Fluorometric Assay to Detect the Germination of Bacillus Anthracis Spores and the Germination Inhibitory Effects of Antibodies
Abstract
A sensitive assay for germination of spores of the anthrax agent was developed using a fluorescence reader. Significant inhibition of germination was detected for spores initially treated with antispore or antitoxin antibodies. Antigens specific to ungerminated spores that stimulate potentially protective antibodies are being identified and their role in inhibition of germination and in immune protection against anthrax will be determined. The germination of spores of Bacillus anthracis is typically detected by alterations in spore refractility, stainability, and absorbance (OD560). We have developed a more quantitative, semi-automated procedure for detecting germination by using a microtiter kinetic reader for fluorescence spectrophotometry (Biotek FL600). The procedure was based on the increase in fluorescence of spores with time during their incubation in germination medium containing Syto-9, a fluorescent nucleic acidbinding dye which stained germinated B. anthracis but not dormant spores. A colorless, defined medium containing equal parts of L-alanine, adenosine, and casamino acids provided low levels of background fluorescence, stimulated germination, and yielded reasonable germination kinetics. Antibodies (Abs) to the protective antigen (PA) component of the anthrax toxins of B. anthracis have been shown to inhibit in vitro germination. Regression analyses of the germination data obtained with the reader yielded parameters (i.e., the difference between the baseline and maximal fluorescence values [a coefficient] and the area under the curve) that gave significant differences between spores pretreated with anti-PA or antispore antibodies and buffer or nonimmune sera. We are testing sera from vaccinated or passively-immunized animals to determine the correlation between serum germination inhibitory activity, titers of serum anti-PA or antispore antibodies, and survival after lethal challenge.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2003
- Accession Number
- ADA483426
Entities
People
- John S. Lee
- Kelly M. Rea
- Paul H. Gibbs
- Stephen F. Little
- Susan L. Welkos
Organizations
- United States Army Medical Research Institute of Infectious Diseases