The use of blue-excitable nucleic-acid dyes for the detection of bacteria in well water using a simple field fluorometer and a flow cytometer.

The blue-excitable nucleic acid dyes Coriphosphine O (CPO), YOYO-1, and YOPRO-1 were evaluated to rapidly detect the presence of bacteria in well water samples using a simple field fluorometer (Turner Designs, Sunnyvale, CA, Model 10-AU-005) and a tabletop flow cytometer (Coulter Epics XL). The dyes were first titrated on the Turner Designs Model 10-AU field fluorometer with log-fold dilutions of Esherichia coli, since this organism is the indicator organism for water contamination. A detection limit of 10(4) Colony Forming Units per ml (CFU/ml) was established for YOPRO-1 and 10(5) CFU/ml for YOYO-1. The detection limit with CPO was determined to be 10(7) CFU/ml due to the high background fluorescence of the dye. The dyes were also evaluated with ragweed pollen to gauge the effect of a biological interferent. Ten well-water samples were subsequently analyzed using the technique. The results showed that only YOYO-1 correctly detected all the samples that were positive according to the reference laboratory. YOPRO-1 correctly detected only one of four positive samples. Analysis with the CPO dye was inconclusive due to high background fluorescence. The samples were then subjected to analysis on the flow cytometer. Results obtained with YOYO-1 compared well to those obtained on the fluorometer and by the reference techniques. YOPRO-1 performed better on the flow cytometer than with the simple fluorometer, correctly detecting three of four positive samples. Although the CPO results showed a very slight increase of green fluorescence with positive samples, they were largely indistinguishable from negative samples. This study suggests YOYO-1 could be useful with either a simple fluorometer or with a tabletop flow cytometer in screening water samples for the presence of bacterial contamination.

The flow cytometry of Bacillus anthracis spores revisited.

BACKGROUND: The potential use of Bacillus anthracis spores as a weapon of terror has rekindled interest in the rapid detection and identification of the spores of these bacteria. Prior efforts to utilize flow cytometry (FCM) for this purpose resulted in tedious and time-consuming protocols. Advances in rapid immunoassays suggest a reinvestigation of the use of FCM because this may allow for the development of a rapid and sensitive system for detection and/or identification of spores in suspect samples. METHODS: In this study, antiserum was raised in goats using three different strains of B. anthracis spores as the immunogen. The resultant antibodies were purified, labeled with fluorescein, and evaluated for use in an immunoassay on a Coulter Epics XL flow cytometer. In the protocol that was developed, fluorescein-labeled antibodies are simply mixed with the sample, allowed to incubate, and then analyzed on the flow cytometer. Washes and centrifugation were eliminated. RESULTS: The results showed that a rapid (5 min) and sensitive immunological analysis was feasible. The detection limit (approximately 10(3) colony-forming units [CFU]/ ml) varied with strain, but there was no difference in the detection limit between live and irradiated spores. In addition, the power of FCM was utilized to minimize false-positive reactions among similar species of Bacillus by placing constraints on scatter and fluorescence intensity. The data also suggest that scatter might be useful to determine spore viability. CONCLUSION: This study shows that FCM may be an effective platform on which to perform immunological analysis for the detection and/or presumptive identification of B. anthracis spores. Published 2000 Wiley-Liss, Inc.

Recombinant antibodies: a new reagent for biological agent detection.

Antibodies are critical reagents used in several biodetection platforms for the identification of biological agents. Recent advances in phage display technology allow isolation of high affinity recombinant antibody fragments (Fabs) that may bind unique epitopes of biological threat agents. The versatility of the selection process lends itself to efficient screening methodologies and can increase the number of antigen binding clones that can be isolated. Pilot scale biomanufacturing can then be used for the economical production of these immunoglobulin reagents in bacterial fermentation systems, and expression vectors with hexahistidine tags can be used to simplify downstream purification. One such Fab reagent directed against botulinum neurotoxin A/B has been shown to be sensitive in a variety of assay formats including surface plasmon resonance (SPR), flow cytometry, enzyme linked immunosorbent assay (ELISA), and hand-held immunochromatographic assay. Recombinant antibodies can provide another source of high quality detection reagents in our arsenal to identify or detect pathogens in environmental samples.

