CN1271409C - Raid quantitative determination of cardiac muscle troponin I by three-anti method - Google Patents
Raid quantitative determination of cardiac muscle troponin I by three-anti method Download PDFInfo
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- CN1271409C CN1271409C CN 03131931 CN03131931A CN1271409C CN 1271409 C CN1271409 C CN 1271409C CN 03131931 CN03131931 CN 03131931 CN 03131931 A CN03131931 A CN 03131931A CN 1271409 C CN1271409 C CN 1271409C
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Abstract
The present invention relates to a novel method for rapidly and quantitatively measuring the content of cardiac troponin I in peripheral blood or tissues of a human body according to the enzyme-linked immune principle. Due to the low content of cardiac troponin I in blood, the measurement sensitivity of the existing common enzyme-linked method is low, and the lowest measurement sensitivity is about 0.2 ng/ml and is difficult to be lower than 0.1 ng/ml. The measuring speed is also low, and measurement needs more than 1 hour. In order to enhance the measuring speed and the measurement sensitivity, the present invention successfully develops a kit for rapidly and quantitatively measuring cardiac troponin I through a three-resistant method, and two monoclonal antibodies are utilized to replace one monoclonal antibody to capture antibodies, so that the antibody capturing capability is enhanced. Simultaneously, a streptavidin-biotin amplification system is combined, so that the detecting time is effectively shortened, the measurement sensitivity is enhanced, and the rapid and quantitative measurement of cardiac troponin I on a common enzyme-linked instrument, etc. can satisfy testing and clinical requirements.
Description
Technical field patent of the present invention relates to a kind of new quantitative determination human peripheral blood of setting up according to the enzyme linked immunological principle or the method for organizing cardiac muscle troponin I content.
Background technology myocardial ischemic injury, particularly acute myocardial infarction (AMI) are one of principal diseases of the human life of prestige association, and clinical chemistry man and clinical science man strive to find always and explore good, the highly sensitive serum index of specificity.Cardiac muscle troponin I (cardiac troponin I cTnI) has the myocardium specificity of height, when the cardiac muscle cell is impaired, in the blood cTnI time of occurrence early, longer duration, closely related with myocardial damage degree and prognosis.Therefore, it can be used as a kind of specific index of myocardial damage, has crucial meaning in cardiopathic diagnosis and prognosis judgement.
Cardiac muscle troponin I is measured and can be divided into two kinds of methods of qualitative and quantitative mensuration, chromatographic theory is mainly used in qualitative determination, the making test-paper is finished, carried out clinical practice at present, its advantage is that speed is fast, easy to operate, but its shortcoming is only can show the positive, negative findings, there is not concrete numerical value, its dynamic change be can not observe and infarct time, area and prognosis judged according to numerical value, and susceptibility is low, be subject to multiple factor and disturb, even improved sxemiquantitative miniature instrument assay method equally also has above-mentioned shortcoming.Quantitative measurement is mainly according to enzyme linked immunological and chemiluminescence principle design, the overwhelming majority has been used two strain monoclonal antibodies (small part is only with a strain monoclonal antibody or a strain polyclonal antibody wherein), one strain monoclonal antibody is a capture antibody, another strain monoclonal antibody is a labelled antibody, forms enzyme connection sandwich method for determining.Rely on Chemiluminescence Apparatus, fluor tester and enzyme connection instrument etc. that robotization and non-automaticization are arranged with auxiliary instrument in the mensuration, wherein enzyme connection instrument price is low, between thousands of units to tens thousand of units, domestic large, medium and small hospital is comparatively universal, and price costlinesses such as Chemiluminescence Apparatus and fluor tester, hundreds thousand of units are to several 1,000,000 yuan, the man major company of external existing number produces the large automatic chemiluminescence or luminoscope is finished quantitative measurement, but it costs an arm and a leg, and need matched reagent, domestic most of hospital still is difficult to apply.So relying on enzyme connection instrument to set up the quantitative enzyme-linked method of cardiac muscle troponin I is determined at domestic very necessary, but the sensitivity that the cardiac muscle troponin I that common enzyme-linked method is set up is measured is lower, lowest detection sensitivity is about 0.2ng/ml, be difficult to reach below the 0.1ng/ml, finding speed is also slower, needs more than 1 hour.
