CN104007256A - Method for manufacturing total PSA and free PSA two-in-one chemiluminescence immunologic diagnosis kit - Google Patents
Method for manufacturing total PSA and free PSA two-in-one chemiluminescence immunologic diagnosis kit Download PDFInfo
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- CN104007256A CN104007256A CN201310055076.XA CN201310055076A CN104007256A CN 104007256 A CN104007256 A CN 104007256A CN 201310055076 A CN201310055076 A CN 201310055076A CN 104007256 A CN104007256 A CN 104007256A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
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- G01N2800/342—Prostate diseases, e.g. BPH, prostatitis
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Abstract
The invention discloses a method for manufacturing a total PSA and free PSA two-in-one chemiluminescence immunologic diagnosis kit. The method includes the steps that an anti-PSA antibody is used for being wrapped into a solid phase coated plate, two sorts of different enzymes are used for marking PSA antibodies on different sites, then different substrates corresponding to different enzymes are used for obtaining respective standard curves, and hence the concentration of a PSA and the concentration of an FPSA can be detected at the same time in the primary immunoreaction process. Before the method is used, total PSA and free PSA detection is conducted through two experiments respectively, so that operation is tedious, and the time period is long. By the adoption of the method, two detection reagents can be combined into one, two project results can be obtained through the primary experiment, the experimental period is shortened, and the experimental procedures are reduced.
Description
Technical field
The present invention relates to a kind of Medical Devices and manufacture method thereof.
Background technology
For the detection of T-PSA (PSA) and free prostate gland specificity antigen (FPSA), carry out respectively often both at home and abroad at present.PSA mainly contains two kinds of existence forms in serum: a kind of is free PSA (FPSA), accounts for 10%～30% of blood-serum P SA total concentration.Another kind is and the PSA (PSA-ACT) of α 1-ACT (ACT) combination, accounts for 70%～90% of blood-serum P SA total concentration.When adopting chemiluminescence immunoassay diagnostic method to detect, often adopt sandwich method, coated with a strain antibody, tracer on another strain antibody mark.When concrete detection PSA and PSA, the same strain antibody of general employing is as coated antibody, this strain antibody can either be with PSA in conjunction with following FPSA combination again, when needs detect be PSA time just with another strain antibody, do sandwich reaction, at this moment require the PSA site of this strain antibody outside can being exposed in PSA-ACT to be combined.If in the time of need to detecting FPSA, contrary, at this moment as this sandwich strain antibody, can not be exposed to outer PSA site combination and should be combined with the PSA site of wrapping in it with PSA-ACT.In this case, sterically hindered due to the Nei Bao site of PSA-ACT, causes only, with the site combination on PSA free in body fluid, so just having reached the object that only detects FPSA.
This is just for the invention provides a kind of possibility, by adopt two strains for the antibody in differential responses site as tracer antibody, wherein a strain is only can be combined with the Nei Bao site of PSA-ACT, another strain only can be combined with the site of exposing of PSA-ACT, and this two strain antibody adopts respectively different tracer agents, just can distinguish the number of the amount of PSA and FPSA.
Summary of the invention
For the defect of prior art existence, the invention provides the preparation method of the two-in-one chemiluminescence immunoassay diagnostic kit of a kind of t-PSA and PSA.Method of the present invention is coated with into solid-phase coating plate with the anti-PSA antibody of a strain, with two kinds of different enzymes, carry out the antibody of mark PSA different loci, then obtain typical curve separately and then realize the concentration that simultaneously detects PSA and two kinds of materials of FPSA in primary immune response course of reaction with different substrates corresponding to different enzymes.Adopt detection t-PSA and PSA before this invention to carry out respectively by twice experiment, complex operation, the time cycle is long.After employing the present invention, can detect reagent by two kinds two-in-one, by once testing the result that obtains two kinds of projects, the experimental period of shortening, reduces experimental procedure.
