CN103777026B - A kind of for diagnosing the kit of hepatocellular carcinoma - Google Patents

A kind of for diagnosing the kit of hepatocellular carcinoma Download PDF

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CN103777026B
CN103777026B CN201410058332.5A CN201410058332A CN103777026B CN 103777026 B CN103777026 B CN 103777026B CN 201410058332 A CN201410058332 A CN 201410058332A CN 103777026 B CN103777026 B CN 103777026B
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CN103777026A (en
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张舒
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Zhongshan Hospital Fudan University
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

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Abstract

The invention provides and a kind ofly to it is characterized in that for diagnosing the kit of hepatocellular carcinoma, comprise Fuc-PON1 half-quantitative detection elisa plate, described Fuc-PON1 half-quantitative detection elisa plate is made up of AAL-ELISA plate and Protein-ELISA plate.Kit of the present invention has high flux property, comparatively high specific, detects and quick and precisely waits outstanding advantages, have important clinical value to the diagnosis of liver cancer.

Description

A kind of for diagnosing the kit of hepatocellular carcinoma
Technical field
The present invention relates to biomedical sector, be associated with application basic medical research and clinical research; Contain oncology, diagnostics, glycobiology.
Background technology
The common cancer that HCC is a kind of polygenes, multifactor, pathomechanism is complicated.In China, hepatitis carrier accounts for 7.18% of population, is whole world HBVer's 2/3.HCC patient populations related to this is huge, about accounting for global half.Most morbidity's concealment, belong to late period when seeking medical advice after there is clinical symptoms, lose the good opportunity for the treatment of, therefore early diagnosis is most important for the survival rate improving patient.Desirable liver cancer marker needs higher specificity, the diseases such as liver cancer and cirrhosis, hepatitis, liver regeneration tubercle can be made a distinction; Also need higher susceptibility simultaneously, namely can point out diagnosis in early days in liver cancer, easy detection should be had simultaneously, can repeat, can through features such as non-invasive procedures mensuration.
Glycoprotein glycan also exists the inhomogeneity of both macro and micro, and its structure function changes and has close contacting with disease development.Glycan plays an important role in a lot of crucial biological process, such as cell adhesion, molecule transport and removing, receptor activation, signal transduction and endocytosis etc.In organismal development process, glycan structures is also in change, and in different differential periods, glycan has different expression.In the generating process of cancer, glycosylated change is ubiquitous, if pay close attention to glycosylated change, can promote specificity and the susceptibility of diagnosis.Therefore, utilize the exception detecting glycoprotein glycan structures to carry out diagnosing malignant tumor and caused suitable attention, the change as AFP-L3 has become the important glycan mark etc. detecting liver cancer.The technical method of current detection glycoprotein glycan structures change has biological mass spectrometry, liquid chromatography and phytolectin imprinting etc., wherein phytolectin is the glycoprotein that a class is present in most plants, its maximum feature to identify the glycan in glycoprotein and glycolipid, the particularly glycan of labyrinth and surface of cell membrane determinant in cell membrane, the combination of they and glycan is non-covalent and reversible.AAL is that a kind of affine α connects fucose (Terminal α Fuc and ± Sia-Le x) phytolectin, fucose is modified and can be given glycan a lot of unique functional characteristics, plays a significant role in the adhesion of its leucocyte at transfusion reaction, lectin-mediated and endothelium, host and microbial interaction and ontogeny etc.In addition, fucose also participates in the glycan structures forming some important adhesion molecule, in close relations with metastases.SialylLewis is found in cancer xand sialylLewis acontent increases, and in many tumours, A and Type B blood group antigens are lost along with H blood group antigens and Lewis yexpress and raise.
