all-in-one immunoassays with time-resolved fluorometry for detection. ..... rate, such as temperature, component concentration, dynamics, and quality of ...
42:8
ClinicalChemistiy 1196-1201
(1996)
One-step all-in-one dry reagent immunoassays with fluorescent europium chelate label and timeresolved fluorometry TIM0
L#{246}vGREN,l,* LIIsA KAJ
MERI#{246},2KATJA BLOMBERG,2
MITRUNEN,1
TOM
PALENIUS,2
all of which
perform
mR.MS:
characteristics of the measurements, at least equivalent to state-of-the-art
chorionic
gonadotropin
.
a-fetoprotein
search
University
of Turku,
Tykistokaru
MAKELA,2
PETTERSSON’
with superior
detection
limits and dynamic
for more
sensitive
detection
technologies.
Factors
such
as
orometry and lanthanide chelate labels originally relied upon the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA#{174})concept [8]. The high specific activity of the label that could be covalently bound to mAbs without any effects on the affinity and specificity [3] proved to be very useful in the design of several noncompetitive assays with improved sensitivity [9-13]. Competitive assays of various design [14-17] were also carried out with good performance characteristics, although the high specific activity of the label does not substantially contribute to the sensitivity in this assay design [2, 7]. Immunoassays performed frequently in the clinical chemistry laboratory have been subject to extensive automation. The analyzers should operate in a fast, random, and continuous access mode. The throughput capacity should be similar to that of general chemistry analyzers, and large on-board supply of reagents should reduce the operator’s hands-on time. Fulfilling the automation requirements has been difficult for many assay
.
Lanthanide chelate labels and time-resolved fluorometry were introduced in the field of immunoassays >10 years ago [1]. The availability of monoclonal antibodies (mAbs) in combination with the high specific activity of the label enabled noncompet-
of Biotechnology,
MIA
detector noise, interference from the sample causing a nonspecific signal, and nonspecific binding of the label reagent reduce the overall assay sensitivity [2, 3]. Other reasons for designing novel immunoassay strategies are to improve the reliability by high-quality key reagents and to simplify the assay by eliminating unnecessary steps to obtain more direct procedures. The immunoassay technology based on time-resolved flu-
progesterone
‘Department
KIM
MAKINEN,2
range to be carried out [2, 3]! Several different label alternatives with high specific activities have since appeared [4-6]. mAbs have to a great extent replaced polyclonal antibodies, and a noncompetitive assay design is preferred whenever possible. The combination of the solid-phase chemistry and the new nonisotopic labels have contributed especially to the present level of automation in the field of immunoassays. Alternative approaches can be selected to improve the sensitivity and reliability of immunoassays [4-7]. As the potential sensitivity of a noncompetitive immunoassay can only be realized by using labels with a high specific activity together with high-affinity antibodies [2, 7], considerable effort is spent on the
assays. The detection limits for hCG, AFP, and progesterone were 0.3 lUlL, 0.1 xglL, and 0.5 nmol/L, respectively. The assay ranges for hCG and AFP were linear to 5000 LU/L and 1200 gzg/L, respectively. The immunoassay format can be readily implemented in a fully automated random-access immunoassay system with optimal performance characteristics and no handling of analyte-specific assay components. INDEXING
and
itive immunoassays
The availability of an intrinsically fluorescent, inert, and stable Eu chelate label made it feasible to design one-step all-in-one immunoassays with time-resolved fluorometry for detection. Roth competitive and noncompetitive immunoassays are performed in microtitration wells containing all assay-specific components in a stable dry form. Only the sample and one assay buffer common for all analytes need to be added. Model assays for human chorionic gonadotropin (hCG), a-fetoprotein (AFP), and progesterone all reached equilibrium in 15 mm or less without compromis-
ing the performance
MAIJA-LIISA
6A, FIN-
20520Turku, Finland.
Wallac Oy, P.O. Box 10, FIN-20101 Turku, Finland. ‘Author for correspondence. Fax 358-21-333 8050; e-mail timo.lovgren@utu. 2
Nonstandard abbreviations: mAb, monoclonal antibody; hCG, human chorionic gonadotropin; AFP, a-feroprotein; BSA,bovine serum albumin;and DTPA, diethylenetriaminepentaacetic acid.
ReceivedMarch 14, 1996;acceptedApril 19,1996.
