CN1502992A - Growth hormone conjunctive protein activity determination kit, preparation and determination method - Google Patents
Growth hormone conjunctive protein activity determination kit, preparation and determination method Download PDFInfo
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- CN1502992A CN1502992A CNA021546193A CN02154619A CN1502992A CN 1502992 A CN1502992 A CN 1502992A CN A021546193 A CNA021546193 A CN A021546193A CN 02154619 A CN02154619 A CN 02154619A CN 1502992 A CN1502992 A CN 1502992A
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- growth hormone
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Abstract
This invention discloses a determination kit box combining with protein testing the growing hormone activity in and animals and its preparation and test method characterizing that the box is composed of a solution prepared by a labeled growing hormone, unlabeled growing hormone, glucose enveloped carbon, general buffer solution and analytical solution and a specification. The method is that apart from setting 4-8 NSB tubes, two correspondent NSB tubes are specially designed for every sample to make the test more accurate. The glucose enveloped carbon preparation and quatily are necessary conditions for the accurate test.
Description
One, technical field
The present invention is a kind of medical science index determining kit and preparation and assay method of measuring the growth hormone binding protein activity in the humans and animals serum.
Two, background technology
(growth hormone binding protein, GHBP) distribution, metabolism and the physiological activity to growth hormone has vital role to growth hormone binding protein in the humans and animals serum.GHBP is a single chain protein of being made up of 238～246 amino acid, and the about 61kDa of molecular weight belongs to acidic protein, isoelectric point about 5.0.The major physiological effect of GHBP is that the growth hormone that produces in conjunction with hypophysis is transported to growth hormone body tissue's cell in blood, growth hormone is effectively stored and prolong the growth hormone activity, GHBP can also be by the physiological function of regulating growth hormone with growth hormone and the increase and decrease that combines with growth hormone receptor.GHBP can be used as the periphery monitoring index of liver growth hormone receptor activity.The source of GHBP and growth hormone receptor are closely related in the human blood, and the amino acid sequence of GHBP and the ectodomain of growth hormone receptor partly are duplicate.The GHBP activity of measuring serum can reflect the performance situation of growth hormone function indirectly, and (activity of measuring growth hormone receptor is very complicated, and the official of retrieve sample damage health for the situation of change of the activity of the growth hormone receptor of reflection health inner tissue organ under different physiology, pathological conditions; As long as and the active relative not damaged of blood that takes a morsel of mensuration GHBP).
In the GHBP assay method, the gel chromatography separation GHBP compound that utilizes that has is previously measured again at present, and this method is more time-consuming loaded down with trivial details; Now utilize radio immunoassay more, particularly utilize radio immunoassay and activated charcoal absorption process to combine and measure the GHBP activity.Owing to measure the method more complicated of GHBP activity,, have only several pieces of relevant reports through searching document so the report of domestic relevant mensuration GHBP activity seldom.In these reports, Zhang Yujin, Wang Defen, Shi Aisheng, Cao Ji give birth to: growth hormone of serum is in conjunction with protein determination method and clinical meaning thereof, Chinese endocrine metabolism magazine, 1994 the 3rd phases, 170～174; This piece paper has only in short their assay method of brief introduction in full, does not have particular content in detail such as the preparation of agents useful for same and determination step.Qiu Ningyan, Zhang Jingxin, Li Huiping, Bao Yintang: the rapid test method and the Preliminary Clinical thereof of human serum growth hormone binding protein activity, labelled immune analysis and clinical, 1998 the 2nd phases, 93～96; This paper has the in short prescription of explanation " incubation buffering liquid " in full, in short introduces the prescription of " separating agent " in addition, does not have particular content in detail such as the preparation of relevant agents useful for same and determination step in addition.The method of the mensuration GHBP activity that these minority papers are mentioned measures the GHBP activity for general hospital and research unit guidance and reference significance, but with reference to these methods concrete enforcement measurement operation is still had any problem.In a word, senior detection technique personnel have gained some understanding to the technology of measuring the GHBP activity, also need to do many experiments and grope and implement to detect, and general detection technique personnel can't be utilized or directly detect according to the technical literature content that is retrieved.In addition, mentioning the content of measuring people GHBP in other minority documents, but do not introduce concrete grammar, generally is the kit that adopts external reagent or measure GHBP abroad.There is the kit supply of measuring growth hormone binding protein to be external product through the more domestic biotech companies of retrieval, do not measure the kit of growth hormone binding protein activity.
