CN113030459A - Production process of high-binding-force enzyme label plate - Google Patents
Production process of high-binding-force enzyme label plate Download PDFInfo
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- CN113030459A CN113030459A CN202110214380.9A CN202110214380A CN113030459A CN 113030459 A CN113030459 A CN 113030459A CN 202110214380 A CN202110214380 A CN 202110214380A CN 113030459 A CN113030459 A CN 113030459A
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- 229920006248 expandable polystyrene Polymers 0.000 claims abstract description 8
- 238000001723 curing Methods 0.000 claims abstract description 5
- 239000004794 expanded polystyrene Substances 0.000 claims abstract description 5
- 230000001502 supplementing effect Effects 0.000 claims abstract description 5
- 239000007789 gas Substances 0.000 claims description 35
- 239000004793 Polystyrene Substances 0.000 claims description 30
- 229920002223 polystyrene Polymers 0.000 claims description 30
- 239000011324 bead Substances 0.000 claims description 25
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 10
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 8
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- 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
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Abstract
The invention relates to a production process of an enzyme label plate with high binding force, which comprises the following steps: s1: and foaming, curing and molding the expandable polystyrene to prepare the expanded polystyrene elisa plate. S2: and placing the ELISA plate in plasma surface treatment equipment, introducing first gas, and performing surface cleaning on the ELISA plate. S3: and supplementing a second gas into the plasma surface treatment equipment, ionizing positive and negative charges in the plasma surface treatment equipment by the second gas, and carrying out surface hydrophilic modification treatment on the ELISA plate to obtain the high-binding-force ELISA plate. The surface of the ELISA plate is modified to generate amino with positive charges, a hydrophobic bond on the surface of the ELISA plate is replaced by a hydrophilic bond, and the surface of the ELISA plate can be combined with protein with negative charges through an ionic bond under proper buffer solution and pH value. The elisa plate after surface hydrophilic modification has high sensitivity to protein in the experimental process, and can reduce the concentration and dosage of the coated protein.
Description
Technical Field
The invention belongs to the technical field of biological instruments, and particularly relates to a production process of a high-binding-force enzyme label plate.
Background
The ELISA plate is mainly used for bearing various required experimental articles in a laboratory, and plays a very key role in the conditions of antigens, antibodies, labeled antibodies, buffer solutions and the like participating in immunological reaction. And the surface of the solid-phase polystyrene as the enzyme label plate carrier plays an important role in the adsorption of antigens, antibodies or compounds thereof. Antigens, antibodies and biomolecules are bound by adsorption to the surface of the microplate, including passive adsorption, covalent binding by introducing other active groups, and binding by hydrophilic bonds.
When the enzyme label plate is used for experimental determination, a detected sample reacts with an antigen or an antibody on the surface of the enzyme label plate, so that an antigen-antibody complex formed on a solid phase carrier is separated from other substances. Then adding enzyme-labeled antigen and antibody, and binding on solid phase carrier by reaction. The enzyme amount and the mass of the detected object in the sample are in a certain proportion. After the enzyme reaction substrate is added, the enzyme is catalyzed to form a colored product, and the product quantity is directly related to the quality of the detected object, so that qualitative or quantitative analysis can be carried out according to the color depth. The results of the immune reaction are indirectly amplified because of the high catalytic efficiency of the enzyme.
The binding force between the common enzyme label plate and the protein is small, so that the loss of the protein is large.
Therefore, there is a need for an elisa plate that has a higher binding capacity to proteins.
Disclosure of Invention
Technical problem to be solved
The invention provides a production process of an ELISA plate with high binding force, aiming at solving the problems that the binding force of the ELISA plate and protein is weak and the amount of the protein needs to be increased in the prior art.
(II) technical scheme
In order to achieve the purpose, the invention adopts the main technical scheme that:
a production process of an ELISA plate with high binding force comprises the following steps:
s1: foaming, curing and molding expandable polystyrene to prepare a foamed polystyrene elisa plate;
s2: placing the ELISA plate prepared in the step S1 in plasma surface treatment equipment, introducing first gas, and performing surface cleaning and activating treatment on the ELISA plate;
s3: and supplementing a second gas into the plasma surface treatment equipment, and ionizing positive and negative charges in the plasma surface treatment equipment by using the reaction gas to generate groups with positive charges on the surface of the ELISA plate so as to realize surface hydrophilic modification treatment on the ELISA plate.
