CN105044327B - A kind of stop buffer of one-component TMB nitrite ion and preparation method thereof - Google Patents
A kind of stop buffer of one-component TMB nitrite ion and preparation method thereof Download PDFInfo
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- CN105044327B CN105044327B CN201510379958.0A CN201510379958A CN105044327B CN 105044327 B CN105044327 B CN 105044327B CN 201510379958 A CN201510379958 A CN 201510379958A CN 105044327 B CN105044327 B CN 105044327B
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- stop buffer
- sulphuric acid
- nitrite ion
- component tmb
- diethylene glycol
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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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Abstract
The invention provides the stop buffer of a kind of one-component TMB nitrite ion, including sulphuric acid and diethylene glycol；The concentration of described sulphuric acid is 0.3 0.5mol/L, and the w/v of described diethylene glycol is 3 5%.The present invention has the advantage that the stop buffer of one-component TMB nitrite ion of the present invention is specific to the design of one-component tmb substrate liquid compared to existing technology, does not produce precipitation after termination, and in 1 hour, reading is still stable；The stop buffer of one-component TMB nitrite ion of the present invention uses the sulphuric acid of low concentration, it is possible to reduce the injury to operator, and can reduce the harm to environment；The stop buffer of one-component TMB nitrite ion of the present invention has anti-freeze design, and the peculiar anti-freeze components contained by it is applied in combination with certain proportion with the sulphuric acid of low concentration, and effect is efficient, and low temperature environment uses and do not freezes in the winter time, more stable and reliable.
Description
Technical field
The present invention relates in vitro diagnostic reagent, particularly relate to stop buffer of a kind of one-component TMB nitrite ion and preparation method thereof.
Background technology
In euzymelinked immunosorbent assay (ELISA) (ELISA) detection technique in external diagnosis reagent field, often use TMB (3,3', 5,5 '-tetramethyl benzidine) as HRP (horseradish peroxidase) chromogenic substrate.Relatively for other chromogenic substrate, TMB becomes the main flow developer of ELISA detection with the features such as efficient, nontoxic, the most carcinogenic, good stability that develop the color.When ELISA detects, HRP enzyme catalysis peroxide discharges oxygen makes TMB be oxidized to the product of blueness, and after adding acid stop buffer termination reaction, blue solution becomes orange-yellow, has maximum absorption band at 450nm wavelength.The principle terminated: as a example by the sulphuric acid stop buffer as main component, after adding stop buffer, on the one hand, sulphuric acid destroys the activity of HRP enzyme, makes the catalysis of enzyme lose；On the other hand, owing to pH reduces, the cation root of blueness can be made to be changed into the diphenoquinone of yellow, diphenoquinone has maximum extinction coefficient at wavelength 450nm.As terminated reaction not in time, background can be deepened simultaneously, and then affects the correct judgement of result, therefore terminates significant to ELISA.
At present in ELISA kit, the stop buffer of TMB mainly has bronsted lowry acids and bases bronsted lowry two class: acids is mainly hydrochloric acid and sulphuric acid, and bases is then NaOH and SDS (sodium lauryl sulphate).NaOH is rotten owing to the carbon dioxide reaction easily and in air produces, and less application；As for 1%SDS (sodium lauryl sulphate) though there being paper to carry out research, but there are stronger decolourization, unstable result after terminating due to it, the most only staying in laboratory and using.For the hydrochloric acid in acids, concentration mostly is 1% or 2M (i.e. mol/l, lower same), terminate effect still can, but owing to hydrochloric acid is volatile, repeatedly uncap and solute can be made to reduce, and then affect the termination effect of stop buffer and detect precision, therefore Commercialization application is few.
Sulphuric acid is as the main component of stop buffer, owing to advantages such as stablizing, easily obtain, be cheap is increasingly becoming main flow.Its sulfuric acid concentration prepared is different, including up to 2M, minimum 0.2M.And the concrete dosage of stop buffer: after general double-component tmb substrate liquid respectively adds A Yu B liquid 50 μ l, then add the stop buffer 50 μ l of 2M；And for one-component tmb substrate liquid, then the lower concentration sulphuric acid of many employing 0.2-0.8M is as stop buffer, and identical with TMB dosage, the tmb substrate liquid of such as 100 μ l, add the stop buffer of 100 μ l.
