CN110196329A - A kind of cancer of the esophagus early stage combined detection kit - Google Patents
A kind of cancer of the esophagus early stage combined detection kit Download PDFInfo
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Abstract
The invention belongs to field of biomedicine technology, more particularly to a kind of cancer of the esophagus early stage combined detection kit, including solid-phase matrix and the cancer of the esophagus tumor-associated antigen protein being coated on solid-phase matrix, wherein, antigen protein is C-myc gene expression object, HER-2 gene expression object, Cyfra21-1 gene expression object and P53 gene expression object, and kit further includes enzyme mark secondary antibody.Kit of the present invention improves the sensibility and specificity of detection by the way of four kinds of antigen joint-detections, and kit of the present invention is up to 62.6% and 89.8% to the sensibility and specificity that the cancer of the esophagus detects respectively；Kit of the present invention need to only acquire blood of human body to be checked, can screening its risk for suffering from the cancer of the esophagus, compared with organizing biopsy, wound is small, Small side effects, easy to use, has good potential applicability in clinical practice.
Description
Technical field
The invention belongs to field of biomedicine technology, and in particular to a kind of cancer of the esophagus early stage combined detection kit.
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 patient with esophageal carcinoma occurs in China, 100 times higher than western countries disease incidence.China be Incidence of esophageal cancer and
The highest country of the death rate, China's about 250,000 newly-increased diagnosed case every year, accounts for whole world cancer of the esophagus new cases according to statistics
More than half of sum.In China, not only disease incidence is high for the cancer of the esophagus, there is also significant areal variation, such as Henan, Hebei and
The Taihang mountain areas that Shanxi San Sheng has a common boundary is Incidence of esophageal cancer and the highest area of the death rate in the world.Advanced esophageal carcinoma is suffered from
5 years survival rates of person only 10% or so, and the early stage cancer of the esophagus is then up to 90% or more.Since patient with esophageal carcinoma is bright in early stage shortage
Aobvious specific symptoms, and shortage is suitable for economic, the efficient and sensitive biomarker of a wide range of people at highest risk's screening, is caused
The patient with esophageal carcinoma clinically gone to a doctor at present is the middle and advanced stage stage making a definite diagnosis Shi Duoyi.Currently, Endoscopy and Mucosa Biopsy disease
Reason inspection is the important screening method of cancer of the esophagus early detection.But because Endoscopic Screening has wound, and at high cost, low efficiency, limit
Popularization of the Endoscopic Screening in the Silent cerebral infarction cancer of the esophagus early discovery is made.Cancer of the esophagus early detection is to improve patient with esophageal carcinoma
The effective means of survival rate, thus screen efficient, special cancer of the esophagus molecular marker for patient with esophageal carcinoma early detection,
Early screening is particularly important, is also a problem to be solved.
In recent years the study found that the immune system of human body can to self tumor related antigen (TAAs) generate humoral immunity
Response, and cause a large amount of autoantibodies in tumour generating process and discharge into serum.More and more evidences show in serum
The existing circulation autoantibody for TAAs, the 3-5 before the appearance of cancer patient's clinical symptoms can be detected, can make
Biological indicator, a CA125 of diagnosing cancer of liver have been used as the new selection markers object of earlier stage cancer patients, such as alpha-fetoprotein
The generaI investigation and screening of oophoroma have been applied to it, carcinomebryonic antigen (CEA) has been used as early diagnosis adenocarcinoma Specific marker.Cause
This, can be the monitoring of early stage asymptomatic cancer patient using detection of the TAAs to autoantibodies, more sensitive and convenient；This
Outside, autoantibody can stablize in serum, lasting presence, become the cancer of the esophagus early diagnosis ideal serologic marker
Object, be based on the studies above background, develop it is a kind of convenient for people at highest risk carry out the kit for suffering from cancer of the esophagus risk supervision have weight
Want meaning.
Summary of the invention
To solve the problems in the background art, the purpose of the present invention is to provide a kind of cancer of the esophagus early stage joint-detections
Kit, convenient for carrying out early screening to cancer of the esophagus risk population.
