CN1924581A - Chemical luminescent analysis reagent kid for prostate specific antigen - Google Patents
Chemical luminescent analysis reagent kid for prostate specific antigen Download PDFInfo
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- CN1924581A CN1924581A CN 200510017942 CN200510017942A CN1924581A CN 1924581 A CN1924581 A CN 1924581A CN 200510017942 CN200510017942 CN 200510017942 CN 200510017942 A CN200510017942 A CN 200510017942A CN 1924581 A CN1924581 A CN 1924581A
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- specific antigen
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Abstract
This invention relates to prostate gland specific antigen chemical specifying agent box in the clinical testing analysis technique field, which comprises the following parts: non-transparent polyphony lacetylene board with the gland antigen, prostate antigen series standard product, enzyme mark gland specific antigen, lighting bottom object A liquid composed of effective light agent, compound strengthening agent and amino acid and B liquid composed of amino acid oxidize and stabilizer. The lighting bottom A and B is of HRP luminal lighting system.
Description
Technical field
The invention belongs to the clinical blood technical field of immunoassay, particularly a kind of chemical luminescence immune analysis reagent box that is used for human serum prostate specific antigen (PSA) detection by quantitative utilizes this kit detection specificity strong, highly sensitive.
Background technology
In recent years, change along with population structure aging, eating habit, the incidence of disease of China's prostatic disorders increases rapidly, and wherein prostate cancer accounts for second of male sex's incidence of disease, the 3rd of mortality ratio, and can the early detection of prostate cancer be the key point that effect a radical cure.Prostate specific antigen (PSA) is the strongest tumor markers of prostatic cancer specific, the detection of PSA helps early detection, supervision disease progression, the result of treatment monitoring of prostate cancer, and PSA plays decisive role in the antidiastole of primary prostate cancer and Secondary cases prostate cancer simultaneously.Detect at present the method for PSA in clinical labororatory, still mostly is radioimmunoassay method that grows up from the sixties and the enzyme-linked immunosorbent assay that rises the eighties.But because radioimmunoassay method must use radioactively labelled substance, the checkout equipment complexity must be measured testing result with special radioactivity seeker.Simultaneously, operating personnel are also had radioactive contamination and injury, particularly as short nucleic of half life period such as iodine-125, iodine-131 and phosphorus-32 commonly used, its retention period is not long, has brought inconvenience for clinical use yet.Though and enzyme is exempted from method and avoided putting problems such as the pollution of the method for exempting from, loaded down with trivial details, storage life be short, shortcoming still can not satisfy clinical demand because its sensing range is narrow, sensitivity is low etc.
20th century the mid-1970s Arakawe reported first is carried out analyzing and testing with luminous signal, promptly utilize luminous chemical reaction (chemiluminescence, CL) and biological respinse (bioluminescence, BL) analyze the ultramicron material, especially for check ultramicron active substance in the clinical immunoassay.(chemiluminescence immunoassay, CLIA) (attomole promptly 10 so that it is highly sensitive for chemiluminescence immune assay
-18Mol), fast do not use harmful reagent in (produce chemiluminescence signal in several seconds, signal is sustainable several hours under the situation about having), easy, the test, therefore do not become in the on-radiation immunoassay one of the most promising method.Chemiluminescence immunoassay is divided into two kinds of direct labelling method and chemiluminescence enzyme immunoassay methods because of the difference of labeling method, and direct labelling method, and what adopt is that acridinium ester directly is marked at and is used on antigen or the antibody detecting.Because its luminous passage of scintillation light often should not be caught, and often only adapts to full-automatic chemical luminescence immunoassay instrument.
Summary of the invention
The object of the invention is to provide a kind of chemiluminescence enzyme immunoassay kit that is used for human serum prostate specific antigen (PSA) detection by quantitative, utilizes this kit to carry out analyzing and testing high specificity, highly sensitive.
For reaching above-mentioned purpose, the present invention adopts following technical scheme: chemical luminescent analysis reagent kid for prostate specific antigen, mainly form by the opaque polystyrene board that is coated with anti-prostate specific antigen antibody, prostate specific antigen series standard product, enzyme labeling prostate specific antigen antibody, luminous substrate A liquid and luminous substrate B liquid, luminous substrate A liquid is made into by efficient luminous agent, composite fortifier, amino acid, and luminous substrate B liquid is made into by amino acid oxidase and stabilizing agent.
