Development of a Digoxigenin-labeled Peptide: Application to ...Document Transcript
F E A T U R E S
Development of a Digoxigenin-labeled
Peptide: Application to a
Chemiluminoenzyme Immunoassay of
Bradykinin in Inflamed Tissues
ANICK DÉCARIE1, GUY DRAPEAU2, JEAN CLOSSET3, RÉJEAN COUTURE1, AND ALBERT ADAM4
Département de Physiologie, Faculté de Médecine, 4Faculté de Pharmacie, Université de Montréal,
Montréal, Québec, Canada, H3C 3J7
Centre de Recherche, Hôtel-Dieu de Québec, Québec, Canada G1R 2J6
Laboratoire d’endocrinologie, CHU, Université de Liège, 4000 Liège, Belgique
Reprints requests should be sent to:
Dr. Albert Adam, Faculté de Pharmacie, Université de Montréal, C.P. 6128, Succursale A, Montréal,
Québec, Canada, H3C 3J7. Email: email@example.com
Editor’s note: This is a modified version of an article originally published in Peptides. Reprinted by
permission of the publisher from “Development of digoxigenin-labeled peptide: Application to chemilu-
minescence enzyme immunoassay of bradykinin in inflamed tissue.” Anick Décarie, Guy Drapeau,
Jean Closset, Réjean Couture, and Albert Adam; Peptides 15(3):511– 518.. Copyright 1994 by Elsevier
Science Inc. This Biochemica article focuses primarily on the generation and application of digoxigenin-
labeled peptides. For more-detailed descriptions of the biological effect of bradykinin in inflamed tissue
and the influence of kininase inhibitors, please refer to the original Peptides article (1).
We have developed an ultrasensitive Introduction
chemiluminoenzyme immunoassay Immunological techniques have partially satisfied the increasing need for specific,
(CLEIA) using digoxigenin-labeled sensitive methods for detecting and quantifying biologically active peptides in body
bradykinin as a tracer for the quanti- fluids and tissues. For example, measurement of peptide levels in blood has been
fication of kinins in tissue samples. greatly simplified by radioimmunoassays (RIA) and enzyme immunoassays (EIA) for
Rabbit polyclonal IgGs directed against peptides. While the sensitivity level of these peptide immunoassays has been some-
the C-terminal end of bradykinin were what increased by using avidin-biotin technology for two-site competitive EIA (2–6),
used for the immunoconcentration step we propose an alternative for competitive EIA of peptides – one that employs digoxi-
along with a dioxetane derivative genin-labeled peptides as a tracer. Although digoxigenin (DIG) has been broadly used
alkaline phosphatase substrate for the to label proteins and cDNA probes (7), here we apply it to the labeling of a tracer pep-
revelation step. This sensitive assay tide for the first time.
could detect bradykinin levels as low Bradykinin (BK) is one of a group of powerful proinflammatory autacoids and is
as 0.1 fmol/ml with ED50 of 0.78 pmol/ml believed to play a role in acute and chronic inflammatory processes. Previous methods
in extracts of normal and carrageenan- of quantifying bradykinin in biological samples have been hindered by the enzymatic
inflamed tissues. lability and the low concentration of BK in tissues and body fluids (8). The digoxi-
genin-labeled bradykinin tracer has been purified, characterized, and used for the
development of a heterogeneous competitive EIA using polyclonal anti-BK antibodies
for the immunoconcentration step and alkaline phosphatase-conjugated anti-DIG Fab
fragments as an intermediate reaction. Immune complexes are then detected using
dioxetane derivatives as substrates for alkaline phosphatase. Previously, these chemilu-
minescent substrates had been almost exclusively applied to molecular biology (9) and
sandwich EIA methods (10). After analytical validation, this method has been applied
to extracts of normal and carrageenan-inflamed tissues.
