Anti-RBD immunomagnetic bead as well as preparation method and application thereof

Abstract

The invention provides an anti-RBD immunomagnetic bead as well as a preparation method and application thereof, and belongs to the technical field of immunodiagnosis. The preparation method of the anti-RBD immunomagnetic beads comprises the following steps: washing aminated magnetic beads with a 1 * PBST solution, and removing supernatant after washing; adding 15% glutaraldehyde, activating in a dark place at room temperature, removing the supernatant after activation, adding 1 * PBST solution to wash the magnetic beads, and removing the supernatant after washing; adding a 28D9 antibody, incubating at room temperature in a dark place, and removing supernatant after incubation is finished; adding a 1 * PBST solution containing 5% of BSA (Bovine Serum Albumin) for sealing at room temperature in a dark place, removing supernatant after sealing is finished, washing the magnetic beads by using the 1 * PBST solution, and removing supernatant after washing is finished; and adding a preserving fluid to obtain the anti-RBD immunomagnetic bead. The anti-RBD immunomagnetic bead provided by the invention can be used for rapidly purifying RBD protein, so that the time is greatly saved for the production of new crown protein vaccines.

Description

Technical Field

[0001] The present invention relates to the technical field of immunodiagnosis, and in particular to an anti-RBD immunomagnetic bead and a preparation method and application thereof. Background Art

[0002] The outer membrane of the new coronavirus SARS-CoV-2 is composed of nucleocapsid protein (N), membrane protein (M), envelope protein (E) and spike protein (S). Among them, the S protein binds to the receptor angiotensin-converting enzyme 2 (ACE2) through the receptor binding domain (RBD), mediating the entry of the virus into the host cell. Current studies have shown that the RBD domain of the SARS-CoV-2 spike protein is immunodominant and is also the target of 90% of neutralizing antibodies present in SARS-CoV-2 immune serum. Therefore, we have also discovered monoclonal antibodies against SARS-CoV-2S1-RBD targeting RBD, including 30G10, 26D8, 28D9 and 6B11 (Chinese invention patent application number: 202111172732.5).

[0003] Nanomagnetic beads are a type of nano-sized particles made of iron oxide and have superparamagnetic properties. Unlike common ferromagnets, superparamagnetism only exhibits magnetism in the presence of an external magnetic field. This characteristic of nanomagnetic beads depends on the size of the magnetic bead particles themselves, which allows the magnetic beads and the substances they are bound to to be suspended and dispersed without attracting each other, and there is no need to worry about agglomeration. In addition, group-modified magnetic beads can be combined with antibodies, proteins, enzymes, drugs or nucleic acids, etc. Therefore, magnetic beads are currently widely used, including nucleic acid extraction, protein purification, cell separation, exosome purification, flow cytometry detection, target identification of biopharmaceuticals, etc. Summary of the invention

[0004] Based on the above, the object of the present invention is to provide an anti-RBD immunomagnetic bead and a preparation method and application thereof, which can be used to quickly purify RBD protein.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing anti-RBD immunomagnetic beads, comprising the following steps:

[0007] (1) Wash the amino-modified magnetic beads with 1× PBST solution and discard the supernatant;

[0008] (2) Add 15% glutaraldehyde and activate at room temperature in the dark. After activation, remove the supernatant and add 1× PBST solution to wash the magnetic beads. After washing, remove the supernatant.

[0009] (3) Add 28D9 antibody and incubate at room temperature in the dark. After the incubation, remove the supernatant;

[0010] (4) Add 1×PBST solution containing 5% BSA and block at room temperature in the dark. After blocking, remove the supernatant. Wash the magnetic beads with 1×PBST solution and remove the supernatant.

[0011] (5) Add preservation solution to obtain anti-RBD immunomagnetic beads.

[0012] Preferably, the particle size of the amino magnetic beads in step (1) is 300 nm; the 1×PBST solution contains 0.05% Tween 20, and the pH of the 1×PBST solution is 7.4; and the number of washings is 2 to 3 times.

[0013] More preferably, the mass volume ratio of the amino-modified magnetic beads to the 1×PBST solution in step (1) is 5-10:1 mg / ml.

