CN101137394A - Anti-addl antibodies and uses thereof - Google Patents

Abstract

The present invention relates to antibodies that differentially recognize multi-dimensional conformations of Ass-derived diffusible ligands, also known as ADDLs. The antibodies of the invention can distinguish between Alzheimer's Disease and control human brain extracts and are useful in methods of detecting ADDLs and diagnosing Alzheimer's Disease. The present antibodies also block binding of ADDLs to neurons, assembly of ADDLs, and tauphosphorylation and are there useful in methods for the preventing and treating diseases associated with soluble oligomers of amyloid ss 1-42.

Description

Anti-addl antibodies and application thereof

The invention brief introduction

The application requires the U.S. Provisional Patent Application series number 60/621 of submission on October 25th, 2004,776, the Nos.60/652 that submitted on February 14th, 2005,538,60/695 of submission on June 30th, 2005,60/695 of submission on June 30th, 526 and 2005,528 priority, it is reference that the content of these provisional application is drawn with it in full at this.

The present invention makes in the research process of partly being subsidized (subsidy NIHRO1-AG18877 and NIH RO1-AG22547) by NIH.U.S. government has some right in the present invention.

Background of invention

The A Cihai Mo's disease (Alzheimer ' s Disease) be a kind of progressive degenerative dementia (Terry etc. (1991) Ann.Neurol.30:572-580; Coyle (1987) In:Encyclopedia of Neuroscience (neuroscience encyclopedia), Adelman (ed.), Bifkh  usef, Boston-Basel-Stuttgart, pp29-31).At its commitment, the A Cihai Mo's disease mainly shows as the degree of depth that forms new memory can not (Selkoe (2002) Science298:789-791), it is reported that this is by causing from the deutero-neurotoxin of amyloid beta (A β).A β is amphipathic peptide, and its abundance is along with the sudden change relevant with the A Cihai Mo's disease and risk factor and increase.The fibril that forms from A β has constituted the core of amyloid plaque, and it is the sign of suffering from A Cihai Mo's disease brain.Similar fibril in external generation is lethal for the brain neuron of cultivating.These discoveries show that the forfeiture of memory is the result of the neuronal death that causes of fibrous A β.

Although for fibrous A β and the strong experiment support of the loss of memory, the dependency very low (Katzman (1988) Ann.Neurol.23:138-144) between dull-witted and amyloid plaque amount.In addition, develop dependent amyloid plaque of age, the more important thing is transgenic hAPP mice (Dodart etc. (2002) Nat.Neurosci.5:452-457 that develops dependent memory dysfunction of age; Kotilinek etc. (2002) J.Neurosci.22:6331-6335), show after the anti-A β monoclonal antibody of inoculation in 24 hours, the forfeiture of memory can be reversed, and the level of amyloid plaque does not change simultaneously.These find to depend on the machine-processed inconsistent of neuronal death that amyloid fibrils causes with the loss of memory.

Propose other and assembled the neuroactive molecule that forms by A β self.These molecules comprise solubility A beta oligomers, are also referred to as deutero-part or the ADDLs of spreading of A β.Oligomer is metastable, forms under low concentration A β 1-42 (Lambert etc. (1998) Proc.Natl.Acad.Sci.USA 95:6448-6453).The A beta oligomers suppresses long-term potentiation (LTP) fast, and this is the classics experiment example of memory and synaptic plasticity.Therefore, the synapse that the loss of memory is derived from before the neuronal death damages, and the synapse damage causes (Hardy and Selkoe (2002) Science 297:353-356) by the A beta oligomers rather than by fibril.The soluble oligomeric body finds in cerebral tissue that (Kayed etc. (2003) Science 300:486-489 significantly raises in the A Cihai Mo's disease; Gong etc. (2003) Proc.Natl.Acad.Sci.USA100:10417-10422), (Kotilinek etc. (2002) J.Neurosci.22:6331-6335 that in hAPP transgenic mice A Cihai Mo's disease model, also significantly raises; Chang etc. (2003) J.Mol.Neurosci.20:305-313).

The treatment that has proposed multiple A Cihai Mo's disease is selected.The vaccine clinical trial shows have strong immunoreactive people to demonstrate aspect cognitive to vaccine and benefit (Hock etc. (2003) Neuron 38:547-554); But frequent CNS inflammation has caused the premature termination (Birmingham and Frantz (2002) Nat.Med.8:199-200) of part test.As the succedaneum of vaccine, proposed at ADDLs and not in conjunction with monomer or fibriilar therapeutic antibodies (Klein (2002) Neurochem.Int.41:345-352).ADDLs has high resistance originality, produces optionally polyclonal antibody (Lambert etc. (2001) J.Neurochem.79:595-605) of oligomer in rabbit when the about 50 μ g/mL of concentration.The result of transgene mouse model illustrates that also antibody can successfully reverse hypomnesis (Dodart etc. (2002) Nat.Neurosci.5:452-457).Therefore, in the present technique field, need ADDL optionally to treat antibody with prevention and treatment A Cihai Mo's disease.The present invention has satisfied this needs.

The invention summary

The present invention is can deutero-isolated antibody or its fragment that spreads the multidimensional conformation of part of one or more A β of difference identification.In specific embodiment, antibody of the present invention mixes mutually with pharmaceutically useful carrier.In other embodiment, antibody of the present invention is included in the test kit.

Provide in addition and used antibody or the antibody fragment that combines with the deutero-multidimensional conformation that spreads part of one or more A β, prevented that the deutero-part that spreads of A β from combining, suppressing that A β is deutero-spreads the gathering of part and block the method for tau protein in the phosphorylation of Ser202/Thr205 position with neuron.

The present invention has also comprised and has used antibody prevention of the present invention or treatment and the deutero-method that spreads the relevant disease of part of A β.Administration antibody of the present invention can prevent that the deutero-part that spreads of A β from combining with neuronic, thus prevention or treatment and the deutero-relevant disease of part that spreads of A β.

The present invention is spread the method that part and the bonded therapeutic agent of neuron are identified to preventing that A β is deutero-.This method of the present invention is included under the situation that has reagent neuron is contacted with the deutero-part that spreads of A β, and uses antibody of the present invention to determine that the deutero-part that spreads of A β combines with neuronic under the situation of reagent existing.

The present invention has also comprised the deutero-method that spreads part of detection A β in sample, and diagnosis and the deutero-method that spreads the relevant disease of part of A β.Such method comprises sample contacted with antibody of the present invention, so that the deutero-part that spreads of A β can be detected, and can be diagnosed with the deutero-relevant disease of part that spreads of A β.

The accompanying drawing summary

Fig. 1 has shown the result that alkali phosphatase is analyzed, wherein anti-addl antibodies difference block nerves unit.

Fig. 2 shows the bonded summary of bADDL when B103 cell and anti-addl antibodies are incubated in advance.

Fig. 3 has shown the summary in conjunction with feature of the antibody of multidimensional conformation that can difference identification ADDLs.

Fig. 4 has shown that antibody disclosed herein suppresses the accumulative summary of ADDL.

Fig. 5 has shown N2A combination and k _ADDLCorrelation figure.

Fig. 6 has shown the nucleotide sequence of heavy chain and variable region of light chain, is respectively mouse-anti ADDL antibody 20C2 (Fig. 6 A and 6B), 5F10 (Fig. 6 C and 6D), 2D6 (Fig. 6 E and 6F), 2B4 (Fig. 6 G and 6H), 4E2 (Fig. 6 I and 6J), 2H4 (Fig. 6 K and 6L), 2A10 (Fig. 6 M and 6N), 3B3 (Figure 60 and 6P), 1F6 (Fig. 6 Q and 6R), 1F4 (Fig. 6 S and 6T), 2E12 (Fig. 6 U and 6V) and 4C2 (Fig. 6 W and 6X).The targeting sequencing of lower case letter representation antibody, upper-case letters is represented the variable region sequences of antibody.The nucleotide of coding complementary determining region (CDRs) is by underscore.

Fig. 7 has shown the comparison of mouse anti ADDL antibody heavy chain variable region CDR1 (Fig. 7 A), CDR2 (Fig. 7 B), CDR3 (Fig. 7 C) sequence and variable region of light chain CDR1 (Fig. 7 D), CDR2 (Fig. 7 E), CDR3 (Fig. 7 F) sequence.

Fig. 8 has shown the aminoacid sequence of heavy chain and variable region of light chain, is respectively by CDR and transplants humanized anti-addl antibodies 20C2 (Fig. 8 A and 8B), 26D6 (Fig. 8 C and 8D), 4E2 (Fig. 8 E and 8F), 3B3 (Fig. 8 G and 8H), 2H4 (Fig. 8 I and 8J) and the 1F6 (Fig. 8 K) that produces.The comparison of the most homologous human sequence, the most homologous people's gene group sequence and humanization sequence that sequence is rendered as the sequence of mice, obtain from the NCBI albumen database.The aminoacid different with the humanization sequence is represented with runic in mice, people and people's gene group sequence.CDRs is by underscore.Important residue is indicated with * for keeping CDR ring conformation.Indicate with # in the conserved residues that the VL/VH intersection is found.Potential glycosylation site is indicated with italic.For the heavy chain of 20C2, produced two humanization sequences (HCVRA and HCVRB), they have an amino acid whose difference at 24.End user's aminoacid in 20C2 HCVRA uses the aminoacid of mice in 20C2 HCVRB.Not for 1F6 designs light chain, because the light chain of it and 4E2 has same sequence.

Fig. 9 has shown the aminoacid sequence of heavy chain and variable region of light chain, is respectively the humanized anti-addl antibodies 20C2 (Fig. 9 A and 9B) and the 26D6 (Fig. 9 C and 9D) that produce by finishing (veneering).The comparison of the most homologous human sequence, the most homologous people's gene group sequence and humanization sequence that sequence is rendered as the sequence of mice, obtain from the NCBI albumen database.The aminoacid different with the humanization sequence is represented with runic in mice, people and people's gene group sequence.CDRs is by underscore.Important residue is indicated with asterisk for keeping CDR ring conformation.Indicate with pound mark in the conserved residues that the VL/VH intersection is found.Potential glycosylation site is indicated with italic.For the heavy chain of 20C2, produced two humanization sequences (HCVRVenA and HCVRVenB), they have an amino acid whose difference at 81.In 20C2 HCVRVenA, use the aminoacid of mice, end user's aminoacid in 20C2 HCVRVenB.For the 26D6 heavy chain, designed 3 kinds of humanization sequences (HCVR Ven1, Ven2 and Ven3) according to finishing, they are different with 118 amino acids 11,23,15,81,89.In HCVR Ven1, the aminoacid of mice is all used in all positions.In Ven2,81 and 118 residues use the aminoacid of mice, 11,13,15 and 89 residue end user's aminoacid.In Ven3, all positions are end user's aminoacid all.For the 26D6 light chain, designed the humanization sequence (LCVR Ven1 and Ven2) of two finishinges, they are different with 105 aminoacid 88.In LCVR Ven1, the aminoacid of mice is all used in two positions, and in Ven2, two positions are end user's aminoacid all.

The heavy chain of humanization anti-addl antibodies and the nucleotide sequence of variable region of light chain (being respectively HCVRs nuclear LCVRs) have been shown among Figure 10.Figure 10 A has shown HCVRs and the LCVRs that the CDR of 20C2,2D6,4E2,3B3,2H4 and IF6 transplants in Figure 10 K respectively.Figure 10 L has shown the HCVRs (VenA and VenB) and the LCVR of the finishing of 20C2 to Figure 10 N, Figure 100 to Figure 10 S shown the HCVRs (Ven1, Ven2, Ven3) of the finishing of 26D6 and LCVRs (Ven1, Ven2).Uppercase is represented the antibody variable region sequence.CDRs is by underscore.Variable region sequences is cloned in complete heavy chain and the light chain antibody expression vector.

Figure 11 has shown the aminoacid sequence of anti-addl antibodies total length IgG1 and IgG2m4 humanization heavy chain and humanization κ light chain.Figure 11 A, the 20C2 HCVRA IgG1 that CDR transplants; Figure 11 B, the 20C2 HCVRB IgG1 that CDR transplants; Figure 11 C, the 20C2HCVRA IgG2m4 that CDR transplants; Figure 11 D, the 20C2 HCVRB IgG2m4 that CDR transplants; Figure 11 E, the 20C2 LCVR κ that CDR transplants; Figure 11 F, the 26D6 HCVR IgG1 that CDR transplants; Figure 11 G, the 26D6 HCVR IgG2m4 that CDR transplants; Figure 11 H, the 26D6LCVR κ that CDR transplants; Figure 11 I, 4E2 HCVR IgG1 Figure 11 J that CDR transplants, the 4E2LCVR κ that CDR transplants; Figure 11 K, the 3B3 HCVR IgG1 that CDR transplants; Figure 11 L, the 3B3 LCVR κ that CDR transplants; Figure 11 M, the 2H4 HCVR IgG1 that CDR transplants; Figure 11 N, the 2H4 LCVR κ that CDR transplants; Figure 11 O, 1F6 HCVR IgG1 Figure 11 p that CDR transplants, the 20C2 HCVR VenA IgG1 of finishing; Figure 11 Q, the 20C2 HCVR VenBIgG1 of finishing; Figure 11 R, the 20C2 HCVR VenB IgG2m4 of finishing; Figure 11 S, the 20C2 LCVR κ of finishing; Figure 11 T, the 26D6 HCVR Ven1 Ig of finishing; Figure 11 U, the 26D6 HCVR Ven1 IgG1 of finishing; Figure 11 V, the 26D6 HCVRVen2 IgG1 of finishing; Figure 11 W, the 26D6 HCVR Ven3 of finishing; Figure 11 X, the 26D6 LCVR Ven1 κ of finishing; And Figure 11 Y, the 26D6 LCVR Ven2 κ of finishing.Underscore is represented variable region sequences, corresponding to the aminoacid of CDRs by double underline.Remaining aminoacid sequence is the constant region sequence.

Figure 12 has shown the aminoacid sequence of people's antibody constant region and the comparison of IgG2m4 sequence.The glycosylation site of Asn297 position represented in asterisk.The FcRn land is indicated.Sequences different with IgG2 among the IgG2m4 are by underscore.

Figure 13 has shown the heavy chain (Figure 13 A) of the mark of 20C2 humanized antibody in Fab Vector for Phage Display pFab3d and the aminoacid sequence of light chain (Figure 13 B).

Figure 14 has described in two LC-CDR3 libraries of preparation, has been called LC3-1 and LC3-2, has been used for producing design and the primer that the sophisticated 20C2 light chain of affinity CDR3 uses.The restriction enzyme enzyme recognition site that is used to clone is indicated with italic.The nucleic acid of upper case presentation code antibody variable region sequence.The nucleic acid of coding CDRs is by underscore.

Detailed Description Of The Invention

Produced now the monoclonal antibody of the multidimensional conformation of spread to the part (being ADDLs) of can difference identification A β deriving. Favourable part is that this monoclonal antibody can be differentiated people's brain extract of A Cihai Mo's disease and contrast, and endogenous oligomer in the hippocampal cell of evaluation A Cihai Mo's disease brain section neutral incubation. In addition, this antibody endogenous and synthetic ADDLs that can in solution, neutralize. So-called " synthesizing ", ADDLs produced in external the mixing under the condition that can produce ADDLs by the amyloid beta 1-42 with purifying. Referring to U.S. Patent No. 6,218,506. Concrete antibody disclosed herein shows the selective of height, minimum detectable monomer A β peptide to the A β peptide of 3-24 monomer. In addition, the antibody selected of the present invention is blocked the small peptide that the identification of ADDLs is not included the linear order of A β 1-42 or A β 1-40. But, in conjunction with being blocked by A β 1-28, show based on the epi-position of the structure of conformation uniqueness and also can in A β 1-28, find. The sketch plan of this antibody epitope shows that these antibody are with similar compatibility and the same core linear order of specific characteristics identification, as being measured to by ELISA. In addition, this antibody otherness blocking-up contains the prepared product of ADDL in conjunction with the ability of the primary culture of neuroblastoma (neuroblastoma) clone of hippocampus of rats and immortality, also can block the gathering of ADDL. Although this discovery has confirmed these antibody and has had identical linear order discernment and compatibility, still had the ability of the multidimensional conformation of difference identification ADDLs. Because the known and neuronic subclass of ADDLs is relevant, and destroys normal neuronal function, therefore a use of the present invention is exploitation and/or identifies the antibody that can stop ADDLs to be combined with neuron. Such antibody is being treated the relevant disease of ADDL, is being comprised it will being useful in the A Cihai Mo's disease. A kind of improvement to this application is humanization and/or the affine sexually matured form that specificity is used these antibody, to prevent that ADDL is combined with neuron and the gathering of ADDLs.

Therefore, the present invention is the antibody of the separation of one or more multidimensional conformations that can difference identification ADDLs. Antibody of the present invention when it appears in the situation of the large biological molecule that is substantially devoid of other same-type, is known as and is separated. Therefore, " antibody of separation " refers to be substantially devoid of the antibody of other antibody; But molecule can contain reagent or the part (moiety) that fundamental property some other, antagonist (such as binding specificity, neutralization active etc.) does not have adverse effect.

The antibody of one or more multidimensional conformations that can specific binding ADDLs, the specific ADDLS that comes in conjunction with deriving from A β 1-42 oligomerization, but cross reaction occurs in A β peptide, for example A β 1-12, A β 1-28, A β 1-40 and the A β 12-28 with other, as disclosed herein determined by the western hybridization analysis; And tend in solution in conjunction with ADDLs (referring to for example embodiment 21). Specific binding between two entities refers to that generally compatibility is at least 10⁶、10 ⁷、10 ⁸、10 ⁹Or 10¹⁰M ^-1 In order to reach specific binding, compatibility is greater than 10⁸M ^-1Wish.

In specific embodiment, the antibody of multidimensional conformation that can one or more ADDLs of specific binding also is (namely the using its immune animal) that the multidimensional conformation by ADDLs causes. In other embodiment, the antibody of multidimensional conformation that can one or more ADDLs of specific binding forms for example A β 1-42[Nle35-Dpro37 of peptide by low n-aggressiveness] cause.

Term " epi-position " refers on the antigen site with B and/or t cell responses, or will produce for it site of antibody on the molecule, or antibody will with the site of its combination. For example, the epi-position antibody that can be defined this epi-position is identified.

Linear epi-position is the epi-position that wherein amino acid whose primary sequence contains the epi-position that is identified.Linear epi-position typically contains at least 3, more often at least 5, about 8 aminoacid sequences to about 10 uniquenesses for example.

Comformational epitope is opposite with linear epitope, is that the amino acid whose primary sequence that wherein contains epi-position is not the epi-position (epi-position that for example wherein amino acid whose primary sequence must not discerned by the antibody of decision epi-position) of unique determiner of the epi-position that is identified.Comformational epitope typically contains the aminoacid of greater number with respect to linear epitope.Aspect the identification of comformational epitope, antibody recognition peptide or proteic three dimensional structure.For example, when protein molecular was folded to form three dimensional structure, some aminoacid and/or polypeptide backbone that forms conformational determinant became (juxtaposed) side by side, thereby makes antibody identification meter position.The method of determining the conformation of epi-position includes but not limited to rotary label and the electron paramagnetic resonance spectroscopy that for example X-radiocrystallography, two dimensional NMR spectroscopy and site are instructed.Referring to " the epitope mapping scheme " in " molecular biology method " (1996) the 66th volume of for example Morris chief editor.

A β is deutero-to spread the oligomer that part or ADDLs are meant the solubility of amyloid beta 1-42, ideally contains the polymer that is less than 8 or 9 amyloid beta 1-42 peptides, is found relevant with the A Cihai Mo's disease.This is opposite with high-molecular weight polymerization intermediate, and they form micellar thorn, causes fibriilar formation.

As exemplifying in this article, at least a multidimensional conformation (referring to for example Fig. 3) of antibodies of the present invention or identification ADDL.In specific embodiment, at least two kinds, at least three kinds or at least four kinds of multidimensional conformations of this antibodies ADDL.The multidimensional conformation of ADDLs has comprised dimer, trimer, the tetramer, pentamer, six aggressiveness, heptamer, eight aggressiveness, nine aggressiveness, ten aggressiveness etc., as determined by the SDS-PAGE analysis.Because title such as trimer, the tetramer can change (referring to (2005) Amyloid 12:88-95 such as for example Bitan) along with used analytical method, the definition of planting the trimer, the tetramer etc. of use at this paper is analyzed according to SDS-PAGE and is carried out.For the difference binding ability of this antibody is described, have been found that some antibody will discern a kind of multidimensional conformation of ADDLs, the tetramer of for example ADDLs (for example antibody 2D6 or 4E2), and the trimer of several multidimensional conformations of other antibody recognition ADDLs, for example ADDLs and the tetramer (for example antibody 2A10,2B4,5F10 or 20C2).Therefore, antibody of the present invention has the special character of oligomer.In specific embodiment, the multidimensional conformation of ADDLs is relevant with specific polypeptide structure, this structure produced by antibody recognition of the present invention comformational epitope.In other embodiment, antibody of the present invention and magnitude range are approximately the multidimensional conformation ADDL specificity that trimer or the tetramer, molecular weight surpass 50kDa and combine.

