Composition and system for separating and detecting alpha-fetoprotein variant and use thereof

Abstract

Disclosed are a composition and system for separating and detecting an alpha-fetoprotein variant, comprising a separation reagent and a detection reagent; a system for separating and detecting an alpha-fetoprotein variant and a use thereof; and a kit for separating and detecting the alpha-fetoprotein variant. By means of the composition and system for separating and detecting the alpha-fetoprotein variant, and the use thereof, primary liver cancer can be indicated early on, the sensitivity is high, and the method is rapid, simple and automated.

Claims

1. A separation and detection kit for an alpha-fetoprotein variant AFP-L3, comprising a separation reagent, a detection reagent and a reaction plate, wherein the separation reagent comprises a magnetic particle coupling a lectin, and an eluent, wherein the surface of the magnetic particle coupling the lectin is covered with an epoxy resin, the magnetic particle coupling the lectin specifically binds to the alpha-fetoprotein variant AFP-L3 in a sample; the detection reagent comprises a magnetic particle coated with an anti-alpha-fetoprotein antibody and an enzyme-labeled anti-alpha-fetoprotein antibody; and the reaction plate is in a form of a card and comprises a reagent tank and a reaction well.

2. The separation and detection kit according to claim 1, wherein the separation reagent further comprises a protective solution, and the detection reagent further comprises a buffer.

3. The separation and detection kit according to claim 2, wherein the protective solution is 0.02 M PBS pH 7.4, 0.5% BSA and 0.1 M D-mannoside.

4. The separation and detection kit according to claim 2, wherein the buffer is 0.02 M PBS, 10% bovine serum and 0.1% proclin-300.

5. The separation and detection kit according to claim 1 wherein the separation reagent or the detection reagent further comprises a cleaning solution.

6. The separation and detection kit according to claim 5, wherein the cleaning solution in the separation reagent is 20 mM Tris-HCl and 0.5M D-mannoside.

7. The separation and detection kit according to claim 5, wherein the cleaning solution in the detection reagent is a 1% Tween 20 solution prepared in PBS pH 7.4.

8. A separation and detection system for alpha-fetoprotein variant, comprising: a magnetic separation module for separating magnetic particle from a liquid; a detection module for detecting an alpha-fetoprotein variant and an alpha-fetoprotein content; a data processing module for calculating a ratio of the alpha-fetoprotein variant to the alpha-fetoprotein; and the separation and detection kit according to claim 1; the magnetic separation module using the separation reagent in the separation and detection kit for accomplishing the separation of alpha-fetoprotein variant; the detection module using the detection reagent in the separation and detection kit for accomplishing the detection of the alpha-fetoprotein variant content and alpha-fetoprotein content.

9. The separation and detection system according to claim 8, wherein, when the separation and detection system is selected to detect the alpha-fetoprotein content, the magnetic separation module does not use the separation reagent, and the detection means detects the content of alpha-fetoprotein in a sample to be detected; when the separation and detection system is selected to detect the alpha-fetoprotein variant content, the magnetic separation module separates the alpha-fetoprotein variant in the sample to be detected, and the detection module detects the content of the alpha-fetoprotein variant in the sample to be detected; when the separation and detection system is selected to detect a percentage of the alpha-fetoprotein variant, the magnetic separation module separates the alpha-fetoprotein variant in the sample to be detected, the detection module detects the content of alpha-fetoprotein variant and the content of the alpha-fetoprotein in the sample to be detected, and the data processing module calculates the percentage of the alpha-fetoprotein variant.

10. A method for separating and detecting an alpha-fetoprotein variant using the separation and detection kit according to claim 1, the method comprising: a separating step of contacting lectin-labeled magnetic particle with the sample so as to bind to an alpha-fetoprotein variant AFP-L3; an eluting step of separating the magnetic particle and eluting the alpha-fetoprotein variant AFP-L3; and a detecting step of detecting the alpha-fetoprotein variant AFP-L3 by using an AFP immunoassay method.

