CN204422547U - A kind of separation detection kit of alpha-fetoprotein variant and device - Google Patents
A kind of separation detection kit of alpha-fetoprotein variant and device Download PDFInfo
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Abstract
The utility model relates to a kind of separation detection composition of alpha-fetoprotein variant, comprise separation agent and detect reagent, and the separation detecting system of alpha-fetoprotein variant and application, also relate to a kind of separation detection kit of alpha-fetoprotein variant simultaneously, the separation detection composition of a kind of alpha-fetoprotein variant provided by the utility model, system and application thereof, can early primary hepatocarcinoma be pointed out, there is high sensitivity, the fast and convenient and robotization of method.
Description
Technical field
The utility model relates to a kind of separation detection kit and device of alpha-fetoprotein variant, belongs to medicine equipment and in-vitro diagnosis field.
Background technology
Hepatocellular carcinoma (hepatocellular carcinoma, HCC) be the 4th common malignant tumour in world wide, nearly 250000 patients die from hepatocellular carcinoma every year, the pathogenesis of hepatocellular carcinoma is unclear yet so far, grade of malignancy is higher clinically for it, mortality ratio ranked third position in malignant tumor of digestive tract, so early detection, early treatment is the key improving survival, and Most patients middle and advanced stage when going to a doctor, lose best occasion for the treatment, hazards comprise the chronic hepatitis and cirrhosis that are caused by hepatitis type B virus and hepatitis C virus.Be effectively treated, the clinical efficiency of examination and generaI investigation depends on early diagnosis.HCC is the important kind in tumor aetiology, and the chronic liver damage that cirrhosis, virus hepatitis, chemical carcinogen and environmental factor etc. cause all can bring out HCC.HCC grade of malignancy is high, easily recurrence and transfer, poor prognosis, and early diagnosis is more difficult, is easy to incur loss through delay the optimal treatment phase.
Alpha-fetoprotein (alphafetoprotein, AFP) is a kind of glycoprotein, and the AFP content detected in blood is the modal means of current diagnosis liver cancer.AFP was synthesized by liver parenchymal cell and yolk sac cell in the mammal embryo phase, derived from endoblastic gastrointestinal tract mucous cell and also can synthesize on a small quantity.Under normal circumstances, AFP is mainly present in fetal circulation, and along with development of fetus is ripe, AFP synthesis reduces gradually.During birth, in bleeding of the umbilicus, AFP concentration can reach 10 ~ 100mg/L, is born and is down to adult normal's level in latter 1 year.If neonate AFP obviously raises prompting neonatal hepatitis, CBA or has the embryonic malignant tumor secreting AFP.AFP is also have suitable ratio to there is positive findings in hepatopathy and cirrhosis disease as the shortcoming of early liver cancer diagnosis index, AFP slightly increases, and (20 ~ 200ng/m1) sees a considerable amount of patients with chronic liver, and in liver cirrhosis patient, 11.7-44%AFP is positive.Therefore, when differentiating optimum hepatopathy and malignancy hepatic tumor, AFP greatly reduces as the value of early liver cancer screening indexes.
The structure of the many sugar chains of AFP heteroplasmon is not yet completely clear.Think at present, usually said AFP heteroplasmon is actual refers to the A FP-L3 be combined with LCA, one of the 4th national liver cancer academic conference hepatocarcinoma mark thing being classified as primary carcinoma of liver clinical criteria in 1999.Within 2011, AFP-L3 is classified as diagnosing cancer of liver specific index by Chinese liver cancer diagnosis and treatment specification; For many years, AFP-L3 is acknowledged as the primary carcinoma of liver index more more special than simple AFP alpha-fetoprotein.
AFP-L3 is the albumen uniquely generated by cancer cell in patient's liver.In Canada and a multicenter of the U.S., perspective, double blinding, long-term clinical trials, this detection method is studied.Result display AFP-L3% raises the patient of (more than 15%), and in ensuing 21 middle of the month, the danger that hepatocellular carcinoma occurs increases by 7 times more than.According to existing hepatocellular carcinoma tumor practice guideline, these Patients ' Hepatocytes carcinogenesis rates extremely increase.
