CN106053812B - A kind of a variety of autoantibody joint-detection ELISA kits for being used for liver cancer early screening and diagnosis - Google Patents
A kind of a variety of autoantibody joint-detection ELISA kits for being used for liver cancer early screening and diagnosis Download PDFInfo
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Abstract
The invention discloses a kind of for liver cancer early screening and a variety of autoantibody joint-detection ELISA kits of diagnosis, the kit includes solid phase carrier and the tumor associated antigen being coated on solid phase carrier, and the tumor associated antigen is CyclinB1, p90, c myc, p53, NPM1, HCC1,14 3 3zeta and p62.Further, the kit also includes Sample dilution, secondary antibody, secondary antibody dilution, positive control serum, negative control sera, nitrite ion, terminate liquid, cleaning solution.The present invention ELISA kit be used for liver cancer early diagnosis there is high sensitivity, stability is good, it is easy to operate the advantages that.In addition, 11 blood samples can be detected simultaneously using the ELISA kit of the present invention, cost is reduced, improves efficiency, liver cancer patient just can be diagnosed before clinical symptoms appearance and treatment can be received.
Description
Technical field
The present invention relates to field of biomedicine technology, and in particular to it is a kind of for liver cancer early screening and diagnosis it is a variety of from
The antibody combined detection ELISA kit of body.
Background technology
Primary carcinoma of liver is one of most common malignant tumour in the world, and annual new hair liver cancer case has more than half
It is in China, serious threat the life and health of people.Liver cancer onset is hidden very much, poor prognosis.The side such as surgery excision and liver transfer operation
Formula is considered as the means of liver cancer radical treatment, but Most patients just have been enter into later period of hepatocarcinoma when by initial diagnosis, because
And lose the chance for the treatment of.Wherein most important reason is the usual non-evident sympton of early liver cancer patient, or volume very little,
Image analysis is more difficult to be discovered, and alpha-fetoprotein (AFP) has the shortcomings that sensitiveness is low as main serum markers again,
So that in hepatopathy EASD of the U.S. (AASLD) issue in 2010《Clinical hepatocellular carcinoma guide update version》In, no longer
Recommend screening indexes of the AFP as liver cancer.Researchers are studied in the molecular level of liver cancer, be liver cancer molecule diagnose with
And formulation of postoperative individualized treatment scheme etc. provides important theoretical foundation.
Malignant tumour early detection method newest at present has multiple tumor markers detection protein chip in diagnosis (C12 cores
Piece), oncogene mutation detection kit, six tumor markers measure kit, human malignant lesion's specificity growth factor
(TSGF) ELISA (ELISA) kit.But multiple tumor markers detection protein chip in diagnosis (C12 chips)
It is higher to detect false positive, and liver cancer detection mark, detection efficiency are low without having targetedly.Oncogene abrupt climatic change tries
Agent easily produces false positive by the so complicated operation of PCR (PCR), is a kind of insecure analysis side
Method.Six tumor markers determine kit and human malignant lesion's specificity growth factor (TSGF) ELISA
(ELISA) kit, can only measure patient has tumour, but can not judge the particular type of tumour.
In recent years, in the research field of human tumor, many researchs, which are had been found that in cancer patient's serum, contains one
The cell protein of the unique induction autoantibody reaction of group, is referred to as tumor associated antigen (tumor-associated
Antigen, TAA), antibody caused by its induction is referred to as anti-TAA- antibody (autoantibody).The it is proposed of this concept is to liver
A new direction has been guided in cancer early diagnosis research.1998, in the U.S., doctor Zhang of Scripps research institutes was found that one kind
New tumor associated antigen p62.This TAA for being referred to as p62 can exciting human produce immune response, produce autoantibody,
Significant is the time that anti-p62 autoantibodies occur in the liver cancer patient circulatory system：Using from development of chronic hepatitis
Arrive the sequence serum of liver cancer again to hepatic sclerosis, it is found that anti-p62 autoantibodies do not occur in the chronic hepatitis stage of patient, in liver
A period of time content before the early stage of hardening, mid-term also do not occur, and the advanced liver cancer in the hepatic sclerosis stage occurs drastically rises
Height, and now not yet there are the clinical symptoms and sign of liver cancer, liver cancer clinically can not be still found, and this phenomenon is multiple
Repeated in the sequence serum of liver cancer patient.This just illustrates, anti-p62 autoantibodies, which are one, potential can early diagnose
The serologic marker thing of liver cancer.Then the liver cancer of small sample, hepatic sclerosis, chronic hepatitis patient and the Virus monitory of normal person are resisted
P62 autoantibodies are detected, and as a result show that the specificity of this autoantibody is very high, but sensitivity can not still reach
20%.Obviously, it is if individually relatively low as the mark of diagnosing liver cancer, diagnostic value using anti-p62 autoantibodies.Researcher is connecing
In the work got off, it is intended to find sensitiveer and special anti-TAA autoantibodies, but result is not ideal enough.In 2003
7 kinds of a use anti-TAA autoantibodies detection liver cancer research in, the sensitivity of single index is no more than 20%, but logical
This 7 kinds of TAA autoantibodies in parallel are crossed, the sensitivity of detection can reach 56.9%, and the specificity diagnosed is still very high.Explanation
Combining multiple TAA autoantibodies has higher diagnostic value, is a kind of strategy for having application potential.
