CN106053812A - Multi-autoantibody joint detection ELISA kit for early screening and diagnosis on liver cancer - Google Patents
Multi-autoantibody joint detection ELISA kit for early screening and diagnosis on liver cancer Download PDFInfo
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Abstract
The invention discloses a multi-autoantibody joint detection ELISA kit for early screening and diagnosis on liver cancer. The kit contains a solid carrier and tumor-associated antigen, wherein the solid carrier is coated by the tumor-associated antigen; the tumor-associated antigen is CyclinB1, p90, c-myc, p53, NPM1, HCC1, 14-3-3zeta and p62. Furthermore, the kit further contains sample diluent, a second antibody, second antibody diluent, positive control serum, negative control serum, a developing liquid, a termination liquid and a washing liquid. The ELIS kit disclosed by the invention is applied to early screening on liver cancer and has the advantages of high sensitivity, good stability, simplicity and convenience in operation and the like. In addition, the ELISA kit disclosed by the invention is capable of detecting 11 blood samples simultaneously, so that the cost can be lowered, the efficiency can be improved, and patients suffering from liver cancer can be diagnosed and treated before occurrence of clinical symptoms.
Description
Technical field
The present invention relates to field of biomedicine technology, be specifically related to a kind of for early hepatocarcinoma examination and diagnosis multiple from
Body antibody combined detection ELISA kit.
Background technology
Primary hepatocarcinoma is one of the most modal malignant tumor, and annual new hepatocarcinoma case of sending out has more than half
Being in China, the life and health of people in serious threat.Hepatocarcinoma onset is hidden very much, poor prognosis.The side such as excision and liver transplantation
Formula is considered as the means of hepatocarcinoma radical treatment, but Most patients is when by ID, just has been enter into later period of hepatocarcinoma, because of
And lose the chance for the treatment of.The most topmost reason is the usual non-evident sympton of early hepatocarcinoma patient, or volume is the least,
Image analysis is more difficult to be discovered, and alpha-fetoprotein (AFP) is as main serum markers, there is again the shortcoming that sensitivity is low,
To such an extent as in " the clinical hepatocarcinoma guide update version " that hepatopathy EASD of the U.S. (AASLD) issues for 2010, the most no longer
Recommend AFP as the screening indexes of hepatocarcinoma.Researchers study at the molecular level of hepatocarcinoma, for hepatocarcinoma molecular diagnosis with
And the formulation of postoperative individualized treatment scheme etc. provides important theoretical foundation.
The most up-to-date malignant tumor detection method in early days has multiple tumor markers detection protein chip in diagnosis (C12 core
Sheet), oncogene mutation detection kit, six tumor markers measure test kit, human malignant lesion's specificity growth factor
(TSGF) enzyme immunoassay (ELISA) test kit.But, multiple tumor markers detection protein chip in diagnosis (C12 chip)
Detection false positive is higher, and does not have the mark of hepatocarcinoma detection targetedly, and detection efficiency is low.Oncogene abrupt climatic change tries
Agent relies on the operation that polymerase chain reaction (PCR) is the most complicated, and easily produces false positive, is a kind of insecure analysis side
Method.Six tumor markers measures test kit and human malignant lesion's specificity growth factor (TSGF) enzyme immunoassay
(ELISA) test kit, can only measure patient has tumor, but can not judge the particular type of tumor.
