CN108508195B - Immunomagnetic bead and preparation method and application thereof - Google Patents
Immunomagnetic bead and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an immunomagnetic bead, and a preparation method and application thereof. The preparation method of the immunomagnetic beads comprises the following steps: mixing adenosine monophosphate or metal salt thereof, aqueous phase buffer solution, ferroferric oxide, paraffin and span 80, and stirring to form an emulsifying system; the volume ratio of the paraffin to the span 80 is 7: 93-100; the mass ratio of the adenosine monophosphate or the metal salt thereof to the ferroferric oxide is 0.1-0.3: 0.2; adding glutaraldehyde into the emulsification system, and carrying out a first-step crosslinking reaction at a constant temperature; then adding antibody and zinc salt to produce second step cross-linking reaction, and separating solid with magnet. The preparation method simultaneously fixes the antibody and the ferroferric oxide in the metal-organogel solid system by utilizing the property of the metal-organogel immobilized protein, the reaction condition is mild, and special operation or complex pretreatment procedures are not needed, so that the preparation method expands favorable conditions for the wide application of the immunomagnetic beads.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to an immunomagnetic bead, and a preparation method and application thereof.
Background
Immunomagnetic beads are conjugates of superparamagnetic substances and antibodies, and can be used to bind to the corresponding antigen or antibody. Because the immunomagnetic beads can move directionally under the attraction of an external magnetic field, the immunomagnetic beads can be used for separating and detecting target objects, and the purpose of purifying genes, proteins, cells, microorganisms and the like is achieved. Due to the rapidity and specificity of the immunoreaction, the immunomagnetic beads have better sensitivity and specificity than the conventional detection or separation method.
The traditional preparation method of the immunomagnetic beads is complex and has harsh operating conditions, special functional groups need to be modified on the surfaces of paramagnetic substances so as to be convenient for combining antibodies or antigens, the modification process is complex and the reaction conditions are harsh.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a preparation method of immunomagnetic beads, which utilizes the property of metal-organic gel immobilized protein to simultaneously immobilize an antibody and ferroferric oxide in a metal-organic gel solid system, the reaction conditions are mild, and special operation or complex pretreatment procedures are not needed, so that the preparation method expands favorable conditions for the wide application of the immunomagnetic beads.
The second object of the present invention is to provide the use of the above immunomagnetic beads, which can be applied to a wide variety of antibodies, and thus can be applied to a wide variety of antigen types, including compounds of different molecular weights or different microorganisms.
In order to achieve the above purpose, the invention provides the following technical scheme:
a preparation method of immunomagnetic beads comprises the following steps:
step A: mixing adenosine monophosphate or metal salt thereof, aqueous phase buffer solution, ferroferric oxide, paraffin and span 80, and stirring to form an emulsifying system; wherein the volume ratio of the paraffin to the span 80 is 7: 93-100; the mass ratio of the adenosine monophosphate or the metal salt thereof to the ferroferric oxide is 0.1-0.3: 0.2;
and B: adding glutaraldehyde into the emulsification system, and carrying out a first-step crosslinking reaction at a constant temperature; then adding antibody and zinc salt to produce second step cross-linking reaction, and separating solid with magnet.
Firstly, connecting adenosine monophosphate or metal salt thereof with ferroferric oxide by using an emulsifier, and forming a water-in-oil uniform stable system; and then, taking glutaraldehyde as a cross-linking agent, cross-linking the antibody with adenosine monophosphate/adenosine monophosphate, and then cross-linking zinc ions in a network structure of the gel to finally form the solid macromolecular immunomagnetic beads. In conclusion, the immunomagnetic beads prepared by the method have the excellent characteristics of metal-organic gel, ferroferric oxide and antibodies, namely the characteristics of high specific surface, porous structure, superparamagnetism, reaction specificity, high selectivity and the like, and have higher antibody load than the traditional immunomagnetic beads, so that the immunomagnetic beads disclosed by the invention have far better performance than the traditional immunomagnetic beads and can be widely applied to separation, purification and detection of antibody action objects.
