CN101101294B - Colour-developing agent preparation for enzyme-linked immunoassay in vitro diagnosis agent - Google Patents
Colour-developing agent preparation for enzyme-linked immunoassay in vitro diagnosis agent Download PDFInfo
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- CN101101294B CN101101294B CN200610028624XA CN200610028624A CN101101294B CN 101101294 B CN101101294 B CN 101101294B CN 200610028624X A CN200610028624X A CN 200610028624XA CN 200610028624 A CN200610028624 A CN 200610028624A CN 101101294 B CN101101294 B CN 101101294B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 238000003745 diagnosis Methods 0.000 title claims description 9
- 238000002965 ELISA Methods 0.000 title description 5
- 238000000338 in vitro Methods 0.000 title 1
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 11
- 230000035945 sensitivity Effects 0.000 claims abstract description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 9
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 7
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 claims description 7
- 230000001900 immune effect Effects 0.000 claims description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 6
- FCPVYOBCFFNJFS-LQDWTQKMSA-M benzylpenicillin sodium Chemical compound [Na+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 FCPVYOBCFFNJFS-LQDWTQKMSA-M 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 3
- 229960000723 ampicillin Drugs 0.000 claims description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 229940056360 penicillin g Drugs 0.000 claims description 3
- WXKPPMQZRGORPB-UHFFFAOYSA-M sodium;2-hydroxypropane-1,2,3-tricarboxylic acid;acetate Chemical compound [Na+].CC([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O WXKPPMQZRGORPB-UHFFFAOYSA-M 0.000 claims description 3
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 2
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 2
- 238000006911 enzymatic reaction Methods 0.000 claims description 2
- 239000007974 sodium acetate buffer Substances 0.000 claims description 2
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 2
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 claims 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 239000003960 organic solvent Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- IYNDLOXRXUOGIU-LQDWTQKMSA-M benzylpenicillin potassium Chemical compound [K+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 IYNDLOXRXUOGIU-LQDWTQKMSA-M 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- CMZYGFLOKOQMKF-UHFFFAOYSA-N 1-(3,5-dimethylphenyl)-3,5-dimethylbenzene Chemical group CC1=CC(C)=CC(C=2C=C(C)C=C(C)C=2)=C1 CMZYGFLOKOQMKF-UHFFFAOYSA-N 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides the preparation method for developer in the outside diagnose reagent of enzyme immune body. The method of the invention change the form of developer in the outside diagnose reagent of enzyme immune body. It makes the whole process of confect predigest, improves the sensitivity of showing color, increases the stability of developer, and debases the background of showing color. It has great appliance vale for clinic.
Description
Technical field:
The invention belongs to biological external diagnosis reagent technical field.Be specifically related to the preparation of developer in a kind of enzyme linked immunological external diagnosis reagent.
Background technology:
Enzyme-linked immune detection method (ELISA) since have quick, responsive, easy, be easy to advantage such as standardization, be widely used in the detection of antigen, antibody, cell factor and biochemical substances etc.The detection method of ELISA reagent has a variety of, but the whole bag of tricks all be unable to do without enzyme conjugates and developer.Most widely used enzyme is horseradish peroxidase (HRP) in ELISA reagent, and the substrate of HRP is a superoxide, and as hydrogen peroxide, and that the developer of this system has is multiple, and at present the most frequently used is 3,3 ', 5,5 '-tetramethyl benzidine (TMB).With respect to other developers, TMB has many advantages, as not carcinogenicity, good stability etc.During detection, HRP catalysis superoxide discharges oxygen, and colourless TMB is oxidized to blue product, and after the stop buffer cessation reaction of adding acidity, blue solution becomes orange-yellow, at the 450nm wavelength maximum absorption band is arranged.By measuring absorbance, just can qualitative or quantitatively judge the content of detected material.
Use TMB as developer, its preparation process more complicated.Common preparation method is that elder generation is dissolved in TMB in the organic solvent (as dimethyl sulfoxide (DMSO), dimethyl formamide etc.), heats then it is fully dissolved, and is mixed with concentrate, slowly joins in the damping fluid for preparing while stirring again.Whole process of preparation is wanted lucifuge, can not have precipitation to produce, and operates more loaded down with trivial details.
Because TMB is water insoluble, promptly uses the organic solvent hydrotropy, the solubleness in water is not high yet, and the concentration of preparation can not be too high, produces otherwise have precipitation, and therefore the developer sensitivity of preparation is not high.
Except sensitivity, the colour developing background also is one of an ELISA reagent very important index, and higher colour developing background not only makes experimental result be difficult to judge, and the testing result that makes the mistake easily.Low colour developing background is the basic demand of high-quality reagent.And directly often background is higher with the developer of commercially available TMB preparation.