Summary of the invention is atomic because of cardiac muscle troponin I content in blood, for improving finding speed and measuring sensitivity, the present invention successfully develops three anti-method cardiac muscle troponin I quantitative determination kits, a strain monoclonal antibody that substitutes in the classic method with two strain monoclonal antibodies is a capture antibody, improved the antibody capture ability, while marriage chain Avidin-biotin amplification system, shortened detection time effectively, improved mensuration sensitivity, made on instruments such as normal enzyme connection instrument fast quantification detect cardiac muscle troponin I and reach check and clinical requirement.
Technical solution of the present invention: the monoclonal antibody that two strains act on cardiac muscle troponin I is a capture antibody, abbreviates Ab1+Ab2 as, behind connection biotin or the long-chain biotin, abbreviates Ab1-Bio+Ab2-Bio as.The monoclonal antibody that another strain acts on cardiac muscle troponin I is a labelled antibody, abbreviates Ab3 as, behind the connection horseradish peroxidase, abbreviates Ab3-POD as, behind connection biotin or the long-chain biotin, abbreviates Ab3-Bio as.Ab1, Ab2, Ab3 require to act on the different loci of cTnI antigen, there are adhesion and higher specificity preferably in Ab1+Ab2 and Ab3 pairing back to cTnI antigen, monoclonal antibody and other raw material can derive from qualified commercial product (as Sigma, Roch company etc.).Its reaction path of utilization enzyme linked immunological principle
Specifically comprise following several scheme:
Scheme one:
(1) .96 hole elisa plate, enzyme bracing or other solid holder have connect streptavidin or Avidin (abbreviating S as), albumin or the sealing of other confining liquid.
(2) .Ab1-Bio+Ab2-Bio combines with S, cardiac muscle troponin I antigen (comprises the cardiac muscle troponin I in known cardiac muscle troponin I standard items, quality-control product and the testing sample, abbreviate cTnI as) combine with Ab1-Bio+Ab2-Bio, cTnI combines with Ab3-POD.Successively form S～Ab1-Bio+Ab2-Bio～cTnI～Ab3-POD reaction chain.
(3) .POD makes chromogenic substrate (as tetramethyl benzidine TMB etc.) colour developing or chemical luminous substrate luminous, measures by corresponding instrument again, calculates testing sample cardiac muscle troponin I content according to typical curve.
Scheme two:
(1) .96 hole elisa plate, enzyme bracing or other solid holder have connect Ab1+Ab2, albumin or the sealing of other confining liquid.
(2) .Ab1+Ab2 combines with cTnI, and cTnI combines with Ab3-POD, successively forms Ab1+Ab2～cTnI～Ab3-POD reaction chain.
(3). with (3) in the scheme one.
Scheme three:
(1). with in the scheme two 1.
(2) .Ab1+Ab2 combines with cTnI, and cTnI combines with Ab3-Bio, and Ab3-Bio combines with streptavidin horseradish peroxidase (being called for short S-POD), successively forms Ab1+Ab2～cTnI～Ab3-Bio～S-POD reaction chain.
(3). with (3) in the scheme one.
After above-mentioned innovation, this law minute can foreshorten to about 35 minutes, its lowest detection sensitivity is 0.05ng/ml, be equal to or surpass external similar kit level, and only need normal enzyme connection instrument to measure, provide testing result fast, flexibly gradation is used for the detection of emergency treatment and conventional different case loads, be more convenient for clinical practice, be suitable for large, medium and small type hospital wide popularization and application, this has immeasurable impetus to the domestic cardiovascular disease diagnostic level of further raising.
Embodiment by above-mentioned three kinds of scheme approach embodiments is:
Scheme one:
(1) .96 hole elisa plate or enzyme bracing have connect streptavidin, and 0.5～1% albumin or other confining liquid have sealed standby.
