Aβ Measurement by Enzyme-Linked Immunosorbent Assay

  • Stephen D. Schmidt
  • Matthew J. Mazzella
  • Ralph A. Nixon
  • Paul M. Mathews
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 849)

Abstract

The neuritic plaque in the brain of Alzheimer’s disease patients consists of an amyloid composed primarily of Aβ, an approximately 4-kDa peptide derived from the amyloid precursor protein. Multiple lines of evidence suggest that Aβ plays a key role in the pathogenesis of the disease, and potential treatments that target Aβ production and/or Aβ accumulation in the brain as β-amyloid are being aggressively pursued. Methods to quantitate the Aβ peptide are, therefore, invaluable to most studies aimed at a better understanding of the molecular etiology of the disease and in assessing potential therapeutics. Although other techniques have been used to measure Aβ in the brains of AD patients and β-amyloid-depositing transgenic mice, the enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used, reliable, and sensitive methods for quantitating the Aβ peptide. Here we describe methods for the recovery of both soluble and deposited Aβ from brain tissue and the subsequent quantitation of the peptide by sandwich ELISA.

Key words

Aβ Amyloid Enzyme-linked immunosorbent assay ELISA Extraction Quantification Quantitation Alzheimer’s disease Amyloid precursor protein APP Formic acid Brain 

References

  1. 1.
    Glenner, G. G., and Wong, C. W. (1984) Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein, Biochem Biophys Res Commun 120, 885–890.PubMedCrossRefGoogle Scholar
  2. 2.
    Masters, C. L., Simms, G., Weinman, N. A., Multhaup, G., McDonald, B. L., and Beyreuther, K. (1985) Amyloid plaque core protein in Alzheimer disease and Down syndrome, Proc Natl Acad Sci USA 82, 4245–4249.PubMedCrossRefGoogle Scholar
  3. 3.
    De Strooper, B., and Annaert, W. (2000) Proteolytic processing and cell biological functions of the amyloid precursor protein, J Cell Sci 113, 1857–1870.PubMedGoogle Scholar
  4. 4.
    Chartier-Harlin, M. C., Crawford, F., Houlden, H., Warren, A., Hughes, D., Fidani, L., Goate, A., Rossor, M., Roques, P., Hardy, J., and et al. (1991) Early-onset Alzheimer’s disease caused by mutations at codon 717 of the beta-amyloid precursor protein gene, Nature 353, 844–846.PubMedCrossRefGoogle Scholar
  5. 5.
    Levy, E., Carman, M. D., Fernandez-Madrid, I. J., Power, M. D., Lieberburg, I., van Duinen, S. G., Bots, G. T., Luyendijk, W., and Frangione, B. (1990) Mutation of the Alzheimer’s disease amyloid gene in hereditary cerebral hemorrhage, Dutch type, Science 248, 1124–1126.PubMedCrossRefGoogle Scholar
  6. 6.
    Tanzi, R. E., and Bertram, L. (2001) New frontiers in Alzheimer’s disease genetics, Neuron 32, 181–184.PubMedCrossRefGoogle Scholar
  7. 7.
    Mullan, M., Houlden, H., Windelspecht, M., Fidani, L., Lombardi, C., Diaz, P., Rossor, M., Crook, R., Hardy, J., Duff, K., and et al. (1992) A locus for familial early-onset Alzheimer’s disease on the long arm of chromosome 14, proximal to the alpha 1-antichymotrypsin gene, Nat Genet 2, 340–342.PubMedCrossRefGoogle Scholar
  8. 8.
    Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D. M., Oshima, J., Pettingell, W. H., Yu, C. E., Jondro, P. D., Schmidt, S. D., Wang, K., and et al. (1995) Candidate gene for the chromosome 1 familial Alzheimer’s disease locus, Science 269, 973–977.PubMedCrossRefGoogle Scholar
  9. 9.
    Rogaev, E. I., Sherrington, R., Rogaeva, E. A., Levesque, G., Ikeda, M., Liang, Y., Chi, H., Lin, C., Holman, K., Tsuda, T., and et al. (1995) Familial Alzheimer’s disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer’s disease type 3 gene, Nature 376, 775–778.PubMedCrossRefGoogle Scholar
  10. 10.
