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flocculation 276 flow cytometry
ppt µg.N
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
PRO-ZONE
50
100
Flocculation.
Tests of supernatants
with Ag + + + – – – – – – – – – – – –
with Ab – – – – – – – – – + + + + + +
E 0.5 cm (280 mµ)
150
Toxin µg N
200
250
Flocculation curve of Pappenheimer and Robinson.
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0
0.20
0.40
0.60
0.80
1.00
1.20
Weight of Thyroglobulin Added (mg)
1.40
300
Flocculation curve with human thyroglobulin and homologous antibody.
flocculation
A variant of the precipitin reaction in which soluble antigens
interact with antibody to produce precipitation over
a relatively narrow range of antigen-to-antibody ratios;
also used in the past to refer to flocculation aggregation of
such lipids as cardiolipin and others in serological tests for
syphilis, but these reactions are more correctly referred
to as agglutination rather than flocculation. Flocculation
differs from the classic precipitin reaction in that insoluble
aggregates are not formed until a greater amount of antigen
is added than would be required in a typical precipitin reaction.
If the antibody (or total protein) precipitated is plotted
against antigen added, the plot does not extrapolate to the
origin. In flocculation reactions, excess antibody and excess
antigen inhibit precipitation. Thus, precipitation occurs only
over a narrow range of antibody-to-antigen ratios. Soluble
antigen–antibody complexes are formed in both antigen and
antibody excess. Horse antisera commonly give flocculation
reactions (for example, antisera to diphtheria toxin
and certain streptococcal toxins). The peculiar aspects of
the flocculation reaction must be attributed to the reacting
antibodies as opposed to the antigen, which gives a typical
precipitin reaction with rabbit antisera. For many years,
this reaction was known as the toxin–antitoxin type of
curve because it was observed with horse antibodies against
diphtheria and tetanus toxins. In recent years, it has been
observed with sera from some patients with Hashimoto’s
thyroiditis. These patients develop autoantibodies against
human thyroglobulin. This antithyroglobulin antibody may
give a classic precipitin curve, but some individuals develop
flocculation types of antibody responses against the antigen.
flow cytometry
An analytical technique to phenotype cell populations. It
requires a special apparatus, termed a flow cytometer, that
can detect fluorescence on individual cells in suspension
and thereby ascertain the number of cells that express the
molecule binding a fluorescent probe. Cell suspensions are
incubated with fluorescent-labeled monoclonal antibodies
or other probes, and the quantity of probe bound by each
cell in the population is assayed by passing the cells one at
a time through a spectrofluorometer with a laser-generated
incident beam. Sample cells flow single file past a narrowly
focused excitation light beam that is used to probe
the cell properties of interest. As the cells pass the focused
excitation light beam, each cell scatters light and may emit
fluorescent light, depending on whether or not it is labeled
with a fluorochrome or is autofluorescent. Scattered light is
measured in both the forward and perpendicular directions
relative to the incident beam. The fluorescent emissions of
the cell are measured in the perpendicular directions by a
photosensitive detector. Measurements of light scatter and
fluorescent emission intensities are used to characterize
each cell as it is processed. Flow cytometry is a fast, accurate
way to measure multiple characteristics of a single cell
simultaneously. These objective measurements are made
one cell at a time, at routine rates of 500 to 4000 particles
per second in a moving fluid stream. A flow cytometer
measures relative size (FSC), relative granularity or internal
complexity (SSC), and relative fluorescence. Three-color
flow cytometry is used to analyze blood cells by size,
cytoplasmic granularity, and surface markers labeled with
different fluorochromes. Flow cytometry serves as the basis