Anti-botulinum toxin antibody capillary immunoelectrophoresis fluorescent 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 capillary immunoelectrophoresis fluorescent fingerprint spectrum construction method and application thereof.According to the construction method, capillary immunoelectrophoresis of the fluorescence technology is used for construction, a fluorescent antibody corresponding to a primary antibody is used as a secondary antibody, and the fluorescent antibody is used as a secondary antibody. The technology for detecting the botulinum toxin antibody through fluorescence capillary immunoelectrophoresis is practically applied, and due to the fact that more signals are generated through fluorescence detection, compared with capillary immunoelectrophoresis based on the chemiluminescence technology, the method has better resolution capacity.

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 capillary immunoelectrophoresis fluorescence fingerprint spectrum and an application thereof. Background Art

[0002] In the process of studying botulinum toxin, it is usually necessary to test for anti-botulinum toxin antibodies, which can be defined as laboratory testing for anti-botulinum toxin antibodies. For botulinum toxin or botulinum neurotoxin for injection, it is generally used in humans or experimental animals during the research and development, clinical trial stages and after marketing. Whether serum antibodies are produced after injection and at what level are important aspects of botulinum toxin research, namely, botulinum toxin serum antibody testing. The data generated by the test is of great significance to product development and drug vigilance, and botulinum toxin serum antibody testing is usually tested using immunological methods.

[0003] At present, the conventional detection technology is mainly the ELISA indirect method, but this method has low sensitivity. Due to the extremely strong toxicity of botulinum toxin, even if antibodies are produced when the injection dose is very low, the antibody level may be lower than the detection limit of the ELISA indirect method, resulting in failure to detect it. Therefore, the ELISA indirect method cannot fully meet the requirements for the accuracy of botulinum toxin detection. In addition, the ELISA method can only perform macroscopic detection of samples, and the results are judged as negative or positive, and cannot perform a more in-depth analysis of the subunits in the sample.

[0004] Botulinum toxin (hereinafter referred to as botulinum toxin complex) is a complex protein that contains multiple subunits such as botulinum neurotoxin, hemagglutinin 70, hemagglutinin 33 and hemagglutinin 70 fragments. Botulinum toxin complex antibodies include monoclonal antibodies and polyclonal antibodies against these specific subunits, as well as polyclonal antibodies against the complete toxin complex. Although the ELISA method can be used to detect botulinum neurotoxin antibodies, due to the difficulty in obtaining antigens, the botulinum neurotoxin needs to be separated from the botulinum toxin complex before testing, and it is also impossible to detect antibodies to other subunits. At the same time, there is a lack of holistic analysis methods for the complete toxin complex and serum antibodies.

[0005] There is also an immunological detection method for antibody detection, capillary immunoelectrophoresis (HPCE). This detection method has high detection accuracy, picogram (pg) level sensitivity, and can detect subunit antibodies of botulinum toxin, which can solve the detection difficulties faced by ELISA. However, HPCE cannot directly detect botulinum toxin antibodies, and still needs to determine the existence of antibodies and their corresponding subunits based on the corresponding immunological reaction fingerprint.

[0006] In summary, it is crucial to design a method for constructing anti-botulinum toxin antibody capillary immunoelectrophoresis fingerprints, which can achieve higher sensitivity and more comprehensive antibody detection. Summary of the invention

[0007] The object of the present invention is to provide a method for constructing an anti-botulinum toxin antibody capillary immunoelectrophoresis fluorescence fingerprint spectrum and its application. This construction method obtains a near-infrared light source spectrum and an infrared light source spectrum of the botulinum neurotoxin and a near-infrared light source spectrum and an infrared light source spectrum of each subunit of the botulinum toxin complex by performing capillary immunoelectrophoresis reaction on botulinum neurotoxin and on each component of the botulinum toxin complex. The spectra obtained in this method can be compared with the detection of the botulinum toxin complex.

[0008] The object of the present invention is to provide a method for constructing an anti-botulinum toxin antibody capillary immunoelectrophoresis fluorescence fingerprint and its application. This construction method obtains infrared spectra and near-infrared spectra of each component of botulinum toxin by performing capillary immunoelectrophoresis reaction on botulinum neurotoxin and hemagglutinin 70, hemagglutinin 33, hemagglutinin 70 fragment, botulinum neurotoxin heavy chain and botulinum neurotoxin light chain, thereby improving the subsequent detection efficiency of anti-botulinum toxin antibodies and thus improving the detection accuracy of anti-botulinum toxin antibodies.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] A method for constructing an anti-botulinum toxin antibody capillary immunoelectrophoresis fluorescence fingerprint, the method comprising performing a capillary immunoelectrophoresis reaction on a botulinum neurotoxin and performing a capillary immunoelectrophoresis reaction on each component of a botulinum toxin complex, wherein the botulinum neurotoxin is purified from the botulinum toxin complex;

[0011] Wherein, the botulinum neurotoxin is subjected to capillary immunoelectrophoresis reaction to obtain a near-infrared light source spectrum and an infrared light source spectrum 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 a fluorescent antibody corresponding to the primary antibody;

[0012] Capillary immunoelectrophoresis is performed on each subunit of the botulinum toxin complex to obtain a near-infrared light source spectrum and an infrared light source spectrum 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 on 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 a fluorescent antibody corresponding to the primary antibody.

