CN1924582A - Chemical luminescent analysis reagent kid for testosterone - Google Patents
Chemical luminescent analysis reagent kid for testosterone Download PDFInfo
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- CN1924582A CN1924582A CN 200510017943 CN200510017943A CN1924582A CN 1924582 A CN1924582 A CN 1924582A CN 200510017943 CN200510017943 CN 200510017943 CN 200510017943 A CN200510017943 A CN 200510017943A CN 1924582 A CN1924582 A CN 1924582A
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- testosterone
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- horseradish peroxidase
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Abstract
This invention relates to testosterone chemical lighting quantitative testing agent box, which belongs to clinical blood test analysis technology field and comprises non-transparent polyphenylacetylene of second antibody, testosterone series standard product, anti-testosterone solution, horse radish peroxidase mark testosterone resolution, lighting bottom a solution and B solution.
Description
Technical field
The invention belongs to clinical blood check and analysis technical field, particularly a kind of kit that detects testosterone (Testosterone) content in the human serum with the enzyme-catalyzed chemical luminescence standard measure.
Background technology
Testosterone is a kind of steroid hormone that extensively is present in the humans and animals body, accurately measures its content in vivo, is used for judging its reproductive function, normal physiological index and pathology concentration playing an important role aspect the diagnosis of disease.Testosterone is produced by interstitial glands in the males, and the testosterone concentration in male sex's serum is to confirm suspicious testicular function disorder, proves that male sex hormone lacks and the most important detected parameters of detection testosterone replacement therapy.Testosterone is mainly synthetic in ovary in women's body, and also synthetic in adrenal cortex, the testosterone concentration in women's serum is used for determining women's hyperandrogenism.
At present the testosterone hormone is detected, be mostly to adopt radioimmunoassay method that grows up from the sixties and the enzyme-linked immunosorbent assay that rises the eighties.Radio immunoassay must use radioactively labelled substance, and the checkout equipment complexity must be measured testing result with special radioactivity seeker.Simultaneously, operating personnel are also had radioactive contamination and injury, need special protection and washer.In addition, because of the radioactive nuclide moment of use radioactively labelled substance is all decaying, 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 enzyme-linked immunosorbent assay has been avoided problems such as the pollution of radio immunoassay, 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.
Conventional steroid hormone detects the immunological method of being set up, and is mostly directly to use the antibody of anti-this hormone, and promptly so-called first antibody wraps quilt to the plate hole of used microwell plate.Because competition law requires to limit the quantity of the bag quilt, often has than evident difference between the effect hole of direct coated, the coefficient of variation is about 5-20%, the precision (being reappearance) of directly influence mensuration; If bag is by the excessive sensitivity that can reduce reagent again.
Chemiluminescence immunoassay is divided into two kinds of direct labelling method and chemiluminescence enzyme immunoassay methods with the difference of labeling method.Wherein, that what adopt is that acridinium ester or luminol directly are marked at and are used on antigen or the antibody detecting to direct labelling method is more.Because it is luminous to be passage of scintillation light, should not catch, and is applicable to full-automatic chemical luminescence immunoassay instrument.Design of the present invention is at Clinical Test Lab, for it provides a kind of both manually actuated, the detectable that can be used for some full-automatic detecting instrument again, therefore, it is solid phase carrier that the present invention adopts 96 hole microwell plates of standard, with the horseradish peroxidase-labeled testosterone, the catalytic luminescence substrate luminous as tracer signal in order to detect.
Summary of the invention
The object of the present invention is to provide a kind of pre-bag of second antibody that adopts to be detected the chemical luminescent analysis reagent kid that testosterone is used by the mode of plate, it is higher to detect precision, sensitivity and stability, and specificity and accuracy are better.
For reaching above-mentioned purpose, the present invention adopts following technical scheme: testosterone (Testosterone) chemical luminescent analysis reagent kid, mainly formed by the opaque polystyrene board of second antibody, testosterone series standard product, anti-testosterone antibody-solutions, horseradish peroxidase-labeled testosterone solution, luminous substrate A liquid and luminous substrate B liquid by bag.
