathological sections (comprising HE, PAS, Masson and PASM dyeing), immunofluorescence and electron microscopic examination.Wherein pathological diagnosis is that all kinds of renal glomerular disease persons are 38 examples (comprising chronic renal failure 7 examples that renal glomerular disease causes), the acute tubular damage (comprises acute tubular necrosis, renal tubules,convoluted cavity change etc.) be 15 examples, chronic renal Tubulointerstitial pathology 7 examples that other causes of disease cause.Aristolochic acid nephropathy patient is 8 examples, and Jing Xiajun shows as chronic renal Tubulointerstitial pathology.

Two) urine NEP detection by quantitative

Adopt the method step of embodiment 1.

Three) statistical analysis

Carry out correlation analysis with SAS6.12 software and Excel, Wilcoxin sum of ranks, signed rank test and descriptive analysis.

Two, result

(1) detection (the results are shown in following table 1) of normal control group urine NEP:

The testing result of table 1 normal control group urine NEP

The routine number NEP median interquartile range of dividing into groups

(μg/mmol?Cr) (μg/mmol?Cr)

Total normal group 100 68.41 68.15

The male sex organizes 54 71.84 69.28

Women's group 46 63.76 61.44

＜60 years old groups 92 70.35 67.57

〉=60 years old groups 8 59.19 57.80

The men and women urinates NEP and detects difference not statistically significant (Z=0.9356, P＞0.05), and＜60 years old group and 〉=60 years old group detect difference not statistically significant (Z=1.5483, P＞0.05).95% credibility interval of normal control group urine NEP is 46.29～89.52 μ g/mmol Cr.

(2) each group urine NEP and with the comparison (the results are shown in following table 2) of normal group:

Each group urine NEP of table 2 reaches the comparative result with normal group

Median and normal control group that sample is respectively organized in grouping compare

(μg/mmol?Cr) P

The acute tubular damage organizes 196.36 0.0001 ^*

The damage of chronic renal tubule organizes 31.64 0.0017 ^*

Aristolochic acid nephropathy group 32.78 0.0076 ^*

Simple renal glomerular disease group 75.49 0.1425

CRF due to the renal glomerular disease organizes 19.40 0.0042 ^*

Acute tubular damage group, in the urine NEP apparently higher than the normal control group ( ^*P＜0.01); Chronic renal tubule damage group and Aristolochic acid nephropathy group and CRF group, in the urine NEP be starkly lower than the normal control group ( ^*P＜0.01); Simple renal glomerular disease group, urine NEP and normal control difference not statistically significant (P＞0.05).

The coincidence rate of (three) two kinds of detection methods relatively

Coincidence rate is one can reflect susceptibility and specific index simultaneously.Effect for ELISA method more of the present invention and fluorescence method detection urine NEP detects urine NEP with fluorescence method simultaneously, and to urinate the NAG activity, to urinate α ₁-MG carries out coincidence rate relatively (result sees following table 3,4 for details) as the reference index of renal tubule damage to two kinds of assay methods.

Coincidence rate=(true positives example number+true negative example number)/total case load.

Wherein, adopt the normal reference value of urine NAG activity:＜18.50u/l; Urine α ₁-MG normal reference value＜1.28mg/ml.

Table 3 is the coincidence rate comparative result of two kinds of methods of standard with urine NAG activity

Grouping ELISA method meets fluorescence method and meets two kinds of method coincidence rates relatively

Rate (%) rate (%) X ²Value P value

The acute tubular damage organizes 77.27 79.54 0.032 0.885

The damage of chronic renal tubule organizes 52.08 54.17 0.213 0.534

Aristolochic acid nephropathy group 61.54 61.54 0.517 0.406

Renal glomerular disease group 38.71 35.48 0.069 0.698

CRF organizes 38.46 32.77 0.018 0.973

Table 4 is with urine α ₁-MG activity is the coincidence rate comparative result of two kinds of methods of standard

Grouping ELISA method meets fluorescence method and meets two kinds of method coincidence rates relatively

Rate (%) rate (%) X ²Value P value

The acute tubular damage organizes 43.45 46.45 0.530 0.467

The damage of chronic renal tubule organizes 72.92 70.83 0.043 0.835

Aristolochic acid nephropathy group 69.23 76.92 0.000 1.000

Renal glomerular disease group 41.94 38.71 0.065 0.799

CRF organizes 69.23 69.23 0.170 0.680

Table 3,4 X ²Check all shows: the coincidence rate that each patient organizes two kinds of method checks does not have significant difference (the P value all＞0.05).