In certain embodiments, except with the multidimensional conformation combines, antibody of the present invention also combines with the selected linear epitope of amyloid beta 1-42.The linear epitope of ADDLs is the peptide that is positioned at 4,5,6 or more a plurality of amino acid residues of 10,11,12,15 or 20 amino acid residues of amyloid beta 1-42N end.In specific embodiment, antibody of the present invention combines with linear epitope specificity among amyloid beta 1-42 residue 1-10,1-8,3-10 or the 3-8.The linear epitope of the example of amyloid beta 1-42 includes but not limited to amino acid residue EFRHDS (SEQ ID NO:177), DAEFRHDS (SEQ ID NO:178) and EFRHDSGY (SEQ ID NO:179).

Although antibody of the present invention may have same linear epitope, this linear epitope also not exclusively shows the binding characteristic (promptly blocking ADDL combines, stops the τ phosphorylation and suppress the accumulative ability of ADDL with neuron) of this antibody, this be because, as one of skill in the art is known, linear epitope may be only corresponding to the part of antigenic epi-position (referring to for example Breitling and D ü bel (1999) In:Recombinant Antibodies, John Wiley ﹠amp; Sons, Inc., NY, pg.115).For example, 20C2 is found can be in conjunction with the polymer of A β 7-42 peptide charge reversal, truncate, and this peptide lacks the linear epitope (being amino acid residue 3-8) of 20C2, and contains the sequence very different with the residue 7-16 of A β.Therefore the comformational epitope of the element in 20C2 and the residue 17-42 that depends on A β combines, but is under the situation of multidimensional conformation.Antibody of the present invention can distinguish with other antibody in this area, because they can discern multidimensional ADDLs in diversity ground, therefore can difference blocking-up ADDL and the neuronic gathering that suppresses ADDL with combining, prevent to diversity τ phosphorylation and diversity.

The antibody of Shi Yonging includes but not limited to (for example from B cell separation), humanized, neutral, bispecific or its single-chain antibody of polyclone or monoclonal antibody and chimeric, people in the present invention.In one embodiment, antibody of the present invention is monoclonal.In order to produce antibody, can comprise goat, rabbit, chicken, rat, mice, people etc. by coming immune various hosts with synthetic or natural ADDLs injection.The method of producing antibody is well-known in the present technique field." antibody: laboratory manual " (Cold SpringHarbor Laboratory, New York (1988)) referring to for example Kohler and Milstein ((1975) Nature256:495-497) and Harlow and Lane.

According to host's kind, can use different adjuvants to react with enhance immunity.Be used for adjuvant of the present invention and wish to strengthen intrinsic reaction, and do not cause the variation of conformation in the immunogen ADDLs, thus the qualitative form of influence reaction.Particularly suitable adjuvant comprises 3-deoxidation acidylate monophosphoryl lipid A (MPL ^TMRIBI ImmunoChem Research Inc., Hamilton, MT; Referring to GB 2220211) and oil-in-water emulsion; for example zamene or Oleum Arachidis hypogaeae semen; can be used in combination with immunostimulant, for example monophosphoryl lipid A (referring to (1997) N.Engl.J.Med.336:86-91 such as Stoute); muramyl peptide (N-acetyl muramyl-L-threonyl-D-isoglutamine (thr-MDP) for example; N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (nor-MDP); the different glutamy of N-acetyl muramyl-L-alanyl-D--L-alanine-2-(1 '-2 ' two palmityls-sn-glycerol-3-hydroxyl phosphoryl oxygen)-ethamine (E-PE); the different glutamy of N-acetyl glucosamine amido-N-acetyl muramyl-L-Al-D--L-alanine-two palmityl propionic acid amide. (DTP-DPP)); or other bacteria cell wall composition.The concrete example of oil-in-water emulsion comprises MF59 (WO90/14837), contains 5% zamene, 0.5%TWEEN ^TM80 and 0.5%SPAN85 (also can contain not commensurability MTP-PE), use micro-fluidizer for example 110Y type trace fluidizer (Microfluidics, Newton MA) make the granule of submicron order; SAF contains 10% zamene, 0.4%TWEEN ^TM80, the polymer L121 and the thr-MDP of 5%PLURONIC  blocking-up can make the granule of submicron order by micro-fluidizer, also can vibrate to produce the emulsion of larger particles yardstick; And RIBI ^TM(RibiImmunoChem, Hamilton MT), contain 2% zamene, 0.2%TWEEN to adjuvant system (RAS) ^TM80 and one or more bacteria cell wall compositions, for example monophosphoryl lipid A, two mould sour trehaloses (TREHALOSE DIMYCOLATE) are (TDM) and cell wall skeleton (CWS).

Another kind of adjuvant is saponin adjuvant, for example STIMULON ^TM(QS-21, Aquila, Framingham, MA) or from the microgranule of its generation for example ISCOMs (immunostimulating complex) and ISCOMATRIX  (CSL Ltd., Parkville, Australia).Other adjuvant that is fit to comprises for example aluminium hydroxide and surfactant LYSOLECITHIN SUNLECITHIN A, PLURONIC  polyhydric alcohol, polyanion, peptide, CpG (WO98/40100), keyhole limpet hemocyanin, dinitrophenol,DNP and cytokine interleukin (IL-1 for example for example of complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), mineral coagulant, IL-2, and IL-12), M-CSF (M-CSF) and tumor necrosis factor (TNF).Be used for people's adjuvant, BCG (bacillus calmette-guerin vaccine) and Corynebacterium (Corynebacterium parvum) are particularly suitable for.

Antibody at multidimensional conformation ADDL produces by using the ADDLs immune animal.In general, ADDLs can synthesize or produce by recombinant fragment expression and purification.Synthetic ADDLs can according in method disclosed herein or the U.S. Patent No. 6,218,506 or the method in co-pending application USSN 60/621,776,60/652,538,60/695,526 and 60/695,528 prepare.In addition, ADDLs can with other albumen for example keyhole limpet hemocyanin merge to produce antibody at chimeric molecule.ADDLs can be restricted on conformation to form such as described herein useful epi-position, in addition, ADDLs also can be with surface-associated, for example to allow generation or to be chemically bonded to the surface by the mode physical connection of the conformation of antibody recognition of the present invention.

Can use at the monoclonal antibody of the multidimensional conformation of ADDLs anyly provides the technology of antibody molecule production to prepare by the successive cell strain in the culture.These technology include but not limited to hybridoma technology, human B cell hybridoma technology and EBV hybridoma technology (Kohler etc. (1975) Nature256:495-497; Kozbor etc. (1985) J.Immunol.Methods81:31-42; Cote etc. (1983) Proc.Natl.Acad.Sci.80:2026-2030; Cole etc. (1984) Mol.Cell Biol.62:109-120).Exemplary monoclonal antibody comprises the murine antibody of called after 2A10,4C2,2D6,4E2,20C2,2B4,5F10,2H4,2E12,1F6,1F4,3B3,5G12,6B7,6B11,11B4,11B5,14A11,15G6,17G4,20C2,3B7,1E3,1A9,1G3,1A7 and 1E5.

In addition, humanized and chimeric antibody can by thereby the splicing of mouse antibodies gene and human immunoglobulin gene is obtained to have suitable antigenic specificity and bioactive molecule produce (referring to (1984) Proc.Natl.Acad.Sci.81 such as Morrison, 6851-6855; Neuberger etc. (1984) Nature 312:604-608; Takeda etc. (1985) Nature314:452-454; Queen etc. (1989) Proc.Natl.Acad.Sci.USA86:10029-10033; WO90/07861).For example, mouse antibodies is selected to be expressed as Fv or Fab fragment in the carrier in phage.Gene of light chain (being the gene of heavy chain in parallel laboratory test) and the exchange of human immunoglobulin gene library.Identify then still can conjugated antigen phage antibody.This method is commonly referred to as chain and resets, the humanized antibody that provides can with the mouse antibodies in its source in conjunction with identical epi-position (Jespers etc. (1994) Biotechnology NY12:899-903).As optional method, chain is reset also can carry out (referring to (1994) J.Mol.Biol.239:68-78 such as Figini) on protein level.

People's antibody also can use the phage display method to obtain.Referring to for example WO91/17271 and WO92/01047.In these methods, the phage library of generation each member is therein showed different antibody on their outer surface.Antibody is shown as Fv or Fab fragment usually.Select to have required specific phage displaying antibody by affinity enrichment to ADDLs.People's antibody at ADDLs also can be from inhuman transgene mammal production, at least a portion of the gene code human normal immunoglobulin site that this mammal changes over to and the endogenous immunoglobulin locus of inactivation.Referring to for example WO93/12227 and WO91/10741, they each draw at this and be reference.People's antibody can be selected by competitive binding experiment, perhaps has same epitope specificity with specific mouse antibodies.Such antibody may be enjoyed the useful functional character of mouse antibodies very much.People's polyclonal antibody also can provide from the philtrum with the immunity of immunogenicity reagent with the form of serum.Optional, such polyclonal antibody also can use ADDLs to concentrate by affinity purification as affinity reagent.

Humanized antibody also can be produced by the finishing or the resurfacing (resurfacing) of murine antibody.Finishing comprises the aminoacid of only replacing surperficial fixed area in murine heavy chain and the variable region of light chain with homologous human antibody sequence.With the surface amino groups acid of the alternative mice of people's residue of position equally among the homologous human sequence, be shown the immunogenicity that can reduce mouse antibodies and kept its part combination simultaneously.The replacement of outside residue generally has only slight influence or not influence (referring to for example U.S. Patent No. 6,797,492) for the contact between internal structure territory or domain.

People or humanized antibody can be designed as have IgG, IgD, IgA, IgM or IgE constant region, and any isotype comprises IgG1, IgG2, IgG3 and IgG4.In specific embodiment, antibody of the present invention is IgG or IgM, or its combination.The constant region that specific combination contains is by forming in the human IgG2's constant region that optionally human IgG 4 sequences is incorporated into standard.The IgG2 Fc of exemplary sudden change is IgG2m4, proposes in the SEQ of this paper IDNO:254.Antibody can be expressed as the tetramer that contains two light chains and two heavy chains, is expressed as isolating heavy chain and light chain, also can be expressed as single-chain antibody, and heavy chain wherein is connected by sept with the light chain variable domain.The technology of manufacture order chain antibody is well-known in this area.

The humanized antibody that passes through CDR transplanting and finishing generation of example is the antibody of called after 4E2 disclosed herein, 26D6,20C2,3B3,2H4 and 1F6.IgG1 and IgG2M4 variable region of heavy chain and transplant and the aminoacid sequence in the kappa light chain variable district of humanization 4E2,26D6,20C2,3B3,2H4 and 1F6 that finishing produces is presented at Figure 11 A in 11Y by CDR, and be published as SEQ ID NOs:152 to 176.

Miniature bifunctional antibody (diabodies) also has been contemplated.Miniature bifunctional antibody is meant the antibody construction thing of through engineering approaches, it is to separate, provide linking group to prepare then by the binding structural domain (heavy chain and light chain) with binding antibody, this linking group with heavy chain and light chain in conjunction with or be operatively coupled on the same polypeptide chain, thereby keep combined function (referring to (1993) Proc.Natl.Acad.Sci.USA 90:6444 such as Holliger; Poljak (1994) Structure 2:1121-1123).This has just formed the antibody of very simplifying in fact, only contains the necessary variable region of conjugated antigen domain.Thereby by using paired joint between very short two domains that can not allow on same the chain, domain be forced to another chain on complementary domain pairing, and produce two antigen binding sites.These disome antibody fragments or miniature bifunctional antibody be bivalence with bispecific.The professional and technical personnel will recognize that any method that can produce miniature bifunctional antibody can be used.In (1996) Biotechnology14:192-196 such as the method that is fit to is the same in (1993) such as Holliger, Poljak (1994) is the same, Zhu and the U.S. Patent No. 6,492,123 description is arranged, draw at this and be reference.

The fragment of isolated antibody of the present invention also comprises in the present invention clearly.The fragment of design comprises the fragment that Fab fragment, F (ab ') 2 fragments, F (ab ') fragment, bispecific scFv fragment, Fd fragment and Fab expression library produce, and the adaptive son of peptide (aptamer).For example, F (ab ') 2 fragments are by producing with pepsin digestion antibody molecule of the present invention, and the Fab fragment produces by reduction F (ab ') 2 segmental disulfide bond bridges.In addition, can make up the Fab expression library to allow fast, easily to identify to have required specific monoclonal Fab fragment (referring to (1989) Science 254:1275-1281 such as Huse).In specific embodiment, antibody fragment of the present invention is the fragment of neutralizing antibody that has kept the variable region binding site of neutralizing antibody.Example is F (ab ') 2 fragments, F (ab ') fragment and Fab fragment.Generally can be referring to " immunology basic skills " (second edition), J.Bellanti chief editor, 95-97 page or leaf.

The adaptive son of peptide of multidimensional conformation that can difference identification ADDLs can reasonably be designed, or screening in adaptive sublibrary (for example Aptanomics SA, Lyon, the library that France provided).In general, the adaptive son of peptide is synthetic identification molecule, and its design is based on the structure of antibody.The adaptive son of peptide is made of variable peptide ring, and its two ends link to each other with the albumen support.This dual structure restriction has greatly increased the binding affinity of the adaptive son of peptide, has reached the level (nanomole scope) that can compare with the binding affinity of antibody.

Being used for of example produces the encoding heavy chain of antibody of the present invention and antibody fragment and the nucleotide sequence of variable region of light chain is disclosed in Fig. 6 and 10 (being SEQ ID NOs:1-24 and SEQID NOs:132-151) in this article.As the professional and technical personnel will be understood that, variable region of heavy chain disclosed herein can be used in combination with any variable region of light chain disclosed herein, has the antibody of the affinity, dissociation constant, epi-position etc. of modification with generation.For example, with variable region of heavy chain (by the SEQ ID NO:13 coding) combination of the variable region of light chain of 2H4 (by SEQ ID NO:12 coding), can discern bigger linear epitope with 2A10.

The heavy chain and the light chain CDRs that are used for producing the example of antibody of the present invention or antibody fragment are disclosed in Fig. 7 A-7F, and they have SEQ ID NOs:25,26 and 28 (heavy chain CDR1), SEQ ID NOs:29,30,31,33,34,35 and 36 (heavy chain CDR2), SEQ ID NOs:38,39,40,41,43,44,45,46,47 and 48 (heavy chain CDR3), SEQ ID NOs:49,50,51 and 53 (light chain CDR1), SEQ ID NOs:54,55,56 and 58 (light chain CDR2), and SEQ ID NOs:59,60,61,62,63, the aminoacid sequence that shows among 64 and 66 (the light chain CDR3).The heavy chain of antibody of the present invention or antibody fragment and the specific embodiment of light chain are as follows.Heavy chain CDR1 has aminoacid sequence Ser-Phe-Gly-Met-His (SEQ IDNO:28) or Thr-Ser-Gly-Met-Gly-Val-Xaa (SEQ ID NO:27), and wherein Xaa is the aminoacid (for example serine, glycine or alanine) that does not have side chain or have little side chain.Heavy chain CDR2 has aminoacid sequence His-Ile-Xaa1-Trp-Asp-Asp-Asp-Lys-Xaa2-Tyr-Asn-Pro-Ser-Le u-Lys-Ser (SEQ ID NO:32), wherein Xaa ₁Be aminoacid (for example phenylalanine, tyrosine or tryptophan) with aromatic series side-chain radical, Xaa ₂Be serine, arginine or tyrosine; Perhaps heavy chain CDR2 has aminoacid sequence Tyr-Ile-Xaa ₁-Xaa ₂-Xaa ₃-Ser-Xaa ₄-Thr-Ile-Tyr-Tyr-Ala-Asp-Thr-Val-Lys-Arg (SEQ ID NO:37), wherein Xaa ₁And Xaa ₂Be aminoacid (for example arginine, serine, glycine, threonine, cysteine, tyrosine, agedoite, glutamine, lysine or histidine) with polar side chain group, Xaa ₃Be glycine or valine, Xaa ₄Be aminoacid (for example glycine, serine, threonine, cysteine, tyrosine, agedoite or glutamine) with polarity and uncharged side-chain radical.Heavy chain CDR3 has aminoacid sequence Arg-Ser-Ile-Xaa ₁-Xaa ₂-Xaa ₃-Xaa ₄-Pro-Glu-Asp-Tyr-Phe-Xaa ₅-Tyr (SEQID NO:42), wherein Xaa ₁Be aminoacid (for example glycine, serine, threonine, cysteine, tyrosine, agedoite or glutamine) with polarity and uncharged side-chain radical, Xaa ₂Be aminoacid (for example serine or threonine) with hydroxyl side-chain radical, Xaa ₃And Xaa ₄Be aminoacid (for example alanine, valine, leucine, isoleucine or proline) with aliphatic lateral chain group, Xaa ₅Be aspartic acid or alanine.Light chain CDR1 has aminoacid sequence Arg-Ser-Ser-Gln-Ser-Xaa ₁-Xaa ₂-His-Ser-Asn-Gly-Asn-Thr-Tyr-Leu-Xaa ₃(SEQ ID NO:52), wherein Xaa ₁And Xaa ₂Be aminoacid (for example alanine, valine, leucine, isoleucine or proline) with aliphatic lateral chain group, Xaa ₃Be aminoacid (for example aspartic acid, glutamic acid, arginine, histidine or lysine) with electrically charged side-chain radical.Light chain CDR ₂Has aminoacid sequence Lys-Xaa ₁-Ser-Asn-Arg-Phe-Xaa ₂(SEQ ID NO:57), wherein Xaa ₁Be aminoacid (for example alanine, valine, leucine, isoleucine or proline) with aliphatic lateral chain group, Xaa ₂Be serine or phenylalanine.Light chain CDR3 has aminoacid sequence Xaa ₁-Gln-Xaa ₂-Xaa ₃-Xaa ₄-Val-Pro-Xaa ₅-Thr (SEQ ID NO:65), wherein Xaa ₁Be serine or phenylalanine, Xaa ₂Be the aminoacid (for example glycine) of not being with side chain or aminoacid (for example serine or threonine) with hydroxyl side-chain radical, Xaa ₃Be aminoacid (for example serine or threonine) with hydroxyl side-chain radical, Xaa ₄Be histidine, tyrosine or leucine, Xaa ₅Be aminoacid (for example alanine, valine, leucine, isoleucine or proline) with aliphatic lateral chain group.As the professional and technical personnel will be understood that, the one or more CDRs in heavy chain of antibody and the variable region of light chain can be replaced by the one or more CDRs in another antibody, thereby produce brand-new antibody or antibody fragment.For example the CDR3 with the 4E2 heavy chain replaces the heavy chain CDR3 (SEQ ID NO:41) of 5F10 can strengthen 5F10 blocking-up ADDLs and the bonded ability of neuronal cell.

Antibody with special properties also has been contemplated.In one embodiment, antibody in conjunction with the 3-8 amino acids epi-position of A β 1-42 contains heavy chain CDR1 aminoacid sequence Thr-Ser-Gly-Met-Gly-Val-Xaa (SEQ ID NO:27), wherein Xaa is the aminoacid (for example serine, glycine or alanine) that does not have side chain or have little side chain, or heavy chain CDR2 aminoacid sequence His-Ile-Xaa ₁-Trp-Asp-Asp-Asp-Lys-Xaa ₂-Tyr-Asn-Pro-Ser-Leu-Lys-Ser (SEQ ID NO:32), wherein Xaa ₁Be aminoacid (for example phenylalanine, tyrosine or tryptophan) with aromatic series side-chain radical, Xaa ₂Be serine, arginine or tyrosine.In another embodiment, for big (〉 50kDa) the ADDL aggregation than little (＜30kDa) ADDL aggregation (promptly being respectively SEC peak 1 and peak 2) has the antibody of moderate affinity, contains heavy chain CDR3 aminoacid sequence Arg-Ser-Ile-Xaa1-Xaa2-Xaa ₃-Xaa ₄-Pro-Glu-Asp-Tyr-Phe-Xaa ₅-Tyr (SEQID NO:42), wherein Xaa ₁Be aminoacid (for example glycine, serine, threonine, cysteine, tyrosine, agedoite or glutamine) with polarity and uncharged side-chain radical, Xaa ₂Be aminoacid (for example serine or threonine) with hydroxyl side-chain radical, Xaa ₃And Xaa ₄Be aminoacid (for example alanine, valine, leucine, isoleucine or proline) with aliphatic lateral chain group, Xaa ₅Be aspartic acid or alanine.

Antibody of the present invention or antibody fragment can be connected with additional group thereon.For example, microsphere or microgranule can be combined on antibody or the antibody fragment, and as U.S. Patent No. 4,493, as described in 825, its content is drawn at this and is reference.