In recent years, in the research field of human tumor, many researchs have been found that in cancer patient's serum containing one
The cell protein of the induction autoantibody reaction that group is unique, is referred to as tumor associated antigen (tumor-associated
Antigen, TAA), the antibody that its induction produces is referred to as anti-TAA-antibody (autoantibody).The proposition of this concept is to liver
A new direction has been guided in the research of cancer early diagnosis.1998, doctor Zhang at U.S.'s Scripps institute was found that one
New tumor associated antigen p62.This TAA being referred to as p62 can produce immunoreation by exciting human, produces autoantibody,
Significant is time that anti-p62 autoantibody occurs in liver cancer patient blood circulation: use from development of chronic hepatitis
Arrive the sequence serum of hepatocarcinoma to liver cirrhosis again, find that anti-p62 autoantibody did not occur, liver in the chronic hepatitis stage of patient
A period of time content before the early stage of hardening, mid-term the most do not occur, and the advanced liver cancer in the liver cirrhosis stage occurs drastically rises
Height, and clinical symptoms and the sign of hepatocarcinoma the most not yet occurs, the most still can not find hepatocarcinoma, and this phenomenon is multiple
The sequence serum of liver cancer patient is repeated.This just explanation, anti-p62 autoantibody be one potential can early diagnosis
The serologic marker thing of hepatocarcinoma.Subsequently the Virus monitory of hepatocarcinoma, liver cirrhosis, chronic hepatitis patient and the normal person of small sample is resisted
P62 autoantibody detects, and result shows that the specificity of this autoantibody is the highest, but sensitivity still can not reach
20%.Obviously, if individually using anti-p62 autoantibody as the mark of diagnosing liver cancer, diagnostic value is relatively low.Researcher is connecing
In the work got off, it is intended to find sensitiveer and special anti-TAA autoantibody, but result is not ideal enough.In 2003
A use 7 kinds of anti-TAA autoantibodys detection hepatocarcinoma research in, the sensitivity of single index is less than 20%, but logical
Crossing these 7 kinds of TAA autoantibodys in parallel, the sensitivity of detection can reach 56.9%, and the specificity diagnosed is the highest.Explanation
Combine multiple TAA autoantibody and there is higher diagnostic value, be a kind of strategy having application potential.
Follow-up study over more than ten years always strives to find the most sensitive special anti-TAA autoantibody of diagnosing liver cancer, excellent
Change the combination of diagnosing liver cancer.Finding valuable TAA autoantibody, conventional method has two kinds: one to be that recombinant cDNA expresses literary composition
Storehouse serological screening (serological analysis of recombinant cDNA expression libraries,
SEREX)；Another kind is proteomic techniques.
Applying eight kinds of TAA combinations that proteomic techniques filters out in this research, these eight kinds of TAA are respectively
CyclinB1, p90, c-myc, p53, NPM1, HCC1,14-3-3zeta and p62, research has shown that it develops with hepatocarcinoma
Closely related.CyclinB1 is a kind of cyclin, is the positivity regulatory factor of cell cycle, enters controlling cell
Playing a significant role during G2/M phase test point, its overexpression may result in cell division proliferation out of control thus induced tumor
Occurring, at the commitment of cancer, the unconventionality expression of CyclinB1 can also be identified by the immune system of human body, and body occurs
Immunoreation, and then produce substantial amounts of B1 cyclin antibody.P90 has another name called CIP2A, the tumor suppression of PP2A
The factor, interferes cancer suppressorfactor protein phosphatase 2A, thus promotes cell proliferation.C-myc transcription factor protein, is a kind of turning
The record factor, promotes cell division and propagation.There are some researches show that c-myc gene is unrestrictedly expressed in tumor, some can be made thin
Born of the same parents are the most dead.P53, tumor suppressor protein, control the startup of cell cycle.P53 gene mutation betides the tumor of majority
In cell, by the p53 albumen of encoding mutant type after sudden change.Mutain may interfere with the function of p53, and its overexpression and accumulation are led
Causing normal p53 albumen and lose function, this will cause the generation of p53 antibody in serum, and induced tumor develops.NPM1 is core
Core Phospoprotein, participates in regulation ARP/p53 path, frequent overexpression in human cell's cancer, sudden change, resets and delete mistake, because of
