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 separation detect reagent box and device of alpha fetoprotein heteroplasmon belongs to medical instrument and external diagnosis field.
Background
Hepatocellular carcinoma (HCC) is the fourth most common malignancy worldwide, approximately 250000 patients die of hepatocellular carcinoma every year, the pathogenesis of hepatocellular carcinoma remains unknown, the clinical malignancy is high, the mortality rate is third in digestive tract malignancies, so early detection and early treatment are the key to improve the survival rate of patients, most patients are diagnosed with chronic hepatitis and cirrhosis caused by hepatitis b virus and hepatitis c virus, and the optimal treatment time is lost in the middle and late stages. To be effective, the clinical efficacy of screening and screening depends on early diagnosis. HCC is an important type of etiology of tumors, and chronic liver damage caused by cirrhosis, viral hepatitis, chemical carcinogens, environmental factors, etc. can induce HCC. HCC is highly malignant, prone to recurrence and metastasis, has a poor prognosis, is difficult to diagnose early, and is prone to delay the optimal treatment period.
Alpha Fetoprotein (AFP) is a glycoprotein, and detection of AFP content in blood is currently the most common means for diagnosing liver cancer. AFP is synthesized in mammalian embryonic stages by liver parenchymal cells and yolk sac cells, and is also synthesized in small amounts by gastrointestinal mucosal cells derived from endoderm. Normally, AFP is predominantly present in the fetal circulation, with gradually decreasing AFP synthesis as the fetus matures. The AFP concentration in cord blood at birth can reach 10-100 mg/L, and is reduced to the level of normal adults one year after birth. If the AFP of the newborn is obviously increased, the newborn is suggested to have hepatitis, congenital biliary atresia or embryonic malignant tumor capable of secreting AFP. The defect of AFP as an early liver cancer diagnosis index is that a considerable proportion of liver diseases and liver cirrhosis diseases have positive results, and a slight increase (20-200 ng/m1) of AFP is seen in a considerable number of chronic liver disease patients, and 11.7-44% of AFP is positive in liver cirrhosis patients. Therefore, the value of AFP as an early liver cancer screening index is greatly reduced when benign liver diseases and malignant liver tumors are distinguished.
The structure of many sugar chains of the AFP heteroplasmon is not completely understood. At present, the AFP heteroplasmon is considered to be one of liver cancer markers listed as clinical diagnosis standard of primary liver cancer in A FP-L3,1999 combined with LCA in the fourth national liver cancer academic conference. In 2011, the Chinese liver cancer diagnosis and treatment standard lists AFP-L3 as a liver cancer diagnosis specificity index; for many years, AFP-L3 has been recognized as a more specific indicator of primary liver cancer than AFP alpha fetoprotein alone.
AFP-L3 is the only protein produced by cancer cells in the liver of a patient. This detection method was studied in a multicenter, prospective, double blind, long-term clinical trial in canada and the united states. The results show that patients with elevated AFP-L3% (more than 15%) had a 7-fold increased risk of developing hepatocellular carcinoma in the next 21 months. According to the existing practical guidelines for hepatocellular carcinoma oncology, these patients have an extremely high incidence of hepatocellular carcinoma.
Clinical significance of AFP heteroplasmon detection:
1) liver cancer and benign liver disease are identified. AFP is often increased in patients with primary liver cancer, but AFP can also be increased in a plurality of benign liver diseases, and good and malignant lesions are sometimes difficult to distinguish by means of AFP results. At the moment, the AFP heteroplasmon detection has good clinical significance, and particularly has good value for AFP between 30 ng/ml and 400 ng/ml. Yozhiaki was a prospective study of 361 cases of liver cirrhosis, and of 53 cases of patients with AFP at 30ug/L or more, 21 cases developed liver cancer 2 years later, and 39% of patients with liver cancer confirmed had AFP at 400ug/L or less. Compared with the AFP measured value of a liver cell liver cancer (HCC) group and a non-HCC group at the beginning of the research, the difference is not found to be significant, the research finds that the types of AFP heteroplasms are different when the disease is changed, the LCA positive rate is 87.12 percent, the false positive rate is 21.5 percent, the ConA positive rate is 89.17 percent and the false positive rate is 17.15 percent for HCC diagnosis. The current research result takes the AFP-L3 content of more than 15 percent as the positive index of liver cancer.
