CN103048453B - Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof - Google Patents
Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof Download PDFInfo
- Publication number
- CN103048453B CN103048453B CN201210550170.8A CN201210550170A CN103048453B CN 103048453 B CN103048453 B CN 103048453B CN 201210550170 A CN201210550170 A CN 201210550170A CN 103048453 B CN103048453 B CN 103048453B
- Authority
- CN
- China
- Prior art keywords
- antibody
- related antigen
- reagent
- carbohydrate related
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a nanometer magnetic particle chemiluminescence detection kit for a carbohydrate antigen CA19-9 as well as a preparation method thereof and a detecting method thereof. The kit comprises a solution, which contains a fluorescein labeled carbohydrate related antigen CA19-9, a suspension, which is coated with a fluorescein antibody, and a solution, which contains an alkaline phosphatase labeled carbohydrate related antigen CA19-9 antibody, wherein the alkaline phosphatase labeled carbohydrate related antigen CA19-9 antibody is obtained by connecting an alkaline phosphatase and the carbohydrate related antigen CA19-9 antibody through cross-linking agents SMCC and 2-IT. According to the invention, the carbohydrate related antigen CA19-9 can be quantitatively detected with lower cost, higher accuracy and higher precision.
Description
Technical field
The invention belongs to biological technical field, be specifically related to nano magnetic particulate chemistry luminescence assays kit of a kind of carbohydrate related antigen CA19-9 that combines immune magnetic particle isolation technics and chemiluminescence immunoassay technology and preparation method thereof and detection method.
Background technology
Sugar antigen CA19-9(Carbohydrate Antigen19-9) be the glycolipid matter on cell membrane, molecular weight is about 1000KD.CA19-9 be 1979 by the colon cancer cell immune mouse such as Koprowski, and with myeloma hybridization gained 116NS19-9 monoclonal antibody and name, be hitherto reported to the highest mark of cancer of pancreas susceptibility.
CA19-9 is mainly used in following aspect clinically:
Malignant tumor of digestive tract
Cancer of pancreas, liver and gall are that the CA 19-9 level of cancer, cancer of the stomach, colorectal cancer is respectively 683,535,279,115 times of normal mean value.And positive rate with cancer of pancreas for the highest, indicate preferably therefore CA19-9 is cancer of pancreas.
Application in diagnosis of pancreatic cancer:
Most of Pancreas cancer patients serum CA 19-9 levels obviously increases.If taking the normal reference range upper limit (37U/mL) as diagnostic criteria, susceptibility and specificity all can reach more than 90%; CA19-9 level is relevant with the stage of tumour, the complexity of the height of content prompting operation in serum; Preoperative CA19-9 level has certain suggesting effect to prognosis, and low person's prognosis is better; Postoperative CA19-9 level is down to normal person and is longer than not descender life cycle; When tumor recurrence, CA19-9 can raise once again, and before betiding imaging diagnosis.Therefore can be used as monitoring the recurrence of tumour.
Other malignant tumours
The positive rate of oophoroma, lymthoma, cancer of the stomach, liver cancer, the cancer of the esophagus, breast cancer is lower.Apply as follows:
The positive rate of cancer of the stomach is about 25%-60%, and relevant with neoplasm staging.For patients with gastric cancer, detect CEA simultaneously and can improve positive rate; Rectum, colorectal cancer patients, positive rate is 18%-58%, relevant with neoplasm staging.Measure CEA simultaneously and can improve susceptibility, if treatment effectively, CA19-9 decline rate is fast compared with CEA; The CA19-9 positive rate of carcinoma of gallbladder, cholangiocarcinoma, cancer of bile ducts is higher, can be used for distinguishing jaundice and the obstructive jaundice of cancer of pancreas, cholangiocarcinoma merging.The latter's CA19-9 level is lower.
Using priciple: CA19-9 and AFP, CEA joint inspection, contribute to improve the diagnosis efficiency of gastroenteric tumor.
Non-neoplastic disease
Low concentration increases, one cross property and increase and be found in chronic pancreatitis, cholelithiasis, cirrhosis, renal insufficiency, diabetes etc.
At present mainly contain enzyme-linked immunosorbent assay, chemiluminescence immunoassay etc. for detection of the immune analysis method of sugar antigen CA19-9.Enzyme-linked immunosorbent assay exists sensitivity low, narrow, the difficult methodology limiting factors such as full-automation that realize of the range of linearity.Chemiluminescence immunoassay is a kind of immunoassay technology growing up on enzyme-linked immunosorbent assay basis, have highly sensitive, detect the advantages such as linear wide ranges, easy and simple to handle, automaticity height.At present chemiluminescence immunoassay technology has above-mentioned be manyly widely used a little because of it.