Based on above-mentioned purpose, the technical solution adopted by the present invention are as follows: a kind of cancer of the esophagus early stage combined detection kit, including
Solid-phase matrix and the cancer of the esophagus tumor-associated antigen protein being coated on solid-phase matrix, the antigen protein are C-myc gene table
Up to object (hereinafter referred to as C-myc), HER-2 gene expression object (hereinafter referred to as HER-2), Cyfra21-1 gene expression object (with
It is referred to as Cyfra21-1 down) and P53 gene expression object (hereinafter referred to as P53)；The kit further includes that enzyme mark second is anti-
Body.
Further, enzyme mark secondary antibody is goat anti-human immunoglobulin's antibody of phycoerythrin label, i.e. PE label
Goat anti-human immunoglobulin's antibody.
Further, solid-phase matrix is the supported matrix that microwell plate, microsphere or perforated membrane are constituted.
Further, kit further includes Sample dilution, cleaning solution, secondary antibody dilution, developing solution and terminate liquid.
Further, Sample dilution is the PBST buffer (w/v) of 1%BSA；The cleaning solution is containing 0.2% polysorbas20
Phosphate buffer (w/v)；The secondary antibody dilution is the PBST buffer (w/v) of 1%BSA；The developing solution is by showing
Color liquid A and developing solution B are mixed in equal volume, and developing solution A is 0.02%TMB(w/v), developing solution B is 0.006% urea peroxide element
(w/v)；The terminate liquid is 2mol/L sulfuric acid solution.
Further, kit is the Multiple detection kit based on Luminex xMAP technology.
By being carried out to specific expressed eight kinds of antibody in patient with esophageal carcinoma body the study found that wherein four kinds i.e. anti-C-
Myc antibody, anti-HER-2 antibody, the expression of anti-Cyfra21-1 antibody and antibodies against P 53 in patients serum are significantly higher than
Cancer of the esophagus benign lesion patient and healthy population, therefore kit of the present invention is with the corresponding C-myc gene expression of above-mentioned four kinds of antibody
Object, HER-2 gene expression object, Cyfra21-1 gene expression object and P53 gene expression object are as antigen combination, by to be checked
Associated antibodies in human serum are detected, and suffer from cancer of the esophagus risk for judging.
Kit of the present invention improves the sensibility and specificity of detection by the way of four kinds of antigen joint-detections, this
Invention kit is up to 62.6% and 89.8% to the sensibility and specificity that the cancer of the esophagus detects respectively.
Compared with organizing biopsy, kit provided by the invention need to only acquire blood of human body to be checked, can screening its trouble
The risk of the cancer of the esophagus, wound is small, Small side effects, easy to use, has good potential applicability in clinical practice.
Detailed description of the invention
Fig. 1 is the testing result that small sample tests eight kinds of autoantibodies levels between each group；
Fig. 2 is the testing result that 818 samples carry out the detection of four kinds of autoantibody markers in training set；
Fig. 3 is detection sensitivity and specificity of four kinds of autoantibodies as detection marker；
Fig. 4 is the sensibility and specificity that all integrated modes of four kinds of antigen proteins carry out EC diagnosis；
Fig. 5 is the sensibility and specificity that four kinds of antigen protein joint-detections diagnose EC；
Fig. 6 is the result that four kinds of antigen protein joint-detections concentrate sample progress EC diagnosis to verifying；
Fig. 7 is the sensibility and specificity that four kinds of antigen protein joint-detections concentrate sample progress EC diagnosis to verifying；
Fig. 8 is the correlation of Luminex method and ELISA method detection result.
