In response to proposed federal regulations calling for increased monitoring of protozoans in raw source waters, several different systems are under evaluation to determine their accuracy and precision for determining protozoan concentrations. The approved method for the detection of Cryptosporidium (EPA Method 1623) under EPA's LT2ESWTR (proposed) has wide method performance acceptance criteria and is extremely labor intensive. The objective of this study was to evaluate the accuracy of oocyst recovery and detection methods using a variety of immunocapture methods (IMS) and labeling systems in conjunction with a new automated rapid detection technology (RBD2100). In contrast to traditional flow cytometry, this new technology provides an easy to use microbial counting platform that would eliminate the need for microscope-based enumeration by a technician, saving time and potentially improving the accuracy of the measurement process. Working with water samples spiked with formaldehyde preserved oocysts of a known concentration (Waterborne), commercially available Cryptosporidium oocyst specific immunomagnetic beads (Dynal and Aureon) were used according to the manufacturers recommended procedures. Following the dissociation of beads/oocyst complex (13.3.3 from 1623), the oocysts were labeled with a small volume of the Cy5-conjugated antibody (Waterborne) and analyzed on the RBD2100 for enumeration. Parallel samples were treated only with the Cy5-conjugated antibody followed by enumeration on the RBD2100 (no IMS performed) for use as a total count used in IMS recovery calculations. The Dynal and Aureon beads respectively provided recoveries of 72% and 75% respectively based upon RBD2100 enumeration. Alternatively, Protein G bound paramagnetic beads (50 nm) were coated with oocyst specific antibody from Biogenesis, mixed with the Cy5 Cryptosporidium antibody (Waterborne) and added to water samples spiked with a wide range of oocysts concentrations (176 to 2832 per ml). The mixture was incubated for 1 hr at 37 degrees C prior to IMS and the captured sample was then analyzed on the RBD2100 (without the bead dissociation and removal step). The flow cytometer counts correlated very well (R2=0.966) with the expected counts within this concentration range. The next phase of this work will involve using matrix-containing water from the 1623 method (through step 13.2) in conjunction with this alternative enumeration technology. The immunocapture procedure described in combination with the RBD2100 technology represents a potential modification to the currently available procedures for the assessment of oocyst contamination in drinking water. Includes 6 references, table, figures.
