CN110187113A - A kind of autoantibody joint-detection ELISA kit for esophageal squamous cell carcinoma early screening - Google Patents
A kind of autoantibody joint-detection ELISA kit for esophageal squamous cell carcinoma early screening Download PDFInfo
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- CN110187113A CN110187113A CN201910532284.1A CN201910532284A CN110187113A CN 110187113 A CN110187113 A CN 110187113A CN 201910532284 A CN201910532284 A CN 201910532284A CN 110187113 A CN110187113 A CN 110187113A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
Abstract
The invention belongs to medical oncology technical fields, specifically disclose a kind of autoantibody joint-detection ELISA kit for esophageal squamous cell carcinoma early screening, the kit includes solid phase carrier and the tumor associated antigen that is coated on solid phase carrier, and the tumor associated antigen is made of P53, TP53, CDKN2B and NPM1.Further, the kit further includes sample diluting liquid, secondary antibody, secondary antibody dilution, positive control serum, negative control sera, developing solution, terminate liquid and cleaning solution.ELISA kit of the invention can effectively detect esophageal squamous cell carcinoma, especially early stage esophageal squamous cell carcinoma, its detection sensitivity is up to 88.8%, specificity has reached 86.3%, the extensive screening that can be used for esophageal squamous cell carcinoma district occurred frequently Silent cerebral infarction is conducive to asymptomatic people at highest risk's esophageal squamous cell carcinoma screening and early detection.
Description
Technical field
The present invention relates to molecular biology and oncology, and in particular to it is a kind of for esophageal squamous cell carcinoma early screening from
The antibody combined detection ELISA kit of body.
Background technique
The cancer of the esophagus is one of most common malignant tumour in the whole world, and the death rate occupies the 6th, the annual new hair about 500,000 in the whole world
More than half in example patients with esophageal squamous cell carcinoma occurs in China, 100 times higher than western countries disease incidence.China is esophageal squamous cell carcinoma morbidity
Rate and the highest country of the death rate, China's about 250,000 newly-increased diagnosed case every year, it is new to account for whole world esophageal squamous cell carcinoma according to statistics
Increase more than half of total cases.In China, not only disease incidence is high for esophageal squamous cell carcinoma, and there is also significant areal variations, such as China
The north, the especially ground such as Linzhou City, Henan Province and its Huixian adjoined, Anyang, Ci County are esophageal squamous cell carcinoma disease incidence and death in the world
The highest area of rate.Since the pathogenesis of esophageal squamous cell carcinoma is unclear, while shortage refers to for the early detection sensitivity of esophageal squamous cell carcinoma
Mark and effective detection methods, for most of patient when being diagnosed, disease has been developed to middle and advanced stage, poor prognosis.Currently, food
Pipe squamous carcinoma is still one of this area's tumour correlation major causes of death, is caused to the Health and Living of local resident huge
It threatens.
Esophageal squamous cell carcinoma research important scientific issues early detection is the key that reduce esophageal squamous cell carcinoma morbidity and mortality；It is early
Phase discovery refers to asymptomatic severe precancerous lesion patient and early carcinoma patient.Currently, clinically 90% patients with esophageal squamous cell carcinoma
The main reason for having belonged to middle and advanced stage when medical, having led to this final result is patient's early stage without specific symptoms, lacks asymptomatic people at highest risk
The molecular target of early warning screening.Traditionally, " district occurred frequently, 40 years old or more, male, smoke, drink, family history positive it is asymptomatic
Crowd " is generally defined as the main object of " esophageal squamous cell carcinoma high-risk or High risk group " and esophageal squamous cell carcinoma early screening.Mesh
Before, Endoscopy and mucous membrane biopsy are the important screening methods of esophageal squamous cell carcinoma early detection.But Endoscopic Screening have wound, at
This height and low efficiency (for example, conventional district occurred frequently Endoscopic Screening people at highest risk, the discovery rate of early carcinoma only 2% or so, about 90% with
On asymptomatic people at highest risk be " accompany check "), these limit popularization and application of the Endoscopic Screening in Silent cerebral infarction.
Thus, effective esophageal squamous cell carcinoma specific molecular diagnosis marker is found for screening esophageal squamous cell carcinoma High risk group, to oesophagus
The early discovery of squamous carcinoma, early treatment, and then the existence and prognosis of raising patients with esophageal squamous cell carcinoma are of great significance.
