CN109387642A - A kind of detection reagent detecting trilute - Google Patents
A kind of detection reagent detecting trilute Download PDFInfo
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- CN109387642A CN109387642A CN201710659111.7A CN201710659111A CN109387642A CN 109387642 A CN109387642 A CN 109387642A CN 201710659111 A CN201710659111 A CN 201710659111A CN 109387642 A CN109387642 A CN 109387642A
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- trilute
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/046—Thyroid disorders
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Abstract
The invention discloses a kind of detection reagents for detecting trilute, comprising: reagent one: the magnetic particle of trilute antigen coat, preservative 0.1%, the TRIS buffer containing protein stabiliser；Reagent two: acridinium ester of the trilute similar to substance markers, preservative 0.1%, the TRIS buffer containing protein stabiliser；Calibration object: 1 2.01ng/mL of antigen containing trilute of level, preservative 0.1%, the phosphate buffer buffer containing protein stabiliser；2 4.13ng/mL of antigen containing trilute of level, preservative 0.1%, the phosphate buffer buffer containing protein stabiliser；Quality-control product: 1 .95ng/mL of antigen 1 containing trilute of quality-control product, preservative 0.1%, the phosphate buffer buffer containing protein stabiliser；Detection reagent sensitivity with higher and accuracy, greatly reduce reagent cost.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of detection examination for detecting trilute
Agent.
Background technique
Trilute be it is a kind of synthesized by thyroid gland and secreted or iodine is taken off by total thyroxin in periphery formed
Hormone, they metabolism adjusting in play a very important role.In the circulating cycle, 99.7% triiodo thyroid gland original ammonia
Acid is reversibly incorporated on carrier protein.Measuring serum trilute is to judge hyperthyroidism first choice index
One of, especially trilute toxaemia patient, serum total thyroxine concentration is normal, and triiodo thyroid gland original ammonia
Acid is significantly raised.Therefore, measurement serum trilute is an important indicator of clinical diagnosis thyroid function.
In general, Patients with Hyperthyroidism serum trilute value can be significantly raised.In most hyperthyroidism cases, serum triiodo first
The raising of shape gland original ammonia acid and serum total thyroxine increase parallel；And trilute toxication, also known as triiodo first
In shape gland original ammonia acid type hyperthyroidism, serum total thyroxine value is in normal range (NR), and only trilute increases.Clinically
It is common to certain patients before developing into typical thyrotoxicosis, first pass through a serum trilute level liter
High-stage illustrates that measurement serum trilute is more meaningful compared with measurement serum total thyroxine for diagnosis of hyperthyroidism.
Furthermore it is carried out in radioiodine or drug treatment in Patients with Hyperthyroidism, serum total thyroxine is down to normally, and serum triiodo
Thyronine is still higher than normally, until clinical thyroid functional rehabilitation is normal, serum trilute side is down to
Normal level；Therefore measurement serum trilute is but also as the reliability index for judging curative effect.Triiodo thyroid gland original ammonia
The concentration variation of acid in blood is faster than total thyroxin and becomes apparent from, so the measurement of blood trilute level
It can also be used for response excitement and inhibit in test to evaluate thyroid function.It is generally believed that under the conditions of thyroid gland is strong excited, three
Iodine thyronine level is to represent a good index of thyroid function.
The method for clinically detecting trilute at present has radioimmunoassay, EIA enzyme immunoassay, is immunized
Fluorescence analysis, Time-resolved Fluoroimmunoassay and chemiluminescence immune assay etc..It is radioimmunoassay high sensitivity, special
Property strong but this method there is radioactive pollution, cumbersome, market is gradually dispirited；EIA enzyme immunoassay is easy to operate, detection when
Between it is short and pollution-free, but sensitivity is not good enough；Immunofluorescence analysis and Time-resolved Fluoroimmunoassay need special instrument
Device equipment, testing cost are more expensive；Chemiluminescence immune assay has high sensitivity, and high specificity, detection range be wide, particularly nothing
The advantages such as radiological hazard are gradually replacing radioimmunoassay and EIA enzyme immunoassay.In medical institutions and clinical labororatory
In, the reagent of better performances has Roche, Abbott Laboratories, Beckman, Siemens, Hitachi etc., the resulting clinical mark of these types of detection method
This detected value can more actually react the morbid state of patient, high specificity, high sensitivity, rate of missed diagnosis and misdiagnosis rate all compared with
Low, often as the ideal method of clinical samples detection, but mentioned reagent must be matched with expensive necessary instrument, greatly
Reagent cost is increased greatly, is primarily adapted for use in large hospital and scientific research institution.It is directed to for national second level and following hospital and adopts
It is very high with import reagent cost, it is not suitable for promoting.The detection reagent of detection trilute domestic at present is with Beijing
Yuan De Bioisystech Co., Ltd is representative, and domestic reagent does not need the instrument of special expensive, and testing cost is lower, is suitable for
Phenomena such as small-middle hospital and some township hospital, but there are certain stability is bad, repeatability is slightly poor.Therefore, it is necessary to
Domestic high performance chemiluminescence immune assay detection reagent at exploitation.