The clinical meaning that AFP heteroplasmon detects:
1) liver cancer and optimum hepatopathy is differentiated.Patients with Primary AFP often raises, but many benign liver d iseases also can have AFP to raise, and is sometimes difficult to distinguish benign and malignant lesions only according to AFP result.Now AFP heteroplasmon detects and just has good clinical meaning, especially has good value for AFP person between 30 ~ 400ng/ml.Yozhiaki has carried out perspective study to 361 routine cirrhosis, and in 53 patients of routine AFP at more than 30ug/L, after 2 years, 21 examples develop into liver cancer, make a definite diagnosis the patient AFP of during liver cancer 39% at below 400ug/L.Hepatocellular carcinoma (HCC) group and non-HCC group AFP measured value when comparative study starts, do not found differences conspicuousness, during research discovery pathology, the heteroplasmonic type of AFP is different, LCA positive rate 87.12% false positive rate 21.5% is diagnosed to HCC, ConA positive rate 89.17%, false positive rate 17.15%.Current result of study is greater than 15% as the positive indication of liver cancer using AFP-L3 content.
2) monitoring after Liver Cancer Operation.Post hepatectomy of liver cancer, Serum AFP content declines thereupon, and its decline rate depends on residual AFP amount and half life period in body, and turn out cloudy in general February, when turning out cloudy, AFP heteroplasmon disappears thereupon.If AFP obviously declines but do not turn out cloudy, heteroplasmon change is not obvious, then prompting operation is not thorough, also may there are margin residual, blood vessel cancer embolus, stellate ganglion or transfer etc.If heteroplasmon drops to less than 25%, AFP and heteroplasmon relative concentration is constant, then may be that patient has caused by hepatitis or cirrhosis.
3) embryo's anormogenesis and fetal congenital illness.In Normal Pregnancy maternal serum, in AFP and embryo, AFP is in equilibrium state, once fetal anomaly or placental barrier occur abnormal, fetal serum can be caused to infiltrate in amniotic fluid or amniotic fluid infiltrates maternal serum, cause maternal amniotic fluid or Serum AFP sharply to raise.But only measure AFP total amount and have certain limitation.Experiment shows neural-tube defect, anencephalus or spina bifida etc.Children Hepatoblastoma, Biliary atresia, gonadal tumor, malignant teratoma etc. can have the heteroplasmonic positive of AFP and/or AFP.
Lectin affinity chromatography method, polyacrylamide gel electrophoresis, imprinting method, affine crossed immunoelectrophoresis is had at present for the heteroplasmonic detection method of AFP, these methods all can directly be isolated AFP-L3 albumen and carry out quantitative estimation, can be divided into according to final detection method:
Coomassie brilliant blue: the sample after electrophoresis directly with Coomassie brilliant blue dyeing, observes peak band after wash-out.Method is simple, but disturbing factor is many, and sensitivity is about 1000ug/L.
Enzyme linked immunosorbent assay: develop the color with diaminobenzidine after incubation, rinsing together with the sample after electrophoresis with the antibody of peroxidase labelling, detection sensitivity can be increased to 50ug/L.
Silver Staining Method: the direct and staphylococcus aureus protein A 2 glue gold incubation by the sample after electrophoresis, then develop the color with silver-colored nitrite ion, the peak band obtained is clear, and sensitivity can reach 32ug/L.
Autoradiography: be the most frequently used up to now detection method of China, sensitivity reaches 31ug/L.Its principle is that sample is carried out separation electrophoresis in containing the gel of agglutinin, after carry out secondary electrophoresis adding in the gel containing 125IAFP, AntiAFP antibody, after electrophoresis terminates, gel is dried, covers X-ray film exposure, be flushed into picture.Whole experimentation complicated operation, for a long time, domestic only have minority clinical labororatory can measure AFP heteroplasmon, and a few cities can meet the heteroplasmonic requirement of clinical assays AFP, and does not have domestic reagent supply.
Current domestic main employing centrifuge tube separation method in addition, the method adopts agarose coupling LCA, adopts centrifugal method to be separated AFP-L3, and then detects with AFP reagent.Centrifuge tube separation method is the method that the approval of current domestic unique acquisition Bureau of Drugs Supervision is applicable to AFP-L3% detection.
During existing method measures alpha-fetoprotein variant accounting, manual procedure is many, complex steps, needs support equipment many, length consuming time, and can not robotization be realized, therefore, cannot realize high-throughout pattern detection, and it is comparatively large to be subject to manual operation impact, finally can cause the deviation of testing result.
