CN103048461A - Nanometer magnetic particle chemiluminescent assay kit for cancer antigen CA15-3, and preparation method and detection method thereof - Google Patents
Nanometer magnetic particle chemiluminescent assay kit for cancer antigen CA15-3, and preparation method and detection method thereof Download PDFInfo
- Publication number
- CN103048461A CN103048461A CN2012105501731A CN201210550173A CN103048461A CN 103048461 A CN103048461 A CN 103048461A CN 2012105501731 A CN2012105501731 A CN 2012105501731A CN 201210550173 A CN201210550173 A CN 201210550173A CN 103048461 A CN103048461 A CN 103048461A
- Authority
- CN
- China
- Prior art keywords
- antibody
- cancer antigens
- reagent
- fluorescein
- magnetic
- 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.)
- Pending
Links
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a nanometer magnetic particle chemiluminescent assay kit for a cancer antigen CA15-3, and a preparation method and a detection method thereof. The kit comprises a solution containing a fluorescein-mark labeled cancer antigen CA15-3 antibody, suspension of fluorescein antibody coated magnetic particles and a solution containing an alkaline phosphatase labeled cancer antigen CA15-3 antibody, wherein the alkaline phosphatase labeled cancer antigen CA15-3 antibody is formed by connecting alkaline phosphatase and the cancer antigen CA15-3 antibody through a crosslinking agent SMCC and 2-IT. According to the invention, the cancer antigen CA15-3 can be quantitatively detected with low cost, high accuracy degree and high precision degree.
Description
Technical field
The invention belongs to biological technical field, be specifically related to nano magnetic particulate chemistry luminescence assays kit and preparation method thereof and the detection method of a kind of cancer antigens c A15-3 that combines immune magnetic particle isolation technics and chemiluminescence immunoassay technology.
Background technology
Cancer antigens c A15-3 (Carbohydrate Antigen 15-3, CA15-3) is a kind of polymorphic Glycoproteins in Epithelial, and molecular weight is about 400kD, and molecular structure still imperfectly understands.CA15-3 is the monoclonal antibody (115-DB) of being made by the glycoprotein MAM-6 from HMFG's film such as Hilkens in 1984, meanwhile Kufu etc. makes monoclonal antibody (DF-3) from the hepatic metastases breast cancer cell membrane, and therefore both are altogether by name CA15-3.
CA15-3 is mainly used in monitoring and the screening of breast cancer, and the index that is used for evaluate patient prognosis situation.Patient with breast cancer 30-50%CA15-3 increased content has metastasis person to increase and can reach 80%; The susceptibility of finding metastasis of cancer is higher than carcinomebryonic antigen and tissue polypeptide antigen, and shifts early than clinical discovery; CA15-3 is monitoring patient postoperative recurrence situation, the particularly important indicator of cancer metastasis patient's monitoring after operation, and change of serum C A15-3 level increases, the part of prompting breast cancer or whole body recurrence, and increase early than nuclide examination and clinical examination; Be used for to treatment with looking into assessment and the early stage metastasis of finding, the increasing of CA15-3 goes out metastasis than clinical diagnosis and wants the Zao some months time; CA15-3 can with CA125 joint inspection, be used for the early diagnosis of Ovarian cancer; During with the CEA joint-detection, can improve susceptibility and the specificity of early diagnosing mammary cancer; Other malignant tumours such as lung cancer, oophoroma, colon cancer, liver cancer etc., change of serum C A15-3 also can occur and increase.The non-malignant tumors disease can cause that such as benign breast disease, disease of ovary etc. the level of CA15-3 increases.
Immune analysis method for detection of CA15-3 mainly contains enzyme-linked immunosorbent assay, chemiluminescence immunoassay etc. at present.The methodology limiting factors such as enzyme-linked immunosorbent assay exists sensitivity low, and narrow, the difficult realization of the range of linearity is full-automatic.Chemiluminescence immunoassay is a kind of immunoassay technology that grows up on the enzyme-linked immunosorbent assay basis, have highly sensitive, detect the advantages such as linear wide ranges, easy and simple to handle, automaticity height.Chemiluminescence immunoassay technology is widely used because it has above-mentioned plurality of advantages at present.