[0013] 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.

[0014] 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:

[0015] Cultivate the type A Clostridium botulinum Hall strain to obtain a type A Clostridium botulinum bacterial liquid:

[0016] The type A botulinum Clostridium botulinum bacterial liquid is collected and purified by chromatography to obtain the type A botulinum toxin complex.

[0017] Furthermore, the botulinum toxin type A complex is purified by ion exchange chromatography to obtain botulinum neurotoxin type A.

[0018] Furthermore, 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.

[0019] Furthermore, the anti-botulinum toxin complex polyclonal antibody is an anti-botulinum toxin type A complex polyclonal antibody obtained by immunizing rabbits with botulinum toxin type A complex, and the corresponding secondary antibody is an anti-rabbit fluorescent antibody.

[0020] 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 fluorescent antibody.

[0021] An anti-botulinum toxin antibody capillary immunoelectrophoresis fluorescence fingerprint spectrum, wherein the characteristic peaks of the type A botulinum neurotoxin near-infrared light source spectrum include a 53kd peak; and the characteristic peaks of the type A botulinum neurotoxin infrared light source spectrum include a 53kd peak.

[0022] Furthermore, the characteristic peaks of the near-infrared light source spectrum of the botulinum neurotoxin type A heavy chain include a 71-72kd peak; the characteristic peaks of the infrared light source spectrum include a 71-72kd peak.

[0023] Furthermore, the characteristic peaks of the near-infrared light source spectrum of the botulinum neurotoxin type A light chain include a 49kd peak; the characteristic peaks of the infrared light source spectrum include a 49kd peak.

[0024] Furthermore, the characteristic peaks of the near-infrared light source spectrum of the hemagglutinin 70 include a 55-56 kd peak; the characteristic peaks of the infrared light source spectrum include a 55-56 kd peak.

[0025] Furthermore, the characteristic peaks of the near-infrared light source spectrum of the hemagglutinin 33 include a 35-36 kd peak; the characteristic peaks of the infrared light source spectrum include a 35-36 kd peak.

[0026] Furthermore, the characteristic peaks of the near-infrared light source spectrum of the hemagglutinin 70 fragment include a 24kd peak; the characteristic peaks of the infrared light source spectrum include a 24kd peak.

[0027] The infrared light source spectrum of hemagglutinin 70, the near-infrared light source spectrum of hemagglutinin 70, the infrared light source spectrum of hemagglutinin 33, the near-infrared light source spectrum of hemagglutinin 33, the infrared light source spectrum of hemagglutinin 70 fragment or the near-infrared light source spectrum of hemagglutinin 70 fragment obtained above can be used to detect the comparison of antibodies against botulinum toxin type B, C, D, E and F complexes.

[0028] The present invention also provides a method for detecting anti-botulinum toxin antibodies, comprising the following steps:

[0029] Prepare antigen, test antibody and secondary antibody;

[0030] 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 a fluorescent antibody corresponding to the antibody to be tested;

[0031] 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;

[0032] After the incubation is completed, capillary immunoelectrophoresis analysis is performed to obtain near-infrared light source spectrum and infrared light source spectrum of the antibody to be tested;

[0033] The near-infrared light source spectrum and infrared light source spectrum of the antibody to be tested are compared with the near-infrared light source spectrum and infrared light source spectrum obtained by the above-mentioned construction method to obtain the test result.

[0034] 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:

[0035] Cultivate the type A Clostridium botulinum Hall strain to obtain a type A Clostridium botulinum bacterial liquid:

[0036] The type A botulinum Clostridium botulinum bacterial liquid is collected and purified by chromatography to obtain the type A botulinum toxin complex.

[0037] Furthermore, after the secondary antibody is fully combined with the antibody to be tested, a reaction gel is added for incubation.

[0038] After the antibody sample to be tested is subjected to capillary immunoelectrophoresis analysis, the following situations can be analyzed by comparing the peak type of the sample with the characteristic peak of the fingerprint spectrum:

[0039] Multiple peaks and the peak shapes are completely consistent with the characteristic peaks of the fingerprint spectrum: This indicates that the sample is a polyclonal antibody against botulinum toxin complex, with high purity and no impurities.