Summary of the invention
The purpose of the present invention is to provide a kind of detection reagent for detecting trilute, which has
Higher sensitivity and accuracy, the Full-automatic chemiluminescence immunoassay analysis meter produced with our company, which matches, to be greatly reduced
Reagent cost.
Technical solution of the present invention includes the following contents: 1, a kind of detection reagent for detecting trilute, packet
It includes: reagent one: the magnetic particle suspension of trilute antigen coat；Reagent two: trilute is similar
The acridine ester solution of substance markers；Calibration object and quality-control product；It is characterized in that by following proportion: one group of reagent becomes triiodo thyroid gland original
The magnetic particle 2.5mg of propylhomoserin antigen coat, 7.5 μ L of preservative, dilution are the trihydroxy methyl amino first containing protein stabiliser
Alkane buffer.Two groups of reagent become acridine ester complexes 125ng of the trilute similar to substance markers, 7.5 μ of preservative
L, dilution are the TRIS buffer containing protein stabiliser.Calibration object group becomes 1 shape of first containing triiodo of level
Gland original ammonia acid antigen 2.01ng, 1 μ L of preservative, the phosphate buffer buffer containing protein stabiliser, 2 shape of first containing triiodo of level
Gland original ammonia acid antigen 4.13ng, 1 μ L of preservative, the phosphate buffer buffer containing protein stabiliser.Quality-control product group becomes matter
Control product 1 .78-2.92ng of antigen 1 containing trilute, 1 μ L of preservative, the phosphate buffer containing protein stabiliser are slow
Fliud flushing；Quality-control product 2 3.69-5.79ng of antigen containing trilute, 1 μ L of preservative, the phosphate containing protein stabiliser
Buffer buffer.
The magnetic particle of the trilute antigen coat has superparamagnetism, and partial size is 1.0-1.2 μm.
The trilute analog is monoclonal antibody or polyclonal antibody, preferably monoclonal antibody.
The detection sample of kit of the present invention is human serum or blood plasma, is also applied for containing ethylenediamine tetra-acetic acid, citric acid
Sodium or the sample of anticoagulant heparin agent.
The lowest detection of kit of the present invention is limited to 0.25ng/mL.
Reagent of the invention uses chemoluminescence method magnetic particle detection method, and stable processing technique is feasible, and production cost is low, right
The system evaluation of the reagent shows that it, with accuracy height, the sensitivity and specificity of height and is not easily susceptible to various chaff interferents
The influence of matter.Reagent is reproducible, and validity period up to 12 months, is suitable for clinical application.
Specific embodiment
A kind of detection reagent detecting trilute, comprising:
Reagent one: the magnetic particle suspension of trilute antigen coat；
Reagent two: the acridine ester solution of trilute antibody label；
Calibration object accuses product.
The preparation method of 1 reagent one of embodiment:
Step 1: the magnetic particle (concentration 1%) for taking 0.5mL to mix well is placed in micro-pipe, is placed in magnetic bases 1 and is divided
Clock removes supernatant.It takes 1mL reaction solution to be added in above-mentioned micro-pipe, mixes well stirring 30 minutes.Magnetic bases 1 are placed in divide
Clock removes supernatant.It takes 1mL combination buffer to be added in above-mentioned micro-pipe, 100 μ g triiodo thyroid gland original ammonia is taken after mixing well
Sour antigen, and micro-pipe is placed in impeller and is stirred 3 hours.1mL confining liquid is added, mixes well stirring 30 minutes, sets
In magnetic bases 1 minute, supernatant is removed.The cleaning buffer solution for adding 1mL, mixes well and removes supernatant.Take 1mL containing egg
The TRIS buffer of white stabilizer, which is added in micro-pipe, mixes well to obtain trilute antigen
Coated magnetic particle suspension.Reagent one is obtained after the phosphate buffer mixing of specified amount is added.
Step 2: the determination of one concentration of reagent
The magnetic particle dilution of the magnetic particle of trilute antigen coat assay approval is diluted, it is dilute
The ratio of releasing is respectively 1: 100,1: 50,1: 25,1: 12.5, by detection by high level Sample Dilution at concentration be respectively 8ng/mL,
The sample and matrix liquid (0 value) of 4ng/mL, 2ng/mL, 1ng/mL, 0.5ng/mL evaluate the dilution ratio of magnetic particle, knot
Fruit is shown in Table 1.