Esophageal squamous cell carcinoma is participated in jointly by multiple tumor suppressor and oncogene, and interactive with environmental factor
The process of multistage evolution.Cancer cell, which can synthesize during its occurrence and development and release one group of its distinctive secretory, to be resisted
Original, i.e. tumor associated antigen (Tumor-associated Antigen, TAA), thus may have this in the serum of patient
The corresponding autoantibody of a little antigens.Some researchs in relation to liver cancer and colon cancer are it has been shown that using Enzyme-linked Immunosorbent Assay reality
The autoantibody for testing (Enzyme-linked Immunosorbent Assay, ELISA) method detection patient's body facilitates height
The early warning and screening of danger crowd.Simultaneously correlative study show tumour have heterogeneity, even if same tumour may also have it is different
Antigen presentation, people not yet find the single molecular marker for a certain tumour at present.Therefore, it is detected with single autoantibody
Method is compared, and with multiple tumor associated antigens come the expression of joint inspection patient's body autoantibody, is conducive to improve needle
To the recall rate of a certain tumour.However, for a variety of autoantibody associated detecting methods and phase of esophageal squamous cell carcinoma early diagnosis
Answer the application of kit also more rare.
This research filters out 4 kinds of tumor associated antigens using using proteomic techniques, and 4 kinds of tumor associated antigens are respectively
P53, TP53, CDKN2B and NPM1, and it was found that the autoantibody of this 4 kinds of tumor associated antigens is in early stage patients with esophageal squamous cell carcinoma
It can detect, and have higher specificity and sensibility.CDKN2B gene with the protein-interacting of comb more than two kinds by drawing
Play other Silenced gene transcriptions.There are three functional domains, the i.e. oligomeric domain of amino terminal, carboxyl terminal nucleic acid binding domain for NPM1 tool
With histone combination intermediate field.Amino-terminal domain is related to chaperone activity, helps to prevent the protein aggregation in kernel, promotees
Into the assembling of histone and nucleosome.TP53 gene is tumor suppressor gene, is divided into wild type and saltant type, when wild type TP53 gene
When point mutation, missing and inactivation occurs, i.e., saltant type is changed by wild type, promotes the occurrence and development of tumour instead.P53 base
Because as a kind of transcription factor, being one of the tumor suppressor gene found earliest, expressed by P53 albumen adjust Apoptosis mistake
It plays an important role in journey, there is apparent cancer suppressing action, research has confirmed, the generation of most of malignant tumours is all with P53's
It is mutated related.It is related with the early diagnosis of esophageal squamous cell carcinoma that the P53 of 4 kinds of TAA has a document report, but TP53, CDKN2B and
NPM1 yet there are no document report in the application of diagnosis esophageal squamous cell carcinoma.Come using this 4 kinds of TAA of P53, TP53, CDKN2B and NPM1
The expression of corresponding autoantibody also has not been reported in detection patients with esophageal squamous cell carcinoma serum.
Summary of the invention
Aiming at the problems and shortcomings existing in the prior art, the object of the present invention is to provide one kind for esophageal squamous cell carcinoma early stage
The autoantibody joint-detection ELISA kit of screening.
To realize goal of the invention, The technical solution adopted by the invention is as follows:
The combination of tumor associated antigen P53, TP53, CDKN2B and NPM1 are in the agent box that preparation is used for esophageal squamous cell carcinoma screening
Application.
A kind of autoantibody joint-detection ELISA kit for esophageal squamous cell carcinoma early screening, the kit include
Solid phase carrier and the tumor associated antigen being coated on solid phase carrier, the tumor associated antigen by P53, TP53, CDKN2B and
NPM1 composition.
According to above-mentioned autoantibody joint-detection ELISA kit, it is preferable that the kit further includes sample dilution
Liquid, secondary antibody, secondary antibody dilution, negative control sera, positive control serum, cleaning solution, developing solution and terminate liquid.More
Add preferably, the sample diluting liquid is PBST (phosphate tween) buffer containing 1% (W/V) BSA；The secondary antibody
Dilution is PBST (phosphate tween) buffer containing 1% (W/V) BSA；The developing solution is by developing solution A and developing solution B
Composition, the developing solution A are 0.02% (W/V) TMB (3,3',5,5'-tetramethylbenzidine), and the developing solution B is 0.006%
(W/V) urea peroxide；The terminate liquid is the sulfuric acid of 2mol/L；The cleaning solution is the PBST (phosphate containing 0.2% polysorbas20
Tween) buffer.
According to above-mentioned autoantibody joint-detection ELISA kit, it is preferable that the secondary antibody is with detectable
Marker.
According to above-mentioned autoantibody joint-detection ELISA kit, it is preferable that the marker is horseradish peroxidase
Enzyme.
According to above-mentioned autoantibody joint-detection ELISA kit, it is preferable that the secondary antibody is RecA albumen.
According to above-mentioned autoantibody joint-detection ELISA kit, it is preferable that the positive control serum is P53 sun
Property control serum, the negative control sera be P53 negative control sera.
