Clinical Chemistry 43: 71-75, 1997;
(Clinical Chemistry. 1997;43:71-75.)
© 1997 American Association for Clinical Chemistry, Inc.
Two-site immunofluorometric assay of intact salmon calcitonin with improved sensitivity
Haiqin Rong,
Leonard J Deftos1,
Hong Ji and
Elisabet Buchta
Department of Molecular Medicine, Endocrine and Diabetes Unit, Karolinska Hospital and Institute, S-171 76 Stockholm, Sweden.
1
Department of Medicine, University of California, San
Diego and the San Diego VA Medical Center, La Jolla, CA 91261.
a Address correspondence to this author at: Department of Molecular Medicine, The Endocrine and Diabetes Unit, Karolinska Hospital, L1:02, S-171 76 Stockholm, Sweden. Fax 46-8-30 34 58; email bucht{at}enk.ks.se
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Abstract
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We recently developed a two-site immunofluorometric assay (IFMA) of
salmon calcitonin (SCT) by DELFIA (dissociation enhancement lanthanide
fluoroimmunoassay) technique using the same polyclonal antibodies both
for "catching" the antigen and for signaling. In the present study
we used a monoclonal antibody to SCT 1–11 as the capture antibody.
This antibody was biotinylated before use in streptavidin-coated
microtitration plates. The polyclonal antibody labeled with Eu chelate
was used as a signaling marker. This combination of antibodies resulted
in an assay that was three to four times more sensitive than the
previous IFMA, with a detection limit of 0.3 pmol/L serum. Intact SCT
1–32 was detected by the assay (recoveries 94–96%), but not the
fragments SCT 1–11 and SCT 10–32 or human calcitonin. Dilutions of
plasma samples containing SCT were parallel to the calibration curve of
SCT 1–32. Pharmacokinetic studies of SCT, 100 IU administered
intramuscularly to 10 men, indicated peak serum concentrations of
32–128 pmol/L within 10–20 min with apparent half-life of 56 ±
18 min (mean ± SD). This new assay will allow study of the
pharmacokinetics of new calcitonin preparations that do not require
injection.
Key Words: indexing terms: fluorometry • lanthanide
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Introduction
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Synthetic salmon calcitonin (SCT), a peptide hormone of 32 amino
acids, is used in the treatment of diseases with increased bone
resorption (1)(2)(3) and hypercalcemia of different origins
(4)(5).1
Many laboratories including ours have developed immunoassays in various
formats for SCT (6)(7)(8). We have recently developed a
two-site immunofluorometric assay (IFMA) with DELFIA (dissociation and
enhancement lanthanide fluoroimmunoassay) technique (8).
In the present study, we have improved the sensitivity of this assay by
introducing a biotinylated monoclonal antibody against SCT fragment
1–11 (7) immobilized on streptavidin-coated plates as
"catching" antibody. For signaling, a Eu-labeled affinity-purified
polyclonal antibody was used as in the former study (8).
The Eu chelates are ideal alternatives to radioactive labels because of
their long half-life and because they can be measured with high
sensitivity by time-resolved fluorometry (9)(10)(11).
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Materials and Methods
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antibody preparation
A polyclonal antibody (309) raised in rabbit against synthetic
SCT coupled to ovalbumin was used as a signaling antibody
(8). Before labeling with the Eu chelate the antibody was
affinity-purified by means of SCT 1–32 covalently coupled to
CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) and dialyzed
against labeling buffer (50 mmol/L NaHCO3, pH 8.5,
containing 9 g/L NaCl). Finally the dialyzed antibody solution was
concentrated by use of Centriflo membrane cones CF 50A (Amicon
Scientific Systems Division, Danvers, MA) to ~1 mg in 500 µL of
labeling buffer for Eu labeling.
