WO2008031288A1

WO2008031288A1 - Pre-filled centrifugal column for detecting hepatocellular carcinoma-specific alpha-fetoprotein variant and test kit containing the column

Info

Publication number: WO2008031288A1
Application number: PCT/CN2006/003210
Authority: WO
Inventors: Changqing Lin
Original assignee: Beijing Hotgen Biotech Co., Ltd
Priority date: 2006-09-13
Filing date: 2006-11-29
Publication date: 2008-03-20
Also published as: CN100588942C; CN1920520A

Abstract

A pre-filled centrifugal column for detecting hepatocellular carcinoma-specific alpha-fetoprotein variant and a test kit containing the column. The centrifugal column comprises an upper separating tube and a lower collecting tube. The upper separating tube contains media coupled with an agglutinin, the bottom of which is provided with filer cloth. A buffer solution is provided in the lower collecting tube. The agglutinin is Lens culinaries agglutinin (LCA) or Canavalia ensiformis agglutinin (Con-A), which can bind sugar chains of the alpha-fetoprotein variant. By eluting and centrifuging the column, the component AFP-L3 is purified. The pre-filled centrifugal column and the test kit of the invention can detect rapidly and simply the amount of hepatocellular carcinoma-specific alpha-fetoprotein variant, by which hepatocellular carcinoma can be early diagnosed.

Description

Pre-packed spin column for detecting hepatocellular carcinoma alpha-fetoprotein-like shield and kit containing the same

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.

Background technique

Hepatocel lular carcinoma (HCC) 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. 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 (AFP) 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. Under normal circumstances, 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. If the neonatal AFP is significantly elevated, it suggests 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 / 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. .

Sumner and Howe 11 first purified the concanavalin-ConA (Con-A) from the seeds of jackbean (Canaval ia ens iformis) in 1936. 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.

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. At present, according to the type of combined sugar, 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.

The most well-identified family of plant lectins is 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.

With the development and application of biochemistry and related analytical techniques, it is found that the affinity of AFP molecules is different from that of exogenous lectins, that is, the heterogeneity of sugar chain is shielded. Different lectin affinity electrophoresis can be used to separate them into several components, and isoelectric focusing techniques can also be used to separate AFP components. 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).

As early as 1970, some scholars found that serum AFP starch gel electrophoresis often showed different mobility in patients with hepatocellular carcinoma, while AFP electrophoretic mobility was the same in neonates and fetuses. At that time, it was thought that the AFP contained different sialic acid content, and the concept of AFP Var iant s AFP-V was proposed. Subsequent studies have shown that the sugar chain structure of AFP has varying degrees of variation, and the so-called heterogeneity is mainly due to the different carbohydrates contained in AFP. The exact mechanism of AFP heterogene formation is not well understood. 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. The sugar chain structure of AFP can interact with various lectins, and the structural part of AFP molecules bound by different lectins are basically the same, but the sugar chain parts are different. Different sugar chains determine their affinity for lectins. Depending on the affinity, 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. If LCA is used, it is named 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.

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). It is currently believed that the so-called 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.

Clinical significance of AFP heterogeneity detection:

1) Identify liver cancer and benign liver disease. AFP is often elevated in patients with primary liver cancer, but many benign liver diseases can also have elevated AFP. It is sometimes difficult to distinguish between benign and malignant lesions by AFP alone. At this time, AFP heterogeneity detection has a good clinical significance, especially for AFP between 30 ~ 400ng / ml. Yozhiaki conducted a prospective study of 361 patients with cirrhosis. In 53 patients with AFP above 30 ug/L, 21 patients developed liver cancer 2 years later, and 39% of patients with AFP were below 400 ug/L. At the beginning of the comparative study, the AFP values of the hepatocellular carcinoma (HCC) group and the non-HCC group were not significantly different. 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.

2) Monitoring of postoperative liver cancer. After liver cancer resection, 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.

3) abnormal embryo development and fetal congenital disorders. 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. However, 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.

Summary of the invention

In order to overcome the deficiencies of the prior art, 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:

1. The source of lentils is preferred in Gansu Province, China;

2, take lentils (dry seeds) 100g, add 0.8% NaCl solution 1000ml, set at 4 °C, soak for 24 hours;

3, high-speed masher 8000r / min, cut into a paste. 4 ° C, 4500 r / min, centrifugation for 20 minutes, take the upper layer of milk;

4. Add solid ammonium sulfate to the milk to 1/3 saturation, mix and let stand for 1 hour at room temperature. Centrifuge according to the above conditions, and collect the supernatant;

5. Add solid ammonium sulfate to the supernatant to 2/3 saturation, mix and let stand for 1 hour at room temperature. Centrifuge according to the above conditions, collect the precipitate;

6. The precipitate was dissolved in 0. 05M PH=8.3 of Tr is-HCl buffer, and dialyzed thoroughly with physiological saline to the non-Li 3+, and then dialyzed against the above buffer;

7. The above dialysis balanced extract is further purified by DEAE Sepharose Fast Flow chromatography to collect a breakthrough peak;

8. Identification by electrophoresis, the molecular weight of the protein was 17KD for LCA, and the activity was identified by rabbit red blood cell agglutination test.