Claims (8)

1, a kind of immunologic detection method of urinating neutral endopeptidase is characterized in that this method adopts enzyme linked immunosorbent assay (ELISA) (ELISA) technology.

2, immunologic detection method according to claim 1 is characterized in that this method is used to urinate the quantitative measurement of neutral endopeptidase.

3, immunologic detection method according to claim 2, the concentration determination scope that it is characterized in that this urine neutral endopeptidase are 20-200 μ g/L.

4, immunologic detection method according to claim 1 is characterized in that this method comprises the following steps:

1) urinate neutral endopeptidase antibody sandwich enzyme reaction plate with the anti-people of 5-15 μ g/ml animal, every hole 50-200 μ l, put 1-40 ℃ 12 hours;

2) discard liquid in the hole, washing dries;

3) 1-40 ℃ was sealed enzyme reaction plate 1-24 hour;

4) discard liquid in the hole, washing dries;

5) add standard items, urine sample, the damping fluid that suitably dilutes, every part all adds multiple hole, and every hole 50-200 μ l was hatched 10 minutes-24 hours for 1-40 ℃;

6) discard liquid in the hole, washing dries;

7) add the monoclonal antibody that the anti-people of 2-10 μ g/ml animal urinates neutral endopeptidase, every hole 50-200 μ l was hatched 1-24 hour for 1-40 ℃;

8) discard liquid in the hole, washing dries;

9) antibody of the animal immune globulin in the contragradience rapid 7 of adding horseradish peroxidase-labeled, every hole 50-200 μ l was hatched 1-24 hour for 1-40 ℃;

10) washing dries;

11) after the colour developing, cessation reaction;

12) microplate reader reads each hole absorbance OD value at λ=450nm place;

13) according to the absorbance of standard series concentration, on semilog coordinate, obtain typical curve;

14) regression equation of trying to achieve according to typical curve calculates corresponding absorbance the concentration of urinating neutral endopeptidase.

5, immunologic detection method according to claim 4 is characterized in that it is that anti-people of rabbit or mouse-anti people urinate neutral endopeptidase antibody that the anti-people of the animal described in this step 1 and 7 urinates neutral endopeptidase antibody.

6, immunologic detection method according to claim 4, the antibody that it is characterized in that the animal immune globulin in the contragradience rapid 7 of the horseradish peroxidase-labeled described in this step 9 are 1: 1000 the goat-anti rabbit of horseradish peroxidase-labeled or the antibody of sheep anti mouse immunoglobulin (Ig).

7, immunologic detection method according to claim 4 is characterized in that the substrate solution that the color condition in this step 11 is now joined for adding, and every hole 50-200 μ l is under the 1-40 ℃ of lucifuge environment.

8,, it is characterized in that the concrete steps of this method are as the described immunologic detection method of the arbitrary claim of claim 4-7:

1) with the polyclonal antibody bag of the neutral endopeptidase of the anti-people of 10 μ g/ml rabbits by enzyme reaction plate, every hole 100 μ l put 4 ℃, 12 hours;

2) discard liquid in the hole, cleansing solution washing 3 times each 3 minutes, dries;

3) seal enzyme reaction plate with confining liquid, every hole 200 μ l put 4 ℃, 18 hours;

4) with step 2;

5) add standard items, urine sample, the damping fluid that suitably dilutes, every hole 100 μ l, every portion of standard items, sample, damping fluid all add multiple hole, hatch 1 hour for 37 ℃;

6) with step 2;

7) monoclonal antibody of adding 6 μ g/ml mouse anti human neutral endopeptidases, every hole 100 μ l were hatched 1 hour for 37 ℃;

8) with step 2;

9) the goat-anti mouse immuning ball protein antibody of 1: 1000 horseradish peroxidase-labeled of adding, every hole 100 μ l were hatched 1 hour for 37 ℃;

10) cleansing solution is washed plate 4 times, dries in each 3 minutes;

11) add the substrate solution of now joining, every hole 100 μ l, 37 ℃ of lucifuge environment colour developings down, after 15 minutes, every hole adds stop buffer 50 μ l cessation reactions;

12) microplate reader reads each hole absorbance OD value at λ=450nm place;

13) according to the absorbance of standard series concentration, on semilog coordinate, obtain typical curve;

14) regression equation of trying to achieve according to typical curve calculates corresponding absorbance the concentration of neutral endopeptidase.

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