Utility model content
In order to overcome the deficiencies in the prior art, the utility model provides a kind of separation detection kit and separation detecting device of alpha-fetoprotein variant, by detecting alpha-fetoprotein variant content and α-Fetoprotein in blood sample, obtain alpha-fetoprotein variant accounting.
The separation detection kit of a kind of alpha-fetoprotein variant that the utility model provides, what comprise reagent card and alpha-fetoprotein variant is separated detection composition, described reagent card comprises sample aperture, separation agent groove, detects reagent trough and reacting hole, the separation detection composition of described alpha-fetoprotein variant, comprise separation agent and detect reagent, described separation agent comprises magnetic-particle and the eluent of coupling agglutinin, and the magnetic-particle of described coupling agglutinin is used for and the AFP-L3 specific binding in detected sample; Described detection reagent comprises by the anti-alpha-fetoprotein antibody of the magnetic-particle of alpha-fetoprotein antibody, marker enzyme.
The utility model additionally provides the optimal technical scheme of above-mentioned separation detection kit.
As preferably, described separation agent also comprises protection liquid, for increasing separation agent stability and improve separation efficiency.
As preferably, described detection reagent also comprises damping fluid, for increasing detect reagent stability, improve detection sensitivity and detection specificity.
As preferably, described separation agent and/or detect reagent and also comprise cleaning fluid, for the sensitivity improving joint efficiency, reduce non-specific adsorption, improve separation and detection.
As preferably, in the magnetic-particle of described coupling agglutinin, agglutinin comprises LcA and/or ConA.
As preferably, in the magnetic-particle of described coupling agglutinin, the macromolecule component of magnetic-particle surface coverage comprises silicide, polysaccharide, albumen, cellulose or resin.
As preferably, described composition provides with cassette or bar formula.
The utility model further provides a kind of separation detection kit of alpha-fetoprotein variant, comprises above-mentioned separation detection composition.
The utility model further provides again a kind of separation detecting device of alpha-fetoprotein variant, comprising:
Magneto separate module, for being separated magnetic particle in liquid, Magneto separate module coordinates separation agent and detects reagent use;
Detection module, for detecting alpha-fetoprotein variant and α-Fetoprotein;
Data processing module, for calculating alpha-fetoprotein variant relative to alpha-fetoprotein ratio, and
The above-mentioned separation detection composition of any one or mentioned reagent box.
Described Magneto separate module matches with the described separation agent be separated in detection composition, for completing the separation to alpha-fetoprotein variant;
Described detection module coordinates with the described detection Reagent evaluation be separated in detection composition, for completing the detection of content to alpha-fetoprotein variant and α-Fetoprotein.
The utility model additionally provides the optimal technical scheme of above-mentioned separation detecting device.
As preferably,
When selecting to measure α-Fetoprotein, described Magneto separate module does not match with separation agent, and described pick-up unit detects the content of alpha-fetoprotein in detected sample,
When selecting to measure alpha-fetoprotein variant content, described Magneto separate module is separated alpha-fetoprotein variant in detected sample, and described detection module detects the content of alpha-fetoprotein variant in detected sample;
When selecting to measure alpha-fetoprotein variant accounting, described Magneto separate module is separated alpha-fetoprotein variant in detected sample, described detection module detects the content of alpha-fetoprotein variant and the content of alpha-fetoprotein in detected sample, described data processing module calculates alpha-fetoprotein variant accounting, i.e. the accounting (AFP-L3%) of alpha-fetoprotein variant (AFP-L3) in total alpha-fetoprotein (AFP).
The utility model finally provides a kind of above-mentioned separation detection kit or above-mentioned separation detecting device is being separated the application detected in alpha-fetoprotein variant.
Compared with prior art, the beneficial effects of the utility model are: operator only needs to add the simple operationss such as sample, in 30 minutes, just can complete detection; The method directly can quantitatively calculate alpha-fetoprotein contained in blood sample and the content of alpha-fetoprotein variant by detecting simultaneously, and obtains AFP-L3% simultaneously; Method is easy, detects quick, and result is accurate, highly sensitive, and robotization, for the prevention of liver cancer, diagnosis, treatment provide support.