PON1 is a kind of glycoprotein synthesized by liver, combines with high-density lipoprotein (HDL) (HDL), is present in blood and liver, when chronic liver disease has activity pathology, liver cell continuous necrotic, synthesis reduces, the PON1 correspondingly entering people's circulation reduces, and causes blood-serum P ON1 activity to reduce.The existing hepatocellular continuous necrotic of posthepatitic cirrhosis, have liver fibrosis again, its pathologic process is very complicated, and blood-serum P ON1 is active significantly to be reduced.Blood-serum P ON1 activity can be used as one of biochemical indicator judging to implant liver survival, works in coordination with the liver function that other biochemical indicators are used for monitoring liver-transplantation patients.It is reported, the inflammation that HCV causes fiberization then, PON1 level meeting continuous decrease, as combining the molecule of many oxidative and anti-oxidatives together as clinical useful monitoring index.The protein content of latest find HDL particulate and some lipids is abnormal, and the glycosylation comprising PON1 all can cause the expression of PON1 in hepatopathy patient serum to reduce.In previous work, find that the affine AAL ability of liver cancer patient blood serum unit PON1 significantly strengthens, illustrate that the unit PON1 fucosylated glycan content of liver cancer patient increases.The present invention's application AAL is to the high-affinity of fucosylated glycan, by preparation and the detection of AAL-ELISA and Protein-ELISA, form PON1 antibody-antigene-biotin mark AAL compound and PON1 antibody-antigene-biotin labeling antibody compound respectively, obtain the absorbance of PON1 in conjunction with AAL and the absorbance ratio Fuc-PON1 of PON1 albumen, by carrying out retrospective study to clinical data, set up tumour Distinguishing diagnosis function, obtain ROC curve and Cutoff value, compare the accuracy rate difference of AFP-L3 and Fuc-PON1 to diagnosing cancer of liver simultaneously.The present invention is applicable to Liver Cancer scene or the extensive examination of hepatopathy crowd, for the diagnosis improving liver cancer, realizes early intervention treatment, reduces liver cancer case fatality rate and have very important significance.
Summary of the invention
The object of this invention is to provide a kind of kit for diagnosing hepatocellular carcinoma (HCC) had compared with high-accuracy.
In order to achieve the above object, the invention provides a kind of for diagnosing the kit of hepatocellular carcinoma, it is characterized in that, comprise Fuc-PON1 half-quantitative detection elisa plate, described Fuc-PON1 half-quantitative detection elisa plate is made up of AAL-ELISA plate and Protein-ELISA plate.
Preferably, described for diagnosing the kit of hepatocellular carcinoma also to comprise HCC forecast model:
P=exp(-0.915+0.275*Fuc-PON1)／[1+exp(-0.915+0.275*Fuc-PON1)]
The computing method of described Fuc-PON1 value are: in each blood serum sample, PON1 is in conjunction with the ratio of the actual OD value of AAL with the actual OD value of PON1 albumen in identical blood serum sample, in described blood serum sample, PON1 is obtained by the detection of AAL-ELISA plate in conjunction with the actual OD value of AAL, in blood serum sample, the actual OD value of PON1 albumen is detected by Protein-ELISA plate and obtains, and the Cutoff value that this model distinguishes liver cancer and cirrhosis is 2.2727.
Preferably, described for diagnosing the kit of hepatocellular carcinoma also to comprise PON1 capture antibody, its concentration is 1 μ g/mL.
Preferably, described for diagnosing the kit of hepatocellular carcinoma also to comprise the first sealer and the second sealer, the first sealer is 3%BSA solution, and for closed AAL-ELISA plate, the second sealer is for containing 1%BSA and 0.05%NaN 3solution, for closed Protein-ELISA plate.
Preferably, described for diagnosing the kit of hepatocellular carcinoma also to comprise the first oxygenant, the first oxygenant be pH4.0 containing 100mMNaIO 4with the solution of 50mM citric acid, for the oxidation of AAL-ELISA plate.
Preferably, described for diagnosing the kit of hepatocellular carcinoma also to comprise biotin labeled AAL and biotin labeled antibody.
Preferably, described for diagnosing the kit of hepatocellular carcinoma also to comprise blood sample collection device and operational manual.
The PON1 albumen mentioned in the present invention is significantly strengthened by the affine AAL ability of proteomic efforts determination in early stage unit PON1, illustrates that the unit PON1 fucosylated glycan content of liver cancer patient increases.
The present invention mainly sets up and optimizes AAL-ELISA and Protein-ELISA method, determines the applied sample amount of optimum capture antibody concentration, blood serum sample, agglutinin concentration and detects antibody concentration; And select suitable AAL-ELISA sealer and oxygenant.Get rid of the cross reaction of PON1 antibody and other antigen, establish AAL-ELISA and Protein-ELISA to the sensing range of PON1 and repeatability, build the Fuc-PON1 half-quantitative detection system that can be used for the examination of clinical serum sample.