The object of the invention provides a kind of medical science index determining kit and comprises its preparation and assay method, can be in hospital with radiocounting instrument or laboratory, and the growth hormone binding protein in the more convenient mensuration humans and animals serum is relatively in conjunction with active.
Three, summary of the invention
The active radioimmunoassay kit of growth hormone binding protein and preparation and assay method, kit is made of 5 parts of solvents and a operation instructions.Its solvent is respectively: the labeling and growing hormone (
125I-hGH), unmarked growth hormone (hGH), glucosan coating charcoal (Dextran charcoal), general damping fluid (general buffer), analysis buffer (testbuffer).Kit also can be made of 3 parts of solvents and a operation instructions, and its solvent is respectively: the labeling and growing hormone (
125I-hGH), unmarked growth hormone (hGH), glucosan coating charcoal (Dextran charcoal); General damping fluid and analysis buffer are then prepared by user oneself to list prescription in the use instructions.
The specification of kit is a packing can measure 50 serum samples generally, can be a packing with the specification of measuring 100～200 samples also.
Principle be blood serum sample and labeling and growing hormone (
125I-hGH) at room temperature hatch a period of time jointly, isolate and combine with GHBP
125I-hGH and do not have combination
125I-hGH.In conjunction with
125I-hGH is with total
125The I-hGH radioactive intensity is than the relative value that can reflect GHBP.
The manufacture method of 5 bottles of reagent is as follows:
(1) the labeling and growing hormone (
125I-hGH):
Labeling method: because of the toluene-sodium-sulfonchloramide method (can be with reference to relevant document such as Davis S.L.Plasma levels of prolactin, growth hormone and insul in sheep following the infusion of arginine, leucine andphenylalanine.Endocrinology.1972,91:773～780) with
125About 15 seconds of I labelling human recombinant human growth hormone, the specific activity scope is at from 90 to 120 μ Ci/ μ g.
Purification process: adopt Biorad AG1 * 8 resins (50～100 order) chromatography, remove free
125I.Cross post with 1 * 50cm BiogelP100 chromatography again, with PBS (containing 0.5%BSA) wash-out under 4 ℃ of conditions, remove cohesion
125I-hGH obtains monomer
125I-hGH.The labeling and growing hormone (
125I-hGH) can directly buy to du pont company, also there is supply in domestic relevant company.When general requirement guarantees that delivering to the user uses
125The specific activity of I-hGH has 60 μ Ci/ μ g approximately.
(2) unmarked growth hormone (hGH): the human growth hormone recombinant who does not have mark to cross.Technical requirement is to divide when being added in each non-specific binding (NSB) test tube after this unmarked growth hormone dilutes with general damping fluid, and keeping hGH concentration in test tube incubation reaction solution is 2 μ g/ml.The incubation reaction overall solution volume is 500 μ l in general each non-specific binding test tube, and therefore adding in each non-specific binding test tube is the hGH of 1 μ g.
(3) glucosan coating charcoal (Dextran charcoal): 2% glucosan and 2% activated charcoal are dissolved in PBS.Method for making: earlier with 2% glucosan (Dextran) stirring and dissolving under 40～70 ℃ of temperature conditions, treat the glucosan dissolving after, add activated charcoal (charcoal, 50～200 orders), stirred under the room temperature 1～5 hour.Method, requirement and the quality of the preparation of glucosan coating charcoal also are to analyze necessary condition accurately.
