CLIN.CHEM.38/3, 327-328 (1992)
Multianalyte
Testing
Traditionally,
immu.noassays
are performed
as discrete tests, i.e., one analyte
per assay tube. A longcherished goal of the clinical chemist is multianalyte
testing (dual assays, simultaneous
assays) in which two
or more analytes
are measured
simultaneously
in a
single assay. The advantages
of multianalyte
testing
are work simplification
(fewer assay tubes, fewer pipetting operations,
etc.), an increase in test throughput,
and possible reduction in the overall cost per test. This
type of testing is most attractive for analytes that are
currently
grouped in panels (e.g., thyroid-function
tests,
allergens) and in situations where a simple positive test
result in a group of tests is significant,
as in testing
units of blood for infectious
agents-hepatitis,
cytomegalovirus, and human immunodeficiency
virus (HN).
The latter situation is the least technically demanding,
because there is little need to distinguish which analyte
gave the positive result. Hence, identical labels can be
used, as in a recent simultaneous
assay for antibodies to
human T-cell leukemia virus (HTLV-I) and HIV-1 (1).
Various
assay formats have been devised in an attempt to realize the goal of simultaneous
multianalyte
testing. Finding combinations
of different labels (one
per analyte)
has proved problematical
and has not
progressed
beyond two labels. For example, two radioisotopic labels, 1251 and 1311 (2) or 125j and 57Co (3) can be
combined in a dual analyte assay, as exemplified by the
Simu1TROPIN#{174}assay for lutropin and follitropin (3).
Fluorophore labels provide a broad range of distinguishable fluorescence signals, and combinations
of europium
and terbium
or of europium
and samarium
can be
effectively combined for dual assays (4, 5).
Efforts to combine two or more enzyme labels have
not resulted
in fruitful avenues of research. Enzymes
commonly used as labels have markedly different requirements
for optimum enzyme activity; e.g., the pH
optima for horseradish
peroxidase,
alkaline phosphatase, and /3-galactosidase
are 5-7,8-10,
and 6-8, respectively. Hence, a simultaneous
assay of two or more
enzyme labels inevitably involves a compromise in the
final assay conditions.
Labels of f3-galactosidase
and
phosphodiesterase
have been combined in a dual assay
for phenobarbital
and phenytoin.
The fluorescent
products generated
from a 4-methylumbelliferyl
galactosyl
derivative
and a phosphodiester
derivative
were measured at staggered times with the use of two different
excitation
wavelengths
(6). /3-Galactosidase
and alkaline phosphatase
labels can also be assayed simultaneously by using kinetic assays monitored
at two wavelengths (7, 8).
The most successful
multianalyte
assays have in-
volved use of sophisticated
solid phases to achieve simultaneous
assay. Early strategies were based on colorcoded beads coated with different antibodies (9). More
recently, arrays of individual reaction zones on a single
solid phase have become the favored format. This has
the advantage of using only one label, but does require
a detection
system capable of serial or simultaneous
quantification
of signals associated
with each individual
zone.
In this issue, Kakabakos
et al. (10) present a “sandwich” time-resolved
fluoroimmunoassay
for quantifying
lutropin, follitropin, choriogonadotropin,
and prolactin
in serum. Four antibody-coated
plastic disks are attached to a stick, two disks on each side. Fluorescent
europium chelate label bound to the disks is detected by
laser-excited
time-resolved
fluorometry in two sequential measurements
(side 1, then side 2 of the stick).
Spatially
separated
reactive
areas can also be
achieved by simply dotting, entrapping,
or chemically
immobilizing
different antigens onto a suitable membrane (11-13). This is exemplified by an immunoassay
system based on a nitrocellulose
strip embossed to form
a 5 x 6 array of small (2.5-mm-diameter)
islands.
Different capture proteins are immobilized
on the embossed areas to form a multianalyte
test array (13).
Another strategy is to form an array of discrete reactive
zones, as in the MASTpette’’
device for allergy testing,
which contains as many as 35 threads, each coated with
a different allergen (14).