The preparation method of the two-in-one chemiluminescence immunoassay diagnostic kit of t-PSA of the present invention and PSA comprises the steps:
Step 1: the carbonic acid buffer of getting the anti-PSA of 0.5～1.5mg 0.05M, pH9.6 for coated antibody is diluted to 4～6 μ g/ml, and every hole adds 100～120 μ l on 96 hole Chemiluminescent plates; 4 standing over night, by plank dabber, add solution 150～250 μ l that contain 1% bovine serum albumin(BSA) next day, 37 ℃ of cappings 0.5～1.5 hour; Plank is patted dry again, be placed in dry house dehumidifier and dry final vacuum packing;
Preferably, in step 1, the carbonic acid buffer of getting the anti-PSA of 1mg 0.05M, pH9.6 for coated antibody is diluted to 5 μ g/ml, and on 96 hole Chemiluminescent plates, every hole adds 110 μ l; 4 ℃ of standing over night, by plank dabber, add the solution 200 μ l that contain 1% bovine serum albumin(BSA) next day, 37 ℃ of cappings 1 hour; Plank is patted dry again, be placed in dry house dehumidifier and dry final vacuum packing;
Step 2: get the anti-PSA monoclonal antibody of 0.5～1.5mg and 0.5～1.5mg horseradish peroxidase and be dissolved in the phosphate buffer of 0.5～1.5ml, 0.1M, pH8.0, add water-soluble carbodiimide 0.05～0.15mg; Stirring at room 0.5～1.5 hour; Take out the phosphate buffer dialysed overnight of reactant liquor to 0.02M, pH7.4, liquid is changed 2～4 times in centre;
Preferably, in step 2, get the anti-PSA monoclonal antibody of 1mg and 1mg horseradish peroxidase and be dissolved in the phosphate buffer of 1ml, 0.1M pH8.0, add water-soluble carbodiimide 0.1mg.Stirring at room 1 hour; Take out the phosphate buffer dialysed overnight of reactant liquor to 0.02M pH7.4, liquid is changed 3 times in centre;
Step 3: get the anti-PSA monoclonal antibody of 0.5～1.5mg and 0.5～1.5mg alkaline phosphatase and be dissolved in the citrate buffer solution of 0.5～1.5ml, 0.1M, pH6.0, add 4% glutaraldehyde 0.05～0.15ml, stirring at room 0.5～1.5 hour, add 0.05～0.15mg lysine cessation reaction, room temperature continues to stir 10～20 minutes, take out the phosphate buffer dialysed overnight of reactant liquor to 0.02M, pH7.4, liquid is changed 2～4 times in centre; With the Tris-HCl damping fluid of the 0.1M that contains 0.2% bovine serum albumin(BSA), pH7.4, by 900～1100 times of step 2 and step 3 reactant liquor mixed dilutings, 10 milliliters of packing are standby;
Preferably, in step 3, getting the anti-PSA monoclonal antibody of 1mg and 1mg alkaline phosphatase is dissolved in the citrate buffer solution of 1ml, 0.1M pH6.0, add 4% glutaraldehyde 0.1ml, stirring at room 1 hour, adds 0.1mg lysine cessation reaction, and room temperature continues to stir 15 minutes, take out the phosphate buffer dialysed overnight of reactant liquor to 0.02M pH7.4, liquid is changed 3 times in centre; With the Tris-HCl damping fluid of the 0.1MpH7.4 that contains 0.2% bovine serum albumin(BSA), by 1000 times of step 2 and step 3 reactant liquor mixed dilutings, 10 milliliters of packing are standby;
Step 4: measure respectively 90～110ml, 4～6 parts of the NBCSs of deactivation, in every part of serum, add t-PSA standard items raw material and PSA standard items raw material respectively, after mixing, the blood serum sample of preparation is demarcated respectively to t-PSA concentration value and PSA concentration value with Roche electrochemical luminescence system, after packing as the serial calibration object of kit;
Preferably, in step 4, measure respectively 100ml 5 parts of the NBCSs of deactivation;
Step 5: adopt respectively commercially available diamantane chemical luminescence substrate system and luminol chemiluminescence substrate system as luminous substrate; 4～8 milliliters of each kits of diamantane cycle chemistry luminous substrate, luminol chemiluminescence substrate system is divided into again A liquid, two components of B liquid, and wherein A liquid is luminol solution, 4～8 milliliters; B liquid is oxidizing agent solution, 4～8 milliliters;
Preferably, 6 milliliters of each kits of diamantane cycle chemistry luminous substrate, luminol chemiluminescence substrate system is divided into again A liquid, two components of B liquid, and wherein A liquid is luminol solution, 6 milliliters; B liquid is oxidizing agent solution, 6 milliliters;
Step 6: the Tris-HCl damping fluid of the 0.01M pH7.4 that preparation contains 0.05%TWEEN20 is cleansing solution.