Follow-up study over more than ten years always strives to find the more sensitive special anti-TAA autoantibodies of diagnosing liver cancer, excellent
Change the combination of diagnosing liver cancer.Valuable TAA autoantibodies are found, conventional method there are two kinds：First, recombinant cDNA expression text
Storehouse serological screening (serological analysis of recombinant cDNA expression libraries,
SEREX)；Another kind is proteomic techniques.
The eight kinds of TAA combinations filtered out in this research using proteomic techniques, this eight kinds of TAA are respectively
CyclinB1, p90, c-myc, p53, NPM1, HCC1,14-3-3zeta and p62, research have shown its occurrence and development with liver cancer
It is closely related.CyclinB1 is a kind of cyclin, is the positivity regulatory factor of cell cycle, is entered in control cell
Played a significant role during G2/M phase test points, its overexpression can cause cell division proliferation out of control so as to induced tumor
Occur, in the early stage of cancer, CyclinB1 unconventionality expression can also be identified by the immune system of human body, and body occurs
Immune response, and then produce substantial amounts of B1 cyclins antibody.P90 also known as CIP2A, the tumor suppression of PP2A
The factor, cancer suppressorfactor protein phosphatase 2A is interfered, so as to promote cell to breed.C-myc transcription factor proteins, it is a kind of turn
The factor is recorded, promotes cell division and propagation.There are some researches show c-myc genes are unrestrictedly expressed in tumour, some can be made thin
Born of the same parents are never dead.P53, tumor suppressor protein, control the startup of cell cycle.P53 gene mutations betide the tumour of majority
In cell, by the p53 albumen of encoding mutant type after mutation.Mutain may interfere with p53 function, and it is over-expressed and accumulation is led
Normal p53 albumen is caused to lose function, this will cause the generation of p53 antibody in serum, the occurrence and development of induced tumor.NPM1 is core
Benevolence Phospoprotein, regulation ARP/p53 paths are participated in, the frequent overexpression in human cell's cancer, mutation, reset and delete mistake, because
This is considered as often tumor markers.HCC1/CAPER α, mRNA suppress oncoprotein enzyme inhibitor, are that Steroid hormone receptors are situated between
The transcription led and alternative splicing factor, play the role of in terms of control Rel/NF-kB carcinogenic sexuality important.14-3-
A member in 3zeta, 14-3-3 family, by combine the protein mediated signal transduction containing phosphoric acid silk ammonia, with cancer starting and
Target protein in progression occurs reciprocation and plays potential carcinogenesis.Its mechanism is also not very clear, still, closely
A little years, importance of the 14-3-3zeta albumen in cancer have become clear day by day.It is many to be closed on hepatocellular carcinoma and 14-3-3zeta
The research of system shows that 14-3-3zeta is played an important role in hepatocellular carcinoma 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 hepatocellular carcinoma.p62/
IMP2, insulin-like growth factor 2mRNA associated proteins 2, p62 genes are 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, is compiled with reference to mRNA
Code insulin-like growth factor II (IGF-II), participates in signal pathway and the insulin secretion of insulin, be one kind it is known
Over-expressed in hepatocellular carcinoma, and carcinogenic growth factor bindin in transgenic animals.Therefore, this research will combine
The eight kinds of TAA autoantibodies filtered out, exploitation it is a kind of with high sensitivity is high and high specific be used for hepatocarcinoma early diagnosis and
The ELISA kit of examination.
