CN104730250A

CN104730250A - Enzyme linked immunosorbent assay kit for detecting human kidney injury molecule-1

Info

Publication number: CN104730250A
Application number: CN201310706630.6A
Authority: CN
Inventors: 张�杰; 颜艳; 黄序
Original assignee: Sino Biological Inc
Current assignee: Sino Biological Inc
Priority date: 2013-12-20
Filing date: 2013-12-20
Publication date: 2015-06-24

Abstract

The invention discloses a kit for detecting human kidney injury molecule-1 by a double antibody sandwich method, which comprises a KIM-1 resistant mice monoclonal coated antibody, an enzyme-labeled HE4 rabbit polyclonal antibody and a recombinant HE4 albumen standard substance. The detection kit provided by the invention can be used for qualitatively or quantitatively detecting the KIM-1 level in sample and has the advantages that the detection method is convenient and practicable, the detection sensitivity and accuracy are high, the specificity is strong, and the detection kit can simultaneously rapidly detect bulk samples and can play a vital role in KIM-1 related foundation and clinical research.

Description

A kind of enzyme linked immunological kit detecting people's kidney injury molecule

Technical field

The present invention relates to a kind of enzyme linked immunological kit detecting people's kidney injury molecule in enzyme-linked immuno assay technical field.

Background technology

Iehimura equals within 1998, to adopt presentation variance analysis (rep-resentational differeneeanalysis, RDA) in ischemia-reper rat kidney cell, a kind of new I type transmembrane protein is identified, called after kidney injury molecule 1 (kidney injury molecule 1, KIM-1), the proximal tubular epithelial cells that its great expression regenerates after Ischemia reperfusion injury, and express very micro-in normal kidney tissue.The mankind KIM-1 assignment of genes gene mapping is in No. 5 chromosome long arm (5q33.2), KIM-1 albumen is made up of 334 amino acid residues, protein structure shows, KIM-1 possibility contactin, its extracellular region comprises 1 signal peptide, 1 Ig territory and 1 mucin territory, performs the function of membrane receptor/adhesion molecule.KIM-1 intracellular region is shorter.Containing a conservative Tyr phosphorylation site, it may be the intracellular signal transduction path of transducing receptor one ligand interaction.The expression of KIM-1 has high tissue specificity.RNA Blot Hybridization Analysis shows, KIM-1 does not express in tire liver, tire kidney, has faint expression at adult normal's liver,kidney,spleen, and expresses in nephridial tissue after ischemic injuries and significantly strengthen.Immunohistochemical staining and RNA in situ hybridization display, KIM-1mRNA and protein molecular are expressed in the proximal convoluted tubule S3 district re-epithelialize cell of outer marrow skin and cortex medullary ray, and Subcellular Localization is mostly at basilar memebrane cell apical.The biological function of KIM-1, may participate in the Injury and reformation process of kidney trouble, adhere to and immunoreaction process damage-resistant.

KIM-1 can be used as sensitive, special, the quantitative biological marker detecting acute injury of kidney clinically: acute injury of kidney (acute kidney injury, AKI) multi-disciplinary a kind of common disease is related to, early detection and in time treatment can prevent the state of an illness from worsening further, stop it to develop into kidney failure.Previously traditional index such as serum creatinine, urea nitrogen cannot meet the clinical early diagnosis needs of AKI.One of KIM 1 mark being considered to AKI, KIM 1 is a kind of new I type transmembrane glycoprotein, express hardly in normal nephridial tissue, but the renal cells of the AKI caused by ischemic, renal toxicity etc. is high expressed, its extracellular is at metallo-matrix proteases (matrixmetallopr-oteinases, MMP) under effect, cleavable is that soluble fragments is discharged into extracellular, and enters in urine.Zooscopy shows, the rising of urine KIM 1 is early than indexs such as serum creatinine, urea nitrogen, Urine proteins, when there is AKI in the mankind, urine KIM-1 raises also more obvious, but KIM 1 can finally generally acknowledge by vast clinician and become the sensitive indexes making a definite diagnosis AKI in early days, also need be confirmed in further clinical research.