In the above-mentioned microplate production process, in step S1, the polystyrene beads and steam are continuously mixed in the image prefoamer to complete the foaming reaction, and the image prefoamer is kept open at normal pressure, and the foamed polystyrene beads overflow from the top of the image prefoamer.
In the above-described process for producing an microplate, after the polystyrene beads are foamed, the polystyrene beads are cured to allow air to be mixed into the cells, thereby obtaining cured pre-foamed beads.
According to the production process of the ELISA plate, the cured pre-foamed beads are placed into a molding device for molding treatment, so that the ELISA plate is obtained.
In the above-described microplate production process, step S3, the second gas is a mixture of oxygen and ammonia.
In the above-mentioned elisa plate production process, in step S2, the first gas is compressed air, the compressed air is introduced into the plasma surface treatment device, and the compressed air is ionized to generate plasma, so as to clean the surface of the elisa plate.
In step S3, a second gas is introduced into the plasma surface treatment device, and the second gas ionizes in the plasma surface treatment device to form active groups, which perform surface hydrophilic modification treatment on the elisa plate on the surface of the elisa plate.
In the above-mentioned elisa plate production process, in step S2, the washing time of the elisa plate is 10-20 min.
In the above-mentioned elisa plate production process, in step S3, the time period of the surface hydrophilic modification treatment is 10-20 min.
The above-mentioned production process of the ELISA plate, in step S2 and step S3, the vacuum degree is 0.02-0.03mbar, the processing temperature is 30-40 ℃, the processing power is 600-.
(III) advantageous effects
The invention has the beneficial effects that:
the invention takes the polystyrene as the raw material to prepare the ELISA plate, the ELISA plate is crystal clear, the plate surface is flat and smooth, the polystyrene is in a head-tail structure, the main chain is a saturated carbon chain, the side group is a conjugated benzene ring, the molecular structure is irregular, and the rigidity of the molecule is increased, so the polystyrene is a non-crystalline linear polymer.
Polystyrene has a high Tg value due to the presence of benzene rings, is transparent and rigid at room temperature, and is susceptible to stress cracking due to the rigidity of the molecular chains. The polystyrene is colorless and transparent, can be freely colored, has high relative density, excellent electrical properties and particularly good high-frequency characteristics. In addition, polystyrene is good in light stability, and radiation resistance is the strongest of all plastics. Polystyrene has excellent thermal stability and fluidity when melted, so that it is easy to mold and process, and in particular, injection molding is easy, the molding shrinkage is small, and the dimensional stability of the molded product is also excellent, and it is suitable for mass production.
In step S2, the plasma generated by the first gas is used to clean the surface of the ELISA plate, and a large amount of active particles such as ions, excited molecules, free radicals and the like in the plasma act on the surface of the ELISA plate to remove the original pollutants and impurities on the surface of the ELISA plate. The particle energy in the plasma is 0-20eV, the material of the ELISA plate is polystyrene, most bonds in the polystyrene are 0-10eV, and therefore, after the plasma acts on the surface of the ELISA plate, the original chemical bonds on the surface of the ELISA plate can be broken, and free radicals in the plasma and the broken chemical bonds form a net-shaped cross-linking structure, so that the surface activity of the ELISA plate is greatly activated.
In the step S3, a second gas is introduced into the plasma surface treatment device, reactive gas ionizes active groups including amino groups in the plasma surface treatment device, the active groups perform surface hydrophilic modification treatment on the elisa plate on the surface of the elisa plate, complex chemical reaction can occur on the surface of the activated material, and new functional groups are introduced, so that the surface activity of the elisa plate can be obviously improved, and the surface hydrophilic capability of the elisa plate can be effectively improved.
The invention adds ionizable gas including oxygen and ammonia gas in the glow discharge area to generate more charges to be processed by plasma surface processing equipment, thereby increasing the hydrophilicity of the surface of the enzyme label plate.
The ELISA plate has positively charged amino groups after surface modification treatment, the hydrophobic bond of the ELISA plate is replaced by a hydrophilic bond, the ELISA plate is suitable for being used as a solid phase carrier of small molecular proteins, and the surface of the ELISA plate can be combined with negatively charged proteins through ionic bonds under the appropriate buffer solution and pH value. The elisa plate after surface hydrophilic modification has high sensitivity to protein in the experimental process, and can reduce the concentration and dosage of the coated protein.