The content of the invention
It is an object of the invention to provide a kind of for liver cancer early screening and a variety of autoantibody joint-detections of diagnosis
ELISA kit, the ELISA kit have higher sensitivity and specificity, and easy to use, cheap, testing cost
It is low, it can be promoted the use of in common lab.
The technical solution adopted by the present invention is：
A kind of a variety of autoantibody joint-detection ELISA kits for being used for liver cancer early screening and diagnosis, the reagent
Box includes solid phase carrier and the tumor associated antigen that is coated on solid phase carrier, the tumor associated antigen be CyclinB1,
P90, c-myc, p53, NPM1, HCC1,14-3-3zeta and p62.
According to above-mentioned a variety of autoantibody joint-detection ELISA kits, the kit also include Sample dilution,
Secondary antibody, secondary antibody dilution, positive control serum, negative control sera, nitrite ion, terminate liquid, cleaning solution.The sample
This dilution is PBST (phosphate tween) buffer solution containing 1% (W/V) BSA；The secondary antibody dilution is to contain 1%
(W/V) BSA PBST (phosphate tween) buffer solution；The nitrite ion is made up of nitrite ion A and nitrite ion B, the nitrite ion A
For 0.02% (W/V) TMB (TMB), the nitrite ion B is 0.006% (W/V) urea peroxide element；
The terminate liquid is 10% concentrated sulfuric acid；The cleaning solution is the 0.01M of the pH7.4 containing 0.05% Tween-20 PBST (phosphoric acid
Salt tween) buffer solution.
According to above-mentioned a variety of autoantibody joint-detection ELISA kits, the secondary antibody carries detectable mark
Remember thing；The detectable label is preferably horseradish peroxidase；Further, the secondary antibody is preferably RecA eggs
(recombinant protein A) in vain.
According to above-mentioned a variety of autoantibody joint-detection ELISA kits, the positive control serum is preferably p53 sun
Property control serum, the negative control sera is preferably p53 negative control seras；Wherein, the p53 positive control serums are to make
It is positive liver cancer patient blood serum with indirect ELISA and Western blot methods detection p53 antibody, the p53 feminine genders are right
It is to detect p53 antibody expressions using indirect ELISA method as the normal of normal population serum antibody average content according to serum
The serum of people.Because research has clearly indicated that p53 antigens participate in the occurrence and development of liver cancer, current lot of documents also reports anti-p53
Antibody has higher positive rate in liver cancer patient blood serum.Therefore in order to improve operating 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 by screening meticulously, because
This, the power of other antigen-antibody reactions of same ELISA kit can be used as reference accordingly, reach the purpose of Quality Control.
According to above-mentioned a variety of autoantibody joint-detection ELISA kits, the solid phase carrier is preferably 96 hole enzyme marks
Plate；96 hole elisa Plates according to well-designed layout (referring to Fig. 2) be coated with CyclinB1, p90, c-myc, p53,
This eight kinds of tumor associated antigens of NPM1, HCC1,14-3-3zeta and p62, where each row are 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
One row of ELISA Plate, it can reach while detect the purpose of the expression of eight kinds of TAA antibody in the blood serum sample.Further
Ground, blank control wells, Positive control wells and negative control hole are additionally provided with 96 hole elisa Plates, is wrapped in the blank control wells
By the coating buffer without antigen, p53 antigens are coated with the Positive control wells and negative control hole.
According to above-mentioned a variety of autoantibody joint-detection ELISA kits, a variety of autoantibody joint-detections
The detection object of ELISA kit is human serum.Same blood serum sample adds a row of 96 hole elisa Plates after dilution, can
Reach while detect the expression of eight kinds of TAA antibody in the blood serum sample.
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 early screening combined for liver cancer and diagnosis, there is higher accuracy rate.
(2) present invention is prepared for a variety of autoantibody joint-detection ELISA kits 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；In addition, testing cost is low, can be promoted the use of in common lab.
(3) a variety of autoantibody joint-detection ELISA kits of the invention use RecA albumen as secondary antibody, its
Can with the Fc section specific bonds in antibody structure, relative to the IgG of traditional horseradish peroxidase-labeled, with RecA albumen
There is the characteristics of high specificity, background value is low as secondary antibody, it is possible to increase the accuracy rate of antibody test, reduce false positive
Rate.
