CN101692087A - Method for preparing streptavidin pre-coated elisa plate and application - Google Patents
Method for preparing streptavidin pre-coated elisa plate and application Download PDFInfo
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- CN101692087A CN101692087A CN200910192897A CN200910192897A CN101692087A CN 101692087 A CN101692087 A CN 101692087A CN 200910192897 A CN200910192897 A CN 200910192897A CN 200910192897 A CN200910192897 A CN 200910192897A CN 101692087 A CN101692087 A CN 101692087A
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Abstract
The invention discloses a streptavidin pre-coated elisa plate, a preparation method thereof and application thereof. The preparation method comprises the following steps of: dissolving streptavidin by using carbonate buffer solution or citric acid phosphate buffer solution to obtain coated buffer solution; and adding the coated buffer solution into an elisa plate, and then drying the elisa plate to obtain the streptavidin pre-coated elisa plate. The preparation method is simple, and the utilization rate of the streptavidin is high so as to reduce the dosage of the streptavidin. The preparation method has low requirement for the quality of a blank elisa plate; and the batch of the obtained streptavidin pre-coated elisa plate is stable, and the hole difference, batch difference and stability of the streptavidin pre-coated elisa plate are higher than those of wet coating, so the streptavidin pre-coated elisa plate is more suitable for long-term storage. The streptavidin pre-coated elisa plate has low cost and good effect, and can be applied in the technical field of in vitro immune detection or the field of nucleic acid detection in molecular biology.
Description
Technical field
The invention belongs to external technical field of immunoassay, particularly a kind of streptavidin pre-coated elisa plate and preparation method thereof and application.
Background technology
Streptavidin (streptavidin) is by a kind of alkaline glycoprotein of streptomycete streptomyces secretion, is easy to combine with the polystyrene plastics microwell plate.A Streptavidin molecular energy is in conjunction with 4 biotin molecules, and the two affinity costant (K) is 10
15L/mol.Because biotin and affinely have a high adhesion, (biotin-avidin system BAS) is widely used in the enzyme linked immunosorbent detection biotin-avidin system.
Streptavidin is fixed on the solid phase carrier, utilizes the micromolecule antigen-antibody of the fixing difficult bag quilt of BAS or the method for nucleic acid to be used widely.Streptavidin pre-coated elisa plate all has application in fields such as enzyme linked immunological, PCR-ELISA.The good result of Streptavidin solid-phase coating is depended in the widespread use of BAS fixation.
The Streptavidin bag of report is realized for passing through conventional wet method method for coating at present.The wet method method for coating is cushioned liquid optimization bag by effect by concentration and the bag of regulating Streptavidin, and its process is loaded down with trivial details, and required time is long, poor between the hole, difference between batch is bigger, poor stability, the biotin adhesion is low, the Streptavidin of bag quilt is fixing unstable, washes the easy flush away of plate process.And the wet method method for coating requires height to the absorption affinity of ELISA Plate, will get rid of most of coating buffer after bag is moved to end, and the utilization factor of Streptavidin is low, the cost height.The dry method method for coating be owing to can influence the activity and the space structure of antigen-antibody etc., therefore the enzyme linked immunological bag by in use seldom.The report that the streptavidin pre-coated elisa plate of dry method method for coating preparation is not arranged up to now, as yet.
Summary of the invention
The wet method bag that primary and foremost purpose of the present invention is to overcome present routine is produced the weak point of streptavidin pre-coated elisa plate, and a kind of preparation method of streptavidin pre-coated elisa plate is provided.
Another object of the present invention is to provide streptavidin pre-coated elisa plate by described preparation method's preparation.
A further object of the present invention is to provide the application of described streptavidin pre-coated elisa plate.
Purpose of the present invention is achieved through the following technical solutions: a kind of preparation method of streptavidin pre-coated elisa plate may further comprise the steps:
With the citric acid phosphoric acid salt buffer dissolving Streptavidin of the carbonate buffer solution of pH9.0～9.8,0.01～0.07mol/L or pH4.5～5.5,0.1～0.2mol/L, the concentration of Streptavidin is 1.0～6.0 μ g/ml, obtains bag and is cushioned liquid; Bag is cushioned liquid adds in the blank ELISA Plate, every hole 100～250 μ l; Place 32～40 ℃ of oven dry afterwards, obtain streptavidin pre-coated elisa plate.