KIM-1 also can be used for the early diagnosis of kidney neoplasms.Have been reported and show, the mucin territory of solubility KIM-1 can to anti-adhesive, and play cytoprotection, the mucin overexpression on tumour cell, also can suppress the adhesion of lymphocyte and tumour cell, thus protection tumour cell, making it shift becomes possibility.Renal cell carcinoma cell strain 769-P energy great expression KIM-1, renal cell carcinoma patients Renal biospy sample is shown by SABC, same height expresses KIM-1, and after nephrectomy, urine KIM-1 level declines rapidly or disappears, show that KIM-1 and soluble component thereof may work in the development of kidney neoplasms and transfer, can be used as the early diagnosis index of kidney neoplasms.

Based on the vital role that KIM-1 may have in clinical research and auxiliary diagnosis, the KIM-1 level in the different urine specimen of Accurate Determining all has great importance for basis and clinical research.Euzymelinked immunosorbent assay (ELISA) due to checkout equipment require low, be easy to realize fast high-flux, quantitatively comparatively accurate, therefore be the Main Means measuring KIM-1 at present, the complete independent development of the present invention has gone out the novel mouse-anti KIM-1 monoclonal antibody for KIM-1 different loci, adopt double-antibody method to constitute KIM-1 detection kit, basis and the clinical research of KIM-1 can be widely used in.

Summary of the invention

The object of this invention is to provide that a kind of detection sensitivity is high, accuracy be strong, the people KIM-1 double-antibody method detection kit of low cost.

Detection kit provided by the present invention comprises specific KIM-1 coated antibody, and enzyme mark KIM-1 detects antibody and people KIM-1 protein standard substance.

Wherein said KIM-1 coated antibody and detection antibody are all mouse resource monoclonal antibody, and described people KIM-1 protein standard substance is the recombined human KIM-1 albumen of eukaryotic expression.

Described monoclonal antibody adopts recombined human KIM-1 protein immune animal, utilizes hybridoma technology to prepare.Enzyme labelled antibody conventionally utilizes horseradish peroxidase (Horseradish peroxidase, HRP) labelled antibody to obtain.Described recombined human KIM-1 albumen is that after eukaryotic expression, purifying obtains.

Technical scheme of the present invention is that preferred pin is to the different epi-position of KIM-1, there is the monoclonal antibody combinations of pairs of high sensitivity, high specific, one strain monoclonal antibody is adsorbed on solid phase carrier as coated antibody, coated antibody specificly can catch KIM-1 and the KIM-1 protein standard substance in sample, after adding enzyme mark detection antibody, form coated antibody, antigen, detection antibody complex, stop after corresponding substrate colour developing, read sample light absorption value, by comparing the content that can draw KIM-1 with typical curve.

The present invention adopts the KIM-1 level in double antibodies sandwich standard measure or qualitative detection sample, the method of inspection is convenient and easy, detection sensitivity and accuracy is high, high specificity, simultaneously can detect large quantities of sample fast, immunogene needed for Dispersal risk and the protein standard substance screened in antigen and kit all adopt recombined human KIM-1 albumen, antibody and the protein standard substance of kit chief component are independent research, therefore cost is low, reliability is strong, and expectation will play a significant role in KIM-1 relevant rudimentary and clinical research.

Accompanying drawing explanation

Fig. 1 recombined human KIM-1 protein standard substance electroresis appraisal figure

Fig. 2 people KIM-1 enzyme linked immunological kit examination criteria curve map

Embodiment

Below in conjunction with specific embodiment, the present invention will be further described and explanation.

