CN104837866A - Binding/neutralizing human monoclonal antibodies against botulinum neurotoxin type B - Google Patents

Abstract

Isolated anti-botulinum neurotoxin type B monoclonal antibody and antigen-binding fragments thereof having a neutralization activity against a botulinum neurotoxin type B are provided, including a human monoclonal antibody. Hybridomas that produce such antibodies or fragments thereof are also provided by the present invention, as well as methods of producing such hybridomas and method of producing antibodies or fragments thereof from such hybridomas. Pharmaceutical compositions and kits including antibodies or fragments thereof for at least one of the prevention, the treatment, and the detection of botulinum neurotoxin type B poisoning are further provided. Methods of inhibiting or treating botulism in a human subject are provided, as are methods of detecting botulinum neurotoxin type B.

Description

Human monoclonal antibodies combine/in and Type B botulic neurotoxin

Technical field

The present invention relates to the antibody of anti-botulic neurotoxin (botulinum neurotoxin) (botulinus toxin (botulin)) and use and manufacture their method.

[related application]

This application claims the rights and interests of the U.S. Provisional Patent Application No.61/695318 that on August 31st, 2012 submits, it is incorporated to by quoting at this by this application in full.

Background technology

Botulic neurotoxin is the most poisonous known material.These toxin can cause sausage poisoning (botulism), are comprised by various bacterium clostridium botulinum (Clostridium botulinum)produce.Botulinogenic clostridium is anaerobic gram positive bacteria.These bacteriums form spore, and usually distribute in the environment.On a molecular scale, sausage poisoning is that the suppression that the vagusstoff by being caused at myoneural junction by toxin discharges causes.7 kinds of toxin serotypes (A, B, C, D, E, F and G) are determined.They have corresponding to comprising the similar structure of 100-kDa heavy chain with the zinc endopeptidase albumen of about 150kD of 50-kDa light chain.Toxin serotypes A, B, E are usually relevant to sausage poisoning in the mankind with F.

Sausage poisoning can be divided into different classes of-food source property, wound infection, baby's enterotoxemia, adult's enterotoxemia, toxin injection (iatrogenic sausage poisoning) and weaponization toxin.Food origin disease is because picked-up is occurred by the toxin in the food of toxigenic bacterial contamination.In wound infection, wound assembles the malicious clostridium of product, and then it grows and produces toxin in wound.In baby's sausage poisoning, baby intestinal assembles the malicious clostridium of product, and then it produces toxin.Adult's enterotoxemia is similar to baby's sausage poisoning.Beauty treatment or treatment injection Toxins, botulin may cause sausage poisoning sometimes.Sausage poisoning also can result from the injury that weaponization toxin causes, such as, and the Toxins, botulin be atomized by inhaling air.

The feature of sausage poisoning is symmetrical parmlysis of cranial nerve, and then voluntary muscle successively decreases symmetrical flaccid paralysis.This paralysis causes losing breathes and death.It is irreversible that toxin combines.Recover the growth relating to new nerve ending.Recovery may be very long, and relates to the outpatient rehabilitation treatment of prolongation.After toxin exposure, the outbreak of this disease can be rapidly.Treatment is usually directed to use toxinicide, and it can help the degree limiting paralysis.Available most of toxinicides are horse source property, and are not necessarily exclusively used in the botulic neurotoxin of particular type.There are the antitoxic needs to form more targetedly.Also have the needs of being free from side effects as the effective toxin neutralizing antibody of serum sickness.Consider that sausage poisoning development is so fast, there are the needs to better diagnostic test further.

Reference listing

Non-patent literature

[non-patent literature 1] Arimitsu etc., Infect.Immun.71:1599-1603,2003

Summary of the invention

(technical problem)

Therefore, a feature of the present invention is to provide the therapeutic antibodies of the anti-botulic neurotoxin with superior efficacy and security.

Another feature of the present invention is to provide the method suppressing and treat sausage poisoning.

Further feature of the present invention is to provide the therapeutical agent of being free from side effects as serum sickness.

Other feature and advantage of the present invention will part be set forth in the following description, and part will be obvious according to specification sheets, or can be learnt by enforcement of the present invention.Key element by particularly pointing out in specification sheets and appended claim and combination realize and reach by object of the present invention and other advantage.

(technical scheme of technical solution problem)

In order to realize these and other advantage, and according to object of the present invention, as here concrete implement with broadly described, the present invention relates to anti-Type B botulic neurotoxin monoclonal antibody or its Fab of separation, there is the Neutralization effect of anti-Type B botulic neurotoxin.Monoclonal antibody can comprise human monoclonal antibodies, Humanized monoclonal antibodies or both.The anti-Type B botulic neurotoxin monoclonal antibody be separated or its Fab can have the Neutralization effect of the precursor toxin of anti-Type B botulic neurotoxin.The anti-Type B botulic neurotoxin monoclonal antibody be separated or its Fab can have the specific binding activity of light chain to Type B botulic neurotoxin (botulinumneurotoxin type B, BoNT/B) or the specific binding activity of the light chain had Type B botulic neurotoxin (BoNT/B) and heavy chain.The present invention also provides the hybridoma (Hybridomas) producing antibody of the present invention or its fragment, and forms the method for such hybridoma, and produces the method for antibody or its fragment from such hybridoma.

The invention still further relates to pharmaceutical composition.Pharmaceutical composition can comprise one or more anti-Type B botulic neurotoxin monoclonal antibody be separated and its Fab and pharmaceutically acceptable carriers.The invention provides for prevent, treat and detect Type B botulic neurotoxin in human experimenter poisoning in the test kit of at least one.Test kit can comprise the anti-Type B botulic neurotoxin monoclonal antibody of separation of the present invention, its Fab or both.The present invention also provides the anti-Type B botulic neurotoxin monoclonal antibody of separation, its Fab or both are for the preparation of the purposes of medicine for suppressing or treat sausage poisoning in human experimenter.

The invention still further relates to the method suppressing or treat sausage poisoning in human experimenter.Method can comprise the anti-Type B botulic neurotoxin monoclonal antibody of the separation to human experimenter's administering therapeutic significant quantity, its Fab or both.It is poisoning that method can also comprise diagnosis patient botulic neurotoxin.Method can comprise the minimizing of at least one symptom of monitoring sausage poisoning.Suppress or in treatment human experimenter the method for sausage poisoning can comprise use separation anti-Type B botulic neurotoxin monoclonal antibody, its Fab or both and one or more other treatments of combining for sausage poisoning.Associating can act synergistically suppress or treat sausage poisoning.

The invention still further relates to the method detecting Type B botulic neurotoxin in human experimenter.Method can comprise by from sample and the anti-Type B botulic neurotoxin monoclonal antibody be separated of human experimenter, its Fab or both contact.Method can also comprise based on antibody, its fragment or both whether detect the presence or absence of Type B botulic neurotoxin in human experimenter in conjunction with Type B botulic neurotoxin.

Should be understood that general description above and detailed description are below all exemplary with illustrative, aim to provide claimed of the present invention further explanation.

Accompanying drawing comprises in this application and forms a application's part, and specific embodiments more of the present invention are described, and is used from specification sheets one and explains principle of the present invention.

Brief Description Of Drawings

Fig. 1 illustrates how to carry out immunity according to volunteer of the present invention and produce the schematic diagram how cell of antibody to carry out being separated.

Fig. 2 is the schema described according to cytogamy of the present invention and clone-time table.

Fig. 3 illustrates nucleotide sequence according to the heavy chain of M-2 antibody of the present invention and aminoacid sequence.

Fig. 4 illustrates nucleotide sequence according to the light chain of M-2 antibody of the present invention and aminoacid sequence.

Fig. 5 illustrates nucleotide sequence according to the heavy chain of M-4 antibody of the present invention and aminoacid sequence.

Fig. 6 illustrates nucleotide sequence according to the light chain of RM-4LC-16 antibody of the present invention and aminoacid sequence.

Fig. 7 illustrates nucleotide sequence according to the light chain of RM-4LC-18 antibody of the present invention and aminoacid sequence.

Fig. 8 illustrates nucleotide sequence according to the light chain of RM-4LC-19 antibody of the present invention and aminoacid sequence.

Fig. 9 comprises the broken line graph of display human monoclonal antibodies according to the present invention in conjunction with BoNT/A or BoNT/B.Human monoclonal antibodies (HuMAb) is analyzed by ELISA in conjunction with BoNT.HuMAbs (about 0.05-1.0 mcg/ml) adds to the plate being coated with BoNT/A or BoNT/B.After washing, in conjunction with HuMAbs detected by the anti-human IgG antibodies of coupling HRP.M-2 and M-4 shows the specific binding to BoNT/B.

Figure 10 comprise according to the west of the epitope mapping for human monoclonal antibodies of the present invention turn stain (Western blot) analyze.BoNT is separated into Hc and Lc by SDS-PAGE, and transfers on nitrocellulose filter.HuMAbs (1.0 mcg/ml) adds to this film, and at room temperature incubation 1 hour.After washing, by the anti-human IgG antibodies of film and coupling HRP at room temperature incubation 1 hour, then specific band is visual by ECL.The light chain of M-2 specific binding BoNT/B.By contrast, M-4 is in conjunction with light chain and heavy chain.

Figure 11 is the histogram of display human monoclonal antibodies according to the present invention in conjunction with BoNT/B, precursor toxin and non-toxic components.HuMAb is analyzed by ELISA in conjunction with BoNT, precursor toxin (12S toxin and 16S toxin) or non-toxic components (NAP-16).HuMAbs adds to the plate being coated with BoNT, precursor toxin or NAP-16.After washing, in conjunction with HuMAbs detected by the anti-human IgG antibodies of coupling HRP.M-2 and M-4 is in conjunction with BoNT and precursor toxin.By contrast, the faint combination of M-2 and M-4 display to NAP-16.These results show that M-2 and M-4 is in conjunction with the BoNT in precursor toxin and independent BoNT, and show M-2 and M-4 specific binding BoNT.

Figure 12 is the broken line graph of combining Neutralization effect to BoNT/B of display according to human monoclonal antibodies of the present invention.10LD ₅₀boNT/B and M-2 and M-4 (often kind 0.05,0.1 or 0.5 microgram) mixture incubation and be expelled in mouse.The mouse of the mixture of all M-2 of acceptance (0.5 microgram) and M-4 (0.5 microgram) all survives and does not have symptom.

Figure 13 be illustrate human monoclonal antibodies according to the present invention use after the broken line graph of the Neutralization effect to precursor toxin (16S toxin).Precursor toxin (16S toxin is used to Mouse oral, 10 nanograms), within 12,24 or 36 hours after Orally administered 16S toxin subsequently, use HuMAbs (M-20.5 microgram+M-4 0.5 microgram) by intraperitoneal injection (i.p.injection).Those are not dead in 72 hours by the control mice of HuMAbs process.By contrast, treat after the exposure of HuMAbs to provide for 12 hours after Orally administered 16S toxin and survive completely, within 24 hours and 36 hours, provide part survival after application.

Figure 14 comprise display combine according to human monoclonal antibodies according to the present invention and in and the column diagram of BoNT/B2 (bacterial strain 111) and BoNT/B6 (bacterial strain Osaka05) and broken line graph.HuMAbs (M-2 and M-4) is analyzed by ELISA in conjunction with BoNT/B2 (bacterial strain 111) and BoNT/B6 (bacterial strain Osaka05).HuMAbs (0.5 mcg/ml) adds to the plate being coated with BoNT.After washing, in conjunction with HuMAbs detected by the anti-human IgG antibodies of coupling HRP.M-2 and M-4 display is except also combining BoNT/B2 and BoNT/B6 brute force except BoNT/B1.In neutralization test, with BoNT/B2 (10 nanogram) and BoNT/B6 (2.5 nanogram) during M-2+M-4 (0.5 microgram+0.5 microgram) is complete.

Figure 15 comprises display recombinant human monoclonal antibody according to the present invention and turns stain analysis and broken line graph in conjunction with the west of BoNT/B.With LIPOFECTAMINE 2000 transfection reagent by pQCXIP-hCH and pQCXIH-hCl expression vector transient transfection HEK293 cell.The anti-human igg of the restructuring HuMAbs coupling HRP in culture supernatant measures.Restructuring M-2 (RM-2) and restructuring M-4 (RM-4) are analyzed by ELISA in conjunction with BoNT/B.Restructuring HuMAb (about 0.01-0.5 mcg/ml) adds to the plate being coated with BoNT/B.After washing, in conjunction with restructuring HuMAbs detected by the anti-human IgG antibodies of coupling HRP.RM-2 with RM-4 in the mode identical with M-2 with M-4 deriving from hybridoma in conjunction with BoNT/B.

