CN106932578A - A kind of hepatocarcinoma early diagnosis kit based on POSTN genes - Google Patents
A kind of hepatocarcinoma early diagnosis kit based on POSTN genes Download PDFInfo
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Abstract
The invention discloses a kind of hepatocarcinoma early diagnosis kit based on POSTN genes, the kit includes Fuc POSTN half-quantitative detections elisa plate, ELISA ELISA Plates, magnetic bead AAL, Hp ELISA ELISA Plates；G1 is filled with Hp ELISA ELISA Plates：5%SDS, G2：2.3 units/μ L PNGase Fs, G3A：110mM trisulfonic acid trisodium salt fluorescent markers, G3B：1M organic matter reducing agents, G4：Sialidase, GW：Deionized water.Method of the present invention and kit have high flux property, compared with high specific, detection quick and precisely waits outstanding advantages, suitable for Liver Cancer scene or the extensive examination of hepatopathy crowd, detection is rapid convenient, for the diagnosis for improving liver cancer, realize that early intervention is treated, reduce liver cancer case fatality rate and have very important significance.
Description
Technical field
The invention belongs to medicine and pharmacology technical field, more particularly to a kind of hepatocarcinoma early diagnosis reagent based on POSTN genes
Box.
Background technology
HCC is the complicated common cancer of a kind of polygenes, multifactor, pathomechanism.In China, HBV carrier
Person accounts for the 7.18% of population, is the 2/3 of whole world HBVer.HCC patient populations related to this are huge, account for global
Half or so.Most morbidities concealment, occurs having belonged to late period when seeing a doctor after clinical symptoms, loses the good opportunity for the treatment of,
Therefore early diagnosis is most important for improving the survival rate of patient.Preferable liver cancer marker needs specificity higher,
The diseases such as liver cancer and cirrhosis, hepatitis, liver regeneration tubercle can be made a distinction；Sensitiveness higher is also needed simultaneously, can
Diagnosis is pointed out in liver cancer early stage, while the features such as there should be easily detection, repeat, can be determined through non-invasive procedures.
Glycoprotein glycan has the inhomogeneity of both macro and micro, and its structure function changes and has with disease development
Close contact.Glycan plays an important role in many crucial biological processes, such as cell adhesion, molecule transport and
Removing, receptor activation, signal transduction and endocytosis etc..During organismal development, glycan structures are also changing, not
Same differential period, glycan has different expression.In the generating process of cancer, glycosylated change is generally existing, such as
Glycosylated change can be paid close attention to, the specificity and sensitiveness of diagnosis can be lifted.Therefore, using detecting glycoprotein glycan structures
Exception carrys out diagnosing malignant tumor and has caused the quite attention, the change of such as AFP-L3 to have turned into the important glycan mark for detecting liver cancer
Deng.The technical method of detection glycoprotein glycan structures change has biological mass spectrometry, liquid chromatogram and the affine print of phytolectin at present
Mark etc., wherein phytolectin are the glycoprotein during a class is present in most plants, and its maximum feature is to be capable of identify that glycoprotein
It is surface of cell membrane determinant with the glycan of labyrinth in the glycan in glycolipid, particularly cell membrane, their knots with glycan
Conjunction is non-covalent and reversible.POSTN is a kind of plant of affine α connections fucose (Terminal α Fuc and ± Sia-Le x)
Agglutinin, fucose modification can assign glycan many unique functional characteristics, and it is in transfusion reaction, the leucocyte of lectin-mediated
The aspects such as adhesion, host and microbial interaction and ontogeny with endothelium play a significant role.Additionally, fucose is also joined
With constitute some important adhesion molecules glycan structures, it is in close relations with metastases.SialylLewis x are found in cancer
Increase with sialyl Lewis a contents, A and Type B blood group antigens are lost along with H blood group antigens and Lewis y in many tumours
Expression is raised.
Periostin (POSTN) is a kind of extracellular matrix secretion that Gegenbaur's cell and osteoblast-like cells system secrete
Albumen, can promote Gegenbaur's cell and its precursor to assemble and break up in periosteum, be earliest by Japanese scholars Takeshita etc. from mouse
A kind of bone adhesion molecule cloned in Gegenbaur's cell system MC3r13-E1cDNA libraries.With the intensification studied it, find
POSTN has expression high in Various Tissues perhaps, especially with malignancy and transfer, the hyperplasia and fibrosis of inflammatory tissue
There is close relationship, be expected to the Testing index of diagnosis and treatment as many tumours and inflammation related disease.
