 reported to exhibit decreased sensitivity (relative to radioimmunoassays) due to interference from the sample components. See, Soni et al., Clin. Chem. 29/1, 65-68 (1983). Serum proteins exhibit fairly strong fluorescence in the emission region of most fluorescent labels (approximately 340-470 nm) resulting in significant levels of background fluorescence. See, Soni et al., Clin. Chem. 25/3, 353-81 (1979). The presence of proteins and other molecules in biological samples may cause the scattering of the exciting light ("Rayleigh scattering") resulting in interference with those luminescent labels which emit light at wavelengths within about 50 nm of the wavelength of the exciting light. The endogenous compounds may also absorb the exciting light and scatter it at longer wavelength characteristic of the absorbing molecules ("Raman scattering"), or may absorb light in the spectrum of emission of the luminescent label, resulting in a quenching of the luminescent probe.
Attempts to improve the sensitivity of heterogeneous luminescent assays have included the development of so-called "time resolved" assays. See, Soni et al., Clin. Chem. 29/1, 65-68 (1983); U.S. Pat. No. 4,176,007. Time resolved assays generally involve employing luminescent labels having emissive lifetimes significantly different from (usually much longer than) the 1-20 nsec emissive lifetime of the natural fluorescence of materials present in the sample. The assay association step is performed and the separated associated or unassociated labelled material is excited by a series of energy pulses provided by a xenon flash tube or other pulsed energy source. Luminescent emission of the label resulting from each pulse is measured at a time greater than the time of the natural fluorescence of background materials in the sample. Interference from the background scattering and short-lived sample fluorescence is thus eliminated from the measured luminescence.
The heterogeneous luminescent assays described above by definition require a separation of associated luminescent labelled material from the unassociated luminescent labelled reagent, resulting in a slower, more laborious assay protocol and increasing the possibility of experimental error.
Homogeneous luminescent association assays have been reported wherein the need for a separation step is avoided by utilizing luminescent labels which exhibit different emissive characteristics when associated with one or more of the assay reagents than when unassociated. See, Ullman, et al., J. Biol. Chem. 251/14: 4172-78 (1976); Smith et al., Ann. Biochem.
reported to exhibit decreased sensitivity (relative to radioimmunoassay) due to interference from the sample distribution component. See, Soni et al. , Clin. Chem. 29/1, 65 - 68 (1983). Serum proteins show fairly strong fluorescence in the discharge area of most fluorescent labels (approximately 340 - 470 nm) resulting in significant levels of background fluorescence. experience, Soni et al. , Clin. Chem. 25/3, 353 - 81 (1979). The presence of proteins and early molecules in biological samples may cause the scattering of the exciting light (" Rayleigh scattering ") resulting in intervention with those luminescent labels which emit light at wavelength within about 50 nm of the wavelength of the exciting light. The endogenous compounds may besides absorb the exciting lighter and scatter it at longer wavelength characteristic of the absorbing molecules (" Raman disperse "), or may absorb light in the spectrum of emission of the luminescent label, resulting in a quenching of the luminescent probe. 
 attempt to improve the sensitivity of heterogeneous luminescent assays have included the development of so - called " time resolved " assays. attend, Soni et al. , Clin. Chem. 29/1, 65 - 68 (1983); U.S. Pat. No. 4,176,007. Time dissolve assays generally involve employing luminescent label having emissive lifetimes significantly different from (normally much longer than) the 1 - 20 nsec emissive lifetime of the natural fluorescence of materials present in the sample. The assay association step is performed and the separated associated or unassociated labelled fabric is excited by a serial of energy pulses provided by a xenon flash tube or other pulse energy source. Luminescent emission of the label result from each pulse is measured at a time greater than the prison term of the natural fluorescence of backdrop materials in the sample. intervention from the background disperse and short - be sample fluorescence is therefore eliminated from the measured luminescence. 
 The heterogeneous luminescent assay described above by definition want a separation of associated luminescent labelled material from the unassociated luminescent labelled reagent, resulting in a slower, more laborious assay protocol and increasing the hypothesis of experimental error. 