The application of the invention, can be so that the kit that can use of medical inspection mechanism detects two kinds of indexs simultaneously, and the running time shortens half, and operation steps reduces half.Patient can obtain assay sooner.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below by specific embodiment, preparation method of the present invention is further described.
Embodiment 1:
Get the anti-PSA coated antibody of 1mg and be diluted to 5 μ g/ml with the carbonic acid buffer of 0.05M pH9.6, on 96 hole Chemiluminescent plates, every hole adds 110 μ l.4 ℃ of standing over night, by plank dabber, add the solution 200 μ l that contain 1% bovine serum albumin(BSA) next day, 37 ℃ of cappings 1 hour.Plank is patted dry again, be placed in dry house dehumidifier and dry final vacuum packing.
Get the anti-PSA of 1mg (PSA-ACT Nei Bao site) monoclonal antibody and 1mg horseradish peroxidase and be dissolved in the phosphate buffer (P.B) of 1ml, 0.1M pH8.0, add water-soluble carbodiimide (EDC) 0.1mg.Stirring at room 1 hour.Take out the P.B dialysed overnight of reactant liquor to 0.02M pH7.4, liquid is changed 3 times in centre.
Get in the citrate buffer solution that the anti-PSA of 1mg (PSA-ACT exposes site) monoclonal antibody and 1mg alkaline phosphatase be dissolved in 1ml, 0.1M pH6.0, add 4% glutaraldehyde 0.1ml, stirring at room 1 hour, add 0.1mg lysine cessation reaction, room temperature continues to stir 15 minutes, take out the P.B dialysed overnight of reactant liquor to 0.02M pH7.4, liquid is changed 3 times in centre.The horseradish enzyme labeling thing reactant liquor that takes out reactant liquor and prepare with back mix and the Tris-HCl damping fluid of using the 0.1M pH7.4 that contains 0.2% bovine serum albumin(BSA) by 1000 times of step 2 and step 3 reactant liquor mixed dilutings, the thing working fluid that serves as a mark after 10 milliliters of packing is standby.
Measure respectively 100ml 5 parts of the NBCSs of deactivation, in every part of serum, add a certain amount of t-PSA standard items raw material and PSA standard items raw material respectively, after mixing, the blood serum sample of preparation is demarcated respectively to t-PSA concentration value and PSA concentration value with Roche electrochemical luminescence system, after 0.5 milliliter of packing as the serial calibration object of kit.
Adopt respectively commercially available diamantane chemical luminescence substrate system and luminol chemiluminescence substrate system as luminous substrate.6 milliliters of each kits of diamantane cycle chemistry luminous substrate, luminol chemiluminescence substrate system is divided into again A liquid, two components of B liquid, and wherein A liquid is luminol solution, 6 milliliters; B liquid is oxidizing agent solution, 6 milliliters.
The Tris-HCl damping fluid of the 0.01M pH7.4 that preparation contains 0.05%TWEEN20 is cleansing solution.
During use, in 96 hole Chemiluminescent plates, add respectively the serial calibration object of having demarcated t-PSA and PSA concentration, and each 25 microlitres of test serum sample; Every hole adds mixed mark thing working fluid 100 microlitres, and 37 degrees Celsius of incubations took out after 1 hour.With after cleansing solution washing 5 times, every hole adds 50 microlitre diamantane chemical luminous substrates, places to be placed on for 30 minutes for 37 degrees Celsius and on chemiluminescent analyzer, detects luminous signal.
T-PSA series calibration object concentration is set up to t-PSA typical curve to its luminous signal, and the luminous signal of sample to be tested goes out corresponding concentration by t-PSA typical curve reverse.