Accompanying drawing explanation
Fig. 1 is that the separation detection composition of alpha-fetoprotein variant of the present utility model provides schematic diagram with cassette,
Wherein, 1-sample aperture, 2-reagent trough of the magnetic-particle of coupling agglutinin is housed, 3-reagent trough of eluent is housed, 4-bag is housed by the reagent trough of the magnetic-particle of alpha-fetoprotein monoclonal antibody, 5-reagent trough of the anti-alpha-fetoprotein antibody of marker enzyme is housed, 6-reacting hole, 7-reagent card;
Fig. 2 is the schematic diagram of the separation detecting system of alpha-fetoprotein variant of the present utility model.
Embodiment
Below in conjunction with drawings and Examples, the utility model is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the utility model, and be not used in restriction the utility model.
Embodiment 1
The separation detection composition of the alpha-fetoprotein variant that the utility model provides, comprises separation agent and detects reagent, for detecting alpha-fetoprotein variant accounting.
Separation agent comprises magnetic-particle and the eluent of coupling agglutinin, and the magnetic-particle of coupling agglutinin is used for and the AFP-L3 specific binding in detected sample; Detect reagent to comprise by the anti-alpha-fetoprotein antibody of the magnetic-particle of alpha-fetoprotein monoclonal antibody, marker enzyme.Key component and the preparation method of mentioned reagent are as follows:
The magnetic-particle of coupling agglutinin:
Wherein,
Agglutinin adopts LcA (LCA), ConA or LcA and ConA.
Magnetic-particle adopts the magnetic-particle being enclosed with epoxy resin of activation and the LCA of Sigma company.
Agglutinin and magnetic-particle adopt following steps coupling:
(1) take 2mgLCA, be dissolved in 7.5mL coupling buffer (0.1mmol/L NaHCO3, pH 8.3,0.5mol/L NaCl), merge with the 1.5g magnetic-particle through washing, with mixing (room temperature, 2h) of turning upside down with the 10mL test tube of plug;
(2) LCA of non-coupling is washed away with 10mL coupling buffer.LCA I content after measured in cleansing solution, counts Conjugate ratio is 98%;
(3) residue activating gene is closed with 0.2mol/L glycocoll;
(4) 10mL 0.1mol/L acetate buffer solution (pH 4, containing 0.5mol/L NaCl) and 0.1mol/L Tris damping fluid (pH 8, containing 0.5mol/L NaCl) is used to wash 3 times successively, then to contain 0.1%BSA and 0.1mmol/L CaCl
2pBS (PBS-BSA) wash 1 time, 4 DEG C are temporary for subsequent use.
The above-mentioned magnetic-particle being enclosed with epoxy resin can also adopt the magnetic-particle being enclosed with the materials such as titanium silicide, polystyrene, glucosan, agarose, sulfonamide resin, bovine serum albumin(BSA), biotin to substitute.
AFP-L3 eluent:
0.02M PBS (pH7.0), 5MD-mannoside,
Or 20mmTris-Hcl, NaCL 150mm, ph7.4 damping fluid, wherein containing 500mm Alpha-Methyl-D-MANNOSE glycosides, 0.1%, Proclin 300.
