CN115032379A - Quick quantitative determination card of type B botulinum toxin - Google Patents

Quick quantitative determination card of type B botulinum toxin Download PDF

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CN115032379A
CN115032379A CN202210621717.2A CN202210621717A CN115032379A CN 115032379 A CN115032379 A CN 115032379A CN 202210621717 A CN202210621717 A CN 202210621717A CN 115032379 A CN115032379 A CN 115032379A
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botulinum toxin
sample
toxin type
pad
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高姗
李岩伟
白雪欣
吕若梅
康琳
王菁
胡宸艺
王景林
辛文文
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Academy of Military Medical Sciences AMMS of PLA
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Abstract

The invention discloses a rapid quantitative detection card for botulinum toxin type B, which comprises a PVC (polyvinyl chloride) bottom plate, a water absorption pad, a nitrocellulose membrane, a marker pad, a sample pad and a diluent pad which are arranged in a shell; the nitrocellulose membrane is fixed on a PVC base plate, a water absorption pad is lapped at the left end of the nitrocellulose membrane, a dilution pad is lapped at the right end of the nitrocellulose membrane, a marker pad is arranged at the middle part of the nitrocellulose membrane close to the right, a sample pad is arranged on the marker pad, and a quality control line and a detection line are arranged between the water absorption pad and the marker pad on the nitrocellulose membrane. The antibody uses a nano antibody designed aiming at the botulinum toxin B, so that the sensitivity and the precision of a detected object are improved, and the characteristics of rapidness and convenience of a quick diagnosis detection method are retained.

Description

Quick quantitative determination card of type B botulinum toxin
Technical Field
The invention relates to the field of biological detection, in particular to a rapid quantitative detection card for botulinum toxin B.
Background
Botulinum toxin (BoNT) is a protein neurotoxin produced by clostridium Botulinum, is a biological toxin which is known to be the most toxic at present, and according to the antigenicity difference of the toxin produced by clostridium Botulinum, A, B, C, D, E, F and seven types G are found, wherein A, B, E can cause botulism in human beings, and the type A, B is the most common. The current methods for botulinum toxin detection are mostly focused on botulinum toxin type A, and methods specific to botulinum toxin type B are rare.
At present, no specific detoxification medicine is available for the botulism B, and only symptomatic treatment can be adopted, or the treatment can be carried out by injecting corresponding antiserum after definite botulinum typing. Therefore, rapid, sensitive and accurate typing of toxin-contaminated samples is the key to preventive control and diagnosis and treatment of poisoned patients. The current detection methods aiming at the botulinum toxin type B mainly comprise: the ELISA method has the sensitivity reaching ng/ml, but is more suitable for laboratory detection rather than on-site rapid detection due to complex operation and long time consumption; the colloidal carbon detection method gets rid of the limitation of complex instruments, has low cost and simple operation, cannot carry out quantitative detection and has lower sensitivity. Therefore, the search for a new method for effectively detecting ricin in express is very important.
The lanthanide immunochromatographic test strip is a detection method established on the basis of immunochromatography by combining a time-resolved fluorescence immunoassay technology, and usually contains lanthanide (such as Eu) 3+ ，Tb 3+ Etc.) as a marker, and effectively eliminates the interference of non-specific background fluorescence by two resolution technologies of wavelength and time by utilizing the characteristics of long fluorescence life of lanthanide and large Stokes shift. Judging the object to be detected by the strength of the fluorescence signalThe concentration of the substance, thereby realizing accurate quantification. Has the advantages of high sensitivity, high stability, no environmental pollution, simple operation, etc.
Nanobodies are a new class of antibodies, first discovered in 1989 in the serum of dromedary camels, in which half of the antibodies naturally lack the first constant regions of the light and Heavy chains (CH1), and are therefore called Heavy chain antibodies (HCAbs). The variable region of heavy chain antibodies was cloned to obtain a single domain antibody consisting of only one heavy chain variable region, called VHH antibody (VHH), which has a small molecular weight of about 15 kda, which is only 1/10 for conventional antibodies, and is called Nanobody (Nb) because its crystal structure is ellipsoidal, 2.5nm in diameter and 4nm in length. The nano antibody has the characteristics of high affinity, strong specificity, easy expression, large-scale production and the like, and is widely concerned.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a test strip for rapidly and quantitatively detecting botulinum toxin type B by using a nano antibody.
