Magnetic bead for labeling and purifying IgG antibody biotin and application method thereof

Abstract

The invention provides a magnetic bead for IgG antibody biotin labeling and purification and an application method, and the magnetic bead is prepared by the following steps: 1) preparing rProtein A and rTurbo ID into a protein solution; (2) mixing the protein solution with the carboxyl magnetic beads to react, so that the protein is coupled to the carboxyl magnetic beads to obtain rProtein A and rTurbo ID protein coated magnetic beads; according to the magnetic bead disclosed by the invention, biotin labeling of an antibody can be realized by applying Turbo ID enzyme, only ATP and common biotin molecules need to be used, and the cost is very low; and the biotin labeling reaction is carried out on the magnetic beads, so that the purification and biotin labeling operation is simplified, and the operation is simple and convenient.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomolecule detection, and particularly relates to a magnetic bead for biotin labeling and purification of IgG antibody and an application method. BACKGROUND

[0002] Antibodies are important raw materials for biomolecule detection, and a conventional detection method uses primary antibodies and corresponding secondary antibodies for detection. The corresponding secondary antibodies need to be selected according to the species of the primary antibodies, and the universality is poor. Moreover, some samples to be detected contain antibody molecules, and the secondary antibodies also have non-specific binding with the antibody molecules, resulting in poor specificity. The binding between streptavidin and biotin has high specificity and high affinity, and the affinity is much higher than that of the binding between secondary antibodies and primary antibodies. The antibody is biotin labeled, then streptavidin coupled with horseradish peroxidase (HRP) is combined with the biotin-labeled antibody, and then a substrate is added, so that the signal can be detected through a color reaction or a chemiluminescence reaction, that is, high specificity and high universality can be achieved. However, the cost of biotin-labeled antibodies is too high at present, and the popularization rate is low. Chemical biotin labeling of antibodies needs to synthesize specific biotin derivatives, and the cost is high. At present, the purification of antibodies and the biotin labeling of antibodies are carried out in steps, and multiple rounds of purification are needed, which is complicated. SUMMARY

[0003] Therefore, the application aims to provide a magnetic bead for biotin labeling and purification of IgG antibody and an application method to overcome the defects of the prior art.

[0004] To achieve the above-mentioned purpose, the technical scheme of the application is as follows:

[0005] A magnetic bead for biotin labeling and purification of IgG antibody, the preparation thereof comprises the following steps,

[0006] A) preparing a protein solution by mixing rProtein A and rTurboID;

[0007] B) mixing the protein solution with carboxyl magnetic beads to react, so that the proteins are coupled to the carboxyl magnetic beads, and rProtein A and rTurboID protein-coated magnetic beads, that is, the magnetic beads for biotin labeling and purification of IgG antibody, are obtained.

[0008] Preferably, the mass ratio of rProtein A to rTurboID is 1:4-4:1, and the total protein amount is 1.5-2.5 mg / mL.

[0009] Preferably, the base sequence of rProtein A is shown in SEQ ID NO: 1, and the base sequence of rTurboID is shown in SEQ ID NO: 2.

[0010] The application also provides a method for preparing a purified biotin-labeled IgG antibody, using the magnetic beads for biotin labeling and purification of IgG antibody as described above, comprising the following steps,

[0011] 1) mixing the magnetic beads for biotin labeling and purification of IgG antibody as described above with animal serum containing IgG antibody to be labeled to obtain magnetic beads combined with IgG antibody;

[0012] 2) biotin labeling of the IgG antibody combined on the magnetic beads combined with IgG antibody in step 1);

[0013] 3) eluting the biotin-labeled IgG antibody obtained in step 2) from the magnetic beads to obtain purified biotin-labeled IgG antibody.

[0014] Preferably, in step 1), the magnetic beads combined with IgG antibody are obtained by the following steps,

[0015] a) placing the magnetic beads for biotin labeling and purification of IgG antibody on a magnetic stand for 5-15 s, and discarding the supernatant;

[0016] b) adding 300-700 μL of buffer to resuspend the magnetic beads, and then placing them on a magnetic stand, after the solution is clarified, discarding the supernatant, and repeating the operation 2-3 times;

[0017] c) diluting the animal serum with buffer, and after dilution, 300-700 μL is added to a centrifuge tube, and placed on a vortex mixer for 30-60 minutes to obtain magnetic beads combined with IgG antibody.

[0018] Preferably, in step 2), the biotin labeling comprises the following steps,

[0019] A) removing the supernatant in the centrifuge tube containing the magnetic beads combined with IgG antibody;

[0020] B) adding 300-700 μL of buffer; resuspending the magnetic beads with a pipette, and then placing them on a magnetic stand, discarding the supernatant, and repeating the step 2-5 times;

[0021] C) preparing a mixture required for biotin labeling, and adding the mixture to the centrifuge tube containing the magnetic beads combined with IgG antibody, and placing it on a vortex mixer for 2-4 hours of reaction at room temperature;

[0022] D) adding 300-700 μL of buffer; resuspending the magnetic beads with a pipette, and then placing them on a magnetic stand, discarding the supernatant, and repeating the step 2-5 times.

[0023] Preferably, the final concentrations of the components in the mixture required for biotin labeling are 0.5-1.5 mM ATP, 3-7 mM biotin, and 3-7 mM MgCl2.

