Anti-botulinum toxin antibody HPCE fingerprint spectrum construction method and application thereof

Abstract

The invention relates to the field of botulinum toxin analysis, in particular to an anti-botulinum toxin antibody HPCE fingerprint construction method and application thereof.According to the method, a capillary immunoelectrophoresis reaction is carried out on botulinum neurotoxin, and a capillary immunoelectrophoresis reaction is carried out on each subunit of a botulinum toxin compound. The capillary immunoelectrophoretogram of the botulinum neurotoxin and the capillary immunoelectrophoretogram of each subunit of the botulinum toxin compound are obtained, the spectrograms obtained through the method can be compared with botulinum toxin compound detection, and the detection precision of the botulinum toxin compound is improved to the pg level.

Description

Technical Field

[0001] The present invention relates to the field of botulinum toxin analysis, and in particular to a method for constructing an anti-botulinum toxin antibody HPCE fingerprint spectrum and an application thereof. Background Art

[0002] During the research of botulinum toxin, laboratory testing of botulinum toxin antibodies is usually required. During the research and development, clinical trials and post-marketing drug monitoring stages, botulinum toxin or botulinum neurotoxin is usually injected into humans or experimental animals. Whether serum antibodies are produced after injection and the level of antibodies are key contents in botulinum toxin research. This type of test can be defined as a botulinum toxin serum antibody test, and the data generated is crucial for product development and post-marketing drug vigilance. Usually, this type of test is performed by immunological methods.

[0003] At present, the conventional detection technology is the ELISA indirect method. However, the defect of the ELISA indirect method is that its sensitivity can only reach the nanogram (ng) level, and it is difficult to detect antibodies at the picogram (pg) level. Due to the extremely strong toxicity of botulinum toxin and the extremely low injection dose, even if antibodies are produced, their levels may be lower than the detection limit of the ELISA indirect method. Therefore, the ELISA method cannot fully meet the needs of botulinum toxin research. In addition, the ELISA method mainly detects samples at the macro level, and the results can only give a positive or negative judgment, and it is impossible to conduct a more in-depth analysis of the antibody subunits in the sample.

[0004] Botulinum toxin is a complex composed of multiple protein subunits, including neurotoxin, hemagglutinin 70, hemagglutinin 33 and hemagglutinin 70 fragments. Accordingly, botulinum toxin antibodies include monoclonal antibodies against specific subunits and polyclonal antibodies against the complete toxin complex. At present, the ELISA method can detect neurotoxin antibodies, but due to the difficulty in obtaining other subunit antigens, it is still difficult to detect other subunit antibodies. At the same time, there is a lack of overall analysis methods for polyclonal antibodies to the complete toxin complex and serum antibodies that may be produced by botulinum toxin injection.

[0005] Capillary immunoelectrophoresis (HPCE) is an immunological detection method with a sensitivity of up to the picogram (pg) level. It can detect various subunit antibodies of botulinum toxin, overcoming the limitations of the ELISA method. However, HPCE technology cannot directly detect botulinum toxin antibodies. Specific indicators are still needed to determine whether the result is a botulinum toxin antibody and which subunit the antibody belongs to. These indicators form a unique immunological reaction fingerprint of botulinum toxin antibodies.

[0006] Therefore, it is particularly important to construct an HPCE fingerprint of anti-botulinum toxin antibodies, which can provide necessary support for more accurate antibody detection and research. Summary of the invention

[0007] The object of the present invention is to provide a method for constructing an HPCE fingerprint spectrum of an anti-botulinum toxin antibody and its application. This method performs a capillary immunoelectrophoresis reaction on botulinum neurotoxin and on each subunit of a botulinum toxin complex to obtain a capillary immunoelectrophoresis spectrum of the botulinum neurotoxin and a capillary immunoelectrophoresis spectrum of each subunit of the botulinum toxin complex. The spectrum obtained by this method can be compared with the detection of the botulinum toxin complex.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] A method for constructing an anti-botulinum toxin antibody HPCE fingerprint, comprising performing a capillary immunoelectrophoresis reaction on a botulinum neurotoxin and performing a capillary immunoelectrophoresis reaction on each subunit of a botulinum toxin complex, wherein the botulinum neurotoxin is purified from the botulinum toxin complex;

[0010] Wherein, a capillary immunoelectrophoresis reaction is performed on botulinum neurotoxin to obtain a capillary immunoelectrophoresis pattern of the botulinum neurotoxin; in the capillary immunoelectrophoresis reaction of the botulinum neurotoxin, the antigen is the botulinum neurotoxin, the primary antibody is an anti-botulinum neurotoxin monoclonal antibody or an anti-botulinum toxin complex polyclonal antibody, and the secondary antibody is an HRP-labeled antibody corresponding to the primary antibody;

[0011] Capillary immunoelectrophoresis is performed on each subunit of the botulinum toxin complex to obtain a capillary immunoelectrophoresis pattern of each subunit of the botulinum toxin complex; the subunits of the botulinum toxin complex include hemagglutinin 70, hemagglutinin 33, hemagglutinin 70 fragment, botulinum neurotoxin heavy chain and botulinum neurotoxin light chain. In the capillary immunoelectrophoresis reaction of each subunit of the botulinum toxin complex, hemagglutinin 70, hemagglutinin 33, hemagglutinin 70 fragment, botulinum neurotoxin heavy chain and botulinum neurotoxin light chain are used as antigens respectively, the primary antibody is an anti-botulinum toxin complex polyclonal antibody, and the secondary antibody is an HRP-labeled antibody corresponding to the primary antibody.

