Methods for Sample Preparation for Direct Immunoassay Measurement of Analytes in Tissue Homogenates: ELISA Assay of Amyloid β‐Peptides

Paul A. Hyslop1, Mark H. Bender1

1 Eli Lilly and Company, Indianapolis, Indiana
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.20
DOI:  10.1002/0471142301.ns0720s18
Online Posting Date:  May, 2002
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Abstract

Use of low abundance analytes in whole tissue homogenates has been realized with the development of assays in which a specific analyte is captured and detected using immunological reagents. One of the many advantages of analyte immunoassay in crude homogenates is its relative simplicity, allowing high throughput analysis of samples. In this unit, some major key determinants in sample and standard preparation and handling are described that have been shown to improve the performance and reliability of these assay systems. The ELISA assay of amyloid peptides from brain tissue is described as an example, since the protocols for this analysis exemplify many of the techniques and problems that are encountered in the development of new assays.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation and Homogenization of Tissues for Analysis of Amyloid β‐Peptides
  • Basic Protocol 2: Example of ELISA Technique for the Analysis of Amyloid Peptide Aβ(1‐x) and Aβ(1‐42) from Brain Homogenates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation and Homogenization of Tissues for Analysis of Amyloid β‐Peptides

  Materials
  • Experimental animal or tissue sample
  • Guanidine buffer (see recipe), ice‐cold
  • Casein buffer with protease inhibitors (see recipe) ice‐cold
  • Tube rotator (e.g., Cole‐Palmer Roto‐Torque)
  • Refrigerated centrifuge
  • MultiScreen‐BV 1.2 µm Durapore PVDF, preassembled 96‐well filtration plates (Millipore)
  • 96‐well polypropylene eluant‐capture plates (Fisher Scientific)
  • Millipore MultiScreen Filtration System vacuum manifold

Basic Protocol 2: Example of ELISA Technique for the Analysis of Amyloid Peptide Aβ(1‐x) and Aβ(1‐42) from Brain Homogenates

  Materials
  • Capture antibodies:
  • 266, prepared by immunizing mice with human Aβ peptide fragment 13‐28
  •  21F12, prepared by immunizing mice with human Aβ peptide fragment 33‐42
  • Plate coating buffer (see recipe)
  • Plate blocking buffer (see recipe)
  • Wash buffer (see recipe)
  • Samples to be analyzed for amyloid β‐peptides (see protocol 1)
  • Amyloid β‐peptide standards (see Critical Parameters)
  • 270 µg/ml de‐salted, biotinylated reporter (detection) antibody 3D6 (Innogenetics). Use as directed by the manufacturer. Use EZ‐Link Sulfo‐NHS‐LC‐Biotin (Pierce) to biotinylate 1.2 mg/ml 3D6 (or other reporter antibody) according to the manufacturer's instructions.
  • Streptavidin‐horseradish peroxidase conjugate (Amersham)
  • TMB substrate kit (Pierce; store at 4°C; once opened, use for a period of no longer than 1 to 2 months)
  • Stop reagent: 2 N H 2SO 4
  • Polystyrene high‐protein‐binding ELISA plates (optimize for assay; coefficients of variation for IgG bound between wells should be <5%, and background ±0.005 AU from mean). It is worthwhile evaluating plates from more than one vendor during assay development, because the optimal plate for a particular assay may vary.
  • Plate sealers
  • Spectrophotometer accommodating microtiter plates
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Figures

Videos

Literature Cited

Literature Cited
   Crowther, J.R. 1995. ELISA theory and practice. In Methods in Molecular Biology, Vol. 42. (J.M. Walker, Series Ed.). Humana Press, Totowa, N.J.
   Engvall, E. and Perlmann, P. 1971. Enzyme‐linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871‐874.
   Gervay, J. and McReynolds, K.D. 1999. Utilization of ELISA technology to measure biological activities of carbohydrates relevant in disease states. Curr. Med. Chem. 6:129‐153.
   Seubert, P., Vigo‐Pelfrey, C., Esch, F., Lee, M., Dovey, H., Davis, D., Sinha, S., Schlossmacher, M.G., Whaley, J., Swindlehurst, C., McCormack, R., Wolfert, R., Selkoe, D.J., Lieberburg, I., and Schenk, D. 1992. Isolation and quantitation of soluble Alzheimer's β‐peptide from biological fluids. Nature 359:325‐327.
   Van Weeman, B.K. and Schuurs, A.H.W.M. 1971. Immunoassay using antigen‐enzyme conjugates. FEBS Lett. 15:232‐236.
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