    Wasco, W., Pettingell, W. P., Jondro, P. D., Schmidt, S. D., Gurubhagavatula, S., Rodes, L., DiBlasi, T., Romano, D. M., Guenette, S. Y., Kovacs, D. M., and et al. (1995) Familial Alzheimer’s chromosome 14 mutations, Nat Med 1, 848.PubMedCrossRefGoogle Scholar
  11. 11.
    Cai, X. D., Golde, T. E., and Younkin, S. G. (1993) Release of excess amyloid beta protein from a mutant amyloid beta protein precursor, Science 259, 514–516.PubMedCrossRefGoogle Scholar
  12. 12.
    Citron, M., Oltersdorf, T., Haass, C., McConlogue, L., Hung, A. Y., Seubert, P., Vigo-Pelfrey, C., Lieberburg, I., and Selkoe, D. J. (1992) Mutation of the beta-amyloid precursor protein in familial Alzheimer’s disease increases beta-protein production, Nature 360, 672–674.PubMedCrossRefGoogle Scholar
  13. 13.
    Borchelt, D. R., Thinakaran, G., Eckman, C. B., Lee, M. K., Davenport, F., Ratovitsky, T., Prada, C. M., Kim, G., Seekins, S., Yager, D., Slunt, H. H., Wang, R., Seeger, M., Levey, A. I., Gandy, S. E., Copeland, N. G., Jenkins, N. A., Price, D. L., Younkin, S. G., and Sisodia, S. S. (1996) Familial Alzheimer’s disease-linked presenilin 1 variants elevate Abeta1-42/1-40 ratio in vitro and in vivo, Neuron 17, 1005–1013.PubMedCrossRefGoogle Scholar
  14. 14.
    Citron, M., Westaway, D., Xia, W., Carlson, G., Diehl, T., Levesque, G., Johnson-Wood, K., Lee, M., Seubert, P., Davis, A., Kholodenko, D., Motter, R., Sherrington, R., Perry, B., Yao, H., Strome, R., Lieberburg, I., Rommens, J., Kim, S., Schenk, D., Fraser, P., St George Hyslop, P., and Selkoe, D. J. (1997) Mutant presenilins of Alzheimer’s disease increase production of 42- residue amyloid beta-protein in both transfected cells and transgenic mice, Nat Med 3, 67–72.PubMedCrossRefGoogle Scholar
  15. 15.
    Lemere, C. A., Lopera, F., Kosik, K. S., Lendon, C. L., Ossa, J., Saido, T. C., Yamaguchi, H., Ruiz, A., Martinez, A., Madrigal, L., Hincapie, L., Arango, J. C., Anthony, D. C., Koo, E. H., Goate, A. M., and Selkoe, D. J. (1996) The E280A presenilin 1 Alzheimer mutation produces increased A beta 42 deposition and severe cerebellar pathology, Nat Med 2, 1146–1150.PubMedCrossRefGoogle Scholar
  16. 16.
    Hsiao, K., Chapman, P., Nilsen, S., Eckman, C., Harigaya, Y., Younkin, S., Yang, F., and Cole, G. (1996) Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice, Science 274, 99–102.PubMedCrossRefGoogle Scholar
  17. 17.
    Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P., Blackwell, C., Carr, T., Clemens, J., Donaldson, T., Gillespie, F., and et al. (1995) Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein, Nature 373, 523–527.PubMedCrossRefGoogle Scholar
  18. 18.
    Sturchler-Pierrat, C., Abramowski, D., Duke, M., Wiederhold, K. H., Mistl, C., Rothacher, S., Ledermann, B., Burki, K., Frey, P., Paganetti, P. A., Waridel, C., Calhoun, M. E., Jucker, M., Probst, A., Staufenbiel, M., and Sommer, B. (1997) Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology, Proc Natl Acad Sci USA 94, 13287–13292.PubMedCrossRefGoogle Scholar
  19. 19.
