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Abstracts of Oak Ridge Posters |
1
Advanced Diagnostics Division, Dade Behring Inc., PO Box 49013, San Jose, CA 95161
a author for correspondence: fax 408-239-2707, e-mail yenping_liu{at}dadebehring.com
Homocysteine (Hcy) is present in plasma primarily bound as
disulfides with itself, Cys, and albumin (
70%) (1)(2)(3)(4).
Total homocysteine (tHcy) in serum or plasma is markedly increased in
patients with cobalamin or folate deficiency (3), and
decreases only when they are treated with the deficient vitamin. tHcy
is therefore of clinical relevance, with reference values in fasting
subjects of 5–15 µmol/L (1). In addition, even a
moderate increase of Hcy (hyperhomocysteinemia) is a risk factor for
premature cardiovascular disease (4). These disorders
justify introduction of the tHcy assay in the routine clinical
chemistry laboratory.
The development of a rapid, homogeneous assay for Hcy in serum or plasma using the luminescent oxygen channeling immunoassay (LOCITM) (5) is described. An ELISA for the determination of tHcy based on the modification of sample tHcy by alkylation and detection of the alkylated product was described previously (6).
The current assay is designed for tHcy determination in serum or plasma at clinically relevant concentrations. Chemical reactions involved in the assay can be divided into three steps:
The first step involves the release of bound Hcy by reduction of
serum disulfides with tris-(2-carboxyethyl)phosphine (TCEP); the second
step involves the derivatization of Hcy and cysteine with CABA to
produce acyclic Hcy-ABA and cyclic Cys-ABA as shown in the alkylation
reaction (Fig. 1A
); the third step involves the selective binding of Hcy-ABA to
anti-Hcy-ABA coated on chemiluminescent latex particles in competition
with binding of Hcy-ABA coated on sensitizer latex particles.
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A two-reagent assay protocol was used in this system: The first reagent
contains TCEP, CABA-phosphate (the enol-phosphate derivative of
chloroacetylbenzoic acid), and latex particles coated with Hcy-ABA (the
product of alkylation of Hcy with CABA). The phosphate protecting group
of CABA is required for compatibility with TCEP and greatly increases
the stability and solubility of CABA. A second reagent contains
alkaline phosphatase (required for the release of CABA) and latex
particles coated with an antibody specific to Hcy-ABA. Disulfide bonds
in the sample are reduced when the sample is combined with the first
reagent. When the second reagent is added, CABA is released and
alkylates the sulfhydryl groups of Hcy and Cys. The amino group of the
alkylated cysteine but not the alkylated Hcy can react internally with
the ketone introduced by CABA to give a cyclic imide (Fig. 1A
). The
alkylated Hcy derivative (Hcy-ABA) then binds to the antibody that is
coated on the chemiluminescent latex particles. The structurally
distinct cyclic cysteine derivative (Cys-ABA) is not recognized by the
antibody. This overcomes the problem of developing antibodies to Hcy
that do not cross-react with cysteine, which usually is present at
25-fold molar excess concentration (7).
The detection step is superficially similar to latex agglutination but
uses a low concentration of particles and a novel photochemically
triggered chemiluminescent detection technique (5). Two
types of latex particles (0.2–0.3 µm in diameter) are used. Both
types of particles have a hydrogel coating that protects the particles
from nonspecific interactions with matrix components and provides a
functionalized surface to which antibodies and analytes can be
covalently attached. Binding of the two particles is mediated by
Hcy-ABA and is detected by measurement of the chemiluminescence that
ensues following brief irradiation of the assay mixture. The
chemiluminescence is generated by reaction of an olefinic acceptor in
the chemiluminescent particles with singlet oxygen that is generated by
a photosensitizer particle in close proximity. The adduct that is
formed has a half-life of 0.6 s and decays with emission of light at
wavelength >600 nm. Because of the short lifetime of singlet oxygen in
water (4 µs), it can only diffuse a few microns. A signal can
therefore be produced only when the particles are closely associated.