Drawings
FIG. 1 is a schematic diagram of the surface hydrophilic treatment of an ELISA plate by using a plasma device in the present invention;
FIG. 1a is a schematic diagram of the enzyme label plate surface cleaning by using the plasma generated by the discharge gas in step S1;
FIG. 1b is a schematic diagram of the chemical reaction on the surface of the microplate generated by the reactive group generated by the reactive gas in step S2;
FIG. 2 is a schematic structural view of the plasma surface treatment apparatus according to the present invention.
[ description of reference ]
1: plasma surface treatment equipment; 2: a reaction gas inlet valve; 3: an oxygen intake valve; 4: an ammonia gas inlet valve; 5: and (4) exhausting the valve.
Detailed Description
For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.
The invention provides a production process of an ELISA plate with high binding force, which comprises the following steps:
s1: foaming, curing and molding expandable polystyrene to prepare a foamed polystyrene elisa plate;
s2: placing the ELISA plate prepared in the step S1 in plasma surface treatment equipment, introducing first gas, and performing surface cleaning and activating treatment on the ELISA plate;
s3: and supplementing a second gas into the plasma surface treatment equipment, and ionizing positive and negative charges in the plasma surface treatment equipment by using the reaction gas to generate groups with positive charges on the surface of the ELISA plate so as to realize surface hydrophilic modification treatment on the ELISA plate.
In the embodiment of the invention, the enzyme label plate is prepared by taking polystyrene as a raw material, the surface is glittering and translucent, the plate surface is flat and smooth, the polystyrene is in a head-tail structure, the main chain is a saturated carbon chain, and the side group is a conjugated benzene ring, so that the molecular structure is irregular, and the rigidity of the molecule is increased, therefore, the polystyrene is an amorphous linear polymer.
Polystyrene has a high Tg value due to the presence of benzene rings, is transparent and rigid at room temperature, and is susceptible to stress cracking due to the rigidity of the molecular chains. The polystyrene is colorless and transparent, can be freely colored, has high relative density, excellent electrical properties and particularly good high-frequency characteristics. In addition, polystyrene is good in light stability, and radiation resistance is the strongest of all plastics. Polystyrene has excellent thermal stability and fluidity when melted, so that it is easy to mold and process, and in particular, injection molding is easy, the molding shrinkage is small, and the dimensional stability of the molded product is also excellent, and it is suitable for mass production.
In the step S2, the plasma generated by the discharge gas is adopted to clean the surface of the ELISA plate, and a large amount of ions, excited molecules, free radicals and other active particles in the plasma act on the surface of the ELISA plate to remove the original pollutants and impurities on the surface of the ELISA plate. The particle energy in the plasma is 0-20eV, the material of the ELISA plate is polystyrene, most bonds in the polystyrene are 0-10eV, and therefore, after the plasma acts on the surface of the ELISA plate, the original chemical bonds on the surface of the ELISA plate can be broken, and free radicals in the plasma and the broken chemical bonds form a net-shaped cross-linking structure, so that the surface activity of the ELISA plate is greatly activated.
In the step S3, reaction gas is introduced into the plasma surface treatment device, the reaction gas ionizes active groups including amino groups and the like in the plasma surface treatment device, the active groups carry out surface hydrophilic modification treatment on the elisa plate on the surface of the elisa plate, complex chemical reactions can occur on the surface of the activated material, new functional groups are introduced, the surface activity of the elisa plate can be obviously improved, and the surface hydrophilic capability of the elisa plate is effectively improved.
The invention improves the atmospheric pressure glow discharge area, and the glow discharge area is supplemented with ionizable gas including oxygen and ammonia gas to generate more charges to be processed by plasma surface processing equipment, thereby increasing the hydrophilicity of the surface of the enzyme label plate.
The ELISA plate has positively charged amino groups after surface modification treatment, and hydrophobic bonds of the ELISA plate are replaced by hydrophilic bonds, so that the ELISA plate is suitable for being used as a solid phase carrier of micromolecular protein. Under appropriate buffer and pH value, the surface of the enzyme label plate can be combined with the protein with negative charge through ionic bonds. The elisa plate after surface hydrophilic modification has high sensitivity to protein in the experimental process, and can reduce the concentration and dosage of the coated protein.