The invention provides a test strip for detecting botulinum toxin type B, which comprises a sample pad, a nitrocellulose membrane, a marker pad, an NC membrane and a water absorption pad; the NC film is provided with a detection line and a quality control line; the marker pad is coated with a marker-labeled chicken IgY antibody and a marker-labeled B-type botulinum toxin nano antibody a; the detection line is coated with a B-type botulinum toxin nano antibody B, and the quality control line is coated with IgG of the goat anti-chicken IgY antibody; the botulinum toxin type B nano antibody a can be combined with botulinum toxin type B and can capture botulinum toxin type B nano antibody B.
The label is at least one of colloidal gold, fluorescein, lanthanide fluorescent microspheres, colored latex, quantum dots, up-conversion fluorescent substances, magnetic fluorescent microspheres, enzyme labels or chemiluminescent substances.
Wherein the marker is lanthanide fluorescent microspheres.
Wherein the sequence of the botulinum toxin type B nano antibody a is QVQLAESGGGLVQSGGSLRLSCAASGSIDSLYHMGWYRQAPGKERELVARVQDGGSTAYKDSVKGRFTISRDFSRSTMYLQMNSLKPEDTAIYYCAAKSTISTPLSWGQGTQVTVSS, and is shown as sequence 1 in the sequence table; the sequence of the botulinum toxin type B nano antibody B is QVQLAESGGGLVQPGGSLRLSCAASILTYDLDYYYIGWVRQAPGKEREGVSCISSTDGATYYADSVKGRFTISRNNAKNTVYLQMNNLKPEDTAIYYCAAAPLAGRYCPASHEYGYWGQGTQVTVSS, and is shown as a sequence 2 in a sequence table.
The kit comprises a sample pad, a nitrocellulose membrane, a marker pad, an NC membrane and a water absorption pad, wherein the sample pad, the nitrocellulose membrane, the marker pad, the NC membrane and the water absorption pad are sequentially fixed on the base plate.
The application of the test strip in detecting the botulinum toxin type B or detecting the content of the botulinum toxin type B in a sample to be detected also falls within the protection scope of the present invention.
The application of the test strip in the preparation of products for detecting botulinum toxin type B or detecting the content of botulinum toxin type B in samples to be detected also falls within the protection scope of the invention.
The invention provides a method for detecting the content of botulinum toxin type B in a sample to be detected, which comprises the following steps: detecting a sample to be detected and a negative control sample by using the test strip to respectively obtain the fluorescence signal ratio of the sample to be detected and the negative control sample in the detection area and the quality control area of the test strip, wherein if the fluorescence signal ratio of the sample to be detected in the detection area and the quality control area of the test strip is more than or equal to 2.1 times of the fluorescence signal ratio of the negative control sample in the detection area and the quality control area of the test strip, the sample to be detected contains botulinum toxin type B; if the ratio of the fluorescence signals of the sample to be detected in the detection area and the quality control area of the test paper is less than 2.1 times of the ratio of the fluorescence signals of the negative control sample in the detection area and the quality control area of the test paper, the sample to be detected does not contain the botulinum toxin B.
The invention provides a method for detecting the content of botulinum toxin type B in a sample to be detected, which comprises the following steps:
(1) detecting the botulinum toxin type B standard substance by using the test strip to obtain the fluorescence signal ratio of the standard substance in the detection area and the quality control area of the test strip, making a unary linear regression curve of the fluorescence signal ratio corresponding to the concentration of the botulinum toxin type B standard substance, and calculating a regression equation;
(2) replacing the standard substance of the botulinum toxin type B with a sample to be detected, detecting the sample to be detected by using the test paper to obtain the ratio of fluorescence signals of the sample to be detected in a detection area and a quality control area of the test paper, and obtaining the content of the botulinum toxin type B in the sample to be detected according to the regression equation in the step (1).
The test strip and the detection method using the test strip provided by the invention have the advantages that a higher minimum detection limit is obtained in the detection of biotoxin, and the test strip is matched with a matched small portable detection instrument for use, so that the test strip has the advantages of high detection speed and capability of remotely transmitting a result. The high-sensitivity fluorescence detection method for the botulinum toxin type B has the advantages of high sensitivity, miniaturization of a detection instrument and low cost, and can solve the defects of the prior art. The method has important significance for a plurality of fields such as emergent public health incidents, food safety and the like, and provides a quick, sensitive and accurate detection method for multi-environment field detection.