11. The method for separating and detecting an alpha-fetoprotein variant according to claim 10, wherein the AFP immunoassay method is a magnetic particle chemiluminescence method.

12. The separation and detection kit according to claim 1, wherein the eluent is 0.02 M PBS (pH 7.0), 5M D-mannoside; and the lectin is lens culinaris agglutinin or concanavalin lectin.

13. The separation and detection kit according to claim 1, wherein the anti-alpha-fetoprotein antibody is a monoclonal antibody against alpha-fetoprotein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of an alpha-fetoprotein variant separating and detecting composition according to the present invention, provided in form of card.

(2) Wherein, 1a sample well; 2a reagent tank containing magnetic particle coupling a lectin; 3a reagent tank containing an eluent; 4a reagent tank containing magnetic particle coated with an anti-alpha-fetoprotein antibody; 5a reagent tank containing an enzyme-labeled anti-alpha-fetoprotein antibody; 6a reaction well; 7a reagent card.

(3) FIG. 2 is a schematic diagram of an alpha-fetoprotein variant separating and detecting system according to the present invention.

(4) FIG. 3 is a schematic diagram of one detailed embodiment of the alpha-fetoprotein variant separating and detecting system according to the present invention. Wherein, 1a sample well; 2a reagent tank for pre-dispensing magnetic particle coupling a lectin; 3a reagent tank for pre-dispensing AFP-L3 cleaning solution; 4a reagent tank for pre-dispensing AFP-L3 eluent; 5a reagent tank for pre-dispensing magnetic particle coated with an anti-alpha-fetoprotein antibody (anti-AFP-1); 6a reaction well; 7a reagent tank for pre-dispensing an enzyme-labeled anti-alpha-fetoprotein antibody (anti-AFP-2); 8a reagent tank for pre-dispensing an AFP cleaning solution; 9a reagent tank for pre-dispensing a Luminol substrate; 10a reagent card.

DETAILED EMBODIMENTS

(5) The present invention will be described in detail with reference to the drawings and examples. It should be understood that the specific examples described here only for illustrating the present invention and are not used for limiting the scope of the present invention.

EXAMPLE 1

(6) A separation and detection composition for an alpha-fetoprotein variant provided by the present invention comprises a separation reagent and a detection reagent for detecting a percentage of the alpha-fetoprotein variant.

(7) The separation reagent comprises magnetic particle coupling a lectin, and an eluent; the magnetic particle coupling the lectin are used to specifically bind to AFP-L3 in a sample to be detected; the detection reagent comprises magnetic particle coated with an anti-alpha-fetoprotein antibody and an enzyme-labeled anti-alpha-fetoprotein antibody. The major components of the aforesaid reagents and preparation methods thereof are as follows.

(8) In the magnetic particle coupling a lectin, the lectin is lens culinaris agglutinin (LCA), concanavalin lectin, or lens culinaris agglutinin (LCA) and concanavalin lectin.

(9) The magnetic particle is activated ferriferrous oxide superparamagnetic nanoparticle that are coated with an epoxy resin and have a size of 1 micron (hereinafter referred to as magnetic particle) and lens culinaris agglutinin (LCA) from Sigma company.

(10) 1. The lectin and the magnetic particle is coupled through the following steps:

(11) 1) Weigh 2 mg of LCA and dissolve it in 7.5 mL of a coupling buffer (0.1 mmol/L NaHCO.sub.3, pH 8.3, 0.5 mol/L NaCl), combine it with 1.5 g of washed magnetic particle, mix them up and down in a plugged 10 mL tube (mix at room temperature for 2 h);

(12) 2) Wash uncoupled LCA with 10 mL of the coupling buffer. Based on the measured LCA content in the washing solution, the coupling rate was 98%;

(13) 3) Block the remaining activated groups with 0.2 mol/L glycine;

(14) 4) Perform washing three times with 10 mL 0.1 mol/L acetic acid buffer (pH 4, containing 0.5 mol/L NaCl) and 0.1 mol/L Tris buffer (pH 8, containing 0.5 mol/L NaCl), followed by washing once with 0.1% BSA and 0.1 mmol/L PBS (PBS-BSA) of CaCl.sub.2, temporarily reserve at 4 C.

