CN106279420B - CD20 antibody immunomagnetic beads and preparation method thereof - Google Patents

CD20 antibody immunomagnetic beads and preparation method thereof Download PDF

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CN106279420B
CN106279420B CN201610780208.9A CN201610780208A CN106279420B CN 106279420 B CN106279420 B CN 106279420B CN 201610780208 A CN201610780208 A CN 201610780208A CN 106279420 B CN106279420 B CN 106279420B
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李静
蔡红东
陈昌岳
刘关
张培培
张祥林
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Shanghai Majorbio Bio Pharm Technology Co ltd
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Abstract

The invention provides a CD20 antibody immunomagnetic bead, which comprises a magnetic microsphere part and an antibody part, wherein the immunomagnetic bead has a structural formula as follows: a-NH-N ═ CH-B, where a represents magnetic microspheres and B represents CD20 antibody, or a represents CD20 antibody and B represents magnetic microspheres. The immunomagnetic beads are used for capturing tumor cells, and have the advantages of good specificity and sensitivity, rapid magnetic response, short enrichment time and high capture efficiency. And the immunomagnetic beads have stable properties, small particle size, good magnetic responsiveness and good dispersibility. And the preparation method is simple and has strong practicability.

Description

CD20 antibody immunomagnetic beads and preparation method thereof
Technical Field
The invention relates to the field of preparation of immunomagnetic beads, in particular to CD20 antibody immunomagnetic beads and a preparation method thereof.
Background
The CD20 molecule is a transmembrane protein on B lymphocytes, consists of 297 amino acids, belongs to non-glycosylated phosphoprotein, is a differentiation antigen on the surface of the B lymphocytes, is mainly involved in regulating the proliferation and differentiation of the B lymphocytes, plays an important role in the immune system, and is often used as a marker for identifying the B cells. CD20 is a specific marker molecule on the surface of B lymphocytes, is highly expressed on the surface of most B lymphocytes, is expressed in more than 95% of normal or malignant B lymphocytes, is not expressed in hematopoietic stem cells, plasma cells and other normal tissues, and is not subjected to obvious internalization and shedding, thus being an ideal biological immune marker of the B lymphocytes.
The immune magnetic bead sorting (IMS) is a method for sorting cells that has recently been developed, in which immunoreactive antibodies are coated on the surfaces of magnetic beads to perform antigen-antibody reaction, thereby forming "antigen-antibody-magnetic bead" immune complexes on the surfaces of cells. Once placed under a strong magnetic field, these cells bound to the magnetic beads move in a targeted manner, grouping the immune complexes with other unbound cells. When the magnetic beads with superparamagnetism are separated from the magnetic field, the magnetism disappears immediately, thereby achieving the purpose of selecting specific cells positively or negatively.
The application of immunomagnetic bead sorting to the sorting of cells is a technology that has emerged in the field in recent years. However, the use of immunomagnetic bead sorting to sort specific cells from complex blood environments requires specific biomarkers for immunomagnetic beads, which are stable, dispersible, and non-agglomerating. Meanwhile, the particle size of the immunomagnetic beads cannot be too large, and the cells can be pressed if the particle size is too large; the particle size of the immunomagnetic beads can not be too small, the magnetic responsiveness is also small when the particle size is too small, and the effect of cell sorting can not be achieved easily.
Disclosure of Invention
The CD20 antibody immunomagnetic bead is small in particle size, good in magnetic responsiveness, high in capture efficiency and short in enrichment time, can be used for capturing B lymphocytes, and can be used for further analysis of the captured B lymphocytes, and meanwhile, the preparation process is simple and the cost is low.
In one aspect of the invention, the invention is realized by the following technical scheme: a CD20 antibody immunomagnetic bead, comprising a magnetic microsphere part and an antibody part, wherein the immunomagnetic bead has a structural formula: a-NH-N ═ CH-B, where a represents magnetic microspheres and B represents CD20 antibody, or a represents CD20 antibody and B represents magnetic microspheres.
Preferably, the magnetic microspheres are inorganic or organic polymer-coated magnetic microspheres with a core-shell structure. Such as magnetic ferroferric oxide wrapped by silicon dioxide or magnetic ferroferric oxide wrapped by glucan and the like. Most preferably, the magnetic microsphere part is magnetic ferroferric oxide coated by silicon dioxide.
Preferably, the CD20 antibody immunomagnetic beads have a particle size of 200-300 nm.
