9 4,931,223 10
cent 1,2-dioxietane compounds tised in the method of cent compound's molecule. In this way ewýrgy transfer
this invention that will accept energy, especially light, is enhanced it= to the two fluorophores being in close
from these light-emitting fluorophores and in rum emit proximity to one mother and by beneficial spatial at
detectable energy, again preferably light, can be used rangemrits provided by the rigidity of the inicroenvir
when practicing this invention. Among such auxiEaiy 5 onment. Auxiliary fluorophores that are insoluble or
fluorophores that can be used, alone or in combination, partially insoluble in aqueous medium can be solubilized
we the following substances whose residues can be by first grafting them onto salubilizing runlectiles, e.g.,
present in known chemiluminescent 1,2-dioxetanes, water soluble oligorner or polymer molecules.
such as those disclosed in the abovementioned copend- And, in all cases, enhe cement of the intensity of the
ing Bronstein, Bronstein at al and Edwards appliudions, 10 light emitted by decomposition of the chemidurnmes
as fluorescent chromophore groups; cent 1,2-dioxetane compounds used in the improved
andiraceme and anthmocne derivatives, e.g., 9.10- methods of this invention earned out in aqueous media
diphenylanthracene, 9-methylauthmoene, 9-arithracene can be achieved by the'methods disclosed in the afbrý
carboxaldehyde, anthryldcohols and 9-phenylanthra- mentioned Voyta et al application.
cene; 15 In order that those skilled in the art can mom fully
rhodammis and rhodamine derivatives, eg., rhDdols6 understand this invention, the following examples are
tcuamethyl rhodamine, tetiaethyl rhodamine, di- set forth. These examples are given solely for purposes
Pb=YldmIdbYl rhrxtlmmný d'Phcnyldlethyl rhodarame of illitatration, and should not be considered as expresi;
and dinaphdiyl rhodaimme; ing limitations unless so set forth in the appended
fluorescein and fluorescein derivatives, e.g., 5- 20 claims All parts and percentages are by weight, unless
iodoacetamido fluorescein, 6-iodoacetaundo fluorescein otherwise stated.
and fluorescem-54nakinaide,
oolmarin and cournarin derivatives, ýg, 7-dialk- EXAMPLEI
ylandrio-4-methyleritimarlia, 4mbromomethyl-7-methox- A dual channel assay for Humari Chorionic Gonado
ycoumarta and 4-bromomethyl-7-hydroxy cournarm; 25
erythrosin and erythrown dcxivativesý trophis (ft-cham), P-HCG, and Human Lutrinizing
eg-, hydroxy Hormonm HLH, is carried out as follows:
erythrosins, erythrosin4-iodoacetanxide and erythrosin
5-malcimide; MATERIALS
aciridine and achridine derivatives, e.g., hydroxy
aciridines and 9-methyl aciridine; 30 A round nylon membrane (approximately I inch in
pyrenc and pyrene derivatives, eg.. N-0-pyrime) diameter) containing two sets of covalently inanobi
iodoncetaraide, hydroxy pyrenes and I-pyrenamethyl lized capture monoclonal aunbodies is used, one set for
iodonodate; jg.HCG available from Medix Biotech. Anti HCG. Cat.
stilbene and stfliene derivatives, e.g., 6,6'-dibromos- No. H298-01, and the ý'Ond for HLH also available
tilbene and hydroxy stilberies; 35 from Medix Biotech, Anti LK Cat No. L461-09. TIUS
naphthalene and naphthalene derivatives, e.g., 5- nylon membrane is stored in a fail pouch until used.
dimethylamino naplithalene-l-suffanic acid and hy- Mouse monoclonal and S-HCG available from
droxy naphthalems; Medix Biotech, Cat. No. B298-12, is conjugated with
nitrobenzmadiazoles and mixotienzomiliazole deriv- alkaline phosphatase using the glataraldebyde coupling
atives, e.g., hydroxy nitrobenzoxaciJazoles, 4-chloro-7. 40 Procedure [VOller, A-, at. id., Bull, World Health Org.,
nitrobenz-2-oxa-1,3-diawle, 2-(7-nitrobenz-2-oxa-1,3- 53, 55 (1976)land used as a detection antibody for 0
dinaul-4-yl) amthylaminoacetaldehyde and 6-(7-mtmb- HCQ.
ý-2-oza-1,3-dmwl-4-yl-aminobexanoic acid; Mouse monoclonal anti HLH available from Media
quincline and quinoline derivatives. e.g., (ýhydrox- Biotech, Cat. No. L461-03, is conjugated to carboxyles
yquinoline and 6-amintiquincifine, 45 texase also using the gluturaldehyde coupling procedure
andme and fidine derivatives, g, N thylacný referenced above, and used as a detection antibody for
dine and N-phenylacridine; HLH.
acidoxcridine and acidoacridine derivatives, e.g., The substrate buffer solution contains 0.05M carbon
9-methylacidonFidine and hydroly.9,0,thylacidoacri. ate, I mM MSC12, al% by weight BSA (pH=0.5) and
dine; so 3-(2spiroadý tý)4-methoxy-4(3-phosphoryloxy)
carbazole and carbawle derivatives, e.g., N-methyl- phenyl-1,2-dioxetane disarlitint salt, (50 Ag/ml), and
carbazole and hydroxy-N-methylearbamole; 3.(2'.spkoadamantane)4-(3"-benzothiazol-2-yi-7"-p
fluorescent cyanines, eg. DCM (a laser dye), hy- galwtýyloxy)benzo-2H.pyran-2'.yl.1,2ýRoxeane, (50
droxy cyanines, 1,6-diphenyl-U.5-hexatriene, 1-(4- lLg/ml) w the chernflumduescent substrates.
dimethyi aminophenyr)-6-phenylbentriene and the cor- 5s The wash buffer contains 0 05M carbonate, I mm
responding 1,3-butedienes; MgCl2=d 2% by weight BSA (pH=9.5).
carbocyanines and carbocyanine derivativesý e.g.,
phany1carbocyanine and hidroxy carbocyanines; ASSAY PROCEDURE
pyridinium. salts, e.g-, 4-(4-dialkyldiaminostyryl)N- Five drops of a previously collected urine sample are
methyl pyridinium iodate and hydroxy-substituted pyri. 60 placed onto the center of the assay membrane and al
duriurn sidtie lowed to soak into the membrane. Next, five drops of a
oxonoX- and solution containing A-HCG and HLH conjugated deý
resorrifins and hydroxy resortifins. tection antibodies at a concentration of 0.01 miHimolar
When such auxiliary fluorophores are bonded to a are added to the assay membrane. The liquid is allowed
chemiluminescient compound, they are preferably 65 to soak in for at least one minute. Six drops of the wash
bonded to the portion of the chemilummescent com- buffer are slowly added and allowed to soak in and
pound that, upon decomposition, forms a fragment con- drain for 30 to 60 seconds. Then, five drops of the buffer
taining the flucirciphore portion of the clictailumines- solution containing cherailumnimccift substrates we
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Last modified: November 3, 2007