Drawings
Fig. 1 is a schematic structural diagram of the test strip of the present invention.
FIG. 2 is a standard curve for botulinum toxin type B.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The chicken IgY in the following examples was purchased from Hangzhou Qitai Biotech, Inc.; IgG of goat anti-chicken IgY was purchased from Kitty Biotech, Hangzhou; two nanobodies a and B of Botulinum toxin type B can be prepared according to the methods in Lam K, Tremblay J M, Vazzez-Cintron E, Perry K, Ondeck C, Webb R P, Mcnutt P M, Shoemaker C B, Jin R.2020.structural antibodies in random Design of Single-Domain Antibody-Based antibodies against infection Bourninum Neurotoxins [ J ]. Cell structures, 30(8):2526-2539. Lanthanide fluorescent microspheres were purchased from kyemitting microbiology co. The detection instrument was purchased from Douglas Biotech, Inc.
EXAMPLE 1 preparation of botulinum toxin type B test card
Preparation of botulinum toxin type B nanobody a
The sequence of the botulinum toxin B nano antibody a is designed to be QVQLAESGGGLVQSGGSLRLSCAASGSIDSLYHMGWYRQAPGKERELVARVQDGGSTAYKDSVKGRFTISRDFSRSTMYLQMNSLKPEDTAIYYCAAKSTISTPLSWGQGTQVTVSS, and is shown as the sequence 1 in the sequence table. The sequence 1 is designed and constructed on a plasmid pET-28a, a His label is contained on the designed plasmid, and the plasmid is optimized and synthesized by Nanjing Kingsler biology company. Then the plasmid is transferred into competent cells E
Figure BDA0003677012490000041
T7-K12 (Midbo Bio Inc., Beijing, China), activated and plated overnight. And selecting single colonies on the plate to continue culturing, sequencing and comparing. Colonies with the correct sequence alignment were selected and expanded to 1L of bacterial suspension, IPTG was added at OD600 of 0.4-0.8 to a final concentration of 0.4mM, 16 ℃ and 180r/min, and induction was carried out overnight. The supernatant was discarded by centrifugation, sonicated, and HisTrap was used TM And (3) an HP column (GE company in the United states) and finally obtaining the high-purity botulinum toxin type B nano antibody a by imidazole gradient elution purification, wherein the amino acid calculation sequence of the botulinum toxin type B nano antibody a is shown as a sequence 1.
Preparation of botulinum toxin type B Nanobody B
The sequence of the botulinum toxin B nano antibody B is QVQLAESGGGLVQPGGSLRLSCAASILTYDLDYYYIGWVRQAPGKEREGVSCISSTDGATYYADSVKGRFTISRNNAKNTVYLQMNNLKPEDTAIYYCAAAPLAGRYCPASHEYGYWGQGTQVTVSS, and is shown as a sequence 2 in a sequence table. The sequence 2 is designed and constructed on a plasmid pET-28a, a His label is contained on the designed plasmid, and the plasmid is optimized and synthesized by Nanjing Kingsler biology company. Coli, then the plasmid was transferred into competent cells e
Figure BDA0003677012490000042
T7-K12 (Midbo Bio Inc., Beijing, China), activated and plated overnight. And selecting single colonies on the plate to continue culturing, sequencing and comparing. Colonies with the correct sequence alignment were selected and expanded to 1L of bacterial suspension, IPTG was added at OD600 of 0.4-0.8 to a final concentration of 0.4mM, 16 ℃ and 180r/min, and induction was carried out overnight. The supernatant was discarded by centrifugation, sonicated, and HisTrap was used TM And (3) an HP column (GE company in the United states) and finally obtaining the high-purity botulinum toxin type B nano antibody B through imidazole gradient elution and purification, wherein the amino acid calculation sequence of the botulinum toxin type B nano antibody B is shown as a sequence 2.
