CN210620670U - Antibody extraction device - Google Patents
Antibody extraction device Download PDFInfo
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- CN210620670U CN210620670U CN201920644992.XU CN201920644992U CN210620670U CN 210620670 U CN210620670 U CN 210620670U CN 201920644992 U CN201920644992 U CN 201920644992U CN 210620670 U CN210620670 U CN 210620670U
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Abstract
The utility model discloses an antibody extraction element. Specifically, the utility model discloses an antibody extraction element includes the preliminary treatment module, incubates dissociation module and neutralization purification module, the utility model discloses a device is applicable to the antibody of extracting cell surface antigen, and is easy and simple to handle, and low cost easily promotes.
Description
Technical Field
The utility model relates to the field of biotechnology, in particular to draw device of anti cell surface antigen's antibody.
Background
The cell membrane is a barrier for preventing extracellular substances from freely entering cells, and ensures the relative stability of the intracellular environment, so that various biochemical reactions can be orderly operated. However, the cells must exchange information, substances and energy with the surrounding environment to perform a specific physiological function, and therefore, the cells must have a substance transport system for obtaining desired substances and discharging metabolic wastes. It is estimated that the proteins on the cell membrane involved in substance transport account for 15-30% of the proteins encoded by nuclear genes, and the energy used by the cells in substance transport amounts to two thirds of the total energy consumed by the cells.
Cell membranes play an important role in maintaining cell permeability, physiological signal transduction and other physiological and pathological states of human bodies. The structure and function of cell membrane molecules are leading hot spots in life science research in recent years. But how to identify cell membrane molecules is also a current difficulty. Antibodies are widely used for screening and identifying target antigen molecules due to their affinity binding type with specificity to antigen receptors. An antibody directed against the cell surface (membrane) is an antibody with a native conformation, which has a unique advantage for recognizing cell membrane antigens. However, the prior art antibody extraction devices have some disadvantages, and there is a need in the art to develop new devices for extracting antibodies against cell surface antigens.
SUMMERY OF THE UTILITY MODEL
An object of the utility model is to provide an extract device of anti cell surface antigen's antibody, the utility model discloses an antibody extraction device includes pretreatment module, incubates dissociation module and neutralization purification module, the utility model discloses a device is applicable to the antibody of extracting cell surface antigen, and is easy and simple to handle, low cost easily promotes.
Compared with the classical method for extracting serum antibodies by using natural protein A/G medium, the device for extracting the antibodies has higher specificity, only adsorbs the antibodies aiming at the molecules on the surface of the cell membrane, and has unique advantages for the subsequent research on the structure and the function of the molecules of the cell membrane. And the price is low, so that the method is a practical antibody purification technology.
Specifically, the utility model relates to a device for extracting anti cell surface antigen's antibody, the device includes:
the pretreatment module comprises a separation bag for separating a serum sample, and a serum storage bag and a platelet storage bag which are connected with an input port of the separation bag through a connecting hose;
the incubation and dissociation module comprises a reaction processing bag for collecting the first supernatant separated from the separation bag and incubating and dissociating the first supernatant, and a mammalian cell storage bag, a rinsing liquid storage bag and an eluent storage bag which are connected with an input port of the reaction processing bag through a connecting hose; and
and the neutralization and purification module comprises an ultrafiltration bottle for performing ultrafiltration on the second supernatant outputted by the reaction treatment bag, and a neutralization solution storage bag connected with an input port of the ultrafiltration bottle through a connecting hose.
In another preferred embodiment, the pretreatment module, the incubation dissociation module and the neutralization purification module are positioned in the shell.
In another preferred embodiment, the output port of the separation bag is connected to the reaction processing bag through a connection hose.
In another preferred example, the output port of the separation bag is provided with a first filter.
In another preferred example, the reaction processing bag is provided with a waste liquid outlet and a supernatant liquid outlet, the supernatant liquid outlet is connected with the input port of the ultrafiltration bottle, and the waste liquid outlet is connected with the waste liquid bottle.
In another preferred embodiment, the waste liquid outlet is provided with a second filter.
In another preferred example, the supernatant outlet is provided with a third filter.