In addition, as mentioned above, all the steps of the preparation method of the present invention are completed at normal temperature and normal pressure (or near normal temperature), and no harsh operation conditions are required, which is more beneficial to protecting the activity of the antibody and easier to popularize.
The metal salt of adenosine monophosphate of the present invention is any salt, for example, sodium salt, potassium salt, etc.
In the invention, the volume ratio of the paraffin and the span 80 is important for the formation of an emulsification system, and any value in the range of 7: 93-100 can be adopted, such as 7:93, 7:94, 7:95, 7:96, 7:97, 7:98, 7:99, 7:100 and the like.
In the invention, the proportion of the adenosine monophosphate or the metal salt thereof to the ferroferric oxide has important influence on the stability of an emulsification system and the magnetism of immunomagnetic beads, and any mass ratio in the range of 0.1-0.3: 0.2 can be adopted, such as 0.1:0.2, 0.15:0.2, 1:1, 0.25:0.2, 0.1:0.3 and the like.
The invention is not limited by the type of the antibody, and the antibody can be antibodies of different action objects (antitoxin, antibacterial antibody, antiviral antibody and cytotropic antibody) or antibodies of different chemical structures (IgG, IgA, IgM, IgE and IgD), and the preparation methods of the immunomagnetic beads prepared by the antibodies are the same.
In addition, the preparation method can be further improved to simplify the process, improve the efficiency, increase the loading capacity or improve the physicochemical property of the immunomagnetic beads, and the like, which is concretely shown in the following.
Preferably, the pH value of the aqueous phase buffer solution is 7-8.
The emulsification reaction of the invention is suitably carried out in a neutral environment, and the buffer solution can be inorganic or organic buffer solution.
Preferably, the aqueous buffer is 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES buffer).
The emulsion under this organic buffer is more stable.
Preferably, the temperature of the first step crosslinking reaction is: 35-45 ℃.
Since the cross-linking of adenosine monophosphate and glutaraldehyde is mainly an amide reaction, the reaction rate is higher at 35-45 ℃ by using glutaraldehyde as a cross-linking agent, such as 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃ and the like.
Preferably, the temperature of the second-step crosslinking reaction is: 20 to 30 ℃.
In order to sufficiently retain the activity of the antibody, the temperature of the second step of the crosslinking reaction is 20 to 30 ℃, for example, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃ and the like.
Preferably, in the step B, the antibody and the zinc salt are added in a manner that: the antibody was added dropwise first, followed by the zinc salt.
The antibody, the zinc salt and the adenosine monophosphate are combined in different modes, the molecular weights of the antibody and the zinc salt are different, from the aspect of influence of steric hindrance on reaction, the zinc salt is added after the antibody is added, the loading capacity of the antibody and the combination capacity of the zinc are higher, and the dropwise addition mode is more favorable for full reaction.
Preferably, in the step B, the antibody is a zearalenone antibody, preferably zearalenone monoclonal antibody.
Preferably, the stirring mode during the formation of the emulsifying system is mechanical stirring, and the rotating speed is 300-1000 r/min. The stirring speed at this time has an important influence on the formation of the internal structure of the metal organic framework material, and influences the physicochemical properties such as the exposure of binding sites, the aperture, the specific surface area and the like.
Preferably, the molar ratio of adenosine monophosphate or a metal salt thereof to zinc ions in the zinc salt is 1: 1.
Preferably, the mass ratio of the zearalenone antibody to the adenosine monophosphate or the metal salt thereof is 19: 0.2 to 0.3.
Preferably, in the step B, the addition manner of glutaraldehyde in the step B is: adding glutaraldehyde solution with volume concentration of 20-30%.
Preferably, in the step B, the antibody and the zinc salt are added in a manner that: firstly, the antibody is dripped at the speed of 0.9-1.1 mg/min, and then Zn is dripped at the speed of 0.045-0.055 mmol/min2+The zinc salt is added dropwise at a rate of (1).
An immunomagnetic bead is prepared by the preparation method.
By SEM representation and infrared spectrum representation, the immunomagnetic beads are macromolecular compounds formed by combining AMP, zinc, antibodies and ferroferric oxide.