In addition, antibody of the present invention or antibody fragment can be suddenlyd change and be screened the dissociation constant to increase antigen affinity, neutralization active (promptly blocking bonded ability of ADDLS and neuronal cell or the accumulative ability of blocking-up ADDL) or to obtain to modify.It is the method for the nucleic acid molecules of encoding antibody of suddenling change being used to of having set up that escherichia coli mutator (mutatorstrains) (Low etc. (1996) J.Mol.Biol.260:359-368), chain are reset (Figini etc. (1994) are the same) and PCR mutation.As an illustration, can be by a large amount of phage antibodies be contacted so that the affinity of increase is screened in the antibody competition combination with a spot of biotin labeled antigen.In this case, the quantity of antigen molecule should surpass the quantity of phage antibody, but antigenic concentration should be a shade below dissociation constant.Therefore, the phage antibody of the sudden change of the affinity with increase of advantage combines with biotin labeled antigen, keeps not combination and major part has the phage antibody of more weak affinity.Streptavidin can help from mixture enrichment to have phage antibody (Schier etc. (1996) J.Mol.Biol.255:28-43) than the sudden change of high-affinity then.Disclose the sophisticated light chain CDR3 of the affinity aminoacid sequence (referring to table 11 and 12) of example herein, in specific embodiment, comprised light chain CDR3 aminoacid sequence Xaa ₁-Gln-Xaa ₂-Thr-Arg-Val-Pro-Leu-Thr (SEQ ID NO:316), wherein Xaa ₁Be phenylalanine or leucine, Xaa ₂Be alanine or threonine.

For using, some treatment may wish to reduce antibody dissociating from the antigen.In order to reach this purpose, phage antibody is combined with biotin labeled antigen, and add excessive not biotin labeled antigen.After after a while, advantageously (Predominantly) can use the phage antibody (Hawkins etc. (1992) J.Mol.Biol.226:889-96) that the streptavidin results have low dissociation constant.

Can use various immune analysis methods to comprise that method disclosed herein screens, have required specific antibody or its fragment to identify multidimensional conformation to ADDLs.Be used for competitively in a large number, use polyclone or monoclonal antibody or its segmental dynamic analysis (BIACORE for example in conjunction with (for example ELISA), latex agglutination analysis, immunoradiometric assay ^TMAnalysis) rules are well-known in the present technique field.Such immunoassay has typically comprised measures the complex that forms between specific antibodies and relevant (cognate) antigen thereof.Use can with the dibit point of the monoclonal antibody of two mutual glitch-free epi-positions reactions, be fit to based on the immune analysis method of monoclonal antibody, but competitive binding analysis also can use.Such analytical method also can be used for the multidimensional conformation of ADDLs in the test sample.

Antibody or antibody fragment also can be used for other biological activity analysis, for example replace ADDL and neuron or cultured rat hippocampal cell to combine or block the gathering of ADDL, with assessment neutralization activity or pharmaceutically active with as prevention or treat the latent effectiveness of reagent.Such analytical method is described in this article, and is well-known in the present technique field.

Antibody and antibody fragment can be used as hybridoma production and keep, also can be in any expression system of having set up recombinant production, these expression systems include but not limited to escherichia coli, yeast (for example Saccharomyces spp. and Pichia spp.), baculovirus, mammalian cell (for example myeloma, CHO, COS), plant or transgenic animal (Breitling and D ü bel (1999) " recombinant antibodies ", John Wiley ﹠amp; Sons, Inc., NY, pp.119-132).Transplanting and the IgG1 of humanized 4E2,26D6,20C2,3B3,2H4 and 1F6 that finishing produces and the variable region of heavy chain of IgG2m4 and the examples of nucleic acid in kappa light chain variable district are presented at Figure 10 A in 10S by CDR, is SEQ ID NOs:132 to 151 by display in this article.For can use any suitable method, include but not limited to affinity chromatograph, immune globulin binding molecule (for example protein A, L, G or H) comes separation antibody and antibody fragment, label (for example His-tag, FLAG -tag, Strep tag, c-myc tag) etc. can be operably connected with antibody or antibody fragment.The same referring to Breitling and D ü bel (1999).

Antibody of the present invention and antibody fragment have various application, comprise the diagnosis, blocking-up of the disease relevant with the accumulation of ADDLs or suppress the treatment reagent that preventative or therapeutic treatment, the evaluation of the gathering that combines, blocks ADDL of ADDLs and neuronal cell, the disease of being correlated with ADLLs can stop ADDLs to combine with neuron and stop tau protein phosphorylation on the Ser202/Thr205 position.

Antibody of the present invention and antibody fragment also can be used for blocking-up or suppress in ADDLs and the neuronic bonded method.The execution of this method of the present invention is by contacting with neuron with antibody of the present invention or antibody fragment in external or body, so that ADDLs is blocked with neuronic the combination.In specific embodiment, antibody of the present invention or antibody fragment make under the combination of ADDLs and the situation that does not have antibody or antibody fragment the combination of ADDLs compare to have reduced at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 97%.The bonded degree of antibody blocking ADDLs and neuron can determine according to method disclosed herein, i.e. immunocytochemistry or based on alkaline phosphatase method of analyzing enzyme or any analytical method that other is fit to of cell.For reducing 20C2,3B3,1F4,1F6,4E2,2B4,2D6 and the 2H4 monoclonal antibody that ADDLs useful especially antibody with neuron combines comprises example.

Antibody of the present invention and antibody fragment also can be used for blocking-up or suppress the accumulative method of ADDLs.This method comprises that the sample that will contain amyloid beta 1-42 peptide contacts so that the gathering of ADDL is suppressed with antibody of the present invention or antibody fragment.The accumulative degree of antibody blocking ADDLs can be measured according to method disclosed herein, i.e. FRET or fluorescence polarization or any analytical method that other is fit to.Useful especially antibody comprises 1F4,20C2,4C2,1F6,2B4,5F10,2A10 and the 2D6 antibody of example for the gathering of blocking-up ADDLs.

Antibody disclosed herein also can be used for stoping the method for tau protein in Ser202/Thr205 site phosphorylation.This method comprises that the sample that will contain tau protein contacts with antibody of the present invention or antibody fragment, so that ADDLs is blocked with neuronic the combination, thus the phosphorylation of prevention tau protein.Antibody stops the degree of tau protein phosphorylation to measure according to method disclosed herein or any analytical method that other is fit to.

Blocking-up or reduce ADDLs with the neuronic gathering that combines, suppresses ADDLs, stop the phosphorylation of tau protein in the Ser202/Thr205 site, can accumulate discovery application in the method for relevant disease at preventative or therapeutic treatment and ADDLs.Therefore, the present invention has also comprised the disease (for example A Cihai Mo's disease or disorder similar and that memory is relevant) of using antibody of the present invention or antibody fragment prevention or treatment relevant with the ADDLs accumulation.Treatable patient includes disease risks but does not show the individuality of symptom and the patient who has shown symptom at present.With regard to the A Cihai Mo's disease, in fact anyone has the risk of suffering from the A Cihai Mo's disease, if he or she live long enough.Therefore, antibody of the present invention or antibody fragment can not need to be carried out any risk assessment with regard to the preventative general population of being used for to trying patient.This method is particularly useful for known individuality with A Cihai Mo's disease genetic risk.These individualities comprise its relatives by diagnosis suffer from this disease, with and the individuality determined of risk by genetic marker or biochemical biomarker.The genetic marker of A Cihai Mo's disease risk comprises the sudden change in the app gene, particularly 717 and 670 and 671 sudden changes that are called as Hardy and Swedish sudden change respectively.Other risk labelling is sudden change, A Cihai Mo's disease family history, hypercholesterolemia or the atherosclerosis among senilism protein gene PS1 and PS2 and the ApoE4.Suffering from the individuality of A Cihai Mo's disease at present can discern according to the existence of distinctive dementia and above-mentioned risk factor.In addition, there are many diagnostic tests to can be used for identifying the individuality of suffering from the A Cihai Mo's disease.This comprises the level of measuring CSF τ and A β 1-42.Suffering from the individuality of A Cihai Mo's disease also can diagnose by ADRDA criterion or method disclosed herein.

In asymptomatic patient, treatment can begin (for example 10,20,30 years old) at any age.But, as a rule patient reach 40,50,60 or 70 years old before must begin treatment.Treatment need be used multidose in one period in typical case.Treatment can be monitored by analyzing in time existence of ADDLs.

In treatment is used, the pharmaceutical composition or the medicament that will contain antibody of the present invention or antibody fragment are administered to the patient who suspects or suffered from the disease relevant with the accumulation of ADDLs, its dosage is enough treated or is stoped the symptom (biochemistry, histology and/or ethological symptom) of disease to small part, comprises its complication and middle pathological manifestations type in the disease progression process.In prophylactic use, to contain the pharmaceutical composition of antibody of the present invention or antibody fragment or medicament is administered to and suspects to suffer from disease relevant with the accumulation of ADDLs or the patient with risk of such disease, its dosage is enough realized passive immunity in patient, thereby eliminate or reduce risks, slow down the outbreak of the state of an illness or delay disease, comprise biochemistry, histology and/or behavioristics's symptom, its complication in the disease progression process and the appearance of middle pathological manifestations type of disease.In some method, also do not developing among the pathological patient of distinctive A Cihai Mo's disease the administration medicament and reduce or eliminated the flesh cognitive impairment.In specific embodiment, the antibody of the present invention of effective dose or antibody fragment are the combination of ADDLs under ADDLs and neuronic combination and the situation of not treating is compared be reduced by at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 97% dosage.The damage that potentiation/memory equally, for a long time forms has also reduced.

The effective dose that is used for the treatment of the compositions of the present invention of above-mentioned disease changes along with many different factors, and mode, patient's physiological status, the patient who comprises administration is that other medicament and the treatment of people or animal, use is preventative or curative.Generally, patient is the people, but for example Canis familiaris L. or transgene mammal also can be treated for inhuman mammal.

Therapeutic dose is general titrated so that safety and effectiveness optimization.For the passive immunity that uses antibody or antibody fragment, dosage range is from about 0.0001 to 100mg/kg host's body weight, is more typically 0.01 and is fit to 5mg/kg host's body weight.For example, dosage can be 1mg/kg body weight or 10mg/kg body weight or in the scope of 1-10mg/kg.Exemplary Therapeutic Method need be administered once per fortnight or January once or per 3 once by 6 months.In some method, the antibody that two or more have different binding specificities of the present invention is by the while administration, and the dosage of the antibody of every kind of administration is positioned at pointed scope in this case.Antibody usually repeatedly by administration, wherein the interval between every dose can be the week, month or year.Also can be irregular at interval, the blood levels at the antibody of ADDLs in measuring patient be shown.In some method, dosage is adjusted to obtain plasma antibody concentration 1-1000 μ g/mL, is 25-300 μ g/mL in some method.In addition, antibody or antibody fragment also can be used as slow releasing preparation by administration, and the administration frequency that needs in this case reduces.Dosage and frequency along with antibody in patient half-life and change.In general, people and humanized antibody are than chimeric antibody and non-human antibody's long half time.As pointing out above, the dosage of administration and frequency can be preventative or curative the variations according to treatment.In prophylactic use, in the longer time with the relative not frequent low relatively dosage of interval administration.Some patient receives treatment in their remaining years continuously.In therapeutic is used, need in short relatively interval, use high relatively dosage sometimes, slowed down or stopped up to advancing of disease, demonstrate the partially or completely improvement of disease symptoms in the preferred case up to patient.Afterwards, patient can be according to the prophylactic methods administration.

Antibody of the present invention and antibody fragment can be used as the composition of pharmaceutical composition thing or medicament by administration.Pharmaceutical composition thing or medicament generally contain active treatment reagent and various other pharmaceutically useful composition.Referring to " the materia medica science with put into practice " of Remington, AlfonsoR.Gennaro chief editor, the 20th edition, Lippincott Williams ﹠amp; Wilkins:Philadelphia, PA, 2000.Preferred form depends on required mode of administration and treatment is used.Pharmaceutical composition depends on required dosage form, and pharmaceutical composition can contain pharmaceutically useful, avirulent carrier or diluent, and they are defined as being generally used for the media that the compounding pharmaceutical compositions is used for the pharmaceutical composition of animal and human's administration.The selected biologic activity that makes it not influence compositions of diluent.The example of such diluent is saline, Ringer's solution, glucose solution and the Hank ' s solution of distilled water, physiology phosphoric acid buffer.

That the pharmaceutical composition thing also can contain is big, metabolism macromole albumen, polysaccharide chitosan, polylactic acid, polyglycolic acid and the copolymer (SEPHAROSE of latex functionalization for example for example for example slowly ^TM, agarose, cellulose etc.), polyamino acid, amino acid copolymer and lipid aggregation (for example oil droplet or liposome).

The administration of pharmaceutical composition of the present invention or medicament can be carried out by all means, includes but not limited to oral, local, pulmonary, rectum, subcutaneous, Intradermal, intranasal, intracranial, intramuscular, ophthalmic or intra-articular injection etc.Most typical route of administration is an intravenous, secondly is subcutaneous, although other approach is also effectively equal.Also can in arm or leg muscle, carry out intramuscular injection.In some method, medicament is injected directly in the particular organization of accumulation of deposits, for example intracranial injection.In certain embodiments, antibody or antibody fragment are as slow releasing composition or install for example MEDIPAD ^TMDevice is by administration.

For parenteral, antibody of the present invention or antibody fragment can be used as the solution of material or the injectable dosage of suspension carries out administration, these substance dissolves or be suspended in the physiology acceptable diluent with pharmaceutical carrier, they can be for example water, oil, saline, glycerol or ethanol of aseptic liquid.In addition, for example wetting agent or emulsifying agent, surfactant, pH buffer substance etc. may reside in the compositions auxiliary substance.Other composition of pharmaceutical composition is the composition in oil source, animal origin, plant origin or synthetic source, for example Oleum Arachidis hypogaeae semen, soybean oil and mineral oil.In general, dihydroxy alcohol for example propylene glycol or Polyethylene Glycol is the liquid-carrier that is fit to, especially for Injectable solution.Antibody can be with bolus infusion liquid (depotinjection) or the form of implanting prepared product by administration, and they can be formulated into the form that allows active component slowly to discharge.The compositions of example contains the antibody of 5mg/mL, is formulated in to contain 50mM L-histidine and 150mM NaCl, adjusts to HCl in the buffer solution of pH6.0.

In typical case, compositions is prepared to injectable, perhaps as solution or as suspension; Before injection, also can prepare the solid form that is fit to be dissolved or suspended in the liquid media.Prepared product also can be emulsified or be wrapped in liposome or microgranule for example discharges to strengthen in polyactide, poly-Acetic acid, hydroxy-, bimol. cyclic ester or the copolymer.

For suppository, conjugate and carrier comprise for example poly alkylene glycol or triglyceride; Such suppository can from contain 0.5% to 10%, the mixture of the active component of better 1% to 2% scope forms.

Oral formulations contains excipient, for example pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose and magnesium carbonate.These compositionss are taked solution, suspension, tablet, pill, capsule, slow releasing preparation or form of powder, and containing 10% to 95%, being more suitable for is 25% to 70% active component.

Topical application can cause the release of transdermal or Intradermal.Topical can be by with the derivant of cholera toxin or its detoxification or its subunit or other similar bacteriotoxin co-administered and judicial convenience (referring to (1998) Nature 391:851 such as Glenn).Co-administered can be realized by composition is used or is expressed as fusion rotein as mixture or by the binding molecule that chemical crosslinking obtains.

In addition, transdermal delivery can use the skin approach or use transmission body (transferosome) to realize (Paul etc. (1995) Eur.J.Immunol.25:3521-24; Cevc etc. (1998) Biochem.Biophys.Acta1368:201-15).

Antibody of the present invention or antibody fragment also can choose wantonly with at least for effectively other agent combination administration of the disease of amyloid source property part.

Antibody of the present invention and antibody fragment also identify to stop ADDLs with neuron (for example hippocampal cell) thus the healing potion that combines the downstream events that prevention ADDLs causes in found application.The execution of this analytical method is by neuron being contacted with ADDLs existing under the situation of medicament, uses antibody of the present invention or antibody fragment to determine that ADDLs combines with neuronic under the situation of medicament existing.As those skilled in the art will recognize that, compare with the neuron that does not contact with medicament, can block ADDLs and neuronic bonded reagent, can reduce the amount that is attached to the ADDLs on the neuron, this amount can be detected in the immunoassay that uses antibody of the present invention or antibody fragment.It is open in this article with the immune analysis method of the bonded ADDLs of neuron to be fit to detection.

The reagent that can use method provided herein to screen comprises a large amount of chemical classes, although they are organic molecules in typical case, be preferably molecular weight greater than 100 less than about 2500 daltonian little organic molecules.Described reagent contains and albumen recurring structure interaction, the particularly necessary functional group of hydrogen bonding, typically comprises amine, carbonyl, hydroxyl or carboxylic group at least, preferably has two chemical functional groups at least.This reagent contains ring-type carbon or heterocycle structure and/or fragrance or many aromatic structures usually, is replaced by one or more above-mentioned functional groups.This reagent also can be found in biomolecule, comprises peptide, antibody, saccharide, fatty acid, steroidal, purine, pyrimidine, its derivant, analog or its combination.Reagent can comprise natural or synthetic library of compounds from source acquisition widely.

For example salt and neutral protein also can be included in during screening analyzes various other reagent.Reagent, for example protease inhibitor, nucleic acid inhibitor, antimicrobial reagent that can improve analysis efficiency etc. also can use.Mixture of ingredients can add with any order, as long as required combination can be provided.

Reagent by screening Analysis and Identification of the present invention will be of value to the disease that is derived from amyloid and/or the treatment of tau protein disease (tauopathy).In addition, the experimental system of having conceived these notions that are used to demonstrate has been represented the research tool that is used to assess, identify the new medicine target relevant with the inductive τ phosphorylation of screening and amyloid beta.

The present invention also provides the method for using antibody of the present invention or antibody fragment to detect the accumulation diseases associated of ADDLs and diagnosis and ADDLs.The accumulation that the disease relevant with the accumulation of ADDLs plans to comprise any wherein ADDLs has caused the damaged disease of physiological of long-term potentiation/memory formation.Such disease includes but not limited to A Cihai Mo's disease and similarly relevant with memory disorder.

According to these methods, will contact with antibody of the present invention or antibody fragment from patient's sample, the associative list of antibody or antibody fragment and sample is understood the existence of ADDLs in the sample.When being used for when of the present invention, sample means any body fluid or tissue of using immune analysis method to analyze of being suitable for.The sample of can the method according to this invention analyzing that is fit to includes but not limited to biopsy samples and the fluid sample from patient's (for example mammal for example people) brain.For external purpose (for example in the analytical method of the formation of monitoring oligomer), sample can be neuronal cell system or tissue sample.For diagnostic purpose, conceived sample and can suffer from the individuality of the disease relevant from suspection with the accumulation of ADDLs, perhaps, for example has the individuality of the family history that makes the individual easily disease that trouble is relevant with the accumulation of ADDLs from the individuality of risk with disease relevant with the accumulation of ADDLs.

The bonded detection of ADDLS in antibody or antibody fragment and the sample can use the immune analysis method (method for example disclosed herein) of any standard to carry out, perhaps when antibody fragment be the adaptive period of the day from 11 p.m. to 1 a.m of peptide for example, in conjunction with can be by for example directly being detected with the detectable labelled protein (for example beta galactosidase, GFP or luciferase) of adaptive sub-fusion.Thereafter, the existence of ADDL-antibody complex or do not exist respectively with the existence or do not exist of ADDLs in the sample is associated, and therefore the existence of relevant with the accumulation of ADDLs respectively disease or do not exist is associated.Having conceived one or more antibody of the present invention or antibody fragment can combine with the imaging technique based on immunity of existing Noninvasive, greatly to strengthen the detection and the early diagnosis of the disease relevant with the accumulation of ADDLs.

For the ease of diagnosis, the present invention also has been equipped with the test kit that contains antibody of the present invention or antibody fragment.Test kit comprises container, wherein hold one or more antibody of the multidimensional conformation that can discern ADDLs or antibody fragment and use antibody in conjunction with ADDLs with form antibody-antigenic compound and detect antibody-antigenic compound formation in case with antibody-antigenic compound whether exist with sample in ADDLs whether have a description that associates.The example of container comprises the porous plate that allows to detect simultaneously ADDLs in a large amount of samples.

By following non-restrictive example, the present invention will be described in more detail.

Embodiment 1: versatile material and method

The preparation of ADDL.(CA) ADDLs in prepares from A β 1-42 according to the method for having set up (Lambert etc. (2001) are the same) for Biosource, Camarillo in the F12 culture medium.In simple terms, with A β 1-42 peptide (American Peptide Co., Sunnyvale, CA or California Peptide Research, Inc., Napa, CA) weigh, be placed on and hold q.s HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) in the vial, make peptide concentration reach 10mg/mL.HFIP is added in the exsiccant peptide, and with the glass cap upper cover, soft vortex oscillation is mixed, and peptide/HFIP solution was stored in room temperature at least 1 hour.With the sample aliquot of peptide solution (50 or 100 μ L, contain respectively 0.5 or 1.0mg) be assigned in the taper centrifuge tube of a series of 1.5mL.Pipe is placed in the SpeedVac (speedvac) spends the night to remove HFIP.The pipe that contains exsiccant peptide thin film is covered lid, in having the sealed container of desiccant in-70 ℃ of preservations.

