Group of specific antibodies for detecting botulinum toxin A and application thereof

Abstract

The invention provides a group of specific antibodies for detecting botulinum toxin A and application thereof, and belongs to the technical field of botulinum toxin detection. The invention provides a group of specific antibodies for detecting botulinum toxin A or antigen binding fragments thereof. The specific antibodies or the antigen binding fragments thereof comprise a rabbit monoclonal antibody 1F11 or an antigen binding fragment thereof and a rabbit monoclonal antibody 2A10 or an antigen binding fragment thereof. The time-resolved immunodetection kit prepared by the invention has good detection sensitivity, specificity, repeatability and sample matrix tolerance, the detection range is 0.02 ng / mL-10 ng / mL, the detection limit is 0.02 ng / mL, cross reaction with other serotype botulinum toxins (B, E and F) does not exist, and the time-resolved immunodetection kit is not influenced by sample matrixes such as ham sausages and thick broad-bean sauce. The detection system established by the invention has a good application prospect in detection of botulinum toxin type A.

Description

Technical Field

[0001] The present invention relates to the technical field of botulinum toxin detection, and in particular to a group of specific antibodies for detecting botulinum toxin type A and applications thereof. Background Art

[0002] Botulinum is a Gram-positive, spore-forming, anaerobic bacillus that produces an inactive 150kD polypeptide, botulinum toxin (BoNT), during its reproduction. This polypeptide is then cleaved by proteases into a 50kD light chain and a 100kD heavy chain, linked by disulfide bonds to form an active two-chain molecule. BoNT can act as a pathogenic agent by enzymatically damaging proteins essential for neuronal vesicle fusion, thereby blocking acetylcholine release.

[0003] BoNT can be divided into seven serotypes (A to G) and over 40 subtypes. An eighth serotype (H) has been discovered, with serotype A being the most toxic. Therefore, rapid and highly sensitive detection of botulinum toxin is essential.

[0004] With the development of detection technology, there are currently many detection methods for BoNT, including animal-based in vivo assays, ELISA, PCR, cell-based assays, flow cytometry, mass spectrometry, and immunochromatography. Among them, immune methods such as ELISA and immunochromatography are easy to operate but require highly sensitive antibody pairs. Therefore, screening out highly sensitive antibody pairs for BoNT is very critical.

[0005] In 1975, molecular biologists Kohler and Milstein identified hybridoma cells that secrete specific antibodies, marking the advent of the monoclonal antibody era. With the advancement of the times and the rapid development of science and technology, monoclonal antibody production technologies such as hybridoma technology, phage display technology, and single B cell technology have emerged, making monoclonal antibody production faster and more accurate. Among them, the recently developed single B cell technology shortens experimental cycles while maintaining high specificity for its target, and therefore has been widely used.

[0006] Compared to mouse monoclonal antibodies, rabbit monoclonal antibodies have higher affinity. Currently, there are no studies using single B cell technology to generate rabbit monoclonal antibodies against BoNT / A. Therefore, the present invention discloses a pair of rabbit monoclonal antibodies against botulinum toxin type A and applies this antibody pair to a time-resolved immunochromatographic assay, hoping to provide a new method for the accurate and rapid detection of botulinum toxin type A. Summary of the Invention

[0007] The object of the present invention is to provide a group of specific antibodies for detecting botulinum toxin type A and their applications.

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

[0009] The present invention provides a group of specific antibodies or antigen-binding fragments thereof for detecting botulinum toxin type A, wherein the specific antibodies or antigen-binding fragments thereof include rabbit monoclonal antibody 1F11 or an antigen-binding fragment thereof, rabbit monoclonal antibody 2A10 or an antigen-binding fragment thereof;

[0010] The rabbit monoclonal antibody 1F11 or its antigen-binding fragment comprises heavy chain CDR1 to CDR3 as shown in amino acid sequences of SEQ ID NO.5 to SEQ ID NO.7 and light chain CDR1 to CDR3 as shown in amino acid sequences of SEQ ID NO.8 to SEQ ID NO.10;

[0011] The rabbit monoclonal antibody 2A10 or its antigen-binding fragment comprises heavy chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.11 to SEQ ID NO.13 and light chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.14 to SEQ ID NO.16.