According to above-mentioned autoantibody joint-detection ELISA kit, it is preferable that the P53 positive control serum is to make
It is positive patients with esophageal squamous cell carcinoma serum with indirect ELISA and Western blot method detection P53 antibody, the P53 yin
Property control serum be using indirect ELISA and Western blot method detection P53 antibody expression be normal population serum
The serum of the normal person of antibody average content.Numerous studies have clearly indicated that P53 antigen in the occurrence and development process of esophageal squamous cell carcinoma
In play highly important adjustment effect, and the expression with higher in patients with esophageal squamous cell carcinoma serum of P53 antibody.Present invention choosing
P53 Positive Sera is selected as positive control, P53 negative antibody serum is as negative control, positive control serum and feminine gender
Control serum is the serum by screening meticulously, the powers of other antigen-antibody reactions of same ELISA kit can be with
It is used as reference accordingly, achievees the purpose that Quality Control.
According to above-mentioned autoantibody joint-detection ELISA kit, it is preferable that the solid phase carrier is ELISA Plate.More
Add preferably, the ELISA Plate is 96 hole elisa Plates (totally 8 rows 12 arrange), and 96 hole elisa Plates are according to well-designed layout
(referring to fig. 2) this 4 kinds of tumor associated antigens of P53, TP53, CDKN2B and NPM1 are coated with, wherein each row is coated with a kind of antigen, often
Kind antigen coat is in 11 reacting holes.It is same that 96 hole elisa Plates are added after dilution in the blood serum sample of same test object
(hole A-D in the such as the 1st column, the hole E-H in the 1st column, the hole A-D in the 2nd column, the E-H in the 2nd column in continuous 4 holes of column
Hole, and so on), the ELISA test agent box can be used to detect simultaneously in 22 test serum samples 4 kinds of tumor associated antigens from
The expression of body antibody can carry out large-scale sample detection.Be equipped in 96 hole elisa Plates the 12nd column blank control wells,
Positive control wells and negative control hole, coating is free of the coating buffer of antigen in the blank control wells, the Positive control wells and
P53 antigen is coated in negative control hole.
According to above-mentioned autoantibody joint-detection ELISA kit, it is preferable that the autoantibody joint-detection
The test object of ELISA kit is human serum
Compared with prior art, the positive beneficial effect that the present invention obtains are as follows:
(1) present invention is combined this 4 kinds of TAA of P53, TP53, CDKN2B and NPM1 as one for the first time, joint-detection people's blood
The antibody expression of above-mentioned 4 kinds of TAA in clear can effectively detect esophageal squamous cell carcinoma, especially early stage esophageal squamous cell carcinoma, detection spirit
Sensitivity is up to 88.8% and (is correctly examined when being diagnosed i.e. in early stage patients with esophageal squamous cell carcinoma using this 4 tumor associated antigens
Breaking as the ratio of early stage esophageal squamous cell carcinoma is that 88.8%), specificity has reached 86.3% (4 kinds of tumours of i.e. non-patients with esophageal squamous cell carcinoma
When related antigen joint-detection, the ratio for being confirmed as being not suffering from esophageal squamous cell carcinoma person is 86.3%), to sieve much higher than existing clinical endoscopic
The recall rate of esophageal squamous cell carcinoma is looked into, therefore, ELISA kit of the invention sensitivity with higher and specificity can be used for eating
The extensive screening of pipe squamous carcinoma district occurred frequently Silent cerebral infarction, can greatly improve the recall rate of early stage esophageal squamous cell carcinoma, be conducive to nothing
Symptom people at highest risk screening and early detection thus greatly reduce the death rate of patients with esophageal squamous cell carcinoma, are patients with esophageal squamous cell carcinoma
Great happiness is brought with family.
(2) ELISA kit prepared by the present invention can detect the expression of 4 kinds of TAA antibody in blood serum sample simultaneously, with
The independent detection of 4 kinds of TAA antibody is compared, 4 kinds of antibody combined detections of TAA, and detection success rate is high, and technique reproducible is good, and consumptive material is few,
It is at low cost, it is easy to operate, it is easy to use, quick, greatly improve the detection efficiency and diagnosis efficiency of clinical esophageal squamous cell carcinoma, energy
It is enough to be promoted the use of in common lab.
Detailed description of the invention
Fig. 1 is Immunofluorescent antibody experimental principle figure.
Fig. 2 is antigen coat layout (wherein, the antigen title representative of 96 hole elisa Plates in ELISA kit of the present invention
The reacting hole has been coated with this antigen, and addition is test serum, for detecting the expression of corresponding antibodies in test serum；
"+" represents Positive control wells, and addition is positive control serum；"-" represents negative control hole, and addition is negative control blood
Clearly；" sky " represents blank control wells, which is added the Sample dilution for being free of serum, all the same, the blank control of other operations
For the background value during reaction experiment).
