Antibody and utilization of the same

An antibody and antibody concentration technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, instruments, etc., can solve problems such as difficulty in obtaining antibodies, difficulties in antibodies, and unclear parts of essential antigens

Inactive Publication Date: 2007-11-28
MITSUBISHI CHEM CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

That is, (a) amyloid spheroids are aggregates of amyloid β proteins, and it is often difficult to obtain antibodies reactive with aggregated proteins, (b) the mechanism of amyloid spheroid aggregation or the relationship between structure and function is unclear , and the essential antigenic portion required for antibody function (1) and (2) is unclear
Therefore, it is particularly difficult to obtain antibodies that specifically react with amyloid spheroids and inhibit the toxicity of the protein to neuronal cells

Method used

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  • Antibody and utilization of the same
  • Antibody and utilization of the same
  • Antibody and utilization of the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1 Preparation of a solution containing amyloid spheroids

[0108] (1) Preparation of amyloid β40 (SEQ ID NO: 1) resin

[0109]Fmoc-Val resin (342 mg, amine content: 0.73 mmol / g resin) was loaded on an A433 automatic peptide synthesizer (Perkin Elmer Applied Biosystems). Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc -Ile-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc- Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Glu( OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg (Pmc)-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, and Fmoc-Asp(OtBu)-OH are added on it, using HBTU[2-(1H-benzene Triazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorop...

Embodiment 2

[0116] Example 2 Preparation of anti-amyloid spheroid antibody

[0117] (1) Preparation of rabbit anti-amyloid spheroid polyclonal antibody

[0118] The amyloid spheroids 40 and 42 prepared in Example 1 were mixed with complete Freund's adjuvant and subcutaneously administered to New Zealand white rabbits as antigens, so that 60 μg of the aforementioned amylospheroids could be administered per New Zealand white rabbit . Then, the same amount of amyloid β protein was mixed with incomplete Freund's adjuvant and administered once every 2 weeks for a total of 8 administrations. Bleeding was performed 10 days after the last immunization.

[0119] After exsanguination, the blood was allowed to remain at 37° C. for 1 hour, the resulting blood clot was removed by centrifugation, and the serum was recovered. Then, the sera were inactivated at 57°C for 30 minutes. Add ProClin300 (Sigma-Aldrich) to make the concentration 1ppm, and save. IgG is isolated from serum in the following ma...

Embodiment 3

[0127] Example 3: Analysis of Antibody Properties

[0128] (1) Solid-phase amyloid spheroid ELISA (to determine the reactivity with amyloid spheroids)

[0129] Amyloid spheroids 40 or 42 (50 μl) diluted to 1 μg / ml in phosphate-buffered saline (without Ca or Mg, pH 7.2, PBS) were added to 96-well ELISA plates (MaxiSorp, Nunc), Plates were coated overnight at 4°C. A PBS solution containing 1% bovine serum albumin (BSA, fraction V; Sigma-Aldrich) was added thereto for at least 1 hour at room temperature to block non-specific binding sites, and the plate was washed with water. Antiserum or hybridoma culture supernatant (50 [mu]l) diluted in 1% bovine serum albumin in PBS was added and the reaction was allowed to continue for at least 1 hour at room temperature. The plate was washed 5 times with normal saline containing 0.05% Tween 20, and the peroxidase-labeled secondary antibody (anti-mouse IgG antibody (Zymed), anti-mouse IgM (Biosource), and anti-mouse immunoglobulin (DAKO))...

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Abstract

It is intended to provide antibodies having a higher reactivity with amylospheroid than a reactivity with amyloid ss-fiber and so on. Some of these antibodies have an activity of inhibiting amylospheroid formation or an activity of inhibiting the induction of nerve cell death by amylospheroid. Such antibodies are usable in remedies / preventives for Alzheimer's disease, screening these drugs, detecting an individual suffering from Alzheimer's disease, reagents and so on.

Description

technical field [0001] The present invention relates to antibodies highly reactive with amylospheroids and their use. Background technique [0002] "Aberrant structural proteins" are now attracting attention as a common mechanism behind many neurodegenerative diseases that occur with age, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and prion diseases, the The molecular properties of proteins are also studied. Deposition of two types of fibrous aggregates in the brain: the deposition of senile plaques mainly composed of amyloid β protein (Aβ) (Selkoe, D.J., Annu.Rev.Neurosci., 12, 463-490, (1989) and Glenner, G.G. and Wong, C.W., Biochem.Biophys.Res.Commun., 120(3), 885-890, (1984)); and deposition of neurofibrillary degeneration with phosphorylated tau as a major component (Paired Helical Fibrils (PHF)) (Ihara, Y. et al., J. Biochem., 99, 1807-1810, (1986); and Grundke-Iqbal, I. et al., Proc. Natl. Acad . Sci. U.S.A., 83, 4913-4917, (1986)) has...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/15A61K39/395G01N33/50A61P25/28G01N33/53A61P43/00
CPCC07K16/18G01N33/6893A61K2039/505G01N2800/2821A61P25/28A61P43/00Y10S530/809A61K39/395C07K16/00
Inventor 星美奈子内藤幸嗣井手野祥次
Owner MITSUBISHI CHEM CORP
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