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Oak Ridge Conference |
Sienna Biotech, Inc., 9115 Guilford Rd., Suite 180, Columbia, MD 21046.
a Author for correspondence. Fax 301-497-8796;
Abstract
Coupled particlelight scattering (CopalisTM) is a homogeneous immunoassay technology that permits simultaneous determination of multiple analytes in serum, plasma, or whole blood. Copalis differentiates monomeric latex microparticles from latex aggregates and cells on the basis of their unique light scatter properties. Copalis readily discriminates small (~0.1 µm) differences in latex microparticle size. Therefore, multiple simultaneous assays are configured by the use of mixtures of different-size latex microparticles. The Copalis research immunoassay for hepatitis B surface antigen (HBsAg) is configured in a sandwich format where the extent of light scatter histogram broadening due to HBsAg-mediated binding of colloidal gold to latex provides the basis for antigen quantification. Simultaneous Copalis forward- and wide-angle light scatter measurements allow discrimination of latex microparticles from the cell components of whole blood. Consequently, direct detection of HBsAg in unprocessed whole-blood samples by Copalis is feasible.
Coupled particle light
scattering (CopalisTM) is a novel homogeneous
immunoassay technology that permits rapid, sensitive, and simultaneous
determination of multiple analytes in serum, plasma, or whole blood
(1). Copalis differentiates monomeric latex microspheres
from latex aggregates and cells on the basis of their unique light
scatter properties as they pass individually through a finely focused
beam of light produced by a semiconductor laser (Fig. 1
). Small differences in latex microsphere size (~0.1 µm) are
readily discriminated by the high-resolution Copalis light scatter
signal. Consequently, multiple simultaneous assays can be configured by
the use of different-size latex particles in the same reaction mixture.
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Copalis assays can be configured in two distinct but complementary homogeneous test formats (2). Latex microsphere self-agglutination mediated by an immunochemical reaction provides the basis for the Copalis latex assay format (3). The extent of latex aggregate formation is correlated with sample analyte concentration during a Copalis latex assay. Light scatter histogram broadening caused by analyte-directed binding of colloidal metallic particles to latex microspheres provides the basis for the second Copalis assay format (4). In this type of assay, the magnitude of histogram broadening is used to quantify sample analyte concentration.
We provide here a brief overview of the Copalis technology and an illustration of its capabilities in the sensitive detection of hepatitis B surface antigen (HBsAg) in serum, plasma, or whole blood.1 The Copalis research immunoassay for HBsAg is configured in a sandwich format where antigen mediates binding of colloidal gold particles to latex microspheres. Formation of the colloidal gold:HBsAg:latex microsphere sandwich causes a change in the latex microparticle light scatter contour plot constructed from simultaneous Copalis forward-angle and wide-angle light scatter measurements. The ability of colloidal gold to modulate the light scatter signature of the latex assay microparticle provides the basis for the Copalis HBsAg immunoassay. Since the Copalis two-channel light scatter measurement allows for the discrimination of the latex assay particles from the red and white cell components of whole blood, we demonstrate here the feasibility of direct Copalis detection of HBsAg in unprocessed whole-blood samples.
Materials and Methods
instrumentation
The basic measurement device is an optical flow particle analyzer
using low forward-angle light scatter as the measurement principle and
a 635-nm low-power (5 mW) semiconductor diode laser as the light source
(2). To obtain maximum resolution of the light scatter
signal, sheath flow is used to create a concentric stream that
constricts the sample stream to a narrow cross-section (Fig. 1
). The
collimated output of the laser is focused at the center of the flow
cell in the highest intensity and most uniform region of the focused
beam. As each particle type passes through the beam, it generates a
unique light scatter signature that is simultaneously detected by a
photodiode positioned to collect only low forward-angle scattered light
and a photomultiplier tube positioned to collect wide-angle light
scatter (Fig. 1
).
reagents
Latex microspheres.
Sulfate latex microspheres were
obtained from Interfacial Dynamics Corp. The microspheres meet
specifications of <2% variation in diameter in the 1.0–1.5 µm
range and <4% diameter variation in the >1.5 µm size range.