Fig. 3 is distribution map of the autoantibody of 4 kinds of tumor associated antigens in early stage esophageal squamous cell carcinoma group serum.
Fig. 4 is distribution map of the autoantibody of 4 kinds of tumor associated antigens in control group serum.
Fig. 5 is positive rate result figure of 4 kinds of TAA autoantibodies in control group and early stage esophageal squamous cell carcinoma group.
Fig. 6 is the ROC curve figure that 4 kinds of TAA autoantibodies detect early stage esophageal squamous cell carcinoma.
Specific embodiment
Below by way of specific embodiment, invention is further described in detail, but does not limit the scope of the invention.
Experimental method described in the following example is unless otherwise specified the art routine techniques, or according to life
Produce condition proposed by manufacturer；Production firm person is not specified in agents useful for same, material and instrument, and being can be by commercially available acquisition
Conventional products.
The present invention, which is prepared for one kind according to the principle of Dot-ELISA, can be used for early stage esophageal squamous cell carcinoma screening and diagnosis
Autoantibody joint-detection ELISA kit.The principle of Dot-ELISA is that antigen is connected on solid phase carrier, sample
In product test antibodies it is in combination at solid phase antigen-by inspection antibody complex, then with ELIAS secondary antibody and solid phase antigen-by examining antibody
Antibody in compound combines, and forms solid phase antigen-by inspection antibody-ELIAS secondary antibody compound, then measures aobvious after adding substrate
Color degree determines test antibodies content (referring to Fig. 1).
Embodiment 1: the preparation of kit
1, experimental material and reagent:
(1) 4 kind of tumor-associated antigen protein (P53, TP53, CDKN2B and NPM1), purchase have in Wuhan Amy victory science and technology
Limit company；
(2) 96 hole elisa Plates: 3590 (U.S. costar.)；
(3) coating buffer: 50mM carbonate buffer solution, pH=9.6；
(4) confining liquid: contain the PBST buffer of 2% (W/V) BSA；
(5) sample diluting liquid: contain the PBST buffer of 1% (W/V) BSA；
(6) secondary antibody dilution: contain the PBST buffer of 1% (W/V) BSA；
(7) enzyme mark secondary antibody: the RecA albumen (Invitrogen company) of horseradish peroxidase-labeled；
(8) cleaning solution: PBST (phosphate tween) buffer containing 0.2% polysorbas20；
(9) positive control serum: P53 positive control serum is detected using indirect ELISA and Western blot method
P53 antibody is positive patients with esophageal squamous cell carcinoma serum；
(10) negative control sera: P53 negative control sera is examined using indirect ELISA and Western blot method
Survey the serum for the normal person that P53 antibody expression is normal population serum antibody average content；
(11) developing solution A:0.02% (W/V) TMB is prepared: being taken methyl biphenyl amine (TMB) 0.005g, be dissolved in 25ml and go
In ionized water；
(12) developing solution B:0.006% (W/V) urea peroxide element is prepared: taking citric acid 4.665g, Na2HPO418.40g,
It is completely dissolved in 400ml deionized water, adds 0.75% urea peroxide 3.2ml, adjust pH value to 5.0-5.5, add deionized water
It is settled to final volume 500ml, mixes 4 DEG C of preservations；
(13) terminate liquid: the sulfuric acid of 2mol/L；
(14) microplate reader: Star Fax 2100 (U.S. Awareness.).
2. preparing the ELISA Plate of antigen coat:
(1) 4 kinds of tumor associated antigen solution are prepared:
4 kinds of tumor-associated antigen proteins are dissolved separately in coating buffer, are mixed well, being configured to concentration is 0.5 μ g/
4 kinds of antigenic solutions of μ L.
(2) coated elisa plate:
Prepare 4 kinds of tumor associated antigen solution are added separately to 96 hole elisa Plates according to layout shown in Fig. 2
In reacting hole, sample-adding amount is 100 holes μ l/；P53 antigenic solution, blank control wells are added in Positive control wells and negative control hole
Be added coating buffer, 37 DEG C of constant incubators are incubated for 1h, 4 DEG C overnight after remove coating buffer, then wash 3 times with cleaning solution, every time
Wash 3min.
(3) it closes:
Confining liquid (sample-adding amount is 300 holes μ l/), incubation at room temperature 2 are added into the reacting hole of 96 hole elisa Plates after coating
Hour, confining liquid is removed, is then washed 3 times with cleaning solution, washs 3min every time.