Described carbonate buffer solution is preferably sodium carbonate-sodium bicarbonate buffer liquid;
Described citrate buffer is preferably citric acid-sodium hydrogen phosphate damping fluid;
Described streptavidin pre-coated elisa plate preferably is placed in the sealed bag in oven dry and preserves down in 2～5 ℃;
Streptavidin in the described streptavidin pre-coated elisa plate is in conjunction with stable, and is strong with the biotin adhesion;
Described streptavidin pre-coated elisa plate is applied to external technical field of immunoassay, also can be used for molecular biology amplifying nucleic acid detection range;
The application of described streptavidin pre-coated elisa plate, comprise following steps: use the described streptavidin pre-coated elisa plate of PBST buffer solution for cleaning at least 5 times earlier, each 1 minute, get rid of after each the cleaning and pat dry, remove unconjugated Streptavidin, the adding test substance carries out Enzyme Linked Immunoadsorbent Assay and gets final product; Described PBST damping fluid is the PBS damping fluid that contains Tween 20, and wherein Tween 20 concentration are percent by volume 0.05%;
Described PBS damping fluid is preferably that the pH value is 7.4, concentration is the PBS damping fluid of 0.01mol/L;
The present invention has following advantage and effect with respect to prior art:
(1) preparation method of streptavidin pre-coated elisa plate is simple among the present invention, Streptavidin utilization factor height, thereby reduced Streptavidin consumption (the Streptavidin coating buffer concentration of prior art needs 10 μ g/ml, and dry method when 2 μ g/ml, can reach compare the more excellent bag of wet method by effect).
(2) streptavidin pre-coated elisa plate that obtains by preparation method of the present invention is batch stable, and promptly the amount of the Streptavidin of solid phase carrier-ELISA Plate combination is comparatively even.In addition, poor between the hole of described streptavidin pre-coated elisa plate, difference between batch and stability all are higher than wet method bag quilt, are more suitable for long preservation.
(3) the streptavidin pre-coated elisa plate Streptavidin of the present invention's preparation is in conjunction with stable, and it is not remarkable that the PBST damping fluid washes the plain binding capacity variation of 20 secondary pollutants, function admirable.Streptavidin pre-coated elisa plate can reflect the quality of wrapping quilt for the determination data of the binding capacity of biotin, and the biotin binding capacity of ELISA Plate is big more, illustrates that the bag of streptavidin pre-coated elisa plate is superior more by effect.The maximum biotin of clean extra-high-speed affinity plate dry method bag quilt is the 10ng/ml (see figure 1) in conjunction with concentration in this experiment, and promptly every hole can be in conjunction with 1ng biotin (4.1pmol/ hole).And the homemade pre-bag that same method records is had only 0.2ng (0.8pmol/ hole) by plate biotin binding capacity, shows that the bag of this experiment is better than homemade pre-bag by plate by effect.
(4) preparation method of the present invention is low to the quality requirements of blank ELISA Plate, difference between homemade plate and the import plate is not remarkable, and the wet method bag is by the quality requirements height to blank ELISA Plate, there were significant differences between homemade plate and the import plate (as shown in table 4), therefore, preparation method's cost of the present invention is lower.
(5) the present invention has adopted carbonate buffer solution (pH 9.6) and two kinds of pH values of citric acid phosphoric acid salt buffer (pH 5.0) to differ great bag and has been cushioned liquid, can be fit to the detection architecture of different pH values in the late detection respectively, has enlarged the scope of application of the present invention.
Description of drawings
Fig. 1 is biotin binding capacity measurement result figure.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
Embodiment 1
Blank ELISA Plate is clean extra-high-speed affinity ELISA Plate.
Use carbonate buffer solution (1.59g sodium carbonate and 2.93g sodium bicarbonate thin up are to 1000mL) dilution Streptavidin to the 2 μ g/ml of pH 9.6,0.05M, obtain bag and be cushioned liquid; Bag is cushioned liquid is added in the blank ELISA Plate, every hole 100 μ l place constant temperature oven to be incubated overnight to bone dry for 37 ℃ afterwards, obtain streptavidin pre-coated elisa plate.Dry ELISA Plate installs in 4 ℃ with sealed bag and preserves down.