Specific embodiment
1: eight kind of autoantibodies detection of embodiment early diagnoses experimental study to the cancer of the esophagus
1. research object
In March, 2015 is collected to 2 months 2018 300 parts of serum samples from the first affiliated hospital, Zhengzhou University, wherein early stage
EC patient 100, EBL patient 100, HC patient 100.100 early stage EC patients combination cell on the basis of scope
It learns or histopathology is made a definite diagnosis, do not receive any antineoplaston such as chemotherapy, radiotherapy, TNM classification 0, I, II phase.100 EBL
Patient carrys out automatic self synchronizing and is hospitalized the just patient that controls in the first affiliated hospital, Zhengzhou University, including benign tumor of esophagus, oesophagus are benign bursts
Ulcer, oesophagus erosion and reflux esophagitis etc..Health of 100 HC patients from the first affiliated hospital, Zhengzhou University medical center
Physical examination is adult, does not show the evidence of any malignant tumour.This research is criticized by the Ethics Committee of the first affiliated hospital, Zhengzhou University
Standard, see Table 1 for details for research object Clinical symptoms.
1 300 research object Clinical symptoms of table
As shown in Table 1, there was no significant difference in age, gender, smoking history between early stage EC group and EBL group, HC group (p>
0.05).
The method that 2.ELISA detects eight kinds of autoantibodies
It is coated with enzyme mark hole respectively with eight kinds of antigen proteins of Koc, HER-2, Cyfra21-1, P53, C-myc, P16, YB-1 and XPC
Plate, the concentration of eight kinds of antigen proteins are 0.5ng/ μ L, and the dosage of eight kinds of antigen proteins is 100 holes μ L/, are coated at 4 DEG C
Overnight；After then being washed three times with the phosphate buffer (hereinafter referred to as PBST) containing 0.2%(v/v) polysorbas20,3%(v/ will be contained
V) the phosphate buffer of bovine serum albumin(BSA) (BSA) is added in enzyme mark hole and is closed, and additive amount is 300 holes μ L/, at 37 DEG C
Lower closing 1 hour；It then washed once with PBST, the serum sample after dilution be then added into enzyme mark hole, wherein serum sample
Originally it is by the PBST buffer containing 1%(w/v) BSA according to 1:500(v/v) it is diluted, the addition of the serum sample after dilution
Amount is 100 holes μ L/, is incubated l hours at 37 DEG C；Horseradish peroxidase after PBST is washed four times, after dilution is added
(HRP) goat anti-human immunoglobulin's antibody is marked (hereinafter referred to as HRP marks goat anti-human igg antibody), wherein HRP marks sheep
Anti-human IgG antibodies are by the PBST buffer containing 1%(w/v) BSA according to 1:40000(v/v) it is diluted, the HRP after dilution
The additive amount for marking goat anti-human igg antibody is 100 holes μ L/, incubates 30min at 37 DEG C；After PBST is washed four times, it is added four
Methyl biphenyl limb (TMB) and each 50 μ L of hydrogen peroxide 3%(v/v) are protected from light colour developing 10min at 37 DEG C；2mol/L is then added
50 μ L color development stopping of sulfuric acid, measures every hole OD value by microplate reader at 450nm wavelength.
ELISA result judgement: the average of normal serum OD value adds two standard deviations (Mean+2SD) as critical value
(Cut-off value) is then positive higher than critical value, is then negative lower than this value.Average with OD value in Positive control wells is
Reference excludes false positive and false negative result.
Testing result is as shown in Table 2 and Fig. 1.
The comparison of eight kinds of autoantibodies positive rates between 2 each group of table
ELISA testing result is as shown in Table 2 and Fig. 1, anti-Koc antibody, anti-P16 antibody, anti-YB- in eight kinds of autoantibodies
1 antibody and anti-XPC antibody (P> 0.05) level and positive rate is not statistically significant in early stage EC group and EBL/HC；And eight kinds of blood
In clear autoantibody anti-HER-2 antibody, anti-Cyfra21-1 antibody, antibodies against P 53 (P < 0.01) and anti-C-myc antibody (P<
0.05) it obviously higher than oesophagus benign lesion (EBL) group (n=100) and is good in the positive rate of early stage EC group (n=100) and level
Health compares (HC) group (n=100).Therefore the present invention is with anti-HER-2 antibody, anti-Cyfra21-1 antibody, antibodies against P 53 and anti-C-myc
The marker that these four autoantibodies of antibody are early diagnosed as EC.