(15) The aforesaid magnetic particle coated with an epoxy resin may be replaced with magnetic particle coated with materials such as titanium silicide, polystyrene, dextran, agarose, sulfonamide resin, bovine serum albumin, biotin.

(16) 2. Magnetic particle coated with alpha-fetoprotein antibody:

(17) Wherein, for the alpha-fetoprotein antibody, an anti-alpha fetoprotein monoclonal antibody with an item number A8452 manufactured by Sigma company was used as anti-alpha fetoprotein antibody 1 (anti-AFP-1). The preparation method is as follows.

(18) 1) Activation of the magnetic particle

(19) a. suck 50 ml magnetic particles (10% W/V);

(20) b. wash the magnetic particles with an equal volume of 50 mM MES buffer;

(21) c. re-suspend the magnetic particles with an equal volume of 100 mM MES buffer;

(22) d. add an activating reagent carbodiimide (EDC) to a final concentration of 0.04 g/ml;

(23) e. vibrate to perform activation at room temperature for 1 h.

(24) 2) Coating of the magnetic particles with an anti-alpha-fetoprotein antibody 1 (anti-AFP-1)

(25) a. after the activation ends, apply a magnetic field and discard the supernatant;

(26) b. wash the activated magnetic particles with 10 times the volume of 50 mM MES;

(27) c. add 0.2 mg of anti-alpha-fetoprotein antibody 1;

(28) d. vibrate to carry out reaction at room temperature for 3 h.

(29) 3) Termination of the coating of the magnetic particles with an anti-alpha-fetoprotein antibody 1 (anti-AFP-1)

(30) a. after the reaction ends, apply a magnetic field and discard the supernatant;

(31) b. add 10 times the volume of a coating terminating solution;

(32) c. vibrate to carry out reaction at room temperature for 3 h.

(33) 4) Cleaning and preservation of the anti-alpha-fetoprotein antibody 1 (anti-AFP-1) magnetic particles

(34) a. after the reaction ends, apply a magnetic field and discard the supernatant;

(35) b. add 10 times the volume of a coating cleaning solution, of which the main ingredients are 0.02 M PBS, 0.5% Tween-20, 100 mM NaCl, repeatedly clean four times;

(36) c. preserve the anti-alpha-fetoprotein antibody 1 (anti-AFP-1) magnetic particles with 10 times the volume of a magnetic particle preservation solution (pH 7.4, 0.02 M PBS, 0.5% BSA, 2% sucrose, 0.2% Tween-20, 0.2% PC-300).

(37) 3. Enzyme-labeled anti-alpha-fetoprotein antibody

(38) A peroxidase (HRP) from Sigma company was coupled with an anti-alpha-fetoprotein antibody with an item number HPA023600 from Sigma company, to form an anti-alpha-fetoprotein antibody 2 (anti-AFP-2). The preparation method is as follows.

(39) 1) Oxidation of enzyme (keep dark during the whole process)

(40) a. weigh 5 mg HRP, dissolve it with 250 l of ddH.sub.2O;

(41) b. weigh 5 mg NaIO.sub.4, dissolve it with 250 l of ddH.sub.2O to a concentration of 20 mg/ml;

(42) c. add the NaIO.sub.4 solution dropwise to the HRP solution while stirring;

(43) d. place the mixed solution at 4 C., stand for 30 minutes;

(44) e. weigh 5 ml of ethylene glycol and dissolve it in 25 l of ddH.sub.2O, and add the resulting solution dropwise to the above mixed solution while stirring;

(45) f. stand for 30 minutes at room temperature;

(46) g. The oxidation of enzyme finished, and the final HRP concentration was 10 mg/ml.

