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Chemiluminescent assay of various enzymes using indoxyl derivatives as substrate and its applications to enzyme immunoassay and DNA probe assay
ANALYTICAL
BIOCHEMISTRY
199,238-242
(1991)
Chemiluminescent Assay of Various Enzymes Using lndoxyl Derivatives as Substrate and Its Applications to Enzyme Immunoassay and DNA Probe Assay Hidetoshi
Arakawa,
School of Pharmaceutical
Received
May
Masako
Maeda,
Sciences, Showa
and Akio University,
Tsujil
Hatanodai,
Press,
Inc.
A sensitive assay of enzyme activity is very important for developing a more sensitive enzyme immunoassay (EIA)’ and nonisotopic DNA probe assay using enzyme as a label. Recently, highly sensitive chemiluminescent (CL) and bioluminescent (BL) methods have become available for detecting the alkaline phosphatase (ALP) labels used in EIA. The CL and BL assays use an adamantyl-1,2dioxetane phosphate (AMPPD) (1) and
1 To whom correspondence should be addressed. * Abbreviations used: ALP, alkaline phosphatase; p-gal, @-D-g&Ztosidase; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; X-Gal, 5bromo-4-chloro-3-indolyl-fi-n-galactopyranoside; BCIG, 5-bromo-4chloro-3-indolyl-P-D-glucoside; AMPPD, adamantyl-1,2dioxetane phosphate; CL, chemiluminescence; EIA, enzyme immunoassay; BL, bioluminescence; HRP, horseradish peroxidase; m-POD, microperoxidase; BSA, bovine serum albumin; AFP, a-fetoprotein.
238
Tokyo 142, Japan
20, 1991
Chemiluminescent assays of various enzymes have been developed using indoxyl derivatives as substrates. The principle of the method is as follows: an enzyme causes hydrolysis of an indoxyl derivative to an intermediate indoxyl that is readily oxidized to indigo dye and simultaneously produces hydrogen peroxide (H,O,). Hydrogen peroxide is detected chemiluminescently using isoluminol-microperoxidase. Alkaline phosphatase (ALP), &D-galactosidase (&gal), and & glucosidase were assayed by this method using 5bromo-4-chloro-3-indolyl phosphate (BCIP), Ei-bromo4-chloro-3-indolyl-/3-D-galactopyranoside (X-Gal), and 5 - bromo - 4 - chloro - 3 - indol yl - @- D - glucoside, respectively, as substrates. Using BCIP and X-Gal substrates, we have been able to detect 10-l’ mol of ALP and &gal, respectively. This assay system can be applied o 1~91 to enzyme immunoassay and DNA probe assay. Academic
Shinagawa,
firefly D-luciferin-O-phosphate (2), respectively, as substrates. Both assays have also been used for photographic detection of ALP label blotted on a nitrocellulose filter (3,4). We have already developed a CL assay of horseradish peroxidase (HRP) (5), glucose oxidase (6), /3-D-galactosidase (@-gal) (7), invertase (8), and ALP (9) and applied these methods to the detection of EIAs and DNA probe assays. In the study reported here, we developed new CL assays of various enzymes using indoxyl derivatives that have been used in enzyme cytochemistry. Indoxyl derivative is hydrolyzed enzymatitally to liberate free indoxyl followed by oxidation to indigo dye and simultaneously produces hydrogen peroxide (H202). Hydrogen peroxide (H,O,) is detected chemiluminescently using isoluminol-microperoxidase (mPOD). ALP, @-gal, and /3-glucosidase were assayed by this method using 5-bromo-4-chrolo-3-indolyl phosphate (BCIP), 5-bromo-4-chrolo-3-indolyl+D-galactopyranoside (X-Gal), and 5-bromo-4-chrolo-3-indolyl+ D-glucoside (BCIG), respectively, as substrates. We have been able to detect 10-l’ mol of ALP and P-gal. The CL assay of ALP was preliminarily applied to EIA and DNA probe assay.
MATERIALS
Alkaline phosphatase (EC 3.1.3.1), P-D-galactosidase (EC 3.2.1.23), and P-glucosidase (EC 3.2.1.21) were purchased from Boehringer-Mannheim-Yamanouchi, Co. (Tokyo, Japan). m-POD was obtained from Sigma Chemical Co. (St. Louis, MO). BCIP, X-Gal, and BCIG were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Isoluminol was obtained from Tokyo Chemical Industry Co. (Tokyo, Japan). ALP-avidin conjugate and biotin hydrazide were obtained from Bioyeda LTD. (Rehout, Israel). Anti-a-fetoprotein (AFP)antibody-coated tube and ALP-anti-AFP antibody con0003-2697/91
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENZYME
ASSAY
USING
INDOXYL
239
DERIVATIVES
Cl
Br
Enzyme t
‘4202
+Br&!;C=
C<;DBr+ Cl
A
H2Qz
FIG. 1. D-glucoside
+
lsoluminol
Principle of the chemiluminescent for /3-glucosidase.
m-POD assay
system
Chemiluminescence for various
jugate were donated by Sankyo Co. (Tokyo, Japan). Other chemicals were of analytical reagent grade.