2 four kinds of autoantibodies detections of embodiment early diagnose experimental study to the cancer of the esophagus
Based on the four kinds of autoantibody markers selected in embodiment 1 by small sample testing sieve, in order to determine these four itself
Antibody marlcers have a preferable early diagnosis value to EC, and the present invention is by the ELISA method of building in the big of 1138 serum
The anti-C-myc antibody of further investigation, anti-HER-2 antibody, anti-Cyfra21-1 antibody and antibodies against P 53 detection are to EC in sample range
The value of early diagnosis, the research queue of development include 818 training sets and the verifying collection of 320 independent matchings of tool.
The model foundation process of the corresponding antigen joint-detection of four kinds of autoantibody markers mainly includes two stages: the
One stage, by 818 training set samples, including 258 early stage EC patients, 260 EBL patients and 300 HC, four kinds of blood
The built-up pattern of the corresponding antigen of autoantibody for EC early diagnosis is established in the ELISA method detection of clear autoantibody；Second
Stage verifies the autoantibody built-up pattern of foundation by the ELISA method detection to 320 verifying collection sample autoantibodies, and
Clear four kinds of autoantibodies distinguish the ability of early stage EC patient group and control group (EBL+HC).Detailed process is as follows.
1. research object
This research is ratified by the Ethics Committee of the first affiliated hospital, Zhengzhou University, collects and comes from 2 months in June, 2018 in 2016
1138 parts of serum samples of the first affiliated hospital, Zhengzhou University, wherein 818 parts of serum samples are as training set, in training set sample
The Clinical symptoms of research object is as shown in table 3, and 320 parts of serum samples collect as verifying, and research object faces in verifying collection sample
Bed feature is as shown in table 4.
The Clinical symptoms of research object in 3 training set sample of table
The Clinical symptoms of research object in the verifying collection sample of table 4
Early stage EC patient 258 in training set, EBL patient 260, HC Patients with 300 Cases.258 early stage EC patients are in scope
On the basis of combination cell or histopathology make a definite diagnosis, do not receive any antineoplaston such as chemotherapy, radiotherapy, TNM classification is
0, I the and II phase.260 EBL patients come the patient that automatic self synchronizing is just controlled in hospital in the first affiliated hospital, Zhengzhou University, including esophageal benign and malignant
Property tumour, oesophagus benign ulcer, oesophagus be rotten to the corn and reflux esophagitis etc..300 HC patients are attached from Zhengzhou University first
The health examination at Hospital Physical Examination center is adult, does not show the evidence of any malignant tumour.
Verifying concentrates early stage EC patient 100, EBL patient 100, HC patient 120.100 early stage EC patients exist
Combination cell or histopathology are made a definite diagnosis on the basis of scope, do not receive any antineoplaston such as chemotherapy, radiotherapy, and TNM divides
Class is 0, I and II phase.100 EBL patients come the patient that automatic self synchronizing is just controlled in hospital in the first affiliated hospital, Zhengzhou University, including food
Pipe benign tumour, oesophagus benign ulcer, oesophagus erosion and reflux esophagitis etc..120 HC patients come from Zhengzhou University first
The health examination of affiliated hospital's medical center is adult, does not show the evidence of any malignant tumour.
