Detection and quantitation of Clostridium thermocellum by enzyme-linked immunosorbent assay

Abstract

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for detection and quantitation of C. thermocellum in mixed populations of anaerobic bacteria. The assay utilizes a polyclonal detector antibody which was produced from rabbit antiserum. The polyclonal antiserum showed a small degree of cross-reactivity with three other species out of number which were examined. This cross-reactivity was removed by absorption of non-specific antibodies from the antiserum by contacting it with cell suspensions of the cross-reactive species. This increased the specificity so that C. thermocellum could be distinguished from the other species. The antiserum was further purified to obtain the IgG fraction using ammonium sulfate precipitation. This increased but the specificity and the sensitivity of the ELISA test. A cell-free antigen solution was prepared by harvesting and washing cells in the sample, resuspending the cells in buffer, autoclaving, and centrifuging. Optimum conditions for the ELISA were to use a 1:1000 dilution of the IgG solution and a pH of 7.2. Alkaline-phosphatase-labelled goat antirabbit IgG was used for detection. The detection range for C. thermocellum antigen was from 0.08 to 2.5 μg/ml (based on soluble protein). This range corresponded to viable cell counts ranging from 5 x 10⁴ to 1.6 x 10⁶ cells/ml as determined by the most probable number technique. This assay can quantify C. thermocellum in mixed cultures at total cell protein concentrations of about 2.5 μlg/ml and can be used as qualitative assay at higher concentrations.