As described above, since the immunomagnetic beads are integrated with AMP, zinc, antibodies and ferroferric oxide, the immunomagnetic beads have the characteristics of specific surface, porous structure, superparamagnetism, reaction specificity, high selectivity and the like, and can be widely applied to separation, purification and detection of antibody action objects.
In summary, compared with the prior art, the invention achieves the following technical effects:
(1) the coupling process of the paramagnetic substance and the antibody is simplified;
(2) paramagnetic substances, antibodies and metal-organic gel are combined into a novel high polymer material, which has the excellent characteristics of all raw materials and has superiority in the fields of separation, detection or purification;
(3) conditions of each reaction step are optimized, so that the antibody loading capacity and the stability (mainly referring to the washing times) are improved.
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, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows Fe provided in the examples of the present invention3O4SEM pictures of nano magnetic cores (× 20);
FIG. 2 shows Fe provided in the examples of the present invention3O4SEM pictures of nano magnetic cores (× 40);
FIG. 3 is a SEM picture (x 20) of immunomagnetic beads provided by an embodiment of the invention;
FIG. 4 is a SEM picture (x 40) of immunomagnetic beads provided by an embodiment of the invention;
fig. 5 is an infrared spectrum of an immunomagnetic bead provided in an embodiment of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
Immunomagnetic bead
The first step is as follows: preparation of Fe3O4Nano magnetic core
20.0g of ferrous sulfate heptahydrate is weighed and dissolved in 140.0mL of deionized water, heated to 90 ℃ in a water bath and reacted for 1h under the condition of mechanical stirring at the rotating speed of 400 r/min. Simultaneously, 1.6g of potassium nitrate and 11.2g of potassium hydroxide are dissolved in 60.0mL of deionized water, and the prepared solution is added into the ferrous ion solution dropwise (1mL/min) by a constant flow pump. Stirring at 90 deg.C for 2 hr to obtain black turbid solution, washing with deionized water for several times, and oven drying to obtain black solid which is ferroferric oxide with SEM pictures shown in figures 1 and 2, wherein Fe3O4The particle size of the nano magnetic core is 100-300 nm, the magnetic core is unstable in balling, and some magnetic cores are irregular. Because the magnetic beads are more active and part of the magnetic beads are magnetized in the process of separating by an external magnetic field, the magnetic beads are easy to agglomerate.
The second step is that: AMP (adenosine monophosphate)&ZnCl2Hydrogel coated magnetic bead
0.196g of disodium adenosine monophosphate salt was weighed and dissolved in 20mL of HEPES buffer (10mM, pH7.4), and 200mg of ferroferric oxide was added thereto and sufficiently stirred. Adding 150mL paraffin mixed solution containing 7% span 80 dropwise under stirring, stirring at 500r/min to form emulsion system, continuously stirring for 30min, heating in water bath to 40 deg.C, and adding 3mL glutaraldehyde solution (C)5H8O2Volume fraction of 25%), and constant-temperature crosslinking reaction for 30 min. ReduceAt the normal temperature, 2mL of 1mg/mL ZEA monoclonal antibody is added into the reaction system by a constant flow pump dropwise (1mL/min), after the mixture is uniformly stirred, 10mL of 50mM ZnCl is added by the constant flow pump dropwise (1mL/min)2And (3) carrying out crosslinking reaction on the solution at normal temperature for 2 hours.
After the reaction is finished, the mixture is washed for a plurality of times by using petroleum ether, ethanol and deionized water in sequence, solid substances are separated by using a magnet, and the magnetic microspheres coated with hydrogel and coupled with the ZEA monoclonal antibody are obtained and sealed in HEPES buffer (10mM, pH 7.4).
The third step: product characterization
Observation of Fe with cold field emission high resolution scanning electron microscope3O4@AMP&ZnCl2ZEA mab magnetic bead (i.e. final immunomagnetic bead) morphology, as shown in fig. 3 and 4; determination of Fe by infrared spectrometer3O4@AMP&ZnCl2The surface functional group property of the ZEA monoclonal antibody magnetic beads: weighing 1-2 mg of sample, adding 200mg of potassium bromide powder, grinding, and determining the infrared spectrum by a tabletting method, as shown in figure 5.