As shown in Figure 1, the present invention is by the preparation of AAL-ELISA and Protein-ELISA, the fucose (Fuc) of Paraoxonase (PON) 1 and the expression of albumen in associating parallel parsing serum, in unit PON1 albumen in liver cancer and patient with liver cirrhosis serum in conjunction with the quantitative detection of the content (Fuc-PON1) of fucose.And according to the detection data of Fuc-PON1 and the retrospective study of clinical data, set up the early diagnosis that tumour Distinguishing diagnosis function can be used for primary hepatocyte hepatocarcinoma.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention is by the preparation of AAL (willow eats agglutinin)-ELISA and Protein-ELISA and detection, form antibody-antigene-biotin mark AAL compound and antibody-antigene-biotin labeling antibody compound respectively, using PON1 in conjunction with the absorbance (OD value) of AAL and the absorbance ratio of PON1 albumen in unit of measurement PON1 albumen in conjunction with the content (Fuc-PON1) of fucose, by carrying out retrospective study to clinical data, set up tumour Distinguishing diagnosis function, obtain receiver operating curves (ROC) and differentiate (Cutoff) value, this function is respectively 60.3% and 71.4% for the diagnostic sensitivity of HCC and specificity, AUC is 0.736, be better than alpha-fetoprotein N-glycan core fucose (AFP-L3).Method of the present invention and kit have high flux property, comparatively high specific, detect and quick and precisely wait outstanding advantages, have important clinical value to the diagnosis of liver cancer.
Accompanying drawing explanation
Fig. 1 is high flux Fuc-PON1 detection system principle schematic;
Fig. 2 a is AAL-ELISA linear detection range figure
Fig. 2 b is Protein-ELISA linear detection range figure.
Fig. 3 is that Fuc-PON1 and AFP-L3 detection system builds ROC curve map respectively;
Embodiment
The present invention is illustrated below in conjunction with embodiment.
Embodiment:
One, for diagnosing a kit for hepatocellular carcinoma, following component is comprised:
1, Fuc-PON1 half-quantitative detection elisa plate, described Fuc-PON1 half-quantitative detection elisa plate is made up of AAL-ELISA plate and Protein-ELISA plate.
2, HCC forecast model:
P=exp(-0.915+0.275*Fuc-PON1)／[1+exp(-0.915+0.275*Fuc-PON1)]
The computing method of described Fuc-PON1 value are: in each blood serum sample, PON1 is in conjunction with the ratio of the actual OD value of AAL with the actual OD value of PON1 albumen in identical blood serum sample, in described blood serum sample, PON1 is obtained by the detection of AAL-ELISA plate in conjunction with the actual OD value of AAL, in blood serum sample, the actual OD value of PON1 albumen is detected by Protein-ELISA plate and obtains, and the Cutoff value that this model distinguishes liver cancer and cirrhosis is 2.2727.
3, PON1 capture antibody, its concentration is 1 μ g/mL.(R&DSystems，Catalog#DYC5816，Part#843480)
4, the first sealer (3%BSAinPBS, pH7.2-7.4).
5, the second sealer (1%BSA, 0.05%NaN 3inPBS, pH7.2-7.4).
6, the first oxygenant (100mMNaIO 4, 50mMCitricacid, pH4.0).
7, biotin labeled AAL.
8, biotin labeled antibody (1%BSAinPBS, pH7.2-7.4 dilute).
9、HRP-streptavidin。(ThermoScientific，Product#N100)。
10, blood sample collection device and operational manual.
Two, detection method:
(1) preprocess method of AAL-ELISA plate and Protein-ELISA plate:
1, get two pieces of elisa plates, be used separately as AAL-ELISA plate and Protein-ELISA plate;
2, capture antibody: add PON1 capture antibody in AAL-ELISA plate and Protein-ELISA plate, in AAL-ELISA, PON1 capture antibody concentration is 1 μ g/mL, 100 μ L/well; In Protein-ELISA, PON1 capture antibody concentration is 1 μ g/mL, 100 μ L/well.Incubated at room is spent the night.
3, wash plate: adopt PBST (0.05%Tween20inPBS, pH7.2-7.4) to wash plate 4 times, each to soak 2 minutes, 300 μ L/well, dry, and pat on thieving paper and patted dry by liquid in hole.
4, close: AAL-ELISA plate adopts the first sealer (3%BSAinPBS, pH7.2-7.4); Protein-ELISA adopts the second sealer (1%BSA, 0.05%NaN 3inPBS, pH7.2-7.4) room temperature closes 1h, 200 μ L/well.
5, wash plate: adopt PBST (0.05%Tween20inPBS, pH7.2-7.4) to wash plate 4 times, each to soak 2 minutes, 300 μ L/well, dry, and pat on thieving paper and patted dry by liquid in hole.
6, be oxidized: AAL-ELISA plate adopts the first oxygenant (100mMNaIO 4, 50mMCitricacid, pH4.0); Protein-ELISA adopts the second sealer (1%BSA, 0.05%NaN 3inPBS, pH7.2-7.4) be oxidized, 4 DEG C of lucifuge reactions 1h, 200 μ L/well.
7, plate is washed: adopt PBST (0.05%Tween20inPBS, pH7.2-7.4) plate is washed 4 times, each immersion 2 minutes, 300 μ L/well, dry, and pat on thieving paper liquid in hole is patted dry, obtain the AAL-ELISA plate after processing and Protein-ELISA plate respectively, for following step.
(2) Fuc-PON1 half-quantitative detection ELISA detection method:
1, application of sample: AAL-ELISA plate adds dilution serum sample 50 μ L/well (adopting PBS1:30 dilution); Protein-ELISA plate adds dilution serum sample 100 μ L/well (0.5 μ L serum adopts 1mMEDTA, 0.5%TritonX-100inPBS, pH7.2-7.4 to be diluted to 100 μ L), room temperature 2h.
2, wash plate: adopt PBST (0.05%Tween20inPBS, pH7.2-7.4) to wash plate 4 times, each to soak 2 minutes, 300 μ L/well, dry, and pat on thieving paper and patted dry by liquid in hole.
3, reagent is detected: in AAL-ELISA plate, add the biotin labeled AAL of 1 μ g/mL (PBST dilution); The biotin labeled antibody of 600ng/mL (1%BSAinPBS, pH7.2-7.4 dilute) is added, 100 μ L/well, room temperature 2h in Protein-ELISA plate.
4, wash plate: adopt PBST (0.05%Tween20inPBS, pH7.2-7.4) to wash plate 4 times, each to soak 2 minutes, 300 μ L/well, dry, and pat on thieving paper and patted dry by liquid in hole.
5, enzyme conjugates working fluid: add HRP-sztreptavidin (adopting sealer 1,1:12500 to dilute) in AAL-ELISA plate; HRP-sztreptavidin (adopting 1%BSAinPBS, 1:12500 dilution) is added, 100 μ L/well, room temperature 30min in Protein-ELISA plate.