Preparation of test paper strip for rapid quantitative detection of B-type botulinum toxin
1) Spraying IgG diluted to 0.3mg/ml of goat anti-chicken IgY on an NC membrane by adopting a special machine for spotting and gold spraying to form a quality control line (C line) and a B type botulinum toxin nano antibody B diluted to 2mg/ml to form a detection line (T line), wherein the spraying amount is 1 mu l/cm, and then baking for 16 hours at the temperature of 37 ℃;
2) preparing lanthanide fluorescent microspheres marked with chicken IgY and lanthanide fluorescent microspheres marked with botulinum toxin type B nano-antibody a, adding 1mL of lanthanide fluorescent microspheres into 5mL of MES (2- (N-morpholine) ethanesulfonic acid) buffer (0.05M, pH7.2), adding 10mg of carbodiimide (EDC) and 10mg of N-hydroxysuccinimide sulfonic acid sodium salt, stirring and dissolving, reacting at room temperature for 30 minutes, centrifuging, redissolving the centrifugal precipitate with 50mM boric acid buffer (pH8.2), adding 2mg of dialyzed chicken IgY, stirring and reacting at room temperature for 24 hours, centrifuging, sealing, and storing in a diluent (the storage environment temperature is 2-8 ℃), thus obtaining the lanthanide fluorescent microspheres marked with chicken IgY; marking lanthanide series fluorescent microspheres of the botulinum toxin type B nano antibody a by the same method;
3) respectively diluting lanthanide series fluorescent microspheres marked with chicken IgY and B type botulinum toxin nano antibody B to the concentrations of 0.1 mu g/ml and 4 mu g/ml, mixing, spraying the lanthanide series fluorescent microspheres on a polyester film by adopting a spot film gold spraying machine to form a marker pad, wherein the spraying amount is 2.5 mu l/cm, and then baking for 8 hours at the temperature of 37 ℃;
4) cutting the marker pad into 8mm long, cutting the sample pad into 8mm long, overlapping and sticking together by double-sided adhesive tape, cutting into 4mm wide sample adding strips by a continuous cutting machine, and assembling the strips under the sample adding hole of the upper cover of the shell of the detection card;
5) and correspondingly adhering a diluting pad with the length of 305mm and the width of 12mm and a water absorption pad with the length of 305mm and the width of 17mm to two ends of a nitrocellulose membrane on a PVC (polyvinyl chloride) base plate to form a large card, cutting the large card into test strips with the width of 4.15mm by using a continuous cutting machine, assembling the test strips in a lower cover of a shell of the detection card, and buckling an upper cover and a lower cover of the detection card together to obtain the botulinum toxin type B detection card.
When the detection is carried out, 100 mu L of sample is added into a sample adding hole, the sample is kept still for 15 minutes, and finally the result is interpreted by a fluorescence analysis instrument.
The test strip for rapidly and quantitatively detecting the botulinum toxin type B prepared by the method is shown in figure 1, and comprises a PVC (polyvinyl chloride) bottom plate 1, a sample pad 2, a nitrocellulose membrane 3, a marker pad 4, an NC (numerical control) membrane 5 and a water absorption pad 6; the overlap width of each part of a detection line (T line) and a quality control line (C line) arranged on the NC film from the marker pad to the absorbent pad is 2 mm.
The marker pad is coated with a marker marked chicken IgY antibody and a marker marked botulinum toxin type B nano antibody a; the detection line is coated with a B-type botulinum toxin nano antibody B, and the quality control line is coated with IgG of the goat anti-chicken IgY antibody.
The marker is suitable for colloidal gold, fluorescein, lanthanide fluorescence, color latex, quantum dots, up-conversion fluorescent substance, magnetic fluorescent microsphere, enzyme marker and chemiluminescent substance; specifically, the fluorescent microsphere can be lanthanide series fluorescent microsphere.
The detection principle (double antibody sandwich method) of the botulinum toxin type B detection card is as follows: as shown in figure 1, IgG of goat anti-chicken IgY is coated on the quality control line, and botulinum toxin type B nano antibody is coated on the detection line. When a sample containing BoNT-B is added and passes through a marker pad, the BoNT-B is combined with lanthanide fluorescent microspheres marked with botulinum toxin type B nano-antibodies a to form complexes, then the complexes are captured by botulinum toxin type B nano-antibodies B coated by a T line to form double-antibody sandwich complexes, the complexes are fixed on the T line, so that the detection line (T) shows fluorescence, and the BoNT-B conjugates captured by the detection line (T) are in direct proportion to the content of marked lanthanide fluorescent substances. When the sample does not contain the BoNT-B antigen, the lanthanide fluorescent microspheres marked with the B-type botulinum toxin nano antibody a cannot be captured by the B-type botulinum toxin nano antibody B on the detection line, so that the detection line does not show fluorescence; the lanthanide fluorescent microspheres labeled with chicken IgY are captured by IgG of goat anti-chicken IgY on the quality control line, so that the quality control line shows fluorescence. Detecting by lanthanide fluorescence immunoassay analyzer to obtain fluorescence ratio of detection line and reference line, and calculating by presetting corresponding "standard curve" to obtain the content of botulinum toxin type B in the sample. Realize the rapid and accurate quantitative detection of the botulinum toxin type B.