The screening of 1 magnetic particle dilution ratio of table
As can be known from the results of Table 1: when the magnetic particle of trilute antigen coat and the volume ratio of dilution are 1
: when 25-50, calibration object curve correlation coefficient is big, and especially when magnetic particle dilution ratio is 1: 25, calibration object curve linear is related
Coefficient r is maximum, and slope is -0.8409, closest -1.Final preferably magnetic particle dilution ratio is 1: 25.
The preparation of 2 reagent two of embodiment:
Step 1: (1) taking trilute antibody 1mg, is dialysed 24 hours with combination buffer, and slow with combining
Fliud flushing is configured to concentration 1mg/mL.(2) it takes acridinium ester 1mg to be dissolved in 1mL dimethyl sulfoxide and obtains concentration as 1mg/mL.(3) it takes
The 80 μ L of acridine ester solution of above-mentioned concentration is added in antibody-solutions, and is sufficiently stirred 2 hours.(4) with storage buffer to step
Suddenly the solution in (3) carries out dialysis 24 hours, mixes well after adding 50% glycerol.The phosphate buffer of specified amount is added
Reagent two is obtained after mixing.
Step 2: the determination of two concentration of reagent:
The acridinium ester that trilute antibody is marked is according to 1: 250,1: 500,1: 1000 and 1: 2000 difference
It is diluted, at concentration is respectively 8ng/mL, 4ng/mL, 2ng/mL, 1ng/mL, 0.5ng/ by detecting by high level Sample Dilution
The sample and matrix liquid (0 value) of mL evaluates the dilution ratio of acridinium ester, and testing result is shown in Table 3.
The dilution ratio of 2 trilute antibody of table label acridinium ester
As can be seen from Table 3, when the volume ratio of trilute antibody label acridinium ester and the dilution is 1
: when 1000-2000, curve linear correlation coefficient r is big, and trilute is dilute 1: 1000 similar to the acridinium ester of substance markers
When releasing, curve linear correlation coefficient r is maximum, and slope is closest to 1.Final preferably trilute antibody label
Acridinium ester ratio is 1: 1000.
The preparation of 3 calibration object of embodiment
It takes trilute antigen to be added to the phosphate buffer buffer containing protein stabiliser to mix well
Afterwards, assignment is carried out with national standard.
The preparation of 4 quality-control product of embodiment
It takes trilute antigen to be added to the phosphate buffer buffer containing protein stabiliser to mix well
Afterwards, assignment is carried out with national standard.
5 specific detection of embodiment
Concentration is the total thyroxin sample of 500.00ng/mL, and the testing result on this reagent is not higher than 2.00ng/
mL。
Concentration is the reverse triiodothyronine sample of 50.00ng/mL, and the testing result on this reagent is not higher than
2.00ng/mL。
6 range of linearity test experience of embodiment
With the buffer system of detection reagent by trilute national standard, according to the range of linearity of this reagent
(0.50ng/mL~8.00ng/mL) dilutes at least five concentration point in proportion, replication 3 times, calculates its average value, will tie
Fruit average value and dilution ratio carry out straight line fitting with least square method, and calculating linearly dependent coefficient r should be not less than 0.9900.
The detection of 3 range of linearity of table
7 sensitivity experiment of embodiment
It is operated in strict accordance with specification, detects zero calibration object, replication 20 times, obtain the luminous value of 20 measurement results
(relative light unit) calculates its average and standard deviation, obtains the sum of average value and twice of standard deviation, according to zero-dose calibration object
Concentration and luminous value (relative light unit) between calibration object level one carry out two o'clock recurrence, obtain linear function, will be averaged
The sum of value and twice of standard deviation are brought into this linear function, obtain corresponding concentration value, as minimum detection limit, as a result Ying Bugao
In 0,.25ng/mL.
4 sensitivity experiment of table
8 stability experiment of embodiment
Under 2-8 DEG C of condition of storage, quality-control product is measured 0 month, 12 months respectively, each sample measures 10
It is secondary, take mean value.The result shows that (see the table below), the coefficient of variation can satisfy requirement, illustrate the detection reagent in embodiment 1,2 in 2-
It can at least stablize 12 months under 8 DEG C of conditions of storage.