[0040] A single peak and the peak shape is consistent with the molecular weight of one of the characteristic peaks in the fingerprint: This indicates that the sample is a monoclonal antibody against botulinum toxin subunit, and it is of high purity and free of impurities.

[0041] Multiple peaks and some peak shapes are consistent with the characteristic peaks of the fingerprint spectrum, but have different peak shapes: This indicates that the sample is a polyclonal antibody against botulinum toxin complex, but there are impurities.

[0042] The molecular weight of a single peak is consistent with one of the characteristic peaks in the fingerprint, and there is another different peak type: this indicates that the sample is a monoclonal antibody against a botulinum toxin subunit, but contains impurities.

[0043] Antibody degradation analysis: Antibodies may slowly degrade during storage, so capillary immunoelectrophoresis analysis should be performed before storage to generate a peak profile. Figure 1 If the peaks are consistent, it means that the antibody has not been degraded; if there are peaks that do not exist in the original peak diagram, it means that the antibody has begun to degrade. Such peaks are called degradation peaks, and usually the molecular weight is slightly smaller than the characteristic peak.

[0044] Peak area change: Each peak in the original peak diagram generated before saving has specific peak area data. After a period of storage, if the peak area of ​​a peak is significantly reduced, it means that the antibody has been degraded, and the degree of peak area reduction reflects the severity of degradation.

[0045] Signs of severe degradation: If a large number of peaks different from the original peak pattern appear in the capillary immunoelectrophoresis analysis of the stored antibody sample, it indicates that the antibody has been severely degraded.

[0046] 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.

[0047] Botulinum toxin complex contains multiple subunits, and anti-botulinum toxin complex antibodies should contain antibody components corresponding to each subunit. Therefore, capillary immunoelectrophoresis analysis of anti-botulinum toxin complex antibodies will show multiple peaks, which should correspond to the characteristic peaks of the spectrum. If peaks inconsistent with the characteristic peaks appear, it indicates that the anti-botulinum toxin complex antibody contains other non-specific antibodies, and the application of such antibodies should be cautious.

[0048] 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 botulinum 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 botulinum neurotoxin heavy chain and light chain. This is a guide to site specificity.

[0049] The present invention has the following beneficial effects:

[0050] Through the construction method of the present invention, the characteristic spectrum of anti-botulinum toxin antibodies in fluorescent capillary immunoelectrophoresis can be determined, making the fluorescent capillary immunoelectrophoresis technology for detecting botulinum toxin antibodies practical. Fluorescence detection generates more signals and has improved resolution compared to capillary immunoelectrophoresis based on chemiluminescence technology.

[0051] By utilizing the characteristic spectrum obtained through the construction method of the present invention, the efficiency of anti-botulinum toxin antibody detection is improved.

[0052] In the construction method of the present invention, capillary immunoelectrophoresis based on dual-channel fluorescence technology is used, which has improved resolution ability compared with capillary immunoelectrophoresis based on chemiluminescence technology.

[0053] In the construction method of the present invention, the composition of botulinum toxin antibodies is analyzed in detail. Through the analysis of each subunit antibody, data can be provided for the development and market application of botulinum toxin products.

[0054] The fingerprint obtained by the construction method of the present invention can guide the detection of the stability of botulinum toxin antibodies, find out whether the antibodies are degraded and the subunits that have been degraded, and the degree of degradation, and can be used for antibody quality control.

[0055] 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 near-infrared light source spectra and infrared light source spectra 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.

[0056] The present invention can guide the detection of the immunological purity and site specificity of botulinum toxin monoclonal antibodies and polyclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] 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.

[0058] 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;

[0059] Figure 2 This is the near-infrared light source spectrum of botulinum neurotoxin type A in Example 1 of the specific embodiment of the present invention;

[0060] Figure 3 This is the infrared light source spectrum of botulinum neurotoxin type A in Example 1 of the specific embodiment of the present invention;

[0061] Figure 4 It is a near-infrared light source reference spectrum of botulinum neurotoxin type A in Example 1 of the specific embodiment of the present invention;

[0062] Figure 5 This is the infrared light source reference spectrum of botulinum neurotoxin type A in Example 1 of the specific implementation mode of the present invention;

[0063] Figure 6 This is the near-infrared spectrum of the heavy chain of botulinum neurotoxin type A in Example 1 of the specific embodiment of the present invention;

[0064] Figure 7This is the infrared light source spectrum of the heavy chain of botulinum neurotoxin type A in Example 1 of the specific embodiment of the present invention;