[0014] Preferably, the mass volume ratio of the washed amino magnetic beads in step (2) to the 15% glutaraldehyde solution is 5-10:1 mg / ml; the activation temperature is 22-27° C., the activation time is 1-2 h; and the number of washings is 2-3 times.

[0015] Preferably, in step (3), the mass ratio of the activated and washed amino magnetic beads to the 28D9 antibody is 25-50:1; the incubation temperature is 22-27° C., and the incubation time is 2.5-3.5 h.

[0016] More preferably, the concentration of the 28D9 antibody is 1-2 μg / μl.

[0017] Preferably, the blocking time in step (4) is 1 to 2 hours, and the number of washings is 2 to 3 times.

[0018] Preferably, the preservation solution in step (5) is a 1×PBS solution containing 0.08-0.12% bovine serum albumin and 0.08-0.12% preservative Proclin 300.

[0019] The present invention also provides an anti-RBD immunomagnetic bead obtained by any of the above preparation methods.

[0020] The present invention also provides the use of the anti-RBD immunomagnetic beads in the rapid purification of RBD protein.

[0021] The beneficial effects of the present invention are:

[0022] The present invention screened several antibodies 30G10, 26D8, 28D9 and 6B11 obtained in previous studies to obtain 28D9, and then compared the magnetic beads with different modifications of carboxyl and amino groups, and found that the amino-modified nano-magnetic beads had better effects. Finally, the 300nm amino-modified magnetic beads were activated and coupled with the anti-RBD antibody 28D9 to prepare the anti-RBD immunomagnetic beads.

[0023] The comparison of the RBD enrichment level of magnetic beads was carried out through IP experiments, which confirmed that the anti-RBD immunomagnetic beads prepared by the present invention have the characteristics of low nonspecific adsorption, easy dispersion, small particle size, expression of anti-RBD antibodies, etc., and can be used for rapid purification of RBD protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of anti-RBD immunomagnetic beads constructed in an embodiment of the present invention;

[0025] Figure 2 This is a graph showing the test results of immunoprecipitation experiment of the immunomagnetic beads coupled with the anti-RBD antibody 30G10 and the RBD protein sample in Test Example 1;

[0026] Figure 3 This is a graph showing the test results of immunoprecipitation experiments performed on the immunomagnetic beads coupled with the anti-RBD antibodies 26D8 and 28D9 and the RBD protein sample in Test Example 2;

[0027] Figure 4 This is a graph showing the test results of repeated experiments on magnetic beads for different batches of RBD samples in Test Example 3. DETAILED DESCRIPTION

[0028] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0029] The main reagents and consumables during the implementation process are as follows:

[0030]

[0031] In the present application, "room temperature" may be 20 to 30°C.

[0032] Comparative Example Carboxylation Magnetic Beads Coupled with Anti-RBD Antibody

[0033] 1. Take 100μl 10mg / ml 300nm carboxyl magnetic beads in a 1.5ml centrifuge tube, and then remove the supernatant by magnetic separation. Then, wash twice with 200μl MEST solution (100mM, pH5.0; containing 0.05% Tween 20). After removing the supernatant, quickly add 100μl 20mg / ml EDC solution and 100μl 20mg / ml NHS solution to the magnetic beads, mix well, and activate at room temperature for 30-60min.

[0034] 2. Comparison of antibody coupling rate: First, remove the supernatant from the activated carboxylated magnetic beads by magnetic separation, and then add 50, 100, 200, 500, and 1000 μg of anti-RBD antibody 30G10, mix gently, and couple at room temperature for 2 hours. After the coupling is completed, the supernatant is collected, and the concentration is determined to calculate the efficiency of antibody coupling to magnetic beads. The calculation formula is as follows: (1-supernatant antibody residue / antibody input amount) × 100%. Test results: As shown in Table 1, after coupling carboxylated magnetic beads with different amounts of anti-RBD antibody 30G10, it was found that when the antibody amount: magnetic bead amount was 1:5, the antibody coupling efficiency was the highest. Next, this application couples the antibody to carboxylated magnetic beads using this ratio.