[0012] The hemagglutinin 70 fragment is the cleavage product of hemagglutinin 70. Under severe environmental conditions, part of hemagglutinin 70 will be cleaved, and the cleavage product is the hemagglutinin 70 fragment. Under electrophoresis conditions, hemagglutinin 70 and the hemagglutinin 70 fragment can be observed.

[0013] Furthermore, the botulinum toxin complex is a botulinum toxin type A complex, and the method for preparing the botulinum toxin type A complex comprises the following steps:

[0014] Cultivate the type A Clostridium botulinum Hall strain to obtain a type A Clostridium botulinum bacterial liquid:

[0015] The type A botulinum Clostridium botulinum bacterial liquid is collected and purified by chromatography to obtain the type A botulinum toxin complex.

[0016] Furthermore, the botulinum toxin type A complex is purified by ion exchange chromatography to obtain botulinum neurotoxin type A.

[0017] Further, the botulinum toxin type A complex is subjected to gel excision purification to obtain hemagglutinin 70, hemagglutinin 33, hemagglutinin 70 fragment, botulinum neurotoxin type A heavy chain and botulinum neurotoxin type A light chain. Further, the anti-botulinum toxin complex polyclonal antibody is an anti-botulinum toxin complex polyclonal antibody obtained by immunizing rabbits with botulinum toxin type A complex, and the corresponding secondary antibody is an anti-rabbit HRP-labeled antibody.

[0018] Furthermore, the anti-botulinum neurotoxin type A monoclonal antibody is an anti-botulinum neurotoxin type A monoclonal antibody obtained by immunizing mice with botulinum neurotoxin type A, and the secondary antibody is an anti-mouse HRP-labeled antibody.

[0019] An anti-botulinum toxin antibody HPCE fingerprint spectrum, wherein the characteristic peak of the botulinum neurotoxin non-reducing capillary immunoelectrophoresis spectrum is a 94-98kd peak.

[0020] Furthermore, the characteristic peak of the botulinum neurotoxin subunit heavy chain capillary immunoelectrophoresis is a 99 kd peak.

[0021] Furthermore, the characteristic peak of the botulinum neurotoxin subunit light chain capillary immunoelectrophoresis is a 52 kd peak.

[0022] Furthermore, the characteristic peak of the capillary immunoelectrophoresis spectrum of hemagglutinin 70 is a 58kd peak.

[0023] Furthermore, the characteristic peak of the capillary immunoelectrophoresis spectrum of hemagglutinin 33 is a 38 kd peak.

[0024] Furthermore, the characteristic peak of the capillary immunoelectrophoresis spectrum of the hemagglutinin 70 fragment is a 24 kd peak.

[0025] The capillary immunoelectrophoresis patterns of hemagglutinin 70, hemagglutinin 33 or hemagglutinin 70 fragments obtained above can be used to detect the comparison of antibodies against botulinum toxin type B, C, D, E and F complexes.

[0026] The present invention also provides a method for detecting anti-botulinum toxin antibodies, comprising the following steps:

[0027] Prepare antigen, test antibody and secondary antibody;

[0028] The antigen is a botulinum toxin complex, the antibody to be tested is an anti-botulinum neurotoxin monoclonal antibody or an anti-botulinum toxin complex polyclonal antibody, and the secondary antibody is an HRP-labeled antibody corresponding to the antibody to be tested;

[0029] The antibody to be tested is added to the antigen. After sufficient binding, the secondary antibody is added. The secondary antibody fully binds to the antibody to be tested and the incubation continues;

[0030] After the incubation is completed, capillary immunoelectrophoresis analysis is performed to obtain a capillary immunoelectrophoresis pattern of the antibody to be tested;

[0031] The capillary immunoelectrophoresis pattern of the antibody to be tested is compared with the capillary immunoelectrophoresis pattern obtained by the above-mentioned construction method of the claim to obtain the test result.

[0032] Furthermore, the antigen is a botulinum toxin type A complex, and the method for preparing the botulinum toxin type A complex comprises the following steps:

[0033] Cultivate the type A Clostridium botulinum Hall strain to obtain a type A Clostridium botulinum bacterial liquid:

[0034] The type A botulinum Clostridium botulinum bacterial liquid is collected and purified by chromatography to obtain the type A botulinum toxin complex.

[0035] Furthermore, after the secondary antibody is fully combined with the antibody to be tested, a reaction gel is added for incubation.

[0036] Furthermore, the obtained capillary immunoelectrophoresis pattern of hemagglutinin 70, the capillary immunoelectrophoresis pattern of hemagglutinin 33 or the capillary immunoelectrophoresis pattern of hemagglutinin 70 fragment is used to detect the comparison of antibodies against botulinum toxin type B, C, D, E and F complexes.