PROCEDURES
1. CL Assay
R-OH
of ALP
A 1Oq.d of sample solution in 0.1 M Tris-HCl buffer (pH 9.5) containing 0.1% BSA was added to a microtest tube (10 X 75 mm), followed by addition of 100 gl of 0.38 mM BCIP solution in 0.1 M Tris-HCl buffer (pH 9.5) containing 0.1 M MgCl,. The reaction mixture was incubated at 37°C for 2 h and then 500 ~1 of a CL reagent solution (1.2 X 1O-4M isoluminol and 5X lop7 M m-POD in 0.4 M carbonate buffer (pH 9.5)) was added. The CL intensity was measured by an Aloka luminescence reader for the 6-s interval from 15 to 21 s after addition of the CL reagent solution.
2. CL Assay of p-gal A 50-~1 sample solution in 0.01 M phosphate buffer (pH 8.0) containing 0.1% BSA and 0.5 mM MgCl, was added to a microtest tube, followed by addition of 100 ~1 of 0.2 mM X-Gal solution in 0.01 M phosphate buffer (pH 8.0) containing 0.5 mM MgCl,. The reaction mixture was incubated at 37°C for 2 h. The CL intensity was measured by adding the CL reagent solution as described above.
3. CL assay of @-Glucosidase A 50-~1 sample solution in 0.05 M acetate buffer (pH 5.4) containing 0.1% BSA was added to a microtest tube, followed by addition of 100 ~1of 0.2 mM BCIG in 0.05 M acetate buffer (pH 5.4). The reaction mixture was incubated at 37°C for 2 h. The CL intensity was measured by adding the CL reagent solution as described above.
enzymes.
R: phosphate
for ALP,
D-galactopyranoside
for P-gal,
and
4. CL Assay of ALP by the One-Step Method A lo-p1 sample solution in 0.1 M Tris-HCl buffer (pH 9.5) was added to a microtest tube, followed by addition of 200 ~1 of the CL mixed solution of substrate and CL reagent containing 2.5 X lop4 M isoluminol, 5 U/ml HRP, 0.1 M MgCl,, and 0.19 mM BCIP in 0.1 M TrisHCl buffer (pH 9.5). The time course of CL intensity was measured by the Aloka luminescence reader for 30 min after addition of the CL mixed solution.
5. CL EIA of a-Fetoprotein The CL EIA of AFP was performed by Sankyo Luminomaster as follows: A 3Oq.d sample solution was pipetted into the antibody-coated test tube, followed by addition of 270 ~1 of 0.01 M phosphate buffer (pH 7.0). After incubation for 15 min at 37°C 300 ~1 of the ALPlabeled antibody solution was added to the test tube followed by washing with 0.01 M phosphate buffer (pH 7.0) (500 ~1, 1X) and incubating for 15 min at 37°C. After the test tube was washed with the same buffer (500 ~&3x), 200 ~1 of 0.38 mM BCIP was added and incubated for 15 min at 37°C and 100 ~1 of the reaction mixture was added to the cuvette containing 200 ~1 of the CL reagent solution described in procedure 1.
6. CL Assay of DNA Probe Nonisotopic hybridization was carried out according to the procedure previously reported by us (9). Preparation of biotin-labeled DNA probe. DNA (50 pg) was dissolved in 50 ~1of H,O and denatured by heating at 95°C for 5 min. After cooling for 5 min in an ice bath, 12.5 ~1 of biotin hydrazide (or biotin X-hydrazide) solution (10 mg/ml in H,O) and 15 ~1 of 5% glutaraldehyde solution were added to 25 ~1 of the heat-denatured DNA solution. The mixture was incubated at 37°C for 10 min, and then 5.83 ~1 of 3 M sodium acetate solution and 146 ~1 of cold ethanol were added. The resultant
240
ARAKAWA,
MAEDA,
to-14
2.
TSUJI
b
a
FIG.
AND
Alkaline
phosphatase
Standard
curves
(M)
10-73 ,&I2 ,0-l,lo-“10-1~ 10-Q
8-D-galactosidase
of (a) alkaline
phosphatase,
solution was cooled at -20°C overnight. After centrifugation for 10 min at room temperature, the precipitated biotin-labeled DNA was washed with cold 80% (v/v) ethanol, dried in uucuo for 2 h, redissolved in 50 ~1 of 0.1 mM EDTA solution, and stored at 4°C until use. Hybridization assay. The hybridization assay was carried out in a well of a microtiter plate. A X-DNA sample was immobilized according to the procedure of Nagata et al. (10) and hybridized with biotin-labeled XDNA probe. The hybridized biotin-labeled probe was detected by the use of ALP-labeled avidin and ALP activity was measured by the CL assay of ALP mentioned in the CL EIA (5).
a
lo-8
/3-glucosidase
(Ml
(b) P-D-galactosidase,
lo-7
(M)
and (c) @glucosidase.