Figure 16 comprises and carries out west to the HuMAbs and restructuring HuMAbs (RM-2 and RM-4) that derive from hybridoma with the combination of BoNT/B (BoNT/B1, BoNT/B2 or BoNT/B6) and turn stain analysis.BoNT/B1, BoNT/B2 or BoNT/B6 are separated into Hc and Lc by SDS-PAGE, and transfer on nitrocellulose filter.HuMAbs (1.0 mcg/ml) adds to this film, and at room temperature incubation 1 hour.After washing, by the anti-human IgG antibodies of film and coupling HRP at room temperature incubation 1 hour, then specific band is visual by ECL.Restructuring HuMAbs display is identical with the HuMAbs deriving from hybridoma in conjunction with profile.The light chain of M-2 and RM-2 specific binding BoNT/B.By contrast, M-4 and RM-4 (RM-4LC16, RM-4LC18, RM-4LC19 have the heavy chain of identical M-4) is attached to light chain and heavy chain.By M-4 and RM-4 antibody, the heavy chain of B2 hypotype detects clearly than the heavy chain of B1.In addition, the heavy chain of B6 hypotype detects clearly than the heavy chain of B2.

Embodiment

(detailed description of the present invention)

According to the present invention, a kind of anti-Type B botulic neurotoxin monoclonal antibody or its Fab of separation is provided, there is the Neutralization effect of anti-Type B botulic neurotoxin.Monoclonal antibody can comprise human monoclonal antibodies, Humanized monoclonal antibodies or be somebody's turn to do both.The anti-Type B botulic neurotoxin monoclonal antibody be separated or its Fab can have the Neutralization effect of the precursor toxin of anti-Type B botulic neurotoxin.The anti-Type B botulic neurotoxin monoclonal antibody be separated or its Fab can have anti-by Type B clostridium botulinumboNT/B1 (bacterial strain Okra), Type B clostridium botulinumboNT/B2 (bacterial strain 111), Type B clostridium botulinumboNT/B6 (bacterial strain Osaka05) or the Neutralization effect of Type B botulic neurotoxin that produces of its arbitrary combination.Therefore the anti-Type B botulic neurotoxin monoclonal antibody be separated or its Fab can have the specific binding activity of the light chain to Type B botulic neurotoxin (BoNT/B).

Antibody or its fragment can use any suitable technology to produce.Such as, the anti-Type B botulic neurotoxin monoclonal antibody be separated can be produced by hybridoma, this hybridoma by merge come the immunity of personal Type B botulic neurotoxin people peripheral blood lymphocytes (peripheral blood mononuclear cell, PBMC) and can effective cell fusion fusion partner cells be prepared.Type B botulic neurotoxin can be Type B clostridium botulinumboNT/B1 (bacterial strain Okra), Type B clostridium botulinumboNT/B2 (bacterial strain 111), Type B clostridium botulinumboNT/B6 (bacterial strain Osaka05) or the product of its arbitrary combination.Any suitable fusion partner cells can be used, such as SPYMEG cell.

The present invention also provides the hybridoma producing antibody of the present invention or its fragment.Such as, hybridoma can have the preserving number of NITEBP-01639 or NITE BP-01640.The monoclonal antibody of separation is also provided, is produced by the hybridoma with NITE BP-01639 or NITE BP-01640 preserving number.The example of the monoclonal antibody of acquisition described above comprises by the monoclonal antibody of the hybridoma generation of " hybridoma M-2 (M1E9) " (" Hybridoma M-2 (M1E9) ") by name (following, referred to as M-2 antibody) and the monoclonal antibody (hreinafter referred to as M-4 antibody) that produced by the hybridoma of " hybridoma M-4 (M4C9) " (" Hybridoma M-4 (M4C9) ") by name.These hybridomas are deposited in independent administrative legal person's products assessment technique basal disc organization's patent Organism Depositary (the NITE Patent Microorganisms Depositary on June 26th, 2013 with preserving number NITEBP-01639 and NITE BP-01640, National Institute of Technology andEvaluation) (total honest and clean sufficient 2-5-8 in Mu Geng Jinshi City, Chiba, Japan, No. 122 room, postcode 292-0818).Then, according to budapest treaty, they shift as international accession with identical preserving number.

The anti-Type B botulic neurotoxin monoclonal antibody be separated or its Fab can be IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgD, IgE, its any fragment or its arbitrary combination.Antibody fragment can comprise such as Fab, Fab ', F (ab ') 2, scFv, dsFv or its arbitrary combination.The anti-Type B botulic neurotoxin monoclonal antibody be separated or its Fab can comprise heavy chain and/or variable region of light chain.Such as, variable region of heavy chain can comprise: have first complementary determining region (complementarity determining region) (CDR1) of the first aminoacid sequence comprising SEQ ID NO:5 or 19, have second complementary determining region (CDR2) of the second aminoacid sequence comprising SEQ ID NO:6 or 20, have the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:7 or 21.Variable region of light chain can comprise: such as, first complementary determining region (CDR1) with the tetramino acid sequence comprising SEQ ID NO:12 or 26, second complementary determining region (CDR2) with the pentaamino acid sequence comprising SEQ ID NO:13 or 27 and have the 3rd complementary determining region (CDR3) of the 6th nitrogen base acid sequence comprising SEQ ID NO:14 or 28.

In another example, variable region of heavy chain can comprise: first complementary determining region (CDR1) with the first aminoacid sequence comprising SEQ ID NO:5, second complementary determining region (CDR2) with the second aminoacid sequence comprising SEQ ID NO:6, have the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:7.Variable region of light chain can comprise: first complementary determining region (CDR1) with the tetramino acid sequence comprising SEQ ID NO:12, second complementary determining region (CDR2) with the pentaamino acid sequence comprising SEQ ID NO:13 and have the 3rd complementary determining region (CDR3) of the 6th aminoacid sequence comprising SEQID NO:14.

In another example, variable region of heavy chain can comprise: first complementary determining region (CDR1) with the first aminoacid sequence comprising SEQ ID NO:19, second complementary determining region (CDR2) with the second aminoacid sequence comprising SEQ ID NO:20 and have the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:21.Variable region of light chain can comprise: such as, first complementary determining region (CDR1) with the tetramino acid sequence comprising SEQ ID NO:26, second complementary determining region (CDR2) with the pentaamino acid sequence comprising SEQ ID NO:27 and have the 3rd complementary determining region (CDR3) of the 6th aminoacid sequence comprising SEQ ID NO:28.In further example, variable region of heavy chain can comprise SEQ ID NO:3 or 17, and variable region of light chain can comprise SEQ ID NO:10 or 24.

The invention provides pharmaceutical composition.Pharmaceutical composition can comprise one or more anti-Type B botulic neurotoxin monoclonal antibody be separated and its Fab and pharmaceutically acceptable carriers.Pharmaceutical composition can comprise anti-Type B botulic neurotoxin monoclonal antibody and pharmaceutically acceptable carrier.Pharmaceutical composition can comprise two kinds be separated anti-Type B botulic neurotoxin monoclonal antibodies, its Fab or both.Pharmaceutical composition can comprise the first and second antibody.First human monoclonal antibodies be separated or Fab can comprise: such as, (1) human monoclonal antibodies be separated or the Fab of variable region of heavy chain and variable region of light chain is comprised, variable region of heavy chain comprises: first complementary determining region (CDR1) with the first aminoacid sequence comprising SEQ ID NO:5, there is second complementary determining region (CDR2) of the second aminoacid sequence comprising SEQ ID NO:6, with the 3rd complementary determining region (CDR3) with the triamino acid sequence comprising SEQ ID NO:7, variable region of light chain comprises: first complementary determining region (CDR1) with the tetramino acid sequence comprising SEQ ID NO:12, there is second complementary determining region (CDR2) of the pentaamino acid sequence comprising SEQ ID NO:13, with the 3rd complementary determining region (CDR3) with the 6th aminoacid sequence comprising SEQ ID NO:14.First human monoclonal antibodies be separated or Fab can comprise: such as, (2) human monoclonal antibodies be separated or the Fab of variable region of heavy chain and variable region of light chain is comprised, variable region of heavy chain comprises SEQ ID NO:3, and variable region of light chain comprises SEQ ID NO:10.First human monoclonal antibodies be separated or Fab can comprise: such as, the monoclonal antibody that (3) are separated, and it is produced by the hybridoma with preserving number NITE BP-01639.First human monoclonal antibodies be separated or Fab can comprise arbitrary combination as escribed above.Second human monoclonal antibodies be separated or Fab can comprise: such as, (4) human monoclonal antibodies be separated or the Fab of variable region of heavy chain and variable region of light chain is comprised, variable region of heavy chain comprises: first complementary determining region (CDR1) with the first aminoacid sequence comprising SEQ ID NO:19, there is second complementary determining region (CDR2) of the second aminoacid sequence comprising SEQ ID NO:20, with the 3rd complementary determining region (CDR3) with the triamino acid sequence comprising SEQ ID NO:21, variable region of light chain comprises: first complementary determining region (CDR1) with the tetramino acid sequence comprising SEQ ID NO:26, there is second complementary determining region (CDR2) of the pentaamino acid sequence comprising SEQ IDNO:27, with the 3rd complementary determining region (CDR3) with the 6th aminoacid sequence comprising SEQ ID NO:28.Second human monoclonal antibodies be separated or Fab can comprise: such as, (5) human monoclonal antibodies be separated or the Fab of variable region of heavy chain and variable region of light chain is comprised, variable region of heavy chain comprises SEQ ID NO:17, and variable region of light chain comprises SEQ ID NO:24.Second human monoclonal antibodies be separated or Fab can comprise: such as, the monoclonal antibody that (6) are separated, and it is produced by the hybridoma with preserving number NITE BP-01640.Second human monoclonal antibodies be separated or Fab can comprise arbitrary combination as escribed above.

The invention provides the method for generation of the anti-Type B botulic neurotoxin monoclonal antibody be separated.The method can comprise by merge come the immunity of personal Type B botulic neurotoxin people peripheral blood lymphocytes (PBMC) and can effective cell fusion fusion partner cells produce hybridoma.The method may further include and obtains Type B botulic neurotoxin monoclonal antibody from hybridoma.Type B botulic neurotoxin, such as, pass through Type B clostridium botulinumboNT/B1 (bacterial strain Okra), Type B clostridium botulinumboNT/B2 (bacterial strain 111), Type B meat poisoning shuttle shape brood cell bacillusboNT/B6 (bacterial strain Osaka05) or its arbitrary combination produce.Fusion partner cells can be such as SPYMEG cell.

The invention provides for prevent, treat and detect Type B botulic neurotoxin in human experimenter poisoning in the test kit of at least one.Test kit can comprise the anti-Type B botulic neurotoxin monoclonal antibody of separation of the present invention, its Fab or both.Test kit can also comprise and be used for the treatment of and/or detect one or more poisoning other reagent of Type B botulic neurotoxin.Test kit can also comprise one or more reagent of the botulic neurotoxin being used for the treatment of and/or detecting one or more other types such as A type.The present invention also provides the anti-Type B botulic neurotoxin monoclonal antibody of separation, its Fab or both are for the preparation of suppressing or the purposes of the medicine of sausage poisoning in treatment human experimenter.

The invention provides the method suppressing or treat sausage poisoning in human experimenter.Method can comprise the anti-Type B botulic neurotoxin monoclonal antibody of the separation to human experimenter's administering therapeutic significant quantity, its Fab or both.It is poisoning that method can also comprise diagnosis patient botulic neurotoxin.Method can comprise the minimizing of at least one symptom of monitoring sausage poisoning.At least one symptom can comprise: such as, paralysis, symmetrical parmlysis of cranial nerve, Rearrangments flaccid paralysis, the paralysis of voluntary muscle symmetry, pharyngeally to subside, breath stopped, cannot suck, cannot swallow, sound reduction, blepharoptosis, facial paralysis, pupil is fixed, platycoria, blurred vision, soft neck, whole-body muscle relaxes, general weakness, diplopia, dysarthria, dysphonia, dysphagia, lossless, erythroplakia, postural hypotension, feel sick, constipation, uroschesis, dizzy, dry, throat pain, underdevelopment (suppressed), pharyngeal reflex, the amyotrophy of whole body reflectivity, other symptom of sausage poisoning, or its arbitrary combination.In human experimenter, can suppress or treat the sausage poisoning of any type, such as, source property sausage poisoning, wound infection sausage poisoning, baby's enterotoxemia sausage poisoning, adult's enterotoxemia sausage poisoning, iatrogenic sausage poisoning, airborne transmission sausage poisoning or its arbitrary combination is eaten.

Suppress or anti-Type B botulic neurotoxin monoclonal antibody that in treatment human experimenter, the method for sausage poisoning can comprise co-administered separation, its Fab or both and for one or more other treatments of sausage poisoning.Associating can act synergistically suppress or treat sausage poisoning.One or more other treatments can comprise: such as, use the anti-Type B botulic neurotoxin monoclonal antibody of the second separation, its Fab or both.One or more other treatments can comprise: such as, use the anti-A type botulic neurotoxin monoclonal antibody of separation, its Fab or both.One or more other treatments can comprise such as one or more microbiotic, such as penicillin or its arbitrary combination.

The invention provides the method detecting Type B botulic neurotoxin in human experimenter.Method can comprise by from sample and the anti-Type B botulic neurotoxin monoclonal antibody be separated of human experimenter, its Fab or both contact.Method can also comprise based on antibody, its fragment or both whether detect the presence or absence of Type B botulic neurotoxin in human experimenter in conjunction with Type B botulic neurotoxin.Method can also comprise the botulic neurotoxin detecting one or more other type, such as A type.