This is considered as often tumor markers.HCC1/CAPER α, mRNA suppress oncoprotein enzyme inhibitor, are that Steroid hormone receptors is situated between
That leads transcribes and alternative splicing factor, has important effect in terms of controlling the carcinogenecity activity of Rel/NF-kB.14-3-
A member in 3zeta, 14-3-3 family, by combining containing the protein mediated signal transduction of phosphoric acid silk ammonia, initial with cancer and
Target protein generation reciprocal action in progression and play potential carcinogenesis.Its mechanism is also not very clear, but, closely
In a little years, 14-3-3zeta albumen importance in cancer becomes clear day by day.Many is closed with 14-3-3zeta about hepatocarcinoma
The research of system shows, 14-3-3zeta plays an important role in hepatocarcinoma process.14-3-3zeta can be used as
One potential tumor associated antigen, the discovery of 14-3-3zeta antibody perhaps can help the early diagnosis of hepatocarcinoma.p62/
IMP2, insulin like growth factor 2mRNA associated proteins 2, p62 gene is initially from patients with hepatocellular carcinoma in cDNA expression library
Serum in autoantibody in immunoscreening and obtain.P62 is a kind of Cytoplasm mRNA associated proteins, compiles in conjunction with messenger RNA
Code insulin-like growth factor II (IGF-II), participates in the signal pathway of insulin and insulin secretion, be one known
Overexpression in hepatocarcinoma, and growth factor bindin carcinogenic in transgenic animal.Therefore, this research will associating
The eight kinds of TAA autoantibodys filtered out, develop a kind of have that high sensitivity is high and high specific for hepatocarcinoma early diagnosis and
The ELISA kit of examination.
Summary of the invention
It is an object of the invention to provide a kind of for early hepatocarcinoma examination with the multiple autoantibody joint-detection of diagnosis
ELISA kit, this ELISA kit has higher sensitivity and specificity, and easy to use, cheap, testing cost
Low, it is possible to promote the use of in common lab.
The technical solution used in the present invention is:
A kind of for early hepatocarcinoma examination with the multiple autoantibody joint-detection ELISA kit of diagnosis, described reagent
Box include solid phase carrier and the tumor associated antigen being coated on solid phase carrier, described tumor associated antigen be CyclinB1,
P90, c-myc, p53, NPM1, HCC1,14-3-3zeta and p62.
According to above-mentioned multiple autoantibody joint-detection ELISA kit, described test kit also include Sample dilution,
Second antibody, second antibody diluent, positive control serum, negative control sera, nitrite ion, stop buffer, cleaning mixture.Described sample
This diluent is PBST (phosphate tween) buffer containing 1% (W/V) BSA；Described second antibody diluent is containing 1%
(W/V) PBST (phosphate tween) buffer of BSA；Described nitrite ion is made up of nitrite ion A and nitrite ion B, described nitrite ion A
Being 0.02% (W/V) TMB (TMB), described nitrite ion B is 0.006% (W/V) urea peroxide element；
Described stop buffer is the concentrated sulphuric acid of 10%；Described cleaning mixture is the PBST (phosphoric acid of the 0.01M of the pH7.4 containing 0.05% tween 20
Salt tween) buffer.
According to above-mentioned multiple autoantibody joint-detection ELISA kit, described second antibody is with detectable mark
Note thing；Described detectable label is preferably horseradish peroxidase；Further, described second antibody is preferably RecA egg
In vain (recombinant protein A).
According to above-mentioned multiple autoantibody joint-detection ELISA kit, described positive control serum is preferably p53 sun
Property control serum, described negative control sera is preferably p53 negative control sera；Wherein, described p53 positive control serum is to make
Detecting p53 antibody by indirect ELISA and Western blot method and be the liver cancer patient blood serum of the positive, described p53 feminine gender is right
According to serum be use indirect ELISA method detection p53 antibody expression be the normal of normal population serum antibody average content
The serum of people.Because research has clearly indicated that p53 antigen participates in hepatocarcinoma and develops, current lot of documents also reports anti-p53
Antibody has higher positive rate in liver cancer patient blood serum.Therefore to raising work efficiency, the present invention selects p53 antibody sun
Property serum is as positive control.Because positive control serum and negative control sera are the serum through meticulously screening, because of
This, the power of other antigen antibody reactions of same ELISA kit can reach the purpose of Quality Control accordingly as reference.
