The potential to use antibodies as research tools has
been known since the early 1890s, when Karl Landsteiner
used antibodies to distinguish between the
ortho and meta configurations of groups on benzene
rings. Concurrently, Emil von Behring and Shibasaburo
Kitasato demonstrated antibody-mediated immunity
against diphtheria and tetanus toxins, culminating in
Paul Ehrlich’s proposal that antibodies could be used as
“magic bullets” to target and defeat human diseases.
Over the next 80 years, much progress was made in
understanding both the structure and function of the
immunoglobulin molecule within the body, but direct
applications to research did not arise until the 1970s.
With the advent of monoclonal antibodies, enzymatic
detection, and fluorescent labeling, antibodies finally
came into their own. While applications like immunohistochemistry,
Western blotting, enzyme-linked
immunosorbent assay (ELISA), and immunoprecipitation
have changed the way we study biological
systems, the commercialization of monoclonal antibodies
for therapeutic use has created new and lucrative
markets within the pharmaceutical and biotech
industries. Today, there are hundreds of companies
around the world that supply antibodies for research,
commercial, and therapeutic use.
Millipore antibody story
Companies such as Upstate, Chemicon, and Linco (all
part of Millipore Corp., Billerica, MA) were founded in
the early days of antibody research in order to license,
develop, and sell these new tools to the scientific community.
Upstate, started by Drs. Gordon Sato and
Wallace
McKeehan from the W. Alton Jones Cell Science
Center in 1987, utilized a novel licensing model to
provide unique cell signaling targets and the first commercially
available kinases. Coupled with
one of the highest validation standards
in the market, Upstate quickly grew into
a boutique antibody business by offering
the most novel and validated antibodies,
proteins, and kinases.
Chemicon’s success was also built on antibodies.
Founded in 1983 by Dave Beckman,
the company started as an OEM service
for scientists who were overburdened
by requests for their home-grown antibodies.
The company soon began producing its
own antibodies and expanded its business
by offering in vitro diagnostic products,
novel targets such as NeuN, and stem cell-related
kits and reagents.
Linco Research, Inc. was founded by
Dr. Ronald Gingerich
in 1977 to provide
unique tools for the diabetes research
market while pioneering the process for
preparing prediluted, ready-to-use primary
and secondary antibodies. In the
early 1990s, Linco began to manufacture
diagnostic kits with bioanalytical service support,
quickly expanding into contract work for clinical trials.
With the addition of new radioimmunoassay (RIA)
and ELISA kits and the introduction of multiplex
assays detecting multiple analytes in a single sample,
2002 saw the formation of Linco Diagnostic Services
Inc., a new company focused exclusively on clinical
and bioanalytical support for discovery research, regulatory
compliance studies, and clinical diagnostic testing.
The success and growth of all three companies led to
their acquisition by Serologicals Corp., which was
in turn acquired by Millipore in 2006. The influx of
new products significantly strengthened Millipore’s
bioscience
portfolio, giving it a leading-edge position
in high-growth life science segments such as drug discovery,
antibodies, cell biology reagents, and stem
cell research. In particular, the addition of antibody
products to the company’s existing immunodetection
portfolio reinforced a synergy between complementary
capabilities. This created the possibility for new technology
platforms as well as innovative and highly differentiated
products in various research areas.
Millipore’s portfolio of antibody-based products
is distinctive among other providers based on its
breadth across major scientific disciplines (neuroscience,
cell signaling, nuclear function, apoptosis,
adhesion, and cell structure) as well as
disease-focused areas (cancer, cardiovascular, neurodegenerative
disease, and inflammation). Within
each of these portfolios is a wide array of products
including individual antibodies, ELISAs, multiplex
assays, and other detection assays. With over
8000 antibody-based products, Millipore is a leading
source for novel targets in multiple disciplines that
use a variety of technology platforms.
Detection research—antibodies in use
Protein researchers know that even the best antibody
is only as good as the immunodetection system. In
addition to enhancements in antibody affinity, stability,
and specificity, new technologies in platforms
used to detect the antibody–antigen interaction have
proved to be crucial in advancing protein research.
Western blotting
Advances in Western blotting, in particular, have
complemented and accelerated antibody-based
research. Western blotting is a powerful tool to detect
which proteins are present and compare their relative
levels without prior purification. Its widespread
appeal is based on its overall simplicity, coupled with
the high resolution of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) prior to membrane transfer.
Although sensitivity and flexibility have increased,
there have been few major advances within this application.
The SNAP i.d.™ protein detection system
(Millipore) seeks to revolutionize Western blotting
by decreasing the immunodetection time (blocking,
washing, and incubations) to less than 30 min while
producing results that are similar to or better than
those using traditional immunodetection protocols.