1196
Clinical Chemistry
technologies, and the performance characteristics of the analytes in the panels have suffered because of unavoidable compromises. In the present work we describe a relatively fast immunoassay technology suitable for automation in which all assays are performed in one-step procedures in microtitration wells containing all the assay-specific reagents in a dry format. An intrinsically fluorescent stable Eu chelate with a high specific activity is used as the label. The performance of the model assays for human chorionic gonadotropin (hCG), a-fetoprotein (AFP), and progesterone is at least state-of-the-art assays.
equivalent
to that
of the
best
AND
INSTRUMENTATION
Special
low-fluorescence Maxisorp microtitration strips made of polystyrene (8 X 12 well format) were purchased from Nunc (Roskilde, Denmark). The 1234 DELFIA Fluorometer, DELFIA Platewash, microtitration plates coated with goat anti-rabbit IgG, DELFIA Enhancement Solution and wash solution, the Eu chelate of 4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6,-bis{[N,N-bis(carboxymethyl)-amino]methyl}pyridine, the Eu-labeled progesterone1 1-glucuronide, the mAbs against hCG and AFP, and the polyclonal anti-progesterone antibody were from Wallac (Turku, Finland). Bovine serum albumin (BSA) was supplied by Intergen (Chicago, IL) and danazol by Sigma Chemical Co. (St Louis, MO). The heterophile blocking reagents HBR-2, MAK33, and mouse IgG were supplied by Scantibodies Lab. (Santee, CA), Boehringer Mannheim (Mannheim, Germany), and Sigma, respectively. The bacteriostatic agent Germall II was obtained from Sutton Labs. (Chatham, NJ). All other chemicals used were of analytical grade. LABELING
OF
OF ALL-IN-ONE
MICROTITRATION
DRY REAGENT
WELLS
bCG. The
capture mAb was immobilized onto microtitration strips through physical adsorption. The wells were coated overnight at 35 #{176}C with 1 jg of the mAb in 100 L of a solution containing 0.2 mol/L NaH2PO4. Coated wells were washed twice with DELFIA wash solution by using DELFIA Platewash, and then saturated for 3 h at room temperature with 300 .L of a solution containing, per liter, 1 g of BSA, 60 g of trehalose, 1 g of Germall II, and 50 mmol of NaH2PO4. After saturation, the wells were aspirated and dried for 2 h at 25 #{176}C. The insulating carbohydrate
layer
was prepared
of bovine gammaglobulin, 50 mmol of Tris-HCI, mg of each of the blockers, i.e., mouse IgG,
by adding
to the coated
pH 7.75, 15 HBR-2, and
MAK3 3, to reduce different heterophile interactions from rum samples. The solution in the well was dried overnight
seat
35 #{176}C. The labeled
on
anti-hCG
mAb (300 ng) was dispensed
layer in 1 .tL of a solution containing, per liter, 5 g of diethylenetriaminepentaacetic acid (DTPA)-treated
top
of the insulating
BSA, 50 g of trehalose,
9 g of NaCl, 0.5 g of NaN3,
and 50 mmol
desiccant.
AFP. The
preparation of the all-in-one dry reagent wells for AFP was carried out identically to the procedure for hCG. Only the composition ent, containing,
of the insulating carbohydrate layer was differper liter, 10 g of casein, 200 g of sorbitol, 9 g of
NaC1, 0.5 g of NaN3, 0.4 g of Tween 20, 2.4 g of bovine gamma globulin, 50 mmol of Tris-HCI, pH 7.75, and 30 mg of each of the blockers (mouse IgG, HBR-2, and MAK33). The volume per well was 10 L.
Progesterone. .tL diluted IgG-coated
The polyclonal 1:50 000) was microtitration
well 20
anti-progesterone antibody (100 preincubated in goat anti-rabbit
wells overnight
at 35 #{176}C in a solution
per liter, 5 g of DTPA-treated BSA, 50 g of 9 g of NaCI, 0.5 g of NaN3, and 50 mmol of pH 7.75. The wells were aspirated and dried for 2 h
containing,
trehalose, Tris-HC1,
at 35 #{176}C. The progesterone
IMMUNOREAGENTS
Labeling with the stable fluorescent Eu chelate was performed overnight at 4#{176}C with a 70-fold (anti-hCG mAb) or 130-fold (anti-AFP mAb) molar excess in a buffer containing 50 mmol/L NaHCO3 (pH 9.3). The labeled antibody was separated from excess free chelate and eventual protein aggregates by gel filtration on a 1.6 x 60cm column of Superdex 200 (Pharmacia, Uppsala, Sweden) by using an elution buffer containing, per liter, 50 mmol of Tris-HC1, 9 g of NaCl, and 0.5 g of NaN3, pH 7.75. The number of Eu chelates covalently bound to each IgG molecule of the pooled antibody fraction was 5-7 and 5-6 for the anti-hCG and anti-AFP mAbs, respectively, when measured against a Eu calibrator. PREPARATION
.tL of a solution containing, per liter, 50 g of BSA, 100 g of sorbitol, 9 g of NaCI, 0.5 g of NaN3, 0.2 g of Tween 20, 1.2 g
of Tris-HC1, pH 7.75. The dispensed solution was dried immediately by blowing air into the well. The all-in-one dry reagent wells were stored at 4 #{176}C in a sealed package with
Matenals and Methods REAGENTS
1197
42, No. 8, 1996
derivative
(0.1 pmol)
the Eu chelate and the blocker danazol the antibody-coated well in a small
labeled
with
(1 p.g) were added onto volume (1 j.iL)of the
solution
used for the anti-progesterone antibody addition. The solution was dried immediately by blowing air into the well. The all-in-one dry reagent wells were stored at 4 #{176}C in dispensed
a sealed
package
with desiccant.