RESULTS AND DISCUSSION Indoxyl derivatives are chromogenic substrates that have been used for the histochemical demonstration of endogenous enzymes. Several enzymes (ALP, fl-D-galactosidase, P-glucosidase, esterase, and sulfatase) localized in tissue can be determined by utilizing each indoxyl derivative as the substrate. The principle of this method is that an appropriate indoxyl derivative produces an unstable intermediate indoxyl by enzymatic hydrolysis, which results in the precipitation of an indigoid dye by aerial oxidation (11). Recently, this method was enhanced by the use of a simultaneously coupled reaction in which nitro blue tetrazolium salt was re-
b 0.5
100
KCPM
0.25
I
25
/ FIG. 3. The kinetics 100 pg ALP).
50
Time of the chemilun&inescence
75
1
obtained
by (a) the two-step
method
a
d
20
Time
(seconds)
signals
t
10
I
30
(minutes)
(at 10 pg ALP)
and (b) the one-step
method
(at
ENZYME
ASSAY USING
INDOXYL
241
DERIVATIVES
b
0
1
10
100
1000
10
a -Fetoprotein (rig/ml) FIG. 4.
Standard curves of a-fetoprotein
1000
10000
A -DNA (pg/well)
and X-DNA obtained by (a) CL EIA and (b) CL DNA probe assay, respectively.
duced to formazan-type dye. The enhanced methods for ALP and P-gal are used in the detection of nonisotopic hybrydization assay (12) and enzyme-linked immunoassay for antigen in cells (13). However, these calorimetric assays of the enzymes immobilized on cellulose or in cells are not quantitative and are not very sensitive. Cotson and Holt (14) reported that aerial oxidation of indoxyls to indigo dyes was a free radical reaction involving the intermediate formation of leucoindigo, in which approximately one hydrogen peroxide molecule was formed for every molecule of indoxyl oxidized. Therefore, we devised a CL assay of various enzymes by using the CL reaction of luminol after enzymatic hydrolysis of indoxyl derivatives. The CL reaction of this system is shown in Fig. 1. CL Assay of ALP
100
lo-l4 M were 4.4, 9.4, 9.6, 1.7, 4.2, and 3.7, respectively. The detection limit (at blank + 3 SD) was 7 X 10-l’ mol/assay, which is 1000 times more sensitive than the value obtained by the spectrophotometric method using BCIP/nitro blue tetrazolium phosphate as the substrate. The sensitivity of this method is also 2.5 times that of the previous CL assay using the nicotinamide adenine dinucleotide phosphate (NADP)/alcohol/alcoho1 dehydrogenase/m-POD/isoluminol system, but 1 order lower in sensitivity than that of the AMPPD method (lo-” mol) reported recently (1). CL Assay of P-gal To establish optimal assay conditions, various factors were examined, and the procedures for the CL assay of P-gal were as described in procedure 2. As shown in Fig. 2b, a working curve was obtained in the range of 1 X lo-i4 to 2.5 X 10-l’ M. The CV percentages (n = 5) at 1 X 10-14, 0.25 X 10-13, 1 x 10-13,0.25 X 10-12, 1 x 10-12,0.25 X lo-“, and 1 X 10-l’ M were 3.5, 6.3, 10.1, 4.0, 8.4, 7.3, and 4.8, respectively. The detection limit (at blank + 3 SD) was 2 X 10-l’ mol/assay. The sensitivity of this method was 25 times that of the CL method using 3-(2’spiroadamantane)-4-methoxy-4-(3”-P-D-galaCtOpyranosyloxyphenyl)-1,2-dioxetane for P-gal reported by Bronstein et al. (1).
The effects of various factors (pH, reaction time, concentrations of the reagents used) on the CL assay of ALP were examined to establish the optimal assay conditions. In the case of BCIP concentration, the highest ratio of signal/noise (signal, S, at 7 X 10-l’ M ALP, noise, N, at 0 M ALP) was obtained at 0.38 mM. The addition of more BCIP decreased the SIN ratio. Therefore, 0.38 mM was determined to be the optimum BCIP concentration. The procedure for the CL assay of ALP was as described in procedure 1. As shown in Fig. 2a, a linear relation between CL and ALP was obtained in the CL Assay of ,&Glucosidase range of 7 X lo-l4 to 3.6 X lo-l1 M. The coefficients of @-Glucosidase has also been used as the label enzyme variation (CV, n = 5) were examined at each point of the in EIA and is usually assayed by a fluorophotometric standard curve. The CV percentages (n = 5) at 7 x 10-17, method. ,&Glucosidase can also be determined by a CL 3.5 X 10-16, 7 X 10-16, 3.5 X 10-15, 7 X 10-15, and 3.5 X method. To establish the optimal assay conditions,
242
ARAKAWA,
MAEDA,
various factors were examined. The procedure for CL assay of P-glucosidase was as described in procedure 3. As shown in Fig. 2c, the measurable range was from 10-l’ to 10v7 M. The CV percentages (n = 5) at lo-“, 2 X lo-“, lo-“, 2 X 10-l’, lo-‘, 2 X lo-‘, 10e8, 2 X 10e8, and 10-l M were 3.6, 4.1, 4.0, 4.3, 3.6, 7.6, 4.6, 6.1, and 3, respectively. The detection limit (at blank + 2 SD) was 10-u M, corresponding to 5 X lOpi6 mol/assay.
