WO2016107576A1 - 一种甲胎蛋白异质体的分离检测组合物、系统及其应用 - Google Patents

一种甲胎蛋白异质体的分离检测组合物、系统及其应用 Download PDF

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WO2016107576A1
WO2016107576A1 PCT/CN2015/099774 CN2015099774W WO2016107576A1 WO 2016107576 A1 WO2016107576 A1 WO 2016107576A1 CN 2015099774 W CN2015099774 W CN 2015099774W WO 2016107576 A1 WO2016107576 A1 WO 2016107576A1
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alpha
fetoprotein
separation
afp
detection
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French (fr)
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罗海峰
李伯安
林长青
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北京热景生物技术有限公司
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Priority to EP15875252.7A priority Critical patent/EP3242133B1/en
Priority to US15/541,178 priority patent/US10329335B2/en
Priority to JP2017533971A priority patent/JP6655622B2/ja
Publication of WO2016107576A1 publication Critical patent/WO2016107576A1/zh

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/065—Preparation using different phases to separate parts of sample

Definitions

  • the invention relates to an isolated detection composition, system, method and application of alpha-fetoprotein heterogeneity, and also relates to an isolation and detection kit for alpha-fetoprotein heterogeneity, belonging to the field of medical instruments and in-vitro diagnosis.
  • Hepatocellular 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. Its clinical malignancy Higher, mortality ranks third in malignant tumors of the digestive tract, so early detection and early treatment are the key to improve the survival rate of patients with hepatocellular carcinoma, and most patients have reached the middle and late stage of treatment, losing the best treatment opportunity. . Risk factors for hepatocellular carcinoma include chronic hepatitis and cirrhosis caused by hepatitis B virus and hepatitis C virus. For effective treatment, the clinical efficiency of screening and screening for hepatocellular carcinoma depends on early diagnosis.
  • HCC is an important type of tumor etiology. 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.
  • Alpha-fetoprotein is a glycoprotein.
  • the detection of AFP in blood is the most common method for diagnosing hepatocellular carcinoma.
  • AFP is synthesized by hepatic parenchymal cells and yolk sac 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 the serum of newborns at birth can reach 10 ⁇ 100ng / ml, and it will drop to normal adult level (less than 20ng / ml) after one year of birth.
  • AFP is significantly elevated, it indicates neonatal hepatitis, congenital biliary atresia, or an embryonic malignant tumor that secretes AFP.
  • the shortcoming of 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/m1) is found in a considerable number of patients with chronic liver disease. 11.7-44% is positive for AFP. 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.
  • AFP heterogeneity actually refers to AFP-L3 combined with lentil lectin (LCA).
  • LCA lentil lectin
  • AFP-L3 is the only protein produced by cancer cells in the liver of a patient.
  • a method for detecting hepatocellular carcinoma using AFP-L3 was studied in a multicenter, prospective, double-blind, long-term clinical trial in Canada and the United States. The study showed that patients with elevated AFP-L3 (more than 15%) had a 7-fold increased risk of developing hepatocellular carcinoma in the next 21 months. According to the existing Guide to Hepatocellular Carcinoma Oncology, the incidence of hepatocellular carcinoma is extremely high in these patients.
  • AFP hepatocellular carcinoma and benign liver disease.
  • AFP is often elevated in patients with primary hepatocellular carcinoma, but many benign liver diseases may also be associated with elevated AFP. It is sometimes difficult to distinguish between benign liver disease and malignant lesions by AFP alone.
  • AFP heterogeneity detection has a good clinical significance, especially for those with AFP content between 30-400 ng/ml. Sato et al. completed a study of 361 cases (Early recognition of hepatocellular carcinoma based on altered pro les of alpha-fetoprotein. Sato, Y., et al., N. Engl. J.
  • AFP-L3% was tested in 361 patients with chronic hepatitis B and hepatitis C and followed for 35 months. During the study period, 24 (73%) of 33 patients with AFP-L3% above baseline had developed liver cancer. AFP-L3% can be used as an indicator to distinguish liver cancer patients from patients with cirrhosis, and can be used as a predictive indicator 3-18 months ahead of imaging diagnosis.
  • AFP 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 neural tube defects, no brain or spina bifida. Childhood hepatoblastoma, biliary atresia, gonadal tumor, malignant teratoma, etc. may be positive for AFP and/or AFP heterogeneity.
  • the detection methods for AFP heterogeneity include plant lectin affinity chromatography, polyacrylamide gel electrophoresis, affinity blotting, affinity cross-immunoelectrophoresis, etc., and these methods can directly separate AFP-
  • the L3 protein is also quantitatively estimated and can be classified according to the final detection method:
  • Coomassie brilliant blue method The sample after electrophoresis was directly stained with Coomassie brilliant blue, and the peak band was observed after elution.
  • the method is simple, but there are many interference factors, and the sensitivity is about 1000 ng/ml.
  • Enzyme labeling method The antibody labeled with peroxidase was incubated with the electrophoresed sample, and after rinsing, the color was developed with diaminobenzidine, and the detection sensitivity was increased to 50 ng/ml.
  • Gold and silver staining method The sample after electrophoresis 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 ng/ml.
  • Autoradiography It is the most commonly used detection method in China, with a sensitivity of 31 ng/ml.
  • the principle is that the sample is separated and electrophoresed in a gel containing lectin, and then electrophoresed by adding AFP containing iodine 125, followed by electrophoresis in a gel of anti-AFP antibody, and the gel is baked after electrophoresis. Dry, cover X-ray film exposure, rinse imaging.
  • the whole experimental procedure is complicated. For a long time, only a few clinical laboratories in China have been able to measure AFP heteroplasms. A few cities can meet the requirements for clinical determination of AFP heterogenes, and there is no relevant domestic reagent supply.
  • AFP-L3 is mainly isolated by centrifugal tube separation method in China.
  • the method uses agarose-coupled LCA to separate AFP-L3 by centrifugation, and then uses AFP detection reagent for detection.
  • the centrifuge tube separation method is currently the only method approved by the Food and Drug Administration for the AFP-L3 percentage detection.
  • the method for determining the percentage of the existing alpha-fetoprotein heterogeneous body has many manual procedures, complicated steps, requires a large number of supporting equipment, takes a long time, and cannot be automated, and therefore, high-throughput sample detection cannot be achieved, and It is greatly affected by manual operation and will eventually cause deviations in the test results.
  • the present invention provides an isolated detection composition, system, method and application of alpha-fetoprotein heterogeneity, by detecting alpha-fetoprotein heterogene content and alpha-fetoprotein content in a blood sample, The percentage of alpha-fetoprotein heterogenes was obtained.
  • the present invention provides an isolated detection composition of alpha-fetoprotein heterogeneous AFP-L3, comprising a separation reagent and a detection reagent, the separation reagent comprising a magnetic particle and an eluent coupled to a lectin, the coupling agglutination
  • the magnetic particles are used to specifically bind to AFP-L3 in the sample to be detected;
  • the detection reagent includes magnetic particles coated with an antibody against alpha-fetoprotein and an enzyme-labeled anti-alphafetoprotein antibody.
  • the anti-alphafetoprotein antibody is a monoclonal antibody against alpha-fetoprotein.
  • the anti-alphafetoprotein antibody is an anti-alphafetoprotein monoclonal antibody manufactured by Sigma Corporation under the number A8452, or an anti-alphafetoprotein antibody manufactured by Sigma Corporation under the accession number HPA023600.
  • the separation reagent further comprises a protective solution for increasing the stability of the separation reagent and improving the separation efficiency.
  • the protective solution is 0.02 M PBS, 0.5% BSA, pH 7.4 and 0.1 M D-mannosidic.
  • the detection reagent further comprises a buffer for increasing the stability of the detection reagent, improving detection sensitivity, and detecting specificity.
  • the buffer is 0.02 M PBS, 10% calf serum, 0.1% proclin-300.