[0024] The present application also provides an antigen detection kit, comprising the magnetic beads for biotin labeling and purification of IgG antibody as described above or the purified biotin-labeled IgG antibody prepared by the preparation method as described above.

[0025] Preferably, the antigen is a lipopolysaccharide antigen of Brucella.

[0026] The present application also provides a method for using the purified biotin-labeled IgG antibody prepared by the preparation method as described above, comprising the following steps:

[0027] (1) Take out the antigen-coated enzyme-labeled plate, add 90-100 μL of the diluted eluate to the hole, incubate at 36-38°C for 20-40 min;

[0028] (2) Shake off the liquid on the plate, fill with PBST (1x), stand for 1-2 min, pat the liquid in the plate, repeat 4-5 times;

[0029] (3) Dilute the HRP-labeled streptavidin with 1x PBST, use a pipette to add to each hole, react at 37°C for 20-40 min;

[0030] (4) Repeat (2), wash the residual liquid in the hole, pat the hole;

[0031] (5) Add 90-100 μL of LTMB color developing solution, develop color in the dark for 3-30 min;

[0032] (6) After color development, add 90-100 μL of LTMB color developing termination solution to terminate the reaction;

[0033] (7) During color development, prepare the enzyme-labeled instrument in advance, measure the 450 mM absorbance after adding the termination solution

[0034] The protein A and TurboID are coupled together on the magnetic beads, which can achieve: 1) capturing IgG antibody on the magnetic beads, making the intermolecular distance of IgG antibody close to TurboID; 2) TurboID can efficiently biotin label IgG antibody under the condition of providing ATP, Mg 2+ and biotin; 3) IgG on the magnetic beads can be efficiently exchanged with magnetic force frame; 4) after labeling, IgG can be eluted from the magnetic beads using antibody elution solution, realizing the integration of antibody purification and biotin labeling operation, without multiple rounds of antibody purification.

[0035] The antibody purification-labeling integrated system has a core functional element of rProteinA and rTurboID, which is a double-component cooperative system, rProtein A plays a role of purifying antibodies in the system, and rTurboID plays a role of biotin labeling in the system. According to the labeling range of rTurboID, and in combination with the fact that the solid-phase labeling ability of rTurboID is better than that of liquid-phase, it can be concluded that when the integrated system is constructed, rProteinA and rTurboID are fixed on the magnetic beads together, rProteinA can combine IgG antibodies, so that the distance between IgG and rTurboID is shortened, and the spatial distance requirement of rTurboID for biotin labeling of target proteins is met; the integrated antibody purification and biotin labeling detection system established by coupling rProteinA and rTurboID on carboxyl magnetic beads is better than the detection method of directly using rTurboID to liquidize biotin label antibodies first and then purifying, and can improve the efficiency of biotin labeling.

[0036] Compared with the prior art, the magnetic beads and kit for purifying biotin-labeled IgG antibodies provided by the application have the following advantages:

[0037] (1) The application of TurboID enzyme for biotin labeling of antibodies only needs to use ATP and ordinary biotin molecules, and the cost is very low.

[0038] (2) The protein A and TurboID for purifying IgG antibodies are coupled on the magnetic beads in the application, and the magnetic beads are used for purification and solid-liquid separation, so that the steps of purification, biotin labeling, and corresponding purification and buffer replacement are more simple. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 It is a pET21a-rProteinA induced expression effect diagram; M: protein marker, Thermo, 26616, 3 muL; UI: rProteinA sample before induction; I: rProteinA sample after induction;

[0040] Figure 2 It is a pET28a-rTurboID induced expression effect diagram; M: protein marker, Thermo, 26616, 3 muL; UI: rTurboID sample before induction; I: rTurboID sample after induction.

[0041] Figure 3Figure for the purification effect of rProteinA protein; M: Protein Marker; H: whole bacteria; S: supernatant after bacteria broken; G: Beads after hanging column; W: Beads after Wash; E1: first eluent (200 mM Imidazole); E2: second eluent (500 mM Imidazole);

[0042] Figure 4 Figure for the purification effect of rTurboID protein; M: Protein Marker; H: whole bacteria; S: supernatant after bacteria broken; G: Beads after hanging column; W: Beads after Wash; E1: first eluent (200 mM Imidazole); E2: second eluent (500 mM Imidazole);

[0043] Figure 5 Figure for the identification effect of rProteinA protein; M: Marker; rA: rProteinA

[0044] Figure 6 Figure for the identification effect of rTurboID protein; M: Marker; rD: rTurboID

[0045] Figure 7 Figure for the comparison of positive serum and negative serum of bovine brucellosis; PC: positive serum; NC: negative serum; ****: p<0.0001; ***: p<0.001; **: p<0.01; *: p<0.1; ns: no statistical significance;

[0046] Figure 8 Figure for the detection of positive serum and negative serum of bovine brucellosis by integrated system; ****: p<0.0001; ***: p<0.001; **: p<0.01; *: p<0.1; ns: no statistical significance;

[0047] Figure 9 Figure for the comparison of HRP-SA and HRP-IgG and biotinylated antibody; ****: p<0.0001; ***: p<0.001; **: p<0.01; *: p<0.1; ns: no statistical significance;

[0048] Figure 10 Figure for the detection of brucellosis antibody by integrated system; ****: p<0.0001; ***: p<0.001; **: p<0.01; *: p<0.1; ns: no statistical significance. DETAILED DESCRIPTION

[0049] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this application belongs. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods used are all conventional methods unless otherwise specified.