Bag is by the magnetic-particle of alpha-fetoprotein antibody:
Wherein,
Alpha-fetoprotein antibody adopts anti-alpha-fetoprotein antibody 1 (anti-AFP-1), and its preparation method is as follows:
(1) activation of magnetic-particle
A. magnetic-particle 50ml (10%W/V) is drawn;
B. equal-volume 50mM MES washs magnetic-particle;
C. the resuspended magnetic-particle of equal-volume 100mM MES is used;
D. activating reagent EDC is added, final concentration 0.04g/ml;
E. room temperature concussion activation 1h
(2) the bag quilt of anti-alpha-fetoprotein antibody 1 (anti-AFP-1) and magnetic-particle
A. activate end, add magnetic field abandoning supernatant;
B.10 times volume 50mM MES washs the rear magnetic-particle of activation;
C. 0.2mg antibody is added
D. room temperature concussion reaction 3h;
(3) termination of anti-alpha-fetoprotein antibody 1 (anti-AFP-1) and magnetic-particle bag quilt
A. react end, add magnetic field abandoning supernatant;
B. add 10 times of body bags and be terminated liquid;
C. normal temperature concussion reaction 3h
(4) cleaning of anti-alpha-fetoprotein antibody 1 (anti-AFP-1) magnetic-particle is preserved
A. react end, add magnetic field abandoning supernatant;
B. 10 times of body bags are added by cleaning fluid, repeated washing four times;
C.10 times volume magnetic bead conserving liquid preserves anti-alpha-fetoprotein antibody 1 (anti-AFP-1) magnetic-particle
The anti-alpha-fetoprotein antibody of marker enzyme:
Adopt the anti-alpha-fetoprotein antibody 2 (anti-AFP-2) of coupling peroxidase (HRP), preparation method is as follows:
(1) oxidation (overall process lucifuge) of enzyme:
A. take HRP 5mg, add ddH
2o 250 μ l dissolves;
B. NaIO is taken
45mg, adds ddH
2o 250 μ l dissolves, and is mixed with the concentration of 20mg/mL;
C. in HRP solution, dropwise NaIO is added
4solution, limit edged stirs;
D. the solution mixed is placed in 4 DEG C, leaves standstill 30 minutes;
E. get 5ml ethylene glycol and be dissolved in 25 μ l ddH
2in O, dropwise add in above-mentioned mixed solution, limit edged stirs;
F. room temperature leaves standstill 30 minutes;
G. oxydasis process completes, and HRP final concentration is 10mg/ml.
(2) preparation of anti-alpha-fetoprotein antibody 2 (anti-AFP-2) and mark (lucifuge):
A. antibody concentration is adjusted to about 5mg/ml (protein concentration is too low then to be concentrated with PEG20000), with 50mmol/L CB (the 1mol/L NaHCO of about pH9.5
3with 1mol/L Na
2cO
3mix in the ratio of 10:1, with distilled water diluting 20 times before using) dialyse and remove glycerine or impurity (as Tris), dialysed overnight at 4 DEG C, wherein change liquid 3 times;
B. mixed by 1:4 with HRP by anti-alpha-fetoprotein antibody 2 (anti-AFP-2), dialyse more than 6 hours in 50mmol/L pH9.5CB, the first two hour changes liquid once;
C. with the 1mg NaBH of fresh configuration
4solution cessation reaction.Shake up, 4 DEG C leave standstill 2 hours, and per half an hour shakes once, NaBH
4the amount that solution adds is suitable;
D. the 10mM PBS (Na of pre-configured 0.01mol/L of pH7.2 is used
2hPO
4and NaH
2pO
4storing solution, both pH value mixings as required become PBS damping fluid) dialysed overnight.Change liquid once.
(3) purifying HRP enzyme mark anti-alpha-fetoprotein antibody 2 (anti-AFP-2)
A. complete in the monoclonal antibody solution of mark and dropwise add saturated ammonium sulfate solution, stir while adding, until saturated ammonium sulfate concentration is reduced to 1/3;
DEG C b.4 1 hour is left standstill;
C.8000rpm centrifugal 10 minutes, supernatant is moved in new pipe, precipitation equal-volume PBS Eddy diffusion;
D. repeat above operation, saturated ammonium sulfate concentration is brought up to 40%, collect upper cleer and peaceful precipitation respectively;
E. repeat above operation, saturated ammonium sulfate concentration is brought up to 50%, collect upper cleer and peaceful precipitation respectively;
F. repeat above operation, saturated ammonium sulfate concentration is brought up to 60%, collect upper cleer and peaceful precipitation respectively;
G. collect each component of separation, SDS-PAGE identifies purity;
The HRP-monoclonal antibody of h. purifying is to PBS dialysed overnight;
I. the HRP-monoclonal antibody of super filter tube centrifugal concentrating purifying, obtains HRP enzyme mark anti-alpha-fetoprotein antibody 2 (anti-AFP-2) of mole ratio close to 1:8.
(4) packing: with the damping fluid dilution containing 10% hyclone by step 3) HRP enzyme mark anti-alpha-fetoprotein antibody 2 (anti-AFP-2) that obtains to suitable working concentration, by the packing of 6ml/ bottle, be stored in 4 DEG C.
