CN104280553B - The ELISA detection kit of a kind of little mouse soluble type CD74 albumen and detection method - Google Patents
The ELISA detection kit of a kind of little mouse soluble type CD74 albumen and detection method Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
The invention belongs to immunology and biological technical field, the invention provides the ELISA detection kit of a kind of little mouse soluble type CD74 albumen and detection method thereof. Can the test kit of the present invention accurately detect the content of the soluble type CD74 albumen in mice body fluid, and result, by microplate reader quantitative analysis, eliminates SABC, immunofluorescence, Western? the subjectivity of the semi-quantitative methods such as blot; And highly sensitive, the CD74 albumen that detects is minimum can to 781.25pg/ml; Do not need complex instrument when the present invention detects, it is easy to popularization and application in research institutions and medical institutions, basic research biological sample can be detected on a large scale, quickly obtain the relevant mass data of mice CD74 albumen and information.
Description
Technical field
The invention belongs to immunology and biological technical field, be specifically related to ELISA detection kit and the detection method thereof of a kind of little mouse soluble type CD74 albumen.
Background technology
CD74 and MHC class ��molecule associated constant chain (majorhistocompatibilitycomplex, MHC-II-associatedinvariantchain, Ii), it is a kind of non-polymorphism II type transmembrane glycoprotein, the outer section of film inner segment, cross-film section and film can be divided into. CD74 is primarily present in cell, as the molecular chaperones of MHC-class ��molecule, promotes endoplasmic reticulum processing release MHC class ��molecule, is transported to endosome, stops MHC class ��molecule to be combined with endogenous polypeptide in endoplasmic reticulum. Additionally, the CD74 of about 2-5% is expressed in cell surface, such as B cell system, epithelial cell, mononuclear cell etc., and not depending on MHC class ��molecule, its function and MHC-class ��molecule are unrelated. Recently research finds that CD74 is macrophage migration inhibition factor (MacrophageMigrationInhibitoryFactor, MIF) high-affinity membrane receptor, signal transduction pathways such as can activating NF-�� B, ERK1/2 and then the secretion of inflammation inducing cytokine is combined with MIF.
Existing research confirms in born of the same parents and on after birth, CD74 take part in the pathogenic process of some diseases associated with inflammation, autoimmune disease, tumor. Having the Ldlr knock out mice of arteriosclerosis plaque formability, as lacked CD74 gene simultaneously, then atherosclerotic degree alleviates.CD74 in the speckle of research discovery Atheromatosis people and peripheral blood lymphocytes expresses and increases, and the expression of CD74 is relevant to blood vessel endothelium thickness, and prompting CD74 can as atherosclerotic biological diagnosis index and therapy target. Additionally, MIF and pulmonary alveolar macrophage film surface C D74 effect, activating ERK1/2 signal path, induction neutrophilic granulocyte is at the gathering of alveolar space, anti-CD74 antibody blocks said process, and prompting CD74 participates in the pulmonary inflammatory reaction of MIF mediation. Under normal physiological conditions, Urothelial cell film surface is not expressed or expresses CD74 on a small quantity, cannot participate in the MIF signal path started. Under inflammatory conditions, neurotransmitter Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 can raise the expression of Urothelial cell MIF and CD74, participates in bladder inflammation reaction. Under the stimulation of cytokine IFN-��, gastric epithelial cell surface C D74 expresses increase, rises with helicobacter pylori amount; After using anti-CD74 antibody or CD74 hydrolytic enzyme, particle-bound bacteria amount then reduces. Research finds Helicobacter pylori infection two weeks after, the expression of MIF and CD74 all increases, MIF is by activating intracellular signalling pathways NF-�� B and ERK1/2 in conjunction with CD74, promote the generation of inflammatory cytokine, accelerate cell proliferation and inhibited apoptosis, Chronic inflammation stimulates to develop becomes chronic gastritis, forms gastric ulcer, and finally causes the malignant change of stomach.
For systemic lupus erythematosus (sle), two kinds of mouse species with lupus tendency, in its blood circulation, the rising of MIF level has close time-dependent relation with advancing of disease and the vasculitic formation of glomerular. Meanwhile, the development also with inflammation of expressing of kidney MIF, CD74, and CD44 (the collaborative molecule of CD74) mRNA and albumen significantly raises. Research recently finds to derive from the B cell of systemic lupus erythematosus (sle) mice, and the expression of its MIF and CD74 is all substantially increased, and as activated the MIF/CD74 path of this kind of B cell, then can extend the time-to-live of systemic lupus erythematosus (sle) mice.