(4) using a variety of autoantibody joint-detection ELISA kits of the present invention, it is possible to achieve while detection 11 is treated
The expression of 8 kinds of TAA antibody in blood serum sample is surveyed, available for large-scale pattern detection, detection is drastically increased and examines
Disconnected efficiency.
Brief description of the drawings
Fig. 1 indirect enzyme-linked immunosorbent test principle figures.
The antigen coat layout of 96 hole elisa Plates in a variety of autoantibody joint-detection ELISA kits of Fig. 2 present invention
(what wherein, antigen title represented is to be coated with this antigen, and what is finally detected is that corresponding antibodies express water in test serum sample
It is flat；"+" is Positive control wells, and it is positive control serum to represent added during serum sample incubation reaction；"-" is negative control
Hole, it is negative control sera to represent added during serum sample incubation reaction；" blank " is blank control wells, i.e. serum sample is incubated
The Sample dilution without serum, other operation all sames are added when educating reaction, blank control reflects the back of the body in experimentation
Scape value.
Distribution map of the 8 kinds of tumor associated antigen autoantibodies of Fig. 3 in control group serum；This 8 kinds of tumour phases as seen from the figure
Closing antigen autoantibody, Average expression level is than relatively low in control group, and mean OD value is much smaller than 0.25, and OD values are more than 0.5
Serum it is also fewer.
Distribution map of the 8 kinds of tumor associated antigen autoantibodies of Fig. 4 in liver cancer group serum；This 8 kinds of tumour phases as seen from the figure
Pass antigen autoantibody Average expression level in liver cancer group is higher, and mean OD value floats near 0.25, and OD values are big
Serum in 0.5 is also relatively more, and the OD values of part serum have been even more than 1.0.
Positive rate result of the 8 kinds of tumor associated antigen autoantibodies of Fig. 5 in liver cancer group and control group.(result shows liver
8 kinds of tumor associated antigen autoantibodies rates are in 20.2%-41.4% in cancer group, and this 8 kinds of tumor associated antigens itself are anti-
Positive rate of the body in control group is no more than 10%.Through statistical test, this 8 kinds of tumor associated antigen autoantibodies are in liver cancer
In positive rate be above control group.)
The ROC curve (Receiver operating curve) of 8 kinds of tumor associated antigen autoantibody detection liver cancer of Fig. 6；It is vertical to sit
True Positive Rate (sensitivity) is designated as, abscissa is false positive rate.
Embodiment
With reference to specific embodiment, the present invention will be further described, but is not intended to limit protection scope of the present invention.
Embodiment 1：The preparation of eight kinds of tumor associated antigens
By using prokaryotic expression system, prokaryotic expression and purifying 8 kinds of tumor associated antigens (CyclinB1, p90, c-
Myc, p53, NPM1, HCC1,14-3-3zeta and p62), to be prepared for the experiment of lower step.Specific antigen preparation process is as follows：
1) using 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) express express target protein：The recombined pronucleus expression plasmid of structure is transformed into Escherichia coli Bl21 (DE3) respectively,
Use the expression of IPTG (isopropylthiogalactoside) induction destination proteins.
3) destination protein is purified：Label according to entrained by destination protein, using traditional corresponding purification schemes to mesh
Albumen purified；Wherein, for carrying histidine-tagged c-myc, p53, p62,14-3-3zeta, NPM1 and HCC1 egg
Purified in vain using Ni-NTA posts, and for carry GST labels Cyclin B1 and p90 albumen then using glutathione S-
Transferase is purified.
4) using Bradford methods measure protein concentration (Suo Laibao kits), using Western blot method to pure
The immunologic competence for changing albumen is identified.
In view of comparative maturity, detailed process are no longer described in detail prokaryotic system expression and purification recombinant protein technology, pass through the above
Preparation process, 8 kinds of active destination proteins (i.e. 8 kinds of tumor associated antigens) are successfully obtained, are prepared for subsequent experimental.