(47) 2) Preparation and labeling of the anti-alpha-fetoprotein antibody 2 (anti-AFP-2) (keep dark):

(48) a. adjust the anti-alpha-fetoprotein antibody concentration to about 5 mg/ml (concentrate with PEG 20000 if the protein concentration was too low), perform dialysis to remove glycerol or impurities (such as Tris) with 50 mmol/L CB at about pH 9.5 (mix 1 mol/L NaHCO.sub.3 with 1 mol/L Na.sub.2CO.sub.3 at a ratio of 10:1, dilute the mixture 20 times with a distilled water before use), perform dialysis overnight at 4 C., while exchanging the liquid three times;

(49) b. mix the anti-alpha-fetoprotein antibody 2 (anti-AFP-2) and HRP at a ratio of 1:4, and subject the mixture to dialysis in 50 mmol/L pH 9.5 CB for 6 hours or more, while exchanging the liquid once in the first two hours;

(50) c. terminate the reaction with 0.2 ml of freshly prepared 5 mg/ml NaBH.sub.4 solution, shake well, and stand at 4 C. for 2 hours, while shaking every half hour;

(51) d. perform dialysis overnight with 10 mM PBS at pH 7.2 (prepare a 0.01 mol/L stock solution of Na.sub.2HPO.sub.4 and NaH.sub.2PO.sub.4, mix them evenly to form a PBS buffer according to the desired pH), while exchanging the liquid once.

(52) 3) Subpackage: dilute the HRP enzyme labeled anti-alpha-fetoprotein antibody 2 (anti-AFP-2) obtained in step 2) to 1 mg/ml (calculated based on the initial antibody concentration) with a buffer containing 10% fetal bovine serum, of which the main ingredients are 0.02M PBS, 10% fetal bovine serum, 3% sucrose, 0.2% Tween-20, etc., subpackage and store at 4 C.

(53) 4. Step of separating the alpha-fetoprotein variant by using the magnetic particles coupling the lectin in this example:

(54) 1) add 50 l of 1% magnetic particles coupling the lectin and 200 l of serum samples to be tested in the same tube, mix well and stand for 5 minutes;

(55) 2) adsorb the magnetic particles at the bottom of the tube with a magnet, and then absorb the supernatant and discard it;

(56) 3) remove the magnet, add 500 l of an AFP-L3 cleaning solution, mix well to ensure that the magnetic particles are completely suspended;

(57) the AFP-L3 cleaning solution: 0.02 M PBS (pH 7.0), 0.5% Tween-20;

(58) 4) adsorb the magnetic particles at the bottom of the tube with a magnet, and then absorb the supernatant and discard it;

(59) 5) repeat steps 3), 4) twice, and wash the magnetic particles;

(60) 6) remove the magnet, add 200 l of an AFP-L3 eluent, mix well to ensure that the magnetic particles are completely suspended, stand for 10 minutes;

(61) AFP-L3 eluent: 0.02 M PBS (pH 7.0), 5 M D-mannoside,

(62) or 20 mm Tris-HCl, NaCl 150 mm, pH 7.4 buffer, which containing 500 mm -methyl-D-mannoside, 0.1% Proclin 300;

(63) 7) adsorb the magnetic particles at the bottom of the tube with a magnet, to obtain the supernatant which is a liquid of the separated alpha-fetoprotein variant AFP-L3.

(64) 5. Step of detecting AFP by using the magnetic particles coated with anti-alpha-fetoprotein antibody (anti-AFP-1) and an HRP enzyme-labeled anti-alpha-fetoprotein antibody 2 (anti-AFP-2) in this example:

(65) 1) add 50 l of 1% magnetic particles coated with anti-alpha-fetoprotein antibody (anti-AFP-1) in an assay tube, add 50 l of a sample to be detected at the same time, followed by addition of 50 l of 1000-fold diluted HRP enzyme-labeled anti-alpha-fetoprotein antibody 2 (anti-AFP-2), mix well, and perform incubation at 37 C. for 5 minutes;

(66) 2) adsorb the magnetic particles at the bottom of the tube with a magnet, and then absorb the supernatant and discard it;