Yet, in the immune detection of reality, owing to impurity component contained in the testing sample is more, detection sensitivity and accuracy have been affected to a certain extent, so from the sample substrate of complexity, separate fast, be purified into the purpose determinand, it is one of difficult problem of facing of clinical examination worker.
The magnetic particle immunoassay technology is to utilize the magnetic solid phase particle of synthesis of polymer material certain particle size size to make carrier, have the various immunologic active materials such as the antibody of specificity affinity or antigen on coated with methods such as physisorption, chemical couplings, have that velocity of separation is fast, efficient is high, the characteristics such as favorable repeatability, simple to operate, the biological character that do not affect separated cell or other biological material and function, adding orientable motion under the magnetic fields, so that some special composition is separated, concentrated or purifying.
The open CN102288766A of Chinese invention patent discloses the immue quantitative detection reagent box of CA15-3 (CA15-3) a kind of, this kit comprises the CA15-3 magnetic separation agent that contains the magnetic microsphere that is marked with anti-CA15-3 monoclonal antibody, the enzyme reaction thing that contains the anti-CA15-3 monoclonal antibody of alkali phosphatase enzyme mark, increased response liquid, dilution, the CA15-3 standard items, CA15-3 quality-control product, cleaning concentrate and substrate solution.Although this kit can be realized detecting quickly and accurately, but wherein, the CA15-3 magnetic separation agent that contains the magnetic microsphere that is marked with anti-CA15-3 monoclonal antibody is very loaded down with trivial details with the preparation process of the enzyme reaction thing of the anti-CA15-3 monoclonal antibody that contains alkali phosphatase enzyme mark, technology stability is bad, and mark rate is low, limits it and detects particularly precision and cause cost to increase of effect.
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 cancer antigens c A15-3 is provided; it can prepare with lower cost, and can realize cancer antigens c A15-3 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 cancer antigens c A15-3 simultaneously, the method process stabilizing, and cost is low, and the precision of gained kit is high.
The kit that cancer antigen is used easy and preparation method cheaply.
The nano magnetic particulate chemistry luminescence assays kit of a kind of cancer antigens c A15-3 is characterized in that, this kit comprises:
The first reagent: the solution that contains fluorescein-labeled cancer antigens c A15-3 antibody;
The second reagent: the solution that contains the cancer antigens c A15-3 antibody of alkaline phosphatase (ALP) mark;
Magnetic separating agent: the suspending liquid that contains the magnetic particle of coated fluorescein antibody;
Wherein, the cancer antigens c A15-3 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 cancer antigens c A15-3 antibody, SMCC) and 2-imino group sulfane hydrochloride (2-Iminothiolane HCl, 2-IT) connect and compose.
Further, in the 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 cancer antigens c A15-3 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 cancer antigens c A15-3 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 such as other reagent such as substrate solutions, therefore, although can comprise these reagent in the 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 cancer antigens c A15-3; it comprises the step for preparing respectively described the first reagent, described the second reagent and magnetic separating agent, and wherein: the preparation process of described the second reagent is as follows:
1. with after room temperature leaves standstill in the cancer antigens c A15-3 antibody adding crosslinking chemical 2-imino group sulfane hydrochloride solution, add glycine solution, room temperature leaves standstill again, collects the cancer antigens c A15-3 antibody after activating, and saves backup under 2 ~ 8 ℃ again;
2. the alkaline phosphatase enzyme solutions is added crosslinking chemical 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester solution, room temperature leaves standstill, and collects the alkaline phosphatase after activating, and saves backup under 2 ~ 8 ℃;
3. with above-mentioned steps 1. the alkaline phosphatase of the cancer antigens c A15-3 antibody after the gained activation after 2. gained activates with step mix, leave standstill reaction, make the cancer antigens c A15-3 antibody that generates alkali phosphatase enzyme mark, after reaction finishes, with reactant liquor Supperdex200 gel-purified post purifying, damping fluid adjustment concentration and the pH value selecting to have proper pH value namely get described the second reagent;
Wherein, the purity of described cancer antigens c A15-3 antibody, described alkaline phosphatase is all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase surpasses 1000u/mg.
