Preparation method of cross-linked heterophilic antibody blocking agent
Through the BS3 method, the restructuring IGG monomer is cross -linking, preparing cross -linking heterosexual antibody blockers, solving the problem that it is difficult to meet a large number of production needs in traditional methods, and achieved stable and efficient antibody production and interference blocking effects.
Patent Information
- Application Number
- CN202411618046.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-13
- Publication Date
- 2025-01-28
AI Technical Summary
Traditional methods require the preparation of antigens before producing antibodies, and it is difficult to meet the needs of mass production.
The BS3 method is used to cross -cross the IGG monomer to prepare the cross -linking heterosexual antibody blocker.
Realize stable and repeated production, small differences between batch, meet a large number of production needs, and improve the stability of cross -linking products and the ability to block heterosexual antibody interference.
Smart Images
Figure CN119372259A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing a cross-linked heterophilic antibody blocker. Background Art
[0002] Heterophilic antibodies are endogenous antibodies in the human body, with a relatively stable chemical structure and generally no clear immunogen. They are usually produced after direct contact with animals, animal serum products, immunotherapy, or vaccination with vaccines derived from animal serum and tissues. Heterophilic antibodies also exist in 3% to 15% of normal people. Heterophilic antibodies can bind to the determinants of the Fc segment and F(ab')2 region of various animal immunoglobulins. In the absence of antigen, they bridge or block the capture antibody and the detection antibody, causing the labeled antibody bound to the solid phase to increase or decrease, causing a false increase or decrease in the test result. The detection method most affected by this interference is the double antibody sandwich method, especially the detection method using mouse monoclonal antibodies.
[0003] When interference is suspected in the immunoassay, the specimen can be pretreated by adding a heterophilic antibody blocker before testing; a heterophilic antibody blocker is a biological agent that, when added to the immunoassay system, can bind to heterophilic antibodies, thereby effectively preventing non-analyte-mediated antibody bridging.
[0004] Non-specific substances such as mouse IgG, mouse serum, non-specific monoclonal antibodies, polymerized cross-linked IgG and other heterophilic antibody blockers can block the binding of human heterophilic antibodies.
[0005] The traditional method of producing mouse IgG antibodies is to stimulate the animal immune system with antigens. This method requires the preparation of antigens first, and it is difficult to produce antibodies in large quantities (it requires a large amount of experimental consumables and time). The method of producing IgG antibodies using mammalian cells can achieve stable long-term production in subsequent experiments, with high batch-to-batch reproducibility and can meet large-scale production needs. The recombinant IgG monomer is highly stable and is not prone to precipitation during the cross-linking process.
[0006] Polymers are particularly effective against interference, and also have a good elimination effect on samples with higher specificity and monovalent interference. The method of converting monomeric IgG into polymeric IgG is mostly chemical cross-linking, that is, using some chemical reagents to react with the carboxyl or amino groups on the amino acid residues of the antibody to cross-link the IgG monomers into polymers by forming intermolecular covalent bonds. Commonly used cross-linking agents include glutaraldehyde, carbodiimide, N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), BS3 (bis(sulfosuccinimidyl) suberate), etc. Summary of the invention
[0007] The present invention adopts BS3 method to cross-link recombinant IgG monomers to obtain a cross-linked heterophilic antibody blocker.
[0008] The purpose of the present application is to provide a method for preparing a cross-linked heterophilic antibody blocker to solve the problem that the traditional method of producing antibodies requires the preparation of antigens first, and the method is difficult to produce antibodies in large quantities.
[0009] To achieve the above object, the present invention provides the following technical solutions: A method for preparing a cross-linked heterophilic antibody blocker comprises the following steps: S1, cationic liposome transfection method plasmid transfected CHO cells to prepare recombinant IgG monomer for purification; The cross-linked heterophilic antibody blocker was prepared by cross-linking with S2 and di(sulfosuccinimidyl) suberate (BS3) cross-linker.
