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directed antibody that causes precipitation and
can be isolated by centrifugation.
8
This
are associated with prostate cancer. This
problem can be circumvented by MS. The great
power of MALISA lies in the ability of antibodies
to rapidly select families of proteins based on
a common structural feature while the MS iden-
ti
“
pull-
down
method is widely used to determine
species that are bound to antigens as discussed
shortly. This old pull-down method is still used
but is not as sensitive.
A second approach is to capture the immune
complex formed in solution by af
”
es and quanti
es those features that are
speci
c to different biological phenomena.
MALISA methods are of three types: (1) those
in which native antigens are immune-selected
and after desorption from the immune complex
identi
nity chroma-
tography, generally with a protein A or G (A/
G) column or with an avidin/streptavidin
column in the case of biotinylated antibodies.
Protein A or protein G columns select IgG and
in the course of doing so capture the immune
complex along with free IgG. This form of
antigen capture is used in immunological assays
where detection is achieved by mass spectrom-
etry. These MALISA methods
9
have much
greater selectivity than the older ELISAmethods,
as will be discussed later in this chapter.
A third strategy is to immobilize the antibody
on a small particle that is dispersed in the sample
prior to immune complex formation, much like
the old radioimmunoassay (RIA) format of Yal-
low
10
with 1
m
m particles. A new twist on this
approach was introduced with the use of
magnetic particles to circumvent centrifugation,
but the advance is not so much the use of
magnetic particles as coupling detection to
mass spectrometry.
11
A good example is stable
isotope standard capture with antipeptide anti-
bodies (SISCAPA) using a magnetic particle
immunosorbent format with MS detection.
12
The fourth antigen capture format is based on
the use of an immobilized antibody in a
ed by MS (
Figure 1
, route 1), (2) assays
in which proteins in a sample are trypsin
digested,
signature peptide fragments
from
a speci
c protein are captured by immune selec-
tion, and the selected peptides identi
ed by
LC-MS/MS (
Figure 1
, route 3), and (3) a combi-
nation of the
first two assay methods in which
immune-selected antigens are trypsin digested
and then identi
ed by LC-MS/MS (
Figure 1
,
route 2). Based on their similarity to proteomic
methods, these three approaches are described
as top-down, bottom-up, and mixed-modes of
MALISA, respectively. A fourth type involving
double af
nity selection achieved by coupling
routes 1 and 3 will not be discussed.
Multiplexing is becoming increasingly impor-
tant, particularly in those cases where large
numbers of biomarkers are being validated or
used in diagnostic assays. Af
nity selection
can be achieved in either solution as with
pull-down methods or with an immobilized
antibody. Solution-based immune complex
formation has inherent advantages in MALISA
type multiplexing based on the following set of
facts: (1) there is no need for antibody immobili-
zation, even when selecting large numbers of
antigens, (2) complex formation occurs faster
and with less antibody in a homogeneous
system, (3) antibodies and their concentration
are easily chosen or altered, (4) a universal
immune complex capture column such as
protein A/G, avidin, or a second antibody can
be used with any combination of antigen capture
antibodies and antigens, (5) this style of antigen
capture easily
ow-
through particle bed,
13
generally in some type
of MALISA format as discussed later in this
chapter.
ELISA has been the gold standard immuno-
logical assay method for half a century. But
ELISA has substantial limitations, the major
one being that many of the protein isoforms
selected by the antibody may not be associated
with the biological phenomenon being assayed.
The prostate-speci
c antigen (PSA) assay is an
example; only a few of the captured isoforms
fits into top-down and mixed-
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