Anti-botulic neurotoxin antibody and the polypeptide containing its Fab are provided, and adopt their method, purposes, composition and test kit.Except as otherwise noted, describing with the claimed monoclonal antibody be separated and Fab thereof in the application is not natural product.These monoclonal antibodies and Fab thereof are the products of hybridoma or the equivalent artificial cell system that uses various experimental technique to produce.Use antibody or its Fab to be used for the treatment of, diagnose or the antibody of the method for other objects, the composition comprising them, the test kit comprising them and/or non-natural generation or antibody fragment are not limited thereto, unless expressly stated.

The method formed the specific antibody of botulic neurotoxin or its polypeptide or fragment is provided.Such method can comprise: the nucleic acid providing coding botulic neurotoxin antigenic peptide or the polypeptide containing its immunologic opsonin epi-position; The polypeptide of antigen aminoacid sequence or the polypeptide containing its immunologic opsonin epi-position is contained from the expression of nucleic acid be separated; With the antibody produced the polypeptid specificity obtained, or the polypeptide containing its Fab.The antibody produced by preceding method or the polypeptide containing its Fab are provided.The antibody of the separation of specific binding botulic neurotoxin antigen or the polypeptide containing its Fab of separation are provided.Any acceptable method as known in the art can be used to produce such antibody.The other side of antibody of the present invention and test kit, method and/or use antibody can comprise one or more following materials: polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, univalent antibody, double antibody and/or humanized antibody.

The antibody structural unit of natural generation comprises the tetramer usually.Each such tetramer can form identical polypeptide chain by two, and often pair has total length " gently " chain (such as, about 1kDa to 25kDa) and total length " weight " chain (such as, about 50kDa-70kDa).The amino terminus portion of every bar chain generally includes about 100 to 110 or more an amino acid whose variable region of being usually responsible for antigen recognition.The carboxy-terminal sections of every bar chain limits the constant region may being responsible for effector function usually.People's light chain is divided into κ and lambda light chain usually.Heavy chain be usually divided into μ, 6, γ, α or ε, and the isotype limiting antibody is respectively IgM, IgD, IgG, IgA and IgE.IgG has several subclass, includes but not limited to IgG1, IgG2, IgG3 and IgG4.IgM has subclass, includes but not limited to IgM1 and IgM2.IgA is divided into subclass similarly, includes but not limited to IgA1 and IgA2.In light chain and heavy chain, variable region and constant region can be connected by about 12 or more amino acid whose " J " district, and heavy chain also comprises about 10 or more individual amino acid whose " D " districts.See such as: basic immunology the 7th chapter (Fundamental Immunology Ch.7.Paul, W., ed., 2nd ed.Raven Press, N.Y (1989)).The variable region that each light/heavy chain is right forms antigen binding site usually.

Variable region shows usually by three hypervariable regions, the identical usual structure of relatively conservative framework region (framework region, FR) that connects also referred to as complementary determining region (complementarity determiningregion) or CDR.CDR from two chains of every a pair is aimed at by framework region usually, and it can make to be attached to specificity epitope.From N-terminal to C-terminal, light chain and variable region of heavy chain comprise structural domain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 usually.Amino acid to the distribution of each structural domain normally according to Kabat sequence (the KabatSequences of Proteins of Immunological Interest.National Institutes of Health of albumen interested in immunology, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J.MoI.Biol.196:901-917 (1987); The restriction of Chothia et al., Nature342:878-883 (1989).

" antibody fragment " comprises a part for complete antibody, and the antigen as complete antibody combines or variable region.The example of antibody fragment comprises: Fab, Fab1, F (ab ') 2, and Fv fragment; Double antibody; Linear antibodies (Zapata et al., Protein Eng.8 (10): 1057-1062 [1995]); Single-chain antibody molecules; With the multi-specificity antibody formed by antibody fragment.Papain digestion of antibodies produces two identical Fabs, is called " Fab " fragment, has an independent antigen binding site separately, and " Fc " fragment that remaining, and name reflects the ability being easy to crystallization.Pepsin produces F (ab ') 2 fragment, and it has two antigen binding sites and still can crosslinking antigen." Fv " is the antibody fragment containing a complete antigen recognition and binding site.This region comprise a heavy-chain variable domains and light variable domains closely, the dimer of Non-covalent binding.Independent variable domains (or the half of Fv only containing 3 antigen-specific CDR) can identify and conjugated antigen." scFv " or " sFv " antibody fragment comprises VH and the VL structural domain of antibody, and wherein these structural domains are present in single polypeptide chain.Fv polypeptide can be included in further between VH and VL structural domain and to make sFv form the peptide linker of the desired structure combined for antigen.For the summary of sFv, see Pluckthun in ThePharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994).

Antibody can be used as probe, therapeutic treatment and other purposes.Antibody can by being prepared the peptide fragment injection mouse of translation product or its synthesis, rabbit, goat or other animal.These antibody are useful in diagnositc analysis or as the activeconstituents in pharmaceutical composition.

The antibody used or polypeptide can be coupled to functional agent to form immune conjugate.Functional agent can be cytotoxic agent such as chemotherapeutics, toxin (such as, the enzymatic activity toxin of bacterium, fungi, plant or animal-origin, or its fragment) or radio isotope (that is, radiating conjugate), microbiotic, nucleolytic enzyme or its arbitrary combination.Chemotherapeutics can be used in the generation of immune conjugate, such as, methotrexate (methotrexate), Zorubicin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine(VCR) (vincristine), vinealeucoblastine(VLB) (vinblastine), Etoposide (etoposide)), Dx (doxorubicin), melphalan (melphalan), ametycin (mitomycin C), Chlorambucil (chlorambucil), daunorubicin (daunorubicin) or other intercalator, enzyme, and/or its fragment, as nucleolytic enzyme (nucleolytic enzyme), microbiotic, with toxin such as small molecule toxins or bacterium, fungi, the enzymatic activity toxin of plant or animal-origin, comprise its fragment and/or variant, and the various antitumour drug disclosed below or anticarcinogen.Operable enzymatic activity toxin and fragment thereof comprise: such as, diphtheria A chain (diphtheria A chain), the nonbinding active fragments of diphtheria toxin, exotoxin A chain (exotoxin A chain) (from Pseudomonas aeruginosa (Pseudomonasaeruginosa)), ricin A chain (ricin A chain), abrin A chain (abrin A chain), modeccin A chain (modeccin A chain), the bent toxalbumin (alpha-sarcin) of α-broom, Aleurites fordii proteins (Aleurites fordiiprotein), oleanolic acid albumen (dianthin protein), dyers' grapes albumen (Phytolacca americana protein) (PAPI, PAPII, and PAP-S), momordica charantia inhibitor (momordica charantia inhibitor), curcin (curcin), crotin (crotin), crystal soda grass inhibitor (saponaria officinalis inhibitor), gelonin (gelonin), mitogillin (mitogellin), Restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and Trichothecenes toxin (trichothecenes).Known in the art or otherwise available any suitable radioactive nucleotides or radioreagent may be used for the antibody producing radioactivity coupling.

The conjugate of antibody and cytotoxic agent can use multiple bifunctional protein coupling agent as N-succinimido-3-(2-pyridine dimercapto) propionic ester (N-succinimidyl-3-(2-pyridyldithiol) propionate, SPDP); Iminothiolane (iminothiolane, IT); The dual-function derivative (such as dimethyl imido-ester hydrochloride (dimethyl adipimidate HCL)) of imido-ester (imidoester); Active ester (such as disuccinimidyl suberate (disuccinimidylsuberate)); Aldehyde (such as glutaraldehyde (glutaraldehyde)); Double azido compound (such as two (to azidobenzoyl) hexanediamine (bis (p-azidobenzoyl) hexanediamine)); Dual azepine derivatives (such as two (to diazo benzoyl)-quadrol (bis-(p-diazoniumbenzoyl)-ethylenediamine)); Vulcabond (such as toluene 2,6-vulcabond (tolyene 2,6-diisocyanate)); Double activated fluorine cpd (such as 1,5-bis-fluoro-2,4-dinitrobenzenes (1,5-difluoro-2,4-dinitrobenzene)); Maleimidocaproyl (maleimidocaproyl, MC); Valine-citrulline, two peptide sites (valine-citrulline, VC) of proteolytic enzyme cleavable joint; The p-amino-benzene methylcarbamoyl (p-aminobenzylcarbamoyl) of 2-amino-5-ureido pentanoic acid (2-amino-5-ureido pentanoic acid) PAB=(" certainly coming off " part of joint) (Citrulene); N-methyl-α-amino-isovaleric acid citrulline (N-methyl-valine citrulline), its center tap peptide bond has been modified to prevent it by cathepsin B's cracking (Me); Maleimidocaproyl-polyoxyethylene glycol, is connected to antibody halfcystine; N-succinimido 4-(2-pyridine dimercapto) valerate (N-Succinimidyl 4-(2-pyridylthio) pentanoate, SPP); Be prepared with N-succinimido 4-(N-maleimidomehyl) hexanaphthene-1 carboxylicesters (N-succinimidyl4-(N-maleimidomethyl) cyclohexane-l carboxylate, SMCC).Such as, ricin immunotoxin can according to Vitetta et al., and the method described in Science, 238:1098 (1987) is prepared.1-isothiocyanatobenzyl-3-methyl diethyl pentetic acid (the l-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaac ctic acid of carbon-14 mark, MX-DTPA) be a kind of exemplary sequestrant for radioactive nucleotides being coupled to antibody, see WO 94/11026.Antibody can be coupled to " acceptor " (as Streptavidin) for tumour target in advance, wherein antibody-receptor conjugate is applied to experimenter, then use scavenging agent from the unconjugated conjugate of circulation removing, then " part " (as the avidin) being coupled to cytotoxic agent (such as, radioactive nucleotides) is used.

Antibody of the present invention directly or indirectly can be coupled to detectable by technology well known in the art.Detectable is that one is such as by the detectable reagent of spectroscopy, photochemistry, biological chemistry, immunochemistry or chemical means.Useful detectable includes but not limited to, fluorescence dye, chemiluminescence compound, radio isotope, electron dense bodies reagent, enzyme, coloured particle, vitamin H or digoxin (digoxigenin).Detectable often produces measurable signal, such as radioactivity, fluorescence, color or enzymic activity.Be coupled to and the antibody of detection reagent can may be used for diagnosis or therapeutic purpose.The example of detection reagent can comprise various enzyme, prothetic group, fluorescent material, luminescent material, bioluminescent material, radio active material, the positron emitting metal of the various positron emission tomography art of use and on-radiation paramagnetic metal ion.Detectable substance can use technology known in the art directly or by intermediate as such as joint known in the art indirectly with antibody coupling or combination.See such as No. 4741900th, United States Patent (USP), describe metallic ion coupled to antibody for diagnostic uses.The example of suitable enzyme comprises horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes and acetylcholinesterase; The example of suitable prosthetic group complexes comprises Streptavidin/vitamin H and avidin/biotin; The example of suitable fluorescent material comprises Umbelliferone (umbelliferone), fluorescein (fluorescein), fluorescein isothiocyanate (fluorescein isothiocyanate), rhodamine (rhodamine), dichlorotriazine ammonia fluorescein (dichlorotriazinylamine fluorescein), dansyl chloride (dansyl chloride) and phycoerythrin (phycoerythrin); An example of luminescent material comprises luminol,3-aminophthalic acid cyclic hydrazide (luminol); The example of bioluminescent material comprises luciferin (luciferin) and aequorin (aequorin).

Following method can be used to prepare in laboratory animal or by recombinant DNA technology putting into practice antibody useful in the present invention.Polyclonal antibody can by repeatedly subcutaneous (subcutaneous in animal, sc) or intraperitoneal (intraperitoneal, ip) inject gene product molecule or its fragment combination adjuvant in parallel such as freund's adjuvant (completely or not exclusively) and cultivate.In order to strengthen immunogenicity, use difunctional or derivative reagent, such as, maleimidobenzoyl thiosuccimide ester (maleimidobenzoyl sulfosuccinimide ester) (by cysteine residues coupling), N-hydroxy-succinamide (passing through lysine residue), glutaraldehyde, succinyl oxide, sulfur oxychloride (socl) etc., first the gene product molecule containing target amino acid sequence or fragment being coupled in the species treating immunity is on immunogenic albumen, such as keyhole limpet hemocyanin (keyhole limpet hemocyanin), serum albumin (serum albumin), bovine thyroid element sphaeroprotein (bovine thyroglobulin), or Trypsin inhibitor SBTI (soybean trypsin inhibitor), may be useful.Selectively, immunogenic conjugate can produce as fusion rotein restructuring.

Can with immunogenic conjugate or derivative (fragment such as containing target amino acid sequence), combine about 1 milligram or about 1 microgram conjugate (respectively for rabbit or mouse) and this solution of multiple intradermal injections by the Freund's complete adjuvant of about 3 times of volumes, carry out immune animal.After about 7 to 14 days, gather the blood of animal and measure the antibody titers of serum.Repeatedly use antigen booster immunization animal until titre arrives titre platform place.Can carry out booster immunization with the identical molecule used with initial immunity or its fragment to animal, but it is from different albumen coupling and/or by different linking agent coupling.In addition, agglutinant such as alum can be used in injection and strengthen immunne response to improve.