Preferably, in step S1, the polystyrene beads and steam are continuously mixed in a pre-foaming machine under stirring in a stirring tank to complete the foaming reaction, and the pre-foaming machine is kept open at normal pressure, and the expanded polystyrene beads overflow from the top of the pre-foaming machine.
Preferably, after the polystyrene beads are expanded, a curing treatment is performed to incorporate air into the cells to obtain cured pre-expanded beads.
Preferably, the cured pre-expanded beads are placed into a molding device for molding treatment, so as to obtain the elisa plate.
For small and complex products, a venturi action device such as a filler gun is used to blow the beads into the mold cavity by means of an air stream. For large products, the mold cavity can be filled by the self gravity. The mold cavity filled with the pellets is closed and heated and the beads are softened by the heat to expand the cells. The beads expand to fill the interstices between them and bond into a uniform foam. This foam is still soft at this point and is subjected to the pressure of hot gases within the cells. Before the product is taken out of the mold, the gas is permeated out of the cells and the temperature is reduced by spraying water to the inner wall of the mold to stabilize the shape of the product. The molding apparatus selects a mold having a specific cavity, in this embodiment, a double-walled layer of aluminum and cast in the shape of a microplate. The size of the inner wall of the mould is the size of the actual product, and the inner wall of the mould is provided with air holes so that steam penetrates through the foam body and hot air is diffused out. The space between the double walls forms a steam chamber into which steam is passed for heating the beads. In this example, the molding pressure of expanded polystyrene was lower than 276 kPa. The expanded polystyrene has low molding pressure and low molding equipment cost, so the method is an economic production method.
Preferably, in step S3, the reaction gas is oxygen, ammonia, or a mixture of oxygen and ammonia.
Preferably, the discharge gas is compressed air, the compressed air is introduced into the plasma surface treatment equipment and ionized to generate plasma, and the surface of the elisa plate is cleaned.
Preferably, in step S3, a reactive gas is introduced into the plasma surface treatment device, the reactive gas ionizes in the plasma surface treatment device to form active groups, and the active groups perform surface hydrophilic modification treatment on the elisa plate on the surface of the elisa plate.
Preferably, in step S2, the washing time of the ELISA plate is 10-20 min. The embodiment of the invention adopts vacuum plasma to carry out surface hydrophilic treatment on the ELISA plate, and compared with some traditional cleaning methods such as ultrasonic cleaning and UV cleaning, the method has the following advantages:
1. the treatment temperature is low: in this embodiment, the temperature can be controlled to be 20-40 ℃. The low treatment temperature ensures that the surface of the polystyrene disposable enzyme label plate is not affected by heat.
2. The whole treatment process is pollution-free: the plasma surface treatment equipment is environment-friendly, and does not produce any pollution, and the treatment process does not produce any pollution. Therefore, the automatic production line can be matched with the original production line, and full-automatic online production is realized, so that the labor cost is saved.
3. The treatment effect is stable: the plasma cleaning treatment effect is very uniform and stable, and the treatment effect is kept good for a long time after the conventional sample is treated.
4. The applicability is wide: for samples of complex shape, plasma cleaning can find a suitable solution. In addition, the vacuum plasma cleaning can also realize the cleaning of specific positions of the solid sample, including the inner wall of the inner hole, and the cleaning is carried out uniformly in all directions.
Preferably, in step S3, the time period of the surface hydrophilic modification treatment is 10-20 min.
Preferably, in steps S2 and S3, the vacuum degree is 0.02-0.03mbar, the processing temperature is 30-40 ℃, the processing power is 600-. In this embodiment, the plasma surface equipment adopts an original imported power supply, and has comprehensive safety protection functions such as a temperature safety protection function, an overload protection function, a short circuit and disconnection alarm protection function, various misoperation protection functions and the like. Meanwhile, the equipment also adopts the design and the manufacturing process of a military-grade high-vacuum-degree vacuum cavity and is provided with an import technology vacuum pump for operation. The gas flow of the equipment is set and accurately monitored and displayed on the touch screen, so that the gas source can be saved, and the whole machine program is automatic.