3.ELISA method detects four kinds of autoantibodies
Be coated with enzyme mark orifice plate respectively with tetra- kinds of antigen proteins of HER-2, Cyfra21-1, P53 and C-myc, four kinds of antigen proteins it is dense
Degree is 0.5ng/ μ L, and the dosage of four kinds of antigen proteins is 100 holes μ L/, is coated with overnight at 4 DEG C；Then washed with PBST
After three times, 3%(v/v will be contained) BSA phosphate buffer be added enzyme mark hole in close, additive amount be 300 holes μ L/, at 37 DEG C
Lower closing 1 hour；Then, it washed once with PBST, the serum sample after dilution be then added into enzyme mark hole, wherein serum
Sample is by the PBST buffer containing 1%(w/v) BSA, according to 1:500(v/v) it is diluted, the serum sample after dilution adds
Dosage is 100 holes μ L/, is incubated for l hours at 37 DEG C；After PBST is washed four times, the HRP after dilution is added marks goat-anti people
IgG antibody, wherein HRP label goat anti-human igg antibody is by the PBST buffer containing 1%(w/v) BSA according to 1:40000(v/
V) it is diluted, the additive amount of the HRP label goat anti-human igg antibody after dilution is 100 holes μ L/, incubates 30min at 37 DEG C；
After PBST is washed four times, tetramethyl biphenyl limb (TMB) and 3%(v/v) each 50 μ L of hydrogen peroxide is added, is protected from light at 37 DEG C aobvious
Color 10min；50 μ L color development stopping of 2mol/L sulfuric acid is then added, every hole OD value is measured at 450nm wavelength by microplate reader.
Testing result based on ELISA method is as shown in Figures 2 and 3, and Fig. 2 carries out four kinds certainly to 818 samples in training set
The testing result of body antibody labels analyte detection, in figure 2 it can be seen that four kinds of autoantibodies are equal in the level of early stage EC group
Be apparently higher than EBL group, HC group (p<0.001)；Fig. 3 is sensitivity and specificity knot of four kinds of autoantibodies as detection marker
Fruit uses the best cut off value of every kind of candidate markers of following criterion evaluation: 1) at least 90% specificity；2) highest area
The ability of dividing；By best cut off value determine verifying collection four kinds of autoantibodies of sample to the diagnostic sensitivity of early stage EC with
Specificity.ELISA testing result based on training set sample draws Receiver operating curve (ROC), calculates area under ROC
(AUC), to assess every kind of candidate markers to the diagnosis performance of early stage EC patient.
Shown in testing result Fig. 3: antibodies against P 53 is respectively 0.682 to the AUC of early stage EC, sensitivity and specificity,
26.3% and 96.4%；Anti- HER-2 antibody is respectively 0.622,30.6% and 97.6% to the AUC of early stage EC, sensitivity and specificity；
Anti- Cyfra21-1 antibody is respectively 0.815,31.4% and 94.9% to the AUC of early stage EC, sensitivity and specificity；Anti- C-myc is anti-
Body is respectively 0.638,19.5% and 97.3% to the AUC of early stage EC, sensitivity and specificity.
Embodiment 3: the composition of kit
The composition of kit of the present invention is as follows:
(1) 96 hole enzyme reaction plates of antigen coat
Preparation method is as follows:
.Luminex microballoon couples antigen protein, detailed process are as follows:
5.0 × 106 raw material Luminex microballoon is transferred in microcentrifugal tube；And 10 μ L 150mg/ are added in centrifuge tube
ML sulfo group-NHS and 10 μ L150mg/mL EDC is incubated 20 minutes at room temperature；Then activation is washed with the MES that 50mM pH is 5.0
Luminex microballoon to remove excessive sulphur group base-NHS and EDC；10 μ g antigen proteins are added in microcentrifugal tube, then plus
Entering the MES that 50mM pH is 5.0 makes total volume to 500 μ L, and rotation is incubated for 2 hours at room temperature；The Luminex microballoon coupled
Through containing 0.1%(v/v) BSA, 0.02%(v/v) Tween-20,0.05%(v/v) and pH be 7.4 Sodium Azide PBS washing two
After secondary, it is kept in dark place at 2-8 DEG C spare.
The preparation of 96 hole enzyme reaction plates of antigen coat: by 4 kinds of antigen proteins, that is, C-myc gene expression object, HER-
2 gene expression objects, Cyfra21-1 gene expression object and P53 gene expression object respectively with coating diluted to 0.5 μ g/ μ L,
Wherein, it is 9.6 that coating dilution, which is pH, and concentration is the carbonate buffer solution of 50mmol, each of on 96 hole enzyme reaction plates
Antigen protein after 100 μ L dilution is added in antigen hole removes more after being incubated for 4h at 37 DEG C or standing overnight at 4 DEG C
Extraction raffinate body；Each antigen hole is washed 3 times through PBST, each wash time is 3min；300 μ L are added then to each antigen hole
Confining liquid, wherein confining liquid is containing 2%(w/v) the PBST buffer of BSA, it is incubated for 40min at 37 DEG C, then removes extra liquid
Body.