But, in actual immune detection, because impurity component contained in testing sample is more, detection sensitivity and accuracy are affected to a certain extent, so separate fast, be purified into object determinand from complicated sample substrate, it is one of difficult problem of facing of clinical examination worker.
Magnetic particle immunoassay technology is to utilize the magnetic solid phase particle of synthesis of polymer material certain particle size size to make carrier, on coated with the method such as physisorption, chemical coupling, there are the various immunologic active materials such as antibody or antigen of specificity affinity, have that velocity of separation is fast, efficiency is high, favorable repeatability, simple to operate, the feature such as biological character and function that do not affect separated cell or other biological material, orientable motion under additional magnetic fields, makes some special composition be separated, concentrate or purifying.
The open CN101373188A of Chinese invention patent discloses the immue quantitative detection reagent box of CA19-9 (CA19-9) a kind of, this kit comprises CA19-9 calibration object, be coated with the solid phase carrier of CA19-9 monoclonal antibody, the CA19-9 monoclonal antibody of horseradish peroxidase-labeled, and chemical luminous substrate A liquid, B liquid, wherein said solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.The CA19-9 monoclonal antibody that is coated with solid phase carrier, the horseradish peroxidase-labeled of CA19-9 monoclonal antibody in this kit forms the immune response reagent that immune response step is used.Although this kit can be realized quickly and accurately and detecting, but wherein, the preparation process that is coated with the solid phase carrier of CA19-9 monoclonal antibody and the CA19-9 monoclonal antibody of horseradish peroxidase-labeled is very loaded down with trivial details, technology stability is bad, limits its detection effect and particularly analyzes a precision and cause cost to increase.
Summary of the invention
Technical matters to be solved by this invention is to overcome the deficiencies in the prior art; the nano magnetic particulate chemistry luminescence assays kit of a kind of sugar antigen CA19-9 is provided; it can prepare with lower cost, and can realize carbohydrate related antigen CA19-9 accurately and high precision quantitative measurement.
The present invention also provides the preparation method of the nano magnetic particulate chemistry luminescence assays kit of a kind of sugar antigen CA19-9 simultaneously, the method process stabilizing, and cost is low, and the precision of gained kit is high.
A nano magnetic particulate chemistry luminescence assays kit of sugar antigen CA19-9, is characterized in that, this kit comprises:
The first reagent: containing the solution of fluorescein-labeled carbohydrate related antigen CA19-9 antibody;
The second reagent: the solution that contains the carbohydrate related antigen CA19-9 antibody of alkaline phosphatase (ALP) mark;
Magnetic separating agent: the suspending liquid that contains the magnetic particle that is being coated with fluorescein antibody.
Preferably, the carbohydrate related antigen CA19-9 antibody of this alkali phosphatase enzyme mark passes through crosslinking chemical 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester (Succinimidyl 4-(N-Maleimidomethyl) Cyclohexane-1-Carbo by alkaline phosphatase and carbohydrate related antigen CA19-9 antibody, SMCC) and 2-imino group sulfane hydrochloride (2-Iminothiolane HCl, 2-IT) connect and compose.
Further, in described magnetic particle reagent, the magnetic particle that is coated with fluorescein antibody passes through the coupling of coupling agent phase chemistry by fluorescein antibody and magnetic particle.
Further, the concentration of the fluorescein-labeled carbohydrate related antigen CA19-9 antibody in described the first reagent is 0.5 ~ 1 μ g/mL, and the pH of described the first reagent is 7-9; The concentration of the carbohydrate related antigen CA19-9 antibody of the alkali phosphatase enzyme mark in described the second reagent is 0.5 ~ 1 μ g/mL, and the pH of described the second reagent is 7-9.
Those skilled in the art should know, and kit of the present invention can further include other and detects required reagent, for example substrate solution.But can buy separately or prepare other reagent such as substrate solution, therefore, although can comprise these reagent in kit, they be not essential for kit of the present invention.
The another technical scheme that the present invention takes is: the preparation method of the nano magnetic particulate chemistry luminescence assays kit of a kind of sugar antigen CA19-9; it comprises the step of preparing respectively described the first reagent, described the second reagent and magnetic separating agent, wherein: the preparation process of described the second reagent is as follows:
1. after carbohydrate related antigen CA19-9 antibody being added in crosslinking chemical 2-imino group sulfane hydrochloride solution room temperature to leave standstill, then add glycine solution, room temperature leaves standstill again, collects the carbohydrate related antigen CA19-9 antibody after activation, at 2 ~ 8 DEG C, saves backup;
2. alkaline phosphatase enzyme solutions is added to crosslinking chemical 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester solution, room temperature leaves standstill, and collects the alkaline phosphatase after activation, at 2 ~ 8 DEG C, saves backup;
3. by above-mentioned steps 1. the carbohydrate related antigen CA19-9 antibody after gained activation and step 2. the alkaline phosphatase of gained after activating mix, leave standstill reaction, make to generate the carbohydrate related antigen CA19-9 antibody of alkali phosphatase enzyme mark, after reaction finishes, reactant liquor is purified with Supp erdex200 gel-purified post, select damping fluid adjustment concentration and the pH value with proper pH value to obtain described the second reagent;
Wherein, described carbohydrate related antigen CA19-9 antibody, the purity of described alkaline phosphatase are all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase exceedes 1000u/mg.
