Rapid Assays for the Detection and Determination of Sparse Populations of Bacteria and Bacteriophage T7 with Radioactively Labelled Homologous Antibodies

Abstract

SUMMARYSmall numbers of several bacterial species were rapidly and specifically detected with the l'Wlabelled antibody method described previously. Multibacterial species detection was achieved in one operation with 12SI-1abelled mixtures of type-specific anti-bacterial globulins but the level and variability of the blank value increased, and sensitivity decreased, with the number of globulin components in the mixture. An indirect radio-assay that involved successive treatments of bacteria with unlabelled rabbit anti-bacterial serum and 1261-labelled immunopurified goat anti-rabbit globulin was less sensitive and reproducible than the direct radio-assay. A modified assay, developed to detect and determine bacteria filtered on to a membrane filter, allowed the detection of small numbers of bacteria in large volumes of aqueous sample.The feasibility of rapidly and specifically detecting small numbers of virus particles with 1261-labelled anti-viral globulin was investigated with bacteriophage T7 as a model particle. Methods for the rapid separation of phage-lzI-labelled globulin complex were developed and a minimum of about 5 x 105 total phage particles was detected.

Methods for the Assessment of Microbial Populations Recovered from Enclosed Aerosols

“…Previous work showed that the relationship between the concentration of bacteria in a sample and the radioactive signal given by the separated 1251-Iabelled immune complex in the radio-assay was usually nearly linear (Strange et al 1971); this also holds for coliphage T7 (Strange & Martin, 1972). For the present work it was shown that a similar linear relationship existed between the Concentration of the bacterial species or coliphage T7 recovered in samples of aerosols and the radioactive signal produced in the radio-assay.…”

“…Purified phage suspensions were prepared from crude lysate (Strange & Martin, 1972 Assay of colony-or plaque-forming units. Samples (0.25 ml) of suitably diluted suspensions of Escherichia coli with or without Bacilhs subtilis spores (as a tracer) were spread on to each of four tryptone glucose agar plates and colonies were counted after incubation for 20 h at 37 "c; when present, the characteristic orange-coloured colonies of B. subtilis var.…”

An Indirect Immunoradiometric Assay of Microbes

“…Immuno-purzjied antibodies. Purified rabbit anti-bacterial globulins were prepared as described previously (Strange et al, 1971 ;Strange & Martin, 1972). lmmuno-purified sheep anti-rabbit (SAR) globulin was prepared by absorbing sheep antiserum (36 ml) with CNBractivated Sepharose (Pharmacja; I g) linked to normal rabbit globulins (50 % saturated ammonium sulphate precipitate of normal rabbit serum ; reprecipitated twice and dialysed until free of detectable ammonium ion) as described in Pharmacia's instruction sheet.…”

Determination of Antibacterial Antibodies in Serum by Immunoradiometric Assays

“…The antibody proteins migrated as y globulins on electrophoresis (cellulose-acetate strips) and had high specific precipitating activity against homologous bacteria (Strange and Martin, 1972) but their actual purity is not known. For calibration purposes, however, antibody preparations were assumed to be pure and hence the sensitivities of all the assay systems tested are probably underestimated.…”

“…Washed live and killed suspensions of Serratia marcescens strain 8UK were prepared as described by Strange and Martin (1972).…”