[0012] Preferably, the amino acid sequence of the heavy chain variable region of the rabbit monoclonal antibody 1F11 or its antigen-binding fragment is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.2;

[0013] The amino acid sequence of the heavy chain variable region of the rabbit monoclonal antibody 2A10 or its antigen-binding fragment is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0014] SEQ ID NO.1: QSVEESGGRLVTPGGSLTLTCTVSGIDLSSNAMSWVRQAPGEGLEWIGIISGAD ATYYATWATGRFTISKTSSTTVDLKATSLTTEDTATYFCARDVSGSYYSLDIWGPGTLVTVSS

[0015] CDR1: SNAMS (SEQ ID NO.5); CDR2: IISGADATYYATWAT (SEQ ID NO.6); CDR3: DVSGSYYSLDI (SEQ ID NO.7)

[0016] SEQ ID NO.2:AYDMTQTPASVDVAVGGTVTIKCQASENIYSSLAWYQQKPGQPPKLLIHGAST LASGVPSRFRGSGSGTEYSLTISGVQCADAATYYCQQGFTVTNVENAFGGGTEVVVK

[0017] CDR1: QASENIYSSLA (SEQ ID NO.8); CDR2: GASTLAS (SEQ ID NO.9); CDR3: QQGFTVTNVENA (SEQ ID NO.10)

[0018] SEQ ID NO.3: QSLEESGGDLVKPGASLTLTCKASGFSFSSSYYMCWVRQAPGKGLEWIACIYG GGSGGTYYASWAIGRFTISKTSSTTVTLELTSLTAADTATYFCGRSVAVSRSWPLNLWGPGTLVTVSSCDR1:SSYYMC(SEQ ID NO.11); CDR2:CIYGGGSGGTYYASWAIG(SEQ ID NO.12); CDR3:SVAVSRSWPLNL(SEQ IDNO.13)

[0019] SEQ ID NO.4: AFELTQTPSSVEAAVGGTVTIKCQASQSISDYLAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTEFTLTISDLECADAATYYCQTYYDSSSTTFGGGTEVVVK

[0020] CDR1: QASQSISDYLA (SEQ ID NO.14); CDR2: DASDLAS (SEQ ID NO.15); CDR3: QTYYDSSSTT (SEQ ID NO.16)

[0021] The present invention provides a related biomaterial of the specific antibody or antigen-binding fragment thereof, wherein the related biomaterial is any one of the following:

[0022] (a) a nucleic acid molecule encoding the specific antibody or antigen-binding fragment thereof;

[0023] (b) nucleic acid molecules encoding the heavy and light chains of the specific antibody or antigen-binding fragment thereof;

[0024] (c) nucleic acid molecules encoding the heavy chain variable region and light chain variable region of the specific antibody or antigen-binding fragment thereof;

[0025] (d) an expression cassette containing the nucleic acid molecule of any one of (a), (b), and (c);

[0026] (e) a recombinant vector containing the nucleic acid molecule of any one of (a), (b), and (c), or a recombinant vector containing the expression cassette of (d);

[0027] (f) A recombinant cell containing the nucleic acid molecule of any one of (a), (b), and (c), or a recombinant cell containing the expression cassette of (d), or a recombinant cell containing the recombinant vector of (e).

[0028] The present invention provides a use of the specific antibody or antigen-binding fragment thereof or the related biological material in preparing a product for detecting botulinum toxin type A.

[0029] Preferably, the product comprises a reagent, a kit or a test strip.

[0030] The present invention provides a test strip for detecting botulinum toxin type A, comprising a PVC base, a nitrocellulose membrane, a fluorescent pad, a sample pad, and a water-absorbing pad; the nitrocellulose membrane contains a quality control line and a test line, the test line is coated with rabbit monoclonal antibody 1F11; the fluorescent pad is coated with rabbit monoclonal antibody 2A10 labeled with time-resolved microspheres;

[0031] The rabbit monoclonal antibody 1F11 comprises heavy chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.5 to SEQ ID NO.7 and light chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.8 to SEQ ID NO.10;

[0032] The rabbit monoclonal antibody 2A10 includes heavy chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.11 to SEQ ID NO.13 and light chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.14 to SEQ ID NO.16.

