7 4,931,223
Mich as cholesterol oxidase or glucose oxidwe, to form Such an assay can, for example, be a simultaneous
a substance, e.g., hydrogen peroxide, capable in combi- sandwich two antibody capture enzyme immuntiasnay
nation with another reagent of causing the chemilumi- iii which a serum or urine sainiph: containing a mixture
nescent compound to decompose. of analytes; HLH, FSH and P-HCG, for example, or
As noted above, by using two or more chemdumines- 3 any two of them, is added to a coated matrix containing
cent 1,2-dioxetanes that each emit light of a different capture antibodies specific for HLII, FSH and A-HCG
wavelength from the others, or by using one or more of and incubated. Next, enzyme labeled antibodies: anti
these different colored fight-emitting cbeinfluramescent HLH labeled with jS-gslactosidase, anti FSH labeled
1,Moxetanes with one or more other cheiniluminesý with allcal ime phosphatase and anti P-HCG labeled with
cent compounds which emit light of yet other wave. 10 acitylesterase, am added, followed by, e.g., a mixture of
lengft each of such compounds being structured so as the aforementioned three chemilinninescent 1,2-cfioxe
to be decomposable by a different means, this invention tane substrates: the acetoxybenzopyrmyl dioxetane
enables multichannel assays be designed in which differ- cleavable with acetylesterase, the phosphoryloxybca
ent cleaving means, and especially two or more differ. mopyranyl dioxelane cleavable with alkaline phospha
ent enzYMM attached to or associated with two or 15 tase and the P-galactosyloxybenzopyranyl dioxetane
more different analytes will, by cleaving different cleav- cleavable with fl-galactosidase. The resulting light
able dwitetane Substiments, induce the emission of light emissions can be detected either with three different
of a different wavelength for each sualyte being as. monochromaton or on black and white photographic
sayed- film with three different color filters, with the intensity
3-(2'-Spiroadamantane)-4-(7"-aCetOxy)benzD-2H- 20 of the light emissions being a function of the various
pynan-T-yl-1,2-ificimetime [dispim(adaxumtane-2)-3'-(l', sunlYte concentration&
2'-dimictime)mW, 2"-(7"-acctcxy-3"-chromcmc)], A homogeneous assay using, e.g., these same three
clacmiluminescent 1,2-dioxetane substrams can be car
0-0 0 ried out by first adding a mixture of the analytes (Agi,
0 H 25 Ag7, Ag3) to a mistum of specific Anti AgI, Anti Agz
O-C-ýM3 and Anti A93 antibodies and small quantities of each of
the three analytes bound to the three different enzymes:
A91-0-gallOcIDO"ise, A92-allicalme phosphatase, A93
acetylesterase, and incubating.
30 Since in anti AgiAllit-fl-galactosidase, Anti A92Ag2
allmfine phosphatase and Anti Ag3 Ag3-acetylestersse
for example, when cleaved with a carboxylestemse, will complexes ft enzyme wiU be mactivated and hence
emit fight of 450 not (blue), 3_(2'_sphoadammt.04_ unable to induce luminescence, only enzyme labeled
(7"-Phosphoryloxy-4"-trifluoromethyl)bcnzo-2H- antigens thin are unbound will cleave the substrates to
pyran-T-YI-42-dioxetaine, 35 emit light. The emitted light cam be detected in the -me
manner as in the above-described sandwich assay. Since
0 there is a competition between native antigens and en
0-0 1 zyme labeled antigens6 the intensity of the light emitted
0 0-F-OH will be a fitaction of unbound hibeled antigens, and thus
OH 40 will cormspond to the concentrations of the analytes.
measured
Ught of various colon emitted when using the im
Proved methods ofthis invention to identify or quantify
various analytes can also be used to make a permanent
when cleaved with an alkaline phosphatme, will emit 45 record of such emissions on color photographic cmul.
light of 480 tim. (cyan, iý., blue greený and 3.(2'. slow as well as on specially sensitized black and white
sPimadamairtane)443" -benzothiazol 2-yl-7"-,6.gWac- high speed filins. And. these improved methods can be
tOsYlOxY)benz(ý-2H-pyran-2'-YI-1,2-(fioxetane, used to achieve a matched response by detectors:
charged coupled devices (CCD's) or sificor phowdi.
CH20H 5D Odes, for example, having maximma sensitivity for a
color other than blue, e.g., green or red. Further, by
HO COH using chcoliluminescenit 1,2dioxeta= together with a
0-0 H light absorbingAight shi g auxiliary fluarophore/
HO light enhancer substance which absorbs light of one
0 H 55 wavelength and in turn emits light of a different wave
OH H length, e.g., a phycobiliprotein (phycobdiproteins are
naturaRY-tioctirring substances in which a fluorophore
Os is bonded to a protein), such as phycDcyanine or phy.
coallocYanme, that Will absorb the green light emitted
N)I[ D101 60 by one such substance that emits light in this region of
the spectrum and reemit this fight as red light, matched
responses by color photographic emulsions that exhibit
when cleaved with 0-gaiactosidase, will emit light of a poor response to blue light, a better response to peen
515 can. (green). A simultaneous assay for HLH, FSH light but the best response to red fight can also be
and j3-HCG, or any two of them, cam hence be designed 65 achieved.
using these three chermiuminescent substances, or any Besides the PhYcolidiproWins. other auxiliary fluoro
two of diem, to produce light emissions of a different Phores extraneous to the light-emitting fluorophom,
color for each of the ansilytes. produced by the decomposition of the chemilumines-
Page: Previous 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Next
Last modified: November 3, 2007