In recent years many sections of bibliographical information CD74 are relevant with the generation of kinds of tumors development. As the researchs such as Ishinami find, the patients with gastric cancer operation prognosis of CD74 expression feminine gender is better than CD74 and expresses positive patient; Nagata etc. find that CD74 can as the biomolecule mark of prediction cancer of pancreas. Additionally, CD74 and part MIF thereof also assists in the generation of kinds of tumors. MIF, in conjunction with CD74, activates NF-�� B, raises Tap63, secretes inflammatory factor IL-8, promotes the survival of chronic lymphocytic leukemia cell. MIF is combined in the CD74 of human embryonic kidney cell 293 (HEK293) with ectopic expression, activates ERK and PI3K/AKT signal path, then activation NF-�� B, raises Vascular endothelial growth fator-D, promotes growth and the transfer of tumor.
The generation of the signal path not only involved in diseases of CD74 mediation, also assists in the internal organs of protection damage. In the lung inflammation later stage, MIF and CD74 combines the propagation that can induce lung II type epithelial cell (AEC-II), promotes the reparation of alveolar epithelial cells. MIF acts on CD74 can activate the protein kinase (adenosinemonophosphate-activatedproteinkinase that myocardial cell AMP activates; AMPK) signal path; promoting the picked-up of glucose, protecting myocardial cell is from ischemical reperfusion injury. Heinrichs et al. finds that MIF acts on CD74 and activates AMPK signal path in hepatic stellate cell, it is suppressed that hepatic stellate cell proliferation, has anti-fibrosis effect.
The studies above shows that CD74 has played important effect in inflammation, autoimmune disease, tumor, it is possible to become the biomarker of these diseases, is also likely to become efficacy determination and the prognostic indicator of relevant disease.
The CD74 research expressed mainly was adopted the semiquantitative methods such as SABC, immunofluorescence, Westernblot and RT-PCR in the past. The applicant's early stage has designed the ELISA detection method of humanized's soluble type CD74 albumen, and Successful utilization is in clinical research (Chinese patent application CN201310103321.X, denomination of invention is " ELISA detection kit of a kind of humanized's soluble type CD74 albumen and detection method ", and publication number is CN103207277A). Assis also reports a kind of ELISA method detecting humanized soluble type CD74 in August, 2013, speculate that soluble type CD74 is the extracellular segment of CD74 on after birth, it is discharge to peripheral circulation or body fluid after being sheared under the effect of protease, and the function of MIF can be neutralized. But the effect of soluble type CD74 is resistant to MIF still collaborative MIF actually, also have a lot of unknown, relevant research is (DavidN.Assis at the early-stage, etal.Theroleofmacrophagemigrationinhibitoryfactor (MIF) inautoimmuneliverdisease, Hepatology.2014,59 (2): 580-91.).
Owing to there is species specificity in little mouse CD74 and humanized CD74, early stage we adopt the ELISA detection method (CN103207277A) of humanized soluble type CD74 to fail to detect little mouse soluble type CD74 albumen. And, there is no any report about the little mouse soluble type CD74 albumen of detection by quantitative both at home and abroad at present, basic research is caused certain obstruction, thus the quantitative detecting method of soluble type CD74 that is necessary to be developed in infrastest biological specimen, the function for studying CD74 albumen further lays the foundation.
Summary of the invention
It is an object of the invention to provide the ELISA detection kit of a kind of little mouse soluble type CD74 albumen, another object of the present invention is to provide the ELISA detection method of a kind of little mouse soluble type CD74 albumen utilizing mentioned reagent box, it is contemplated that based on study medium and small mouse soluble type CD74 protein content accurate, easy, the highly sensitive and detection means that can be widely used be provided.
A first aspect of the present invention, it is provided that the ELISA detection kit of a kind of little mouse soluble type CD74 albumen, this test kit includes:
It is coated the little mouse CD74 antibody of ELISA ELISA Plate, for sheep antimouse CD74 antibody;
Detect the antibody of little mouse soluble type CD74, for rat anti-mouse CD74 monoclonal antibody;
ELIAS secondary antibody, for the mountain goat anti rat IgG of HRP (horseradish peroxidase) labelling;
Standard protein, for restructuring Mus CD74 albumen.
Preferably, described sheep antimouse CD74 antibody is R&DSystems, AF7478, sheepanti-mouseCD74polyclonalantibody, and best effort concentration is 0.25ug/ml (extension rate 1:800).