[0037] The commonly used biochemical analysis method for detecting and analyzing proteins is SDS-PAGE (denaturing polyacrylamide gel electrophoresis) technology. This method is mature and widely used, but its sensitivity is low and the sample consumption is large. It cannot fully meet the requirements of current protein analysis. Therefore, the microanalysis technology that has emerged is capillary electrophoresis. The technology combined with SDS-PAGE and immunological technology is western blotting technology, which is used for further immunological analysis of biochemical analysis. This method also has low sensitivity and large sample consumption. The capillary immunoelectrophoresis technology produced by combining capillary electrophoresis with immunological technology is essentially a highly sensitive ultra-micro western blotting technology. The basic principle of this technology is to first carry a protein (here equivalent to an antigen) after the action of SDS and thus swim in a specific electric field. The swimming device is a tiny capillary that stays at a specific position. Then, the antibody of the protein enters the capillary. If it is a specific antibody corresponding to the protein (referred to as a primary antibody, which is a sample to be tested), the antibody binds to the antigen to form a complex. If it is a non-specific antibody, the antigen and antibody do not bind. The third component entering the capillary is the antibody of the specific antibody (referred to as the secondary antibody). If the antigen is combined with the primary antibody to form a complex, the secondary antibody can combine with the complex to form an antigen-primary antibody-secondary antibody complex. Since the secondary antibody is coupled with HRP (horseradish peroxidase), the antigen-primary antibody-secondary antibody complex can show a chemiluminescent effect after being treated with a specific chemiluminescent colorimetric solution. The generated light signal is received by the capillary electrophoresis instrument and output in graphical and digital form. The specific antigen-primary antibody-secondary antibody complex will show a peak shape at a specific position of the coordinate, and there will be a specific number as the basis for judging the peak.

[0038] When botulinum toxin complex or botulinum toxin subunit is used as the antigen, the sample to be tested (including botulinum toxin complex polyclonal antibody / botulinum toxin serum antibody, botulinum toxin subunit polyclonal antibody, botulinum toxin subunit monoclonal antibody) is the primary antibody, and the anti-primary antibody coupled to HRP is the secondary antibody, different peak shapes can be presented through capillary immunoelectrophoresis analysis.

[0039] Using botulinum toxin type A complex or botulinum neurotoxin type A subunit as the antigen, anti-botulinum neurotoxin type A subunit monoclonal antibody as the primary antibody (the primary antibody here is not the sample to be tested but a known high-purity antibody used to lock the neurotoxin, usually from mouse origin), and HRP-coupled anti-mouse antibody as the secondary antibody, capillary immunoelectrophoresis analysis can be used to lock the characteristic peaks of botulinum neurotoxin type A subunits in capillary immunoelectrophoresis.

[0040] Using the purified single components of the heavy chain and light chain of the botulinum neurotoxin type A subunit as antigens (because of the lack of monoclonal antibodies against the heavy chain and light chain, the antigen must be a single component, that is, at least one of the antigen or antibody in the antigen-antibody reaction must be a single component in order to lock the characteristic peak), using anti-botulinum neurotoxin type A heavy chain and anti-botulinum neurotoxin type A light chain polyclonal antibodies as primary antibodies (rabbit source), and HRP-coupled anti-rabbit antibody as secondary antibody, through capillary immunoelectrophoresis analysis, the heavy chain and light chain characteristic peaks of the botulinum neurotoxin type A subunit in capillary immunoelectrophoresis can be locked as auxiliary reference peaks of the characteristic peaks of the botulinum neurotoxin type A subunit.

[0041] Using purified botulinum toxin type A hemagglutinin 70 subunit as antigen, anti-botulinum toxin type A complex polyclonal antibody as primary antibody (rabbit source), and HRP-coupled anti-rabbit antibody as secondary antibody, capillary immunoelectrophoresis analysis was performed to lock in the characteristic peak of botulinum toxin type A hemagglutinin 70 subunit in capillary immunoelectrophoresis.

[0042] Using purified botulinum toxin type A hemagglutinin subunit 33 as antigen, anti-botulinum toxin type A complex polyclonal antibody as primary antibody (rabbit source), and HRP-coupled anti-rabbit antibody as secondary antibody, capillary immunoelectrophoresis analysis was performed to lock the characteristic peak of botulinum toxin type A hemagglutinin subunit 33 in capillary immunoelectrophoresis.

[0043] Using the purified hemagglutinin 70 fragment subunit of botulinum toxin type A as the antigen, anti-botulinum toxin type A complex polyclonal antibody as the primary antibody (rabbit source), and HRP-coupled anti-rabbit antibody as the secondary antibody, capillary immunoelectrophoresis analysis was performed to lock the characteristic peak of the hemagglutinin 70 fragment subunit of botulinum toxin type A in capillary immunoelectrophoresis.

[0044] Combining the above peaks, the characteristic peaks of botulinum neurotoxin type A subunits in capillary immunoelectrophoresis, the characteristic peaks of heavy and light chains of botulinum neurotoxin type A subunits in capillary immunoelectrophoresis, as the auxiliary reference peaks of the characteristic peaks of botulinum neurotoxin type A subunits, the characteristic peaks of botulinum toxin type A hemagglutinin 70 subunits in capillary immunoelectrophoresis, the characteristic peaks of botulinum toxin type A hemagglutinin 33 subunits in capillary immunoelectrophoresis, and the characteristic peaks of botulinum toxin type A hemagglutinin 70 fragment subunits in capillary immunoelectrophoresis, a total of 6 peaks, form the fingerprint spectrum of anti-botulinum toxin type A antibody capillary immunoelectrophoresis, which is the basis for judgment of all anti-botulinum toxin type A antibody capillary immunoelectrophoresis.

[0045] Perform capillary immunoelectrophoresis analysis on the antibody sample to be tested. Sample peaks appear and the characteristic peaks are compared with the sample peaks:

[0046] If the sample shows multiple peaks and they completely match the characteristic peaks, it means that the sample is a highly pure anti-botulinum toxin polyclonal antibody.

[0047] If a single peak appears and matches a characteristic peak, it means that the sample is a highly pure anti-botulinum toxin subunit monoclonal antibody, and its specificity can be determined by the molecular weight.

[0048] If multiple peaks appear but only partially match the characteristic peak, it indicates that the sample is a contaminated polyclonal antibody.