  • the separation reagent and/or detection reagent further comprises a cleaning solution for increasing binding efficiency, reducing non-specific adsorption, and improving sensitivity of separation and detection.
  • the separation reagent washing solution is 20 mM Tris-HCl and 0.5 MD-mannosin
  • the detection reagent cleaning solution is a 1% Tween 20 solution prepared in PBS pH 7.4.
  • the lectin in the lectin-coupled magnetic particles, comprises lentil lectin and/or concanava lectin.
  • the polymer component covered by the surface of the magnetic particle comprises a silicide, a polysaccharide, a protein, a cellulose or a resin.
  • the separation detection composition is provided in a cassette or strip.
  • the present invention further provides an isolated detection kit for alpha-fetoprotein heteroplasm, comprising the above-described separation detection composition.
  • the invention still further provides a separation and detection system for alpha-fetoprotein heterogeneous bodies, comprising:
  • a magnetic separation module for separating magnetic particles from a liquid, the magnetic separation module being used with a separation reagent and a detection reagent;
  • a detection module for detecting alpha-fetoprotein heterogene and alpha-fetoprotein content
  • a data processing module for calculating the ratio of alpha-fetoprotein heterogeneous to alpha-fetoprotein
  • the magnetic separation module cooperates with the separation reagent in the separation detection composition to complete the separation of the alpha-fetoprotein heterogeneity
  • the detection module cooperates with the detection reagent in the separation detection composition to complete the detection of the alpha-fetoprotein heterogene content and the alpha-fetoprotein content.
  • the magnetic separation module when the alpha-fetoprotein content is selected for determination, does not cooperate with a separation reagent that detects the alpha-fetoprotein content of the sample to be tested,
  • the magnetic separation module cooperates with the separation reagent to separate the alpha-fetoprotein heterogene in the sample to be detected, and the detection module detects the alpha-fetoprotein heterogene in the sample to be detected.
  • the magnetic separation module separates the alpha-fetoprotein heterogene in the sample to be detected, and the detection module detects the alpha-fetoprotein heterogeneous content in the sample to be detected, respectively. And alpha fetoprotein content, the data processing module calculates the percentage of alpha-fetoprotein heterogenes, ie the percentage of alpha-fetoprotein heterogene (AFP-L3) in total alpha-fetoprotein (AFP) ( AFP-L3%).
  • the present invention finally provides a method for separating and detecting alpha-fetoprotein heterogeneous bodies, and a separation detection group as described above
  • the method of isolating the alpha-fetoprotein heterogeneity comprises the steps of:
  • the alpha-fetoprotein heterogeneous AFP-L3 is isolated from the alpha-fetoprotein by an isolation method, and the isolated alpha-fetoprotein heterogeneous AFP-L3 is detected, wherein the separation method is mainly by labeling a lectin
  • the magnetic particles are bound to the alpha-fetoprotein heterogeneous AFP-L3 in the sample; then the isolated alpha-fetoprotein heterogeneous AFP-L3 is obtained by magnetic particle separation and corresponding elution method, and an immunoassay method for AFP is adopted. AFP detection was performed on the separated samples, and the obtained AFP result was the result of AFP-L3.
  • the magnetic particles labeled with lectin are agarose-coated magnetic particles labeled with lentil lectin (LCA), and can be combined with alpha-fetoprotein heterogeneous agarose-coated paramagnetic particles. .
  • LCA lentil lectin
  • the immunodetection method for AFP is a magnetic particle chemiluminescence method.
  • the invention has the beneficial effects that the operator only needs to add a simple operation such as a sample, and the detection can be completed within 30 minutes; at the same time, the method can quantitatively calculate the content contained in the blood sample by directly detecting the sample.
  • Figure 1 is a schematic view showing the separation detection composition of the alpha-fetoprotein heterogeneous body of the present invention in a card form
  • 1 - sample well 1 - sample well, 2 - reagent tank containing magnetic particles coupled with lectin, 3 - reagent tank containing eluent, 4 - reagent tank containing magnetic particles coated with anti-alpha-fetoprotein antibody , 5 - reagent tank containing enzyme-labeled anti-alpha-fetoprotein antibody, 6-reaction well, 7-reagent card;
  • Figure 2 is a schematic illustration of the separation detection system for alpha-fetoprotein heterogenes of the present invention.
  • Figure 3 is a schematic illustration of one embodiment of a separation detection system for alpha-fetoprotein heterogenes of the present invention.
  • the separation detection composition of the alpha-fetoprotein heterogeneous body comprises a separation reagent and a detection reagent for detecting the percentage of alpha-fetoprotein heterogeneous bodies.
  • the separation reagent includes a magnetic particle and an eluate coupled to the lectin, and the magnetic particle coupled to the lectin is specifically used for binding to AFP-L3 in the sample to be detected; the detection reagent includes the magnetic particle coated with the anti-alphafetoprotein antibody And enzyme-labeled anti-alpha-fetoprotein antibodies.
  • the main components and preparation methods of the above reagents are as follows:
  • a magnetic particle coupled to a lectin wherein the lectin is lentils lectin (LCA), concanavalin or lentil lectin, and concanavalin.
  • the lectin is lentils lectin (LCA), concanavalin or lentil lectin, and concanavalin.
  • the magnetic particles were activated with an iron oxide-coated triiron tetroxide superparamagnetic nanoparticle (hereinafter referred to as magnetic particles) and an lentil lectin (LCA) derived from Sigma.
  • magnetic particles iron oxide-coated triiron tetroxide superparamagnetic nanoparticle
  • LCA lentil lectin
  • Lectin and magnetic particles are coupled using the following steps:
  • the magnetic particles coated with the epoxy resin may also be replaced by magnetic particles coated with materials such as titanium silicide, polystyrene, dextran, agarose, sulfonamide resin, bovine serum albumin, biotin and the like.
  • the alpha-fetoprotein antibody is an anti-alphafetoprotein monoclonal antibody (anti-AFP-1), which is an anti-alpha-fetoprotein monoclonal antibody from A Sigma, and is prepared as follows:
  • the main component is 0.02M PBS, 0.5% Tween-20, 100 mM NaCl, repeated washing four times;
  • the anti-alphafetoprotein antibody No. HPA023600 from Sigma was coupled to anti-alpha-fetoprotein antibody 2 (anti-AFP-2) using peroxidase (HRP) from Sigma.
  • the preparation method is as follows:
  • the mixed solution was placed at 4 ° C and allowed to stand for 30 minutes;
  • anti-alpha-fetoprotein antibody 2 (anti-AFP-2) (protected from light):
  • Anti-AFP-2 (anti-AFP-2) and HRP were mixed 1:4, dialyzed in 50mmol/L pH9.5CB for more than 6 hours, and changed for the first two hours;
  • AFP-L3 cleaning solution 0.02M PBS (pH7.0), 0.5% Tween-20;
  • Magnets are adsorbed at the bottom of the tube by a magnet, and then the supernatant is taken up and discarded;
  • AFP-L3 eluate 0.02 M PBS (pH 7.0), 5MD-mannose glycoside,
  • Magnetic particles were adsorbed at the bottom of the tube by a magnet to obtain a supernatant, which was a alpha-fetoprotein heterogeneous AFP-L3 separation liquid.
  • AFP cleaning solution 0.02M PBS (pH7.0), 0.5% Tween-20;
  • the intensity of the luminescent signal was measured on a chemiluminescence measuring instrument, and the content of AFP in the sample was calculated by a standard curve.
  • This embodiment performs the AFP-L3 detection step:
  • AFP-L3 cleaning solution 0.02M PBS (pH7.0), 0.5% Tween-20;
  • Magnets are adsorbed at the bottom of the tube by a magnet, and then the supernatant is taken up and discarded;
  • AFP-L3 eluate 0.02 M PBS (pH 7.0), 5MD-mannose glycoside,
  • Magnetic particles were adsorbed at the bottom of the tube by a magnet to obtain a supernatant, which was a alpha-fetoprotein heterogeneous AFP-L3 separation liquid.