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:

(1) Soaking and washing the hydrogen bromide-activated agarose gel Sepharose 4B, or sephrose 6B, or sephrose FF with 1 mmol/L HC1;

(2) Weighing LCA, dissolving in the coupling buffer, and mixing with the washed agarose gel Sepharose 4B, or sephrose 6B, or sephrose FF;

(3) The unconjugated LCA was washed away with a coupling buffer, and the LCA content in the washing solution was measured, and the coupling ratio was 98%;

(4) The remaining activating gene was blocked with glycine, washed, and stored at 4 °C for later use.

The agarose gel may preferably be Pharmacia hydrogen bromide-activated Sepharose 4B using the following specific steps:

(1) After activation of 1. 5 g of Sepharose 4B with hydrogen bromide, immersed in 1 mmol / L of HC1 to swell, transferred into the funnel, and washed with 300 mL of 1 mmol / L HC1 for about 30 min;

(2) Weigh 2 mg of LCA, dissolved in 7.5 mL of coupling buffer (0.1 mmol/L NaHC03 and 0.5 mol/L NaCl, pH 8. 3), and combined with washed Sepharose 4B. Mix the 10 mL tube with stoppers upside down (room temperature, 2 h);

(3) The unconjugated LCA was washed away with 10 mL of coupling buffer, and the LCA content in the washing solution was determined, and the coupling ratio was 98%;

(4) blocking the remaining activating gene with 0.2 mol/L glycine;

(5) 0. 5 mol / L Tri s buffer (pH 8 , containing 0.5. 5 mol / L of acetic acid buffer (pH 4, containing 0.5 mol / L NaCl) and 0.1 mol / L Tri s buffer (pH 8, with 0.5 Washed 3 times with mol/L NaCl), washed once with PBS (PBS-BSA) containing 0.1% BSA, 1 mmol/L CaCl ₂ and 0.1 mmol/L MnCl ₂ , and stored at 4 °C for later use. .

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.

l°。 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 ΙμΙ, 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;

(2) Preparation of positive and negative controls:

Positive control: collect ascites from liver cancer patients, filter and remove bacteria;

Negative control: collect normal human serum, filter sterilization, and dispense;

(3) 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) Oxidation of HRP by NaI0 ₄ -ethylene glycol method to a final concentration of 10 mg/ml;

b) Monoclonal antibody and HRP were dialyzed in alkaline carbonate buffer for 6 hours to achieve labeling of monoclonal antibody by HRP. After the reaction, the reaction was terminated with NaBH ₄ solution, and then dialyzed against PBS overnight;

c) Precipitating with saturated ammonium sulfate to obtain a purified HRP-labeled anti-AFP-L3 monoclonal antibody;

(4) 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 %);

b) coloring solution: luminol 5.0xl0~ ⁴ Mol/L;

c) AFP-L3 Wash Buffer: 20 匪 ol/LTris- Hcl, pH 7.4, containing 0. roclin 300 preservative;

l%Proclin。 The AFP-L3 eluate: 20 mmol / L Tris-Hcl, pH 7.4, 150 mmol / L NaCl and 500 leg ol / La-mercapto-D-mannosidase, eluent containing 0. l% Proclin 300 preservatives;

e) concentrated washing solution (20 times concentrated solution, 20x): 0.05% Tween 20 solution prepared in PBS (pH 7.4);

(5) Pre-packed spin column, chemiluminescent substrate labeled with alpha-fetoprotein monoclonal antibody, positive control, negative control, enzyme-labeled anti-fetal protein monoclonal antibody, substrate solution, color development The solution, the AFP-L3 washing buffer, the AFP-L3 eluent, the concentrated washing solution, and the AFP standard are packaged together to obtain a kit.