(67) 3) remove the magnet, add 500 l of an AFP cleaning solution, mix well to ensure that the magnetic particles are completely suspended;

(68) the AFP cleaning solution: 0.02 M PBS (pH 7.0), 0.5% Tween-20;

(69) 4) adsorb the magnetic particles at the bottom of the tube with a magnet, and then absorb the supernatant and discard it;

(70) 5) repeat steps 3), 4) twice, and wash the magnetic particles;

(71) 6) remove the magnet, add 100 l of a Luminol substrate, mix well to ensure that the magnetic particles are completely suspended, perform incubation at 37 C. for 1 minute;

(72) 7) measure the intensity of a luminescence signal on a chemiluminescence analyzer, and calculate the content of AFP in the sample according to a standard curve.

(73) 6. Step of detecting AFP-L3 in this example:

(74) 1) add 50 l of 1% magnetic particles coupling the lectin and 200 l of serum samples to be tested in the same tube, mix well and stand for 5 minutes;

(75) 2) adsorb the magnetic particles at the bottom of the tube with a magnet, and then absorb the supernatant and discard it;

(76) 3) remove the magnet, add 500 l of an AFP-L3 cleaning solution, mix well to ensure that the magnetic particles are completely suspended;

(77) the AFP-L3 cleaning solution: 0.02 M PBS (pH 7.0), 0.5% Tween-20;

(78) 4) adsorb the magnetic particles at the bottom of the tube with a magnet, and then absorb the supernatant and discard it;

(79) 5) repeat steps 3), 4) twice, and wash the magnetic particles;

(80) 6) remove the magnet, add 200 l of an AFP-L3 eluent, mix well to ensure that the magnetic particles are completely suspended, stand for 10 minutes;

(81) AFP-L3 eluent: 0.02 M PBS (pH 7.0), 5 M D-mannoside,

(82) or 20 mm Tris-HCl, NaCl 150 mm, pH 7.4 buffer, which containing 500 mm -methyl-D-mannoside, 0.1% Proclin 300;

(83) 7) adsorb the magnetic particles at the bottom of the tube with a magnet, to obtain the supernatant which is a liquid of the separated alpha-fetoprotein variant AFP-L3;

(84) 8) add 50 l of 1% magnetic particles coated with anti-alpha-fetoprotein antibody (anti-AFP-1) in an assay tube, add 50 l of the liquid obtained in 7) at the same time, followed by addition of 50 l of 1000-fold diluted HRP enzyme-labeled anti-alpha-fetoprotein antibody 2 (anti-AFP-2), mix well, and perform incubation at 37 C. for 5 minutes;

(85) 9) adsorb the magnetic particles at the bottom of the tube with a magnet, and then absorb the supernatant and discard it;

(86) 10) remove the magnet, add 500 l of an AFP cleaning solution, mix well to ensure that the magnetic particles are completely suspended;

(87) the AFP cleaning solution: 0.02 M PBS (pH 7.0), 0.5% Tween-20;

(88) 11) adsorb the magnetic particles at the bottom of the tube with a magnet, and then absorb the supernatant and discard it;

(89) 12) repeat steps 3), 4) twice, and wash the magnetic particles;

(90) 13) remove the magnet, add 100 l of a Luminol substrate, mix well to ensure that the magnetic particles are completely suspended, perform incubation at 37 C. for 1 minute;

(91) 14) measure the intensity of a luminescence signal on a chemiluminescence analyzer, and calculate the content of AFP in the detected sample according to a standard curve.

(92) 7. Step of detecting a percentage (AFP-L3%) of the alpha-fetoprotein variant in this example:

(93) 1) detect the alpha-fetoprotein AFP content in the sample according to the above item 5;

(94) 2) detect the alpha-fetoprotein variant AFP-L3 content in the sample according to the above item 6;

(95) 3) divide the alpha-fetoprotein variant AFP-L3 content calculated in 2) by the alpha-fetoprotein AFP content, to obtain the percentage (AFP-L3%) of the alpha-fetoprotein variant.