Before the use, A β 1-42 peptide thin film is taken out from-70 ℃ storage, be warmed to room temperature.Add fresh DMSO (44 μ L/mg peptide thin film; 5mM), peptide/DMSO mixture was vibrated 10 minutes with possible minimum speed on turbine mixer.Add F12 culture medium (2mL/mg peptide) in every pipe DMSO/ peptide, the lid upper tube cap is put upside down mixing.With the preparation liquid of 100 μ M be stored in 2-8 ℃ 18 to 24 hours.Sample 2-8 ℃ with 14, centrifugal 10 minutes of 000xg.Supernatant is transferred in the new pipe, be stored in 2-8 ℃ standby.

Biotin labeled ADDL prepared product (bADDLs) to be to be used for the preparation of the identical method of ADDL prepared product with above-mentioned, use the terminal biotin labeled amyloid beta peptide of 100%N-(American Peptide Company, Sunnyvale, CA).

The preparation of ADDL fibril.Every milligram of peptide adds 2mL 10mM hydrochloric acid in room temperature ADDL peptide thin film.Solution was mixed 5 to 10 minutes with possible minimum speed in turbine mixer, and the prepared product that obtains is stored in 37 ℃ 18 to 24 hours before use.

The monomer preparation.The prepared product of amyloid beta (1-40) peptide (A β 1-40) that evaporates HFIP is according to the preparation of the method for A β (1-42) peptide.The peptide Film Fractionation in the borate buffer (pH8.5) of every milligram of peptide 2mL 25mM, is divided into sample aliquot, be chilled in-70 ℃ standby.

The fibriilar preparation of people.The sample that obtains from refrigerated people's cortex have protease inhibitor (COMPLETE , Roche Diagnostics Corporation, Indianapolis, homogenate is 1 minute in ice-cold 20X F12 culture medium IN).Then with sample 4 ℃ with 10, centrifugal 1 hour of 000xg.After washing twice with F12, will precipitate and be suspended in again among the 2%SDS/F12, be incubated 30 minutes on ice.Then with sample 4 ℃ with 220, centrifugal 1 hour of 000xg.Precipitation is suspended among the cold F12 again, then with 15 seconds impulse ultrasound 1 minute.Use COOMASSIE PLUS ^TM(Pierce Biotechnology, Rockford IL) measure albumen to test kit.

Immunity.The solubility A β oligomer that obtains, be referred to herein as " synthetic " ADDLs, mix with 1: 1 with complete Freund's adjuvant (first and second inoculations) or incomplete Freund's adjuvant (inoculation that all are follow-up), in subcutaneous then (preceding twice inoculation) or peritoneal injection to three mice, cumulative volume is every about 1mL of mice.Per injection is made up of the ADDLs of the purification that is equal to 194 ± 25 μ g total proteins.The approximately per three weeks injection of mice once.After six injections, a dead mouse, its spleen is frozen.To have the spleen of mice of highest serum titre and SP2/0 myeloma cell then and merge existing under the situation of Polyethylene Glycol, be layered in 6 96 orifice plates.Cell is selected in the culture medium in 37 ℃ and 5%CO at the HAT of 200 μ L ₂Under cultivated 10 days, this culture medium is to have added 10% hyclone (FBS), 1 μ g/mLHYBRIMAX  (azaserine-hypoxanthine, Sigma-Aldrich, St.Louis, MO) and the ISCOV culture medium of 30% conditioned medium of from the SP2/0 cell culture fluid, collecting.Culture with the ISCOV culture medium feed supplement that is supplemented with 10%FBS once, took out culture supernatant and screen positive hole in ELISA in the time of the 10th day in the time of the 14th day.Positive culture is by further cloning for the Method of Limited Dilution of each 0.3 cell in hole with probability.Positive colony confirms in ELISA, and further breeds.

The screening of supernatant comprises 5 kinds of analyses: Dot blot and western immunoblotting (Lambert etc. (2001) are the same), use the non-degeneration immunoblotting of synthetic ADDLs, and use endogenous fibriilar Dot blot and the western trace of organizing acquisition from the people.These analytical tests combine (Dot blot) of antibody with ADDLs, and identified oligomer (western) with maximum affinity.In Dot blot, use 5pmole ADDLs test all antibody (576 supernatant that merge for the first time and merge for the second time in 1920 supernatant).Use the western trace further to screen to being tested as male supernatant then with the ADDLs of 10-20pmole.Screening is repeated to identify low positive son or false positive.The supernatant in the supernatant in 10 holes of first mice and 45 holes of second mice is amplified.Then with the freezing or sub-clone in hole that increases.

In female balb/c mice, use standard schedule (the modern handbook of molecular biology) to produce the ascites that contains monoclonal antibody.In simple terms, make mouse sensitization (primed) by peritoneal injection norphytane (pristane).A week after the sensitization is with the about 5x10 in the 1mL phosphate buffer (PBS) ⁶Individual hybridoma is by the peritoneal injection mice.Collect ascites after 10 to 14 days.Use BIO-RAD  AFFI-GEL  Protein protein A MAPS  II test kit, carry out the purification of IgG according to the rules of manufacturer.In each run, 3mL ascites is carried out desalination by desalting column, be eluted in the 4mL binding buffer liquid.Then with sample protein A post on the sample.After the flushing of 40mL binding buffer liquid,, collect the fraction of 5mL with elution buffer eluting pillar.Adding 60 μ L 10N NaOH in sample neutralizes.For buffer is transformed into PBS,, use the PBS eluting with second desalting column of sample on the sample.

Control antibodies.Polyclonal antibody M71/2 and M90/1 are from Bethyl Laboratories, and (Montgomery TX) obtains Inc..(Dedham MA) obtains from Signet Labs for anti-A β monoclonal antibody 6E10 (at residue 1-17) and 4G8 (at residue 17-24).In the present technique field, can discern 1-40 and 1-42 by the known monoclonal antibody WO-2 of western engram analysis (Ida etc. (1996) J.Biol.Chem.271:22908-22914).(Cambridge MA) obtains monoclonal antibody BAM-10 (at A β 1-40) from ABCAM .Can discern the amino acid/11-12 (Lu etc. (2000) Nat.Med.6:397-404) of A β sequence at monoclonal antibody 26D6 well know in the art.

Immunoblotting assay.SDS-PAGE (SDS-PAGE) uses existing method to carry out (Lambert etc. (2001) are the same), and (BIO-RAD , Hercules CA) with at 120V separates except using the 10-20%Tris-Tricine gel.Gel shifts according to standard method, and with 1: 40,000 dilution factor used second antibody according to routine.

For initial screening, 2.7 μ g ADDLs are equivalent to approximately every road 16-20pmol, on two dimension (2D) gel of 4-20%, separate.The method of electrophoresis and transfer as mentioned above.Use tracer dye as guidance, nitrocellulose filter is put into Surf-blot device (IdeaScientific, Minneapolis, MN), add 200 μ L and the blended hybridoma supernatant of blocking-up buffer in each hole in 20-21 hole, the blocking-up buffer is the TWEEN that has that contains 5% defatted milk powder ^TM20 tris salt buffer saline (TBS-T; Lambert etc. (2001) are the same).Shake insulation after 1.5 hours in room temperature, remove supernatant, with 200 μ L blocking-up buffer solution for cleaning hole.Then film is taken out from the Surf-blot device, in TBS-T, clean 3 times each 15 minutes.Then with second antibody (with the link coupled anti-mice IgG of HRP, 1: 40,000 dilution factor; Molecular Probes, Eugene OR) is incubated 1 hour with film in room temperature.After cleaning (3 times, each 15 minutes), (Pierce, Rockland IL) make oligomer visual to use the SUPERSIGNAL  of a half intensity.The fibriilar western immunoblotting of end user carries out in the same way, uses about 64 μ g people fibril tissues in each 2D SDS-PAGE immunoblotting.

Native polyacrylamide gel electrophoresis carries out (Chromy etc. (2003) Biochemistry42:12749-12760) according to existing method, except separating under 120V.

The Western trace.Separated albumen is transferred on the nitrocellulose filter.Hybridization is used in TBS-T and (contains 0.1%TWEEN ^TM20 TBS) 5% defatted milk powder in or the sealing of 1% bovine serum albumin (BSA) solution are spent the night, with first antibody insulation 1.5 hours, clean, with the bonded second antibody of horseradish peroxidase (HRP) (Amersham Biosciences Corp., Piscataway, NJ) insulation is 1 hour.After last cleaning, albumen West Femto chemical luminescence reagent kit (Pierce Biotechnology, Rockford, IL) and KODAK  image workstation 440CF or with film (HYPERFILMTM, Amersham Biosciences Corp., Piscataway, NJ) carry out visual.

The Hippocampus culture.Culture is from rules (Brewer (1997) J.Neurosci.Methods71:143-155 of E18 embryo according to standard; Stevens etc. (1996) J.Neurosci.Res.46:445-455) preparation.Living cells is counted, be layered on the coverslip with polylysine (200 μ g/mL) bag quilt, concentration is from 1.5x10 ⁴To 10 ⁶Individual cell/cm ²Also use interpolation NEUROBASAL by the culture medium of removing half ^TMCulture medium replace, culture medium is changed.

Former generation neuron.Former generation the Hippocampus culture from refrigerated, dissociated, neonate rat hippocampal cell (Cambrex, Corp., East Rutherford, NJ) preparation, this cell is melted and is layered in the 96 hole COSTAR  plates, concentration is 20,000 cells in each hole.Cell is maintained at the NEUROBASAL that does not contain L-glutaminate ^TM(GIBCO-BRL ^TM, Gaithersburg, MD), and additional B27 (GIBCO-BRL ^TM, Gaithersburg in culture medium MD), cell culture fortnight, is used in conjunction with research then.

The B103 cell.B103 neuroblast tumor cell strain (Schubert and Behl (1993) Brain Res.629:275-82) is cultured in and does not contain (GIBCO-BRL among the phenol red DMEM ^TM, Gaithersburg, MD), wherein exist 10%FBS (Hyclone, Logan, UT) and 1%Pen-Strep (GIBCO-BRL ^TM, Gaithersburg, MD).With the B103 cell separation of exponential growth, be layered in the 96 hole CORNING  plates, concentration is each hole 5,000 cell.Behind the bed board 24 hours, cell was used to assess the combination of ADDL and bADDL, and business-like and new anti-addl monoclonal antibody is carried out property research.

Dot blot is analyzed.(carry out, and ADDLs (each speckle 5pmole) or fibril are added on the nitrocellulose filter by (2001) the same method according to Lambert etc. for Dot blot.In order to carry out Dot blot subsequently, use and ADDLs is added on the exsiccant nitrocellulose filter each concentration two increment with picomole concentration different among 0.5 μ L from the deutero-template of Surf-blot equipment.Then with sample drying 15 minutes, with sealing buffer sealing 1 hour, with the antibody insulation that adds or do not add peptide 1.5 hours, wherein antibody in the room temperature pre-incubation at least 1 hour.From Surf-blot equipment, remove solution,, take out film in the slave unit with sealing buffer solution for cleaning hole.Nitrocellulose filter is cleaned in description according to the front, handles also visual with second antibody.

Immunocytochemistry.(Lambert etc. ((2001) are the same) carry out immunocytochemistry, except second antibody and ALEXAFLUOR  588 (Molecular Probes, Eugene, OR) combination according to ready-made method.Before being applied to 21 days hippocampal cell culture, antibody and ADDLs are with antibody: ADDLs ratio 1: 4 is incubated 1 hour in advance in room temperature.For endogenous ADDLs, clean at pair cell as previously mentioned, fixing and visual before, with HBP's (according to the same method preparation such as Lambert (2001)) and cell insulation 1 hour.

Will be from slight fixed frozen section (4% paraformaldehyde is fixed 30 hours at 4 ℃, and low-temperature protection is in 40 μ m sucrose) and the antibody (being diluted in phosphate buffer (PBS) at 1: 1000) of A Cihai Mo's disease and contrast Hippocampus 4 ℃ of incubated overnight.After removing antibody, section is washed 3 times with PBS, is incubated in room temperature with second antibody.Use DAB (SIGMA then ^TM, St.Louis is MO) to visual in conjunction with carrying out.To cut into slices with haematoxylin redyeing, sealing then, have SPOT ^TMINSIGHT ^TMImaging under the NIKON  ECLIPSE  E600 optical microscope of digital camera (v.3.2).

Quantitative immunocytochemistry.Cultured rat hippocampal cell and 500nM ADDLs were 37 ℃ of insulations 1 hour.Remove ADDLs by cleaning, with cell 3.7% formaldehyde fixed.Cell with contain 0.1%TRITON ^TMThe PBS-NGS of X-100 (PBS that contains 10% normal goats serum) insulation 30 minutes is cleaned once, with required first antibody (diluting) with PBS-NGS 4 ℃ of incubated overnight.Sample is cleaned, with the second antibody that is fit to for example ALEXAFLUOR  488 or 594 anti-mices and anti-rabbit igg s (Eugene is OR) 37 ℃ of insulations 2 hours for Molecular Probes, Inc..Coverslip cleaned and is locked in (Eugene OR), uses LEICA  TCS SP2 confocal scanning DMRXE7 microscope imaging for Molecular Probes, Inc. in the anti-fluorescent quenching mounting of the PROLONG  liquid (Antifade Mounting Medium).

ELISA。The anti-ADDLs IgG of polyclone (M90/1; Bethyl Laboratories, Inc., Montgomery, TX) amount with each hole 0.25mg is laid down on IMMULON ^TM3REMOVAWELL ^TMBatten (Dynatech Labs, Chantilly, VA) in, room temperature was placed 2 hours, with the TBS blind hole that contains 2%BSA.In the hole, add sample with the F12 dilution that contains 1%BSA, allow its 4 ℃ in conjunction with 2 hours, clean 3 times in room temperature with BSA/TBS.The monoclonal antibody of diluting in BSA/TBS uses the VECTASTAIN  ABC test kit at mice IgG to detect room temperature insulation 90 minutes.Use BIO-RAD  peroxidase substrate to make the HRP labelling visual, read on the plate device at the 405nm reading at Dynex MRX-TC.

Embodiment 2: the exploitation of anti-addl antibodies and property research

To inoculating once in three per three weeks of mice, inoculate altogether six times with ADDLs (per injection 194 ± 25 μ g albumen).The hybridoma that obtains from spleen cell and the SP2 cell fusion of these mices is cultured in 96 orifice plates.Supernatant in these holes is screened in Dot blot, use synthetic ADDLs to identify positive colony, they can compare to pick out difference with endogenous fibriilar Dot blot.Only seek with synthetic ADDLs not and the bonded hybridoma of endogenous fibril.For the product of further understanding hybridoma in conjunction with what and be combined under what condition and take place, each positive colony is carried out 3 kinds of western trace: ADDLs SDS-PAGE, ADDLs native gel and have endogenous fibriilar SDS-PAGE.Approximately filtering out 40 clones further checks.Tested each be cloned under the different condition identification to solubility A Cihai Mo's disease people brain extract, with evaluation and the bonded ability of blocking-up ADDL of the bonded ADDLs of cultured rat hippocampal cell.From culture medium, collect the antibody that is selected, use Protein G SEPHAROSE ^TMBe further purified.

Screen one group of hybridoma by Dot blot, about 30% produces positive supernatant at every turn.Wherein, have only one or two hybridoma in conjunction with synthetic ADDLs not in conjunction with endogenous fibril.When measuring, account for greatly under the low ADDL concentration of being cloned in of initial quantity 2% in conjunction with synthetic ADDLs not in conjunction with monomer with the western engram analysis.Clone 3B7 can not in conjunction with fibril, be left further and analyze in the western trace in conjunction with synthetic ADDLs.

Identify binding ratio being combined to trimer/tetramer oligomer of 1 to 2 clone to the material (12-24 monomer) of higher molecular weight.Identifying 2 to 3, be cloned under the non-degeneration condition can be in conjunction with natural ADDLs, but in that exist can not be in conjunction with ADDLs under the situation of SDS.

The result of this analysis shows that ADDLs is good antigen in mice, can produce binding ratio and the much bigger monoclonal antibody of monomeric binding affinity with synthetic ADDLs.

Embodiment 3: with the immunohistochemical analysis of the bonded endogenous and synthetic ADDLs of cultured rat hippocampal cell

The cultured rat hippocampal cell is also analyzed to determine whether the monoclonal antibody that can distinguish A Cihai Mo's disease and contrast brain extract can be identified with the bonded ADDLs of cultured cells (endogenous or synthetic).The Hippocampus culture prepares according to existing rules, allows growth 3-4 week.Synthetic ADDLs is according to standard schedule preparation (for example United States Patent (USP) U.S. patent 6,218,506).Endogenous ADDLs method according to ((2003) are the same) such as Gong from A Cihai Mo's disease people's brain is extracted.With ADDLs (100nM is in F12, or the 2mg total protein is in F12) and cell insulation 1 hour, then according to standard method cleaning and fixing.After the cleaning, with cell and 20C2,3B7, M94,2A10,4E2,2D6,4C2,2B4,5F10 or the insulation of 5G12 monoclonal antibody, then with the anti-mice second antibody insulation that combines ALEXAFLUOR  488.Use has COOLSNAP ^TMThe NIKON  DIAPHOT of HQ camera ^TMMETAMORPH is used in the fluorescence microscope imaging ^TM(Universal Imaging, Downingtown PA) analyzes software.

When using 20C2 visual, endogenous and synthetic ADDLs shows the focus pattern of standard in cultured cell.Therefore, can both identify with the bonded synthetic and endogenous ADDLs of cultured rat hippocampal cell for monoclonal antibody 20C2 pair.And 3B7 can not be in conjunction with fibril, high molecular oligomer and monomer, and 3B7 is the ADDLs of oligomerization to ADDLs's in conjunction with focus.Other antibody shows identification and is attached to different epi-positions on the ADDLs of cell, from bumps focus of its scope (M94,2A10) to the cyton specificity in conjunction with (4E2) and other intermediateness (2D6,4C2,2B4,5F10,5G12).

Embodiment 4: use mouse-anti ADDL antibody to suppress ADDL and combine with neuronic

For whether the monoclonal antibody of the brain extract of determining to distinguish A Cihai Mo's disease and contrast also can block combining of ADDLs and cultured cell, with cultured rat hippocampal cell and the pre-incubation of 20C2 antibody, by the combination of immunocytochemical determination ADDL.The Hippocampus culture is according to ready-made method preparation (for example referring to U.S. Patent No. 6,218,506 etc.).Endogenous ADDLs method according to ((2003) are the same) such as Gong from A Cihai Mo's disease people's brain is extracted.With ADDLs (100nM is in F12, or the 2mg total protein is in F12) 37 ℃ with 20C2 antibody pre-incubation 1 hour, add cell insulation 1 hour then.Cell is cleaned, fixes, with the anti-mice second antibody insulation that combines ALEXAFLUOR  488.

Can block combining of endogenous and synthetic ADDL and cultured cell with the 20C2 pre-incubation.Media and do not have the image of the contrast of second antibody to deceive.

Embodiment 5: use biotin labeled ADDLs to detect ADDL and combine with neuronic

The immunofluorescence method of use standard has detected ADDLs or bADDLs (biotin labeled ADDLs) combines with neuronic.Former generation hippocampal neuron (cultivating 14 days) or B103 cell (bed board 24 hours) are incubated 1 hour with 5-25 μ m ADDLs or bADDLs at 37 ℃, with warm culture medium cell are cleaned 3 to 4 times to remove unconjugated ADDLs or bADDLs then.Then cell is fixed 10 minutes in room temperature with 4% paraformaldehyde, it is paraformaldehyde (Electron Microscopy Sciences, Fort Washington, the PA) preparation with PBS dilution from 16%.Remove solution then, add fresh fixative, continue to place 10 minutes in room temperature.Cell is carried out penetrating processing in 10 minutes (contain 0.1%TRITON ^TM4% paraformaldehyde solution of-X 100; SIGMA, St.Louis MO), washes 6 times with PBS, with the sealing buffer (PBS that contains 10%BSA; Sigma, St.Louis is MO) 37 ℃ of insulations 1 hour.From this moment, the rules that detect bonded ADDLs and bADDLs begin different.For detect ADDL in conjunction with, (with 1: 1,000 was diluted among the PBS that contains 1%BSA with cell and 4G8; Signet Labs, Dedham, MA), 6E10 (1: 1,000 the dilution; Signet Labs, Dedham, a kind of (with 1: 1,000 dilution) MA) or in the anti-addl monoclonal antibody disclosed herein is 37 ℃ of incubated overnight.In addition, and the polyclonal antiserum that produces at τ (1: 1,000 dilution; Sigma, St.Louis MO) is used to make cell process visual.Second day, cell is cleaned 3 times with PBS, (diluted with 1: 500 in the anti-mice second antibody of room temperature and ALEXA  594 labellings with the PBS that contains 1%BSA; MolecularProbes, Eugene, OR) and the anti-rabbit second antibody of ALEXA  488 labellings (with 1: 1,000 dilution; Molecular Probes, Eugene, OR) insulation is 1 hour, washes in PBS 3 times, uses the microscopic examination combination with fluorescence ability.For detecting the bADDL combination, with cell and τ antibody incubated overnight.With PBS cell is cleaned 3 times then, (diluted with 1: 500 in the anti-rabbit second antibody (the same) of room temperature and ALEXA  488 labellings and the streptavidin of ALEXA  594 labellings; Molecular Probes, Eugene, OR) insulation is 1 hour, washes 5-6 time in PBS, uses the fluorescence microscope combination.Pair cell nuclear dyes if desired, nuclear is dyeed according to standard schedule with DAPI (1: 1000).