As Kakabakos
et al. (10) point out, several practical
hurdles stand in the way of routine implementation
of
simultaneous
multianalyte
assays. These include the
possibility of cross-reactions,
leading to false-positive
or
false-negative
results
on multiantibody-coated
solid
phases, and difficulties
in optimizing the assay ranges
for the individual
analytes. Quality control is another
problem. If one analyte in the multianalyte
array fails
quality control, is the analytical
performance
of the
whole device in question? The resulting repeat assays
could undermine any savings in time and money to the
laboratory. Also, as the number of analytes in the array
increases,
so may the chances
of analytical
failure,
leading to a diminishing
return.
References
1. Yamamoto
K, Higashimoto
K, Minagawa
H, Okada M, Kasahara Y. Simultaneous
detection of antibodies to HTLV-I and
H1V-1 by chemiluminescent
enzyme
immunoassay
[Abstract].
Clin Chem 1991;37:1031.
2. Morgan CR. Inimunoassay
of human insulin and growth hormone simultaneously using ‘‘I and 1251 tracers. Proc Soc Exp Biol
Med 1966;123:230-3.
3. Wians FH, Dev J, Powell MM, Heald JI. Evaluation of simulCLINICAL
CHEMISTRY,Vol.38, No.3, 1992 327
taneous measurement of lutropin and follitropin with the SimulTROPIN’ radioimmunoasaay
kit. Clin Chem 1986;32:887-90.
4. Hemmila I, Holttinen S, Petterson K, Lovgren T. Double-label
time-resolved
immunofluorometry
of lutropin and follitropin in
serum. Clin Chem 1987;33:2281-3.
5. Saarma M, Jarvekulg L, Hemmilfi I, Siitari H, Sinijarv R.
Simultaneous
quantification of two plant viruses by double-label
time-resolved
immunofluorometric
assay. J Virol Methods 1989;
23:47-54.
6. Dean LI, Thompson SG, Burd JF, Buckler RT. Simultaneous
of phenytoin and phenobarbital in serum or plasma
by substrate-labeled
fluorescent immunoassay. Clin Chem 1983;
29:1051-6.
7. Blake C, Al Bassam MN, Gould BJ, et al. Simultaneous
enzyme
immunoassay
of two thyroid hormones. Clin Chem 1982;28: 1469determination
73.
8. Bates DL, Bailey WR. Dual enzyme assay. International
Patent
Appl. 1989;W089/06802.
9. Streefkerk JG, Kors N, Boden D. Principle of a reaction for
simultaneous
detection of various antibodies using coloured antigen-coupled agaroae beads. Protides Biol Fluids 1976;24:811-4.
10. Kakabakos SE, Christopoulos
TK, Diamandis EP. Multiana-
328 CLINICALCHEMISTRY,Vol.38, No.3, 1992
lyte immunoassay-based
spatially distinct fluorescent areas quantified by laser-excited solid-phase time-resolved fluorometry. Clin
Chem 1992;38:338-42.
11. Ekins R, Chu F, Biggart E. Fluorescence spectroscopy and its
application to a new generation of high sensitivity, multi-microspot, multianalyte
immunoassay.
Clin Chim Acta 1990;194:91114.
12. Pappas MG. Dot enzyme-linked immunosorbent
assays. In:
Collins WP, ed. Complementary immunoasaays.
Chichester, U.K.:
Wiley, 1988:113-34.
13. Donohue J, Bailey M, Gray R, et al. Enzyme immunoassay
system for panel testing. Clin Chem 1989;35:1874-7.
14. Brown CR, Higgins KW, Frazer K, et al. Simultaneous determination of total IgE and allergen-specific IgE in serum by the
MAST chemiluminescent
assay system. Clin Chem 1985;31:
1500-S.
J. Kricka
Medicine
Larry
Department
University
Philadelphia,
of Pathology and Laboratory
of Pennsylvania
PA 19104