  • AFP cleaning solution 0.02M PBS (pH7.0), 0.5% Tween-20;
  • AFP-L3% The percentage of alpha-fetoprotein heterogeneous body (AFP-L3%) was obtained by dividing the content of alpha-fetoprotein heterogeneous AFP-L3 calculated in (2) by the alpha-fetoprotein AFP content.
  • the AFP content was detected by using the separation detecting composition described in the present example:
  • test results showed that the sensitivity and specificity of the present example reached 100% among the 452 samples tested.
  • the AFP-L3 percentage detection was performed using the separation detection composition described in this example:
  • the alpha-fetoprotein heterogeneous separation tube produced by Beijing Thermal Biotechnology Co., Ltd., product registration certificate number: National Food and Drug Administration (quasi) from 2014 No. 3401646, with Roche AFP detection kit (electrochemiluminescence method)
  • the accuracy of the AFP-L3 percentage test results was compared.
  • the cutoff value of the AFP-L3 percentage is 10%.
  • Table 2 The test results are shown in Table 2:
  • control groups were tested as AFP-L3 percentage positive samples, this example also detected The percentage of AFP-L3 was positive; 172 samples were tested as AFP-L3 percentage negative samples, 169 were negative in this example, and the other 3 samples were clinically diagnosed and found to be early primary hepatocellular carcinoma. patient.
  • the test results showed that the separation detection composition described in the present example was more sensitive than the detection performance of the control group. The reason should be that the separation efficiency of AFP-L3 protein is improved, and the percentage of AFP-L3 in the sample can be more accurately detected.
  • the separation detecting composition of the alpha-fetoprotein heterogeneous body provided by the present invention may further include a protective liquid.
  • the detection reagent may also include a buffer.
  • the separation reagent and the detection reagent may each include a cleaning solution.
  • Buffer 0.02 M PBS, 10% calf serum, 0.1% proclin-300,
  • Separation reagent cleaning solution 20 mM Tris-HCl, 0.5 MD-mannose,
  • Detection reagent cleaning solution 1% Tween 20 solution prepared in PBS pH 7.4.
  • the above D-mannosidic acid can be replaced by a saccharide such as fucose, fructose, sucrose or trehalose.
  • the invention provides an separation detection system for alpha-fetoprotein heteroplasms for detecting the percentage of alpha-fetoprotein heterogeneous bodies, as shown in FIG. 2 .
  • a magnetic separation module for isolating alpha-fetoprotein heterogenes a detection module for detecting alpha-fetoprotein heterogene content and alpha-fetoprotein content, for calculating a ratio of alpha-fetoprotein heterogene to alpha-fetoprotein Data processing module.
  • the detection and separation system may further comprise a sampling module, and the data processing module may further comprise an optical signal reading device, preferably a photon counting probe manufactured by Hamamatsu Photonics Co., Ltd., model H10682-110, or a photomultiplier tube, Model R1166.
  • the reagent card includes the separation detecting composition described in Example 1, and the separation detecting composition is pre-packaged in a plurality of reagent tanks on the reagent card for performing a reaction, and each reagent has at least one reagent tank.
  • the reagent card includes a sample well, a separation reagent tank, a detection reagent tank and a reaction well.
  • the reagent card of this embodiment as shown in FIG. 3, has the following composition:
  • the pore positions b, c, and d together constitute a magnetic separation module, in which all reagents can be used to complete the separation of the alpha-fetoprotein heterogeneity; the pore positions e, f, g, h, i together constitute a detection module. All of these reagents can be used The detection of the AFP concentration in the added sample is completed.
  • the detection module can be used alone to determine the AFP content in the sample; the separation module and the detection module cooperate to complete the separation of the alpha-fetoprotein heterogeneous AFP-L3 in the sample, and determine the content of AFP-L3; Subsequent to the division of AFP-L3 content and AFP content, the proportion of alpha-fetoprotein heterogenes was obtained.
  • the experimental results show that the positive rate of detection of primary hepatocellular carcinoma in the present invention is 92%, the specificity for healthy people is 100%, and the specificity for liver cirrhosis and hepatitis is 95% and 97%, respectively, for other cancers. The specificity reached 0%.
  • the separation detection system for alpha-fetoprotein heterogenes further comprising a detection setting module comprising a alpha-fetoprotein content determination unit, a alpha-fetoprotein heterogene content determination unit, and alpha-fetoprotein a unit for determining the percentage of plastids,
  • the magnetic separation module does not participate in processing the sample to be detected, and the detecting device detects the alpha-fetoprotein content of the sample to be detected,
  • the magnetic separation module separates the alpha-fetoprotein heterogene in the sample to be detected, and the detecting module detects the content of the alpha-fetoprotein heterogene in the sample to be detected;
  • the magnetic separation module separates the alpha-fetoprotein heterogene in the sample to be detected when the measurement unit of the percentage of the alpha-fetoprotein heterogeneity is selected, and the detection module detects the alpha-fetoprotein heterogene in the sample to be detected.
  • the content and alpha-fetoprotein content the data processing module calculates the percentage of alpha-fetoprotein heterogeneity.
  • Example 3 According to the separation detection system for alpha-fetoprotein heterogenes provided in Example 3 or 4, the fractions described in Example 2 were used.
  • the separation detecting composition described in the first embodiment was replaced by the detection composition.
  • the method for separating and detecting alpha-fetoprotein heterogeneity adopts the above separation detection system for detecting the percentage of alpha-fetoprotein heterogeneous body, including the following steps,
  • the alpha-fetoprotein heterogeneous eluate was added to a cuvette containing magnetic particles coated with alpha-fetoprotein monoclonal antibody, and an enzyme-labeled anti-alpha-fetoprotein antibody was added thereto and incubated.
  • enrichment and cleaning enrich the magnetic particles with a magnetic separation module, discard the liquid, and add the washing liquid in the reaction cup to mix, and then enrich the magnetic particles with the magnetic separation module, and discard the liquid;
  • the method for separating and detecting alpha-fetoprotein heterogeneity adopts the above separation detection system for detecting the percentage of alpha-fetoprotein heterogeneous body, including the following steps,
  • the magnetic separation module enriches the magnetic particles and discards the liquid
  • the washed magnetic particles are added to the reagent tank containing the eluent, mixed, and the magnetic particles are enriched by the magnetic separation module to obtain an alpha-fetoprotein heterogeneous eluate;
  • the alpha-fetoprotein heterogeneous eluate was added to a reagent tank containing magnetic particles coated with alpha-fetoprotein monoclonal antibody, and an anti-alpha-fetoprotein antibody pre-loaded in another reagent tank was added and incubated.
  • the magnetic separation module enriches the magnetic particles and discards the liquid
  • the system adds the sample to the reagent tank containing the magnetic particles coated with the alpha-fetoprotein monoclonal antibody, and repeats steps (3) to (6) to determine the concentration of alpha-fetoprotein;
  • Logical algorithm for the percentage of data processing module to alpha-fetoprotein heterogeneity judged by sample number, the instrument will retrieve the alpha-fetoprotein concentration and alpha-fetoprotein heterogene concentration for the same sample, then calculate AFP - The percentage of L3 in the AFP, thereby calculating the AFP-L3 content, ie AFP-L3%.