Embodiment 2
Blank ELISA Plate is clean extra-high-speed affinity ELISA Plate.
Use citric acid phosphoric acid salt buffer (1030ml 0.1M citric acid and the 970ml0.2M Na of pH 5.0,0.15M
2HPO
4Mix and obtain) dilution Streptavidin to 2 μ g/ml, obtain bag and be cushioned liquid; Bag is cushioned liquid is added in the blank ELISA Plate, every hole 100 μ l place constant temperature oven to be incubated overnight to bone dry for 37 ℃ afterwards, obtain streptavidin pre-coated elisa plate.Dry ELISA Plate installs in 4 ℃ with sealed bag and preserves down.
Embodiment 3
Blank ELISA Plate is the Nunc ELISA Plate.
Use citric acid phosphoric acid salt buffer (1030ml 0.1M citric acid and the 970ml0.2M Na of pH 5.0,0.15M
2HPO
4Mix and obtain) dilution Streptavidin to 2 μ g/ml, obtain bag and be cushioned liquid; Bag is cushioned liquid is added in the blank ELISA Plate, every hole 100 μ l place constant temperature oven to be incubated overnight to bone dry for 37 ℃ afterwards, obtain streptavidin pre-coated elisa plate.Dry ELISA Plate installs in 4 ℃ with sealed bag and preserves down.
Embodiment 4
Blank ELISA Plate is clean extra-high-speed affinity ELISA Plate.
Use sodium carbonate-sodium bicarbonate buffer liquid dilution Streptavidin to 6 μ g/ml of pH 9.0,0.01M, obtain bag and be cushioned liquid; Bag is cushioned liquid is added in the blank ELISA Plate, every hole 200 μ l place constant temperature oven to be incubated overnight to bone dry for 40 ℃ afterwards, obtain streptavidin pre-coated elisa plate.Dry ELISA Plate installs in 4 ℃ with sealed bag and preserves down.
Embodiment 5
Blank ELISA Plate is the Nunc ELISA Plate.
Use sodium carbonate-sodium bicarbonate buffer liquid dilution Streptavidin to 1 μ g/ml of pH 9.8,0.07M, obtain bag and be cushioned liquid; Bag is cushioned liquid is added in the blank ELISA Plate, every hole 250 μ l place constant temperature oven to be incubated overnight to bone dry for 32 ℃ afterwards, obtain streptavidin pre-coated elisa plate.Dry ELISA Plate installs in 4 ℃ with sealed bag and preserves down.
Embodiment 6
Blank ELISA Plate is the Nunc ELISA Plate.
Use citric acid-sodium hydrogen phosphate damping fluid dilution Streptavidin to 6 μ g/ml of pH 4.5,0.2M, obtain bag and be cushioned liquid; Bag is cushioned liquid is added in the blank ELISA Plate, every hole 250 μ l place constant temperature oven to be incubated overnight to bone dry for 40 ℃ afterwards, obtain streptavidin pre-coated elisa plate.Dry ELISA Plate installs in 4 ℃ with sealed bag and preserves down.
Embodiment 7
Blank ELISA Plate is clean extra-high-speed affinity ELISA Plate.
Use citric acid-sodium hydrogen phosphate damping fluid dilution Streptavidin to 1 μ g/ml of pH 5.5,0.1M, obtain bag and be cushioned liquid; Bag is cushioned liquid is added in the blank ELISA Plate, every hole 150 μ l place constant temperature oven to be incubated overnight to bone dry for 32 ℃ afterwards, obtain streptavidin pre-coated elisa plate.Dry ELISA Plate installs in 4 ℃ with sealed bag and preserves down.