[0010] Preferably, the IgG monomer purification in step S1 comprises the following steps: S11. Sample preparation: The collected transfected cell fluid was centrifuged at 4000 rpm for 10 min, the supernatant was collected, filtered through a 0.45 um filter, and the supernatant volume was measured using a measuring cylinder; Column preparation: Fill 500 mL of Protein A filler and wash 5-10 column volumes with purified water to remove ethanol; S12, balance: Protein A column is balanced with 10mM PBS (pH7.4) balance buffer for 5 times column volume. After the column is balanced, the A280 value displayed by the protein UV detector is adjusted to 0 as the baseline; S13, sample loading: dilute the supernatant with 1 / 5 times the volume of the supernatant with 100 mM PBS (pH 7.4) dilution buffer and slowly load the sample at a flow rate of 3-5 ml / min; S14, balance: after loading, use 10mM PBS (pH7.4) as balance buffer for 5-10 column volumes until no protein flows out, i.e. the protein UV detection value A280 shows 0; S15, dissociation: after equilibrium is completed, use 0.1M citric acid (pH3.5) buffer for dissociation, collect samples with absorbance of 0.2-0.5 and greater than 0.5 in sections, and continue to wash the column with 0.1M citric acid after dissociation until the detection value of the nucleic acid detector A280 is stable; S16, equilibration: equilibrate the column with 5-10 column volumes of equilibration buffer 10 mM PBS (pH 7.4); S17, dialysis: use 10mM PBS (pH7.4) dialysis buffer to dialyze the sample, the dialysis ratio is 1:50, the solution is changed 3 times, and the dialysis interval is not less than 4 hours; Preferably, the cross-linking agent used in step S2 is BS3, ie, di(sulfosuccinimidyl) suberate, which is a homobifunctional, water-soluble, non-cleavable and membrane-impermeable amine-amine cross-linking agent.
[0011] Preferably, in step S2, preparing the cross-linked heterophilic antibody blocking agent comprises the following steps: S21 and BS3 cross-linkers were used to obtain cross-linked heterophilic antibody blockers; S22, handling and storage of cross-linked heterophilic antibody blocking agents; Preferably, obtaining a cross-linked heterophilic antibody blocking agent using a BS3 cross-linking agent comprises the following steps: S211, collecting the IgG monomers purified in the above S1 and measuring the volume; S212, place the beaker on a magnetic stirrer, stir and add a corresponding volume of 0.2M BS3 (bis(sulfosuccinimidyl) suberate) using a 1 mL pipette to a final concentration of 2 mM at a speed of 50 s / ml; S213, continue stirring at room temperature for 1h; S214, 4℃ overnight for 16-20h; S215, evenly add 2M Tris to a final concentration of 0.02M, at a speed of 50s / ml, stir with a magnetic stirrer for 30min, and take a sample for HPLC detection; S216. The sample was dialyzed using 10 mM PBS (pH 7.4) buffer at a dialysis ratio of 1:50. The solution was changed 3 times with an interval of no less than 4 hours.
[0012] Preferably, the processing and storage of the cross-linked heterophilic antibody blocking agent comprises the following steps: S221, unpack the dialysis bag and measure the volume of the antibody; S222, add 4% of the antibody volume of sucrose and stir until uniform; S223, filter using a 0.22 μm filter membrane, and measure and record the antibody concentration using a Nano300 UV spectrophotometer; S224. Add 0.1% of the antibody volume of Proclin 300 preservative to the antibody treated in step S22, mix well, and store at 4° C. to avoid freezing and thawing.
[0013] Preferably, the cross-linked heterophilic antibody blocking agent has the appearance of a colorless transparent liquid without obvious precipitation and floccules.
[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention prepares recombinant IgG monomers, can be stably and repeatedly produced, has small batch-to-batch differences, can meet large production needs, and is conducive to batch production. In addition, the recombinant IgG monomers have high stability, are not prone to precipitation during the cross-linking process, and can greatly improve the stability of the cross-linked product.
[0015] The polymer cross-linker used is BS3. The cross-linked IgG polymer has a high polymer content and a colorless and transparent appearance, which can avoid color interference in subsequent immunoassays.