Embodiment 2：The preparation of kit
The present invention according to the principle of Dot-ELISA be prepared for it is a kind of available for liver cancer early screening and diagnosis it is more
Kind autoantibody joint-detection ELISA kit.The principle of Dot-ELISA is that antigen is connected on solid phase carrier, sample
Test antibodies are in combination into solid phase antigen-by inspection antibody complex in product, then with ELIAS secondary antibody and solid phase antigen-by inspection antibody
Antibody binding in compound, formation solid phase antigen-by inspection antibody-ELIAS secondary antibody compound, then determine aobvious after adding substrate
Color degree, determine test antibodies content.
1st, 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 coat liquid：50mM carbonate buffer solutions, pH=9.6；
(3) confining liquid：PBST buffer solutions containing 2% (W/V) BSA；
(4) enzyme mark secondary antibody：The RecA albumen of horseradish peroxidase-labeled, purchased from Invitrogen companies；
(5) 96 hole elisa Plates：Purchased from Jet Biofil companies；
(6) nitrite ion A：0.02% (W/V) TMB (TMB), prepare：Weigh methyl biphenyl amine
(TMB) 0.005g, it is dissolved in 25ml deionized waters.
(7) nitrite ion B：0.006% (W/V) urea peroxide element, is prepared：Weigh citric acid 4.665g, Na2HPO4·12H2O
18.40g, after being completely dissolved in 400ml deionized waters, add 0.75% carbamide peroxide element 3.2ml, adjustment pH value to 5.0~
5.4, add deionized water to be settled to final volume 500ml, mix 4 DEG C of preservations.
2nd, the ELISA Plate of antigen coat is prepared
(1) eight kinds of tumor associated antigen solution are prepared：
Eight kinds of tumor correlated albumens are dissolved separately in coating buffer, fully mixed, are configured to the antigen of 8 kinds of various concentrations
Solution, wherein, the concentration of NPM1 solution is that the concentration of 0.125 μ g/ml, p53 solution is that the concentration of 0.25 μ g/ml, p90 solution is
0.25μg/ml；The concentration of p62 solution is that the concentration of 0.5 μ g/ml, HCC1 solution is 0.5 μ g/ml；The concentration of CyclinB1 solution
Concentration for 1.0 μ g/ml, 14-3-3zeta solution is that the concentration of 1.0 μ g/ml, c-myc solution is 1.0 μ g/ml.
(2) coated elisa plate：Point sample is carried out by prepare eight kinds of tumor associated antigen solution according to the layout shown in Fig. 2
It is added separately in the loading wells of 96 hole elisa Plates, sample-adding amount is 100 μ l/ holes；Added in Positive control wells and negative control hole
P53 antigenic solutions, blank control wells add coating buffer, coating buffer are removed after 4 DEG C of overnight incubations.
(3) close：Confining liquid is added into the loading wells of 96 hole elisa Plates after coating, is incubated at room temperature 2 hours, then removes
Deblocking agent.
(4) will be packed, 4 DEG C of preservations are standby after 96 hole elisa Plates after step (3) processing place 37 DEG C of drying baker drying
With.
3rd, the composition of kit of the present invention is as follows：
(1) 96 hole elisa Plates of antigen coat；
(2) Sample dilution：PBST buffer solutions containing 1% (W/V) BSA；
(3) secondary antibody dilution：For the PBST buffer solutions containing 1% (W/V) BSA；
(4) enzyme mark secondary antibody：The RecA albumen of horseradish peroxidase-labeled, purchased from Invitrogen companies；
(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 are 0.006% (W/V) urea peroxide element；
(6) terminate liquid：10% concentrated sulfuric acid；
(7) cleaning solution：The 0.01M of pH7.4 containing 0.05% Tween-20 PBST (phosphate tween) buffer solution.
Embodiment 3：The detection method of kit of the present invention
1st, serum sample is incubated：
Serum sample to be detected is pressed 1 with 1%BSA solution：100 ratio is diluted, then by the blood after dilution
Final proof sheet is added in the loading wells of 96 hole elisa Plates of envelope antigen according to the layout shown in Fig. 2, and sample-adding amount is 100 μ l/
Hole, it is placed in 37 DEG C of constant incubators and is incubated 1h, then discard liquid in loading wells, washed 5 times with cleaning solution.