The LOCI Hcy assay is performed on an automated instrument (Tecan), which was modified with a pulsed diode laser and a luminometer (5). The patient sample is incubated by mixing 5 µL of serum or EDTA-treated plasma with 50 µL of the first reagent, which contains 2 mmol/L TCEP, 5 mmol/L CABA-phosphate, and 5 µg of Hcy-ABA-coated sensitizer particles. After a 7-min incubation at 37 °C and the addition of 50 µL of the second reagent, which contains 50 µg of alkaline phosphatase and 12.5 µg of anti Hcy-ABA monoclonal antibody-coated chemiluminescent particles. After the addition of 145 µL of 0.1 mol/L borate buffer, pH 9.2, the mixture is incubated an additional 2.6 min. The chemiluminescent signal is then measured by repetitively irradiating at 680 nm for 1 s and reading at 600–620 nm for 1 s. The assay signal is inversely related to the amount of tHcy present in the serum sample. Concentrations are determined using pooled serum calibrators.
Calibrators were prepared from a pooled serum sample
supplemented with known amounts of Hcy and verified by a HPLC
method (3). Signal modulation of 65–75% over the range of
0–60 µmol/L Hcy was demonstrated. Cross-reactivity of
L-cysteine and
L-methionine, each at 10 mmol/L in assay buffer,
was assayed with Hcy LOCI. The observed relative luminescent units
corresponded to 0.87 and 0.67 µmol/L tHcy, respectively. The
intraassay CVs obtained by assaying five replicates on the same
carousel were 5.9%, 2.7%, and 3.4% for 10, 30, and 60
µmol/L Hcy. This process was repeated twice to determine the
interassay CVs, which were 5.3%, 2.7%, and 3.9% for 10, 30, and 60
µmol/L Hcy, respectively. The recovery of Hcy from patient samples
supplemented with exogenous Hcy (n = 2 at five concentrations) was
91–106% with a mean of 97.3%. Regression analyses of the results
obtained using 97 serum or 50 plasma clinical samples analyzed by a
single LOCI measurement (y) and by an established HPLC
method (x) gave the following equations: for serum,
y = 1.00x - 0.50 (r =
0.98; slope = 1.00; n = 97), as shown in Fig. 1B
; and for
plasma, y = 0.86x + 1.24 (r
= 0.96; slope = 0.90; n = 50). Total Hcy in the majority of
the plasma samples was <20 µmol/L, and mostly in the range of 5–15
µmol/L. Samples with results >100 µmol/L tHcy had to be diluted
and reassayed and were excluded from this study.
In conclusion, we have demonstrated that LOCI is applicable to an antibody-based assay for tHcy quantification. This unique method is simple, rapid, and highly robust and suitable for routine determinations of serum or plasma tHcy concentrations in clinical laboratories.
Acknowledgments
We thank Dr. Frederick Van Lente and Ingrid Raulinaitis at the Cleveland Clinical Foundation, Department of Clinical Pathology, Section of Biochemistry (Cleveland, OH) who provided all of the samples and HPLC analyses for this study. We also thank Drs. A. Dafforn and S. Rose for reviewing this manuscript.
References
The following articles in journals at HighWire Press have cited this article:
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  J. F. Glickman, X. Wu, R. Mercuri, C. Illy, B. R. Bowen, Y. He, and M. Sills A Comparison of ALPHAScreen, TR-FRET, and TRF as Assay Methods for FXR Nuclear Receptors J Biomol Screen, February 1, 2002; 7(1): 3 - 10. [Abstract] [PDF]
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 N. Matsuyama, M. Yamaguchi, M. Toyosato, M. Takayama, and K. Mizuno New Enzymatic Colorimetric Assay for Total Homocysteine Clin. Chem., December 1, 2001; 47(12): 2155 - 2156. [Full Text] [PDF]
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