In previous work, it is found that the liver cancer patient blood serum unit affine AAL abilities of POSTN are significantly increased, illustrate that liver cancer is suffered from
The unit POSTN fucosylated glycan contents of person increase.Present invention application POSTN to the high-affinity of fucosylated glycan,
By the preparation and detection of POSTN-ELISA and Protein-ELISA, POSTN antibody-antigenes-biotin mark is formed respectively
POSTN compounds and POSTN antibody-antigenes-biotin labeling antibody compound, obtain POSTN combination POSTN absorbance and
The absorbance ratio Fuc-POSTN of POSTN albumen, retrospective study is carried out by clinical data, sets up tumour Distinguishing diagnosis
Function, obtains ROC curve and Cut off values, while comparing the accuracy rate difference of AFP-L3 and Fuc-POSTN to diagnosing cancer of liver.
The present invention is applied to Liver Cancer scene or the extensive examination of hepatopathy crowd, for the diagnosis for improving liver cancer, realizes early stage
Therapeutic intervention, reduces liver cancer case fatality rate and has very important significance.
Compared to the various imaging techniques of current diagnosis liver cancer, such as ultrasonic examination, computed tomography, magnetic resonance into
As etc., these technologies can not differentiate benign liver change, such as area of the major tubercle and liver cancer of hypogenetic tubercle and hardening
Not.It is main in the industry to select blood serum tumor markers to detect malignant tumour, and serum alpha-fetoprotein (AFP) is used as uniquely extensive
Be applied to diagnose and check the blood serum designated object of liver cancer, the sensitivity and specificity of nearest analysis display AFP detections have compared with
Big fluctuation range, and these fluctuations are not fully due to having used the threshold effect of different cutoff levels.At present, facing
Lack sensitive, special early liver cancer diagnosis marker on bed, the early liver cancer case that can be detected constitutes about all liver cancer diseases
The 25% of example.The detection product that therefore, it can fast and convenient accurate detection liver cancer and tracing detection liver cancer treatment effect has ten
Divide wide market.
The content of the invention
It is an object of the invention to provide a kind of hepatocarcinoma early diagnosis kit based on POSTN genes, it is intended to solve mesh
Preceding clinically to lack sensitive, special early liver cancer diagnosis marker, detection liver cancer and tracing detection liver cancer treatment effect are not
Good problem.
The present invention is achieved in that a kind of hepatocarcinoma early diagnosis kit based on POSTN genes, described to be based on
The hepatocarcinoma early diagnosis kit of POSTN genes comprising Fuc-POSTN half-quantitative detections elisa plate, ELISA ELISA Plates, magnetic bead-
AAL, Hp-ELISA ELISA Plate；Described Fuc-POSTN half-quantitative detections elisa plate is by POSTN-ELISA plates, Protein-
Elisa plate is constituted；
G1 is filled with the Hp-ELISA ELISA Plates：5%SDS, G2：2.3 units/μ L PNGase Fs, G3A：110mM tri-
Sulfonic acid trisodium salt fluorescent marker, G3B：1M organic matter reducing agents, G4：Sialidase, GW：Deionized water.
Further, the HCC forecast models of the hepatocarcinoma early diagnosis kit based on POSTN genes：
P=exp (- 0.915+0.275*Fuc-POSTN)/[1+exp (- 0.915+0.275*Fuc-POSTN)]
The computational methods of described Fuc-POSTN values are：The actual OD values of POSTN combinations POSTN in each blood serum sample
With the ratio of the actual OD values of POSTN albumen in identical blood serum sample, the reality of POSTN combinations POSTN in described blood serum sample
Border OD values detect and obtain that the actual OD values of POSTN albumen pass through Protein- in blood serum sample by POSTN-ELISA plates
Elisa plate detection is obtained, and it is 2.281 that the model distinguishes liver cancer and the Cut off values of cirrhosis；
Hepatocarcinoma early diagnosis kit based on POSTN genes also captures antibody comprising POSTN, and its concentration is 1 μ g/mL；
The first sealer and the second sealer are also included, the first sealer is 3.5%BSA solution, for closing POSTN-
Elisa plate, the second sealer is to contain 1.5%BSA and 0.06%NaN3Solution, for closing Protein-ELISA plates；
The first oxidant is also included, the first oxidant contains 100mM NaIO for pH4.0's4It is molten with 50mM citric acids
Liquid, for the oxidation of POSTN-ELISA plates；
Also include the POSTN of biotin labeling and the antibody of biotin labeling；
Also comprising collection blood sample device and operational manual.
Further, magnetic bead and the mass ratio of POSTN are 170~230 in the magnetic bead-POSTN:1；Magnetic bead-the POSTN
Be split as magnetic bead, POSTN and magnetic bead-POSTN prepares related reagent.
Further, the volume of the G1 is that the volume of 100 μ L, G2 is that the volume of 60 μ L, G3A is that the volume of 20 μ L, G3B is
The volume of 20 μ L, G4 is that the volume of 45 μ L, GW is 4.5mL.
