WO2008031288A1 - Colonne centrifuge préremplie servant à dépister un variant d'alpha-fétoprotéine spécifique de l'hépatocarcinome, et trousse d'analyse contenant ladite colonne - Google Patents

Colonne centrifuge préremplie servant à dépister un variant d'alpha-fétoprotéine spécifique de l'hépatocarcinome, et trousse d'analyse contenant ladite colonne Download PDF

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WO2008031288A1
WO2008031288A1 PCT/CN2006/003210 CN2006003210W WO2008031288A1 WO 2008031288 A1 WO2008031288 A1 WO 2008031288A1 CN 2006003210 W CN2006003210 W CN 2006003210W WO 2008031288 A1 WO2008031288 A1 WO 2008031288A1
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afp
solution
buffer
monoclonal antibody
protein
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PCT/CN2006/003210
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Chinese (zh)
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Changqing Lin
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Beijing Hotgen Biotech Co., Ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5021—Test tubes specially adapted for centrifugation purposes
    • G—PHYSICS
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/047—Additional chamber, reservoir
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • B01L3/50825—Closing or opening means, corks, bungs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/471—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein

Definitions

  • the invention relates to a pre-packed spin column for detecting hepatic alpha-fetoprotein heterogeneous body, and relates to a chemiluminescence detecting kit containing the pre-packed spin column and an enzyme-linked immunosorbent assay containing the pre-packed spin column
  • the quantitative detection kit and the preparation method of the kits belong to the technical field of medical products and are used for early diagnosis of liver cancer.
  • Hepatocel lular carcinoma is the fourth most common malignant tumor in the world. About 250 000 patients die of hepatocellular carcinoma every year. The pathogenesis of hepatocellular carcinoma is still unclear. The malignancy is higher, and the mortality rate ranks third among malignant tumors of the digestive tract. Therefore, early detection and early treatment are the key to improve the survival rate of patients. Most patients have reached the middle and late stage of treatment and lost the best treatment opportunity. Risk factors include chronic hepatitis and cirrhosis caused by hepatitis B virus and hepatitis C virus. For effective treatment, the clinical efficiency of screening and screening depends on early diagnosis. HCC is an important type of tumor etiology.
  • HCC chronic liver damage caused by cirrhosis, viral hepatitis, chemical carcinogens and environmental factors can induce HCC.
  • HCC has high malignancy, is prone to recurrence and metastasis, has a poor prognosis, and is difficult to diagnose early, which is prone to delay the optimal treatment period.
  • Alphafetoprotein is a glycoprotein.
  • the detection of AFP in blood is the most common method for diagnosing liver cancer.
  • AFP is synthesized by hepatic parenchymal cells and yolk sputum cells in mammalian embryonic stage, and gastrointestinal mucosal cells derived from endoderm can also be synthesized in small amounts.
  • AFP mainly exists in the fetal circulation, and as the fetus matures, AFP synthesis gradually decreases.
  • the concentration of AFP in cord blood at birth can reach 10 ⁇ 100mg / L, and it will drop to normal adult level one year after birth.
  • AFP as a diagnostic indicator for early liver cancer is that there is a significant proportion of liver disease and cirrhosis diseases. AFP mild increase (20 ⁇ 200 ng / ml) is seen in a considerable number of chronic liver diseases. The patient was 11. 7-44% AFP positive in patients with cirrhosis. Therefore, when distinguishing between benign liver disease and malignant liver tumor, the value of AFP as an early liver cancer screening index is greatly reduced.
  • Lectin is a large class of non-immune-derived proteins or glycoproteins widely found in nature. It can be reversibly combined with sugars, non-covalently, and has agglutinated blood cells. It is called lectin. .
  • ConA concanavalin-ConA
  • jackbean Canaval ia ens iformis
  • ConA can agglutinate glycogen and starch in solution, and the hemagglutination of ConA can be inhibited by sucrose, so it is speculated that the hemagglutination of ConA is the result of cell surface sugar action.