[0049] If a single peak matches the characteristic peak but there are other impurity peaks, it means that the sample is a subunit monoclonal antibody with impurities.

[0050] Before storage, antibodies should be subjected to capillary immunoelectrophoresis analysis to generate an original peak pattern. Subsequent testing can determine whether the antibody has been degraded by comparing the original peak pattern; changes in peak area reflect antibody degradation, and a decrease in peak area indicates a decrease in antibody titer.

[0051] Peak area can also be used to evaluate antibody titer, and capillary immunoelectrophoresis can help select the monoclonal antibody with the highest titer and strong specificity as the standard antibody.

[0052] If a large number of peaks different from the original peak shapes appear after the antibody is stored, it means that the antibody has been severely degraded and should be discarded and re-prepared.

[0053] If a monoclonal antibody has good purity as determined by SDS-PAGE or HPLC and forms one peak in capillary immunoelectrophoresis analysis, it only indicates that the biochemical purity of the antibody is consistent with its immunological purity. If multiple peaks are formed in capillary immunoelectrophoresis analysis and only one peak is consistent with the characteristic peak, although the biochemical purity determined by SDS-PAGE or HPLC is high, the immunological purity is low, indicating the presence of other nonspecific antibodies in the sample and the use of this antibody should be cautious.

[0054] The botulinum toxin complex contains multiple subunits, and anti-botulinum toxin polyclonal antibodies should contain antibody components corresponding to each subunit. Therefore, capillary immunoelectrophoresis analysis of anti-botulinum toxin polyclonal antibodies will show multiple peaks, which should correspond to the characteristic peaks of the spectrum. If peaks that do not match the characteristic peaks appear, it indicates that the anti-botulinum toxin polyclonal antibodies contain other non-specific antibodies, and the use of such antibodies should be cautious.

[0055] The antibodies produced by botulinum neurotoxin are divided into heavy chain antibodies and light chain antibodies according to site specificity. For example, if the antibody sample is an anti-botulinum neurotoxin polyclonal antibody, it will contain heavy chain antibodies and light chain antibodies. For such antibodies, the ratio of heavy chain antibodies to light chain antibodies needs to be determined by capillary immunoelectrophoresis analysis based on the characteristic peaks of the neurotoxin heavy chain and light chain. If the antibody sample is an anti-botulinum neurotoxin monoclonal antibody, it can only be a heavy chain antibody or a light chain antibody. For such antibodies, the type of antibody needs to be determined by capillary immunoelectrophoresis analysis based on the characteristic peaks of the neurotoxin heavy chain and light chain. This is a site-specific guide.

[0056] The present invention has the following beneficial effects:

[0057] The method of the present invention can obtain the characteristic peak of botulinum toxin antibody in capillary immunoelectrophoresis, so that the technology of detecting botulinum toxin antibody by capillary immunoelectrophoresis can be realized and the detection sensitivity is improved from nanogram level to picogram level.

[0058] The characteristic peak of botulinum toxin antibody in capillary immunoelectrophoresis can be obtained by the method of the present invention, so that the detection efficiency and resolution of the antibody are better than those of ELISA detection.

[0059] The present invention analyzes in detail the composition of botulinum toxin antibodies and the proportion of each subunit antibody, providing data for the research and development and market application of botulinum toxin products.

[0060] The characteristic peak of botulinum toxin antibody obtained by the method of the present invention in capillary immunoelectrophoresis is used for capillary immunoelectrophoresis detection of botulinum toxin antibody, and it can be found whether the antibody is degraded and the subunit that has been degraded, and the degree of degradation, which is used for antibody quality control.

[0061] The present invention can guide the detection of the immunological purity and site specificity of botulinum toxin monoclonal antibodies and polyclonal antibodies.

[0062] Since the difference between botulinum toxin types A, B, C, D, E, and F complexes lies only in the botulinum neurotoxin heavy chain and light chain, and there is no difference in other subcomponents, the capillary immunoelectrophoresis patterns of hemagglutinin 70, hemagglutinin 33, and hemagglutinin 70 fragments obtained by the construction method of the present invention can be used for comparison of the detection of botulinum toxin types A, B, C, D, E, and F complexes. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0064] Figure 1 This is the SDS-PAGE diagram in Example 1 of the specific implementation method of this application. Figure 1 a is the SDS-PAGE image of botulinum toxin type A. Figure 1 b is the SDS-PAGE image of botulinum toxin type A complex;

[0065] Figure 2 The capillary immunoelectrophoresis pattern of botulinum neurotoxin type A in Example 1 of the specific implementation method of this application;

[0066] Figure 3 The capillary immunoelectrophoresis pattern of the heavy chain of the subunit of botulinum neurotoxin in Example 1 of the specific embodiment of the present application;

[0067] Figure 4 The capillary immunoelectrophoresis pattern of the botulinum neurotoxin subunit light chain in Example 1 of the specific implementation method of this application;

[0068] Figure 5 The capillary immunoelectrophoresis pattern of the hemagglutinin 70 subunit of the botulinum toxin in Example 1 of the specific implementation method of the present application;

[0069] Figure 6 The capillary immunoelectrophoresis pattern of the hemagglutinin 33 subunit of the botulinum toxin in Example 1 of the specific implementation method of the present application;

[0070] Figure 7 This is the capillary immunoelectrophoresis pattern of the hemagglutinin 70 fragment subunit of the botulinum toxin in Example 1 of the specific implementation method of this application;

[0071] Figure 8 This is the capillary immunoelectrophoresis profile with a sample load of 1 ng in Experimental Example 1 of the specific implementation method of this application;