Further, the preparation method of the magnetic bead-POSTN includes step：
1) magnetic bead is placed in EDC solution and is reacted；
2) after the completion of reacting, remove supernatant and use buffer solution for cleaning magnetic bead；
3) take a certain amount of POSTN to be placed in coupling liquid, add magnetic bead reaction；
4) after the completion of reacting, unreacted activated group is closed.
Further, the hepatocarcinoma early diagnosis kit based on POSTN genes also includes：
Sample diluting liquid, shown sample diluting liquid is 0.15M Tris-HCl, 0.18M NaCl, 1.5mM CaCl2、
1.5mM MgCl2, pH 7.5 solution；
Also include the Hp antibody of HPR marks.
Further, the hepatocarcinoma early diagnosis kit application method based on POSTN genes, comprises the following steps：
The serum of sample containing POSTN of 2.5 μ L is first added in different test tubes, then is separately added into 5 μ L G1, AAL, use whirlpool
Whirlpool oscillator is fully mixed, and carries out within 5 minutes high-temperature denatured treatment and be cooled back to 4 DEG C in 98 DEG C of PCR heating to obtain sample；
The G2 of 3.5 μ L is added in sample tube containing G1, the biotin mark of 3.5 μ L is added in sample tube containing POSTN
The antibody of note and 3.5 μ L POSTN capture antibody；Centrifugation is mixed, 37 DEG C keep being cooled back within 4 hours 4 DEG C in incubator, right
Sample after cooling is separately added into -20 DEG C of preservations of deionized water GW terminating reactions of 60 μ L；
The sample for taking 15 μ L Cord bloods respectively is dried 100 minutes in metal bath, 4 DEG C is cooled back to, to dried
Containing adding 2.5 μ L by volume 1 in G1, G2 sample tube:The mixed liquor of the G3A and G3B of 1 configuration, centrifugal treating, and in training
37 DEG C of holdings carry out fluorescence labeling for 16 hours and are cooled back to 4 DEG C in foster case, and adding the GW of 120 μ L carries out terminating reaction, mixes
- 20 DEG C of preservations after centrifugation；Sample adds 2.5 μ LG4 after taking 2.5 μ L terminating reactions, mixes centrifugation, 37 DEG C in incubator, keeps
20 hours, the GW terminating reactions of 50 μ L are added, sample 15 μ L after mixing centrifugation carries out N- oligonucleotide chain pieces by gene sequencer
Section separation detection, -20 DEG C of preservations of extraction raffinate；
In the sample tube of the antibody of the biotin labeling containing POSTN and 3.5 μ L after the drying, predicted using HCC
Model, calculates Fuc-POSTN values, and the computational methods of Fuc-POSTN values are the reality of POSTN combinations POSTN in each blood serum sample
The ratio of border OD values and the actual OD values of POSTN albumen in identical blood serum sample, the reality of POSTN combinations POSTN in blood serum sample
Border OD values detect and obtain that the actual OD values of POSTN albumen pass through Protein- in identical blood serum sample by POSTN-ELISA plates
Elisa plate detection is obtained, and the Cut off values of liver cancer and cirrhosis are distinguished using HCC forecast models.
The present invention by magnetic bead-POSTN-Hp-ELISA detect and conventional H p-ELISA detect, respectively formed magnetic bead-
POSTN- antigens-HRP marks Hp antibody and Hp antibody-antigenes-HRP mark Hp antibody complexes, obtains the absorbance of Hp combinations POSTN
With the absorbance ratio Fuc-Hp (index) of Hp albumen, retrospective study is carried out by clinical data, set up tumour differentiation and examine
Disconnected function, obtains receiver operating curves (ROC) and discrimination threshold, diagnostic sensitivity and specificity of the function for HCC
Respectively 76.1% and 85.3%, accuracy 80%, AUC is 0.852.
Method of the present invention and kit have high flux property, and compared with high specific, detection quick and precisely waits protrusion excellent
Point, has important clinical value to the diagnosis of liver cancer, it is adaptable to Liver Cancer scene or the extensive examination of hepatopathy crowd, inspection
Survey rapid convenient, for the diagnosis for improving liver cancer, realize that early intervention is treated, reduction liver cancer case fatality rate has very important
Meaning.
Preparation and detection that the present invention passes through POSTN-ELISA and Protein-ELISA, form antibody-antigene-life respectively
Thing element mark POSTN compounds and antibody-antigene-biotin labeling antibody compound, with the absorbance (OD of POSTN combinations POSTN
Value) with the absorbance ratio of POSTN albumen as the content (Fuc-POSTN) in unit of measurement POSTN albumen with reference to fucose,
Carry out retrospective study by clinical data, set up tumour Distinguishing diagnosis function, obtain receiver operating curves (ROC) and
Differentiate (Cut off) value, diagnostic sensitivity of the function for HCC and specificity be respectively 60.3% and 71.4%, AUC be
0.736, better than alpha-fetoprotein N- glycan cores fucose (AFP-L3).Method of the present invention and kit have high flux
Property, compared with high specific, detection quick and precisely waits outstanding advantages.