Three monoclonal antibodies were raised against SCT peptides, 5G9 and
8A4 against SCT 1–11 and 3G4 against SCT 10–32 (7). They
were purified from ascites by Staphylococcus protein A (SPA)
chromatography before biotinylation and (or) Eu labeling.
antibody labeling
The affinity-purified polyclonal antibody (309) was labeled
with a Eu chelate as follows: 1 mg of the antibodies was incubated at
room temperature overnight with 0.2 mg of Eu-labeling reagent (Wallac,
Turku, Finland) at a final volume of 0.5 mL of labeling buffer. The
Eu-labeled antibodies were purified from excess Eu-labeling reagent and
aggregated antibodies on a Sepharose 6B column with Sephadex G50 on the
top as earlier described (8). The labeled antibody was
subaliquoted and kept frozen at -80 °C. Before use, diluted labeled
antibody solution was passed through a 0.2-µm filter unit
Minisart® N (Sartorius, Göttingen, Germany) to
exclude any aggregated antibodies. The monoclonal antibody (3G4) was
also labeled in the same way.
antibody biotinylation
One mg of antibody in PBS was reacted with biotin-7-NHS
(D-biotinoyl-
-aminocaproic
acid-N-hydroxysuccinimide ester) for 2 h at room
temperature by using a biotin labeling kit (Boehringer Mannheim
Biochemica, Mannheim, Germany). The amount of biotin-7-NHS added was 30
µg for the monoclonal antibodies 5G9, 8A4, and 3G4 and 150 µg for
the polyclonal 309; this gave a molar ratio of biotin-7-NHS to
antibody of 10 for the monoclonal and 50 for the polyclonal. Nonbound
biotin-7-NHS was removed by gel filtration. The biotinylated antibodies
were divided into 100-µg portions in PBS and lyophilized. Before use,
the antibodies were dissolved in 100 µL of water and diluted 1:1000
in assay buffer (50 mmol of Tris-HCl, 0.15 mmol of NaCl, 0.5 g of
NaN3, 5 g of bovine serum albumin, 10 mg of
diethylenetriaminepentaacetic acid, and 0.1 mL of Tween 40 per liter).
ifma
Biotinylated antibody solution (1 mg/L) was incubated in
streptavidin-coated microtitration plates (Wallac) (200 ng/well)
overnight at 4 °C. The plate was washed three times with 0.3 mL of
washing solution [25-fold dilution of 225 g/L NaCl, 125 mmol/L
Tris-HCl, 25 g/L Germall II (Sutton Labs., Chatham, NJ), 1.25 g/L Tween
20, pH 7.75]. Calibrator (synthetic SCT 1–32) (Sandoz, E. Hanover,
NJ) or samples (0.2 mL) were incubated at 4 °C for 24 h. For
dilution studies, plasma samples were diluted with plasma without SCT
1:1, 1:2, and 1:4. After the plate was washed three times, 0.2 mL of
the Eu-labeled antibodies was added. After shaking for 2 h at room
temperature, the plate was washed six times and 0.2 mL of enhancement
solution (Wallac) was added; and after another 5 min of shaking, the
fluorescence was measured with a DELFIA 1234 plate fluorometer
(Wallac).
subjects and sampling
By use of the IFMA we have studied the pharmacokinetics of SCT in
10 healthy men, ages 19–75, within 15% of ideal body weight for
height and body frame (Metropolitan Insurance Co.). After an overnight
fast, they were given 100 IU of SCT (Miacalcin®,
Sandoz) intramuscularly. Blood samples were collected at the indicated
time points and the serum kept frozen for SCT assay. The protocol was
approved by the Institutional Review Board of the University of
California, San Diego. The individual elimination half-life, i.e., the
time when the serum concentration of SCT attains half of the peak
values after injection, was determined by their respective
concentration–time curves.
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Results
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tests of antibody combinations
Biotinylated antibodies 309, 8A4, 5G9, and 3G4 (B-309, B-8A4,
B-5G9, and B-3G4) and Eu-labeled antibodies 309 and 3G4 (Eu-309 and
Eu-3G4) were respectively tested to determine the best combination
(Fig. 1
). The combination B-5G9/Eu-309 resulted in the best
sensitivity, and the background was low. The fluorescence responses of
various SCT concentrations in the calibration curves assayed by
B-309/Eu-309 and B-8A4/Eu-309 increased gradually but not linearly.