[0035] Table 1. Comparison of the efficiency of coupling anti-RBD antibody 30G10 to carboxylated magnetic beads

[0036] Serial number Antibody amount: Magnetic beads amount Antibody amount (μg) Magnetic beads amount (μg) System (μl) <![CDATA[Coupling rate a (%)]]> 1 1:1 1000 1000 100 72.76±3.37 2 1:2 500 1000 100 80.60±1.83 3 1:5 200 1000 100 95.35±2.80 4 1:10 100 1000 100 91.24±2.21 5 1:20 50 1000 100 83.75±2.76

[0037] a Mean ± SD.

[0038] 3. Remove the supernatant from the activated carboxylated magnetic beads by magnetic separation, then add 200 μg of anti-RBD antibody 30G10, 26D8 or 28D9 (adjust the volume to 100 μl with 1×PBS), mix gently, and couple at room temperature for 2 hours. After the coupling is completed, remove the supernatant, add 200 μl of PBST solution containing 1% bovine serum albumin (1×PBS, pH7.2; containing 0.01% Tween 20), and block at room temperature for 1 hour. After the blocking is completed, remove the supernatant, wash 3 times with 200 μl of 1×PBS, and finally add 100 μl of preservation solution (1×PBS containing 0.1% bovine serum albumin and 0.1% Proclin300), that is, prepare anti-RBD immunomagnetic beads, and store at 4°C.

[0039] The negative control was a conjugate using mouse IgG instead, and the preparation process was the same as above to obtain mouse IgG magnetic beads.

[0040] 1. Take 100 μl of 10 mg / ml 300 nm amino magnetic beads in a 1.5 ml centrifuge tube and remove the supernatant using a magnetic separator. Then wash the magnetic beads twice with 200 μl of 1×PBST solution, remove the supernatant, add 100 μl of 15% glutaraldehyde, mix thoroughly, and activate at room temperature in the dark for 1 hour. The "1×PBST solution" used in each step of this application refers to a 1×PBS solution with a pH of 7.4 and containing 0.05% Tween 20.

[0041] 2. Comparison of antibody coupling rate: First, the activated amino magnetic beads were magnetically separated to remove the supernatant, and the magnetic beads were washed twice with 200μl 1×PBST solution to remove the supernatant, and then 50, 100, 200, 500, and 1000μg of anti-RBD antibody 30G10 were added respectively, mixed gently, and coupled at room temperature for 2.5 hours. After the coupling is completed, the supernatant is collected, and the concentration is determined to calculate the efficiency of antibody coupling magnetic beads. The calculation formula is as follows: (1-supernatant antibody residue / antibody input amount) × 100%. Test results: As shown in Table 2, after coupling amino magnetic beads with different amounts of anti-RBD antibody 30G10, it was found that when the antibody amount: magnetic bead amount was 1:5, the antibody coupling efficiency was the highest. Next, this application couples the antibody with amino magnetic beads using this ratio.

[0042] Table 2. Comparison of the efficiency of coupling anti-RBD antibody 30G10 to amino-modified magnetic beads

[0043] Serial number Antibody amount: Magnetic beads amount Antibody amount (μg) Magnetic beads amount (μg) System (μl) <![CDATA[Coupling rate a (%)]]> 1 1:1 1000 1000 100 68.42±2.77 2 1:2 500 1000 100 77.37±2.43 3 1:5 200 1000 100 94.20±2.16 4 1:10 100 1000 100 90.42±1.82 5 1:20 50 1000 100 81.03±2.41

[0044] a Mean ± SD.

[0045] 3. Use magnetic separation to remove the supernatant of the activated amino magnetic beads, and add 1×PBST solution to wash the magnetic beads twice. Then, after removing the supernatant, add 200μg of anti-RBD antibody 30G10, 26D8 or 28D9 (adjust the volume to 100μl with 1×PBS), mix gently, and couple at room temperature for 2.5 hours. After the coupling is completed, remove the supernatant, add PBST solution containing 5% BSA and block at room temperature in the dark for 1 hour. After the blocking is completed, remove the supernatant and wash the magnetic beads 3 times with 1×PBST solution. Remove the supernatant and add 100μl of preservation solution (1×PBS containing 0.1% bovine serum albumin and 0.1% Proclin300) to prepare anti-RBD immunomagnetic beads, which are stored at 4°C. The schematic diagram is as follows Figure 1 shown.