CL Assay of ALP by the One-Step Method Detection of nucleic acid and antigen by Southern and Western blotting techniques via the use of chromogenic substrates and antibody-enzyme conjugates is a widely used method in molecular biology. Recently, photographic detection has often been used for these blotting assays; the bound antibody or avidinenzyme conjugate on nitrocellulose filter is visualized by the addition of a light-emitting substrate, luminol (15) or AMPPD (3), and the resulting light emission is detected by the use of either Polaroid film or X-ray film. We performed the following preliminary experiment to develop photographic detection of this CL reaction. The effect of various factors on CL was examined to establish the optimal assay conditions. The procedure for the CL assay of ALP by the one-step method was as described in procedure 5. The decay time of light emission obtained by the one-step method was different from that of two-step method (procedure l), which results in rapid decay of the light emission. Figure 3 illustrates the kinetics of the CL signals. The CL can be recorded without loss of intensity over at least 30 min. This method (procedure 4) for ALP was less sensitive (l/100) than the two-step method (procedure 1). However, the sensitivity can be enhanced by using a photon counting and imaging camera system (Argus 100 from Hamamatsu Photonics) which can capture weak light emission by using a long exposure time. We have obtained preliminary results in which 7 X 10-l’ mol of ALP could be detected. This study is underway in our laboratory and will be reported elsewhere.
Application of CL Assay of ALP in the Immunoassay and DNA Probe Assay The CL assay of ALP using BCIP as a substrate can be used to detect alkaline phosphatase conjugates in EIA and DNA probe assay. We developed a sandwichtype EIA for AFP. The assay involves two monoclonal antibodies, one of which is labeled with ALP and one of which is coated on a polystyrene tube. Various factors were examined to establish the optimal standard method of EIA for AFP. The procedure of EIA for AFP was as described in procedure 6. The measurable range of AFP is 1 to 1000 rig/ml and the detection limit is 1 rig/ml. Intraassay CVs (n = 5) at 1 and 100 rig/ml were
AND
TSUJI
3.7 and 3.9%, respectively. We also developed a nonisotopic DNA probe assay for X-phage DNA. Biotin-labeled DNA probes were prepared by coupling biotin hydrazide to X-phage DNA using glutaraldehyde as coupling reagent. Hybridization assay was carried out in the well of a microtiter plate. A hybrydized biotin-labeled probe was detected by the use of ALP-labeled avidin, and ALP activity was measured by the CL assay using BCIP as a substrate. By this method, 10 pg of A-DNA can be detected, which corresponds to 3 X 10-l’ mol. Figure 4 illustrates the standard curves of a-fetoprotein and X-DNA obtained by CL EIA and CL DNA probe assay, respectively. CONCLUSION Highly sensitive CL assays of ALP, @-gal, and @-glucosidase have been developed using indoxyl derivatives as substrates. CL assays of esterase and sulfatase will be developed using 5-bromo-4-chloro-3-indolyl acetate and 5-bromo-4-chloro-3-indolyl sulfate, respectively, as substrates. The CL assay of ALP by these methods has been applied to the EIA and DNA hybridization assay. Other CL assays of P-gal and @-glucosidase will be applied to EIA. The assay systems reported here are sensitive, simple, and inexpensive. Thus, this CL assay of enzymes would be useful for the determination of enzyme in EIA and nonisotopic DNA hybridization assay. REFERENCES 1. Bronstein, I., Edwards, B., and Voyta, C. (1989) J. Biolumin. Chemilumin. 4,99-111. 2. Miska, W., and Geiger, R. (1987) J. Clin. Chem. Clin. Biochem. 25, 23-30. 3. Bronstein, I., Voyata, J. C., and Edwards, them. 180.95-98. 4. Hauber, R., and Geiger, R. (1989) J. Clin. 27,361-363. 5. Arakawa, H., Maeda, 248-254. 6. Arakawa, H., Maeda, 430-434. 7. Maeda, M., Arakawa, lumin. 4, 140-148.
M., and Tsuji, M.,
and
8. Shimizu, S., Arakawa, H., Maeda, seki Kuguku 37, 123-127. 9. Arakawa, H., Ckem. Pharm.
Maeda, M., Tsuji, Bull. 37,1831-1833.
Chem.
A. (1979)
Tsuji,
H., and Tsuji,
B. (1989)
A. (1989) M.,
Biochem.
97,
Chem.
31,
Clin.
Chemi-
A. (1988)
Takahashi,
Bun-
T.