[0072] Fig. 9 This is the capillary immunoelectrophoresis profile with a sample load of 100 ng in Experimental Example 1 of the specific implementation method of this application;

[0073] Fig.10 This is the capillary immunoelectrophoresis profile with a sample load of 50 pg in Experimental Example 1 of the specific implementation method of this application;

[0074] Fig.11 This is the capillary immunoelectrophoresis profile with a sample load of 10 pg in Experimental Example 1 of the specific implementation method of this application;

[0075] Fig.12 This is the capillary immunoelectrophoresis profile with a sample load of 5 pg in Experimental Example 1 of the specific implementation method of this application;

[0076] Fig.13 This is the capillary immunoelectrophoresis profile with a sample load of 1 pg in Experimental Example 1 of the specific implementation method of this application;

[0077] Fig.14 This is the ELISA test diagram in Experimental Example 2 of the specific implementation method of this application;

[0078] Fig.15 This is the capillary immunoelectrophoresis profile of the No. 1 antibody to be tested in Experimental Example 3 of the specific implementation method of this application;

[0079] Fig.16 This is the capillary immunoelectrophoresis profile of the No. 2 antibody to be tested in Experimental Example 3 of the specific implementation method of this application;

[0080] Fig.17 This is the capillary immunoelectrophoresis profile of the No. 3 antibody to be tested in Experimental Example 3 of the specific implementation method of this application;

[0081] Fig.18 This is the capillary immunoelectrophoresis spectrum of the antibody to be detected in Experimental Example 4 of the specific implementation method of this application. DETAILED DESCRIPTION

[0082] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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.

[0083] Example 1

[0084] The botulinum toxin type A complex prepared by Hall strain of Clostridium botulinum type A and purified by chromatography has a concentration of approximately 1 mg / mL, is present in 0.05 mol / L, pH 7.0 PB (phosphate) buffer, does not contain glycerol, and is stored at -80°C.

[0085] A portion of the botulinum toxin type A complex was purified by ion exchange chromatography to obtain a purified botulinum neurotoxin type A with a concentration of about 1 mg / mL in 0.05 mol / L, pH 7.0 PB (phosphate) buffer without glycerol and stored at -80°C.

[0086] The remaining part of the botulinum toxin type A complex was subjected to SDS-PAGE and then gel-cutting purification to obtain purified hemagglutinin 70, hemagglutinin 33, hemagglutinin 70 fragment, neurotoxin heavy chain and neurotoxin light chain. The SDS-PAGE image of botulinum toxin type A and the SDS-PAGE image of botulinum toxin type A complex can be found at Figure 1 .

[0087] The specific gel cutting and purification steps are as follows: the botulinum toxin type A is subjected to SDS-PAGE, the gel is stained with Coomassie brilliant blue R250, and decolorized with methanol-glacial acetic acid. The various components of the botulinum toxin show blue bands. The bands of hemagglutinin 70, hemagglutinin 33, hemagglutinin 70 fragment, neurotoxin heavy chain and neurotoxin light chain components are cut with a blade, as shown in the red frame in the figure. The cut gel blocks are rinsed with purified water twice, frozen at -80℃ for at least 3h, and ground without loss with a specific grinder developed by this laboratory until the gel blocks are ground into a homogenate, and recovered and purified with a gel purification recovery kit (Shenggong). Each component can be recovered and purified to obtain about 200μL, with a concentration of 0.5mg / mL. It is present in 0.05mol / L, pH7.0 PB (phosphate) buffer, without glycerol, and stored at -80℃.

[0088] The anti-botulinum neurotoxin type A monoclonal antibody was obtained by immunizing mice with botulinum neurotoxin type A. The concentration is 1 mg / mL, present in pH 7.2 PBS buffer, without glycerol, and stored at -80°C.

[0089] The anti-botulinum toxin type A complex polyclonal antibody was obtained by immunizing rabbits with botulinum toxin type A complex at a concentration of 1 mg / mL in pH 7.2 PBS buffer without glycerol and stored at -80°C.

[0090] The polyclonal antibodies against heavy and light chains of botulinum neurotoxin type A are products of PrimeBio, with a concentration of 1 mg / mL, in pH 7.2 PBS buffer containing 50% glycerol and stored at -20°C.

[0091] The capillary electrophoresis instrument is ProteinsimpleJess. The capillary immunoelectrophoresis experimental operations are all fixed operating procedures supporting ProteinsimpleJess, which are not described in detail here.

[0092] The capillary immunoelectrophoresis reaction of botulinum neurotoxin type A was performed, the antigen was botulinum neurotoxin type A complex, the primary antibody was anti-botulinum neurotoxin type A monoclonal antibody, and the secondary antibody was HRP-labeled anti-mouse antibody, to obtain the immunoreactive characteristic pattern of botulinum neurotoxin type A. The immunoreactive characteristic pattern of botulinum neurotoxin type A can be found in Figure 2 .

[0093] Depend on Figure 2 It can be seen that the characteristic peak of botulinum neurotoxin type A is the 94-98kd peak, and the characteristic peak area is 6778288.9.

[0094] Capillary immunoelectrophoresis of other components of botulinum toxin type A was performed. The antigens were hemagglutinin 70, hemagglutinin 33, hemagglutinin 70 fragment, neurotoxin heavy chain and neurotoxin light chain components. The primary antibody was an anti-botulinum toxin type A polyclonal antibody, and the secondary antibody was an HRP-labeled anti-rabbit antibody to obtain the characteristic immunoreaction pattern of botulinum neurotoxin type A.