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 Å / 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 ΙΟΟμΙ, 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;

(2) Preparation of positive and negative controls:

a) oxidation of HRP by NaI0 ₄ -ethylene glycol method to a final concentration of 10 mg / ml;

b) Monoclonal antibody and HRP were dialyzed in alkaline carbonate buffer for 6 hours to achieve labeling of monoclonal antibody by HRP. After the reaction, the reaction was terminated with NaBiU solution, and then dialyzed against PBS overnight; c) saturated ^ Ammonium precipitation, obtaining purified HRP enzyme-labeled anti-AFP-L3 monoclonal antibody; (4) Preparation of auxiliary reagents is 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;

c) AFP-L3 wash buffer: 20 mmol / LTris-Hcl, pH 7.4, containing 0. l ° /. Proclin 300 preservative;

l%Procl in 300, the eluent of the AFP-L3 eluate: 20 mmol / L Tris-Hcl, pH 7.4, 150 mmol / L NaCl and 500 mmol / La-mercapto-D-mannosidase, the eluent contains 0. l% Procl in 300 Preservative

e) Concentrated washing solution (20 times concentrated solution, 20x): 0.05 ° / PBS (pH 7.4). Tween 20 solution;

f) 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.

DRAWINGS

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.

detailed description

The invention is further illustrated by the following examples. It is to be understood that the embodiments of the present invention are intended to be illustrative of the invention and are not intended to

Referring to Figure 1, 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. Usually, 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. Cloth, 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.

Example 1: Preparation and use of prepacked spin columns:

1. 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.

- Heart column.

2. Preparation of the spin column packing material: Using Pharmacia's hydrogen bromide-activated Sepharose 4B and Sigma LCA, coupled as follows:

(1) Soak 1.5 g of Sepharose 4B in 1 leg ol / L HC1 to swell, transfer to the gravel funnel, and wash with 300 mL of 1 mmol / L HC1 for about 30 min;

(2) Weigh 50 mg of LCA, dissolve in 7.5 mL of coupling buffer (0.1 mmol/L NaHC03 and 0.5 mol/L NaCl, H 8.3), and combine with washed Sepharose 4B to invert the lOmL tube with stopper. Mix (room temperature, 2 h);

(3) Unconjugated LCA was washed away with 10 mL of coupling buffer. After determining the LCA content in the washing liquid, the coupling ratio was 98%;

(4) blocking the remaining activating gene with 0.2 mol/L glycine;

(5) Wash 10 times with 10 mL of 0.1 mol/L acetate buffer (pH 4, containing 0.5 mol/L NaCl) and 0.1 mol/L Tris buffer (pH 8, containing 0.5 mol/L NaCl). The cells were washed once with PBS (PBS-BSA) containing 0.1% BSA, 1 mmol/L CaCl ₂ and 0.1 mmol/L MnC, and stored at 4 °C for later use.

3. Add a filter cloth to the bottom of the upper separation tube and fix it with a plastic gasket. The pore size of the filter cloth is smaller than that of agarose, so the agarose can not pass, but the protein and liquid can flow. 300 ul of sephrose 4B to which lectin has been coupled is added to a centrifuge tube in the upper part of the spin column. 4. Add 1ml - 2ml lectin storage buffer, the buffer is filled with the medium present, and most of it is present in the lower collection tube. 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:

1. 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;

2, sample dilution: Pipette 250ul serum in a test tube, and add 350ul cleaning solution to dilute and shake;

3. Loading: Aspirate 450 ul of diluted sample into the upper part of the centrifuge tube. The 37 ° C incubator is allowed to stand. Do not cover the centrifuge tube cover. The serum dilution will flow into the lower collection tube within 15 minutes. The remaining 150 ul of diluted serum (sample 1) in the test tube is ready for control.

4. Discard the liquid in the collection tube;

5. Add 600 ul of cleaning solution to the upper centrifuge tube, wait for the cleaning solution to flow into the lower collection tube (about 3 minutes), cover the centrifuge tube cap, and centrifuge at room temperature for 2 minutes at 2000 rpm (or 3000 rpm).

6. Discard the liquid in the lower collection tube;

7. Add 600 ul of cleaning solution to the upper centrifuge tube, wait for the cleaning solution to flow into the lower collection tube (about 3 minutes), cover the centrifuge tube cover, centrifuge at 2000 rpm (or 3000 rpm) for 2 minutes at room temperature;

8. Discard the liquid in the lower collection tube;

9. Add 450 ul of eluent. When a few drops of the eluent are present in the lower collection tube, cover the centrifuge tube cap, place it in a 37 °C incubator, and incubate for 30 minutes.

10. Remove the centrifuge tube and centrifuge at room temperature for 2000 minutes at 2000/min (or 3000 rpm).

11. Collect the liquid flowing into the lower collection tube (sample 2) for testing.

Example 2: Preparation of an enzyme-linked immunoassay kit for detecting hepatocellular carcinoma alpha-fetoprotein heterogeneity: The kit (96 servings) consists of:

1 bottle of AFP-L3 negative and positive control;

AFP monoclonal antibody coated plate (96 wells) 1 block;

Horseradish peroxidase (HRP)-labeled AFP monoclonal antibody 1 bottle, 6ml/bottle;

AFP standard 1 bottle;

1 bottle of substrate solution and color developing solution, 5ml/bottle;

Reaction stop solution 1 bottle, 5ml / bottle;

AFP-L3 washing buffer 1 bottle, 25ml/bottle;

AFP-L3 eluent 1 bottle, 15ml/bottle;

Concentrated washing solution (20 times concentrated solution, 20x) 1 bottle, 2 Oml / bottle.