Preferably, step 1. in, get cancer antigens c A15-3 antibody, add the dissolving of coupling agent 2-IT solution, room temperature leaves standstill 10min ~ 30min, adds glycine solution, and room temperature leaves standstill 2 ~ 10min, with G-25 gel-purified post desalination, collect the cancer antigens c A15-3 antibody after activating, save backup in 2-8 ℃.
Preferably, step 2. in, get concentration more than or equal to the alkaline phosphatase enzyme solutions of 5mg/mL, add and use SMCC solution, room temperature leaves standstill 20 ~ 40min, with G-25 gel column desalination, collects the rear alkaline phosphatase of activation, saves backup in 2-8 ℃.
Preferably, step 3. in, be that 1:1 ~ 1:2 mixes with the alkaline phosphatase of the cancer antigens c A15-3 antibody of above-mentioned activation and activation by the molecule mol ratio, under 2-8 ℃ of condition, leave standstill 12-24h.
Wherein the damping fluid of proper pH value can be for for example containing the TRIS damping fluid of 0.5% bovine serum albumin(BSA), pH8.0.
Further, the preparation method of described the first reagent is as follows: the pH that preparation contains fluorescein is 9 ~ 10 damping fluid, then the molecular proportion according to fluorescein and cancer antigens c A15-3 antibody is the ratio of 20 ~ 200:1, be 9 ~ 10 damping fluid with the described pH that contains fluorescein with the pH of cancer antigens c A15-3 antibody be that 9 ~ 10 damping fluid mixes, behind the mixing, room temperature leaves standstill reaction, then reactant liquor is separated by the G-25 gel column, remove free fluorescein, obtain containing the solution of fluorescein-labeled anticancer antigens c A15-3 antibody, then adjust concentration and pH with the damping fluid with proper pH value, and get final product.Wherein the damping fluid of proper pH value can be for for example containing the TRIS damping fluid of 0.5% bovine serum albumin(BSA), pH8.0.
Further, the preparation method of described magnetic separating agent is as follows: will contain the magnetic particle of carboxyl reactive group and fluorescein antibody in the presence of the 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, namely get described magnetic separating agent.Wherein said magnetic particle has superparamagnetism, and its diameter is 0.5 ~ 2 μ m, on every gram 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 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 commonly used have for example fluorescein isothiocynate, RB 200, a TRITC etc.
The present invention also provides simultaneously a kind of and has adopted above-mentioned kit to be applied to the detection method that cancer antigens c A15-3 quantitatively detects, and it is characterized in that, may further comprise the steps:
(1) immune response: add the sample to be tested dilution in detector tube, add successively the first reagent and the second reagent, mixing carries out the incubation first time under 25 ~ 40 ℃, then add the magnetic separation agent, and mixing carries out the incubation second time under 25～40 ℃;
(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 detect luminous intensity values behind the suspendible.
Further, the time of incubation described first time can be 20 ~ 40min, is generally 30min; The time of incubation can be 2 ~ 15min for the second time, is generally 10min.
Because the enforcement of above technical scheme, the present invention compared with prior art has following advantage:
1. adopt kit of the present invention can realize accurate, the accurate detection of cancer antigens c A15-3, its three kinds of reagent all can prepare by stable preparation technology, and production cost is low, and because preparation technology's stability, it is little that kit is analyzed differences between batches, and precision improves between the analysis of detection;
2. the preparation method of the solution of the cancer antigens c A15-3 antibody that contains alkali phosphatase enzyme mark in the kit of the present invention, can be effectively with cancer antigens c A15-3 antibody and alkaline phosphatase coupling, coupling efficiency is high, further reduces the cost of kit low and guarantee the detection effect 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 need not pre-dilution, simple to operate saving time compared with the method that adopts the import reagent box to detect, and detection method of the present invention has significant advantage at cost.