After 96 hole enzyme reaction plates after treatment being placed 37 DEG C of drying box drying, packaging is standby in 4 DEG C of preservations
With.Layout of the microballoon lotus root associated antigen albumen in 96 hole enzyme reaction plates is as shown in table 5.
Layout of the 5 microballoon lotus root associated antigen albumen of table in 96 hole enzyme reaction plates
Note:Positive control serum: P53 positive control serum；Negative control sera: P53 negative control sera.
.+: positive control serum is added in the hole；: negative control sera is added in the hole.
(2) Sample dilution, Sample dilution are the PBST buffer (w/v) containing 1%BSA.
(3) enzyme mark secondary antibody, enzyme mark secondary antibody are goat anti-human immunoglobulin's antibody of PE label.
(4) secondary antibody dilution, secondary antibody dilution are the PBST buffer (w/v) containing 1%BSA.
(5) developing solution, developing solution include developing solution A and developing solution B, and developing solution A is 0.02%TMB(w/v), developing solution B is
0.006% urea peroxide element (w/v)；Terminate liquid is 2mol/L sulfuric acid, and developing solution A and developing solution B are according to bodies such as 1:1 when in use
It is used after product mixing.
(6) cleaning solution, cleaning solution are the phosphate buffer (w/v) containing 0.2% polysorbas20.
(7) terminate liquid, terminate liquid are the sulfuric acid solution of 2mol/L.
96 hole enzyme reaction plates, Sample dilution, secondary antibody dilution, the enzyme mark of antigen coat in kit of the present invention
Secondary antibody, developing solution A, developing solution B, cleaning solution and terminate liquid be independent packaging.
Embodiment 4: the comparison of four kinds of antigen joint-detection modes in kit
The all possible integrated mode of the corresponding antigen of four kinds of autoantibodies includes any one, two kinds any, three kinds any
And the mode of four kinds of autoantibodies combination, as shown in table 6.
The combined code table of the corresponding antigen of 6 four kinds of autoantibodies of table
Under all possible combinations mode of four kinds of antigens of ELISA Analysis of test results based on training set sample, EC early stage is examined
Disconnected performance is assessed, the specific steps are as follows: the actually detected signal value of every kind of marker is converted to 1 or 0,1 expression and is believed
Number best cut off value, is otherwise 0.2, the summation of the n kind marker combination binary system score value of setting is calculated, as corresponding blood
The total score of final proof sheet.If the total score of sample is greater than k (1≤k≤n), for the positive: recording the sensibility of every kind of combination and special
Property.With the combination of best separating capacity appraisal mark object and its k value, the mode of optimal combination is obtained.
EC detection is carried out according to four kinds of antigen protein integrated modes that table 6 provides, as a result as shown in figure 4, comparison is found, this
The mode diagnosis performance with higher for four kinds of antigen joint-detections that invention kit uses.Four kinds of antigen protein joint-detections
There is highest diagnosis performance to early stage EC, the sensibility and specificity that four kinds of antigen protein joint-detections diagnose getting up early EC is such as
Shown in Fig. 5, the sensitivity and specificity of diagnosis is respectively 62.6%, 89.8%.
The application of 5 kit of embodiment
The effect that the kit is verified using 320 samples that table 4 provides, wherein 100 early stage EC patients, 100 EBL patients
With 120 HC.
, ELISA method is to four kinds of antigen joint-detections
320 samples are concentrated to detect above-mentioned verifying using four kinds of antigen joint-detection modes based on ELISA method,
Testing result is as shown in Figure 6, it can be seen that four kinds of autoantibodies are in the horizontal obviously higher than EBL group and HC of early stage EC group
Group (P< 0.01).Four kinds of antigen joint-detections are to diagnostic sensitivity and the specific outcome of early stage EC as shown in fig. 7, four kinds of antigens
Joint-detection is respectively 58.0%, 90.0% to the diagnostic sensitivity and specificity of early stage EC.