Preferably, step 1. in, get carbohydrate related antigen CA19-9 antibody, add coupling agent 2-IT solution to dissolve, room temperature leaves standstill 10min ~ 30min, adds glycine solution, room temperature leaves standstill 2 ~ 10min, with G-25 gel-purified post desalination, collect the carbohydrate related antigen CA19-9 antibody after activation, save backup in 2-8 DEG C.
Preferably, step 2. in, get the alkaline phosphatase enzyme solutions that concentration is more than or equal to 5mg/mL, add and use SMCC solution, room temperature leaves standstill 20 ~ 40min, with G-25 gel column desalination, collects alkaline phosphatase after activation, saves backup in 2-8 DEG C.
Preferably, step 3. in, be that 1:1 ~ 1:2 mix with the alkaline phosphatase of activation by molecule mol ratio by the carbohydrate related antigen CA19-9 antibody of above-mentioned activation, under 2-8 DEG C of condition leave standstill 12-24h.
Wherein the damping fluid of proper pH value can be the TRIS damping fluid that for example contains 0.5% bovine serum albumin(BSA), pH8.0.
Further, the preparation method of the first described reagent is as follows: the damping fluid that the pH that preparation contains fluorescein is 9 ~ 10, then the ratio that is 20 ~ 200:1 according to the molecular proportion of fluorescein and carbohydrate related antigen CA19-9 antibody, the damping fluid that is 9 ~ 10 by the described pH that contains fluorescein mixes with the damping fluid that the pH of carbohydrate related antigen CA19-9 antibody is 9 ~ 10, after mixing, room temperature leaves standstill reaction, then reactant liquor is separated by G-25 gel column, remove free fluorescein, obtain the solution that contains fluorescein-labeled anti-carbohydrate related antigen CA19-9 antibody, then adjust concentration and pH with the damping fluid with proper pH value, obtain.Wherein the damping fluid of proper pH value can be the TRIS damping fluid that for example contains 0.5% bovine serum albumin(BSA), pH8.0.
Further, the preparation method of described magnetic separating agent is as follows: by the magnetic particle that contains carboxyl reactive group and fluorescein antibody under the existence of coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction finishes, magnetic separates, remove supernatant, adjust pH and concentration with the damping fluid with proper pH value, obtain described magnetic separating agent.Wherein said magnetic particle has superparamagnetism, and its diameter is 0.5 ~ 2 μ m, on every gram of magnetic particle with the content of carboxyl reactive group be not less than 0.4mmol; Described fluorescein antibody is monoclonal antibody or polyclonal antibody, and purity is more than or equal to 90wt%, and dilution is tired and is greater than 1:100 ten thousand.
Preferably, the above-mentioned damping fluid with proper pH value is the TRIS damping fluid that contains 0.5% bovine serum albumin(BSA), pH8.0.
Fluorescein of the present invention can be known various fluoresceins, and conventional have for example fluorescein isothiocynate, RB 200, a TRITC etc.
The present invention also provides a kind of simultaneously and has adopted above-mentioned kit to be applied to the detection method that tumor-associated antigen 125 quantitatively detects, and it is characterized in that, comprises the following steps:
(1) immune response: add sample to be tested stoste in detector tube, add successively the first reagent and the second reagent, mix, carry out incubation for the first time at 25 ~ 40 DEG C, then add magnetic separation agent, mix, carry out incubation for the second time at 25～40 DEG C;
(2) washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add cleaning fluid, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, and then magnetic separates, and removes supernatant;
(3) add substrate solution and detect luminous intensity: in detector tube, add the chemical luminous substrate of alkaline phosphatase enzymatic, remove magnetic field, fully after suspendible, detect luminous intensity values.
Further, described in step (1), the time of incubation can be 10 ~ 20min for the first time, is generally 15min; The time of incubation can be 2 ~ 15min for the second time, is generally 5min.