[0033] Preferably, the nitrocellulose membrane is fixed on a PVC base plate, the fluorescent pad and the absorbent pad are respectively overlapped at both ends of the nitrocellulose membrane and overlapped by 1 to 2 mm, and the sample pad is overlapped at the other end of the fluorescent pad and overlapped by 1 to 2 mm;

[0034] The quality control line is coated with protein A, and the concentration of the protein A is 0.8-1.2 mg / mL; the detection line is coated with rabbit monoclonal antibody 1F11 at a concentration of 0.8-1.2 mg / mL.

[0035] Preferably, the mass ratio of the time-resolved microspheres to the rabbit monoclonal antibody 2A10 is 400-600:40-60; the rabbit monoclonal antibody 2A10 labeled with the time-resolved microspheres and the time-resolved microsphere storage solution are diluted in a volume ratio of 0.8-1.2:3.5-4.5 and then coated on a fluorescent pad.

[0036] Preferably, the amino acid sequence of the heavy chain variable region of the rabbit monoclonal antibody 1F11 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2;

[0037] The amino acid sequence of the heavy chain variable region of the rabbit monoclonal antibody 2A10 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0038] The present invention provides a kit for detecting botulinum toxin type A, wherein the kit contains the test strip.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention provides a pair of specific antibodies for detecting botulinum toxin type A, including rabbit monoclonal antibody 1F11 and rabbit monoclonal antibody 2A10. The present invention utilizes rabbit monoclonal antibody 1F11 as the coating antibody to coat NC membranes, and rabbit monoclonal antibody 2A10 as the detection antibody to label time-resolved immunofluorescent microspheres, thereby establishing a rapid time-resolved immunochromatographic detection method capable of rapidly and accurately detecting botulinum toxin type A. This method combines the high specificity of high-affinity rabbit monoclonal antibodies with the high sensitivity of time-resolved immunofluorescent microspheres, combined with the convenient and rapid detection of immunochromatographic reagent strips. The resulting detection method is accurate, rapid, specific, and sensitive, making it suitable for on-site rapid testing of food hygiene inspections and patient clinical samples. It is of great significance for ensuring food safety and providing timely and accurate guidance for drug use.

[0041] The time-resolved immunoassay kit prepared in this invention exhibits excellent sensitivity, specificity, reproducibility, and sample matrix tolerance. Its detection range is 0.02 ng / mL to 10 ng / mL, with a detection limit of 0.02 ng / mL. It exhibits no cross-reactivity with other serotypes of botulinum toxin (B, E, and F) and is unaffected by sample matrices such as ham and broad bean paste. This detection system has promising application prospects in the detection of botulinum toxin type A. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] 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 merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0043] Figure 1 The recombinant vector of the heavy chain variable region of the rabbit monoclonal antibody 1F11 in Example 1;

[0044] Figure 2 The recombinant vector of the light chain variable region of the rabbit monoclonal antibody 1F11 in Example 1;

[0045] Figure 3 The recombinant vector of the heavy chain variable region of the rabbit monoclonal antibody 2A10 in Example 1;

[0046] Figure 4 The recombinant vector of the light chain variable region of the rabbit monoclonal antibody 2A10 in Example 1;

[0047] Figure 5 The titer test results of the rabbit monoclonal antibody 1F11 in Example 1 are as follows;

[0048] Figure 6 The titer test results of the rabbit monoclonal antibody 2A10 in Example 1 are as follows;

[0049] Figure 7 The SDS-PAGE electrophoresis diagram of the rabbit monoclonal antibody 1F11 and the rabbit monoclonal antibody 2A10 in Example 1;

[0050] Figure 8 The results of affinity determination of rabbit monoclonal antibody 1F11 and rabbit monoclonal antibody 2A10 in Example 1 are shown;

[0051] Figure 9 The detection range of the time-resolved immunoassay kit in Example 3;

[0052] Figure 10 The specific detection results of the time-resolved immunoassay kit in Example 3;

[0053] Figure 11 These are the results of detecting the ham and bean paste sample matrices using the time-resolved immunoassay kit in Example 3. DETAILED DESCRIPTION

[0054] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1 Preparation and Identification of Botulinum Toxin A Rabbit Monoclonal Antibody