, multiple detection method based on Luminex xMAP technology is to four kinds of antigen joint-detections
Since ELISA method is the common detection methods of individual event marker, but in terms of the combine detection of a variety of autoantibodies,
ELISA method is cumbersome, and clinical value is limited.Compared to ELISA method, Luminex xMAP technology has the excellent of high throughput
Gesture can be applied to the combine detection of autoantibodies combination.Therefore the present invention is based on Luminex xMAP technologies to establish four kinds
The detection method of antigen joint-detection, detailed process is as follows.
1) Luminex microballoon couples
The specification provided according to Sai Mofei company carries out microballoon and couples, the specific steps are as follows: by 5.0 × 106 raw material microballoon
It is transferred in microcentrifugal tube；Sulfo group-the NHS and 10 μ L 150mg/mL of 10 μ L 150mg/mL are added in the microcentrifugal tube
EDC, at room temperature incubate 20 minutes；The microballoon of activation is washed with the MES that 50mM pH is 5.0 to remove excessive sulfo group-NHS
And EDC；10 μ g antigen proteins are added in microcentrifugal tube, adding the MES that 50mM pH is 5.0 makes total volume to 500 μ L,
And rotation is incubated for 2 hours at room temperature；It is 7.4 that the microballoon coupled, which is passed through containing 0.1%BSA, 0.02%Tween-20,0.05% and pH,
Sodium Azide PBS wash twice after, be kept in dark place at 2-8 DEG C spare.
2) building of the multiple detection method based on Luminex xMAP technology platform
The application method of kit is as follows: the method for the Luminex xMAP technology platform joint-detection based on building carries out sample
Detection, steps are as follows:
It after the diluted blood serum sample of 10 μ L and 25 μ L antigen proteins being coupled microballoon mixing, is added in 96 hole microtiter plates,
37 DEG C of oscillator plates are incubated for 15 minutes, and concussion revolving speed is l000rpm/min；Wherein, blood serum sample is by containing 1%(w/v) BSA
PBST buffer according to 1:20(v/v) be diluted.
.PBST it washs three times, goat anti-human immunoglobulin's antibody of PE label is added, is incubated on 37 DEG C of oscillator plates
It educates 15 minutes, concussion revolving speed is 1000rpm/min, is then washed three times with PBST；
100 μ L measurement buffer is added in 96 hole microtiter plates, microballoon is resuspended, examined in 200 streaming fluorescence of Luminex
It surveys on instrument and detects the fluorescence signal of microballoon, the median fluorescence intensity of every kind of fluorescent microsphere is calculated with Luminex xPONENT software
(MFI) value, MFI are greater than 3 for the positive.
3) sample is collected into detection to verifying using the multiple detection method of Luminex xMAP technology platform
Using the Luminex xMAP multiple detection method of building, four kinds of autoantibodies levels in verifying collection sample are carried out
Detection.
320 samples are concentrated to carry out the detection performance of four kinds of antigen joint-detection modes by verifying using above-mentioned model
Verifying finds that four kinds of antigen joint-detections have very high diagnosis performance to early stage EC, and sensibility can be improved to 60% left side
The right side, specificity to 90% or so.
By related to the verifying collection result progress of pattern detection to ELISA method to Luminex xMAP multiple detection method
Property analysis, as a result as shown in Figure 8, it is seen then that between Luminex method testing result and ELISA method detection result have apparent phase
Guan Xing shows that the multiple detection method based on Luminex xMAP technology that constructs of the present invention has stronger reliability, also into
One step confirms four kinds of anti-C-myc antibody, anti-HER-2 antibody, anti-Cyfra21-1 antibody and antibodies against P 53 autoantibody marks
Object is worth EC with extraordinary early diagnosis.Further, since present invention building is based on the more of Luminex xMAP technology
Re-detection method, can be realized the detection of four kinds of autoantibodies of expansion simultaneously in same a serum sample, therefore reagent of the present invention
Box is combined and is detected using the corresponding four kinds of antigen of four kinds of autoantibody markers, and kit of the present invention for based on
The Multiple detection kit of Luminex xMAP technology.