IMMUNOASSAY
PROCEDURES
The immunoassays reagent
otherwise
wells
were performed
according
stated.
A seruin
to
the
sample
in one-step following
or calibrator
all-in-one
procedures
dry if not
(10 jLL for hCG
and AFP,
25 tL for progesterone) was added to each well, followed by assay buffer (50 L for hCG and AFP, 100 L for progesterone) containing, per liter, 50 mmol of HEPES, pH 7.4, 9 g of NaC1, and 0.5 g of NaN3. Incubations were carried out for 15 nun at 36 #{176}C with continuous shaking. Six (hCG, AFP) or four (progesterone) washings in the DELFIA Platewash were carried out with the DELFIA wash solution. To each well, 200 L of the LANFIA solution, pH 10, containing, per liter, 50 mmol of glycine-NaOH, 1.75 mol of NaSCN, 2 mmol of Na2CO3,
50 mL of glycerol,
5 mg of Tween
40, 4.5 mol
of
and 200 mL of 1-propanol, was added. The wells were shaken for 3 mm and the signals were measured with the DELFIA 1234 Fluorometer. DTPA,
Lovgren
1198
et al.: One-step
-
immunoassays
-
Compstltlve
Fig. 1. Principal design of the one-step all-in-one dry reagent noncompetitive and competitive immunoassay formats. Tracer layer contains either the Eu chelate-labeled antibody (noncompetitive assay) or hapten (competitive assay).
Results ONE-STEP
ALL-IN-ONE
DRY REAGENT
IMMUNOASSAY
Concept. The immunoassay format allows both noncompetitive and competitive assays to be carried out (Fig. 1). In the noncompeutive assay format, the analyte-specific capture antibody is immobilized onto the surface of the well. An insulating carbohydrate layer is dried on top of the capture antibody in the bottom of the well. Finally, the labeled antibody is added in a small volume on top of the insulating layer and dried. The competitive assay format, on the other hand, contains a secondary antibody and the analyte-specific antibody immobilized onto the surface of the well. An insulating carbohydrate layer is used, containing
assay-specific
protein
additives
as well
as blocking
agents. The labeled analyte is dried in a small volume on top of the insulating layer. Alternatively, the insulating layer is omitted and the labeled competing analyte is added in a small volume directly on the top of the specific antibody and dried. To start the immunoassay, only the sample (or calibrator) and a common assay buffer need to be added (see Materials and Methods). Specific activity of the label. The detection fluorescent
Eu
chelate
of
limit of the intrinsically
4-[2-(4-isothiocyanatophenyl)ethy-
nyl]-2,6,-bis{[N,N-bis(carboxymethyl)amino]methyl}pyridine was measured. In contrast to the DELFIA enhancement principle for the measurement of Eu, the lanthanide is all the time bound to the original chelate and the excitation energy is efficiently absorbed by the energy-mediating moiety of the molecule. The detection limit (background +2SD) for the fluorometer was 5 X 10 14 mol/L when the fluorescence from the Eu chelate was measured, which corresponds to the detection limit of Eu in the DELFIA enhancement solution [8].
