CN110095605A - A kind of kit for screening of lung cancer - Google Patents

A kind of kit for screening of lung cancer Download PDF

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Publication number
CN110095605A
CN110095605A CN201910347649.3A CN201910347649A CN110095605A CN 110095605 A CN110095605 A CN 110095605A CN 201910347649 A CN201910347649 A CN 201910347649A CN 110095605 A CN110095605 A CN 110095605A
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Prior art keywords
phosphorylation
reagent
lung cancer
blk
expression
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CN201910347649.3A
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CN110095605B (en
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李丽
杨瑛
张立
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West China Hospital of Sichuan University
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West China Hospital of Sichuan University
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84Systems specially adapted for particular applications
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531Production of immunochemical test materials
    • G01N33/532Production of labelled immunochemicals
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57423Specifically defined cancers of lung
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484Immunoassay; 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/302Stain compositions

Abstract

The invention discloses a kind of kit for screening of lung cancer, it includes optional for detecting the reagent of phosphorylation BLK expression.The invention also discloses the reagents of detection phosphorylation BLK expression to prepare the purposes in screening lung cancer reagent.Kit of the present invention can effectively judge the risk that crowd to be checked suffers from lung cancer, can be used for the auxiliary diagnosis of clinical lung cancer by the expression of detection phosphorylation BLK, take relevant remedy measures or decision to provide effective foundation for patient, potential applicability in clinical practice is good.