2) Monitoring after liver cancer operation. After the hepatoma resection, the content of serum AFP is reduced, the reduction speed of the serum AFP is determined by the amount and half-life period of the residual AFP in vivo, the serum becomes negative within 2 months generally, and the AFP heteroplasmon disappears when the serum becomes negative. If AFP is obviously reduced but not turned negative, and the heterogeneity is not obviously changed, the operation is not thorough, and marginal residues, vascular cancer emboli, satellite nodules or metastasis and the like can exist. If the heteroplasmon drops below 25% and the AFP and heteroplasmon concentration are relatively constant, it may be the result of a patient having hepatitis or cirrhosis.
3) Abnormal development of embryo and congenital diseases of fetus. The AFP in maternal serum of normal pregnancy and the AFP in embryo are in balance state, once the fetus is abnormal or the placenta barrier is abnormal, the fetus serum can be caused to permeate into amniotic fluid or the amniotic fluid permeates into maternal serum, and the maternal amniotic fluid or the serum AFP is increased rapidly. There are limitations to determining the total amount of AFP only. Experiments show that the nerve tube is defective, has no brain or spinal fissure, etc. Children hepatoblastoma, biliary atresia, gonadal tumor, malignant teratoma, etc. may be AFP and/or AFP heteroplasmon positive.
The current detection methods for AFP heteroplasmon include lectin affinity chromatography, polyacrylamide gel electrophoresis, affinity blotting, and affinity cross-immunoelectrophoresis, which can be used to directly isolate AFP-L3 protein and perform quantitative estimation, and can be classified into the following methods according to the final detection method:
coomassie brilliant blue method: the electrophoresed specimen was directly stained with Coomassie Brilliant blue, and the peak band was observed after elution. The method is simple, but has a plurality of interference factors and the sensitivity of about 1000 ug/L.
Enzyme labeling: the peroxidase-labeled antibody and the sample after electrophoresis are incubated together, rinsed and developed by diaminobenzidine, and the detection sensitivity can be improved to 50 ug/L.
Gold and silver staining method, which comprises directly incubating electrophoresed specimen with Staphylococcus aureus protein A2 colloidal gold, and developing with silver developing solution to obtain clear peak band with sensitivity up to 32 ug/L.
The autoradiography is the most common detection method in China so far, and the sensitivity reaches 31 ug/L. The principle is that the specimen is separated and electrophoresed in gel containing agglutinin, and then is electrophoresed for the second time in gel containing 125IAFP and AFP antibody, after electrophoresis, the gel is dried, covered with X-ray film for exposure, and washed and imaged. The whole experimental process is complex in operation, only a few clinical laboratories in China can measure the AFP heteroplasmon for a long time, a few cities can meet the requirements of clinical measurement of the AFP heteroplasmon, and no domestic reagents are supplied.
In addition, a centrifugal tube separation method is mainly adopted at home at present, the method adopts agarose coupling LCA, adopts a centrifugal method to separate AFP-L3, and then uses an AFP reagent to carry out detection. The centrifugal tube separation method is the only method which is approved by the drug administration and is suitable for AFP-L3% detection in China at present.
In the existing method for determining the proportion of alpha fetoprotein heteroplasmons, the manual operation procedures are multiple, the steps are complex, the number of matched devices is multiple, the time consumption is long, and the automation cannot be realized, so that the high-flux sample detection cannot be realized, the influence of manual operation is large, and the deviation of the detection result can be caused finally.
SUMMERY OF THE UTILITY MODEL
In order to overcome the not enough of prior art, the utility model provides a separation detect reagent box and separation detection device of alpha fetoprotein heteroplasmon obtains the alpha fetoprotein heteroplasmon proportion through alpha fetoprotein heteroplasmon content and alpha fetoprotein content in detecting the blood sample.
The utility model provides a pair of separation detect reagent box of alpha fetoprotein heteroplasmon, including the separation detection composition of reagent card and alpha fetoprotein heteroplasmon, the reagent card includes sample hole, separation reagent groove, detect reagent groove and reaction hole, the separation detection composition of alpha fetoprotein heteroplasmon, including separation reagent and examination testing agent, separation reagent includes the magnetic particle and the eluant of coupling lectin, the magnetic particle of coupling lectin is used for with the AFP-L3 specific binding in waiting to detect the sample; the detection reagent comprises magnetic particles coated with alpha-fetoprotein antibodies and enzyme-labeled anti-alpha-fetoprotein antibodies.
