CN114560933B

CN114560933B - Monoclonal antibody specifically binding to ricin and application thereof

Info

Publication number: CN114560933B
Application number: CN202210086552.3A
Authority: CN
Inventors: 许宏瑞; 王德鸿; 魏颖; 由赛男; 王福; 崔贝贝; 邓鹏�; 应天翼; 李岩松; 余涛
Original assignee: Beijing Hongjin Jiuan Biotechnology Co ltd
Current assignee: Beijing Hongjin Jiuan Biotechnology Co ltd
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2023-01-31
Anticipated expiration: 2042-01-25
Also published as: CN114560933A

Abstract

The invention discloses an antibody or an active fragment thereof specifically binding to ricin, colloidal gold immunochromatographic assay test paper or a kit comprising the antibody, and application of the test paper or the kit. The test paper or the kit has the advantages of convenient production, stable effect, no non-specific reaction and low coating amount, and is beneficial to mass production. The method is simple and quick to operate, good in reproducibility and extremely convenient to use on site, and the establishment of the method not only enriches the existing ricin detection method, but also lays a foundation for the treatment and emergency prevention of ricin poisoning.

Description

Monoclonal antibody specifically binding to ricin and application thereof

Technical Field

The invention relates to the field of immunization, in particular to an antibody specifically combined with ricin, and detection test paper and a kit for detecting ricin.

Background

Ricin (RT) is derived from the seeds of the euphorbiaceae plant castor (Ricin communis), a lethal and extremely toxic biotoxin that can cause accidental poisoning and can also be used in criminal attempts. One molecule of RT, which is introduced into the cell, is sufficient to stop the synthesis of whole cellular proteins and die, with 6000 times the toxicity of cyanide. Toxicity of castor seeds was studied at the end of the 19 th century beginning at Schmiedeberg laboratories in stusburg and subsequently at the dorpatt medical institute (now talr diagram), a highly toxic protein was purified from castor seeds or from the filter cake and named RT. RT has a molecular weight of about 60 kDa and is a type II Ribosome Inactivating Protein (RIP) consisting of an A chain (RTA) and a B chain (RTB) linked by a disulfide bond. The extraction of high purity RT is the basis for verifying its biological characteristics and preparing RT monoclonal antibody.

Therefore, the establishment of a rapid, convenient, high-sensitivity and high-specificity RT detection method is very urgent.

Disclosure of Invention

It is a first object of the present invention to provide antibodies and active fragments thereof that specifically bind ricin.

In particular, the invention provides an antibody or active fragment comprising at least one heavy chain variable region and at least one light chain variable region, wherein:

the heavy chain variable region has CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 4 and/or CDR3 shown in SEQ ID NO. 5; or, the heavy chain variable region has CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14 and/or CDR3 shown in SEQ ID NO. 15;

the light chain variable region has CDR1 shown in SEQ ID NO. 8, CDR2 shown in SEQ ID NO. 9 and/or CDR3 shown in SEQ ID NO. 10; alternatively, the light chain variable region has CDR1 shown in SEQ ID NO. 18, CDR2 shown in SEQ ID NO. 19 and/or CDR3 shown in SEQ ID NO. 20.

In a preferred embodiment of the present invention, the heavy chain variable region of the antibody has CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 4 and/or CDR3 of SEQ ID NO. 5; the light chain variable region has CDR1 shown in SEQ ID NO. 8, CDR2 shown in SEQ ID NO. 9 and/or CDR3 shown in SEQ ID NO. 10.

In a preferred embodiment of the present invention, in the antibody, the heavy chain variable region has an amino acid sequence shown in SEQ ID No. 1, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID No. 1; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 6, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 6.

In a preferred embodiment of the present invention, the heavy chain variable region of the antibody has CDR1 of SEQ ID NO. 13, CDR2 of SEQ ID NO. 14 and/or CDR3 of SEQ ID NO. 15; the light chain variable region has CDR1 shown in SEQ ID NO. 18, CDR2 shown in SEQ ID NO. 19 and/or CDR3 shown in SEQ ID NO. 20.

In a preferable embodiment of the invention, in the antibody, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 11, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO. 11; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 16, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 16.