Preferably, described rat anti-mouse CD74 monoclonal antibody is R&DSystems, MAB7478, ratanti-mouseCD74monoclonalantibody, and best effort concentration is 1 �� g/ml (extension rate 1:500).
Preferably, the anti-rat Ab of goat of described HRP (horseradish peroxidase) labelling is CellSignaling, #7077, goatanti-ratHRP-linkedantibody, optimum diluting multiple 1:2000.
Preferably, described restructuring Mus CD74 albumen is R&DSystems, 7478-CD, recombinantmouseCD74, for Criterion curve, set concentration is: 25ng/ml, 12.5ng/ml, 6.25ng/ml, 5.208ng/ml, 4.557ng/ml, 3.125ng/ml, 2.604ng/ml, 0.913ng/ml, 0.651ng/ml.For detecting the sensitivity of ELISA kit, arranging concentration is: 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.125ng/ml, 1.5625ng/ml, 0.78125ng/ml, 0.390625ng/ml, 0ng/ml.
The ELISA detection kit of described a kind of little mouse soluble type CD74 albumen, also includes:
Sample diluting liquid, it is coated buffer, confining liquid, ELISA ELISA Plate eluent, antibody diluent, nitrite ion and stop buffer.
Preferably, described sample diluting liquid: when sample is serum or bronchoalveolar lavage fluid, sample diluting liquid is 1 �� PBS, pH:7.4.
Preferably, described confining liquid, for the SuperBlock Block buffer of Thermo company.
Preferably, the described buffer that is coated is 1 �� PBS, pH:7.4;
Preferably, described ELISA ELISA Plate eluent is 1 �� PBS solution containing 0.05%Tween-20;
Preferably, described antibody diluent is 1 �� PBS, pH:7.4;
Preferably, described nitrite ion is the TMBsubstratekit of Thermo company;
Preferably, described stop buffer is the sulfuric acid solution of 2mol/L.
Preferably, described ELISA ELISA Plate is the Maxisorp series ELISA ELISA Plate of NUNC company of Denmark, 96 holes.
A second aspect of the present invention, it is provided that the ELISA detection method of a kind of little mouse soluble type CD74 albumen, comprises the steps:
It is coated ELISA ELISA Plate with CD74 protein polyclone antibody, after confining liquid is closed, it is separately added into the diluent of standard restructuring Mus CD74 albumen and testing sample, add CD74 protein monoclonal antibody, then add ELIAS secondary antibody, add substrate colour developing after washing, under 450nm wavelength, measure OD value by microplate reader, the standard curve of the standard restructuring Mus CD74 albumen of system, calculates the concentration of soluble type CD74 albumen in testing sample by standard curve.
Preferably, described CD74 protein polyclone antibody is purchase the sheep antimouse CD74 antibody (R&DSystems in R&DSystems company, AF7478, sheepanti-mouseCD74polyclonalantibody), best effort concentration is 0.25ug/ml (extension rate 1:800).
Preferably, described CD74 protein monoclonal antibody is purchase the rat anti-mouse CD74 antibody (R&DSystems in R&DSystems company, MAB7478, ratanti-mouseCD74monoclonalantibody), best effort concentration is 1 �� g/ml (extension rate 1:500).
Described restructuring Mus CD74 albumen is for purchasing in R&DSystems company (R&DSystems, 7478-CD, recombinantmouseCD74).
Preferably, described ELIAS secondary antibody is purchase the goat anti-rat Ab (CellSignaling in CellSignaling company HRP (horseradish peroxidase) labelling, #7077, goatanti-ratHRP-linkedantibody), optimum diluting multiple 1:2000.
Preferably, described confining liquid is the PBS solution containing 1% bovine serum albumin+1% sucrose, containing the PBS solution of 5% bovine serum albumin+1% sucrose, or the SuperBlock Block buffer of Thermo company.
Preferably, selecting room temperature off-period 1 hour, room temperature 2 hours, 4 DEG C overnight.
It is further preferable that the SuperBlock Block buffer 37 DEG C that described confining liquid is Thermo company is closed 2 hours.
Another aspect of the present invention, it is provided that the ELISA detection kit of a kind of little mouse soluble type CD74 albumen, except mentioned reagent, also includes the sample diluting liquid of routine, is coated buffer, ELISA ELISA Plate eluent, antibody diluent, nitrite ion and stop buffer.