[0016] In addition, the cross-linked heterophilic antibody blocker has a good ability to block the interference of heterophilic antibodies. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is a comparison of the appearance of IgG obtained by the two processes of Example 1 and Comparative Example 1 after repeated freezing and thawing (the left side is the recombinant IgG purified by the protein A method, and the right side is the mouse IgG purified by the protein G method); Figure 2 This is a high-performance liquid chromatography diagram of the cross-linked heterophilic antibody blocking agent obtained in Example 2; Figure 3 This is a high-performance liquid chromatography diagram of the cross-linked heterophilic antibody blocking agent obtained in Comparative Example 2; Figure 4 This is a comparison of the appearances of the two cross-linking processes (the left side is BS3 cross-linked recombinant IgG, and the right side is glutaraldehyde cross-linked recombinant IgG); DETAILED DESCRIPTION
[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example
[0019] Recombinant IgG Purification Sample preparation: Centrifuge the collected transfected cell solution at 4000 rpm for 10 min, collect the supernatant, filter with a 0.45 um filter, and measure the supernatant volume with a measuring cylinder; Column preparation: Fill 500 mL of Protein A filler and wash 5-10 column volumes with purified water to remove ethanol; Balance: The Protein A column was balanced with 10 mM PBS (pH 7.4) for 5 times the column volume. After the column was balanced, the A280 value displayed by the protein UV detector was adjusted to 0 as the baseline. Sample loading: dilute the supernatant with 1 / 5 times the volume of the supernatant with 100 mM PBS (pH 7.4) dilution buffer and slowly load the sample at a flow rate of 3-5 ml / min; Balance: After loading, use 10mM (pH 7.4) PBS to balance the column for 5-10 times the volume until no protein flows out, i.e. the protein UV detection value A280 shows 0; Dissociation: After equilibrium is completed, use 0.1M citric acid (pH3.5) buffer for dissociation, and collect samples with absorbance of 0.2-0.5 and greater than 0.5 in sections. After dissociation, continue to wash the column with 0.1M citric acid until the detection value of the nucleic acid detector A280 is stable; Equilibration: Equilibrate the column with 5-10 column volumes of equilibration buffer 10 mM PBS (pH 7.4); Dialysis: Use 10mM PBS (pH7.4) dialysis buffer to dialyze the sample at a dialysis ratio of 1:50, change the solution 3 times, and the dialysis interval should not be less than 4 hours.
[0020] After the above steps, recombinant IgG monomers were obtained. Figure 1 Shown on the left Example
[0021] Monomer cross-linking Collect the recombinant IgG monomer purified in Example 1 and measure the volume; Place the beaker on a magnetic stirrer, stir and add the corresponding volume of 0.2M BS3 (bis(sulfosuccinimidyl) suberate) with a 1mL pipette to a final concentration of 2mM at a speed of 50s / ml; Continue stirring at room temperature for 1 h; 4℃ overnight for 16-20h; 2M Tris was evenly added to a final concentration of 0.02M, at a speed of 50s / ml, and stirred for 30min using a magnetic stirrer; The sample was dialyzed with 10 mM PBS (pH 7.4) buffer at a dialysis ratio of 1:50, with the solution changed 3 times. The dialysis interval was not less than 4 hours.
[0022] After the above steps, a cross-linked heterophilic antibody blocking agent is obtained, which has an appearance as shown in Figure 3 Left picture.