In a second aspect of the present invention, there is provided a method for preparing the above CD20 antibody immunomagnetic beads, which comprises the steps of:
s1. preparing magnetic nanoclusters;
s2, preparing amino-modified magnetic microspheres;
s3. preparation of hydrazine modified part A;
s4. preparing aldehyde group modified B part;
s5. preparation of immunomagnetic beads: and (4) mixing the hydrazine modified part A obtained in the step s3 with the aldehyde modified part B obtained in the step s4, and performing coupling reaction at 4-25 ℃ for 2-24 hours to obtain the immunomagnetic beads.
In one embodiment of the invention, the amino-modified magnetic microspheres are aldehyde-modified and the CD20 antibody is hydrazine-modified. Or hydrazine group modification is carried out on the amino modified magnetic microsphere, and aldehyde group modification is carried out on the CD20 antibody, so that the immunomagnetic bead with the structure can be realized.
Further, the hydrazine modified a moiety in step s3 is modified by: the amino-modified magnetic microsphere or CD20 antibody is obtained by performing hydrazine modification on SANH with the molar equivalent of 10-50 times. Most preferably, the molar equivalent of SANH is 25 times that of amino-modified magnetic microspheres or CD20 antibody. The SANH is p-propyl hydrazone pyridine carboxylic acid N-hydroxysuccinimide ester (CAS: 362522-50-7), and can mildly convert amino into hydrazine group at room temperature. The SANH is generally dissolved in DMF solution for reaction, and the concentration can be adjusted according to the concentration of the magnetic microspheres or CD20 antibody, so that the reaction result is not influenced. The reaction process can be carried out at the room temperature of 15-25 ℃, the reaction time is judged according to the conventional detection technology of a person skilled in the art, the modification reaction time of the magnetic microspheres is 16-24 hours, and the modification reaction time of the CD20 antibody is 2-4 hours.
Further, the aldehyde modified B moiety in step s4 is obtained by: the amino-modified magnetic microsphere or CD20 antibody is obtained by performing aldehyde group modification on SFB with the molar equivalent of 5-20 times. Most preferably, the SFB is 10 times the molar equivalent of the amino-modified magnetic microsphere or CD20 antibody. The SFB is 4-formylbenzoic acid N-succinimidyl ester (CAS: 60444-78-2), and can mildly convert amino into aldehyde group under room temperature. The SFB is generally dissolved in DMF solution for reaction, and the concentration can be adjusted according to the concentration of the magnetic microspheres or CD20 antibody, so that the reaction result is not influenced. The reaction process can be carried out at the room temperature of 15-25 ℃, the reaction time is judged according to the conventional detection technology of a person skilled in the art, the modification reaction time of the magnetic microspheres is 16-24 hours, and the modification reaction time of the CD20 antibody is 2-4 hours.
The magnetic nanoclusters in the step s1 are prepared by a hydrothermal method, a solvothermal method or a coprecipitation method, and commercially available products can also be adopted. The method for preparing the magnetic nano-cluster is a technique well known by the technical personnel in the field, and the obtained product only needs to have good magnetism and can form a core-shell structure with inorganic or organic macromolecules.
Preferably, the magnetic nanoclusters in step s1 are prepared by the following method:
1) adding ammonia water into the aqueous solution of ferrous salt in the air, and stirring to turn the solution into black to obtain black Fe3O4Particles;
2) adding oleic acid into the step 1), uniformly mixing, transferring to a closed reaction kettle, and heating and reacting at 60-130 ℃ for 3-5 hours to obtain the magnetic nanocluster.
Further, the amino-modified magnetic microspheres in step s2 are magnetic microspheres wrapped with silica of a core-shell structure, and the effect is better than that of magnetic microspheres obtained by directly modifying amino on the surface of magnetic nanocluster particles, and the magnetic microspheres can be prepared by commercially available products or conventional methods known by those skilled in the art, without affecting the result of the present invention. The nano-clusters can be gathered in the silicon dioxide to form a core-shell structure, the particle size is increased, and the magnetism and the stability are increased. Preferably, the method for preparing the magnetic microsphere coated with the amino-modified silicon dioxide comprises the following steps: adding ammonia water, a silanization reagent and an aminosilane coupling agent into a solution containing the magnetic nanoclusters, and reacting for 1-3 days to obtain an amino-modified magnetic microsphere part; the mass ratio of the magnetic nano-cluster to the ammonia water to the silanization reagent to the aminosilane coupling agent is as follows: 1 (12.5-40), (2-8) and (0.5-3). Wherein the mass percentage concentration of the ammonia water is 25-28%. Wherein, the silanization reagent can be tetraethyl orthosilicate (CAS: 562-90-3), and the aminosilane coupling agent can be (3-aminopropyl) triethoxysilane (CAS: 919-30-2). The proportion of the reaction raw materials can be adjusted by those skilled in the art when selecting other silylation agents and aminosilane coupling agents, without departing from the scope of the present invention.