[0065] Figure 8 This is the near-infrared light source spectrum of the botulinum neurotoxin type A light chain in Example 1 of the specific embodiment of the present invention;

[0066] Fig. 9 This is the infrared light source spectrum of the botulinum neurotoxin type A light chain in Example 1 of the specific embodiment of the present invention;

[0067] Fig.10 The near infrared light source spectrum of botulinum toxin type A hemagglutinin 70 in Example 1 of the specific embodiment of the present invention;

[0068] Fig.11 This is the infrared light source spectrum of botulinum toxin type A hemagglutinin 70 in Example 1 of the specific implementation mode of the present invention;

[0069] Fig.12 This is the near-infrared light source spectrum of botulinum toxin type A hemagglutinin 33 in Example 1 of the specific implementation mode of the present invention;

[0070] Fig.13 This is the infrared light source spectrum of botulinum toxin type A hemagglutinin 33 in Example 1 of the specific implementation mode of the present invention;

[0071] Fig.14 This is the near-infrared light source spectrum of the hemagglutinin 70 fragment of type A botulinum toxin in Example 1 of the specific implementation mode of the present invention;

[0072] Fig.15 This is the infrared light source spectrum of the hemagglutinin 70 fragment of botulinum toxin type A in Example 1 of the specific implementation method of the present invention.

[0073] Fig.16 This is the infrared spectrum of the 1 ng / mL sample in Experimental Example 1 of the specific embodiment of the present invention;

[0074] Fig.17 This is the infrared spectrum of the 100 pg / mL sample in Experimental Example 1 of the specific embodiment of the present invention;

[0075] Fig.18 This is the infrared spectrum of the 10 μg / mL sample in Experimental Example 1 of the specific embodiment of the present invention;

[0076] Fig.19 This is the infrared spectrum of the 100 μg / mL sample in Experimental Example 1 of the specific embodiment of the present invention;

[0077] Fig. 20 This is the ELISA test diagram in Experimental Example 2 of the specific embodiment of the present invention;

[0078] Fig.21This is an infrared spectrum of botulinum toxin complex No. 1 in Experimental Example 3 of the specific embodiment of the present invention;

[0079] Fig. 22 This is a near infrared spectrum of botulinum toxin complex No. 1 in Experimental Example 3 of the specific embodiment of the present invention;

[0080] Fig.23 This is an infrared spectrum of botulinum toxin complex No. 2 in Experimental Example 3 of the specific embodiment of the present invention;

[0081] Fig.24 This is a near-infrared spectrum of botulinum toxin complex No. 2 in Experimental Example 3 of the specific implementation example of the present invention. 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 from the Hall strain of Clostridium botulinum type A and purified by chromatography has a concentration of approximately 1 mg / mL and is stored in 0.05 mol / LPB (phosphate) buffer, pH 7.0, without glycerol, and stored at -80°C.

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

[0086] The botulinum toxin type A complex was subjected to SDS-PAGE followed by gel purification to obtain purified hemagglutinin 70, hemagglutinin 33, hemagglutinin 70 fragment, botulinum neurotoxin heavy chain and botulinum neurotoxin light chain.

[0087] The anti-botulinum neurotoxin type A monoclonal antibody obtained by the above-mentioned purified botulinum neurotoxin type A and immunization of mice was stored in PBS at pH 7.2 without glycerol at -80°C at 1 mg / mL.

[0088] Anti-botulinum toxin type A complex polyclonal antibody, anti-botulinum neurotoxin type A heavy chain polyclonal antibody and anti-botulinum neurotoxin type A light chain polyclonal antibody are all PrimeBio products, 1 mg / mL, stored in PBS at pH 7.2, containing 50% glycerol, and stored at -20°C.

[0089] The capillary electrophoresis instrument was ProteinsimpleJess, the fluorescent antibody was ProteinsimpleJess, and the capillary immunoelectrophoresis experimental operations were all fixed operating procedures supporting ProteinsimpleJess, which are not described in detail here.

[0090] The botulinum toxin type A complex was subjected to SDS-PAGE, the gel was stained with Coomassie Brilliant Blue R250, and decolorized with methanol-glacial acetic acid. The components of botulinum toxin showed blue bands. The bands of hemagglutinin 70, hemagglutinin 33, hemagglutinin 70 fragment, botulinum neurotoxin heavy chain and botulinum neurotoxin light chain components were cut out with a blade. Figure 1 In the red frame, the cut gel block was rinsed twice with purified water, frozen at -80℃ for at least 3h, and ground without damage until the gel block was ground into a homogenate, and recovered and purified using a gel purification recovery kit (Sangong). Each component can be recovered and purified to obtain about 200μL, with a concentration of 0.5mg / mL. It was stored in 0.05mol / L, pH7.0 PB (phosphate) buffer, without glycerol, and stored at -80℃. The SDS-PAGE electrophoresis of the botulinum toxin type A complex can be found in Figure 1 .