[0046] The negative control was a conjugate using mouse IgG instead, and the preparation process was the same as above to obtain mouse IgG magnetic beads.

[0047] Test Example 1

[0048] 1. Preparation of samples expressing His-RBD protein

[0049] 1. Construction of RBD recombinant plasmid

[0050] The gene sequence of S1-RBD was obtained by gene synthesis technology (Suzhou Jinkairui), and the S1-RBD gene was cloned into the modified Bacmid vector (Addgene) containing the GP64 signal peptide sequence, i.e., His-tagged protein, using EcoRI and HindIII as double restriction sites, and named pFastBac Dual-GP64-RBD. The GP64 signal peptide sequence is: MVSAIVLYVLLAAAAHSAFA. The primers used to amplify the RBD sequence are (underlined for restriction sites): pFast-dual-RBD-EcoRI-F, 5'-CG GAATTC ATGAACATCACAAACTTGTGCC-3';pFast-dual-RBD-HindIII-R,5'-CC AAGCTT TTAGAAATTGACGCACTTATTTTTG-3'.

[0051] 2. Expression of RBD protein

[0052] Using Lipofectamine TM 2000 According to the instructions, the pFastBac Dual-GP64-RBD plasmid was transfected into SF9 cells attached to a six-well plate. Then, after culturing the cells at 27°C for 5 hours, the supernatant containing the transfection complex was removed, 2 mL of complete culture medium containing antibiotics was added, and the cells were cultured in an incubator at 27°C for 72 hours. After 72 hours, the cell supernatant was collected and filtered using a 0.22 μm filter membrane (Millipore) to obtain a P1 generation baculovirus containing S1-RBD integrated into the viral genome. Then, the obtained P1 generation baculovirus was infected with Hi-5 cells at a volume ratio of 1 / 5, and the cell supernatant was collected after 72 hours of serum-free culture, and filtered using a 0.22 μm filter membrane to obtain a P2 generation baculovirus. Finally, the P1 and P2 generation baculovirus supernatants were collected, which were samples expressing the His-RBD protein. In the following experimental examples, samples prepared by this method and steps were used.

[0053] 2. Take the carboxylated magnetic beads coupled to the anti-RBD antibody 30G10 immunomagnetic beads (i.e., 30G10-conjugated Mag COOH beads), the amino magnetic beads coupled to the anti-RBD antibody 30G10 immunomagnetic beads (i.e., 30G10-conjugated Mag NH2 beads), and the negative control magnetic beads coupled to mouse IgG (i.e., Mag NH2 beads, Mag COOH beads) constructed in the above comparative examples and embodiments.

[0054] First, gently mix the above magnetic beads, then take 25μl of magnetic beads in a 1.5ml centrifuge tube, place it on a magnetic stand, and discard the preservation solution. Then, add 1ml 1×PBS (pH7.4) to wash 1 to 2 times, and remove the supernatant by magnetic separation. Next, add 500μl of the sample solution containing His-RBD protein to the washed magnetic beads, mix gently, place on a shaker, and incubate overnight at 4℃ (or incubate at room temperature for 2 hours). After the incubation, collect the supernatant by magnetic separation, which is the Output sample; then add 1ml 1×PBS to the magnetic beads and wash the magnetic beads 2 to 3 times. After washing, remove the supernatant by magnetic separation, then add 25μl 2×loading buffer, boil it at 95℃ in a metal bath for 5 minutes, and the supernatant is the RBD protein immunoprecipitated (IP). And take 40μl of the Output sample, add 10μl 5×loading buffer, boil it at 95℃ in a metal bath for 5 minutes to obtain a control sample. Finally, 10% SDS-PAGE gel electrophoresis was used to detect the purification effect of RBD to evaluate the effect of the constructed magnetic beads. 15 μl of IP was loaded and 15 μl of Output was loaded.

[0055] The results are as follows Figure 2 As shown: Both amino- and carboxyl-coupled magnetic beads coupled with 30G10 antibody failed to successfully pull down RBD protein.