20 BIOCHEMICA s NO. 4 
F E A T U R E S
Materials and Methods with a Vydac™ C18 15–20 µm resin pre- the BK standard or 50 µl diluted biologi-
Preparation and purification equilibrated with 0.05% trifluoroacetic cal sample and 50 µl DIG-BK. The plates
of antibodies acid (TFA)/water. The incubation mix- were incubated for 16 h at 4°C under
Antibodies against BK were produced ture was separated using a gradient of agitation to allow the immunological
in albinos rabbits (Charles River, St. 5–70% acetonitrile/water containing reaction to occur. After removal of
Constant, Québec, Canada) immunized 0.05% TFA; a total volume of 600 ml elu- unbound material by a careful wash
with BK that had been covalently linked ent was pumped through the column at a cycle, the bound DIG-BK was reacted
to bovine serum albumin by the glutar- rate of 6.5 ml/min and collected in with alkaline phosphatase-conjugated
aldehyde method (11). After four boost- 6–8 ml fractions. Fractions containing anti-DIG Fab fragments for 2 h at 37°C.
ings, we obtained antiserum exhibiting a the pure product (DIG-BK) were pooled Finally, 100 µl of a chemiluminescent
titer of 1/150,000 when assayed in RIA and lyophilized. Purity of the final prod- alkaline phosphatase substrate was in-
(30% binding). For the EIA method, IgGs uct was evaluated by analytical HPLC, cubated in the plate wells for 30 min at
were purified by affinity chromatography and its identity was confirmed by FAB 37°C to measure alkaline phosphatase
for protein G. The concentration of the mass spectrometry. activity and reveal the presence of
purified material was measured by its Separations were achieved with a immune complexes. The intensity of
absorption at 278 nm. The purity of this Vydac 10 µm (3.9 X 300 mm) reverse light emission was measured at 540 nm
material was verified using the A278/252 phase C18 column using a linear gradient on a luminometer, and the results were
nm ratio (12). of 5–65% acetonitrile/0.05% TFA/water expressed in Relative Light Units (RLU).
at 2 ml/min over a period of 20 min. The
Preparation of the DIG-labeled absorbance was measured at 214 nm.
Five milligrams of Digoxigenin-3-0- Immunoreactivity of DIG-BK
methylcarbonyl-ε-aminocaproic acid-N- Immunoreactivity of DIG-labeled BK
hydroxysuccinimide ester (DIG-NHS was tested by radioimmunoassays using
ester) was added to 16 mg bradykinin successive dilutions of this synthesized
dissolved in 5 ml of DMF (pH adjusted to material. The incubation medium (0.5 ml)
9.0 with N-methylmorpholine). After contained standard BK (500 pmol–
adding 1.5 mg 1-hydroxybenzotriazole 5 fmol /ml) or DIG-BK dilutions (100 µl),
to the mixture, the reaction was moni- plus 100 µl [3H]-BK (10,000 dpm), Figure 2 Schematic structure of DIG-labeled BK.
tored by analytical HPLC as described 100 µl antiserum (final dilution,
below. After 5 h, the reaction mixture 1/150,000), and 200 µl assay buffer Application of DIG-BK in
was loaded on a preparative Michel- (200 mM Tris-HCl, pH 7.4, containing an animal model
Miller column (22 X 130 mm) packed 2 g/l lysozyme). After an overnight in- The preparation of an animal model
cubation at 4°C, the separation of bound of acute inflammation, tissue sampling
from free fraction was achieved by ad- and processing, immunoreactivity pro-
ding 500 µl of a charcoal mixture (1 g/l files of the inflamed tissue extract, and
charcoal, 0.1 g/l dextran T70 in water). statistical data analysis are beyond the
The tubes were then vortexed, allowed to scope of this Biochemica article and were
stand for 15 min at 10°C, and centri- performed as described in the original
fuged at 3,000 rpm for 15 min at 4°C. Peptides paper (1).
The radioactivity of the supernatant was
then counted in a beta counter. Results
Characterization of the DIG-BK
phosphatase substrate CLEIA of BK Mass spectrometry of DIG-BK corrob-
Figure 1 summarizes the different orated the calculated molecular weight
steps of this enzyme immunoassay. (1603.4 g). The DIG-labeled BK corre-
Unless otherwise indicated, all washing, sponds to the structure represented in
dilution, and incubation steps were per- Figure 2. The immunoreactivity of the
formed in the same buffer: 50 mM Tris- DIG-BK derivative was tested by radio-
HCl (pH 7.4) containing 100 mM NaCl immunoassay using successive dilutions
Figure 1 Schematic representation of the dif-
and 0.5 ml/l Tween®20. (from 100 to 0.004 pmol/ml) of this
ferent steps of chemiluminescent enzyme
immunoassay: 1-Addition of BK ( ) and BK-DIG to Microtiter plates (96-well) were coat- derivative (Figure 3). The calibration
wells coated with polyclonal anti-BK IgG; ed with 100 µl polyclonal anti-BK IgG, curve for BK and the displacement curve
2-Competition between BK and BK-DIG for polyclon- diluted in 100 µM carbonate coating obtained with DIG-BK display ED50s of
al anti-BK IgG (–〈); 3-Bound DIG-BK is let to react buffer (pH 9.5), for 24 h at 4°C. The 1.60 and 1.67 pmol/ml, respectively.