2nd, ELIAS secondary antibody is incubated：
The RecA albumen of horseradish peroxidase-labeled is pressed 1 with 1%BSA solution：8000 ratio is diluted, then
The RecA albumen of horseradish peroxidase-labeled after dilution is added in the loading wells of 96 hole elisa Plates, sample-adding amount is 100 μ l/
Hole, it is placed in 37 DEG C of constant incubators and is incubated 1h, then discard liquid in loading wells, washed 5 times with cleaning solution.
3rd, colour developing and terminating reaction
By nitrite ion A and nitrite ion B according to 1:1 is isometric well mixed, is then rapidly added mixed nitrite ion
In the loading wells of 96 hole elisa Plates, sample-adding amount is 100 μ l/ holes, 37 DEG C of water-bath lucifuge colour developing 15min is placed in, then to each point
50 μ l terminate liquids are added in sample hole, color development stopping reaction, OD values are then read at 450nm and 595nm with automatic ELIASA,
And returned to zero with blank well.
4th, result judgement：
Two standard deviations (Mean+2SD) are added to be used as cutoff value (Cut- using the average of the OD values of all Normal groups
Off values), it is determined as the positive higher than this value, is determined as feminine gender less than this value.
Embodiment 4：With the detection liver cancer patient of kit and the serum sample of normal person of the embodiment of the present invention 2
Sample prepares：According to epidemiological analysis, this research have collected the serum (liver cancer of 84 Patients with Primary
Group), and the serum (control group) of 89 normal persons.In 84 Patients with Primary, male 56, women 28, averagely
Age is 57.0 ± 11.3 years old, and the range of age is 23-77 year；In 89 normal persons, male 59, women 30, average age
For 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 liver cancer in patient not yet to connect
Collected during by any chemicotherapy, the time being diagnosed is in October, 2011 in October, 2013.Normal human serum is from participation
Annual health examination, the Check-up crowd without any malignant tumour relevant disease.
84 liver cancer are suffered from respectively using the kit described in the embodiment of the present invention 2 and the detection method described in embodiment 3
The content of 8 kinds of tumor associated antigen autoantibodies is examined in person's serum (liver cancer group) and 89 normal human serums (control group)
Survey.Two standard deviations are added to divide as cutoff value using the average value of 8 kinds of tumor associated antigen autoantibody contents in control group serum
Not Ji Suan in liver cancer group and control group 8 kinds of tumor associated antigen autoantibodies positive rate.Using 8 kinds of MedCalc Software on Drawing
Average expression level distribution map (result be shown in Fig. 3 and Fig. 4) of the tumor associated antigen autoantibody in liver cancer group and control group, with
And its column diagram in two groups of positives rates (result is shown in Fig. 5).This research also carries out statistical test using SPSS21.0 softwares,
Compare liver cancer group and control group antibody positive rate using two independent sample Chi-square Test methods, inspection level α takes 0.05, then adopted
With the diagnostic value (the results are shown in Table 1 and Fig. 6) of the evaluation method evaluation autoantibody detection liver cancer of Screen test.
The different TAAs (tumor associated antigen) of table 1 combine detection result sets Authentic Assessment in parallel
Note：Youden index：Youden index is sensitivity and specificity sum subtracts 1, and overall merit this method is used for liver
The diagnostic value of cancer, the numerical value is more than 0 less than 1, bigger closer to 1 diagnostic value.
From Fig. 3 and Fig. 4, in control group, 8 kinds of tumor associated antigen autoantibody Average expression levels are relatively low, put down
Equal OD values are less more than 0.5 serum much smaller than 0.25, OD values；And in liver cancer group, in serum 8 kinds of tumor associated antigens from
Body antibody average water is higher, and mean OD value floats near 0.25, and OD values are more more than 0.5 serum, some serum
Detection tumor associated antigen autoantibody OD values have been even more than 1.0；Thus illustrate that this 8 kinds of tumor associated antigen autoantibodies can
For the diagnosis of liver cancer.As shown in Figure 5,8 kinds of tumor associated antigen autoantibodies rates of liver cancer group are in 20.2%-
41.4%, and this 8 kinds of tumor associated antigen autoantibodies are no more than 10% in control group positive rate, and examined through statistics
Test, thus this positive rate of 8 kinds of tumor associated antigen autoantibodies in liver cancer proves that this 8 kinds are swollen obviously higher than control group
Knurl related antigen autoantibody can be as the Testing index of hepatocarcinoma early diagnosis, the early diagnosis for liver cancer.