The specific operations are as follows:

1. Preparation of AFP-negative, positive control:

1) 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;

2) Packing:

5毫升。 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毫升。 Each tube was filled into a 5. 5ml eppendorf tube, each tube 0. 5ml. Store at 4. C.

2. Make AFP monoclonal antibody coated plates:

a) Coating:

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 ΙμΙ, 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;

b) Preparation of a spicy 4-purine peroxidase (HRP)-labeled anti-AFP monoclonal antibody:

Anti-AFP monoclonal antibodies are commercially available.

3. Labeling of monoclonal antibodies:

1) Oxidation of the enzyme (the whole process is protected from light):

a) Weigh 5mg of HRP, add ddH ₂ 0 250μ1 to dissolve;

b) Weigh NaI0 ₄ 5mg, add ddH ₂ 0 250μ1 to dissolve, and prepare a concentration of 20mg/mL; c) Add NaI0 ₄ solution dropwise to HRP solution, while stirring;

d) The mixed solution is placed at 4 ° C and allowed to stand for 30 minutes;

e) taking 5ml of ethylene glycol dissolved in 25μ1 ddH ₂ 0, adding dropwise to the above mixed solution, and stirring while adding;

f) Allow to stand at room temperature for 30 minutes;

g) The enzymatic oxidation process is completed with a final HRP concentration of 10 mg/ral.

2) Preparation and labeling of monoclonal antibodies (protected from light):

a) Adjust the antibody concentration to about 5mg/ml (concentrate with PEG20000 when the protein concentration is too low), and use 50mmol/L CB (1mol/L NaHC0 ₃ and 1mol/L Na ₂ C0 _{3 at} 10: 1) with a pH of about 9.5. Mix in proportion, dilute 20 times with distilled water before use, dialyze off glycerin or impurities (such as Triis), dialyze overnight at 4 °C, and change the solution 3 times;

b) Mixing the monoclonal antibody with HRP at 1:4, dialysis for more than 6 hours at 50 mmol/L pH 9.5 CB, and changing the solution for the first two hours;

c) Stop the reaction with freshly prepared ₁ mg NaBH ₄ solution. Shake well, let stand at 4 ° C for 2 hours, shake once every half hour, the amount of NaBIU solution should be appropriate;

d) dialyzed overnight with pH 7.2 of 10 mM PBS (pre-configured with 0. Olmol/L of Na ₂ HP0 ₄ and NaH ₂ P0 ₄ stock solution, mix the two into PBS buffer according to the required pH). The liquid can be used once. 3) Purification of HRP-labeled anti-AFP monoclonal antibody:

a) 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;

b) standing at 4 ° C for 1 hour;

c) Centrifuge at SOOOrpm for 10 minutes, transfer the supernatant to a new tube, and resuspend the pellet with an equal volume of PBS;

d) Repeat the above operation, increase the saturated sulfuric acid to 40% according to the concentration, separately collect the supernatant and precipitate; e) repeat the above operation, increase the saturated ammonium sulfate concentration to 50%, separately collect the supernatant and precipitate; f) repeat the above operation, The saturated ammonium sulfate concentration was increased to 60%, and the supernatant and the precipitate were separately collected; g) the separated components were collected, and the purity was identified by SDS-PAGE;

h) purified HRP-monoclonal antibody was dialyzed against PBS overnight;

i) Purification of purified HRP-monoclonal antibody by ultrafiltration tube to obtain an anti-monoclonal antibody with a molar ratio close to 1:8.

4) Packing: Dilute the enzyme-labeled anti-AFP - L3 monoclonal antibody obtained in step 3) to a suitable working concentration with a buffer containing 10% fetal bovine serum, dispense in 6 ml/bottle, and store at 4 °C. .