The component preparation of embodiment 1 enzyme linked immunological kit

1. the preparation of people KIM-1 albumen mouse monoclonal antibody:

1) animal immune

Adopt Balb/c mouse as immune animal, the recombined human KIM-1 albumen produced with Yi Qiao Divine Land Bioisystech Co., Ltd is for immunogene, immunizing dose is 50 μ g/, the complete Freund's adjuvant of immunogene and equivalent is made emulsifying agent by first immunisation, subcutaneous abdomen multi-point injection, At intervals of two to three weeks gets same dose immunogene and equivalent incomplete Freund's adjuvant makes emulsifying agent, booster immunization twice, serum titer is measured after three immunity, get the high mouse peritoneal booster immunization of serum titer once, after 4 days, extracting spleen cell merges.

2) Fusion of Cells and cloning

Get immune mouse spleen cell, mix with SP2/0 myeloma cell in 4: 1 ratios, utilize polyethylene glycol method to merge, obtain hybridoma.Adopt recombined human KIM-1 albumen as envelope antigen, measure cell conditioned medium liquid by ELISA method, choose positive hole and carry out cloning by limiting dilution, until obtain the hybridoma cell strain of stably excreting KIM-1 monoclonal antibody specific.

3) production of monoclonal antibody and purifying

Choose hybridoma cell strain, utilize culture flask or bio-reactor to carry out cell chulture, collecting cell culture supernatant, utilize albumin A to carry out affinity purification, qualification antibody purity and concentration after packing, in-20 DEG C of Cord blood.

2. the preparation of enzyme labelled antibody:

1) take 5mg HRP to be dissolved in 0.5ml distilled water.

2) in upper liquid, add the 0.1M NaIO that 0.5ml newly joins ₄solution, under room temperature, lucifuge stirs 20 minutes.

3) loaded in bag filter by above-mentioned solution, dialyse to the sodium-acetate buffer (pH4.4) of 1mM, 4 DEG C are spent the night

4) add 0.2M carbonate buffer solution (pH9.5), make the pH of above hydroformylation HRP be elevated to 9.0 ~ 9.5, then add 5mg IgG immediately in 1ml0.01M carbonate buffer solution, room temperature lucifuge stirs 2 hours gently.

5) the 4mg/ml NaBH that 0.2ml newly joins is added ₄liquid, mixing, places 2 hours in 4 DEG C.

6) loaded in bag filter by above-mentioned liquid, dialyse in the PBS of pH7.4,0.15M, 4 DEG C are spent the night.

Embodiment 2 detects the establishment of the enzyme linked immunological kit of people KIM-1

Set up the enzyme linked immunological kit detecting people KIM-1, comprise following reagent:

1) mouse anti human KIM-1 monoclonal coated antibody;

2) mouse monoclonal antibody of HRP mark;

3) recombined human KIM-1 protein standard substance;

4) bag is buffered the phosphate buffer that liquid is pH7.4;

5) confining liquid is the phosphate buffer containing 2% bovine serum albumin(BSA);

6) sample diluting liquid is the phosphate buffer containing 0.1% bovine serum albumin(BSA);

7) concentrated cleaning solution is the phosphate buffer cleansing solution containing 2% tween;

8) nitrite ion A and nitrite ion B forms, and nitrite ion A is hydrogen peroxide or urea peroxide, and nitrite ion B is tetramethyl biphenyl;

9) stop buffer is the sulfuric acid of 2mol/L

Embodiment 3 detects the preparation of the enzyme linked immunological kit of people KIM-1

1. utilize orthogonal test to grope the optimum antibody concentration of enzyme linked immunological kit

According to ultraviolet spectrophotometer method, measure the concentration of antibody and antigen.Adopt orthogonal test method, anti-KIM-1 monoclonal antibody being diluted to concentration is 4 μ g/ml, 2 μ g/ml, 1 μ g/ml, restructuring KIM-1 protein concentration is diluted to 4000pg/ml, 400pg/ml, 0pg/ml, anti-KIM-1 cloned mouse antibody dilution concentration to the 4 μ g/ml of HRP mark, 2 μ g/ml, 1 μ g/ml, 0.5 μ g/ml.Consider the absorbance value of background and recombinant protein, the bag selecting antibody is 2 μ g/ml by concentration, and the working concentration of HRP labelled antibody is 0.5 μ g/ml.