According to above-mentioned multiple autoantibody joint-detection ELISA kit, described solid phase carrier is preferably 96 hole enzyme marks
Plate；Described 96 hole ELISA Plate according to well-designed layout (seeing Fig. 2) be coated CyclinB1, p90, c-myc, p53,
These eight kinds of tumor associated antigens of NPM1, HCC1,14-3-3zeta and p62, where each row is coated with a kind of antigen, every kind of antigen bag
By in 11 loading wells.The blood serum sample of same detection object adds 96 holes of ELISA kit of the present invention after dilution
The string of ELISA Plate, can reach and detect the purpose of the expression of eight kinds of TAA antibody in this blood serum sample simultaneously.Further
Ground, described 96 hole ELISA Plate are additionally provided with blank control wells, Positive control wells and negative control hole, wrap in described blank control wells
By the liquid that is coated without antigen, described Positive control wells and negative control hole are all coated p53 antigen.
According to above-mentioned multiple autoantibody joint-detection ELISA kit, described multiple autoantibody joint-detection
The detection object of ELISA kit is serum human.Same blood serum sample adds the string of 96 hole ELISA Plate after dilution, can
Reach to detect the expression of eight kinds of TAA antibody in this blood serum sample simultaneously.
The positive beneficial effect that the present invention obtains:
(1) present invention uses CyclinB1, p90, c-myc, p53, NPM1, HCC1,14-3-3zeta and p62 conduct first
One combination is used for early screening and the diagnosis of hepatocarcinoma, has higher accuracy rate.
(2) present invention is prepared for multiple autoantibody joint-detection ELISA kit according to Dot-ELISA, should
ELISA kit has higher sensitivity and specificity, and easy to use, and operating procedure is simple, quick, is greatly enhanced
Clinical detection efficiency；Additionally, testing cost is low, it is possible to promote the use of in common lab.
(3) the multiple autoantibody joint-detection ELISA kit use RecA albumen of the present invention is as second antibody, its
Can be with the Fc section specific bond in antibody structure, relative to the IgG of traditional horseradish peroxidase-labeled, with RecA albumen
As second antibody, there is high specificity, feature that background value is low, it is possible to increase the accuracy rate of antibody test, reduce false positive
Rate.
(4) the multiple autoantibody joint-detection ELISA kit of the present invention is used, it is possible to achieve detection 11 simultaneously is treated
Survey the expression of 8 kinds of TAA antibody in blood serum sample, can be used for large-scale pattern detection, drastically increase detection and examine
Disconnected efficiency.
Accompanying drawing explanation
Fig. 1 indirect enzyme-linked immunosorbent test principle figure.
The antigen coated layout of 96 hole ELISA Plate in Fig. 2 present invention multiple autoantibody joint-detection ELISA kit
(wherein, what antigen title represented is to be coated this antigen, and final detection is that in test serum sample, corresponding antibodies expresses water
Flat；"+" i.e. Positive control wells, add when representing serum sample incubation reaction is positive control serum；"-" i.e. negative control
Hole, add when representing serum sample incubation reaction is negative control sera；" blank " i.e. blank control wells, i.e. serum sample are incubated
Adding the Sample dilution without serum when educating reaction, other operation is the most identical, and blank reflects the back of the body in experimentation
Scape value.
The 8 kinds of tumor associated antigen autoantibodys of Fig. 3 scattergram in matched group serum；These 8 kinds of tumor phases as seen from the figure
Closing antigen autoantibody Average expression level ratio in matched group relatively low, mean OD value is all much smaller than 0.25, and OD value is more than 0.5
Serum the most fewer.
The 8 kinds of tumor associated antigen autoantibodys of Fig. 4 scattergram in hepatocarcinoma group serum；These 8 kinds of tumor phases as seen from the figure
Closing antigen autoantibody Average expression level in hepatocarcinoma group higher, mean OD value floats near 0.25, and OD value is big
Serum in 0.5 also compares many, and the OD value of part serum has been even more than 1.0.