Bag is that bag is by the opaque polystyrene board of goat-anti rabbit/rat immune globulin G antibody by the opaque polystyrene board of second antibody; Testosterone series standard product spend hormone serum as matrix, add formulated through the pure product of testosterone of anhydrous alcohol solution; Anti-testosterone antibody-solutions, adds anti-testosterone polyclonal antibody of certain density rabbit or mouse-anti testosterone monoclonal antibody and is made into as matrix with analysis buffer; Horseradish peroxidase-labeled testosterone solution as matrix, adds certain density horseradish peroxidase-labeled testosterone with analysis buffer; Luminous substrate A liquid is made up of efficient luminous agent, composite fortifier, amino acid; Luminous substrate B liquid is made up of amino acid oxidase and stabilizing agent.
Analysis buffer is formed pH7.5 by 0.05Mtris-HCl salt solusion and 1% bovine serum albumin(BSA) (BSA); Working concentration when the horseradish peroxidase-labeled testosterone uses is 1: 300000-500000; Luminous substrate A formula of liquid is: luminol 0.15mM, Hydroxycoumarin 0.59mM, gallic acid 0.35mM, Tris-Hcl damping fluid 0.2M, and pH 9.4; Luminous substrate B formula of 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.
Testosterone of the present invention (Testosterone) chemical luminescent analysis reagent kid is made up of immune response titration microwell plate and detectable two parts.Immune response titration microwell plate wherein adopts the opaque polystyrene white microwell plate in 96 holes of standard; Detectable comprises anti-testosterone antibody, testosterone standard items, the reagent such as enzyme mark testosterone, luminous substrate A and luminous substrate B as first antibody.Opaque polystyrene white microwell plate is that bag is by the microwell plate of second antibody in advance, 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.
Bag is by used second antibody among the present invention, and the method for available present routine prepares voluntarily, also can select commercial goat anti-rabbit immunoglobulin G or sheep anti mouse immunoglobulin G for use.The purity that improves used second antibody helps guaranteeing and improves and test effect.
The anti-testosterone polyclonal antibody of rabbit used in the detectable can adopt conventional method to prepare voluntarily, as: as antigen, rabbit is carried out immunity with testosterone-3-ethyloic-BSA, can obtain the antiserum of high specificity.Also can adopt commercial finished product.The titre that improves the anti-testosterone antibody of used rabbit can improve the specificity and the accuracy of detection.
Used mouse-anti testosterone monoclonal antibody in the detectable as antigen, adopts conventional quadroma technology preparation with testosterone-3-ethyloic-BSA.Also can adopt commercial finished product.
Enzyme used in the detectable is marked testosterone, can adopt mixed anhydride method with horseradish peroxidase testosterone-3-ethyloic oxime to be carried out mark, and the working concentration of the enzyme mark testosterone that the method obtains is 1: 30 ten thousand-500,000.
The present invention adopts chemiluminescence immune analysis method, utilizes the horseradish peroxidase enzyme catalytic luminous substrate, and 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.
Characteristics such as that chemiluminescence immunoassay technology has is highly sensitive, sensing range is wide, easy and simple to handle, "dead" pollution now enter the routine clinical diagnostic application from laboratory study.
The use running program of detection kit of the present invention is as follows:
(1) prepares before the experiment
1, guarantees operating environment at room temperature (18-25 ℃), take out kit and testing sample.All reagent and sample are returned to room temperature (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 50 μ l, can be needed mixed luminous substrate 0.5ml to mix by plate (8 hole) according to every bag, and the rest may be inferred);
(2) experimental procedure
1, take out a certain amount of bag and numbered by the hole, every hole adds 25 μ l testosterone standard items or patients serum's samples respectively;
2, every hole adds anti-testosterone antibody-solutions 50 μ l (green liquid) respectively;
3, every hole adds enzyme mark testosterone 50 μ l (red solution) respectively;
4, vibration fully mixed it in 30 seconds on micro oscillator;
5, seal film after, room temperature condition (18-25C) incubation 60 minutes;
6, get rid of potpourri in the hole, wash micropore 5 times, after each flushing moisture is got rid of to the greatest extent, on thieving paper, pat dry at last, be sure not wiping reacting hole inwall with the working concentration washing lotion;
7, every hole adds mixed substrate 50 μ l, room temperature (18-25 ℃) reaction 10 minutes;
8, chemiluminescence detector detects the luminous intensity in each hole, sets up typical curve, calculates the content of testosterone in patients serum's sample.