The present invention is regulated and controled by the reaction time to sialidase, concentration, temperature, and time-consuming cost is shortened
Whole reaction time, improve production efficiency；Raw material are economic and practical, produced beneficial to popularization metaplasia；AFP can effectively be reduced
The rate of missed diagnosis of (alpha-fetoprotein) for liver cancer, it is ensured that considerable accuracy rate.
Brief description of the drawings
Fig. 1 is the stream of the hepatocarcinoma early diagnosis kit application method based on POSTN genes provided in an embodiment of the present invention
Cheng Tu.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is described in detail below in conjunction with the accompanying drawings.
The hepatocarcinoma early diagnosis kit based on POSTN genes of inventive embodiments offer is unable to, comprising Fuc-POSTN half
Quantitative determination elisa plate, ELISA ELISA Plates, magnetic bead-AAL, Hp-ELISA ELISA Plates；Described Fuc-POSTN half-quantitative detections
Elisa plate is made up of POSTN-ELISA plates, Protein-ELISA plates；
G1 is filled with the Hp-ELISA ELISA Plates：5%SDS, G2：2.3 units/μ L PNGase Fs, G3A：110mM tri-
Sulfonic acid trisodium salt fluorescent marker, G3B：1M organic matter reducing agents, G4：Sialidase, GW：Deionized water.
Further, the HCC forecast models of the hepatocarcinoma early diagnosis kit based on POSTN genes：
P=exp (- 0.915+0.275*Fuc-POSTN)/[1+exp (- 0.915+0.275*Fuc-POSTN)]
The computational methods of described Fuc-POSTN values are：The actual OD values of POSTN combinations POSTN in each blood serum sample
With the ratio of the actual OD values of POSTN albumen in identical blood serum sample, the reality of POSTN combinations POSTN in described blood serum sample
Border OD values detect and obtain that the actual OD values of POSTN albumen pass through Protein- in blood serum sample by POSTN-ELISA plates
Elisa plate detection is obtained, and it is 2.281 that the model distinguishes liver cancer and the Cut off values of cirrhosis；
Hepatocarcinoma early diagnosis kit based on POSTN genes also captures antibody comprising POSTN, and its concentration is 1 μ g/mL；
The first sealer and the second sealer are also included, the first sealer is 3.5%BSA solution, for closing POSTN-
Elisa plate, the second sealer is to contain 1.5%BSA and 0.06%NaN3Solution, for closing Protein-ELISA plates；
The first oxidant is also included, the first oxidant contains 100mM NaIO for pH4.0's4It is molten with 50mM citric acids
Liquid, for the oxidation of POSTN-ELISA plates；
Also include the POSTN of biotin labeling and the antibody of biotin labeling；
Also comprising collection blood sample device and operational manual.
Further, magnetic bead and the mass ratio of POSTN are 170~230 in the magnetic bead-POSTN:1；Magnetic bead-the POSTN
Be split as magnetic bead, POSTN and magnetic bead-POSTN prepares related reagent.
Further, the volume of the G1 is that the volume of 100 μ L, G2 is that the volume of 60 μ L, G3A is that the volume of 20 μ L, G3B is
The volume of 20 μ L, G4 is that the volume of 45 μ L, GW is 4.5mL.
Further, the preparation method of the magnetic bead-POSTN includes step：
1) magnetic bead is placed in EDC solution and is reacted；
2) after the completion of reacting, remove supernatant and use buffer solution for cleaning magnetic bead；
3) take a certain amount of POSTN to be placed in coupling liquid, add magnetic bead reaction；
4) after the completion of reacting, unreacted activated group is closed.
Further, the hepatocarcinoma early diagnosis kit based on POSTN genes also includes：
Sample diluting liquid, shown sample diluting liquid is 0.15M Tris-HCl, 0.18M NaCl, 1.5mM CaCl2、
1.5mM MgCl2, pH 7.5 solution；
Also include the Hp antibody of HPR marks.
As shown in figure 1, the hepatocarcinoma early diagnosis kit application method based on POSTN genes, comprises the following steps：
S101：The serum of sample containing POSTN of 2.5 μ L is first added in different test tubes, then is separately added into 5 μ L G1, AAL, made
Fully mixed with vortex oscillator, carry out within 5 minutes high-temperature denatured treatment and be cooled back to 4 DEG C in 98 DEG C of PCR heating to obtain sample.