The 8 kinds of tumor associated antigen autoantibodys of Fig. 5 positive rate result in hepatocarcinoma group and matched group.(result display liver
In cancer group, 8 kinds of tumor associated antigen autoantibodies rates are at 20.2%-41.4%, and these 8 kinds of tumor associated antigens self resist
Body positive rate in matched group is all less than 10%.Through statistical test, these 8 kinds of tumor associated antigen autoantibodys are in hepatocarcinoma
In positive rate be above matched group.)
The ROC curve (Receiver operating curve) of 8 kinds of tumor associated antigen autoantibody detection hepatocarcinoma of Fig. 6；Vertical seat
Being designated as True Positive Rate (sensitivity), abscissa is false positive rate.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described, but is not limiting as protection scope of the present invention.
The preparation of 1: eight kind of tumor associated antigen of embodiment
By using prokaryotic expression system, prokaryotic expression and purification 8 kinds of tumor associated antigens (CyclinB1, p90, c-
Myc, p53, NPM1, HCC1,14-3-3zeta and p62), in order to prepare for the experiment of lower step.Concrete antigen preparation process is as follows:
1) use gene clone technology build eight kinds of tumor correlated albumens (CyclinB1, p90, c-myc, p53, NPM1,
HCC1,14-3-3zeta and p62) recombined pronucleus expression plasmid.
2) destination protein is expressed: be transformed into respectively in escherichia coli Bl21 (DE3) by the recombined pronucleus expression plasmid of structure,
Use the expression of IPTG (isopropylthiogalactoside) induction destination protein.
3) purification destination protein: according to the label entrained by destination protein, uses traditional corresponding purification schemes to mesh
Albumen be purified；Wherein, for carrying histidine-tagged c-myc, p53, p62,14-3-3zeta, NPM1 and HCC1 egg
White use Ni-NTA post to be purified, the Cyclin B1 and p90 albumen for carrying GST label then use glutathione S-
Transferring enzyme is purified.
4) use Bradford method to measure protein concentration (Suo Laibao test kit), use the method for Western blot to pure
The immunologic competence changing albumen is identified.
In view of prokaryotic system expression and purification recombiant protein technology comparative maturity, detailed process no longer describes in detail, by more than
Preparation process, successfully obtains 8 kinds of activated destination proteins (i.e. 8 kinds tumor associated antigens), prepares for subsequent experimental.
Embodiment 2: the preparation of test kit
The present invention is prepared for a kind of can be used for early hepatocarcinoma examination and diagnosis is many according to the principle of Dot-ELISA
Plant autoantibody joint-detection ELISA kit.The principle of Dot-ELISA is to be connected on solid phase carrier by antigen, sample
Test antibodies one-tenth in combination solid phase antigen in product-by inspection antibody complex, then with ELIAS secondary antibody and solid phase antigen-by examining antibody
Antibodies in complex, formation solid phase antigen-by inspection antibody-ELIAS secondary antibody complex, then measure showing after adding substrate
Color degree, determines test antibodies content.
1, reagent and material
(1) eight kind of tumor correlated albumen (CyclinB1, p90, c-myc, p53, NPM1, HCC1,14-3-3zeta and p62
Albumen)；
(2) antigen coated liquid: 50mM carbonate buffer solution, pH=9.6；
(3) confining liquid: containing the PBST buffer of 2% (W/V) BSA；
(4) enzyme mark second antibody: the RecA albumen of horseradish peroxidase-labeled, purchased from Invitrogen company；
(5) 96 hole ELISA Plate: purchased from Jet Biofil company；
(6) nitrite ion A:0.02% (W/V) TMB (TMB), preparation: weigh methyl biphenyl amine
(TMB) 0.005g, is dissolved in 25ml deionized water.