As shown in Table 1, as the increase of antigen combination number, the sensitivity of diagnosis are consequently increased, when 8 kinds of tumour correlations
During antigen combination, the sensitivity of diagnosis has been finally reached 86.9%, is correctly to be examined using this kind of method in liver cancer patient
It is 86.9% to break as the percentage of liver cancer.And although specificity reduces, 73% can be reached, this demonstrate non-liver cancer patient
The percentage for being correctly diagnosed as being not suffering from liver cancer using this kind of method is 73%.Use this 8 kinds of tumor associated antigen combinations pair
Liver cancer is diagnosed, the sensitivity of raising diagnosis that can be on the premise of ensureing to diagnose specificity by a relatively large margin, the combination pair
Object to be measured whether be liver cancer patient judgement accuracy it is very high, be 79.8%, thus can also prove this 8 kinds of tumour correlations
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 a variety of autoantibody joint-detections
ELISA kit has higher judgement correctness and higher diagnostic value for liver cancer, further demonstrate that we are opened
This variety of autoantibody joint-detection ELISA kit sent be ideal hepatocarcinoma early diagnosis and screening method and
Means.
Claims (6)
- It is 1. a kind of for liver cancer early screening and a variety of autoantibody joint-detection ELISA kits of diagnosis, it is characterised in that The kit includes solid phase carrier and the tumor associated antigen being coated on solid phase carrier, and the tumor associated antigen is CyclinB1, p90, c-myc, p53, NPM1, HCC1,14-3-3zeta and p62；The kit also include Sample dilution, Secondary antibody, secondary antibody dilution, positive control serum, negative control sera, nitrite ion, terminate liquid, cleaning solution；Described Two antibody are RecA albumen.
- 2. a variety of autoantibody joint-detection ELISA kits according to claim 1, it is characterised in that the solid phase Carrier is 96 hole elisa Plates.
- 3. a variety of autoantibody joint-detection ELISA kits according to claim 1, it is characterised in that described second Antibody carries detectable label.
- 4. a variety of autoantibody joint-detection ELISA kits according to claim 3, it is characterised in that described to examine The label of survey is horseradish peroxidase.
- 5. a variety of autoantibody joint-detection ELISA kits according to claim 1, it is characterised in that the positive Control serum is p53 positive control serums, and the negative control sera is p53 negative control seras；Wherein, the p53 is positive The liver cancer patient blood serum that it is the positive using indirect ELISA and Western blot methods detection p53 antibody that control serum, which is, institute It is to be averaged using indirect ELISA method detection p53 antibody expressions for normal population serum antibody to state p53 negative control seras The serum of the normal person of content.
- 6. a variety of autoantibody joint-detection ELISA kits according to claim 1, it is characterised in that described a variety of The detection object of autoantibody joint-detection ELISA kit is human serum.
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| CN109116024B (en) * | 2018-06-14 | 2021-04-23 | 郑州大学第一附属医院 | Lung cancer marker anti-ACTR 3 autoantibody and application thereof |
| CN109374897B (en) * | 2018-11-22 | 2021-11-02 | 大连大学 | Kit for liver cancer detection |
| CN110187109B (en) * | 2019-05-31 | 2020-02-11 | 郑州大学第一附属医院 | Autoantibody joint detection ELISA kit for early screening of cardia adenocarcinoma |
| CN110187113B (en) * | 2019-06-19 | 2020-02-21 | 郑州大学第一附属医院 | Autoantibody joint detection ELISA kit for early screening of esophageal squamous cell carcinoma |
| CN110286235B (en) * | 2019-07-10 | 2022-07-15 | 郑州大学 | Combined detection serum marker, kit and detection method for early screening and diagnosis of liver cancer |
| CN111308090B (en) * | 2020-02-27 | 2020-11-06 | 郑州大学第一附属医院 | Esophageal cancer multi-joint rapid detection ELISA 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 |
| 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 |
| CN114113611B (en) * | 2021-12-13 | 2023-07-14 | 郑州大学 | Biomarker for liver cancer diagnosis and detection kit |
| CN116413430B (en) * | 2023-03-17 | 2024-07-05 | 杭州凯保罗生物科技有限公司 | Autoantibody/antigen combination and detection kit for early prediction of liver cancer |
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