Measurement of Glial Fibrillary Acidic Protein

James P. O'Callaghan1

1 Center for Disease Control and Prevention NIOSH, Morgantown, West Virginia
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.8
DOI:  10.1002/0471140856.tx1208s11
Online Posting Date:  May, 2002
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Toxicant‐induced injury of the adult or developing central nervous system results in hypertrophy of astrocytes at the site of injury. The hallmark of this response is enhanced expression of the major intermediate filament protein of astrocytes, glial fibrillary acid protein (GFAP). A protocol is provided for assaying GFAP levels in detergent homogenates of brain tissue using a sandwich ELISA.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: GFAP Sandwich ELISA
  • Support Protocol 1: Brain Tissue Preparation
  • Support Protocol 2: Assay for Total Protein
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: GFAP Sandwich ELISA

  Materials
  • Bovine GFAP (American Research Products)
  • PBS/Triton: PBS containing 0.5% (v/v) Triton X‐100 (Bio‐Rad)
  • Sample: brain homogenate from treated animal ( protocol 2)
  • Rabbit anti‐glial fibrillary acidic protein antibody (see recipe; Dako, cat. #Z0334)
  • Phosphate‐buffered saline (PBS; e.g., Pierce; appendix 2A; see recipe)
  • Blocking solution (see recipe)
  • Detection antibody solution (see recipe) consisting of mouse anti‐glial fibrillary acidic protein monoclonal antibody (clone GA5; Oncogene Research Products) and alkaline phosphatase–conjugated anti‐mouse IgG (Jackson Immuno Research, cat. #315‐055‐003)
  • Blocking solution containing Triton
  • Alkaline phosphatase substrate kit (e.g., Bio‐Rad) containing:
  •  Alkaline phosphatase substrate: p‐nitrophenylphosphate tablets (see recipe)
  •  5× diethanolamine buffer
  • 0.4 N NaOH (e.g., Labchem)
  • Vortex mixer or ultrasonic cell disruptor (e.g., PGC with 2‐mm probe)
  • 96‐well flat‐bottom, microtiter plates (e.g., Immulon 2, Dynatech)
  • Microtiter plate reader, 405 nm
NOTE: Detergents act as wetting agents, therefore, more than a single use of a pipet tip with SDS‐ or Triton X‐100‐containing samples can lead to carry‐over errors. Thus, it is recommended to use a single pipet tip per sample and to withdraw the sample only a single time per tip.

Support Protocol 1: Brain Tissue Preparation

  Materials
  • Control or treated animal
  • 1% (w/v) SDS, 85° to 95°C
  • Dissecting instruments
  • Ultrasonic cell disruptor (e.g., PGC with 2‐mm probe)