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Abstract

一种甲胎蛋白异质体的分离检测组合物，包括分离试剂和检测试剂，以及甲胎蛋白异质体的分离检测系统和应用，甲胎蛋白异质体的分离检测试剂盒。通过甲胎蛋白异质体的分离检测组合物、系统及其应用，可以提示早期原发性肝细胞癌，具有极高的灵敏度，方法快速简便而且自动化。

Description

一种甲胎蛋白异质体的分离检测组合物、系统及其应用 技术领域
本发明涉及一种甲胎蛋白异质体的分离检测组合物、系统、方法及应用，同时还涉及了一种甲胎蛋白异质体的分离检测试剂盒，属于医疗器械及体外诊断领域。
背景技术
肝细胞癌(hepatocellular carcinoma，HCC)是世界范围内第四位常见的恶性肿瘤，每年大约有250000名患者死于肝细胞癌，肝细胞癌的发病机制至今仍不清楚，其在临床上恶性度较高，死亡率在消化道恶性肿瘤中排第三位，所以早期发现和早期治疗是提高肝细胞癌患者生存率的关键，而大多数患者就诊时已届中晚期，失去了最佳治疗时机。肝细胞癌的危险因素包括由乙型肝炎病毒和丙型肝炎病毒导致的慢性肝炎和肝硬化。要得到有效治疗，肝细胞癌的筛查和普查的临床效率依赖于早期诊断。HCC是肿瘤病因学中的重要类型，肝硬化、病毒性肝炎、化学致癌物及环境因素等所造成的慢性肝脏损害都可诱发HCC。HCC恶性度高，容易复发及转移，预后差，而且早期诊断较困难，易于延误最佳治疗期。
甲胎蛋白(alphafetoprotein，AFP)是一种糖蛋白，检测血液中的AFP含量是目前诊断肝细胞癌最常见的手段。AFP在哺乳动物胚胎期由肝脏实质细胞和卵黄囊细胞合成，来源于内胚层的胃肠道粘膜细胞也可少量合成。正常情况下，AFP主要存在于胎儿循环中，随着胎儿发育成熟，AFP合成逐渐减少。出生时新生儿血清中AFP浓度可达10～100ng/ml，出生一年后降至正常成人水平(小于20ng/ml)。如果新生儿AFP明显升高，则表明患有新生儿肝炎、先天性胆道闭锁或能分泌AFP的胚胎性恶性肿瘤。AFP作为早期肝癌诊断指标的缺点是在肝病和肝硬化疾病中也有相当的比例存在阳性结果，AFP轻度增高(20～200ng/m1)见于相当数量的慢性肝病患者中，在肝硬化患者中有11.7-44％为AFP阳性。因此在分辨良性肝病和恶性肝肿瘤时，AFP作为早期肝癌筛查指标的价值大为降低。
AFP异质体的许多糖链的结构目前尚不完全清楚。目前认为,通常所说的AFP异质体实际是指与小扁豆凝集素(LCA)结合的AFP-L3。1999年的第四届全国肝癌学术会议将AFP-L3列为原发性肝细胞癌临床诊断标准的肝细胞癌标记物之一。2011年的《中国肝癌诊疗规范》将AFP-L3列为肝细胞癌诊断的特异性指标。多年来，AFP-L3被公认为是比单纯的AFP甲胎蛋白更为特异性的原发性肝细胞癌指标。
AFP-L3是唯一一种由患者肝脏中的癌细胞生成的蛋白。在加拿大和美国的一项多中心、前瞻性、双盲且长期临床试验中，对使用AFP-L3检测肝细胞癌的方法进行了研究。研究结果显示AFP-L3升高(15％以上)的患者，在接下来的21个月中患上肝细胞癌的危险增加7倍之多。根据已有的《肝细胞癌肿瘤学实践指南》，这些患者中肝细胞癌发生率极端增高。
AFP异质体检测的临床意义：
1)鉴别肝细胞癌与良性肝病。原发性肝细胞癌患者中AFP常升高，但许多良性肝脏疾病也可伴有AFP升高，单凭AFP检测结果有时很难区分良性肝病和恶性病变。此时AFP异质体检测就具有良好的临床意义，尤其对于AFP含量在30-400ng/ml之间者具有较好的价值。Sato等人完成了361例病例的前瞻性研究(Early recognition of hepatocellular carcinoma based on altered pro les of alpha-fetoprotein.Sato,Y.,et al.,N.Engl.J.Med.,328,1802-1806,1993)，对于361位罹患慢性乙型肝炎及丙型肝炎的患者进行了AFP-L3％的检测并且进行了为期35个月的追踪。在研究期间33例AFP-L3％高于基准的患者中有24位(73％)发展为肝癌。AFP-L3％可以作为将肝癌患者从肝硬化患者中区分的指标，并且可以作为比影像学诊断提前3-18个月的一种预测性指标。
2)肝细胞癌术后的监测。肝细胞癌切除术后，血清AFP含量随之下降，其下降速度取决于体内残留的AFP量及半衰期，一般2月内转阴，转阴时AFP异质体随之消失。如果AFP明显下降但不转阴，异质体变化不明显，则提示手术不彻底，可能还存在边缘残留、血管癌栓、卫星结节或转移等。如果AFP总含量中AFP异质体所占的百分比下降至25％以下，AFP和异质体浓度相对恒定，则可能是患者有肝炎或肝硬化所致。
3)胚胎异常发育及胎儿先天性疾患。正常妊娠期母体血清中AFP与胚胎中AFP处于平衡状态，一旦胎儿畸形或胎盘屏障发生异常，可导致胎儿血清渗入羊水中或羊水渗入母体血清，造成母体羊水或血清AFP急剧升高。但仅仅测定AFP总量有一定的局限性。实验表明神经管缺损、无脑儿或脊柱裂等。儿童肝母细胞瘤、胆道闭锁、性腺肿瘤、恶性畸胎瘤等可有AFP和/或AFP异质体的阳性。
目前用于AFP异质体的检测方法有植物凝集素亲和层析法、聚丙烯酰胺凝胶电泳法、亲和印迹法、亲和交叉免疫电泳法等，这些方法均可以直接分离出AFP-L3蛋白并且进行定量估算，依据最终检测方法可以分为：
考马斯亮兰法：电泳后的样本直接用考马斯亮兰染色,洗脱后观察峰带。方法简单，但干扰因素多，灵敏度约1000ng/ml。
酶标法：用过氧化物酶标记的抗体与电泳后的样本一起温育、漂洗后用二氨基联苯胺显色，检测灵敏度可提高至50ng/ml。
金银染色法:将电泳后的样本直接与金黄色葡萄球菌蛋白A2胶金温育,再用银显色液显色,得到的峰带清晰,灵敏度可达32ng/ml。
放射自显影法:是我国迄今为止最常用的检测方法,灵敏度达31ng/ml。其原理是将样本在含凝集素的凝胶中进行分离电泳,后在添加含碘125标记的AFP进行电泳，后加入抗AFP抗体的凝胶中进行二次电泳,电泳结束后将凝胶烘干,覆盖X线胶片爆光,冲洗成像。整个实验过程操作复杂，长期以来，国内只有少数临床实验室能够测定AFP异质体，少数城市能满足临床测定AFP异质体的要求，并且没有相关国产试剂供应。
另外目前国内主要采用离心管分离方法分离AFP-L3，该方法采用琼脂糖偶联LCA，采用离心方法分离AFP-L3，然后再用AFP检测试剂进行检测。离心管分离方法是目前国内唯一获得药监局批准适用于AFP-L3百分比检测的方法。
现有的甲胎蛋白异质体所占的百分比的测定法，手工操作程序多，步骤繁琐，需要配套设备多，耗时长，且不能实现自动化，因此，无法实现高通量的样本检测，并且受到人工操作影响较大，最终会引起检测结果的偏差。
发明内容