(4) dry, packaging:
After 96 hole elisa Plates after Seal treatment are placed 37 DEG C of drying box drying, packaging obtains 96 holes of antigen coat
ELISA Plate, 4 DEG C save backup.
3. the composition of kit of the present invention is as follows:
(1) 96 hole elisa Plates of antigen coat prepared by step 2；
(2) sample diluting liquid: contain the PBST buffer of 1% (W/V) BSA；
(3) secondary antibody dilution: contain the PBST buffer of 1% (W/V) BSA；
(4) enzyme mark secondary antibody: the RecA albumen (Invitrogen company) of horseradish peroxidase-labeled；
(5) developing solution: developing solution is made of developing solution A and developing solution B, wherein and developing solution A is 0.02% (W/V) TMB,
Developing solution B is 0.006% (W/V) urea peroxide；Developing solution A and developing solution B are uniformly mixed in equal volume according to 1:1 when use；
(6) terminate liquid: the sulfuric acid of 2mol/L；
(7) cleaning solution: PBST (phosphate tween) buffer containing 0.2% polysorbas20；
(8) positive control serum: P53 positive control serum；
(9) negative control sera: P53 negative control sera.
Reagent (2)-(9) with 96 hole elisa Plates of antigen coat constitute kit after packing respectively.
Embodiment 2: the application method of kit
1. serum sample is incubated for:
Serum sample to be detected is diluted with sample diluting liquid in the ratio of 1:500, then by the blood after dilution
In the reacting hole of 96 hole elisa Plates of envelope antigen, sample-adding amount is 100 holes μ l/ for final proof this addition, is placed in 37 DEG C of constant temperature incubations
Case is incubated for 1h, then discards liquid in reacting hole, is washed 3 times with cleaning solution, wash 3min every time.
2. ELIAS secondary antibody is incubated for:
The RecA albumen of horseradish peroxidase-labeled is carried out in the ratio of 1:40000 with secondary antibody dilution dilute
It releases, then the RecA albumen of the horseradish peroxidase-labeled after dilution is added in the reacting hole of 96 hole elisa Plates, sample-adding amount
For 100 holes μ l/, it is placed in 37 DEG C of constant incubators and is incubated for 50min, then discard liquid in reacting hole, washed 3 times with cleaning solution,
Each 3min.
3. colour developing and termination reaction:
Developing solution A and developing solution B are uniformly mixed in equal volume according to 1:1, are then rapidly added mixed developing solution
In the reacting hole of 96 hole elisa Plates, sample-adding amount is 100 holes μ l/, is placed in 37 DEG C and is protected from light 15min, then to each reacting hole
In add 50 μ l terminate liquids, reaction is terminated, in using microplate reader device to read OD450 OD value in 20 minutes, and with blank pair
It returns to zero according to hole.
4. result judgement:
Add two standard deviations (Mean+2SD) for cutoff value (cut-off with the average of the surveyed OD value of negative control hole
Value), it is the positive that OD value reading, which is more than or equal to the judgement of cutoff value, in reacting hole, and OD value reading is less than sentencing for cutoff value in reacting hole
Break as feminine gender.
Embodiment 3: the value of diagnosis of kit of the present invention
The serum sample of detection the early stage patients with esophageal squamous cell carcinoma and normal person of the kit described in the embodiment of the present invention 1,
To assess and analyze value of the kit of the present invention for the screening of early stage esophageal squamous cell carcinoma and diagnosis.
1. samples sources
It collects and comes from 160 parts of cancer of the esophagus emphasis open laboratory, Henan Province, the first affiliated hospital, Zhengzhou University serum sample,
Wherein 80 parts of normal human serum (control group), 80 parts of early stage patients with esophageal squamous cell carcinoma serum (early stage esophageal squamous cell carcinoma group).80 normal
People taking physical examination of the human serum from the laboratory chain hospital medical center, without the relevant evidence of any tumour.80 just
In ordinary person, male 43, women 37, average age 58.9 ± 6.3 years old, the range of age 40-70 years old.80 parts of early stage esophageal squamous cell carcinomas
Patients serum derives from early stage (the 0 phase phase+I) patients with esophageal squamous cell carcinoma confirmed through histopathology, does not receive radiotherapy or chemotherapy
Treatment.In 80 patients with esophageal squamous cell carcinoma, male 51, women 29, average age 58.5 ± 7.0 years old, the range of age 45-75
Year.
2. prepared by serum
Limosis vein blood 5ml is extracted into centrifuge tube, stands 30 minutes in room temperature, is centrifuged (2000 turns/min), in absorption
Layer serum packing, every 100 μ l of pipe, -80 DEG C of refrigerator storages.