(4) general damping fluid (general buffer): PBS (pH 7～7.5)
(5) analysis buffer (test buffer): PBS contains 0.5%BSA and 75mMgCl2.
This kit requires to preserve under 4 ℃ of conditions.
The assay method of this kit: 4～8 total " non-specific binding " are set (NSB) measure pipe, purpose is accurate for making non-specific binding measure the average radiocounting of counting, and investigates the accuracy of application of sample operation; Except that this total NSB, each measures " non-specific binding (NSB) " mensuration pipe that blood sample specially is provided with 2 correspondences in addition, makes measurement result more accurate.
This kit is formed simple in structure, carries out time-and-motion study according to its operation instructions, and step is clearly demarcated, and is easy to use, and measurement result accurately and reliably.Generally have the hospital or the laboratory of radiocounting instrument, determination techniques personnel can measure.
Four, embodiment
Below in conjunction with table 1 and table 2 and embodiment the present invention is described in further detail, wherein table 1 is the signal of embodiment of the present invention step, and table 2 is the mensuration application of sample order that must list in the kit operation instructions and the inventory of quantity.
Dilute with general buffer earlier during measurement operation
125I-hGH gets 100 μ l's while dilute
125I-hGH tests to the radiocounting instrument, up to what diluted
125I-hGH reaches till the about 20000cpm of counting of per 100 μ l.
All add 100 μ l's in all each test tubes
125I-hGH.
6 tale pipes are set, and purpose is for making the average radiocounting of tale pipe accurate, and investigates the accuracy of application of sample operation.Each tale pipe removes and adds 100 μ l
125Outside the I-hGH, add 400 μ l again and analyze buffer, total incubation reaction overall solution volume is 500 μ l.
6 total " non-specific binding " are set (NSB) measure pipe, purpose is accurate for making non-specific binding measure the average radiocounting of counting, and investigates the accuracy of application of sample operation; These 6 non-specific binding are measured each pipe of pipe except that adding 100 μ l
125Outside the I-hGH, add pooled serum (promptly take from respectively measure the serum equal amount of mixture be called pooled serum) 50 μ l, add the unmarked growth hormone of 50 μ l again, add the general buffer of 300 μ l, total incubation reaction overall solution volume is 500 μ l.In addition, be the accuracy of guaranteeing to measure, each determination of serum sample also be provided with 2 " non-specific binding " (NSB) measure pipe that each pipe of the determination of serum sample NSB of these corresponding serum samples removes and adds 100 μ l
125Outside the I-hGH, add the serum of the surveying 50 μ l of institute, add the unmarked growth hormone of 50 μ l again, add the general buffer of 300 μ l, always the incubation reaction overall solution volume is 500 μ l.When concrete calculations incorporated is active, be as the criterion with these 2 averages corresponding to the NSB of surveyed serum sample.
Each sample determination pipe is set to 2 same test tubes (parallel pipe), to reduce the error of application of sample and mensuration.Remove in every mensuration pipe
125Outside the I-hGH, add serum 50 μ l to be determined, add 350 μ l again and analyze buffer, the incubation reaction overall solution volume is 500 μ l.
All test tubes add according to the program of application of sample inventory
125Behind the reagent such as I-hGH, unmarked GH, test sample book serum, general buffer or analysis buffer, each pipe all will pass through strong suspendible, hatches 8 hours overnight incubation under room temperature when temperature is low when temperature is high under the room temperature.
(Dextran charcoal) is placed on precooling under 4 ℃ of conditions with glucosan coating charcoal.This suspending liquid is as easy as rolling off a log to be precipitated, and for the quantity that guarantees to join glucosan coating charcoal in each pipe is even, need adds at it and keeps this solution to be in process that drips to each pipe ceaselessly swaying suspension.Each pipe adds glucosan coating charcoal solution 500 μ l.
After all test tubes add glucosan coating charcoal, through strong suspendible, carry out centrifuging under 2000g and the 4 ℃ of conditions.
Each tale pipe is removed supernatant, reclaim precipitation; Each sample determination pipe and each non-specific binding are measured pipe recovery supernatant separately, remove precipitation.