S102：The G2 of 3.5 μ L is added in sample tube containing G1, the biotin of 3.5 μ L is added in sample tube containing AAL
The antibody of mark and 3.5 μ L POSTN capture antibody；Centrifugation is mixed, 37 DEG C of holdings are cooled back to 4 DEG C in 4 hours in incubator,
- 20 DEG C of preservations of deionized water GW terminating reactions of 60 μ L are separately added into the sample after cooling.
S103：The sample for taking 15 μ L Cord bloods respectively is dried 100 minutes in metal bath, 4 DEG C is cooled back to, to drying
Afterwards containing adding 2.5 μ L by volume 1 in G1, G2 sample tube:The mixed liquor of the G3A and G3B of 1 configuration, centrifugal treating, and
37 DEG C of holdings carry out fluorescence labeling for 16 hours and are cooled back to 4 DEG C in incubator, and the GW for adding 120 μ L carries out terminating reaction,
Mix -20 DEG C of preservations after centrifugation；Sample adds 2.5 μ LG4 after taking 2.5 μ L terminating reactions, mixes centrifugation, 37 DEG C in incubator,
Kept for 20 hours, add the GW terminating reactions of 50 μ L, sample 15 μ L after mixing centrifugation carries out N- oligosaccharides by gene sequencer
Chain fragment separation detection, -20 DEG C of preservations of extraction raffinate.
S104：In the sample tube of the antibody of the biotin labeling containing POSTN and 3.5 μ L after the drying, using HCC
Forecast model, calculates Fuc-POSTN values, and the computational methods of Fuc-POSTN values are POSTN combinations POSTN in each blood serum sample
Actual OD values and the actual OD values of POSTN albumen in identical blood serum sample ratio, POSTN combinations POSTN in blood serum sample
Actual OD values by POSTN-ELISA plates detect obtain, the actual OD values of POSTN albumen pass through in identical blood serum sample
The detection of Protein-ELISA plates is obtained, and the Cut off values of liver cancer and cirrhosis are distinguished using HCC forecast models.
The present invention by magnetic bead-POSTN-Hp-ELISA detect and conventional H p-ELISA detect, respectively formed magnetic bead-
POSTN- antigens-HRP marks Hp antibody and Hp antibody-antigenes-HRP mark Hp antibody complexes, obtains the absorbance of Hp combinations POSTN
With the absorbance ratio Fuc-Hp (index) of Hp albumen, retrospective study is carried out by clinical data, set up tumour differentiation and examine
Disconnected function, obtains receiver operating curves (ROC) and discrimination threshold, diagnostic sensitivity and specificity of the function for HCC
Respectively 76.1% and 85.3%, accuracy 80%, AUC is 0.852.
Method of the present invention and kit have high flux property, and compared with high specific, detection quick and precisely waits protrusion excellent
Point, has important clinical value to the diagnosis of liver cancer, it is adaptable to Liver Cancer scene or the extensive examination of hepatopathy crowd, inspection
Survey rapid convenient, for the diagnosis for improving liver cancer, realize that early intervention is treated, reduction liver cancer case fatality rate has very important
Meaning.
Preparation and detection that the present invention passes through POSTN-ELISA and Protein-ELISA, form antibody-antigene-life respectively
Thing element mark POSTN compounds and antibody-antigene-biotin labeling antibody compound, with the absorbance (OD values) of POSTN combinations AAL
With the absorbance ratio of POSTN albumen as the content (Fuc-POSTN) in unit of measurement POSTN albumen with reference to fucose, lead to
Cross carries out retrospective study to clinical data, sets up tumour Distinguishing diagnosis function, obtains receiver operating curves (ROC) and sentences
Not other (Cut off) value, diagnostic sensitivity of the function for HCC and specificity be respectively 60.3% and 71.4%, AUC be
0.736, better than alpha-fetoprotein N- glycan cores fucose (AFP-L3).Method of the present invention and kit have high flux
Property, compared with high specific, detection quick and precisely waits outstanding advantages.
The present invention is regulated and controled by the reaction time to sialidase, concentration, temperature, and time-consuming cost is shortened
Whole reaction time, improve production efficiency；Raw material are economic and practical, produced beneficial to popularization metaplasia；
Rates of missed diagnosis of the AFP (alpha-fetoprotein) for liver cancer can effectively be reduced, it is ensured that considerable accuracy rate.
Application principle of the invention is further described with reference to specific embodiment.
Detection method：
(1) preprocess method of POSTN-ELISA plates and Protein-ELISA plates：
1st, two pieces of elisa plates are taken, POSTN-ELISA plates and Protein-ELISA plates is used separately as；
2nd, antibody is captured：POSTN capture antibody is added in POSTN-ELISA plates and Protein-ELISA plates,
POSTN captures AC is 1 μ g/mL, 100 μ L/well in POSTN-ELISA；In Protein-ELISA
POSTN capture ACs are 1 μ g/mL, 100 μ L/well.Incubation at room temperature is overnight.