    Duff, K., Eckman, C., Zehr, C., Yu, X., Prada, C. M., Perez-tur, J., Hutton, M., Buee, L., Harigaya, Y., Yager, D., Morgan, D., Gordon, M. N., Holcomb, L., Refolo, L., Zenk, B., Hardy, J., and Younkin, S. (1996) Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin 1, Nature 383, 710–713.PubMedCrossRefGoogle Scholar
  20. 20.
    Holcomb, L., Gordon, M. N., McGowan, E., Yu, X., Benkovic, S., Jantzen, P., Wright, K., Saad, I., Mueller, R., Morgan, D., Sanders, S., Zehr, C., O’Campo, K., Hardy, J., Prada, C. M., Eckman, C., Younkin, S., Hsiao, K., and Duff, K. (1998) Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes, Nat Med 4, 97–100.PubMedCrossRefGoogle Scholar
  21. 21.
    Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St George-Hyslop, P., and Westaway, D. (2000) A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer’s disease, Nature 408, 979–982.PubMedCrossRefGoogle Scholar
  22. 22.
    Refolo, L. M., Pappolla, M. A., LaFrancois, J., Malester, B., Schmidt, S. D., Thomas-Bryant, T., Tint, G. S., Wang, R., Mercken, M., Petanceska, S. S., and Duff, K. E. (2001) A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer’s disease, Neurobiol Dis 8, 890–899.PubMedCrossRefGoogle Scholar
  23. 23.
    Rozmahel, R., Huang, J., Chen, F., Liang, Y., Nguyen, V., Ikeda, M., Levesque, G., Yu, G., Nishimura, M., Mathews, P., Schmidt, S. D., Mercken, M., Bergeron, C., Westaway, D., and St George-Hyslop, P. (2002) Normal brain development in PS1 hypomorphic mice with markedly reduced gamma-secretase cleavage of betaAPP, Neurobiol Aging 23, 187–194.PubMedCrossRefGoogle Scholar
  24. 24.
    Rozmahel, R., Mount, H. T., Chen, F., Nguyen, V., Huang, J., Erdebil, S., Liauw, J., Yu, G., Hasegawa, H., Gu, Y., Song, Y. Q., Schmidt, S. D., Nixon, R. A., Mathews, P. M., Bergeron, C., Fraser, P., Westaway, D., and George-Hyslop, P. S. (2002) Alleles at the Nicastrin locus modify presenilin 1-deficiency phenotype, Proc Natl Acad Sci USA 99, 14452–14457.PubMedCrossRefGoogle Scholar
  25. 25.
    Pfeifer, M., Boncristiano, S., Bondolfi, L., Stalder, A., Deller, T., Staufenbiel, M., Mathews, P. M., and Jucker, M. (2002) Cerebral hemorrhage after passive anti-Abeta immunotherapy, Science 298, 1379.PubMedCrossRefGoogle Scholar
  26. 26.
    Phinney, A. L., Drisaldi, B., Schmidt, S. D., Lugowski, S., Coronado, V., Liang, Y., Horne, P., Yang, J., Sekoulidis, J., Coomaraswamy, J., Chishti, M. A., Cox, D. W., Mathews, P. M., Nixon, R. A., Carlson, G. A., St George-Hyslop, P., and Westaway, D. (2003) In vivo reduction of amyloid-beta by a mutant copper transporter, Proc Natl Acad Sci USA 100, 14193–14198.PubMedCrossRefGoogle Scholar
  27. 27.
    Herzig, M. C., Winkler, D. T., Burgermeister, P., Pfeifer, M., Kohler, E., Schmidt, S. D., Danner, S., Abramowski, D., Sturchler-Pierrat, C., Burki, K., van Duinen, S. G., Maat-Schieman, M. L., Staufenbiel, M., Mathews, P. M., and Jucker, M. (2004) Abeta is targeted to the vasculature in a mouse model of hereditary cerebral hemorrhage with amyloidosis, Nat Neurosci 7, 954–960.PubMedCrossRefGoogle Scholar
  28. 28.
    Lacombe, P., Mathews, P. M., Schmidt, S. D., Breidert, T., Heneka, M. T., Landreth, G. E., Feinstein, D. L., and Galea, E. (2004) Effect of anti-inflammatory agents on transforming growth factor beta over-expressing mouse brains: a model revised, J Neuroinflammation 1, 11.PubMedCrossRefGoogle Scholar
  29. 29.