Description

A kind of kit for screening of lung cancer
Technical field
The present invention relates to a kind of kit for screening of lung cancer.
Background technique
Lung cancer is that morbidity and mortality growth is most fast, to one of population health and the maximum malignant tumour of life threat. Many countries all report that the morbidity and mortality of lung cancer obviously increase in the past 50 years, and male lung cancer morbidity and mortality are equal First of all malignant tumours is accounted for, women disease incidence accounts for second, and the death rate accounts for second.Non-small cell type lung cancer is lung cancer In one kind, account for about the 80% of all lung cancer, when about 75% Finding case has been in middle and advanced stage, and survival rate is very low within 5 years.Cause This, it is extremely important to lung cancer progress early stage, quick diagnosis.
The screening of lung cancer, referring to does not have the related indication crowd of lung cancer to carry out routine examination those, before there is symptom It was found that lung cancer.If the lung cancer molecular marker inside blood can be found, for prompting clinician to take patient early stage Relevant remedy measures or decision have great importance.
Currently, the diagnosis of lung cancer all relies on greatly In vivo detection lung tissue, but such detection method is complicated for operation, It is very big to the injury of lungs of patient, and it is likely to result in lung cancer diffusion, there are great risks.Searching is more easy, wound is small, The detection method of Small side effects is urgently to be resolved.
Phosphorylation BLK is the bone-marrow-derived lymphocyte tyrosine kinases of phosphorylation.It now there are no document report and pass through detection phosphoric acid Change BLK to detect lung cancer.
Summary of the invention
To solve the above-mentioned problems, small, Small side effects that the present invention provides a kind of wounds, lung cancer detection examination easy to use Agent box.
The kit for screening of lung cancer of the present invention, it includes optional for detecting the reagent of phosphorylation BLK expression.
Wherein, the reagent is the reagent for detecting phosphorylation BLK expression in serum and/or lung tissue.
Wherein, the reagent of the detection phosphorylation BLK expression is ELISA detection reagent.
Wherein, the reagent of the detection phosphorylation BLK expression is Western-blot detection method reagent.
The present invention also provides the reagents of detection phosphorylation BLK expression to prepare the use in screening lung cancer reagent On the way.
Wherein, the reagent of the detection phosphorylation BLK expression is ELISA detection reagent.
Wherein, the reagent of the detection phosphorylation BLK expression is Western-blot detection method reagent.
Wherein, the reagent is the reagent for detecting phosphorylation BLK expression in serum and/or lung tissue.
The expression that kit of the present invention passes through detection phosphorylation BLK, it can be determined that crowd to be checked suffers from the risk of lung cancer, Can be used for the auxiliary diagnosis of clinical lung cancer, kit of the present invention only needs the blood of acquisition sample to be examined, so that it may screening its Suffer from the risk of lung cancer, wound is small, Small side effects, easy to use, and potential applicability in clinical practice is good.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention The technology realized all belongs to the scope of the present invention.
Specific embodiment
The expression of 1 phosphorylation BLK of embodiment and the relationship of lung cancer
One, experimental method
1, clinical data
Patients with lung cancer paraffin section 30 are chosen, distal end control 30 (refers to the distal end normal lung tissue paraffin of patients with lung cancer Slice, is confirmed as normal tissue apart from cancerous tissue 5cm, and through pathology), essential information is shown in Table 1.
1 clinical elementary information of table
*Note: other include large cell carcinoma, adenosquamous carcinoma, small cell carcinoma.
Paraffin section is compareed to patients with lung cancer paraffin section, remote organization, is carried out using phosphorylation BLK as the immune group of index Change (IHC).
Phosphorylation BLK antibody: U.S. Affinity company, article No. AF8133 are purchased from;
EnVisionTMHRP secondary antibody, antibody diluent, DAB color developing agent, antigen retrieval buffers, Wash Buffer, immune group Change pen: being purchased from Dako company, Denmark.
The expression of phosphorylation BLK is detected as follows:
(1) it closes peroxidase: paraffin section being taken to dewax, (dewaxing process is as follows: dimethylbenzene to aquation for dewaxing I10min → dimethylbenzene II10min → the ethyl alcohol of dehydrated alcohol 5min → 95% ethyl alcohol of 5min → 85% ethyl alcohol of 5min → 75% After 5min), 3%H is used2O2Solution closes endogenous peroxydase in dark place, is incubated at room temperature 15min;
(2) distilled water rinses: slice is placed in distilled water, is washed 5min/ times on shaking table, altogether three times;
(3) antigen retrieval: slice is put into the EDTA antigen retrieval solution of pH9.0, the water-bath 40min in 98 DEG C of water-baths, Carry out antigen retrieval;
(4) distilled water rinses: taking out and is sliced, after cooled to room temperature, is washed three times with distillation, then use Wash Buffer rinsing is primary;
(5) it is incubated for primary antibody: being enclosed tissue with immunohistochemistry pen, phosphorylation BLK antibody working solution (phosphoric acid is added dropwise in circle Change BLK to mix with antibody diluent 1:150), 4 DEG C of overnight incubations;
(6) distilled water rinses: with distillation washing three times, Wash Buffer rinses primary;
(7) secondary antibody is added dropwise: the secondary antibody working solution of Dako HRP label is added dropwise, is incubated at room temperature 60min;
(8) distilled water rinses: distillation washing 3 times, each 5min;
(9) DAB develops the color: chromogenic substrate DAB is added dropwise, observation colour developing situation, terminates reaction in distilled water under the microscope;
(10) haematoxylin is redyed: slice is put into haematoxylin dye liquor and dyes 2min, after the differentiation of 1% hydrochloride alcohol, and flowing water Rinse 10min;
(11) it is dehydrated transparent: being sliced, Yu Tongfeng transparent through 75%, 85%, 95%, 100% gradient alcohol dehydration, dimethylbenzene It is dry in cupboard;
(12) it mounting: with neutral Instant cement mounting, observes under an optical microscope, DP Controller Image Acquisition system System adopts figure.After adopting figure, immunohistochemistry is given according to the tumour cell staining power and positive area of patients with lung cancer and remote organization Scoring.
3, immunohistochemistry standards of grading
First according to the form below provides the scoring of tumour cell staining power, positive area:
@Note: it is independently scored tumour cell staining power result by two veteran pathologists
Calculate the product (0-9 point) of the scoring of tumour cell staining power and positive area scoring again, it is specified that:
Phosphorylation BLK expression is negative: 0 point;
Phosphorylation BLK expression is low positive: 1-3 points;
Phosphorylation BLK expression High positive > 3 divides.
4, interpretation of result
Statistical analysis is carried out using SPSS17.0 cancerous lung tissue group and remote organization's control group.
Two, experimental result
Cancerous lung tissue compareed with remote organization in the expression testing result of phosphorylation BLK be shown in Table 2.
The expression of 2 phosphorylation BLK of table
As can be seen from Table 2, the positive expression rate of phosphorylation BLK is 6.7% in the normal tissue of distal end, High positive expression rate is 0%;Phosphorylation BLK positive expression rate in cancerous lung tissue is 93.3%, and High positive expression rate is 60%;Phosphorylation BLK exists It is significantly increased in cancerous lung tissue, compared with the normal tissue of distal end, phosphorylation BLK expression difference has statistical significance (P < 0.0001)。
As can be seen from the above results, compared with Ai Pang normal lung tissue, the phosphorylation BLK expression of cancerous lung tissue is aobvious It writes and increases (P < 0.0001), illustrate that lung cancer is positively correlated with phosphorylation BLK expression, the high expression of phosphorylation BLK can be significant A possibility that lung cancer, is suffered from raising.Since the phosphorylation BLK level of Ai Pang normal lung tissue can reflect the phosphoric acid of Normal Lung tissue It is horizontal to change BLK, therefore, can be gone out Susceptible population's screening of lung cancer by the expression of the phosphorylation BLK of detection person to be checked Come.
2 screening lung cancer kit of embodiment and its application method
One, kit forms
Detection kit (50 person-portion):
Component Volume
Phosphorylation BLK antibody 100μl
EnVisionTMHRP secondary antibody 10ml
Antibody diluent 10ml
DAB color developing agent 10ml
Antigen retrieval buffers 500ml
Wash Buffer* 500ml
Immunohistochemistry pen 1
Note: it is based on 100 μ l that all kinds of experiment reagents, which are added dropwise, by every slice in this kit, and specific dosage can be according to tissue size Appropriate increase and decrease.
Two, the application method of kit
By sample to be examined lung tissue, paraffin section is prepared, as detection sample, detects phosphorylation as follows The expression of BLK:
(1) it closes peroxidase: paraffin section being taken to dewax, (dewaxing process is as follows: dimethylbenzene to aquation for dewaxing I10min → dimethylbenzene II10min → the ethyl alcohol of dehydrated alcohol 5min → 95% ethyl alcohol of 5min → 85% ethyl alcohol of 5min → 75% After 5min), endogenous peroxydase is closed in dark place with 3%H2O2 solution, is incubated at room temperature 15min;
(2) distilled water rinses: slice is placed in distilled water, is washed 5min/ times on shaking table, altogether three times;
(3) antigen retrieval: slice is put into the EDTA antigen retrieval solution of pH9.0, the water-bath 40min in 98 DEG C of water-baths, Carry out antigen retrieval;
(4) distilled water rinses: taking out and is sliced, after cooled to room temperature, is washed three times with distillation, then use Wash Buffer rinsing is primary;
(5) it is incubated for primary antibody: being enclosed tissue with immunohistochemistry pen, phosphorylation BLK antibody working solution (phosphoric acid is added dropwise in circle Change BLK to mix with antibody diluent 1: 150), 4 DEG C of overnight incubations;
(6) distilled water rinses: with distillation washing three times, Wash Buffer rinses primary;
(7) secondary antibody is added dropwise: the secondary antibody working solution of Dako HRP label is added dropwise, is incubated at room temperature 60min;
(8) distilled water rinses: distillation washing 3 times, each 5min;
(9) DAB develops the color: chromogenic substrate DAB is added dropwise, observation colour developing situation, terminates reaction in distilled water under the microscope;
(10) haematoxylin is redyed: slice is put into haematoxylin dye liquor and dyes 2min, after the differentiation of 1% hydrochloride alcohol, and flowing water Rinse 10min;
(11) it is dehydrated transparent: being sliced, Yu Tongfeng transparent through 75%, 85%, 95%, 100% gradient alcohol dehydration, dimethylbenzene It is dry in cupboard;
(12) it mounting: with neutral Instant cement mounting, observes under an optical microscope, DP Controller Image Acquisition system System adopts figure.After adopting figure, immunohistochemistry is given according to the tumour cell staining power and positive area of patients with lung cancer and remote organization Scoring, methods of marking are shown in embodiment 1.
To pulmonary abnormalities person, abnormal position, normal portions tissue can be taken respectively, compare phosphorylation BLK expression, in turn A possibility that it suffers from lung cancer is evaluated, the auxiliary diagnosis means as clinical lung cancer.
To sum up, kit of the present invention, can be with screening crowd's trials and tribulations lung cancer to be checked by the expression of detection phosphorylation BLK Risk, can be used for the auxiliary diagnosis of clinical lung cancer, for patient take relevant remedy measures or decision provide effectively according to According to potential applicability in clinical practice is good.