Precipitation and each sample determination pipe to the tale pipe carry out radiocounting, every pipe counting 1 minute with each non-specific binding mensuration pipe supernatant separately.According to each pipe counting cpm of record, calculate the specific binding activity of each serum sample with following formula:
SB(％)＝[(TB-NSB)/TC]×100％
In the formula:
The SB=specific binding activity
The cpm of TB=sample determination pipe
The NSB=non-specific binding is measured the cpm of pipe
The cpm of TC=tale pipe
Table 1 " the active radioimmunoassay kit of growth hormone binding protein " embodiment step principle
| Tale pipe (TC) | Non-specific binding is measured pipe (NSB) | Sample determination pipe (TB) | |
| Labeling and growing hormone (125I-hGH) (being diluted in general beffer, per 100 μ l about 20000) | ????100μl | 100μl | ????100μl |
| Test serum | 50 μ l (preceding spy is provided with the NSB pipe and adds pooled serum, and the corresponding NSB pipe of each sample determination pipe adds test serum) | ????50μl | |
| Unmarked growth hormone (hGH) | 50μl | ||
| General beffer | 300μl | ||
| Analyze buffer | ????400μl | ????350μl |
Below respectively manage strong suspendible, hatched under the room temperature 8 hours when temperature is high, overnight incubation under room temperature when temperature is low
| The glucosan coating charcoal of precooling | ????500μl | ????500μl | ????500μl |
Below respectively manage strong suspendible, centrifugal under 4 ℃ of conditions of 2000g
| Radiocounting | Remove supernatant, precipitation was counted 1 minute | Reclaim about 1000 μ l supernatants, counted 1 minute | Reclaim about 1000 μ l supernatants, counted 1 minute |
Table 2 " the active radioimmunoassay kit of growth hormone binding protein " operation application of sample inventory
| The labeling and growing hormone | Serum | Unmarked growth hormone | General buffer | Analyze buffer | Suspendible is hatched | Cold glucosan coating charcoal | Suspendible, centrifugal | Counting | |
| Tale pipe 1 | ?100μl | ?400μl | ?500μl | ||||||
| Tale pipe 2 | ?100μl | ?400μl | ?500μl | ||||||
| Tale pipe 3 | ?100μl | ?400μl | ?500μl | ||||||
| Tale pipe 4 | ?100μl | ?400μl | ?500μl | ||||||
| Tale pipe 5 | ?100μl | ?400μl | ?500μl | ||||||
| Tale pipe 6 | ?100μl | ?400μl | ?500μl | ||||||
| Non-specific binding pipe 1 | ?100μl | 50 μ l mix | 50μl | ?300μl | ?500μl | ||||
| Non-specific binding pipe 2 | ?100μl | 50 μ l mix | 50μl | ?300μl | ?500μl | ||||
| Non-specific binding pipe 3 | ?100μl | 50 μ l mix | 50μl | ?300μl | ?500μl | ||||
| Non-specific binding pipe 4 | ?100μl | 50 μ l mix | 50μl | ?300μl | ?500μl | ||||
| Non-specific binding pipe 5 | ?100μl | 50 μ l mix | 50μl | ?300μl | ?500μl | ||||
| Non-specific binding pipe 6 | ?100μl | 50 μ l mix | 50μl | ?300μl | ?500μl | ||||
| Sample determination pipe 1a | ?100μl | ?50μl | ?350μl | ?500μl | |||||
| Sample determination pipe 1b | ?100μl | ?50μl | ?350μl | ?500μl | |||||
| Non-specific binding pipe 1a | ?100μl | ?50μl | 50μl | ?300μl | ?500μl | ||||
| Non-specific binding pipe 1b | ?100μl | ?50μl | 50μl | ?300μl | ?500μl | ||||
| Sample determination pipe 2a | ?100μl | ?50μl | ?350μl | ?500μl | |||||
| Sample determination pipe 2b | ?100μl | ?50μl | ?350μl | ?500μl | |||||
| Non-specific binding pipe 2a | ?100μl | ?50μl | 50μl | ?300μl | ?500μl | ||||
| Non-specific binding pipe 2b | ?100μl | ?50μl | 50μl | ?300μl | ?500μl | ||||
| Sample determination pipe 3a .... | ?100μl | ?50μl | ?350μl | ?500μl |
Claims (4)
1. one kind is used for humans and animals growth hormone binding protein determination of activity kit, it is characterized in that, kit constitutes solvent and a operation instructions that comprise 5 kinds of preparations, the solvent of 5 kinds of specific preparations is: (1) labeling and growing hormone, (2) unmarked growth hormone, (3) glucosan coating activated charcoal, (4) analysis buffer, (5) general damping fluid.