As the inventionIn a preferred embodiment, the antibody or active fragment is a monoclonal antibody and/or a genetically engineered antibody; the genetic engineering antibody is selected from one of a single-chain antibody, a single-chain antibody fragment, a chimeric monoclonal antibody fragment, a modified monoclonal antibody and a modified monoclonal antibody fragment. Preferably, the antibody or active fragment includes, but is not limited to, a monoclonal antibody, F (ab') ₂ Fab', fab, fv, scFv, dsFv and dAb.

It is a second object of the invention to provide a nucleotide sequence encoding said antibody or active fragment that specifically binds ricin.

In the present invention, the nucleotide sequence shown by SEQ ID NO. 2 encodes the amino acid sequence shown by SEQ ID NO. 1; the nucleotide sequence shown in SEQ ID NO. 7 encodes the amino acid sequence shown in SEQ ID NO. 6; the nucleotide sequence shown as SEQ ID NO. 12 codes the amino acid sequence shown as SEQ ID NO. 11; the nucleotide sequence shown in SEQ ID NO. 17 encodes the amino acid sequence shown in SEQ ID NO. 16.

The third purpose of the invention is to provide a kit for detecting ricin, which comprises a detection test paper, wherein the test paper contains the antibody or the active fragment of the invention.

Preferably, the test paper is a colloidal gold immunochromatographic assay test paper.

As a preferred embodiment of the present invention, the test strip comprises a binding pad, a detection line (T line), and a control line (C line); the conjugate of the antibody or the active fragment and colloidal gold is coated on the bonding pad, the antibody or the active fragment is coated on the detection line, and the goat anti-mouse IgG antibody is coated on the control line.

Wherein, in the antibody or active fragment coated on the binding pad coupled with colloidal gold, the heavy chain variable region has CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 4 and/or CDR3 shown in SEQ ID NO. 5, and the light chain variable region has CDR1 shown in SEQ ID NO. 8, CDR2 shown in SEQ ID NO. 9 and/or CDR3 shown in SEQ ID NO. 10. Preferably, in the antibody or active fragment coated on the binding pad coupled with colloidal gold, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 1, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids of the amino acid sequence shown in SEQ ID NO. 1, and the light chain variable region has an amino acid sequence shown in SEQ ID NO. 6, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids of the amino acid sequence shown in SEQ ID NO. 6.

Wherein, in the antibody or active fragment coated on the detection line, the heavy chain variable region has CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14 and/or CDR3 shown in SEQ ID NO. 15, and the light chain variable region has CDR1 shown in SEQ ID NO. 18, CDR2 shown in SEQ ID NO. 19 and/or CDR3 shown in SEQ ID NO. 20. Preferably, in the antibody or active fragment coated on the detection line, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 11, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO. 11, and the light chain variable region has an amino acid sequence shown in SEQ ID NO. 16, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO. 16.

As a preferred scheme of the invention, the colloidal gold immunochromatographic test strip is prepared by a method comprising the following steps:

the antibody or the active fragment provided by the invention is coupled with colloidal gold to obtain a solution, and the solution is sprayed on a bonding pad; after being dissolved by coating buffer solution, the antibody provided by the invention is coated on a detection line of a nitrocellulose membrane; dissolving an anti-mouse IgG antibody by using a coating buffer solution, and coating the anti-mouse IgG antibody on a control line of a nitrocellulose membrane; and assembling the combination pad and the nitrocellulose membrane on a back plate to prepare a large plate, and cutting the large plate into a bare strip, namely the test paper.

The fourth purpose of the invention is to provide the application of the antibody or the active fragment, or the test paper or the kit, in the detection of ricin in a sample.

A fifth object of the present invention is to provide the use of the antibody or active fragment of the present invention for preparing a reagent for detecting ricin.

The sixth purpose of the present invention is to provide a method for detecting ricin in a sample by using the antibody or active fragment of the present invention, or the test paper or kit.

As a preferred embodiment of the present invention, the method comprises the steps of: and dropwise adding the solution of the sample to be detected onto the colloidal gold immunochromatography detection test paper or into a sample adding hole of a kit internally provided with the test paper, standing, and observing the color development conditions of the T line and the C line on the test paper.

As a preferable scheme of the invention, if the T line and the C line are both developed, the sample contains ricin with the concentration of more than or equal to 10 ng/mL; if the line C is colored and the line T is not colored, the sample contains ricin with the concentration less than 10ng/mL or does not contain ricin; if the C line does not develop color, the detection reagent is invalid.