Claims (8)

1. a kind of screening lung cancer kit, it is characterised in that: it includes optional for detecting the examination of phosphorylation BLK expression Agent.
2. kit for screening according to claim 1, it is characterised in that: the reagent is for detecting serum and/or lung The reagent of phosphorylation BLK expression in tissue.
3. kit for screening according to claim 1 or 2, it is characterised in that: the detection phosphorylation BLK expression Reagent is ELISA detection reagent.
4. kit for screening according to claim 1 or 2, it is characterised in that: the detection phosphorylation BLK expression Reagent is Western-blot detection method reagent.
5. the reagent of detection phosphorylation BLK expression is preparing the purposes in screening lung cancer reagent.
6. purposes according to claim 5, it is characterised in that: it is described detection phosphorylation BLK expression reagent be Reagent is used in ELISA detection.
7. purposes according to claim 5, it is characterised in that: it is described detection phosphorylation BLK expression reagent be Western-blot detection method reagent.
8. according to the described in any item purposes of claim 5~7, it is characterised in that: the reagent be for detect serum and/or The reagent of phosphorylation BLK expression in lung tissue.
CN201910347649.3A 2018-04-27 2019-04-26 Lung cancer screening kit Active CN110095605B (en)

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CN201810395698X 2018-04-27

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012125712A2 (en) * 2011-03-14 2012-09-20 Respira Health, Llc Lung tumor classifier for current and former smokers
CN106918698A (en) * 2015-12-25 2017-07-04 广州瑞博奥生物科技有限公司 A kind of phospho-AB chip agent box for detecting human receptor tyrosine kinase enzyme

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012125712A2 (en) * 2011-03-14 2012-09-20 Respira Health, Llc Lung tumor classifier for current and former smokers
CN106918698A (en) * 2015-12-25 2017-07-04 广州瑞博奥生物科技有限公司 A kind of phospho-AB chip agent box for detecting human receptor tyrosine kinase enzyme

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HONG-REN YU ET AL: "Distinct expressions of immunoglobulin signal related molecules between human Kawasaki disease and BALB/c mice treated with Lactobacillus casei cell wall extract", 《INT J CLIN EXP MED》 *

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