4. Preparation of auxiliary reagents:

1) Substrate solution: 3% hydrogen peroxide solution prepared by phosphoric acid-citric acid buffer (pH 5.0), packed in 5ml/bottle;

2) Coloring solution: TMB (0. lmg/ml) sterol solution, divided into 5ml / bottle;

3) Reaction stop solution: 2mol/L H ₂ S0 ₄ , packed in 3ml/bottle;

4) 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防腐液: 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:

1 bottle of AFP-L3 negative and positive control;

AFP monoclonal antibody-coated plate ⁽⁹⁶ wells) 1;

Horseradish peroxidase (HRP) labeled AFP monoclonal antibody 1 bottle, ⁶ ml / bottle;

AFP standard 1 bottle;

1 bottle of substrate solution and color developing solution, 5ml/bottle;

AFP-L3 Wash Buffer 1 bottle, 25ml/bottle;

AFP-L3 eluent 1 bottle, 15ml/bottle;

Concentrated washing solution (20 times concentrated solution, 20x) 2 Oml/bottle.

The specific operations are as follows:

1. Preparation of AFP-negative, positive control:

1) Collecting serum: Obtain normal human serum from the hospital or blood station, ascites from liver cancer patients, and keep at -70 °C for use;

2) Packing:

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毫升。 Each tube was filled into a 5. 5ml eppendorf tube, each tube 0. 5ml. Store at 4 °C.

2. Make AFP monoclonal antibody coated plates:

" 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, ΙΟΟμΙ 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.

3. Labeling of monoclonal antibodies:

1) Enzyme oxidation (all processes protected from light):

a) Weigh 5mg of HRP and dissolve it with ddH ₂ 0 250μ1;

b) Weigh NaI0 ₄ 5rag, add ddH ₂ 0 250μ1 to dissolve, and prepare a concentration of 20mg/mL; c) Add NaI0 ₄ solution dropwise to HRP solution, while stirring;

d) The mixed solution is placed at 4 ° C and allowed to stand for 30 minutes;

e) take 5ml of ethylene glycol dissolved in 25μ1 ddH ₂ 0, add dropwise to the above mixed solution, and add while mixing;

f) Allow to stand at room temperature for 30 minutes;

g) The enzymatic oxidation process is completed with a final HRP concentration of 10 mg/ml.

2) Preparation and labeling of monoclonal antibodies (protected from light):

a) Adjust the antibody concentration to about 5mg/ml (concentrate with PEG20000 when the protein concentration is too low), and use 50mmol/L CB (1mol/L NaHC0 ₃ and 1mol/L Na ₂ C0 _{3 at} 10: 1) with a pH of about 9.5. Proportionate mixing, dialysis against glycerin or impurities (such as Tris) before use, dialysis overnight at 4 ° C, which changed the liquid 3 times;

b) Mix the monoclonal antibody with HRP at 1:4, dialysis for more than 6 hours at 50 awake ol/L pH9.5 CB, and change the solution for the first two hours;

c) Stop the reaction with freshly prepared ₁ mg NaBH ₄ solution. Shake well, let stand at 4 °C for 2 hours, shake once every half hour, the amount of NaBH ₄ solution should be appropriate; d) Dialysis overnight with 10 mM PBS of H7.2 (pre-configured with 0. Olmol/L of Na ₂ HP0 ₄ and NaH ₂ P0 ₄ stock solutions, mixing the two into PBS buffer according to the desired pH). Change it once.

3) Purification HRP-labeled anti-AFP-L3 monoclonal antibody:

a) adding the saturated ammonium sulfate solution to the labeled monoclonal antibody solution, stirring while adding, until the saturated ammonium sulfate concentration is reduced to 1/3;

b) standing at 4 ° C for 1 hour;

c) Centrifuge at 8000 rpm for 10 minutes, transfer the supernatant to a new tube, and resuspend the pellet with an equal volume of PBS;

d) Repeat the above operation to increase the saturated ammonium sulfate concentration to 40%, separately collect the supernatant and precipitate; e) repeat the above operation, increase the saturated sulfuric acid to 50% according to the concentration, separately collect the supernatant and precipitate; f) repeat the above operation, The saturated ammonium sulfate concentration was increased to 60%, and the supernatant and the precipitate were separately collected; g) the separated components were collected, and the purity was identified by SDS-PAGE;

h) purified HRP-monoclonal antibody was dialyzed against PBS overnight;

i) Purification of purified HRP-monoclonal antibody by ultrafiltration tube to obtain an enzyme-labeled anti-monoclonal antibody with a molar ratio close to 1:8.

4) 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.

4. Preparation of auxiliary reagents:

1) Substrate ^{solution: 1.0ml EDTA (1.0x10- 2 M)} , 1.0ml H 2 0 2 (7.5x10- 3 M), 0.4ml HC1 (1.0x10 a ² M) and 0.2ml T een20 (1%)

2) Coloring solution: luminol 5.0xl0 ^_ Mol/L;

3) AFP-L3 washing buffer: 20 awake ol/LTris-Hcl, ΡΗ7·4, containing 0. l% Procl in 300 preservative;

4%Proclin 300腐腐。 AFP-L3 eluent: 20 mmol / L Tris-Hcl, pH 7.4, 150 mmol / L NaCl and 500mmol / La-mercapto-D-mannose glycosides, the eluate containing 0. l% Proclin 300 antiseptic Agent 55%吐温20的溶液。 5) Concentrated washing solution (20 times concentrated solution, 20x): PBS (pH 7.4) prepared 0. 05% Tween 20 solution.