Due to the enforcement of above technical scheme, the present invention compared with prior art tool has the following advantages:
1. adopt kit of the present invention can realize accurate, the accurate detection of carbohydrate related antigen CA19-9, its three kinds of reagent all can prepare by stable preparation technology, production cost is low, and due to preparation technology's stability, it is little that kit is analyzed differences between batches, and between the analysis of detection, precision improves;
2. the preparation method of the solution of the carbohydrate related antigen CA19-9 antibody that contains alkali phosphatase enzyme mark in kit of the present invention, can be effectively by carbohydrate related antigen CA19-9 antibody and alkaline phosphatase coupling, coupling efficiency is high, further reduces the low detection effect with guaranteeing kit of cost of kit;
3. take kit of the present invention to detect, accuracy is good, and precision is high, highly sensitive, sensing range is wide, and sample is without pre-dilution, simple to operate saving time, compared with the method that adopts import reagent box to detect, detection method of the present invention has significant advantage on cost.
Brief description of the drawings
Fig. 1 is detection calibration product typical curves;
Fig. 2 is that matched curve is evaluated in sensitivity;
Fig. 3 is serum sample testing result correlativity (wherein horizontal ordinate x is the kit sample measured value that embodiment 4 is prepared into, and concentration unit is U/mL, and ordinate y is ROCHE company kit sample measured value, and concentration unit is U/mL).
Embodiment
The preparation of embodiment 1 first reagent
(1) material and instrument: the carbohydrate related antigen CA19-9(CA19-9 preserving with phosphate buffer) monoclonal antibody (purity exceedes 95wt%, and concentration is 2mg/mL); Fluorescein isothiocynate (FITC), the reagent such as sodium carbonate should reach chemical pure; G-25 gel-purified post is purchased from GE company.
(2) preparation process:
1. use the FITC solution of the carbonate buffer solution preparation 0.5mg/mL of 0.1 ~ 0.2mol/L pH 9.0 ~ 10.0;
2. the ratio that is 1:20 according to CA19-9 antibody and FITC molecular proportion adds step 1. to be joined FITC solution in antibody-solutions, mixes, and room temperature leaves standstill 12h hour, and reaction generates CA19-9 antibody-FITC connector;
3. by separating by G-25 gel column through step reactant liquor 2., remove unreacted FITC, obtain the solution that contains CA19-9 antibody-FITC connector (being the CA19-9 antibody of FITC mark);
4. the step solution that 3. gained contains CA19-9 antibody-FITC connector being diluted to CA19-9 antibody-FITC connector concentration with the TRIS damping fluid of the 0.1mol/L containing 0.5% bovine serum albumin(BSA) (BSA) pH 8.0 is 0.5 ~ 1 μ g/mL, is the first reagent.
The preparation of embodiment 2 second reagent
(1) material and instrument: the CA19-9 monoclonal antibody (purity exceedes 95wt%, and concentration is 2mg/mL) of preserving with phosphate buffer; The alkaline phosphatase preserved with phosphate buffer (ALP solution, ALP purity is approximately 99%, specific activity is about 1500U/mg, concentration is 10mg/mL); Crosslinking aid S MCC, 2-IT is purchased from THERMO company, and the chemical reagent such as TRIS should reach chemical pure; G-25 gel-purified post is GE company product.
(2) preparation process:
1. get 1mg CA19-9 antibody, add the coupling agent 2-IT solution 2-4 μ L of 10mg/mL, room temperature leaves standstill 20min, the glycine solution 10 μ L that add 0.1mol/L, room temperature leaves standstill 5min, with G-25 gel column desalination, collect the rear CA19-9 antibody of activation, 2-8 DEG C saves backup;
2. get the ALP solution of 1.5mg, add the SMCC solution 10-20 μ L of 5mg/mL, room temperature leaves standstill 30min, with G-25 gel column desalination, collects the rear ALP of activation, and 2-8 DEG C saves backup;
3. the cancer antigens c A19-9 antibody of above-mentioned activation is mixed with the ALP of activation, under 2-8 DEG C of condition, leave standstill 12-24h, purify conjugate with Supperdex200 gel-purified post, acquisition CA19-9 antibody-ALP connector strong solution, 2-8 DEG C saves backup;
4. CA19-9 antibody-ALP connector strong solution is diluted to 0.5-1 μ g/mL with the TRIS damping fluid of the 0.1mol/L containing 0.5% bovine serum albumin(BSA) (BSA) pH8.0, completes the preparation of the second reagent.
The preparation of embodiment 3 magnetic separation agents
(1) material and instrument:
The suspending liquid of magnetic particle: magnetic particle content 5wt%, magnetic particle is containing the active group of carboxyl (COOH), and every gram of (g) magnetic particle (dry weight) carboxyl-content is not less than 0.4 mM (mmol), has superparamagnetism, and diameter is between 0.5-2 μ m.
Anti-FITC mAb: can be polyclonal antibody, can be also monoclonal antibody, and purity is more than 90wt%, dilution is tired and is exceeded 1:100 ten thousand;
MES (MES), carbodiimide (EDC), TRIS and other reagent should reach chemical pure.