Summary of the invention:
Technical matters to be solved by this invention is to overcome above-mentioned weak point, and research improves the preparation of developer in the enzyme linked immunological external diagnosis reagent.
The present invention simplifies the process for preparation of developer by the prescription that changes the developer in the enzyme linked immunological external diagnosis reagent and improves its sensitivity and improve the colour developing background.
The present invention changes the composition of the developer in the enzyme linked immunological external diagnosis reagent, uses 3,3 ', 5,5 '-tetramethyl biphenyl amine hydrochlorate (3,3 ', 5,5 '-Tetramethylbenzidlinedihydrochloride, the TMB hydrochloride) replaces 3,3 ', 5,5 '-tetramethyl benzidine (TMB) is simplified whole process for preparation.The TMB hydrochloride can directly be dissolved in the aqueous solution, and does not need hydrotropy with an organic solvent.
The solubleness of TMB hydrochloride in water can reach 1%, is easy to just can be mixed with the higher solution of concentration according to the reagent needs, obtains higher sensitivity.And TMB even with an organic solvent the solubleness of hydrotropy in water is also lower, usually below 0.1%, in order to obtain high slightly solubleness, need to increase consumption of organic solvent, consumption of organic solvent strengthens has conversely influenced developing sensitivity again.
The damping fluid that the present invention uses is 0.01-0.1M Tris-HCl damping fluid, PH is 2.0～5.0, or other buffer systems, as 0.01-0.1M citric acid-sodium acetate, PH is 2.0～5.0, in this buffer system,, can increase the stability of developer, the storage life of developer is prolonged by adding the EDTA disodium salt.
The present invention has added Benzylpenicillin sodium salt (or benzylpenicillin potassium or ampicillin) and azanol and has reduced the colour developing background and increase its stability simultaneously in developer.
The invention provides the preparation method of developer in a kind of enzyme linked immunological external diagnosis reagent.
Developer preparation of the present invention comprises the following steps:
(1) preparation 0.01-0.1M Tris-HCl or citric acid-sodium acetate buffer, the pH value is 2.0～5.0;
(2) add disodium EDTA in this buffer system, final concentration is 0.06g/L～0.32g/L, stirs, and makes abundant dissolving;
(3) add Benzylpenicillin sodium salt or benzylpenicillin potassium or ampicillin, final concentration is 0.02g/L～0.1g/L, stirs, and makes abundant dissolving;
(4) add azanol, final concentration is 0.01ml/L～0.06ml/L, stirs, and makes abundant dissolving;
(5) add the TMB hydrochloride, final concentration is 0.3g/L～2.4g/L, and the concrete concentration during use needs sensitivity to decide according to reagent place, stirs, and makes abundant dissolving.
Developer of the present invention is applicable to the superoxide to be in the Color Appearance System of enzymatic reaction substrate.Wherein said superoxide is hydrogen peroxide or urea peroxide, is that the enzyme of substrate is horseradish peroxidase (HRP) with this type of material.
Beneficial effect of the present invention:
(1) uses TMB hydrochloride, developing sensitivity height.
(2) the colour developing background is low.
(3) process for preparation is simple, has removed the process of organic solvent dissolution in the classic method and preparation concentrate, need not heat and dissolve and dilute.
(4) under the storage temperature (2～8 ℃) of kit, stability increases.
Developer of the present invention and traditional developer comparing data
Detecting HBsAg with the double-antibody sandwich elisa method is example, the antigen that in the microwell plate of coated antibody, adds serial dilution, test according to the popular response process, contrast traditional developer and developer of the present invention respectively, after 2M sulfuric acid solution cessation reaction, colorimetric on microplate reader, the result is as follows:
Embodiment 1:
Preparation 1L developer, TMB hydrochloride concentration is 0.04%:
1, in volumetric flask, adds about 500ml distilled water, add following material more successively, add a kind of Tris2.42g in back before treating after a kind of dissolving again; HCl (36%-38%) 2.4ml; EDTA two receives salt 0.186g; Benzylpenicillin sodium salt 0.05g; Azanol 0.03ml; TMB hydrochloride 0.4g.Supply distilled water to 1L after waiting to dissolve.
2, adjusting the pH value with HCl is 2.5.
Embodiment 2:
Preparation 1L developer, TMB hydrochloride concentration is 0.06%:
1, in volumetric flask, adds about 500ml distilled water, add following material more successively, add a kind of Tris2.42g in back before treating after a kind of dissolving again; HCl (36%-38%) 2.4ml; EDTA two receives salt 0.186g; Benzylpenicillin sodium salt 0.05g; Azanol 0.03ml; TMB hydrochloride 0.6g.Supply distilled water to 1L after waiting to dissolve.
