CN1379245A - Process for selectively staining water-soluble protein with reactive stain - Google Patents
Process for selectively staining water-soluble protein with reactive stain Download PDFInfo
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- CN1379245A CN1379245A CN 01110355 CN01110355A CN1379245A CN 1379245 A CN1379245 A CN 1379245A CN 01110355 CN01110355 CN 01110355 CN 01110355 A CN01110355 A CN 01110355A CN 1379245 A CN1379245 A CN 1379245A
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Abstract
A process for selectively dyeing water-soluble protein with reactive dye used in textile industry features that the dye molecules are respectively bonded to the hydroxy, amino, or thiol radicals of amino acid molecules in protein. For antibody, the dyeing is controlled in acidic environment. For immunoanalysis, the protein is dyed in slightly alkaline solution for dark color. The dyed protein has high stability.
Description
The present invention relates to a kind of method of protein staining, and be particularly related to a kind of chemically-reactive dyes process for selectively staining water-soluble protein that utilizes.
Various immunoassays are used more and more widely in clinical diagnosis, food QC, prevention and cure of pollution and each field of scientific research.But no matter that type immunoassay all must indicate protein, as the metering purposes in the analytic process.As measured object Z is antigen A g, or antibody A b, then can be according to the price of antigen, antibody and the factors such as complexity that obtain, and just like the give an example analysis logic design of (1) and (2) of Fig. 1.At this moment, Ab1 and Ab2 are two kinds of antibody of different nature of anti-measured object Z, and L promptly is the so-called part (marker ligand) that indicates.When L was radioelement, this method was called as radio immunoassay; When L is enzyme, be called enzyme immunoassay (EIA); When L is fluorescent thing or chemical cold light thing, be called fluorescent immunoassay and cold light immunoassay.In a word, along with the difference of L, immunoassay has different metering methods, and hat has different titles.More than these four kinds of methods are main flows of practical immunoassay of present stage.
Since the fifties radio immunoassay practicability, human society has been had major contribution, sensitivity is cheap again, but operator's safety concerns and offal treatment have limited its development.The enzyme immunoassay (EIA) of the seventies invention has replaced the most of market of radio immunoassay smoothly.The shortcoming of enzyme immunoassay (EIA) is that stability and sensitivity are relatively poor, and the cultivation time is long.Fluorescent immunoassay benefit is highly sensitive, has further replaced the application of some radiommunoassays, and shortcoming is the instrument and equipment matching problem, makes to be difficult for the frequent application of this use of clinical examination so as to doing daily chemical examination.The benefit of chemistry cold light immunoassay is identical with the fluorescent immunoassay, but in case be adapted as at a high speed, robotization immunoassay instrument, become bulky unavoidably and cost an arm and a leg, and be unfavorable for immunoassay applying in people's livelihood welfare society.
In the eighties, human diffusivity dyestuff is arranged, make 200～300 nanometers (nanometer) particulate and adsorb antibody, develop so-called dyestuff immunoassay (dye immunoassay, DIA).Difficulty, different antibodies have different adsorptive poweies, different diffusivity dyestuff same protein to be had technological factors such as different adsorption reactions again to same diffusivity dyestuff to the method because of particle size, shape and distribution control, add the product factors such as dyestuff particulate less stable of absorption antibody, and be not able to practicality always.
Chemically-reactive dyes (reactive dye) is called reactive dye (active dye) again, releases in 1956, and purpose of design is bonded on dye molecule on the hydroxyl of cotton fiber for by the covalent bonds mode.Basically be divided into (1) nucleophilicity substitution reaction (substitution), dyestuff is as six ring benzene (triazine) chemically-reactive dyes series and (2) nucleophilic addition reactions (addition) of three nitrogen, dyestuff such as vinyl sulfone (vinyl sulfone) series two big classes.Though the former dyed cotton yarn that is designed to, because of above two kinds of nucleophillic attacks (nucleophilic reaction), again can with amino and mercapto effect, so often be used for the dyeing of wool and silk for many years again.Chemically-reactive dyes generally when the dyeing of continuous yarn, wool and silk material, generally all takes the pH value more than 10.5, and temperature of reaction adds a large amount of salts simultaneously between 60～100 ℃.Purpose is as follows: (1) impels various textile materials to expand, and easily makes uniform coloring; (2) accelerate dyeing kinetics; (3) dye coloring rate height, few, the compliance with environmental protection requirements of waste material; (4) in alkaline environment, hydroxyl, freeization of mercapto, and amino is neutralized, the not sharing electron of above-mentioned each functional group all greatly increases the electronegativity of (lone paired electron) in the amino acid, and helps the nucleophillic attack to dye molecule.