(2) preparation process:
1. the suspending liquid of getting 100mg magnetic particle, magnetic divides the supernatant of leaving away, and with 0.05mol/L, 10mL is resuspended for pH 4.5 ~ 5MES damping fluid;
2. add the anti-FITC mAb of 2 ~ 4mg, room temperature suspendible 30 ~ 60min;
3. add the EDC aqueous solution of the freshly prepared 10mg/mL of 0.5 ~ 1mL, room temperature suspendible 2 ~ 12h;
4. magnetic separates, and removes supernatant, uses the TRIS damping fluid of the 0.1mol/L that contains 0.5% bovine serum albumin(BSA) (BSA) pH 8.0 to be resuspended to 1mg/mL, and pH 8.0, is magnetic separation agent.
The nano magnetic particulate chemistry luminescence assays kit of embodiment 4 sugar antigen CA19-9
This kit comprises:
The first reagent (concentration is 0.75 μ g/mL) of preparing according to embodiment 1 method, 50mL;
The second reagent (concentration is 0.75 μ g/mL) of preparing according to embodiment 2 methods, 50mL;
The magnetic separation agent 50mL preparing according to embodiment 3 methods.
The nano magnetic particulate chemistry luminescence assays kit of embodiment 5 sugar antigen CA19-9
This kit comprises:
The first reagent (concentration is 0.5 μ g/mL) of preparing according to embodiment 1 method, 50mL;
The second reagent (concentration is 0.5 μ g/mL) of preparing according to embodiment 2 methods, 50mL;
The magnetic separation agent 50mL preparing according to embodiment 3 methods.
The nano magnetic particulate chemistry luminescence assays kit of embodiment 6 sugar antigen CA19-9
This kit comprises:
The first reagent (concentration is 1 μ g/mL) of preparing according to embodiment 1 method, 50mL;
The second reagent (concentration is 1 μ g/mL) of preparing according to embodiment 2 methods, 50mL;
The magnetic separation agent 50mL preparing according to embodiment 3 methods.
Embodiment 7 takes the kit of embodiment 4 to carry out the quantitative detection of carbohydrate related antigen CA19-9
(1) detecting step:
1. immune response: add 20 μ L sample to be tested (serum or blood plasma) stostes in detector tube, then add 50 μ L the first reagent, 50 μ L the second reagent, mix, incubation 15min under 37 ± 1 DEG C of conditions; Add 50 μ L magnetic separation agents, mix incubation 5min under 37 ± 1 DEG C of conditions;
2. washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add the cleaning fluid of 300 μ L, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, and then magnetic separates, and removes supernatant; This step repeats 3 times;
3. add substrate solution and detect luminous intensity: in detector tube, add 100 μ L alkaline phosphatase chemical luminous substrate solution (APCL-of Beijing Apis Biotechnology Co., Ltd. I), concussion makes the abundant suspendible of magnetic particle, detects luminous intensity in 5min.
(2) draw calibration object typical curve
Calibration object typical curve is referring to Fig. 1.
(3) sensitivity evaluation
Detect " 0 " concentration samples, duplicate detection 20 times, calculate mean value (M) and the standard deviation (SD) of relative luminous intensity (RLU), and calculate M+2SD value, carry out 2 regression fits according to the concentration-RLU between zero-dose calibration object and adjacent calibration object and draw linear function, M+2SD value is brought in above-mentioned equation, obtain corresponding concentration value, be lowest detectable limit.The sensitivity of this method is not more than 2U/mL.Wherein: A point luminous value is respectively referring to table 1:
Table 1
The luminous average X=679 of A point
SD=43.6
X+2SD=766.2
B point luminous value is respectively referring to table 2.
The luminous average X=27042 of B point
Table 2
| CA19-9-STD-B(RLU) |
| 28486 |
| 25597 |
A, B point connects some matched curve referring to Fig. 2.Sensitivity=0.041U/mL.
(4) precision evaluation
1. precision in analyzing
By a collection of the kit of embodiment 4, measure respectively the serum of basic, normal, high three kinds of variable concentrations, 10 hole replicate determinations, result, referring to table 3, show that variation within batch coefficient is 3.69%～6.25%.
Table 3 is analyzed interior precision test
| Measure serum-concentration (U/mL) | Measure number of times | CV (%) in analyzing |
| 40 | 10 | 6.25 |
| 80 | 10 | 3.69 |
| 150 | 10 | 4.35 |
2. precision between analyzing
The kit of embodiment 4 is got to three batches, and every batch of kit is all measured the serum of basic, normal, high three kinds of variable concentrations, 10 hole replicate determinations.Every part of serum obtains 30 concentration measured values, and referring to table 4, the coefficient of variation between statistical study, is 5.65%～7.98%.