为了克服现有技术的不足，本发明提供了一种甲胎蛋白异质体的分离检测组合物、系统、方法及应用，通过检测血液样本中甲胎蛋白异质体含量与甲胎蛋白含量，得到甲胎蛋白异质体百分比。
本发明提供的一种甲胎蛋白异质体AFP-L3的分离检测组合物，包括分离试剂和检测试剂，所述分离试剂包括偶联凝集素的磁性颗粒和洗脱液，所述偶联凝集素的磁性颗粒用于与待检测样品中的AFP-L3特异性结合；所述检测试剂包括包被抗甲胎蛋白的抗体的磁性颗粒和经酶标记的抗甲胎蛋白抗体。
在一个优选的实施方案中，所述抗甲胎蛋白的抗体为针对甲胎蛋白的单克隆抗体。在一个更优选的实施方案中，所述抗甲胎蛋白的抗体为：Sigma公司生产的货号为A8452的抗甲胎蛋白单克隆抗体，或者Sigma公司生产的货号为HPA023600的抗甲胎蛋白抗体。
在一个优选的实施方案中，所述分离试剂还包括保护液,用于增加分离试剂的稳定性及提高分离效率。优选地，所述保护液为0.02M PBS，0.5％BSA，pH7.4和0.1M D-甘露糖苷。
在一个优选的实施方案中，所述检测试剂还包括缓冲液，用于增加检测试剂的稳定性、提高检测灵敏度以及检测特异性。优选地，所述缓冲液为0.02M PBS，10％小牛血清，0.1％proclin-300。
在一个优选的实施方案中，所述分离试剂和/或检测试剂还包括清洗液，用于提高结合效率、降低非特异性吸附、提高分离和检测的灵敏度。优选地，所述分离试剂清洗液为20mM Tris-HCl和0.5MD-甘露糖苷，所述检测试剂清洗液为PBSpH7.4配制的1％吐温20溶液。
在一个优选的实施方案中，所述偶联凝集素的磁性颗粒中，所述凝集素包括小扁豆凝集素和/或刀豆凝集素。
在一个优选的实施方案中，所述偶联凝集素的磁性颗粒中，所述磁性颗粒表面覆盖的高分子成分包括硅化物、多聚糖、蛋白、纤维素或树脂。
在一个优选的实施方案中，所述分离检测组合物是以卡式或条式提供。
本发明进一步提供了一种甲胎蛋白异质体的分离检测试剂盒，包括上述分离检测组合物。
本发明又进一步提供了一种甲胎蛋白异质体的分离检测系统，包括：
磁分离模块，用于自液体中分离磁颗粒，磁分离模块配合分离试剂和检测试剂使用；
检测模块，用于检测甲胎蛋白异质体和甲胎蛋白含量；
数据处理模块，用于计算甲胎蛋白异质体相对于甲胎蛋白比值和
任一种的上述分离检测组合物或上述试剂盒。
所述磁分离模块与所述分离检测组合物中的分离试剂相配合，用于完成对甲胎蛋白异质体的分离；
所述检测模块与所述分离检测组合物中的检测试剂相配合，用于完成对甲胎蛋白异质体的含量和甲胎蛋白含量的检测。
在一个优选的实施方案中，当选择测定甲胎蛋白含量时，所述磁分离模块不与分离试剂相配合，所述检测装置检测待检测样品中甲胎蛋白的含量，
当选择测定甲胎蛋白异质体含量时，所述磁分离模块与分离试剂相配合来分离待检测样品中甲胎蛋白异质体，所述检测模块检测待检测样品中甲胎蛋白异质体的含量；
当选择测定甲胎蛋白异质体所占的百分比时，所述磁分离模块分离待检测样品中甲胎蛋白异质体，所述检测模块分别检测待检测样品中甲胎蛋白异质体的含量和甲胎蛋白的含量，所述数据处理模块计算甲胎蛋白异质体所占的百分比，即甲胎蛋白异质体(AFP-L3)在总甲胎蛋白(AFP)中所占的百分比(AFP-L3％)。
本发明最后提供了一种分离检测甲胎蛋白异质体的方法，以及一种上述分离检测组 合物或上述分离检测系统在分离检测甲胎蛋白异质体中的应用。
在一个优选的实施方案中，所述分离检测甲胎蛋白异质体的方法，包括如下步骤：
通过分离方法将甲胎蛋白异质体AFP-L3从甲胎蛋白中分离出来，并且对于分离出的甲胎蛋白异质体AFP-L3进行检测，其中所述分离方法主要是通过标记有凝集素的磁性颗粒与样本中的甲胎蛋白异质体AFP-L3结合；然后通过磁性颗粒分离及相应洗脱方法得到分离出的甲胎蛋白异质体AFP-L3，并且采用针对AFP的免疫检测方法对于分离出的样本进行AFP的检测，得到的AFP结果就是AFP-L3的结果。
在一个优选的技术方案中，所述标记有凝集素的磁性颗粒为标记小扁豆凝集素(LCA)的琼脂糖包裹的磁性颗粒，可以结合甲胎蛋白异质体的琼脂糖包裹的顺磁颗粒。
在一个优选的实施方案中，所述的针对AFP的免疫检测方法为磁颗粒化学发光方法。
与现有技术相比，本发明的有益效果是：操作者只需要加入样本等简单操作，在30分钟内就可以完成检测；同时该方法可以直接通过检测而定量计算出血样样本中所含的甲胎蛋白和甲胎蛋白异质体的含量，并同时获得AFP-L3％；方法简便，检测快捷，结果准确，灵敏度高，且自动化，为肝癌的预防、诊断、治疗提供了支持。
附图说明
图1是本发明的甲胎蛋白异质体的分离检测组合物以卡式提供示意图，
其中，1—样本孔，2—装有偶联凝集素的磁性颗粒的试剂槽，3—装有洗脱液的试剂槽，4—装有包被抗甲胎蛋白抗体的磁性颗粒的试剂槽，5—装有经酶标记的抗甲胎蛋白抗体的试剂槽，6—反应孔，7—试剂卡；
图2是本发明的甲胎蛋白异质体的分离检测系统的示意图。
图3是本发明的甲胎蛋白异质体的的分离检测系统的一个具体实施方案的示意图。其中，1—样本孔；2—预分装的偶联凝集素的磁性颗粒的试剂槽；3—预分装AFP-L3清洗液的试剂槽；4—预分装AFP-L3洗脱液的试剂槽；5—预分装的包被抗甲胎蛋白抗体1(anti-AFP-1)的磁性颗粒的试剂槽；6—反应孔；7—预分装的标记酶的抗甲胎蛋白抗体(anti-AFP-2)的试剂槽；8—预分装AFP清洗液的试剂槽；9—预分装鲁米诺底物的试剂槽；10—试剂卡。
具体实施方式
以下结合附图和实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明的范围。
实施例1
本发明提供的甲胎蛋白异质体的分离检测组合物，包括分离试剂和检测试剂，用于检测甲胎蛋白异质体所占的百分比。
分离试剂包括偶联凝集素的磁性颗粒和洗脱液，偶联凝集素的磁性颗粒用于与待检测样品中的AFP-L3特异性结合；检测试剂包括抗甲胎蛋白抗体包被的磁性颗粒和酶标记的抗甲胎蛋白抗体。上述试剂的主要组分及制备方法如下：
偶联凝集素的磁性颗粒，其中所述凝集素采用小扁豆凝集素(LCA)、刀豆凝集素或小扁豆凝集素和刀豆凝集素。
磁性颗粒采用活化的包裹有环氧树脂的大小为1微米的四氧化三铁超顺磁纳米颗粒(以下简称磁性颗粒)和来源自Sigma公司的小扁豆凝集素(LCA)。
1.凝集素与磁性颗粒采用以下步骤偶联：
(1)称取2mgLCA，溶于7.5mL偶联缓冲液(0.1mmol/L NaHCO3，pH8.3，0.5mol/L NaCl)，与经洗涤的1.5g磁性颗粒合并，以带塞的10mL试管上下颠倒混匀(室温下混合2h)；
(2)以10mL偶联缓冲液洗去未偶联的LCA。经测定洗涤液中的LCA含量，计得偶联率为98％；
(3)用0.2mol/L甘氨酸封闭剩余活化基因；
(4)依次用10mL 0.1mol/L醋酸缓冲液(pH 4，含0.5mol/L NaCl)和0.1mol/L Tris缓冲液(pH 8，含0.5mol/L NaCl)洗涤3次，再以含0.1％BSA和0.1mmol/L CaCl2的PBS(PBS-BSA)洗涤1次，4℃暂存备用。