[0056] 1. Expression of rabbit monoclonal antibody against botulinum toxin A

[0057] The nucleotide sequences of the heavy and light chain variable regions (VH and VL, SEQ ID NO. 1 to SEQ ID NO. 4) of rabbit monoclonal antibody 1F11 and rabbit monoclonal antibody 2A10 were inserted into a eukaryotic vector (pCDNA3.4 plasmid backbone, the antibody variable regions were connected between the plasmid constant regions and the IL-2 signal peptide by homologous recombination) containing the IgG antibody heavy chain constant region and the kappa light chain constant region, and the obtained vectors were as follows: Figures 1 to 4 The recombinant vector shown was used to transform Trans1-T1 competent cells (Beijing Quanshijin Biotechnology Co., Ltd.), and the positive monoclonal clones were picked from plates smeared with ampicillin (100 μg / mL). The cells were cultured by shaking, and the antibody plasmid was extracted using an endotoxin-free plasmid extraction kit (Beijing Quanshijin Biotechnology Co., Ltd.). The cells were transfected into Expi293F cells (Thermo Fisher Scientific), cultured for 1 week, centrifuged at 8000 rpm for 10 min, and the supernatant was collected.

[0058] The nucleotide sequence of the heavy chain variable region of the rabbit monoclonal antibody 1F11 is shown in SEQ ID NO. 17, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO. 18. The nucleotide sequence of the heavy chain variable region of the rabbit monoclonal antibody 2A10 is shown in SEQ ID NO. 19, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO. 20. The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 21, and the nucleotide sequence is shown in SEQ ID NO. 22; the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 23, and the nucleotide sequence is shown in SEQ ID NO. 24.

[0059] 2. Determination of Antibody Titer in Cell Supernatant

[0060] Antibody titer was assessed using ELISA. BoNT / A recombinant antigen (amino acid sequence shown in SEQ ID NO. 25) was diluted to 1 μg / mL using 1× coating buffer (Beijing Solaibao Technology Co., Ltd.). The diluted antigen was added to an ELISA strip at a volume of 100 μL / well and coated overnight at 4°C. The plate was washed three times with PBST (phosphate-buffered saline-Tween) at a volume of 200 μL / well. The plate was patted dry and blocking buffer (3% BSA) was added at a volume of 150 μL / well. The plate was blocked at 37°C for 2 h. The blocking buffer was discarded and the plate was patted dry. The cell supernatant collected above was diluted 1000-fold, 10,000-fold, and 100,000-fold with PBST, respectively. The diluted supernatant was added to the ELISA strip. The dilutions served as blank controls at a volume of 100 μL / well. The plate was incubated at 37°C for 30 min. The plate was washed three times with PBST at a volume of 200 μL / well. Dilute the goat anti-rabbit HRP antibody (Beijing Quanshijin Biotechnology Co., Ltd.) 8000 times with PBST, add the diluted goat anti-rabbit HRP antibody to the enzyme-linked strip, 100 μL / well, and incubate at 37°C for 15 minutes. Wash the plate 5 times with PBST, 200 μL / well / time. Add TMB (3,3',5,5'-tetramethylbenzidine) colorimetric solution to the enzyme-linked strip, 100 μL / well, react at room temperature in the dark for 10 minutes, stop the reaction with ELISA stop solution (Beijing Solebold Technology Co., Ltd.), 50 μL / well, and measure the absorbance at 450 nm. The average value of the blank control plus 3 times the standard deviation is the Cut off value. The antibody dilution factor with an OD value higher than the Cut off value is the antibody titer. The test results are as follows: Figure 5 and Figure 6 As shown, the titer of 1F11 antibody is 10 -5 , the titer of 2A10 antibody is 10 -4 .

[0061] 3. Purification of Botulinum toxin A rabbit monoclonal antibody

[0062] Antibodies were purified using an AKTA protein purifier. Column A was equilibrated with 20 mM PB (phosphate buffer), 0.15 M sodium chloride, pH 7.4. The collected cell supernatant was bound to the column and eluted with 20 mM sodium acetate (pH 3.5) after equilibration. After elution, the pH of the antibody solution was adjusted to 7.0 with a neutralizing solution (1 M Tris-HCl, pH 9.0). The collected antibodies were subjected to SDS-PAGE electrophoresis, and the purified antibodies were placed in PBS, dialyzed overnight at 4 ° C, and quantified using a nucleic acid protein quantifier (NanoDrop 2000). The SDS-PAGE electrophoresis is shown in the figure below. Figure 7 shown.

