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.
| Edition : | Vol. - No. |
| File Size : | 1
file
, 420 KB |
| Note : | This product is unavailable in Ukraine, Russia, Belarus |
| Number of Pages : | 13 |
| Published : | 11/01/2002 |