Because of the design of the instrument, smaller volumes
of antibodies can be used for reactions on multiple
blots of varying sizes during a single run. The
system is compatible with fluorescent, chemiluminescent,
or chromogenic detection methods, such as the
fluorescent detection experiment described in Figure
1, and effectively detects a wide range of proteins over
a broad range of sensitivity, as shown in Figure 2.
Figure 1 - Upper panel: Scanned images of blots developed with standard immunodetection (standard, left panel) and with SNAP
i.d. protein detection system (right panel). The blot was scanned on a Fuji FLA-5100 imaging system (Fujifilm Life Science USA,
Stamford, CT) after drying the blot for 3 hr under vacuum. Lower panel: Quantification of GAP43 protein bands. Fluorescent band
intensity (y-axis) is plotted for indicated input tissue lysate amount (x-axis) from corresponding blots in the upper panel. R2 value is indicated
for each experiment.
MILLIPLEX™MAP
Bead-based multiplexed immunoassays represent one
type of microarray in which antibodies are utilized
for analyzing complex biological responses. The utility
of multiplexed assays derives from small sample
volumes, specificity, and sensitivity with simultaneous
detection of analytes of interest in a reproducible
platform. Multiplexed measurements are important
in the postgenomic era, in which proteins are known
to function in multiple biological pathways and complexes.
Bead-based immunoassays such as those based
on Luminex® xMAP® technology (Luminex Corp.,
Austin, TX) not only detect and quantify proteins
of interest, but report the effects of protein–protein
interaction among up to 100 unique analytes.
Figure 2 - Immunodetection after optimization of primary and secondary antibody:
A variety of proteins from different lysate sources (rat liver, cancer cell, and
stem cell) with a wide range of molecular weights (18–220 kDa) and relative abundance
in the cell (depending on the sample type and amount loaded in the gel), were
detected by the SNAP i.d. system.
The underlying strength of these assays is the unique
antibody pairs bound to the beads. Validated analyte
panels utilize an open architecture allowing for multiple
targets of interest within a specific disease state, expression profile, therapeutic area, or biological process
(cytokine biology). These assays are being readily
used as tools for biomarker discovery and profiling,
detecting early-stage cancers, injury and healing, and
other pathological conditions with numerous commercial
providers offering a multitude of panels.
Figure 3 - Concentrations for each biomarker in the healthy and cancer groups are shown. Serum from patients with ovarian cancer
showed elevated levels of prolactin, OPN, MIF, and CA-125, and lower levels of leptin and IGF-II compared to the healthy group.
One such kit, the MILLIPLEX MAP Human Cancer
Biomarker Panel (Millipore), was developed
in conjunction with Dr. Gil Mor of Yale University
(Departments of Obstetrics and Gynecology
and Reproductive Science, Yale University School
of Medicine, New Haven, CT). The assay simultaneously
measures five serum biomarkers: macrophage
migration inhibitory factor (MIF), prolactin,
CA-125, leptin, and osteopontin (OPN), and a
single plex IGF-II assay, representing a signature for
screening and detecting ovarian cancers. The serum
levels of these biomarkers change with respect to the
body’s response to the presence of cancer cells, as
seen in Figure 3. The Biomarker Panel can detect a
wide range of serum levels, as shown by the standard
curves in Figure 4.
Figure 4 - Human Cancer Biomarker Panel standard
curves.
The proteome challenge—one protein, one antibody
Proteomics promises to hold one of the brightest
futures for antibodies, with its requirements for
specific reagents that enable the understanding of
protein function and its correlation to a vast range
of post-translational modifications (phosphorylation,
glycosylation, methylation, ubiquitination,
etc.). Antibody-based proteomic approaches such
as microarrays, multiplexed arrays, ELISAs, and
enrichment/depletion prior to mass spectrometry
have become routine methods, creating an even
greater need for novel and unique protein targets to identify relevant biomarkers and other disease models.
Although various consortia, organizations, and
commercial companies are working to develop novel
antibody targets for use in proteomics and protein
research protocols, the challenge remains in creating
a complete protein profile library based on the post-translational
modifications of known protein species.
With the potential for millions of different protein
species within the human proteome, the need for
unique antibodies recognizing each protein species
becomes paramount, especially in therapeutic
areas and disease research. The need for increased
quantitative results and clinical-grade requirements
promises to drive advances in antibody-dependent
research, using proven technologies such as magnetic
beads, mass spectrometry, flow cytometry, and various
microseparation techniques. The expanding opportunities
for drug development, research, and clinical
uses of antibodies will continue to demand increasing
researcher productivity in the understanding of how
proteins interact in their natural environments.
Ms. Armin is Group Product Manager, Antibody Technologies,
Millipore Corp., 28820 Single Oak Dr.,
Temecula, CA 92590, U.S.A.; tel.: 951-514-4355;
e-mail:[email protected].