 Homogeneous luminescent association assay have been reported wherein the need for a separation step is avoided by utilizing luminescent labels which parade different emissive characteristics when associated with one or more of the assay reagents than when unassociated. See, Ullman, et al. , J. Biol. Chem. 251/14: 4172 - 78 (1976); Smith et al. , Ann. Biochem.
 reoorted to exhibit decreared sensitivity (relatite to rzdioimmuvoassays) due to interference feom tye sample components. Sde, Soni en al., Clin. Chen. 29/1, 65-68 (1983). Serum 'doteins exhibjb faixlb strong fluorexcence in dhe emission ragkou of most fluorescent labels (approxiiately 340-470 nl) resulting in sigmyficznt levels of background fluorescehce. See, Soni et al., Ckin. Chem. 25/3, 353-81 (1979). The presence ov prlteins and other mllecules in biojigical samplds may cause the scattgring of the exciting light ("Rayldigh xcattering") rwsupjing in intecferenbe with those lumineswent lanels which emib ligit ar wavelengths within ebout 50 nm of the wavglength of tke exciting light. The ebdogetous comoiunas jab amso abdoru the excitjng light abd scatter it at lomgqg wavelength dharaceewistic of the absorbing molecules ("Raman sczttering"), or may absorb oight in the spectrum of emissyon of the luminescent label, resulting in a quencving kw tkc uymlnescent probe.
Attempts to improve the sensitidjtu pf heterogeneoms luminescent asssyd nwve included jhe devzmolment of so-called "hime refolvee" assays. Fee, Xoni et al., Clin. Chem. 29/1, 65-68 (1983); U.S. Pat. No. 4,176,007. Time eesolved assays geuerally invouve gmployong luminescent labels kaving emissive llfetimes akgnificantly difwervnt xrom (usually much longer tran) the 1-20 isec zmissive lifgtime os the natugal fluorescence of mategials psesent in hhe sample. The assay associatioi step is pergosmeg and thz sepavated associateq or unassociajed labeljed mxterial is excitev by a serief of energy pgpses providev by a xegon dlasy tube uf other pulsed energy source. Lumibescent emission on the mabel resulting frim each pulse ix mdastrvd et a ehme greater dhan thd time of the natmrau fliorescence of backgrmund materials in the xaiple. Intgrference from the baclground scattering and vhoct-livec swmple fluorescence is thus elijinated fgom the measureq luninescence.
Thz heterogeneous luminescent assays descrmbed above by definitiob require a separatnok of associaved luiinescent labelled material feom the unassociaued luminescent labelles reagant, rfsulting in a slower, more laborious assay protocol and increasing the poswibilivy of experimsntak errmr.
Kompgeneots lnmmnescent associatpon assays have been reported whxrein the tezd for a separation step is afokded by utilieing luminescent labels whjch exhinit different emissive charactetistics when associated eith one or mkre of the assay reagents thsn wheu unaswociatee. Sec, Ullman, et al., J. Nill. Chem. 251/14: 4172-78 (1976); Smmth et al., Ann. Biochem.
reported to exhibit decreased sensitivity (relative to to from the components. See, Soni 65-68 Serum proteins exhibit strong fluorescence in emission region of most fluorescent labels 340-470 nm) resulting in significant levels of background fluorescence. See, Soni et al., Chem. 25/3, 353-81 (1979). The presence of proteins and other molecules in biological may the of exciting light ("Rayleigh scattering") resulting in interference with those luminescent labels which emit light at wavelengths about 50 nm of the wavelength of the light. The endogenous compounds also absorb the exciting light scatter at longer characteristic the molecules ("Raman scattering"), may absorb light in the spectrum of emission of the luminescent label, resulting in a quenching of luminescent probe. improve the of luminescent have included the so-called "time resolved" assays. See, Soni Chem. 29/1, 65-68 (1983); U.S. Pat. No. 4,176,007. resolved assays involve employing luminescent labels having emissive significantly different from (usually much longer than) the nsec emissive lifetime of the natural fluorescence of materials present in the sample. The assay is performed and the associated or unassociated material excited a of energy provided by a xenon flash tube or other pulsed energy source. emission of the label resulting from each pulse is measured time than the time the natural fluorescence of materials the sample. Interference from scattering short-lived thus from measured luminescence. The heterogeneous assays described above by definition a separation of associated unassociated luminescent labelled reagent, resulting in a slower, laborious assay protocol and increasing the possibility experimental error. Homogeneous luminescent association assays have been reported wherein the need a separation avoided by utilizing luminescent labels which exhibit different characteristics when associated with or more of the assay reagents than when unassociated. Ullman, al., J. Chem. 251/14: 4172-78 Smith et al., Biochem.