The antibody used can comprise chimeric antibody.The antibody used can comprise humanized antibody.The antibody used can comprise the antibody of full-length human.Antibody can be humanized or part-humanised.Non-human antibody can use any applicable method as known in the art to carry out humanization.Humanized antibody can use immunity system partly or the transgenic animal of full-length human produce.Any antibody of the present invention or its fragment can be partially or completely humanized.Chimeric antibody can use any known technology in this area to produce.See such as No. 5169939th, United States Patent (USP), No. 5750078, No. 6020153, No. 6420113, No. 6423511, No. 6632927 and No. 6800738.

The antibody used can comprise monoclonal antibody, that is, can be the of the present invention anti-botulic neurotoxin antibody of monoclonal antibody.Monoclonal antibody can use hybridoma method those methods as described by Kohler and Milstein, Nature, 256:495 (1975) to be prepared.In hybridoma method, mouse, hamster or other suitable host animal carry out immunity with immunoreagent usually, maybe can produce the lymphocyte of the antibody of specific binding immunoreagent to cause generation.Selectively, lymphocyte can carry out immunity in vitro.Monoclonal antibody can as such as at Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988); Screen as described in Goding, Monoclonal Antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986).Monoclonal antibody can be tested to the specific immune response of translation product with lack with corresponding prototype gene product immunoreactivity.

Monoclonal antibody can by reclaim from immune animal splenocyte and in a usual manner, such as by making cell infinite multiplication to be prepared with myeloma cell fusion.Preferably, peripheral blood lymphocytes (PBMC) and the myeloma cell fusion of the people of personal Clostridium botulinum toxoid immunity is made.Then clone is screened to obtain those and express the clone expecting antibody.Monoclonal antibody preferably not with other gene product generation cross reaction.After the hybridoma expected obtains determining, clone can carry out subclone by limited dilution method, and is grown by standard method.Suitable substratum for this object comprises: such as, DMEM (Dulbecco ' s Modified Eagle ' s Medium) and RPMI-1640 substratum.Selectively, hybridoma can grow as ascites in mammalian body.The monoclonal antibody of being secreted by subclone can by conventional immune globulins purification process as such as Protein A-agarose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography carry out isolated or purified from substratum or ascites.

Monoclonal antibody can also pass through recombinant DNA method, and those methods as described in No. 4816567th, United States Patent (USP) are prepared.The DNA of monoclonal antibody of the present invention of encoding can use ordinary method (such as, by use can the heavy chain of specific binding encoding murine antibody and the oligonucleotide probe of the gene of light chain) easily to carry out being separated and checking order.Hybridoma of the present invention can as the preferred source of this DNA.Once be separated, DNA can be placed in expression vector, then be transfected into otherwise can not produce immunoglobulin (Ig) host cell as in monkey COS cell, Chinese hamster ovary (CHO) cell or myeloma cell, to obtain the synthesis of monoclonal antibody in recombinant host cell.DNA also can such as, by the encoding sequence of people's heavy chain and light chain constant domain is replaced homology mouse sequence or by all or part of immunoglobulin coding sequence that is covalently bond to of the encoding sequence being used for NIg polypeptide is modified.This NIg polypeptide can replace with the constant domain of antibody of the present invention, maybe can replace with the variable domains of an antigen binding site of antibody of the present invention, to form chimeric bivalent antibody.Use recombinant DNA method such as phage display method Dispersal risk can use commercially available test kit, such as, from Pharmacia (Uppsala, Sweden) available restructuring phagemid antibody forming system, or SurfZAP ^tMphage display system (Stratagene Inc., La Jolla, Califorinia) has come.The present invention uses mouse-people's mosaic fusion partner cells system, and SPYMEG by name, provides the human monoclonal antibodies (HuMAbs) of anti-A type BoNT (BoNT/A) and Type B BoNT (BoNT/B).

Also comprise in the present invention: produce the hybridoma cell line of monoclonal antibody of the present invention, the B cell system of conversion and host cell; The B cell system of these hybridomas, conversion and the filial generation of host cell or derivative; And equivalent or similar hybridoma, the B cell system of conversion and host cell.

Antibody can be double antibody.Term " double antibody " refers to the little antibody fragment with two antigen binding sites, and fragment comprises the heavy-chain variable domains (VH) of the light variable domains (VL) be connected in identical polypeptide chain (VH-VL).Not allowing by using too short joint that same chain matches between two structural domains, the complementary domain of structural domain and another chain can be forced to match, and form two antigen binding sites.Double antibody is more completely described in such as EP404097, W093/11161 and Hollinger et al., in Proc.Natl.Acad.Sci.USA, 90:6444-6448 (1993).

The antibody used can comprise single-chain antibody.Antibody can be univalent antibody.The method preparing univalent antibody is well known in the art.Such as, a kind of method relates to recombinant expressed light chain immunoglobulin and modifies heavy chain.Heavy chain can block by any point in Fc district usually, thus prevents heavy chain to be cross-linked.Selectively, relevant cysteine residues replaces to other amino-acid residue or removal, thus prevents from being cross-linked.In vitro method is also suitable for preparing univalent antibody.Antibody digestion is to produce its fragment, and especially Fab fragment, can use routine techniques as known in the art.

Antibody can be dual specific.Use and described standard method generation in the literature, separated island form specific binding albumen specific binding and pathology and/or the bi-specific antibody for the treatment of other relevant antigen.(see such as: Pluckthun & Pack, Immunotechnology, 3:83-105 (1997); Carter, et al., J.Hematotherapy, 4:463-470 (1995); Renner & Pfreundschuh, Immunological Reviews, 1995, No.145, pp.179-209; No. 5643759th, Pfreundschuh United States Patent (USP); Segal, et al., J.Hematotherapy, 4:377-382 (1995); Segal, et al., Immunobiology, 185:390-402 (1992); And Bolhuis, et al., Cancer Immunol.Immunother., 34:1-8 (1991)).

Antibody disclosed here can make immunoliposome.Liposome containing antibody is prepared by methods known in the art, as at Epstein et al., and Proc.Natl.Acad.Sci.USA, 82:3688 (1985); Hwang et al., Proc.Natl.Acad.Sci.USA77:4030 (1980); And describe in United States Patent (USP) the 4485045th and No. 4544545.The liposome that cycling time extends is disclosed in No. 5013556th, United States Patent (USP).Useful especially liposome can be produced by the lipid composition of reverse phase evaporation containing phosphatidylcholine, cholesterol and PEG derivatization phospholipid acyl thanomin (PEG-PE).Liposome can be extruded by the filter limiting aperture, to obtain the liposome with desired diameter.Fab ' the fragment of antibody of the present invention can be coupled to liposome, as being described in Martin et al., in J.Biol.Chem., 257:286-288 (1982) through disulfide exchange reaction.Chemotherapeutics (as Dx) is included in liposome alternatively.See Gabizon et al, J.National Cancer Inst., 81 (19): 1484 (1989).

The invention provides and suppress or the method for sausage poisoning in treatment human experimenter, comprise the of the present invention anti-botulic neurotoxin monoclonal antibody to human experimenter's administering therapeutic significant quantity, its Fab or both.It is poisoning that method can also comprise diagnosis patient botulic neurotoxin.Anti-botulic neurotoxin antibody of the present invention or its Fab can diagnosis patient have botulic neurotoxin poisoning before, central and/or be applied to experimenter afterwards.Method can also comprise the minimizing of the poisoning at least one symptom of monitoring botulic neurotoxin.

According to the present invention, two or more botulic neurotoxin antagonists can be used.At least one botulic neurotoxin antagonist can comprise botulic neurotoxin antagonist.At least one botulic neurotoxin antagonist can combine one or more other botulic neurotoxin antagonists.At least one botulic neurotoxin antagonist can be used by combined needle one or more other medicines that are poisoning to botulic neurotoxin and/or Clostridium botulinum infection.Use the medicine of two or more, comprise one or more botulic neurotoxin antagonists, can be simultaneously, order or associating.Therefore, when using two or more medicines, they do not need simultaneously or use in the same manner or with same dose.When using simultaneously, two or more medicines can be used with same composition or different compositions.Two or more medicines can use identical route of administration or different route of administration and use.When using in the different time, medicine can be used before or after each other.The order of administration of two or more medicines can replace.The respective dosage of one or more medicines can change in time.The type of one or more medicines can change in time.When using splitting time, the separation of using for twice or more time can be cycle any time.If repeatedly used, the length of time cycle can change.Separation between the using of two or more medicines can be 0 second, 1 second, 5 seconds, 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hour, 2 hours, 2.5 hour, 3 hours, 4 hours, 5 hours, 7.5 hour, 10 hours, 12 hours, after 15 hours, after 18 hours, 21 hours, 24 hours, 1.5 my god, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 6 weeks, 8 weeks, 3 months, 6 months, 1 year or longer.

Two or more botulic neurotoxin antagonists can act synergistically to treat or reduce poisoning or its symptom of botulic neurotoxin.The symptom of sausage poisoning comprises: such as, paralysis, symmetrical parmlysis of cranial nerve, Rearrangments flaccid paralysis, the paralysis of voluntary muscle symmetry, pharyngeally to subside, breath stopped, cannot suck, cannot swallow, sound reduction, blepharoptosis, facial paralysis, pupil is fixed, platycoria, blurred vision, soft neck, whole-body muscle relaxes, general weakness, diplopia, dysarthria, dysphonia, dysphagia, lossless, erythroplakia, postural hypotension, feel sick, constipation, uroschesis, dizzy, dry, throat pain, underdevelopment, pharyngeal reflex, with the amyotrophy of whole body reflectivity.Sausage poisoning can in the lab by proving that the toxin in the sample of clinical sample or food intake confirms.Wound is cultivated and produced organism is highly point out Symptomatic situation.Sausage poisoning approves to be undertaken by Mouse bioassay really.Show that toxin serotypes exists in clinical sample by paralysing in specific toxinicide and in mouse.

Botulic neurotoxin antagonist can be one or more independent anti-botulic neurotoxin antibody or combine one or more other botulic neurotoxin antagonists, such as, little pharmaceutical preparation, or the medicine of other anti-botulic neurotoxin.The example of little pharmaceutical preparation comprises microbiotic, as penicillin.Other anti-botulinal medicine comprises septivalency Clostridium botulinum toxinicide (heptavalent botulinum antitoxin, HBAT).The antibody fragment that HBAT can originate containing the horse serum for neurotoxin A-G, and can comprise: such as, be less than the complete immunoglobulin (Ig) of about 2.0%, and be more than or equal to Fab and F (ab ') 2 antibody fragment of about 90%.The antibody of two or more anti-botulic neurotoxins, or the antibody of the anti-botulic neurotoxin of at least one and one or more other medicines can act synergistically, to treat or to reduce botulic neurotoxin poisoning.Two or more medicines, comprise one or more anti-botulic neurotoxin antibody, can use with collaborative dosage.Therefore, no matter using of two or more medicines, be simultaneously, sequentially or with any combined administration, can have synergy in the minimizing of poisoning one or more symptoms of botulic neurotoxin.First medicine can increase effect of the second medicine, be used alone, or the second medicine increases effect of the first medicine if be greater than the second medicine, or both.The effect using two or more medicines can be that such so that in one or more symptoms that minimizing botulic neurotoxin is poisoning effect is greater than often kind of addition effect used separately.When giving with collaborative dosage, medicine can strengthen the effect of one or more other medicines in the minimizing of poisoning one or more symptoms of botulic neurotoxin, even if one or more symptoms poisoning to botulic neurotoxin are not had remarkable efficacy separately by the amount of one or more medicines.Synergistic measurement and calculation, can as at Teicher, " Assays for In Vitro and In VivoSynergy; " in Methods in Molecular Medicine, vol.85:Novel Anticancer Drug Protocols, described in pp.297-321 (2003) and/or by use CalcuSyn computed in software association index (combination index, CI) carry out.

Definite preparation, route of administration and dosage can be selected by the state of an illness of solo practitioner depending on patient.(see such as: Fingl et.al., in The Pharmacological Basis ofTherapeutics, 1975, Ch.1 p.I.) attending doctor can determine when stop due to toxicity or organ dysfunction, interrupt or adjust administration.On the contrary, if get rid of toxicity, clinical response is not enough, and attending doctor also can adjust treatment to higher level.In the management of interested disease, the amplitude of dosage changes with the severity of disease to be treated and route of administration.The severity of disease can such as part be evaluated by standard prognostic evaluation method.Dosage and administration frequency can change according to the age of individual patient, body weight and reaction.With above similar program is discussed and can be used in veterinary science.

Using pharmaceutically acceptable carrier to be mixed with for putting into practice compound of the present invention the formulation being suitable for Formulations for systemic administration by disclosed here, is within the scope of the present invention.By suitably selecting carrier and suitable manufacturing practice, composition related to the present invention, particularly those are mixed with the composition of solution, can administered parenterally, such as, used by intravenous injection.These compounds can use pharmaceutically acceptable carrier well known in the art to be easily mixed with the formulation being suitable for oral administration.Such carrier makes the compound relevant with the present invention be formulated as tablet, pill, capsule, liquid, gelifying agent, syrup, paste, dragee, solution, suspension etc., for patient's orally ingestible to be treated.