The quality detection method of the kit of the present invention is:

1. 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.

2. Accuracy: The test results of 10 normal negative control serum (including specific control serum) reference products, no false positives appeared. The results of the reference products of 5 liver cancer AFP positive control serums showed no false negative appearance.

3. Precision: Randomly extract 20 boxes of different batches of kits, and use the same liver cancer positive control serum to repeat the measurement according to the instructions. Calculate the results of each measurement and find the mean, SD, and coefficient of variation CV. The precision test results show that the inter-batch CV is less than 10°/. .

4. Detection sensitivity: According to the dilution test results of AFP standard, the detection sensitivity of this kit is 5ng/ml.

5. Specificity: Take four samples to be tested and mix them into four mixed serum samples, each 1ml. Add 50ng dose of tissue plasminogen activator (tPA), plasmin

(Plasmin), or fibronectin (FN), were made into interference test serum samples #1, #2, #3, and #4 mixed serum samples without any interfering substances were used as the base samples. Perform the measurement according to the instructions and calculate the result. Then calculate the interference rate according to the interference test calculation formula. The interference errors of specimens #1, #2, and #3 are less than 5%.

Example 4: Method for detecting alpha fetoprotein heterogeneous AFP-L 3 content using the chemiluminescence detection kit provided by the present invention:

1. Specimen processing and AFP-L3 purification:

1) completely centrifuge the serum to be tested without hemolysis; take out the pre-packed micro-centrifugation column and discard the liquid in the lower collection tube; 2) Sample dilution: Pipette 250 ul of serum into a test tube, and add 35 Qui cleaning solution to dilute and shake;

3) Loading: Aspirate 450 ul of the diluted sample into the upper part of the centrifuge tube. The 37 ° C incubator is allowed to stand. Do not cover the centrifuge tube cover. The serum dilution will flow into the lower collection tube within 15 minutes. The remaining 150 ul of diluted serum (sample 1) in the test tube is ready for control.

4) Discard the liquid in the collection tube;

5) Add 600 ul of cleaning solution to the upper centrifuge tube, wait for the cleaning solution to flow into the lower collection tube (about 3 minutes), cover the centrifuge tube cover, centrifuge at 2000 rpm (or 3000 rpm) for 2 minutes at room temperature; 6) Discard The liquid in the lower collection tube;

7) Add 600 ul of cleaning solution to the upper centrifuge tube, wait for the cleaning solution to flow into the lower collection tube (about 3 minutes), cover the centrifuge tube cap, and centrifuge at room temperature for 2 minutes at 2000 rpm (or 3000 rpm).

8) discard the liquid in the lower collection tube;

9) Add 450 ul of eluent, and when a few drops of the eluent are present in the lower collection tube, cover the centrifuge tube cap, place it in a 37 ° C incubator, and incubate for 30 minutes;

10) Remove the centrifuge tube, centrifuge at 2000 (or 3000 rpm) for 2 minutes at room temperature;

11) Collect the liquid flowing into the lower collection tube (sample 2) for testing.

2. Quantitative detection AFP- L3°/. :

1) Design the position and quantity of the coated board, the unused cover sheets are sealed, and put into the refrigerator at 2-8 °C;

2) Add 50 μl pre-pretreatment sample Φ (detect total AFP), processed sample 2 (detect AFP-L3) to the corresponding coated plate microwell. Each pre-pretreatment specimen and post-treatment specimen are double-well detection;

3) Incubate at 37 ° C for 30 minutes;

4) Washing 5 times, deducting the residual liquid on the board;

5) sequentially adding 100 μl of the enzyme conjugate to each well;

6) Incubate at 37 ° C for 30 minutes; 7) Washing 5 times, deducting the residual liquid on the board;

8) Mix the substrate solution and the color developing solution in equal volume, add 50 μl of this mixture to each well; or add 2 μl of the substrate solution to each well and add 25 μl of the coloring solution. 5 seconds;

9) Detecting with a chemiluminometer within 10 minutes after adding the substrate solution and the color developing solution. 3. Result judgment:

Make a standard curve: Take the standard concentration as the abscissa, the RLU value of the standard is the ordinate, and make a standard curve; Calculate the standard curve regression coefficient R ² . When R ² >0.95, the measurement is valid. The detected readings were calculated for the AFP content according to the reagent standard curve, and then the ratio of AFP-L3 was calculated. The specific calculation method is:

1) Sample 2 contains AFP-L3, and sample 1 contains total AFP. When the test result shows that AFP-L3 in specimen 2 satisfies 3⁄4 2ug/L, the following formula is calculated:

AFP-L3%= (sample 2 AFP content ÷ sample 1 AFP content) X 100°/.