The opaque polystyrene board that is coated with anti-prostate specific antigen antibody is the opaque polystyrene board that is coated with anti-prostate specific antigen monoclonal or polyclonal antibody; Prostate specific antigen series standard product are matrix with the analysis buffer, and the pure product of adding prostate specific antigen are formulated; Enzyme labeling prostate specific antigen antibody is the antibody of the prostate specific antigen of horseradish peroxidase-labeled; Luminous substrate A, B are HRP-luminol luminescence system.
The working concentration of horseradish peroxidase-labeled prostate specific antigen antibody is 1: 5000-9000; The opaque polystyrene board that is coated with anti-prostate specific antigen antibody adopts direct physical absorption method bag quilt.
Luminous substrate A liquid is made up of following ingredients: luminol 0.15mM, Hydroxycoumarin 0.59mM, gallic acid 0.35mM, Tris-Hcl damping fluid 0.2M; Luminous substrate B liquid is made up of following ingredients: amino acid oxidase 0.85mM, Tween-20 0.8%, V/V, DTPA 0.5mM, vitamin C 0.12mM, acetate-acetate buffer 0.2M.
In the kit of the present invention, analysis buffer is formed Ph7.2 by sodium chloride and 1% bovine serum albumin(BSA) (BSA) of 0.05M sodium hydrogen phosphate-sodium dihydrogen phosphate, 0.01M; The PSA monoclonal antibody that used antibody adopts conventional quadroma technology to prepare, after adopting sad-ammonium sulfate salting-out process that the antibody for preparing is carried out purifying, carry out Screening and Identification again, select a pair of preferably monoclonal antibody of pairing and be used for detecting, standby in-20 ℃ of preservations; Opaque polystyrene board adopts direct physical absorption method Sheet clonal antibody, and coating buffer is the carbonate buffer solution of 0.05M pH9.6, and bag is 2-5ug/ml by concentration, package amount 100ul/ hole, 0 ℃ of-4 ℃ of overnight incubation; Discard liquid in the hole, wash plate hole twice with the PBS-Tween washing lotion; Use pH7.4PBS-1%BSA, the sealing of 150ul/ hole, incubated at room 3 hours; Discard liquid in the hole, use pH7.4PBS-2.5% sucrose, the protection of 150ul/ hole; Discard liquid in the hole, sealing is preserved in the aluminium foil bag of the damp proof insulation of packing into after drying.
Used PSA series standard product in the kit of the present invention are matrix with the analysis buffer, adopt to concentrate refining and form with the dilution that is as the criterion of national standard product, are serial dried frozen aquatic products, redissolve with distilled water before the use; The antibody of used enzyme labeling prostate specific antigen adopts the sodium periodate method of improvement to carry out mark.Its principle is: horseradish peroxidase is a kind of glycoprotein, contains about 18% sugar, and is irrelevant with enzymatic activity.The glycosyl irrelevant with enzymatic activity is oxidized to aldehyde radical by sodium periodate (NaIO4), and amino and the aldehyde radical with antibody protein forms Schiff ' s alkali again.For self coupling takes place in the amino that prevents zymoprotein and aldehyde radical reaction, before mark earlier with 2, remaining α in 4-dinitrofluorobenzene (DNFB) the sealase albumen-and epsilon-amino.Behind the association reaction of enzyme and antibody, add sodium borohydride (NaHB again
4) be reduced into stable bond.Geometric ratio adding glycerine is deposited standby in-20 ℃ of bags in the good antibody-solutions of mark.Test shows, the working concentration that used enzyme labelled antibody uses be 1: 8000 best.Used luminous substrate system adopts the efficient stable enzyme-catalyzed chemical luminescence substrate system, and A liquid is: luminol 0.15mM, Hydroxycoumarin 0.59mM, gallic acid 0.35mM, Tris-Hcl damping fluid 0.2M, pH 9.4; B liquid is: amino acid oxidase 0.85mM, Tween-20 0.8% (V/V), DTPA 0.5mM, vitamin C 0.12mM, acetate-acetate buffer 0.2M, pH 6.5.
The present invention is directed to Clinical Test Lab and provide a kind of both manually actuated,, set up the quantitative detecting method that detects human serum PSA again applicable to the detection means of the full-automatic detecting instrument of some standard.It adopts standard 96 hole microwell plates is solid phase carrier, with horseradish peroxidase-labeled antibody, the catalytic luminescence substrate luminous as tracer signal in order to detect.Utilize the horseradish peroxidase enzyme catalytic luminous substrate, luminous substrate generation chemical reaction also discharges lot of energy, produces the excited state intermediate.This excited state intermediate when it gets back to stable ground state, can be launched photon simultaneously, utilizes the luminous signal surveying instrument can the measuring light quantum yield, and the amount of the test substance in this quantum yield of luminscence and the sample is directly proportional.Can set up the content of test substance in typical curve and the calculation sample thus.