(7) nitrite ion B:0.006% (W/V) urea peroxide element, preparation: weigh citric acid 4.665g, Na2HPO4·12H2O
18.40g, after being completely dissolved in 400ml deionized water, adds 0.75% carbamide peroxide element 3.2ml, adjust pH value to 5.0～
5.4, add deionized water and be settled to final volume 500ml, mix 4 DEG C of preservations.
2, antigen coated ELISA Plate is prepared
(1) eight kinds of tumor associated antigen solution are prepared:
Eight kinds of tumor correlated albumens are dissolved separately in and are coated in liquid, fully mix, be configured to the antigen of 8 kinds of variable concentrations
Solution, wherein, the concentration of NPM1 solution is 0.125 μ g/ml, and the concentration of p53 solution is 0.25 μ g/ml, and the concentration of p90 solution is
0.25μg/ml；The concentration of p62 solution is 0.5 μ g/ml, and the concentration of HCC1 solution is 0.5 μ g/ml；The concentration of CyclinB1 solution
Being 1.0 μ g/ml, the concentration of 14-3-3zeta solution is 1.0 μ g/ml, and the concentration of c-myc solution is 1.0 μ g/ml.
(2) coated elisa plate: carry out eight kinds of tumor associated antigen solution that point sample will prepare according to the layout shown in Fig. 2
Being added separately in the loading wells of 96 hole ELISA Plate, sample-adding amount is 100 μ l/ holes；Positive control wells and negative control hole add
P53 antigenic solution, blank control wells addition is coated liquid, removes and be coated liquid after 4 DEG C of overnight incubation.
(3) close: the loading wells of 96 hole ELISA Plate after being coated adds confining liquid, incubated at room 2 hours, then removes
Deblocking agent.
(4) will be after 96 hole ELISA Plate after step (3) processes to place 37 DEG C of drying bakers drying, packaging, 4 DEG C of preservations are standby
With.
3, the composition of test kit of the present invention is as follows:
(1) 96 antigen coated hole ELISA Plate；
(2) Sample dilution: containing the PBST buffer of 1% (W/V) BSA；
(3) second antibody diluent: for the PBST buffer containing 1% (W/V) BSA；
(4) enzyme mark second antibody: the RecA albumen of horseradish peroxidase-labeled, purchased from Invitrogen company；
(5) nitrite ion: nitrite ion is made up of nitrite ion A and nitrite ion B, wherein, nitrite ion A is 0.02% (W/V) TMB
(TMB), nitrite ion B is 0.006% (W/V) urea peroxide element；
(6) stop buffer: the concentrated sulphuric acid of 10%；
(7) cleaning mixture: PBST (phosphate tween) buffer of the 0.01M of the pH7.4 containing 0.05% tween 20.
Embodiment 3: the detection method of test kit of the present invention
1, serum sample is hatched:
Serum sample 1%BSA solution to be detected is diluted in the ratio of 1:100, then by the blood after dilution
Final proof originally adds according to the layout shown in Fig. 2 in the loading wells of 96 hole ELISA Plate of envelope antigen, and sample-adding amount is 100 μ l/
Hole, is placed in 37 DEG C of constant incubators and hatches 1h, then discard liquid in loading wells, washs 5 times with cleaning mixture.
2, ELIAS secondary antibody is hatched:
The RecA albumen 1%BSA solution of horseradish peroxidase-labeled is diluted in the ratio of 1:8000, then
Being added by the RecA albumen of the horseradish peroxidase-labeled after dilution in the loading wells of 96 hole ELISA Plate, sample-adding amount is 100 μ l/
Hole, is placed in 37 DEG C of constant incubators and hatches 1h, then discard liquid in loading wells, washs 5 times with cleaning mixture.
3, develop the color and terminate reaction
By nitrite ion A and nitrite ion B according to 1:1 equal-volume mix homogeneously, then mixed nitrite ion is rapidly added
In the loading wells of 96 hole ELISA Plate, sample-adding amount is 100 μ l/ holes, is placed in 37 DEG C of water-bath lucifuge colour developing 15min, then to each point
Adding 50 μ l stop buffers in sample hole, color development stopping is reacted, and then reads OD value at 450nm and 595nm by automatic microplate reader,
And return to zero by blank well.