It is very short that above-mentioned detection kit of the present invention is measured the used time by said procedure, and a collection of mensuration generally only needed to finish in more than one hour, fast convenient.
As previously mentioned, during with anti-testosterone specific antibody direct coated, the coefficient of variation that is fixed on the antibody molecule quantity variance in each plate hole is bigger, can reach 5-20%, is a key factor that influences precision of measurement.What pre-bag of the present invention was adopted by second antibody is excessive bag quilt, even the second antibody molecular number that is adsorbed in each hole has certain difference, also influence is little.As long as the volume of the first antibody solution that adds is identical in follow-up operation in each hole, and the molecular amounts of the first antibody that is fixed on hole wall is just basic identical, and the difference in each space reduces greatly, actual measurement show its coefficient of variation generally equal＜5%.Thereby can significantly improve the precision of mensuration.Meanwhile, second antibody is inexpensive, and the consumption of the first antibody that price is higher only needs the 3-4% with first antibody method for coating consumption, but saves this a kind of active material greatly, significantly reduces the cost of whole kit.
Through experimental results demonstrate, the methodology appraising datum of the above-mentioned chemiluminescence detection by quantitative of the present invention reagent when being used for testosterone concentration mensuration can reach following index:
Sensitivity-minimum detectable activity is 0.1ng/ml;
Standard curve range-0-20ng/ml;
Precision average out to 5.53% (n=10) in precision-analysis, precision average out to 9.65% (n=10) between analysis far above national standard, illustrates that kit of the present invention has good repeatability in test experience;
Specificity-be with the cross reacting rate of inhomogeneity steroid hormone: testosterone 100%, dihydrotestosterone 5.6%, androsterone 0.1%, progesterone, estradiol, estriol and cortisol are all＜0.1%;
The recovery that accuracy-in serum is added behind the standard testosterone is 98-110%.
Experiment shows that practical application the present invention measures the testosterone in the human serum, and the sample that only need take a morsel can carry out, to the detected person without any injury.The second antibody bag that the present invention adopts has been significantly improved the precision of measuring by method, has significantly reduced the higher first antibody consumption of price.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 for clinical provide a kind of testosterone more cheap, accurately, method easily and efficiently, can satisfy clinical needs better.
Embodiment
Testosterone (Testosterone) chemical luminescent analysis reagent kid, mainly wrap the opaque polystyrene board of coated goat-anti rabbit/rat immune globulin G antibody by (1), (2) spend hormone serum as matrix, adding is through the formulated testosterone series standard product of the pure product of testosterone of anhydrous alcohol solution, (3) use analysis buffer as matrix, add the anti-testosterone antibody-solutions that anti-testosterone polyclonal antibody of certain density rabbit or mouse-anti testosterone monoclonal antibody are made into, (4) use analysis buffer as matrix, add the horseradish peroxidase-labeled testosterone solution that certain density horseradish peroxidase-labeled testosterone is made into, (5) luminous substrate A liquid and (5) luminous substrate B liquid are formed.
Analysis buffer is formed pH7.5 by 0.05Mtris-HCl salt solusion and 1% bovine serum albumin(BSA) (BSA);
Working concentration when the horseradish peroxidase-labeled testosterone uses is 1: 30 ten thousand;
Luminous substrate A formula of liquid is: luminol 0.15mM, Hydroxycoumarin 0.59mM, gallic acid 0.35mM, Tris-Hcl damping fluid 0.2M, and pH 9.4;
Luminous substrate B formula of 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.