  • lectin activity Nearly a thousand plants have been tested for lectin activity. In plants, not only lectins are present in seeds, but lectins are also found in roots, stems, leaves, skins, and fruit juices. In addition to plants, other organisms, such as various fungi, certain viruses, ridgeless pushers, spinal pushers, and various tissues and organs to the human body, are present in lectins.
  • Lectins can specifically bind to sugars.
  • lectins can be divided into six categories: D-mannose or D-glucose; N-acetylglucosamine; N-acetylgalactosamine; D-galactose; L-fucose; Lectin (WGA) can specifically bind sialic acid.
  • leguminous including concanavalin ConA, lentil lectin LCA, and the like.
  • Lentils The famous peas, the lectin LCA (Lens cul inar is lect in ) extracted from lentils, can specifically bind fucosylated glycans.
  • AFP can be divided into three parts by LCA affinity electrophoresis: AFP-L1 (LCA unbound), AFP-L2 (LCA weakly bound), AFP-L3 LCA strong binding).
  • AFP-L1 from benign liver disease, is the main component of AFP; AFP-L2 is from pregnant women; AFP-L3 is unique to HCC cells. Therefore, the AFP heterogeneous body containing the fucosylated sugar chain synthesized and secreted by the liver cancer cells is called liver cancer-specific AFP (AFP-L3).
  • the process may be as follows: Proliferating hepatocytes and liver cancer cells synthesize AFP in G1 and S phases and distribute them in the perinuclear, endoplasmic reticulum and Golgi. In secreted sputum, AFP located in the rough endoplasmic reticulum and Golgi is secreted into the circulation by different glycosylation modifications with the participation of glycosyltransferase. Due to the difference in glycosylation, the difference in AFP heterogeneity is caused. Another possible cause is a change in the conformation of AFP after glycosylation due to abnormal glycosylation.
  • AFP heterogenes can be classified into different types, such as lentil lectin (LCA) affinity, pea lectin incompatibility, concan agglutinin (ConA) affinity and ConA. Affinity type. Another classification method is to name the bands obtained by electrophoresis from the anode starting with 1, 2, 3, and combining lectin.
  • LCA lentil lectin
  • ConA concan agglutinin
  • AFP-L1 (LCA unbound type)
  • AFP-L2 (LCA weakly bound type)
  • AFP-L3 (LCA strong binding type)
  • domestic and foreign studies have proved that AFP-L3 is specifically produced by liver cancer cells. of.
  • AFP heterogenes The structure of many sugar chains in AFP heterogenes is not fully understood.
  • the group that binds to LCA is AFP sugar chain fucosylation (LCA has strong affinity with fucose). Since the embryonic AFP has almost no fucose component, it can be considered that the abnormality of this molecular sugar chain is A reflection of abnormal glycosylation.
  • the binding site of AFP-L3 to LCA is asparagine-linked fucosylated N-acetylglucosamine.
  • AFP heterologues containing fucosylated sugar chains are specifically secreted by hepatocytes and are referred to as liver cancer-specific AFP (AFP-L3).
  • AFP heterogeneity actually refers to A FP-L3 combined with LCA. It was listed as one of the liver cancer markers of primary liver cancer clinical diagnostic criteria in the Fourth National Conference on Liver Cancer in 1999. It is recognized as a primary liver cancer indicator that is more specific than AFP aflatase alone.
  • AFP hepatocellular carcinoma
  • the type of AFP heterogeneity was different in the study.
  • the positive rate of LCA in the diagnosis of HCC was 87.12%.
  • the false positive rate was 21.5%, the ConA positive rate was 89.17%, and the false positive rate was 17.15%.
  • the current study results in a content of AFP-L3 greater than 15% as a positive indicator of liver cancer.
  • the serum AFP content decreases, and the rate of decline depends on the amount of residual AFP and half-life in the body. Generally, it turns negative in 2 months, and the AFP heterogeneity disappears when it turns negative. If the AFP drops significantly but does not turn negative, the heterogeneous changes are not obvious, suggesting that the surgery is not complete, there may be marginal residual, vascular tumor thrombus, satellite nodules or metastasis. If the heterogeneity falls below 25% and the AFP and heterogene concentrations are relatively constant, it may be due to hepatitis or cirrhosis in the patient.