In conclusion the present invention by being carried out to specific expressed eight kinds of antibody in patient with esophageal carcinoma body the study found that
The wherein four kinds i.e. table of anti-C-myc antibody, anti-HER-2 antibody, anti-Cyfra21-1 antibody and antibodies against P 53 in patients serum
Be significantly higher than cancer of the esophagus benign lesion patient and healthy population up to level, thus the present invention use for the first time C-myc gene expression object,
The early stage of HER-2 gene expression object, Cyfra21-1 gene expression object and P53 gene expression object as a combination for EC examines
It is disconnected, sensibility and specificity with higher；In addition, the cohort study that first passage of the present invention is carried out, within the scope of large sample
Deeply illustrate the group of the anti-c-myc antibody of four kinds of autoantibodies, anti-HER-2 antibody, anti-Cyfra21-1 antibody and antibodies against P 53
Closing detection has ideal early diagnosis advantage to EC；Finally, the present invention establishes four by Luminex xMAP technology platform
The multiple detection method of kind autoantibody combination, facilitates the serological screening quality for being further improved EC early diagnosis, in turn
Promote the raising of patient's cure rate and the extension of survival rate.
Kit of the present invention improves the sensibility and specificity of detection by the way of four kinds of antigen joint-detections；This
Invention kit need to only acquire blood of human body to be checked, can screening its risk for suffering from the cancer of the esophagus, compared with organizing biopsy, wound
Small, Small side effects, it is easy to use, there is good potential applicability in clinical practice.
Claims (6)
1. a kind of cancer of the esophagus early stage combined detection kit, which is characterized in that including solid-phase matrix and be coated on solid-phase matrix
Cancer of the esophagus tumor-associated antigen protein, the antigen protein be C-myc gene expression object, HER-2 gene expression object,
Cyfra21-1 gene expression object and P53 gene expression object；The kit further includes enzyme mark secondary antibody.
2. cancer of the esophagus early stage combined detection kit according to claim 1, which is characterized in that the enzyme mark secondary antibody
For goat anti-human immunoglobulin's antibody of phycoerythrin label.
3. cancer of the esophagus early stage combined detection kit according to claim 2, which is characterized in that the solid-phase matrix is micro-
The supported matrix that orifice plate, microsphere or perforated membrane are constituted.
4. cancer of the esophagus early stage combined detection kit according to claim 3, which is characterized in that the kit further includes
Sample dilution, cleaning solution, secondary antibody dilution, developing solution and terminate liquid.
5. cancer of the esophagus early stage combined detection kit according to claim 4, which is characterized in that the Sample dilution is
The PBST buffer (w/v) of 1%BSA；The cleaning solution is the phosphate buffer (w/v) containing 0.2% polysorbas20；Described second is anti-
Body dilution is the PBST buffer (w/v) of 1%BSA；The developing solution is mixed in equal volume by developing solution A and developing solution B,
Developing solution A is 0.02%TMB(w/v), developing solution B is that 0.006% urea peroxide is plain (w/v)；The terminate liquid is 2mol/L sulfuric acid
Solution.
6. cancer of the esophagus early stage combined detection kit according to claim 5, which is characterized in that the kit be based on
The Multiple detection kit of Luminex xMAP technology.
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| CN112595849A (en) * | 2020-06-01 | 2021-04-02 | 郑州大学第一附属医院 | Serological detection marker for early esophageal cancer screening and application thereof |
| CN112595849B (en) * | 2020-06-01 | 2023-05-26 | 郑州大学第一附属医院 | Serological detection marker for screening early esophageal cancer and application thereof |
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| CN114397454A (en) * | 2021-12-03 | 2022-04-26 | 新疆医科大学第三附属医院 | Rapid human c-myc gene monitoring kit and use method thereof |
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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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Application publication date: 20190903 |