A seventh object of the present invention is to provide a method for purifying ricin, the method comprising the steps of: pulverizing semen Ricini, leaching with phosphate buffer solution, centrifuging, collecting supernatant, adding saturated ammonium sulfate solution, standing, centrifuging, collecting precipitate, dissolving in phosphate buffer solution, dialyzing, centrifuging to obtain supernatant as coarse toxin of semen Ricini; adsorbing the castor crude toxin by a Sepharose 4B column, eluting by galactose, and performing Sephacryl S-100 molecular sieve chromatography to prepare the castor toxin with the purity of more than 90 percent.

After castor crude toxin is extracted from castor seeds, RT and castor agglutinin are adsorbed by Sepharose 4B column by using special affinity of RT to galactose, eluted by 150 mM galactose, and separated by Sephacryl S-100 molecular sieve chromatography to prepare RT with purity higher than 90%, and monoclonal antibody is prepared by immunizing mice. Then, the prepared monoclonal antibodies are randomly paired, and the two successfully paired monoclonal antibodies (the cell strain numbers are 401 and 901 respectively) are used for colloidal gold labeling and coating respectively, so that a colloidal gold immune lateral chromatography test strip detection method capable of specifically detecting RT is established. The method is simple and quick to operate, good in reproducibility and extremely convenient to use on site, and the establishment of the method not only enriches the existing RT detection method, but also lays a foundation for the treatment and emergency prevention of RT poisoning.

Drawings

FIG. 1 shows a photograph of a colloidal gold immuno-lateral chromatography test card.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 extraction of crude RT toxin

Taking 50 g of castor beans, shelling, putting into a pulverizer, adding liquid nitrogen, pulverizing and taking out. Then 100 mL of 10 mM phosphate buffer (pH 7.2) was added, and the mixture was extracted at 4 ℃ for 24 hours, 17000 g, and centrifuged at 4 ℃ for 40 minutes to remove the precipitate and the upper fat, and the supernatant was collected and filtered with a three-layer filter paper. Then adding saturated ammonium sulfate solution, standing at 4 deg.C overnight, 17000 g, centrifuging at 4 deg.C for 40 min, and collecting precipitate. Dissolving the obtained precipitate in 10 mM phosphate buffer solution (pH 7.2), dialyzing with 10 mM phosphate buffer solution (pH 7.2) overnight, and centrifuging at 17000 g at 4 deg.C for 15 min to obtain supernatant as crude toxin of Ricinus communis.

Example 2 purification of crude RT toxin

(1) Affinity chromatography

Adding a proper amount of castor crude toxin into a well-balanced Sepharose 4B medium, uniformly mixing and adsorbing for 1.5 h, then loading the medium into a gravity column, connecting a constant flow pump, controlling the flow rate at 70 r/min, and recording the chromatographic profile by using an HD-A chromatographic profile acquisition analyzer. The hybrid protein was washed with 10 mM phosphate buffer (pH 7.2), and after the UV curve was washed flat, 150 mM galactose-PBS (pH 7.2) was added to elute the protein, and the elution peak was collected.

(2) Molecular sieve chromatography

The affinity collected liquid was applied to Sephacryl S-100 HR pre-packed column at a flow rate of 0.5 mL/min, eluted with 10 mM phosphate buffer (pH 7.2) and the eluted peak was collected.

Example 3 RT identification and protein concentration determination

And (3) boiling the collected RT protein by using a loading buffer solution containing beta mercaptoethanol and a loading buffer solution not containing beta mercaptoethanol respectively, and performing SDS-PAGE electrophoresis. Staining with Coomassie brilliant blue R-250, decolorizing the decolorized solution on a shaker, and analyzing the gel image to determine the relative molecular weights of RT, RTA, and RTB. The concentration of RT was determined by the BCA method.

Example 4 preparation of RT monoclonal antibodies

Selecting 5 Balb/c healthy female mice with the age of 8 to 10 weeks, and carrying out back subcutaneous injection immunization on the purified RT according to 100 micrograms/0.2 mL each time at an interval of 2 weeks; and (3) collecting blood from the orbit of the mouse on the 7 th day after 3 times of immunization, obtaining the serum of the mouse, detecting the binding titer of the RT monoclonal antibody in the serum of the mouse by an ELISA method, and if the titer is less than 10000, performing boosting immunization for 1 to 2 times until the antibody titer is more than 10000.