Preferably, in step s5, the mass ratio of the magnetic microspheres to the CD20 antibody is 1: (0.01-1). The higher the mass ratio of the CD20 antibody to the magnetic microsphere is, the more favorable the coupling of the CD20 antibody on the surface of the magnetic microsphere is, but considering the cost factor of the CD20 antibody, the mass ratio of the magnetic microsphere to the CD20 antibody adopted by the invention is 1: (0.01-0.2).
In a third aspect of the present invention, there is provided a kit for capturing B cells, wherein the kit comprises the CD20 antibody immunomagnetic beads.
The invention has the beneficial effects that:
(1) the immunomagnetic beads are used for capturing B cells, and have the advantages of good specificity and sensitivity, rapid magnetic response, short enrichment time and high capture efficiency. Captured B cells are avoided from damage and can be used for further analysis and culture.
(2) According to the immunomagnetic bead, the magnetic microsphere part and the CD20 antibody part are connected through a hydrazone bond structure, so that the obtained immunomagnetic bead is stable in property under a weak alkaline condition (in blood), small in particle size, good in magnetic responsiveness and good in dispersibility.
(3) The preparation method is simple, the reaction conditions are mild, the modification processes of amino, aldehyde and hydrazine groups can be carried out at room temperature, the CD20 antibody is not easy to deteriorate, degrade and the like, and meanwhile, a reducing agent is avoided, so that the CD20 antibody can be incubated with cells at low temperature, and the biological activities of the CD20 antibody and the cells are maintained.
(4) The invention has the advantages of simple and easily obtained raw materials, low cost, simple process steps and strong practicability.
Drawings
FIG. 1 is a B cell immunofluorescence map of CD20 antibody immunomagnetic bead sorting of the present invention.
Detailed Description
The invention is further illustrated by the following specific examples: the experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1 preparation of CD20 antibody immunomagnetic beads
(1) Preparing a magnetic nano cluster:
a. in air, 7g FeCl2·4H2Adding O into 50mL of deionized water to obtain FeCl with the concentration of 0.14g/mL2An aqueous solution. To 50mL FeCl2Adding 30mL of ammonia water into the aqueous solution, stirring for 20min, gradually changing the color into light green, then changing the color into dark green, and finally changing the color into black;
b. adding 1.1g of oleic acid into the step a, uniformly mixing, placing the mixed solution into a closed reaction kettle, heating and reacting for 4 hours at 110 ℃, then alternately washing with deionized water and ethanol once, dispersing in n-hexane after magnetic separation, and obtaining black magnetic nano cluster Fe3O4 1。
(2) Preparation of amino-modified magnetic microspheres: to 10mg of magnetic nanocluster Fe3O41, adding 125mg of ammonia water, 30mg of tetraethyl orthosilicate and 30mg of (3-aminopropyl) triethoxysilane, reacting for 1 day, performing magnetic separation, alternately washing with ethanol and water twice, and slowing with 0.1M phosphoric acid with pH of 7-8And (5) obtaining the amino modified magnetic microsphere 1 with the concentration of 5mmol/L after washing and dispersing.
(3) Preparation of hydrazine-modified CD20 antibody: mu.L of SANH in DMF (at a concentration of 5mmol/L) was added to 100. mu.L of a CD20 antibody solution (at a concentration of 10. mu. mol/L), reacted at room temperature for 2 hours, and then purified by an ultrafiltration column to give hydrazino-modified CD20 antibody (abbreviated as CD20-SANH) 1.
Detection of the concentration of CD 20-SANH: the concentration of the modified CD20-SANH was calculated to be 0.7mg/mL by the BCA method.
Detecting the hydrazine modification rate: and (3) detecting the hydrazino modification rate by using a quantitative 2-formylbenzenesulfonyl sodium salt solution. Adding the purified CD20-SANH into the 2-formyl benzenesulfonyl sodium salt solution, uniformly mixing by vortex, reacting at 37 ℃ for 1h, and detecting the light absorption value at 348nm by Nanodrop to be 0.15. The hydrazine modification ratio of CD20-SANH was calculated to be 2.6 from the absorbance at 348nm and the concentration of CD 20-SANH.
(4) Preparing aldehyde group modified magnetic microspheres: adding 5 mu L of the amino modified magnetic microsphere 1 into 50 mu L of DMF solution (with the concentration of 5mmol/L) of SFB, reacting for 20h at room temperature, and performing magnetic separation and purification to obtain the aldehyde modified magnetic microsphere 1.