3. experimental method
The application method of the kit and kit as described in example 2 that are prepared using the embodiment of the present invention 1 is to 80
In normal population serum (control group) and 80 patients with esophageal squamous cell carcinoma serum (esophageal squamous cell carcinoma group) content of 4 kinds of TAA autoantibodies into
Row detection.Using 4 kinds of tumor associated antigen autoantibody being averaged in esophageal squamous cell carcinoma group and control group of MedCalc Software on Drawing
Expression distribution map (referring to Fig. 3 and Fig. 4)；Using the result judgment criteria in 2 step 4 of embodiment as standard, food is calculated separately
Positive rate (the positive object number of cases detected in every group of 4 kinds of tumor associated antigen autoantibodies in pipe squamous carcinoma group and control group
It is positive rate divided by this group of detected object total number of cases) (referring to table 1), and application 4 kinds of tumour correlations of Excel Software on Drawing are anti-
The column diagram of former autoantibody positive rate in early stage esophageal squamous cell carcinoma group and control group (referring to Fig. 5)；It is soft using SPSS22.0
Part carries out statistical test, compares esophageal squamous cell carcinoma group and control group antibody positive rate using two independent sample Chi-square Test methods,
As a result inspection level α=0.05 has statistical significance as P < 0.05, then evaluated using the evaluation method of Screen test
The diagnostic value of autoantibody detection esophageal squamous cell carcinoma (result is referring to table 2 and Fig. 6).
4. interpretation of result
Fig. 3 is distribution map of 4 kinds of tumor associated antigen autoantibodies in 80 esophageal squamous cell carcinoma group serum, can from the figure
To find out, this 4 kinds of tumor associated antigen autoantibodies Average expression level in early stage esophageal squamous cell carcinoma group is relatively high.Fig. 4 is 4 kinds
Distribution map of the tumor associated antigen autoantibody in 80 control group serum, it will be apparent from this figure that this 4 kinds of tumour correlations
Antigen autoantibody Average expression level in control group is relatively low.As can be seen from figs. 3 and 4 early stage esophageal squamous cell carcinoma group patient's blood
4 kinds of TAA average levels prompt 4 kinds of TAA autoantibodies to can be used for early stage esophageal squamous cell carcinoma obviously higher than control group in clear
Screening.
The expression of 4 kinds of TAA autoantibodies in 1 control group of table and esophageal squamous cell carcinoma group
Note: n representative sample sum, N represent antibody positive number, and % represents antibody positive rate.
By table 1 and Fig. 5 it is found that in control group, the positive expression rate of 4 kinds of TAA autoantibodies is relatively low, and in early stage
In esophageal squamous cell carcinoma group, the positive expression rate of 4 kinds of TAA autoantibodies is relatively high, and passes through statistical test, 4 kinds of TAA itself
Thus positive expression rate of the antibody in early stage esophageal squamous cell carcinoma group prove obviously higher than control group, P53, TP53, CDKN2B and
This 4 kinds of TAA autoantibodies of NPM1 can be used for the detection of early stage esophageal squamous cell carcinoma, have the value of early diagnosis.
The different tumor associated antigen TAA autoantibody combinatorial association testing results of table 2
As shown in Table 2, with the increase of antigen number, in early stage patients with esophageal squamous cell carcinoma serum, tumor associated antigen itself
The positive rate of antibody increases to 88.8% by 52.5%, and the susceptibility of detection is increased to 88.8% by 52.5%, as 4 kinds of TAA
When combination, the susceptibility of detection has reached 88.8%, that is to say, that can correctly examine in patients with esophageal squamous cell carcinoma using this method
Breaking as the percentage of esophageal squamous cell carcinoma is 88.8%.Although detection specificity gradually decreases, when 4 with the increase of antigen number
When kind tumor associated antigen combination, specificity still is able to reach 86.3%, this is the result shows that non-patients with esophageal squamous cell carcinoma is adopted
When with 4 kinds of tumor associated antigen joint-detections, the percentage for being correctly diagnosed as being not suffering from esophageal squamous cell carcinoma is 86.3%；Moreover,
When being diagnosed using this 4 kinds of tumor associated antigen combinations to esophageal squamous cell carcinoma, sensitivity is to be detected with single index P53
1.7 times；Therefore, using P53, TP53, CDKN2B and NPM1, this 4 kinds of tumor associated antigen combination progress early stage esophageal squamous cell carcinomas are examined
It is disconnected, it can be in the sensitivity for guaranteeing that diagnosis is greatly improved under the premise of diagnosing specificity.In addition, youden index is in statistics
In refer to that the sum of susceptibility and specificity subtract 1, numberical range is 0-1, and youden index closer to 1, get over by diagnostic value
Greatly, show that the application value of this method is higher.With the increase of antigen number, youden index is increasing and is gradually intended to
1, show that 4 kinds of tumor associated antigen combinations have preferable diagnostic value with the method for screening early stage esophageal squamous cell carcinoma for diagnosing.