[0091] The capillary immunoelectrophoresis reaction of botulinum neurotoxin type A was performed, the antigen was the above-mentioned botulinum neurotoxin type A, the primary antibody was an anti-botulinum toxin type A complex polyclonal antibody, and the secondary antibody was an anti-rabbit fluorescent antibody, and the near-infrared light source spectrum and infrared light source spectrum of botulinum neurotoxin type A were obtained. The near-infrared light source spectrum can be found in Figure 2 , infrared spectrum see Figure 3 ,Depend on Figure 2 and Figure 3 It can be seen that the effective peaks of the near-infrared light source spectrum of botulinum neurotoxin type A are 53kd peak and 95kd peak, and the corresponding peak areas are 2347.4 and 1274.3; the effective peaks of the infrared light source spectrum of botulinum neurotoxin type A are 39kd peak, 53kd peak, 59kd peak, 95kd peak, 123kd peak and 141kd peak, and the corresponding peak areas are 509174.4, 1125124.8, 721120.8, 791499.9, 292161.2 and 306195.2.

[0092] A capillary immunoelectrophoresis reaction of botulinum neurotoxin type A was performed, the antigen was botulinum neurotoxin type A, the primary antibody was anti-botulinum neurotoxin type A monoclonal antibody, and the secondary antibody was anti-mouse fluorescent antibody. A near-infrared light source reference spectrum and an infrared light source reference spectrum of botulinum neurotoxin type A were obtained. The near-infrared light source reference spectrum can be found in Figure 4 , infrared reference spectrum see Figure 5 ,Depend on Figure 4 and Figure 5 It can be seen that the effective peak of the near-infrared light source reference spectrum of botulinum neurotoxin type A is the 53kd peak, with a peak area of ​​1454.8, and the effective peak of the infrared light source reference spectrum of botulinum neurotoxin type A is the 53kd peak, with a peak area of ​​614942.5;

[0093] The capillary immunoelectrophoresis reaction of botulinum neurotoxin type A heavy chain was performed. The antigen was the botulinum neurotoxin type A heavy chain purified by SDS-PAGE gel cutting. The primary antibody was an anti-botulinum neurotoxin type A heavy chain polyclonal antibody. The secondary antibody was an anti-rabbit fluorescent antibody. The near-infrared light source spectrum and infrared light source spectrum of the botulinum neurotoxin type A heavy chain were obtained. The near-infrared light source spectrum can be found in Figure 6 , infrared spectrum see Figure 7 ,Depend on Figure 6 and Figure 7 It can be seen that the effective peaks of the near-infrared light source spectrum of the botulinum neurotoxin type A heavy chain are 71kd peak and 189kd peak, and the corresponding peak areas are 5975.5 and 309.1; the effective peaks of the infrared light source spectrum of the botulinum neurotoxin type A heavy chain are 72kd peak, 101kd peak, 140kd peak, 171kd peak and 205kd peak, and the corresponding peak areas are 2599002.9, 467894.7, 861578.0, 333595.1 and 1085968.4.

[0094] The capillary immunoelectrophoresis reaction of botulinum neurotoxin type A light chain was performed. The antigen was botulinum neurotoxin type A light chain purified by SDS-PAGE gel cutting. The primary antibody was anti-botulinum neurotoxin type A light chain polyclonal antibody. The secondary antibody was anti-rabbit fluorescent antibody. The near-infrared light source spectrum and infrared light source spectrum of the fluorescent immunoreaction of botulinum neurotoxin type A light chain were obtained. The near-infrared light source spectrum can be found in Figure 8 , infrared spectrum see Fig. 9 ,Depend on Figure 8 and Fig. 9 It can be seen that the effective peaks of the near-infrared light source spectrum of the botulinum neurotoxin type A light chain are 49kd peak and 230kd peak, and the corresponding peak areas are 9092.2 and 505.3. The effective peaks of the infrared light source spectrum of the anti-botulinum neurotoxin type A light chain antibody are 49kd peak and 92kd peak, and the corresponding peak areas are 3382254.4 and 587858.6.