[0063] 4. Determination of affinity of rabbit monoclonal antibody to botulinum toxin A

[0064] Antibody affinity was determined using biolayer interferometry (BLI) experiments, and data analysis was performed using Gator software. The purified antibodies were captured by Protein A biosensors and then immersed in wells containing 100nM BoNT / A recombinant antigen to evaluate the association rate (association rate constant, kon), and then immersed in wells containing only binding buffer (PBS buffer containing 0.02% Tween 20 and 0.2% bovine serum albumin) to evaluate the dissociation rate (dissociation rate constant, koff). The binding affinity constant (dissociation constant, KD) was calculated as koff / kon. The KD value of the 1F11 antibody was 7.26E-10 and the KD value of the 2A10 antibody was <1E-12 ( Figure 8 ).

[0065] Example 2 Establishment of time-resolved immunochromatographic detection method

[0066] 1. Conjugation of 2A10 antibody to time-resolved fluorescent microspheres

[0067] 500 μg of time-resolved fluorescent microspheres (Suzhou Weidu Biotechnology Co., Ltd.) were mixed and diluted with 1 mL of 50 mM MES buffer (pH 6.0). 35 μg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 350 μg of NHS (N-hydroxysuccinimide) were added to the diluted microspheres and incubated at room temperature for 20 minutes. After the incubation was completed, the suspension was centrifuged at 20,000 g for 15 minutes, and the supernatant was discarded. The precipitate was washed twice with 1 mL of 50 mM MES buffer (pH 6.0). The suspension was homogenized by ultrasound until it became clear and free of visible particles. Subsequently, 50 μg of botulinum toxin A rabbit monoclonal antibody 2A10 was added to the microsphere suspension to obtain an antibody-microsphere mixture. The antibody-microsphere mixture was incubated at room temperature for 2 hours. To block unbound sites on the microparticles, 25 μL of a 20% (w / v) bovine serum albumin (BSA) solution (dissolved in 50 mM Tris-HCl) was added to the suspension. The blocking reaction was incubated at room temperature for 1 hour. The microsphere-bound antibody was then washed twice with 50 mM Tris-HCl (pH 8.0) and resuspended in 500 μL of 50 mM Tris-HCl (pH 8.0) to obtain time-resolved fluorescent microspheres labeled with the 2A10 antibody. The suspension was stored at 4°C until use in subsequent experiments.

[0068] 2. Preparation of time-resolved immunoassay kit

[0069] The kit consists of 5 parts: a PVC base plate, a nitrocellulose (NC) membrane, a fluorescent pad, a sample pad and an absorbent pad.

[0070] Protein A (SPA) and rabbit monoclonal antibody 1F11 against botulinum toxin A were diluted to 1 mg / mL in PBS (1×, pH 7.4). 1 mg / mL of SPA was applied to the control line (line C) of the NC membrane, and 1 mg / mL of 1F11 antibody was applied to the test line (line T) of the NC membrane.

[0071] The 2A10 antibody labeled with the time-resolved fluorescent microspheres prepared above was diluted with the time-resolved microsphere storage solution (50 mM Tris-HCl, 1% BSA, 5% sucrose, pH 8.0) at a volume ratio of 1:4 and evenly coated on the fluorescent pad, and then dried in an oven for 30 minutes.

[0072] A nitrocellulose membrane (NC membrane) was fixed in the middle of a PVC baseplate. A fluorescent pad was attached to the lower end of the NC membrane, overlapping it by 1.5 mm. A sample pad was attached to the lower end of the fluorescent pad, overlapping it by 1.5 mm. An absorbent pad was attached to the upper end of the NC membrane, also overlapping it by 1.5 mm. The membranes were tightly fitted to avoid air bubbles or gaps, forming a continuous chromatography path. This ensured that the sample liquid could flow smoothly from the sample pad, through the fluorescent pad and NC membrane, to the absorbent pad, completing the chromatography process. After attachment, the membranes were cut into 3.6 mm wide strips and packaged in a reagent card to obtain a time-resolved immunoassay kit.