  • AFP in maternal serum during normal gestation is in equilibrium with AFP in the embryo. Once fetal malformation or abnormal placental barrier occurs, fetal serum may infiltrate into amniotic fluid or amniotic fluid may penetrate into maternal serum, resulting in a sharp increase in maternal amniotic fluid or serum AFP.
  • AFP only measuring the total amount of AFP has certain limitations. Experiments have shown that the neural tube defect, no brain or spina bifida. Childhood hepatoblastoma, biliary atresia, gonadal tumor, malignant teratoma, etc. may be positive for AFP and/or AFP heterogeneity.
  • AFP-L3 is the only protein produced by cancer cells in the liver of a patient.
  • the test method was studied in a multicenter, prospective, double-blind, long-term clinical trial in Canada and the United States. The results showed that patients with elevated AFP-L 3% (15% or more) had a 7-fold increased risk of developing hepatocellular carcinoma in the next 21 months. According to existing guidelines for hepatocellular carcinoma oncology, the incidence of hepatocellular carcinoma in these patients is extremely high.
  • the current detection methods for AFP heterogeneous bodies include plant lectin affinity chromatography, polyacrylamide gel electrophoresis, affinity blotting, and affinity cross-immunoelectrophoresis. Affinity imprinting is used in France, and domestic cross-immunoelectrophoresis technology is used. According to the different staining methods, it can be divided into:
  • Coomassie Brilliant Blue The electrophoresed specimens were directly stained with Coomassie brilliant blue, and the peak bands were observed after elution. The method is simple, but there are many interference factors, and the sensitivity is about 1000ug/L.
  • Enzyme labeling method The antibody labeled with peroxidase is incubated with the electrophoresis sample, rinsed, and developed with diaminobenzidine, and the detection sensitivity can be increased to 50 ug/L.
  • Gold and silver staining method The electrophoresis specimen is directly incubated with Staphylococcus aureus protein A2 gel gold, and then developed with silver coloring solution, and the peak band is clear, and the sensitivity is up to 32 ug/L.
  • Autoradiography It is the most commonly used detection method in China, with a sensitivity of 31ug/L.
  • the principle is that the specimen is separated and electrophoresed in a gel containing lectin, and then subjected to secondary electrophoresis in a gel containing 125 IAFP and anti-AFP antibody. After electrophoresis, the gel is dried and covered with X-ray film. , rinse the image.
  • the whole experimental procedure is complicated. For a long time, only a few clinical laboratories in China have been able to measure AFP heterogeneous bodies. A few cities can meet the requirements for clinical determination of AFP heterogeneous bodies, and there is no domestic reagent supply.
  • the present invention provides a prepacked spin column for detecting a hepatoma alpha-fetoprotein heterogeneous body and a diagnostic kit containing the prepacked column.
  • These pre-packed spin columns and kits can truly detect the level of alpha-fetoprotein heterogene in serum and accurately diagnose early primary liver cancer. It has extremely high sensitivity and is quick and easy.
  • the technical scheme of the present invention is: a pre-assembled spin column for detecting hepatic alpha-fetoprotein heterogeneous body, comprising an upper separating tube and a lower collecting tube, wherein the upper separating tube is provided with a medium coupled with a lectin, the lower portion
  • the collection tube is provided with a buffer solution, and a filter cloth is disposed at the bottom of the separation tube so that the medium cannot pass through the filter cloth.
  • the lectin is lentil lectin LCA or concanavalin con-Con.
  • the lentil lectin LCA can be extracted by the following method:
  • the source of lentils is preferred in Gansuzhou, China;
  • the above dialysis balanced extract is further purified by DEAE Sepharose Fast Flow chromatography to collect a breakthrough peak;
  • the medium is preferably coupled to an agglutinin affinity medium, such as agarose microspheres, and other media, such as polystyrene microspheres, cellulose, chitosan, which are capable of blocking the coupling of lectins.
  • the medium passes through a filter cloth that does not block the passage of liquids and proteins.
  • the filter cloth refers to various forms of filter materials, and the buffer solution is an active protective solution of lectin, for example, containing 1 mmol/L of CaCl 2 , 1 mmol/L MnCl 2 and 50 mmol/L Tr is-HCL, pH 7.5.