And (2) mixing the spleen cells of the mice with the antibody titer of more than 10000 with myeloma SP2/0 cells (spleen cells: SP2/0 cells 5.

The autoclaved paraffin oil is injected into the abdominal cavity of female mice at a rate of 0.5 mL/female mouse, and is injected into the female mice 10 days after one week ⁶ And (3) after 7 to 10 days, extracting ascites when the abdomen of the mouse obviously swells, and purifying by using a Protein A/G antibody purification column, wherein the ELISA titer of the purified antibody is more than 1, 128000, and the purity is more than 90%.

Example 5 amplification and sequencing of the CDR region sequences of RT monoclonal antibodies

Resuscitating hybridoma cell lines 401 and 901, culturing, and collecting about 1 × 10 cells when the cells grow to logarithmic growth phase ⁷ And (4) cells. Total RNA of hybridoma cells was extracted according to the TaKaRaMiniBEST Universal RNA Extraction Kit, the reaction system was added as shown in Table 1, and first Strand (1 st-Strand) cDNA synthesis was performed according to the reaction system shown in Table 2.

PCR amplification of the variable region nucleotide sequences of the light and heavy chains of the monoclonal antibodies using the primer sequences shown in Table 3 and the reverse transcribed cDNA as template, with reference to pMD ^TM 19-T Vector Cloning Kit, ligation of amplified nucleic acid fragments to pMD ^TM And (2) transforming the 19-T vector into TOP10 competent cells, identifying the cells by using PCR as a bacterial liquid, selecting positive bacterial colonies for amplification culture, extracting a pMD19-T plasmid connected with a variable region sequence of the monoclonal antibody according to the specification of a small root plasmid kit, determining the plasmid concentration, and sending the plasmid concentration to a sequencing company for sequencing.

Example 6 alignment analysis of variable regions of RT monoclonal antibodies

The nucleic acid sequences of the variable regions of the 401 and 901 monoclonal antibodies are subjected to homology analysis in a GenBank database, and the results show that the amplified sequences have the highest homology with immunoglobulin variable region genes of Balb/c mice in GenBank, which indicates that the sequenced gene sequences are mouse antibody sequences. The amino acid sequences of the variable regions of the light chain and the heavy chain of the antibody and the division of the CDR regions are obtained by utilizing the IMGT/V-QUEST and ABYSIS software analysis. The sequencing results are as follows.

(1) Sequencing results of variable region heavy chain of RT monoclonal antibody strain 401 (see Table 4)

The amino acid sequence is as follows:

LTMECSWVMLFLVATASGVHSQVQLQQPGSELVRPGASVKLSCKASGYAFTSYWIQWVKQRPGQGLERIGNIYPASGRINYDEMFKPKATLTVDTSSNTAYMQLSNLTSEDSAVYYCTRFIYYGSHGYAIDYWGQGTSVTVSSAKTTPPSVYPLVPVC（SEQ ID NO: 1）

nucleotide sequence (total 475 bp):

CTCACCATGGAATGCAGCTGGGTAATGCTCTTCTTGGTAGCAACAGCCTCAGGTGTCCACTCCCAGGTCCAACTGCAGCAACCTGGGTCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAACTGTCCTGCAAGGCTTCTGGCTACGCATTCACCAGCTATTGGATCCAGTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGCGGATTGGAAATATTTATCCTGCTAGTGGTCGTATTAATTACGATGAGATGTTCAAGCCCAAGGCCACACTGACTGTGGACACATCCTCTAACACAGCCTACATGCAGCTCAGCAACCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGATTCATCTACTATGGTTCCCACGGATATGCTATCGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGTCCCTGTGTGTG（SEQ ID NO: 2）

(2) Sequencing results of variable region light chain of RT monoclonal antibody Strain 401 (see Table 5)

Amino acid sequence:

DIQLTQSPAIMSASPGEKVTISCSARSGLSFTYWYQQKAGSSPKPWIYRTSIMASGVPARFSGSGSGTSYSLTVSSMEAEDAATYYCQQYHTYAPTFGGGTKLEI（SEQ ID NO: 6）

nucleotide sequence (total 320 bp):

GACATTCAGCTGACCCAGTCTCCAGCAATCATGTCTGCTTCTCCAGGGGAGAAGGTCACCATATCCTGCAGTGCCAGATCAGGTCTAAGTTTCACGTACTGGTACCAGCAGAAGGCAGGATCCTCCCCCAAACCCTGGATTTATCGCACATCCATCATGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAGTCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTATCATACTTACGCACCCACGTTCGGAGGGGGGACCAAGCTGGAGATCTAACA（SEQ ID NO: 7)