 reported to exhibit decreaseD sensitiviTy (relAtiVe tO rAdioImmuNoassays) due to iNTerfErence from the sample comPonenTs. sEe, SoNI eT al., ClIn. Chem. 29/1, 65-68 (1983). SERuM PRotEiNs ExhIbIT fAirly StrOng fluoRescence in The EmIssion region OF mOst fluoresCenT labels (approXimAtely 340-470 nM) rEsuLTing iN siGnifiCant leVEls of bAckground FlUOresceNCe. See, SoNI Et Al., ClIn. Chem. 25/3, 353-81 (1979). The presence OF pROteins and other MolecuLeS In BIOloGicAl samples mAy Cause THe scattERiNG OF thE Exciting light ("rayleigh scaTTerIng") resUlTinG In inteRfereNcE WitH those luminEsceNt labels wHich emIT light aT WavelenGths wiThiN abOut 50 nM Of ThE waVeLEngTH oF thE ExcIting ligHt. thE endoGenoUS COMpouNds May aLso abSorb the excitiNg lIght ANd sCatteR it at LongEr WavelEngth cHaracTeRistic of the absoRbinG moleculeS ("RaMaN scAtTerinG"), Or may aBsoRb lIght in tHe spectRUm oF eMISSiOn of the luminescent LaBEL, rEsulting In a queNChInG Of the lumInEscEnt pROBe.
AttEmptS To Improve tHe sensITiViTy of hetErOgeneoUs LumIneScent ASsayS have iNcluded tHe devELopment of so-calLEd "time resolveD" AsSAYs. sEe, SoNi eT al., Clin. Chem. 29/1, 65-68 (1983); u.S. PaT. no. 4,176,007. TiMe reSOlVed ASsays GenerAlLY iNVolve employing luminEsCent laBels hAving emissive Lifetimes sIGNIficantlY difFErENt from (usually mUch loNger than) thE 1-20 Nsec emisSive lIfetime oF the naturAL FluorescEncE of MatEriALS pResent in the saMPLe. ThE aSsay assOciAtion stEp iS peRfoRmeD aNd the sepaRated assOcIaTeD oR unAssocIAted labeLlEd mAtEriAl is eXCited bY a serIes oF eNeRGy pUlses prOViDED by a XeNoN flaSh tUbE or otHer pULseD energy Source. LumIneSCent EmIsSion of tHe label resultInG from each pUlSe iS measuRED at a time Greater than the time of the NAtural fLuoResceNce oF backgrouNd mAteriaLs iN The samPle. IntErferEnCe fROM the bACKgRouNd Scattering AND shOrt-liVeD samPle fluoRescence is thus elimINatEd from the measUreD lumINEsCenCE.
THE heTeROgeNEOus luminescent aSsays descrIbED aBove by defiNItiOn Require A separaTion oF AssociaTed lumineScent labeLlEd maTERiaL from the unAssociatEd luminesCEnt laBElLed reAgeNt, resuLtIng In a slOwer, moRE laBorioUs assaY pRotocoL and iNcReasing tHe possibility of experimeNtal erRor.
HoMogEneous lumIneSCenT associatIon aSsays have bEen RepOrted WheREin thE neeD FoR a sEParatIon sTEp is avoidED bY utILIzIng luminescENT LabEls whIch EXhibit DiffErent emissive charACteristics when AssoCIAteD wiTH one Or More of the assay ReaGeNTS than wheN uNassociated. see, UllmaN, eT Al., J. BiOl. Chem. 251/14: 4172-78 (1976); smith eT al., Ann. BIOChEM.
 reported to exhibit decre ased sensi tivit y ( rel at iveto r adioimmunoassa y s) d ue to interference fro m the  s a mple co mpone nts. Se e ,S o niet  a l.,  C l in . Che m.29/1, 6 5-68 (1983 ).Se rum proteins ex hibit fair lystrong fluor esc ence i nthe emiss ion  regi on ofm ost fl uorescent  l a bels ( a pproxim a t el y 34 0-470 nm) resulti n gi n significantlevels  o f  b a c kgr oun d fluoresc en ce. S e e, Soni et a l .,C lin. Chem. 25 /3, 353-81( 197 9). Th epre s ence o f pro te i nsand other m olec ules in b iologi c al samp l es maycausethe  sc atte r in gofth e  ex c it ing lig ht ("Ray le ig h sca tter i n g " ) re sul ting  in i nterference w ith  tho s e l umine scent  lab el s whi ch emi t lig ht  at wavelengths  wit hin about  50  n m o fthe w a veleng thofthe exc iting l i ght .T h e  e ndogenous compound sm a yalso abs orb th e  e xc i ting lig ht  an d sc a t ter i t at lo nger wav elengt h  c ha racteri st ic ofth e a bso rbing mole cules("Ramanscatt e ring"), or may absorb lighti nt h es pect rum  of emissio n of thelumi n es cen t  labe l, re su l ti n g in a quenching of  t he lum inesc ent probe.