The comparative example 1
Adopt the wet method bag by Streptavidin, use clean extra-high-speed affinity ELISA Plate:
Use citric acid phosphoric acid salt buffer (1030ml 0.1M citric acid and the 970ml0.2M Na of pH 5.0,0.15M
2HPO
4Mix and obtain) dilution Streptavidin to 4 μ g/ml, obtain bag and be cushioned liquid; Bag is cushioned liquid is added in the blank ELISA Plate, every hole 100 μ l place 4 ℃ of bags of constant temperature oven by 16h afterwards, dry coating buffer, at 37 ℃ of following shrouding 2h, are stored in 4 ℃ after sealed bag installs.
The comparative example 2
Operation is with comparative example 1, and difference only is that the blank ELISA Plate of using is the Nunc ELISA Plate.
Effect compares:
Embodiment 1～3 is compared by the streptavidin pre-coated elisa plate that wet method prepares by streptavidin pre-coated elisa plate and the comparative example 1～2 that dry process obtains:
Table 1 is that the bag of various ELISA Plate is compared by effect.(concentration of PBS damping fluid is 0.01mol/L to various ELISA Plate with the PBST damping fluid that contains percent by volume 0.05%Tween 20 before use, the pH value is 7.4) wash 5 times, each 1min to wash off in conjunction with unstable Streptavidin, improves the biotin adhesion.Then, add biotin labeled HRP 100 μ l (0.01M PBS dilution in 1: 1000), measure the OD value after TMB (4, the 5-TMB) develops the color.Can be got by data in the table, these four kinds of mode bags are not had significant difference (P＜0.01) by effect.Because the dry method bag is few by the Streptavidin consumption, only be half of wet method, illustrate that preparation method of the present invention utilizes the efficient height of Streptavidin.In addition, the ELISA Plate difference that embodiment 1～3 uses illustrates that for preparation method of the present invention the quality of blank ELISA Plate is little for the streptavidin pre-coated elisa plate quality influence that finally obtains, and this helps the reduction of cost.
Table 1: four kinds of embodiment bags by effect relatively
Annotate: A, a are the symbols in the statistics, and be alphabetical up and down identical, difference not significantly (0.05) or the difference utmost point not significantly (0.01) be described, down together.
Table 2 be poor between hole between the ELISA Plate of different method for coating bag quilts, difference between batch relatively.With the PBST damping fluid flushing that contains percent by volume 0.05%Tween 20 5 times, each 1min to wash off in conjunction with unstable Streptavidin, improves the biotin adhesion to various ELISA Plate before use.Then, add biotin labeled HRP 100 μ l (0.01M PBS dilution in 1: 1000), measure the OD value after TMB develops the color.The dry method bag of wherein clean special plate, Nunc plate is met national standard (GB CV value＜15%).
Table 2: two kinds of ELISA Plate bags by the back relatively
Table 3 is the desorption test of the ELISA Plate of embodiment 1 preparation.Wash ELISA Plate respectively 5,10 and 20 times with washing lotion PBST (0.05%Tween 20), scavenging period is 1min/ time, the biotin binding ability that adds biotin labeled HRP 100 μ l (0.01M PBS dilution in 1: the 1000) ELISA Plate that wash number is different does not have significant difference (P＜0.01), illustrates that the dry method bag by the Streptavidin combination firmly.
Table 3: clean special plate dry method desorption relatively
The bag that table 4 is respectively import ELISA Plate and homemade ELISA Plate for preparation method's of the present invention blank ELISA Plate is by effect, and operating conditions is with embodiment 1.Preparation method of the present invention is low to the quality requirements of blank ELISA Plate, difference between the domestic and imported plate is not remarkable, the wet method bag is by then there were significant differences (as shown in table 4), that is to say that the wet method bag is by the requirement height to blank ELISA Plate, therefore, preparation method's cost of the present invention is lower.
Fig. 1 is for carrying out case study on implementation 1 gained streptavidin pre-coated elisa plate, the biotin solution that adds variable concentrations, be respectively 0,1.0,2.0,4.0,6.0,8.0,12.0,16.0,24.0,32.0ng/ml, 37 ℃ in conjunction with 20min, dry biotin solution, add 1: develop the color gained OD value such as Fig. 1 behind the 1000HRP incubation 1h.When the OD value is reduced to the biotin capacity that lowest part is ELISA Plate, be 10ng/ml as the maximum biotin of Fig. 1 case study on implementation 1 gained ELISA Plate in conjunction with concentration, promptly every hole can be in conjunction with 1ng biotin (4.1pmol/ hole).And the homemade pre-bag that same method records is had only 0.2ng (0.8pmol/ hole) by plate biotin binding capacity, shows that the bag of this experiment is better than homemade pre-bag by plate by effect.