[0050] The present application will be described in detail below with reference to the examples.

[0051] Table 1 Solutions used in the experiment

[0052]

[0053] Table 2 Reagents and consumables used in the experiment

[0054]

[0055]

[0056] A magnetic bead for biotin labeling and purification of IgG antibody, comprising the following steps,

[0057] A. rProteinA and rTurboID were prepared into protein solutions of different proportions with 1x Reaction Buffer, and the total protein amount was 2 mg / mL. The groups are shown in Table 3.

[0058] Table 3 rProteinA and rTurboID protein solution groups

[0059]

[0060] B. The carboxyl magnetic beads were combined with the grouped protein solutions, and the rProteinA and rTurboID protein-coated magnetic beads were obtained. Protein solutions before and after combination were taken and prepared into samples. Then blocking was performed, and finally Protective Buffer was added for storage in a 4°C refrigerator for standby.

[0061] The coupling / coating step is,

[0062] (1) Reagent preparation: dilute 10x Activation Buffer and 10x Reaction Buffer to 1x with ultrapure water;

[0063] (2) Magnetic bead preparation: resuspend the carboxyl magnetic beads, take 100 μL of the magnetic bead suspension into a centrifuge tube with a pipette, and stand on a magnetic stand for 10 s. When the solution is clear, discard the supernatant;

[0064] (3) Resuspend the magnetic beads with 500 μL of Activation Buffer (1x);

[0065] (4) Resuspend the beads by pipetting, place the tube on the magnetic stand for 10 seconds to remove the supernatant, repeat this operation twice;

[0066] (5) Add the same volume of 1 x Activation Buffer as the initial volume of the bead suspension;

[0067] (6) Activation of the carboxyl group on the surface of the carboxyl magnetic beads: weigh an appropriate amount of EDC and NHS to prepare EDC Solution and NHS Solution with a concentration of 10 mg / mL in Activation Buffer (1 x) (EDC Solution and NHS Solution must be prepared immediately before use);

[0068] (7) Add 100 μL of the prepared EDC Solution and NHS Solution to the resuspended magnetic beads in (5), place the centrifuge tube in the vortex mixer, and activate at room temperature for 30-60 minutes. The magnetic beads should be kept in suspension during activation (the activated magnetic beads should be immediately subjected to the coupling reaction);

[0069] (8) Coupling of protein to carboxyl magnetic beads: after activation of the magnetic beads, place the tube on the magnetic stand for 10 seconds to remove the supernatant;

[0070] (9) Prepare a protein solution (concentration 0.5-2 mg / mL) with 1 x Reaction Buffer, and take a sample;

[0071] (10) Add 100 μL of the protein solution to the centrifuge tube, resuspend with a pipette, and transfer to the vortex mixer for coupling at room temperature for 2 hours or first at room temperature for 1 hour and then at 4°C overnight. The magnetic beads should be kept in suspension during coupling;

[0072] (11) After coupling, place the magnetic beads on the magnetic stand, collect the supernatant, and take a sample;

[0073] (12) Resuspend the magnetic beads with 500 μL of Reaction Buffer (1 x), gently pipette the magnetic beads, place the tube on the magnetic stand for 10 seconds to remove the supernatant, and repeat this operation twice.

[0074] A method for preparing a purified biotin-labeled IgG antibody, comprising the following steps:

[0075] 1. Combine the rProteinA and rTurboID protein-coated magnetic beads prepared in step B with an equal volume of animal serum;

[0076] (1) Place the carboxyl magnetic bead system on the magnetic stand for 10 seconds, and discard the supernatant;

[0077] (2) After adding 500 μL PBS buffer solution to resuspend the magnetic beads, place them on the magnetic stand, and after the solution is clarified, discard the supernatant and repeat the operation 2-3 times;

[0078] (3) Dilute the animal serum with PBS buffer solution, and after dilution, add 500 μL to the centrifuge tube and place it on the mixing instrument for 30-60 minutes to obtain IgG-bound magnetic beads;

[0079] 2. Biotin labeling of the combined magnetic beads prepared in step 1, the magnetic beads have been coupled with rTurboID and rProtein A, and the solution required for biotin labeling is added.

[0080] (1) After binding, place the centrifuge tube on the magnetic stand and discard the supernatant;

[0081] (2) Add 500 μL PBS buffer solution. Resuspend the magnetic beads with a pipette and then place them on the magnetic stand. Discard the supernatant and repeat this step 3 times;

[0082] (3) Prepare the solution required for biotin labeling, and the final concentration of each component in the mixed solution is 1 mM ATP, 5 mM biotin, and 5 mM MgCl2. Take 100 μL of the mixed solution into the centrifuge tube and place it on the mixing instrument for 2-4 hours at room temperature;

[0083] (4) Repeat operation (2).

[0084] 3. Elute the labeled antibodies on the magnetic beads with an acidic eluent and neutralize with a neutralizing solution, and take samples; the acidic eluent is 100 mM glycine, 0.1% Tween-20, pH 5.0; the neutralizing solution is 1.0 M Tris-HCl, pH 9.0. The mutant protein A (Q10H / N11H) can be eluted in a more neutral pH environment to minimize the damage to IgG antibodies caused by low pH.