(96) The separation and detection composition described in this example was used to perform detection of the AFP content:

(97) An AFP detection kit (electrochemiluminescence method) manufactured by Roche company was used as a control group, to compare the accuracy of AFP content detection results. The cutoff value of AFP content is 20 g/L, so results higher than 20 g/L were positive results, and results lower than 20 g/L were negative results. The detection results are shown in Table 1:

(98) TABLE-US-00001 Positive Negative samples samples 198 254 Roche AFP detection kit 198 254 This example 198 254

(99) The detection results show that the sensitivity and specificity of this example reached 100% in 452 samples detected.

(100) The separation and detection composition described in this example was used to detect a percentage of AFP-L3:

(101) An alpha-fetoprotein variant separation tube manufactured by Beijing Hotgen Biotech CO., Ltd., of which the product registration certificate number is No. 3401646 from 2014 (approved) by State Food and Drug Administration, was used while using an AFP detection kit (electrochemiluminescence method) manufactured by Roche company was used as a control group, to compare the accuracy of AFP-L3 percentage detection results. The cutoff value of AFP-L3 percentage is 10%. The detection results are shown in Table 2:

(102) TABLE-US-00002 TABLE 2 Positive Negative samples samples 107 172 Roche AFP detection kit 107 172 (electrochemiluminescence method) This example 107 169

(103) As compared with the control group, AFP-L3 percentage of 107 samples, of which the AFP-L3 percentage was positive in the detection of the control group, was also positive in the detection of this example; among 172 samples of which the AFP-L3 percentage was negative in the detection of the control group, 169 samples had negative results in the detection of this example. For the other three samples, after comparing the clinical diagnosis, it was found that the patients were patients with early primary hepatocellular carcinoma. As shown by the detection results, the separation and detection composition described in this example was more sensitive than the detection performance of the control group, for which the reason should be that the AFP-L3 protein separation efficiency is improved so that it is possible to more accurately detect AFP-L3 percentage in the sample.

EXAMPLE 2

(104) A separation and detection composition for an alpha-fetoprotein variant provided by the present invention according to the separation and detection composition for an alpha-fetoprotein variant provided in Example 1, wherein the separation reagent may further comprise a protective solution, and the detection reagent may further comprise a buffer.

(105) The separation reagent and the detection reagent may comprise a cleaning solution, respectively.

(106) Major ingredients of the aforesaid reagents are as follows:

(107) the protective solution: 0.02 M PBS, 0.5% BSA, pH 7.4, 0.1 M D-mannoside,

(108) the buffer: 0.02 M PBS, 10% bovine serum, 0.1% proclin-300,

(109) the cleaning solution in the separation reagent: 20 mM Tris-HCl, 0.5 M D-mannoside,

(110) the cleaning solution in the detection reagent: 1% Tween 20 solution prepared by PBS pH 7.4.

(111) The above-mentioned D-mannoside can be replaced with carbohydrates such as fucose, fructose, sucrose and trehalose.

EXAMPLE 3

(112) A separation and detection system for an alpha-fetoprotein variant provided by the present invention is used for detecting a percentage of the alpha-fetoprotein variant. As shown by FIG. 2,

(113) it comprises a magnetic separation module for separating the alpha-fetoprotein variant, a detection module for detecting an alpha-fetoprotein variant content and an alpha-fetoprotein content, and a data processing module for calculating a ratio of the alpha-fetoprotein variant to the alpha-fetoprotein. Preferably, the separation and detection 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 with model number H10682-110 or a photomultiplier tube with model number R1166 manufactured by Nippon Hamamatsu Photonics Co. Ltd.