[0023] Comparative Example 1 The difference from Example 1 is that in Comparative Example 1, the mouse IgG ascites was purified by protein G column method to obtain mouse IgG monomer. The specific process is as follows; Sample preparation: Frozen mouse IgG ascites was thawed at 10-15°C, first quickly filtered with absorbent cotton, and then filtered with 20-micron pore size rapid filter paper to remove oil, precipitated protein and tissue fragments; Column preparation: Fill 500 mL of Protein G filler and wash 5-10 column volumes with purified water to remove ethanol; Balance: The Protein G column was balanced with 10 mM PBS (pH 7.2) for 5 column volumes. After the column was balanced, the A280 value displayed by the protein UV detector was adjusted to 0 as the baseline. Sample loading: dilute the ascites with two volumes of equilibration buffer 10 mM PBS (pH 7.2) as sample loading, use a peristaltic pump to load the sample at 20-25 °C, with a flow rate of 5-6 ml / min; Balance: After loading, use 10 mM PBS (pH 7.2) as the balance buffer for 5-10 column volumes until no protein flows out, i.e. the protein UV detection value A280 shows 0; Dissociation: After equilibrium is completed, use 0.1M glycine buffer (pH 2.7) for dissociation. Start collecting protein when the protein UV detector displays a value greater than 0.3, and stop collecting protein when the protein UV detector displays a value less than 0.3. Pre-add 0.1 times the collection volume of 2M Tris (pH 8.0) and 0.03 times the collection volume of 5M NaCl to the collection tube.
[0024] After the above steps, mouse IgG monomers were obtained. After repeated freeze-thaw verification of stability, the appearance was as follows Figure 1 As shown on the right, after the same number of freeze-thaw cycles (the left side shows the freeze-thaw cycle of the recombinant IgG monomer, and the right side shows the freeze-thaw cycle of the mouse IgG monomer). By comparison, it can be seen that after repeated freeze-thaw cycles for the same number of times, the mouse IgG monomer has already been degraded and denatured protein aggregation has occurred. This shows that the recombinant IgG monomer has high stability and is more conducive to the stability of the cross-linked heterophilic antibody blocker.
[0025] Comparative Example 2 It is substantially the same as Example 2, except that glutaraldehyde is used to crosslink the monomer in Comparative Example 2. The specific process is as follows: Collect the recombinant IgG monomer purified in Example 1 and measure the volume; Place the beaker on a magnetic stirrer, stir and add the corresponding volume of 2.5 M glutaraldehyde with a 1 mL pipette to make the final concentration 4 mM at a speed of 50 s / ml; Continue stirring at room temperature for 3 h; 2 M glycine was added evenly to a final concentration of 0.02 M at a speed of 50 s / ml and stirred for 30 min using a magnetic stirrer; The sample was dialyzed using 10 mM PBS (pH 7.4) buffer at a dialysis ratio of 1:50, with the solution changed 3 times, and the dialysis interval was no less than 4 hours.
[0026] After the above steps, a cross-linked heterophilic antibody blocking agent is obtained, which has an appearance as shown in Figure 3 The figure on the right shows a comparison Figure 2-3 It can be seen that the heterophilic antibody blocker obtained by cross-linking with BS3 has more aggregates and a higher aggregate content.
[0027] The appearance of the heterophilic antibody blocker obtained after cross-linking the recombinant IgG monomers with BS3 and glutaraldehyde is as follows: Figure 4 (BS3 cross-linking on the left, glutaraldehyde cross-linking on the right) As shown, it can be seen that the appearance after BS3 cross-linking is clear and transparent, which is more conducive to subsequent immunoassays.
[0028] The comparison results of the blocking ability of the blocker obtained by this preparation method and the control blocker are shown in Table 1: Table 1 Comparison of the blocking ability of the blockers obtained by the present preparation method and the control blockers
[0029] Data analysis: The ability of this example-20240521 to eliminate heterophilic antibody interference is slightly better than that of blocker-1 and blocker-2.
[0030] It can be seen from the table that the cross-linked heterophilic antibody blocker obtained in the embodiment of the present invention has a good ability to block the interference of heterophilic antibodies.
[0031] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-limiting from any point of view, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention, and any reference numerals in the claims should not be regarded as limiting the claims involved.
Claims
1. A method for preparing a cross-linked heterophilic antibody blocking agent, characterized in that: The following steps are involved: S1, cationic liposome transfection method plasmid transfected CHO cells to prepare recombinant IgG monomer for purification; The cross-linked heterophilic antibody blocker was prepared by cross-linking with S2 and di(sulfosuccinimidyl) suberate (BS3) cross-linker.
