Rapid preparation kit and method for antibody digestion fragment

Abstract

The invention provides a kit and a method for rapidly preparing an antibody digestion fragment. The kit comprises an antibody digestion basic solution, an antibody digestion system agent, a digestion targeting agent, an antibody digestion treating agent and an antibody digestion refining device. The method disclosed by the invention has relatively strong compatibility to the antibody, and the whole antibody digestion fragment product is complete in activity preservation, strong in specificity, high in yield and rapid, simple and convenient to operate. A fragment treated by the antibody enzyme digestion refining device can be applied to the fields of immunodetection, immunoassay tracking and imaging, purification and capture, chemical modification, treatment and in-vivo diagnosis, vaccines, immunoregulation and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a kit and method for rapidly preparing antibody enzyme-cleaved fragments. Background Art

[0002] Antibodies are proteins, and their structure is crucial to their biological functions. Antibodies are widely used in immunoassay and tracking imaging, purification and capture, chemical modification, treatment and in vivo diagnosis, as well as vaccines and immune regulation. Under normal circumstances, intact antibodies can meet the above applications. Under special circumstances, antibody enzyme fragments have a variety of unique application values: For example, in the field of immunoassay, the use of antibody enzyme fragments for the design and development of immunoassay products or systems helps to solve the high background problem of immunoassay reagents, and at the same time has significant effects in improving the specificity of the detection reagents, improving stability, and improving sensitivity; For example, in the field of antibody structure research, antibody enzyme fragments can be used to study the relationship between the structure and function of antibodies, so as to gain a deeper understanding of the mechanism of action of antibodies; For example, in the field of antibody drugs, enzyme fragments can be used to prepare antibody fragment drugs, which have smaller molecular weights and better tissue penetration ability, and can better play a therapeutic role; For example, in the field of immune research, the Fc region in intact antibodies is the main part that causes immune responses. Antibody fragments, especially those that do not contain the Fc region (such as Fab), usually have lower immunogenicity. This reduces the risk of nonspecific immune activation and antibody-mediated adverse reactions; such as in the field of genetic engineering: using recombinant DNA technology, antibody fragments can be genetically modified relatively easily to optimize their affinity, stability or other desired properties. This provides great flexibility for customizing therapeutic antibody fragments. In addition, the production of antibody fragments is generally more cost-effective than that of complete antibodies. This is because they can be expressed in bacteria, yeast or mammalian cells at high yields at lower costs.

[0003] At present, there is no standard process for the commonly used methods of preparing antibody enzyme fragments. Most of the relevant personnel rely on past experience to explore. The operations in antibody treatment, enzyme system solidification, enzyme material concentration setting, purification and other links are complicated and cumbersome, and often require expensive equipment such as chromatographs. It is unlikely to be implemented in laboratories under general conditions. The entire enzyme cleavage cycle is long, and it takes 2-3 days to complete. There is no standard operating procedure, which leads to the inability to effectively purify and refine after enzyme cleavage, and the purity of antibody enzyme fragments is not high, which affects subsequent use and the batch difference of preparation is large. Summary of the invention

[0004] Purpose of the invention: In view of the deficiencies of the prior art and actual needs, the present invention provides a kit and method for rapid preparation of antibody enzymatic fragments, which adopts an antibody enzymatic refining device for purification and recovery, and preformulates the enzymatic targeting agent, so as to achieve standard, efficient and high-purity preparation of antibody enzymatic fragments, so as to solve the technical problems of non-standard operation, cumbersome process, long cycle, large batch difference and high equipment requirements in the preparation of antibody enzymatic fragments in the prior art.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] The invention provides a rapid preparation kit for antibody enzymatic fragments, comprising: an antibody enzymatic cleavage base solution, an antibody enzymatic cleavage system agent, an enzymatic cleavage targeting agent, an antibody enzymatic cleavage treatment agent and an antibody enzymatic cleavage refining device.

[0007] Preferably, the antibody enzymatic cleavage base solution contains a neutral sodium phosphate buffer at a concentration of 10 mmol / L-30 mmol / L. More preferably, the concentration of the sodium phosphate buffer is 20 mmol / L, and the pH is 7.5.

[0008] Preferably, the antibody enzymatic cleavage system contains hydrochloric acid at a concentration of 0.1 mol / L-1 mol / L, which is used to adjust the optimal protein state of the antibody. More preferably, the hydrochloric acid concentration is 1 mol / L.

[0009] Preferably, the enzymatic targeting agent is papain, pepsin or ficin.

[0010] Preferably, the antibody enzymatic cleavage treatment agent contains sodium hydroxide at a concentration of 0.1 mol / L-1 mol / L. More preferably, the concentration of sodium hydroxide is 1 mol / L.

[0011] Preferably, the antibody enzymatic cleavage and purification device is a gel column and a concentration column.

[0012] In the present invention, the enzymatic targeting agent is prefabricated and combined with the antibody enzymatic purification device to achieve standard, efficient and high-purity preparation of antibody enzymatic fragments.