  • the agarose sphere may be agarose gel sephalose 4B, agarose gel sephrose 6B or agarose gel sephrose FF.
  • the agarose gel coupled to the lentil lectin can be obtained by the following method:
  • the agarose gel may preferably be Pharmacia hydrogen bromide-activated Sepharose 4B using the following specific steps:
  • the invention provides a chemiluminescence kit containing the above pre-packed spin column and a preparation method thereof.
  • the chemiluminescence kit comprises a prepacked spin column for detecting hepatocellular protein adenoma; a chemiluminescent plate labeled with a monoclonal antibody against adenine; a positive control, a negative control; an enzyme-labeled anti-A fetus Protein monoclonal antibody; substrate solution, color developing solution, AFP-L3 washing buffer, AFP-L3 eluent, concentrated washing solution, AFP standard.
  • the AFP-L3 washing buffer is 20 mmol/L Tris-HCl, pH 7.4, and contains 0.1% Procl in 300 preservative.
  • the AFP-L3 eluate is 20 mmol / L Tri s-Hcl, PH7. 4, 150 mmol / L NaCl and 500mmol / L a - mercapto-D - mannoside, the eluent contains 0. l ° /»Procl in 300 preservative.
  • the kit is made as follows:
  • (1) coating The common high titer of the fetus protein monoclonal antibody (commercially available) was diluted with 0.05 M citric acid buffer and added to each well of the microtiter plate, each well ⁇ , adsorbed overnight, with The plate was washed with Tween phosphate buffer, blocked with Tween phosphate buffer containing bovine serum albumin overnight, dried and dried to obtain a monoclonal antibody coated ELISA plate.
  • the chemiluminescent ELISA plate can be made of domestic or imported plates; the specification can be 96-well plate or 12x8, 12X4 detachable plate;
  • Positive control collect ascites from liver cancer patients, filter and remove bacteria
  • Negative control collect normal human serum, filter sterilization, and dispense
  • Enzyme-labeled monoclonal antibody The enzyme-labeled monoclonal antibody in the kit is prepared by labeling a monoclonal antibody with horseradish peroxidase (HRP), and the steps are as follows:
  • auxiliary reagent The preparation method of the auxiliary reagent is as follows: a) Substrate solution: 1. Oml EDTA (1.0x10- 2 M ), 1.0ml H 2 Q 2 (7.5x10- 3 M), 0.4ml HC1 (1. (MO- 2 M) and 0.2ml Tween20 (1 %);
  • the invention also provides an enzyme-linked immunoassay kit comprising the above pre-packed spin column and a preparation method thereof.
  • the kit comprises a prepacked spin column for detecting liver cancer adenine protein heterogeneity; a microplate coated with adenine protein monoclonal antibody; a positive control, a negative control; an enzyme-labeled anti-fetal protein monoclonal antibody ; substrate solution, color developing solution, reaction stop solution, AFP-L3 washing buffer, AFP-L3 eluent, concentrated washing solution, AFP standard.
  • the AFP-L3 washing buffer is 20 mmol/L Tris-Hcl, pH 7.4, and contains 0.1% Proclin 300 preservative.
  • lWProcl in The EFP eluate is 20 mmol / L Tris-Hcl, pH 7.4, 150 mmol / L NaCl and 500 ⁇ / La-mercapto-D-mannosidase, the eluent contains 0. lWProcl in 300 preservatives.
  • the kit is made as follows: (1) Coating: The common high titer alpha-fetoprotein monoclonal antibody (commercially available) was diluted with 0.05 M carbonate buffer and added to each well of the enzyme-labeled plate, each well ⁇ , adsorbed overnight, with Tween Phosphate The plate was washed with a buffer, and then blocked with Tween phosphate buffer containing bovine serum albumin overnight, dried and dried to obtain a monoclonal antibody-coated ELISA plate.