Described test kit, ELISA ELISA Plate is commercially available ELISA ELISA Plate, it is preferable that can be selected for the Maxisorp series ELISA ELISA Plate (96 holes, #44-2404) of NUNC company of Denmark.
It is coated buffer, it is therefore preferable to 1 �� PBS, pH:7.4;
ELISA ELISA Plate eluent, it is therefore preferable to 1 �� PBS solution containing 0.05%Tween-20
Sample diluting liquid: when sample is serum or bronchoalveolar lavage fluid, it is therefore preferable to 1 �� PBS, pH:7.4.
Antibody diluent, it is therefore preferable to 1 �� PBS, pH:7.4;
Nitrite ion, it is therefore preferable to the TMBsubstratekit (#34021) of Thermo company;
Stop buffer, it is preferred that for the sulfuric acid solution of 2mol/L.
The double-antibody sandwich elisa detection method of described little mouse soluble type CD74 albumen, comprises the steps:
Being coated buffer and AF7478 is diluted to 0.25 �� g/ml, add 100 �� l in every hole of ELISA ELISA Plate, shrouding is placed on 4 DEG C of overnight incubation; With the ELISA ELISA Plate eluent of fresh configuration, every hole 300 �� l, automatic plate washer Elx50 (BioTek) washs 3-5 time, and absorbent paper pats dry; Add ELISA ELISA Plate confining liquid, every hole 200 �� l, incubated at room 2 hours; The same method ELISA ELISA Plate eluent, every hole 300 �� l, washs 3-5 time, pats dry; It is put in 4 DEG C, standby.
Serum sample or BALF sample, dilute with 1:2 with sample diluting liquid, with the volume application of sample in 100 �� l/ holes, incubated at room 2 hours; The same method ELISA ELISA Plate eluent, every hole 300 �� l, washs 3-5 time, pats dry; CD74 antibody MAB7478 is diluted to 1 �� g/ml, every hole 100 �� l, incubated at room 2 hours; The same method ELISA ELISA Plate eluent, every hole 300 �� l, washs 3-5 time, pats dry; Add the mountain goat anti rat IgG of HRP labelling, every hole 100 �� l, incubated at room 1 hour; The same method ELISA ELISA Plate eluent, every hole 300 �� l, washs 5-7 time, pats dry; Adding substrate tetramethyl benzidine H2O2, every hole 100 �� l, room temperature lucifuge is hatched 10-15 minute, adds 2mol/L sulfuric acid solution and terminates reaction, every hole 50 �� l; In microplate reader, (450nm) measures OD value.
Compared with prior art, beneficial effects of the present invention is as follows:
1) accurate: the ELISA detection kit of the little mouse soluble type CD74 albumen of commercial-free in the market, the method not having the little mouse soluble type CD74 protein content of detection by quantitative by literature search, therefore cannot accurate quantification for the soluble type CD74 albumen in the body fluid (serum, BALF) of mice. This ELISA kit can accurately detect the content of the soluble type CD74 albumen in mice body fluid, and result, by microplate reader quantitative analysis, eliminates the subjectivity of the semi-quantitative methods such as SABC, immunofluorescence, Westernblot.
2) highly sensitive: the CD74 albumen that detects by the method is minimum can to 781.25pg/ml, and sensitivity is apparently higher than semi-quantitative methods such as common westernblot and SABC.
3) simple and convenient: in this method, agents useful for same and experiment consumptive material are commercially available commercially produced product, it is easy to obtain; Only needing pipettor and microplate reader to carry out application of sample and reading in detection, common laboratory all can carry out this detection.
ELISA kit provided by the invention is simple to operation, the soluble type CD74 protein content in the body fluid of mice can be detected accurately, in high sensitivity, based on research provide a kind of new means and method.
The test kit of the present invention can be used for the detection by quantitative of soluble type CD74 albumen in the humoral sample of the models such as basic research small mouse autoimmune disease, various inflammation, tumor and the detection of the CD74 albumen in various biological sample (such as cell culture supernatant, cell pyrolysis liquid).
Complex instrument is not needed when the present invention detects, it is prone to popularization and application in research institutions and medical institutions, biological sample can be detected on a large scale, quickly obtain the relevant mass data of mice CD74 albumen and information, the basis relevant for CD74 and clinic study are added fuel to the flames, and have wide market prospect and bigger economical, societal benefits.