Highly selective acridine and ethidium staining of bacterial DNA and RNA.

The acridine dyes acridine orange (AO) and coriphosphine O (CPO) and ethidium bromide (EtBr) were used to stain bacterial digests after electrophoresis in native and denaturing (SDS) polyacrylamide gels and were shown to stain DNA and RNA preferentially over other subcellular components in the gels. Vegetative cell digests of Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus showed intense staining of DNA with AO and CPO near the top of the gel, but little or no staining of other cellular constituents. EtBr stained both DNA and RNA in the gels. Protein standards and non-nucleic acid cellular constituents stained faintly with high concentrations (> or = 100 microM) of AO, lower concentrations (13.9 microM) of CPO, and did not stain with 0.5 microgram/ml EtBr in denaturing gels. The complete set of cellular biochemicals was visualized by silver staining, while the protein subset was detected by Coomassie blue staining. The highest concentrations of AO (120 microM) and CPO (13.9 microM) were shown to detect purified DNA in gels with a sensitivity in the range of 25-50 ng per band. This work demonstrates the specificity of acridine and ethidium dyes for nucleic acids, while illustrating the level of non-nucleic acid-specific interactions with other cellular components by staining of electrophoretically separated cellular components in a gel matrix.

Comparison of seven enzyme immunoassay systems for measurement of cytomegalovirus.

The relative sensitivities of seven different enzyme immunoassay (EIA) systems for the measurement of cytomegalovirus (CMV) were compared. Methods which used two separate antisera to CMV provided the greatest degree of sensitivity. Equivalent sensitivity was noted with the use of either enzyme-labeled antiglobulin or unlabeled staphylococcal protein A and rabbit enzyme-antienzyme complex to measure the second anti-CMV antibody bound to the solid phase. Single-antibody methods were less sensitive than the double-antibody methods but were more sensitive than an inhibition EIA. However, the sensitivity of the inhibition EIA was improved when CMV-antibody complexes were separated from unreacted antibody by means of precipitation with polyethylene glycol. Double-antibody EIA systems are preferable when antisera prepared in two different animal species are obtainable. However, a number of single-antibody EIA systems can be formulated for use in situations where only a single antiserum is available.

Analysis of nonspecific reactions in enzyme-linked immunosorbent assay testing for human rotavirus.

Solid-phase enzyme immunoassays can be utilized to detect antigens directly in clinical specimens. However, a small number of stools which we tested for human rotavirus by enzyme-linked immunosorbent assay (ELISA) were found to have nonspecific activity in the absence of rotaviral antigen. Similar nonspecific activity was found in eight of eight sera which contained rheumatoid factor. This nonspecific activity was markedly reduced by pretreatment of the specimens with reducing agents, normal goat serum, and anti-human immunoglobulin M (IgM). Thus, it is likely that these specimens contain an IgM antibody capable of reacting nonspecifically with the other components of the assay. Although pretreatment with the mild reducing agent N-acetylcysteine markedly reduced this nonspecific activity, such treatment did not reduce the specific ELISA activity due to rotavirus. Other treatments did produce a reduction in specific activity. Thus pretreatment with N-acetylcysteine offers a practical means to increase the specificity of ELISA systems without reducing their sensitivity.

Enzyme-linked fluorescence assay: Ultrasensitive solid-phase assay for detection of human rotavirus.

Enzyme-linked immunosorbent assay (ELISA) has proven to be a useful assay system for the direct detection of infectious agents. However, when the usual color-producing substrates are employed, relatively large amounts of substrate must be hydrolyzed by the bound enzyme before detection can be achieved. We attempted to improve the sensitivity of ELISA by utilizing a substrate that yields a fluorescent product on enzyme action. The enzyme-linked fluorescence assay (ELFA) based on this principle was approximately 100 times more sensitive than the corresponding ELISA or radioimmunoassay for the detection of human rotavirus in a standard stool suspension. In addition, the ELFA for human rotavirus was capable of detecting antigen in six specimens that were negative by ELISA. Five of these specimens were obtained late in the course of confirmed rotavirus infections. ELFA provides a simple, reliable, ultrasensitive method for the rapid detection of viral antigen.