13 4,931,223 14
0-galarAosyloxy)ben7.ý2H-pyrm.2'-yl-1,2-diaxetane chemiluminescent 1,2-dioxetane compinuids, at least
(50 mg/ml) as the chemilummescent substrates. one of said 1,2-dioxetane compounds being an enzymat
ASSAY PPOCEDURF ically decomposable 1,2-dioxetme compound, said 1,2
dioxetame compounds being configured, by means of
Samples (50 pl) containing DNA are denatured by 5 the inclusion of a different fight emitting fluorophore in
incubation for 10 minutes at room temperature in 200;&1 each of said 1,2-dioxetane compounds, to each emit
of Denaturation Buffer. Ice cold Neutralizatort Buffer upon decomposition light of said different wavelengths,
(250 ILI) is then added, and the samples placed on ice. which comprises decomposing each of said 1,2-dioxe
Nylon membrane is soaked for 15 minutes in Wetting tane compounds by means of one of two or more differ.
Buffer and then inserted into a vacumn manifold device 10 ent enzyme, chemical or electrochemical decomposing
producing 2 cm dian, ter spots. Loading Buffer, (200 means,,each of saW decomposing means being specific
pl) is then aspirated through each Well. The denatured to one of said 1,2-dioxetme compounds, at least one of
DNA samples are then added to each well and aspirated said decomposing means being an enzyme decomposing
through the membrane. 71te manifold is then disasseni- nueang,
bled and the DNA fixed to the membrane using a LTV 15 2. The process of claim I in which each of said com
Transfilummator for 5 munnes. The membrane is then pounds is represented by the general formula,
incubated in Prehybridizatirin Buffer at 70' C. for I .
hour. 0-0
Dots of membrane from the region to which the RI
sample DNA is applied are punched out and inserted 20
into tubes for the remaining steps of the assay. The T )--ý P2
following enzyme labeled probes are used: probe for
HSV labeled with all-Mine phosphatow, probe for HPV
labeled with ft-galactaudaw, probe for CMV labeled Wherein RI represents hydrogen, Or a bond winch, to
with carboxylesterase. 25 gether with the indicated bond between R2 and the
The enzyme labeled probes (5D u of each probe in 200 4-carbon atom of the dioxetane fing, bonds a substituent
ILI of Hybridization Buffer per tube) am added to each represented by R2 to the 4-cartion atom through a spiro
tube and incubated for 2 hours at MO C. Ile Hybridiza- linkage, or an organic substituent that does not interfere
tion Buffer is removed and 400 W of Wash Buffer I with the production of fight and that satisfies the va
added and the tubes agitated for 10 minutes at room 30 lence of the dioxemme ring carbon atom to which it is
temperature. Washing is continued by first washing attached, or a light-emitting fluorophore-forming fluo
with 400 jil of Wash BmTer II at 50- C. for 30 minutes; rescent chromophore group bonded to the dioxetane
then With 40() Pj of Wash Buffer I], at room teurpera_ ring through a single bond or a spiro linkage, to winch
ture for 10 minutes; and then With 200 pA of Wash an enzymatically, chemically or electrochemically de
Buffer IV at room temperature. 35 comPOsable group 'a bonded, R2 represents hydrogen'
The membrane is subsequently rmsed with Wash or a bond whick together with the indicated bond
Buffm V at pH 6.0 and placed on a piece of transparent between RI and the 4-carbon atom of the dioxetftne
Mylar polyester film. Then, 200 F41 of the Substrate ring, bonds a substituent represented by Ri to the 4-car
Buffer is added and allowed to saak in. bon atom through a spiro linkage, or a fight-emitting
The assay tatutbrarie is placed in a camera luminomý 40 flninriPhOre'lorm"19 fluorescent chromophore group
ter &,ice equipped with preexposed calibraWn scales bonded to the dioxetane ring through a single bond or a
for HSV, HIYV and CMV. spiro linkage, to which an enzymatically, chemically or
The chemilaminescent light emission generated as a electrochemically decomposable group is bonded, at
function of the cnzymes--elkaline phosphatase, cax- least one of Ri and R2 being such light-emitting fluoro
boxyl esterm and p-gidectoddase -is imaged through 45 phore-forming fluorescent chromophore group, and T
a mask contimM three narrow bandpass Kodak Wmt- represents a stabilizing group that prevents the dioxe
tan gelatin filters (approximately I cm in diameter), tone compound from decomposing before the bond in
which isolaw the blue emission from the phenyl phos- the enzymatically, chemically or electrochemically
phate dioxetane, the cyan ernigodiin from the plies. decomposable group is intentionally cleaved.
phoyImytdflumomibyIbmxcpy.yI dioxetane and so 3. The process of claim 2 in which the process carried
the green emniasion from the galactosylozybenzopyra. out is a step in an immunomay.
ryl dioxetane, respectively. 4. ne process of claim 3 in which the hrurrunoassay
The relative levels of HSV, HPV and CMV present is for the detection of specific binding pain comprising
in the sample are determined by a comparison of the an antigen and an antibody.
appropriate unage brightness with the relevant calibra- 55 5. The process of claim 3 in which the labels used in
tion scale. the assay are enzymes.
The above discussion of this invention is directed 6. Ile process of claim 3 in which the labels used in
primarily to preferred embodiments and practices the assay are the ch6niluminescent 1,2-diomtane com
thereof. It Will be readily apparent to those skilled in the pounds.
art that further changes and modifications in the actual 6o 7. TUe process of claim 3 in which the immunciamay
implementation of the concepts described herein can is for the detection of an enzyme.
easily be made without departing from the spirit and 8. The process of claim 3 in which the immunoassay
scope of the invention as derined by the following is for the detection of hormones.
claims. 9. The process of claim 2 in which the process carried
We claim: 65 out is a step in a chemical assay.
1. A process in which light of different wavelengths is 10. Ile process of claim 9 in which the chemical
simultaneously released from two or more enzymati- assay is for the detection of chemical substances which,
cally, chemically or electrochemically decomposable during the assay, are caused to decompose to form
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