CA1166133A - Chemical luminescence amplification substrate system for immuno chemistry - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A system for the detection of a biological analyte of interest is disclosed which comprises contacting a sample with a fluorescer which has been conjugated to an immuno-logical specie specific to the biological analyte of interest, in the presence of an energy source which is capable of activating the fluorescer. A method for the qualitative and/or quantitative detection of a biological of interest is disclosed, which comprises:
(a) labeling an immunological specie specific to the analyte of interest with a fluorescer material which is biologically compatible with such specie:
(b) contacting the fluorescer labeled specie and the biological of interest:
(c) separating the fluorescer labeled specie/bio-logical complex:
(d) contacting the separated fluorescer labeled specie/biological complex of (c) with an energy source which is capable of activating the fluorescer label: and (e) determining the presence of and/or measuring the quantum of chemiluminescent light emitted.

Description

This invention relates to a system for the detection of a biological analyte of interest which comprises contacting a sample with a fluorescer which has been conjugated to an immunological specie specific to the biological analyte of interest, in the presence of an energy source which is cap-able of activating the fluorescer.
This invention also relates to a novel class of fluorescer materials which may be conjugated to an immuno-logical specie specific to a biological of interest in order to provide for the detection of such biological.
This invention also relates to novel fluorescer and conjugated fluorescer/immunological specie compositions useful in the detection of various biological analytes of interest.

- 2 - ~ 7 ~ le clinici~l is coI~c~nlad with cletectiIlg the pres~lce of, and quantitatively mea Æ illg a variety of substance5 via th~ use of many different analytical techniques. The most commonly used techniques employ absorbtiometry, both at visible and ultraviolet wavelengths, how~ver, emission, flame photometry and radioactivity are also ccmmonly used. A novel technique, thus far relatively unexplored in chemistry, is that employing the phenomenon of lumunescence.
Analyses based on the measurement of emitted light offer several distinct advantages over conventionally employed techniques, including high sensitivity, wqde lin~r range, low cost per test, and relatively simple and inexpensive equipment.
It has been predicted that the phenomenon of luminescence, and more particularly chemiluminescence, could have a ~ajor impact in two main areas of clinical analysis. First, it may have an important role as a replacement for conventional colorimetric or spectrophotometric indicator reactions in assays for substrates of oxidases and dehydrogenases. In this type of assay the sensitivity of the luminescence indicator reaction may be used to quantitate su~strates not easily measured by conventional techniques (e.g.
prostaglandins and vitamins).
m e second major clinical application of luminescence might be in the utilization of luminescent molecules as replacements for radioactive or enzyme labels in imnunoassay.

l In each of these major clinical application areas, chemiluminescent reactions can provide a means to achieve a

3 high level of analytical sensitivity.
S Chemiluminescence may be simply defined as the chemical 6 production of light. In the literature it is often confused 7 with fluorescence. The difference between these two phenomena 8 lies in the source of the energy which promotes molecules to 9 an excited state. In chemiluminescence this source is the energy ¦
yielded as the result of a chemical reaction. The subsequent decay 11 of molecules from the excited state back to the ground state is 12 accompanied by emission of light, which is called luminescence.
i3 ! In contrast, in fluorescence, incident radiation is the source 14 of the energy which promotes molecules to an excited state.
16 ¦¦ . . From an analytical point of vie~7, the types of 17 ¦l luminescence that have engendered the most interest are chemi-18 ¦1 luminescence and bioluminescence. The latter being the name 19 ~ givèn to a special form of chemiluminescence found in biological -20 1 systems, in which a catalytic protein increases the efficiency 21 ¦ of the luminescent reaction. Bioluminescent reactions such as 22 the enzymatic firefly process,have been very useful analytically 23 and convert chemical energy to light with a quantum efficiency 24 of ~8%.
~5 26 In contrast to bioluminescence with the longevity 2nd 27 efficiency of the firefly, the history of chemiluminescence 28 (hereinafter referred to as CL), especially that occurring in the ¦
29 non-aqueous phase, is remarkably short. The important aqueous C~. ¦
substances luminol and lucigenin were discovered in 192~ and 1935,¦
: . . I

6~3- l l 1 respectively. A series of organic soluble CL ~aterials were developed in the early 1960's based upon a study of the 3 luminescent reactions of a number of oxalate c3mpounds. A

4 typical organic system useful for CL was disclosed by Bollyka et al., U.S. Patent No. 3,597,362,and claimed to exhibit a ~uantum 6 efficiency of about 23% compared with about 3% for the best kno~m 7 available aqueous systems.