Comparative Fe3O4The SEM picture of the nanometer magnetic core shows that the edges and corners of the irregular magnetic core are passivated, the coated magnetic beads tend to be spherical in figures 3 and 4, and the surfaces of the magnetic beads are also rough from smooth. By comparing and analyzing the SEM pictures before and after the coating of the magnetic beads, the existence of the coating on the surfaces of the magnetic beads in the images 3 and 4 can be seen.
As can be seen in FIG. 5, 3440cm-1Is represented by-NH21648cm of antisymmetric telescopic vibration peak of-1、1606cm-1The vicinities thereof are respectively C-C, C-N stretching vibration absorption peak on AMP carbon nitrogen hybridization ring, 1109cm-1The absorption band is C-O-C asymmetric stretching vibration, 1295cm-1The absorption peak of the stretching vibration is P ═ O, 1017cm-1The left and right sides are asymmetric stretching vibration of P-O, 582cm-1The absorption peak is Fe-O. The combination of SEM characterization and infrared spectroscopy proves that the method successfully converts AMP&ZnCl2Hydrogel coating on Fe3O4And (4) the surface of the magnetic beads.
The fourth step: measurement of antibody Loading
Take 1mL100mg/mL Fe3O4@AMP&ZnCl2The ZEA monoclonal antibody magnetic beads were placed in a 50mL centrifuge tube, washed twice with ultrapure water, and the water wash was removed by collecting the magnetic beads with a magnet. 3mL of 1.2 mu g/mL ZEN toxin standard solution is added, mixed uniformly and reacted for 30min by shaking on a shaking table. After the reaction was completed, the magnetic beads were collected with a magnet, and the supernatant was taken out and filtered with a microfiltration membrane, and then the amount of ZEA therein was measured. Washing the magnetic beads twice with 3mL of ultrapure water, collecting the magnetic beads with a magnet, combining the water washing solutions of the two times, filtering the washing solution with a microfiltration membrane, and measuring the amount of ZEA in the water washing solution. Washing the magnetic beads with 3mL of methanol (3 mL/time) for three times, shaking for 10min after adding methanol each time, eluting the ZEA toxin adsorbed on the magnetic beads, collecting the magnetic beads with a magnet, taking out methanol eluent, filtering with a microfiltration membrane, not combining the three eluents, and respectively determining the amount of ZEA in the methanol eluent. The calculation formula is shown as formula (1) and formula (2).
ZEA negative amount ZEA addition amount-amount of ZEA in supernatant-amount of ZEA in water rinse (1)
The results are shown in Table 1 and are found to be per 100mg of Fe3O4@AMP&ZnCl2The ZEA toxin loading capacity of the ZEA monoclonal antibody magnetic beads is about 3000ng, and the loading capacity gradually decreases with the increase of the number of times of use. This is because on the one hand, the eluent methanol can destroy the antibody structure, so that part of the antibody activity is reduced or even inactivated, and part of the antigen-antibody binding capacity is weakened or disappeared; on the other hand, part of ZEA toxin can not be eluted from the antibody, so that part of binding sites of the antigen antibody are occupied, the sites of the toxin capable of binding the antibody are reduced, and the loading capacity is reduced.
By analyzing the effect of three times of repeated use, Fe can be found3O4@AMP&ZnCl2The ZEA monoclonal antibody magnetic beads have high-efficiency adsorption capacity on ZEA toxin, the toxin desorption effect is good, high recovery rate can be realized, and the ZEA monoclonal antibody magnetic beads have the advantage of being repeatedly used for many times.
TABLE 1 Fe3O4@AMP&ZnCl2Adsorption and recovery rate of ZEA monoclonal antibody magnetic beads repeatedly used for 3 times
Note: the detection method of the ZEA toxin comprises the following steps:
in the experiment, a 1200 high performance liquid chromatography-6410 triple quadrupole mass spectrometer is used for determining the content of the extracted ZEA toxin in the sample. ZEA liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established.