In another preferred example, the output port of the ultrafiltration bottle is connected with the product storage bottle through a connecting hose.
In another preferred example, the device further comprises a numerical control pump for driving the liquid to flow.
In another preferred example, the connecting hose is provided with an electromagnetic valve for controlling the liquid flow.
In another preferred embodiment, the serum storage bag is used for infusing serum into the separation bag.
In another preferred embodiment, the platelet storage bag is used for transfusion of platelets into the separation bag.
In another preferred embodiment, the mammalian cell storage bag is used for infusing mammalian cells, preferably human umbilical cord vascular endothelial cells, more preferably human umbilical cord venous vascular endothelial cells into the reaction processing bag.
In another preferred example, the rinsing liquid storage bag is used for infusing rinsing liquid into the reaction processing bag.
In another preferred embodiment, the eluent storage bag is used for infusing antibody eluent into the reaction processing bag.
In another preferred embodiment, the neutralizing solution storage bag is used for infusing the neutralizing solution into the ultrafiltration bottle.
In another preferred embodiment, the ultrafiltration bottle is used for neutralizing the second supernatant and removing the small molecules in the mixed solution by ultrafiltration.
In another preferred embodiment, the product storage bottle is used for storing extracted antibodies against cell surface antigens.
In another preferred embodiment, the reaction processing bag is disposed in an oscillator device.
Compared with the prior art, the utility model has the advantages of:
antibody extraction element operating method simple and convenient, low cost not only can avoid human factor and environmental factor to the pollution of purification process, can high-efficient quick purification antibody moreover. The utility model discloses convenient and fast gets rid of the velocity of flow of artificial interference purification, and antibody purification process can be stabilized to the flow, improves antibody purification efficiency. The antibody specificity of the device separation of the utility model is high, and only the antibody aiming at the cell membrane surface molecule is absorbed.
It is understood that within the scope of the present invention, the above-mentioned technical features of the present invention and those specifically described below (e.g. in the examples) can be combined with each other to constitute new or preferred technical solutions. Not to be reiterated herein, but to the extent of space.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
Fig. 1 is a schematic structural diagram of a preferred embodiment of the present invention.
FIG. 2 shows the results of ELISA assays for the degree of enrichment of cell membrane surface antibodies.
Detailed Description
The utility model relates to a device for extracting anti-cell surface antigen's antibody. Antibody extraction element operating method simple and convenient, low cost not only can avoid human factor and environmental factor to the pollution of purification process, can high-efficient quick purification antibody moreover. The utility model discloses convenient and fast gets rid of the velocity of flow of artificial interference purification, and antibody purification process can be stabilized to the flow, improves antibody purification efficiency. The antibody specificity of the device separation of the utility model is high, and only the antibody aiming at the cell membrane surface molecule is absorbed. On this basis, the utility model discloses has been accomplished.
Term(s) for
As used herein, the terms "preferred," "optimal," "exemplary," or "optionally" do not limit the scope of the invention or its embodiments.
When a series of values is recited, it is merely for convenience or brevity and includes all possible data ranges and individual values within or around the limits of that range. Any numerical value, unless otherwise specified, also includes actual approximations, and integer values do not exclude fractional values. Subranges and actual approximations should not be construed as specifically disclosed values.
Reference to an object in the singular does not exclude the plural unless the context indicates otherwise.
Principle of operation
After specific antibodies (IgG antibodies are the main) in human serum are incubated with mammalian cells, certain antibodies can be specifically combined with antigens on the surface of cell membranes, after centrifugation and cell rinsing liquid are used for rinsing the cells, only the antibodies are firmly combined on the surface of the cell membranes, the antibodies and the antigens on the surface of the cell membranes are combined under a weak acid condition to form a separation state, eluent can be added into neutralizing liquid after centrifugation, and the pH value of an antibody solution is recovered to be neutral. Adding the mixed solution containing the antibody into an ultrafiltration centrifugal tube (the centrifugal tube has the function of a molecular sieve, the antibody molecules can be intercepted, impurities such as small molecules and the like can be filtered out through centrifugation, and adding Phosphate Buffer Solution (PBS) with pH of 7.0-7.4 to repeatedly wash for 2-3 times to achieve the purpose of purifying the antibody.