[0085] The method for purifying biotin-labeled IgG antibodies using magnetic beads comprises the following steps:

[0086] (1) Take out the Brucella LPS-coated enzyme plate, dilute the purified biotin-labeled IgG antibodies to the appropriate concentration, and add 100 μL to different wells, respectively, and incubate at 37°C for 30 min; the Brucella LPS-coated enzyme plate is provided by Shijiazhuang Shengbo Biological Technology Co., Ltd.

[0087] (2) Shake off the liquid on the plate, fill it with PBST (1×), and let it stand for 1-2 min, then pat the liquid in the plate dry, and repeat 3 times;

[0088] (3) Dilute the HRP-labeled streptavidin with 1×PBST, and add it to each well with a pipette, and react at 37°C for 30 min;

[0089] (4) Repeat (2), wash away the residual liquid in the hole, and pat dry the hole;

[0090] (5) Add 100 μL TMB color developing solution, and develop color in the dark for 3-30 min;

[0091] (6) After color development, add 100 μL TMB color developing termination solution to terminate the reaction;

[0092] (7) During color development, open the enzyme marker in advance, and measure the 450 mM absorbance after adding the termination solution.

[0093] Design and optimization of rProteinA protein

[0094] To achieve more efficient elution, the embodiment selects an optimized ProteinA (Q10H / N11H), which is named rProteinA. In the selection of the expression vector, pET21a is selected. The replicon type of the vector is ColE1, a T7 strong promoter is used, and the transcription efficiency is extremely high, which is beneficial to the expression of the target gene and thus the protein yield is high. The vector carries a 6×His tag at the C terminal, which facilitates subsequent protein purification by affinity chromatography. The cloning and construction methods used mainly include Gibson Assembly and GoldenGate DNA seamless assembly technology.

[0095] The base sequence of the rProteinA protein after codon optimization used in the embodiment is shown in SEQ ID NO: 1:

[0096]

[0097] Construction of rTurboID prokaryotic expression system

[0098] For the optimized rTurboID, pET28a is selected as the expression vector. The reason for selection is that pET28a is a high-copy plasmid, and its replicon is ColE1 type, which can balance the copy number pressure through host regulation. In addition, the vector uses a T7 promoter, which can promote the transcription of the target gene during induction and expression, which is beneficial to the expression of the target protein in the later stage. At the same time, the 6×His tag at the N terminal of the vector is replaced with 10×His to optimize the purification. The synthesized gene is used to construct the protein expression vector of rTurboID by GoldenGate seamless cloning technology.

[0099] The base sequence of the rTurboID protein after codon optimization used in the embodiment is shown in SEQ ID NO: 2:

[0100]

[0101] Transformation

[0102] Transform pET21a-rProteinA or pET28a-rTurboID into BL21(DE3) expression competent cells.

[0103] (1) Take the prepared competent cells from -80℃ refrigerator, put them into ice box and melt on ice;

[0104] (2) Add the reacted product into the melted cells (the volume of the product should not exceed 1 / 10 of the volume of the competent cell suspension), put them into ice box and ice bath for 30 minutes;

[0105] (3) During the ice bath, preheat the water bath to 42℃;

[0106] (4) Put the competent cells with the added product in (2) into the preheated water bath for about 60-90 seconds;

[0107] (5) After the heat shock, quickly put the tube into the ice box and ice bath for 2-3 minutes. Do not shake the centrifuge tube during the ice bath.

[0108] (6) Add 0.9 mL of SOC medium into the centrifuge tube after the ice bath;

[0109] (7) Transfer the centrifuge tube with the added medium to the shaker and incubate at 37℃, 180 rpm for about 1 hour;

[0110] (8) During the incubation, take the prepared solid medium plate with added antibiotic from the refrigerator and put it into the 37℃ incubator;

[0111] (9) Use the pipette to take an appropriate amount of the incubated cell solution from the tube and spread it evenly on the plate using a spreader;

[0112] (10) After the plate is dried, put it into the 37℃ incubator and incubate it upside down for 12-16 hours.

[0113] Induced expression of recombinant protein

[0114] 1) Pick a single colony from the incubated plate, send it for testing, compare the sequencing results, extract the correct plasmid from the correct bacteria, and then transfer it to competent cells BL21(DE3);

[0115] 2) Pick a single colony in the clean bench and put it into 5 mL of LB medium with added antibiotic, incubate it at 37℃, 180 rpm overnight;

[0116] 3) In the super-clean bench, take the liquid in 2) to the new 5 mL LB medium with antibiotics by pipette, transfer to the shaker, 37℃, 180 rpm, culture for about 1-2 h, measure the OD value, when the OD value is about 0.6, take the sample before induction;

[0117] 4) After sampling, put the bottle into ice bath, add inducer IPTG, so that the final concentration is 1 mM;

[0118] 5) After adding the inducer, transfer to the shaker (200 rpm) for culture, 20℃, induce expression for 16 h, observe the induction effect the next day;

[0119] 6) After the induction is completed, take the sample for standby;

[0120] 7) Prepare SDS-PAGE gel, the SDS-PAGE gel uses commercially available precast gel, which can be operated according to the instructions;

[0121] 8) After the gel is completely solidified, prepare the sample for SDS-PAGE electrophoresis;

[0122] Through SDS-PAGE, analyze the whole bacterial lysate, and the results show that Figure 1 、 Figure 2 SDS-PAGE analysis shows that rProteinA and rTurboID are successfully expressed. The target band is located at the position of 35 kDa Marker, and the deviation from the theoretical molecular weight (34.8 kDa and 35.2 kDa) is relatively small.