Detect and completely with cleansing solution washing luminous plaque 5 times, add respectively luminol chemiluminescence substrate A liquid 50 microlitres and B liquid 50 microlitres, within 1 minute, be placed on and on chemiluminescent analyzer, detect luminous signal.PSA series calibration object concentration is set up to PSA typical curve to its luminous signal, and the luminous signal of sample to be tested goes out corresponding concentration by PSA typical curve reverse.
Embodiment 2:
The preparation method of the two-in-one chemiluminescence immunoassay diagnostic kit of t-PSA of the present invention and PSA comprises the steps:
Step 1: get the anti-PSA coated antibody of 1mg and be diluted to 5 μ g/ml with the carbonic acid buffer of 0.05M pH9.6, every hole adds 110 μ l on 96 hole Chemiluminescent plates.4 ℃ of standing over night, by plank dabber, add the solution 200 μ l that contain 1% bovine serum albumin(BSA) next day, 37 ℃ of cappings 1 hour.Plank is patted dry again, be placed in dry house dehumidifier and dry final vacuum packing.
Step 2: get the anti-PSA of 1mg (PSA-ACT Nei Bao site) monoclonal antibody and 1mg horseradish peroxidase and be dissolved in the phosphate buffer (P.B) of 1ml, 0.1M pH8.0, add water-soluble carbodiimide (EDC) 0.1mg.Stirring at room 1 hour.Take out the P.B dialysed overnight of reactant liquor to 0.02M pH7.4, liquid is changed 3 times in centre.
Step 3: get in the citrate buffer solution that the anti-PSA of 1mg (PSA-ACT exposes site) monoclonal antibody and 1mg alkaline phosphatase be dissolved in 1ml, 0.1M pH6.0, add 4% glutaraldehyde 0.1ml, stirring at room 1 hour, add 0.1mg lysine cessation reaction, room temperature continues to stir 15 minutes, take out the P.B dialysed overnight of reactant liquor to 0.02MpH7.4, liquid is changed 3 times in centre.With the Tris-HCl damping fluid of the 0.1MpH7.4 that contains 0.2% bovine serum albumin(BSA), by 1000 times of step 2 and step 3 reactant liquor mixed dilutings, 10 milliliters of packing are standby.
Step 4: measure respectively 100ml 5 parts of the NBCSs of deactivation, in every part of serum, add t-PSA standard items raw material and PSA standard items raw material respectively, after mixing, the blood serum sample of preparation is demarcated respectively to t-PSA concentration value and PSA concentration value with Roche electrochemical luminescence system, after packing as the serial calibration object of kit.
Step 5: adopt respectively commercially available diamantane chemical luminescence substrate system and luminol chemiluminescence substrate system as luminous substrate.6 milliliters of each kits of diamantane cycle chemistry luminous substrate, luminol chemiluminescence substrate system is divided into again A liquid, two components of B liquid, and wherein A liquid is luminol solution, 6 milliliters; B liquid is oxidizing agent solution, 6 milliliters.
Step 6: the Tris-HCl damping fluid of the 0.01M pH7.4 that preparation contains 0.05%TWEEN20 is cleansing solution.
Feature of the present invention be by current chemiluminescence immunoassay diagnostic field relatively conventional two kinds of labels---alkaline phosphatase (AP) and horseradish peroxidase (HRP) mark are for two strain antibodies of PSA different loci.By primary immune response, react, adopt two kinds of enzymes respectively corresponding substrate carry out input, the typical curve separately of asking and then calculate respectively corresponding PSA and FPSA concentration is come.By this way, be prepared into kit and just become two-in-one kit.
More than described the preferred embodiments of the present invention, but it will be understood by those skilled in the art that and do not departing under the prerequisite of design philosophy of the present invention, its various modification or combination are all included in the protection domain of claim of the present invention.