No significant responses were found in the assays with B-309/Eu-3G4,
B-5G9/Eu-3G4, and B-3G4/Eu-309. In addition, the assays with Eu-3G4 as
a signal had a high background.
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Figure 1. IFMA of SCT with various combinations of biotinylated
(B-309#, B-5G9, B-3G4, and B-8A4) and Eu-labeled (Eu-3G4 and Eu-309#)
antibodies.
cps, counts per second.
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effects of coating conditions
The incubation time, temperature after addition of biotinylated
antibody solution, and concentration of biotinylated antibodies were
compared (Fig. 2
). The coating periods of 2, 15, and 72 h had no obvious
effect on the calibration curves. Overnight incubation at 4 °C,
25 °C, and 37 °C showed different fluorescence responses, but the
sensitivity was not affected. Two hours of incubation at 4 °C and
25 °C resulted in similar curves. At higher concentrations of SCT,
the wells coated with 400 ng of biotinylated antibody showed a slightly
higher fluorescence response than those coated with 200 ng of antibody.
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Figure 2. Effect on SCT IFMA of different coating conditions of the
biotinylated monoclonal antibody 5G9 when detected with the Eu-labeled
309# antibody.
In all conditions except of panel C ( ),
the concentration of the biotinylated antibodies was 200 ng/well.
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effects of incubation time
Incubation of SCT with the immobilized antibodies for 24 and
72 h at 4 °C is demonstrated in Fig. 3
. The prolonged incubation resulted in lower fluorescence
responses, and the sensitivity was not improved.
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Figure 3. IFMA of SCT with incubation of 4 °C for 24 h (•)
and 72 h ().
The combination of B-5G9 and Eu-309# was used.
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b-5g9/eu-309 assay validation
Polyclonal antibody 309 and monoclonal antibody 5G9 gave the best
assay results. The sensitivity and specificity of the assay when using
the polyclonal antibody 309 for signaling and the monoclonal antibody
5G9 for catching are demonstrated in Fig. 4
. The fluorescence responses were linear to various SCT
concentrations. The dilutions of human serum samples supplemented with
SCT were parallel to the calibration curve. There was no cross-reaction
with the synthetic SCT fragments 1–11 and 10–32 or with human
synthetic calcitonin (Peninsula, Belmont, CA) up to a concentration of
9.4 nmol/L. The detection limit based on 2 SD above the background was
0.15 pmol/L in assay buffer and 0.3 pmol/L in serum. The recovery of
SCT added to serum was 93.9 ± 5.6% (n = 4), 94.2 ±
7.3% (n = 12), and 95.5 ± 10% (n = 8) at 2.9, 5.9,
and 23.5 pmol/L, respectively. The intraassay variations were 7.8%,
4.9%, 2.6%, and 1.0% (n = 12) for SCT concentrations of 0.6,
1.2, 2.4, and 4.7 pmol/L, respectively. The interassay variations were
5.9% (n = 4), 6.9% (n = 8), and 3.7% (n = 8) for 2.9,
5.9, and 23.5 pmol/L, respectively. The binding capacity of the plate
when coated with 200 ng of biotinylated antibody was up to 300 pmol/L.
The calibration curve is linear until at least 300 pmol/L (data not
shown).
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Figure 4. Calibration curve of SCT 1–32 (•) with biotinylated 5G9
and Eu-labeled 309#.
Synthetic human calcitonin (— — —) and salmon calcitonin fragments
1–11 (——-) and 10–32 (— - — - —) and dilutions of plasma samples
( , ) supplemented with SCT at 1:1, 1:2, and 1:4 in plasma without
SCT.
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pharmacokinetic studies
Figure 5
shows the individual pharmacokinetic profiles of serum SCT
after intramuscular administration of 100 IU in 10 healthy men. In most
of the subjects, serum SCT attained peak concentrations at 10 min and
in some at 20 min. The range of peak values was 32–128 pmol/L. At ~1
h, the serum SCT concentration declined to half of the peak values. The
mean (± SD) elimination half-life was 56 ± 18 min (range 34–85
min). By 4 h, the serum SCT concentrations were 1.4 ± 0.9
pmol/L (mean ± SD, range 0.3–3.1 pmol/L) in seven subjects and
the remaining three subjects had undetectable concentrations (<0.3
pmol/L).