(5) Preparing immunomagnetic beads: and (3) mixing 1mg of the aldehyde-modified magnetic microspheres obtained in the step (4) with 0.01mg of hydrazine-modified CD20 antibody obtained in the step (3), mixing the aldehyde-modified magnetic microspheres and the hydrazine-modified CD20 antibody in PBS buffer solution with the pH value of 6.0 at 25 ℃ for 2 hours, and carrying out magnetic separation to obtain CD20 antibody immunomagnetic beads of the CD20 antibody-coupled magnetic microspheres. The percentage of antibody coated per mg of CD20 antibody immunomagnetic bead 1 was calculated to be 83% (ratio of consumed antibody to total antibody added).
Example 2 sensitive detection of CD20 antibody immunomagnetic beads
Culturing Jurkat cells (human T lymphocyte leukemia cells, sourced from cell bank of Chinese academy of sciences), adding B lymphocyte NK-92 cell suspension (sourced from cell bank of Chinese academy of sciences) of human malignant non-Hodgkin lymphoma patient into Jurkat at a ratio of Jurkat to NK-92 cells of 103:1、104:1、105:1、106:1. Then adding immunomagnetic beads into the mixed cell suspensions in sequenceIn solution, incubation was carried out at 4 ℃ for 30 minutes. Magnetic sorting and washing with PBS 2-3 times over 1 minute gave NK-92 cells captured and recovered with immunomagnetic beads. And the magnetic sorting is completed within 1 minute, which shows that the magnetic responsiveness of the immunomagnetic beads is good.
The experiment under each concentration is repeated, and the result shows that NK-92 cells can be detected under all concentrations, which indicates that the sensitivity of the immunomagnetic beads is good.
The incubated beads were resuspended in Ca-Mg-free PBS buffer, and the eluted bead-cells were purified of total RNA using a PicoPure RNA Isolation Kit (Thermo Cat No: KIT0214) using PrimeScriptTMRT reagent Kit with gDNA Eraser (TAKARA, Cat No: RR047A), following the instructions, genomic DNA was removed and cDNA strand synthesis was performed. And then, related to corresponding Taqman primers, carrying out qPCR detection, and comparing qPCR detection results by taking GAPDH as an internal standard, wherein the qPCR detection results are consistent with those of NK-92 cells under the same conditions. The immunomagnetic beads of the invention are used for capturing tumor cells without damaging the cells, and can be used for subsequent cell analysis. Also, it is shown that the cells captured by the immunomagnetic beads of the present invention are substantially free of PBMCs cells, which are substantially free of binding to CD20 antibody immunomagnetic beads.
Example 3CD20 antibody immunomagnetic bead Forward screening and identification Capture
The peripheral blood of healthy people is taken to capture tumor cells in the blood. The conventional leukocyte value of the blood of the subject is required to be 2X 106～1.2×107Between one/mL, no hemolysis or clot coagulation of the whole blood sample occurred. The method comprises the following steps of obtaining relevant information of a subject, collecting and storing samples, and carrying out experiment operation according to the relevant information: 3ml of blood of different healthy persons is taken, red blood cell lysate is added respectively, and white blood cell samples are obtained after treatment according to the method of the instruction. CD20 antibody immunomagnetic beads were added to each mixed cell suspension in sequence, incubated at 4 ℃ for 30 minutes, then magnetically sorted within 1 minute and washed 2-3 times with PBS. Recovering the positive and negative screened cells of the magnetic beads, respectively staining with CD19 antibody, fixing on a glass slide, then performing DAPI staining of cell nucleus, and mountingThen, the cell membrane surface of the sample subjected to the forward screening showed fluorescence and green fluorescence under the fluorescence microscope, as shown in fig. 1, indicating that the cell surface captured by the forward screening was substantially 100% positive for CD19 and B cells. The positive rate of CD19 in the negative screening sample is very low, and is only about 2%. Considering that the surface of B cells had substantially all CD19 and only a portion of the surface of B cells had CD20, the 2% residual ratio was not due to insufficient sensitivity of the CD20 beads themselves, but rather was a negative selection of cells with a portion of CD19+ CD 20-in the sample.
The above-described embodiment is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the principle of the embodiment of the present invention and the claims, and these modifications and improvements are also considered to be within the scope of the embodiment of the present invention.