[0056] Test Example 2

[0057] Take the immunomagnetic beads of carboxylation magnetic beads coupled to anti-RBD antibodies 26D8 and 28D9 (i.e., 26D8-conjugated Mag COOH beads and 28D9-conjugated Mag COOH beads), the immunomagnetic beads of amino magnetic beads coupled to anti-RBD antibodies 26D8 and 28D9 (i.e., 26D8-conjugated Mag NH2 beads and 28D9-conjugated Mag NH2 beads), and negative control magnetic beads coupled to mouse IgG (Mag NH2 beads, MagCOOH beads) constructed in the above comparative examples and embodiments; and incubate the above magnetic beads with the sample solution containing His-RBD respectively according to the method of Experimental Example 1, and finally use 10% SDS-PAGE gel electrophoresis to detect the purification effect of RBD, so as to evaluate the effect of the constructed magnetic beads. Among them, 15μl is loaded for IP and 15μl is loaded for Output.

[0058] The results are as follows Figure 3 As shown: 28D9-conjugated Mag NH2 beads and 26D8-conjugated Mag NH2 beads can both pull down RBD protein, but the amount of RBD protein pulled down by 28D9-conjugated Mag NH2 beads is significantly more than that by 26D8-conjugated Mag NH2 beads; and the non-specific adsorption of negative control Mag NH2 beads is much weaker than that of carboxylated Mag NH2 beads. Therefore, we can preliminarily know that 28D9-conjugated Mag NH2 beads can specifically pull down RBD protein.

[0059] Test Example 3

[0060] According to the method in Test Example 1, 2 batches of RBD protein samples were re-expressed to test the effect of the anti-RBD immunomagnetic beads constructed in the embodiment. According to the detection method in Test Example 2, repeat test 1 and repeat test 2 were performed.

[0061] The results are as follows Figure 4 As shown: 28D9-conjugated Mag NH2beads can specifically pull down the RBD protein in 2 batches of test samples, which once again suggests that the performance of 28D9-conjugated Mag NH2beads is relatively stable.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing anti-RBD immunomagnetic beads, comprising the following steps: (1) Wash the amino-modified magnetic beads with 1× PBST solution and discard the supernatant; (2) Add 15% glutaraldehyde and activate at room temperature in the dark. After activation, remove the supernatant and add 1× PBST solution to wash the magnetic beads. After washing, remove the supernatant. (3) Add 28D9 antibody and incubate at room temperature in the dark. After the incubation, remove the supernatant; (4) Add 1×PBST solution containing 5% BSA and block at room temperature in the dark. After blocking, remove the supernatant and wash the magnetic beads with 1×PBST solution. After washing, remove the supernatant. (5) Add preservation solution to obtain anti-RBD immunomagnetic beads.

2. The preparation method according to claim 1, characterized in that: The particle size of the amino magnetic beads in step (1) is 300 nm; the 1×PBST solution contains 0.05% Tween 20, and the pH of the 1×PBST solution is 7.4; the number of washings is 2 to 3 times.

3. The preparation method according to claim 2, characterized in that: The mass volume ratio of the amino magnetic beads to the 1×PBST solution in step (1) is 5-10:1 mg / ml.

4. The preparation method according to claim 1, characterized in that: The mass volume ratio of the washed amino magnetic beads in step (2) to the 15% glutaraldehyde solution is 5-10:1 mg / ml; the activation temperature is 22-27° C., the activation time is 1-2 h; and the number of washings is 2-3 times.

5. The preparation method according to claim 1, characterized in that: In step (3), the mass ratio of the activated and washed amino magnetic beads to the 28D9 antibody is 25-50:1; the incubation temperature is 22-27° C., and the incubation time is 2.5-3.5 h.

6. The preparation method according to claim 5, characterized in that: The concentration of the 28D9 antibody is 1-2 μg / μl.

7. The preparation method according to claim 1, characterized in that: The blocking time in step (4) is 1 to 2 hours, and the number of washings is 2 to 3 times.

8. The preparation method according to claim 1, characterized in that: The preservation solution in step (5) is a 1×PBS solution containing 0.08-0.12% bovine serum albumin and 0.08-0.12% preservative Proclin 300.

9. An anti-RBD immunomagnetic bead obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the anti-RBD immunomagnetic beads according to claim 9 in the rapid purification of RBD protein.

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