3rd, board-washing：Using PBST (0.05%Tween20in PBS, pH7.2-7.4) board-washing 4 times, immersion 2 minutes every time,
300 μ L/well, dry, and patted on blotting paper and pat dry liquid in hole.
4th, close：POSTN-ELISA plates use the first sealer (3%BSA in PBS, pH7.2-7.4)；Protein-
ELISA closes 1h, 200 μ L/well using the second sealer (1%BSA, 0.05%NaN 3in PBS, pH7.2-7.4) room temperature.
5th, board-washing：Using PBST (0.05%Tween20in PBS, pH7.2-7.4) board-washing 4 times, immersion 2 minutes every time,
300 μ L/well, dry, and patted on blotting paper and pat dry liquid in hole.
6th, aoxidize：POSTN-ELISA plates using the first oxidant (100mM NaIO 4,50mM Citric acid,
pH4.0)；Protein-ELISA carries out oxygen using the second sealer (1%BSA, 0.05%NaN 3in PBS, pH7.2-7.4)
Change, 4 DEG C of lucifuges react 1h, 200 μ L/well.
7th, board-washing：Using PBST (0.05%Tween20in PBS, pH7.2-7.4) board-washing 4 times, immersion 2 minutes every time,
300 μ L/well, dry, and are patted on blotting paper and pat dry liquid in hole, respectively obtain the POSTN-ELISA plates after treatment
With Protein-ELISA plates, for following step.
(2) Fuc-POSTN half-quantitative detections ELISA detection method：
1st, it is loaded：POSTN-ELISA plates add the dilution μ L/well of serum sample 50 (to use PBS1：30 dilutions)；
Protein-ELISA plates add dilution serum sample 100 μ L/well, and (0.5 μ L serum uses 1mMEDTA, 0.5%Triton
X-100in PBS, pH7.2-7.4 are diluted to 100 μ L), room temperature 2h.
2nd, board-washing：Using PBST (0.05%Tween20in PBS, pH7.2-7.4) board-washing 4 times, immersion 2 minutes every time,
300 μ L/well, dry, and patted on blotting paper and pat dry liquid in hole.
3rd, detection reagent：The POSTN (PBST dilutions) of 1 μ g/mL biotin labelings is added in POSTN-ELISA plates；
Antibody (1%BSA the in PBS, pH7.2-7.4 of 600ng/mL biotin labelings are added in Protein-ELISA plates
Dilution), 100 μ L/well, room temperature 2h.
4th, board-washing：Using PBST (0.05%Tween20in PBS, pH7.2-7.4) board-washing 4 times, immersion 2 minutes every time,
300 μ L/well, dry, and patted on blotting paper and pat dry liquid in hole.5th, enzyme conjugates working solution：POSTN-ELISA
HRP-sztreptavidin is added (to use sealer 1,1 in plate：12500 dilutions)；In Protein-ELISA plates
HRP-sztreptavidin is added (using 1%BSA in PBS, 1：12500 dilutions), 100 μ L/well, room temperature 30min.
6th, board-washing：Using PBST (0.05%Tween20in PBS, pH7.2-7.4) board-washing 4 times, immersion 2 minutes every time,
300 μ L/well, dry, and patted on blotting paper and pat dry liquid in hole.
7th, substrate solution is added：By substrate solution according to TMB：H 2O 2=1：2 mixing, 100 μ L/well, room temperature 20min.
8th, terminate liquid is added：100 μ L Stop Solution/well, the addition sequence of terminate liquid and the addition of substrate solution
It is sequentially identical.
9th, detect：Immediately with NanoQuant infinite M200 (TECAN) ELIASAs in each hole of 450nm wavelength measurements
OD values.ELIASA power supply should be in advance opened, preheater apparatus set detection program.
(3) Fuc-POSTN half-quantitative detections elisa assay method：
1st, data：The OD values that the OD values of each blood serum sample should subtract blank well are actual OD values.Each blood serum sample
The actual OD values of actual OD values (POSTN-ELISA) blood serum sample POSTN albumen identical with this of POSTN combinations POSTN
(Protein-ELISA) ratio is Fuc-POSTN values.
P values are calculated according to above-mentioned HCC forecast models, P values are compared with Cut off values, ＞ 2.2727 is liver cancer, ＜
2.281 is cirrhosis.