Example 2
Preparation of botulinum toxin type B
Performing toxigenic culture and enrichment culture of Clostridium botulinum type B with a Pachyrhizus culture medium, and then performing filtration on the A pump and HiPrep Sephacryl with deionized water TM S-200 column at 1mL/min flow rate washing 1 times column volume, using the filtered working solution (sodium citrate buffer +150mM NaCl) on the A pump and HiPrep Sephacryl TM Washing S-200 column at 1.0mL/min flow rate, balancing 1 column volume, sampling 2.1 samples with sampling ring at flow rate of 1mL/min, and purifying with A 280 Monitoring and collecting A 280 And (3) continuously purifying the sample with the ultraviolet peak which has obvious absorption by using a DEAE FF column, washing the A pump and the DEAE FF column by using the filtered sodium citrate buffer solution at the flow rate of 2.0mL/min, loading the sample at the flow rate of 2mL/min after balancing 2 column volumes, eluting the hybrid protein by using 1M NaCl, and collecting the protein peak to obtain the botulinum toxin type B.
Preparation of botulinum toxin type A
Performing toxigenic culture and enrichment culture of A type Clostridium botulinum with meat culture medium, and mixing with A pump and HiPrep Sephacryl by using filtered deionized water TM S-200 column at 1mL/min flow rate washing 1 times the column volume, use the filtered working solution (sodium citrate buffer +150mM NaCl) on the A pump and HiPrepSephacryl TM Washing an S-200 column at the flow rate of 1.0mL/min, balancing the volume of 1 column, taking a sample of 2.1, loading the sample by adopting a sample injection ring, setting the flow rate at 1mL/min, monitoring by using A280 in the purification process, collecting a peak sample with obvious absorption of A280 ultraviolet, continuously purifying by using a DEAE FF column, washing an A pump and the DEAE FF column by using a filtered sodium citrate buffer solution at the flow rate of 2.0mL/min, loading the sample at the flow rate of 2mL/min after balancing the volume of 2 columns, eluting the foreign protein by using 1M NaCl, and collecting the protein peak to obtain the botulinum toxin type A.
3. Sensitivity test
Using PBS as a diluent to prepare botulinum toxin type B solutions with the concentrations of 0, 0.1, 1, 5, 10, 50 and 100ng/mL respectively; adding 80uL of the standard solutions with different concentrations into sample adding holes of an assembled detection card respectively, carrying out chromatography immunoreaction for 15 minutes, detecting by a lanthanide fluorescence immunoassay analyzer, calculating the fluorescence signal intensity ratio of a detection line and a quality control line corresponding to a standard product, and carrying out linear regression according to the data to prepare a standard curve of the botulinum toxin type B, wherein the equation of the standard curve is that y is 0.036x +0.0585, and R2 is 0.997, as shown in figure 2 and table 1. The detection limit determination criterion is that when the T/C value of the detection sample is more than 2.1 times of the T/C value of the negative control, the detection sample contains the botulinum toxin type B.
As can be seen from FIG. 2, the detection limit is 5ng/mL, i.e., the minimum concentration detectable by the test strip of the present invention is 5 ng/mL.
TABLE 1 detection T/C values of botulinum toxin type B solutions at different concentrations
Concentration (ng/ml) T/C
0 0.089
0.1 0.0732
1 0.0802
5 0.2143
10 0.4956
50 1.7107
100 3.7255
4. Experiment of specificity
Taking PBS as a diluent, and preparing botulinum toxin type A solutions with the concentrations of 0, 1, 10, 100 and 1000ng/mL respectively; and (3) respectively adding 80uL of the standard solution with different concentrations into sample adding holes of the assembled detection card, carrying out chromatography immunoreaction for 15 minutes, and detecting by using a lanthanide fluorescence immunoassay analyzer. The result is shown in table 2, and the botulinum toxin type A T/C at each concentration is not different from the negative control, which shows that the test strip cannot detect botulinum toxin type A and has good specificity.