The separation detection composition that application the present embodiment is recorded carries out AFP content detection:
The AFP detection kit (Electrochemiluminescince) adopting Roche company as a control group, compares the accuracy of AFP content detection result.The cutoff value of AFP content is 20 μ g/L, and being positive findings higher than 20 μ g/L, is negative findings lower than 20 μ g/L.Testing result is as shown in table 1:
Table 1
Testing result shows: in 452 increments bases of detection, sensitivity, the specificity of the present embodiment reach 100%.
The separation detection composition that application the present embodiment is recorded carries out AFP-L3% detection:
The alpha-fetoprotein variant separator tube adopting Beijing Re Jing biotech firm to produce coordinates the AFP detection kit (Electrochemiluminescince) of Roche company as a control group, compares the accuracy of AFP-L3% testing result.The cutoff value of AFP-L3% is 10%.Testing result is as shown in table 2:
Table 2:
Compared with control group: 107 parts of control groups be detected as AFP ?sample the present embodiment of the L3% positive detect also for AFP ?L3% positive; 172 parts of control groups are detected as the sample of AFP ?L3% feminine gender, and the present embodiment detects has 169 parts for negative, wherein 3 increments this contrast through clinical diagnosis, find that patient is early primary hepatocarcinoma patient.Testing result shows: the separation detection composition that this enforcement is recorded is sensitiveer than the detection perform of control group.Reason should be AFP ?L3 Protein Separation efficiency improve, can more accurately detect AFP in sample ?L3%.
Embodiment 2
The separation detection composition of the alpha-fetoprotein variant that the utility model provides, according to the separation detection composition of the alpha-fetoprotein variant provided of embodiment 1, separation agent can also comprise protection liquid.Detect reagent and can also comprise damping fluid.
Separation agent and detection reagent can comprise cleaning fluid respectively.
The key component situation of mentioned reagent is as follows:
Protection liquid: 0.02M PBS, 0.5%BSA, pH7.4,0.1M D-MANNOSE glycosides,
Damping fluid: 0.02M PBS, 10% calf serum, 0.1%proclin-300,
Separation agent cleaning fluid: 20mM Tris-HCl, 0.5MD-mannoside,
Detect reagent cleaning fluid: the 1% polysorbas20 solution that PBS pH7.4 prepares.
Above-mentioned D-MANNOSE glycosides can adopt the glucides such as fucose, fructose, sucrose, trehalose to substitute.
Embodiment 3
The separation detecting device of the alpha-fetoprotein variant that the utility model provides, for detecting alpha-fetoprotein variant accounting, as shown in Figure 2,
Comprise the Magneto separate module for separating of alpha-fetoprotein variant, for detecting the detection module of alpha-fetoprotein variant content and α-Fetoprotein, for calculating alpha-fetoprotein variant relative to the data processing module of alpha-fetoprotein ratio and reagent card.Detect piece-rate system and can also comprise sampling module, data processing module can also comprise light signal reading device.
Reagent card comprises the separation detection composition that embodiment 1 is recorded, and be separated detection composition and be sub-packed in the multiple reagent troughs on reagent card in advance, for reacting, often kind of reagent has at least one reagent trough.Reagent card comprises sample aperture, separation agent groove, detects reagent trough and reacting hole, the reagent card of the present embodiment, as shown in Figure 1, composed as follows:
A. sample aperture; B. the reagent trough of the magnetic-particle of pre-packing coupling agglutinin; C., the reagent trough of eluent is housed; D. pre-packing bag is by the reagent trough of the magnetic-particle of anti-alpha-fetoprotein antibody 1 (anti-AFP-1); E. the reagent trough of the anti-alpha-fetoprotein antibody (anti-AFP-2) of pre-packing marker enzyme; F. reacting hole.
Magneto separate module matches with the separation agent be separated in detection composition, for completing the separation to alpha-fetoprotein variant;
Separation module, detection module coordinate with detection Reagent evaluation, for completing the detection to alpha-fetoprotein variant content and α-Fetoprotein.The detection of described alpha-fetoprotein variant content and α-Fetoprotein can adopt magnetic granule chemoluminescence reagent.
Described detection module can only for detecting α-Fetoprotein.
Described data processing module can show α-Fetoprotein data, alpha-fetoprotein variant content data and alpha-fetoprotein variant accounting data.
Adopt the method recorded in the present embodiment to detect sample to be tested, testing result is as shown in table 3:
Table 3
Experimental result shows, the utility model reaches 92% for the Positive rate of primary carcinoma of liver, specificity for Healthy People reaches 100%, and the specificity for cirrhosis and hepatitis reaches 95% and 97% respectively, and the specificity for other cancers reaches 0%.