  • the ELISA plate is a polystyrene ELISA plate, which can be made of domestic or imported plates; the specification can be 96-well plate or 12x8, 12X4 detachable plate;
  • Positive control collect ascites from liver cancer patients, filter and remove bacteria
  • Negative control collect normal human serum, filter sterilization, and dispense
  • Enzyme-labeled monoclonal antibody The enzyme-labeled monoclonal antibody in the kit is prepared by labeling a monoclonal antibody with horseradish peroxidase (HRP), and the steps are as follows:
  • a The solution of the solution: a 3% hydrogen peroxide solution prepared by a phosphate-citrate buffer (pH 5. Q); b) a color developing solution: a tetradecylbenzidine (TMB) sterol solution, a concentration of 0. lmg / Ml;
  • TMB tetradecylbenzidine
  • reaction stop solution 2 mol / L ⁇ acid; (5) pre-packed spin column, ELISA plate coated with adenine protein monoclonal antibody, positive control, negative control, enzyme-labeled anti-fetal protein monoclonal antibody, substrate solution, color developing solution, The AFP-L 3 washing buffer, AFP-L3 eluent, reaction stop solution, concentrated washing solution, and AFP standard are packaged together to obtain a kit.
  • the invention overcomes the shortcomings of the operation steps of the prior detection method, such as cumbersome, time-consuming, and requiring a large number of supporting equipments.
  • the operator only needs simple operations such as centrifugation and incubation, and can complete the detection within 2 hours, and directly calculate the blood sample sample.
  • the content of abortion protein and abortion protein heterogeneity is simple, rapid and accurate, and provides support for the prevention, diagnosis and treatment of liver cancer.
  • FIG. 1 is a schematic cross-sectional structural view of a pre-assembled spin column of the present invention
  • Fig. 2 is a distribution diagram of AFP-L3 detection results of primary liver cancer specimens and chronic hepatitis Sanyang specimens using the chemiluminescent detection kit of the present invention.
  • the present invention provides a pre-packed spin column for detecting a liver cancer abortin protein heterogeneous body, which is composed of a separation tube 1 located at the upper portion and a collection tube 3 located at the lower portion.
  • the separation tube can be inserted into the collection tube.
  • the connection between the two is realized by means of a tube, and the upper separation tube is provided with a medium 4 for coupling a lectin (for the sake of clarity of the drawing, the inside of the tube for loading the medium is marked in the figure, and the physical substance is not drawn)
  • the lower collecting tube is provided with a buffer solution 5 (for the sake of clarity of the drawing, the inside of the tube for the buffer solution is marked in the drawing, the solid substance is not drawn), and the bottom of the separating tube is mainly a filter.
  • the filter cloth is a filter cloth that blocks the passage of a medium coupled with a lectin without blocking the passage of liquid and protein, so that when separated, the separated liquid and protein can pass through the filter cloth into the lower Collect Tube.
  • the filter cloth uses a filter material of any structure having the above functions, and is provided with a clamp plastic gasket at the bottom of the separation tube to fix the filter cloth.
  • the pre-assembled spin column consists of a spin column and a filling medium.
  • the spin column consists of an upper centrifuge tube and a lower collection tube. The lower end of the centrifuge tube is inserted from the collection tube nozzle, and the two are sleeved together.
  • the buffer in the lower collection tube of the pre-filled spin column of the present invention contains 1 wake-up ol /L of CaCl 2 , 1 mmol/L MnCl 2 and 50 mmol/L Tr is-HCL, pH 7.5.
  • the pre-installed spin column is used as follows:
  • the serum to be tested is completely centrifuged without hemolysis; the pre-packed micro-centrifugation column is taken out, and the liquid in the lower collecting tube is discarded;
  • sample dilution Pipette 250ul serum in a test tube, and add 350ul cleaning solution to dilute and shake;
  • Example 2 Preparation of an enzyme-linked immunoassay kit for detecting hepatocellular carcinoma alpha-fetoprotein heterogeneity:
  • the kit (96 servings) consists of:
  • HRP Horseradish peroxidase
  • Concentrated washing solution (20 times concentrated solution, 20x) 1 bottle, 2 Oml / bottle.
