Chapter 12
Immunological Methods
Scot E. Dowd, Marilyn J. Halonen, and Raina M. Maier
1.
Describe an immunological assay for the accurate quantitation of Rhizobium around
root nodules.
The assay of choice would be immunofluorescent microscopy. The antigen (Rhizobium in this
case) is detected using an antibody to which a fluorescent signal molecule is conjugated.
The Rhizobium cells surrounding root nodules can then be observed and counted using an
epifluorescent microscope. An example of immunofluorescent antibody detection of
Rhizobium is shown in Fig. 9.13.
2.
Describe an immunological assay for detection of Giardia in water samples.
The assay of choice would again be immunofluorescent microscopy as for question 1. In this
case Giardia is the antigen. An example of immunofluorescent antibody detection of
Giardia is shown in Fig. 8.10.
3.
Immunomagnetic purification methods are becoming popular in environmental
microbiology. Sediment slurries can contain paramagnetic particles, which are
copurified along with target antigens, making subsequent assay procedures difficult.
Describe the problems that would arise with immunomagnetic analysis in this case,
and devise potential methods for solving these issues.
In this type of assay, a fluorescently labeled antiglobulin is normally used to detect the antigen
that is captured on the magnetic beads. The presence of contaminating paramagnetic
particles may interfere with the fluorescence assay for the immunomagnetic bead-antigen
complex by nonspecifically binding the fluorescently labeled antiglobulin. One way to
solve this problem would be to do a pre-separation on the sample prior to adding the
immunomagnetic beads. Thus the contaminating paramagnetic particles would be
removed using a magnet, then the pre-cleaned sample would be assayed using the
immunomagnetic beads.
4.
How could you determine whether an enzyme is intracellular or extracellular using
immunoassay techniques?
Centrifuge the sample containing the cells and collect the supernatant. Wash the cells several
times, and after each wash centrifuge and collect the supernatant. Combine all
supernatants together as the extracellular sample.
Take the cell pellet and lyse the cells, this is the intracellular sample.
Run an immunoassay (for example an immunoaffinity chromatography assay) on both samples
using a monoclonal antibody for the enzyme to determine whether the enzyme is inside
or exterior to the cell.
5.
You have two monoclonal antibodies available from two different commercial
companies. Both cost the same and both are specific for the rhizobia you are
studying in question 1. After ordering both antibodies, how would you determine
which antibody was the best for the assay you designed in question 1? How would
you perform these evaluations?
Run a series of evaluations to determine the sensitivity of each antibody. In this case, root
samples would be inoculated with Rhizobium at different cell concentrations. Then the
immunofluorescent microscopy assay would be performed to compare which antibody
can 1) detect the lowest number of Rhizobium cells, and 2) which antibody has the least
nonspecific binding to things in the sample other than the Rhizobium cells.
6.
Rapid detection of biological warfare agents is an emerging area of research
(Chapter 5). Design a rapid detection method using an immunoassay that can aid in
the detection of Bacillus anthracis.
One approach would be to develop an immunoaffinity chromatography based assay similar to
those available for other pathogenic microorganisms. In this case, the antibody is fixed to
the solid phase in a small cartridge or test strip. The sample that may or may not contain
the antigen (Bacillus anthracis) is applied to the cartridge or test strip. A color reaction
occurs if the antigen is present. This qualitative test takes 10-20 min. to perform.
7.
What immunoassays described offer quantitative results?
Fluorescence immunolabeling, ELISA, immunoprecipitation assays
8.
What immunoassays described allow for purification of antigens from
heterogeneous samples?
Immunomagnetic separation assays, immunoaffinity chromatography.