Using kit and detection method in embodiment, 86 hepatopathy blood serum samples of joint parallel parsing (cirrhosis and
Each 43 of liver cancer patient, through clinical definite) in POSTN fucose and the expression of albumen, be calculated Fuc-POSTN
Content.The above-mentioned HCC forecast models set up using SPSS software logistic regression analyses carry out detection differentiation liver cancer and liver is hard
The Cut off values of change are 2.281.(AFP-L3 detection kits are purchased from Abnova companies (Catalog#KA1187),
According to AFP-L3 contents in the kit operating procedure above-mentioned 86 hepatopathy blood serum samples of detection, obtained using SPSS softwares
Obtain ROC curve.), compare the accuracy and specificity of kit of the present invention and AFP-L3 to diagnosing cancer of liver, kit of the present invention
Sensitivity is 60.3%, specificity 71.4%, and accuracy is that 64%, AUC is 0.736, and diagnosis of the AFP-L3 to HCC is sensitive
It is 0.612 that degree, specificity and accuracy are 60.5%, AUC.Liver cirrhosis patient AFP-L3 detection averages are 0.8466, standard
Difference is 0.32；Liver cancer patient AFP-L3 detection averages are 0.9779, and standard deviation is 0.36；P value is 0.0393.Liver cirrhosis patient
Fuc-POSTN detection averages are 2.6315, and standard deviation is 2.18；Liver cancer patient Fuc-POSTN detection averages are 4.3609, standard
Difference is 3.08；P value is 0.002.
Using Fuc-POSTN levels in 40 test set serum of kit assay of the present invention, with reference to Cut off values, as a result
Show and successfully predict 27 patients, accuracy rate is 67.5%.Therefore, the present invention has specificity higher and accuracy, to liver
The prediction of cancer has important clinical value.
Take the POSTN- in 1 pooled serum sample detection kit for diagnosing hepatocellular carcinoma of the invention
ELISA whether there is non-specific cross-reaction, as a result as shown in table 2, POSTN- in kit of the present invention with other albumen
ELISA detects that the OD values of other albumen are consistent with PBS, and the OD values of blood-serum P OSTN are much larger than other albumen.
3 pooled serum sample detections are taken for diagnosing the repeatability of the kit of hepatocellular carcinoma：
Every Virus monitory 3 times, calculates every the average value of serum Fuc-POSTN, standard deviation and CV values and meets the requirements.
Doubling dilution is carried out using the serum of known POSTN protein concentrations, it is determined that POSTN-ELISA in kit of the present invention
POSTN protein concentrations determine to use ELISA immue quantitative detection reagent boxes in linear detection range, wherein serum, carry out doubling dilution,
Determine Protein-ELISA linear detection ranges.As a result Protein-ELISA linear detection ranges are more than POSTN-ELISA lines
Property detection range, therefore the detection range of Fuc-POSTN detecting systems is POSTN-ELISA linear detection ranges.It is of the invention
The quantitative linearity detection range of kit is 0.71~7.1ng/mL.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (7)
1. a kind of hepatocarcinoma early diagnosis kit based on POSTN genes, it is characterised in that the liver based on POSTN genes
Cancer early diagnosis kit includes Fuc-POSTN half-quantitative detections elisa plate, ELISA ELISA Plates, magnetic bead-AAL, Hp-ELISA
ELISA Plate；Described Fuc-POSTN half-quantitative detections elisa plate is made up of POSTN-ELISA plates, Protein-ELISA plates；
G1 is filled with the Hp-ELISA ELISA Plates：5%SDS, G2：2.3 units/μ L PNGase Fs, G3A：110mM trisulfonic acids
Trisodium salt fluorescent marker, G3B：1M organic matter reducing agents, G4：Sialidase, GW：Deionized water.
2. the hepatocarcinoma early diagnosis kit of POSTN genes is based on as claimed in claim 1, it is characterised in that based on POSTN
The HCC forecast models of the hepatocarcinoma early diagnosis kit of gene：
P=exp (- 0.915+0.275*Fuc-POSTN)/[1+exp (- 0.915+0.275*Fuc-POSTN)]
The computational methods of Fuc-POSTN values are the actual OD values and identical serum sample of POSTN combinations POSTN in each blood serum sample
The ratio of the actual OD values of POSTN albumen in product, the actual OD values of POSTN combinations POSTN pass through in described blood serum sample
The detection of POSTN-ELISA plates is obtained, and the actual OD values of POSTN albumen are examined by Protein-ELISA plates in identical blood serum sample
Measure, it is 2.281 that the model distinguishes liver cancer and the Cut off values of cirrhosis；
Hepatocarcinoma early diagnosis kit based on POSTN genes also captures antibody comprising POSTN, and its concentration is 1 μ g/mL；
The first sealer and the second sealer are also included, the first sealer is 3.5%BSA solution, for closing POSTN-ELISA
Plate, the second sealer is to contain 1.5%BSA and 0.06%NaN3Solution, for closing Protein-ELISA plates；
The first oxidant is also included, the first oxidant contains 100mM NaIO for pH4.0's4With the solution of 50mM citric acids, it is used for
The oxidation of POSTN-ELISA plates；
Also include the POSTN of biotin labeling and the antibody of biotin labeling；
Also comprising collection blood sample device and operational manual.