Chromatographic conditions are as follows: an Agilent ZORBAX Bonus-RP chromatographic column (50mm multiplied by 2.1mm, 3.5 μm) with a column temperature of 45 ℃ and a sample injection amount of 50 μ L; the mobile phase is 10mM ammonium acetate water solution (A) and acetonitrile (B), and the gradient condition is as follows: 0-0.1 min, wherein B is 10%; b is increased from 10% to 50% in 0.1-2 min; 2-10 min, B is increased from 50% to 80%; 10-15 min, wherein B is 80%; reducing B from 80% to 10% in 15-16 min; and (3) keeping B for 10% for 16-20 min. Ionization mode, monitor ion pair (m/z) and other parameters are shown in table 2.
Mass spectrum conditions: ionization mode adopts electrospray ionization positive ion mode (ESI)+) And negative ion mode (ESI)－) (ii) a Selecting Multiple Reaction Monitoring (MRM) by a mass spectrum scanning mode; positive ion mode (ESI)+) The spraying voltage is 4500V; negative ion mode (ESI)－) The spraying voltage is 2500V; the ion source temperature is 450 ℃; the air curtain air is 8L/min.
TABLE 2 Mass Spectrometry parameters of ZEA toxin
Note:※quantifying the ions.
The ZEA toxin was isolated using the immunomagnetic beads of this example and compared with the method of immunoaffinity column isolation.
The experimental method comprises the following steps:
firstly, extracting ZEA toxin in actual sample
Weighing 25.0g of corn flour sample (accurate to 0.1g, 6 types of corn flour on the market are selected in the experiment and numbered as a-f) into a 500mL conical flask with a plug, adding 100mL of 70% methanol solution (v/v), soaking for 15min, ultrasonically oscillating for 40min, and filtering to obtain a sample extracting solution.
Second, immunoaffinity column separation of ZEA toxin
Taking 16mL of sample extract, passing through immunoaffinity column at a speed of 2mL/min, and then passing through 16mL of H2O washing the column at a rate of 5 mL/min; eluting the column with 1mL of 100% methanol solution (v/v) at a rate of 1d/s, collecting the eluate in a 2mL sample bottle, and adding 1mL of H2O washes the immunoaffinity column at 5mL/min, and collects in the sample solution bottle as well. The sample is detected by an Agilent 1200-ESI 6410 liquid chromatography-mass spectrometer, and the sample injection amount is 50 mu L.
Thirdly, separating ZEA toxin by immunomagnetic beads
1mL of 100mg/mL Fe3O4@AMP&ZnCl2The ZEA monoclonal antibody magnetic beads were placed in a 50mL centrifuge tube, washed twice with ultrapure water, and the water wash was removed by collecting the magnetic beads with a magnet. Then, 30mL of the sample extract was added, the reaction was shaken for 30min, the magnetic beads were collected with a magnet, the supernatant was removed, and 20mL of ultrapure water was added thereto to wash the magnetic beads twice at a rate of 10 mL/time. And finally, adding 30mL of methanol, washing 3 times by 10 mL/time, collecting three times of alcohol washing liquid, and respectively measuring ZEA toxin in three times of methanol elution by using an Agilent 1200-ESI 6410 liquid chromatography-mass spectrometer.
The experiment also tested the rate of recovery of the immunomagnetic beads, and the results are shown in table 3. The results are shown in Table 3, the addition recovery rate of 3 levels of corn flour of the c sample is 84.61-90.00%, the relative standard deviation is 4.86-9.44%, the trace analysis requirement is met, and the prepared Fe is proved3O4@AMP&ZnCl2ZEA monoclonal antibody magnetic beads can be used for purification of actual samples.
TABLE 3 recovery of ZEA toxin addition from immunomagnetic bead purified c sample
Note: the addition levels were 30, 60, 90. mu.g/kg, and 6 replicates of each level were performed.
After the extraction of the ZEA toxin in the 6 corn flour a-f, the ZEA toxin in the 6 sample extracting solutions is purified by the method described above, and each experiment is repeated 3 times for detection. The results are shown in Table 4, and compared with the purification results of the practical sample by the immunoaffinity column, the purification results of the immunomagnetic beads are slightly larger, but are consistent with the purification effect of the immunoaffinity column as a whole. The reason is probably that the immunoaffinity column can purify a plurality of toxins simultaneously, and the prepared immunomagnetic beads are used for specifically purifying the ZEA toxin, so that the detection result is more efficient.