Description of drawings
Fig. 1 is detection calibration product typical curves;
Fig. 2 is that matched curve is estimated 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: with the cancer antigens c A15-3(CA15-3 of phosphate buffer preservation) monoclonal antibody (purity surpasses 95wt%, and concentration is 2mg/mL); Fluorescein isothiocynate (FITC), the reagent such as sodium carbonate should reach chemical pure; The buying of G-25 gel-purified post is 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. add step according to CA15-3 antibody and FITC molecular proportion by the ratio of 1:20 in antibody-solutions and 1. joined FITC solution, mix, room temperature left standstill 12h hour, and reaction generates CA15-3 antibody-FITC connector;
3. will separate by the G-25 gel column through step reactant liquor 2., remove unreacted FITC, obtain containing the solution of CA15-3 antibody-FITC connector (being the CA15-3 antibody of FITC mark);
4. with step 3. the gained solution that contains CA15-3 antibody-FITC connector to be diluted to CA15-3 antibody-FITC connector concentration with the TRIS damping fluid of the 0.1mol/L that contains 0.5% bovine serum albumin(BSA) (BSA) pH 8.0 be 0.5 ~ 1 μ g/mL, be the first reagent.
The preparation of embodiment 2 second reagent
(1) material and instrument: with the CA15-3 monoclonal antibody (purity surpasses 95wt%, and concentration is 2mg/mL) of phosphate buffer preservation; The alkaline phosphatase of preserving with phosphate buffer (ALP solution, ALP purity are approximately 99%, and specific activity is about 1500U/mg, and concentration is 10mg/mL); Crosslinking aid S MCC, 2-IT is available 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 CA15-3 antibody, add the coupling agent 2-IT solution 2-4 μ L of 10mg/mL, room temperature leaves standstill 20min, add the glycine solution 10 μ L of 0.1mol/L, room temperature leaves standstill 5min, with G-25 gel column desalination, collect the rear CA15-3 antibody of activation, 2-8 ℃ 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 ℃ saves backup;
3. the cancer antigens c A15-3 antibody with above-mentioned activation mixes with the ALP of activation, leaves standstill 12-24h under the 2-8 ℃ of condition, with Supperdex200 gel-purified post purifying conjugate, obtains CA15-3 antibody-ALP connector strong solution, and 2-8 ℃ saves backup.
4. use the TRIS damping fluid of the 0.1mol/L that contains 0.5% bovine serum albumin(BSA) (BSA) pH8.0 to be diluted to 0.5-1 μ g/mL CA15-3 antibody-ALP connector strong solution, finish 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 contain the active group of carboxyl (COOH), and every gram (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, also can be monoclonal antibody, and purity is more than the 90wt%, and dilution is tired above 1:100 ten thousand;
MES (MES), carbodiimide (EDC), TRI S and other reagent should reach chemical pure.
(2) preparation process:
1. get the suspending liquid of 100mg magnetic particle, magnetic divides the supernatant of leaving away, and uses 0.05mol/L, and 10mL is resuspended for pH 4.5 ~ 5MES damping fluid;
2. the anti-FITC mAb that adds 2 ~ 4mg, room temperature suspendible 30 ~ 60min;
3. the EDC aqueous solution that adds 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, are the magnetic separation agent.
The nano magnetic particulate chemistry luminescence assays kit of embodiment 4 cancer antigens c A15-3
This kit comprises:
According to first reagent (concentration is 0.75 μ g/mL) of embodiment 1 method preparation, 50mL;
According to second reagent (concentration is 0.75 μ g/mL) of embodiment 2 methods preparation, 50mL;
Magnetic separation agent 50mL according to the preparation of embodiment 3 methods.
The nano magnetic particulate chemistry luminescence assays kit of embodiment 5 cancer antigens c A15-3
This kit comprises:
According to first reagent (concentration is 0.5 μ g/mL) of embodiment 1 method preparation, 50mL;
According to second reagent (concentration is 0.5 μ g/mL) of embodiment 2 methods preparation, 50mL;
Magnetic separation agent 50mL according to the preparation of embodiment 3 methods.
The nano magnetic particulate chemistry luminescence assays kit of embodiment 6 cancer antigens c A15-3
This kit comprises:
According to first reagent (concentration is 1 μ g/mL) of embodiment 1 method preparation, 50mL;
According to second reagent (concentration is 1 μ g/mL) of embodiment 2 methods preparation, 50mL;
Magnetic separation agent 50mL according to the preparation of embodiment 3 methods.