[0123] Protein purification

[0124] (1) According to the method of 2.3, after obtaining the activated bacterial solution, take 500 μL into a 50 mL conical flask containing LB medium by pipette;

[0125] (2) Transfer the conical flask to the shaker for culture, 37℃, 180 rpm, culture to the logarithmic phase, that is, OD600 is in the range of 0.6-0.8, take the sample;

[0126] (3) Then put the conical flask into ice bath, and induce according to the optimized induction conditions of 2.3;

[0127] (4) After the induction is completed, take the induced sample;

[0128] (5) Divide the culture solution into 50 mL centrifuge tubes, and balance each other. Centrifuge at 12000 rpm for 1 min;

[0129] (6) After centrifugation, discard the supernatant and collect the bacterial pellet;

[0130] (7) Add Binding Buffer to the centrifuge tube, resuspend the collected pellet (Binding Buffer is added at a volume of 10 mL per 1 g of pellet);

[0131] (8) After the pellet is resuspended, the cells are lysed by sonication (the breaking condition is generally set at 100 W, running for 3 s and stopping for 8 s), and the time is not more than 15 min;

[0132] (9) After the cells are lysed by sonication, centrifugation is performed at 12,000 rpm for 30 min;

[0133] (10) After centrifugation, the supernatant is collected and stored.

[0134] (11) During centrifugation, magnetic beads can be prepared. The magnetic beads are shaken uniformly, and 1 mL of the magnetic bead suspension is taken with a pipette and added to the centrifuge tube;

[0135] (12) The centrifuge tube is placed on a magnetic stand, and after the solution is clear, the solution is aspirated with a pipette and discarded;

[0136] (13) 1.5 mL of Binding Buffer is added, and the solution is shaken up and down, and then placed on a magnetic stand, and after the solution is clear, the solution is discarded, and the operation is repeated multiple times to achieve the purpose of balancing the magnetic beads;

[0137] (14) After balancing, the supernatant collected in (10) is combined with the magnetic beads, and placed in a vortexer for 1 hour at 4°C;

[0138] (15) After binding, the centrifuge tube is placed on a magnetic stand, and after the solution is clear, the solution is discarded;

[0139] (16) Resuspend with the balancing Buffer, place on the magnetic stand, and after the solution is clear, discard the solution, and repeat multiple times. Before the solution is discarded for the last time, take a sample;

[0140] (17) Add Wash Buffer, shake up and down uniformly, and then place on a magnetic stand, and after the solution is clear, discard the solution, and repeat 2-3 times to achieve the purpose of washing away impurities;

[0141] (18) Finally, elution is performed. Before elution, the operation of (16) is repeated, and then elution is performed. During elution, the target protein concentration can be adjusted by changing the elution volume. Add 0.5 mL of Elution Buffer 1, shake up and down uniformly, place on a magnetic stand, collect the target protein solution, and take a sample;

[0142] (19) Secondary elution, add 0.5 mL of Elution Buffer 2, shake up and down, and collect the target protein solution;

[0143] (20) After obtaining the target protein, the magnetic beads were treated, 1.5 mL of Elution Buffer 2 was added to the centrifugal tube, shaken uniformly, placed on the magnetic stand, and the solution was discarded. Finally, Elution Buffer 2 was replaced with deionized water, and repeated washing was performed 3-5 times. Finally, 20% ethanol solution was added, and stored in a 4°C refrigerator.

[0144] (21) The sample was prepared, SDS-PAGE was performed, and the purified product was analyzed. The purification solution used in this experiment is shown in the following table.

[0145] Table 4 Purification solution used in protein purification experiment

[0146]

[0147] Figure 3 In the middle, it can be seen from the lane that a small amount of host impurities exists in the rProteinA sample in lane 4. After washing with WashBuffer, the impurities remaining on the magnetic beads are significantly reduced. After elution, the elution results of ElutionBF1 and ElutionBF2 are compared, and it is found that after elution with ElutionBF1, the protein on the magnetic beads is not completely eluted, and subsequent elution with ElutionBF2 is required.

[0148] Figure 4 In the middle, after rTurboID protein binds to the magnetic beads, a small amount of impurities still exists, which needs to be washed. After elution with ElutionBF1 and ElutionBF2, it is found that after elution with the former, a small amount of protein remains on the magnetic beads and is not completely eluted. In order to recover as much target protein as possible, subsequent elution with ElutionBF2 is required.

[0149] In order to ensure that the obtained protein is the target protein, Western-blot was used to identify the product. Western Blot is a method that uses the specific recognition and binding of antigens and antibodies. In addition, the target protein is a protein expressed in prokaryotes and has a His tag. Based on these characteristics, His tag was selected for identification to verify whether the purified protein is the target protein rProteinA and rTurboID.

[0150] (1) rProteinA and rTurboID proteins were subjected to SDS-PAGE gel electrophoresis, respectively;

[0151] (2) After electrophoresis, the gel was peeled off the mold with a plastic plate, and the gel block containing the target protein was cut off;

[0152] (3) Measure the size of the gel block, and cut the PVDF membrane to the appropriate size. Cut a corner from the upper left corner of the membrane to facilitate orientation of the membrane later.