(1989)
10. Nagata, Y., Yokota, H., Kosuda, O., Yokoo, K., Takemura, and Kikuchi, T. (1985) FEBS Lett. 183,379-382. B. 142. 160-169. 11. Holt, S. J. (1954) Proc. R. Sot. London 12. Leary, Acad.
J. J., Brigati, D. J., and Sci. USA 80,4045-4049.
13. Nibbering, R. (1986)
Ward,
D. C. (1983)
P. H., Marijnen, J. G. J., Raap, Histochemistry 84,538-543.
Bio-
Biockem.
J. Biolumin.
and Tsuji,
A., and
Clin.
Anal.
A. (1985)
Anal.
K.,
Proc.
Natl.
A. K., and Van Furth,
14. Cotson, S., and Holt, S. J. (1958) Proc. R. Sot. London 506-519. 15. Leong, M. M. L., and Fox, G. R. (1988) Anal. Biochem. 150.
B
148,
172,145-
BIOCHEMISTRY
199,238-242
(1991)
Chemiluminescent Assay of Various Enzymes Using lndoxyl Derivatives as Substrate and Its Applications to Enzyme Immunoassay and DNA Probe Assay Hidetoshi
Arakawa,
School of Pharmaceutical
Received
May
Masako
Maeda,
Sciences, Showa
and Akio University,
Tsujil
Hatanodai,
Press,
Inc.
A sensitive assay of enzyme activity is very important for developing a more sensitive enzyme immunoassay (EIA)’ and nonisotopic DNA probe assay using enzyme as a label. Recently, highly sensitive chemiluminescent (CL) and bioluminescent (BL) methods have become available for detecting the alkaline phosphatase (ALP) labels used in EIA. The CL and BL assays use an adamantyl-1,2dioxetane phosphate (AMPPD) (1) and
1 To whom correspondence should be addressed. * Abbreviations used: ALP, alkaline phosphatase; p-gal, @-D-g&Ztosidase; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; X-Gal, 5bromo-4-chloro-3-indolyl-fi-n-galactopyranoside; BCIG, 5-bromo-4chloro-3-indolyl-P-D-glucoside; AMPPD, adamantyl-1,2dioxetane phosphate; CL, chemiluminescence; EIA, enzyme immunoassay; BL, bioluminescence; HRP, horseradish peroxidase; m-POD, microperoxidase; BSA, bovine serum albumin; AFP, a-fetoprotein.
238
Tokyo 142, Japan
20, 1991
Chemiluminescent assays of various enzymes have been developed using indoxyl derivatives as substrates. The principle of the method is as follows: an enzyme causes hydrolysis of an indoxyl derivative to an intermediate indoxyl that is readily oxidized to indigo dye and simultaneously produces hydrogen peroxide (H,O,). Hydrogen peroxide is detected chemiluminescently using isoluminol-microperoxidase. Alkaline phosphatase (ALP), &D-galactosidase (&gal), and & glucosidase were assayed by this method using 5bromo-4-chloro-3-indolyl phosphate (BCIP), Ei-bromo4-chloro-3-indolyl-/3-D-galactopyranoside (X-Gal), and 5 - bromo - 4 - chloro - 3 - indol yl - @- D - glucoside, respectively, as substrates. Using BCIP and X-Gal substrates, we have been able to detect 10-l’ mol of ALP and &gal, respectively. This assay system can be applied o 1~91 to enzyme immunoassay and DNA probe assay. Academic
Shinagawa,
firefly D-luciferin-O-phosphate (2), respectively, as substrates. Both assays have also been used for photographic detection of ALP label blotted on a nitrocellulose filter (3,4). We have already developed a CL assay of horseradish peroxidase (HRP) (5), glucose oxidase (6), /3-D-galactosidase (@-gal) (7), invertase (8), and ALP (9) and applied these methods to the detection of EIAs and DNA probe assays. In the study reported here, we developed new CL assays of various enzymes using indoxyl derivatives that have been used in enzyme cytochemistry. Indoxyl derivative is hydrolyzed enzymatitally to liberate free indoxyl followed by oxidation to indigo dye and simultaneously produces hydrogen peroxide (H202). Hydrogen peroxide (H,O,) is detected chemiluminescently using isoluminol-microperoxidase (mPOD). ALP, @-gal, and /3-glucosidase were assayed by this method using 5-bromo-4-chrolo-3-indolyl phosphate (BCIP), 5-bromo-4-chrolo-3-indolyl+D-galactopyranoside (X-Gal), and 5-bromo-4-chrolo-3-indolyl+ D-glucoside (BCIG), respectively, as substrates. We have been able to detect 10-l’ mol of ALP and P-gal. The CL assay of ALP was preliminarily applied to EIA and DNA probe assay.