Utilize the present invention to detect, highly sensitive, sensing range is wide, easy and simple to handle, "dead" pollution; The kit production cost is low, has alleviated the doctor and patient burden greatly.Therefore the present invention is more suitable for the use of China's clinical laboratory.
The use running program of detection kit of the present invention is as follows:
(1) prepares before the experiment
1, all detectable and serum specimen are returned to room temperature 18-25 ℃ (needing 30 minutes approximately);
2, constant temperature oven or water-bath are transferred to temperature of reaction;
3, luminous substrate A, B liquid equal proportion are mixed to this test volume required (every hole needs 100 μ l, can be needed mixed luminous substrate 1ml to mix by plate (8 hole) according to every bag, and the rest may be inferred).
Immune response microwell plate of the present invention is fixed on plate hole with PSA antibody, can directly use, and on-the-spot bag quilt is very convenient before needn't using.
(2) experimental implementation step
1, the coated slab with institute's expense is placed on the support;
2, in wrapping, add 50 μ l standard items, blood serum sample respectively by the hole;
3, every hole adds enzyme mark antibody solution 100 μ l more respectively.Vibration mixed it in 30 seconds on the micro oscillator;
4, put 37 ℃ of incubations 60 minutes;
5, get rid of potpourri in the hole, fill with each hole, get rid of, repeat 5 times, on thieving paper, pat dry at last with distilled water;
6, every hole adds mixed luminous substrate 100 μ l, room temperature (18-25 ℃) reaction 5 minutes;
7, chemiluminescence detector detects luminous intensity values;
8, being horizontal ordinate (X-axis) with the standard items concentration value, is ordinate (Y-axis) with the standard items luminous intensity values, sets up typical curve, calculates measurement result.
It is shorter to measure the used time with the above-mentioned detection kit of the present invention by said procedure, and a collection of mensuration generally only needed to finish in more than one hour, fast convenient.
Through experimental results demonstrate, the methodology appraising datum of the above-mentioned detectable of the present invention when being used for prostatic special antigen content mensuration can reach following index:
Sensitivity-minimum detectable activity is 0.3ng/ml;
Standard curve range is 0-100ng/ml;
Average 4.5% (n=10) of precision in precision-analysis, precision average out to 9.6% (n=10) between analysis far above national standard, illustrates that kit of the present invention has good repeatability in test experience;
Accuracy---the recovery in serum behind the known PSA standard items of interpolation is 98%-109%.
Experiment shows, uses the present invention the prostate specific antigen in the human serum is measured, and the sample that only need take a morsel can carry out, to the detected person without any injury.Simultaneously, in These parameters, all be better than present widely used enzyme-linked immune detection method, also do not have the shortcomings such as radioactive contamination, term of validity weak point, complicated operation of radioimmunology simultaneously because the present invention has adopted the chemiluminescence enzyme immunoassay detection method.Therefore, the present invention provides a kind of more accurate, method easily and efficiently that detects prostate specific antigen for clinical, can satisfy clinical needs better.
Embodiment
Chemical luminescent analysis reagent kid for prostate specific antigen, be coated with the opaque polystyrene board in 96 holes of anti-prostate specific antigen monoclonal antibody by (1), (2) with the analysis buffer be matrix, add the formulated prostate specific antigen series standard product of the pure product of prostate specific antigen, (3) antibody of the prostate specific antigen of horseradish peroxidase-labeled, its working concentration is 1: 8000, (4) luminous substrate A liquid, and (5) luminous substrate B liquid is formed.Luminous substrate A liquid is made into by efficient luminous agent, composite fortifier, amino acid, and luminous substrate B liquid is made into by amino acid oxidase and stabilizing agent.
The opaque polystyrene board that is coated with anti-prostate specific antigen antibody adopts direct physical absorption method bag quilt.
Luminous substrate A liquid is made up of following ingredients: luminol 0.15mM, Hydroxycoumarin 0.59mM, gallic acid 0.35mM, Tris-Hcl damping fluid 0.2M; Luminous substrate B liquid is made up of following ingredients: amino acid oxidase 0.85mM, Tween-20 0.8% (V/V), DTPA 0.5mM, vitamin C 0.12mM, acetate-acetate buffer 0.2M.
Claims (4)
1, chemical luminescent analysis reagent kid for prostate specific antigen, it is characterized in that, mainly form by the opaque polystyrene board that is coated with anti-prostate specific antigen antibody, prostate specific antigen series standard product, enzyme labeling prostate specific antigen antibody, luminous substrate A liquid and luminous substrate B liquid, luminous substrate A liquid is made into by efficient luminous agent, composite fortifier, amino acid, and luminous substrate B liquid is made into by amino acid oxidase and stabilizing agent.