[0153] (4) Soak the cut PVDF membrane in anhydrous ethanol for 1 min before use to activate the positive groups on the membrane, making it easier to bind to the negatively charged proteins.

[0154] (5) Soak the cut gel, activated PVDF membrane, filter paper, and sponge in a culture dish containing transfer buffer for about 5 min. This step is to balance the purpose.

[0155] (6) Place the transfer sandwich flat with the transparent side up and the black side down. Place the sponge, 4 filter papers, cut gel, PVDF membrane, 4 filter papers, and sponge in order from bottom to top. Roll the stack of filter papers with a roller to remove air bubbles.

[0156] (7) Place the assembled transfer sandwich in the electrophoresis tank. Prepare an ice brick and place it in a plastic bag. Then place the plastic bag containing the ice brick in the gap of the electrophoresis tank.

[0157] (8) Connect the electrophoresis tank and the electrophoresis instrument, and perform electrophoresis. Choose constant current mode, 300 mA, and run for 25-30 min.

[0158] (9) After the transfer is complete, turn off the power and remove the PVDF membrane. Identify the orientation based on the corner cut earlier, and transfer the membrane to a box containing blocking solution. Place the box on a decolorizing shaker and block at room temperature for 2 h.

[0159] (10) Wash the membrane: After blocking is complete, add 1x PBST to cover the PVDF membrane. Then place the membrane on a decolorizing shaker and shake at low speed for 5 min. Repeat 3-5 times.

[0160] (11) Dilute the primary antibody to the appropriate concentration according to the instructions. Prepare a plastic bag similar in size to the membrane, and transfer the diluted primary antibody to the bag. Incubate on a decolorizing shaker for 1 h at room temperature or overnight at 4°C.

[0161] (12) After incubation of the primary antibody, transfer the membrane to a small box and add 1x PBST to cover the membrane. Wash the membrane according to step (10).

[0162] (13) Prepare the secondary antibody dilution according to the instructions. Incubate the secondary antibody according to step (11) on a decolorizing shaker for 30 min to 1 h at room temperature.

[0163] (14) After incubation of the secondary antibody, wash the membrane according to step (10).

[0164] (15) Use the DAB color developing kit or ECL luminescent kit for color development.

[0165] (16) After color development according to the description, an image is collected.

[0166] Figure 5 In the rProtein A and rTurboID corresponding positions on the PVDF membrane, specific bands appear, and the molecular weight size is consistent with the expectation, so it can be confirmed that the obtained protein product is rProtein A.

[0167] Figure 6 In the rProtein A and rTurboID corresponding positions on the PVDF membrane, specific bands appear, and the molecular weight size is consistent with the expectation, so it can be confirmed that the obtained protein product is rProtein A.

[0168] The effect of the application is verified through the detection experiment of Brucella LPS antigen.

[0169] Table 5 Reagents used in the experiment

[0170]

[0171] A method for preparing a purified biotin-labeled IgG antibody, comprising the following steps,

[0172] (1) Dilute the Brucellosis positive serum and negative serum of cattle according to the same dilution ratio;

[0173] (2) The diluted serum is combined with the magnetic beads for IgG antibody biotin labeling and purification, resuspended by blowing and sucking with a pipette, transferred to a vortex mixer, combined at room temperature for 30 minutes, and the magnetic beads are kept in suspension during the combination;

[0174] The preparation of the magnetic beads for IgG antibody biotin labeling and purification includes the following steps:

[0175] A, rProtein A and rTurboID are prepared into a protein solution with 1xReactionBuffer, and the total protein amount is 2mg / mL of the protein solution, the mass fraction of rTurboID is 80%, and the mass fraction of rProtein A is 20%.

[0176] B, combine the carboxyl magnetic beads with the grouped protein solution to obtain the magnetic beads for IgG antibody biotin labeling and purification; take the protein solution before and after combination to prepare samples. Then, blocking is performed, and finally, Protective Buffer is added for storage in a 4℃ refrigerator for standby.

[0177] (3) After combination, stand on the magnetic stand for 10s, and discard the supernatant;

[0178] (4) Add 500 μL buffer to resuspend the magnetic beads, place on the magnetic stand, discard the supernatant, and repeat this step 3 times;

[0179] (5) Biotin labeling: prepare the solution required for biotin labeling, the final concentration of each component in the mixed solution is 1 mM ATP, 5 mM Biotin, 5 mM MgCl2; take 100 μL of the mixed solution into a centrifuge tube, place it in a homogenizer, keep the magnetic beads in a suspended state, and react at room temperature for 2-4 hours;

[0180] (6) After incubation, place it on the magnetic stand, discard the supernatant, and wash away the residual liquid according to (4);

[0181] (7) Add acid eluent, shake during the period, and the time should not exceed 15 minutes;

[0182] (8) Place on the magnetic stand, collect the clear supernatant, add neutralizing solution, and mix well;

[0183] (9) Dilute the eluent in (8) by gradient dilution for ELISA;

[0184] (10) Take out the Brucella LPS coated enzyme labeled plate, take 100 μL of the diluted eluent into different wells, incubate at 37°C for 30 min;

[0185] (11) Shake off the liquid on the plate, fill it with PBST (1x), stand for 1-2 min, pat the liquid in the plate, and repeat at least 3 times;

[0186] (12) Dilute HRP-streptavidin with 1x PBST, add 100 μL to each well, and react at 37°C for 30 min;

[0187] (13) Repeat step (11) to wash the wells;

[0188] (14) Add 100 μL of TMB color developing solution, develop color in the dark for 3-30 min;

[0189] (15) Take an equal volume of TMB color developing termination solution to terminate the reaction, and measure the 450 nm absorbance value with an enzyme labeled instrument.