MATERIALS
Alkaline phosphatase (EC 3.1.3.1), P-D-galactosidase (EC 3.2.1.23), and P-glucosidase (EC 3.2.1.21) were purchased from Boehringer-Mannheim-Yamanouchi, Co. (Tokyo, Japan). m-POD was obtained from Sigma Chemical Co. (St. Louis, MO). BCIP, X-Gal, and BCIG were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Isoluminol was obtained from Tokyo Chemical Industry Co. (Tokyo, Japan). ALP-avidin conjugate and biotin hydrazide were obtained from Bioyeda LTD. (Rehout, Israel). Anti-a-fetoprotein (AFP)antibody-coated tube and ALP-anti-AFP antibody con0003-2697/91
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENZYME
ASSAY
USING
INDOXYL
239
DERIVATIVES
Cl
Br
Enzyme t
‘4202
+Br&!;C=
C<;DBr+ Cl
A
H2Qz
FIG. 1. D-glucoside
+
lsoluminol
Principle of the chemiluminescent for /3-glucosidase.
m-POD assay
system
Chemiluminescence for various
jugate were donated by Sankyo Co. (Tokyo, Japan). Other chemicals were of analytical reagent grade.
PROCEDURES
1. CL Assay
R-OH
of ALP
A 1Oq.d of sample solution in 0.1 M Tris-HCl buffer (pH 9.5) containing 0.1% BSA was added to a microtest tube (10 X 75 mm), followed by addition of 100 gl of 0.38 mM BCIP solution in 0.1 M Tris-HCl buffer (pH 9.5) containing 0.1 M MgCl,. The reaction mixture was incubated at 37°C for 2 h and then 500 ~1 of a CL reagent solution (1.2 X 1O-4M isoluminol and 5X lop7 M m-POD in 0.4 M carbonate buffer (pH 9.5)) was added. The CL intensity was measured by an Aloka luminescence reader for the 6-s interval from 15 to 21 s after addition of the CL reagent solution.
2. CL Assay of p-gal A 50-~1 sample solution in 0.01 M phosphate buffer (pH 8.0) containing 0.1% BSA and 0.5 mM MgCl, was added to a microtest tube, followed by addition of 100 ~1 of 0.2 mM X-Gal solution in 0.01 M phosphate buffer (pH 8.0) containing 0.5 mM MgCl,. The reaction mixture was incubated at 37°C for 2 h. The CL intensity was measured by adding the CL reagent solution as described above.
3. CL assay of @-Glucosidase A 50-~1 sample solution in 0.05 M acetate buffer (pH 5.4) containing 0.1% BSA was added to a microtest tube, followed by addition of 100 ~1of 0.2 mM BCIG in 0.05 M acetate buffer (pH 5.4). The reaction mixture was incubated at 37°C for 2 h. The CL intensity was measured by adding the CL reagent solution as described above.
enzymes.
R: phosphate
for ALP,
D-galactopyranoside
for P-gal,
and
4. CL Assay of ALP by the One-Step Method A lo-p1 sample solution in 0.1 M Tris-HCl buffer (pH 9.5) was added to a microtest tube, followed by addition of 200 ~1 of the CL mixed solution of substrate and CL reagent containing 2.5 X lop4 M isoluminol, 5 U/ml HRP, 0.1 M MgCl,, and 0.19 mM BCIP in 0.1 M TrisHCl buffer (pH 9.5). The time course of CL intensity was measured by the Aloka luminescence reader for 30 min after addition of the CL mixed solution.
5. CL EIA of a-Fetoprotein The CL EIA of AFP was performed by Sankyo Luminomaster as follows: A 3Oq.d sample solution was pipetted into the antibody-coated test tube, followed by addition of 270 ~1 of 0.01 M phosphate buffer (pH 7.0). After incubation for 15 min at 37°C 300 ~1 of the ALPlabeled antibody solution was added to the test tube followed by washing with 0.01 M phosphate buffer (pH 7.0) (500 ~1, 1X) and incubating for 15 min at 37°C. After the test tube was washed with the same buffer (500 ~&3x), 200 ~1 of 0.38 mM BCIP was added and incubated for 15 min at 37°C and 100 ~1 of the reaction mixture was added to the cuvette containing 200 ~1 of the CL reagent solution described in procedure 1.
6. CL Assay of DNA Probe Nonisotopic hybridization was carried out according to the procedure previously reported by us (9). Preparation of biotin-labeled DNA probe. DNA (50 pg) was dissolved in 50 ~1of H,O and denatured by heating at 95°C for 5 min. After cooling for 5 min in an ice bath, 12.5 ~1 of biotin hydrazide (or biotin X-hydrazide) solution (10 mg/ml in H,O) and 15 ~1 of 5% glutaraldehyde solution were added to 25 ~1 of the heat-denatured DNA solution. The mixture was incubated at 37°C for 10 min, and then 5.83 ~1 of 3 M sodium acetate solution and 146 ~1 of cold ethanol were added. The resultant
240
ARAKAWA,
MAEDA,
to-14
2.
TSUJI
b
a
FIG.