Passive adsorption is the latex coating method of choice
(5). This method involves dialyzing the protein to be
bound into a low ionic strength buffer that is 1–2 pH units above the
isoelectric point of the protein. The latex microspheres are prewashed
extensively in the coating buffer and brought to a final concentration
of 5 g/L latex solids. Antibody or antigen (20 to 150 mg/L latex) is
added and incubated overnight at 4 °C with continuous mixing. The
coated beads are postcoated with 10 g/L bovine serum albumin (BSA) and
can be stored for several months at 4 °C until subsequent use in the
Copalis reagent drying process.
Gold colloid preparation.
Colloidal gold
(
max = 560 nm) is prepared by the chemical reduction of
hydrogen tetrachloroaurate(III), HAuCl4 (6).
Gold particle size analysis by electron microscopy reveals the mean
particle size to be 84 ± 15 nm. To bind proteins, the pH of the
colloidal gold suspension is typically adjusted to one pH unit above
the isoelectric point of the protein to be bound (7).
Soluble protein is added at room temperature at a range of
concentrations to optimize the specific activity of the preparation.
After 30 min, the preparation is stabilized to salt flocculation by
postcoating (7) and can be stored for up to several months
at 4 °C until subsequent use in the reagent drying process.
Copalis dry reagent preparation.
Rapid air drying is
used to convert the three components of the Copalis research HBsAg
reagent, which include an internal reference particle (1.4 µm), a
HBsAg antibody-coated latex assay particle (1.9 µm), and colloidal
gold particles (~0.08 µm) coated with a second HBsAg antibody to a
dry reagent format. A similar rapid air drying procedure is used to
convert the microparticle reagent components of the simultaneous
Copalis latex test for Toxoplasma gondii, rubella, and
cytomegalovirus total antibodies to a dry reagent format.
assay procedures
Multiplex Copalis test for
T. gondii, rubella, and
cytomegalovirus total antibodies. This latex assay is configured
to react at relatively high concentrations of latex microparticles
(1012/L) in a 0.1 mol/L glycine pH 9 buffer containing a
chaotropic salt and 10 g/L BSA. In the automated test procedure, the
dried latex microparticle reagent is reconstituted in reaction buffer,
serum (10% of total reaction volume) is added to the rehydrated
reagent, and after 10 min of mechanical agitation at room temperature
the reaction mix is subjected to Copalis analysis. Data is collected
for ~40 000 particles at a data acquisition rate near 3 kHz.
Quantification of the consumption of monomeric assay particles relative
to the number of invariant reference particles is used to analyze the
extent of reaction.
Copalis HBsAg reagent.
This gold assay is carried out at
relatively low concentrations of latex microspheres (109/L)
in the presence of a large excess (1012/L) of colloidal
gold particles in a 0.05 mol/L glycine pH 9 buffer containing 0.6 mol/L
potassium chloride and 6 g/L BSA. In the automated HBsAg test
procedure, the prototype Copalis analyzer reconstitutes the dried HBsAg
microparticle reagent and transfers an aliquot (5% of total assay
volume) of whole blood, serum, or plasma to the reconstituted reagent.
After a 30-min static incubation at 37 °C, the instrument completes
the Copalis analysis of the reaction mix (~10 000 total events)
within 30 s. The Copalis HBsAg assay response parameter quantifies
the proportion of latex assay particles that move out of an analysis
window because of antigen-mediated binding of colloidal gold. The
boundaries of this window were originally set to encompass the light
scatter contour of a reference HBsAg-negative serum sample. The Copalis
response index is obtained by calculating the ratio of test sample
assay response to that of the HBsAg-negative serum reference sample and
multiplying by 100.
Whole-blood sample handling.
EDTA- or acid citrate
dextrose-anticoagulated whole-blood samples were analyzed within
36 h after the blood draw. Whole-blood samples were centrifuged at
1000g for 10 min to prepare linked plasma samples.