TABLE 2 measured T/C values of botulinum toxin type A solutions at different concentrations
Concentration (ng/ml) T/C
0 0.1025
1 0.0999
10 0.091
100 0.1919
1000 0.1003
5. Reagent sample detection
Serum samples of suspected botulism patients provided by national hospitals of Shijiazhuang city (3 cases), Bai Cai Yi Hospital of Jilin university (1 case), and Children's Hospital affiliated to the first school of research (2 cases) were used as test samples, serum samples of healthy persons were used as negative controls, and the samples were diluted 2-fold, 5-fold, and 10-fold with PBS, respectively, and then tested according to the method of example 1, and the content of botulinum toxin type B was calculated according to the standard curve equation, and the test results are shown in Table 3. The determination standard is that if the ratio of the fluorescence signals of the sample to be detected in the detection area and the quality control area of the test paper is more than or equal to 2.1 times of the ratio of the fluorescence signals of the negative control sample in the detection area and the quality control area of the test paper, the sample to be detected contains the botulinum toxin type B and is a positive sample; if the ratio of the fluorescence signals of the sample to be detected in the detection area and the quality control area of the test paper is less than 2.1 times of the ratio of the fluorescence signals of the negative control sample in the detection area and the quality control area of the test paper, the sample to be detected does not contain the botulinum toxin B and is a negative sample. As can be seen from Table 3, among 6 cases of suspected botulinum toxin patients, 4 cases of positive samples of botulinum toxin type B and 2 cases of negative samples of non-botulinum toxin type B were obtained, and the results of the determination of the cause of the toxicity were determined by combining the patient history and clinical symptoms, which indicates that the concordance rate between the method and the final clinical result determination was 100%.
TABLE 3 clinical specimen test results
2 times of dilution 5 times dilution 10 times dilution Determination of results
Negative control 0.179 0.0558 0.0383
Test sample 1 2.1963 0.2512 0.2073 Positive male notebook
Test sample
2 0.1954 0.0929 0.0458 Negative sample
Test sample
3 1.3847 0.2551 0.0703 Positive male notebook
Test sample
4 0.4337 0.1876 0.0694 Positive yang book
Test sample
5 0.1581 0.0506 0.0432 Negative sample
Test sample
6 0.939 0.2244 0.1339 Positive sample
The invention establishes a lanthanide fluorescence immunochromatographic assay method based on visible light excitation by utilizing the botulinum toxin type B quantitative detection card, obtains higher minimum detection limit in the detection of biotoxin, is matched with a matched small portable detection instrument for use, and has the advantages of rapid detection and capability of remotely transmitting results. The high-sensitivity fluorescence detection method for the botulinum toxin type B has the advantages of high sensitivity, miniaturization of a detection instrument and low cost, and can solve the defects of the prior art. The method has important significance for a plurality of fields such as emergent public health incidents, food safety and the like, and provides a quick, sensitive and accurate detection method for multi-environment field detection.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> military medical research institute of military science institute of people's liberation force of China
<120> quick quantitative detection card for botulinum toxin type B
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gln Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Asp Ser Leu Tyr
20 25 30
His Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Arg Val Gln Asp Gly Gly Ser Thr Ala Tyr Lys Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Phe Ser Arg Ser Thr Met Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Ala Lys Ser Thr Ile Ser Thr Pro Leu Ser Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser
115
<210> 2
<211> 127
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gln Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Ile Leu Thr Tyr Asp Leu Asp
20 25 30
Tyr Tyr Tyr Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Gly Val Ser Cys Ile Ser Ser Thr Asp Gly Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Lys Asn Thr
65 70 75 80
Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Ala Ala Pro Leu Ala Gly Arg Tyr Cys Pro Ala Ser His
100 105 110
Glu Tyr Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125

Claims (9)

  1. The test paper strip for detecting the botulinum toxin type B is characterized by comprising a sample pad, a nitrocellulose membrane, a marker pad, an NC membrane and a water absorption pad; the NC membrane is provided with a detection line and a quality control line; the marker pad is coated with a marker-labeled chicken IgY antibody and a marker-labeled B-type botulinum toxin nano antibody A; the detection line is coated with a botulinum toxin type B nano antibody, and the quality control line is coated with IgG of the goat anti-chicken IgY antibody; the botulinum toxin type B nano antibody a and the botulinum toxin type B nano antibody B can be combined with botulinum toxin type B.