[0190] Comparison of dual-function system purified-biotin labeled antibody with traditional secondary antibody

[0191] In order to compare the effects of biotin labeled antibody, HRP-SA and HRP-goat anti-bovine IgG on ELISA detection, the ELISA detection system based on Brucella LPS antigen was optimized, the purified-biotin labeled antibody was combined with the plate coated with Brucella LPS, and the experiment was as follows:

[0192] (1) Take out the enzyme-labeled plate coated with Brucella LPS, add the diluted antibody respectively, 37℃, 30min;

[0193] (2) Discard the liquid in the plate, fill each well with PBST (1x), wait for 1-2min, shake off the liquid in the plate, repeat the operation 3 times;

[0194] (3) Dilute HRP-labeled streptavidin and goat anti-cow IgG with 1x PBST according to the instructions;

[0195] (4) Take 100μL of the diluted antibody into the well respectively, incubate at 37℃ for 30min;

[0196] (5) Repeat (2) to wash away the residual liquid in the well and pat dry the liquid in the air;

[0197] (6) Add 100μL of TMB color developing liquid, develop color for 3-30min in the dark;

[0198] (7) Take an equal volume of TMB color developing termination liquid to terminate the reaction, and measure the absorbance at 450nm with an enzyme-labeled instrument.

[0199] Application of integrated system to establish indirect ELISA for detection of Brucella antibody

[0200] (1) Purify the antibody in the positive serum of bovine brucellosis with the constructed magnetic bead system and biotin label;

[0201] (2) Use gradient dilution to perform ELISA with the antibody purified and biotin-labeled by the integrated system. This experiment designs several groups including the experimental group (antibody concentration after gradient dilution 1μg / mL, 5μg / mL, 10μg / mL, 50μg / mL, 100μg / mL), the control group one (without adding primary antibody), the control group two (without adding secondary antibody), and the blank group;

[0202] (3) Take out the enzyme-labeled plate coated with Brucella LPS, add the diluted antibody respectively, 37℃, 30min;

[0203] (4) Discard the liquid in the plate, fill each well with PBST (1x), wait for 1-2min, shake off the liquid in the plate, repeat the operation 3 times;

[0204] (5) Dilute HRP-streptavidin with 1x PBST according to the instructions;

[0205] (6) Take 100μL of the diluted antibody into the well respectively, incubate at 37℃ for 30min;

[0206] (7) Repeat (2) and pat dry the liquid in the air;

[0207] (8) Add 100 μL TMB color developing solution, develop color in dark for 3-30 min;

[0208] (9) Take equal volume of TMB color developing termination solution to terminate reaction, and measure absorbance at 450 nm by enzyme labeling instrument.

[0209] Compare ELISA results of positive serum group and negative serum group of bovine brucellosis, analyze whether the selected antibodies are suitable for detection, and results are shown in Figure 7 It is found that there is obvious difference between two groups, and the selected HRP-goat anti-bovine IgG can be used as secondary antibody for detection in the experiment.

[0210] The bifunctional system is combined with positive serum and negative serum respectively and biotin labeled, and ELISA detection is performed, and absorbance of two groups is compared, and results are shown in Figure 8 It is found that OD450 of the integrated system combined with positive group is higher than that of negative group, that is, the purified-biotin labeled antibody of the positive group of the system can be used as primary antibody of indirect ELISA, and it is preliminarily determined that the system can be used for detection of bovine brucellosis.

[0211] The primary antibody of the integrated system (antibody obtained by combining positive serum of the integrated system, purification and biotin labeling) is reacted with HRP-streptavidin and HRP-goat anti-bovine IgG, and difference between detection results of two groups. Results are shown in 9, and it can be seen that, when reacted with the same concentration of labeled primary antibody, absorbance of the HRP-SA group is generally higher than that of the HRP-goat anti-bovine IgG group, that is, the detection sensitivity of the method established in the application is higher than that of the traditional ELISA. It can be proved that the indirect ELISA of the integrated system established in the application can be used for detection of brucella antibody.

[0212] The system constructed is applied to detection of brucella antibody, and the antibody is gradient diluted for indirect ELISA. Experimental groups are set: experimental group (1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL), control group (no primary antibody group, no secondary antibody group, no antigen group), in order to ensure accuracy of the experiment, each group has 3 repeats. Results are shown in Figure 10 Absorbance changes with antibody concentration, and change trend is obvious, and absorbance of the experimental group is obviously higher than that of the control group.