The utility model also provides an optimal technical scheme of above-mentioned separation detect reagent box.
Preferably, the separation reagent further comprises a protective solution for increasing the stability of the separation reagent and improving the separation efficiency.
Preferably, the detection reagent further comprises a buffer solution for increasing the stability of the detection reagent, improving the detection sensitivity and the detection specificity.
Preferably, the separation reagent and/or the detection reagent further comprises a washing solution for improving the binding efficiency, reducing non-specific adsorption, and improving the sensitivity of separation and detection.
Preferably, the lectin in the lectin-coupled magnetic particles comprises lentil lectin and/or canavalin.
Preferably, the polymer component coated on the surface of the magnetic particle in the magnetic particle coupled with the lectin comprises silicide, polysaccharide, protein, cellulose or resin.
Preferably, the composition is provided in a card format or in a stick format.
The utility model further provides a separation and detection kit of alpha fetoprotein heteroplasmon, including above-mentioned separation and detection composition.
The utility model also further provides a separation detection device of alpha fetoprotein heteroplasmon, include:
the magnetic separation module is used for separating magnetic particles from liquid and is matched with a separation reagent and a detection reagent for use;
the detection module is used for detecting the content of alpha-fetoprotein heteroplasmons and alpha-fetoprotein;
a data processing module for calculating the ratio of alpha-fetoprotein heteroplasmon to alpha-fetoprotein, and
any of the above-described separation detection compositions or the above-described kits.
The magnetic separation module is matched with a separation reagent in the separation detection composition and is used for completing the separation of the alpha fetoprotein heteroplasmon;
the detection module is matched with a detection reagent in the separation detection composition and is used for completing the detection of the content of alpha-fetoprotein heteroplasmon and the content of alpha-fetoprotein.
The utility model also provides an above-mentioned separation detection device's preferred technical scheme.
As a preference, the first and second liquid crystal compositions are,
when the determination of the alpha fetoprotein content is selected, the magnetic separation module is not matched with the separation reagent, the detection device detects the alpha fetoprotein content in the sample to be detected,
when the content of the alpha-fetoprotein heteroplasmon is selected to be measured, the magnetic separation module separates the alpha-fetoprotein heteroplasmon in the sample to be measured, and the detection module detects the content of the alpha-fetoprotein heteroplasmon in the sample to be measured;
when the proportion of the alpha-fetoprotein heteroplasmons is selected to be determined, the magnetic separation module separates the alpha-fetoprotein heteroplasmons in the sample to be detected, the detection module detects the content of the alpha-fetoprotein heteroplasmons and the content of the alpha-fetoprotein in the sample to be detected, and the data processing module calculates the proportion of the alpha-fetoprotein heteroplasmons, namely the proportion of the alpha-fetoprotein heteroplasmons (AFP-L3) in the total alpha-fetoprotein (AFP) (AFP-L3%).
The utility model finally provides an above-mentioned separation detect reagent box or the application of above-mentioned separation detect device in the separation detects alpha fetoprotein heteroplasmon.
Compared with the prior art, the beneficial effects of the utility model are that: the operator only needs to add a sample and other simple operations, and the detection can be completed within 30 minutes; meanwhile, the method can directly quantitatively calculate the content of alpha fetoprotein and alpha fetoprotein heteroplasmon contained in the blood sample through detection, and simultaneously obtain AFP-L3%; the method is simple, convenient, rapid in detection, accurate in result, high in sensitivity and automatic, and provides support for prevention, diagnosis and treatment of liver cancer.
Drawings
FIG. 1 is a schematic representation of the isolated detection composition of the alpha-fetoprotein variant of the present invention provided in cartridge form,
wherein, 1-sample hole, 2-reagent groove containing magnetic particle coupled with agglutinin, 3-reagent groove containing eluent, 4-reagent groove containing magnetic particle coated with alpha fetoprotein monoclonal antibody, 5-reagent groove containing anti-alpha fetoprotein antibody of labeled enzyme, 6-reaction hole, 7-reagent card;
FIG. 2 is a schematic diagram of the system for detecting the separation of alpha-fetoprotein heteroplasmon of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the invention.