According to the regulations of the national food safety standard GB 2761-2017, the limit index of zearalenone ZEA in the corn flour is 60 mu g/kg, so that the ZEA content of the b sample in 6 samples exceeds the standard.
Table 4 detection results of ZEA toxin after 6 kinds of corn flour are purified by two methods of immunoaffinity column and immunomagnetic beads
Note: x is an average value; and y is the standard deviation.
Example 2
It differs from example 1 in that AMP&ZnCl2The reaction conditions of the hydrogel-coated magnetic beads are different, and the specific conditions are as follows.
0.3g of disodium adenosine monophosphate salt was weighed and dissolved in 20mL of HEPES buffer (10mM, pH7.4), and 200mg of ferroferric oxide was added thereto and sufficiently stirred. Adding 150mL paraffin mixed solution containing 6.5% span 80 dropwise under stirring, stirring at 500r/min to form emulsion system, stirring for 30min, heating in water bath to 40 deg.C, and adding 3mL glutaraldehyde solution (C)5H8O2Volume fraction of 25%), and constant-temperature crosslinking reaction for 30 min. Reducing the temperature, dropwise adding 2mL of 1mg/mL ZEA monoclonal antibody into the reaction system by a constant flow pump (1mL/min) at normal temperature, uniformly stirring, and dropwise adding 10mL of 50mM ZnCl into the reaction system by the constant flow pump (1mL/min)2And (3) carrying out crosslinking reaction on the solution at normal temperature for 2 hours.
After the reaction is finished, the mixture is washed for a plurality of times by using petroleum ether, ethanol and deionized water in sequence, solid substances are separated by using a magnet, and the magnetic microspheres coated with hydrogel and coupled with the ZEA monoclonal antibody are obtained and sealed in HEPES buffer (10mM, pH 7.4).
Characterization of immunomagnetic beads was performed in the same manner as in example 1, and the results were in accordance with example 1 and per 100mg of Fe3O4@AMP&ZnCl2The ZEA toxin loading of the ZEA mab magnetic beads was about 3200ng (first assay).
Example 3
It differs from example 1 in that AMP&ZnCl2The reaction conditions of the hydrogel-coated magnetic beads are different, and the specific conditions are as follows.
0.3g of disodium adenosine monophosphate salt was weighed and dissolved in 20mL of HEPES buffer (10mM, pH7.4), and 200mg of ferroferric oxide was added thereto and sufficiently stirred. Adding 150mL paraffin mixed solution containing 6.5% span 80 dropwise under stirring, stirring at 500r/min to form emulsion system, stirring for 30min, heating in water bath to 45 deg.C, and adding 3mL glutaraldehyde solution (C)5H8O2Volume fraction of 25%), and constant-temperature crosslinking reaction for 25 min. Reducing the temperature, dropwise adding 2mL of 1mg/mL ZEA monoclonal antibody into the reaction system by a constant flow pump (1mL/min) at normal temperature, uniformly stirring, and dropwise adding 10mL of 50mM ZnCl into the reaction system by the constant flow pump (1mL/min)2And (3) carrying out crosslinking reaction on the solution at normal temperature for 2 hours.
After the reaction is finished, the mixture is washed for a plurality of times by using petroleum ether, ethanol and deionized water in sequence, solid substances are separated by using a magnet, and the magnetic microspheres coated with hydrogel and coupled with the ZEA monoclonal antibody are obtained and sealed in HEPES buffer (10mM, pH 7.4).
Characterization of immunomagnetic beads was performed in the same manner as in example 1, and the results were in accordance with example 1 and per 100mg of Fe3O4@AMP&ZnCl2The ZEA toxin loading of the ZEA mab magnetic beads was about 3000ng (first assay).
Example 4
It differs from example 1 in that AMP&ZnCl2The reaction conditions of the hydrogel-coated magnetic beads are different, and the specific conditions are as follows.