Therapeutical agent can be prepared with sustained release drug (depot) form, to allow to be released in the health that it uses (see such as No. 4450150th, United States Patent (USP)) relative to the position control in time and health.The sustained release drug form of therapeutical agent can be: such as, and containing therapeutical agent and porous or non-porous material as the implanted composition of polymkeric substance, wherein therapeutical agent is by the degraded encapsulation of material and/or non-porous material or diffusion.Then sustained release drug is implanted to the desired location in health, and therapeutical agent discharges from implant with predetermined speed.

Use therapeutical agent in the present invention can be formed as composition, as the pharmaceutical composition containing carrier and therapeutic compound.Pharmaceutical composition containing therapeutical agent can comprise more than one therapeutical agent.Pharmaceutical composition selectively can comprise therapeutical agent and combine other forms of pharmacologically active agents or medicine.

Carrier can be any suitable carrier.Such as, carrier can be pharmaceutically acceptable carrier.Relative to pharmaceutical composition, carrier can be consider chemical physics Consideration, such as solvability and lack the reactivity with active compound, and by those carriers that route of administration routine uses.Except drug regimen beyond the region of objective existence described below, or selectively, the therapeutic compound of the inventive method can be mixed with inclusion compound, such as cyclodextrin inclusion compound, or liposome.

Pharmaceutically acceptable carrier described here, such as mediator (vehicle), adjuvant, vehicle and thinner, know for those skilled in the art, and be easy to obtain for the public.Pharmaceutically acceptable carrier can be the carrier of inertia, under conditions of use with no harmful side-effects or toxicity to chemically promoting agent.The selection of carrier, can partly determine by specific therapeutical agent and by the ad hoc approach for administering therapeutic compound.There is the suitable preparation of multiple pharmaceutical composition of the present invention.For oral, spraying, parenteral, subcutaneous, through skin, in mucous membrane, intestines, intramedullary injection, directly ventricle, in intravenously, nose, in intraocular, intramuscular, intra-arterial, sheath, the following preparation of intraperitoneal, rectum and vagina administration is exemplary, and be never restriction.More than one approach can be used for administering therapeutic agent, and in some cases, a kind of specific approach can provide quicker and more effective response than another kind of approach.According to the concrete disease for the treatment of, these medicaments can be prepared and whole body or topical.Can at Remington ' s Pharmaceutical Sciences with the technology of administration for preparing, 18th ed., Mack Publishing Co., Easton, Pa. find in (1990).

The preparation being suitable for oral administration can comprise: (a) liquor, such as, be dissolved in the inhibitor of thinner as the significant quantity of water, salt solution or orange juice; B () capsule, sachet, tablet, lozenge (lozenges) and lozenge (troches), contain the activeconstituents of predetermined amount separately with solid or particle form; (c) powder; (d) suspension in suitable liquid; And the emulsion that (e) is suitable.Liquid preparation can comprise thinner, as water and alcohol such as ethanol, phenylcarbinol and polyvinyl alcohol, adds simultaneously or does not add pharmaceutically acceptable tensio-active agent.Capsule form can be containing such as tensio-active agent, lubricant and inert filler, as the common hard or soft-shelled gelatin type of lactose, sucrose, calcium phosphate and W-Gum.Tablet form can comprise one or more of vehicle of lactose, sucrose, mannitol, W-Gum, yam starch, alginic acid, Microcrystalline Cellulose, gum arabic, gelatin, guar gum, silica colloidal, croscarmellose sodium, talcum powder, Magnesium Stearate, calcium stearate, Zinic stearas, stearic acid and other vehicle, tinting material, thinner, buffer reagent, disintegrating agent, wetting agent, sanitas, seasonings and other pharmaceutically compatible.Lozenge (lozenges) form can containing the inhibitor be generally at seasonings in sucrose and gum arabic or tragacanth, and in inert base is as gelatin and glycerine or sucrose and Sudan Gum-arabic, emulsion, gel etc., comprise the pastille (pastille) of inhibitor, it is except inhibitor, also comprises vehicle known in the art.

The pharmaceutical preparation that can orally use comprises the capsule of slippaging (push-fit capsule) be made up of gelatin, and the capsule of the soft sealing of being made up as glycerine or Sorbitol Powder of gelatin and softening agent.Capsule of slippaging can comprise the activeconstituents mixed as talcum powder or Magnesium Stearate and optional stablizer as starch and/or lubricant as lactose, tackiness agent with filler.In soft capsule, active compound can be dissolved or suspended in suitable liquid as in fatty oil, whiteruss or liquid macrogol.In addition, stablizer can be added.

Therapeutical agent individually or combine other suitable component, can be made via inhalation aerosol formulation.These aerosol formulations can be placed in acceptable propelling agent such as Refrigerant 12, propane, the nitrogen etc. of pressurization.They also can be mixed with for non-pressurised preparation as the medicine in atomizer or spraying gun.This spray agent can also be used for spray mucosa.Topical formulations is known for those of ordinary skill in the art.Such preparation is particularly suitable for being applied to skin in context of the present invention.

Injectable formulation is according to the invention.For the parameter of the active drug carrier of Injectable composition, for those of ordinary skill in the art be know (see such as: Pharmaceutics and Pharmacy Practice, J.B.Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238250 (1982), with ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622630 (1986)).During for injecting, reagent of the present invention can be prepared in aqueous, and the damping fluid of preferred PHYSIOLOGICALLY COMPATIBLE is as in Han Keshi solution (Hanks ' ssolution), Ringer's solution (Ringer ' s solution) or normal saline buffer solution.For such mucosal, use in the formulation for the suitable permeate agent of barrier to be infiltrated.Such permeate agent is well known in the art usually.

The preparation being suitable for administered parenterally can comprise water-based and without water-based isotonic sterile injection solution, its solute that can contain antioxidant, buffer reagent, fungistat and make the blood of preparation and target recipient isotonic, and can comprise suspending agent, solubilizing agent, thickening material, stablizer and sanitas water-based and without water-based sterile suspensions.Therapeutical agent can be used in pharmaceutical carrier in physiologically acceptable thinner, this thinner is as sterile liquid or comprise water, salt solution, D/W and relevant sugar soln, alcohol is as ethanol or hexadecanol, dibasic alcohol is as propylene glycol or polyoxyethylene glycol, PEG 400, glycerine, dimethyl sulfoxide (DMSO), ketal is as 2, 2-dimethyl-1, 3-dioxolane-4-methyl alcohol, ether, oil, lipid acid, fatty acid ester or glyceryl ester, or the mixture of the liquid of acetylated fatty acid glyceride, add or do not add pharmaceutically acceptable tensio-active agent, as soap class (soap) or washing composition, suspending agent is as pectin, carbomer, methylcellulose gum, Vltra tears or carboxymethyl cellulose, or emulsifying agent and other medicinal adjuvant.

Oil can be used in parenteral administration, comprises oil, animal oil, vegetables oil or synthetic oil.The object lesson of oil comprises peanut oil, soybean oil, sesame oil, Oleum Gossypii semen, Semen Maydis oil, sweet oil, paraffin oil and mineral oil.Suitable lipid acid for parenteral administration comprises oleic acid, stearic acid and Unimac 5680.Ethyl oleate and Isopropyl myristate are the examples of suitable fatty acid ester.

Fatty alkali metal salt is comprised for the suitable soap class be used in parenteral administration, ammonium salt, and triethanolamine salt, suitable washing composition comprises: (a) cationic detergent, as such as dimethyl dialkyl ammonium halide and alkylpyridinium halides, (b) teepol, as such as alkyl, aryl and alkene sulfonate, alkyl, alkene, ether and monoglyceride sulfates, and sulfosuccinate, (c) non-ionic octoxynol detergent, as such as fatty amine oxide, fatty acid alkyl amide, with polyoxyethylene polypropylene multipolymer, (d) zwitter-ion washing agent, as such as alkyl-β-alanine ester, with 2-alkyl imidazoline quaternary ammonium salt, and (e) its mixture.

Parenteral administration in the solution can containing have an appointment 0.5 % by weight to about 25 % by weight medicine.Sanitas and damping fluid can be used.In order to reduce or eliminate the stimulation on injection site to greatest extent, this based composition can containing one or more nonionogenic tensides of Hydrophilic Lipophilic Balance (HLB) with about 12 to about 17.The amount of the tensio-active agent in such preparation by usual in the scope of about 5 % by weight to about 15 % by weight.Suitable tensio-active agent comprises polyoxyethylene glycol sorbitan fatty acid esters, as dehydrated sorbitol mono-fatty acid ester and oxyethane and the high molecular weight adducts of hydrophobic matrix that formed by propylene oxide and propylene glycol condensation.Parenteral administration may reside in unitary dose or multiple doses sealed vessel as in ampoule and bottle, and under can being stored in lyophilize (freeze-drying) condition, during for injecting, relating to and adding sterile liquid excipient such as water before use immediately.Interim injection solution and suspension can be prepared from the sterilized powder of previously described kind, particle and tablet.

Therapeutical agent can by mixing with various matrix, as emulsifying base or water-soluble base and make suppository.The preparation being suitable for vagina administration can be rendered as vaginal suppository, suppository, ointment, gelifying agent, paste, foaming agent or sprays, in addition to the active ingredient (s, also containing, for example these carriers suitably known in the art.

The technology that the reagent being intended to use in cell can use those of ordinary skill in the art to know is used.Such as, such reagent can be encapsulated in liposome.Liposome is the spherical lipid bilayer with aqueous interior.The molecule be present in the aqueous solution enters aqueous interior when liposome is formed.Liposomal contents is both protected from external microenvironment, and because liposome and cell membrane fusion, liposomal contents is delivered in tenuigenin effectively.In addition, due to their hydrophobicity, organic molecule can directly be used in cell.The materials and methods described for one aspect of the present invention also may be used for other side of the present invention.Such as, describe material such as nucleic acid or the antibody be used in screening strength and also can be used as therapeutical agent, vice versa.

The present invention includes the following aspect/specific embodiment/feature of random order and/or any combination:

1, the anti-Type B botulic neurotoxin monoclonal antibody of separation or its Fab, comprise the Neutralization effect of anti-Type B botulic neurotoxin, wherein said monoclonal antibody comprise human monoclonal antibodies, Humanized monoclonal antibodies or both.

2, the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below or its Fab, wherein said monoclonal antibody or its Fab also comprise the Neutralization effect of the precursor toxin of anti-Type B botulic neurotoxin.

3, the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below or its Fab, wherein said monoclonal antibody or its Fab comprise anti-by Type B meat poisoning shuttle shape bud born of the same parents bacillusboNT/B1 (bacterial strain Okra), Type B clostridium botulinumboNT/B2 (bacterial strain 111), Type B meat poisoning clostridiumboNT/B6 (bacterial strain Osaka05) or the Neutralization effect of Type B botulic neurotoxin that produces of its arbitrary combination.

4, the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below or its Fab, wherein said monoclonal antibody or its Fab have the specific binding activity of the light chain to Type B botulic neurotoxin (BoNT/B).

5, the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below or its Fab, wherein said human monoclonal antibodies is by the peripheral blood lymphocytes (PBMC) by merging the people carrying out personal Clostridium botulinum toxoid immunity and can the hybridoma generation that is prepared of the fusion partner cells that merges of effective cell.

6, the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below or its Fab, wherein said Type B botulic neurotoxin is Type B clostridium botulinumboNT/B1 (bacterial strain Okra), Type B clostridium botulinumboNT/B2 (bacterial strain 111), Type B meat poisoning shuttle shape brood cell bacillusboNT/B6 (bacterial strain Osaka05) or the product of its arbitrary combination.

7, the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below or its Fab, wherein said fusion partner cells is SPYMEG cell.

8, the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below or its Fab, comprise IgG, Fab, Fab ', F (ab ') 2, scFv, dsFv or its arbitrary combination.

9, the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below or its Fab, comprising:

Variable region of heavy chain, comprising:

There is first complementary determining region (CDR1) of the first aminoacid sequence comprising SEQ ID NO:5 or 19,

There is second complementary determining region (CDR2) of the second aminoacid sequence comprising SEQ ID NO:6 or 20, and

There is the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:7 or 21; With

Variable region of light chain, comprising:

There is first complementary determining region (CDR1) of the tetramino acid sequence comprising SEQ ID NO:12 or 26,

There is second complementary determining region (CDR2) of the pentaamino acid sequence comprising SEQ ID NO:13 or 27, and

There is the 3rd complementary determining region (CDR3) of the 6th aminoacid sequence comprising SEQ ID NO:14 or 28.

10, the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below or its Fab, comprising:

Variable region of heavy chain, comprising:

There is first complementary determining region (CDR1) of the first aminoacid sequence comprising SEQ ID NO:5,

There is second complementary determining region (CDR2) of the second aminoacid sequence comprising SEQ ID NO:6, and

There is the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:7; With

Variable region of light chain, comprising:

There is first complementary determining region (CDR1) of the tetramino acid sequence comprising SEQ ID NO:12,

There is second complementary determining region (CDR2) of the pentaamino acid sequence comprising SEQ ID NO:13, and

There is the 3rd complementary determining region (CDR3) of the 6th aminoacid sequence comprising SEQ ID NO:14.