(3) RT monoclonal antibody strain 901 variable region heavy chain sequencing results (see Table 6)

The amino acid sequence is as follows:

LTMDFGLSLVFLPTLPHGIQCQVKLVESGGGLVQPGGSLRLSCATSGFTFSHLNMEWVRQPPGKRLEWLAASRNKANDYIIEYNVSVKGRFTVSRDTSQGTLNPQMYALRAQDTAIYYCARDHYGTDGPRRGAWLLGPRHHSHSLLSQNDTPICLSTGPCVWR（SEQ ID NO: 11）

nucleotide sequence (491 bp altogether):

CTCACCATGGACTTCGGGCTGAGCTTGGTTTTTCTTCCAACACTTCCACATGGTATCCAGTGTCAGGTGAAGCTGGTGGAATCTGGAGGAGGCTTGGTACAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCAACTTCTGGGTTCACCTTCAGTCATTTGAACATGGAGTGGGTCCGCCAGCCTCCAGGCAAGAGACTGGAGTGGCTGGCTGCAAGTAGAAACAAAGCTAATGATTATATAATAGAATACAATGTCTCTGTGAAGGGTCGGTTCACCGTCTCCAGAGACACTTCCCAAGGCACCCTCAACCCTCAGATGTATGCCCTGAGAGCTCAGGACACTGCCATTTATTATTGTGCAAGAGATCACTATGGTACCGACGGTCCTCGACGAGGGGCTTGGCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGTGTGTGGAGATA（SEQ ID NO: 12）

(4) RT monoclonal antibody strain 901 variable region light chain sequencing results (see Table 7)

The amino acid sequence is as follows:

DIVLTQSPASLAVSLGQRATISYRASNSVSTPGYTYMHWNQQKPGQPPRLLIYLASHLASGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQDISELTRSEGGPSWK（SEQ ID NO: 16）

nucleotide sequence (total 324 bp):

GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAACAGTGTCAGTACACCTGGCTATACTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGCATCCCACCTAGCATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGGACATTAGCGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAA（SEQ ID NO: 17）

example 7 RT colloidal gold Immunochromatographic assay kit

Coupling an antibody purified from ascites corresponding to the cell strain number 401 with colloidal gold with the particle size of 40 nm, dissolving the antibody in a complex solution, and spraying the antibody on a pretreated combination pad by using a gold-labeled three-dimensional membrane-scribing spraying point instrument; dissolving an antibody purified from ascites corresponding to a cell line number 901 by using a coating buffer solution, and coating the antibody on a TEST line (T line) of a pretreated Nitrocellulose (NC) membrane by using a gold-labeled three-dimensional membrane-scribing dot-spraying instrument; dissolving an outsourced goat anti-mouse IgG antibody with a coating buffer solution, and coating the dissolved antibody on a CONTROL line (C line) of a pretreated nitrocellulose membrane by using a gold-labeled three-dimensional membrane-scribing dot spraying instrument; assembling the bonding pad subjected to metal spraying treatment, the NC membrane subjected to coating treatment and the pretreated bonding pad on a back plate to manufacture a large plate. And cutting the large plate manufactured in the previous step into a bare strip with the width of 4 mm by using a slitter, assembling the bare strip into a matched card shell, and preparing the colloidal gold detection reagent for detecting RT.

Preparing RT sample liquid (solvent is phosphate buffer solution with pH7.4 and 0.01M) with different concentrations, respectively transferring 80 mu L of RT sample liquid with different concentrations by a pipette, dripping the RT sample liquid in parallel to a sample adding hole of a colloidal gold detection reagent, observing the color development conditions of a T line and a C line when 10 min is carried out, and if the T line and the C line are both developed, determining that the RT sample liquid is positive; c line color development, T line color development is negative; the line C does not develop color, and the detection reagent is invalid. As shown in fig. 1, wherein 1 is negative; 2. ricin sample solution with concentration of 10 ng/mL; 3. ricin sample solution with concentration of 1. Mu.g/mL.