Accompanying drawing explanation
Fig. 1 is the result of double-antibodies sandwich ELISA detection restructuring mouse CD74 albumen, and wherein A figure is block diagram, and B figure is scatterplot (being linearly correlated with);
Fig. 2 is soluble type CD74 albumen in double-antibody sandwich elisa and Dotblot method detection mice with acute lung injury serum specimen, and wherein A figure is ELISA result, and B figure is Dotblot result;
Fig. 3 is double-antibody sandwich elisa and Dotblot method detection mice with acute lung injury bronchoalveolar lavage fluid
(BALF) soluble type CD74 albumen in specimen, wherein A figure is ELISA result, and B figure is Dotblot result;
Fig. 4 is the sensitivity results of double-antibodies sandwich ELISA;
Fig. 5 is the result of soluble type CD74 albumen in the double-antibodies sandwich ELISA detection LPS mice with acute lung injury serum induced and BALF specimen, and wherein A figure is soluble type CD74 albumen result in serum, and B figure is soluble type CD74 albumen result in BALF;
Fig. 6 is the optium concentration of screening coated antibody AF7478;
Fig. 7 is the optium concentration of selective mechanisms antibody MAB7478.
Detailed description of the invention
In conjunction with embodiment and accompanying drawing, the invention will be further described, but the enforcement of the present invention is not limited to that.
Embodiment 1: experimental technique
The preparation of detection ELISA ELISA Plate: being coated buffer and AF7478 is diluted to 0.25 �� g/ml, add 100 �� l in every hole of ELISA ELISA Plate, shrouding is placed on 4 DEG C of overnight incubation; With the ELISA ELISA Plate eluent of fresh configuration, every hole 300 �� l, automatic plate washer Elx50 (BioTek) washs 3-5 time, and absorbent paper pats dry; Add ELISA ELISA Plate confining liquid, every hole 200 �� l, incubated at room 2 hours; The same method ELISA ELISA Plate eluent, every hole 300 �� l, washs 3-5 time, pats dry; It is put in 4 DEG C, standby.
Serum sample or BALF sample, dilute with 1:2 with sample diluting liquid, with the volume application of sample in 100 �� l/ holes, incubated at room 2 hours; The same method ELISA ELISA Plate eluent, every hole 300 �� l, washs 3-5 time, pats dry; CD74 antibody MAB7478 is diluted to 1 �� g/ml, every hole 100 �� l, incubated at room 2 hours; The same method ELISA ELISA Plate eluent, every hole 300 �� l, washs 3-5 time, pats dry; Add the mountain goat anti rat IgG of HRP labelling, every hole 100 �� l, incubated at room 1 hour; The same method ELISA ELISA Plate eluent, every hole 300 �� l, washs 5-7 time, pats dry; Adding substrate tetramethyl benzidine H2O2, every hole 100 �� l, room temperature lucifuge is hatched 10-15 minute, adds 2mol/L sulfuric acid solution and terminates reaction, every hole 50 �� l; In microplate reader, (450nm) measures OD value.
Embodiment 2: condition optimizing
One, the optimization of coated antibody concentration:
Select coated antibody AF7478 (the 1 �� g/ml of variable concentrations respectively, 0.5 �� g/ml, 0.25 �� g/ml, 0.125 �� g/ml) it is coated ELISA ELISA Plate, detect according to the operating procedure in embodiment 1, add known variable concentrations CD74 protein standard substance.According to the OD value obtained, selecting blank group OD value minimum, what linear relationship between OD value (deduct blank organize OD value) and standard protein concentration was the closest is coated concentration as optimum antibody. It is 0.25 �� g/ml that the optimum antibody of AF7478 is coated concentration.
The optium concentration of screening coated antibody AF7478 refers to Fig. 6.
Two, the optimization of confining liquid:
Confining liquid selects the PBS solution containing 1% bovine serum albumin+1% sucrose, containing the PBS solution of 5% bovine serum albumin+1% sucrose, the SuperBlock Block buffer of Thermo company; Selecting room temperature off-period 1 hour, room temperature 2 hours, 4 DEG C overnight.
Detect according to the operating procedure in embodiment 1, add known variable concentrations CD74 protein standard substance, select blank group OD value minimum, and the closest conduct the best confining liquid of the linear relationship between OD value and standard protein concentration and off-period.
The final SuperBlock Block buffer room temperature with Thermo company closes 2 hours for best of breed.