For the immunocytochemical assay of the ADDLs that uses the ADDL monoclonal antibody specific to carry out, cell with the ADDLs insulation after clean, fix, penetrating processing and sealing.In order to detect bonded bADDLs with monoclonal antibody, with a kind of incubated overnight in cell and 4G8,6E10 or the anti-addl monoclonal antibody of the present invention, the anti-mice second antibody with ALEXA  488 labellings detects immunoreactivity then.It is visual that bonded bADDLs uses the streptavidin of ALEXA  594 labellings to carry out, with DAPI to nuclear staining.Dyeing back bADDL is in conjunction with using fluorescence microscope to detect with the immunoreactive location (co localization) altogether of ADDL.

For every kind of evaluated monoclonal antibody (being 20C2,2H4,2B4 and 2A10) all observed to ADDLs insulation former generation hippocampal cell special immunoreactivity.Bonded ADDLs shows as the point-like stain along neurite and cyton.This pattern is only observed on a kind of neuron of subclass, and it is consistent with the former bonded report that uses commercialization and non-commercialization antibody to describe ADDL and primary neuronal.The pattern of stain and the result of many comparative studies have confirmed the specificity of these antibody.

Use bADDLs that the method for simplifying is provided, can detect bonded ADDLs and assessment bonded blocking-up ADDL and monoclonal antibody.When bADDLs is added in former generation hippocampal cell and with fluorescently-labeled streptavidin assessment combination, a kind of neurite of subclass cell can be seen the specificity combination in the culture.If subsequently with cell fixation and carry out immunocytochemistry and handle, use anti-addl antibodies, can observe the stain of same pattern to visual in conjunction with carrying out.In addition, stacks of these dyeing patterns show antibody staining and perfect overlapping in conjunction with bADDLs, so have proved that bADDLs and ADDLs are of equal value on function, and in binding analysis the use of bADDLs.

Embodiment 6: detect and measure mouse-anti ADDL monoclonal antibody to bADDL and the bonded difference displacement of neuron

The immunocytochemistry antagonist described herein that use has carried out revising at several places is blocked the bonded ability of ADDLs or bADDLs and neuron culture (primary neuronal or B103 cell) and has been carried out property research.(antibody: bADDLs) mix, (Miltenyi Biotec, Auburn is CA) in 37 ℃ of insulations 1 hour on the slow-speed shaking table in the microcentrifugal tube of silanization with the mol ratio of 1: 1,1: 5 or 1: 10 with monoclonal antibody and 1-10 μ m bADDLs.Then, antibody/bADDL mixture is joined in the cell, continue insulation 1 hour at 37 ℃.After the insulation, with cell clean, fixing, penetrating processing, sealing, then with at the polyclonal antiserum incubated overnight of τ so that cell process is visual.Second day, cell is cleaned, with the anti-rabbit second antibody of ALEXA  488-labelling and the streptavidin insulation of ALEXA  594 labellings, cell is examined so that detect with DAPI dyeing.After the dyeing, bonded degree can use fluorescence microscope to carry out visual assessment.

For the bonded degree of qualitative assessment bADDL and anti-addl antibodies reduce this interactional ability, developed alkaline phosphatase method of analyzing enzyme based on cell.Monoclonal antibody or PBS and 2.5-10 μ m (final concentration) bADDLs are mixed with the molar ratio of 1: 1 (B103 cell) or 1: 5 (former generation neuron), on the slow-speed shaking table in 37 ℃ of insulations 1 hour.After pre-incubation, antibody/bADDL prepared product is joined in B103 or the former generation neuron culture, be incubated 1 hour again in 37 ℃.Insulation is removed the bADDLs/ mixtures of antibodies after finishing, and washes plate 6 times with culture medium.Cell is fixed 10 minutes in room temperature in 4% paraformaldehyde, remove solution, adds fresh fixative, and cell is fixed 10 minutes again.With cell with containing 0.1%TRITON ^TM4% the paraformaldehyde of X-100 carries out penetrating processing (twice, each room temperature was carried out 10 minutes), washes in PBS 6 times, handles 1 hour at 37 ℃ with the PBS that contains 10%BSA.(with 1: 1,500 diluted with 1%BSA with the bonded streptavidin of alkali phosphatase; Molecular Probes, Eugene OR) joins in the cell, room temperature insulation 1 hour.Cell cleans 6 times with PBS, adds alkaline phosphatase substrate (CDP-STAR  and SAPPHIRE-II in cell ^TMApplied Biosystems, Foster City CA), is incubated 30 minutes, then at LJL illumination meter (Analyst AD; LJL Bio Systems, Sunnyvale, CA) upward measurement is luminous.

When assessing bADDLs, observed the dyeing pattern of antibody dependent with neuronic the combination.Some antibody that is studied has reduced the combination of bADDLs significantly, and other antibody is not too effective.Be that third order antibody demonstrates and strengthened bADDLs and combine with neuronic unexpectedly.Although the result of these researchs is qualitatively rather than quantitative in itself, they show that still antibody diversity blocking-up bADDL combines with neuronic.Qualitative assessment has shown similar trend (Fig. 1).That is to say that some antibody has reduced bADDL and combined with neuronic, some has faint or does not almost have effect, and several the enhancing in conjunction with (being 5F10 and 4C2) arranged.In addition, the Fab of mice can not block the combination of bADDL, has further confirmed the specificity of monoclonal antibody in this analytical method.

In neuroblast tumor cell strain B103, analyze combination and the blocking-up of bADDL, show that bADDL combines with the specificity of B103 cell, but do not combine with ovary cell line (CHO) specificity with monoclonal antibody.In addition, before bADDLs is joining in the B103 cell during with the anti-addl monoclonal antibody pre-incubation, in conjunction with sharply weakening.Demonstrate with the bonded qualitative assessment of monoclonal antibody blocking-up bADDL and B103 cell that monoclonal antibody is blocked bADDL and the bonded ability of cell is unequal (Fig. 2).As use former generation hippocampal cell viewed, some antibody is good at blocking combination very much, other then not too effective.In addition, antibody 4C2 also strengthens the bonded ability of B103 cell in bADDLs and the culture.

In order to show that bADDLs also in conjunction with the zone of Hippocampus participation in learning and memory, uses rat hippocampus slice culture thing to carry out a series of researchs.Can be in conjunction with the neuron in the dentate gyrus zone that studies show that out CA1-3 and Hippocampus in conjunction with bADDLs, and the neuron in other zone can not.When bADDLs and anti-addl monoclonal antibody pre-incubation, the bonded degree of bADDL weakens in the mode of dose dependent.These results demonstrate monoclonal antibody also can weaken combining of bADDLs and subgroup hippocampal neuron, and these neurons are crucial for learning and memory.

Embodiment 7: anti-addl antibodies and combining from A Cihai Mo's disease people and the endogenous ADDLs that contrasts brain

In order further to study the character of monoclonal antibody disclosed herein, determined whether monoclonal antibody can identify ADDLs (endogenous ADDLs) from the soluble extract of A Cihai Mo's disease human brain, and whether can with the contrast brain extract in making a distinction.Synthetic ADDLs and the people's brain extract for preparing in F12 dilute in F12, point sample (1 pmoleADDLs; 0.5 μ g brain extract) double is at exsiccant HYBOND ^TMECL ^TMOn the nitrocellulose filter.Having the corresponding C ERAD rank (setting up the association of record for A Cihai Mo's disease people) and the cerebral tissue in Braak stage obtains from NU brain center bank (NU Brain Bank Core).With dry 20 minutes of speckle, containing 3%H then ₂O ₂TBS (20mM Tris-HCl, pH7.5,0.8%NaCl) in room temperature insulation 20 minutes.Speckle is cut into inch strips, (contain 0.1%TWEEN with the TBS-T that contains 5% milk ^TM-20 TBS) room temperature sealing 1 hour.Rabbit polyclonal antibody M71/2 (dilution in 1: 2500,0.4 μ g; Bethyl Laboratories, Inc., Montgomery, TX), monoclonal antibody 6E10 (1: 500 the dilution, 3 μ g; Signet Labs, Dedham, MA) and monoclonal antibody 20C2 (1.52mg/mL disclosed herein, 5 μ g), 11B5 (2.54mg/ml, 5 μ g), 2B4 (1.71mg/mL, 5 μ g) and 2A10 (1.93mg/mL, 7.5 μ g) (Fig. 3) be diluted among milk/TBS-T of 1.5mL, room temperature insulation 1 hour.Speckle cleans 3 times with TBS-T, each 10 minutes.The second antibody that speckle is connected with horseradish peroxidase (HRP) (1: 40,000 is diluted among milk/TBS-T; Amersham Life Science, Inc., Arlington Heights is IL) room temperature insulation 1 hour.Speckle cleans 3 times with TBS-T, and each 10 minutes, wash 3 times with dH2O, use SUPERSIGNAL ^TMSubstrate is (with distilled water dilution in 1: 1; Pierce, Rockland, IL) colour developing is exposed to HYPERFILM ^TMECL ^TM(Amersham Life Science, Inc., Arlington Heights, IL) in.

Even except also only having the more weak bonded 2A10 in test under the higher protein concentration, the antibody of all tests can both be with intensive in conjunction with identifying synthetic ADDLs.Polyclonal antibody M71/2 and monoclonal antibody 20C2 and 11B5 still only demonstrate the combination of very faint similar background strongly in conjunction with two kinds of A Cihai Mo's disease human samples in the contrast brain.On the contrary, monoclonal antibody 6E10,2B4 and 2A10 demonstrate faint combination the with A Cihai Mo's disease human brain.

The result of this analysis shows that the monoclonal antibody of two kinds of tests can differentiate the brain of A Cihai Mo's disease people and contrast, wherein has the specificity of height with combining of endogenous oligomer.In addition, these data show that detection can finish at the commitment of A Cihai Mo's disease.

Embodiment 8: the immunohistochemical analysis of A Cihai Mo's disease people and contrast brain section

Use monoclonal antibody disclosed herein to carry out immunohistochemical analysis determining whether to use the monoclonal antibody that to distinguish A Cihai Mo's disease people and contrast brain extract in brain section that ADDLs is visual, and confirm the essence (for example spread, around neuron, mottled etc.) and the distribution in people's tissue thereof of ADDL labelling.The fixed section (40 μ m) of A Cihai Mo's disease people and contrast brain prepares according to standard method.To cut into slices with several monoclonal antibodies and a kind of polyclonal antibody labelling, examine with recognizing cells with haematoxylin redyeing then.Use has SPOT ^TMINSIGHT ^TMThe NIKON  ECLIPSE  E600 optical microscope of digital camera (v.3.2) obtains image.

Immunohistochemical analysis shows that ADDL dyeing appears in Hippocampus, entorhinal cortex and the middle frontal gyrus of A Cihai Mo's disease human brain.In serious A Cihai Mo's disease case, abundant optics ADDLs dyeing is arranged, mainly be shown as the speckle shape and distribute.In a A Cihai Mo's disease case, it is around neuronic that some optics ADDLs dyeing is observed.On the contrary, use any antibody all not have dyeing in any zone of control sample, even the neuron that is centered on by the point-like immunostaining is not seldom observed yet.

These data show that polyclonal antibody and monoclonal antibody can be used for identifying the ADDLs in the people's tissue that is fixed, labelling wherein is variable, comprises mottled zone, the tubular area of individual cells and some cell mass (clusters) and centers on neuronic labelling.In addition, observed the labelling of ADDLs in the A Cihai Mo's disease human brain at least 3 brain zones, and do not observed in the contrast brain, they are Hippocampus, entorhinal cortex and middle frontal gyrus.

The immunostaining of embodiment 9:A β 1-40 monomer sample contrast

A β 1-40 compares in DMSO/F12 oligomerization with ADDLs slower.Therefore, determined whether A β 1-40 can contrast as the monomer sample.ADDLs is carried out aperture exclusion chromatography (SEC) (ADDL063) on SUPERDEX  75 posts, it is divided into two peaks.With A β 1-40 (45.5mM) preparation in DMSO/F12, freezing, thawing then.Sample is diluted with F12, and (BIO-RAD , Waltham is MA) with about 2: 1 mixed with the Tricine sample buffer.(BIO-RAD , Waltham carry out on MA) SDS-PAGE, and (BIO-RAD , Waltham MA) carried out under room temperature and 120V 80 minutes to use the Tris/Tricine/SDS buffer at the 10-20%Tris-Tricine gel.With gel SILVERXPRESS ^TM(INVITROGEN ^TM, Carlsbad, CA) silver dyes (60 pmolesA β 1-40 or ADDLs; 40pmoles peak 1 or peak 2).In addition, with gel (20 pmolesA β 1-40 or ADDLs; 30 pmoles peaks 1 or peak 2) electricity is transferred to HYBOND ^TMECL ^TMOn the nitrocellulose filter, use 25mM Tris-192mM glycine, 20%v/v methanol, pH8.3,0.02%SDS carried out 1 hour under 8 ℃ and 100V.Speckle (is contained 0.1%TWEEN with the TBS-T that contains 5% milk ^TM-20 20mM Tris-HCl, pH7.5 0.8%NaCl) spends the night 8 ℃ of sealings.With monoclonal antibody 6E10 (dilution in 1: 2000; Signet Labs, Dedham, MA), monoclonal antibody 20C2 (1.52mg/mL, 1: 2000 the dilution; Fig. 3) or polyclonal antibody M71/2 (dilution in 1: 4000, Bethyl Laboratories, Inc., Montgomery, TX) with the dilution of milk/TBS-T, with speckle room temperature insulation 90 minutes.Speckle is washed 3 times with TBS-T, each 10 minutes, then with the second antibody of horseradish peroxidase (1: 40,000 is diluted among the TBS-T; Amersham Life Science, Inc., Arlington Heights is IL) room temperature insulation 1 hour.After cleaning 3 times, each 10 minutes with TBS-T, speckle is washed 3 times with dH2O, (dilute at 1: 1 with SUPERSIGNAL  West Femto maximum sensitivity substrate with distilled water; Pierce, Rockland, IL) colour developing is exposed to HYPERFILM ^TMECL ^TM(Amersham Life Science, Inc., ArlingtonHeights, IL).

Silver dyes to analyze and shows that A β 1-40 is dark monomer band.On the contrary, ADDLs and peak 1 are shown as monomer, trimer and the tetramer, although the tetramer is less.The silver at peak 2 dyes to analyze and demonstrates dark monomer and more shallow trimer and very shallow tetramer band.

A β 1-40 only demonstrates shallow monomer band with the immunostaining of 6E10.The immunostaining of ADDLs and peak 1 usefulness 6E10 demonstrates monomer, trimer, the tetramer and 12-14mer.2 usefulness 6E10 dyeing in peak demonstrates dark monomer and some shallow trimer and tetramers, does not have 12-24mer.There is not monomer with 20C2 or M71/2 dyeing A β 1-40.Although 20C2 and M71/2 only show to ADDLs and peak 1 or do not have monomer dyeing that these samples have trimer, the tetramer and 12-24mer to dye with 20C2 and M71/2 minimum.Peak 2 usefulness 20C2 and M71/2 dyeing demonstrate shallow monomer, trimer and the tetramer, do not observe 12-24mer.A β 1-40 compares with the 6E10 immunostaining more shallow with the ADDL monomer, darker although silver dyes.

These results show that opposite with the 6E10 antibody that demonstrates the good identification of monomer, the gel that shifts with the transfering buffering liquid that contains 0.02%SDS demonstrates minimum monomer detection with the special antibody of oligomer.The immunostaining of SEC level part demonstrates peak 2 and mainly contains monomer and a spot of trimer and the tetramer, do not have 12-24mer, and there are monomer, trimer, the tetramer and 12-24mer in peak 1.

In order further to study the character of monoclonal antibody, developed the sandwich ELISA that uses polyclonal antibody M90 to detect ADDLs as capture antibody in conjunction with peak 1 and peak 2.SEC peak 1 and peak 2 in this article refer to ADDLs at SEPHADEX ^TMTwo main levels that 75 column fractionations form part have the oligomer of potential source biomolecule activity or non-activity with difference.Native gel electrophoresis confirms to be separated into big (〉 50kDa) and little (＜30kDa) aggregation, they are stablized down at 37 ℃.These peaks are used separately as the detection material of clone's supernatant.In conjunction with visual with VECTASTAIN  test kit.All observed the difference that two peaks are discerned with all antibody.The ratio (Fig. 3) of comparison peak 1 and peak 2 antagonist 2B4 and 20C2 for example.Have only an antibody to reflect to the preferred property of the control antibodies at peak 2 (6E10).

Embodiment 10: detect from A β 1-42 and form ADDL

That has used in Dot blot that polyclonal antibody shows time dependence forms ADDL from A β 1-42.Therefore, confirmed that the monoclonal antibody 20C2 of preferred combination oligomer also can demonstrate the signal that increases in time when from A β 1-42 formation ADDLs.The HFIP Film Fractionation of A β 1-42, about 750 pmoles in 1.5mL DMSO (0.5mM), is diluted to final volume 100 μ L with 2 μ L sample aliquot with F12 (10nM), in insulation on ice.At specific time point 2 μ L (20fmol) reactant mixtures are transferred to exsiccant HYBOND ^TMECL ^TMOn the nitrocellulose filter (Amersham Life Science, Inc., Arlington Heights, IL).Nitrocellulose filter is used TBS-T (20mM Tris-HCl, pH7.5,0.8%NaCl, the 0.1%TWEEN that contains 5% defatted milk powder ^TM-20) room temperature sealing 1 hour.With polyclonal antibody M90/1 (Bethyl Laboratories, Inc., Montgomery, TX) or monoclonal antibody 20C2 (1.52mg/mL) in milk powder/TBS-T with 1: 2000 the dilution, at room temperature, clean 3 times each 10 minutes then with TBS-T with speckle insulation 90 minutes.With the bonded second antibody of HRP (Amersham Life Science, Inc., Arlington Heights, IL) in milk powder/TBS-T with 1: 40,000 dilution room temperature insulation 60 minutes, is cleaned speckle then with above-mentioned.After simple the cleaning, speckle and SUPERSIGNAL  West Femto maximum sensitivity substrate (are diluted with distilled water at 1: 1 with distilled water; Pierce, Rockland, IL) insulation is 60 seconds, is exposed to HYPERFILM ^TMECL ^TM(Amersham Life Science, Inc., ArlingtonHeights, IL) in.With Dot blot scanning, use ADOBE  PHOTOSHOP  to measure the intensity of speckle.

What two kinds of antibody had all detected time dependence forms ADDL from A β 1-42, and wherein the result of 20C2 demonstrates signal and concordance preferably.There is not antibody can under the concentration suitable, detect A β 1-40 with ADDL.These data have further confirmed the oligomer specificity of this antibody, because monomer always exists, and oligomer forms in time.In addition, M90/1 and 20C2 demonstrate minimum identification to A β 1-40 monomer, even under the concentration higher 100 times than ADDLs.

Embodiment 11: competitive Dot blot analysis

Whether can use synthetic ADDLs, 20C2 and A β 1-40 to carry out competitive Dot blot analysis in order to determine monoclonal antibody disclosed herein in conjunction with monomer.ADDLs is added on the exsiccant nitrocellulose filter with the concentration of 10pmol/0.5 μ L.Nitrocellulose filter was sealed in 5%NDM/TBS-T 1 hour, then the 20C2 among each 200 μ L5%NDM/TBS-T (final concentration 1.5 μ g/mL) and the ADDLs of variable concentrations and fresh A β 1-40 are incubated 1 hour.Use the SURF-BLOT device to be added on the nitrocellulose filter these solution then, shake insulation 1.5 hours in room temperature.Make speckle visual with anti-mice IgG-HRP and chemiluminescence agent then.Use KODAK  IMAGESTATION  440 and EXCEL  to carry out quantitatively.

The result of these analyses show synthetic ADDLs in the solution can be effectively and block 20C2 specifically and be fixed on nitrocellulose filter on the combining of ADDLs, to observed half maximum inhibition of ADDLs less than 50nM.On the contrary, the A β 1-40 in the solution can not block combining of 20C2 and fixed ADDLs.