[0095] Capillary immunoelectrophoresis of botulinum toxin type A hemagglutinin 70 was performed, the antigen was botulinum toxin type A hemagglutinin 70 purified by SDS-PAGE gel cutting, the primary antibody was anti-botulinum toxin type A complex polyclonal antibody, the secondary antibody was anti-rabbit fluorescent antibody, and the near-infrared light source spectrum and infrared light source spectrum of the fluorescent immunoreaction of botulinum toxin type A hemagglutinin 70 were obtained. The near-infrared light source spectrum can be found in Fig.10 , infrared spectrum see Fig.11 ,Depend on Fig.10 and Fig.11 It can be seen that the effective peak of the near-infrared light source spectrum of botulinum toxin type A hemagglutinin 70 is the 55kd peak, and the peak area is 6114.2. The effective peaks of the infrared light source spectrum of botulinum toxin type A hemagglutinin 70 are the 47kd peak, 56kd peak and 118kd peak, and the corresponding peak areas are 442016.7, 2898231.1 and 696958.4.

[0096] Capillary immunoelectrophoresis of botulinum toxin type A hemagglutinin 33 was performed. The antigen was botulinum toxin type A hemagglutinin 33 purified by SDS-PAGE gel cutting. The primary antibody was an anti-botulinum toxin type A complex polyclonal antibody. The secondary antibody was an anti-rabbit fluorescent antibody. The near-infrared light source spectrum and infrared light source spectrum of botulinum toxin type A hemagglutinin 33 were obtained. The near-infrared light source spectrum can be found in Fig.12 , infrared spectrum see Fig.13 ,Depend on Fig.12 and Fig.13 It can be seen that the effective peak of the near-infrared light source spectrum of anti-botulinum toxin type A hemagglutinin 33 antibody is the 35kd peak, and the peak area is 6403.6. The effective peaks of the infrared light source spectrum of botulinum toxin type A hemagglutinin 33 are the 36kd peak and the 61kd peak, and the corresponding peak areas are 3231975.3 and 498364.6.

[0097] Capillary immunoelectrophoresis of botulinum toxin type A hemagglutinin 70 fragment was performed, the antigen was botulinum toxin type A hemagglutinin 70 fragment purified by SDS-PAGE gel cutting, the primary antibody was anti-botulinum toxin type A complex polyclonal antibody, the secondary antibody was anti-rabbit fluorescent antibody, and the near-infrared light source spectrum and infrared light source spectrum of botulinum toxin type A hemagglutinin 70 fragment were obtained. The near-infrared light source spectrum can be found in Fig.14 , infrared spectrum see Fig.15 ,Depend on Fig.14 and Fig.15 It can be seen that the effective peaks of the near-infrared light source spectrum of the botulinum toxin type A hemagglutinin 70 fragment are 24kd peak, 34kd peak and 222kd peak, and the corresponding peak areas are 4264.2, 3227.7 and 517.0; the effective peaks of the infrared light source spectrum of the botulinum toxin type A hemagglutinin 70 fragment are 24kd peak and 41kd peak, and the corresponding peak areas are 2720534 and 88780.1.

[0098] Experimental Example 1: Using fingerprints to guide qualitative detection of anti-botulinum toxin type A complex antibodies

[0099] Experimental instruments and reagents:

[0100] Instruments: Jess capillary immunoelectrophoresis instrument, reaction plate, reaction kit and ultra-sensitive fluorescent anti-rabbit kit are all Protensimple brand;

[0101] Antigen: Botulinum toxin type A complex (made by our company), the concentration of each reaction well is 1 mg / mL;

[0102] Primary antibody (test sample): anti-botulinum toxin type A complex antibody, concentrations are 10 μg / mL, 100 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, 1 pg / mL;

[0103] Experimental steps:

[0104] Prepare antigen and antibody samples:

[0105] Add sample:

[0106] Botulinum toxin type A complex (1 mg / mL) was used as an antigen and added to each reaction well in a prescribed amount.

[0107] The anti-botulinum toxin type A complex antibodies to be tested were prepared and added according to a concentration gradient (10 μg / mL, 100 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, 1 pg / mL).

[0108] Prepare Stellar secondary antibody: Prepare Stellar Anti-Rabbit Secondary according to the prescribed amount and add it to each reaction well;

[0109] Prepare ANNEAL: Prepare the prescribed amount of IR Anneal and add it to each reaction well;

[0110] Prepare LABEL: Prepare the IR Label in the specified amount and add it to each reaction well;

[0111] Centrifugation: After the sample is loaded, place the reaction plate in a centrifuge and centrifuge at 4000 rpm for 5 minutes to ensure that the sample is evenly distributed and fully in contact with the antigen.

[0112] Add reaction gel: After centrifugation, add the reaction gel provided by the kit to each reaction well and prepare for the reaction.

[0113] Perform capillary immunoelectrophoresis analysis: Place the reaction plate in the Jess capillary immunoelectrophoresis instrument and start the analysis. The instrument will detect the reaction of antibodies and antigens at various concentrations through electrophoretic separation and fluorescent labeling to generate a fingerprint.