2. the batch preparation of kit

1) anti-KIM-1 antibody dilution to concentration is 2 μ g/ml by coated elisa plate: wrap quilt with phosphate buffer (PBS), and 100 μ L/ holes, wrap by 96 hole ELISA Plate, 4 DEG C of overnight incubation.

2) close: remove the liquid not wrapping quilt, with PBS (PBST) the 200 μ l/ hole containing 0.1%Tween, wash plate 1 time.Add 300 μ l confining liquids and close nonspecific binding site, incubated at room 1 hour.PBST washes plate 2 times, and pat dry rear vacuum packing machine packaging, 4 DEG C save backup.

3) preparation of protein standard substance: with the KIM-1 albumen of the PBS dilution restructuring containing 0.5%BSA, freeze-drying after packing, 4 DEG C save backup.

4) preparation of enzyme labelled antibody: using as detect the anti-KIM-1 of antibody monoclonal antibody-purified after, HRP marks, and add 50% glycerine, after packing ,-20 DEG C save backup.

The mensuration of embodiment 4 kit major parameter

The range of linearity, specificity, sensitivity, precision, linear, the recovery etc. are comprised on the major parameter affecting kit quality, its measure concrete grammar and result as follows:

1. the range of linearity of kit:

1) be diluted to by the PBST of KIM-1 albumen containing 0.1%BSA, 10,000pg/mL, 1000pg/mL, 0.1pg/mL, 0pg/ml, 100 μ l/ holes join above-mentioned bag by good ELISA Plate, incubated at room 2 hours.PBST washes plate 3 times.

2) PBST of HPR labelled antibody containing 0.1%BSA is diluted to 1 μ g/ml, 100 μ l/ holes add in reacting hole, incubated at room 1 hour.PBST washes plate 3 times.

3) add the tmb substrate of the fresh configuration of 200 μ l, room temperature lucifuge develops the color 20 minutes, adds 50 μ l2M sulfuric acid cessation reactions, reads value under microplate reader 450nm wavelength.

4) test findings shows, when KIM-1 albumen is saturated when concentration is 10,000pg/mL, when 1000pg/mL, 450nm light absorption value is in the range of linearity.

5) revision test step 1), 2), 3); The typical curve each point of adjustment KIM-1 albumen is: 4000pg/mL, 2000pg/mL, 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 0pg/mL. make its linearly dependent coefficient more than 0.99; Namely the range of linearity of this kit is 62.5pg/mL-4000pg/mL.

2. the specificity of kit: the mouse KIM-1 of restructuring, rat KIM-1, dog KIM-1 and monkey KIM-1 are diluted to 1250ng/ml, employment KIM-1 enzyme-linked immunologic detecting kit is tested, experiment results proved, mouse KIM-1, the rat KIM-1 of this kit and 1250ng/ml, dog KIM-1 and monkey KIM-1 no cross reaction.

3. the sensitivity of kit

Add three times of standard deviations with the average light absorption value of 20 blank samples, calculate corresponding K IM-1 content, the minimum detectability and the sensitivity that obtain kit are 0.008pg/mL; Illustrate that this kit has higher detection sensitivity.

4. the precision of kit measures

1) mensuration of the interior precision of test

Select 4 parts of urine samples containing different K IM-1 concentration, the repetition of 20, each sample, detect in same ELISA Plate, calculate the coefficient of variation (C.V) of each testing result respectively, average coefficient of variation is 3.1% (see table 1).

Precision Experiment in the test of table 1KIM-1ELISA kit

2) mensuration of test bay precision

Select 4 parts of urine samples containing different K IM-1 concentration, the repetition of 2, each sample, duplicate detection 5 times, standard control curve is set in each plate, calculate the coefficient of variation (C.V) between same increment product 5 testing results, the coefficient of variation average out to 7.7% (see table 2) of 4 increment product, 5 testing results.