Example 1
The utility model provides a separation detection composition of alpha fetoprotein heteroplasmon, including separating reagent and detect reagent for detect the alpha fetoprotein heteroplasmon and account for the ratio.
The separation reagent comprises magnetic particles coupled with agglutinin and eluent, the magnetic particles coupled with agglutinin are used for specifically combining with AFP-L3 in a sample to be detected; the detection reagent comprises magnetic particles coated with the alpha-fetoprotein monoclonal antibody and an anti-alpha-fetoprotein antibody of a marker enzyme. The main components of the reagent and the preparation method are as follows:
magnetic particle coupled with lectin:
wherein,
the agglutinin is selected from lentil agglutinin (LCA), Canavalia ensiformis agglutinin or lentil agglutinin and Canavalia ensiformis agglutinin.
The magnetic particles used were activated epoxy coated magnetic particles and LCA from Sigma.
The lectin is coupled to the magnetic particles by the following steps:
(1) weighing 2mg LCA, dissolving in 7.5mL coupling buffer (0.1mmol/L NaHCO3, pH 8.3, 0.5mol/L NaCl), combining with washed 1.5g magnetic particles, and mixing by turning upside down in a stoppered 10mL tube (room temperature, 2 h);
(2) unconjugated LCA was washed off with 10mL of coupling buffer. The coupling rate is 98 percent by measuring the content of LCA I in the washing liquid;
(3) blocking the rest of the activated genes by using 0.2mol/L glycine;
(4) washing with 10mL of 0.1mol/L acetate buffer solution (pH 4, containing 0.5mol/L NaCl) and 0.1mol/L Tris buffer solution (pH 8, containing 0.5mol/L NaCl) for 3 times, and adding 0.1% BSA and 0.1mmol/L CaCl2The resulting solution was washed 1 time with PBS (PBS-BSA) and stored at 4 ℃ until use.
The magnetic particles coated with the epoxy resin can also be replaced by magnetic particles coated with titanium silicide, polystyrene, dextran, agarose, sulfonamide resin, bovine serum albumin, biotin and other materials.
AFP-L3 eluent:
0.02M PBS (pH7.0), 5 MD-mannoside,
or 20mm Tris-HCl, NaCL 150mm, ph7.4 buffer solution, which contains 500mm alpha-methyl-D-mannoside, 0.1% and Proclin 300.
Magnetic particles coated with alpha-fetoprotein antibodies:
wherein,
the alpha-fetoprotein antibody adopts an anti-alpha-fetoprotein antibody 1(anti-AFP-1), and the preparation method comprises the following steps:
(1) activation of magnetic particles
a. Sucking 50ml (10% W/V) of magnetic particles;
b. washing the magnetic particles with an equal volume of 50mM MES;
c. resuspend the magnetic particles with an equal volume of 100mM MES;
d. adding an activating reagent EDC to a final concentration of 0.04 g/ml;
e. shaking and activating for 1h at room temperature
(2) Coating of anti-alpha-fetoprotein antibody 1(anti-AFP-1) and magnetic particles
a. After activation, adding a magnetic field and removing supernatant;
b.10 volumes of 50mM MES washed activated magnetic particles;
c. adding 0.2mg of antibody
d. Oscillating and reacting for 3 hours at room temperature;
(3) termination of anti-alpha-fetoprotein antibody 1(anti-AFP-1) with magnetic particle coating
a. After the reaction is finished, adding a magnetic field and removing a supernatant;
b. adding 10 times of coating stop solution;
c. shaking reaction for 3h at normal temperature
(4) Cleaning and preservation of anti-alpha-fetoprotein antibody 1(anti-AFP-1) magnetic particles
a. After the reaction is finished, adding a magnetic field and removing a supernatant;
b. adding 10 times of coating cleaning solution, and repeatedly cleaning for four times;
c.10 times volume of magnetic bead preservation solution for preserving anti-alpha fetoprotein antibody 1(anti-AFP-1) magnetic particles
Enzyme-labeled anti-alpha-fetoprotein antibody:
an anti-alpha-fetoprotein antibody 2(anti-AFP-2) coupled with peroxidase (HRP) is adopted, and the preparation method is as follows:
(1) oxidation of the enzyme (whole process protected from light):
a. weighing HRP 5mg, adding ddH2Dissolving in 250 μ l of O;
b. weighing NaIO45mg, plus ddH2Dissolving O250 mul to prepare the concentration of 20 mg/mL;
c. adding NaIO into HRP solution dropwise4Adding the solution while stirring;
d. placing the mixed solution at 4 ℃ and standing for 30 minutes;
e. 5ml of ethylene glycol was dissolved in 25. mu.l of ddH2Adding the mixture into the mixed solution dropwise while stirring;
f. standing for 30 minutes at room temperature;
g. the enzymatic oxidation process was complete and the final concentration of HRP was 10 mg/ml.