    Pawlik, M., Sastre, M., Calero, M., Mathews, P. M., Schmidt, S. D., Nixon, R. A., and Levy, E. (2004) Overexpression of human cystatin C in transgenic mice does not affect levels of endogenous brain amyloid Beta Peptide, J Mol Neurosci 22, 13–18.PubMedCrossRefGoogle Scholar
  30. 30.
    Yao, J., Petanceska, S. S., Montine, T. J., Holtzman, D. M., Schmidt, S. D., Parker, C. A., Callahan, M. J., Lipinski, W. J., Bisgaier, C. L., Turner, B. A., Nixon, R. A., Martins, R. N., Ouimet, C., Smith, J. D., Davies, P., Laska, E., Ehrlich, M. E., Walker, L. C., Mathews, P. M., and Gandy, S. (2004) Aging, gender and APOE isotype modulate metabolism of Alzheimer’s Abeta peptides and F-isoprostanes in the absence of detectable amyloid deposits, J Neurochem 90, 1011–1018.PubMedCrossRefGoogle Scholar
  31. 31.
    Mastrangelo, P., Mathews, P. M., Chishti, M. A., Schmidt, S. D., Gu, Y., Yang, J., Mazzella, M. J., Coomaraswamy, J., Horne, P., Strome, B., Pelly, H., Levesque, G., Ebeling, C., Jiang, Y., Nixon, R. A., Rozmahel, R., Fraser, P. E., St George-Hyslop, P., Carlson, G. A., and Westaway, D. (2005) Dissociated phenotypes in presenilin transgenic mice define functionally distinct gamma-secretases, Proc Natl Acad Sci USA 102, 8972–8977.PubMedCrossRefGoogle Scholar
  32. 32.
    Gandy, S., Zhang, Y. W., Ikin, A., Schmidt, S. D., Bogush, A., Levy, E., Sheffield, R., Nixon, R. A., Liao, F. F., Mathews, P. M., Xu, H., and Ehrlich, M. E. (2007) Alzheimer’s presenilin 1 modulates sorting of APP and its carboxyl-­terminal fragments in cerebral neurons in vivo, J Neurochem 102, 619–626.PubMedCrossRefGoogle Scholar
  33. 33.
    Mi, W., Pawlik, M., Sastre, M., Jung, S. S., Radvinsky, D. S., Klein, A. M., Sommer, J., Schmidt, S. D., Nixon, R. A., Mathews, P. M., and Levy, E. (2007) Cystatin C inhibits amyloid-beta deposition in Alzheimer’s disease mouse models, Nat Genet 39, 1440–1442.PubMedCrossRefGoogle Scholar
  34. 34.
    Trinchese, F., Fa, M., Liu, S., Zhang, H., Hidalgo, A., Schmidt, S. D., Yamaguchi, H., Yoshii, N., Mathews, P. M., Nixon, R. A., and Arancio, O. (2008) Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease, J Clin Invest 118, 2796–2807.PubMedCrossRefGoogle Scholar
  35. 35.
    Choi, J. H., Berger, J. D., Mazzella, M. J., Morales-Corraliza, J., Cataldo, A. M., Nixon, R. A., Ginsberg, S. D., Levy, E., and Mathews, P. M. (2009) Age-dependent dysregulation of brain amyloid precursor protein in the Ts65Dn Down syndrome mouse model, J Neurochem 110, 1818–1827.PubMedCrossRefGoogle Scholar
  36. 36.
    Morales-Corraliza, J., Mazzella, M. J., Berger, J. D., Diaz, N. S., Choi, J. H., Levy, E., Matsuoka, Y., Planel, E., and Mathews, P. M. (2009) In vivo turnover of tau and APP metabolites in the brains of wild-type and Tg2576 mice: greater stability of sAPP in the beta-­amyloid depositing mice, PLoS One 4, e7134.PubMedCrossRefGoogle Scholar
  37. 37.