Claims (8)
1. a preparation method for the two-in-one chemiluminescence immunoassay diagnostic kit of t-PSA and PSA, is characterized in that, described preparation method comprises the steps:
Step 1: the carbonic acid buffer of getting the anti-PSA of 0.5～1.5mg 0.05M, pH9.6 for coated antibody is diluted to 4～6 μ g/ml, and every hole adds 100～120 μ l on 96 hole Chemiluminescent plates; 4 ℃ of standing over night, by plank dabber, add solution 150～250 μ l that contain 1% bovine serum albumin(BSA) next day, 37 ℃ of cappings 0.5～1.5 hour; Plank is patted dry again, be placed in dry house dehumidifier and dry final vacuum packing;
Step 2: get the anti-PSA monoclonal antibody of 0.5～1.5mg and 0.5～1.5mg horseradish peroxidase and be dissolved in the phosphate buffer of 0.5～1.5ml, 0.1M, pH8.0, add water-soluble carbodiimide 0.05～0.15mg; Stirring at room 0.5～1.5 hour; Take out the phosphate buffer dialysed overnight of reactant liquor to 0.02M, pH7.4, liquid is changed 2～4 times in centre;
Step 3: get the anti-PSA monoclonal antibody of 0.5～1.5mg and 0.5～1.5mg alkaline phosphatase and be dissolved in the citrate buffer solution of 0.5～1.5ml, 0.1M, pH6.0, add 4% glutaraldehyde 0.05～0.15ml, stirring at room 0.5～1.5 hour, add 0.05～0.15mg lysine cessation reaction, room temperature continues to stir 10～20 minutes, take out the phosphate buffer dialysed overnight of reactant liquor to 0.02M, pH7.4, liquid is changed 2～4 times in centre; With the Tris-HCl damping fluid of the 0.1M that contains 0.2% bovine serum albumin(BSA), pH7.4, by 900～1100 times of step 2 and step 3 reactant liquor mixed dilutings, 10 milliliters of packing are standby;
Step 4: measure respectively 90～110ml, 4～6 parts of the NBCSs of deactivation, in every part of serum, add t-PSA standard items raw material and PSA standard items raw material respectively, after mixing, the blood serum sample of preparation is demarcated respectively to t-PSA concentration value and PSA concentration value with Roche electrochemical luminescence system, after packing as the serial calibration object of kit;
Step 5: adopt respectively commercially available diamantane chemical luminescence substrate system and luminol chemiluminescence substrate system as luminous substrate; 6 milliliters of each kits of diamantane cycle chemistry luminous substrate, luminol chemiluminescence substrate system is divided into again A liquid, two components of B liquid, and wherein A liquid is luminol solution, 6 milliliters; B liquid is oxidizing agent solution, 6 milliliters;
Step 6: the Tris-HCl damping fluid of the 0.01M pH7.4 that preparation contains 0.05%TWEEN20 is cleansing solution.
2. preparation method as described in claim 1, is characterized in that, in step 1, the carbonic acid buffer of getting the anti-PSA of 1mg 0.05M, pH9.6 for coated antibody is diluted to 5 μ g/ml, and on 96 hole Chemiluminescent plates, every hole adds 110 μ l; 4 ℃ of standing over night, by plank dabber, add the solution 200 μ l that contain 1% bovine serum albumin(BSA) next day, 37 ℃ of cappings 1 hour; Plank is patted dry again, be placed in dry house dehumidifier and dry final vacuum packing.
3. preparation method as described in claim 1, is characterized in that, in step 2, gets the anti-PSA monoclonal antibody of 1mg and 1mg horseradish peroxidase and is dissolved in the phosphate buffer of 1ml, 0.1M pH8.0, adds water-soluble carbodiimide 0.1mg.Stirring at room 1 hour; Take out the phosphate buffer dialysed overnight of reactant liquor to 0.02M pH7.4, liquid is changed 3 times in centre.
4. preparation method as described in claim 1, it is characterized in that, in step 3, get the anti-PSA monoclonal antibody of 1mg and 1mg alkaline phosphatase and be dissolved in the citrate buffer solution of 1ml, 0.1M pH6.0, add 4% glutaraldehyde 0.1ml, stirring at room 1 hour, add 0.1mg lysine cessation reaction, room temperature continues to stir 15 minutes, takes out the phosphate buffer dialysed overnight of reactant liquor to 0.02M pH7.4, and liquid is changed 3 times in centre; With the Tris-HCl damping fluid of the 0.1M pH7.4 that contains 0.2% bovine serum albumin(BSA), by 1000 times of step 2 and step 3 reactant liquor mixed dilutings, 10 milliliters of packing are standby.