(2). add a certain amount of Ab1+Ab2-Bio, hatch certain hour (preferred 60 minutes) or 4 ℃ for 37 ℃ and spend the night, be packaged in after the sealing in the kit, open application when sample is measured.
(3). (known cardiac muscle troponin I standard items also can be chilled in the gauge orifice of setting in advance to survey cardiac muscle troponin I standard items known in the timing hole or testing sample, adding the dissolving of standard dilution during detection gets final product), add a certain amount of Ab3-POD reactant liquor again, gently shake back 37 ℃ and hatch certain hour (preferred 20 minutes).
(4). liquid in the hole of inclining, washing lotion (PBS contains 0.05%Tween-20) are washed for several times, and it is 250～300 μ l that each every hole adds the washing lotion amount the suitableeest.
(5). add colour developing liquid tetramethyl benzidine (TMB), hatch certain hour (preferred 15 minutes) for 37 ℃.
(6). every hole adds stop buffer (0.5～2M H
2SO
4) 50 μ l or instil, the even 10 minutes endoenzyme connection instrument 450nm that shake gently read the OD value.
(7). calculate testing sample cardiac muscle troponin I content according to typical curve.
Scheme two:
(1) add a certain amount of Ab1 in .96 hole elisa plate or the enzyme bracing and Ab2 wraps quilt in advance, hatch certain hour (preferred 60 minutes) or 4 ℃ for 37 ℃ and spend the night, 0.5～1% albumin or other confining liquid have sealed standby.
(2). following steps with (3) in the scheme one to (7).
Scheme three:
(1). with (1) in the scheme two.
(2). (known cardiac muscle troponin I standard items also can be chilled in the gauge orifice of setting in advance to survey cardiac muscle troponin I standard items known in the timing hole or testing sample, adding the dissolving of standard dilution during detection gets final product), the mixed reaction solution that adds a certain amount of Ab3 and S-POD gentlier shakes back 37 ℃ and hatches certain hour (preferred 20 minutes).
(3). following steps with (3) in the scheme one to (7).
Reactant liquor PH is preferable over 6.8～7.2 in the above-mentioned described method between 6.5～7.5.Used washing lotion PH6.5～7.5, preferred 6.8～7.2; Temperature preferably is lower than 15 ℃ at 2～37 ℃; Washing lotion is washed 3～6 times, preferably washes 5 times.
Three anti-method cardiac muscle troponin I quantitative determination kits by scheme one processing are formed and operational manual:
Kit is formed
1, enzyme connection frame is 1.
2, the enzyme bracing is 12, and wherein the standard bar is 6,6 of sample strip (trial product standard bar and sample strip respectively are 2).Standard bar first hole is a blank, and 2～6 holes are standard point (cardiac muscle troponin I concentration is respectively 3.2,1.6,0.8,0.4,0.2ng/ml, and freeze drying is in the hole), and the 7th hole is a sample well, can be used as emergency treatment or conventional case and detects.Sample strip the 2nd～7 hole is the test sample hole.Standard bar and sample strip quantitative proportion can be ordered especially according to user's needs, are certificate to measure number of perforations still, do not charge additional cost.
3, standard dilution 10ml (the blue lid of white bottle).
4,4. washing lotion 200ml (big bottle).
5, reactant liquor 10ml (the red lid of white bottle).
6,6. colour developing liquid A (the black lid of white bottle).
7, colour developing liquid B 20ml (the purple lid of white bottle).
8, stop buffer 10ml (drop bottle).
9, instructions is 1 part.
Operational manual
1. principle
With three plant height specificitys and hypersensitivity monoclone antibody against human cardiac troponin I, wherein two strains are capture antibody, one strain is a labelled antibody, marriage chain Avidin-biotin amplification system, use the content that the enzyme linked immunological principle detects cardiac muscle troponin I (cTnI) in human blood or other sample, be used for the diagnosis that multiple disease causes myocardial damage.