3. the hepatocarcinoma early diagnosis kit of POSTN genes is based on as claimed in claim 1, it is characterised in that based on POSTN
The hepatocarcinoma early diagnosis kit of gene also includes：
Sample diluting liquid, shown sample diluting liquid is 0.15M Tris-HCl, 0.18M NaCl, 1.5mM CaCl2、1.5mM
MgCl2, pH 7.5 solution；
Also include the Hp antibody of HPR marks.
4. the hepatocarcinoma early diagnosis kit of POSTN genes is based on as claimed in claim 1, it is characterised in that the magnetic bead-
Magnetic bead and the mass ratio of POSTN are 170~230 in POSTN:1；Magnetic bead-the POSTN be split as magnetic bead, POSTN and magnetic bead-
POSTN's prepares related reagent.
5. the hepatocarcinoma early diagnosis kit of POSTN genes is based on as claimed in claim 1, it is characterised in that the G1's
Volume is that the volume of 100 μ L, G2 is that the volume of 60 μ L, G3A is that the volume of 20 μ L, G3B is that the volume of 20 μ L, G4 is 45 μ L, GW
Volume be 4.5mL.
6. the hepatocarcinoma early diagnosis kit of POSTN genes is based on as claimed in claim 1, it is characterised in that the magnetic bead-
The preparation method of POSTN includes step：
1) magnetic bead is placed in EDC solution and is reacted；
2) after the completion of reacting, remove supernatant and use buffer solution for cleaning magnetic bead；
3) take a certain amount of POSTN to be placed in coupling liquid, add magnetic bead reaction；
4) after the completion of reacting, unreacted activated group is closed.
7. the hepatocarcinoma early diagnosis kit of POSTN genes is based on as claimed in claim 1, it is characterised in that based on POSTN
The hepatocarcinoma early diagnosis kit application method of gene, comprises the following steps：
The serum of sample containing POSTN of 2.5 μ L is first added in different test tubes, then is separately added into 5 μ L G1, AAL, shaken using whirlpool
Swing device fully to mix, carry out within 5 minutes high-temperature denatured treatment and be cooled back to 4 DEG C in 98 DEG C of PCR heating to obtain sample；
The G2 of 3.5 μ L is added in sample tube containing G1, the biotin labeling of 3.5 μ L is added in sample tube containing POSTN
Antibody and 3.5 μ L POSTN capture antibody；Centrifugation is mixed, 37 DEG C of holdings are cooled back to 4 DEG C in 4 hours in incubator, to cooling
Sample afterwards is separately added into -20 DEG C of preservations of deionized water GW terminating reactions of 60 μ L；
Take the sample of 15 μ L Cord bloods respectively to be dried 100 minutes in metal bath, be cooled back to 4 DEG C, to it is dried containing
2.5 μ L by volume 1 are added in G1, G2 sample tube:The mixed liquor of the G3A and G3B of 1 configuration, centrifugal treating, and in incubator
In 37 DEG C keep carrying out within 16 hours fluorescence labeling and being cooled back to 4 DEG C, adding the GW of 120 μ L carries out terminating reaction, mixes centrifugation
- 20 DEG C of preservations afterwards；Sample adds 2.5 μ LG4 after taking 2.5 μ L terminating reactions, mixes centrifugation, and 37 DEG C in incubator, holding 20 is small
When, the GW terminating reactions of 50 μ L are added, sample 15 μ L after mixing centrifugation carries out N- oligonucleotide chains fragment point by gene sequencer
From detection, -20 DEG C of preservations of extraction raffinate；
In the sample tube of the antibody of the biotin labeling containing POSTN and 3.5 μ L after the drying, using HCC forecast models,
Fuc-POSTN values are calculated, the computational methods of Fuc-POSTN values are the actual OD values of POSTN combinations POSTN in each blood serum sample
With the ratio of the actual OD values of POSTN albumen in identical blood serum sample, the actual OD values of POSTN combinations POSTN in blood serum sample
Detected by POSTN-ELISA plates and obtained, the actual OD values of POSTN albumen pass through Protein-ELISA in identical blood serum sample
Plate detection is obtained, and the Cut off values of liver cancer and cirrhosis are distinguished using HCC forecast models.
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| CN109633176A (en) * | 2019-01-11 | 2019-04-16 | 广东医科大学附属医院 | A kind of nephrosis gene therapy diagnostic kit |
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| CN109633176A (en) * | 2019-01-11 | 2019-04-16 | 广东医科大学附属医院 | A kind of nephrosis gene therapy diagnostic kit |
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Application publication date: 20170707 |