[0073] 3. How to use the time-resolved immunoassay kit

[0074] Add 100 μL of the sample to be tested to the sample well of the kit and let it stand for 15 minutes. Place the kit in the fluorescent immunoassay analyzer, detect the fluorescence intensity of the T line and C line, and calculate the T / C value. The T / C value is proportional to the antigen concentration. Draw a standard curve based on the antigen concentration and T / C value to perform quantitative analysis on the test sample.

[0075] Example 3 Performance Test of Time-Resolved Immunoassay Kit

[0076] 1. Sensitivity and detection range

[0077] 10 ng / mL BoNT / A recombinant antigen (amino acid sequence shown in SEQ ID NO. 25) was serially diluted 2-fold in a buffer solution (50 mM PBS, 2% BSA, 0.1% Tween-20, pH 7.4) to evaluate the sensitivity of the assay. A standard curve was drawn using a four-parameter fit of concentration and signal values. The results showed that the limit of detection (LOD) of the time-resolved immunoassay kit for botulinum toxin A was 0.02 ng / mL, and the linear range was 0.02 ng / mL to 10 ng / mL. Figure 9 ).

[0078] 2. Specificity

[0079] The recombinant antigens of BoNT / A (amino acid sequence as shown in SEQ ID NO.25), BoNT / B (amino acid sequence as shown in SEQ ID NO.26), BoNT / E (amino acid sequence as shown in SEQ ID NO.27) and BoNT / F (amino acid sequence as shown in SEQ ID NO.28) were diluted to 100 ng / mL, 50 ng / mL and 10 ng / mL respectively using a buffer solution (50 mM PBS, 2% BSA, 0.1% Tween-20, pH 7.4). The dilutions were used as blank controls (0 ng / mL). 100 μL of botulinum toxin recombinant antigens of different concentrations were dripped onto the sample wells of the kit and allowed to stand for 15 minutes. The kit was placed in a fluorescence immunoassay instrument to detect the fluorescence intensity of the T line and C line, and the T / C value was calculated. The cut-off value is the average value of the blank control plus 3 times the standard deviation. The time-resolved immunoassay method for BoNT / A does not have any cross-reaction with the other three botulinum toxins ( Figure 10 ), indicating that the time-resolved immunoassay kit prepared by the present invention has excellent specificity.

[0080] 3. Repeatability

[0081] BoNT / A recombinant antigen was diluted to 0.1, 1, and 10 ng / mL in a buffer solution (50 mM PBS, 2% BSA, 0.1% Tween-20, pH 7.4). 100 μL of each concentration of botulinum toxin recombinant antigen was dripped onto the sample wells of the kit. The solution was allowed to stand for 15 minutes. The kit was then placed in a fluorescence immunoassay analyzer, and the fluorescence intensities of the T and C lines were measured, and the T / C ratio was calculated. The assay was repeated 12 times for each BoNT / A concentration. The results showed that the CV values of the T / C ratios for the time-resolved immunochromatographic assay for the three concentrations of botulinum toxin recombinant antigen were all <10% (Table 1), demonstrating the good reproducibility and uniformity of the assay.

[0082] Table 1 Repeatability test results

[0083] Concentration (ng / mL) Mean ± SD (n = 12) Coefficient of variation (CV%) 10 4.57±0.41 9.02 1 0.51±0.05 9.86 0.1 0.05±0.00 7.35

[0084] 4. Sample matrix tolerance

[0085] In order to evaluate the effect of different matrices on time-resolved immunochromatographic detection, ham sausage and bean paste were purchased from the supermarket, 0.4 g of each sample was weighed into a 1.5 mL EP tube, 1 mL of buffer (50 mM PBS, 2% BSA, 0.1% Tween-20, pH 7.4) was added, and homogenized at 70 Hz for 180 s. Centrifuged at 8000 rpm for 10 min, the supernatant was taken, and the preparation of 40% (w / v) samples of ham sausage and bean paste was completed. Buffer was added again and diluted to 10% and 20% (w / v), respectively. BoNT / A recombinant antigen was added to the 10% and 20% (w / v) samples to prepare simulation samples of different concentrations. The test results showed that ( Figure 11 ), 20% and 10% ham sausage and bean paste made of BoNT / A recombinant antigen simulation samples, the detection limit was 0.02 ng / mL, consistent with that in the buffer solution, indicating that the time-resolved immunochromatographic detection kit prepared with the high-affinity antibody pair 1F11 and 2A10 is not affected by sample matrices such as ham sausage and bean paste, has good matrix tolerance, and is suitable for the detection of a variety of samples.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A group of specific antibodies or antigen-binding fragments thereof for detecting botulinum toxin type A, characterized in that: The specific antibodies or antigen-binding fragments thereof include rabbit monoclonal antibody 1F11 or an antigen-binding fragment thereof, rabbit monoclonal antibody 2A10 or an antigen-binding fragment thereof; The rabbit monoclonal antibody 1F11 or its antigen-binding fragment comprises heavy chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.5 to SEQ ID NO.7 and light chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.8 to SEQ ID NO.10; The rabbit monoclonal antibody 2A10 or an antigen-binding fragment thereof comprises heavy chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.11 to SEQ ID NO.13 and light chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.14 to SEQ ID NO.