Table 4: import, homemade ELISA Plate bag are by effect relatively
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (8)
1. the preparation method of a streptavidin pre-coated elisa plate, it is characterized in that: may further comprise the steps: the citric acid phosphoric acid salt buffer dissolving Streptavidin of the carbonate buffer solution of usefulness pH9.0～9.8,0.01～0.07mol/L or pH4.5～5.5,0.1～0.2mol/L, the concentration of Streptavidin is 1.0～6.0 μ g/ml, obtains bag and is cushioned liquid; Bag is cushioned liquid adds in the blank ELISA Plate, every hole 100～250 μ l; Place 32～40 ℃ of oven dry afterwards, obtain streptavidin pre-coated elisa plate.
2. according to the preparation method of the described streptavidin pre-coated elisa plate of claim 1, it is characterized in that: described carbonate buffer solution is sodium carbonate-sodium bicarbonate buffer liquid.
3. according to the preparation method of the described streptavidin pre-coated elisa plate of claim 1, it is characterized in that: described citrate buffer is citric acid-sodium hydrogen phosphate damping fluid.
4. according to the preparation method of the described streptavidin pre-coated elisa plate of claim 1, it is characterized in that: described streptavidin pre-coated elisa plate is placed in the sealed bag in oven dry and preserves down in 2～5 ℃.
5. streptavidin pre-coated elisa plate, each described preparation method obtains by claim 1～4.
6. the application of the described streptavidin pre-coated elisa plate of claim 5 is characterized in that: described streptavidin pre-coated elisa plate is applied to external technical field of immunoassay or molecular biology amplifying nucleic acid detection range.
7. application according to claim 6, it is characterized in that: use the described streptavidin pre-coated elisa plate of PBST buffer solution for cleaning at least 5 times earlier, each 1 minute, get rid of after each the cleaning and pat dry, remove unconjugated Streptavidin, the adding test substance carries out Enzyme Linked Immunoadsorbent Assay and gets final product; Described PBST damping fluid is the PBS damping fluid that contains Tween 20, and wherein Tween 20 concentration are percent by volume 0.05%.
8. application according to claim 7 is characterized in that: described PBS damping fluid is that the pH value is 7.4, concentration is the PBS damping fluid of 0.01mol/L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102226808A (en) * | 2011-03-28 | 2011-10-26 | 北京永瀚星港生物技术有限公司 | Trypsinogen-2 chemiluminescent immunoassay kit and preparation method thereof |
| CN103048456A (en) * | 2012-10-08 | 2013-04-17 | 武汉康珠生物技术有限公司 | Hepatitis B virus PreS1 antigen enzyme-linked immunoassay kit employing one-step method |
| CN105759055A (en) * | 2016-03-31 | 2016-07-13 | 广州菲康生物技术有限公司 | P1NP detection kit and preparation method thereof |
| CN107807235A (en) * | 2017-11-01 | 2018-03-16 | 郑州欧柯奇仪器制造有限公司 | Clenbuterol ELISA kit, its preparation method and its application |
-
2009
- 2009-09-30 CN CN200910192897A patent/CN101692087A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102226808A (en) * | 2011-03-28 | 2011-10-26 | 北京永瀚星港生物技术有限公司 | Trypsinogen-2 chemiluminescent immunoassay kit and preparation method thereof |
| CN103048456A (en) * | 2012-10-08 | 2013-04-17 | 武汉康珠生物技术有限公司 | Hepatitis B virus PreS1 antigen enzyme-linked immunoassay kit employing one-step method |
| CN105759055A (en) * | 2016-03-31 | 2016-07-13 | 广州菲康生物技术有限公司 | P1NP detection kit and preparation method thereof |
| CN107807235A (en) * | 2017-11-01 | 2018-03-16 | 郑州欧柯奇仪器制造有限公司 | Clenbuterol ELISA kit, its preparation method and its application |
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Application publication date: 20100407 |