2) When the test result shows that the AFP-L3 content in sample 2 is < 2ug/L, AFP-L3 is negative, regardless of the reading value of sample 1, the ratio is calculated as 1%:

3) 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.

Table 1. Comparison of AFP-V detection rates in different specimens

Positive proportion of serum source cases AFP positive AFP- V content

(greater than 20ng) ( > 15% )

50 primary liver cancer positive specimens 50 100% 91 %

83 healthy people 83 0 0

100 cirrhosis 100 100% 7%

100 hepatitis 100 100% 6%

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.

Experiment 3: A group of specimens obtained from Changzhou Liver Disease Research Institute of Jiangsu Province were tested for AFP-L3 and the development results were followed. It was found that multiple AFP-L3 positive patients were confirmed as liver cancer several months later, showing AFP-L3 index as early stage. Early warning of liver cancer has important value. The test results are shown in Table 2.

Table 2. Results of AFP-L3 test on liver cancer patients and patients diagnosed with liver cancer after discharge (sample from Jiangsu Changzhou Liver Disease Research Institute) Patient living AFP-L3 (ng/ral

Age discharge master diagnosis

No. t-AFP (ng/ral) ) AFP-L3% Pathology Verification

129382 52 Hepatocellular carcinoma 547. 06 61. 95 11%

128726 52 Hepatocellular carcinoma 8. 69 0. 31 4% After liver transplantation

127981 34 Hepatocellular carcinoma 359. 30 9. 19 3%

129412 56 Hepatocellular carcinoma 542. 44 423. 56 78%

128769 54 Hepatocellular carcinoma 537. 82 485. 38 90%

128072 59 Hepatocellular carcinoma 466. 47 83. 29 18%

127976 40 Hepatocellular carcinoma 62. 33 9. 81 16%

128656 38 Hepatocellular carcinoma 510. 88 431. 08 84%

128643 64 Hepatocellular carcinoma 279. 04 76. 75 28%

129925 51 Hepatocellular carcinoma 345. 79 72%

127185 59 Hepatic cirrhosis after hepatitis 107. 81 17. 77 16% Confirmed hepatocellular carcinoma after 3 months

127374 38 Hepatic cirrhosis after hepatitis 64. 58 15. 69 24% Hepatocellular carcinoma confirmed after 12 months

Oo

128750 32 Chronic active hepatitis B (severe) 564. 28 450. 58 80% Confirmation of hepatocellular carcinoma death after 6 months

127789 54 Hepatitis B subacute severe 51. 68 7. 57 15% Follow-up confirmation of hepatocellular carcinoma

127921 54 Hepatic cirrhosis after hepatitis 526. 43 409. 70 78% 3 months shadow! ^Check for hepatocellular carcinoma

128592 60 Chronic active hepatitis B (severe) 472. 62 446. 23 94% Check for confirmed hepatocellular carcinoma

129903 55 Hepatic cirrhosis after hepatitis 400. 62 97. 01 24% After 9 months, hemodialysis confirmed hepatocellular carcinoma

129613 53 cirrhosis with esophageal varices bleeding 462. 66 444. 61 96% image confirmed hepatocellular carcinoma

Claims

Rights request

A pre-filled spin column of a liver 3' fetal protein heterogeneous body, characterized in that it consists of an upper separation tube and a lower collection tube, the upper separation tube being provided with a medium coupled to a lectin, and having a bottom portion a filter cloth, wherein the lower collection tube is provided with a buffer solution.

2. The prepacked spin column for detecting a liver cancer sputum protein heterogeneity according to claim 1, wherein: the lectin is lentil lectin LCA or concanavalin condensate Con-A, and the medium is An agglutinin-coupled affinity medium, polystyrene microspheres, cellulose or chitosan, which is a filter cloth capable of blocking the passage of a medium coupled with a lectin without blocking the passage of liquid and protein. The buffer solution is an active protective solution of lectin.

3. The prepacked spin column for detecting hepatocellular carcinoma alpha-fetoprotein heterogeneity according to claim 2, wherein: the agglutinin-binding affinity medium is agarose microspheres, and the agarose microspheres are used. Agarose gel sephrose 4B, or agarose gel sephrose 6B, or agarose gel sephrose FF, the buffer solution contains 1 ol / L of CaCl ₂ , 1 mmol / L of MnCl ₂ and 50 mmol / L of Tr i s- HCL, PH7. 5.