  2. 2. The botulinum toxin type B test strip of claim 1, wherein the label is at least one of colloidal gold, fluorescein, lanthanide fluorescent microspheres, colored latex, quantum dots, upconversion fluorescent substance, magnetofluorescent microspheres, enzyme label, or chemiluminescent substance.
  3. 3. The botulinum toxin type B test strip of claim 2, wherein the marker is a lanthanide fluorescent microsphere.
  4. 4. The test paper strip for detecting botulinum toxin type B according to claim 1, wherein the sequence of botulinum toxin type B nano antibody a is shown as sequence 1 in the sequence table; the sequence of the botulinum toxin type B nano antibody B is shown as a sequence 2 in a sequence table.
  5. 5. The botulinum toxin type B test strip of claim 1, further comprising a base plate, wherein the sample pad, the nitrocellulose membrane, the marker pad, the NC membrane and the absorbent pad are sequentially immobilized on the base plate.
  6. 6. Use of a test strip according to any one of claims 1 to 5 for the detection of botulinum toxin type B or for the detection of the botulinum toxin type B content of a sample to be tested.
  7. 7. Use of a test strip according to any one of claims 1 to 5 in the manufacture of a product for detecting botulinum toxin type B or for detecting the amount of botulinum toxin type B in a sample to be tested.
  8. 8. A method for detecting whether a sample to be detected contains botulinum toxin type B or not comprises the following steps:
    (1) detecting a sample to be detected and a negative control sample by using the test strip of any one of claims 1 to 5, and respectively obtaining the ratio of the fluorescence signals of the sample to be detected and the negative control sample in the detection area and the quality control area of the test strip, wherein if the ratio of the fluorescence signals of the sample to be detected in the detection area and the quality control area of the test strip is more than or equal to 2.1 times of the ratio of the fluorescence signals of the negative control sample in the detection area and the quality control area of the test strip, the sample to be detected contains botulinum toxin type B; if the ratio of the fluorescence signals of the sample to be detected in the detection area and the quality control area of the test paper is less than 2.1 times of the ratio of the fluorescence signals of the negative control sample in the detection area and the quality control area of the test paper, the sample to be detected does not contain the botulinum toxin B.
  9. 9. A method for detecting the content of botulinum toxin type B in a sample to be detected comprises the following steps:
    (1) detecting a botulinum toxin type B standard substance by using the test strip of any one of claims 1 to 5 to obtain a fluorescence signal ratio of the standard substance in a detection area and a quality control area of the test strip, making a unitary linear regression curve of the concentration of the botulinum toxin type B standard substance corresponding to the fluorescence signal ratio, and calculating a regression equation;
    (2) replacing the standard substance of the botulinum toxin type B with a sample to be detected, detecting the sample to be detected by using the test paper to obtain the ratio of fluorescence signals of the sample to be detected in a detection area and a quality control area of the test paper, and obtaining the content of the botulinum toxin type B in the sample to be detected according to the regression equation in the step (1).
CN202210621717.2A 2022-06-02 2022-06-02 Quick quantitative determination card of type B botulinum toxin Pending CN115032379A (en)

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Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210621717.2A CN115032379A (en) 2022-06-02 2022-06-02 Quick quantitative determination card of type B botulinum toxin

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CN115032379A true CN115032379A (en) 2022-09-09

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Country Link
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116925213A (en) * 2023-09-12 2023-10-24 中国人民解放军军事科学院军事医学研究院 Nanometer antibody for neutralizing botulinum toxin type A
CN117736318A (en) * 2024-02-18 2024-03-22 中国人民解放军军事科学院军事医学研究院 Anti-botulinum toxin nano antibody and application thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116925213A (en) * 2023-09-12 2023-10-24 中国人民解放军军事科学院军事医学研究院 Nanometer antibody for neutralizing botulinum toxin type A
CN116925213B (en) * 2023-09-12 2024-03-15 中国人民解放军军事科学院军事医学研究院 Nanometer antibody for neutralizing botulinum toxin type A
CN117736318A (en) * 2024-02-18 2024-03-22 中国人民解放军军事科学院军事医学研究院 Anti-botulinum toxin nano antibody and application thereof
CN117736318B (en) * 2024-02-18 2024-05-07 中国人民解放军军事科学院军事医学研究院 Anti-botulinum toxin nano antibody and application thereof

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