[0013] The present invention also provides a method for rapidly preparing antibody enzyme-cleaved fragments, comprising:

[0014] (1) Use antibody enzymatic digestion base solution to rinse the concentration and desalting column and clean the gel column.

[0015] (2) Add the antibody to the concentration and desalting column, add the antibody enzyme cleavage system reagent, and use a pipette to mix.

[0016] (3) Add the enzyme targeting agent to the antibody, mix well by pipetting, and react at room temperature for 20-30 minutes.

[0017] (4) Add the antibody cleavage agent to the antibody and mix by pipetting.

[0018] (5) Centrifuge the concentrated desalting column to fully remove free enzymatic targeting agents.

[0019] (6) Recover the antibody and transfer it to a gel column to purify the antibody cleavage fragments.

[0020] Preferably, in step (3), the enzymatic targeting agent is added dropwise to the antibody, and is gently shaken while being added dropwise.

[0021] Preferably, in step (5), the centrifugation operation is performed using a temperature-controlled low-temperature centrifuge, and the centrifugation parameters are set to 10000g for 10 min.

[0022] Preferably, in step (6), the purification operation can be performed by a peristaltic pump pressurization method, a purifier pressurization method, a centrifugal method or a gravity method.

[0023] The beneficial effects of the present invention are:

[0024] (1) The components of the present invention are stable, the storage conditions are loose, the enzymatic cleavage effect is stable and uniform, and it is reproducible.

[0025] (2) The use of the prefabricated enzymatic targeting agent of the present invention can achieve standard and efficient preparation of antibody enzymatic fragments.

[0026] (3) The present invention adopts an antibody enzyme cleavage purification device to simplify the operation in the preparation of antibody enzyme cleavage fragments, shorten the cycle, reduce the dependence on high-end equipment, and ensure the high purity of antibody enzyme cleavage fragments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The present invention is used to compare the reaction ratio and sensitivity of the antibody before and after enzyme cleavage.

[0028] Figure 2 The present invention was experimentally compared with antibody enzyme-digested fragments prepared using an imported commercially available enzyme-digested raw material.

[0029] Figure 3 Different batches of antibody enzyme-cleaved fragments prepared by the present invention were experimentally compared.

[0030] Figure 4 Comparison of stability tests of the antibody enzyme-cleaved fragments prepared by the present invention.

[0031] Figure 5 The shelf stability verification result of the kit of the present invention. DETAILED DESCRIPTION

[0032] In order to more clearly illustrate the present invention, the present invention is further described in detail below in conjunction with embodiments. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0033] The embodiment of the present invention includes: a rapid preparation kit for antibody enzymatic fragments, including: an antibody enzymatic base solution, an antibody enzymatic system agent, an enzymatic targeting agent, an antibody enzymatic treatment agent and an antibody enzymatic refining device.

[0034] The antibody enzymatic cleavage base solution contains a sodium phosphate buffer with a concentration of 20 mmol / L and a pH of 7.5.

[0035] The antibody enzymatic cleavage system agent contains hydrochloric acid with a concentration of 1 mol / L.

[0036] The enzymatic targeting agent is 10 mg / mL pepsin.

[0037] The antibody enzymatic cleavage treatment agent contains sodium hydroxide with a concentration of 1 mol / L.

[0038] The antibody enzyme cleavage and purification device comprises a gel column and a concentration column.

[0039] The present invention also provides a method for rapidly preparing antibody enzyme fragments. The operation steps take Aβ1-42 antibody (concentration 1 mg / mL) as an example and include the following steps:

[0040] (1) Use 30 mL of antibody enzymatic digestion base solution to rinse the concentration and desalting column and clean the gel column.

[0041] (2) Add 5 mg (concentration 1 mg / mL, volume 5 mL) of antibody to the concentrating desalting column, add 200 μL of antibody enzyme cleavage system, and mix well by pipetting.

[0042] (3) Add 50 μL of the enzyme targeting agent to the antibody, mix well by pipetting, and react at room temperature for 20-30 minutes.

[0043] (4) Add 250 μL of antibody enzyme cleavage agent to the antibody and mix by pipetting.

[0044] (5) Place the concentration and desalting column in a centrifuge and centrifuge at 10,000 g for 10 min to fully remove the free enzymatic targeting agent.

[0045] (6) After the recovered antibody is diluted to 2 mL, it is transferred to the gel column and 4 mL of antibody enzymatic cleavage base solution is added to purify the antibody enzymatic cleavage fragments and recover them.

[0046] Using this kit, we prepared Aβ1-42 antibody cleavage fragments. We first used magnetic particle chemiluminescence to detect the reaction ratio and sensitivity before and after antibody cleavage. The results are as follows: Figure 1 As shown in Table 1, it can be seen from the figure that the enzyme-cut fragments prepared by this kit have higher sensitivity and reaction ratio.

[0047] Table 1. Comparison of fragment reaction ratio and sensitivity before and after enzyme digestion using the present invention.

[0048]

[0049] The reaction ratio and sensitivity show that the antibody enzyme-cleaved fragments prepared by the present invention have obvious sensitivity and reaction ratio advantages compared with the intact antibodies that have not been enzyme-cleaved.