5 stability experiment of table
It is provided for the embodiments of the invention a kind of detection reagent for detecting trilute above, has carried out in detail
Thin to introduce, used herein a specific example illustrates the principle and implementation of the invention, and above embodiments are said
It is bright to be merely used to help understand method and its core concept of the invention；At the same time, for those skilled in the art, foundation
Thought of the invention, there will be changes in the specific implementation manner and application range, in conclusion the content of the present specification is not
It is interpreted as limitation of the present invention.
Claims (3)
1. a kind of detection reagent for detecting trilute, comprising: reagent one: trilute antigen coat
Magnetic particle suspension；Reagent two: acridine ester solution of the trilute similar to substance markers；Calibration object and quality-control product:
It is characterized in that by following proportion: one group of the reagent magnetic particle 2.5mg as trilute antigen coat, preservative
7.5 μ L, dilution are the TRIS buffer containing protein stabiliser.Two groups of reagent become triiodo thyroid gland original
Acridine ester complexes 125ng of the propylhomoserin similar to substance markers, 7.5 μ L of preservative, dilution is the three hydroxyl first containing protein stabiliser
Base aminomethane buffer solution.Calibration object group becomes 1 2.01ng of antigen containing trilute of level, 1 μ L of preservative, contains egg
The phosphate buffer buffer of white stabilizer, level 2 4.13ng of antigen containing trilute, 1 μ L of preservative contain egg
The phosphate buffer buffer of white stabilizer.Quality-control product group becomes 1 .78- of antigen 1 containing trilute of quality-control product
2.92ng, 1 μ L of preservative, the phosphate buffer buffer containing protein stabiliser；Quality-control product 2 is anti-containing trilute
Former 3.69-5.79ng, 1 μ L of preservative, the phosphate buffer buffer containing protein stabiliser.
2. a kind of detection reagent for detecting trilute according to claim 1, it is characterized in that triiodo first shape
The magnetic particle of gland original ammonia acid antigen coat has superparamagnetism, and partial size is 1.0-1.2 μm.
3. a kind of detection reagent for detecting trilute according to claim 1, it is characterized in that the triiodo
Thyronines are monoclonal antibody or polyclonal antibody, preferably monoclonal antibody like object.
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| CN201710659111.7A CN109387642A (en) | 2017-08-04 | 2017-08-04 | A kind of detection reagent detecting trilute |
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| CN201710659111.7A CN109387642A (en) | 2017-08-04 | 2017-08-04 | A kind of detection reagent detecting trilute |
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Citations (5)
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|---|---|---|---|---|
| CN101949942A (en) * | 2010-08-03 | 2011-01-19 | 郑州安图绿科生物工程有限公司 | Kit for quantitatively testing free triiodothyronine and preparation method thereof |
| CN101949945A (en) * | 2010-08-03 | 2011-01-19 | 郑州安图绿科生物工程有限公司 | Kit for detecting free thyroxin by using magnetic particles as solid-phase carriers and preparation method thereof |
| CN103063851A (en) * | 2012-12-25 | 2013-04-24 | 苏州浩欧博生物医药有限公司 | Free triiodothyronine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof |
| CN104730257A (en) * | 2015-02-10 | 2015-06-24 | 深圳市新产业生物医学工程股份有限公司 | RT3 chemiluminiscence immunodetection kit as well as detection method and application thereof |
| WO2016127301A1 (en) * | 2015-02-10 | 2016-08-18 | 深圳市新产业生物医学工程股份有限公司 | Rt3 chemiluminescent immunological detection reagent kit, and detection method and application therefor |
-
2017
- 2017-08-04 CN CN201710659111.7A patent/CN109387642A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101949942A (en) * | 2010-08-03 | 2011-01-19 | 郑州安图绿科生物工程有限公司 | Kit for quantitatively testing free triiodothyronine and preparation method thereof |
| CN101949945A (en) * | 2010-08-03 | 2011-01-19 | 郑州安图绿科生物工程有限公司 | Kit for detecting free thyroxin by using magnetic particles as solid-phase carriers and preparation method thereof |
| CN103063851A (en) * | 2012-12-25 | 2013-04-24 | 苏州浩欧博生物医药有限公司 | Free triiodothyronine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof |
| CN104730257A (en) * | 2015-02-10 | 2015-06-24 | 深圳市新产业生物医学工程股份有限公司 | RT3 chemiluminiscence immunodetection kit as well as detection method and application thereof |
| WO2016127301A1 (en) * | 2015-02-10 | 2016-08-18 | 深圳市新产业生物医学工程股份有限公司 | Rt3 chemiluminescent immunological detection reagent kit, and detection method and application therefor |
Non-Patent Citations (1)
| Title |
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| 汪阿恋: "三碘甲状腺原氨酸及真菌毒素的化学发光免疫分析方法研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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Application publication date: 20190226 |