Claims (6)

1. A CD20 antibody immunomagnetic bead, which comprises a magnetic microsphere part and an antibody part, and is characterized in that: the structural formula of the immunomagnetic beads is as follows: a-NH-N ═ CH-B, wherein a represents magnetic microspheres and B represents CD20 antibody, or a represents CD20 antibody and B represents magnetic microspheres;
the magnetic microspheres are inorganic or organic polymer-coated magnetic microspheres with core-shell structures;
the preparation method of the CD20 antibody immunomagnetic bead comprises the following steps:
s1. preparation of magnetic nanoclusters:
a. in air, 7g FeCl2·4H2Adding O into 50mL of deionized water to obtain FeCl with the concentration of 0.14g/mL2Aqueous solution to 50mL FeCl2Adding 30mL of ammonia water into the aqueous solution, stirring for 20min, gradually changing the color into light green, then changing the color into dark green, and finally changing the color into black;
b. adding 1.1g of oleic acid into the step a, uniformly mixing, placing the mixed solution into a closed reaction kettle, heating and reacting for 4 hours at 110 ℃, then alternately washing with deionized water and ethanol once, dispersing in n-hexyl alcohol after magnetic separationIn alkane, black magnetic nano cluster Fe can be obtained3O4；
S2, preparing amino-modified magnetic microspheres;
s3. preparation of hydrazine modified part A;
s4. preparing aldehyde group modified B part;
s5. preparation of immunomagnetic beads: mixing the hydrazine modified part A obtained in the step s3 with the aldehyde modified part B obtained in the step s4, and performing coupling reaction at 4-25 ℃ for 2-24 hours to obtain the immunomagnetic beads;
the amino-modified magnetic microsphere part in the step s2 is a magnetic microsphere wrapped by silicon dioxide with a core-shell structure, and is prepared by the following method: adding ammonia water, a silanization reagent and an aminosilane coupling agent into a solution containing the magnetic nanoclusters, and reacting for 1-3 days to obtain an amino-modified magnetic microsphere part; the mass ratio of the magnetic nano-cluster to the ammonia water to the silanization reagent to the aminosilane coupling agent is as follows: 1 (12.5-40), (2-8) and (0.5-3).
2. The CD20 antibody immunomagnetic bead of claim 1, wherein: the CD20 antibody immunomagnetic beads have the particle size of 200-300 nm.
3. A method of preparing CD20 antibody immunomagnetic beads according to claim 1 or 2, comprising the steps of:
s1. preparation of magnetic nanoclusters:
a. in air, 7g FeCl2·4H2Adding O into 50mL of deionized water to obtain FeCl with the concentration of 0.14g/mL2Aqueous solution to 50mL FeCl2Adding 30mL of ammonia water into the aqueous solution, stirring for 20min, gradually changing the color into light green, then changing the color into dark green, and finally changing the color into black;
b. adding 1.1g of oleic acid into the step a, uniformly mixing, placing the mixed solution into a closed reaction kettle, heating and reacting for 4 hours at 110 ℃, then alternately washing with deionized water and ethanol once, dispersing in n-hexane after magnetic separation, and obtaining black magnetic nano cluster Fe3O4；
S2, preparing amino-modified magnetic microspheres;
s3. preparation of hydrazine modified part A;
s4. preparing aldehyde group modified B part;
s5. preparation of immunomagnetic beads: mixing the hydrazine modified part A obtained in the step s3 with the aldehyde modified part B obtained in the step s4, and performing coupling reaction at 4-25 ℃ for 2-24 hours to obtain the immunomagnetic beads;
the amino-modified magnetic microsphere part in the step s2 is a magnetic microsphere wrapped by silicon dioxide with a core-shell structure, and is prepared by the following method: adding ammonia water, a silanization reagent and an aminosilane coupling agent into a solution containing the magnetic nanoclusters, and reacting for 1-3 days to obtain an amino-modified magnetic microsphere part; the mass ratio of the magnetic nano-cluster to the ammonia water to the silanization reagent to the aminosilane coupling agent is as follows: 1 (12.5-40), (2-8) and (0.5-3).
4. The method for preparing CD20 antibody immunomagnetic beads according to claim 3, wherein: the hydrazine modified part a of step s3 is prepared by: the amino-modified magnetic microsphere or CD20 antibody is obtained by performing hydrazine modification on SANH with the molar equivalent of 10-50 times.
5. The method for preparing CD20 antibody immunomagnetic beads according to claim 3, wherein: the aldehyde modified part B in step s4 is prepared by: the amino-modified magnetic microsphere or CD20 antibody is obtained by performing aldehyde group modification on SFB with the molar equivalent of 5-20 times.
6. A kit for capturing B cells, comprising: the kit contains CD20 antibody immunomagnetic beads according to claim 1.
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