0.195g of disodium adenosine monophosphate salt was weighed and dissolved in 20mL of HEPES buffer (10mM, pH7.4), and 200mg of ferroferric oxide was added thereto and sufficiently stirred. Adding 150mL paraffin mixed solution containing 6.5% span 80 dropwise under stirring, stirring at 500r/min to form emulsion system, stirring for 30min, heating in water bath to 35 deg.C, and adding 3mL glutaraldehyde solution (C)5H8O230 percent of volume fraction), and carrying out constant-temperature crosslinking reaction for 40 min. Reducing the temperature, dropwise adding 3mL of 1mg/mL ZEA monoclonal antibody into the reaction system by a constant flow pump (0.9mL/min) under the condition of normal temperature, uniformly stirring, and then dropwise adding 10mL of 50mM ZnCl by the constant flow pump (0.55mL/min)2And (3) carrying out crosslinking reaction on the solution at normal temperature for 2 hours.
After the reaction is finished, the mixture is washed for a plurality of times by using petroleum ether, ethanol and deionized water in sequence, solid substances are separated by using a magnet, and the magnetic microspheres coated with hydrogel and coupled with the ZEA monoclonal antibody are obtained and sealed in HEPES buffer (10mM, pH 7.4).
Characterization of immunomagnetic beads was performed in the same manner as in example 1, and the results were in accordance with example 1 and per 100mg of Fe3O4@AMP&ZnCl2The ZEA toxin loading of the ZEA mab magnetic beads was about 3200ng (first assay).
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (14)
1. A preparation method of immunomagnetic beads is characterized by comprising the following steps:
step A: mixing adenosine monophosphate or metal salt thereof, aqueous phase buffer solution, ferroferric oxide, paraffin and span 80, and stirring to form an emulsifying system; wherein the volume ratio of the paraffin to the span 80 is 7: 93-100; the mass ratio of the adenosine monophosphate or the metal salt thereof to the ferroferric oxide is 0.1-0.3: 0.2;
and B: adding glutaraldehyde into the emulsification system, and carrying out a first-step crosslinking reaction at a constant temperature; then adding antibody and zinc salt to produce second step cross-linking reaction, and separating solid with magnet.
2. The method according to claim 1, wherein the pH of the aqueous buffer is 7 to 8.
3. The method according to claim 2, wherein the aqueous buffer is 4-hydroxyethylpiperazine ethanesulfonic acid buffer.
4. The method according to claim 1, wherein the first step crosslinking reaction is carried out at a temperature of: 35-45 ℃;
the temperature of the second step of crosslinking reaction is as follows: 20 to 30 ℃.
5. The method according to claim 1, wherein in step B, the antibody and the zinc salt are added in a manner that: the antibody was added dropwise first, followed by the zinc salt.
6. The preparation method according to claim 1, wherein in the step B, the stirring manner during the formation of the emulsification system is mechanical stirring, and the rotation speed is 300-1000 r/min.
7. The method according to claim 1, wherein the antibody in step B is a zearalenone antibody.
8. The method according to claim 1, wherein the antibody in step B is zearalenone monoclonal antibody.
9. The method according to claim 1, wherein the molar ratio of adenosine monophosphate or a metal salt thereof to zinc ions in the zinc salt is 1: 1.
10. The method according to claim 7, wherein the mass ratio of the zearalenone antibody to the adenosine monophosphate or a metal salt thereof is 0.2 to 0.3: 19.
11. The preparation method according to claim 1, wherein in the step B, glutaraldehyde is added in a manner that: adding glutaraldehyde solution with volume concentration of 20-30%.
12. The method according to claim 1 or 7, wherein in step B, the antibody and the zinc salt are added in a manner that: firstly, the antibody is dripped at the speed of 0.9-1.1 mg/min, and then Zn is dripped at the speed of 0.045-0.055 mmol/min2+The zinc salt is added dropwise at a rate of (1).
13. An immunomagnetic bead produced by the production method according to any one of claims 1 to 12.
14. Use of immunomagnetic beads according to claim 13 for isolating, purifying or detecting antigens.
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