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