上述包裹有环氧树脂的磁性颗粒还可以采用包裹有硅化钛、聚苯乙烯、葡聚糖、琼脂糖、磺胺树脂、牛血清白蛋白、生物素等材料的磁性颗粒替代。
2.包被甲胎蛋白抗体的磁性颗粒：
其中，甲胎蛋白抗体采用来自于Sigma公司的货号为A8452的抗甲胎蛋白单克隆抗体作为抗甲胎蛋白抗体1(anti-AFP-1)，其制备方法如下：
(1)磁性颗粒的活化
a.吸取磁性颗粒50ml(10％W/V)；
b.等体积50mM MES缓冲液洗涤磁性颗粒；
c.用等体积100mM MES缓冲液重悬磁性颗粒；
d.加入活化试剂碳化二亚胺(EDC)，终浓度0.04g/ml；
e.室温震荡活化1h
(2)抗甲胎蛋白抗体1(anti-AFP-1)与磁性颗粒的包被
a.活化结束，施加磁场并弃去上清液；
b. 10倍体积50mM MES洗涤活化后磁性颗粒；
c.加入0.2mg抗甲胎蛋白抗体1
d.室温震荡反应3h；
(3)抗甲胎蛋白抗体1(anti-AFP-1)与磁性颗粒包被的终止
a.反应结束，施加磁场并弃去上清液；
b.加入10倍体及包被终止液；
c.常温震荡反应3h
(4)抗甲胎蛋白抗体1(anti-AFP-1)磁性颗粒的清洗保存
a.反应结束，加磁场弃去上清液；
b.加入10倍体积的包被清洗液，其主要组成成分为0.02M PBS，0.5％吐温-20，100mM NaCl，重复清洗四次；
c.10倍体积的磁性颗粒保存液(pH7.4，0.02M PBS,0.5％BSA,2％蔗糖，0.2％吐温-20，0.2％PC-300)保存抗甲胎蛋白抗体1(anti-AFP-1)磁性颗粒
3.经酶标记的抗甲胎蛋白抗体：
采用来自于Sigma公司的过氧化物酶(HRP)偶联来自于Sigma公司的货号为HPA023600的抗甲胎蛋白抗体作为抗甲胎蛋白抗体2(anti-AFP-2)，制备方法如下：
(1)酶的氧化(全过程避光)：
a.称取HRP 5mg，加ddH2O 250μl溶解；
b.称取NaIO45mg，加ddH2O 250μl溶解，配制成20mg/mL的浓度；
c.往HRP溶液中逐滴加入NaIO4溶液，边加边搅拌；
d.将混合好的溶液置于4℃，静置30分钟；
e.取5ml乙二醇溶于25μl ddH2O中，逐滴加入上述混合溶液中，边加边搅拌；
f.室温静置30分钟；
g.酶氧化过程完成，HRP终浓度为10mg/ml。
(2)抗甲胎蛋白抗体2(anti-AFP-2)的准备及标记(避光)：
a.调整抗甲胎蛋白抗体浓度到5mg/ml左右(蛋白浓度过低则用PEG20000浓缩)，用pH9.5左右的50mmol/L CB(1mol/L NaHCO3与1mol/L Na2CO3按10:1的比例混合，使用前以蒸馏水稀释20倍)透析去甘油或杂质(如Tris)，4℃下透析过夜，其中换液3次；
b.将抗甲胎蛋白抗体2(anti-AFP-2)与HRP按1：4混合，于50mmol/L pH9.5CB中透析6小时以上，前两个小时换液一次；
c.用新鲜配置的5mg/mlNaBH4溶液0.2ml终止反应。摇匀，4℃静置2小时，每半小时摇一次；
d.用pH7.2的10mM PBS(预配置0.01mol/L的Na2HPO4和NaH2PO4储备液，根据需要的pH值混匀二者成PBS缓冲液)透析过夜。换液一次即可。
(3)分装：用含10％胎牛血清的缓冲液，主要成分为0.02M PBS，10％胎牛血清，3％蔗糖，0.2％吐温-20等，稀释由步骤(2)获得的HRP酶标抗记的甲胎蛋白抗体2(anti-AFP-2)至1mg/ml(以初始抗体浓度计算)，分装并贮存于4℃。
4.使用本实施例中偶联了凝集素的磁性颗粒进行甲胎蛋白异质体分离步骤：
(1)将50ul 1％浓度的偶联凝集素的磁性颗粒与200ul待检测血清样本加入同一管中，充分混匀，并且静置5分钟；
(2)采用磁铁在管子底部吸附磁性颗粒，然后吸取上清，弃去；
(3)移去磁铁，在管子中加入AFP-L3清洗液500ul，充分混匀，确保磁性颗粒完全悬浮；
AFP-L3清洗液：0.02M PBS(pH7.0)，0.5％吐温-20；
(4)采用磁铁在管子底部吸附磁性颗粒，然后吸取上清，弃去；
(5)重复步骤(3)、(4)2次，进行磁性颗粒清洗；
(6)移去磁铁，在管子中加入AFP-L3洗脱液200ul，充分混匀，确保磁性颗粒完全悬浮，静置10分钟；
AFP-L3洗脱液：0.02M PBS(pH7.0)，5MD-甘露糖苷，
或20mmTris-Hcl,NaCl 150mm,pH7.4缓冲液，其中含有500mmα-甲基-D-甘露糖苷,0.1％Proclin 300；
(7)采用磁铁在管子底部吸附磁性颗粒，得到上清，该上清为甲胎蛋白异质体AFP-L3分离液体。
5.使用本实施例中包被抗甲胎蛋白抗体1(anti-AFP-1)的磁性颗粒与标记HRP酶的抗甲胎蛋白抗体2(anti-AFP-2)进行AFP检测步骤：
(1)在检测管中加入50ul 1％浓度的包被抗甲胎蛋白抗体1(anti-AFP-1)的磁性颗粒，同时加入50ul待检测样本，随后加入50ul 1000倍稀释的标记HRP酶的抗甲胎蛋白抗体2(anti-AFP-2)，充分混匀，在37℃环境下孵育5分钟；
(2)采用磁铁在管子底部吸附磁性颗粒，然后吸取上清，弃去；
(3)移去磁铁，在管子中加入AFP清洗液500ul，充分混匀，确保磁性颗粒完全悬 浮；
AFP清洗液：0.02M PBS(pH7.0)，0.5％吐温-20；
(4)采用磁铁在管子底部吸附磁性颗粒，后吸去上清，弃去；
(5)重复步骤(3)、(4)2次，进行磁性颗粒清洗；
(6)移去磁铁，在管子中加入鲁米诺底物100ul，充分混匀，保证磁性颗粒完全悬浮，在37℃环境下孵育1分钟；
(7)在化学发光测定仪上测定发光信号强度，通过标准品曲线计算得知样本中AFP的含量。
6.本实施例进行AFP-L3检测步骤：
(1)将50ul 1％浓度的偶联凝集素的磁性颗粒与200ul待检测血清样本加入同一管中，充分混匀，并且静置5分钟；
(2)采用磁铁在管子底部吸附磁性颗粒，然后吸取上清，弃去；
(3)移去磁铁，在管子中加入AFP-L3清洗液500ul，充分混匀，确保磁性颗粒完全悬浮；
AFP-L3清洗液：0.02M PBS(pH7.0)，0.5％吐温-20；
(4)采用磁铁在管子底部吸附磁性颗粒，然后吸取上清，弃去；
(5)重复步骤(3)、(4)2次，进行磁性颗粒清洗；
(6)移去磁铁，在管子中加入AFP-L3洗脱液200ul，充分混匀，确保磁性颗粒完全悬浮，静置10分钟；
AFP-L3洗脱液：0.02M PBS(pH7.0)，5MD-甘露糖苷，
或20mmTris-Hcl,NaCl 150mm,pH7.4缓冲液，其中含有500mmα-甲基-D-甘露糖苷,0.1％Proclin 300；
(7)采用磁铁在管子底部吸附磁性颗粒，得到上清，该上清为甲胎蛋白异质体AFP-L3分离液体。