Therefore fundamental purpose of the present invention is to provide a kind of chemically-reactive dyes process for selectively staining water-soluble protein that utilizes exactly.When being applied on the immunoassay, it has safety, short, simple for production, economic and sensitive advantage of reaction time.
The object of the present invention is achieved like this, and the dye molecule of chemically-reactive dyes is combined in respectively on hydroxyl, amino or the mercapto of the amino acid molecular of constitutive protein matter.When dyeing to as if during antibody, be controlled in sour environment, be beneficial to dye molecule and antibody and form amino acid or antibody molecule constant region and hang down hydroxyl effect on the carbohydrate carbohydrates.In immunoassay, when not needing to make when indicating part, in micro alkaline solution, dye of antibody, protein can dye to be had than dark shade.Protein separates the not dye molecule of complete reaction after reactive dyeing, the shades of colour protein stability is very high behind the purifying.
For above and other objects of the present invention, feature and advantage can be become apparent, a preferred embodiment cited below particularly, and conjunction with figs. elaborate:
The drawing explanation:
Fig. 1 is a heterogeneous body formula immunoassay synoptic diagram;
Fig. 2 is the precipitation line chart that produces on the agar substrate through the myoglobins antibody of chemically-reactive dyes procion red MX-8R dyeing and myoglobins;
Fig. 3 is through the myoglobins antibody of amino black dyeing and decolouring and the precipitation line chart that myoglobins produces on the agar substrate;
Fig. 4 is the visible light collection of illustrative plates of bovine serum albumin(BSA) after procion red MX-8R dyeing;
Fig. 5 is the bovine serum albumin(BSA) visible light collection of illustrative plates of no dyeing, and its concentration is 7.8mg/ml.
Embodiment:
Antibody is played the part of key role in immunoassay, and with immunoglobulin G, on the constant region of its molecular structure and heavy chain the oligosaccharides molecule that accounts for molecular weight 2～3% is arranged.And the antigen-binding site on the antibody molecule, the aminoterminal of antibody molecule just unfortunately.The chemically-reactive dyes reaction at random if leave, then this aminoterminal is very easily lost this antibody molecule antigen-binding energy in conjunction with a dye molecule.The chemically-reactive dyes dyeing of general non-antibody protein when being applied in immunoassay, does not then have these misgivings.
This antibody staining and the invention on immunoassay is used thereof, be the chemically-reactive dyes that uses textile industry to use, as be colored the three nitrogen six ring benzene series row that thing carries out the nucleophilicity substitution reaction, and be colored the vinyl sulfone series that thing carries out the nucleophilic addition reaction.In the mode of control potential of hydrogen, reaction is concentrated on the hydroxyl of antibody protein and the conjugation compound sugar on it, and make dyeing antibody keep its former strong valency and immune property.Protein after the staining reaction through separation and purification, is removed the unreacted dyestuff, can be used in the various uses in the immunoassay, become a kind of dyestuff immunoassay of novelty, can be called dissolubility dyestuff analytic approach (solubledye immunoassay, SDIA).Compare with radio immunoassay, safety benefit is easily arranged; Than enzyme immunoassay (EIA) sensitivity, quick, stable; More advantage easy-to-use and with low cost is arranged than fluorescent or cold light immunoassay.