with anti-DIG-labeled Fab fragments coupled with plates were washed and saturated with Moreover, the slopes of these curves were
alkaline phosphatase (ALP); 4-Immune complexes incubation buffer containing 5 g/l gelatin not statistically different from each other
are revealed by measuring the ALP activity with a
for 2 h at 37°C. After another washing (F[1,28] = 2.95, p>0.05) with values of
chemiluminescent alkaline phosphatase substrate.
Intensity of light emission is measured at 540 nm. step, the plates were incubated with 1.01 and 0.93, respectively.
100 µl of a mixture containing 50 µl of
BIOCHEMICA s NO. 4  21
F E A T U R E S
original Peptides article (1). To briefly
7 summarize the results detailed there, this
sensitive chemiluminoenzyme immuno-
assay was capable of detecting a 7-fold
CPM (x 1000) 5 increase in immunoreactive kinins in
carrageenan-inflamed tissues. It also per-
mitted the detection of significantly
3 enhanced kinin tissue content when a mix-
ture of inhibitors of kininase I (mergepta)
and kininase II (captopril) was coinjected
1 with carrageenan. Thus, this assay provides
biochemical evidence that kinins may act as
0 pro-inflammatory mediators, and it high-
10–3 10–2 10–1 1.0 101 102 103
LOG concentration (pmol/mL) lights a compensatory increase of kininase I
and II activities in inflamed tissues.
Figure 3 Calibration curve of BK ( ) and curve displacement obtained with immunoreactive DIG-BK
( ) measured by radioimmunoassay.
This article describes the use of DIG-
labeled peptides as tracers for com-
petitive immunoassays. Here we used
600 DIG-labeled bradykinin to apply this
new analytical approach to the measure-
Light emission (RLU)
500 ment of bradykinin in tissue. Because
digoxigenin occurs exclusively in Digi-
talis plants, this bioanalytical indicator
300 system prevents non-specific reactions
with endogenous substances found in
200 animal materials (7). This property is
particularly advantageous when com-
pared with biotin, which is present in
0 various mammalian tissues (16, 17).
10–4 10–3 10–2 10–1 1.0 101 102 Different digoxigenin derivatives are
LOG concentration (pmol/mL) commercially available for the labeling of
Figure 4 Calibration curve of BK ( ) and competition curve with serial dilutions (1:1 to 1:4096) of an proteins and cDNA. Depending on the
inflamed paw extract ( ) measured by chemiluminoenzyme immunoassay. The light emission of the zero
mean concentration (B0) is represented by (v).
nature of the probe to be identified or
quantified, either hydrocarbon or amino-
terminal groups of proteins can be used
as coupling residues. In our example, the
Analytical characteristics of CLEIA detectable concentration of BK, calcu- DIG-NHS ester was bound specifically to
for BK lated as the light emission of the zero the amino-terminal arginine. After purifi-
Figure 4 shows a typical calibration mean concentration (597 RLU) minus 2 cation of the tracer, mass spectrometry
curve obtained in the conditions opti- standard deviations (0.48 RLU), was confirmed the specificity of this cou-
mized by coating IgG at a concentration 0.1 fmol/ml (15). pling. The specificity of this DIG-BK
of 2.5 µg/ml and incubating a fixed Intra- and inter-assay precision was binding was also apparent from its effec-
amount (0.61 pmol/ml) of DIG-labeled assessed on blank (non-inflamed) tissue tiveness in RIA, in which the DIG-labeled
BK with increasing concentrations of BK extract spiked with BK at concentrations BK exhibited the same immunoreactive
ranging from 0.023 to 23.8 pmol/ml. The of 6.0, 1.5, and 0.35 pmol/ml in tripli- profile as the native BK. Once character-
calibration curve is characterized by an cate. The samples were analyzed on five ized, the tracer can be stored at –20°C for
ED50 of 0.78 pmol/ml. The minimal different plates. The intra- and inter-assay as long as two years without losing its
coefficients of variation are provided in properties. This high stability, coupled
Bradykinin Intra-assay Inter-assay Table 1. with the DIG-labeled tracer’s well-
concentration coefficient of coefficient of defined activity, provides obvious advan-
variation (%) variation (%) Tissue content and biological effect of tages over radioactive and other non-
6.0 pmol/ml 2.2 4.1 BK and influence of kininase inhibitors radioactive tracers.