  • Collecting serum Obtaining healthy normal human serum from the hospital or blood station, and ascites from liver cancer patients, and storing at -70 °C for use;
  • AFP-L3 positive control liver cancer-positive patients with ascites, under sterile conditions, 1. 5ml eppendorf tube, each tube 0. 5ml. Stored at 4 ° C ;
  • Negative control normal human serum, determined to be AFP negative. Take more than one serum and combine them into a batch. After treatment at 60 ° C for 1 hour, filter and sterilize. 5 ⁇ Each tube was filled into a 5. 5ml eppendorf tube, each tube 0. 5ml. Store at 4. C.
  • the ELISA plate uses imported or domestic 12x8 detachable strips.
  • the AFP monoclonal antibody was diluted to 0 ( ⁇ g/ml) with 0. 05mol/L carbonate buffer, and then added to each well of the microplate, each well ⁇ , adsorbed overnight, with The buffer was washed and washed, and then blocked with 2% BSA of the blocking solution overnight, dried and dried to obtain a monoclonal antibody coated ELISA plate. Packed in aluminum foil bag at 96 holes/block, vacuum sealed;
  • Anti-AFP monoclonal antibodies are commercially available.
  • the labeled monoclonal antibody solution is added dropwise to the saturated ammonium sulphate solution, and stirred while adding, until the saturated ammonium sulfate concentration is reduced to 1/3;
  • Substrate solution 3% hydrogen peroxide solution prepared by phosphoric acid-citric acid buffer (pH 5.0), packed in 5ml/bottle;
  • AFP-L3 washing buffer 20 legs ol/LTris- Hcl, pH 7.4, containing 0. l% Proclin 300 antiseptic, divided into 25ml/bottle;
  • 5%Proclin 300 ⁇ AFP-L3 eluent: 20 mmol / L Tris-Hcl, pH 7.4, 150 mmol / L NaCl and 500 mmol / Lct-methyl-D-mannosidase, eluent containing 0. l% Proclin 300 antiseptic Agent, divided into 15ml / bottle; 6) Concentrated washing solution (20 times concentrated solution, 2Qx): 0. 05% Tween 20 solution prepared in PBS (pH 7.4), packed in 2Qml/bottle.
  • Example 3 Preparation of a Chemiluminescence Detection Kit for Detection of Hepatoma Abortion Protein Isomers:
  • the kit (48 servings) consists of:
  • HRP Horseradish peroxidase
  • Collecting serum Obtain normal human serum from the hospital or blood station, ascites from liver cancer patients, and keep at -70 °C for use;
  • AFP-L3 positive control Ascites of liver cancer-positive patients, under sterile conditions, into a 1.5 ml eppendorf tube, 0.5 ml per tube. Stored at 4 ° C ;
  • Negative control serum Normal human serum, which was confirmed to be AFP negative. More than one serum was combined and batched, and treated at 60 ° C for 1 hour, and then sterilized by filtration. 5 ⁇ Each tube was filled into a 5. 5ml eppendorf tube, each tube 0. 5ml. Store at 4 °C.
  • a ) Coating The chemiluminescent ELISA plate uses imported or domestic 12x8 detachable strips.
  • Step 1) AFP monoclonal antibody was diluted with 0. 05mol/L citrate buffer to 2 (g/ml, then added to each well of the microtiter plate, ⁇ per well, adsorbed overnight, washed with washing buffer, then The solution was blocked overnight with the blocking solution buffer, dried and dried to obtain a monoclonal antibody coated ELISA plate, packaged in an aluminum foil pouch at 96 wells/block, vacuum sealed; b) horseradish peroxidase (prepared) HRP )-labeled anti-AFP monoclonal antibody:
  • Anti-AFP monoclonal antibodies are commercially available.
  • Dispensing Dilute the enzyme-labeled anti-AFP monoclonal antibody obtained in step 3) to a suitable working concentration with a buffer containing 10% fetal bovine serum, and dispense at 6 ml/bottle and store at 4 °C.
  • AFP-L3 washing buffer 20 awake ol/LTris-Hcl, ⁇ 7 ⁇ 4, containing 0. l% Procl in 300 preservative;
  • the quality detection method of the kit of the present invention is:
  • Protein loading The content of coupled LCA was calculated by subtracting the unconjugated protein content from the pre-conjugation protein content. The requirement is not less than 7 mg/ml, and good results can be obtained at 10 mg/ml to 20 mg/ml.