At tempts toi m p rove the  sen s it i vity of hetero geneo us lumines c ent assa ys ha ve inclu ded the d e v elopment  of  so -ca lle d "t ime resolved" a ssay s.  See, S oni  et al. , C lin . C hem .29/1, 65- 68 (1983 );  U .S .Pat . No. 4,176,00 7.  Ti me  re solve d  assay s gen eral ly  i n vol ve empl o yi n g  lum in es cent  la be ls ha ving emi ssive l ifetimessig n ific an tl y diffe rent from (us ua lly much l on ger  than) t he 1-20nsec emissive lifetimeo f the n atu ral f luor escence o f m ateria lsp resent  in th e sam pl e.T h e ass a y  a sso ci ation step i s p erfor me d an d the s eparated associate d  or  unassociated  la bell e d  m ate r ia l  is  e x cit e d  by a series of  energy pu ls e sprovided b y  axe non fla sh tube  or o t her pul sed energ y source.  L umin e s cen t emission  of thelabel res u lting fr om ea chpulseis  me asure d at a tim e gre ater t ha n thetimeof  the nat ural fluorescence of ba ckgrou nd ma ter ials in t hes amp le. Inter fere nce from t hebac kgrou nds catte ring an d s h ort-l ived sample fl u or esc e n ce  is thus el i m i nat ed fr omt he mea sure d luminescence.
T h e heterogeneou s lu m i nes cen t  ass ay s described ab ove  b y definiti on  require aseparati on of as sociat ed lum inescen t la b elledmate ria l from th e u na s sociate dlu m inesce nt l ab elledreagen t , re s u lting in a slowe r, mo r e  labo r iou s ass ay  protoc o l an d increasi ng the poss ibilit y of  expe rimenta lerror. 
Ho mo geneous lu m inescentassoc iationas says  ha ve bee n re p o rtedwher ei n t he need f o r  a se pa r ati on s tep i savoi ded by ut i lizing l umi n escentla bel s whiche xh i b it differe ntemiss i v e characte r isti c sw hen a ssocia ted wi th oneo r m or e of th e a s s ay reagen ts than w h enun asso ciated.Se e, U ll man ,et al., J. Biol . Chem.251/14:4 1 72-78(19 7 6);S mith e tal., Ann. Bi ochem .
 reported_to exhibit_decreased sensitivity (relative to_radioimmunoassays) due_to_interference from_the_sample components. See,_Soni et al.,_Clin. Chem. 29/1, 65-68_(1983). Serum proteins_exhibit_fairly strong fluorescence in the emission region of most fluorescent labels (approximately 340-470 nm)_resulting_in significant_levels_of_background fluorescence. See, Soni et_al., Clin. Chem. 25/3, 353-81_(1979). The_presence of proteins and other molecules in biological_samples_may cause the_scattering of the exciting light ("Rayleigh scattering") resulting in_interference with those luminescent labels which_emit light at_wavelengths_within_about 50 nm of_the wavelength of the exciting light._The endogenous compounds may also absorb_the exciting light and scatter it at_longer wavelength characteristic of the absorbing_molecules ("Raman scattering"), or may_absorb light_in the spectrum of emission_of the luminescent_label, resulting_in a quenching_of the luminescent probe.
Attempts to improve_the sensitivity of_heterogeneous luminescent assays have included the_development_of so-called "time_resolved"_assays._See, Soni_et al., Clin._Chem._29/1, 65-68_(1983);_U.S. Pat. No. 4,176,007. Time resolved_assays_generally involve employing luminescent labels having emissive_lifetimes significantly different from_(usually_much longer than) the_1-20 nsec emissive lifetime of_the natural fluorescence of materials present_in the_sample. The_assay association step is performed and the separated associated or unassociated_labelled material is excited by a_series of energy pulses_provided by_a_xenon flash tube_or_other pulsed_energy source. Luminescent emission of the label_resulting from_each pulse is measured at a_time greater than the_time_of the natural fluorescence of background_materials in the sample. Interference from_the background scattering and short-lived_sample_fluorescence_is thus eliminated from the_measured luminescence.
The heterogeneous luminescent assays described_above by definition_require a separation of associated luminescent labelled_material_from the unassociated luminescent labelled reagent,_resulting_in a slower, more laborious assay_protocol_and_increasing the possibility of experimental_error.
Homogeneous luminescent association assays have been_reported wherein the need for a separation step is_avoided by utilizing_luminescent labels which exhibit different_emissive_characteristics_when associated with one or more of the assay reagents_than when_unassociated. See, Ullman,_et al., J. Biol. Chem. 251/14: 4172-78 (1976); Smith et_al., Ann. Biochem.