Therefore, using phase in the autoantigen joint-detection test serum of this 4 kinds of tumor associated antigens of P53, TP53, CDKN2B and NPM1
The method for answering autoantibodies level had not only been able to maintain higher specificity but also can improve the susceptibility of diagnosis, to be measured to assessing
The risk that object suffers from esophageal squamous cell carcinoma has diagnosis and application value well.
It will be appreciated from fig. 6 that using the autoantibody in 4 kinds of tumor associated antigen joint-detection early stage patients with esophageal squamous cell carcinoma serum
When, with the increase of antigen combination number, area rises to 0.8750 by 0.7375 under ROC curve, illustrates to use the ELISA reagent
Box and detection method to the diagnosis of early stage esophageal squamous cell carcinoma sensitivity with higher and specificity, the kit and method be suitble into
The diagnosis and screening of row early stage esophageal squamous cell carcinoma.
The foregoing is merely illustrative of the preferred embodiments of the present invention, but is not limited only to examples detailed above, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. the combination of tumor associated antigen P53, TP53, CDKN2B and NPM1 are in the agent box that preparation is used for esophageal squamous cell carcinoma screening
Using.
2. a kind of autoantibody joint-detection ELISA kit for esophageal squamous cell carcinoma early screening, which is characterized in that the examination
Agent box includes solid phase carrier and the tumor associated antigen that is coated on solid phase carrier, the tumor associated antigen by P53, TP53,
CDKN2B and NPM1 composition.
3. autoantibody joint-detection ELISA kit according to claim 2, which is characterized in that the kit is also
Including sample diluting liquid, secondary antibody, secondary antibody dilution, negative control sera, positive control serum, cleaning solution, colour developing
Liquid and terminate liquid.
4. autoantibody joint-detection ELISA kit according to claim 3, which is characterized in that the secondary antibody
With detectable marker.
5. autoantibody joint-detection ELISA kit according to claim 4, which is characterized in that the marker is
Horseradish peroxidase.
6. according to any autoantibody joint-detection ELISA kit of claim 3~5, which is characterized in that described the
Two antibody are RecA albumen.
7. autoantibody joint-detection ELISA kit according to claim 6, which is characterized in that the positive control
Serum is P53 positive control serum, and the negative control sera is P53 negative control sera.
8. autoantibody joint-detection ELISA kit according to claim 7, which is characterized in that the P53 is positive
It is positive patients with esophageal squamous cell carcinoma blood that control serum, which is using indirect ELISA and Western blot method detection P53 antibody,
Clearly, the P53 negative control sera is to be using indirect ELISA and Western blot method detection P53 antibody expression
The serum of the normal person of normal population serum antibody average content.
9. autoantibody joint-detection ELISA kit according to claim 8, which is characterized in that the solid phase carrier
For ELISA Plate.
10. autoantibody joint-detection ELISA kit according to claim 9, which is characterized in that the autoantibody
The test object of joint-detection ELISA kit is human serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910532284.1A CN110187113B (en) | 2019-06-19 | 2019-06-19 | Autoantibody joint detection ELISA kit for early screening of esophageal squamous cell carcinoma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201910532284.1A CN110187113B (en) | 2019-06-19 | 2019-06-19 | Autoantibody joint detection ELISA kit for early screening of esophageal squamous cell carcinoma |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110716044A (en) * | 2019-10-23 | 2020-01-21 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of esophageal squamous carcinoma |
| CN111323588A (en) * | 2020-02-28 | 2020-06-23 | 郑州大学第一附属医院 | Application of esophageal cancer related antigen-protein combination or specific antibody thereof in esophageal cancer detection kit |
| CN111323586A (en) * | 2020-02-27 | 2020-06-23 | 郑州大学第一附属医院 | ELISA kit for early diagnosis of esophageal squamous cell carcinoma |
| CN113075413A (en) * | 2021-03-29 | 2021-07-06 | 郑州大学第一附属医院 | Early esophageal squamous carcinoma screening kit based on group of tumor-associated antigens |