(2) Preparation and labeling (protected from light) of anti-alpha-fetoprotein antibody 2 (anti-AFP-2):
a. adjusting antibody concentration to about 5mg/ml (concentrating with PEG20000 if protein concentration is too low), and adding 50mmol/L CB (1mol/L NaHCO) with pH of about 9.53With 1mol/L Na2CO3Mixing at ratio of 10:1, diluting with distilled water 20 times before use), dialyzing to remove glycerol or impurities (such as Tris), dialyzing at 4 deg.C overnight, and changing the solution for 3 times;
b. anti-alpha-fetoprotein antibody 2(anti-AFP-2) was mixed with HRP at a ratio of 1: 4, mixing, dialyzing in 50mmol/L pH9.5CB for more than 6 hours, and changing the solution once in the first two hours;
c. with freshly prepared 1mg NaBH4The solution stops the reaction. Shaking up, standing for 2 hours at 4 ℃, shaking once every half an hour, NaBH4The amount of solution added is appropriate;
d. using 10mM PBS (0.01 mol/L pre-configured Na) at pH7.22HPO4And NaH2PO4Stock solution, mix the two into PBS buffer solution according to the required pH) overnight. The liquid is changed once.
(3) Purified HRP enzyme-labeled anti-alpha fetoprotein antibody 2(anti-AFP-2)
a. Dropwise adding a saturated ammonium sulfate solution into the labeled monoclonal antibody solution, and stirring while adding until the concentration of the saturated ammonium sulfate is reduced to 1/3;
b.4 ℃ standing for 1 hour;
c.8000rpm for 10 minutes, transferring the supernatant to a new tube, and resuspending the pellet with an equal volume of PBS;
d. repeating the above operations, increasing the concentration of saturated ammonium sulfate to 40%, and respectively collecting the supernatant and the precipitate;
e. repeating the above operations, increasing the concentration of saturated ammonium sulfate to 50%, and respectively collecting supernatant and precipitate;
f. repeating the above operations, increasing the concentration of saturated ammonium sulfate to 60%, and respectively collecting the supernatant and the precipitate;
g. collecting separated components, and identifying the purity by SDS-PAGE;
h. dialyzing the purified HRP-monoclonal antibody against PBS overnight;
i. and (3) centrifugally concentrating the purified HRP-monoclonal antibody by using an ultrafiltration tube to obtain an HRP enzyme-labeled anti-alpha-fetoprotein antibody 2(anti-AFP-2) with a molar ratio close to 1: 8.
(4) Subpackaging: diluting the HRP enzyme-labeled anti-alpha-fetoprotein antibody 2(anti-AFP-2) obtained in the step 3) to a proper working concentration by using a buffer solution containing 10% fetal calf serum, subpackaging according to 6 ml/bottle, and storing at 4 ℃.
The AFP content was measured using the separation assay composition described in this example:
an AFP detection kit (electrochemical luminescence method) of Roche company is adopted as a control group to compare the accuracy of the AFP content detection result. The cutoff value of the AFP content is 20 mug/L, positive results are obtained when the cutoff value is higher than 20 mug/L, and negative results are obtained when the cutoff value is lower than 20 mug/L. The results are shown in table 1:
TABLE 1
The detection result shows that: in 452 samples tested, the sensitivity and specificity of this example reached 100%.