AND
Alkaline
phosphatase
Standard
curves
(M)
10-73 ,&I2 ,0-l,lo-“10-1~ 10-Q
8-D-galactosidase
of (a) alkaline
phosphatase,
solution was cooled at -20°C overnight. After centrifugation for 10 min at room temperature, the precipitated biotin-labeled DNA was washed with cold 80% (v/v) ethanol, dried in uucuo for 2 h, redissolved in 50 ~1 of 0.1 mM EDTA solution, and stored at 4°C until use. Hybridization assay. The hybridization assay was carried out in a well of a microtiter plate. A X-DNA sample was immobilized according to the procedure of Nagata et al. (10) and hybridized with biotin-labeled XDNA probe. The hybridized biotin-labeled probe was detected by the use of ALP-labeled avidin and ALP activity was measured by the CL assay of ALP mentioned in the CL EIA (5).
a
lo-8
/3-glucosidase
(Ml
(b) P-D-galactosidase,
lo-7
(M)
and (c) @glucosidase.
RESULTS AND DISCUSSION Indoxyl derivatives are chromogenic substrates that have been used for the histochemical demonstration of endogenous enzymes. Several enzymes (ALP, fl-D-galactosidase, P-glucosidase, esterase, and sulfatase) localized in tissue can be determined by utilizing each indoxyl derivative as the substrate. The principle of this method is that an appropriate indoxyl derivative produces an unstable intermediate indoxyl by enzymatic hydrolysis, which results in the precipitation of an indigoid dye by aerial oxidation (11). Recently, this method was enhanced by the use of a simultaneously coupled reaction in which nitro blue tetrazolium salt was re-
b 0.5
100
KCPM
0.25
I
25
/ FIG. 3. The kinetics 100 pg ALP).
50
Time of the chemilun&inescence
75
1
obtained
by (a) the two-step
method
a
d
20
Time
(seconds)
signals
t
10
I
30
(minutes)
(at 10 pg ALP)
and (b) the one-step
method
(at
ENZYME
ASSAY USING
INDOXYL
241
DERIVATIVES
b
0
1
10
100
1000
10
a -Fetoprotein (rig/ml) FIG. 4.
Standard curves of a-fetoprotein
1000
10000
A -DNA (pg/well)
and X-DNA obtained by (a) CL EIA and (b) CL DNA probe assay, respectively.
duced to formazan-type dye. The enhanced methods for ALP and P-gal are used in the detection of nonisotopic hybrydization assay (12) and enzyme-linked immunoassay for antigen in cells (13). However, these calorimetric assays of the enzymes immobilized on cellulose or in cells are not quantitative and are not very sensitive. Cotson and Holt (14) reported that aerial oxidation of indoxyls to indigo dyes was a free radical reaction involving the intermediate formation of leucoindigo, in which approximately one hydrogen peroxide molecule was formed for every molecule of indoxyl oxidized. Therefore, we devised a CL assay of various enzymes by using the CL reaction of luminol after enzymatic hydrolysis of indoxyl derivatives. The CL reaction of this system is shown in Fig. 1. CL Assay of ALP
100
lo-l4 M were 4.4, 9.4, 9.6, 1.7, 4.2, and 3.7, respectively. The detection limit (at blank + 3 SD) was 7 X 10-l’ mol/assay, which is 1000 times more sensitive than the value obtained by the spectrophotometric method using BCIP/nitro blue tetrazolium phosphate as the substrate. The sensitivity of this method is also 2.5 times that of the previous CL assay using the nicotinamide adenine dinucleotide phosphate (NADP)/alcohol/alcoho1 dehydrogenase/m-POD/isoluminol system, but 1 order lower in sensitivity than that of the AMPPD method (lo-” mol) reported recently (1). CL Assay of P-gal To establish optimal assay conditions, various factors were examined, and the procedures for the CL assay of P-gal were as described in procedure 2. As shown in Fig. 2b, a working curve was obtained in the range of 1 X lo-i4 to 2.5 X 10-l’ M. The CV percentages (n = 5) at 1 X 10-14, 0.25 X 10-13, 1 x 10-13,0.25 X 10-12, 1 x 10-12,0.25 X lo-“, and 1 X 10-l’ M were 3.5, 6.3, 10.1, 4.0, 8.4, 7.3, and 4.8, respectively. The detection limit (at blank + 3 SD) was 2 X 10-l’ mol/assay. The sensitivity of this method was 25 times that of the CL method using 3-(2’spiroadamantane)-4-methoxy-4-(3”-P-D-galaCtOpyranosyloxyphenyl)-1,2-dioxetane for P-gal reported by Bronstein et al. (1).