Three, the optimization of antibody is detected:
Detection antibody selects MAB7478, Ii-1 (BDPharmingen, #555317, ratanti-mouseCD74antibody), and detectable concentration selects (2.5 �� g/ml, 1 �� g/ml, 0.5 �� g/ml).
Detect according to the operating procedure in embodiment 1, add known variable concentrations CD74 protein standard substance, select blank group OD value minimum, linear relationship between OD value (deduct blank organize OD value) and standard protein concentration the closest as optimum detection antibody and working concentration.
The working concentration 1 �� g/ml of MAB7478 is best compatibility.
The optium concentration of selective mechanisms antibody MAB7478, refers to Fig. 7.
Four, the optimization of ELIAS secondary antibody:
ELIAS secondary antibody selects the IgG antibody of the anti-rat HRP labelling of horse, the IgG antibody of the anti-rat HRP labelling of donkey, the IgG antibody of mountain goat anti rat HRP labelling, and antibody extension rate selects 1:1000,1:2000,1:3000.
Detect according to the operating procedure in embodiment 1, add known variable concentrations CD74 protein standard substance, select blank group OD value minimum, and the closest conduct the best ELIAS secondary antibody of the linear relationship between OD value and standard protein concentration and diluted concentration.
The IgG antibody of mountain goat anti rat HRP labelling, 1:2000 dilution is the best.
Five, the optimization of sample diluting liquid:
During mice with acute lung injury detection, serum and BALF sample diluting liquid select 1 �� PBS (pH:7.4) or containing 0.1%BSA, 1 �� TBS solution of 0.5%Tween-20, detect according to the operating procedure in embodiment 1, being added with the positive sample of soluble type CD74 albumen, what after selection diluted sample, extension rate was the closest with the linear relationship of OD value is best sample diluent.
1 �� PBS (pH:7.4) is better than the 1 �� TBS solution containing 0.1%BSA, 0.5%Tween-20, for more excellent sample diluting liquid.
Embodiment 3:ELISA test kit detection restructuring mouse CD74 albumen, Criterion curve
Restructuring mouse CD74 standard protein is started dilution from 25ng/ml, dilutes 9 concentration, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 5.208ng/ml, 4.557ng/ml, 3.125ng/ml, 2.604ng/ml, 0.913ng/ml, 0.651ng/ml, each sample arranges 3 multiple holes, 100 �� l/ holes, according to the step duplicate detection 3 times of embodiment 1, result is similar, as it is shown in figure 1, the reaction of restructuring mouse CD74 albumen and antibody has good concentration-dependent relation, and become obvious linear correlation.
Embodiment 4: the specificity of the ELISA kit of little mouse soluble type CD74 albumen and sensitivity assessment
Mouse model of acute lung injury set up list of references (XinqiPeng, etal.Protectiveeffectsofsphingosine1-phosphateinmurineen dotoxin-inducedinflammatorylunginjury.AmJRespirCritCareM ed.2004,169 (11): 1245-51.) described in, blood extracting method: with 1ml syringe through heart puncturing extracting blood (0.5-0.8ml) after mouse anesthesia, room temperature is placed the centrifugal 10min of 1h, 1500g and is taken serum. Bronchoalveolar lavage fluid acquisition method: after mice takes blood, row tracheotomy, inject 0.8ml normal saline through air flue, pumpback after repeatedly aspirating 3 times, 2000r4 DEG C of centrifugal 10min associated protein to be detected.
ELISA experimental procedure is (embodiment 1: experimental technique) as previously mentioned;
Dotblot experimental procedure:
1. soaking nitrocellulose filter 1 minute with formaldehyde, 1 �� TBST (1 �� TBS is containing 1 �� Tween-20) washs 3min, and room temperature is placed air-dry;
2. PBSPH7.4 is diluted the sample liquid 2ul point of (2 ��) on nitrocellulose filter, put in 37 DEG C of incubators and dry 20��30 minutes;
3. close 30min, 1 �� TBST by confining liquid (1 �� TBST containing 5% defatted milk powder) room temperature to wash 3 times;
4.5%BSA (1 �� TBST containing 5%BSA) dilutes 4 DEG C of overnight incubation of primary antibodie (Ii-1, extension rate 1:500);
5.1 �� TBST washs 3 times, anti-(goatanti-ratHRP-linkedantibody, extension rate 1:2000) the incubated at room 1h of 5%BSA dilution two;
6.1 �� TBST washs 3 times;
7. robotics light-emitting appearance development (ECLWesternBlotting detectable).