For the sulphuric acid stop buffer of traditional 2mol/l, it is used for double-component TMB, less is applied in one-component TMB.Reason has: low compared with in double-component of the TMB content in (1) one-component TMB, therefore the sulphuric acid being not required to up to 2M terminates, and the sulphuric acid of excess can make background deepen on the contrary, causes yin and yang attribute contrast poor, final reading instability.(2) sulphuric acid of 2M has stronger corrosivity, and operator is easily caused injury.(3) use remaining stop buffer that environment is created harm.Therefore more of use now the sulphuric acid of low concentration as stop buffer, such as 0.2-0.8M.
Sulphuric acid based on low concentration i.e. dilute sulfuric acid can stably preserve under normal temperature condition, therefore it can directly be stored in room temperature, time in the winter time, the particularly province beyond Guangdong, Hainan and Taiwan Province, winter is many below 0 DEG C when laboratory preserves or transports, and easily makes the stop buffer of room temperature preservation produce and freezes；Additionally, at the refrigerator cold-storage layer of temperature control shakiness, stop buffer is likely to be occurred freezing.The stop buffer freezed has the disadvantage that: thaws and needs the consuming time；As thawing the most completely, uneven meeting cause the concentration of sulphuric acid to produce change, impact experiment precision.
One-component TMB nitrite ion becomes commercial ELISA kit main flow increasingly, seldom has the stop buffer being specifically designed for one-component TMB nitrite ion.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of stop buffer being specifically designed for one-component TMB nitrite ion, its effect efficiently and can be stable be stored in four seasons room temperature.
The invention provides the stop buffer of a kind of one-component TMB nitrite ion, including sulphuric acid and diethylene glycol.
The concentration of described sulphuric acid is 0.3-0.5mol/L, and the w/v of described diethylene glycol is 3-5%.
The concentration of described sulphuric acid is 0.4mol/L, and the w/v of described diethylene glycol is 3%.
Present invention also offers a kind of containing sulphuric acid with the purposes of the compositions of diethylene glycol, for the preparation of the stop buffer of one-component TMB nitrite ion.
The concentration of sulphuric acid described in compositions is 0.3-0.5mol/L, and the w/v of described diethylene glycol is 3-5%.
The concentration of sulphuric acid described in compositions is 0.4mol/L, and the w/v of described diethylene glycol is 3%.
The present invention has the advantage that compared to existing technology
(1) stop buffer of one-component TMB nitrite ion of the present invention is specific to the design of one-component tmb substrate liquid, does not produce precipitation after termination, and in 1 hour, reading is still stable.
(2) stop buffer of one-component TMB nitrite ion of the present invention uses the sulphuric acid of low concentration, it is possible to reduce the injury to operator, and can reduce the harm to environment.
(3) stop buffer of one-component TMB nitrite ion of the present invention has anti-freeze design, the peculiar anti-freeze components contained by it is applied in combination with certain proportion with the sulphuric acid of low concentration, effect is efficient, and low temperature environment uses and do not freezes in the winter time, more stable and reliable.
Detailed description of the invention
The invention provides the stop buffer of a kind of one-component TMB nitrite ion, including each component of following concentration:
Sulphuric acid 0.3-0.5mol/L
Diethylene glycol 3-5% (w/v)
The concentration of described sulphuric acid be 0.3M and more than, it can be ensured that enough sulphuric acid is used for terminating, and the most also maintains a stronger sour environment, the growth of suppression microorganism simultaneously.And the concentration of sulphuric acid is when being more than 0.5M, the stability after termination is inadequate, easily causes the change of reading.During such as high level sample, partly precipitated may be brought, therefore select 0.3-0.5M.
Described diethylene glycol, also referred to as diglycol, diethylene glycol ether or diethylene glycol.Can be miscible with ethanol, ether, acetone and ethylene glycol, insoluble in benzene and carbon tetrachloride, it is dissolved in water.Acrid in the mouth is peppery and micro-sweet, has hygroscopicity.Owing to part antifreeze composition easily causes the interference terminating reacting.Therefore needing prudent especially in the selection in the type and concentration thereof of antifreeze, time as big in material amounts such as glycerol, viscosity increases, and is unfavorable for terminating efficiently carrying out of reaction.The termination of ELISA then will not be interfered by diethylene glycol, has the cryoprotective effects suitable with glycerol, can meet the freeze proof situation of common nearly zero degrees celsius, such as-5 DEG C to 0 DEG C；Its safety is preferable simultaneously；Its concentration is chosen as during 3-5% (w/v) being more beneficial for terminating efficiently carrying out of reaction.