Results
The Copalis reagent for the simultaneous detection of T.
gondii, rubella, and cytomegalovirus total antibodies in serum
contains four different sizes of latex microspheres that span the 1–2
µm diameter range (Fig. 2
). These particles include an internal reference particle (1.1
µm) and the three monomeric latex assay particles that have been
coated with antigens for T. gondii (1.4 µm), rubella (1.7
µm), and cytomegalovirus (1.9 µm), respectively. This reagent,
which is available for commercial use, illustrates a multiplex Copalis
latex assay where serum antibodies mediate formation of dimeric and
multimeric aggregates of the antigen-coated latex microspheres. We have
found that the most accurate and precise measure of sample antibody
presence is the consumption of the respective monomeric assay particles
relative to the reference particle (Fig. 2
, lower trace). The reference
particle verifies proper optical alignment and detects the rare
clinical incidence (~0.25%) of nonspecific microparticle
agglutination.
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The Copalis research immunoassay for HBsAg is configured in a sandwich
format where antigen mediates binding of colloidal gold to latex
microspheres (Fig. 3
). Components of the Copalis HBsAg reagent include an internal
reference particle (1.4 µm), a HBsAg antibody-coated latex assay
particle (1.9 µm), and colloidal gold particles (~0.08 µm) that
have been coated with a second HBsAg antibody. The present assay is an
automated procedure where the Copalis analyzer rehydrates the dried
reagent; transfers an aliquot of serum, plasma, or whole blood to the
reconstituted reagent; and after a 30-min incubation performs the
Copalis measurement on the reaction mix.
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Utilization of the commercially available single-channel Copalis One
Immunoassay System showed that HBsAg-mediated binding of colloidal
gold causes broadening of the Copalis forward-angle light scatter
histogram associated with the HBsAg antibody-coated latex microparticle
(Fig. 4
). Electron microscopy confirmed specific antigen-mediated
binding of colloidal gold particles to the assay particle in samples
displaying analyte-broadened histograms (Fig. 5
).
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Subsequent HBsAg work involved a two-channel research prototype
instrument capable of performing simultaneous forward-angle and
wide-angle (side scatter) Copalis light scatter measurements (Fig. 6
). The advantages of using simultaneous forward and side light
scatter data collection in the Copalis HBsAg assay system include
enhanced assay sensitivity and the ability to perform direct HBsAg
measurements in unprocessed whole-blood samples. Simultaneous
comparison of the HBsAg dose-dependent broadening characteristics of
the Copalis forward and side scatter signals showed that side scatter
displays greater sensitivity at low HBsAg concentrations (Fig. 7
). The enhanced spectral resolution (i.e., 106
vs 4000 channels) provided by the addition of side scatter channel
facilitates the spatial discrimination of latex microspheres in the
1–2 µm diameter range from other sample particulate matter such as
blood cells (Fig. 8
) or lipemia concentrations up to 20 g/L (data not shown).
Consequently, interference from the cell components of whole blood
(Fig. 8
) can be gated out electronically during Copalis two-channel
data acquisition.
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To assess the clinical utility of the Copalis HBsAg assay, we tested
255 HBsAg-negative sera and 100 confirmed HBsAg-positive sera obtained
from Gulf Coast Regional Blood Center in Houston, TX (Fig. 9
). The screening method used at Gulf Coast was the Ortho
Antibody to HBsAg ELISA Test System 2. Samples repeatedly reactive by
the screening method were subjected to further confirmatory testing at
Gulf Coast by the Abbott Auszyme EIA. The current Copalis HBsAg
research assay clearly distinguishes the negative serum population from
the population of HBsAg-confirmed positive sera (Fig. 9
). The current
estimate of the sensitivity of the Copalis HBsAg research reagent is
0.8 µg/L and 0.6 µg/L for the ad and
ay forms of HBsAg, respectively. We anticipate that we
will able to match the sensitivity claims (0.20–0.40 µg/L) of other
commercially available HBsAg tests through standard immunochemical
approaches for immunoassay performance enhancement. In addition, no
significant hook effect was observed during examination of samples with
very high (~200 000 µg/L) antigen concentrations (data not shown).
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Reproducibility panel testing consisting of 13 runs of four replicates
each for negative, low-positive, mid-positive, and high-positive
samples indicated that the Copalis HBsAg reagent system was stable and
precise over the duration of the 14-day stability study (Table 1
).