Support Protocol 2: Assay for Total Protein

  Materials
  • Bovine Serum Albumin (BSA), RIA grade, fraction V (Sigma)
  • 1% (w/v) SDS (Bio‐Rad)
  • Brain homogenates ( protocol 2)
  • BCA Protein Kit (Pierce) containing:
  •  Solution A
  •  Solution B
  • 96‐well microtiter plates
  • Microtiter plate reader, 562 nm
NOTE: Detergents act as wetting agents, therefore, more than a single use of a pipet tip with SDS‐containing samples can lead to carry‐over errors. Thus, it is recommended to use a single pipet tip per sample and to withdraw the sample only a single time per tip.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Butler, J.E., Spradling, J.E., Suter, M., Dierks, S.E., Heyermann, H., and Peterman, J.H. 1986. The immunochemistry of sandwich ELISAs‐I. The binding characteristic of immunoglobulins to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and asymmetrical antibody‐enzyme conjugates.Mol. Immunol. 23:971‐982.
   Eng, L.F. 1988. Regulation of glial intermediate filaments in astrogliosis. In Biochemical Pathology of Astrocytes (M.D. Norenberg, L. Hertz and A. Schousboe eds.) pp. 79‐90. A.R. Liss, New York.
   Eng, L.F., Stöcklin, E., Lee, Y.‐L., Shiurba, R.A., Coria, F., Halks‐Miller, M., Mozsgai, C., Fukayama, G., and Gibbs, M. 1986. Astrocyte culture on nitrocellulose membranes and plastic: Detection of cytoskeletal proteins and mRNAs by immunocytochemistry and in situ hybridization. J. Neurosci. Res. 16:239‐250.
   Fix, A.S., Wightman, K.A., and O'Callaghan, J.P. 1995. Reactive gliosis induced by MK‐801 in the rat posterior cingulate/retrosplenial cortex: GFAP evaluation by sandwich ELISA and immunocytochemistry. Neurotoxicology 16:239‐237.
   Kretzschmar, H.A., DeArmond, S.J., and Forno, L.S. 1985. Measurement of GFAP in hepatic encephalopathy by ELISA and transblots. J.Neuropathol. Exp. Neurol. 44:459‐471.
   Norenberg, M.D. 1994. Astrocyte responses to CNS injury. J. Neuropathol. Exp. Neurol. 53:213‐220.
   Norton, W.T., Aquino, D.A., Hozumi, I., Chiu, F.‐C., and Brosnan, C.F. 1992. Quantitative aspects of reactive gliosis: A review. Neurochem.Res. 17:877‐885.
   O'Callaghan, J.P. 1991. Quantification of glial fibrillary acidic protein: Comparison of slot‐immunobinding assays with a novel sandwich ELISA. Neurotoxicol. Teratol. 13:275‐281.
   O'Callaghan, J.P. 1993. Quantitative features of reactive gliosis following toxicant‐induced damage of the CNS. Ann. N.Y. Acad. Sci. 679:195‐210.
   O'Callaghan, J.P., Miller, D.B., and Reinhard, J.F., Jr. 1990. Characterization of the origins of astrocyte response to injury using the dopaminergic neurotoxicant, 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydro‐pyridine. Brain Res. 521:73‐80.
   O'Callaghan, J.P., Jensen, K.F., and Miller, D.B. 1995. Quantitative aspects of drug‐ and toxicant‐induced astrogliosis. Neurochem. Int. 26:115‐124.
   O'Callaghan, J.P., Imai, H., Miller, D.B., and Minter, A. 1999. Quantitative immunoblots of proteins resolved from brain homogenates: Underestimation of specific protein concentration and of treatment effects. Anal. Biochem. 274:181‐26.
   Rosengren, L.E., Wikkelsø, C., and Hagberg, L. 1994. A sensitive ELISA for glial fibrillary acidic protein: application in CSF of adults. J. Neurosci. Methods 51:197‐204.
   Schmidt, G.R., Hossner, K.L., Yemm, R.S., Gould, D.H., and O'Callaghan, J.P. 1999. An enzyme‐linked immunosorbent assay for glial fibrillary acidic protein as an indicator of the presence of brain or spinal cord in meat. J. Food Protect. 62:394‐397.
   Schmued, L.C. and Hopkins, K.J. 2000. Fluoro‐Jade: Novel fluorochromes for detecting toxicant‐induced neuronal degeneration. Toxicol. Pathol. 28:91‐99.
   Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., and Klenk, D.C. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76‐85.
   Streit, W.J., Walter, S.A., Pennell, N.A. 1999. Reactive microgliosis. Prog. Neurobiol. 57:563‐581.
   Switzer, R.C. 2000. Application of silver degeneration stains for neurotoxicity testing. Toxicol. Pathol. 28:70‐83.
   U.S. Environmental Protection Agency. Pesticide Assessment Guidelines, Subdivision F. Hazard Evaluation: Human and Domestic Animals. Addendum 10, Series 81, 82, 83, Neurotoxicity. EPA‐540/09‐ 91‐123. National Technical Information Service. Springfield, VA.
   Wang, S., Rosengren, L.E., Karlsson, J.‐E., Stigbrand, T., and Haglid, K.G. 1990. A simple quantitative dot‐immunobinding assay for glial and neuronal marker proteins in SDS‐solubilized brain tissue extracts. J. Neurosci. Methods 33:219‐227.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library