11, the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below or its Fab, comprising:

Variable region of heavy chain, comprising:

There is first complementary determining region (CDR1) of the first aminoacid sequence comprising SEQ ID NO:19,

There is second complementary determining region (CDR2) of the second aminoacid sequence comprising SEQ ID NO:20, and

There is the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:21; With

Variable region of light chain, comprising:

There is first complementary determining region (CDR1) of the tetramino acid sequence comprising SEQ ID NO:26,

There is second complementary determining region (CDR2) of the pentaamino acid sequence comprising SEQ ID NO:27, and

There is the 3rd complementary determining region (CDR3) of the 6th aminoacid sequence comprising SEQ ID NO:28.

12, the Type B botulic neurotoxin monoclonal antibody of any specific embodiment/feature/aspect above or below or its Fab, comprising:

Variable region of heavy chain, comprises SEQ ID NO:3 or 17, and

Variable region of light chain, comprises SEQ ID NO:10 or 24.

13, the Type B botulic neurotoxin monoclonal antibody of any specific embodiment/feature/aspect above or below, wherein said antibody is IgG1.

14, a hybridoma, has the preserving number of NITE BP-01639 or NITE BP-01640.

15, a monoclonal antibody for separation, is produced by the hybridoma of the preserving number with NITE BP-01639 or NITE BP-01640.

16, a pharmaceutical composition, comprises anti-Type B botulic neurotoxin monoclonal antibody or its Fab of the separation of one or more any specific embodiment/feature/aspects or below above, and pharmaceutically acceptable carrier.

17, the pharmaceutical composition of any specific embodiment/feature/aspect above or below, comprises anti-Type B botulic neurotoxin monoclonal antibody and pharmaceutically acceptable carrier.

18, the pharmaceutical composition of any specific embodiment/feature/aspect above or below, comprise two kinds of anti-Type B botulic neurotoxin monoclonal antibodies be separated, its Fab or both, comprising:

First human monoclonal antibodies be separated or its Fab, comprising:

(1) human monoclonal antibodies be separated or Fab, comprising:

Variable region of heavy chain, comprise: first complementary determining region (CDR1) with the first aminoacid sequence comprising SEQ ID NO:5, second complementary determining region (CDR2) with the second aminoacid sequence comprising SEQ ID NO:6 and there is the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:7, and

Variable region of light chain, comprise: first complementary determining region (CDR1) with the tetramino acid sequence comprising SEQ ID NO:12, second complementary determining region (CDR2) with the pentaamino acid sequence comprising SEQ ID NO:13 and there is the 3rd complementary determining region (CDR3) of the 6th aminoacid sequence comprising SEQ ID NO:14

(2) human monoclonal antibodies be separated or Fab, comprise variable region of heavy chain and variable region of light chain, and described variable region of heavy chain comprises SEQ ID NO:3, and described variable region of light chain comprises SEQ ID NO:10,

(3) monoclonal antibody be separated, is produced by the hybridoma with preserving number NITE BP-01639,

Or its arbitrary combination; With

Second human monoclonal antibodies be separated or its Fab, comprising:

(4) human monoclonal antibodies be separated or Fab, comprising:

Variable region of heavy chain, comprise: first complementary determining region (CDR1) with the first aminoacid sequence comprising SEQ ID NO:19, second complementary determining region (CDR2) with the second aminoacid sequence comprising SEQ ID NO:20 and there is the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:21, and

Variable region of light chain, comprise: first complementary determining region (CDR1) with the tetramino acid sequence comprising SEQ ID NO:26, second complementary determining region (CDR2) with the pentaamino acid sequence comprising SEQ ID NO:27 and there is the 3rd complementary determining region (CDR3) of the 6th aminoacid sequence comprising SEQ ID NO:28

(5) human monoclonal antibodies be separated or Fab, comprise variable region of heavy chain and variable region of light chain, and described variable region of heavy chain comprises SEQ ID NO:17, and described variable region of light chain comprises SEQ ID NO:24,

(6) monoclonal antibody be separated, is produced by the hybridoma with preserving number NITE BP-01640,

Or its arbitrary combination.

19, for generation of a method for the anti-Type B botulic neurotoxin monoclonal antibody be separated, comprising:

By merge come personal Clostridium botulinum toxoid immunity people peripheral blood lymphocytes (PBMC) and can effective cell merge fusion partner cells generation hybridoma; With

Type B botulic neurotoxin monoclonal antibody is obtained from described hybridoma.

20, the method for generation of the anti-Type B botulic neurotoxin monoclonal antibody be separated of any specific embodiment/feature/aspect above or below, wherein said Type B botulic neurotoxin passes through Type B clostridium botulinumboNT/B1 (bacterial strain Okra), Type B clostridium botulinumboNT/B2 (bacterial strain 111), Type B meat poisoning shuttle shape brood cell bacillusboNT/B6 (bacterial strain Osaka05) or its arbitrary combination produce.

21, the method for generation of anti-Type B botulic neurotoxin monoclonal antibody of any specific embodiment/feature/aspect above or below, wherein said fusion partner cells is SPYMEG cell.

22, for prevent, treat and detect Type B botulic neurotoxin in human experimenter poisoning in the test kit of at least one, comprise the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below, its Fab or both.

23, to suppress or the method for sausage poisoning in treatment human experimenter, comprise the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below to human experimenter's administering therapeutic significant quantity, its Fab or both.

24, the method for sausage poisoning in the suppression of any specific embodiment/feature/aspect above or below or treatment human experimenter, also comprises diagnosis patient botulic neurotoxin poisoning.

25, the method for sausage poisoning in the suppression of any specific embodiment/feature/aspect above or below or treatment human experimenter, also comprises the minimizing of at least one symptom of monitoring sausage poisoning.

26, the method of sausage poisoning in the suppression of any specific embodiment/feature/aspect above or below or treatment human experimenter, wherein said at least one symptom comprises paralysis, symmetrical parmlysis of cranial nerve, Rearrangments flaccid paralysis, the paralysis of voluntary muscle symmetry, pharyngeally to subside, breath stopped, cannot suck, cannot swallow, sound reduction, blepharoptosis, facial paralysis, pupil is fixed, platycoria, blurred vision, soft neck, whole-body muscle relaxes, general weakness, diplopia, dysarthria, dysphonia, dysphagia, lossless, erythroplakia, postural hypotension, feel sick, constipation, uroschesis, dizzy, dry, throat pain, underdevelopment, pharyngeal reflex, the amyotrophy of whole body reflectivity, or its arbitrary combination.

27, the method for sausage poisoning in the suppression of any specific embodiment/feature/aspect above or below or treatment human experimenter, wherein said sausage poisoning comprises food source property sausage poisoning, wound infection sausage poisoning, baby's enterotoxemia sausage poisoning, adult's enterotoxemia sausage poisoning, iatrogenic sausage poisoning, airborne transmission sausage poisoning or its arbitrary combination.

28, the method for sausage poisoning in the suppression of any specific embodiment/feature/aspect above or below or treatment human experimenter, wherein, the anti-Type B botulic neurotoxin monoclonal antibody of the separation of described any specific embodiment/feature/aspect above or below, its Fab or both, combined needle is used one or more other treatments of sausage poisoning.

29, the method for sausage poisoning in the suppression of any specific embodiment/feature/aspect above or below or treatment human experimenter, wherein said joint synergy is to suppress or treatment sausage poisoning.

30, the method for sausage poisoning in the suppression of any specific embodiment/feature/aspect above or below or treatment human experimenter, one or more other treatments wherein said comprise use the second separation anti-Type B botulic neurotoxin monoclonal antibody, its Fab or both.

31, the method for sausage poisoning in the suppression of any specific embodiment/feature/aspect above or below or treatment human experimenter, one or more other treatments wherein said comprise use separation anti-A type botulic neurotoxin monoclonal antibody, its Fab or both.

32, the anti-Type B botulic neurotoxin monoclonal antibody of the separation of any specific embodiment/feature/aspect above or below, its Fab or both are for the preparation of the purposes of medicine for suppressing or treat sausage poisoning in human experimenter.

33, detect a method for Type B botulic neurotoxin in human experimenter, comprising:

By the anti-Type B botulic neurotoxin monoclonal antibody be separated of sample and any specific embodiment/feature/aspect above or below from human experimenter, its Fab or both contact; With

Based on antibody, its fragment or both whether detect the presence or absence of Type B botulic neurotoxin in human experimenter in conjunction with Type B botulic neurotoxin.

The present invention is by by being intended to exemplary instead of limiting following example of the present invention and illustrate further.These examples prove the surprising of antibody required for protection and beyond thought characteristic, comprise such as in them and the ability that infects of botulism and treatment sausage poisoning.These examples prove the exploitation according to the human monoclonal antibodies of anti-botulic neurotoxin of the present invention.Digital proof treatment of the present invention and diagnostic uses.

These examples prove the light chain of M-2 antibodies specific in conjunction with BoNT/B, and the Neutralization effect demonstrating brute force in Mouse bioassay (is about more than or equal to 100LD ₅₀/ milligram).In addition, the associating of M-2 and M-4 can be greater than 10,000LD ₅₀the effect of/milligram completely in and BoNT/B.This effect is the most effective, exceedes for the anti-BoNT antiserum(antisera) of commercially available BABYBIG people (540LD ₅₀/ milligram) in the BoNT/B that designs and standard 18 times.Treat in model after exposure, when when toxin oral administration 12 hours internal therapies, HuMAbs (M-2+M-4) provides the protection completely to lethal dose BoNT/B.Except BoNT/B1, HuMAbs (M-2+M-4) also neutralize other hypotype, BoNT/B2 and BoNT/B6.The data obtained from the experiment of the human monoclonal antibodies with M-2 and M-4 represent usually according to antibody of the present invention and generation and their method of use.

Embodiment

Embodiment 1

Type B meat poisoning shuttle shape brood cell liver bacteriumbacterial strain Okra uses cellophane tube program (cellophane-tube procedure) to cultivate, and obtains culture supernatant.Precursor toxin (16S toxin and 12S toxin) adopts (Arimitsu et al., Infect.Immun.71:1599-1603,2003) to carry out purifying from culture supernatant.What describe as (Arimitsu et al., Infect.Immun.71:1599-1603,2003) prepares BoNT/B1 and NAP-16 from 16S toxin.BoNT/A1 (bacterial strain 62A), BoNT/B2 (bacterial strain 111) and BoNT/B6 (bacterial strain Osaka05) are provided by doctor S.Kozaki.Tetravalence Clostridium botulinum toxoid vaccine is provided by doctor M.Takahashi.12S toxin carries out purifying from the respective culture supernatant of A, B, E, F type clostridium botulinum and uses formalin detoxification.In detoxicated employing mouse peritoneal, (i.p.) detects.The toxoid prepared product of Four types mixes, and then mixes with aluminium (adjuvant) and Thiomersalate (sanitas).Toxoid prepared product every milliliter is containing Four types each 0.1 milligram, 0.2 milligram aluminium, 0.001% Thiomersalate, and 0.0006% formalin.

Immunization schedule follow as in FIG describe.Tetravalence Clostridium botulinum toxoid (A, B, E and F type) is inoculated in two healthy adult human's (4 or 5 times), and it has been submitted Written informed consent and has received physical examination.Toxoid only with O.5ml dosage intramuscularly to healthy individuals.After 9 and 18 days, from each volunteer's peripheral blood sample (10 milliliters).Blood sample, from each volunteer, uses ELISA method to test the antibody titers of anti-BoNT/A or BoNT/B of plasma sample.This experimental program is ratified by the human trial council of Osaka University.

Enzyme linked immunosorbent assay (ELISA) is carried out as follows.With the BoNT/A of 100 microlitres or (with) BoNT/B wraps by 96 hole flat board (Falcon) 2 hours with the ultimate density of 3 micrograms in PBS/ml at 37 DEG C.Hole is washed with PBS (PBS-T) containing 0.05% tween, closedly to spend the night at 4 DEG C with 0.2%BSA (Sigma)/PBS-T.The serial dilution of human serum sample's twice, often kind of diluent 50 microlitre to be joined in hole and at 37 DEG C incubation 2 hours.After washing, add the coupling horseradish peroxidase (HRP) of 50 microlitres anti-human igg (BIO-RAD) and at 37 DEG C incubation 2 hours.After washing, by plate substrate solution (O-Phenylene Diamine, nacalai tesque) incubation 30 minutes at 37 DEG C, be determined at the absorbance at OD492 place.ELISA titre represents with most highly diluted multiple, and display is greater than the absorbancy of the twice of negative control sera.9 or 18 days antibody titerss rising (table 1) in two volunteers after inoculation.

Table 1

The table 1. ELISA titre of the blood plasma of the people volunteer of Clostridium botulinum tetravalence toxoid inoculation

ELISA titre represents with most highly diluted multiple, and display is greater than the absorbancy of the twice of negative control sera.Antibody titers rising in two volunteers in 9 or 18 days after inoculation.