2. method with this claim 1 described kit measurement humans and animals growth hormone binding protein activity, it is characterized in that: (NSB) measure the pipe except that 4～8 total " non-specific binding " are set, each measures " non-specific binding (NSB) " mensuration pipe that blood sample specially is provided with 2 correspondences in addition.
3. compound method to reagent in this claim 1 described kit, it is characterized in that: " glucosan coating activated charcoal " preparing solvent, earlier with 2% glucosan (Dextran) stirring and dissolving under 30～70 ℃ of conditions, after treating the glucosan dissolving, add activated charcoal (charcoal, 50～100 orders), stirred under the room temperature 1～5 hour.
4. according to this claim 1 described kit, it is characterized in that: kit also can be made of 3 parts of solvents and a operation instructions.Its solvent is respectively: labeling and growing hormone, unmarked growth hormone, glucosan coating charcoal; General damping fluid and analysis buffer are then prepared by user oneself to list prescription in the use instructions.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021546193A CN1502992A (en) | 2002-11-26 | 2002-11-26 | Growth hormone conjunctive protein activity determination kit, preparation and determination method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021546193A CN1502992A (en) | 2002-11-26 | 2002-11-26 | Growth hormone conjunctive protein activity determination kit, preparation and determination method |
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| CN1502992A true CN1502992A (en) | 2004-06-09 |
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| CNA021546193A Pending CN1502992A (en) | 2002-11-26 | 2002-11-26 | Growth hormone conjunctive protein activity determination kit, preparation and determination method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101344466B (en) * | 2007-07-11 | 2010-06-23 | 中国科学院海洋研究所 | Sample preparation method for measuring sex hormone level of Japanese flounder larvae |
| CN102253196A (en) * | 2011-04-21 | 2011-11-23 | 江苏昶迅生物科技有限公司 | Method for preparing small-molecule estradiol immunoassay standard substance |
| CN106442084A (en) * | 2016-08-31 | 2017-02-22 | 上海科华生物工程股份有限公司 | Method for removing small-molecule substance from serum |
| US10098722B2 (en) | 2005-04-26 | 2018-10-16 | Poly-Med, Inc. | Absorbable/biodegradable composite yarn constructs and applications thereof |
-
2002
- 2002-11-26 CN CNA021546193A patent/CN1502992A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10098722B2 (en) | 2005-04-26 | 2018-10-16 | Poly-Med, Inc. | Absorbable/biodegradable composite yarn constructs and applications thereof |
| CN101344466B (en) * | 2007-07-11 | 2010-06-23 | 中国科学院海洋研究所 | Sample preparation method for measuring sex hormone level of Japanese flounder larvae |
| CN102253196A (en) * | 2011-04-21 | 2011-11-23 | 江苏昶迅生物科技有限公司 | Method for preparing small-molecule estradiol immunoassay standard substance |
| CN106442084A (en) * | 2016-08-31 | 2017-02-22 | 上海科华生物工程股份有限公司 | Method for removing small-molecule substance from serum |
| CN106442084B (en) * | 2016-08-31 | 2020-08-04 | 上海科华生物工程股份有限公司 | Method for removing small molecular substances in serum |
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