For determine which partly constitute A β 1-42 molecule in conjunction with epi-position, use ADDLs, 20C2 and peptide to carry out competitive Dot blot analysis.ADDLs with four kinds of concentration among per 0.5 μ L (each 1,0.5,0.25 and 0.125pmole) point sample to nitrocellulose filter.Nitrocellulose filter sealed 2 hours in 5%NDM/TBS-T after, 50,100 and the peptide of 200pmol be added into (1.52 μ g/mL, final concentration 1.9pmol is in 5%NDM/TBS-T) among the 200 μ L 20C2, shake in room temperature.Then solution is used SURF-BLOT equipment and nitrocellulose filter room temperature insulation 1.5 hours.Make in conjunction with visual with anti-mice IgG-HRP and chemiluminescence agent.

The result of this analysis shows with combining by ADDLs itself and A β 1-28 of ADDLs and blocks, but do not blocked by the combination of other peptide.Therefore, need some A β 1-28 to realize in conjunction with epi-position but the conformation that in A β 1-12 and A β 12-28 or its combination, can not provide.In addition, A β 1-28 forms dimer, by the combination of sterically hindered blocking-up ADDLs.

In order to determine whether A β 1-28 assembles (similar with A β 1-42) to such an extent as to or folding blocking-up for the epi-position that combines of 20C2, the SDS-PAGE gel is carried out silver dyes, and carry out the western engram analysis.Use 10-20%Tris-Tricine SDS-PAGE that ADDLs and A β 1-28 (using 60pmol, other situation 20pmol for every when silver dyes in two roads) are separated.The road of 60pmol is cut, uses SILVERXPRESS ^TM(INVITROGEN ^TM, Carlsbad, CA) dyeing; In addition, gel (20pmoles ADDLs and A β 1-28) electricity is transferred to HYBOND ^TMECL ^TMOn the nitrocellulose filter, use 25mM Tris-192mM glycine, 20%v/v methanol, pH8.3,0.02%SDS carried out 1 hour under 8 ℃ and 100V.Speckle (contains 0.1%TWEEN with the TBS-T that contains 5% milk powder ^TM-20 20mM Tris-HCl, pH7.5,0.8%NaCl) sealing.Sample and 20C2 (1: 1000,1.52mg/mL) or 20C2+A β 1-28 (2nmol, pre-incubation 2 hours) in above-mentioned sealing buffer in room temperature insulation 1.5 hours.Make in conjunction with visual with anti-mice IgG-HRP (1: 40,000 is diluted among the TBS-T) and chemiluminescence agent.

Silver dyes monomer, trimer and the tetramer that demonstrates in the ADDL road, and is a kind of but A β 1-28 road has only, and operates in about dimeric position.ADDLs rather than A β 1-28 are visual by 20C2, and 20C2 is blocked by A β 1-28 with combining of all ADDL types.In addition, when 20C2 was blocked by A β 1-28 in conjunction with epi-position, 20C2 can not discern A β 1-28 peptide in the western trace.

Embodiment 12: the isotype analysis of anti-addl antibodies

In order further to study the character of monoclonal antibody disclosed herein, use the SIGMA IMMUNOTYPE that contains mouse monoclonal antibody isotype typing reagent ^TMTest kit, (Sigma-Aldrich Co., St.Louis MO) have carried out the isotype analysis according to the description of manufacturer.The result of this analysis is presented among Fig. 3.

Embodiment 13: the linear epitope mapping of the core of anti-addl antibodies

The specificity that has detected anti-addl monoclonal antibody and amyloid beta peptide in the standard ELISA analysis interacts.In simple terms, synthetic peptide or be that ADDL or fibril are used as antigen in some cases is coated on NUNC with the concentration of 4 μ g/mL (about 800 to 1200nM) ^TMMAXISORB ^TMOn the plate.Except as otherwise noted, peptide is coated in the 5mM sodium bicarbonate buffer liquid (pH9.6), spends the night in 4 ℃.With containing 0.05%TWEEN ^TMThe PBS closure plate of 20 and 3% (w/v) defatted milk powder is after 1 hour, and the concentration titration monoclonal antibody to determine in the sealing buffer is incubated 1 hour at ambient temperature with plate then, softly shakes.After cleaning, will join in the plate with the bonded goat anti-mouse IgG of HRP (H+L) of sealing buffer dilution.After removing unconjugated HRP conjugate, in plate, add chromogenic substrate TMB through a large amount of cleanings.Reading on the plate device in 450nm wavelength measurement absorbance.

Map for core linear epitope, synthesized one group of eclipsed, as to have covered A β 1-42 10 amino acid whose peptide (table 1) anti-addl monoclonal antibody.Three 14 amino acid whose peptides with aminoacid sequence that A β 1-42 puts upside down also are synthesized, as nonspecific control peptide.

Table 1

All peptides all are dissolved among the DMSO, and concentration is approximately 400 to 500 μ M (1mg/mL), are divided into a plurality of sample aliquot and are stored in-20 ℃.Peptide is used to elisa assay to determine the core epi-position of anti-addl monoclonal antibody.Every kind of monoclonal antibody (3,1,0.3 and 0.1 μ g/mL) under four kinds of concentration is tested at the group (from residue 1 to 25) of N-terminal peptide or the group (from residue 17 to 42) and the control peptide of C-terminal peptide.The core linear epitope of monoclonal antibody group is listed in the table 2.Several commercialization monoclonal antibodies (6E10, BAM-10,4G8 and WO-2) are also contained in the test form with check analysis, and that reports in the core linear epitope that the result confirms them and the document of delivering is identical.

Table 2

* the position in A β 1-42. ^aIgG1, ^bIgG2b, ^cIgG2a.nd, undetermined.

The map N-terminal zone of A β 1-42 of 9 quilts in 12 evaluated ADDL monoclonal antibody specifics, 7 in them are plotted on amino acid residue 3-8.Two monoclonal antibody 2H4 and 2E12 tend to bigger a little epi-position.Three kinds of monoclonal antibody 1F4,1F6 and 3B3 can not combine with the group of eclipsed peptide, even under the high concentration of 3 μ g/mL, but their epi-position is estimated to be positioned at the N-terminal of A β 1-42, because they can be in conjunction with A β 1-20 peptide, this peptide is used as positive control in experiment.

Embodiment 14: the affinity of mice anti-addl antibodies and specificity

Developed based on the binding analysis of solution and determined affinity and the specificity of anti-addl antibodies different amyloid beta peptide prepared products (ADDL, fibril, A β 1-40, A β 1-20).Set up quantitative ELISA, it can catch monoclonal antibody at being coated on NUNC ^TMThe range of linearity of the dose response of the ADDL on the plate.On the basis of this information, select the monoclonal antibody of fixed concentration, under this concentration, can in ELISA, produce the consistent all the time OD signal that just has been higher than analysis noise (reading of OD450nm is approximately 0.2 to 0.5).In solution, surely descend incubated overnight to reach balance with the different amyloid beta peptide substrate of 20 titre points (ADDL, fibril, A β 1-40, A β 1-20) IgG of this fixed concentration then in the chamber.The amount of free IgG is determined at second day in the mixture, uses quantitative ELISA to be incubated 1 hour on conventional elisa plate and carries out.(ErithacusSoftware, Surrey UK) calculate combined IgG part, and the dependency of the titre of bonded IgG and free ligand (substrate) is used to calculate K to use the GraFit program _DTherefore, every kind of antibody is represented as intrinsic affinity values (K to the preferred property of the substrate of different amyloid beta peptide prepared products _D).

Use this analytical form that several advantages are arranged.At first, the interaction of antibody and substrate is to carry out in mutually at solution, therefore without any at for example conventional elisa assay or BIACORE ^TMThe restriction of the sort of solid phase surface in the experiment must be considered into during at decryption to the interactional potential impact of monoclonal antibody and substrate from the solid phase surface of elisa plate or sensor chip in those analyses or experiment.The second, allowing to interact reaches balance.Therefore, the interaction of IgG and substrate can take place under the limited concentration of two kinds of compositions, therefore need not consider because the precipitation of the IgG that high experimental concentration causes or the oligomerization of additional amyloid beta peptide.The 3rd, the reading of analysis does not rely on the antigen in the solution; Therefore, any heterologous of amyloid beta peptide will not influence the explanation of data and the foundation of mathematical model in the different peptide prepared products (for example ADDL or fibril).Sensitivity for analysis is limited to the limit that elisa assay detects, and allows this analytical method to use K in the scope of nanomole _DValue assessment monoclonal antibody.Conceived alternate substrate for example fluorometric reagent improve the range of sensitivity.It is believed that in quantitative ELISA in 1 hour temperature retention time of catching free IgG, immune complex is minimumly destroyed.

The amount of free IgG is determined by standard curve, and the titre of different substrates is mapped.Mapped with the amount of the bonded IgG of different substrates, information is used to use among the GraFit appropriate mathematical model to carry out curve fitting.The K of monoclonal antibody group disclosed herein _DSummary be presented in the table 3, represent with the nM scope.

Table 3

* all antibody all is IgG.

Value with the italic tabulation has the high standard error, and match is bad.

IC: lame data.

N/T: not test.

Embodiment 15: the detection of τ phosphorylation and measurement

The τ of hyperphosphorylation (pTau) is the sign of A Cihai Mo's disease, although understand seldom for the incident that causes this hyperphosphorylation.Do not wish to be bound by any theory, it is believed that ADDLs may play a significant role in this phosphorylation event.For this is studied, cultivated neuron culture (former generation neuron and B103 cell) according to the description of front, in culture medium, add 1 μ m bADDLs or media, cultivation is proceeded 1,6 or 24 hour then.When every kind cultivate to be finished, with cell clean, fixing, penetrating processing, blocking-up, then with monoclonal anti serum (AT8,1: 500 at pTau; Pierce, Rockland, IL) incubated overnight.Second day, cell is cleaned, be incubated with the anti-mice second antibody of ALEXA  488 labellings and the streptavidin of ALEXA  594 labellings, cell is examined to allow to detect with DAPI dyeing.Use the pTau dye levels of fluorescence microscope assessment cell then, noticed bonded dependency with bADDL at each time point.

The result of this analysis shows that the combination of bADDL and B103 cell is compared with the cell that media is handled has increased the level of pTau in the cell process.In former generation hippocampal cell, also noticed similar variation.When cellular exposure during, also observing the painted increase of pTau in the cell subsets in conjunction with bADDL in bADDLs6 hour.Further studied bADDLs by the adjusting of pTau with the time course research that the B103 cell carries out.Adding bADDLs has caused the limit of pTau to increase in 1 hour.But pTau dyeing sharply increases after adding bADDLs6 hour, and keeps raising after 24 hours.Therefore, these data show that ADDL combines the cascade that may start incident in the cell with neuronic, has caused the hyperphosphorylation of τ, the accumulation of neurofibrillary tangles and final cell death.So those skilled in the art will recognize that blocking-up ADDLs combines with neuronic, will stop the incident in these downstreams conversely, thus for the disease that is derived from amyloid and/or with the treatment of tau protein diseases associated be useful.In addition, also may can illustrate other approach to ADDL in conjunction with the better understanding that the signal that starts and cause pTau to produce conducts incident, they may can be used as the target that is fit to of the new Therapeutic Method of exploitation.

The interaction of embodiment 16:A β peptide/ADDL and antibody and to accumulative inhibition

By fluorescence resonance energy transmission (FRET) and fluorescence polarization (FP), use 1: 4 fluorescein-labeled A β 1-42 monomer and the monomeric mixture of native peptides, under the situation that has selected monoclonal antibody disclosed herein, observed the variation of ADDL kinetics of aggregation and oligomerization size.Because FRET, self cancellation of fluorescein emission had caused fluorescence intensity to reduce by 3 to 5 times in the short time of hour level span when monomer was incorporated among the ADDLs.In addition, the increase of size has caused FP to increase twice when monomer aggregation becomes oligomer ADDL.Have accumulative FRET of ADDL and a FP kinetics progress curve under various new and the situations business-like anti-ADDL and anti-A β peptide antibody, demonstrated antibody suppress that ADDL assembles and/or the ability of binding peptide oligomer in difference (Fig. 4).

Analysis is carried out in 384 hole CORNING  Non-Binding Surface black non transparent microwell plates.Analysis buffer contains 50mM MOPS-Tris (pH8.0) and 100mM MgCl ₂Analysis volume is 50 μ L, comprises 0.2 μ M FITC-A β 1-42 and 0.8 μ M A β 1-42, and analysis temperature is 37 ℃.The gathering of ADDL is read the monitoring of plate device with Tecan GENios Pro, and excitation wavelength is 485nm, and emitting detection wave is long to be 515nm.The kinetics trace is collected by per 5 minutes records fluorescence intensity and polarization readings in 6 hours time course.The negative control reaction that during this period of time obviously is not gathered into ADDLs lacks MgCl ₂, but contain all other buffer and peptide components.The positive control reaction contains all buffer compositions, but does not contain the monoclonal antibody reagent of interpolation.For combination and the accumulative inhibition of testing ADDL, antibody and peptide mixer are incubated being reduced to from 500nM under 8 kinds of concentration of 5nM.

This analysis can be used for ADDL is classified in conjunction with the different overviews (profile) of behavior and the accumulative inhibition of ADDL.Bigger ADDL type can be used as feasible therapeutic strategy by combining and neutralization of taking place with the interaction of specific epi-position of ADDL and/or comformational epitope.In addition, assemble the specific and/or comformational epitope of the ADDL that exists in the aggregation type (non-monomer) by medium (intermediate) ADDL that is combined in temporary transient (transient) and suppress to aggregate into bigger ADDL, for anti-ADDL therapy provides another kind of optional strategy.Show the FP progress curve that has significantly difference between antibody, indicated the combination of this intermediate form or stability types.With other function, cell proofreaied and correct the FP/FRET behavior of monoclonal antibody with external effect after, allow to select required immunotherapy binding mode.

The result of analysis disclosed herein shows that 1F6,2A10,5F10,2D6 and 2B4 show potential gathering and suppress, and 20C2,1F4 and 4C2 show medium gathering inhibition, and 2H4,3B3 and 4E2 demonstrate faint gathering and suppress (Fig. 4).As in table 4 general introduction with Fig. 5 in illustrated, 20C2,4E2,3B3 and 5F10 demonstrate different biochemistry behaviors.

Table 4

	Suppress with the detected potential gathering of FP/FRET	Detected faint or assemble and suppress with FP/FRET
			FP order in the time of 30 minutes	20C2	4E2
Lower or do not have a FP order in the time of 30 minutes	5F10，1A9	3B3

In addition, at the low peptide A β 1-42[Nle35-Dpro37 that forms number of monomers] the antibody 1A9 of one of 5 kinds of antibody purified (being 1A9,1E3,1G3,1A7 and 1E5) of producing, assemble according to it and to suppress and FP behavior and 5F10 branch are opened.

In addition, when measuring with SEC/ICC, find that 20C2 can combine with A β 7-42 peptide aggregation aggregation oppositely charged, truncate, show the linear epitope that lacks with the bonded routine of peptide of A β 7-42 oppositely charged, this peptide has very different sequences in the position corresponding to the residue 7-16 of A β, i.e. A β (7-42) [Orn ₇Orn ₁₁D ₁₃D ₁₄E ₁₆Nle ₃₅].Therefore, the comformational epitope of the element among 20C2 and the residue 17-42 that depends on A β combines, but only is after gathering.

Embodiment 17: separate the mouse antibodies variable region sequences

After using specially designed primer to carry out polymerase chain reaction (PCR), for the CDNAs of mouse antibodies variable domains coding is cloned and is checked order, these primers can with 5 of mice constant region '-Mus targeting sequencing terminal and upstream, V district hybridizes.Guaranteed like this mice variable region sequences that obtains be complete sum accurately.In simple terms, use QIAGEN  OLIGOTEX  Direct mRNA Mini Kit to extract mRNA, use the first chain cDNA synthetic agent box to be transformed into cDNA then from mouse hybridoma cell strain.Then cDNA is obtained the antibody variable region sequence as template in the PCR reaction.

In order to obtain the light chain variable region sequence, use each and 3 ' PCR primer MKC-1 (table 5) in 11 light chains, 5 ' the PCR primer (MKV-1 is to MKV-11) to set up 11 independently PCR reactions.

Table 5

5 ' primer	Sequence	SEQ IDNO：
			MKV-1MKV-2MKV-3MKV-4MKV-5MKV-6MKV-7MKV-8MKV-9MKV-10MKV-11	GAT C TC TAG ATG AAG ATT GCC TGT TAG GCT GTT GGTGCT GGAT C TC TAG ATG GAG WCA GAC ACA CTC CTG YTATGG GTGGAT C TC TAG ATG AGT GTG CTC ACT CAG GTC CTG GSGTTGGAT C TC TAG ATG AGG RCC CCT GCT CAG WTT YTTGGM WTC TTGGAT C TC TAG ATG GAT TTW CAG GTG CAG ATT WTCAGC TTCGAT C TC TAG ATG AGG TKC YYT GYT SAY CTY CTCTGR GGGAT C TC TAG ATG GGC WTC AAA GAT GGA GTC ACAKWY YCW GGGAT C TC TAG ATG TGG GGA YCT KTT TYC MMT TTTTCA ATGGAT CTC TAG ATG GTR TCC WCA SCT CAG TTC CTT GGAT CTC TAG ATG TAT ATA TGT TTG TTG TCT ATT TCTGAT CTC TAG ATG GAA GCC CCA GCT CAG CTT CTC TTCC	223224225226227228229230231232333
3 ' primer	Sequence	SEQ IDNO：
			MKC-1	GAT C GA GCT CAC TGG ATG GTG GGA AGA TGG	234

Underscore and italic sequence are represented XbaI and SacI restriction site respectively.W=A or T, M=A or C, K=G or T, Y=C or T, and R=A or G.

In order to obtain weight chain variabl area sequence, use each and the isotype specificity 3 ' PCR primer (MHCG-1 that is fit in 12 heavy chains, 5 ' the PCR primer (MHV-1 is to MHV-12), MHCG-2A, MHCG-2B, MHCG-3) (table 6) set up 12 independently PCR reactions.

Table 6

5 ' primer	Sequence	SEQIDNO：
			MHV-1MHV-2MHV-3MHV-4MHV-5MHV-6MHV-7MHV-8MHV-9MHV-10MHV-11MHV-12	GAT C TC TAG ATG AAA TGC AGC TGG GGC ATS TTC TTCGAT C TC TAG ATG GGA TGG AGC TRT ATC ATS YTC TTGAT C TC TAG ATG AAG WTG TGG TTA AAC TGG GTT TTTGAT C TC TAG ATG RAC TTT GGG YTC AGC TTG RTT TGAT C TC TAG ATG GGA CTC CAG GCT TCA ATT TAG TTT TCC TTGAT C TC TAG ATG GCT TGT CYT TRG SGC TRC TCT TCT GCGAT C TC TAG ATG GRA TGG AGC KGG RGT CTT TMT CTTGAT C TC TAG ATG AGA GTG CTG ATT CTT TTG TGGA T CTC TAG ATG GMT TGG GTG TGG AMC TTG CTT ATT CCT GGAT CTC TAG ATG GGC AGA CTT ACC ATT CTC ATT CCT GGAT CTC TAG ATG GAT TTT GGG CTG ATT TTT TTT ATT GGAT C TC TAG ATG ATG GTG TTA AGT CTT CTG TAC CTG	235236237238239240241242243244245246
3 ' primer	Sequence	SEQIDNO：
			MHCG-1MHCG-2AMHCG-2BMHCG-3	GCATC GAG CTC CAG TGG ATA GAC AGA TGG GGGGCATC GAG CTC CAG TGG ATA GAC CGA TGG GGGGCATC GAG CTC CAG TGG ATG AGC TGA TGG GGGGCATC GAG CTC CAA GGG ATA GAC AGA TGG GGC	247248249250

Underscore and italic sequence are represented XbaI and SacI restriction site respectively.W=A or T, M=A or C, K=G or T, Y=C or T, and R=A or G.

Each light chain PCR reaction contains 46 μ L INVITROGEN ^TMOne of PLATINUM  PCRSuper Mix, 1.0 μ L, 100 μ M5 ' primers (MKV-1 is to MKV-11), 1.0 μ L100 μ M3 ' primers (MKC-1) and 2.0 μ L hybridoma cDNA.Similarly the PCR reaction is used to clone the murine heavy chain variable region sequences.Reaction is placed in the DNA thermal cycler, after through 97 ℃ of initial denaturing steps 2.0 minutes, carries out 30 following circulations: 95 ℃ 30 seconds, 55 ℃ of 45 seconds and 72 ℃ 90 seconds.In the end after circulation, use last extension step 72 ℃ 10 minutes.In order to determine which PCR reaction has produced product, get 5 μ L aliquot sample from each reaction and separate at 1.5% (w/v) agarose/1XTAE buffer gel, contain 0.5 μ g/mL ethidium bromide in the glue.From the segmental reaction that produces expection size (420 to 500bp), pass through gel-purified PCR product then, with XbaI and SacI digestion, (New England Biolabs, Beverly is among the XbaI and SacI site in polyclone district MA) to be connected to plasmid pNEB193.In addition, the PCR product also can use INVITROGEN ^TMTACLONING  test kit is directly connected among the plasmid pCR  2.1.To connect product then is transformed in the XL-1 cell, with the colibacillary aliquot that transforms be layered on contain 50 μ g/mL ampicillin and be coated with 40 μ L X-Ga1 liquid storages (50mg/mL) and the LB agar plate of 40 μ L IPTG (100mM) solution on, screen to carry out indigo plant/white macula.37 ℃ of incubated overnight, potential clone is accredited as the bacterium colony of white with flat board.Use is used for general forward and the reverse primer of pNEB193 and pCR  2.1, from least 24 of each PCR product independently the DNA that obtain of clone two chains are checked order.Then the sequence that obtains is gathered into contig (contig), to produce the consensus sequence of each light chain of antibody and variable region of heavy chain.Use this method to measure the sequence (Fig. 6 A-6X) of light chain and the antibody heavy chain variable region of hybridoma 20C2,5F10,2D6,2B4,4E2,2H4,2A10,3B3,1F6,1F4,2E12 and 4C2.

