Sample Processing - Waterborne Pathogens: Detection Methods and Applications - page 89

accumulation of particles within the pores. Warkiani fabricated filters using
a “dissolving mold technique” and report a recovery rate during elution
from the filter of 97% for tap water spiked samples. A high flow rate was
maintained throughout the operation and the filters could potentially be
reusable. No cost comparison was presented in the paper, which could be a
barrier to adoption of this type of technology; although if genuinely reus-
able, a higher price might not be such a problem.
4.4.4. Immunomagnetic separation
IMS both purifies and concentrates microorganisms. It is an established
technique used for recovery of microorganisms, other types of cells, pro-
teins etc. using specific antibodies immobilized on paramagnetic beads.
The process works by incubating the beads and sample, allowing time for
reaction to occur between the antibodies and the pathogen of interest.
Mixing enhances this binding step. Subsequently, the beads are pulled to
one side of the container using a magnet, isolating the pathogen of interest
from the rest of the sample, which is removed and discarded. This bead-
microorganism complex can be used for direct plating on selective media
or used for enrichment of a bacterium or for nucleic acid isolation. How-
ever, many protocols include dissolution of the bead-pathogen complex,
often accomplished by adding acid. Next, the now dissociated beads are
removed from the pathogen sample by using the magnet to once again
concentrate them at the side of the container, and removing the solu-
tion now containing the isolated pathogen. IMS offers high recovery rates,
though the drawbacks are the expensive reagents required, the time neces-
sary for binding and unbinding, and problems with unspecific binding or
aggregation of nontarget cells within the bead-pathogen clusters.
IMS is used in the above mentioned standard methods for isolation of
Cryptosporidium oocysts and Giardia cysts, and are commercially available
from several different suppliers. This protocol includes several IMS steps
where the first reduces the sample volume from 10 to 1 mL and is followed
by two rinses in 1 mL buffer. Thereafter, the parasites are eluted in HCl for
10 min and transferred to a microscope slide with NaOH for neutralization
and subsequent immobilization and straining. Manufacturers report high
recovery rates for IMS isolation of (oo)cysts, quoting over 95%.
To the best of our knowledge, beads conjugated with beads against other
parasites are not available today. During method development, Hoffman
et al. recovered E. intestinalis spores, labeled with FITC-conjugated rabbit
polyclonal antibodies using two different commercially available magnetic