(8)在一个新的检测管中加入50ul 1％浓度的包被抗甲胎蛋白抗体1(anti-AFP-1)的磁性颗粒，同时加入50ul(7)中得到的分离液体，随后加入50ul 1000倍稀释的标记标记HRP酶的抗甲胎蛋白抗体2(anti-AFP-2)，充分混匀，在37℃环境下孵育5分钟；
(9)采用磁铁在管子底部吸附磁性颗粒，后吸去上清，弃去；
(10)移去磁铁，在管子中加入AFP清洗液500ul，充分混匀，确保磁性颗粒完全悬浮；
AFP清洗液：0.02M PBS(pH7.0)，0.5％吐温-20；
(11)采用磁铁在管子底部吸附磁性颗粒，后吸去上清，弃去；
(12)重复步骤(3)、(4)2次，进行磁性颗粒清洗；
(13)移去磁铁，在管子中加入鲁米诺底物100ul，充分混匀，保证磁性颗粒完全悬浮，在37℃环境下孵育1分钟；
(14)在化学发光测定仪上测定发光信号强度，通过标准品曲线计算得知检测样本中甲胎蛋白异质体AFP-L3的含量。
7.本实施例进行甲胎蛋白异质体所占的百分比(AFP-L3％)检测步骤
(1)按照5中所示检测样本中的甲胎蛋白AFP含量；；
(2)按照6中所示检测样本中的甲胎蛋白异质体AFP-L3含量；
(3)利用(2)中计算的甲胎蛋白异质体AFP-L3的含量除以甲胎蛋白AFP含量就得到了甲胎蛋白异质体所占的百分比(AFP-L3％)。
应用本实施例记载的分离检测组合物进行AFP含量检测：
采用Roche公司的AFP检测试剂盒(电化学发光法)作为对照组，比较AFP含量检测结果的准确性。AFP含量的cutoff值为20μg/L，高于20ng/ml为阳性结果，低于20ng/ml为阴性结果。检测结果如表1所示：
表1
Figure PCTCN2015099774-appb-000001
检测结果表明：检测的452份样本中，本实施例的灵敏度和特异性达到100％。
应用本实施例记载的分离检测组合物进行AFP-L3百分比检测：
采用北京热景生物公司生产的甲胎蛋白异质体分离管，产品注册证编号：国食药监械(准)自2014第3401646号，配合Roche公司的AFP检测试剂盒(电化学发光法)作为对照组，比较AFP-L3百分比检测结果的准确性。AFP-L3百分比的cutoff值为10％。检测结果如表2所示：
表2：
Figure PCTCN2015099774-appb-000002
与对照组相比较：107份对照组检测为AFP-L3百分比阳性的样本，本实施例检测也 为AFP-L3百分比阳性；172份对照组检测为AFP-L3百分比阴性的样本，本实施例检测有169份为阴性，另外3份样本经过临床诊断对照，发现患者为早期原发性肝细胞癌患者。检测结果表明：本实施例记载的分离检测组合物比对照组的检测性能更加灵敏。原因应是AFP-L3蛋白分离效率提高，可以更加准确检测样本中的AFP-L3百分比。
实施例2
本发明提供的甲胎蛋白异质体的分离检测组合物，根据实施例1的提供的甲胎蛋白异质体的分离检测组合物，分离试剂还可以包括保护液。检测试剂还可以包括缓冲液。
分离试剂和检测试剂分别可以包括清洗液。
上述试剂的主要组分情况如下：
保护液：0.02M PBS，0.5％BSA，pH7.4，0.1M D-甘露糖苷，
缓冲液：0.02M PBS，10％小牛血清，0.1％proclin-300，
分离试剂清洗液：20mM Tris-HCl，0.5MD-甘露糖苷，
检测试剂清洗液：PBSpH7.4配制的1％吐温20溶液。
上述D-甘露糖苷可以采用岩藻糖、果糖、蔗糖、海藻糖等糖类物质替代。
实施例3
本发明提供的甲胎蛋白异质体的分离检测系统，用于检测甲胎蛋白异质体所占的百分比，如图2所示，
包括用于分离甲胎蛋白异质体的磁分离模块，用于检测甲胎蛋白异质体含量和甲胎蛋白含量的检测模块，用于计算甲胎蛋白异质体相对于甲胎蛋白比值的数据处理模块。优选地，检测分离系统还可以包括取样模块，数据处理模块还可以包括光信号读取装置，优选的采用日本滨松光子学株式会社生产的光子计数探头，型号H10682-110，或者光电倍增管，型号R1166。
试剂卡包括实施例1记载的分离检测组合物，分离检测组合物预分装于试剂卡上的多个试剂槽中，用于进行反应，每种试剂有至少一个试剂槽。试剂卡包括样本孔、分离试剂槽、检测试剂槽和反应孔，本实施例的试剂卡，如图3所示，组成如下：
1.样本孔；2.预分装的偶联凝集素的磁性颗粒的试剂槽；3.预分装AFP-L3清洗液的试剂槽；4.预分装AFP-L3洗脱液的试剂槽；5预分装的包被抗甲胎蛋白抗体1(anti-AFP-1)的磁性颗粒的试剂槽；6.反应孔；7.预分装的标记酶的抗甲胎蛋白抗体(anti-AFP-2)的试剂槽；8.预分装AFP清洗液的试剂槽；9.预分装鲁米诺底物的试剂槽。
其中孔位b，c，d共同组成了磁分离模块，其中的所有试剂可以用于完成对甲胎蛋白异质体的分离；孔位e，f，g，h，i共同组成了检测模块，其中的所有试剂可以用于 完成对于加入的样本中AFP浓度的检测。
单独使用检测模块，可以完成对于样本中的AFP含量的测定；分离模块、检测模块相配合，可以完成对于样本中的甲胎蛋白异质体AFP-L3的分离，并且测定AFP-L3的含量；随后通过AFP-L3含量与AFP含量的相除，得到甲胎蛋白异质体的占比。
采用本实施例中记载的方法对待测样本进行检测，检测结果如表3所示：
表3
Figure PCTCN2015099774-appb-000003
实验结果表明，本发明对于原发性肝细胞癌的检测阳性率达到92％，对于健康人的特异性达到100％，对于肝硬化以及肝炎的特异性分别达到95％和97％，对于其他癌症的特异性达到了0％。
实施例4
根据实施例3提供的甲胎蛋白异质体的分离检测系统，还包括检测设定模块，检测设定模块包括甲胎蛋白含量测定单元、甲胎蛋白异质体含量测定单元和甲胎蛋白异质体所占的百分比的测定单元，
当选择甲胎蛋白含量测定单元时，所述磁分离模块不参加处理待检测样品，所述检测装置检测待检测样品中甲胎蛋白的含量，
当选择甲胎蛋白异质体含量测定单元时，所述磁分离模块分离待检测样品中甲胎蛋白异质体，所述检测模块检测待检测样品中甲胎蛋白异质体的含量；
当选择甲胎蛋白异质体所占的百分比的测定单元时，所述磁分离模块分离待检测样品中甲胎蛋白异质体，所述检测模块检测待检测样品中甲胎蛋白异质体的含量和甲胎蛋白的含量，所述数据处理模块计算甲胎蛋白异质体所占的百分比。
实施例5
根据实施例3或4提供的甲胎蛋白异质体的分离检测系统，采用实施例2记载的分 离检测组合物替代实施例1记载的分离检测组合物。
实施例6
本发明提供的甲胎蛋白异质体的分离检测方法，采用上述分离检测系统，用于检测甲胎蛋白异质体所占的百分比，包括如下步骤,
(1)加样:
将无溶血的血清、血浆或者全血的样本加入到分离检测系统，或加入到上述试剂卡提供的样本孔；
(2)分离甲胎蛋白异质体：
将样本加入到装有偶联凝集素的磁性颗粒的反应杯中，混匀；
用磁分离模块富集磁性颗粒，弃去液体；