Based on the above technical solutions, the present invention provides the following embodiments.
Example 1
The embodiment provides a production process of an ELISA plate with high binding force, which comprises the following steps:
s1: the polystyrene beads and steam were continuously mixed in a pre-expander like mixer with stirring in a stirred tank to complete the foaming reaction. The image prefoamer is kept open at normal pressure, and the polystyrene beads which are completely foamed overflow from the top end of the image prefoamer. After the polystyrene beads were expanded, curing treatment was performed to incorporate air into the cells, thereby obtaining cured pre-expanded beads. And placing the cured pre-expanded beads into a molding device for molding treatment to obtain the 12 x 8 enzyme label plate with 96 holes.
S2: and (3) placing the elisa plate prepared in the step (S1) in plasma surface treatment equipment, opening a reaction gas inlet valve 2 and an exhaust valve 5, introducing compressed air into the plasma surface treatment equipment, ionizing the compressed air to generate plasma, keeping the vacuum degree at 0.02mbar, the treatment temperature at 35 ℃, the treatment power at 800W, the gas flow at 200sccm and the exhaust gas volume at 380000Mt, and carrying out surface cleaning on the elisa plate for 15 min. A large amount of active particles such as ions, excited molecules, free radicals and the like in the plasma act on the surface of the ELISA plate, and original pollutants and impurities on the surface of the ELISA plate are removed. After the plasma acts on the surface of the enzyme label plate, the original chemical bonds on the surface of the enzyme label plate are broken, and free radicals in the plasma and the broken chemical bonds form a net-shaped cross-linking structure, so that the surface activity is activated.
S3: and opening an oxygen gas inlet valve 3 and an ammonia gas inlet valve 4, supplementing oxygen gas and ammonia gas into the plasma surface treatment equipment 1, keeping the vacuum degree at 10mbar, the treatment temperature at 21.5 ℃, the treatment power at 800W, the gas flow at 150sccm and the exhaust gas at 453883.1Mt, and carrying out surface hydrophilic treatment on the ELISA plate for 15 min. Active groups, including amino and the like, are ionized by oxygen and ammonia gas, the active groups carry out surface hydrophilic modification treatment on the ELISA plate on the surface of the ELISA plate, complex chemical reaction can occur on the surface of the activated material, new functional groups are introduced, the surface activity of the ELISA plate can be obviously improved, and the surface hydrophilic capability of the ELISA plate is effectively improved.
The enzyme-linked immunosorbent assay is carried out on the high-binding-force ELISA plate prepared in example 1, and the assay method specifically comprises the following steps:
the experimental method comprises the following steps: the same human tumor necrosis factor alpha antibody is used, and a high-binding-force enzyme label plate is used as a solid phase carrier for enzyme-linked immunosorbent assay.
The experimental materials used: the kit comprises a human tumor necrosis factor alpha antibody, a special enzyme-linked immunosorbent coating solution, a confining solution, a special enzyme-linked immunosorbent washing solution-10 PBST, a special enzyme-linked immunosorbent TMB single component, a special enzyme-linked immunosorbent stop solution and an enzyme-linked microplate reader.
And (3) experimental operation:
antigen coating: the antigen coating concentration was determined to be 1. mu.g/ml, and 100. mu.l was added to each well of a 96-well plate and stored at 37 ℃ for 1 hour. And (3) sealing: the supernatant was discarded, washed three times with PBST, and 200. mu.l of blocking solution was added to each well, followed by standing at 37 ℃ for 1-2 hours.
Washing: washed three times with PBST and patted dry.
Adding a primary antibody: serum was taken and treated with PBS 1: 625, and (4) diluting. After adding 100. mu.l of PBS to each well from the second row in the microplate, 200. mu.l of each diluted serum was added to the first row and 100. mu.l was pipetted into the second well, and 100. mu.l was pipetted into the next well after 8 times of repeated blowings with a gun. By analogy, dilute to 12 th well and discard 100. mu.l from 12 th well, after completion, work in 37 ℃ incubator for 1-2 h.
Fourthly, washing: washed three times with PBST and patted dry.
Adding an enzyme-labeled secondary antibody: and taking a secondary anti-mouse antibody and an HRP-labeled goat anti-mouse antibody. According to the following steps: 5000 dilution, 100. mu.l per well, 1h at 37 ℃.