(114) The reagent card comprises the separation and detection composition described in Example 1, which is pre-dispensed into a plurality of reagent tanks on the reagent card, to carry out the reaction, each reagent having at least one reagent tank. The reagent card comprises a sample well, a separation reagent tank, a detection reagent tank and a reaction well, and the reagent card in this example, as shown in FIG. 3, has a constitution as follows:

(115) 1. a sample well; 2. a reagent tank for pre-dispensing magnetic particles coupling a lectin; 3. a reagent tank for pre-dispensing AFP-L3 cleaning solution; 4. a reagent tank for pre-dispensing AFP-L3 eluent; 5. a reagent tank for pre-dispensing magnetic particles coated with an anti-alpha-fetoprotein antibody (anti-AFP-1); 6. a reaction well; 7. a reagent tank for pre-dispensing an enzyme-labeled anti-alpha-fetoprotein antibody (anti-AFP-2); 8. a reagent tank for pre-dispensing an AFP cleaning solution; 9. a reagent tank for pre-dispensing a Luminol substrate.

(116) Wherein, wells b, c, d together compose a magnetic separation module, and all the reagents therein may be used for accomplishing the separation of alpha-fetoprotein variant; wells e, f, g, h, I together compose a detection module, and all the reagents therein may be used for accomplishing the detection of the concentration of AFP added to the sample.

(117) The use of the detection module alone can accomplish detection of AFP content in the sample; the use of the separation module and the detection module in combination can accomplish separation of alpha-fetoprotein variant AFP-L3 in the sample and detection of the AFP-L3 content. Then, a percentage of the alpha-fetoprotein variant is obtained by dividing the AFP content by the AFP-L3 content.

(118) The sample to be detected was detected in the method described in this example, and the detection results are shown in Table 3:

(119) TABLE-US-00003 Positive rate AFP AFP-L3% Sample background number (>=20 ng/ml) (>=10%) Primary hepatocellular carcinoma 216 100% 92% Healthy people 324 0 0 liver cirrhosis 105 100% 5% liver hepatitis 174 100% 3% other cancers* 63 8% 0% *Other cancers include esophageal cancer, lung cancer, leukemia.

(120) As shown by the results, the present invention achieved a positive rate of 92% to primary hepatocellular carcinoma, a specificity of 100% to healthy people, and a specificity of 95% and 97% respectively to liver cirrhosis and liver hepatitis, and a specificity of 0% to other cancers.

EXAMPLE 4

(121) The separation and detection system for an alpha-fetoprotein variant provided according to Example 3 further comprises a detection setting module, which comprises an alpha-fetoprotein content measurement unit, an alpha-fetoprotein variant content measurement unit, and a unit for measuring percentage of alpha-fetoprotein variant.

(122) When the alpha-fetoprotein content measurement unit was selected, the magnetic separation module did not take part in the treatment of a sample to be detected, and the detection means detected the content of alpha-fetoprotein in the sample to be detected.

(123) When the alpha-fetoprotein variant content measurement unit was selected, the magnetic separation module separated the alpha-fetoprotein variant in the sample to be detected, the detection module detects the content of alpha-fetoprotein variant and the content of alpha-fetoprotein in the sample to be detected, and the data processing module calculates a percentage of the alpha-fetoprotein variant.

EXAMPLE 5

(124) The separation and detection system for an alpha-fetoprotein variant provided according to Example 3 or 4 employs the separation and detection composition described in Example 2 instead of the separation and detection composition described in Example 1.

EXAMPLE 6

(125) The method for separating and detecting an alpha-fetoprotein variant provided by the present invention employs the aforesaid separation and detection system to detect a percentage of an alpha-fetoprotein variant, comprising the following steps:

(126) 1) adding sample

(127) add a sample of hemolytic serum, plasma, or whole blood to the separation and detection system or added to the sample wells provided by the aforesaid reagent card;

(128) 2) separating the alpha-fetoprotein variant

(129) add the sample to a reaction cup containing magnetic particle coupling a lectin, shake well;

(130) concentrate the magnetic particle by a magnetic separation module and discard the liquid;

(131) add an eluent to the reaction cup in which the magnetic particle has been concentrated, shake well, and concentrate the magnetic particles by the magnetic separation module, to obtain an alpha-fetoprotein variant eluent;