In Fig. 6 A-6X, 6 complementarity-determining regions (CDRs) are by underscore, and they have formed and the complementary structure of antigen.After having analyzed CDRs and corresponding antigen epi-position (table 2), observed sequence similarity.The antibody of the 3-8 amino acids epi-position of sharing A β 1-42 (being 2A10,4C2,2D6,4E2,20C2,2B4 and 5F10) has been shared the homologous CDR1 of height (Fig. 7 A) and CDR2 (Fig. 7 B) sequence of heavy chain.Antibody 2H4 is found the 1-8 amino acids epi-position that can discern A β 1-42, demonstrates to have unique heavy chain CDR3 (Fig. 7 C) sequence and unique light chain CDR1 (Fig. 7 D), CDR2 (Fig. 7 E) and CDR3 (Fig. 7 F) sequence.Similarly, antibody 2E12 is found the 3-10 amino acids epi-position that can discern A β 1-42, has unique heavy chain CDR3 sequence (Fig. 7 C).In addition, antibody 2A10,2B4,4C2 and 4E2 have similar affinity (referring to Fig. 3) to SEC peak 1 with peak 2ADDLs, share the homologous CDR3 sequence of height (Fig. 7 C) of heavy chain.In addition, aminoacid replacement among the heavy chain CDR3 of antibody 4E2 demonstrates to have strengthened ADDLs blocking-up is combined with neuronal cell, because 4E2 compares with antibody 2D6 and can block ADDL more effectively and combine with neuronic, and be identical 3 amino acid residues of the sequence of the heavy chain of 4E2 and 2D6 and light chain in heavy chain CDR3, they are respectively Ser to Asn, Thr to Ser and Ile to Val (Fig. 7 C) for 2D6 and 4E2.

Embodiment 18: the humanization of mouse anti ADDL antibody variable region sequence

From mouse hybridoma cell is that mouse antibodies heavy chain and the light chain variable domain nucleic acid that 20C2,26D6,4E2,3B3,2H4 and 1F6 obtain uses the CDR implantation method to carry out humanization, is undertaken by the finishing strategy under the situation of 20C2 and 26D6.Those skilled in the art will be understood that, thereby the humanization of mouse antibodies sequence can reduce the treatment potentiality that antiglobulin reaction makes antibody and obtains maximization by improving its serum half-life and Fc effector functions.

By CDR transplanting carrying out humanization is to implement by people's light chain and the variable region of heavy chain selecting to have highest homology with the mice variable domains from the NCBI albumen database.Use the basic local comparison research tool of protein-protein (BLAST) in the data base, mice variable region sequences and everyone variable region sequences to be compared.Then, the CDRs of mice is incorporated in people's framework region, preliminary aminoacid sequence is analyzed.Institute in the framework region between mice and the human sequence has any different evaluated, if particularly they be circulus type sequence a part or be positioned at the VL/VH interface residue (O ' Brien and Jones (2001) " antibody engineering ", Kontermann and Dubel chief editor, the Springer laboratory manual).Also framework region is scanned with searching and compare common or not rare aminoacid and potential glycosylation site with the consensus sequence of people's subclass.Be not included in the type sequence or when not being positioned on the VL/VH interface, when the aminoacid sequence difference that exists between mice and the people's framework region sequence is found at this choice of location people's residue.When having the difference of Key residues, the variable region sequences that produces two kinds of versions is used for assessment.CDR transplants strategy people's framework region has been carried out the change of minimum number, makes reaching the good bonded while of antigen, can keep people's framework region near the sequence that is matched with from natural human antibody.Use the design of the humanization aminoacid sequence of CDR transplanting to be presented among Fig. 8.

For 20C2 and 26D6, also use finishing strategy (referring to for example U.S. Patent No. 6,797,492) to design humanized sequence.Humanization by from the NCBI albumen database, select with the mice variable domains and with immediate people's antibody kind be that family is (referring to (1991) such as Kabat " the proteic sequence with immune importance ", the 5th edition, U.S. sanitary and people service department, NIH, Washington DC) people's light chain and the variable region of heavy chain with highest homology implemented.Use protein-protein BLAST in the data base, mice variable region sequences and everyone variable region sequences to be compared.Mus variable region sequences and their immediate people's congener are moulded by crystalline people's antibody according to immediate, and described antibody is determined by the computer simulation of carrying out in the present technique field.According to the model construction of Mus VH and VL sequence surperficial collection of illustrative plates, it has described the amino acid whose solvent accessibility in murine heavy chain and the variable region of light chain.In order to confirm this model, the comparison of position one by one can be carried out (referring to for example Padlan (1994) Mol.Immunol.31 (3): 169-217) near residue in the residue of these exposures and known surface.According to existing method (referring to U.S. Patent No. 6,797,492) each residue in the sequence is given a mark, with it be assigned as exposure, most of that expose, part is that bury, that major part is buried and bury.In the mice framework residue by marking for that expose and the residue different that expose or most of with homologous human sequence, on this position, be changed adult's residue.The sequence of finishing of design kept mice CDRs, be contained in residue in the type sequence, be arranged in the residue at VL/VH interface and be positioned at murine heavy chain and the residue of light chain N-terminal sequence near residue, the known packets of CDRs.Known N-terminal sequence and CDR surface are adjacency, and may participate in the part combination.Similarly, carefully limited the change of Pro, Gly or charged residue.In case the sequence of finishing is done, they are simulated again to seek any possible tangible structure problem.In some cases, the sequence that has produced more than one finishing is analyzed.Use the design of surface modification method humanization aminoacid sequence to be presented among Fig. 9.

In case selected the humanization aminoacid sequence, with this sequence reverse translation to obtain corresponding DNA sequence.(Lathe (1985) J.Mol.Biol.183 (1): 1-12) codon to DNA sequence carries out optimization, and is used for being cloned into people's antibody expression vector in design limit enzyme site, both sides to use the existing technology in present technique field.Synthetic DNA sequence is presented at Figure 10 A in 10S.For the 20C2 humanized antibody of transplanting with CDR and finishing designs, made up human IgG1/κ and IgG2m4/ κ version, wherein IgG2m4 is illustrated in the sequence that has optionally added human IgG 4 in human IgG2's constant region of standard.For the CDR grafted antibody of 26D6, IgG1/ κ and IgG2m4/ κ version have also been made.For all other antibody, only made IgG1/ κ version.The complete aminoacid sequence of the antibody that obtains is presented among Figure 11 A-11Y.

By in 293 EBNA cells, making antibody obtain expressing isolating light chain and the common transient transfection of heavy chain expression plasmid.Under the situation that has designed an above humanization heavy chain or sequence of light chain for given antibody, all heavy chains and light chain combination are combined to produce corresponding antibody.After transfection, from culture supernatant, use protein A column purification antibody in 7-10 days, be used for the analysis of back.

Embodiment 19:IgG2m4 production of antibodies

Prepared of the formation of IgG2m4 antibody derivatives, kept the half-life and the pharmacokinetic property of the length of typical people's antibody simultaneously with combination, unwanted cells toxicity or the immune complex of the joint that reduces the Fc receptor, C1q.The basic antibody form of IgG2m4 is the form of IgG2, and IgG2 has demonstrated has the good half-life (Zuckier etc. (1994) Cancer Suppl.73:794-799) in experimental model.By the sequence that optionally adds IgG4 the structure of IgG2 is modified, thereby eliminated the combination of C1q, kept typical low-level Fc γ R simultaneously in conjunction with (Canfield and Morrison (1991) J.Exp.Med.173:1483-1491).This is by using wherein the cross point of the sequence something in common of IgG2 and IgG4 to realize, thereby has produced the antibody of the sequence that contains natural Fc sequence rather than any artificial mutation.

The IgG2m4 form of people's antibody constant region is to form by optionally add human IgG 4 sequences in human IgG2's constant region of standard, as shown in Figure 12.From conceptive, IgG2m4 comes from a pair of chain exchange in the CH2 domain that shows among Figure 12.Compare with the IgG4 sequence and to have produced 4 single sudden changes.The Fc residue that suddenlys change in IgG2 comprises His268Gln, Val309Leu, Ala330Ser and Pro331Ser, and they make the minimizing possibility of new epi-position.The concrete IgG4 amino acid residue of putting into the IgG2 constant region is presented at table 7, has shown other replacement in the basic structure simultaneously.

Table 7

Residue (Kabat numbering)	Residue among the IgG2	Residue among the IgG4	Residue among the IgG2m4	Replacement residue among the IgG2m4	Note
						189	Pro or Thr *	Pro	Thr	Pro	The main polymorphism of IgG2; The Pro residue appears in the IGHG*01 allotype, and the Thr residue appears in the IGHG2*02 allotype A, b
268	His	Gln	Gln	--	Change in the B/C ring is known to have participated in Fc γ RII combination c
						309	Val	Leu orVal	Leu	Val	The FcRn binding structural domain
330	Ala	Ser	Ser	--	The bonded Key residues of Clq dAlso may participate in the combination of Fc γ RII and Fc γ RIII e
						331	Pro	Ser	Ser	--	The Clq combination D, fWith Fc γ RI combination gKey residues; Also may participate in the combination of Fc γ RII and Fc γ RIII e
397	Met or Val *	Val	Met	Val	The Val residue appears in the IGHG*01 allotype, and the Met residue appears in the IGHG2*02 allotype a

* allelic variation has taken place in the position of asterisk labelling.

^aHoHgs etc. (2001) Immunogenetics52 (3-4): 242-8.

^bWO97/11971.

^cMedgyesi etc. (2004) Eur.J.Immunol.34:1127-1135.

^dTao etc. (1991) J.Exp.Med.173:1025-1028.

^eArmour etc. (1999) Eur.J.Immunol.29:2613.

^fXu etc. (1994) J.Biol.Chem.269:3469-3474.

^gCanfield and Morrison (1991) J.Exp.Med.173:1483.

Embodiment 20: the binding affinity of humanized anti-addl antibodies

In order to assess the ADDL binding affinity of humanized antibody, carried out titre ELISAs according to disclosing of this paper.(Sigma, St.Louis is MO) with 10% biotin labeled ADDL antigen (1 μ M) bag quilt for 96 hole microwell plates of streptavidin bag quilt.The antibody purification of a series of 2 times of dilutions that begin from 500ng/mL is added into the plate that ADDL catches, with plate 25 ℃ of insulations 2 hours.Wash the plate machine (Bio-Tek, Winooski, clean 5 times with PBS solution in VA) after, (Biomeda, Foster City CA), are incubated 1 hour in room temperature to add the anti-people k of polyclone goat light chain antibody with 2000 times of 3% defatted milk sealer dilutions.(Montgomery TX) detected antibody, room temperature insulation 1 hour for BethylLaboratories, Inc. to add the anti-goat IgG of the bonded rabbit of HRP (H+L) with 2000 times of lock solution dilutions then.After cleaning with PBS, substrate 3,3 ', 5 ' the 5-tetramethyl benzidine that adds HRP is (with the TMB of need with usefulness; Sigma, St.Louis MO), uses 0.5N H after 10 minutes ₂SO ₄Cessation reaction.At plate reading machine (model VICTOR V; Perkin Elmer, Boston, EXCEL  work sheet deal with data is used in the absorption of reading the 450nm wavelength in MA).Analysis difference estimation between the different plates is in 20%.

In different experiments, compared not humanized antibody on the same group.All antibody that comparison shows that of transplanting humanized IgG1 antibody 20C2A, 20C2B, 3B3,4E2,1F6 and 2H4 by CDR can both be in conjunction with ADDLs, wherein the binding ratio of 1F6 most a little less than, the combination of 20C2A is the strongest.4 kinds of different humanization versions of 20C2 IgG1 antibody (version of two kinds of CDR transplanting and the version of two kinds of finishinges) also are compared, find that they show closely similar ADDL binding curve, all combinations are all better a little than chimeric 20C2 antibody.7 kinds of different humanization versions of 26D6 IgG1 (a kind of CDR transplants version and 6 kinds of finishing versions) also are compared.All all are found has the ADDL binding curve similar to the 26D6 of chimeric form.IgG1 and the IgG2m4 antibody of transplanting humanized two kinds of 20C2 versions by CDR are also analyzed, find to have comparable binding curve with IgG1 and the IgG2m4 isotype of transplanting humanized 26D6 by CDR.

Embodiment 21: use humanized anti-addl antibodies to suppress ADDL and combine with neuronic

Use method disclosed herein further to assess humanization anti-addl antibodies blocking-up ADDL and the bonded ability of former generation hippocampal neuron.Relevant antibody or PBS are in contrast mixed with the bADDLs of 2.5-10 μ m (final concentration) with the molar ratio of 1: 1 (B103 neuroblast oncocyte) or 1: 5 (former generation hippocampal neuron), on slow shaking table, be incubated 1 hour at 37 ℃.After pre-incubation, antibody/bADDL prepared product is joined in B103 or the former generation neuron culture, continue insulation 1 hour at 37 ℃.Insulation is removed the bADDLs/ mixtures of antibodies after finishing, and plate is cleaned 6 times with culture medium.Then cell is at room temperature fixed 10 minutes with 4% paraformaldehyde, remove solution, add fresh fixative, cell is fixed 10 minutes again.Use contains 0.1%TRITON ^TM4% the paraformaldehyde of X-100 makes cell penetrating (room temperature twice, each 10 minutes), washes in PBS 6 times, handles 1 hour at 37 ℃ with the PBS that contains 10%BSA then.In cell, add the bonded streptavidin of alkali phosphatase then (with 1,500 times of 1%BSA dilution; Molecular Probes, Eugene, OR), room temperature was placed 1 hour.Cell cleans 6 times with PBS, adds alkaline phosphatase substrate (CDP-STAR  and SAPPHIRE-II in cell ^TMApplied Biosystems, Foster City CA), is incubated 30 minutes, then at LJL illumination meter (Analyst AD; LJL Biosystems, Sunnyvale CA) goes up mensuration luminosity.When using murine antibody, humanized 26D6,20C2,4E2,3B3,2H4 and 1F6 version can suppress ADDL prepared product and B103 neuroblast oncocyte and former generation neuronic the combination.

Embodiment 22: the affinity maturation of humanization anti-addl antibodies

Only encode humanized 20C2 version A variable region of heavy chain, variable region of light chain or for heavy chain and variable region of light chain simultaneously nucleic acid molecules encoding be cloned among the Fab Vector for Phage Display pFab3d.Nucleic acid sequence analysis has confirmed sequence and the direction among the pFab3d.The 20C2 Fab sequence of note is presented among Figure 13 in pFab3d, and heavy chain is shown as SEQ IDNO:255 in this article, and light chain is shown as SEQ ID NO:256.Utilize in the present technique field existing phage display Fab library method that 3 kinds of constructs are used for the ripe program of 20C2.

In simple terms, having designed suddenly change 9 wild-type amino acids of CDR3 of 20C2 light chain (κ) of two libraries (is Phe-Gln-Gly-Ser-Leu-Val-Pro-Leu-Thr; SEQ IDNO:60).These libraries are named as LC3-1 and LC3-2, represent light chain CDR3 sequence X aa-Xaa-Xaa-Xaa-Xaa-Val-Pro-Leu-Thr (SEQ ID NO:257) and Phe-Gln-Gly-Ser-Xaa-Xaa-Xaa-Xaa-Xaa (SEQ ID NO:258) respectively.Biotin labeled reverse primer 20C2LC3-1 (SEQ ID NO:259) and 20C2LC3-2 (SEQ IDNO:260) and forward primer 20C2LC3F (SEQ ID NO:261) combine, and are used to produce LC3-1 and LC3-2 library (referring to Figure 14).Primer is by the polyacrylamide gel electrophoresis purification, and carrier DNA comes purification by gel electrophoresis and electroelution.Two light chain libraries are designed to random mutation.Three 10G5H6 LC ₃The final multiformity in library is respectively 4.76x10 ⁸And 7.45x10 ⁸(table 8).About 100 clones' that choose from the library sequence analysis demonstrates mutant clon has 100% multiformity at the amino acid position of design.

Table 8

* obtained higher titre by concentrated or phage rescue.

Finished of the solubility elutriation of two 20C2 light chain libraries to high molecular bADDL.In simple terms, use biotin labeled high molecular ADDL (bADDL) to carry out 4 and take turns elutriation.Preceding 3 take turns the antigen concentration (input=1x10 that uses about 1.5 μ M ¹⁰To 1x10 ¹¹) carry out.After finishing third round, the output merging with two libraries is divided into three groups, analyzes to increase the stringency of elutriation with the antigen of 10nM, 100nM and about 1.5 μ M.Like this, in analyzing, phage E LISA tested 58 output boards altogether, i.e. two plates in each library (4 plates altogether) in the first round, 6 plates in each library in second takes turns (12 plates altogether), 8 plates in LC3-1 library, 10 plates in LC3-2 library (18 plates altogether) in third round, 8 plates of every kind of antigen concentration (24 plates altogether) in the four-wheel.

Elutriation has produced 1000 points (bit), wherein 436 quilt order-checkings (table 9).

Table 9

^a20C2LC3-1 is to high-molecular weight 10%bADDL.

^b20C2LC3-2 is to high-molecular weight 10%bADDL.

^c20C2LC3-1+20C2LC3-2 is to high-molecular weight 10%bADDL.

* the point of each total plate count.

Highly enriched clone's sequence and frequency are displayed in the table 10.

Table 10

Clone's title	CDR3	SEQ ID NO：	The 2nd takes turns	The 3rd takes turns	The 4th takes turns	Altogether
							Hu20C2LCSJ-p1-31SJ-p1-144P2-12-E3SJ-p1-384P3-59SJ-p2-144P3-114P3-1SJ-p2-10SJ-p2-11SJ-p2-60SJ-p1-18SJ-p3-51SJ-p3-16SJ-p8-8FSJ-p3-26SJ-p3-15SJ-p2-70SJ-p3-24SJ-p3-33SJ-p3-14SJ-p2-1F4P1-22SJ-p2-44SJ-p1-564P1-40SJ-p2-20SJ-p1-41SJ-p2-134P1-26SJ-p1-33SJ-p2-27SJ-p2-624P2-26-E54P1-32SJ-p2-37SJ-p2-644P1-20SJ-p2-39SJ-p2-52SJ-p2-6L4P1-77SJ-p1-59SJ-p2-23SJ-p1-9MSJ-p2-28SJ-p1-214P1-17SJ-p2-66SJ-p1-49SJ-p2-244P1-41SJ-p2-514P1-64SJ-p2-55SJ-p1-25	FQGSLVPLTADTTHVPLTAHSTFVPLTAQASFVPLTAQATKVPLTAQSSKVPLTAQSTLVPLTFAASSVPLTFESTYVPLTFESSRVPLTFNATWVPLTFQASRVPLTFQATRVPLTFQGSFIGLSFQGSFIPGTFQGSFLPPSFQGSFLPQLFQGSLFPPVFQGSLFSPSFQGSRIPISFQGSRLPVSFQGSRVPLVFQSSFVPLTFQSSRVPLTGQTTLVPLTHESTLVPLTHQSSKVPLTIQTSLVPLTIQAALVPLTLQSSFVPLTLETSRVPLTLASSHVPLTLNSTTVPLTLQSKSVPLTLQSVRVPLTLQSSLVPLTLQTGRVPLTLQTSFVPLTLQTSNVPLTLQTTRVPLTLS STFVPLTLSSTHVPLTLTSSAVPLTLVSSLVPLTMETANVPLTMQSSFVPLTMQSSLVPLTMQTSKVPLTSQARMVPLTSQASRVPLTTQSTQVPLTVCATFVPLTVQSSAVPLTVQTSLVPLTVQTSVVPLTVQTTAVPLTLQTARVPLT	60262263264265266267268269270271272273274275276277278279280281282283284285286287288289290291292293294295296297298299300301302303304305306307308309310311312313314315316317	6111111	15111112112122512261221422223232221111211221	1422212222155131131815314131435256112321321123133	3534222322226625233625314154342253362354358432224323332243324

Fab fragment according to preceding 10 clones of enrichment frequency permutation is produced, and 15 clones are converted to IgG1 humanization A version altogether, and two clone 20C2-6 and 20C2-8 are converted into IgG1 humanization B version.Use biotin-β 1-20 (table 11) and bADDL (table 12) to pass through BIACORE as antigen ^TMCalculated these clones' KD value.When comparing with 20C2B and mice 20C2 antibody, observed the rapid increase of affinity with male parent humanization 20C2A.Particularly use light chain CDR3 sequence X aa ₁-Gln-Xaa ₂During-Thr-Arg-Val-Pro-Leu-Thr (SEQ ID NO:318), can obtain low from nanomole to the KDs that is lower than picomole, wherein Xaa ₁Be Phe or Leu, Xaa ₁Be Ala or Thr.In addition, use biotin-A β 1-20 and bADDL to pass through BIACORE ^TMThe for example multidimensional conformation of 20C2 preferred combination ADDLs of anti-addl antibodies rather than monomer A β peptide have more further been confirmed between the KD value that obtains.