[0050] We also verified other properties of the antibody enzyme fragments prepared by this kit and method.

[0051] The antibody cleavage fragments prepared using the kit and method of the present invention were compared with the antibody cleavage fragments prepared using an imported commercially available cleavage raw material (traditional preparation method). The test results are shown in Table 2.

[0052] Table 2. Comparison of enzyme-cut fragments prepared from the present invention and imported commercial raw materials.

[0053]

[0054] The reaction ratio and sensitivity show that the antibody enzyme fragments prepared by the present invention have good correlation with the commercially available imported raw materials and have certain sensitivity advantages. Compared with the commercially available imported raw materials, the present invention also has obvious price advantages and cycle advantages.

[0055] The different batches of antibody enzyme fragments prepared by the kit and method were experimentally compared, and the test results are shown in Table 3.

[0056] Table 3. Comparison of different batches of antibody enzyme fragments prepared by the present invention.

[0057]

[0058] The reaction ratio and sensitivity showed that there was no significant difference between the three different batches of antibody enzyme fragments prepared by the present invention, and the batch reproducibility was good. CV The low concentration of enzyme fragments ensures the reproducibility of enzyme-cut fragments between different preparation batches, and provides a research basis without batch differences for subsequent applications in different fields.

[0059] The stability of the antibody enzyme fragments prepared by the kit and method was verified, and the test results are shown in Table 4.

[0060] Table 4. Stability test of the antibody enzyme fragments prepared by the present invention.

[0061]

[0062] The reaction ratio and sensitivity show that the antibody cleavage fragments prepared by the present invention are stored at 2-8°C for 0, 6 and 12 months without obvious changes in activity, with a high residual activity rate and stable preservation of the cleavage effect; the cleavage fragments have strong stability, which is convenient for subsequent research and use in clinical and scientific research application fields, and convenient for laboratory storage of the cleavage fragment products.

[0063] The shelf stability of this kit was verified, and the test results are shown in Table 5.

[0064] Table 5. The shelf stability verification results of the kit of the present invention.

[0065]

[0066] The reaction ratio and sensitivity show that the enzyme cleavage effect of the present invention does not change significantly when the kit is stored at 2-8°C for 0, 6, and 12 months, the storage conditions are loose, and the enzyme cleavage effect is stable; the enzyme cleavage effect is not affected by the storage environment. In clinical and scientific research applications, it is convenient for laboratory storage and stocking, and the enzyme cleavage effect is not affected by the storage environment within the 12-month validity period.

[0067] The preferred embodiments of the present invention disclosed above are only examples to help explain the present invention more clearly, rather than limiting the implementation methods of the present invention. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Antibody enzyme fragment rapid preparation kit, including: Antibody enzymatic cleavage base liquid, antibody enzymatic cleavage system agent, enzymatic cleavage targeting agent, antibody enzymatic cleavage treatment agent and antibody enzymatic cleavage refining device.

2. The rapid preparation kit for antibody enzyme fragments according to claim 1, characterized in that: The antibody enzymatic cleavage base solution contains a neutral sodium phosphate buffer solution with a concentration of 10 mmol / L-30 mmol / L.

3. The rapid preparation kit for antibody enzyme fragments according to claim 1, characterized in that: The antibody enzymatic cleavage system contains hydrochloric acid with a concentration of 0.1 mol / L-1 mol / L.

4. The rapid preparation kit for antibody enzyme fragments according to claim 1, characterized in that: The enzymatic targeting agent is papain, pepsin or ficin.

5. The rapid preparation kit for antibody enzyme fragments according to claim 1, characterized in that: The antibody enzymatic cleavage treatment agent contains sodium hydroxide with a concentration of 0.1 mol / L-1 mol / L.

6. The rapid preparation kit for antibody enzyme fragments according to claim 1, characterized in that: The antibody enzyme cleavage and purification device comprises a gel column and a concentration column.

7. A rapid preparation method for antibody enzymatic fragments is as follows: the antibody enzymatic base solution is used to rinse the concentrated desalting column and clean the gel column, and then the antibody is added to the concentrated desalting column, the antibody enzymatic system agent is added, and the enzymatic targeting agent is added to the antibody, and the pipette is used to mix, and the reaction is carried out at room temperature for 20-30 minutes, and the antibody enzymatic treatment agent is added to the antibody, and the pipette is used to mix, and the concentrated desalting column is centrifuged to fully remove the free enzymatic targeting agent, and the antibody is recovered and transferred to the gel column to purify the antibody enzymatic fragments.

8. The method for rapidly preparing antibody enzyme fragments according to claim 7, characterized in that: The enzyme targeting agent is added dropwise to the antibody, and gently shaken while adding.

9. The method for rapidly preparing antibody enzyme fragments according to claim 7, characterized in that: The centrifugation operation was performed in a temperature-controlled low-temperature centrifuge, and the centrifugation parameters were set to 10000g for 10 min.

10. The method for rapidly preparing antibody enzyme fragments according to claim 7, characterized in that: The purification operation can be performed by peristaltic pump pressurization method, purifier pressurization method, centrifugation method or gravity method.

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