Precision test between table 4 analysis
| Measure serum-concentration (U/mL) | Measure number of times | CV between analysis (%) |
| 40 | 30 | 7.98 |
| 80 | 30 | 5.65 |
| 150 | 30 | 5.89 |
(5) accuracy estimating
In 2 routine pooled serum samples, add different amount CA19-9 standard items, the serum that forms 3 concentration levels adds sample, and additive volume is less than 10% of cumulative volume.Detect concentration of specimens, by following formula calculate recovery rate.This method serum matrix recovery is between 85-115%.Data are referring to table 5.
R: the recovery;
V: the volume that adds standard solution;
V
0: the volume of people source sample;
C: people source sample adds the detectable concentration after standard solution;
C
0: the detectable concentration of people source sample;
Cs: the concentration of standard solution.
Table 5 accuracy estimating-interpolation recovery experiment data
(6) kit Evaluation on specificity
To kit specificity, inspection is to choose with carbohydrate related antigen CA19-9 to have the alpha-fetoprotein (AFP) of similar structures, carcinomebryonic antigen (CEA) to be mixed with the sample that is greater than physiological concentration, measures with this method.The results are shown in Table 6, this law and AFP, the equal no cross reaction of CEA.
The experiment of table 6 specificity
(7) correlativity evaluation
100 parts of human serum samples are detected simultaneously with the chemical luminescence reagent kit of kit and ROCHE company.Its testing result is referring to accompanying drawing 3, taking the change of serum C A19-9 concentration of the survey of the inventive method as horizontal ordinate, result taking Perkin Elmer company kit measurement is done regretional analysis as ordinate, and dependent equation is: y=-1.2726+0.9962x, related coefficient is: 0.9832.Learn by statistics result and show, this method is good with external kit clinical sample measured value correlativity.
(8) Evaluation of Thermal Stability
Kit is carried out respectively to 4 DEG C of 12 months and 37 DEG C of stability experiments of 7 days, result show kit standard items luminous intensity variation, batch in and the index such as betweenrun precision, accuracy all within normal range, the kit term of validity can reach 12 months.
Above-described embodiment is only explanation technical conceive of the present invention and feature; its object is to allow person skilled in the art can understand content of the present invention and implement according to this; can not limit the scope of the invention with this; all equivalences that Spirit Essence is done according to the present invention change or modify, within all should being encompassed in protection scope of the present invention.
Claims (8)
1. a nano magnetic particulate chemistry luminescence assays kit of sugar antigen CA19-9, described kit comprises:
The first reagent: containing the solution of fluorescein-labeled carbohydrate related antigen CA19-9 antibody;
The second reagent: the solution that contains the carbohydrate related antigen CA19-9 antibody of alkali phosphatase enzyme mark;
Magnetic separating agent: the suspending liquid that contains the magnetic particle that is being coated with fluorescein antibody;
The carbohydrate related antigen CA19-9 antibody of described alkali phosphatase enzyme mark is connected and composed by crosslinking chemical 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester and 2-imino group sulfane hydrochloride by alkaline phosphatase and carbohydrate related antigen CA19-9 antibody;
The concentration of the fluorescein-labeled carbohydrate related antigen CA19-9 antibody in described the first reagent is 0.5 ~ 1 μ g/mL; The pH of described the first reagent is 7-9; The concentration of the carbohydrate related antigen CA19-9 antibody of the alkali phosphatase enzyme mark in described the second reagent is 0.5 ~ 1 μ g/mL, and the pH of described the second reagent is 7-9.
2. the preparation method of the nano magnetic particulate chemistry luminescence assays kit of a sugar antigen CA19-9 as claimed in claim 1; it comprise respectively the solution that contains fluorescein-labeled carbohydrate related antigen CA19-9 antibody described in preparation, described in contain alkali phosphatase enzyme mark carbohydrate related antigen CA19-9 antibody solution and described in be coated with the step of the suspending liquid of the magnetic particle of fluorescein antibody, it is characterized in that: described in contain alkali phosphatase enzyme mark the preparation process of solution of carbohydrate related antigen CA19-9 antibody as follows:
1. after carbohydrate related antigen CA19-9 antibody being added in crosslinking chemical 2-imino group sulfane hydrochloride solution room temperature to leave standstill, then add glycine solution, room temperature leaves standstill again, collects the carbohydrate related antigen CA19-9 antibody after activation, at 2 ~ 8 DEG C, saves backup;
2. alkaline phosphatase enzyme solutions is added to crosslinking chemical 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester solution, room temperature leaves standstill, and collects the alkaline phosphatase after activation, at 2 ~ 8 DEG C, saves backup;
3. by above-mentioned steps 1. the carbohydrate related antigen CA19-9 antibody after gained activation and step 2. the alkaline phosphatase of gained after activating mix, leave standstill reaction, make to generate the carbohydrate related antigen CA19-9 antibody of alkali phosphatase enzyme mark, after reaction finishes, reactant liquor is purified with Supperdex200 gel-purified post, select damping fluid adjustment concentration and the pH value with proper pH value to obtain described the second reagent;
Wherein, described carbohydrate related antigen CA19-9 antibody, the purity of described alkaline phosphatase are all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase exceedes 1000u/mg.