[0213] The feasibility and sensitivity advantage of the integrated indirect ELISA system were verified by systematic experiments. First, by comparing the ELISA results of the positive serum group and the negative serum group of bovine brucella, it was found that the absorbance difference between the two groups was significant when HRP-goat anti-bovine IgG was used as the secondary antibody, indicating that it was suitable for the experimental system. Second, after the bifunctional system was combined with positive and negative serum and biotin labeled, ELISA detection showed that the OD450 of the positive group was significantly higher than that of the negative group ( Figure 8 ), indicating that the purified-biotin labeled positive serum antibody could be used as the primary antibody of indirect ELISA. Then, by comparing the detection effect of HRP-streptavidin (HRP-SA) and HRP-goat anti-bovine IgG, it was found that the absorbance value of HRP-SA group was generally higher ( Figure 9 ), confirming that the sensitivity of the integrated system was better than that of the traditional ELISA. Finally, through the antibody gradient dilution experiment (1-100 μg / mL) and the control group setting (no primary antibody, no secondary antibody, no antigen), the results showed that the absorbance value was obviously dose-dependent with the antibody concentration, and the OD value of the experimental group was significantly higher than that of the control group, thus confirming that the system was stable and reliable.

[0214] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A magnetic bead for biotinylation and purification of IgG antibodies, characterized in that: Its preparation includes the following steps, A) Prepare a protein solution by mixing rProteinA and rTurboID; B) The protein solution is mixed with carboxyl magnetic beads to couple the protein to the carboxyl magnetic beads, resulting in magnetic beads coated with rProteinA and rTurboID proteins, which are used for biotinylate labeling and purification of IgG antibodies.

2. The magnetic beads for biotinylation and purification of IgG antibodies as described in claim 1, characterized in that: The mass ratio of rProteinA to rTurboID is 1:4 to 4:1, and the total protein content is 1.5 to 2.5 mg / mL.

3. The magnetic beads for biotinylation and purification of IgG antibodies as described in claim 1, characterized in that: The base sequence of rProteinA is shown in SEQ ID NO:1; the base sequence of rTurboID is shown in SEQ ID NO:

2.

4. A method for preparing purified biotin-labeled IgG antibody, characterized in that, Using the magnetic beads for biotinylation and purification of IgG antibodies as described in any one of claims 1 to 3, the method includes the following steps: 1) The magnetic beads for biotin labeling and purification of IgG antibodies according to any one of claims 1 to 3 are mixed and reacted with animal serum containing IgG antibodies to be labeled to obtain magnetic beads bound with IgG antibodies. 2) Biotin label the IgG antibody bound to the magnetic beads in step 1) with IgG antibody; 3) Elute the biotin-labeled IgG antibody obtained in step 2) off the magnetic beads to obtain purified biotin-labeled IgG antibody.

5. The method for preparing purified biotin-labeled IgG antibody according to claim 4, characterized in that: Step 1) involves obtaining magnetic beads bound to IgG antibodies, including the following steps: a) Place the magnetic beads used for biotin labeling and purification of IgG antibodies on a magnetic rack for 5–15 seconds, then discard the supernatant; b) After resuspending the magnetic beads in 300-700 μL of buffer, place them on a magnetic rack. After the solution becomes clear, discard the supernatant and repeat the operation 2-3 times. c) Dilute animal serum with buffer solution. After dilution, add 300-700 μL to a centrifuge tube and place it on a mixer for 30-60 minutes to obtain magnetic beads bound with IgG antibodies.

6. The method for preparing purified biotin-labeled IgG antibody according to claim 4, characterized in that: Step 2) biotin labeling includes the following steps: A) Remove the supernatant from the centrifuge tube containing the magnetic beads that bind IgG antibodies; B) Add 300-700 μL of buffer solution; resuspend the magnetic beads by pipetting, then place them on a magnetic rack, discard the supernatant, and repeat this step 2-5 times. C) Prepare the mixture required for biotin labeling, add the mixture to a centrifuge tube containing magnetic beads that bind IgG antibodies, place it in a mixer, and react at room temperature for 2-4 hours; D) Add 300-700 μL of buffer solution; resuspend the magnetic beads by pipetting, then place them on a magnetic rack, discard the supernatant, and repeat this step 2-5 times.

7. The method for preparing purified biotin-labeled IgG antibody according to claim 6, characterized in that: The final concentrations of each component in the mixture required for biotin labeling are 0.5–1.5 mM ATP, 3–7 mM biotin, and 3–7 mM MgCl2.

8. An antigen detection kit, characterized in that: The IgG antibody contains magnetic beads for biotin labeling and purification of IgG antibodies as described in any one of claims 1 to 3, or purified biotin-labeled IgG antibodies prepared by the preparation method described in any one of claims 4 to 7.

9. The antigen detection kit according to claim 8, characterized in that: The antigen is the lipopolysaccharide antigen of Brucella.

10. A method for preparing purified biotin-labeled IgG antibodies using the preparation method according to any one of claims 4 to 7, characterized in that: Includes the following steps: (1) Take out the antigen-coated ELISA plate, add 90-100 μL of diluted elution buffer to the wells, and incubate at 36-38°C for 20-40 min; (2) Shake off the liquid on the plate, fill it with PBST (1×), let it stand for 1-2 minutes, pat the liquid in the plate dry, and repeat 4-5 times. (3) Dilute HRP-labeled streptavidin with 1×PBST, pipette the solution into each well, and incubate at 37°C for 20–40 min. (4) Repeat (2) to wash away the residual liquid in the hole and pat the hole dry; (5) Add 90-100 μL of LTMB colorimetric solution and develop the color in the dark for 3-30 min; (6) After color development, add 90-100 μL of LTMB color development stop solution to terminate the reaction; (7) During the color development period, use an enzyme-linked immunosorbent assay (ELISA) reader in advance, add the stop solution and measure the absorbance at 450 mM.

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