[0114] Data analysis: Compare the fingerprints of anti-botulinum toxin type A complex antibodies at different concentrations and analyze the qualitative results of the antibodies in the samples.

[0115] The experimental results can be found in Figure 16-Figure 19 ,Depend on Figure 16-Figure 19 It can be seen that the infrared spectrum of the antibody produces 9 obvious peak signals, 6 of which are consistent with the fingerprint spectrum, among which the 37kd peak has the highest proportion. Fig.13 The characteristic peaks of hemagglutinin 33 are the same, so it is determined that the antibody is a polyclonal antibody mainly composed of anti-hemagglutinin 33 antibodies. The sample can still be detected when the dilution reaches 1ng / mL, and the minimum limit of qualitative detection of anti-botulinum toxin type A polyclonal antibody by fluorescent capillary immunoelectrophoresis is 1ng / mL.

[0116] Experimental Example 2: ELISA experiment

[0117] Enzyme-linked immunosorbent assay (ELISA) was used to detect botulinum toxin type A antibody samples and observe the reaction of anti-botulinum toxin type A complex antibodies at different concentrations.

[0118] Experimental instruments and reagents: ELISA reader (Thermo brand), plate washer (Beijing Putian Xinbiao Company), water bath (Shanghai Hetian Company), color development solution and stop solution (Maokang Company), PBST washing solution (Shenggong Company), skimmed milk powder (PanReacApplinChem brand), coating solution (Solabo Company).

[0119] Antigen: Botulinum toxin type A complex (1 mg / mL), diluted to a working concentration of 5 μg / mL

[0120] Experimental steps:

[0121] Coating antigen: Dilute the botulinum toxin type A complex (1 mg / mL) to 5 μg / mL with coating solution. Coat a 96-well ELISA plate, add 100 μL of antigen solution to each well, and incubate overnight at 4°C.

[0122] Washing: Use a plate washer to wash the ELISA plate to remove unbound antigens.

[0123] Blocking: Add 100 μL of blocking solution (0.5% skim milk powder in PBST) to each well and incubate at 37°C for 1 hour to block the wells not occupied by antigens. After blocking, wash the ELISA plate again using a plate washer.

[0124] Add the antibody to be tested: take type A anti-botulinum toxin complex antibody (1 mg / mL) and dilute it 10-fold in series with PBST to construct a simulated positive sample; add 100 μL of antibody solution of different concentrations to each well, the concentrations are 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL; incubate at 37°C for 1 hour.

[0125] Washing: Use a plate washer to wash the ELISA plate to remove unbound antibodies.

[0126] Add secondary antibody: Add 100 μL of HRP rabbit anti-mouse secondary antibody to each well, diluted 8000 times, and incubate at 37°C for 1 hour.

[0127] Washing: Use a plate washer to wash the ELISA plate to remove unbound secondary antibody.

[0128] Color reaction: Add 100 μL of color development solution to each well and incubate at 37°C for 5 minutes.

[0129] Stop reaction: Add 100 μL of stop solution to each well to stop the color development reaction.

[0130] The experimental results can be found in Fig. 20 , Fig. 20 The microplate reader readings for well 1 are 10 μg / mL, well 2 is 1 μg / mL, well 3 is 100 ng / mL, well 4 is 10 ng / mL, well 5 is 1 ng / mL, well 6 is 100 pg / mL, and well 7 is 10 pg / mL

[0131] Hole No. 1: 1.317, Hole No. 2: 1.244, Hole No. 3: 0.867, Hole No. 4: 0.344, Hole No. 5: 0.091, Hole No. 6: 0.056, Hole No. 7: 0.045.

[0132] Depend on Fig. 20 It can be seen that wells 5 to 7 are negative.

[0133] Conclusion: The lowest qualitative detection level of anti-botulinum neurotoxin type A monoclonal antibody by ELISA is 10 ng / mL, and the ELISA results cannot provide a detailed analysis of the antibody.

[0134] Experimental Example 3: Using fingerprints to guide the detection of the quality and specificity of two strains (divided into No. 1 and No. 2) of anti-botulinum toxin type A complex antibody components and the identification and comparison of the two antibodies;

[0135] Experimental instruments and reagents: Instruments: Jess capillary immunoelectrophoresis instrument, reaction plate, reaction kit and ultrasensitive fluorescent anti-rabbit kit (secondary antibody) are all Protensimple brand;

[0136] Primary antibody: anti-botulinum toxin type A complex antibody No. 1 and anti-botulinum toxin type A complex antibody No. 2, with a sample concentration of 1 μg / μL.