Embodiment 2

Cytogamy, screening and Cloning processes carry out as described in fig. 2.Latter 9 or 18 days of immunity the last time, peripheral blood sample is from volunteer, and peripheral blood lymphocytes (PBMC) carries out purifying by Ficoll (GE Healthcare) gradient centrifugation.PBMC and SPYMEG cell merges with polyoxyethylene glycol (Roche) with the ratio of 10 to 1.The cell merged cultivates about 10-14 days in 96 orifice plates in supplemented with the DMEM (DMEM, GIBCO) of 15%FBS under xanthoglobulin-aminopterin-induced syndrome-thymidine (HAT, GIBCO) exists.Select through HAT, carry out substratum with ELISA and the first time of the specific antibody of BoNT/A or BoNT/B is screened.Consequently, from the blood sample that inoculation gathers for latter 9 days, obtain 27 positive holes, from the blood sample that inoculation gathers for latter 18 days, obtain 8 positive holes.Cell clone is then carried out by limiting dilution in the positive hole of specific antibody, obtains the cell of cloning.Also carry out programmed screening by ELISA.Obtain 8 stable hybridomas clone (table 2).

Table 2

The result of table 2. first time and programmed screening

Isotype analysis shows that M-1, M-2, M-4 and S-1 are IgG, M-3, M-5 and M-6 be IgM, M-7 is IgA (table 3).

Table 3

The determination of the isotype of table 3.HuMAbs

Obtain 8 hybridoma clones from two volunteers, 4 hybridoma clones produce IgG, and 3 hybridoma clones produce IgM, and 1 hybridoma clone produces IgA.

M-1, M-2, M-4 and S-1 are IgG, M-3, M-5 and M-6 be IgM, M-7 is IgA.

Each stable hybridoma is cultivated in serum free medium (GIBCO), then collects supernatant liquor.The anti-human IgA (Invitrogen) of the anti-human igg (BIO-RAD) of the HuMAbs isotype coupling HRP in culture supernatant, the anti-human IgM (BIOSOURCE INTERNATIONAL) of coupling HRP or coupling HRP turns in stain analysis in west and measures.IgG antibody carries out purifying by Protein G post (GE Healthcare) from supernatant liquor.The subclass of each IgG antibody uses IgG subclass people's ELISA kit (IgG subclass human ELISA kit, Invitrogen) to measure.

Embodiment 3

Carry out the Cloning and sequencing of the variable region gene of monoclonal antibody.The heavy chain determined and the sequence of variable region of light chain are presented in Fig. 3 ~ Fig. 8.Total serum IgE uses RNeasy Mini test kit (Qiagen) to extract from hybridoma according to the explanation of manufacturers, uses SUPERSCRIPT ^(R)vILOTM cDNA synthetic agent box (Invitrogen) is produced by RT-PCR.Increased by PCR KOD-Pus-Neo (Toyobo) and following primer in the coding region of H-and the L-chain of M-2 and M-4 antibody: 5 '-ATGGACTGGACCTGGAGGATCCTC-3 ' (M-2-H-chain sense primer) (SEQ ID NO:35), 5 '-ATGAAACACCTGTGGTTCTTCCTCCT-3 ' (M-4-H-chain sense primer) (SEQ ID NO:36), and 5 '-CTCCCGCGGCTTTGTCTTGGCATTA-3 ' (H-chain antisense primer) (SEQ ID NO:38); With 5 '-ATGGCCTGGWYYCCTCTCYTYCTS-3 ' (M-4-16-L-chain sense primer) (SEQ ID NO:38), 5 '-ATGSCCTGGGCTCYKCTSCTCCTS-3 ' (M-2-and M-4-18-L-chain sense primer) (SEQ ID NO:39), 5 '-ATGGCCTGGRYCYCMYTCYWCCTM-3 ' (M-4-19-L-chain sense primer) (SEQ ID NO:40), and 5 '-TGGCAGCTGTAGCTTCTGTGGGACT-3 ' (L-chain antisense primer) (SEQ ID NO:41).With TaKaRa Ex Taq ^(R)(Takara) after incubation, PCR primer is connected to pGEM-T Easy carrier (Promega), their sequence uses BigDye Terminator v3.1 cycle sequencing test kit and ABI Prism 3100 genetic analyzer (Applied Biosystems) to analyze.IgG subclass analysis display M-2 and M-4 is made up of IgG1 heavy chain and light (λ) chain.

Embodiment 4

BoNT/A or BoNT/B (600 nanogram) is separated into Hc and Lc by SDS-PAGE, and transfers on nitrocellulose filter (BIO-RAD).Film is closed in the Tris buffer saline containing 0.05%Tween with containing 5% skim-milk.HuMAbs (1.0 mcg/ml) joins this film, and at room temperature incubation 1 hour.After washing, by the anti-human IgG antibodies of film and coupling HRP at room temperature incubation 1 hour, then specific band ECL (PIERCE) visual (Fig. 9 A and 9B).

Embodiment 5

In order to determine the epi-position of HuMAbs, carrying out west and turning stain analysis.Result shows the light chain (Figure 10) of M-2 specific binding BoNT/B.In contrast, M-4 is attached to light chain and heavy chain (Figure 10).

Embodiment 6

HuMAb is analyzed by ELISA in conjunction with Type B BoNT, precursor toxin or non-toxic components (NAP-16).HuMAbs joins bag by the plate of BoNT, precursor toxin or NAP-16.HuMAbs (0.01 mcg/ml ~ 0.5 mcg/ml) joins bag by the plate of BoNT/A or BoNT/B.After washing, in conjunction with HuMAbs detected by the anti-human IgG antibodies of coupling HRP.M-2 and M-4 is attached to BoNT and precursor toxin.By contrast, M-2 and M-4 demonstrates the faint combination to NAP-16.These results show that M-2 and M-4 is attached to the BoNT in precursor toxin, and independent BoNT, and M-2 and M-4 specific binding BoNT.M-2 and M-4 shows specific binding BoNT/B (Figure 11).

Embodiment 7

The Neutralization effect of HuMAbs is tested by Mouse bioassay.Before intraperitoneal injection (cumulative volumes with 500 microlitres) to mouse (ddY, female, 4 weeks, SLC), HuMAbs and BoNT/B (10LD ₅₀) at room temperature incubation 1 hour.10LD ₅₀boNT/B and 100 microgram HuMAb or PBS (Cnt) incubation 1 hour, and peritoneal injection is in mouse.Observe mouse invasion rate and mortality ratio 3 weeks.The mouse of injection BoNT/B+PBS (Cnt) is dead in 12 hours.In contrast, the mouse of injection BoNT/B+M-4 is partly protected, and as compared with control mice, proved by the death time increased.On the other hand, protect completely injection BoNT/B+M-2 mouse in observe (table 4).

Table 4

The Neutralization effect of the anti-BoNT/B of table 4. HuMAb in Mouse bioassay

The Neutralization effect of the anti-BoNT/B of HuMAbs is determined by Mouse bioassay.10LD ₅₀boNT/B and 100 microgram HuMAb or PBS (Cnt) incubation 1 hour, and peritoneal injection is in mouse.The mouse of injection BoNT/B+PBS (Cnt) is dead in 12 hours.In contrast, the mouse of injection BoNT/B+M-4 is partly protected, and as compared with control mice, proved by the death time increased.On the other hand, protect completely injection BoNT/B+M-2 mouse in observe.

Embodiment 8

Synergy test has been carried out in combining of HuMAbs.10LD ₅₀boNT/B and M-2 and mixture (often kind of 0.5 microgram, in the volume of the 500 microlitres) incubation of M-4, and to be expelled in mouse.The mouse survival of all HuMAbs of acceptance and do not have symptom (Figure 12).

Embodiment 9

Treat after exposure in model, Mouse oral uses Type B precursor toxin (16S toxin, 10 nanograms are in 300 microlitre volumes), use HuMAbs (M-2+M-4) by intraperitoneal injection in 12,24 or 36 hours after Orally administered 16S toxin subsequently.Observe mouse invasion rate and mortality ratio 3 weeks.Treat after exposure in model, after Orally administered 16S toxin 12 hours, mouse produces the symptom of sausage poisoning.Not dead in 72 hours by the control mice of HuAbs treatment.In contrast, treat after the exposure of HuMAbs and provide whole survival in 12 hours after Orally administered 16S toxin, within 24 and 36 hours, provide part to survive (Figure 13) after application.

Embodiment 10

HuMAbs (M-2 and M-4) is analyzed by ELISA in conjunction with BoNT/B2 (bacterial strain 111) and BoNT/B6 (bacterial strain Osaka05).HuMAbs (0.5 mcg/ml) joins bag by the plate of BoNT.After washing, in conjunction with HuMAbs detected by the anti-human IgG antibodies of coupling HRP.Except BoNT/B1, M-2 and M-4 display goes back brute force in conjunction with BoNT/B2 and BoNT/B6.In neutralization test, with BoNT/B2 (10 nanogram) and BoNT/B6 (2.5 nanogram) (Figure 14) during M-2+M-4 (0.5 microgram+0.5 microgram) is complete.

Embodiment 11

In order to express restructuring HuMAbs and test them in mammalian cell (HEK293 cell), IgG expression vector builds as follows.PGEM-T Easy carrier with the variable region gene of H-and L-chain carries out PCR to add restriction endonuclease sites and Kozak sequence with following primer pair (restriction endonuclease sites underscore represents): 5 '-ATTT gC gGCCGCcATGGACTGGACCTGGAGG-3 ' (M2-H-chain sense primer) (SEQ ID NO:42), 5 '-ATTT gCGGCCGCcATGAAACACCTGTGGTTCTTC-3 ' (M-4-H-chain sense primer) (SEQ IDNO:43) and 5 '-ATA cTCGAGgGTGCCAGGGGGAAGACCGATG-3 ' (H-chain antisense primer) (SEQ ID NO:44); With 5 '-ATTT gCGGCCGCcATGGCCTGGTTTCCTCTCTTC-3 ' (M-4-16-L-chain sense primer) (SEQ ID NO:45), 5 '-ATTT gCGGCCGCcATGGCCTGGGCTCTGCT-3 ' (M-2-and M-4-18-L-chain sense primer) (SEQ ID NO:46), 5 '-ATTT gCGGCCGCcATGGCCTGGGTCTCATT-3 ' (M-4-19-L-chain sense primer) (SEQ ID NO:47), and 5 '-ATA cTCGAGgGCGGGAACAGAGTGACCGTGG-3 ' (L-chain antisense primer) (SEQ ID NO:48).PCR primer restriction enzyme Not I and the Xho I enzyme in H-and L-chain encoding district are cut, and are then connected in expression vector pQCXIP-hCH and pQCXIH-hC, and it has the human normal immunoglobulin constant region (MBL) of γ and λ chain respectively.

HEK293 cell is at 5%C0 ₂in at 37 DEG C containing 10%FBS MEM in cultivate.Explanation according to manufacturers uses LIPOFECTAMINE2000 transfection reagent (Invitrogen), and the cell pQCXIP-hCH that 100 millimeters of culture dish grow and pQCXIH-hC expression vector carry out transient transfection.After transfection, substratum is removed, washed cell at 5%C0 in Opti-Pro serum free medium (Gibco) ₂in at 37 DEG C incubation 10 days.The anti-human igg (BIO-RAD) of the restructuring HuMAbs coupling HRP in culture supernatant measures.Restructuring HuMAbs carries out purifying by Protein G post (GEHealthcare) from supernatant liquor.

Restructuring HuMAbs (RM-2 and RM-4; Be depicted as RM-4LC16, RM-4LC18, RM-4LC19, there is the heavy chain identical with M-4 and light chain, but there is the unlike signal sequence of the light chain be presented at respectively in Fig. 6,7,8) analyzed by ELISA in conjunction with BoNT/B.RM-2 and RM-4 (about 0.01 mcg/ml ~ 0.5 mcg/ml) joins plate.After washing, in conjunction with restructuring HuMAbs detected by the anti-human IgG antibodies of coupling HRP.Consequently, RM-2 with RM-4 is attached to BoNT/B (Figure 15) in the mode identical with M-2 with M-4 deriving from hybridoma.In neutralization test, with BoNT/B1 (10LD in RM-2+RM-4 (0.5 microgram+0.5 microgram) ₅₀) (table 5A and 5B).

Table 5A

The Neutralization effect of the anti-BoNT/B of restructuring HuMAb

The Neutralization effect of the anti-BoNT/B of restructuring HuMAbs is determined by Mouse bioassay.10LD ₅₀boNT/B and M-2 (RM-2) and the mixture incubation of M-4 (RM-4) (each 0.5 microgram), and to be expelled in mouse.In all combinations all of M-2, RM-2, M-4 and RM-4 and BoNT/B.

Table 5B

The Neutralization effect of the anti-BoNT/B of restructuring HuMAb

The Neutralization effect of the anti-BoNT/B of restructuring HuMAbs is determined by Mouse bioassay.10LD ₅₀boNT/B and M-2 (RM-2) and the mixture incubation of M-4 (RM-4) (each 0.5 microgram), and to be expelled in mouse.In all combinations all of M-2, RM-2, M-4 and RM-4 and BoNT/B.