2. sample collecting and processing
Drying tube or anticoagulant heparin blood sampling tube 2ml.Drying tube needs room temperature to place after 30 minutes centrifugal (2000rpm, 5 minutes), gets serum and is used for detecting.The anticoagulant heparin pipe can centrifugal at once (the same) after, get blood plasma and be used for detecting, sample such as need long preservation need separated plasma or serum to put below-20 ℃ and preserve.
3. instrument
37 ℃ of constant water bath box of enzyme connection instrument micro sample adding appliance
4. operation steps
(1) in the recent period used kit can be carried and puts 2～8 ℃ the previous day and thaw or the room temperature quick-thawing.Take out used enzyme bracing before detecting, open and remove and be pressed onto on the enzyme connection frame, after film is sealed in standard article the 7th hole in order to detecting.Getting washing lotion 6ml adds in the reactant liquor bottle (red lid) and abundant mixing (being sure not freeze thawing again, with rearmounted 2～8 ℃ of preservations).
Attention: place 2～8 ℃ of preservations after 1. kit thaws, use up in 2～3 weeks, avoid multigelation;
2. do not seal off as the enzyme bracing of this detection;
3. reagent is still put 2～8 ℃ of preservations after using.
(2) standard bar 1～6 hole adds standard dilution 100 μ l.
Standard article the 7th hole and sample strip the 2nd～7 detect the hole and add testing sample blood plasma or serum 100 μ l.
(3) add reactant liquor 50 μ l in every hole, vibrated 10～30 seconds, put 37 ℃ of water-baths 20 minutes (the enzyme bracing bottom contact water surface or part immerse in the water).Attention: (2) (3) the step application of sample time interval＜3 minute.
(4) liquid in the hole that inclines, washing lotion is washed 5 times, and it is 250～300 μ l that each every hole adds the washing lotion amount the suitableeest, more as the washing lotion consumption, uses (not recommending to use, because of various places water quality can not guarantee) after can diluting 1 times.
(5) get required colour developing liquid A and colour developing liquid B and abundant mixing with 1: 20 volume ratio, every hole adds 150 μ l, 37 ℃ of water-baths 15 minutes.
(6) every hole adds stop buffer 50 μ l or instils one, is blank with standard bar first hole, and the even 10 minutes endoenzyme connection instrument 450nm that shake gently read the OD value.
5. result of calculation
Criterion calculation: be respectively X-axis and Y-axis according to gauge orifice 2～6 hole OD values and corresponding cTnI concentration (3.2,1.6,0.8,0.4,0.2ng/ml), calculate linear regression equation Y=aX ± b, Y is a cTnI concentration, X is the OD value), formula substitution sample OD value thus, calculate the cTnI concentration (ng/ml) of sample, the person's of having ready conditions available computers or the robotization of enzyme connection instrument are handled.
Shortcut Calculation: select the close person of testing sample directly relatively to calculate with standard point OD
Sample cTnI concentration (ng/ml)=(standard point concentration * testing sample OD value)/standard point OD value.
6. the result judges
200 routine healthy people measure statistical study and show, normal reference value＜0.15ng/ml, and 〉=0.15ng/ml prompting cTnI raises, and 0.15～0.5ng/ml points out slight myocardial damage, 〉=0.5ng/ml prompting acute myocardial infarction AMI or serious myocardial damage.＜0.15ng/ml is normal.Sensing range 0.05～4.0ng/ml, minimum detected value is 0.05ng/ml, as result＞4.0ng/ml, needs with detecting after the negative serum dilution suitable multiple.
Claims (3)
1, a kind of is the method that quantitative determination cardiac muscle troponin I content is set up on the basis with the anti-human body cardiac muscle troponin I of three strains monoclonal antibody, the monoclonal antibody that it is characterized in that acting on two strains cardiac muscle troponin I is a capture antibody, the monoclonal antibody that acts on cardiac muscle troponin I with another strain is a labelled antibody, marriage chain Avidin-biotin amplification system, utilization enzyme linked immunological principle is set up the method that the cardiac muscle troponin I fast quantification detects.