Table 1. Performance
and stability
of the one-step
No layer Label
Storage 2 weeks Background8 10 000 lU/L signal8 Signal/background
Component optimization. The effect of the composition of the insulating layer in the dry reagent well on the performance of the immunoassay was investigated. The influence of several additives such as carbohydrates, proteins, and blocker components was tested by using different amounts, volumes, and drying temperatures. In a noncompetitive immunoassay, the labeled component must be dispensed on top of the insulating layer in a small volume and dried immediately. In a competitive immunoassay, the labeled component can alternatively be added directly on top of the specific antibody. The performance of the all-in-one dry reagent concept was always compared with an immunoassay with the corresponding components in solution. Table 1 shows a typical result obtained for the noncompeutive immunoassay of hCG when the performance as well as the stability of the all-in-one dry reagent format were tested with and without an insulating layer containing carbohydrate or protein or both. An insulating layer made of protein and (layer 1 in Table 1) provided the lowest background and highest signal-to-noise ratio when compared with the other alternatives. With a proper insulating layer, an assay performance at least equivalent to the one obtained with all assay components in solution was achieved. Somewhat different performance of the specific assays was obtained depending on the carbohydrate and protein used in the layer. carbohydrate
bCG. The
aim of the hCG determination was a fast one-step equilibrium. It should be sensitive, have a wide dynamic range, and the high-dose hook effect should not interfere with the upper working range of the assay. The requirements were taken into consideration when the assay conditions were optimized and the all-in-one dry reagents were assay that reaches
all-In-one Layer I
dry reagent
noncompetltive Layer 2
immunoassay
of hCG. Layer 3
Uquld
4 ‘C 100
100 2087
4 ‘C 25 ‘C 35 ‘C 4 ‘C 25 ‘C 35 ‘C 4 ‘C 25 ‘C 35 ‘C 4 ‘C 25 ‘C 35 ‘C 95 119 122 78 88 99 107 154 260 126 177 225 99 101 88 106 105 90 103 105 96 98 104 97 2176 1777 1509 2815 2499 1895 2106 1076 811 1693 1289 941
The dry reagentwells were preparedwith no insulating layer (no layer) or the layer contained In additionto 50 mmol/L Tris-HCIbuffer, pH 7.8.9 g/L NaCI,and 0.5 g/L NaN3 either 200 g/L sorbitol and50 g/L BSA(layer1), 200 g/L sorbitol (layer2), or 50 g/L BSA(layer3). The dry reagentwells weretested after storagefor 2 weeks at 4 ‘C, 25 ‘C, and 35 ‘C. In the referenceassay (liquid)the componentswere stored separatelyfor 2 weeks at 4 ‘C. The backgroundand the 10 000 lU/L signal for the referenceassay was 1530 counts (100%)and 3 189 000 counts (100%). respectively. #{149} %of referenceassay (liquid) results.
Clinical Chemistry
42, No. 8, 1996
1199
20%
Table 2. Within- and between-assay Imprecision and total variation of hCG, AFP, and progesterone assays.
iOO%
CV, % No. of
80% 0
E E
Within
Between
Mean
run
run
5
30
3.9
7.4
8.3
5
773
1.4
2.7
3.1
2.0
2.7
3.4
runs
Analyte
8)
Total
hCG, lU/L
80%
8)
1 2 3
/,
0
/ /
-
20%
/
5
AFP, p.g/L :1.
0% 0
to
5
is Time (mm)
25
20
35
30
Fig. 2. Kinetics of the optimized one-step all-in-one dry reagent noncompetitive immunoassay of hCG with two calibration concentrations, 1000 (#{149}) and 10 000 (#{149}) lU/L, and two serum samples (A, U). prepared. Particularly the factors affecting the reaction rate, such as temperature, component concentration, dynamics, and quality of antibodies, were investigated [18]. With an incubation temperature of 36 #{176}C, a total volume of 60 jL, a sample volume of 10 L, and 300 ng of the labeled mAb in a predried format in the well, the reaction proceeded to equilibrium in 15 mm (Fig. 2). About 80% of the maximum signal was reached in 5 mm. Under these assay conditions the lowest detection limit, when defined as the background signal + 2SD, was 0.3 lU/L. The calibration curve was linear up to 5000 lU/L and the signal level at 1.2 x l0 lU/L was still above the linear range of the calibrator (Fig. 3). The reproducibility of the assay calculated by analysis of variance [19] is given in Table 2. The recoveries were in the range 91-102% with a mean value of 96.3%. The one-step all-in-one dry chemistry hCG assay correlated well with the commercially available DELFIA hCG assay (y =
1.208x
1.10, r
-
=
0.995,
n
=
55).
AFP. Performance requirements similar to those of the hCG determination were set for the AFP assay. The reaction rate was improved by the use of high-quality mAb and especially by the increased amount of labeled antibody that did not affect the level of nonspecific binding in the assay. The AFP assay reached IE*08
5073
2 3 Progesterone, nmol/L 1 2 3
4 4 4
1.8 34.5 68.1
2.2 1.7 3.5
6.3 4.8 4.4
6.7 5.1 5.6
3 3 3
3.0 11.7 56.9
7.6 4.4 4.7
15.2 1.0 1.8
17.0 4.5 5.0
The runs were performed on separate days. Replicates per run: HCG, 4: AFP, 2; progesterone,
12.
equilibrium in