The effects of various factors (pH, reaction time, concentrations of the reagents used) on the CL assay of ALP were examined to establish the optimal assay conditions. In the case of BCIP concentration, the highest ratio of signal/noise (signal, S, at 7 X 10-l’ M ALP, noise, N, at 0 M ALP) was obtained at 0.38 mM. The addition of more BCIP decreased the SIN ratio. Therefore, 0.38 mM was determined to be the optimum BCIP concentration. The procedure for the CL assay of ALP was as described in procedure 1. As shown in Fig. 2a, a linear relation between CL and ALP was obtained in the CL Assay of ,&Glucosidase range of 7 X lo-l4 to 3.6 X lo-l1 M. The coefficients of @-Glucosidase has also been used as the label enzyme variation (CV, n = 5) were examined at each point of the in EIA and is usually assayed by a fluorophotometric standard curve. The CV percentages (n = 5) at 7 x 10-17, method. ,&Glucosidase can also be determined by a CL 3.5 X 10-16, 7 X 10-16, 3.5 X 10-15, 7 X 10-15, and 3.5 X method. To establish the optimal assay conditions,
242
ARAKAWA,
MAEDA,
various factors were examined. The procedure for CL assay of P-glucosidase was as described in procedure 3. As shown in Fig. 2c, the measurable range was from 10-l’ to 10v7 M. The CV percentages (n = 5) at lo-“, 2 X lo-“, lo-“, 2 X 10-l’, lo-‘, 2 X lo-‘, 10e8, 2 X 10e8, and 10-l M were 3.6, 4.1, 4.0, 4.3, 3.6, 7.6, 4.6, 6.1, and 3, respectively. The detection limit (at blank + 2 SD) was 10-u M, corresponding to 5 X lOpi6 mol/assay.
CL Assay of ALP by the One-Step Method Detection of nucleic acid and antigen by Southern and Western blotting techniques via the use of chromogenic substrates and antibody-enzyme conjugates is a widely used method in molecular biology. Recently, photographic detection has often been used for these blotting assays; the bound antibody or avidinenzyme conjugate on nitrocellulose filter is visualized by the addition of a light-emitting substrate, luminol (15) or AMPPD (3), and the resulting light emission is detected by the use of either Polaroid film or X-ray film. We performed the following preliminary experiment to develop photographic detection of this CL reaction. The effect of various factors on CL was examined to establish the optimal assay conditions. The procedure for the CL assay of ALP by the one-step method was as described in procedure 5. The decay time of light emission obtained by the one-step method was different from that of two-step method (procedure l), which results in rapid decay of the light emission. Figure 3 illustrates the kinetics of the CL signals. The CL can be recorded without loss of intensity over at least 30 min. This method (procedure 4) for ALP was less sensitive (l/100) than the two-step method (procedure 1). However, the sensitivity can be enhanced by using a photon counting and imaging camera system (Argus 100 from Hamamatsu Photonics) which can capture weak light emission by using a long exposure time. We have obtained preliminary results in which 7 X 10-l’ mol of ALP could be detected. This study is underway in our laboratory and will be reported elsewhere.
Application of CL Assay of ALP in the Immunoassay and DNA Probe Assay The CL assay of ALP using BCIP as a substrate can be used to detect alkaline phosphatase conjugates in EIA and DNA probe assay. We developed a sandwichtype EIA for AFP. The assay involves two monoclonal antibodies, one of which is labeled with ALP and one of which is coated on a polystyrene tube. Various factors were examined to establish the optimal standard method of EIA for AFP. The procedure of EIA for AFP was as described in procedure 6. The measurable range of AFP is 1 to 1000 rig/ml and the detection limit is 1 rig/ml. Intraassay CVs (n = 5) at 1 and 100 rig/ml were
AND
TSUJI
3.7 and 3.9%, respectively. We also developed a nonisotopic DNA probe assay for X-phage DNA. Biotin-labeled DNA probes were prepared by coupling biotin hydrazide to X-phage DNA using glutaraldehyde as coupling reagent. Hybridization assay was carried out in the well of a microtiter plate. A hybrydized biotin-labeled probe was detected by the use of ALP-labeled avidin, and ALP activity was measured by the CL assay using BCIP as a substrate. By this method, 10 pg of A-DNA can be detected, which corresponds to 3 X 10-l’ mol. Figure 4 illustrates the standard curves of a-fetoprotein and X-DNA obtained by CL EIA and CL DNA probe assay, respectively. CONCLUSION Highly sensitive CL assays of ALP, @-gal, and @-glucosidase have been developed using indoxyl derivatives as substrates. CL assays of esterase and sulfatase will be developed using 5-bromo-4-chloro-3-indolyl acetate and 5-bromo-4-chloro-3-indolyl sulfate, respectively, as substrates. The CL assay of ALP by these methods has been applied to the EIA and DNA hybridization assay. Other CL assays of P-gal and @-glucosidase will be applied to EIA. The assay systems reported here are sensitive, simple, and inexpensive. Thus, this CL assay of enzymes would be useful for the determination of enzyme in EIA and nonisotopic DNA hybridization assay. REFERENCES 1. Bronstein, I., Edwards, B., and Voyta, C. (1989) J. Biolumin. Chemilumin. 4,99-111. 2. Miska, W., and Geiger, R. (1987) J. Clin. Chem. Clin. Biochem. 25, 23-30. 3. Bronstein, I., Voyata, J. C., and Edwards, them. 180.95-98. 4. Hauber, R., and Geiger, R. (1989) J. Clin. 27,361-363. 5. Arakawa, H., Maeda, 248-254. 6. Arakawa, H., Maeda, 430-434. 7. Maeda, M., Arakawa, lumin. 4, 140-148.
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