The test bay Precision Experiment of table 2KIM-1 kit

5. the linear determination of kit:

For assessing the linear of kit, have the urine specimen of high level KIM-1 to carry out a series of coefficient to 4 parts, each dilutability recovery of each sample is between 80%-120%, and illustrate that this kit has well linear, concrete outcome is in table 3.

Show the linear of 3KIM-1 kit

6. the mensuration of the kit recovery:

Added to respectively in 4 parts of urine specimens by 3 variable concentrations restructuring KIM-1 albumen and test, calculate each concentration recovery, the average recovery rate of 3 variable concentrations in 4 parts of urine specimens is 92%, and scope is: 80%-105%, has the good recovery.

The detecting step of embodiment 5KIM-1 enzyme linked immunological kit

1. the collection of sample, process and preservation

The collection of urine specimen: collect stage casing urina sanguinis with sterile chamber, 1000 × g removes particle and polymkeric substance in centrifugal 10 minutes, and get supernatant packing ,-20 DEG C save backup.

2. application of sample

1) take out bag by the standard items of good ELISA Plate and freeze-drying, add 1ml and containing the PBST of 0.1%BSA, standard items are dissolved.Ambient temperatare puts 20 minutes.By standard items from 4000pg/ml, do the doubling dilution of 2 times, dilute 7 points, dilution is respectively got 100u.Add in 96 hole ELISA Plate according to the order of following table.

2) get urine specimen to be measured, 100 μ L/ holes add in reacting hole.

3) incubated at room 2 hours.Washing lotion 200 μ L/ washes plate 3 times in hole, pats dry ELISA Plate.

3. add detection antibody

The PBST of HRP labelled antibody 0.1%BSA is diluted to 0.5ug/ml, and 100 μ l/ holes add in reacting hole.Incubated at room 1 hour.Washing lotion 200 μ l/ washes plate 3 times in hole, pats dry ELISA Plate.

4. develop the color

Add the freshly prepared TMB nitrite ion of 200 μ L, room temperature reaction 20 minutes.Add 50 μ L2M sulfuric acid cessation reactions.Value is read under microplate reader 450nm wavelength.

5. the foundation of typical curve

With the concentration of standard items for horizontal ordinate, OD value sets up logarithmic curve (Fig. 2) for ordinate.The sample OD value recorded is substituted into formula, calculates the KIM-1 content in sample.

Finally, it is also to be noted that enumerate above be only specific embodiments of the invention son.Obviously, the invention is not restricted to above examples of implementation, many modification can also be had.All modification that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.

Claims

1. detect a detection kit for people's kidney injury molecule, it is characterized in that, comprise identifying that the monoclonal antibody of the different epi-position of people's kidney injury molecule is respectively as capture antibody and detection antibody, using recombinant expressed people's kidney injury molecule as antigen standard.

2. antigen standard according to claim 1 is the recombined human kidney injury molecule that mammalian cell is expressed.

3. antigen standard according to claim 2 is the recombined human kidney injury molecule of 293 cellular expressions.

4. capture antibody according to claim 1 is mouse monoclonal antibody or rabbit monoclonal antibodies.

5. capture antibody according to claim 1 is fixed on microtiter plate or other solid supports.

6. the amount of capture antibody according to claim 1 is 2 ~ 4ug/mL.

7. detection antibody amount according to claim 1 is 0.5 ~ 1ug/mL.

8. kit according to claim 1, is characterized in that, comprises bag and is buffered liquid, confining liquid, concentrated cleaning solution, sample diluting liquid, nitrite ion, reaction terminating liquid.

9. bag according to claim 8 is buffered liquid is phosphate buffer; Described confining liquid is the phosphate buffer containing 2% bovine serum albumin(BSA); Described concentrated cleaning solution is the phosphate buffer containing 2% tween; Described sample diluting liquid is the phosphate buffer containing 0.1% bovine serum albumin(BSA) and 0.1% tween; Described nitrite ion is made up of nitrite ion A and nitrite ion B, and nitrite ion A is hydrogen peroxide or urea peroxide, and nitrite ion B is tetramethyl benzidine; Described stop buffer is the sulfuric acid of 2M.