AFP-L3% assay was performed using the isolation assay composition described in this example:
the accuracy of the AFP-L3% detection result was compared by using an alpha-fetoprotein heteroplasmon separation tube produced by Beijing Thermopathy organism company in combination with an AFP detection kit (electrochemiluminescence method) of Roche company as a control group. The cutoff value of AFP-L3% was 10%. The results are shown in table 2:
table 2:
comparison with the control group: 107 control groups tested AFP-L3% positive samples this example test was also AFP-L3% positive; 172 samples of the control group tested negative for AFP-L3%, 169 samples of the control group tested negative for the present example, 3 samples of the control group were tested against clinical diagnosis, and the patients were found to be early stage primary liver cancer patients. The detection result shows that: the separation detection composition described in this example is more sensitive than the detection performance of the control group. The AFP-L3 protein separation efficiency is improved, and the AFP-L3% in the sample can be more accurately detected.
Example 2
The utility model provides a separation detection composition of alpha fetoprotein heteroplasmon, according to the separation detection composition of the alpha fetoprotein heteroplasmon that provides of embodiment 1, the separation reagent can also include the protection liquid. The detection reagent may also include a buffer.
The separation reagent and the detection reagent may each comprise a wash solution.
The main components of the reagent are as follows:
protection solution: 0.02M PBS, 0.5% BSA, pH7.4, 0.1M D-mannoside,
buffer solution: 0.02M PBS, 10% calf serum, 0.1% proclin-300,
separating reagent cleaning solution: 20mM Tris-HCl, 0.5 MD-mannoside,
detecting a reagent cleaning solution: 1% Tween 20 solution prepared in PBS pH7.4.
The D-mannoside can be replaced by saccharides such as fucose, fructose, sucrose, trehalose, etc.
Example 3
The device for separating and detecting alpha fetoprotein heteroplasmon provided by the utility model is used for detecting the proportion of the alpha fetoprotein heteroplasmon, as shown in figure 2,
the kit comprises a magnetic separation module for separating alpha-fetoprotein heteroplasmons, a detection module for detecting the content of the alpha-fetoprotein heteroplasmons and the content of the alpha-fetoprotein, a data processing module for calculating the ratio of the alpha-fetoprotein heteroplasmons to the alpha-fetoprotein, and a reagent card. The detection separation system can also comprise a sampling module, and the data processing module can also comprise an optical signal reading device.
The reagent card comprises the separation detection composition described in example 1, which is pre-dispensed into a plurality of reagent wells on the reagent card for reaction, each reagent having at least one reagent well. The reagent card comprises a sample hole, a separation reagent groove, a detection reagent groove and a reaction hole, and the reagent card of the embodiment, as shown in fig. 1, comprises the following components:
a. a sample well; b. a reagent tank for pre-dispensing magnetic particles coupled with lectin; c. a reagent tank containing an eluent; d. a reagent tank for pre-packaging magnetic particles coated with anti-alpha fetoprotein antibody 1 (anti-AFP-1); e. a reagent tank for pre-distributing an anti-alpha fetoprotein antibody (anti-AFP-2) of a labeled enzyme; f. and (4) reaction holes.
The magnetic separation module is matched with a separation reagent in the separation detection composition and is used for completing the separation of the alpha fetoprotein heteroplasmon;
the separation module and the detection module are matched with the detection reagent and used for completing the detection of the alpha fetoprotein heteroplasmon content and the alpha fetoprotein content. The content of alpha-fetoprotein heteroplasmon and the content of alpha-fetoprotein can be detected by using a magnetic particle chemiluminescence reagent.
The detection module may be for detecting alpha-fetoprotein content only.
The data processing module can display the alpha fetoprotein content data, the alpha fetoprotein heteroplasmon content data and the alpha fetoprotein heteroplasmon proportion data.
The method described in this example was used to test the sample to be tested, and the test results are shown in table 3:
TABLE 3
The experimental result shows that the utility model discloses the positive rate of detection to primary liver cancer reaches 92%, reaches 100% to healthy people's specificity, reaches 95% and 97% respectively to the specificity of cirrhosis and hepatitis, has reached 0% to the specificity of other cancers.