Present invention also offers the preparation method of the stop buffer of above-mentioned one-component TMB nitrite ion, comprise the steps:
Take the sulphuric acid of 16.0-26.8ml (i.e. 0.3-0.5mol), add the deionized water of 800ml, add the diethylene glycol of 26.9-44.8ml (i.e. 30-50g) the most wherein, it is settled to 1L with deionized water, mix homogeneously, it is contained in plastics reagent bottle sealing and is stored in room temperature, be the stop buffer of one-component TMB nitrite ion.
Embodiment 1
Present embodiments provide the stop buffer of a kind of one-component TMB nitrite ion, including each component of following concentration:
Sulphuric acid 0.4mol/L
Diethylene glycol 3% (w/v)
The present embodiment additionally provides the preparation method of the stop buffer of above-mentioned one-component TMB nitrite ion, comprises the steps:
Take the sulphuric acid of 21.4ml (i.e. 0.4mol), add the deionized water of 800ml, add the diethylene glycol of 26.9ml (i.e. 30g) the most wherein, it is settled to 1L with deionized water, mix homogeneously, it is contained in plastics reagent bottle sealing and is stored in room temperature, be the stop buffer of one-component TMB nitrite ion.
Wherein concentrated acid and diethylene glycol are domestic analytical reagent, and cost is relatively low.
The stop buffer of the present embodiment one-component TMB nitrite ion can stably preserve more than 1 year at ambient temperature.
Embodiment 2
Present embodiments provide the stop buffer of a kind of one-component TMB nitrite ion, including each component of following concentration:
Sulphuric acid 0.3mol/L
Diethylene glycol 3% (w/v)
The present embodiment additionally provides the preparation method of the stop buffer of above-mentioned one-component TMB nitrite ion, comprises the steps:
Take the sulphuric acid of 16.1ml (i.e. 0.3mol), add the deionized water of 800ml, add the diethylene glycol of 26.9ml (i.e. 30g) the most wherein, it is settled to 1L with deionized water, mix homogeneously, it is contained in plastics reagent bottle sealing and is stored in room temperature, be the stop buffer of one-component TMB nitrite ion.
Other is all with embodiment 1.
Embodiment 3
Present embodiments provide the stop buffer of a kind of one-component TMB nitrite ion, including each component of following concentration:
Sulphuric acid 0.5mol/L
Diethylene glycol 5% (w/v)
The present embodiment additionally provides the preparation method of the stop buffer of above-mentioned one-component TMB nitrite ion, comprises the steps:
Take the sulphuric acid of 26.8ml (i.e. 0.5mol), add the deionized water of 800ml, add the diethylene glycol of 55.8ml (i.e. 50g) the most wherein, it is settled to 1L with deionized water, mix homogeneously, it is contained in plastics reagent bottle sealing and is stored in room temperature, be the stop buffer of one-component TMB nitrite ion.
Other is all with embodiment 1.
Embodiment 4
Present embodiments provide the stop buffer of a kind of one-component TMB nitrite ion, including each component of following concentration:
Sulphuric acid 0.4mol/L
Diethylene glycol 5% (w/v)
The present embodiment additionally provides the preparation method of the stop buffer of above-mentioned one-component TMB nitrite ion, comprises the steps:
Take the sulphuric acid of 21.4ml (i.e. 0.4mol), add the deionized water of 800ml, add the diethylene glycol of 55.8ml (i.e. 50g) the most wherein, it is settled to 1L with deionized water, mix homogeneously, it is contained in plastics reagent bottle sealing and is stored in room temperature, be the stop buffer of one-component TMB nitrite ion.
Other is all with embodiment 1.
Embodiment 5
Present embodiments provide stop buffer in euzymelinked immunosorbent assay (ELISA) test kit with the application example being used in combination of one-component TMB, its detection method comprises the steps:
The cytomegalovirus IgG antibody (following component unless otherwise indicated, is all the composition of this test kit) in serum or plasma specimen is detected with " cytomegalovirus IgG antibody detection kit (euzymelinked immunosorbent assay (ELISA)) " of DIA.PRO company:
(1) in the microwell plate being coated CMV antigen, sample 100u1 is added, sample is respectively negative control (CAL10IU/mL), positive control (CAL68IU/mL), critical comparison (CAL20.5IU/mL), and high level sample L1, intermediate value sample L2, low value sample L3 (are the lowest detectable limit reference material in " cytomegalovirus IgG antibody reference material ", derive from National Institute for Food and Drugs Control, standard substance numbered 360004), respectively do 2 multiple holes, additionally set blank 2 hole, it is not added with sample, puts 37 DEG C of insulation 30min.
