Development of Antibody- and Phage-Based Lateral Flow Test Strips for the Detection of the Bovine Meat and Bone Meal in Animal Feeds and the Bacillus anthracis Spores in Water and Foods

Abstract

The outbreak of bovine spongiform encephalopathy (BSE) in 1980s and 1990s, peaked in 1992, mainly took place in most European countries, especially in the United Kingdom, caused widespread panic in the world. It was generally accepted that the most likely route of infection of cattle with BSE is by consumption of feeds containing low levels of processed animal proteins (PAPs), including bovine meat and bone meal (MBM). At present, classical microscopy is the only official method for the detection of PAPs in animal feeds in the European Union. Nevertheless, the method is laborious and time-consuming, and the analytical results depend largely on the experience of the microscopist. The need for a simple, rapid, specific, sensitive, accurate and inexpensive method to detect Bacillus anthracis (BA) spores became obvious after the Fall 2001 anthrax attacks which caused public fears. Immunoassay and polymerase chain reaction (PCR) are generally used for the detection of BA spores. PCR requires considerable effort in sample processing prior to analysis. Therefore, better detection methods need to be developed. The research described in this dissertation focused on developing antibody- and phage-based lateral flow test strip (LFTS) assay to detect the bovine MBM in animal feeds and the BA spores in water and foods, respectively. Bovine MBM proteins (bMBMP) were extracted from bovine MBM using a special extraction protocol, i.e., bovine MBM was pretreated in 1.0 M NaOH solution under the constant agitation at 37 OC for 48 h, and after pH adjustment and centrifugation, the precipitate was extracted by sterile deionized water. The anti-bMBMP and anti-pBA13 (BA 13 phage) antibodies were produced, and antibodies were purified by saturated ammonium sulfate precipitation followed by Protein A affinity column. Anti-bMBMP antibodies were conjugated to colloidal gold and form gold-anti-bMBMP antibody complex, which was deposited in the conjugate pad of LFTS. In addition, the anti-bMBMP antibody was also used as capture agents on the test line of LFTS, which can detect the bovine MBM in animal feeds. The anti-bMBMP antibody was successfully utilized to construct a LFTS which is sensitive and specific to bovine MBM in animal feeds. The detection limit of anti-bMBMP antibody is less than 0.1% (w/w), which reaches the requirement of industry use. The cross-reaction of the anti-bMBMP antibody with the proteins in the animal feeds can be eliminated or significantly reduced after immunoabsorption. For the development of the phage-based LFTS, B. anthracis 13 phage (pBA13) or JRB7 phage (pJRB7) was conjugated to colloidal gold and form gold-pBA13 or gold-pJRB7 complex which was used as recognition element to detect BA spores in water and foods. This complex was deposited in conjugate pad to bind the BA spores in the sample. In the LFTS, the pBA13 or pJRB7 was immobilized on glutaraldehyde-modified nitrocellulose membrane (NCM) as test line and anti-pBA13 antibody was directly immobilized on NCM as control line for detection. The detection limit of the developed phage-based LFTS is 107 spores/ml.