| CN113265463A (en) * | 2021-04-15 | 2021-08-17 | 山西医科大学 | Application of FAM84B in preparation of esophageal squamous cell carcinoma prognosis evaluation reagent and screening of drugs for targeted therapy of esophageal squamous cell carcinoma |
| CN113671180A (en) * | 2021-09-16 | 2021-11-19 | 郑州大学 | Application of PAIP1 autoantibody in auxiliary diagnosis of esophageal squamous cell carcinoma |
| CN113777311A (en) * | 2021-09-16 | 2021-12-10 | 郑州大学 | ELISA kit for auxiliary diagnosis of esophageal squamous cell carcinoma |
| CN114167059A (en) * | 2021-11-03 | 2022-03-11 | 郑州大学 | Biomarker for diagnosing esophageal squamous carcinoma and detection kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149369A (en) * | 2013-03-20 | 2013-06-12 | 江苏元化生命科技有限公司 | Protein chip for detecting esophageal squamous carcinoma marker and kit box of protein chip |
| CN106053812A (en) * | 2016-06-29 | 2016-10-26 | 厦门生迪生物技术有限公司 | Multi-autoantibody joint detection ELISA kit for early screening and diagnosis on liver cancer |
| CN109342727A (en) * | 2018-10-15 | 2019-02-15 | 汕头大学医学院附属肿瘤医院 | Esophageal squamous cell carcinoma autoantibody molecular marker model and its application |
-
2019
- 2019-06-19 CN CN201910532284.1A patent/CN110187113B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149369A (en) * | 2013-03-20 | 2013-06-12 | 江苏元化生命科技有限公司 | Protein chip for detecting esophageal squamous carcinoma marker and kit box of protein chip |
| CN106053812A (en) * | 2016-06-29 | 2016-10-26 | 厦门生迪生物技术有限公司 | Multi-autoantibody joint detection ELISA kit for early screening and diagnosis on liver cancer |
| CN109342727A (en) * | 2018-10-15 | 2019-02-15 | 汕头大学医学院附属肿瘤医院 | Esophageal squamous cell carcinoma autoantibody molecular marker model and its application |
Non-Patent Citations (2)
| Title |
|---|
| HIDEAKI SHIMADA等: "Serum p53 Antibody Is a Useful Tumor Marker in Superficial Esophageal Squamous Cell Carcinoma", 《CANCER》 * |
| 张群等: "核磷蛋白与肿瘤的发生", 《中国医师进修杂志》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110716044A (en) * | 2019-10-23 | 2020-01-21 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of esophageal squamous carcinoma |
| CN110716044B (en) * | 2019-10-23 | 2023-04-18 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of esophageal squamous carcinoma |
| CN111323586A (en) * | 2020-02-27 | 2020-06-23 | 郑州大学第一附属医院 | ELISA kit for early diagnosis of esophageal squamous cell carcinoma |
| CN111323588A (en) * | 2020-02-28 | 2020-06-23 | 郑州大学第一附属医院 | Application of esophageal cancer related antigen-protein combination or specific antibody thereof in esophageal cancer detection kit |
| CN111323588B (en) * | 2020-02-28 | 2020-09-29 | 郑州大学第一附属医院 | Application of esophageal cancer related antigen-protein combination or specific antibody thereof in esophageal cancer detection kit |
| CN113075413A (en) * | 2021-03-29 | 2021-07-06 | 郑州大学第一附属医院 | Early esophageal squamous carcinoma screening kit based on group of tumor-associated antigens |
| CN113265463A (en) * | 2021-04-15 | 2021-08-17 | 山西医科大学 | Application of FAM84B in preparation of esophageal squamous cell carcinoma prognosis evaluation reagent and screening of drugs for targeted therapy of esophageal squamous cell carcinoma |
| CN113265463B (en) * | 2021-04-15 | 2023-02-28 | 山西医科大学 | Application of FAM84B in preparation of esophageal squamous carcinoma prognosis evaluation reagent and screening of drugs for targeted therapy of esophageal squamous carcinoma |
| CN113671180A (en) * | 2021-09-16 | 2021-11-19 | 郑州大学 | Application of PAIP1 autoantibody in auxiliary diagnosis of esophageal squamous cell carcinoma |
| CN113777311A (en) * | 2021-09-16 | 2021-12-10 | 郑州大学 | ELISA kit for auxiliary diagnosis of esophageal squamous cell carcinoma |
| CN113777311B (en) * | 2021-09-16 | 2023-08-01 | 郑州大学 | ELISA kit for auxiliary diagnosis of esophageal squamous carcinoma |
| CN114167059A (en) * | 2021-11-03 | 2022-03-11 | 郑州大学 | Biomarker for diagnosing esophageal squamous carcinoma and detection kit |
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Address after: 450052 State Key Laboratory of esophageal cancer prevention and treatment, the First Affiliated Hospital of Zhengzhou University, No.40, University Road, Erqi District, Zhengzhou City, Henan Province Applicant after: First Affiliated Hospital of Zhengzhou University Address before: 450052 Construction No. 1 East Road, 27 District, Henan, Zhengzhou Applicant before: First Affiliated Hospital of Zhengzhou University |
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