3. preparation method according to claim 2, it is characterized in that: step 1. in, get carbohydrate related antigen CA19-9 antibody, add coupling agent 2-IT solution to dissolve, room temperature leaves standstill 10min ~ 30min, adds glycine solution, room temperature leaves standstill 2 ~ 10min, with G-25 gel-purified post desalination, collect the carbohydrate related antigen CA19-9 antibody after activation, save backup in 2-8 DEG C.
4. according to the preparation method described in claim 2 or 3, it is characterized in that: step 2. in, get the alkaline phosphatase enzyme solutions that concentration is more than or equal to 5mg/mL, add 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester solution, room temperature leaves standstill 20 ~ 40min, with G-25 gel column desalination, collect the rear alkaline phosphatase of activation, save backup in 2-8 DEG C.
5. preparation method according to claim 2, it is characterized in that: the preparation method of described the first reagent is as follows: the damping fluid that the pH that preparation contains fluorescein is 9 ~ 10, then the ratio that is 20 ~ 200:1 according to the molecular proportion of fluorescein and anti-carbohydrate related antigen CA19-9 antibody, the damping fluid that is 9 ~ 10 by the described pH that contains fluorescein mixes with the damping fluid that the pH of anti-carbohydrate related antigen CA19-9 antibody is 9 ~ 10, after mixing, room temperature leaves standstill reaction, then reactant liquor is separated by G-25 gel column, remove free fluorescein, obtain the solution that contains fluorescein-labeled anti-carbohydrate related antigen CA19-9 antibody, then adjust concentration and pH with the damping fluid with proper pH value, obtain the first reagent.
6. preparation method according to claim 2, it is characterized in that: the preparation method of described magnetic separating agent is as follows: the magnetic particle that makes to contain carboxyl reactive group and fluorescein antibody are under the existence of coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction finishes, magnetic separates, remove supernatant, adjust pH and concentration with the damping fluid with proper pH value, obtain described magnetic separating agent; Wherein said magnetic particle has superparamagnetism, and its diameter is 0.5 ~ 2 μ m, on every gram of magnetic particle with the content of carboxyl reactive group be more than or equal to 0.4mmol; Described fluorescein antibody is monoclonal antibody or polyclonal antibody, and purity is more than or equal to 90wt%, and dilution is tired and is greater than 1:100 ten thousand.
7. according to the preparation method described in claim 2 or 5 or 6, it is characterized in that: the described damping fluid with proper pH value is the TRIS damping fluid that contains 0.5% bovine serum albumin(BSA), pH8.0.
8. adopt the detection method of kit claimed in claim 1 for carbohydrate related antigen CA19-9 is quantitatively detected, it is characterized in that, comprise the following steps:
(1) immune response: add sample to be tested stoste in detector tube, add successively the first reagent and the second reagent, mix, carry out incubation for the first time at 25 ~ 40 DEG C, then add magnetic separation agent, mix, carry out incubation for the second time at 25 ~ 40 DEG C;
(2) washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add cleaning fluid, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, and then magnetic separates, and removes supernatant;
(3) add substrate solution and detect luminous intensity: in detector tube, add the chemical luminous substrate of alkaline phosphatase enzymatic, remove magnetic field, fully after suspendible, detect luminous intensity values.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210550170.8A CN103048453B (en) | 2012-12-18 | 2012-12-18 | Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210550170.8A CN103048453B (en) | 2012-12-18 | 2012-12-18 | Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103048453A CN103048453A (en) | 2013-04-17 |
| CN103048453B true CN103048453B (en) | 2014-11-26 |
Family
ID=48061167
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210550170.8A Active CN103048453B (en) | 2012-12-18 | 2012-12-18 | Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103048453B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105092834A (en) * | 2014-05-05 | 2015-11-25 | 江苏泽成生物技术有限公司 | Carbohydrate antigen 19-9 (CA 19-9) quantitative assay kit, preparation method and detection method thereof |
| CN106124776A (en) * | 2016-06-30 | 2016-11-16 | 深圳市亚辉龙生物科技股份有限公司 | CA724 chemiluminescence immune detection reagent kit and preparation method thereof |
| CN106501515A (en) * | 2016-12-07 | 2017-03-15 | 普菲特益斯生物科技（北京）有限公司 | CA215 detection kit and preparation method thereof and using method |
| CN107389946A (en) * | 2017-08-25 | 2017-11-24 | 北京健安生物科技有限公司 | Sugar antigen CA19 9 determines kit and its detection method |