[0095] Depend on Figure 3-4 It can be seen that the characteristic peak of the heavy chain of the botulinum neurotoxin subunit is the 99kd peak with a peak area of ​​389532.0; the characteristic peak of the light chain of the botulinum neurotoxin subunit is the 52kd peak with a peak area of ​​389532.0

[0096] Depend on Figure 5-7 It can be seen that the characteristic peak of hemagglutinin 70 is a 58kd peak with a peak area of ​​1385324.8; the characteristic graph of hemagglutinin 33 is a 38kd peak with a peak area of ​​941411.6; the characteristic graph of the hemagglutinin 70 fragment is a 24kd peak with a peak area of ​​1552754.6.

[0097] Experimental Example 1

[0098] Capillary immunoelectrophoresis and ELISA were used in parallel experiments to guide the qualitative detection of monoclonal antibodies against botulinum neurotoxin type A using fingerprints.

[0099] Capillary immunoelectrophoresis equipment: Jess capillary immunoelectrophoresis instrument, reaction plates and reagent kits are all protensimple brand.

[0100] Antigen: Botulinum toxin type A complex at a concentration of 1 mg / mL.

[0101] Primary antibody: Type A anti-botulinum neurotoxin monoclonal antibody to be detected, with a concentration of 1 mg / mL. Use 1640 medium for dilution to simulate different positive samples, with the loading amounts of 1 ng, 100 pg, 50 pg, 10 pg, 5 pg, and 1 pg, respectively.

[0102] Secondary antibody: Borcion HRP rabbit anti-mouse antibody, diluted 1:80.

[0103] The experimental results can be found in Figure 8-Figure 13 .

[0104] Depend on Figure 8-Figure 13 and Figure 2 and Figure 3Comparison shows that the main peak of the monoclonal antibody sample to be tested is consistent with the characteristic peak of neurotoxin, and it is determined to be an anti-botulinum neurotoxin type A heavy chain monoclonal antibody. When the sample amount is above 10pg, it presents a single peak, and when the sample amount is below 5pg, other peaks except the neurotoxin peak appear, indicating that the antibody sample has high purity in reactions above the 10pg level, and impurities in reactions below the 5pg level. The reaction peak areas are: 1ng: 2995789.6, 100pg: 430879.4, 50pg: 191028.9, 10pg: 52244.4, 5pg: 21283.5, 1pg: 9505.8.

[0105] Peak area linear regression r 2 =0.998, t=48.916, P<0.05.

[0106] in conclusion:

[0107] Capillary immunoelectrophoresis has a sensitivity of 1 pg for antibody detection, which is a good qualitative detection method. However, the resulting graph cannot be used to make qualitative judgments and has no practical value. Using the graph of the present invention as a guide, accurate qualitative judgments can be made on the result graph, making capillary immunoelectrophoresis for botulinum toxin antibody detection practical.

[0108] The experimental results show that the peak area is proportional to the sample amount, and the determination coefficient of the regression analysis is 0.998, which has good linearity and can be used as a method for quantitative detection of botulinum toxin antibodies. However, due to the lack of standard products for quantitative detection of botulinum toxin antibodies, it has no practical value. Using the map guidance of the present invention, standard products or reference products can be prepared, creating the necessary conditions for further developing quantitative detection methods for botulinum toxin antibodies.

[0109] Experimental Example 2

[0110] ELISA experiment

[0111] Experimental equipment: Thermo brand microplate reader, Beijing Putian Xinbiao Company's plate washer, and Shanghai Hetian Company's water bath. The color development solution and stop solution were purchased from Maokang Company, PBST washing solution was provided by Shenggong Company, skimmed milk powder was PanReacApplinChem brand, and coating solution was purchased from Solebao Company.

[0112] Experimental procedures

[0113] Antigen preparation: The concentration of botulinum toxin type A complex is 1 mg / mL, and it is diluted to the optimal working concentration of 5 μg / mL using coating solution. Coat the antigen on the ELISA plate, add 100 μL of antigen solution to each well, and incubate overnight at 4°C;

[0114] Blocking step: The next day, the ELISA plate was washed using a plate washer, and then 100 μL of blocking solution (0.5% skim milk powder in PBST) was added to each well, and incubated at 37°C for 1 hour to block nonspecific binding sites. The plate washer was then used for washing again.

[0115] Antibody incubation: The type A anti-botulinum neurotoxin monoclonal antibody to be tested (initial concentration 1 mg / mL) was serially diluted 10 times in PBST solution to construct a simulated positive sample. 100 μL of antibody solution of different concentrations (concentrations were 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL) was added to each well, incubated at 37°C for 1 hour, and then the ELISA plate was washed again using a plate washer.

[0116] Secondary antibody incubation: Add 100 μL of HRP-labeled rabbit anti-mouse secondary antibody (Borxi HRP rabbit anti-mouse antibody, 8000-fold dilution) to each well, incubate at 37°C for 1 hour, and then wash the ELISA plate again.

[0117] Color development and termination: Add 100 μL of color development solution to each well, incubate at 37°C for 5 minutes, and then add 100 μL of stop solution.

[0118] Detection: Use an ELISA reader to detect the absorbance of each well and determine the binding of the antibody. For the test results, see Fig.14 .

[0119] Fig.14 Well 1 contained 10 μg / mL, well 2 contained 1 μg / mL, well 3 contained 100 ng / mL, well 4 contained 10 ng / mL, well 5 contained 1 ng / mL, well 6 contained 100 pg / mL, and well 7 contained 10 pg / mL.