5. preparation method as described in claim 1, is characterized in that, in step 4, measures respectively 100ml 5 parts of the NBCSs of deactivation.
6. preparation method as described in claim 1, is characterized in that, 6 milliliters of each kits of diamantane cycle chemistry luminous substrate, and luminol chemiluminescence substrate system is divided into again A liquid, two components of B liquid, and wherein A liquid is luminol solution, 6 milliliters; B liquid is oxidizing agent solution, 6 milliliters.
7. a preparation method for the two-in-one chemiluminescence immunoassay diagnostic kit of t-PSA and PSA, is characterized in that, described preparation method comprises the steps:
Step 1: get the anti-PSA coated antibody of 1mg and be diluted to 5 μ g/ml with the carbonic acid buffer of 0.05M pH9.6, every hole adds 110 μ l on 96 hole Chemiluminescent plates; 4 ℃ of standing over night, by plank dabber, add the solution 200 μ l that contain 1% bovine serum albumin(BSA) next day, 37 ℃ of cappings 1 hour.Plank is patted dry again, be placed in dry house dehumidifier and dry final vacuum packing;
Step 2: get the anti-PSA monoclonal antibody of 1mg and 1mg horseradish peroxidase and be dissolved in the phosphate buffer of 1ml, 0.1M pH8.0, add water-soluble carbodiimide 0.1mg; Stirring at room 1 hour.Take out the phosphate buffer dialysed overnight of reactant liquor to 0.02M pH7.4, liquid is changed 3 times in centre;
Step 3: get the anti-PSA monoclonal antibody of 1mg and 1mg alkaline phosphatase and be dissolved in the citrate buffer solution of 1ml, 0.1M pH6.0, add 4% glutaraldehyde 0.1ml, stirring at room 1 hour, add 0.1mg lysine cessation reaction, room temperature continues to stir 15 minutes, take out the P.B dialysed overnight of reactant liquor to 0.02M pH7.4, liquid is changed 3 times in centre; With the Tris-HCl damping fluid of the 0.1M pH7.4 that contains 0.2% bovine serum albumin(BSA), by 1000 times of step 2 and step 3 reactant liquor mixed dilutings, 10 milliliters of packing are standby;
Step 4: measure respectively 100ml 5 parts of the NBCSs of deactivation, in every part of serum, add t-PSA standard items raw material and PSA standard items raw material respectively, after mixing, the blood serum sample of preparation is demarcated respectively to t-PSA concentration value and PSA concentration value with Roche electrochemical luminescence system, after packing as the serial calibration object of kit;
Step 5: adopt respectively commercially available diamantane chemical luminescence substrate system and luminol chemiluminescence substrate system as luminous substrate; 6 milliliters of each kits of diamantane cycle chemistry luminous substrate, luminol chemiluminescence substrate system is divided into again A liquid, two components of B liquid, and wherein A liquid is luminol solution, 6 milliliters; B liquid is oxidizing agent solution, 6 milliliters;
Step 6: the Tris-HCl damping fluid of the 0.01M pH7.4 that preparation contains 0.05%TWEEN20 is cleansing solution.