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To assess the feasibility of whole-blood Copalis HBsAg testing, we
conducted a preclinical investigation at Providence Laboratory
Associates in Rockville, MD, on a patient population at high risk for
hepatitis B infection. Parallel tests of whole blood and linked plasma
on 62 patients showed excellent agreement between whole-blood and
plasma results for 61 of 62 of the sample pairs (Fig. 10
). This clearly demonstrates feasibility of Copalis HBsAg
testing in unprocessed whole-blood samples. The single
whole-blood/linked plasma pair discordant result observed during
initial testing resolved (i.e., the plasma for patient 55 retested
negative) upon repeat testing. Variation of hematocrit from 18% to
58% had no effect on the ability of the Copalis HBsAg assay to
discriminate a negative whole-blood sample from low- and
mid-concentration positive whole-blood samples (Fig. 11
).
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Discussion
Copalis technology provides numerous advantages for the clinical laboratory over existing methodology. These include (a) a simple automated homogeneous test format; (b) the ability to make direct measurements in unprocessed whole-blood samples; and (c) the ability to perform simultaneous assays on multiple analytes, providing the opportunity to group assays related by a clinical diagnosis.
In the Copalis automated HBsAg test procedure, the analyzer reconstitutes the stable dried microparticle reagents just before use, transfers an aliquot of patient sample to the reconstituted reagent, and after a 30-min incubation period performs the Copalis measurement on the reaction mix. This is in contrast to common HBsAg ELISA microtiter plate procedures, which take from 2 h to overnight to complete. The HBsAg reagent system stability and reproduciblity results reported here are very competitive with ELISA and other automated EIA systems.
The enhanced spectral resolution provided by the addition of side scatter channel facilitates the spatial discrimination of latex assay microparticles in the 1–2 µm diameter range from the red and white cell components of whole blood. Consequently, we were able to demonstrate here that Copalis detection of HBsAg in whole blood is feasible. The ability to make direct measurements in unprocessed whole-blood samples should have future applicability to analytes requiring stat capability (e.g., cardiac marker testing) or very small analysis volumes (e.g., neonatal sample testing).
Small differences in latex microsphere size (~0.1 µm) are readily discriminated by the high-resolution Copalis light scatter signal. Consequently, multiple simultaneous assays can be configured by the use of different-size latex particles in the same reaction mixture. Analytes in future multiplex Copalis tests will be configured in groups related to a single diagnosis. For example, Copalis tests for acute or chronic hepatitis B could be configured by grouping the present HBsAg assay with an IgM antibody test to hepatitis B core antigen (anti-HBc IgM) or hepatitis B e antigen (HBeAg) and total antibody to hepatitis B core antigen (anti-HBc total) tests, respectively.
Acknowledgments
We thank Martha Martin, Jill Burkoff, Nancy Hooper, Benjamin Imus, Kimberli Wanionek, and Providence Laboratory Associates (Anita Mattero and D. Michael Kouns) for expert technical assistance, James DiOrio (Baxter Healthcare Corp., Round Lake, IL) for performing the electron microscopy work, and Sridhar Ganapathy and Reinaldo Gonzales for numerous helpful discussions.
Footnotes
1 Nonstandard abbreviations: HBsAg, hepatitis B virus surface antigen; BSA, bovine serum albumin; and HBc, hepatitis B virus core (antigen). 
References
The following articles in journals at HighWire Press have cited this article:
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  J. Baudry, C. Rouzeau, C. Goubault, C. Robic, L. Cohen-Tannoudji, A. Koenig, E. Bertrand, and J. Bibette Acceleration of the recognition rate between grafted ligands and receptors with magnetic forces PNAS, October 31, 2006; 103(44): 16076 - 16078. [Abstract] [Full Text] [PDF]
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 M. J. Benecky, K. L. McKinney, K. M. Peterson, and J. Q. Kamerud Simultaneous Detection of Multiple Analytes Using Copalis Technology: A Reduction to Practice Clin. Chem., September 1, 1998; 44(9): 2052 - 2054. [Full Text] [PDF]
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, linked plasma.