Embodiment 7 takes the kit of embodiment 4 to carry out the quantitative detection of cancer antigens c A15-3
(1) detecting step:
1. immune response: in detector tube, add 10 μ L sample to be tested (serum or blood plasma) dilutions, then add 50 μ L the first reagent, 50 μ L the second reagent, mixing, incubation 30min under 37 ± 1 ℃ of conditions; Add 50 μ L magnetic separation agents, mixing, incubation 10min under 37 ± 1 ℃ 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: add 100 μ L alkaline phosphatase chemical luminous substrate solution (Beijing Ah APCL-of this Bioisystech Co., Ltd I) in detector tube, concussion makes the abundant suspendible of magnetic particle, detects luminous intensity in 5min.
(2) draw the calibration object typical curve
The 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 calculating M+2SD value, carry out 2 regression fits according to the concentration-RLU between zero-dose calibration object and the adjacent calibration object and draw linear function, the M+2SD value is brought in the above-mentioned equation, obtain corresponding concentration value, be lowest detectable limit.The sensitivity of this method is not more than 1U/mL.Wherein: A point luminous value is respectively referring to table 1:
Table 1
The luminous average X=684 of A point
SD=45
X+2SD=858
B point luminous value is respectively referring to table 2.
The luminous average X=28316 of B point
Table 2
| CA15-3-STD-B(RLU) |
| 29108 |
| 27524 |
A, B point connect the some matched curve referring to Fig. 2.Sensitivity=0.033U/mL.
(4) precision evaluation
1. precision in analyzing
The kit of embodiment 4 is a collection of, measure respectively the serum of basic, normal, high three kinds of variable concentrations, 10 hole replicate determinations, the result is referring to table 3, and drawing the variation within batch coefficient is 3.54%～5.85%.
Precision test in table 3 is analyzed
| Measure serum-concentration (U/mL) | Measure number of times | CV (%) in analyzing |
| 30 | 10 | 5.85 |
| 90 | 10 | 4.90 |
| 150 | 10 | 3.54 |
2. precision between analyzing
The kit of embodiment 4 is got 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 4.65%～8.98%.
Precision test between table 4 is analyzed
| Measure serum-concentration (U/mL) | Measure number of times | CV between analysis (%) |
| 30 | 30 | 8.98 |
| 90 | 30 | 4.65 |
| 150 | 30 | 6.89 |
(5) accuracy estimating
Add different amount CA15-3 standard items in 2 routine pooled serum samples, the serum that forms 3 concentration levels adds sample, and the 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 90-110%.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 behind the standard solution;
C
0: the detectable concentration of people source sample;
C
S: the concentration of standard solution.
Table 5 accuracy estimating-interpolation recovery experiment data
(6) kit Evaluation on specificity
Check is to choose with cancer antigens c A15-3 to have the alpha-fetoprotein (AFP) of similar structures, carcinomebryonic antigen (CEA) to be mixed with the sample greater than physiological concentration to the kit specificity, 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
Chemical luminescence reagent kit with kit and Abbott detects simultaneously to 100 parts of human serum samples.Its testing result is referring to accompanying drawing 3, take the change of serum C A15-3 concentration of the survey of the inventive method as horizontal ordinate, does regretional analysis take the result of ROCHE company kit measurement as ordinate, and dependent equation is: y=0.5011+0.9750x, related coefficient is: 0.9828.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 4 ℃ of 12 months and 37 ℃ of stability experiments of 7 days, the result show kit standard items luminous intensity variation, batch in and the indexs such as betweenrun precision, accuracy all within normal range, the kit term of validity can reach 12 months.
Above-described embodiment only is explanation technical conceive of the present invention and characteristics; its purpose is to allow the personage who is familiar with technique can understand content of the present invention and according to this enforcement; can not limit protection scope of the present invention with this; all equivalences that Spirit Essence is done according to the present invention change or modify, and all should be encompassed within protection scope of the present invention.
Claims (10)
1. the nano magnetic particulate chemistry luminescence assays kit of a cancer antigens c A15-3, described kit comprises:
The first reagent: the solution that contains fluorescein-labeled cancer antigens c A15-3 antibody;
The second reagent: the solution that contains the cancer antigens c A15-3 antibody of alkali phosphatase enzyme mark;
Magnetic separating agent: the suspending liquid that contains the magnetic particle of coated fluorescein antibody;
Wherein, the cancer antigens c A15-3 antibody of this 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 cancer antigens c A15-3 antibody.