The detection limit of the RT colloidal gold detection reagent is 10ng/mL through determination.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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<210> 13

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gly Phe Thr Phe Ser His Leu Asn

1 5

<210> 14

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Ser Arg Asn Lys Ala Asn Asp Tyr Ile Ile

1 5 10

<210> 15

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ala Arg Asp His Tyr Gly Thr Asp Gly Pro Arg Arg Gly Ala Trp Leu

1 5 10 15

<210> 16

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Asn Ser Val Ser Thr Pro

20 25 30

Gly Tyr Thr Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser His Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Asp Ile Ser

85 90 95

Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys

100 105

<210> 17

<211> 324

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60

atctcataca gggccagcaa cagtgtcagt acacctggct atacttatat gcactggaac 120

caacagaaac caggacagcc acccagactc ctcatctatc ttgcatccca cctagcatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagg acattagcga gcttacacgt 300

tcggaggggg gaccaagctg gaaa 324

<210> 18

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Asn Ser Val Ser Thr Pro Gly Tyr Thr Tyr

1 5 10

<210> 19

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Leu Ala Ser

1

<210> 20

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Gln Asp Ile Ser Glu Leu Thr Arg

1 5

<210> 21

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ctcaccatgg agacagacac actcctgcta t 31

<210> 22

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

agcccgtttk atttccarct t 21

<210> 23

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ctcaccatgg ratgsagctg kgtmats 27

<210> 24

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gttttggctg mrgagacdgt ga 22

Claims

1. An antibody and antigen-binding fragment thereof that specifically binds ricin, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region as follows;

the heavy chain variable region has CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 4 and CDR3 shown in SEQ ID NO. 5; the light chain variable region has CDR1 shown in SEQ ID NO. 8, CDR2 shown in SEQ ID NO. 9 and CDR3 shown in SEQ ID NO. 10; alternatively, the first and second electrodes may be,

the heavy chain variable region has CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14 and CDR3 shown in SEQ ID NO. 15; the light chain variable region has CDR1 shown in SEQ ID NO. 18, CDR2 shown in SEQ ID NO. 19 and CDR3 shown in SEQ ID NO. 20.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region has CDR1 of SEQ ID No. 3, CDR2 of SEQ ID No. 4 and CDR3 of SEQ ID No. 5;

the light chain variable region has CDR1 shown in SEQ ID NO. 8, CDR2 shown in SEQ ID NO. 9 and CDR3 shown in SEQ ID NO. 10.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO 1;

the light chain variable region has an amino acid sequence shown in SEQ ID NO. 6.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region has CDR1 of SEQ ID No. 13, CDR2 of SEQ ID No. 14 and CDR3 of SEQ ID No. 15;

the light chain variable region has CDR1 shown in SEQ ID NO. 18, CDR2 shown in SEQ ID NO. 19 and CDR3 shown in SEQ ID NO. 20.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 11;

the variable region of the light chain has an amino acid sequence shown as SEQ ID NO. 16.

6. The antibody or antigen-binding fragment of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is a monoclonal antibody, F (ab') ₂ Fab', fab, fv, scFv or dsFv.

7. A nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1 to 6.

8. A kit for detecting ricin, comprising a test strip comprising the antibody or antigen-binding fragment of any one of claims 1 to 6.

9. The kit according to claim 8, wherein the test paper is a colloidal gold immunochromatographic test paper.

10. The kit of claim 8, wherein the test strip comprises a binding pad, a detection line, and a control line; the combination pad is coated with a conjugate of the antibody or the antigen binding fragment of claim 2 and colloidal gold, the detection line is coated with the antibody or the antigen binding fragment of claim 4, and the control line is coated with a goat anti-mouse IgG antibody.

11. Use of the antibody or antigen-binding fragment of any one of claims 1 to 6 in the preparation of a reagent for the detection of ricin.

12. Use of the antibody or antigen-binding fragment of any one of claims 1 to 6 in the preparation of a test strip or kit for the detection of ricin in a sample.

13. Use according to claim 12, characterized in that said detection comprises the following steps: dripping the solution of a sample to be detected on the colloidal gold immunochromatography detection test paper or dripping the solution of the sample to be detected into a sample adding hole of a kit internally provided with the test paper, standing, and observing the color development conditions of a detection line and a control line on the test paper;

if the detection line and the control line are both colored, the sample contains ricin with the concentration of more than or equal to 10 ng/mL; if the control line is colored and the detection line is not colored, the sample contains ricin with the concentration less than 10ng/mL or does not contain ricin; if the control line does not develop color, the detection reagent is disabled.