[0120] ELISA reader reading:

[0121] Hole No. 1: 1.736, 2: 1.226, 3: 0.329, 4: 0.097, 5: 0.067, 6: 0.063, 7: 0.054.

[0122] Depend on Fig.14 It can be seen that wells 4 to 7 are negative.

[0123] Conclusion: The lowest qualitative detection limit of anti-botulinum neurotoxin type A monoclonal antibody by ELISA is 100 ng / mL, and the ELISA results cannot provide a detailed analysis of the antibody.

[0124] Experimental Example 3

[0125] Using fingerprints to guide site-specific detection of monoclonal antibodies against botulinum neurotoxin type A

[0126] Capillary immunoelectrophoresis equipment: Jess capillary immunoelectrophoresis instrument, reaction plates and reagent kits are all protensimple brand.

[0127] Antigen: Botulinum toxin type A complex (homemade), with a sample concentration of 0.5 mg / mL in each reaction well

[0128] Primary antibody: anti-botulinum neurotoxin type A monoclonal antibody to be tested, a total of 3 strains, divided into No. 1 test antibody, No. 2 test antibody and No. 3 test antibody, the sample addition concentration is 20μg / μL.

[0129] Secondary antibody: HRP-labeled rabbit anti-mouse antibody (purchased from Borsei), diluted 50 times before addition.

[0130] Experimental steps: After adding the sample, centrifuge the reaction plate at 4000 rpm for 5 min, add the reaction gel provided by the kit, and place it in the capillary immunoelectrophoresis instrument to start the reaction.

[0131] The experimental results can be found in Figure 15-17 ,Will Figure 15-17 and Figure 2-Figure 7 By comparison, we can see the reaction peak of antibody No. 1 and the fingerprint spectrum Figure 3 The peak shape and peak position (99kd) are the same, and the reaction peaks of antibodies No. 2 and No. 3 are similar to the fingerprints. Figure 4 The peak shape and peak position (54kd) are the same.

[0132] Conclusion: Antibody No. 1 is a monoclonal antibody against the heavy chain of botulinum neurotoxin type A, and antibodies No. 2 and No. 3 are monoclonal antibodies against the light chain of botulinum neurotoxin type A. All three antibodies have good purity, but antibody No. 1 only detected one peak, which has the highest purity. The peak area of ​​antibody No. 1 is 389532, the peak area of ​​antibody No. 2 54kd is 6932036, and the peak area of ​​antibody No. 3 54kd is 642181. Antibody No. 2 has the strongest specificity. Antibodies No. 1 and No. 2 can be used for botulinum toxin detection, because the two antibodies have different binding sites (binding to the heavy chain and light chain respectively), and are alternative antibodies for sandwich detection of botulinum toxin type A.

[0133] The above information can only be obtained through capillary immunoelectrophoresis and guided by the fingerprint map. ELISA testing with the same antigen and antibody can only conclude that the antibody reaction is positive and cannot make the above analysis.

[0134] Through the above steps, the site-specific detection of anti-botulinum neurotoxin type A monoclonal antibody is performed using capillary immunoelectrophoresis technology.

[0135] Experimental Example 3: Using fingerprints to guide the detection of the components, quality and specificity of polyclonal antibodies against botulinum toxin type A.

[0136] Capillary immunoelectrophoresis equipment: Jess capillary immunoelectrophoresis instrument, reaction plates and reagent kits are all protensimple brand.

[0137] Antigen: Botulinum toxin type A complex (homemade), with a sample concentration of 0.5 mg / mL in each reaction well

[0138] Primary antibody: anti-botulinum toxin type A polyclonal antibody to be detected, with a sample concentration of 1 μg / μL.

[0139] Secondary antibody: HRP-labeled goat anti-rabbit antibody (Sigma), diluted 50 times before addition.

[0140] Experimental steps: After adding the sample, centrifuge the reaction plate at 4000 rpm for 5 min, add the reaction gel provided by the kit, and place it in the capillary immunoelectrophoresis instrument to start the reaction.

[0141] The experimental results can be found in Fig.18 ,Depend on Fig.18 It can be seen that the antibody to be tested has 6 peaks, and the 96kd peak is consistent with this spectrum. Figure 2 The characteristic peaks of anti-botulinum neurotoxin type A antibodies in the spectrum are the same; the 56kd peak is the same as that in this spectrum Figure 5 The characteristic peaks are basically the same as those of anti-hemagglutinin 70 antibody; the 52kd peak is Figure 4 The characteristic peaks are the same, which is the anti-Botulinum neurotoxin light chain antibody; the 36kd peak is the same as this spectrum Figure 6 The characteristic peaks are basically the same as those of the anti-hemagglutinin 33 antibody; the 24kd peak is similar to that of this spectrum. Figure 7 The characteristic peak is the same as that of , which is an anti-hemagglutinin 70 fragment antibody.

[0142] Conclusion: The antibody contains anti-Botulinum neurotoxin type A antibody, anti-hemagglutinin 70 antibody, anti-Botulinum neurotoxin type A light chain antibody, anti-hemagglutinin 33 antibody, and anti-hemagglutinin 70 fragment antibody. According to the peak area, the hemagglutinin 33 antibody is the main one, and there is no peak less than 24kd and no peak greater than 96kd, indicating that this polyclonal antibody does not contain anti-non-toxin non-hemagglutinin (NTNH) antibody and anti-hemagglutinin 17 antibody. The 75kd peak of the antibody does not match this spectrum, and there may be impurities or degradation of anti-neurotoxin antibodies. If it is necessary to confirm, anti-Botulinum neurotoxin type A monoclonal antibody can be used as the primary antibody and HRP-labeled rabbit anti-mouse antibody as the secondary antibody for further capillary immunoelectrophoresis. If a single anti-neurotoxin antibody characteristic peak appears, the 75kd peak can be determined as an impurity. If the anti-neurotoxin antibody peak and the 75kd peak appear at the same time, it indicates that the anti-neurotoxin antibody is partially degraded.

