CN105866430A - Method for detecting chitinase-3-like protein 1 in human serum and application of method - Google Patents
Method for detecting chitinase-3-like protein 1 in human serum and application of method Download PDFInfo
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- CN105866430A CN105866430A CN201610305748.1A CN201610305748A CN105866430A CN 105866430 A CN105866430 A CN 105866430A CN 201610305748 A CN201610305748 A CN 201610305748A CN 105866430 A CN105866430 A CN 105866430A
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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Abstract
The invention discloses a method for detecting chitinase-3-like protein 1 in human serum and application of the method. The method includes: washing Dynabeads Protein G with ultrapure water, rewashing with EBC lysis buffer, dissolving in the EBC lysis buffer to obtain two parts, incubating the two parts of Dynabeads Protein G solution respectively, mixing incubation products, removing supernate from a product, washing three times with EBC lysis solution to obtain precipitate, resuspending the precipitate with EBC lysis buffer, adding SDS sampling buffer, boiling, and detecting chitinase-3-like protein 1 (CHI3L1) by means of Western Blot process. The method of the invention is simple and effective, high in specificity and very high in accuracy; therefore, the defects such as high background and low accuracy in traditional ELISA process for detecting proteins in serum are avoided.
Description
Technical field
The present invention relates to detection method and the application thereof of chitinase 3 sample albumen 1 in human serum, belonged in a kind of serum
Albumen is as the application process of one of diagnosing tumor index.
Background technology
In serum, the content of some albumen can be as the index of medical diagnosis on disease, such as alpha-fetoprotein etc..And present stage is normal
Detection serum in the method for albumen be mainly ELISA double-antibody method, it is mainly by by the antibody of protein-specific
It is fixed on orifice plate, serum is added orifice plate and hatches, add that special one is anti-makes it be combined with each other with antigen afterwards, finally lead to
Cross the method reading numerical values of colour developing, calculate albumen content in serum.Another kind of method is directly serum to be carried out Western
Blot tests, testing goal albumen.
The antibody being fixed on orifice plate in above first method also can by with two anti-bindings, form colored product
Thing, therefore can cause Experimental Background value high, and accuracy is low.Due to albuminous existence a large amount of in serum in second method,
Cause the albumen in serum to be difficult to carry out polyacrylamide gel electrophoresis, and require that destination protein content in serum is higher.But
That the protein content in serum is the most relatively low, extensively application in this way receive certain restriction.There is presently no one
The method of chitinase 3 sample albumen 1 (CHI3L1) in detection serum that kind can be the most accurate and sensitive, and CHI3L1 albumen could
Marker protein as diagnosing tumor is also unknown.
Summary of the invention
In order to solve problem present in background technology, it is an object of the invention to overcome present in prior art above-mentioned
Deficiency, and provide detection method and the application thereof of chitinase 3 sample albumen 1 in a kind of human serum, not only can detect exactly
The content of CHI3L1 albumen in serum, it is possible to using the content of CHI3L1 albumen in human serum as one of the index of diagnosing tumor.
The present invention solves the technical scheme that the problems referred to above are used and comprises the following steps:
1) 40～60 μ l Dynabeads Protein G are taken, after ultra-pure water cleans three times, with the EBC lysate of 1ml again
Cleaning twice, the Dynabeads Protein G after cleaning is dissolved in the EBC lysate of 200 μ l and is divided into two parts；
2) two parts of Dynabeads Protein G solution are hatched process respectively, hatch acquisition product in mixing；
3) by step 2) product that obtains abandons supernatant, cleans three times with EBC lysate, and the precipitation obtained is with 50～100 μ l
EBC lysate is resuspended, adds SDS sample-loading buffer and boils 10min at 100 DEG C；
4) with Western blot method detecting step 3) product that obtains, detects CHI3L1 albumen.
Described step 2) all processes hatched all carry out at 4 DEG C, it is ensured that the activity of albumen；All systems hatched
In all contain protease inhibitor, it is ensured that albumen is not degraded.
Described step 2) particularly as follows:
2.1) take 50～100 μ l fresh or the frozen normal person in-80 DEG C of refrigerators or affinity antibody to SpA, with 900 μ
L EBC lysate mixes, and mixes with a copy of it Dynabeads Protein G solution, 4 DEG C of jogs 1～2h；
2.2) another part of Dynabeads Protein G solution adds EBC lysate polishing to 1ml, afterwards with CHI3L1 egg
White antibody mixing, 4 DEG C of jogs 1～2h；
2.3) collect step 2.1) supernatant fraction and step 2.2) sediment fraction, by both mix, 4 DEG C of jog mistakes
Night.