Application method
The method for extracting the antibody aiming at the surface of the cell membrane by using the device of the utility model comprises the following steps:
(a) pretreatment: providing a plurality of (such as 10) serum samples, mixing and incubating the serum samples with a 20% concentration healthy human platelet solution to obtain a first mixed solution, and centrifuging the first mixed solution to obtain a first supernatant;
(b) and (3) incubation: mixing and incubating the first supernatant with mammalian cells (such as human umbilical vein endothelial cells) to obtain a second mixed solution, and centrifuging the second mixed solution to obtain a cell precipitate;
(c) rinsing: rinsing the cell sediment with a cell rinsing solution to obtain cells with antibody bound on the surface of the cell membrane;
(d) dissociation: mixing the cells obtained in the last step with the antibody eluent to obtain a third mixed solution, and centrifuging the third mixed solution to obtain a second supernatant;
(e) neutralizing: mixing the second supernatant with a neutralizing solution to obtain a fourth mixed solution; and
(f) and (3) purification: and (4) purifying (ultra-filtration and centrifugation) the fourth mixed solution to remove small molecules in the fourth mixed solution, thereby obtaining the antibody aiming at the cell membrane surface.
Key reagent
The utility model discloses a key reagent that device used when using as follows:
1. the cell rinsing solution 0.01M, pH 7.4.4 Phosphate Buffer Solution (PBS) and 2% calf serum solution, the reagent is used for suspending and rinsing living mammalian cells (such as human umbilical cord vascular endothelial cells), and can ensure that the isolated cells are kept in fresh and living state during centrifugation, standing and the like.
2. Cell antibody eluent 0.1M, pH 3.0 citric acid solution
The reagent is used for separating and eluting the specific antibody adsorbed on the surface of the cell membrane from the antigen on the cell membrane under the acidic condition.
3. Neutralization pH 9.0Tris/HCl solution
The reagent has the effect that an antibody eluted by 0.1M citric acid solution with the pH of 3.0 can keep the biological activity of the antibody only by returning to a neutral condition (namely about pH 7.0) as soon as possible, and the antibody solution eluted under the acidic condition is added into a Tris/Hcl solution with the pH of 9.0 of a neutralization solution, so that the antibody is placed in a neutral environment to keep the biological activity of the antibody.
Identification of antibody concentration and purity
1. Determination of antibody concentration
Since antibodies are also proteins, the concentration of the antibodies is determined by a conventional protein determination method, generally by the Bradford method, and the concentration is calculated by measuring the absorbance value by a visible ultraviolet spectrometer.
2. Determination of antibody purity
Antibody purity can be characterized with reference to protein electrophoretic separation methods, since IgG antibodies typically have 2 heavy chains, 50kD (kilodaltons) per chain; 2 light chains, each chain 25kD (kilodaltons). After separation by polyacrylamide gel electrophoresis (SDS-PAGE), under denaturing conditions, a clear band at the 50kD position (antibody heavy chain) and a clear band at the 25kD position (antibody light chain) of the separation gel are respectively seen after Coomassie brilliant blue staining, and no other miscellaneous bands or weaker miscellaneous bands are found.
HLA-class I antibodies
The HLA-I antibody is a human leukocyte differentiation antigen class I antibody, and because the HLA-I antigen is expressed on the surface of human nucleated cells, the HLA-I antibody is used as a positive reference control when detecting cell membrane surface antibodies by flow cytometry. If an antigen specifically bound to the cell membrane surface needs to be separated and identified, in general, a mixed human platelet solution (with a concentration of 20%) is incubated with human serum to be detected, and the HLA-I antibody in the human serum is adsorbed and then the experiment is carried out.