Claims (3)
1, chemical luminescent analysis reagent kid for testosterone, it is characterized in that, mainly formed by the opaque polystyrene board of second antibody, testosterone series standard product, anti-testosterone antibody-solutions, horseradish peroxidase-labeled testosterone solution, luminous substrate A liquid and luminous substrate B liquid by bag.
2, kit as claimed in claim 1 is characterized in that, bag is that bag is by the opaque polystyrene board of goat-anti rabbit/rat immune globulin G antibody by the opaque polystyrene board of second antibody; Testosterone series standard product spend hormone serum as matrix, add formulated through the pure product of testosterone of anhydrous alcohol solution; Anti-testosterone antibody-solutions, adds anti-testosterone polyclonal antibody of certain density rabbit or mouse-anti testosterone monoclonal antibody and is made into as matrix with analysis buffer; Horseradish peroxidase-labeled testosterone solution as matrix, adds certain density horseradish peroxidase-labeled testosterone with analysis buffer; Luminous substrate A liquid is made up of efficient luminous agent, composite fortifier, amino acid; Luminous substrate B liquid is made up of amino acid oxidase and stabilizing agent.
3, kit as claimed in claim 2 is characterized in that, analysis buffer is formed pH7.5 by 0.05Mtris-HCl salt solusion and 1% bovine serum albumin(BSA) (BSA); Working concentration when the horseradish peroxidase-labeled testosterone uses is 1: 300000-500000; Luminous substrate A formula of liquid is: luminol 0.15mM, Hydroxycoumarin 0.59mM, gallic acid 0.35mM, Tris-Hcl damping fluid 0.2M, and pH 9.4; Luminous substrate B formula of liquid is: amino acid oxidase 0.85mM, Tween-20 0.8% (V/V), DTPA0.5mM, vitamin C 0.12mM, acetate-acetate buffer 0.2M, pH 6.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510017943 CN1924582A (en) | 2005-08-30 | 2005-08-30 | Chemical luminescent analysis reagent kid for testosterone |
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| CN 200510017943 CN1924582A (en) | 2005-08-30 | 2005-08-30 | Chemical luminescent analysis reagent kid for testosterone |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103954779A (en) * | 2014-03-12 | 2014-07-30 | 长春迪瑞医疗科技股份有限公司 | Testosterone detection reagent based on microparticle chemiluminescence immunoassay technology |
| CN106199010A (en) * | 2016-06-29 | 2016-12-07 | 无锡市第四人民医院 | A kind of immunochemiluminescence method quickly detects the method for urine steroid hormone |
| CN107015010A (en) * | 2017-06-09 | 2017-08-04 | 深圳大学 | It is a kind of for kit of testosterone residue detection and preparation method thereof and detection method |
| CN114324875A (en) * | 2021-12-13 | 2022-04-12 | 何骏杰 | Cell chip and detection method and application thereof for bacterial infection |
-
2005
- 2005-08-30 CN CN 200510017943 patent/CN1924582A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103954779A (en) * | 2014-03-12 | 2014-07-30 | 长春迪瑞医疗科技股份有限公司 | Testosterone detection reagent based on microparticle chemiluminescence immunoassay technology |
| CN103954779B (en) * | 2014-03-12 | 2015-09-16 | 长春迪瑞医疗科技股份有限公司 | A kind of testosterone based on microparticle chemiluminescence immunoassay technology detects reagent |
| CN106199010A (en) * | 2016-06-29 | 2016-12-07 | 无锡市第四人民医院 | A kind of immunochemiluminescence method quickly detects the method for urine steroid hormone |
| CN107015010A (en) * | 2017-06-09 | 2017-08-04 | 深圳大学 | It is a kind of for kit of testosterone residue detection and preparation method thereof and detection method |
| CN114324875A (en) * | 2021-12-13 | 2022-04-12 | 何骏杰 | Cell chip and detection method and application thereof for bacterial infection |
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