Described step 3) in resuspended after the volume ratio of solution and SDS sample-loading buffer be 4:1.
Described EBC lysate is with water as solvent, and solute includes Tris-HCl (pH 8.0), the NaCl of 120mM of 50mM
PMSF with 2mM.
The Dynabeads Protein G amount that the present invention needs is 40～60 μ l, and the as easy as rolling off a log configuration of EBC lysate.
The present invention needs 50～100 human serums a small amount of for μ l can accurately detect in normal person and affinity antibody to SpA
The difference of CHI3L1 protein content, and serum can be stored in-80 DEG C of refrigerators for a long time.
The antibody of described destination protein is easily prepared, and measures big and low cost.
Described step 2.1) in eliminate the immunoglobulin that human serum is endogenous, therefore in the final sample obtained, component is more
Adding simple (only comprising the antibody of destination protein and destination protein), electrophoretic band becomes apparent from, and data are more accurate.
The present invention compared with prior art, has the advantage that
The inventive method is simply effective, and specificity is good, eliminates the interference of the endogenous immunoglobulin of serum and serves
The effect of the CHI3L1 albumen of trace in enrichment serum, accuracy rate is high.Therefore the present invention not only solves existing traditional use
Method of protein detection in serum and the shortcoming such as reasons for its use is high, and accuracy is low, also can examine CHI3L1 albumen as tumor
Disconnected mark.
Accompanying drawing explanation
Fig. 1 is the testing result histogram of the Western blot method of the embodiment of the present invention 1 product.
Fig. 2 is the testing result histogram of the Western blot method of the embodiment of the present invention 2 product.
Detailed description of the invention
Below in conjunction with the accompanying drawings and the present invention is described in further detail by specific embodiment, following example are to the present invention
Explanation and the invention is not limited in following example.
First embodiments of the invention take the normal person collected on a small quantity or affinity antibody to SpA is diluted in buffer, with
Dynabeads Protein G is hatched under cryogenic.Collect the supernatant after hatching, the CHI3L1 protein antibodies with coupling
Dynabeads Protein G mixes, overnight incubation again under cryogenic conditions.Clean Dynabeads Protein G afterwards, will
Foreign protein cleans up, and the CHI3L1 albumen one that the product finally obtained comprises Dynabeads Protein G and coupling thereof resists
With CHI3L1 albumen.This product is carried out the Western blot detection of CHI3L1 albumen, gets final product comparison of tumor patient with normal
The difference of the content of CHI3L1 albumen in human serum sample.
Embodiment 1
Step is as follows:
1) 40 μ l Dynabeads Protein G are taken, after ultra-pure water cleans three times, with EBC lysate (system: 50mM
Tris-HCl (pH 8.0), 120mM NaCl and 2mM PMSF) balance twice, it is divided into two parts, standby；
2) taking 50 normal persons fresh for μ l or Patients with Gastric Cancer serum, human serum is provided by hospital, with 900 μ l EBC lysates
Mixing, with step 1) in a Dynabeads Protein G mixing, 4 DEG C of jog 1h；
3) step 1) in another part of Dynabeads Protein G add EBC lysate polishing to 1ml, afterwards with
The antibody mixing of CHI3L1 albumen, 4 DEG C of jog 1h；
4) collect step 2) upper cleer and peaceful step 3) sediment fraction, by both mix, 4 DEG C of jogs are overnight；
5) by step 4) product that obtains abandons supernatant, and cleans three times with EBC lysate, and the precipitation finally obtained is with 50 μ l
EBC lysate is resuspended, adds 5 × SDS sample-loading buffer, and 100 DEG C are boiled 10min；
6) with Western blot method detecting step 5) product that obtains, used one resists for CHI3L1 antibody.
Result is as it is shown in figure 1, the CHI3L1 albumen that i.e. indicates of band therein.
In figure visible, calibration ordinary person in Patients with Gastric Cancer serum is high for CHI3L1 albumen, and illustrating can be by serum
CHI3L1 albumen is as the marker protein of diagnosing gastric cancer.
Embodiment 2
Step is as follows:
1) 60 μ l Dynabeads Protein G are taken, after ultra-pure water cleans three times, with EBC lysate (system: 50mM
Tris-HCl (pH 8.0), 120mM NaCl and 2mM PMSF) balance twice, it is divided into two parts, standby；
2) taking 100 normal person fresh for μ l or Serum of Patients With Breast Cancers, human serum is provided by hospital, splits with 900 μ l EBC
Solution liquid mix, with step 1) in a Dynabeads Protein G mixing, 4 DEG C of jog 1h；
3) step 1) in another part of Dynabeads Protein G add EBC lysate polishing to 1ml, afterwards with
The antibody mixing of CHI3L1 albumen, 4 DEG C of jog 1h；
4) collect step 2) upper cleer and peaceful step 3) sediment fraction, by both mix, 4 DEG C of jogs are overnight；
5) by step 4) product that obtains abandons supernatant, and cleans three times with EBC lysate, and the precipitation finally obtained is with 50 μ l
EBC lysate is resuspended, adds 5 × SDS sample-loading buffer, and 100 DEG C are boiled 10min；
6) with Western blot method detecting step 5) product that obtains, used one resists for CHI3L1 antibody.