One, specific test
The ELISA method set up by the present invention detects the mice with acute lung injury (after air flue instills LPS, 6h, 12h draw materials) of 6 LPS inductions and the serum sample of 4 blank mices, and result is Fig. 2 A such as. Being verified by Dotblot method further, detect with Ii-1, the Virus monitory of mice with acute lung injury goes out CD74 albumen relatively blank group is obvious as shown in Figure 2 B. Western blot method is similar to ELISA method result.
Detect the mice with acute lung injury (after air flue instills LPS, 6h, 12h draw materials) of 6 LPS inductions and the BALF sample of 6 blank mices by the ELISA method set up, result is Fig. 3 A such as. Being verified by Dotblot method further, detect with Ii-1, the BALF of mice with acute lung injury can detect that CD74 albumen, blank group fail to detect obvious CD74 albumen as shown in Figure 3 B. Western blot method is similar to ELISA method result.
Two, sensitivity tests
The CD74 standard protein (initial concentration 25ng/ml) of restructuring is done 2 multiple proportions serial dilutions with 1 �� PBS, is arranged side by side 5 multiple holes, show that the mean OD value of each dilution point of standard protein has the most highly diluted multiple of significant difference with blank OD value of organizing. As shown in Figure 4, the OD value after CD74 standard protein dilution 256 times still has significant difference with blank group OD value, it was shown that detectable soluble type CD74 albumen least concentration is 781.25pg/ml.
Soluble type CD74 protein concentration in the serum of the mice with acute lung injury of embodiment 5:ELISA test kit detection LPS induction and BALF
The foundation of Mouse model of acute lung injury, serum, bronchoalveolar lavage fluid collection, method of protein detection ditto described in.
Brood C57B/6 mice is randomly divided into blank group, LPS acute lung injury 6h group, LPS acute lung injury 12h group (5/group), blood and BALF is taken at corresponding time point, according to the operating procedure of embodiment 1, soluble type CD74 protein concentration in detection serum and BALF.
Result as it is shown in figure 5, in mice with acute lung injury serum and BALF soluble type CD74 content be above matched group, and in serum the content 12h group of soluble type CD74 higher than 6 groups.
The ultimate principle of the present invention, principal character and advantages of the present invention have more than been shown and described. Skilled person will appreciate that of the industry; the present invention is not restricted to the described embodiments; described in above-described embodiment and description is that principles of the invention is described; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements both fall within the claimed scope of the invention. Claimed scope is defined by appending claims and equivalent thereof.
Claims (9)
1. the ELISA detection kit of one kind little mouse soluble type CD74 albumen, it is characterised in that this test kit includes:
It is coated the little mouse CD74 antibody of ELISA ELISA Plate, for sheep antimouse CD74 protein polyclone antibody;
Detect the antibody of little mouse soluble type CD74, for rat anti-mouse CD74 protein monoclonal antibody;
ELIAS secondary antibody, for the mountain goat anti rat IgG of horseradish peroxidase-labeled;
Standard protein, for recombined small-mouse CD74 albumen;
The described AF7478 that sheep antimouse CD74 protein polyclone antibody is R&DSystems company, working concentration is 0.25 �� g/ml;
The described MAB7478 that rat anti-mouse CD74 protein monoclonal antibody is R&DSystems company, working concentration is 1 �� g/ml.
2. the ELISA detection kit of a kind of little mouse soluble type CD74 albumen according to claim 1, it is characterised in that the #7077 that mountain goat anti rat IgG is CellSignaling company of described horseradish peroxidase-labeled.
3. the ELISA detection kit of a kind of little mouse soluble type CD74 albumen according to claim 1, it is characterised in that the described 7478-CD that recombined small-mouse CD74 albumen is R&DSystems company.
4. the ELISA detection kit of a kind of little mouse soluble type CD74 albumen according to any one of claims 1 to 3, it is characterized in that, described test kit, also include: sample diluting liquid, be coated buffer, confining liquid, ELISA ELISA Plate eluent, antibody diluent, nitrite ion and stop buffer.
5. the ELISA detection kit of a kind of little mouse soluble type CD74 albumen according to claim 4, it is characterised in that described sample diluting liquid: when sample is serum or bronchoalveolar lavage fluid, sample diluting liquid is 1 �� PBS, pH:7.4.
6. the ELISA detection kit of a kind of little mouse soluble type CD74 albumen according to claim 4, it is characterised in that described confining liquid, for the SuperBlock Block buffer of Thermo company.