向已经富集了磁性颗粒的反应杯中加入洗脱液，混匀，通过磁分离模块富集磁颗粒，得到甲胎蛋白异质体洗脱液；
(3)反应孵育：
将甲胎蛋白异质体洗脱液加入到装有包被甲胎蛋白单抗的磁性颗粒的反应杯中，同时加入酶标记的抗甲胎蛋白抗体，孵育。
(4)富集及清洗：用磁分离模块富集磁性颗粒，弃去液体，并且在反应杯中加入洗液进行混匀，后再次用磁分离模块富集磁性颗粒，弃去液体；
(5)显色：将富集得到的磁性颗粒加入鲁米诺底物，通过数据处理模块得到甲胎蛋白异质体的浓度；
(6)与(2)同时，将样本加入到反应杯中，并且加入包被甲胎蛋白单抗的磁性颗粒混匀，重复步骤(3)至(5)得到甲胎蛋白的浓度；
(7)通过数据处理模块得到甲胎蛋白异质体所占的百分比。
实施例7
根据实施例6提供的甲胎蛋白异质体的分离检测方法，仅包括步骤(1)和(6)，可以直接测得甲胎蛋白的浓度。
实施例8
本发明提供的甲胎蛋白异质体的分离检测方法，采用上述分离检测系统，用于检测甲胎蛋白异质体所占的百分比，包括如下步骤,
(1)加样:
将无溶血的血清、血浆或者全血的样本加入到分离检测系统，或加入到上述试剂卡提供的样本孔；
(2)分离甲胎蛋白异质体：
将样本加入到装有偶联凝集素的磁性颗粒的试剂槽中，混匀；
磁分离模块富集磁性颗粒，弃去液体；
将富集得到的磁性颗粒加入到装有清洗液的试剂槽中，混匀；
将清洗后的磁性颗粒加入到装有洗脱液的试剂槽中，混匀，通过磁分离模块富集磁颗粒，得到甲胎蛋白异质体洗脱液；
(3)反应孵育：
将甲胎蛋白异质体洗脱液加入到装有包被甲胎蛋白单抗的磁性颗粒的试剂槽中，同时加入预装在其他试剂槽中的标记酶的抗甲胎蛋白抗体，孵育。
(4)富集：磁分离模块富集磁性颗粒，弃去液体；
(5)清洗：将富集得到的磁性颗粒加入到装有清洗液的试剂槽中，混匀，重复步骤(4)；
(6)显色：将富集得到的磁性颗粒加入预装在其他试剂槽中的鲁米诺底物，通过数据处理模块得到甲胎蛋白异质体的浓度；
(7)与(2)同时，系统将样本加入到装有包被甲胎蛋白单抗的磁性颗粒的试剂槽中，重复步骤(3)至(6)测得到甲胎蛋白的浓度；
(8)通过数据处理模块得到甲胎蛋白异质体所占的百分比。
数据处理模块对甲胎蛋白异质体所占的百分比的逻辑算法：通过样本编号判断，仪器会对于同样一份样本的甲胎蛋白浓度和甲胎蛋白异质体浓度进行调取，后计算AFP-L3在AFP中所占的百分比，从而计算出AFP-L3含量，即AFP-L3％。
上述说明示出并描述了本发明的优选实施例，如前所述，应当理解本发明并非局限于本文所披露的形式，不应看作是对其他实施例的排除，而可用于各种其他组合、修改和环境，并能够在本文所述发明构想范围内，通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离本发明的精神和范围，则都应在本发明所附权利要求的保护范围内。

Claims (10)

  1. 一种甲胎蛋白异质体AFP-L3的分离检测组合物，包括分离试剂和检测试剂，其特征在于，所述分离试剂包括偶联凝集素的磁性颗粒和洗脱液，所述检测试剂包括包被抗甲胎蛋白抗体的磁性颗粒以及经酶标记的抗甲胎蛋白抗体；
    优选地，所述洗脱液为0.02M PBS(pH7.0)，5M D-甘露糖苷；
    优选地，所述凝集素为小扁豆凝集素或刀豆凝集素；
    优选地，所述抗甲胎蛋白抗体为针对甲胎蛋白的单克隆抗体；
    优选地，所述磁性颗粒为琼脂糖包裹的顺磁颗粒。
  2. 根据权利要求1所述的分离检测组合物，其特征在于，所述分离试剂还包括保护液,和/或所述检测试剂还包括缓冲液；
    优选地，所述保护液为0.02M PBS pH7.4、0.5％ BSA和0.1M D-甘露糖苷；
    优选地，所述缓冲液为0.02M PBS、10％小牛血清和0.1％ proclin-300。
  3. 根据权利要求1或2任一项所述的分离检测组合物，其特征在于，所述分离试剂和/或检测试剂还包括清洗液；
    优选地，所述分离试剂的清洗液为20mM Tris-HCl、0.5MD-甘露糖苷；
    优选地，所述检测试剂的清洗液为PBS pH7.4配制的1％吐温20溶液。
  4. 根据权利要求1所述的分离检测组合物，其特征在于，所述偶联凝集素的磁性颗粒中磁性颗粒表面覆盖有高分子成分，所述高分子成分包括硅化物、多聚糖、蛋白、纤维素或树脂。
  5. 根据权利要求1-4任一项所述的分离检测组合物，其特征在于，所述组合物以卡式或条式提供。
  6. 一种甲胎蛋白异质体的分离检测试剂盒，其特征在于，包括权利要求1-5任一项所述的分离检测组合物。
  7. 一种甲胎蛋白异质体的分离检测系统，包括：
    磁分离模块，用于自液体中分离磁颗粒；
    检测模块，用于检测甲胎蛋白异质体和甲胎蛋白含量；
    数据处理模块，用于计算甲胎蛋白异质体相对于甲胎蛋白比值，和
    权利要求1-7任一项所述分离检测组合物或权利要求8所述试剂盒，
    所述磁分离模块与所述分离检测组合物中的分离试剂相配合，用于完成对甲胎蛋白异质体的分离；
    所述检测模块与所述分离检测组合物中的检测试剂相配合，用于完成对甲胎蛋白异质体的含量和甲胎蛋白含量的检测。
  8. 根据权利要求7所述的分离检测系统，其特征在于，
    当选择测定甲胎蛋白含量时，所述磁分离模块不与分离试剂相配合，所述检测装置检测待检测样品中甲胎蛋白的含量，
    当选择测定甲胎蛋白异质体含量时，所述磁分离模块分离待检测样品中甲胎蛋白异质体，所述检测模块检测待检测样品中甲胎蛋白异质体的含量；
    当选择测定甲胎蛋白异质体所占的百分比时，所述磁分离模块分离待检测样品中甲胎蛋白异质体，所述检测模块检测待检测样品中甲胎蛋白异质体的含量和甲胎蛋白的含量，所述数据处理模块计算甲胎蛋白异质体所占的百分比。
  9. 权利要求1-5任一项所述的分离检测组合物、权利要求6所述试剂盒或者权利要求7或8所述的分离检测系统在分离检测甲胎蛋白异质体中的应用。
  10. 一种分离检测甲胎蛋白异质体的方法，其特征在于使用权利要求1-5任一项所述的分离检测组合物、权利要求6所述试剂盒或者权利要求7或8所述的分离检测系统来分离检测样品中的甲胎蛋白异质体，所述方法包括如下步骤：
    分离步骤：使凝集素标记的磁性颗粒与所述样品接触以结合甲胎蛋白异质体AFP-L3；
    洗脱步骤：分离所述磁性颗粒并洗脱出甲胎蛋白异质体AFP-L3；以及
    检测步骤：使用针对AFP的免疫检测方法检测甲胎蛋白异质体AFP-L3；
    优选地，所述凝集素为小扁豆凝集素(LCA)；
    优选地，所述磁性颗粒为琼脂糖包裹的磁性颗粒；更优选地，所述所述磁性颗粒为琼脂糖包裹的顺磁颗粒；
    优选地，所述的针对AFP的免疫检测方法为磁颗粒化学发光方法。
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