Example 4
The device for separating and detecting alpha-fetoprotein heteroplasmons provided in embodiment 3, further comprising a detection setting module, wherein the detection setting module comprises an alpha-fetoprotein content measuring unit, an alpha-fetoprotein heteroplasmon content measuring unit, and an alpha-fetoprotein heteroplasmon proportion measuring unit,
when the alpha fetoprotein content determination unit is selected, the magnetic separation module does not participate in processing the sample to be detected, the detection device detects the content of the alpha fetoprotein in the sample to be detected,
when the alpha fetoprotein heteroplasmon content determination unit is selected, the magnetic separation module separates the alpha fetoprotein heteroplasmon in the sample to be detected, and the detection module detects the content of the alpha fetoprotein heteroplasmon in the sample to be detected;
when the alpha-fetoprotein heteroplasmon proportion determination unit is selected, the magnetic separation module is used for separating the alpha-fetoprotein heteroplasmons in the sample to be detected, the detection module is used for detecting the content of the alpha-fetoprotein heteroplasmons and the content of the alpha-fetoprotein in the sample to be detected, and the data processing module is used for calculating the alpha-fetoprotein heteroplasmon proportion.
Example 5
According to the apparatus for separating and detecting an alpha-fetoprotein heteroplasmon provided in example 3 or 4, the separation detection composition described in example 2 is used instead of the separation detection composition described in example 1.
Example 6
The utility model provides a separation detection method of alpha fetoprotein heteroplasmon, which adopts the separation detection device to detect the proportion of the alpha fetoprotein heteroplasmon, and comprises the following steps,
(1) sample adding:
adding a sample of serum, plasma or whole blood without hemolysis into a separation detection system or into a sample hole provided by the reagent card;
(2) isolation of alpha-fetoprotein heteroplasmons:
adding a sample into a reagent tank filled with magnetic particles of coupling agglutinin by the system, and uniformly mixing;
the magnetic separation module enriches magnetic particles and discards liquid;
adding the enriched magnetic particles into a reagent tank filled with eluent, uniformly mixing, and enriching the magnetic particles through a magnetic separation module to obtain an alpha-fetoprotein heteroplasmon eluent;
(3) and (3) reaction incubation:
adding the alpha-fetoprotein heteroplasmon eluent into a reagent tank filled with magnetic particles coated with the alpha-fetoprotein monoclonal antibody, simultaneously adding anti-alpha-fetoprotein antibodies of labeling enzymes preloaded in other reagent tanks, and incubating.
(4) Enrichment: the magnetic separation module enriches magnetic particles and discards liquid;
(5) color development: adding the enriched magnetic particles into a color developing agent preinstalled in other reagent tanks, and obtaining the concentration of the alpha fetoprotein heteroplasmon through a data processing module;
(6) simultaneously with the step (2), the system adds the sample into a reagent tank filled with the magnetic particles coated with the alpha-fetoprotein monoclonal antibody, and repeats the steps (3) to (5) to obtain the concentration of the alpha-fetoprotein;
(7) and obtaining the alpha fetoprotein heteroplasmon ratio through a data processing module.
Example 7
According to the method for separating and detecting the alpha-fetoprotein heteroplasmon provided in example 6, which only comprises the steps (1) and (6), the concentration of the alpha-fetoprotein can be directly measured.
Example 8
The utility model provides a separation detection method of alpha fetoprotein heteroplasmon, which adopts the separation detection device to detect the proportion of the alpha fetoprotein heteroplasmon, and comprises the following steps,
(1) sample adding:
adding a sample of serum, plasma or whole blood without hemolysis into a separation detection system or into a sample hole provided by the reagent card;
(2) isolation of alpha-fetoprotein heteroplasmons:
adding a sample into a reagent tank filled with magnetic particles of coupling agglutinin by the system, and uniformly mixing;
the magnetic separation module enriches magnetic particles and discards liquid;
adding the enriched magnetic particles into a reagent tank filled with cleaning liquid, and uniformly mixing;
adding the cleaned magnetic particles into a reagent tank filled with eluent, uniformly mixing, and enriching the magnetic particles through a magnetic separation module to obtain an alpha-fetoprotein heteroplasmon eluent;
(3) and (3) reaction incubation:
adding the alpha-fetoprotein heteroplasmon eluent into a reagent tank filled with magnetic particles coated with the alpha-fetoprotein monoclonal antibody, simultaneously adding anti-alpha-fetoprotein antibodies of labeling enzymes preloaded in other reagent tanks, and incubating.