Embodiment 12

The HuMAbs and restructuring HuMAbs (RM-2 and RM-4) that derive from hybridoma are analyzed by SDS-PAGE in conjunction with BoNT/B (BoNT/B 1, BoNT/B2 or BoNT/B6).BoNT/B1, BoNT/B2 or BoNT/B6 are separated into Hc and Lc by SDS-PAGE, and transfer on nitrocellulose filter.HuMAbs (1.0 mcg/ml) joins this film, and at room temperature incubation 1 hour.After washing, by the anti-human IgG antibodies of film and coupling HRP at room temperature incubation 1 hour, then specific band is visual by ECL.Restructuring HuMAbs display is identical with the HuMAbs deriving from hybridoma in conjunction with profile.The light chain of M-2 and RM-2 specific binding BoNT/B.By contrast, M-4 and RM-4 is attached to light chain and heavy chain.By M-4 and RM-4 antibody, the heavy chain of B2 hypotype detects clearly than the heavy chain of B1.In addition, the heavy chain of B6 hypotype detects clearly (Figure 16) than the heavy chain of B2.

The full content of the reference of all references is merged in this disclosure by applicant clearly.In addition, when quantity, concentration or other values or parameter provide as the list of scope, preferable range or a row preferred upper limit value and preferred lower limit value, be appreciated that any all scopes formed for a pair for specifically disclosing by any range limit or preferred value and any range lower limit or preferred value, no matter whether scope is open separately.When numerical range quoted in this literary composition, unless otherwise mentioned, this scope is intended to comprise its end points and all integers within the scope of this and mark.But and do not mean that the particular value that limit of the present invention is enumerated when confining spectrum.

Consider from this specification sheets disclosed here and enforcement of the present invention, other embodiment of the present invention it will be apparent to those skilled in the art that.Mean that this specification sheets and embodiment are only considered as exemplary, the true scope and spirit of the invention is represented by following claim and its equivalent.

PCT/RO/134 shows

Claims (33)

1. be separated anti-Type B botulic neurotoxin monoclonal antibody or its Fab, comprise the Neutralization effect of anti-Type B botulic neurotoxin, wherein said monoclonal antibody comprise human monoclonal antibodies, Humanized monoclonal antibodies or both.

2. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 1 or its Fab, wherein said monoclonal antibody or its Fab also comprise the Neutralization effect of the precursor toxin of anti-Type B botulic neurotoxin.

3. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 1 or its Fab, wherein said monoclonal antibody or its Fab comprise anti-by Type B clostridium botulinumboNT/B1 (bacterial strain Okra), Type B clostridium botulinumboNT/B2 (bacterial strain 111), Type B meat poisoning shuttle shape bacillusboNT/B6 (bacterial strain Osaka05) or the Neutralization effect of Type B botulic neurotoxin that produces of its arbitrary combination.

4. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 1 or its Fab, wherein said monoclonal antibody or its Fab have the specific binding activity of the light chain to Type B Clostridium botulinum Nervous toxicity rope (BoNT/B).

5. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 1, wherein said human monoclonal antibodies by by merge come personal Clostridium botulinum toxoid immunity people peripheral blood lymphocytes (PBMC) and can effective cell merge fusion partner cells be prepared hybridoma generation.

6. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 5, wherein said Type B botulic neurotoxin is Type B clostridium botulinumboNT/B1 (bacterial strain Okra), Type B meat poison clostridiumboNT/B2 (bacterial strain 111), Type B clostridium botulinumboNT/B6 (bacterial strain Osaka05) or the product of its arbitrary combination.

7. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 5, wherein said fusion partner cells is SPYMEG cell.

8. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 1 or its Fab, comprise IgG, Fab, Fab ', F (ab ') 2, scFv, dsFv or its arbitrary combination.

9. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 1 or its Fab, comprising:

Variable region of heavy chain, comprising:

There is first complementary determining region (CDR1) of the first aminoacid sequence comprising SEQ ID NO:5 or 19,

There is second complementary determining region (CDR2) of the second aminoacid sequence comprising SEQ ID NO:6 or 20, and

There is the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:7 or 21; With

Variable region of light chain, comprising:

There is first complementary determining region (CDR1) of the tetramino acid sequence comprising SEQ ID NO:12 or 26,

There is second complementary determining region (CDR2) of the pentaamino acid sequence comprising SEQ ID NO:13 or 27, and

There is the 3rd complementary determining region (CDR3) of the 6th aminoacid sequence comprising SEQ ID NO:14 or 28.

10. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 1 or its Fab, comprising:

Variable region of heavy chain, comprising:

There is first complementary determining region (CDR1) of the first aminoacid sequence comprising SEQ ID NO:5,

There is second complementary determining region (CDR2) of the second aminoacid sequence comprising SEQ ID NO:6, and

There is the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:7; With

Variable region of light chain, comprising:

There is first complementary determining region (CDR1) of the tetramino acid sequence comprising SEQ ID NO:12,

There is second complementary determining region (CDR2) of the pentaamino acid sequence comprising SEQ ID NO:13, and

There is the 3rd complementary determining region (CDR3) of the 6th aminoacid sequence comprising SEQ ID NO:14.

The anti-Type B botulic neurotoxin monoclonal antibody of 11. separation according to claim 1 or its Fab, comprising:

Variable region of heavy chain, comprising:

There is first complementary determining region (CDR1) of the first aminoacid sequence comprising SEQ ID NO:19,

There is second complementary determining region (CDR2) of the second aminoacid sequence comprising SEQ ID NO:20, and

There is the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:21; With

Variable region of light chain, comprising:

There is first complementary determining region (CDR1) of the tetramino acid sequence comprising SEQ ID NO:26,

There is second complementary determining region (CDR2) of the pentaamino acid sequence comprising SEQ ID NO:27, and

There is the 3rd complementary determining region (CDR3) of the 6th aminoacid sequence comprising SEQ ID NO:28.

12. Type B botulic neurotoxin monoclonal antibody according to claim 1 or its Fabs, comprising:

Variable region of heavy chain, comprises SEQ ID NO:3 or 17, and

Variable region of light chain, comprises SEQ ID NO:10 or 24.

13. Type B botulic neurotoxin monoclonal antibodies according to claim 1, wherein said antibody is IgG1.

14. 1 kinds of hybridomas, have the preserving number of NITE BP-01639 or NITE BP-01640.

15. 1 kinds of monoclonal antibodies be separated, are produced by the hybridoma of the preserving number with NITE BP-01639 or NITE BP-01640.

16. 1 kinds of pharmaceutical compositions, comprise anti-Type B botulic neurotoxin monoclonal antibody or its Fab of one or more separation according to claim 1, and pharmaceutically acceptable carrier.

17. pharmaceutical compositions according to claim 16, comprise anti-Type B botulic neurotoxin monoclonal antibody and pharmaceutically acceptable carrier.

18. pharmaceutical compositions according to claim 16, comprise two kinds be separated anti-Type B botulic neurotoxin monoclonal antibodies, its Fab or both, comprising:

First human monoclonal antibodies be separated or its Fab, comprising:

(1) human monoclonal antibodies be separated or its Fab, comprising:

Variable region of heavy chain, comprise: first complementary determining region (CDR1) with the first aminoacid sequence comprising SEQ ID NO:5, second complementary determining region (CDR2) with the second aminoacid sequence comprising SEQ ID NO:6 and there is the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:7, and

Variable region of light chain, comprise: first complementary determining region (CDR1) with the tetramino acid sequence comprising SEQ ID NO:12, second complementary determining region (CDR2) with the pentaamino acid sequence comprising SEQ ID NO:13 and there is the 3rd complementary determining region (CDR3) of the 6th aminoacid sequence comprising SEQ ID NO:14

(2) human monoclonal antibodies be separated or Fab, comprise variable region of heavy chain and variable region of light chain, and described variable region of heavy chain comprises SEQ ID NO:3, and described variable region of light chain comprises SEQ ID NO:10,

(3) monoclonal antibody be separated, is produced by the hybridoma with preserving number NITE BP-01639,

Or its arbitrary combination; With

Second human monoclonal antibodies be separated or its Fab, comprising:

(4) human monoclonal antibodies be separated or its Fab, comprising:

Variable region of heavy chain, comprise: first complementary determining region (CDR1) with the first aminoacid sequence comprising SEQ ID NO:19, second complementary determining region (CDR2) with the second aminoacid sequence comprising SEQ ID NO:20 and there is the 3rd complementary determining region (CDR3) of the triamino acid sequence comprising SEQ ID NO:21, and

Variable region of light chain, comprise: first complementary determining region (CDR1) with the tetramino acid sequence comprising SEQ ID NO:26, second complementary determining region (CDR2) with the pentaamino acid sequence comprising SEQ ID NO:27 and there is the 3rd complementary determining region (CDR3) of the 6th aminoacid sequence comprising SEQ ID NO:28

(5) human monoclonal antibodies be separated or Fab, comprise variable region of heavy chain and variable region of light chain, and described variable region of heavy chain comprises SEQ ID NO:17, and described variable region of light chain comprises SEQ ID NO:24,

(6) monoclonal antibody be separated, is produced by the hybridoma with preserving number NITE BP-01640,

Or its arbitrary combination.

19. 1 kinds, for generation of the method for the anti-Type B botulic neurotoxin monoclonal antibody be separated, comprising:

By merge come personal Clostridium botulinum toxoid immunity people peripheral blood lymphocytes (PBMC) and can effective cell merge fusion partner cells generation hybridoma; With

Type B botulic neurotoxin monoclonal antibody is obtained from described hybridoma.

The method of the 20. anti-Type B botulic neurotoxin monoclonal antibodies for generation of being separated according to claim 19, wherein said Type B botulic neurotoxin passes through Type B clostridium botulinumboNT/B 1 (bacterial strain Okra), Type B clostridium botulinumboNT/B2 (bacterial strain 111), Type B meat poisoning shuttle shape brood cell bar bacteriumboNT/B6 (bacterial strain Osaka05) or its arbitrary combination produce.

21. methods for generation of anti-Type B botulic neurotoxin monoclonal antibody according to claim 19, wherein said fusion partner cells is SPYMEG cell.

22. 1 kinds for prevent, treat and detect Type B botulic neurotoxin in human experimenter poisoning in the test kit of at least one, comprise the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 1, its Fab or both.

23. 1 kinds are suppressed or the methods of sausage poisoning in treatment human experimenter, comprise the anti-Type B botulic neurotoxin monoclonal antibody of the separation according to claim 1 to human experimenter's administering therapeutic significant quantity, its Fab or both.

The method of sausage poisoning in 24. suppression according to claim 23 or treatment human experimenter, also comprises diagnosis patient botulic neurotoxin poisoning.

The method of sausage poisoning in 25. suppression according to claim 23 or treatment human experimenter, also comprises the minimizing of at least one symptom of monitoring sausage poisoning.

The method of sausage poisoning in 26. suppression according to claim 25 or treatment human experimenter, wherein said at least one symptom comprises paralysis, symmetrical parmlysis of cranial nerve, Rearrangments flaccid paralysis, the paralysis of voluntary muscle symmetry, pharyngeally to subside, breath stopped, cannot suck, cannot swallow, sound reduction, blepharoptosis, facial paralysis, pupil is fixed, platycoria, blurred vision, soft neck, whole-body muscle relaxes, general weakness, diplopia, dysarthria, dysphonia, dysphagia, lossless, erythroplakia, postural hypotension, feel sick, constipation, uroschesis, dizzy, dry, throat pain, underdevelopment, pharyngeal reflex, the amyotrophy of whole body reflectivity, or its arbitrary combination.

The method of sausage poisoning in 27. suppression according to claim 23 or treatment human experimenter, wherein said sausage poisoning comprises food source property sausage poisoning, wound infection sausage poisoning, baby's enterotoxemia sausage poisoning, adult's enterotoxemia sausage poisoning, iatrogenic sausage poisoning, airborne transmission sausage poisoning or its arbitrary combination.

The method of sausage poisoning in 28. suppression according to claim 23 or treatment human experimenter, wherein, the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 1, its Fab or both combined needles are used one or more other treatments of sausage poisoning.

The method of sausage poisoning in 29. suppression according to claim 28 or treatment human experimenter, wherein said joint synergy is to suppress or treatment sausage poisoning.

The method of sausage poisoning in 30. suppression according to claim 28 or treatment human experimenter, one or more other treatments wherein said comprise use the second separation anti-Type B botulic neurotoxin monoclonal antibody, its Fab or both.

The method of sausage poisoning in 31. suppression according to claim 28 or treatment human experimenter, one or more other treatments wherein said comprise use separation anti-A type botulic neurotoxin monoclonal antibody, its Fab or both.

The anti-Type B botulic neurotoxin monoclonal antibody of 32. 1 kinds of separation according to claim 1, its Fab or both are for the preparation of the purposes of medicine for suppressing or treat sausage poisoning in human experimenter.

33. 1 kinds of methods detecting Type B botulic neurotoxin in human experimenter, comprising:

The anti-Type B botulic neurotoxin monoclonal antibody that sample from human experimenter is separated with according to claim 1, its Fab or both contact; With

Based on antibody, its fragment or both whether detect the presence or absence of Type B botulic neurotoxin in human experimenter in conjunction with Type B botulic neurotoxin.