    Yang, D. S., Stavrides, P., Mohan, P. S., Kaushik, S., Kumar, A., Ohno, M., Schmidt, S. D., Wesson, D., Bandyopadhyay, U., Jiang, Y., Pawlik, M., Peterhoff, C. M., Yang, A. J., Wilson, D. A., St George-Hyslop, P., Westaway, D., Mathews, P. M., Levy, E., Cuervo, A. M., and Nixon, R. A. (2011) Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer’s disease ameliorates amyloid pathologies and memory deficits, Brain 134, 258–277.PubMedCrossRefGoogle Scholar
  38. 38.
    Mathews, P. M., Guerra, C. B., Jiang, Y., Grbovic, O. M., Kao, B. H., Schmidt, S. D., Dinakar, R., Mercken, M., Hille-Rehfeld, A., Rohrer, J., Mehta, P., Cataldo, A. M., and Nixon, R. A. (2002) Alzheimer’s disease-related overexpression of the cation-dependent mannose 6-phosphate receptor increases Abeta secretion: role for altered lysosomal hydrolase distribution in beta-amyloidogenesis, J Biol Chem 277, 5299–5307.PubMedCrossRefGoogle Scholar
  39. 39.
    Grbovic, O. M., Mathews, P. M., Jiang, Y., Schmidt, S. D., Dinakar, R., Summers-Terio, N. B., Ceresa, B. P., Nixon, R. A., and Cataldo, A. M. (2003) Rab5-stimulated up-regulation of the endocytic pathway increases intracellular beta-cleaved amyloid precursor protein carboxyl-terminal fragment levels and Abeta ­production, J Biol Chem 278, 31261–31268.PubMedCrossRefGoogle Scholar
  40. 40.
    Gravina, S. A., Ho, L., Eckman, C. B., Long, K. E., Otvos, L., Jr., Younkin, L. H., Suzuki, N., and Younkin, S. G. (1995) Amyloid beta protein (A beta) in Alzheimer’s disease brain. Biochemical and immunocytochemical analysis with antibodies specific for forms ending at A beta 40 or A beta 42(43), J Biol Chem 270, 7013–7016.PubMedCrossRefGoogle Scholar
  41. 41.
    Savage, M. J., Trusko, S. P., Howland, D. S., Pinsker, L. R., Mistretta, S., Reaume, A. G., Greenberg, B. D., Siman, R., and Scott, R. W. (1998) Turnover of amyloid beta-protein in mouse brain and acute reduction of its level by phorbol ester, J Neurosci 18, 1743–1752.PubMedGoogle Scholar
  42. 42.
    Chishti, M. A., Yang, D. S., Janus, C., Phinney, A. L., Horne, P., Pearson, J., Strome, R., Zuker, N., Loukides, J., French, J., Turner, S., Lozza, G., Grilli, M., Kunicki, S., Morissette, C., Paquette, J., Gervais, F., Bergeron, C., Fraser, P. E., Carlson, G. A., George-Hyslop, P. S., and Westaway, D. (2001) Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695, J Biol Chem 276, 21562–21570.PubMedCrossRefGoogle Scholar
  43. 43.
    Mathews, P. M., Jiang, Y., Schmidt, S. D., Grbovic, O. M., Mercken, M., and Nixon, R. A. (2002) Calpain activity regulates the cell surface distribution of amyloid precursor protein: inhibition of calpains enhances endosomal generation of beta-cleaved C-terminal APP fragments, J Biol Chem 277, 36415–36424.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Stephen D. Schmidt
    • 1
    • 2
  • Matthew J. Mazzella
    • 1
  • Ralph A. Nixon
    • 1
    • 3
  • Paul M. Mathews
    • 1
    • 4
  1. 1.Center for Dementia ResearchNathan S. Kline Institute for Psychiatric ResearchOrangeburgUSA
  2. 2.Vaccine Research CenterNational Institute of Allergy and Infectious Diseases , National Institutes of HealthBethesdaUSA
  3. 3.Departments of Psychiatry and Cell BiologyNew York University School of MedicineNew YorkUSA
  4. 4.Department of PsychiatryNew York University School of MedicineNew YorkUSA

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