| CN107957495B (en) * | 2017-11-17 | 2020-10-09 | 南通伊仕生物技术股份有限公司 | CK-MB detection kit and using method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101324579A (en) * | 2007-06-13 | 2008-12-17 | 清华大学 | Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method |
| CA2760366A1 (en) * | 2009-05-29 | 2010-12-02 | F.Hoffmann-La Roche Ag | Secernin-1 as a marker for cancer |
| CN101937000A (en) * | 2010-08-05 | 2011-01-05 | 北京倍爱康生物技术有限公司 | Magnetic particle separation chemiluminescence immunoassay method of human cystatin C |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB201021289D0 (en) * | 2010-12-15 | 2011-01-26 | Immatics Biotechnologies Gmbh | Novel biomarkers for a prediction of the outcome of an immunotherapy against cancer |
| EP2675485A4 (en) * | 2011-02-15 | 2014-10-15 | Immunomedics Inc | Anti-mucin antibodies for early detection and treatment of pancreatic cancer |
-
2012
- 2012-12-18 CN CN201210550170.8A patent/CN103048453B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101324579A (en) * | 2007-06-13 | 2008-12-17 | 清华大学 | Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method |
| CA2760366A1 (en) * | 2009-05-29 | 2010-12-02 | F.Hoffmann-La Roche Ag | Secernin-1 as a marker for cancer |
| CN101937000A (en) * | 2010-08-05 | 2011-01-05 | 北京倍爱康生物技术有限公司 | Magnetic particle separation chemiluminescence immunoassay method of human cystatin C |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103048453A (en) | 2013-04-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103063851B (en) | Free triiodothyronine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof | |
| CN103048452B (en) | Nanometer magnetic particle chemiluminiscence determination kit for antigen CA125 relating to tumor, as well as preparation method and determining method of same | |
| CN103901203B (en) | Chemical luminescent analysis reagent kid of a kind of Procalcitonin and preparation method thereof and detection method | |
| CN104634980B (en) | The super quick detection kit of cardiac muscle troponin I and super quick detection method | |
| CN103389381B (en) | Human epididymal secretory protein E4 chemiluminescence detection kit and preparation method thereof | |
| CN102998467B (en) | β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| CN103048453B (en) | Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof | |
| CN103364558A (en) | Human tumor marker carcinoembryonic antigen (CEA) magnetic particle chemiluminiscence immunoassay kit and detection method | |
| CN103063845A (en) | Nanometer magnetic particle chemiluminiscence kit, preparation method and detection method of hepatitis B virus surface-antibody | |
| CN102901820A (en) | Immunoassay kit and immunodetection method for magnetic particle chemiluminescence of human tumor marker carbohydrate antigen 50 (CA50) | |
| CN104034892A (en) | Magnetic particle chemiluminescence immune assay kit of tumor marker AFP (alpha fetal protein) and detection method thereof | |
| CN103076455B (en) | Quantitative detection serum amyloid A protein kit and Synthesis and applications thereof | |
| CN103592445A (en) | Kit for detecting procalcitonin | |
| CN104198721B (en) | The preparation of a kind of silica-based magnetic bead bond of GP73 (GP73) antigen and application | |
| CN101398431A (en) | Tumor-associated antigen 125 magnetic microparticle chemiluminescence immune assay determination kit and method for making same | |
| Lyu et al. | Automatic label-free immunoassay with high sensitivity for rapid detection of SARS-CoV-2 nucleocapsid protein based on chemiluminescent magnetic beads | |
| CN103344634A (en) | Chemiluminescence immune assay kit and method for magnetic particle of human alpha fetoprotein (AFP) | |
| CN103048477B (en) | Nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as preparation method and detecting method of same | |
| CN101514991A (en) | Alpha-fetoprotein magnetic particle chemoluminescence immunoassay kit and method for preparing same | |
| CN103048476A (en) | Thyroxine nanometer magnetic particle chemiluminiscence determinstion kit as well as preparation method and detection method thereof | |
| CN103018465A (en) | Human cystatin C chemiluminescence quantitative detection method | |
| CN111044724A (en) | Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof | |
| CN102608316B (en) | Kit or test strip for detecting quinoxaline compound | |
| CN106324254A (en) | Anti-insulin antibody detection kit and detection method thereof | |
| CN101398430A (en) | Tumor-associated antigen 153 magnetic microparticle chemiluminescence immune assay determination kit and method for making same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: Xinghu Street Industrial Park of Suzhou city in Jiangsu province 215123 building C6 No. 218 Patentee after: Jiangsu Hao Bo biomedical Limited by Share Ltd Address before: Xinghu Street Industrial Park of Suzhou city in Jiangsu province 215123 building C6 No. 218 Patentee before: Suzhou Haooubo Biopharmaceutical Co., Ltd. |