2, according to the described method of claim 1, two strain capture antibodies abbreviate Ab1 and Ab2 respectively as, behind connection biotin or the long-chain biotin, abbreviate Ab1-Bio and Ab2-Bio respectively as; Another strain labelled antibody abbreviates Ab3 as, behind the connection horseradish peroxidase, abbreviates Ab3-POD as, behind connection biotin or the long-chain biotin, abbreviates Ab3-Bio as; Streptavidin or Avidin abbreviate S as; The streptavidin horseradish peroxidase is called for short S-POD; Cardiac muscle troponin I antigen comprises the cardiac muscle troponin I in known cardiac muscle troponin I standard items, quality-control product and the testing sample, abbreviates cTnI as; The reaction path of three anti-method quantitative determination cardiac muscle troponin I methods specifically comprises following several scheme:
Scheme one:
(1) 96 hole elisa plate, enzyme bracing or other solid holder have connect S, albumin or the sealing of other confining liquid;
(2) Ab1-Bio and Ab2-Bio combine with S, and cTnI combines with Ab1-Bio and Ab2-Bio, and cTnI combines with Ab3-POD, form
Reaction chain;
(3) POD makes the chromogenic substrate colour developing or makes chemical luminous substrate luminous, measures by corresponding instrument again, calculates testing sample cardiac muscle troponin I content according to typical curve;
Scheme two:
(1) 96 hole elisa plate, enzyme bracing or other solid holder have connect Ab1 and Ab2, albumin or the sealing of other confining liquid;
(2) Ab1 and Ab2 combine with cTnI, and cTnI combines with Ab3-POD, form
Reaction chain;
(3) POD makes the chromogenic substrate colour developing or makes chemical luminous substrate luminous, measures by corresponding instrument again, calculates testing sample cardiac muscle troponin I content according to typical curve;
Scheme three:
(1) 96 hole elisa plate, enzyme bracing or other solid holder have connect Ab1 and Ab2, albumin or the sealing of other confining liquid;
(2) Ab1+Ab2 combines with cTnI, and cTnI combines with Ab3-Bio, and Ab3-Bio combines with S-POD, forms
Reaction chain;
(3) POD makes the chromogenic substrate colour developing or makes chemical luminous substrate luminous, measures by corresponding instrument again, calculates testing sample cardiac muscle troponin I content according to typical curve.
3, method according to claim 2 is characterized in that chromogenic substrate is tetramethyl diamine TMB.
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| CN103353531B (en) * | 2006-04-04 | 2019-03-08 | 神谷来克斯公司 | Highly Sensitive System and method for analysis of troponin |
| US7838250B1 (en) | 2006-04-04 | 2010-11-23 | Singulex, Inc. | Highly sensitive system and methods for analysis of troponin |
| CA3086149A1 (en) | 2006-04-04 | 2007-10-11 | Singulex, Inc. | Highly sensitive system and methods for analysis of troponin |
| CN101105497B (en) * | 2006-07-13 | 2011-10-19 | 上海凯创生物技术有限公司 | Cardiac muscle troponin I detection reagent kit and its preparation method |
| CN103940986B (en) * | 2014-03-24 | 2015-10-21 | 安徽省煦棠医疗科技有限公司 | The preparation of Troponin I specific site antibody and detection kit thereof |
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| CN110161250B (en) * | 2018-02-11 | 2023-07-11 | 科美博阳诊断技术(上海)有限公司 | Homogeneous phase human cardiac troponin I rapid detection kit, system, detection method and application |
| CN113125742B (en) * | 2019-12-30 | 2022-12-27 | 广东唯实生物技术有限公司 | Detection method and kit for hypersensitive cardiac troponin I |
| CN115963274A (en) * | 2022-09-30 | 2023-04-14 | 中国科学院长春应用化学研究所 | Detection reagent and method for cardiac troponin I |
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