Embodiment 4
According to the separation detecting device of the alpha-fetoprotein variant that embodiment 3 provides, also comprise detection setting module, detect setting module and comprise α-Fetoprotein determination unit, alpha-fetoprotein variant assay unit and alpha-fetoprotein variant accounting determination unit,
When selecting α-Fetoprotein determination unit, described Magneto separate module does not participate in process detected sample, and described pick-up unit detects the content of alpha-fetoprotein in detected sample,
When selecting alpha-fetoprotein variant assay unit, described Magneto separate module is separated alpha-fetoprotein variant in detected sample, and described detection module detects the content of alpha-fetoprotein variant in detected sample;
When selecting alpha-fetoprotein variant accounting determination unit, described Magneto separate module is separated alpha-fetoprotein variant in detected sample, described detection module detects the content of alpha-fetoprotein variant and the content of alpha-fetoprotein in detected sample, and described data processing module calculates alpha-fetoprotein variant accounting.
Embodiment 5
According to the separation detecting device of the alpha-fetoprotein variant that embodiment 3 or 4 provides, the separation detection composition that the separation detection composition alternate embodiment 1 adopting embodiment 2 to record is recorded.
Embodiment 6
The method for separating and detecting of the alpha-fetoprotein variant that the utility model provides, adopts above-mentioned separation detecting device, for detecting alpha-fetoprotein variant accounting, comprises the steps,
(1) application of sample:
Sample without the serum of haemolysis, blood plasma or whole blood is joined separation detecting system, or joins the sample aperture that mentioned reagent card provides;
(2) alpha-fetoprotein variant is separated:
Sample joins in the reagent trough of the magnetic-particle that coupling agglutinin is housed by system, mixing;
Magneto separate module rich magnetic particle, discards liquid;
Magnetic-particle enrichment obtained joins and is equipped with in the reagent trough of eluent, and mixing, by Magneto separate module enrichment magnetic particle, obtains alpha-fetoprotein variant eluent;
(3) reaction is hatched:
Alpha-fetoprotein variant eluent is joined and bag is housed by the reagent trough of the magnetic-particle of alpha-fetoprotein monoclonal antibody, add the anti-alpha-fetoprotein antibody of the marker enzyme be contained in advance in other reagent troughs simultaneously, hatch.
(4) enrichment: Magneto separate module rich magnetic particle, discards liquid;
(5) develop the color: magnetic-particle enrichment obtained adds the developer be contained in advance in other reagent troughs, is obtained the concentration of alpha-fetoprotein variant by data processing module;
(6) with (2) simultaneously, sample joins and bag is housed by the reagent trough of the magnetic-particle of alpha-fetoprotein monoclonal antibody by system, repeats step (3) and obtains the concentration of alpha-fetoprotein to (5);
(7) alpha-fetoprotein variant accounting is obtained by data processing module.
Embodiment 7
According to the method for separating and detecting of the alpha-fetoprotein variant that embodiment 6 provides, only comprise step (1) and (6), directly can record the concentration of alpha-fetoprotein.
Embodiment 8
The method for separating and detecting of the alpha-fetoprotein variant that the utility model provides, adopts above-mentioned separation detecting device, for detecting alpha-fetoprotein variant accounting, comprises the steps,
(1) application of sample:
Sample without the serum of haemolysis, blood plasma or whole blood is joined separation detecting system, or joins the sample aperture that mentioned reagent card provides;
(2) alpha-fetoprotein variant is separated:
Sample joins in the reagent trough of the magnetic-particle that coupling agglutinin is housed by system, mixing;
Magneto separate module rich magnetic particle, discards liquid;
Magnetic-particle enrichment obtained joins and is equipped with in the reagent trough of cleaning fluid, mixing;
Being joined by magnetic-particle after cleaning is equipped with in the reagent trough of eluent, and mixing, by Magneto separate module enrichment magnetic particle, obtains alpha-fetoprotein variant eluent;
(3) reaction is hatched:
Alpha-fetoprotein variant eluent is joined and bag is housed by the reagent trough of the magnetic-particle of alpha-fetoprotein monoclonal antibody, add the anti-alpha-fetoprotein antibody of the marker enzyme be contained in advance in other reagent troughs simultaneously, hatch.