The dyeing of water soluble protein chemically-reactive dyes, elevator muscle red eggs white (myoglobin) and bovine serum albumin(BSA) are example, carry out according to following condition:
(1) myoglobins antibody staining reaction.
10 milligrams of anti-human muscle hemoglobin antibody of rabbit are dissolved in the sodium acetate salt buffer solution of pH4.8,0.01M, add 5 milligrams of procion red MX-8R, behind ice bath temperature lucifuge stirring reaction 24 hour, inject one 1.1 * 30 centimetres Sephadex G-25 tubing string, with pH6.8,0.05M phosphate buffer, 20 milliliters of speed swashes of wave per hour, and use the classification gatherer, every pipe is collected one milliliter.Through covalent bond dyeing myoglobins antibody, because of volume is big, do not enter the hole of Sephadex G-25 molecular sieve micelle, be not adsorbed, therefore flow out very fast; The dye molecule of unreacted and condense (aggregate) then enters the micelle mesoporosity and goes out than slug flow because of being adsorbed.Each pipe of the redness of going out is earlier preserved after concentrating freeze drying, uses for further experiment.
(2) myoglobins antibody mediated immunity power valency is measured.
1% agar is formulated in barbital (barbital), and (pH8.2 0.05M) in the damping fluid, 50 ℃ of dissolvings, prepares the two-way immunodiffusion thin plate of Ouchterlony.Antibody 1.32 mg/ml of getting dyeing are made the dilution of 2,4,8,16,32,64,128,256,512,1024,2048,4096 equimultiples.No dyeing antibody is got same concentration, and dilution also together, is the contrast experiment.
Get the human muscle hemoglobin of 15 microlitres, 2.1 mg/ml, be positioned over the middle hole of the Ouchterlony agar plate for preparing, respectively get the peripheral hole that different extension rate dyeing of 15 microlitres and undyed myoglobins antibody inject the middle hole of agar plate respectively in addition, add a cover, room temperature left standstill six hours.Observation finds to produce the red precipitate line between the dyeing myoglobins antibody of 2 to 64 times of dilutions and center myoglobins.And the antibody that is unstained only has the turbidity sediment line in 2 to 16 times of dilutions and antigen.Through leaving standstill six hours, the red precipitate line of dyeing antibody and antigen does not increase (see figure 2) again.On the antibody directional diffusion of the being unstained agar plate,, be unstained between antibody and antigen, also dye precipitation line, referring to Fig. 32 to 64 times of dilutions through with amino black (Amido black) dyeing and decolouring.
This description of test, myoglobins antibody through in the pH4.8 environment with the chemically-reactive dyes effect after, not but still keep its activity, its power valency is not compared with the antibody that is unstained, and produces to change.
(3) chemically-reactive dyes covalent bonds position on antibody.
A. the pre-treatment of nerve amines enzyme (neuramidase).
The nerve amines enzyme bought is through at pH8.0, and at one night of 4 ℃ of dialysis of 0.05M acetate buffer solution, ox myosin (fetuin) tubing string that the one 5 milliliters of Sepharose that reinject are fixing washes to the 210nm absorption value with damping fluid then and reduces to below 0.05.Use SB (bicarbonate) damping fluid that neural amino acid enzyme is gone out then, after the neutralization, standby through the dialysis of pH8.0 phosphate buffer.
B. the Oligosaccharide of myoglobins antibody is removed.
Get one milliliter of neural amino acid enzyme of removing protein decomposition enzyme, add the myoglobins antibody of 50 microlitres, 7.5 mg/ml, add 0.05% streptomysin again, cultivated 48 hours at 40 ℃ through aforementioned processing.Inject 1.1 * 45 centimetres Sephadex G-150 tubing string then, wash, collect each pipe of light flesh pink and be myoglobins, after centrifugal concentrating, be made for following dyeing experiment with 10 milliliters of velocity flow per hour.
(4) go the myoglobins antibody staining of Oligosaccharide to react.
The concentration of aforementioned processing antibody is adjusted to 10 mg/ml, and all the other staining procedures are all described identical with (one).