2, adjusting the pH value with HCl is 2.5.
Embodiment 3:
With concentration is that 0.06%TMB hydrochloride solution is developer, detects the HBsAg in serum or the plasma specimen.
(1) in wrapping, adds sample 50ul successively by the microwell plate of anti-HBs, do two hole negative controls, a hole positive control, a hole blank simultaneously, every then hole (except that the blank hole) adds anti-HBs-HRP enzyme conjugates solution 50ul, puts 37 ℃ of insulations 30 minutes.
(2) discard liquid in the hole, wash plate 5 times with cleansing solution.
(3) each hole adds substrate solution 50ul, and the developer 50ul of the embodiment of the invention 2 preparations puts 37 ℃ of insulations 15 minutes.
(4) each hole adds stop buffer 50ul, cessation reaction.
(5) reaction plate is put enzyme mark tintmeter 450nm wavelength place with blank well school zero, read each hole OD value.
(6) calculate, the average OD value of Cut Off value=negative control * 2.1, the average OD value of negative control is pressed 0.05 less than 0.05 and is calculated, and calculates by actual OD value more than or equal to 0.05.
(7) result judges: sample OD value is positive more than or equal to Cut Off value, and is negative less than the CutOff value.
Claims (2)
1. the preparation method of developer in the enzyme linked immunological external diagnosis reagent is characterized in that this method comprises the following steps:
(1) preparation 0.01-0.1M Tris-HCl or citric acid-sodium acetate buffer, the pH value is 2.0～5.0;
(2) add disodium EDTA in this buffer system, final concentration is 0.06g/L～0.32g/L, stirs, and makes abundant dissolving;
(3) add Benzylpenicillin sodium salt or benzylpenicillin potassium or ampicillin, final concentration is 0.02g/L～0.1g/L, stirs, and makes abundant dissolving;
(4) add oxammonium hydrochloride, final concentration is 0.4-0.6g/L, stirs, and makes abundant dissolving;
(5) add the TMB hydrochloride, final concentration is 0.3g/L～2.4g/L, and the concrete concentration during use needs sensitivity to decide according to reagent place, stirs, and makes abundant dissolving;
The developer that makes is applicable to the superoxide to be in the Color Appearance System of enzymatic reaction substrate.
2. superoxide according to claim 1 is hydrogen peroxide or urea peroxide, is that the enzyme of substrate is a horseradish peroxidase with described superoxide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610028624XA CN101101294B (en) | 2006-07-05 | 2006-07-05 | Colour-developing agent preparation for enzyme-linked immunoassay in vitro diagnosis agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610028624XA CN101101294B (en) | 2006-07-05 | 2006-07-05 | Colour-developing agent preparation for enzyme-linked immunoassay in vitro diagnosis agent |
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| Publication Number | Publication Date |
|---|---|
| CN101101294A CN101101294A (en) | 2008-01-09 |
| CN101101294B true CN101101294B (en) | 2011-07-13 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN200610028624XA Expired - Fee Related CN101101294B (en) | 2006-07-05 | 2006-07-05 | Colour-developing agent preparation for enzyme-linked immunoassay in vitro diagnosis agent |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103063661B (en) * | 2012-12-21 | 2015-09-09 | 杭州茂天赛科技有限公司 | A kind of TMB nitrite ion and preparation method thereof |
| CN103884833B (en) * | 2014-02-17 | 2015-07-08 | 江苏硕世生物科技有限公司 | ELISA enzyme-free color developing agent for increasing color development duration time and preparation method and application thereof |
| CN103913566B (en) * | 2014-04-11 | 2016-01-06 | 苏州浩欧博生物医药有限公司 | A kind of enzyme linked immunological chromogenic substrate and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0411945A2 (en) * | 1989-08-04 | 1991-02-06 | BEHRINGWERKE Aktiengesellschaft | Heterogeneous immunoassays |
| EP1312923A2 (en) * | 2001-11-16 | 2003-05-21 | Randox Laboratories Ltd. | Method and kit for detecting, or determining the quantity of, metabolites of fentanyl and metabolites of fentanyl analogs |
| CN1766628A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting diazepam residue and detection method thereof |
-
2006
- 2006-07-05 CN CN200610028624XA patent/CN101101294B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0411945A2 (en) * | 1989-08-04 | 1991-02-06 | BEHRINGWERKE Aktiengesellschaft | Heterogeneous immunoassays |
| EP1312923A2 (en) * | 2001-11-16 | 2003-05-21 | Randox Laboratories Ltd. | Method and kit for detecting, or determining the quantity of, metabolites of fentanyl and metabolites of fentanyl analogs |
| CN1766628A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting diazepam residue and detection method thereof |
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| CN101101294A (en) | 2008-01-09 |
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