  • Detection sensitivity According to the dilution test results of AFP standard, the detection sensitivity of this kit is 5ng/ml.
  • Example 4 Method for detecting alpha fetoprotein heterogeneous AFP-L 3 content using the chemiluminescence detection kit provided by the present invention:
  • pre-pretreatment sample ⁇ detect total AFP
  • processed sample 2 detect AFP-L3
  • AFP-L3% (sample 2 AFP content ⁇ sample 1 AFP content) X 100°/.
  • Ratio judgment AFP-L3% 10% is positive for liver cancer or extremely high risk of liver cancer, and AFP-L3% ⁇ 10% is benign liver disease.
  • Example 5 Detection results of positive samples of liver cancer patients using the kit provided by the present invention: In order to judge the coincidence rate between the chemiluminescence detection kit and the enzyme-linked immunosorbent kit of the present invention and the clinical liver cancer detection results, two types of the present invention are taken. The kit was tested for comparison.
  • Experiment 1 Using the specimens provided by the Beijing Ditan Hospital Virus Research Laboratory for comparative testing: 50 known primary liver cancer positive specimens, 83 physical examination healthy serum, 100 cirrhosis serum, 100 hepatitis, liver disease specimens were AFP positive specimen. The chemiluminescence detection kit and the enzyme-linked immunoassay kit were used for detection, and the results are shown in Table 1.
  • the results of this experiment show that the coincidence rate of the present invention for primary liver cancer is as high as 91%, the specificity in liver cirrhosis is as high as 93%, and the specificity in hepatitis is as high as 94%.
  • Experiment 2 A total of 28 primary liver cancer samples collected from Beijing 301 Hospital and 95 chronic hepatitis Sanyang specimens were tested by AFP-L3 using the chemiluminescence detection kit provided by the present invention. The results are shown in Fig. 2.
  • AFP-L3 quantitative detection was performed on the two types of specimens of the above-mentioned test 1 and test 1, and the results showed that AFP-L3 between the three positive chronic hepatitis and the primary liver cancer can be clearly distinguished when the AFP-L3 of the present invention is quantitatively detected. The difference in content.

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Abstract

Colonne centrifuge préremplie servant à dépister un variant d'alpha-fétoprotéine spécifique de l'hépatocarcinome, et trousse d'analyse contenant ladite colonne. La colonne centrifuge comprend un tube de séparation supérieur et un tube collecteur inférieur. Le tube de séparation supérieur contient des milieux associés à une agglutinine, et son fond est muni d'un tissu de dépôt. Une solution tampon est placée dans le tube collecteur inférieur. L'agglutinine est l'agglutinine Lens culinaris (LCA) ou l'agglutinine Canavalia ensiformis (Con-A), qui peut lier des chaînes glucidiques du variant d'alpha-fétoprotéine. L'élution et la centrifugation de la colonne permettent de purifier la constituante AFP-L3. La colonne centrifuge préremplie et la trousse d'analyse permettent de dépister rapidement et simplement le taux du variant d'alpha-fétoprotéine spécifique de l'hépatocarcinome et, partant, de diagnostiquer de manière précoce un hépatocarcinome.
PCT/CN2006/003210 2006-09-13 2006-11-29 Colonne centrifuge préremplie servant à dépister un variant d'alpha-fétoprotéine spécifique de l'hépatocarcinome, et trousse d'analyse contenant ladite colonne WO2008031288A1 (fr)

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CN111273027B (zh) * 2020-02-24 2023-03-28 安徽医科大学 一种用于检测肝癌血清糖链的spdp修饰凝集素芯片及其制备与应用
CN113009135A (zh) * 2021-02-19 2021-06-22 山东省大健康精准医疗产业技术研究院 一种检测cd47的管式磁微粒化学发光免疫定量试剂盒及其制备方法与应用
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CN115236334A (zh) * 2022-08-03 2022-10-25 武汉生之源生物科技股份有限公司 一种afp-l3的检测试剂、检测试剂盒及其制备方法
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