4. The prepacked spin column for detecting a liver cancer abortin isoform according to claim 3, wherein: the agarose gel coupled to the lentil lectin is obtained by the following steps:

5. A chemiluminescence detection kit for detecting a heterogeneous body of liver cancer adenine, comprising the following components:

(1) pre-packed spin columns for detecting hepatic alpha-fetoprotein heterogeneous bodies;

(2) a chemiluminescent ELISA plate coated with a monoclonal antibody of adenine protein; (3) positive control, negative control;

(4) an enzyme-labeled anti-fetal protein monoclonal antibody;

(5) substrate solution, color developing solution, AFP-L3 washing buffer, AFP-L3 eluent, concentrated washing solution, AFP standard,

The pre-assembled spin column for detecting a liver cancer abortin protein heterogeneity consists of an upper separation tube and a lower collection tube, the upper separation tube is provided with a medium coupled with a lectin, and a filter cloth is arranged at the bottom, and the lower collection tube A buffer solution is placed therein.

6. The kit of claim 5, the method of making the method comprising the steps of:

(1) high titer of adenine protein monoclonal antibody coated onto the plate for adsorption overnight;

(2) Washing the plate with Tween phosphate buffer, blocking with Tween phosphate buffer containing bovine serum albumin overnight, drying and drying;

(3) Collect ascites from liver cancer patients, filter and sterilize, and dispense to obtain a positive control or as a positive quality control serum. Collect normal human serum, filter and sterilize, and dispense, to obtain a negative control;

(4) obtaining an enzyme-labeled anti-fetal protein monoclonal antibody;

(5) Pre-filled spin column, chemiluminescent plate labeled with adenine protein monoclonal antibody, positive control, negative control, enzyme-labeled anti-alpha-fetoprotein monoclonal antibody, substrate solution, color development The solution, the AFP-L3 washing buffer, the AFP-L3 eluent, the concentrated washing solution, and the AFP standard are packaged together to obtain a kit.

The kit according to claim 5 or 6, wherein the AFP-L3 washing buffer is 20 匪ol/L Tris-HcL, pH 7.4; the AFP-L3 eluent is 20 mmol. /L Tris- HcL, PH7.4, 150 leg ol/L NaCl and 500 ol/La-mercapto-D-mannosidic; concentrated washing solution (20 times concentrated solution, 20x) prepared in PBS (pH 7.4) 0.05% Tween 20 solution.

8. An enzyme-linked immunoassay kit for detecting alpha-fetoprotein heterogeneity of liver cancer, comprising the following components:

(1) a pre-packed spin column for detecting a hepatic protein abortion protein heterogeneous body;

(2) an ELISA plate coated with a monoclonal antibody to adenine; (3) positive control, negative control;

(4) an enzyme-labeled anti-fetal protein monoclonal antibody;

(5) substrate solution, color developing solution, AFP-L3 washing buffer, AFP-L3 eluent, concentrated washing solution, reaction stop solution, AFP standard,

9. The kit of claim 8 wherein the method comprises the steps of:

(2) Wash the plate with Tween phosphate buffer, and then block it overnight with Tween phosphate buffer containing bovine serum albumin, dry and dry;

(3) Collecting ascites from patients with liver cancer, filtering and sterilizing, and dispensing, that is, obtaining a positive control. Normal human serum is collected, filtered and sterilized, and a negative control is obtained;

(4) obtaining an enzyme-labeled anti-alphafetoprotein monoclonal antibody;

(5) pre-packed spin column, enzyme-labeled plate containing alpha-fetoprotein monoclonal antibody, positive control, negative control, enzyme-labeled anti-fetal protein monoclonal antibody, substrate solution, color developing solution, The AFP-L3 washing buffer, AFP-L3 eluent, concentrated washing solution, reaction stop solution, and AFP standard are packaged together to obtain a kit.

The kit according to claim 8 or 9, wherein the substrate solution is 3 ° / in a phosphate-citrate buffer (pH 5.0). Hydrogen peroxide solution; the color developing solution is tetradecylbenzidine

(TMB) methanol solution, the concentration is 0. lmg / ml; AFP-L3 washing buffer is 20 sec ol / L Tris-HcL, PH7.4; AFP-L3 eluent is 20mmol / L Tris-HcL, PH7. 4, 150 sec ol / L NaCl and 500 匪ol / L α-mercapto-D-mannosidase; reaction stop solution is 2mol / L sulfuric acid; concentrated washing solution (20 times concentrated solution, 20x) is PBS (pH 7.4) Prepared 0.05% Tween 20 solution.