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Figure 5. Pharmacokinetic profile of serum SCT in 10 healthy men who
received 100 IU of the drug intramuscularly.
The control samples drawn before administration of salmon calcitonin
are indicated as C.
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Discussion
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Immunoassays for SCT were first developed to study the physiology
of the hormone in fish (6)(12). When SCT
became an injectable drug (13), immunoassays were used to
study its pharmacokinetics. The development of transmucosal
preparations of SCT such as the nasal form has required assays of
improved specificity and sensitivity for pharmacokinetic studies, since
lower serum concentrations are achieved (14). To improve
assay sensitivity, we have modified the two-site IFMA by use of a
SPA-purified monoclonal antibody for solid immobilization and an
affinity-purified polyclonal antibody for signaling. In addition, for
antibody immobilization, biotin conjugation technique and
streptavidin-coated microtitration plates have been used. Compared with
a previously developed IFMA (8) in which only one
affinity-purified polyclonal antibody was used both for catching and
for signaling and the antibody was immobilized by physical adsorption
on the plate, the sensitivity of the current assay has been increased
3–4 times. The assay detects only intact SCT 1–32. However, when a
monoclonal antibody for SCT 10–32 was substituted for the monoclonal
antibody to SCT 1–11, there was no reactivity of SCT 1–32, or any of
the other SCT peptides. This result indicates that the polyclonal
antibody recognizes primarily the carboxyl terminus of the peptide.
It was not possible to use SPA-purified antibodies for Eu labeling,
because that resulted in an extremely high background fluorescence.
However, for biotinylation, SPA purification was sufficient. On the
basis of tests of various coating conditions, we chose the
concentration of 200 ng of biotinylated antibody per well, which had
sufficient binding capacity and could be incubated either overnight at
4 °C or 2 h at room temperature to produce similar assay
results.
Our studies (Fig. 5
) demonstrate that the assay has clinical
applications. We can readily monitor the serum concentrations of SCT in
patients who have received the drug. Since the assay is linear over the
wide range of 0.3 to 300 pmol/L, high and low concentrations can be
measured in the same assay and without the need for sample dilution.
Both of these assay characteristics improve its accuracy.
In earlier pharmacokinetic studies, synthetic SCT was administered
subcutaneously to healthy volunteers (15) and patients
(16) and measured with RIA. The mean elimination
half-lives were 87 and 88.2 min respectively and the peak-reaching time
was ~1 h, which was similar to our previous study (8) in
which SCT was also administered subcutaneously. However, when
administered intramuscularly in the present study, the peak was reached
within 10–20 min and the elimination half-life was diminished to 56
min, which was similar to the data of Beveridge et al., who injected
SCT intramuscularly to patients and measured by RIA (16).
Compared with our previous RIAs (6), IRMAs
(7), and IFMAs (8), this two-site immunoassay
has several advantages. It avoids the use of radioactivity and produces
results in hours rather than days. More importantly, the assay can be
used for measuring low concentrations of intact SCT. Thus, this new
assay should be useful for studying the pharmacokinetics of the
relatively low concentration of serum SCT that occurs with new,
noninjectable preparations of the drug.
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Acknowledgments
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The study was supported by grants from the Swedish Cancer Society
(3192), the Swedish Medical Research Council (5992), the Funds of
the Karolinska Institute, the Memory Foundation of Clas Groschinsky,
the Foundation of "Syskonen" Svensson and Åke Wiberg, the National
Institutes of Health, and the Department of Veterans Affairs.
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Footnotes
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1 Nonstandard abbreviations: SCT, salmon calcitonin;
DELFIA, dissociation and enhancement lanthanide fluoroimmunoassay;
IFMA, immunofluorometric assay; SPA, Staphylococcusprotein A; and NHS, N-hydroxysuccinimide. 
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Abstract
Introduction