2, kit as claimed in claim 1 is characterized in that, the opaque polystyrene board that is coated with anti-prostate specific antigen antibody is the opaque polystyrene board that is coated with anti-prostate specific antigen monoclonal or polyclonal antibody; Prostate specific antigen series standard product are matrix with the analysis buffer, and the pure product of adding prostate specific antigen are formulated; Enzyme labeling prostate specific antigen antibody is the antibody of the prostate specific antigen of horseradish peroxidase-labeled; Luminous substrate A, B are HRP-luminol luminescence system.
3, kit as claimed in claim 2 is characterized in that, the working concentration of horseradish peroxidase-labeled prostate specific antigen antibody is 1: 5000-9000; The opaque polystyrene board that is coated with anti-prostate specific antigen antibody adopts direct physical absorption to wrap by the method bag by anti-prostate specific antigen monoclonal or polyclonal antibody.
4, kit as claimed in claim 3 is characterized in that, luminous substrate A liquid is made up of following ingredients: luminol 0.15mM, Hydroxycoumarin 0.59mM, gallic acid 0.35mM, Tris-Hcl damping fluid 0.2M; Luminous substrate B liquid is made up of following ingredients: amino acid oxidase 0.85mM, Tween-20 0.8% (V/V), DTPA 0.5mM, vitamin C 0.12mM, acetate-acetate buffer 0.2M.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510017942 CN1924581A (en) | 2005-08-30 | 2005-08-30 | Chemical luminescent analysis reagent kid for prostate specific antigen |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510017942 CN1924581A (en) | 2005-08-30 | 2005-08-30 | Chemical luminescent analysis reagent kid for prostate specific antigen |
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| CN1924581A true CN1924581A (en) | 2007-03-07 |
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| CN 200510017942 Pending CN1924581A (en) | 2005-08-30 | 2005-08-30 | Chemical luminescent analysis reagent kid for prostate specific antigen |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101368961B (en) * | 2008-04-02 | 2012-11-14 | 北京科美生物技术有限公司 | Chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen and preparation method thereof |
| CN103197077A (en) * | 2013-03-20 | 2013-07-10 | 郑州伊美诺生物技术有限公司 | Assay kit for detecting trace bovine immunoglobulin G |
| CN103645320A (en) * | 2013-11-19 | 2014-03-19 | 洛阳莱普生信息科技有限公司 | Furaltadone metabolite chemiluminescent enzyme linked immune sorbent rapid analysis kit |
| CN104007256A (en) * | 2013-02-21 | 2014-08-27 | 林斯 | Method for manufacturing total PSA and free PSA two-in-one chemiluminescence immunologic diagnosis kit |
| CN104614519A (en) * | 2015-02-02 | 2015-05-13 | 东南大学 | Soybean peroxidase labelled chemiluminescence immunoassay kit, as well as use method and application thereof |
| CN114184789A (en) * | 2021-12-23 | 2022-03-15 | 云南大学 | Prostate specific antigen detection probe and prostate specific antigen detection kit |
-
2005
- 2005-08-30 CN CN 200510017942 patent/CN1924581A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101368961B (en) * | 2008-04-02 | 2012-11-14 | 北京科美生物技术有限公司 | Chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen and preparation method thereof |
| CN104007256A (en) * | 2013-02-21 | 2014-08-27 | 林斯 | Method for manufacturing total PSA and free PSA two-in-one chemiluminescence immunologic diagnosis kit |
| CN103197077A (en) * | 2013-03-20 | 2013-07-10 | 郑州伊美诺生物技术有限公司 | Assay kit for detecting trace bovine immunoglobulin G |
| CN103645320A (en) * | 2013-11-19 | 2014-03-19 | 洛阳莱普生信息科技有限公司 | Furaltadone metabolite chemiluminescent enzyme linked immune sorbent rapid analysis kit |
| CN104614519A (en) * | 2015-02-02 | 2015-05-13 | 东南大学 | Soybean peroxidase labelled chemiluminescence immunoassay kit, as well as use method and application thereof |
| CN114184789A (en) * | 2021-12-23 | 2022-03-15 | 云南大学 | Prostate specific antigen detection probe and prostate specific antigen detection kit |
| CN114184789B (en) * | 2021-12-23 | 2023-06-16 | 云南大学 | Prostate specific antigen detection probe and kit for detecting prostate specific antigen |
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