1.5 pmol/ml 3.3 5.0
As this Biochemica article focuses solely Various immunological methods, in-
on the development of the sensitive cluding RIA, have been used to quantify
0.35 pmol/ml 4.0 7.4
chemiluminoenzyme immunoassay using BK in biological samples (18–20). BK has
Table 1. Intra- and inter-assay precision of CLEIA DIG-labeled bradykinin, more in-depth also been detected by EIA (13, 21, 22),
with DIG-labeled bradykinin. investigations into the role of BK and and one of these methods used biotin-
kininase inhibitors can be found in the labeled BK as a tracer in a non-competi-
22 BIOCHEMICA s NO. 4 
F E A T U R E S
tive approach (13). Compared to these 7. Kessler, C. (1991) The digoxigenin:anti-digoxigenin 23. Barabé, J., Huberdeau, D. and Bernoussi, A. (1988)
assays, the new CLEIA demonstrates a (DIG) technology – a survey on the concept and real- Influence of sodium balance on urinary excretion of
higher sensitivity. In fact, our assay ization of a novel bioanalytical indicator system. immunoreactive kinins. Am. J. Physiol. 254:F484–F491.
Molecular and Cellular Probes 5:161–205. 24. Scicli, A. G., Mindroiu, T., Scicli, G. and Carretero, O.
allows us to detect amounts as low as 8. Bhoola, K. D., Figueroa, C. D. and Worthy, K. (1992) A. (1982) Blood kinins, their concentration in normal
0.1 fmol/ml of immunoreactive BK. Bioregulation of kinins: kallikreins, kininogens, and subjects and in patients with congenital deficiency
Earlier studies show considerable varia- kininases. Pharmacol. Rev. 44:1–80. in plasma prekallikrein and kininogen. J. Lab. Clin.
tion in sensitivity, ranging from 9. Schaap, A. P., Akhavan, H. and Romano, L. J. (1989) Med. 100:81–93.
58 fmol/ml (23) to 2 fmol/ml (24). Chemiluminescent substrates for alkaline phos- 25. Shimamoto, K. and Iimura, O. (1987) “Measurement
Shimamoto and Iimura (25) reported a phatase: applications to ultrasensitive enzyme- of circulating kinins, their changes by inhibition of
detection limit of 0.25 fmol/tube without linked immunoassays and DNA probes. Clin. Chem. kininase II and their possible blood pressure lower-
35:1863–1864. ing effect” in Vasodepressor Hormones, p.
any further specification. The analytical 10. Legris, F., Martel-Pelletier, J., Pelletier, J. P., Colman, 297–307, Birkäuser Verlag, Basel.
performance of the new assay is somewhat R. and Adam, A. (1994) An ultrasensitive chemi- 26. Bronstein, I., Voyta, J. C., Thorfe, G. H., Kricka, L. J.
a result of the affinity of the polyclonal IgG luminoenzyme immunoassay for the quantification and Armstrong, G. (1989) Chemiluminescent assay
used for the immunoconcentration step of human tissue kininogens: application to synovial of alkaline phosphatase applied in an ultrasensitive
but is mainly an outcome of the high membrane and cartilage. J. Immunol. Methods enzyme immunoassay of thyrotropin. Clin. Chem.
specific activity (one molecule of DIG for 168:111–121. 35:1441–1446.
one molecule of peptide) without loss of 11. Avrameas, S. (1969) Coupling of enzymes to pro- 27. Bronstein, I., Edwards, B. and Voyta, J. C. (1989) 1,2-
teins with glutaraldehyde, use of conjugates for the dioxetanes: novel chemiluminescent enzyme sub-
imunoreactivity, the chemiluminescence detection of antigens and antibody. Immuno- strates. Applications to immunoassays. J. Biolum.
detection method, and the low back- chemistry 6:43. Chemilum. 4:99–111.