Sixthly, washing: washing: wash 3 times with PBST and pat dry.
And color development: 100 mu l of TMP developing solution is added into each hole of the enzyme label plate, the incubator is operated for 7-8min at 37 ℃, and a positive result is displayed as blue.
And stopping the step of: 30. mu.l of 2M H was added to each well of the microplate2SO4Upon termination, the solution turned yellow.
Ninthly, determination: and (3) measuring the OD value at 450nm/620nm by using a microplate reader, and judging the test hole/blank hole to be positive if the value is more than 2.1 and the value is not less than 0.2. The concentration of tnf α antibody in each well of a 12 × 8 96 well plate is shown in table 1.
Table 1 concentration of tnf α antibody in each well of the high binding 96-well plate prepared in example 1
The average concentration of the tnf α antibody in each well of the above 96-well plate was: 594.20pg/ml, standard deviation: 625.34pg/ml, CV value mean concentration ÷ standard deviation ÷ 0.95.
Comparative example 1
The comparison example performs enzyme-linked immunosorbent assay on a common enzyme label plate, and comprises the following specific steps:
the experimental method comprises the following steps: the same human tumor necrosis factor alpha antibody is used and the common enzyme label plate in the prior art is used as a solid phase carrier to carry out enzyme-linked immunosorbent assay reaction.
The experimental materials used: the kit comprises a human tumor necrosis factor alpha antibody, a special enzyme-linked immunosorbent coating solution, a confining solution, a special enzyme-linked immunosorbent washing solution-10 PBST, a special enzyme-linked immunosorbent TMB single component, a special enzyme-linked immunosorbent stop solution and an enzyme-linked microplate reader.
And (3) experimental operation:
antigen coating: the antigen coating concentration was determined to be 1. mu.g/ml, and 100. mu.l was added to each well of a 96-well plate and stored at 37 ℃ for 1 hour. And (3) sealing: the supernatant was discarded, washed three times with PBST, and 200. mu.l of blocking solution was added to each well, followed by standing at 37 ℃ for 1-2 hours.
Washing: washed three times with PBST and patted dry.
Adding a primary antibody: serum was taken and treated with PBS 1: 625, and (4) diluting. After adding 100. mu.l of PBS to each well from the second row in the microplate, 200. mu.l of each diluted serum was added to the first row and 100. mu.l was pipetted into the second well, and 100. mu.l was pipetted into the next well after 8 times of repeated blowings with a gun. By analogy, dilute to 12 th well and discard 100. mu.l from 12 th well, after completion, work in 37 ℃ incubator for 1-2 h.
Fourthly, washing: washed three times with PBST and patted dry.
Adding an enzyme-labeled secondary antibody: and taking a secondary anti-mouse antibody and an HRP-labeled goat anti-mouse antibody. According to the following steps: 5000 dilution, 100. mu.l per well, 1h at 37 ℃.
Sixthly, washing: washing: wash 3 times with PBST and pat dry.
And color development: 100 mu l of TMP developing solution is added into each hole of the enzyme label plate, the incubator is operated for 7-8min at 37 ℃, and a positive result is displayed as blue.
And stopping the step of: 30. mu.l of 2M H was added to each well of the microplate2SO4Upon termination, the solution turned yellow.
Ninthly, determination: and (3) measuring the OD value at 450nm/620nm by using a microplate reader, and judging the test hole/blank hole to be positive if the value is more than 2.1 and the value is not less than 0.2. The concentration of tnf α antibody in each well of a 12 × 8 96 well plate is shown in table 2.