Comparison with the prior art
The comparison of the antibodies separated with the device of the present invention with the antibodies separated with the native protein a and G resin (protein a/G resin) of the prior art is as follows:
utilize the utility model discloses a device, with living mammal cell (like human vein vascular endothelial cell), adsorb patient's serum antibody, can obtain a class of antibody on specificity adsorption cell membrane surface, this type of antibody is mostly the antibody that is natural conformation, can pass through flow cytometry appraisal, can further can appraise the cell membrane molecule through co-immunoprecipitation technique binding protein mass spectrum method to can accurately appraise autoimmune disease, organ transplantation immunological rejection's cell membrane antigen molecule. And the protein A/G resin is used for adsorbing the antibody in the human serum, and the antibody in the human serum contains the denatured antibody in the natural conformation because of more types, so that accurate information is difficult to obtain if the marker in the serum needs to be identified.
The utility model discloses a main advantage includes:
(a) the device of the utility model has simple and convenient operation method and low cost.
(b) The antibody specificity of the device separation of the utility model is high, and only the antibody aiming at the cell membrane surface molecule is absorbed.
The present invention will be further described with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Example 1
Antibody extraction device
As shown in fig. 1, the device of the present invention comprises a housing 1, and a pretreatment module, an incubation dissociation module, and a neutralization purification module located in the housing 1.
The pretreatment module comprises a separation bag 2 for separating a serum sample, and a serum storage bag 3 and a platelet storage bag 4 which are connected with an input port of the separation bag 2 through connecting hoses. And a first filter is arranged at the output port of the separation bag 2.
And the incubation and dissociation module comprises a reaction processing bag 5 for collecting, incubating and dissociating the first supernatant separated from the separation bag 2, and a mammalian cell storage bag 6, a rinsing liquid storage bag 7 and an eluent storage bag 8 which are connected with an input port of the reaction processing bag 5 through connecting hoses. Wherein, the reaction processing bag 5 is also provided with a waste liquid outlet and a supernatant outlet, and the waste liquid outlet is connected with the waste liquid bottle 9. And the waste liquid discharge port is provided with a second filter, and the supernatant discharge port is provided with a third filter.
And the neutralization and purification module comprises an ultrafiltration bottle 10 for performing ultrafiltration on the second supernatant outputted by the reaction treatment bag 5 and a neutralization solution storage bag 11 connected with an input port of the ultrafiltration bottle 10 through a connecting hose, wherein the input port of the ultrafiltration bottle 10 is connected with a supernatant discharge port of the reaction treatment bag 5, and an output port of the ultrafiltration bottle 10 is connected with a product storage bottle 12 through a connecting hose.
Example 2
Use of the device
(A) Material
The kit comprises a human umbilical vein endothelial cell line, patient serum of autoimmune liver disease, Tris/HCl solution, sodium citrate solution, an ultrafiltration centrifugal tube, HLA-I antibodies, an ELISA microporous plate, PE-labeled goat anti-human IgG antibodies, a protein vertical electrophoresis device and related reagents.
(II) method
1. Taking 6 cases of serum of patients with autoimmune liver diseases, placing the 6 cases of serum into a serum storage bag 3 of the device for multiple times, and removing HLA antibodies by utilizing a pretreatment module of the device;
2. culturing human umbilical vascular endothelial cells, taking the cells to be placed in PBS (containing 2% calf serum) after logarithmic growth phase, and placing the cells in a mammalian cell storage bag 6 of the device;
3. by 5X 106Mixing the vascular endothelial cells and 100 μ l of serum (i.e. supernatant without HLA class antibody) of autoimmune liver disease patient in reaction treatment bag 5, incubating at room temperature (15-25 deg.C) for 1 hr under shaking, and removing supernatant in reaction treatment bag 5 with filter to obtain cells adsorbed with serum antibody;
4. adding pre-cooled PBS (0.01M, pH 7.4) stored in a rinsing solution storage bag 7 into the reaction processing bag 5, rinsing the cells for 3 times, and washing away non-specifically bound impurities to obtain rinsed cells adsorbed with serum antibodies;
5. adding a citric acid solution (0.01M, pH 3.0) stored in an eluent storage bag 8 into a reaction treatment bag 5, incubating with the cells adsorbed with the serum antibody, gently mixing uniformly for 5 minutes at room temperature, filtering by using a filter, conveying the supernatant into an ultrafiltration bottle 10, adding a Tris/HCl solution with pH 9.0 stored in a neutralization solution storage bag 11 into the ultrafiltration bottle 10, and neutralizing to neutrality to obtain a neutral mixed solution;
6. carrying out ultrafiltration on the neutral mixed solution in the ultrafiltration bottle 10 to remove small molecular impurities so as to obtain a purified antibody;
7. the purified antibody is stored in a medium product storage bottle 12, and the purity of the extracted antibody is identified by SDS-polyacrylamide gel electrophoresis; measuring the concentration of the antibody by using an ultraviolet spectrophotometer; measuring the enrichment degree of the antibody by an ELISA method; the specificity of the antibody for binding of cell surface molecules was determined by flow cytometry.