(4) enrichment: Magneto separate module rich magnetic particle, discards liquid;
(5) clean: magnetic-particle enrichment obtained joins and is equipped with in the reagent trough of cleaning fluid, mixing, repeat step (4);
(6) develop the color: magnetic-particle enrichment obtained adds the developer be contained in advance in other reagent troughs, is obtained the concentration of alpha-fetoprotein variant by data processing module;
(7) with (2) simultaneously, sample joins and bag is housed by the reagent trough of the magnetic-particle of alpha-fetoprotein monoclonal antibody by system, repeats step (3) and records the concentration of alpha-fetoprotein to (6);
(8) alpha-fetoprotein variant accounting is obtained by data processing module.
Data processing module is to the logical algorithm of alpha-fetoprotein variant accounting: judged by sample number, instrument can be transferred for the alpha-fetoprotein concentration of same a sample and alpha-fetoprotein variant concentration, the accounting of rear calculating AFP-L3 in AFP, thus calculate AFP-L3 content, i.e. AFP-L3%.
Above-mentioned explanation illustrate and describes preferred embodiment of the present utility model, as previously mentioned, be to be understood that the utility model is not limited to the form disclosed by this paper, should not regard the eliminating to other embodiments as, and can be used for other combinations various, amendment and environment, and can in utility model contemplated scope described herein, changed by the technology of above-mentioned instruction or association area or knowledge.And the change that those skilled in the art carry out and change do not depart from spirit and scope of the present utility model, then all should in the protection domain of the utility model claims.
Claims (7)
1. a separation detection kit for alpha-fetoprotein variant, is characterized in that, comprise
Reagent card is separated detection composition with alpha-fetoprotein variant,
Described reagent card comprises sample aperture, separation agent groove, detects reagent trough and reacting hole,
The separation detection composition of described alpha-fetoprotein variant, comprises separation agent and detects reagent,
Described separation agent comprises magnetic-particle and the eluent of coupling agglutinin, and the magnetic-particle of described coupling agglutinin is used for and the AFP-L3 specific binding in detected sample; Described detection reagent comprises by the anti-alpha-fetoprotein antibody of the magnetic-particle of alpha-fetoprotein antibody, marker enzyme.
2. separation detection kit according to claim 1, is characterized in that, described separation agent also comprises protection liquid, and/or described detection reagent also comprises damping fluid.
3. separation detection kit according to claim 1, is characterized in that, described separation agent and/or detection reagent also comprise cleaning fluid.
4. separation detection kit according to claim 1, is characterized in that, in the magnetic-particle of described coupling agglutinin, agglutinin comprises LcA and/or ConA.
5. separation detection kit according to claim 1, is characterized in that, in the magnetic-particle of described coupling agglutinin, the macromolecule component of magnetic-particle surface coverage comprises silicide, polysaccharide, albumen, cellulose or resin.
6. a separation detecting device for alpha-fetoprotein variant, comprising:
Magneto separate module, for being separated magnetic particle in liquid;
Detection module, for detecting alpha-fetoprotein variant and α-Fetoprotein;
Data processing module, for calculating alpha-fetoprotein variant relative to alpha-fetoprotein ratio, and
Kit described in any one of claim 1-5,
Described Magneto separate module matches with the described separation agent be separated in detection composition, for completing the separation to alpha-fetoprotein variant;
Described detection module coordinates with the described detection Reagent evaluation be separated in detection composition, for completing the detection of content to alpha-fetoprotein variant and α-Fetoprotein.
7. separation detecting device according to claim 6, is characterized in that,
When selecting to measure α-Fetoprotein, described Magneto separate module does not match with separation agent, and described pick-up unit detects the content of alpha-fetoprotein in detected sample;
When selecting to measure alpha-fetoprotein variant content, described Magneto separate module is separated alpha-fetoprotein variant in detected sample, and described detection module detects the content of alpha-fetoprotein variant in detected sample;
When selecting to measure alpha-fetoprotein variant accounting, described Magneto separate module is separated alpha-fetoprotein variant in detected sample, described detection module detects the content of alpha-fetoprotein variant and the content of alpha-fetoprotein in detected sample, and described data processing module calculates alpha-fetoprotein variant accounting.
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