This section and (one) joint products obtained therefrom takes by weighing with the dilution of weight dried frozen aquatic products, through with BeckmanDU-65 spectrometer scanning result, as table one:
Table one: remove Oligosaccharide/do not go Oligosaccharide antibody staining table
| Sample | Maximum absorption, nanometer (nm) | |
| ????520 | ????550 | |
| Handle myoglobins antibody through removing Oligosaccharide | ????1.09 | ????1.10 |
| Handle myoglobins antibody without removing Oligosaccharide | ????2.09 | ????2.10 |
Table one explanation, obviously expression part procion red MX-8R links to each other former the branch with myoglobins antibody oligosaccharide moiety in the covalent bonds mode, after the Oligosaccharide excision of nerve amines enzyme with antibody constant region and heavy chain part, the chemically-reactive dyes molecule becomes to subtracting to the hydroxyl effective object in sour environment and causes results of comparison as table one.
(5) bovine serum albumin(BSA) staining reaction.
Get the staining reaction in vain of Cohn V level bovine serum albumin,, avoid aminoterminal to be occupied during not as antibody staining and influence the misgivings of antigen/antibody activity by 600～700 molecular weight dye molecules because be general protein staining.So the reaction acid base number is transferred in alkaline range, and that more suitable is about pH10.5, wish amino on the gal4 amino acid, mercapto, and hydroxyl all can participate in reaction, increase the degree of depth and the sensitivity of future in the immunoassay application of dyeing.All the other dyeing and purification step are described with (one).Through the scanning of ultraviolet light visible spectrometry, it the results are shown in Figure 4, and the same concentration bovine serum albumin(BSA) visible light collection of illustrative plates of no dyeing is seen Fig. 5.
One to five result can prove by experiment, when the staining reaction object is any protein of non-antibody, then can select alkaline environment, does not use high temperature, need be with salt or electrolyte.Operate under the easy situation at this, can make hydroxyl, amino, the mercapto of amino acid side chain be the electron rich state, to help necleophilic reaction to dye molecule, generate the dyeing protein molecule conjugate of covalent bond, therefore obtain the higher protein of dyeability.When the staining reaction object is antibody, select sour environment for use, dye molecule is acted on the hydroxyl of antibody protein amino acid, branched hydroxy group and structure constant region and heavy chain part.Avoided the antibody molecule aminoterminal by dye molecule in conjunction with after last, to the influence of antigen active, thereby improve sensitivity on immunoassay is used.
Protein by method provided by the present invention dyeing is in following application, as indicating part.For example be applied to the Analysis of Plate that enzyme connects immunoassay (enzyme linked immunoassay), be applied to histology (histology), cytology (cell cytology), various immune agglutination method (immunoagglutination), various electrophoresis method, various immune chromatography (immunochromatography), be applied to various liquid chromatography (LC) (liquidchromatography), be applied to various sensors (sensor), be applied to the dyeing and the metering of various nucleic acid probes, or be applied to other various analytical chemistry purposes.
Though present embodiment separates the white matter molecule of free dye molecule with dyeing with the tubing string separation method.Yet for those skilled in the art, can understand protein after chemically-reactive dyes dyeing, can also be with molecular sieve, filtering membrane, saltout or the organic solvent mode is separated the protein molecule of free dye molecule with dyeing.Protein separates, removes unreacted dye molecule completely by said method after reactive dyeing, the shades of colour protein stability behind the purifying is very high, and can further promote its storage characteristics through vacuum freeze drying.
It is example that importance of the present invention is lifted Fig. 1, when the L ligand is radioactive isotope or enzyme or fluorescent thing or chemical cold light object, various application such as radio immunoassay, enzyme immunoassay (EIA), fluorescent immunoassay and cold light immunoassay is just arranged respectively.As L, in like manner, (soluble dyeimmunoassay DIA) occurs, most probably the substituted enzyme immunoassay so-called dissolubility dyestuff immunoassay with chemically-reactive dyes in combination the present invention.Its advantage is as follows:
(1),, dye molecule designs because all emphasizing high molar absorption coefficient than enzyme immunoassay (EIA) sensitivity.