8. a two-in-one chemiluminescence immunoassay diagnostic kit, is characterized in that, according to the method preparation described in claim 1 or 7.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105785017A (en) * | 2016-05-11 | 2016-07-20 | 江苏省原子医学研究所 | Double-labeling time-resolved fluorescence immunoassay kit based on PSA magnetic particles |
| CN112014573A (en) * | 2020-08-25 | 2020-12-01 | 武汉生之源生物科技股份有限公司 | Preparation method of high-sensitivity determination kit for troponin I in human whole blood sample and kit |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030219840A1 (en) * | 2002-05-24 | 2003-11-27 | Mikolajczyk Stephen D. | Method of analyzing proenzyme forms of prostate specific antigen in serum to improve prostate cancer detection |
| CN1924581A (en) * | 2005-08-30 | 2007-03-07 | 郑州安图绿科生物工程有限公司 | Chemical luminescent analysis reagent kid for prostate specific antigen |
| US20070254317A1 (en) * | 2004-03-03 | 2007-11-01 | Biomerieux | Method for Detecting the Activatable Free Form of Psa and the Use Thereof for Diagnosing Benign Pathologies of the Prostate and Adenocarcinoma of the Prostate |
| CN101210927A (en) * | 2006-12-30 | 2008-07-02 | 中国科学院上海生命科学研究院 | Prostate cancer diagnosis reagent kit |
| CN101329343A (en) * | 2007-06-19 | 2008-12-24 | 天津迪爱盟生物技术有限公司 | New generation early diagnosis prostate gland cancer reagent kit and preparation method and detection process thereof |
| CN101377500A (en) * | 2007-08-31 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Free prostate gland specificity antigen chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| CN101377498A (en) * | 2007-08-30 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Prostate gland specificity antigen chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| CN101750497A (en) * | 2008-12-17 | 2010-06-23 | 北京科美东雅生物技术有限公司 | Enzymatic chemical luminous immunoassay quantitative determination reagent kit for simultaneously detecting various tumor markers |
-
2013
- 2013-02-21 CN CN201310055076.XA patent/CN104007256A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030219840A1 (en) * | 2002-05-24 | 2003-11-27 | Mikolajczyk Stephen D. | Method of analyzing proenzyme forms of prostate specific antigen in serum to improve prostate cancer detection |
| US20070254317A1 (en) * | 2004-03-03 | 2007-11-01 | Biomerieux | Method for Detecting the Activatable Free Form of Psa and the Use Thereof for Diagnosing Benign Pathologies of the Prostate and Adenocarcinoma of the Prostate |
| CN1924581A (en) * | 2005-08-30 | 2007-03-07 | 郑州安图绿科生物工程有限公司 | Chemical luminescent analysis reagent kid for prostate specific antigen |
| CN101210927A (en) * | 2006-12-30 | 2008-07-02 | 中国科学院上海生命科学研究院 | Prostate cancer diagnosis reagent kit |
| CN101329343A (en) * | 2007-06-19 | 2008-12-24 | 天津迪爱盟生物技术有限公司 | New generation early diagnosis prostate gland cancer reagent kit and preparation method and detection process thereof |
| CN101377498A (en) * | 2007-08-30 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Prostate gland specificity antigen chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| CN101377500A (en) * | 2007-08-31 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Free prostate gland specificity antigen chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| CN101750497A (en) * | 2008-12-17 | 2010-06-23 | 北京科美东雅生物技术有限公司 | Enzymatic chemical luminous immunoassay quantitative determination reagent kit for simultaneously detecting various tumor markers |
Non-Patent Citations (3)
| Title |
|---|
| KIM PETTERSSON ET AL.: "Free and Complexed Prostate-Specific Antigen (PSA): In Vitro Stability, Epitope Map, and Development of Immunofluorometric Assays for Specific and Sensitive Detection of Free PSA and PSA-a1-Antichymotrypsin Complex", 《CLINICAL CHEMISTRY》 * |
| SUSANN ERIKSSON, ET AL.,: "Dual-Label Time-resolved Immunofluorometric Assay of Free and Total Prostate-specific Antigen Based on Recombinant Fab Fragments", 《CLINICAL CHEMISTRY》 * |
| 熊异平: "化学发光法联合检测PSA和fPSA在***疾病中的诊断应用", 《万方数据企业知识服务平台》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105785017A (en) * | 2016-05-11 | 2016-07-20 | 江苏省原子医学研究所 | Double-labeling time-resolved fluorescence immunoassay kit based on PSA magnetic particles |
| CN112014573A (en) * | 2020-08-25 | 2020-12-01 | 武汉生之源生物科技股份有限公司 | Preparation method of high-sensitivity determination kit for troponin I in human whole blood sample and kit |
| CN112014573B (en) * | 2020-08-25 | 2023-08-08 | 武汉生之源生物科技股份有限公司 | Preparation method of high-sensitivity assay kit for troponin I in human whole blood sample and kit |
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