7. the ELISA detection kit of a kind of little mouse soluble type CD74 albumen according to claim 4, it is characterised in that the described buffer that is coated is 1 �� PBS, pH:7.4; Described ELISA ELISA Plate eluent is 1 �� PBS solution containing 0.05%Tween-20; Described antibody diluent is 1 �� PBS, pH:7.4; Described nitrite ion is the TMBsubstratekit of Thermo company; Described stop buffer is the sulfuric acid solution of 2mol/L.
8. the detectable of a little mouse soluble type CD74 albumen application in preparing ELISA detection kit as claimed in claim 1, it is characterised in that ELISA detection method comprises the steps:
It is coated ELISA ELISA Plate with CD74 protein polyclone antibody, after confining liquid is closed, it is separately added into the diluent of standard recombined small-mouse CD74 albumen and testing sample, add CD74 protein monoclonal antibody, then add ELIAS secondary antibody, add substrate colour developing after washing, under 450nm wavelength, measure OD value by microplate reader, the standard curve of preparation standard recombined small-mouse CD74 albumen, calculates the concentration of soluble type CD74 albumen in testing sample by standard curve.
9. the detectable of little mouse soluble type CD74 albumen according to claim 8 application in preparing ELISA detection kit as claimed in claim 1, it is characterised in that ELISA detection method is:
Being coated buffer and AF7478 is diluted to 0.25 �� g/ml, add 100 �� l in every hole of ELISA ELISA Plate, shrouding is placed on 4 DEG C of overnight incubation; With the ELISA ELISA Plate eluent of fresh configuration, every hole 300 �� l, automatic plate washer Elx50 wash 3-5 time, and absorbent paper pats dry; Add ELISA ELISA Plate confining liquid, every hole 200 �� l, incubated at room 2 hours; The same method ELISA ELISA Plate eluent, every hole 300 �� l, washs 3-5 time, pats dry; It is put in 4 DEG C, standby;
Serum sample or BALF sample, dilute with 1:2 with sample diluting liquid, with the volume application of sample in 100 �� l/ holes, incubated at room 2 hours; The same method ELISA ELISA Plate eluent, every hole 300 �� l, washs 3-5 time, pats dry; MAB7478 is diluted to 1 �� g/ml, every hole 100 �� l, incubated at room 2 hours; The same method ELISA ELISA Plate eluent, every hole 300 �� l, washs 3-5 time, pats dry; Add the mountain goat anti rat IgG of HRP labelling, every hole 100 �� l, incubated at room 1 hour; The same method ELISA ELISA Plate eluent, every hole 300 �� l, washs 5-7 time, pats dry; Add substrate tetramethyl benzidine and H2O2, every hole 100 �� l, room temperature lucifuge is hatched 10-15 minute, adds 2mol/L sulfuric acid solution and terminates reaction, every hole 50 �� l; In microplate reader, 450nm measures OD value.
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| CN1649902A (en) * | 2002-03-01 | 2005-08-03 | 免疫医疗公司 | Internalizing anti-CD74 antibodies and methods of use |
| CN103207277A (en) * | 2013-03-28 | 2013-07-17 | 中国人民解放军第二军医大学 | ELISA test kit of human-derived soluble CD74 protein and detection method thereof |
| CN103458930A (en) * | 2011-02-01 | 2013-12-18 | 根马布股份公司 | Human antibodies and antibody-drug conjugates against CD74 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1649902A (en) * | 2002-03-01 | 2005-08-03 | 免疫医疗公司 | Internalizing anti-CD74 antibodies and methods of use |
| CN103458930A (en) * | 2011-02-01 | 2013-12-18 | 根马布股份公司 | Human antibodies and antibody-drug conjugates against CD74 |
| CN103207277A (en) * | 2013-03-28 | 2013-07-17 | 中国人民解放军第二军医大学 | ELISA test kit of human-derived soluble CD74 protein and detection method thereof |
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| The Role of Macrophage Migration Inhibitory Factor in Autoimmune Liver Disease;David N. Assis et al;《HEPATOLOGY》;20131220;第59卷(第2期);580-591 * |
| V. Rebmann et al.Biochemical analysis of plasma-soluble invariant chains and their complex formation with soluble HLA-DR.《Tissue antigens》.1997,第49卷(第5期), * |
| 检测血浆 sCD74 有助于肺癌诊断;祁永健等;《南京医科大学学报(自然科学版)》;20080531;第28卷(第5期);667-669 * |
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