Improving Sensitivity, Precision, and Repeatability of the GFAP-ELISA for Detecting CNS Contamination in AMR-generated Tissues Compared to the Immunohistochemical Protocol used by USDA-FSIS

Lead PIs

Hossner KL, Belk KE, Yemm RS, Mason GL, Cummings BA,BeefUSAlogo

Co-PIs

Sonnenshein S, Reddy MCS, Sofos JN, Scanga JA, Tatum JD, Smith GC

Funding Agency

National Cattlemen’s Beef Association (NCBA)

Grant Number

53-5003

Funding Period

2002 – 2003

Lab Personnel

Keith Belk

Abstract

The presence of brain or spinal cord as an inadvertent contaminant of meat may result from the stunning of livestock, the splitting of the carcass or the preparation of advanced meat recovery (AMR) products from the vertebral column (Kelley et al., 2000). In light of current consumer concern about bovine spongiform encephalopathy (BSE), a disease transmitted by consumption of CNS tissue, a reliable analytical test for CNS tissue in meat products is essential to ensure consumer confidence and allay consumer fears of BSE agents in meat products. A method to detect the presence of CNS tissue in meat products has been developed in our laboratory (Schmidt et al., 2001). The method uses a fluorimetric enzyme-linked immunosorbent assay (F-ELISA) for the detection of glial fibrillary acidic protein (GFAP), an antigen which is highly, but not completely, restricted to astrocytes and satellite cells in the CNS. The F-ELISA was sensitive to 0.2 ng GFAP, had an intra-assay coefficient of variation (CV) of 2.0% and an inter-assay CV of 14.1%. Bovine spinal cord and brain demonstrated dose-response curves which were parallel to GFAP standards while peripheral sciatic nerve and cervical ganglia also cross-react at very high concentrations. Using this assay, we measured levels of GFAP in bovine tissues of approximately 2,000 ng/mg in spinal cord, 600 ng/mg in brain, 12 ng/mg in sciatic nerve and 2-4 ng/mg in cervical dorsal root ganglia. A comparison of this method with the Ridascreen ELISA and an earlier colorimetric GFAP ELISA has been published (Schmidt, et al., 2002). The CSU F-ELISA provides a method to detect small amounts of CNS tissue in meat products which is simple, cost-effective and efficient. This assay forms the basis for a commercially available kit for the detection of CNS tissue in meat products, the 10/5 Ridascreen assay developed by R-Biopharm AG, Darmstadt, Germany. The objective of the current study was to compare the sensitivity and repeatability of the F-ELISA with the Ridascreen 10/5 assay and to examine alterations in the Ridascreen assay which might increase its sensitivity and repeatability. The stated objectives for this work were: This study was designed to compare three methods for detecting CNS tissue in AMRgenerated comminuted beef – that differ in either sampling protocol or laboratory testing methodology – for their sensitivity, repeatability and precision. These methods are: 1. The Colorado State University F-ELISA for GFAP; 2. The R-biopharm GFAP-ELISA; and 3. The USDA Eastern Laboratory immunohistochemical procedure. In addition, we examined modifications of the commercial R-Biopharm protocol to improve its sensitivity, repeatability and precision.