[0143] Using the same reaction system for ELISA testing can only yield a positive conclusion for anti-botulinum toxin type A antibodies, and the above analysis cannot be performed.

Claims

1. A method for constructing an anti-botulinum toxin antibody HPCE fingerprint, characterized in that: The construction method comprises performing capillary immunoelectrophoresis reaction on botulinum neurotoxin and performing capillary immunoelectrophoresis reaction on each subunit of a botulinum toxin complex, wherein the botulinum neurotoxin is purified from the botulinum toxin complex; Wherein, a capillary immunoelectrophoresis reaction is performed on botulinum neurotoxin to obtain a capillary immunoelectrophoresis pattern of the botulinum neurotoxin; in the capillary immunoelectrophoresis reaction of the botulinum neurotoxin, the antigen is the botulinum neurotoxin, the primary antibody is an anti-botulinum neurotoxin monoclonal antibody or an anti-botulinum toxin complex polyclonal antibody, and the secondary antibody is an HRP-labeled antibody corresponding to the primary antibody; Capillary immunoelectrophoresis is performed on each subunit of the botulinum toxin complex to obtain a capillary immunoelectrophoresis pattern of each subunit of the botulinum toxin complex; the subunits of the botulinum toxin complex include hemagglutinin 70, hemagglutinin 33, hemagglutinin 70 fragment, botulinum neurotoxin heavy chain and botulinum neurotoxin light chain. In the capillary immunoelectrophoresis reaction of each subunit of the botulinum toxin complex, hemagglutinin 70, hemagglutinin 33, hemagglutinin 70 fragment, botulinum neurotoxin heavy chain and botulinum neurotoxin light chain are used as antigens respectively, the primary antibody is an anti-botulinum toxin complex polyclonal antibody, and the secondary antibody is an HRP-labeled antibody corresponding to the primary antibody.

2. The construction method according to claim 1, characterized in that: The botulinum toxin complex is a botulinum toxin type A complex, and the method for preparing the botulinum toxin type A complex comprises the following steps: Cultivate the type A Clostridium botulinum Hall strain to obtain a type A Clostridium botulinum bacterial liquid: The type A botulinum Clostridium botulinum bacterial liquid is collected and purified by chromatography to obtain the type A botulinum toxin complex.

3. The construction method according to claim 2, characterized in that: The botulinum toxin type A complex is purified by ion exchange chromatography to obtain botulinum neurotoxin type A.

4. The construction method according to claim 2, characterized in that: The botulinum toxin type A complex is subjected to gel excision purification to obtain hemagglutinin 70, hemagglutinin 33, hemagglutinin 70 fragment, botulinum neurotoxin type A heavy chain and botulinum neurotoxin type A light chain.

5. The construction method according to claim 3, characterized in that: The anti-botulinum toxin complex polyclonal antibody is an anti-botulinum toxin complex polyclonal antibody obtained by immunizing rabbits with botulinum toxin complex type A, and the corresponding secondary antibody is an anti-rabbit HRP-labeled antibody.

6. The construction method according to claim 3, characterized in that: The anti-botulinum neurotoxin type A monoclonal antibody is an anti-botulinum neurotoxin type A monoclonal antibody obtained by immunizing mice with botulinum neurotoxin type A, and the secondary antibody is an anti-mouse HRP-labeled antibody.

7. A method for detecting anti-botulinum toxin antibodies, comprising the following steps: Prepare antigen, test antibody and secondary antibody; The antigen is a botulinum toxin complex, the antibody to be tested is an anti-botulinum neurotoxin monoclonal antibody or an anti-botulinum toxin complex polyclonal antibody, and the secondary antibody is an HRP-labeled antibody corresponding to the antibody to be tested; The antibody to be tested is added to the antigen. After sufficient binding, the secondary antibody is added. The secondary antibody fully binds to the antibody to be tested and the incubation continues; After the incubation is completed, capillary immunoelectrophoresis analysis is performed to obtain a capillary immunoelectrophoresis pattern of the antibody to be tested; The capillary immunoelectrophoresis pattern of the antibody to be tested is compared with the capillary immunoelectrophoresis pattern obtained by the construction method of any one of claims 1 to 6 to obtain the test result.

8. The detection method according to claim 7, characterized in that: The antigen is a botulinum toxin type A complex, and the method for preparing the botulinum toxin type A complex comprises the following steps: Cultivate the type A Clostridium botulinum Hall strain to obtain a type A Clostridium botulinum bacterial liquid: The type A botulinum Clostridium botulinum bacterial liquid is collected and purified by chromatography to obtain the type A botulinum toxin complex.

9. The detection method according to claim 8, characterized in that: After the secondary antibody is fully combined with the antibody to be tested, add the reaction gel for incubation.

10. The detection method according to claim 8, characterized in that: The obtained capillary immunoelectrophoresis pattern of hemagglutinin 70, the capillary immunoelectrophoresis pattern of hemagglutinin 33 or the capillary immunoelectrophoresis pattern of hemagglutinin 70 fragment is used to detect the comparison of antibodies against botulinum toxin type B, C, D, E and F complexes.

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