16.

2. The specific antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the rabbit monoclonal antibody 1F11 or its antigen-binding fragment is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; The amino acid sequence of the heavy chain variable region of the rabbit monoclonal antibody 2A10 or its antigen-binding fragment is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

3. A biological material related to the specific antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The relevant biological material is any one of the following: (a) a nucleic acid molecule encoding the specific antibody or antigen-binding fragment thereof according to claim 1 or 2; (b) a nucleic acid molecule encoding the heavy and light chains of the specific antibody or antigen-binding fragment thereof according to claim 1 or 2; (c) a nucleic acid molecule encoding the heavy chain variable region and light chain variable region of the specific antibody or antigen-binding fragment thereof according to claim 1 or 2; (d) an expression cassette containing the nucleic acid molecule of any one of (a), (b), and (c); (e) a recombinant vector containing the nucleic acid molecule of any one of (a), (b), and (c), or a recombinant vector containing the expression cassette of (d); (f) A recombinant cell containing the nucleic acid molecule of any one of (a), (b), and (c), or a recombinant cell containing the expression cassette of (d), or a recombinant cell containing the recombinant vector of (e).

4. Use of the specific antibody or antigen-binding fragment thereof according to claim 1 or 2, or the related biological material according to claim 3, in the preparation of a product for detecting botulinum toxin type A.

5. The use according to claim 4, characterized in that The products include reagents, test kits or test strips.

6. A test strip for detecting botulinum toxin type A, characterized in that: The test strip comprises a PVC base, a nitrocellulose membrane, a fluorescent pad, a sample pad and a water-absorbing pad; the nitrocellulose membrane contains a quality control line and a test line, and the test line is coated with a rabbit monoclonal antibody 1F11; the fluorescent pad is coated with a rabbit monoclonal antibody 2A10 labeled with a time-resolved microsphere; The rabbit monoclonal antibody 1F11 comprises heavy chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.5 to SEQ ID NO.7 and light chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.8 to SEQ ID NO.10; The rabbit monoclonal antibody 2A10 includes heavy chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.11 to SEQ ID NO.13 and light chain CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO.14 to SEQ ID NO.

16.

7. The test strip according to claim 6, wherein Fix the nitrocellulose membrane on the PVC base plate, overlap the fluorescent pad and the absorbent pad at both ends of the nitrocellulose membrane with 1-2 mm overlap, and overlap the sample pad at the other end of the fluorescent pad with 1-2 mm overlap; The quality control line is coated with protein A, and the concentration of the protein A is 0.8-1.2 mg / mL; the detection line is coated with rabbit monoclonal antibody 1F11 at a concentration of 0.8-1.2 mg / mL.

8. The test strip according to claim 6, wherein The mass ratio of the time-resolved microspheres to the rabbit monoclonal antibody 2A10 is 400-600:40-60; the rabbit monoclonal antibody 2A10 labeled with the time-resolved microspheres and the time-resolved microsphere storage solution are diluted in a volume ratio of 0.8-1.2:3.5-4.5 and then coated on a fluorescent pad.

9. The test strip according to claim 6, wherein The amino acid sequence of the heavy chain variable region of the rabbit monoclonal antibody 1F11 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; The amino acid sequence of the heavy chain variable region of the rabbit monoclonal antibody 2A10 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

10. A kit for detecting botulinum toxin type A, characterized in that: The kit contains the test strip according to any one of claims 6 to 9.

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