TABLE 2 concentration of TNF-. alpha.antibody in each well of a common 96-well plate
| A | B | C | D | E | F | G | H | |
| 1 | 2052.99 | 385.6 | 370.79 | 351.43 | 341.18 | 352.57 | 419.77 | 348.01 |
| 2 | 2006.3 | 425.46 | 417.49 | 368.52 | 424.32 | 351.43 | 406.1 | 366.24 |
| 3 | 906.09 | 369.65 | 341.18 | 377.63 | 371.93 | 367.88 | 361.68 | 432.3 |
| 4 | 935.7 | 386.74 | 381.04 | 369.65 | 403.82 | 353.71 | 360.54 | 407.24 |
| 5 | 499.49 | 395.85 | 357.13 | 345.74 | 417.49 | 434.57 | 351.43 | 379.9 |
| 6 | 504.05 | 407.24 | 355.99 | 349.15 | 398.13 | 436.85 | 345.74 | 365.1 |
| 7 | 289.93 | 363.96 | 386.74 | 365.1 | 382.18 | 419.77 | 415.21 | 424.32 |
| 8 | 280.82 | 433.43 | 351.43 | 436.85 | 433.43 | 365.1 | 366.24 | 346.88 |
| 9 | 136.17 | 365.1 | 400.41 | 367.38 | 412.93 | 394.71 | 420.91 | 362.82 |
| 10 | 138.45 | 350.29 | 409.52 | 365.1 | 341.18 | 342.32 | 345.74 | 423.18 |
| 11 | -1.64 | 395.85 | 385.6 | 394.71 | 348.01 | 396.99 | 428.88 | 393.57 |
| 12 | 1.78 | 389.02 | 419.77 | 395.4 | 410.66 | 341.18 | 393.57 | 344.6 |
The average concentration of the tnf α antibody in each well of the above 96-well plate was: 415.98pg/ml, standard deviation 201.34pg/ml, CV value mean concentration ÷ standard deviation 2.06.
As can be seen from the analysis of table 1 and table 2: compared with the comparative example 1, the CV value of the high-binding-force ELISA plate prepared in the embodiment 1 of the invention and protein is smaller, and the binding is more stable, so that the high-binding-force ELISA plate has higher protein binding force and more stable binding force than the common ELISA plate.
The above embodiments are merely illustrative, and not restrictive, of the scope of the invention, and those skilled in the art will be able to make various changes and modifications within the scope of the appended claims without departing from the spirit of the invention.
Claims (10)
1. The production process of the high-binding-force ELISA plate is characterized by comprising the following steps of:
s1: foaming, curing and molding expandable polystyrene to prepare a foamed polystyrene elisa plate;
s2: placing the ELISA plate prepared in the step S1 in plasma surface treatment equipment, introducing first gas, and performing surface cleaning and activating treatment on the ELISA plate;
s3: and supplementing a second gas into the plasma surface treatment equipment, and ionizing positive and negative charges in the plasma surface treatment equipment by using the reaction gas to generate groups with positive charges on the surface of the ELISA plate so as to realize surface hydrophilic modification treatment on the ELISA plate.
2. The microplate production process of claim 1, wherein in step S1, polystyrene beads and steam are continuously mixed in an image prefoamer to complete the foaming reaction, the image prefoamer is kept open at normal pressure, and the expanded polystyrene beads overflow from the top of the image prefoamer.
3. The process for producing an microplate according to claim 2, wherein after the polystyrene beads are foamed, a curing treatment is performed to incorporate air into the cells to obtain cured pre-foamed beads.
4. The elisa plate production process according to claim 1 or 3, wherein the cured pre-expanded beads are placed into a molding device for molding treatment to obtain the elisa plate.
5. The microplate production process of claim 1, wherein in step S3, the second gas is a mixture of oxygen and ammonia.
6. The elisa plate production process of claim 1, wherein in step S2, the first gas is compressed air, and the compressed air is introduced into the plasma surface treatment device, and the compressed air is ionized to generate plasma, thereby performing surface cleaning on the elisa plate.
7. The process of claim 5, wherein in step S3, a second gas is introduced into the plasma surface treatment device, the second gas ionizes in the plasma surface treatment device to form active groups, and the active groups perform surface hydrophilic modification treatment on the ELISA plate on the surface of the ELISA plate.
8. The process for producing an ELISA plate according to claim 1 wherein in step S2, the washing time of the ELISA plate is 10-20 min.
9. The microplate production process of claim 1, wherein in step S3, the surface hydrophilic modification treatment time is 10-20 min.
10. The process of claim 1, wherein in steps S2 and S3, the vacuum degree is 0.02-0.03mbar, the processing temperature is 30-40 ℃, the processing power is 600-1000W, the gas flow rate is 100-300sccm, and the exhaust gas amount is 350000-400000 Mt.
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| CN114559677A (en) * | 2022-03-01 | 2022-05-31 | 无锡国盛生物工程股份有限公司 | ELISA plate for preventing cross contamination of ELISA plate holes and production process thereof |
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