(2) the immunoassay time shorter, because of not needing to measure the time of enzymatic activity.
(3) protein, is preserved/is taken and should be noted that unlike enzyme conjugation thing and do not lose enzymatic activity with more stable after dye molecule combines through covalent bond.
(4) simple for production, produce in batches easily, only need control pH-value, add simple separation and purification, practical antibody/antigen just can be arranged.
(5) chemically-reactive dyes is used for textile industry in a large number, and the meter ton is sold, and the part of the more any immunoassay of price is cheap.
(6) range of choice of dye colour is promoted, and has enlarged dyeing protein and has used in the analytical chemistry field.
Though the present invention with preferred embodiment explanation as above; but it is not in order to limit the present invention; any those skilled in the art; without departing from the spirit and scope of the present invention; when can doing simple improve and modify, but protection scope of the present invention should be as the criterion with claim institute restricted portion.
Claims (28)
1. the colouring method of a water soluble protein, it is characterized in that: utilize a chemically-reactive dyes to allow this water soluble protein carry out a staining reaction, this chemically-reactive dyes can be selected from the group that the used three nitrogen six ring benzene of textile industry and vinyl sulfone serial reaction dyestuff are formed, and this staining reaction is to carry out under the environment of acidity or an alkalescence, wherein when this water soluble protein is an antibody, this staining reaction is to carry out under the environment of acidity, and dye molecule is acted on the hydroxyl of this antibody protein amino acid side chain, on the hydroxyl of structure constant region and heavy chain part, and with the hydroxyl of the compound sugar of this antibody conjugation on.
2. method according to claim 1 is characterized in that: the pH value about 10.5 of required alkaline environment is carried out in this staining reaction.
3. method according to claim 1 is characterized in that: when this antibody was a myoglobins antibody, this staining reaction was to carry out under the sour environment of pH value about 4.8.
4. method according to claim 1, it is characterized in that: utilizing this chemically-reactive dyes to dye after this water soluble protein, also comprising and utilize tubing string separation method, molecular sieve, filtering membrane, saltout or the protein molecule of this dye molecule that the organic solvent mode will be dissociated and this dyeing separates.
5. method according to claim 1 is characterized in that: this water soluble protein by this method dyeing can be applicable to the Analysis of Plate that enzyme connects immunoassay.
6. method according to claim 1 is characterized in that: by this water soluble protein of this method dyeing, can be applicable to histological subject matter colour developing and metering.
7. method according to claim 1 is characterized in that: by this water soluble protein of this method dyeing, can be applicable to colour developing of cytology subject matter and metering.
8. method according to claim 1 is characterized in that: by this water soluble protein of this method dyeing, can be applicable to the colour developing and the metering of various immune agglutination methods.
9. method according to claim 1 is characterized in that: by this water soluble protein of this method dyeing, can be applicable to the colour developing and the metering of various electrophoresis methods.
10. method according to claim 1 is characterized in that: by this water soluble protein of this method dyeing, can be applicable to the colour developing and the metering of various immune chromatographies.
11. method according to claim 1 is characterized in that:, can be applicable to the application of various liquid chromatography (LC) by this water soluble protein of this method dyeing.
12. method according to claim 1 is characterized in that:, can be applicable to the application of various sensors by this water soluble protein of this method dyeing.
13. method according to claim 1 is characterized in that:, can be applicable to the dyeing and the metering of various nucleic acid probes by this water soluble protein of this method dyeing.
14. method according to claim 1 is characterized in that: this water soluble protein by this method dyeing can be applicable to various analytical chemistry purposes.