6, wash plate: adopt PBST (0.05%Tween20inPBS, pH7.2-7.4) to wash plate 4 times, each to soak 2 minutes, 300 μ L/well, dry, and pat on thieving paper and patted dry by liquid in hole.
7, substrate solution is added: by substrate solution according to TMB:H 2o 2=1:2 mixes, 100 μ L/well, room temperature 20min.
8, stop buffer is added: 100 μ LStopSolution/well, the addition sequence of stop buffer is identical with the addition sequence of substrate solution.
9, detect: use NanoQuantinfiniteM200 (TECAN) microplate reader in the OD value in each hole of 450nm wavelength measurement immediately.Microplate reader power supply should be opened in advance, preheater apparatus, set trace routine.
(3) Fuc-PON1 half-quantitative detection elisa assay method:
1, data: the OD value that the OD value of each blood serum sample should deduct blank well is actual OD value.Each blood serum sample PON1 is Fuc-PON1 value in conjunction with actual OD value (Protein-ELISA) ratio of actual OD value (AAL-ELISA) the identical blood serum sample PON1 albumen therewith of AAL.
Calculate P value according to above-mentioned HCC forecast model, P value compared with Cutoff value, > 2.2727 is liver cancer, and < 2.2727 is cirrhosis.
Adopt the kit in embodiment and detection method, the fucose of PON1 and the expression of albumen in associating parallel parsing 86 routine hepatopathy blood serum sample (cirrhosis and each 43 examples of liver cancer patient, all through clinical definite), calculate Fuc-PON1 content.It is 2.2727 that the above-mentioned HCC forecast model adopting SPSS software logistic regretional analysis to set up carries out detecting the Cutoff value distinguishing liver cancer and cirrhosis.(AFP-L3 detection kit, purchased from Abnova company (Catalog#KA1187), detects AFP-L3 content in above-mentioned 86 routine hepatopathy blood serum samples according to kit operation steps, adopts SPSS software to obtain ROC curve.), relatively kit of the present invention and AFP-L3 are to the accuracy of diagnosing cancer of liver and specificity, result as shown in Figure 3, the sensitivity of kit of the present invention is 60.3%, specificity 71.4%, accuracy is 64%, AUC is 0.736, and AFP-L3 is 60.5%, AUC to the diagnostic sensitivity of HCC, specificity and accuracy is 0.612.As shown in table 1, it is 0.8466 that liver cirrhosis patient AFP-L3 detects average, and standard deviation is 0.32; It is 0.9779 that liver cancer patient AFP-L3 detects average, and standard deviation is 0.36; P value is 0.0393.It is 2.6315 that liver cirrhosis patient Fuc-PON1 detects average, and standard deviation is 2.18; It is 4.3609 that liver cancer patient Fuc-PON1 detects average, and standard deviation is 3.08; P value is 0.002.
Table 1:Fuc-PON1 and AFP-L3 testing result compares mutually
Utilize Fuc-PON1 level in kit assay 40 of the present invention routine test set serum (serum specimen comes from Guangxi Medical University's attached First Hospital liver surgery and GI Medicine inpatient), with reference to Cutoff value, result shows and successfully predicts 27 routine patients, and accuracy rate is 67.5%.Therefore, the present invention has higher specificity and accuracy, has important clinical value to the prediction of liver cancer.
Get (the 10 example mixing of 1 routine pooled serum sample, serum specimen comes from Guangxi Medical University's attached First Hospital liver surgery and GI Medicine inpatient) detect of the present invention for diagnosing the AAL-ELISA in the kit of hepatocellular carcinoma and other albumen whether to there is non-specific cross-reaction, result is as shown in table 2, in kit of the present invention, to detect the OD value of other albumen consistent with PBS damping fluid for AAL-ELISA, and the OD value of blood-serum P ON1 is much larger than other albumen.
In table 2:Fuc-PON1 detection system, AAL-ELISA has no cross reaction:
(every 1 example is mixed by 10 routine serum to get 3 routine pooled serum samples, serum specimen comes from Guangxi Medical University's attached First Hospital liver surgery and GI Medicine inpatient) detect the every routine Virus monitory of repeatability 3 times for the kit diagnosing hepatocellular carcinoma, calculate the mean value of every routine serum Fuc-PON1, standard deviation and CV value.Result is as shown in table 3.
Table 3:Fuc-PON1 detection system three duplicate detection results
The serum of known PON1 protein concentration is adopted to carry out doubling dilution, determine AAL-ELISA linear detection range in kit of the present invention, wherein in serum, PON1 protein concentration is determined to adopt ELISA immue quantitative detection reagent box (R & DSystems, Catalog#DYC5816), result as shown in Figure 2 a, adopt recombinantPON1 standard items (R & DSystems, Catalog#DYC5816, Part#843482) carry out doubling dilution, determine Protein-ELISA linear detection range.As shown in Figure 2 b, Protein-ELISA linear detection range is greater than AAL-ELISA linear detection range to result, therefore the sensing range of Fuc-PON1 detection system and AAL-ELISA linear detection range.The quantitative linearity sensing range of kit of the present invention is 0.71 ~ 7.1ng/mL.