ground values. Like others (26, 27), we 12. Peterson, G. L. (1983) Determination of total protein
have shown that using such dioxetane Methods in Enzymology 91:95–119. Product Cat. No. Pack Size
derivatives as chemiluminescent alkaline 13. Anumula, K. R., Schulz, R. and Back, N. (1990) Digoxigenin-3-0- 1 333 054 5 mg
phosphatase substrates leads to 5–10-fold Immunologic methods for quantitative estimation of methylcarbonyl-ε-
increases in the sensitivity level of sand- small peptides and their application to bradykinin. aminocaproic acid-N-
J. Immunol. Meth. 135:199–208. hydroxy-succinimide ester
wich immunoassays using a visible light 14. Regoli, D. and Barabé, J. (1980) Pharmacology of
absorption (10). (DIG-NHS ester)
bradykinin and related kinins. Pharmacol. Rev.
32:1–46. Anti-Digoxigenin-AP, 1 093 274 150 U
Acknowledgements 15. Chard, T. (1987) “Characteristics of binding assays- Fab fragments (200 µl)
This work was supported by a grant sensitivity” in Laboratory techniques in biochemistry Lysozyme 107 255 1g
from the Fonds de la Recherche en Santé and molecular biology, volume 6, part 2, An intro- 1 243 004 5g
du Québec (FRSQ) to A.A. and R.C. R.C. duction to radioimmunoassay and related tech- 837 059 10 g
niques, (Burdon, R. H. and van Knippenberg, 1 585 657 25 g
and G.D. are scholars of the FRSQ; A.D. P. H., eds.). pp. 161–174, Elsevier Press, Amsterdam.
holds a studentship from the Formation 16. LeVine, S. M. and Macklin, W. B. (1988) Biotin Also Available Cat. No. Pack Size
de Chercheurs et l’Aide à la recherche. enrichment in oligodendrocytes in the rat brain. CSPD® chemi- 1655 884 1 ml
The authors are grateful for the secret- Brain Res. 444:199–203. luminescent substrate
arial assistance of Christiane Laurier and 17. Naritoku, W. Y. and Taylor, C. R. (1982) A competitive
the graphic work of Claude Gauthier. study of the use of monoclonal antibodies using
three different immunohistochemical methods: an
evaluation of monoclonal and polyclonal antibodies
References against human protatic and acid phosphatase.
1. Décarie, A., Drapeau, G., Closset, J., Couture, R. and
J. Histochem. Cytochem. 30:253–260.
Adam, A. (1994) Development of digoxigenin-
18. Goodfriend, T. L. and Ball, D. L. (1969) Radio-
labeled peptide: Application to chemiluminoenzyme
immunoassay of bradykinin: chemical modification
immunoassay of bradykinin in inflamed tissues.
to enable use of radioactive iodine. J. Lab. Clin. Med.
2. Diamandis, E. P. and Christopoulos, T. K. (1991) The
19. Talamo, R. C., Haber, E. and Austen, K. F. (1968)
Biotin-(strept)avidin system: principles and appli-
Antibody to BK: effect of carrier and method of
cations in biotechnology. Clin. Chem. 37:625–636.
coupling on specificity and affinity. J. Immunol.
3. Strasburger, C. J. and Kohen, F. (1990) Two-site and
competitive chemiluminescent immunoassays.
20. van Leeuwen, B. H., Millar, J. A., Hammat, M. T. and
Methods in Enzymology 184:481–491.
Johnston, C. I. (1983) Radioimmunoassay of blood
4. Ternynck, T. and Avrameas, S. (1990) Avidin-biotin
bradykinin: purification of blood extracts to prevent
system in enzyme immunoassays. Methods in
cross-reaction with endogenous kininogen. Clin.
Chim. Acta 127:343–351.
5. Wilchek, M. and Bayer, E. A. (1990) Introduction to
21. Geiger, R. and Miska, W. (1986) Determination of
avidin-biotin technology. Methods in Enzymology
bradykinin by enzyme immunoassay. Adv. Exp. Med.
6. Wilchek, M. and Bayer, E. A. (1990) Avidin-biotin
22. Ueno, A., Ohishi, S., Kitagawa, T. and Katori, M.
mediated immunoassays: overview. Methods in
(1979) Enzyme immunoassay of bradykinin. Adv.
Exp. Med. Biol. 120A:195–202.
BIOCHEMICA s NO. 4  23