2. the nano magnetic particulate chemistry luminescence assays kit of cancer antigens c A15-3 according to claim 1 is characterized in that: in the described magnetic separation agent, and phase chemistry coupling between fluorescein antibody and the magnetic particle.
3. the nano magnetic particulate chemistry luminescence assays kit of cancer antigens c A15-3 according to claim 1 and 2, it is characterized in that: the concentration of the fluorescein-labeled cancer antigens c A15-3 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 cancer antigens c A15-3 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.
4. the preparation method of the nano magnetic particulate chemistry luminescence assays kit of a cancer antigens c A15-3 as claimed in claim 3; the step that it comprises the suspending liquid of the solution for preparing respectively the described solution that contains fluorescein-labeled cancer antigen-antibody, the described cancer antigens c A15-3 antibody that contains alkali phosphatase enzyme mark and the described magnetic particle that is coated with fluorescein antibody is characterized in that: the preparation process of the solution of the described cancer antigens c A15-3 antibody that contains alkali phosphatase enzyme mark is as follows:
1. with after room temperature leaves standstill in the cancer antigens c A15-3 antibody adding crosslinking chemical 2-imino group sulfane hydrochloride solution, add glycine solution, room temperature leaves standstill again, collects the cancer antigens c A15-3 antibody after activating, and saves backup under 2 ~ 8 ℃ again;
2. the alkaline phosphatase enzyme solutions is added crosslinking chemical 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester solution, room temperature leaves standstill, and collects the alkaline phosphatase after activating, and saves backup under 2 ~ 8 ℃;
3. with above-mentioned steps 1. the alkaline phosphatase of the cancer antigens c A15-3 antibody after the gained activation after 2. gained activates with step mix, leave standstill reaction, make the cancer antigens c A15-3 antibody that generates alkali phosphatase enzyme mark, after reaction finishes, with reactant liquor Supperdex200 gel-purified post purifying, damping fluid adjustment concentration and the pH value selecting to have proper pH value namely get described the second reagent;
Wherein, the purity of described cancer antigens c A15-3 antibody, described alkaline phosphatase is all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase surpasses 1000u/mg.
5. preparation method according to claim 4, it is characterized in that: step 1. in, get cancer antigens c A15-3 antibody, add the dissolving of coupling agent 2-IT solution, room temperature leaves standstill 10min ~ 30min, adds glycine solution, room temperature leaves standstill 2 ~ 10min, with G-25 gel-purified post desalination, collect the cancer antigens c A15-3 antibody after activating, save backup in 2-8 ℃.
6. according to claim 4 or 5 described preparation methods, it is characterized in that: step 2. in, get concentration more than or equal to the alkaline phosphatase enzyme solutions of 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 ℃.
7. preparation method according to claim 4, it is characterized in that: the preparation method of described the first reagent is as follows: the pH that preparation contains fluorescein is 9 ~ 10 damping fluid, then the molecular proportion according to fluorescein and anticancer antigens c A15-3 antibody is the ratio of 20 ~ 200:1, be 9 ~ 10 damping fluid with the described pH that contains fluorescein with the pH of anticancer antigens c A15-3 antibody be that 9 ~ 10 damping fluid mixes, behind the mixing, room temperature leaves standstill reaction, then reactant liquor is separated by the G-25 gel column, remove free fluorescein, obtain containing the solution of fluorescein-labeled anticancer antigens c A15-3 antibody, then adjust concentration and pH with the damping fluid with proper pH value, namely get the first reagent.
8. preparation method according to claim 4, it is characterized in that: the preparation method of described magnetic separating agent is as follows: make the magnetic particle that contains the carboxyl reactive group and fluorescein antibody in the presence of the 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, namely get described magnetic separating agent; Wherein said magnetic particle has superparamagnetism, and its diameter is 0.5 ~ 2 μ m, on every gram magnetic particle with the content of carboxyl reactive group 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 greater than 1:100 ten thousand.
9. according to claim 4 or 7 or 8 described preparation methods, it is characterized in that: described damping fluid with proper pH value is the TRIS damping fluid that contains 0.5% bovine serum albumin(BSA), pH8.0.