Table 11

Table 12

Sequence table

＜110〉Merck ﹠ Co., Inc. (Mcrck ﹠ Co., lnc.)

Northwest University (Northwestern University)

＜120〉anti-addl antibodies and application thereof (ANTI-ADDL ANTIBODTES AND USES THEREOF)

<130>SCT071719-47

<150>US 60/621.776

<151>2004-10-25

<150>US 60/652，538

<151>2005-02-14

<150>US 60/695，528

<151>2005-06-30

<150>US 60/695，526

<151>2005-06-30

<160>322

<170>PatentIn version 3.3

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<223>Heavy chain CDR2

<400>29

<210>30

<211>16

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR2

<400>30

<210>31

<211>16

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR2

<400>31

<210>32

<211>16

<212>PRT

<213>Artificial Sequence

<220>

<223>Consensus heavy chain CDR2

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223>Xaa denotes an amino acid with an aromatic side chain group

<220>

<221>MISC_FEATURE

<222>(9)..(9)

<223>Xaa denotes Ser，Arg or Tyr

<400>32

<210>33

<211>17

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR2

<400>33

<210>34

<211>17

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR2

<400>34

<210>35

<211>17

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR2

<400>35

<210>36

<211>17

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR2

<400>36

<210>37

<211>17

<212>PRT

<213>Artificial Sequence

<220>

<223>Consensus heavy chain CDR2

<220>

<221>MISCF_FATURE

<222>(3)..(4)

<223>Xaa denotes amino acids with a polar side chain group

<220>

<221>MISC_FEATURE

<222>(5)..(5)

<223>Xaa denotes Gly or Val

<220>

<221>MISC_FEATURE

<222>(7)..(7)

<223>Xaa denotes an amino acid with a polar and uncharged side group

<400>37

<210>38

<211>14

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR3

<400>38

<210>39

<211>14

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR3

<400>39

<210>40

<211>14

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR3

<400>40

<210>41

<211>14

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR3

<400>41

<210>42

<211>14

<212>PRT

<213>Artificial Sequence

<220>

<223>Consensus heavy chain CDR3

<220>

<221>MISC_FEATURE

<222>(4)..(4)

<223>Xaa denotes an amino acid with a polar and uncharged side group

<220>

<221>MISC_FEATURE

<222>(5)..(5)

<223>Xaa denotes an amino acid with hyroxyl side chain group

<220>

<221>MISC_FEATURE

<222>(6)..(7)

<223>Xaa denotes an amino acid with an aliphatic side chain group

<220>

<221>MISC_FEATURE

<222>(13)..(13)

<223>Xaa denotes Asp or Ala

<400>42

<210>43

<211>13

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR3

<400>43

<210>44

<211>11

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR3

<400>44

<210>45

<211>12

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR3

<400>45

<210>46

<211>12

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR3

<400>46

<210>47

<211>13

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR3

<400>47

<210>48

<211>8

<212>PRT

<213>Artificial Sequence

<220>

<223>Heavy chain CDR3

<400>48

<210>49

<211>16

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR1

<400>49

<210>50

<211>16

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR1

<400>50

<210>51

<211>16

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR1

<400>51

<210>52

<211>16

<212>PRT

<213>Artificial Sequence

<220>

<223>Consensus light chain CDR1

<220>

<221>MISC_FEATURE

<222>(6)..(7)

<223>Xaa denotes an amino acid with an aliphatic side chain group

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223>Xaa denotes an amino acid with a charged side chain group

<400>52

<210>53

<211>11

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR1

<400>53

<210>54

<211>7

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR2

<400>54

<210>55

<211>7

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR2

<400>55

<210>56

<211>7

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR2

<400>563

<210>57

<211>7

<212>PRT

<213>Artificial Sequence

<220>

<223>Consensus light chain CDR2

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223>Xaa denotes an amino acid with an aliphatic side chain group

<220>

<221>MISC_FEATURE

<222>(7)..(7)

<223>Xaa denotes Ser or Phe

<400>57

<210>58

<211>7

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR2

<400>58

<210>59

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR3

<400>59

<210>60

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR3

<400>60

<210>61

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR3

<400>61

<210>62

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR3

<400>62

<210>63

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR3

<400>63

<210>64

<211>9

<212>PRT

<213>Artificial Scquencc

<220>

<223>Light chain CDR3

<400>64

<210>65

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Consensus light chain CDR3

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>Xaa denotes Ser or Phe

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223>Xaa denotes an amino acid with no side chain or hyroxyl sidechain group

<220>

<221>MISC_FEATURE

<222>(4)..(4)

<223>Xaa denotes an amino acid with a hyroxyl side chain group

<220>

<221>MISC_FEATURE

<222>(5)..(5)

<223>Xaa denotes His，Tyr or Leu

<220>

<221>MISC_FEATURE

<222>(8)..(8)

<223>Xaa denotes an amino acid with an aliphatic side chain group

<400>65

<210>66

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Light chain CDR3

<400>66

<210>67

<211>123

<212>PRT

<213>Mus musculus

<400>67

<210>68

<211>130

<212>PRT

<213>Homo sapiens

<400>68

<210>69

<211>100

<212>PRT

<213>Homo sapiens

<400>69

<210>70

<211>124

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>70

<210>71

<211>123

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>71

<210>72

<211>105

<212>PRT

<213>Mus musculus

<400>72

<210>73

<211>115

<212>PRT

<213>Homo sapiens

<400>73

<210>74

<211>100

<212>PRT

<213>Homo sapiens

<400>74

<210>75

<211>114

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>75

<210>76

<211>123

<212>PRT

<213>Mus musculus

<400>76

<210>77

<211>123

<212>PRT

<213>Homo sapiens

<400>77

<210>78

<211>100

<212>PRT

<213>Homo sapiens

<400>78

<210>79

<211>122

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>79

<210>80

<211>115

<212>PRT

<213>Mus muscu1us

<400>80

<210>81

<211>115

<212>PRT

<213>Homo sapiens

<400>81

<210>82

<211>100

<212>PRT

<213>Homo sapiens

<400>82

<210>83

<211>114

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>83

<210>84

<211>124

<212>PRT

<213>Mus musculus

<400>84

<210>85

<211>130

<212>PRT

<213>Homo sapiens

<400>85

<210>86

<211>100

<212>PRT

<213>Homo sapiens

<400>86

<210>87

<211>124

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>87

<210>88

<211>115

<212>PRT

<213>Mus musculus

<400>88

<210>89

<211>115

<212>PRT

<213>Homo sapiens

<400>89

<210>90

<211>100

<212>PRT

<213>Homo sapiens

<400>90

<210>91

<211>114

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>91

<210>92

<211>117

<212>PRT

<213>Mus musculus

<400>92

<210>93

<211>117

<212>PRT

<213>Homo sapiens

<400>93

<210>94

<211>98

<212>PRT

<213>Homo sapiens

<400>94

<210>95

<211>117

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>95

<210>96

<212>PRT

<213>Mus musculus

<400>96

<2l0>97

<211>115

<2l2>PRT

<213>Homo sapiens

<400>97

<210>98

<211>l00

<212>PRT

<213>Homo sapiens

<400>98

<210>99

<211>114

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>99

<210>100

<211>121

<212>PRT

<213>Mus musculus

<400>100

<210>101

<211>121

<212>PRT

<213>Homo sapiens

<400>101

<210>102

<211>98

<212>PRT

<213>Homo sapiens

<400>102

<210>103

<211>121

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>103

<210>104

<211>110

<212>PRT

<213>Mus musculus

<400>104

<210>105

<211>110

<212>PRT

<213>Homo sapiens

<400>105

<210>106

<211>95

<212>PRT

<213>Homo sapiens

<400>106

<210>107

<211>110

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>107

<210>108

<211>122

<212>PRT

<213>Mus musculus

<400>108

<210>109

<211>122

<212>PRT

<213>Homo sapiens

<400>109

<210>110

<211>98

<212>PRT

<213>Homo sapiens

<400>110

<210>111

<211>122

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>111

<210>112

<211>123

<212>PRT

<213>Mus musculus

<400>112

<210>113

<211>130

<212>PRT

<213>Homo sapiens

<400>113

<210>114

<211>100

<212>PRT

<213>Homo sapiens

<400>114

<210>115

<211>123

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variablc region

<400>115

<210>116

<211>123

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>116

<210>117

<211>113

<212>PRT

<213>Mus musculus

<400>117

<210>118

<211>113

<212>PRT

<213>Homo sapiens

<400>118

<210>119

<211>100

<212>PRT

<213>Homo sapiens

<400>119

<210>120

<211>112

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>120

<210>121

<211>113

<212>PRT

<213>Mus musculus

<400>121

<210>122

<211>123

<212>PRT

<213>Homo sapiens

<400>122

<210>123

<211>100

<212>PRT

<213>Homo sapiens

<400>123

<210>124

<211>123

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>124

<210>125

<211>123

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>125

<210>126

<211>122

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>126

<210>127

<211>93

<212>PRT

<213>Mus musculus

<400>127

<210>128

<211>112

<212>PRT

<213>Homo sapiens

<400>128

<210>129

<211>100

<212>PRT

<213>Homo sapiens

<400>129

<210>130

<211>112

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>130

<210>131

<211>111

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>131

<210>132

<211>463

<212>DNA

<213>Articial Sequence

<220>

<223>Humanized heavy chain variable region

<400>132

<210>133

<211>463

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>133

<210>134

<211>389

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>134

<210>135

<211>463

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>135

<210>136

<211>389

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>136

<210>137

<211>466

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>137

<210>138

<211>389

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>138

<210>139

<211>445

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>139

<210>140

<211>389

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>140

<210>141

<211>457

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>141

<210>142

<211>374

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>142

<210>143

<211>460

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>143

<210>144

<211>463

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>144

<210>145

<211>463

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>145

<210>146

<211>389

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>146

<210>147

<211>447

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>147

<210>148

<211>447

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>148

<210>149

<211>447

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized heavy chain variable region

<400>149

<210>150

<211>371

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>150

<210>151

<211>371

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized light chain variable region

<400>151

<210>152

<211>453

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgGl heavy chain variable region

<400>152

<210>153

<211>453

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgGl heavy chain variable region

<400>153

<210>154

<211>449

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgG2M4 heavy chain variable region

<400>154

<210>155

<211>449

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgG2M4 heavy chain variable region

<400>155

<210>156

<211>219

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized kappa light chain variable region

<400>156

<210>157

<211>453

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgGl heavy chain variable region

<400>157

<210>158

<211>449

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized lgG2M4 heavy chain variable region

<400>158

<210>159

<211>219

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized kappa light chain variable region

<400>159

<210>160

<211>454

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgGl heavy chain variable region

<400>160

<210>161

<211>219

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized kappa light chain variable region

<400>161

<210>162

<211>447

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgGl heavy chain variable region

<400>162

<210>163

<211>219

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized kappa light chain variale region

<400>163

<210>164

<211>451

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgGl heavy chain variable region

<400>164

<210>165

<211>214

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized kappa light chain variable region

<400>165

<210>166

<211>452

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgGl heavy chain variable region

<400>166

<210>167

<211>453

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgGl heavy chain variable region

<400>167

<210>168

<211>452

<212>PRT

<213>Artifical Sequence

<220>

<223>Humanized IgGl heavy chain variable region

<400>168

<210>169

<211>449

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized TgG2M4 heavy chain variable region

<400>169

<210>170

<211>449

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgG2M4 heavy chain variable region

<400>170

<210>171

<211>219

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized kappa light chain variable region

<400>171

<210>172

<211>453

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgGl heavy chain variable region

<400>172

<210>173

<211>453

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized IgGl heavy chain variable region

<400>173

<210>174

<211>453

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanied IgGl heavy chain variable region

<400>174

<210>175

<211>219

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized kappa light chain variable region

<400>175

<210>176

<211>219

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized kappa light chain variable region

<400>176

<210>177

<211>6

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>177

<210>178

<211>8

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>178

<210>179

<211>8

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>179

<210>180

<211>42

<212>PRT

<213>Homo sapiens

<400>180

<210>181

<211>7

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>181

<210>182

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>182

<210>183

<211>10

<212>PRT

<213>Artificia1 Sequence

<220>

<223>Synthetic peptide

<400>183

<210>184

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>184

<210>185

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>185

<210>186

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>186

<210>187

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>187

<210>188

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>188

<210>189

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>189

<210>190

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>190

<210>191

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>191

<210>192

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>192

<200>193

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>193

<210>194

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>194

<210>195

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>195

<210>196

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>196

<210>197

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>197

<210>198

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>198

<210>199

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>199

<210>200

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>200

<210>201

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>201

<210>202

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>202

<210>203

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>203

<210>204

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>204

<210>205

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>205

<210>206

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>206

<210>207

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>207

<210>208

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>208

<210>209

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>209

<210>210

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>210

<210>211

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>211

<210>212

<211>10

<242>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>212

<210>213

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>213

<210>214

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>214

<210>215

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>215

<210>216

<211>14

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>216

<210>217

<211>14

<212>PRT

<213> Artificial Sequence

<220>

<223>Synthetic peptide

<400>217

<210>218

<211>16

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>218

<210>219

<211>7

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>219

<210>220

<211>6

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>220

<210>221

<211>11

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>221

<210>222

<211>8

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>222

<210>223

<211>40

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>223

<210>224

<211>39

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>224

<210>225

<211>39

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>225

<210>226

<211>42

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>226

<210>227

<211>39

<212>DNA

<213>Artificil Sequence

<220>

<223>Synthetic oligonucleotide

<400>227

<210>228

<211>38

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>228

<210>229

<211>41

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>229

<210>230

<211>39

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>230

<210>231

<211>34

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>231

<210>232

<211>36

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>232

<210>233

<211>37

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>233

<210>234

<211>30

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>234

<210>235

<211>36

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>235

<210>236

<211>35

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>236

<210>237

<211>36

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>237

<210>238

<211>34

<212>DNA

<213>Artificial Sequence

<220>

<223>Syntheti coligonucleotide

<400>238

<210>239

<211>41

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>239

<210>240

<211>38

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>240

<210>241

<211>36

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>241

<210>242

<211>32

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>242

<210>243

<211>40

<212>DNA

<213>Artificial Scquence

<220>

<223>Synthetic oligonucleotide

<400>243

<210>244

<211>37

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>244

<210>245

<211>37

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>245

<210>246

<211>36

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>246

<210>247

<211>32

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>247

<210>248

<211>32

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonuucleotide

<400>248

<210>249

<211>32

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>249

<210>250

<211>32

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic oligonucleotide

<400>250

<210>251

<211>330

<212>PRT

<213>Homo sapiens

<400>251

<210>252

<211>326

<212>PRT

<213>Homo sapiens

<400>252

<210>253

<211>327

<212>PRT

<213>Homo sapiens

<400>253

<210>254

<211>326

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic immunoglobulin constant region

<400>254

<210>255

<211>492

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic protein

<400>255

<210>256

<211>243

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic protein

<400>256

<210>257

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<220>

<221>MISC_FEATURE

<222>(1)..(5)

<223>Xaa denotes any amino acid residue

<400>257

<210>258

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<220>

<221>MISC_FEATURE

<222>(5)..(9)

<223>Xaa denotes any amino acid residue

<400>258

<210>259

<211>82

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic primer

<220>

<221>misc_feature

<222>(57)..(58)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(60)..(61)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(63)..(64)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(66)..(67)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(69)..(70)

<223>n denotes any nucleotide

<400>259

<210>260

<211>82

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic primer

<220>

<221>misc_feature

<222>(45)..(46)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(48)..(49)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(51)..(52)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(54)..(55)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(57)..(58)

<223>n denotes any nucleotide

<400>260

<210>261

<211>26

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic primer

<400>261

<210>262

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>262

<210>263

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>263

<210>264

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>264

<210>265

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>265

<210>266

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>266

<210>267

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>267

<210>268

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>268

<210>269

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>269

<210>270

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>270

<210>271

<211>9

<212>PRT

<213>Artficial Sequence

<220>

<223>Synthetic peptide

<400>271

<210>272

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>272

<210>273

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>273

<210>274

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>274

<210>275

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>275

<210>276

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>276

<210>277

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>277

<210>278

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>278

<210>279

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>279

<210>280

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>280

<210>281

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>281

<210>282

<211>9

<212>PRT

<213>Artificial Sequcnce

<220>

<223>Synthetic peptide

<400>282

<210>283

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>283

<210>284

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>284

<210>285

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>285

<210>286

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>286

<210>287

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>287

<210>288

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>288

<210>289

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>289

<210>290

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>290

<210>291

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>291

<210>292

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>292

<210>293

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>293

<210>294

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>294

<210>295

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>295

<210>296

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>296

<210>297

<211>9

<212> PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>297

<210>298

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>298

<210>299

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>299

<210>300

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>300

<210>301

<211>9

<212>PRT

<213>Articial Sequence

<220>

<223>Synthetic peptide

<400>301

<210>302

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>302

<210>303

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>303

<210>304

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>304

<210>305

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>305

<210>306

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>306

<210>307

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>307

<210>308

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>308

<210>309

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>309

<210>310

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>310

<210>311

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>311

<210>312

<211>9

<212>PRT

<213>Articfial Sequence

<220>

<223>Synthetic peptide

<400>312

<210>313

<211>9

<212>PRT

<213>Artificial Sequcnce

<220>

<223>Synthetic peptide

<400>313

<210>314

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>314

<210>315

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>315

<210>316

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>316

<210>317

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthetic peptide

<400>317

<210>318

<211>9

<212>PRT

<213>Arificial Sequence

<220>

<223>Synthetic peptide

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>Xaa denotes Phe or Leu

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223>Xaa denotes Ala or Thr

<400>318

<210>319

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Synthctic peptide

<400>319

<210>320

<211>398

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic nuclcic acid

<400>320

<210>321

<211>398

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic nucleic acid

<220>

<221>misc_feature

<222>(329)..(330)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(332)..(333)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(335)..(336)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(338)..(339)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(341)..(342)

<223>n denotes any nucleotide

<400>321

<210>322

<211>398

<212>DNA

<213>Artificial Sequence

<220>

<223>Synthetic nucleic acid

<220>

<221>misc_feature

<222>(341)..(342)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(344)..(345)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(347)..(348)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(350)..(351)

<223>n denotes any nucleotide

<220>

<221>misc_feature

<222>(353)..(354)

<223>n denotes any nucleotide

<400>322

Claims (10)

1. isolated antibody or its fragment, it can the deutero-multidimensional conformation that spreads part of one or more A β of difference identification.

2. contain isolated antibody or its segmental pharmaceutical composition, this antibody or its fragment can the deutero-multidimensional conformations that spreads part of one or more A β of difference identification, and mix with pharmaceutically useful carrier.

3. stop deutero-part and the bonded method of neuron of spreading of A β, comprise neuron is contacted with the antibody of claim 1, thereby stop the deutero-part that spreads of A β to combine with neuronic.

4. suppress the deutero-accumulative method of part that spreads of A β, comprise that the sample that will contain amyloid beta 1-42 peptide contacts with the antibody of claim 1, thereby suppress the deutero-gathering of spreading part of A β.

5. the blocking-up tau protein comprises that in the method for the phosphorylation in Ser202/Thr205 site the sample that will contain tau protein contacts with the antibody of claim 1, thereby the blocking-up tau protein is in the phosphorylation in Ser202/Thr205 site.

6. preventative or therapeutic treatment and the deutero-method that spreads the relevant disease of part of A β comprise the pharmaceutical composition of the claim 2 of effective dosage.

7. identify and stop deutero-part and the bonded treatment compositions and methods of neuron of spreading of A β, comprise the deutero-part that spreads of neuron and A β is contacted existing under the situation of reagent, and use the antibody of claim 1 to determine that the deutero-part that spreads of A β combines with neuronic under the situation that has this reagent.

8. the deutero-method that spreads part of A β in the test sample comprises sample is contacted with the antibody of claim 1, thereby detects the deutero-part that spreads of A β.

9. diagnosis and the deutero-method that spreads the relevant disease of part of A β comprise sample is contacted with the antibody of claim 1, thus diagnosis and the deutero-relevant disease of part that spreads of A β.

10. be used to detect the deutero-test kit that spreads part of A β, contain isolated antibody or its fragment of claim 1.

Images