[0137] After the sample addition, the reaction plate was centrifuged at 4000 rpm for 5 min, the reaction gel provided with the kit was added, and the plate was placed in a capillary immunoelectrophoresis instrument to start the reaction.

[0138] The experimental results can be found in Figure 21-24 , antibody No. 1 produces 8 obvious infrared peaks, among which the 103kd peak is consistent with the spectrum Figure 7 The 101kd peak is similar to Fig.13 The 61kd peak is similar to Fig.11 The 56kd peak is the same as that of Fig.11 The 47kd peak is the same as Fig.13 The 36kd peak is the same as Fig.15 The 24kd peak is the same as the 27kd peak of the spectrum. No obvious near-infrared peak appears.

[0139] Antibody No. 2 produced 9 obvious infrared peaks, of which the 147kd peak and the 118kd peak were consistent with Spectrum 3, and the 98kd peak was consistent with Figure 2 The 75 kd peak is consistent with Figure 7 The 64kd peak is consistent with Fig.13 The 58kd peak is consistent with Fig.11 The 38kd peak is consistent with Fig.13 The 24 kd peak is consistent with Fig.15 Two near-infrared peaks were detected, and the 37kd peak was consistent with Fig.12 The 184kd peak is consistent with Figure 6 Match.

[0140] in conclusion:

[0141] Antibody No. 1 includes anti-botulinum neurotoxin type A heavy chain antibody, anti-botulinum toxin type A hemagglutinin 70 antibody, anti-botulinum toxin type A hemagglutinin 33 antibody, and anti-botulinum toxin type A hemagglutinin 70 fragment antibody. Among them, anti-botulinum toxin type A hemagglutinin 70 antibody, anti-botulinum toxin type A hemagglutinin 33 antibody, and anti-botulinum toxin type A hemagglutinin 70 fragment antibody account for a relatively high and balanced proportion, while anti-botulinum neurotoxin type A heavy chain antibody accounts for a slightly lower proportion. This polyclonal antibody is mainly composed of hemagglutinin antibodies, and only one component is an impurity compared with the spectrum comparison.

[0142] Antibody No. 2 includes anti-botulinum neurotoxin type A antibody, anti-botulinum neurotoxin type A heavy chain antibody, anti-botulinum toxin type A hemagglutinin 70 antibody, anti-botulinum toxin type A hemagglutinin 33 antibody, and anti-botulinum toxin type A hemagglutinin 70 fragment antibody. Among them, hemagglutinin 33 antibody accounts for the highest proportion in the infrared image, and the near-infrared image detects higher signals of anti-botulinum toxin type A hemagglutinin 33 antibody and anti-botulinum neurotoxin type A heavy chain antibody, indicating that this strain of polyclonal antibodies is a polyclonal antibody with anti-botulinum toxin type A hemagglutinin 33 antibody as the main component and anti-botulinum neurotoxin type A heavy chain antibody as the auxiliary component.

[0143] ELISA reaction with the same antigen and antibody can only conclude that the antibody reaction is positive, and the above analysis cannot be made. The amount of signal generated by fluorescent capillary immunoassay antibodies is greater than that generated by chemiluminescent capillary immunoassay antibodies, so the ability to distinguish antibodies is stronger and suitable for quality control of antibodies from different sources or batches, provided that they are guided by fingerprints.

Claims

1. A method for constructing an anti-botulinum toxin antibody capillary immunoelectrophoresis fluorescence 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, the botulinum neurotoxin is subjected to capillary immunoelectrophoresis reaction to obtain a near-infrared light source spectrum and an infrared light source spectrum 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 a fluorescent antibody corresponding to the primary antibody; Capillary immunoelectrophoresis is performed on each subunit of the botulinum toxin complex to obtain a near-infrared light source spectrum and an infrared light source spectrum 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 on 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 a fluorescent 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 fluorescent 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 fluorescent 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 a fluorescent 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 near-infrared light source spectrum and infrared light source spectrum of the antibody to be tested; The near-infrared light source spectrum and infrared light source spectrum of the antibody to be tested are compared with the near-infrared light source spectrum and infrared light source spectrum obtained by the construction method of any one of claims 1 to 6 to obtain a 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 infrared light source spectrum of hemagglutinin 70, the near-infrared light source spectrum of hemagglutinin 70, the infrared light source spectrum of hemagglutinin 33, the near-infrared light source spectrum of hemagglutinin 33, the infrared light source spectrum of hemagglutinin 70 fragment or the near-infrared light source spectrum of hemagglutinin 70 fragment obtained are used to detect the comparison of antibodies against botulinum toxin type B, C, D, E and F complexes.

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