10. the described kit of each claim is used for the quantitative detection method that detects to cancer antigens c A15-3 in the employing claim 1 ~ 3, it is characterized in that, may further comprise the steps:
(1) immune response: add the sample to be tested dilution in detector tube, add successively the first reagent and the second reagent, mixing carries out the incubation first time under 25 ~ 40 ℃, then add the magnetic separation agent, and mixing carries out the incubation second time under 25 ~ 40 ℃;
(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 detect luminous intensity values behind the suspendible.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012105501731A CN103048461A (en) | 2012-12-18 | 2012-12-18 | Nanometer magnetic particle chemiluminescent assay kit for cancer antigen CA15-3, and preparation method and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012105501731A CN103048461A (en) | 2012-12-18 | 2012-12-18 | Nanometer magnetic particle chemiluminescent assay kit for cancer antigen CA15-3, and preparation method and detection method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103048461A true CN103048461A (en) | 2013-04-17 |
Family
ID=48061175
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012105501731A Pending CN103048461A (en) | 2012-12-18 | 2012-12-18 | Nanometer magnetic particle chemiluminescent assay kit for cancer antigen CA15-3, and preparation method and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103048461A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105891193A (en) * | 2016-06-30 | 2016-08-24 | 深圳市亚辉龙生物科技股份有限公司 | Chemiluminescent immune detection kit for respiratory syncytial virus and preparation method thereof |
| CN105929166A (en) * | 2016-04-20 | 2016-09-07 | 北京中航赛维生物科技有限公司 | Magnetic particle-based quantitative chemiluminescent assay kit for anti-LKM-1 antibody IgG, and preparation and detection methods thereof |
| CN112400113A (en) * | 2018-07-13 | 2021-02-23 | 3M创新有限公司 | Specific binding chemiluminescence assay |
| CN116482369A (en) * | 2023-06-13 | 2023-07-25 | 天津市协和医药科技集团有限公司 | Carcinoembryonic antigen detection kit |
Citations (7)
| 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 |
| CN101639481A (en) * | 2009-08-27 | 2010-02-03 | 清华大学 | Magnetic particle chemiluminescence immunoassay kit of free thyroxine |
| CN101762697A (en) * | 2009-06-24 | 2010-06-30 | 北京科美东雅生物技术有限公司 | Magnetic immuno-chromatographic test paper strip for quantitatively detecting tumor associated antigen 15-3 in blood and preparation method thereof |
| WO2010135431A2 (en) * | 2009-05-19 | 2010-11-25 | The Regents Of The University Of California | Compositions, devices, and methods related to prostate-specific membrane antigen |
| CN101975859A (en) * | 2010-09-13 | 2011-02-16 | 北京倍爱康生物技术有限公司 | Magnetic microparticle separation chemiluminescent immunoassay detection method for hepatitis B virus surface antigen |
| CN102288766A (en) * | 2011-08-16 | 2011-12-21 | 内蒙古科慧生物科技有限责任公司 | Carbohydrate antigen 15-3 (CA15-3) quantitative measurement kit and detection method thereof |
| US20120202217A1 (en) * | 2009-12-03 | 2012-08-09 | Abbott Laboratories | Autoantibody enhanced immunoassays and kits |
-
2012
- 2012-12-18 CN CN2012105501731A patent/CN103048461A/en active Pending
Patent Citations (7)
| 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 |
| WO2010135431A2 (en) * | 2009-05-19 | 2010-11-25 | The Regents Of The University Of California | Compositions, devices, and methods related to prostate-specific membrane antigen |
| CN101762697A (en) * | 2009-06-24 | 2010-06-30 | 北京科美东雅生物技术有限公司 | Magnetic immuno-chromatographic test paper strip for quantitatively detecting tumor associated antigen 15-3 in blood and preparation method thereof |
| CN101639481A (en) * | 2009-08-27 | 2010-02-03 | 清华大学 | Magnetic particle chemiluminescence immunoassay kit of free thyroxine |
| US20120202217A1 (en) * | 2009-12-03 | 2012-08-09 | Abbott Laboratories | Autoantibody enhanced immunoassays and kits |