(2) discard liquid in hole, wash plate 4 times with work cleaning mixture.Adding anti-human igg-HRP enzyme conjugates solution 100u1, wherein blank control wells is not added with anti-human igg-HRP enzyme conjugates solution.Put 37 DEG C of insulation 30min.
(3) discard liquid in hole, use work cleaning mixture, wash plate 4 times.
(4) the one-component chromogenic substrate 100u1 during each hole adds test kit, puts 37 DEG C of insulation 15min.
(5) each hole is separately added into 0.3M, and the sulphuric acid of 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, as the stop buffer in stop buffer, and embodiment 1-4, each 100u1, terminates reaction.
(6) Sptting plate is put in microplate reader, with blank well school zero at 450nm wavelength, read each hole OD value.After terminating, and respectively reading a number in 30min, 45min and 60min, result is as shown in Table 1 and Table 2.
(7) result judges: be wherein critical control value (Cut Off value) with the average of CAL2.Sample readings is higher than critical control value, then be positive；Sample readings is lower than critical control value, then be negative.
Table 1
During from table 1 it follows that the concentration of stop buffer sulphuric acid is 0.4M, terminating completely, product can stably maintain 60min, for high, in and low value pattern detection stable reading.
Table 2
From Table 2, it can be seen that add diethylene glycol not interfere with detection reading；Meanwhile, after 0.4M sulphuric acid adds 3% (w/v) diethylene glycol group termination, reading is the most stable, for optimal choice.
Embodiment 6
Present embodiments provide the one-component TMB nitrite ion of embodiment 1 stop buffer freeze test.
Method of testing: by the stop buffer of the one-component TMB nitrite ion in embodiment 1, be respectively placed in-5 DEG C ,-8 DEG C and-10 DEG C each 12 hours, sees whether to freeze.
Test is shown in Table 3, within 12 hours, does not freezes-5 DEG C and-8 DEG C holdings, keeps 12 hours then partly freezing at-10 DEG C.
Table 3
| Storage condition/time | -10 DEG C/12 hours | -8 DEG C/12 hours | -5 DEG C/12 hours |
| Freeze situation | Part is freezed | Do not freeze | Do not freeze |
Claims (4)
1. the stop buffer of an one-component TMB nitrite ion, it is characterised in that include sulphuric acid and diethylene glycol；
The concentration of described sulphuric acid is 0.3-0.5mol/L, and the w/v of described diethylene glycol is 3-5%.
The stop buffer of one-component TMB nitrite ion the most according to claim 1, it is characterised in that described
The concentration of sulphuric acid is 0.4mol/L, and the w/v of described diethylene glycol is 3%.
3. the purposes of the compositions containing sulphuric acid and diethylene glycol, it is characterised in that for one-component
The preparation of the stop buffer of TMB nitrite ion, the concentration of described sulphuric acid is 0.3-0.5mol/L, described diethyl two
The w/v of alcohol is 3-5%.
The most according to claim 3 containing sulphuric acid with the purposes of the compositions of diethylene glycol, its feature exists
In, the concentration of described sulphuric acid is 0.4mol/L, and the w/v of described diethylene glycol is 3%.
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| EP0125893A3 (en) * | 1983-05-12 | 1986-10-15 | Sumitomo Chemical Company, Limited | The quantitative analysis of antigen by the enzyme-antibody bridge method |
| CN1381328A (en) * | 2001-04-18 | 2002-11-27 | 中国科学院化工冶金研究所 | Process for preparing superfine (nano) metal powder by hydrogen reduction of polyol |
| CN102353777B (en) * | 2011-07-05 | 2014-07-02 | 深圳市卫光生物制品股份有限公司 | Kit and method for testing human cytomegalovirus IgG antibody |
| CN104459110B (en) * | 2014-11-25 | 2016-01-13 | 成都威尔诺生物科技有限公司 | A kind of one-component TMB nitrite ion |
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