Claims (5)

1. for diagnosing a kit for hepatocellular carcinoma, it is characterized in that, comprising Fuc-PON1 half-quantitative detection elisa plate, described Fuc-PON1 half-quantitative detection elisa plate is made up of AAL-ELISA plate and Protein-ELISA plate; Wherein, the computing method of Fuc-PON1 value are: in each blood serum sample, PON1 is in conjunction with the ratio of the actual OD value of AAL with the actual OD value of PON1 albumen in identical blood serum sample, in described blood serum sample, PON1 is obtained by the detection of AAL-ELISA plate in conjunction with the actual OD value of AAL, and in blood serum sample, the actual OD value of PON1 albumen is detected by Protein-ELISA plate and obtains; Also comprise HCC forecast model:
P=exp(-0.915+0.275*Fuc-PON1)/[1+exp(-0.915+0.275*Fuc-PON1)]
The Cutoff value that this model distinguishes liver cancer and cirrhosis is 2.2727.
2. as claimed in claim 1 for diagnosing the kit of hepatocellular carcinoma, it is characterized in that, also comprise PON1 capture antibody, its concentration is 1 μ g/mL.
3. as claimed in claim 1 for diagnosing the kit of hepatocellular carcinoma, it is characterized in that, described for diagnosing the kit of hepatocellular carcinoma also to comprise the first sealer and the second sealer, first sealer is 3%BSA solution, for closed AAL-ELISA plate, the second sealer is for containing 1%BSA and 0.05%NaN 3solution, for closed Protein-ELISA plate.
4. as claimed in claim 1 for diagnosing the kit of hepatocellular carcinoma, it is characterized in that, described for diagnosing the kit of hepatocellular carcinoma also to comprise the first oxygenant, the first oxygenant be pH4.0 containing 100mMNaIO 4with the solution of 50mM citric acid, for the oxidation of AAL-ELISA plate.
5. as claimed in claim 1 for diagnosing the kit of hepatocellular carcinoma, it is characterized in that, described for diagnosing the kit of hepatocellular carcinoma also to comprise biotin labeled AAL and biotin labeled antibody.
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Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104483484A (en) * 2014-12-19 2015-04-01 复旦大学附属中山医院 GP73 (Golgi protein 73) variant kit for HCC (hepatocellular carcinoma) serological prediction
CN105181971A (en) * 2015-09-06 2015-12-23 复旦大学附属中山医院 Haptoglobin kit for liver cancer serology prediction
CN105785052B (en) * 2016-05-11 2017-10-10 复旦大学附属中山医院 Hoptoglobin heteroplasmon diagnosing liver cancer kit based on agglutinin magnetic bead
CN108196064A (en) * 2017-12-26 2018-06-22 黑龙江八农垦大学 The double crush syndrome detection kit and detection method of a kind of ox PON1 albumen
CN108267578A (en) * 2017-12-29 2018-07-10 大连医科大学 The detection method of serum IgG core fucosylation level
CN111239412A (en) * 2020-01-20 2020-06-05 复旦大学附属中山医院 Preparation method of protein microchip focusing on liver cell and liver cancer