4, result judges:
Two standard deviations (Mean+2SD) are added as cutoff value (Cut-using the average of the OD value of all Normal groups
Off value), it is judged to the positive higher than this value, is judged to feminine gender less than this value.
Embodiment 4: with detection liver cancer patient and the serum sample of normal person of the test kit of the embodiment of the present invention 2
Sample prepares: according to epidemiological analysis, this research have collected the serum (hepatocarcinoma of 84 example Patients with Primary
Group), and the serum (matched group) of 89 example normal persons.In 84 example Patients with Primary, male 56 example, women 28 example, averagely
Age is 57.0 ± 11.3 years old, and the range of age is 23-77 year；In 89 example normal persons, male 59 example, women 30 example, the mean age
Being 57.2 ± 10.9, the range of age is 25-79 year.All liver cancer patient blood serums are all initially to be diagnosed as hepatocarcinoma patient not yet to connect
Collecting during by any chemicotherapy, the time being diagnosed is that in October, 2011 is in October, 2013.Normal human serum is from participation
Annual health examination, without the Check-up crowd of any malignant tumor relevant disease.
The test kit described in the embodiment of the present invention 2 and the detection method described in embodiment 3 is used respectively 84 example hepatocarcinoma to be suffered from
In person's serum (hepatocarcinoma group) and 89 example normal human serums (matched group), the content of 8 kinds of tumor associated antigen autoantibodys is examined
Survey.In matched group serum, the meansigma methods of 8 kinds of tumor associated antigen autoantibody content adds two standard deviations as cutoff value, point
Ji Suan the positive rate of 8 kinds of tumor associated antigen autoantibodys in hepatocarcinoma group and matched group.Application MedCalc Software on Drawing 8 kinds
Tumor associated antigen autoantibody Average expression level scattergram (result is shown in Fig. 3 and Fig. 4) in hepatocarcinoma group and matched group, with
And the bar diagram (result is shown in Fig. 5) two groups of positives rates.This research also applies SPSS21.0 software to carry out statistical test,
Using two independent sample X 2 test methods to compare hepatocarcinoma group and matched group antibody positive rate, inspection level α takes 0.05, then adopts
The diagnostic value (the results are shown in Table 1 and Fig. 6) of autoantibody detection hepatocarcinoma is evaluated with the evaluation methodology of Screen test.
Table 1 different TAAs (tumor associated antigen) combination detection result sets Authentic Assessment in parallel
Note: youden index: youden index is sensitivity and specificity sum deducts 1, overall merit the method is for liver
The diagnostic value of cancer, this numerical value less than 1, is closer to 1 diagnostic value the biggest more than 0.
From Fig. 3 and Fig. 4, in matched group, 8 kinds of tumor associated antigen autoantibody Average expression level are relatively low, flat
All OD values are all much smaller than 0.25, and the OD value serum more than 0.5 is less；And in hepatocarcinoma group, in serum, 8 kinds of tumor associated antigens are certainly
Body antibody average water is the highest, and mean OD value floats near 0.25, and the serum that OD value is more than 0.5 is more, some serum
Detection tumor associated antigen autoantibody OD value has been even more than 1.0；Thus illustrate that these 8 kinds of tumor associated antigen autoantibodys can
Diagnosis for hepatocarcinoma.As shown in Figure 5,8 kinds of tumor associated antigen autoantibodies rates of hepatocarcinoma group are at 20.2%-
41.4%, and these 8 kinds of tumor associated antigen autoantibodys at matched group positive rate all less than 10%, and through statistics examine
Testing, this 8 kinds of tumor associated antigen autoantibodys positive rate in hepatocarcinoma, obviously higher than matched group, thus proves that these 8 kinds are swollen
Tumor related antigen autoantibody can be as the Testing index of hepatocarcinoma early diagnosis, for the early diagnosis of hepatocarcinoma.