2. The method for preparing a cross-linked heterophilic antibody blocking agent according to claim 1, characterized in that: The IgG monomer purification in step S1 includes the following steps: S11. Sample preparation: The collected transfected cell fluid was centrifuged at 4000 rpm for 10 min, the supernatant was collected, filtered through a 0.45 um filter, and the supernatant volume was measured using a measuring cylinder; Column preparation: Fill 500 mL of Protein A filler and wash 5-10 column volumes with purified water to remove ethanol; S12, balance: Protein A column is balanced with 10mM PBS (pH7.4) balance buffer for 5 times column volume. After the column is balanced, the A280 value displayed by the protein UV detector is adjusted to 0 as the baseline; S13, sample loading: dilute the supernatant with 1 / 5 times the volume of the supernatant with 100 mM PBS (pH 7.4) dilution buffer and slowly load the sample at a flow rate of 3-5 ml / min; S14, balance: after loading, use 10mM PBS (pH7.4) as balance buffer for 5-10 column volumes until no protein flows out, i.e. the protein UV detection value A280 shows 0; S15, dissociation: after equilibrium is completed, use 0.1M citric acid (pH3.5) buffer for dissociation, collect samples with absorbance of 0.2-0.5 and greater than 0.5 in sections, and continue to wash the column with 0.1M citric acid after dissociation until the detection value of the nucleic acid detector A280 is stable; S16, equilibration: equilibrate the column with 5-10 column volumes of equilibration buffer 10 mM PBS (pH 7.4); S17. Dialysis: Use 10 mM PBS (pH 7.4) dialysis buffer to dialyze the sample at a dialysis ratio of 1:50, change the solution 3 times, and the dialysis interval should not be less than 4 hours.
3. The method for preparing a cross-linked heterophilic antibody blocking agent according to claim 1, characterized in that: The cross-linking agent used in step S2 is BS3, ie, di(sulfosuccinimidyl) suberate, which is a homobifunctional, water-soluble, non-cleavable and membrane-impermeable amine-amine cross-linking agent.
4. The method for preparing a cross-linked heterophilic antibody blocking agent according to claim 1, characterized in that: In the step S2, preparing the cross-linked heterophilic antibody blocking agent comprises the following steps: S21 and BS3 cross-linkers were used to obtain cross-linked heterophilic antibody blockers; S22. Handling and storage of cross-linked heterophilic antibody blocking agents.
5. The method for preparing a cross-linked heterophilic antibody blocking agent according to claim 1, characterized in that: The BS3 cross-linker for obtaining a cross-linked heterophilic antibody blocking agent comprises the following steps: S211, collecting the IgG monomers purified in the above S1 and measuring the volume; S212, place the beaker on a magnetic stirrer, stir and add a corresponding volume of 0.2M BS3 (bis(sulfosuccinimidyl) suberate) using a 1 mL pipette to a final concentration of 2 mM at a speed of 50 s / ml; S213, continue stirring at room temperature for 1h; S214, 4℃ overnight for 16-20h; S215, evenly add 2M Tris to a final concentration of 0.02M, at a speed of 50s / ml, stir with a magnetic stirrer for 30min, and take samples for HPLC detection; S216. The sample was dialyzed using 10 mM PBS (pH 7.4) buffer at a dialysis ratio of 1:
50. The solution was changed 3 times with an interval of no less than 4 hours.
6. The method for preparing a cross-linked heterophilic antibody blocking agent according to claim 1, characterized in that: The processing and storage of the cross-linked heterophilic antibody blocking agent comprises the following steps: S221, unpack the dialysis bag and measure the volume of the antibody; S222, add 4% of the antibody volume of sucrose and stir until uniform; S223, filter using a 0.22 μm filter membrane, and measure and record the antibody concentration using a Nano300 UV spectrophotometer; S224. Add 0.1% of the antibody volume of Proclin 300 preservative to the antibody treated in step S22, mix well, and store at 4° C. to avoid freezing and thawing.
7. The method for preparing a cross-linked heterophilic antibody blocking agent according to claim 1, characterized in that: The appearance of the cross-linked heterophilic antibody blocking agent is a colorless transparent liquid without obvious precipitation and floccules.