15. the colouring method of a water soluble protein; it is characterized in that: utilize a chemically-reactive dyes to allow this water soluble protein carry out a staining reaction; this chemically-reactive dyes can be selected from the group that the used three nitrogen six ring benzene of textile industry and vinyl sulfone serial reaction dyestuff are formed; and this staining reaction is to carry out under the environment of acidity or an alkalescence; wherein when this water soluble protein is the protected water soluble protein of aminoterminal need; this staining reaction is carried out under the environment of acidity, and making dye molecule act on this aminoterminal needs on the hydroxyl of protected water soluble protein amino acid side chain; and on the hydroxyl of conjugation compound sugar on it.
16. method according to claim 15 is characterized in that: the pH value about 10.5 of required alkaline environment is carried out in this staining reaction.
17. method according to claim 15 is characterized in that: when this aminoterminal needed protected water soluble protein to be a myoglobins antibody, this staining reaction was carried out under the sour environment of pH value about 4.8.
18. method according to claim 15, it is characterized in that: utilizing this chemically-reactive dyes to dye after this water soluble protein, also comprising and utilize tubing string separation method, molecular sieve, filtering membrane, saltout or the protein molecule of this dye molecule that the organic solvent mode will be dissociated and this dyeing separates.
19. method according to claim 15 is characterized in that: this water soluble protein by this method dyeing can be applicable to the Analysis of Plate that enzyme connects immunoassay.
20. method according to claim 15 is characterized in that:, can be applicable to histological subject matter colour developing and metering by this water soluble protein of this method dyeing.
21. method according to claim 15 is characterized in that:, can be applicable to colour developing of cytology subject matter and metering by this water soluble protein of this method dyeing.
22. method according to claim 15 is characterized in that:, can be applicable to the colour developing and the metering of various immune agglutination methods by this water soluble protein of this method dyeing.
23. method according to claim 15 is characterized in that:, can be applicable to the colour developing and the metering of various electrophoresis methods by this water soluble protein of this method dyeing.
24. method according to claim 15 is characterized in that:, can be applicable to the colour developing and the metering of various immune chromatographies by this water soluble protein of this method dyeing.
25. method according to claim 15 is characterized in that:, can be applicable to the application of various liquid chromatography (LC) by this water soluble protein of this method dyeing.
26. method according to claim 15 is characterized in that:, can be applicable to the application of various sensors by this water soluble protein of this method dyeing.
27. method according to claim 15 is characterized in that:, can be applicable to the dyeing and the metering of various nucleic acid probes by this water soluble protein of this method dyeing.
28. method according to claim 15 is characterized in that: this water soluble protein by this method dyeing can be applicable to each sharp analytical chemistry purposes.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7365849B2 (en) | 2001-12-27 | 2008-04-29 | Taiwan Unison Biotechnology, Inc. | Portable, scanning and analyzing apparatus |
| CN105891208A (en) * | 2016-04-13 | 2016-08-24 | 华中科技大学 | Method for obtaining cytoarchitectonic information for establishing standard map data set |
| CN106483295A (en) * | 2016-06-13 | 2017-03-08 | 苏州泽科生物科技有限公司 | A kind of protide biomacromolecule is quickly coupled labelling pigment group reagent box |
| CN107354155A (en) * | 2017-08-25 | 2017-11-17 | 苏州泽科生物科技有限公司 | Three primary colours nucleic acid molecular weight standard reagent box and its application method |
-
2001
- 2001-04-06 CN CNB011103558A patent/CN1141581C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7365849B2 (en) | 2001-12-27 | 2008-04-29 | Taiwan Unison Biotechnology, Inc. | Portable, scanning and analyzing apparatus |
| CN105891208A (en) * | 2016-04-13 | 2016-08-24 | 华中科技大学 | Method for obtaining cytoarchitectonic information for establishing standard map data set |
| CN106483295A (en) * | 2016-06-13 | 2017-03-08 | 苏州泽科生物科技有限公司 | A kind of protide biomacromolecule is quickly coupled labelling pigment group reagent box |
| CN107354155A (en) * | 2017-08-25 | 2017-11-17 | 苏州泽科生物科技有限公司 | Three primary colours nucleic acid molecular weight standard reagent box and its application method |
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| CN1141581C (en) | 2004-03-10 |
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