As shown in Table 1, along with the increase of antigen combined number, the sensitivity of diagnosis is consequently increased, when 8 kinds of tumors are correlated with
During antigen combination, the sensitivity of diagnosis has been finally reached 86.9%, is i.e. to apply this kind of method to be examined by correct in liver cancer patient
The percentage ratio for hepatocarcinoma that breaks is 86.9%.Although and specificity reduces, but 73% can be reached, this demonstrate non-liver cancer patient
Applying this kind of method is 73% by the correct percentage ratio being diagnosed as being not suffering from hepatocarcinoma.Use these 8 kinds of tumor associated antigen combinations right
Hepatocarcinoma diagnoses, can ensure diagnosis specificity on the premise of by a relatively large margin improve diagnosis sensitivity, this combination is right
Whether object to be measured is that the judgement accuracy of liver cancer patient is the highest, is 79.8%, thus can also prove that these 8 kinds of tumors are correlated with
Antigen combination is ideal hepatocarcinoma early diagnosis and screening method and means.
It will be appreciated from fig. 6 that area is 0.800 under the ROC curve of this method, illustrate to use this multiple autoantibody joint-detection
ELISA kit has higher judgement correctness and higher diagnostic value for hepatocarcinoma, further demonstrate that we are opened
This multiple autoantibody joint-detection ELISA kit sent be ideal hepatocarcinoma early diagnosis and screening method and
Means.
Claims (8)
1. one kind is used for early hepatocarcinoma examination and the multiple autoantibody joint-detection ELISA kit of diagnosis, it is characterised in that
Described test kit includes solid phase carrier and the tumor associated antigen being coated on solid phase carrier, and described tumor associated antigen is
CyclinB1, p90, c-myc, p53, NPM1, HCC1,14-3-3zeta and p62.
Multiple autoantibody joint-detection ELISA kit the most according to claim 1, it is characterised in that described solid phase
Carrier is 96 hole ELISA Plate.
Multiple autoantibody joint-detection ELISA kit the most according to claim 1, it is characterised in that described reagent
Box also include Sample dilution, second antibody, second antibody diluent, positive control serum, negative control sera, nitrite ion,
Stop buffer, cleaning mixture.
Multiple autoantibody joint-detection ELISA kit the most according to claim 3, it is characterised in that described second
Antibody is with detectable label.
Multiple autoantibody joint-detection ELISA kit the most according to claim 4, it is characterised in that described examine
The label surveyed is horseradish peroxidase.
Multiple autoantibody joint-detection ELISA kit the most according to claim 3, it is characterised in that described second
Antibody is RecA albumen.
Multiple autoantibody joint-detection ELISA kit the most according to claim 3, it is characterised in that the described positive
Control serum is p53 positive control serum, and described negative control sera is p53 negative control sera；Wherein, described p53 is positive
Control serum is the liver cancer patient blood serum using indirect ELISA and Western blot method detection p53 antibody to be the positive, institute
State p53 negative control sera be use indirect ELISA method detection p53 antibody expression be that normal population serum antibody is average
The serum of the normal person of content.
Multiple autoantibody joint-detection ELISA kit the most according to claim 1, it is characterised in that described multiple
The detection object of autoantibody joint-detection ELISA kit is serum human.
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| CN110187109A (en) * | 2019-05-31 | 2019-08-30 | 郑州大学第一附属医院 | A kind of autoantibody joint-detection ELISA kit for Grades of Gastric Cardia Adenocarcinoma early screening |
| CN110187113A (en) * | 2019-06-19 | 2019-08-30 | 郑州大学第一附属医院 | A kind of autoantibody joint-detection ELISA kit for esophageal squamous cell carcinoma early screening |
| CN110286235A (en) * | 2019-07-10 | 2019-09-27 | 郑州大学 | A kind of joint-detection blood serum designated object, kit and detection method for liver cancer early screening and diagnosis |
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