Result is as in figure 2 it is shown, the CHI3L1 albumen that i.e. indicates of band therein.
In figure visible, calibration ordinary person in Serum of Patients With Breast Cancer is high for CHI3L1 albumen, and illustrating can be by serum
CHI3L1 albumen is as the marker protein of breast cancer diagnosis.
It will thus be seen that the inventive method is simply effective, specificity is good, and accuracy rate is high, and can be made by CHI3L1 albumen
Marker protein for diagnosing tumor.This provides diagnosis and the another strategy plan for the treatment of for a global tumor difficult problem.
Although the present invention is open as above with embodiment, but it is not limited to protection scope of the present invention, any ripe
Know the technical staff of this technology, in the change made without departing from the spirit and scope of the invention and retouching, all should be belonged to this
The protection domain of invention.
Claims (6)
1. a detection method for chitinase 3 sample albumen 1 in human serum, its feature is to comprise the following steps:
1) take 40～60 μ l Dynabeads Protein G, after ultra-pure water cleans three times, clean again with the EBC lysate of 1ml
Twice, the Dynabeads Protein G after cleaning is dissolved in the EBC lysate of 200 μ l, is divided into two parts；
2) two parts of Dynabeads Protein G solution are hatched process respectively, remix and hatch acquisition product；
3) by step 2) product that obtains abandons supernatant, cleans three times with EBC lysate, and the precipitation obtained is with 50～100 μ l EBC
Lysate is resuspended, adds SDS sample-loading buffer and boils 10min at 100 DEG C；
4) with Western blot method detecting step 3) product that obtains, detection chitinase 3 sample albumen 1 (CHI3L1).
The detection method of chitinase 3 sample albumen 1 in human serum the most according to claim 1, its feature is: described step
Rapid 2) all processes hatched all are carried out at 4 DEG C, all contain protease inhibitor in all systems hatched.
The detection method of chitinase 3 sample albumen 1 in human serum the most according to claim 1, its feature is: described step
Rapid 2) particularly as follows:
2.1) take 50～100 μ l normal person or Patients with Various Cancers, mix with 900 μ l EBC lysates, with a copy of it
Dynabeads Protein G solution mixes, 4 DEG C of jogs 1～2h；
2.2) another part of Dynabeads Protein G solution adds EBC lysate polishing to 1ml, afterwards with CHI3L1 albumen
Antibody mixes, 4 DEG C of jogs 1～2h；
2.3) collect step 2.1) supernatant fraction and step 2.2) sediment fraction, by both mix, 4 DEG C of jogs are overnight.
4., according to the detection method of chitinase 3 sample albumen 1 in the arbitrary described human serum of claims 1 to 3, its feature exists
In described step 3) in resuspended after the volume ratio of solution and SDS sample-loading buffer be 4:1.
5., according to the detection method of chitinase 3 sample albumen 1 in the arbitrary described human serum of claims 1 to 3, its feature exists
In: described EBC lysate is with water as solvent, and solute includes Tris-HCl (pH 8.0), NaCl and 2mM of 120mM of 50mM
PMSF.
6. an application for chitinase 3 sample albumen 1 in human serum, its feature is: for the finger as medical science diagnosing tumor
Mark.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111197040A (en) * | 2020-01-21 | 2020-05-26 | 福建亿彤生物科技有限公司 | Chitinase 3-like protein 1(CHI3L1) epitope peptide, antigen, antibody, application and kit |
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| CN1987464A (en) * | 2006-09-29 | 2007-06-27 | 浙江大学 | Method for using magnetic catching technology in NF-kB protein separation and detection |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111197040A (en) * | 2020-01-21 | 2020-05-26 | 福建亿彤生物科技有限公司 | Chitinase 3-like protein 1(CHI3L1) epitope peptide, antigen, antibody, application and kit |
| CN111197040B (en) * | 2020-01-21 | 2023-08-08 | 福建亿彤生物科技有限公司 | Chitinase 3-like protein 1 (CHI 3L 1) epitope peptide, antigen, antibody, application and kit |
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Application publication date: 20160817 |