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102625915A (en) * 2009-07-14 2012-08-01 独立行政法人产业技术综合研究所 Method for measurement of glycoprotein, method for detection of hepatic diseases, reagent for quantification of glycoprotein, and sugar chain marker glycoprotein as measure of disease conditions of hepatic diseases

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2174132A2 (en) * 2007-07-20 2010-04-14 National Institute for Bioprocessing Research and Training Limited Glycosylation markers for cancer and chronic inflammation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102625915A (en) * 2009-07-14 2012-08-01 独立行政法人产业技术综合研究所 Method for measurement of glycoprotein, method for detection of hepatic diseases, reagent for quantification of glycoprotein, and sugar chain marker glycoprotein as measure of disease conditions of hepatic diseases

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Development of an antibody-lectin enzyme immunoassay for fucosylated α-fetoprotein;Hiroaki Korekane;《Biochimicaet Biophysica Acta》;20111231;第1820卷;第1405-1411页 *
Discrimination of liver cirrhosis from chronic hepatitis by measuring the ratio of Aleuria aurantia ectin-reactive serum cholinesterase to immunoreactive protein;Toshikazu Hada;《Clinica Chimica Acta》;19991231;第281卷;第39页第2.4-2.6节，第42页第1段，第38页第2段，第44页最后一段 *
Enzyme-linked immunosorbent assay (ELISA) for Aleuria aurantia lectin-reactive serum cholinesterase to differentiate liver cirrhosis and chronic hepatitis;Masahide Kondo;《Clinica Chimica Acta 》;19951231;第243卷;第1-9页 *
Integrated Glycoproteomics Demonstrates Fucosylated Serum Paraoxonase1 Alterations in Small Cell Lung Cancer;Jung-Mo Ahn et al;《Molecular & CellularProteomics》;20131001;第13卷;第33页右栏第1-2段 *
Serum paraoxonase 1 heteroplasmon, afucosylated, and sialylated glycoprotein in distinguishing early hepatocellular carcinoma from liver cirrhosis patients;Chun Sun et al;《Acta Biochim Biophys Sin》;20120701;第44卷;摘要，第769页右栏第1-2段 *

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