(4) Enrichment: the magnetic separation module enriches magnetic particles and discards liquid;
(5) cleaning: adding the enriched magnetic particles into a reagent tank filled with cleaning liquid, uniformly mixing, and repeating the step (4);
(6) color development: adding the enriched magnetic particles into a color developing agent preinstalled in other reagent tanks, and obtaining the concentration of the alpha fetoprotein heteroplasmon through a data processing module;
(7) simultaneously with the step (2), the system adds the sample into a reagent tank filled with the magnetic particles coated with the alpha-fetoprotein monoclonal antibody, and repeats the steps (3) to (6) to obtain the concentration of the alpha-fetoprotein;
(8) and obtaining the alpha fetoprotein heteroplasmon ratio through a data processing module.
The data processing module adopts a logic algorithm for the proportion of the alpha fetoprotein heteroplasmons: the sample number judgment is carried out, the instrument can call the alpha fetoprotein concentration and the alpha fetoprotein heteroplasmon concentration of the same sample, and then the proportion of AFP-L3 in the AFP is calculated, so that the content of AFP-L3, namely AFP-L3%, is calculated.
While the foregoing description shows and describes the preferred embodiments of the present invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not intended to be exhaustive or to exclude other embodiments and may be used in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. But that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention, which is to be limited only by the claims appended hereto.
Claims (7)
1. A kit for separating and detecting alpha-fetoprotein heteroplasmon is characterized by comprising
A separation detection composition of a reagent card and an alpha fetoprotein heteroplasmon,
the reagent card comprises a sample hole, a separation reagent groove, a detection reagent groove and a reaction hole,
the separation detection composition of the alpha fetoprotein heteroplasmon comprises a separation reagent and a detection reagent,
the separation reagent comprises magnetic particles coupled with agglutinin and eluent, and the magnetic particles coupled with agglutinin are used for specifically binding with AFP-L3 in a sample to be detected; the detection reagent comprises magnetic particles coated with alpha-fetoprotein antibodies and enzyme-labeled anti-alpha-fetoprotein antibodies.
2. The isolation test kit of claim 1, wherein the isolation reagent further comprises a protective solution, and/or the test reagent further comprises a buffer solution.
3. The isolation test kit of claim 1, wherein the isolation reagent and/or the detection reagent further comprises a wash solution.
4. The isolation detection kit of claim 1, wherein the lectin-coupled magnetic particle comprises lentil lectin and/or canavalin.
5. The separation detection kit according to claim 1, wherein the polymer component coated on the surface of the magnetic particle in the lectin-coupled magnetic particle comprises a silicide, a polysaccharide, a protein, a cellulose, or a resin.
6. An apparatus for separating and detecting alpha-fetoprotein heteroplasmon, comprising:
the magnetic separation module is used for separating magnetic particles from liquid;
the detection module is used for detecting the content of alpha-fetoprotein heteroplasmons and alpha-fetoprotein;
a data processing module for calculating the ratio of alpha-fetoprotein heteroplasmon to alpha-fetoprotein, and
the kit according to any one of claims 1 to 5,
the magnetic separation module is matched with a separation reagent in the separation detection composition and is used for completing the separation of the alpha fetoprotein heteroplasmon;
the detection module is matched with a detection reagent in the separation detection composition and is used for completing the detection of the content of alpha-fetoprotein heteroplasmon and the content of alpha-fetoprotein.
7. The separation detection apparatus according to claim 6,
when the determination of the alpha fetoprotein content is selected, the magnetic separation module is not matched with the separation reagent, and the detection device detects the alpha fetoprotein content in the sample to be detected;
when the content of the alpha-fetoprotein heteroplasmon is selected to be measured, the magnetic separation module separates the alpha-fetoprotein heteroplasmon in the sample to be measured, and the detection module detects the content of the alpha-fetoprotein heteroplasmon in the sample to be measured;
when the proportion of the alpha-fetoprotein heteroplasmons is selected to be determined, the magnetic separation module is used for separating the alpha-fetoprotein heteroplasmons in the sample to be detected, the detection module is used for detecting the content of the alpha-fetoprotein heteroplasmons and the content of the alpha-fetoprotein in the sample to be detected, and the data processing module is used for calculating the proportion of the alpha-fetoprotein heteroplasmons.
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