| CN101975859A (en) * | 2010-09-13 | 2011-02-16 | 北京倍爱康生物技术有限公司 | Magnetic microparticle separation chemiluminescent immunoassay detection method for hepatitis B virus surface antigen |
| CN102288766A (en) * | 2011-08-16 | 2011-12-21 | 内蒙古科慧生物科技有限责任公司 | Carbohydrate antigen 15-3 (CA15-3) quantitative measurement kit and detection method thereof |
Non-Patent Citations (4)
| Title |
|---|
| XIAOYING GUO ET AL: "Enzyme Chemiluminescence Immuno Assay of Free hCGβ in Serum Based on Superparamagnetic Polymer Microbeads", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》, vol. 7, 31 December 2006 (2006-12-31), pages 274 - 288 * |
| 代峰等: "卵巢癌患者血清IGF-1、TGF-α和CA125联检的临床价值", 《放射免疫学杂志》, vol. 19, no. 3, 31 December 2006 (2006-12-31), pages 200 - 201 * |
| 李智勇等: "板式磁颗粒化学发光免疫分析测定人血清中糖类抗原125", 《化学学报》, vol. 68, no. 2, 31 December 2010 (2010-12-31), pages 162 - 168 * |
| 郑铁生等: "磁分离酶联免疫测定男性生殖激素方法性能评价", 《江苏大学学报（医学版）》, vol. 12, no. 4, 31 August 2002 (2002-08-31), pages 326 - 328 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105929166A (en) * | 2016-04-20 | 2016-09-07 | 北京中航赛维生物科技有限公司 | Magnetic particle-based quantitative chemiluminescent assay kit for anti-LKM-1 antibody IgG, and preparation and detection methods thereof |
| CN105891193A (en) * | 2016-06-30 | 2016-08-24 | 深圳市亚辉龙生物科技股份有限公司 | Chemiluminescent immune detection kit for respiratory syncytial virus and preparation method thereof |
| CN112400113A (en) * | 2018-07-13 | 2021-02-23 | 3M创新有限公司 | Specific binding chemiluminescence assay |
| CN116482369A (en) * | 2023-06-13 | 2023-07-25 | 天津市协和医药科技集团有限公司 | Carcinoembryonic antigen detection kit |
| CN116482369B (en) * | 2023-06-13 | 2023-08-25 | 天津市协和医药科技集团有限公司 | Carcinoembryonic antigen detection kit |
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 | |
| CN103389381B (en) | Human epididymal secretory protein E4 chemiluminescence detection kit and preparation method thereof | |
| CN104698184B (en) | The test kit of detection sugar antigen | |
| CN103063845A (en) | Nanometer magnetic particle chemiluminiscence kit, preparation method and detection method of hepatitis B virus surface-antibody | |
| CN110726841B (en) | Method for detecting veterinary drug residues based on enzymatic click reaction signal amplification magnetic relaxation time immunosensor | |
| CN102998467B (en) | β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| CN105181680B (en) | Magnetic bead separation chemiluminescence immunoassay method for melamine | |
| CN103592445A (en) | Kit for detecting procalcitonin | |
| CN105510604A (en) | Method for improving sensitivity and linearity of latex reagent | |
| 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 | |
| CN103364558A (en) | Human tumor marker carcinoembryonic antigen (CEA) magnetic particle chemiluminiscence immunoassay kit and detection method | |
| CN103048453B (en) | Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof | |
| CN109580946A (en) | Test strips and preparation method a kind of while that detect A type and influenza B virus | |
| CN103575902B (en) | A kind of time-resolved fluorescence method four comprehensive detection oophoroma kits and application thereof | |
| CN103018445B (en) | CA50 magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| CN103344634A (en) | Chemiluminescence immune assay kit and method for magnetic particle of human alpha fetoprotein (AFP) | |
| CN104820102A (en) | Kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, and method and application thereof | |
| CN103048477B (en) | Nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as preparation method and detecting method of same | |
| CN103048476A (en) | Thyroxine nanometer magnetic particle chemiluminiscence determinstion kit as well as preparation method and detection method thereof | |
| CN105891463A (en) | Beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence | |
| CN108152507A (en) | A kind of I type precollagen amino terminal peptides detection kit and its detection method | |
| CN104880564A (en) | Kit for detecting resistin as well as preparation method and detection method of kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130417 |