The results of the ELISA assay for the degree of enrichment of cell membrane surface antibodies are shown in FIG. 2.
Analysis of the binding capacity of the adsorbed and eluted antibodies to the membrane surface of the target cells (data measured by flow cytometry) is shown in the following table.
| Serum | Mean Fluorescence Intensity (MFI) |
| Human serum for health | 6.21 |
| HLA-I positive reference serum | 122.98 |
| Adsorbing serum for 1 time for patients with chronic immunological liver diseases | 44.11 |
| Adsorbing serum for 2 times for patients with chronic immunological liver diseases | 72.34 |
| Eluting for 1 time after serum adsorption for patients with chronic immunological liver diseases | 49.58 |
| 2 nd time elution after serum adsorption for patients with chronic immunological liver diseases | 74.99 |
All documents mentioned in this application are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims.
Claims (10)
1. An antibody extraction device, comprising:
a pre-processing module comprising a separation bag (2) for separating a serum sample, and a serum storage bag (3) and a platelet storage bag (4) connected to an input port of the separation bag (2) by a first connection hose;
the incubation and dissociation module comprises a reaction processing bag (5) for collecting and incubating and dissociating the first supernatant separated from the separation bag (2), a mammalian cell storage bag (6) connected with an input port of the reaction processing bag (5) through a third connecting hose, a rinsing liquid storage bag (7) connected with an input port of the reaction processing bag (5) through a fourth connecting hose and an eluent storage bag (8) connected with an input port of the reaction processing bag (5) through a fifth connecting hose; and
and the neutralization and purification module comprises an ultrafiltration bottle (10) for performing ultrafiltration on the second supernatant outputted by the reaction treatment bag (5), and a neutralization solution storage bag (11) connected with an input port of the ultrafiltration bottle (10) through an eighth connecting hose.
2. The device according to claim 1, wherein the pretreatment module, the incubation dissociation module, and the neutralization purification module are located in the housing (1).
3. The device according to claim 1, wherein the outlet port of the separation bag (2) is connected to the reaction processing bag (5) by a second connecting hose.
4. A device according to claim 3, wherein the output port of the separation bag (2) is provided with a first filter.
5. The apparatus according to claim 1, wherein the reaction processing bag (5) is provided with a waste discharge port and a supernatant discharge port, and the supernatant discharge port is connected to the input port of the ultrafiltrating bottle (10) through a sixth connecting hose, and the waste discharge port is connected to the waste liquid bottle (9) through a seventh connecting hose.
6. The apparatus of claim 5, wherein said waste drain is provided with a second filter.
7. The apparatus of claim 5, wherein said supernatant discharge port is provided with a third filter.
8. The device according to claim 1, characterized in that the outlet port of the ultrafiltration bottle (10) is connected to a product storage bottle (12) by a ninth connecting hose.
9. The apparatus of claim 1, further comprising a digitally controlled pump for driving the flow of the liquid.
10. The apparatus of claim 1, wherein each of said connecting hoses is provided with a solenoid valve for controlling the flow of liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201920644992.XU CN210620670U (en) | 2019-05-07 | 2019-05-07 | Antibody extraction device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201920644992.XU CN210620670U (en) | 2019-05-07 | 2019-05-07 | Antibody extraction device |
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| CN210620670U true CN210620670U (en) | 2020-05-26 |
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| CN201920644992.XU Active CN210620670U (en) | 2019-05-07 | 2019-05-07 | Antibody extraction device |
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