(132) 3) incubation

(133) add the alpha-fetoprotein variant eluent to a reaction cup containing magnetic particle coated with alpha-fetoprotein monoclonal antibody, and meanwhile add an enzyme-labeled anti-alpha-fetoprotein antibody to the reaction cup, to undergo incubation;

(134) 4) concentration and cleaning

(135) concentrate the magnetic particle by the magnetic separation module, and discard the liquid; add a cleaning solution to the reaction cup, shake well, and then concentrate the magnetic particle by the magnetic separation module once again, and discard the liquid;

(136) 5) color development

(137) add the concentrated magnetic particle to a luminol substrate, and obtain a concentration of alpha-fetoprotein heterosomes by a data processing module;

(138) 6) at the same time with 2), add a sample to the reaction cup, and add magnetic particle coated with alpha-fetoprotein monoclonal antibody, and shake well; repeat steps 3) to 5) to obtain a concentration of alpha-fetoprotein;

(139) 7) obtain a percentage of alpha-fetoprotein variant by the data processing module.

EXAMPLE 7

(140) The method for separating and detecting an alpha-fetoprotein variant provided by Example 6 only comprises steps 1) and 6), and can directly detect the concentration of alpha-fetoprotein.

EXAMPLE 8

(141) The method for separating and detecting an alpha-fetoprotein variant provided by the present invention employs the aforesaid separation and detection system to detect a percentage of an alpha-fetoprotein variant, comprising the following steps:

(142) 1) adding sample

(143) add a sample of hemolytic serum, plasma, or whole blood to the separation and detection system or added to the sample wells provided by the aforesaid reagent card.

(144) 2) separating the alpha-fetoprotein variant

(145) add the sample to a reagent tank containing magnetic particle coupling a lectin, shake well;

(146) concentrate the magnetic particle by a magnetic separation module and discard the liquid;

(147) add the concentrated magnetic particle to a reagent tank containing a cleaning solution, shake well;

(148) add the cleaned magnetic particle to a reagent tank containing an eluent, shake well, and concentrate the magnetic particle by the magnetic separation module, to obtain an alpha-fetoprotein variant eluent;

(149) 3) incubation

(150) add the alpha-fetoprotein variant eluent to a reaction cup containing magnetic particle coated with alpha-fetoprotein monoclonal antibody, and meanwhile add an enzyme-labeled anti-alpha-fetoprotein antibody pre-packed in another reagent tank, to undergo incubation.

(151) 4) concentration

(152) concentrate the magnetic particle by the magnetic separation module, and discard the liquid;

(153) 5) cleaning

(154) add the concentrated magnetic particle to a reagent tank containing a cleaning solution, shake well, and repeat step 4);

(155) 6) color development

(156) add the concentrated magnetic particle to a luminol substratepre-packed in another reagent tank, and obtain a concentration of alpha-fetoprotein heterosomes by a data processing module.

(157) 7) at the same time with 2), the system adds a sample to a reagent tank containing magnetic particles coated with alpha-fetoprotein monoclonal antibody, repeat steps 3) and 6) to obtain the concentration of alpha-fetoprotein;

(158) 8) obtain a percentage of alpha-fetoprotein variant by the data processing module.

(159) Logical algorithm of the percentage of alpha-fetoprotein variant by the data processing module: after determining the serial number of a sample, the instrument will retrieve concentration of alpha-fetoprotein and concentration of alpha-fetoprotein variant for the same sample, and then calculate the percentage of AFP-L3 in AFP, thereby calculating the AFP-L3 content, i.e., AFP-L3%.

(160) The foregoing description shows and describes preferred embodiments of the present invention. As described above, it should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as an exclusion of other embodiments but may be used in a variety of other combinations, modifications and the environment, and may be modified within the scope of the inventive concept described herein, by the teachings or techniques or knowledge of the related art. Besides, modifications and changes made by one skilled in the art without departing from the spirit and scope of the present invention should be within the scope of the appended claims.