9 Chemiluminescence has become increasingly attractive for its potential in the clinical laboratory, especially for use in 11 the analysis of a number of biologically assoc1ated materials, and 12 its known appIications have been the subject of thorough reviews, - 13 see for example ~ 7hitehead et al. (1~79) Analytical Luminescence 14 ¦ Its potential In The Clinical Laboratory, Clin. Chem., 25, 9 1531-, lS 1546, Gorus et al. (1979) Applications Of Bio- And Chemilu~inescence 16 In The Clinical Laboratory, Clin. Chem., 25, 4 512-519; Isacsson 17 et al. (1974) Chemiluminescence In Analytical Chemistry, 18 Analytical Chemica Acta, 68, 339-362.
19 ` l With few exceptions, most published CL clinical ~;
21 analytical applications have made use of the less efficient but 22 well known diacylhydrazides, acridinium salts, pyrogallol, or 23 lophine structures. It is important to appreciate that due to 24 the nature of the chemical decomposition of the above chemi-luminescent structures in the presence of hydrogen-peroxide, or 26 generators of H2O2, as compared to that of the oxidation reaction 27 of diaryloxalate structures, the latter has over 20 times the 28 quantum yield of chemiluminescence, although its requirement 29 for hydrogen peroxide is greater than the former.
/l . .
,1 ll~ÇlENLI-l 1 Hydrogen peroxide, an essential component in the chemilumli nescent reaction, has usually been the species selected for use in detecting the analyte of interest. Eor example, in the determinatio 4 of glucose - Auses et al.(1975), Chemiluminescent Enzyme Method For Glucose. Analytical Chemistry, 47, No. 2, 244-248 employed 6 the oxidation of glucose in the presence of glucose oxidase as 7 the source of H22 which, in turn, was reacted with luminol to 8 1 produce chemiluminescence in proportion to the initial glucose 9 concentration. A limit of detection of 8 x 10 9M peroxide was obtained with this system. Williams et al. (1976), F.valuation 11 Of Peroxyoxalate Chemil-~minescence ~or Determination Of Enzyme 12 Generated Peroxide. Anal. Chem., 48, 7 1003-1006 in a similar 13 reaction concluded the limit of sensitivity of the peroxyoxalate 14 system is an order of magnitude poorer than that of the luminol system.

17 Therefore, until now the oxalic ester system (oxalate 18 system) was generally thought to have little utility for analyti- ' 19 cal purposes due to its inefficient conversion of hydro~en peroxide 21 The present invention overcomes this deficiency of 22 ~22 dependence by making use of the large chemiluminescent 23 reservoir of energy in the oxalate system's chemistry. By 24 using a suitable quantity of hydrogen peroxide and oxalate, a vast amount of energy may be concentrated in a form which is 26 then released as chemiluminescence upon the introduction of a 27 conjugated fluorescer.

29 Thus, the oxalate, acting in a fashion which can be ~isualized as analogous to a charged chemical battery, releases ..' the stored energy to the fluorescer-conjugate in the same manner as an electrical switch in a circuit releases the energy of a battery to a lamp. This l'switch" action causes chemilumine-sclence and, by conjugating the fluorescer to a detector of the analyte of interest, one can employ the reaction to trigger a detection system both qualitatively and quantitatively related to the analyte to be measured.
In one aspect of the invention there is provided a system for the detection of a biological analyte of interest comprising a fluorescer-catalyst which has been conjugated to an immunological species specific to the biological analyte of interest, in the presence of an energy source other than electro-magnetic radiation which is capable of activating the fluorescer-catalyst.
In another aspect of the invention there is provided a qualitative method for the detection of a biological analyte of interest comprising:
(a) labeling an immunological species specific to the analyte of interest with a fluorescer-catalyst material which is biologically compatible with such species, (b) contacting the fluorescer-catalyst labeled species and the biological of interest, (c) separating the fluorescer-catalyst labeled species/-biological complex, (d) contacting the separated fluorescer-catalyst labeled species/biological complex of (c) with an er.ergy source other than electromagnetic radiation which is capable of activating the fluorescer label, and (e) determining the presence or absence of light emitted from the activated fluorescer-catalyst.
In a particular embodiment of this aspect of the invention the fluorescer-catalyst labeled species/biological A~

complex is not separated prior to activating the fluorescer-catalyst label.
In a further aspect of the invention there is provided a Iquantitative method for measuring the amount of a biological analyte of interest comprising:
(a) labeling an immunological species specific to the analyte of interest with a fluorescer-catalyst material which is biologically compatible with such specie;
(b) contacting the fluorescer-catalyst labeled species and the biological of interest, (c) separating the fluorescer-catalyst labeled species/-biological complex:
(d) contacting the separated fluorescer-catalyst labeled species/biological complex of (c) with an energy source other than electromagnetic radiation which is capable of activating the fluorescer label; and (e) determining the amount of light emitted from the activated fluorescer-catalyst.
In an embodiment of this latter aspect of the invention the fluorescer-catalyst labeled species/biological complex is not separated prior to activating the fluorescer-catalyst label.
T~e invention also provides a novel class of fluores-cer-catalyst materials which may be conjugated to an immuno-logical species specific to a biological of interest in order to provide for the detection of such biological.
Thus in accordance with another aspect of the invention invention there is provided a fluorescer-catalyst composition useful in the labeling of an immunological species specific to and for the detection of a biological of interest, such fluorescer-catalyst having a chemical structure which possesses one or more functional groups capable of chemical reaction with the immunological species without adverse effect on the specificity of such species to the biological of int:erest.
The invention also provides a novel class of con-jugated fluorescer-catalyst/biological compositions useful in the detection of various biologicals of interest.
Thus in accordance with yet another aspect of the invention there is provided a conjugated fluorescer-catalyst/-immunological species composition useful in the detection of a biological of interest which has been formed via reacting an immunological species with a fluorescer-catalyst having a chemical structure which possesses one or more functional groups capable of chemical reaction with the im~unological species without adverse effect on the specificity of such species to the biological of interest.
Additionally, there is provided for novel fluorescer and conjugated fluorescer/immunological specie compositions.
With respect to Charts I, II and III, Rauhut et al.

_ 9 _ Ai`
-., il33 E'.~I-l 1 (1969), Chemiluminescence From Concerted Peroxide Decomposition Reactions, Accounts of Chemical Research, Vol. 2, 80-87, it can 3 be seen that one mole of H202 is necessary to convert one mole 4 of luminol into one mole of the energized or excited molecule.
This excited molecule then revèrts to its ground state and emits 6 light. Of interest is the fact that the CL compound, in Chart I, !
7 luminol or its derivatives, is also capable of converting the 8 chemical energy of the system to light. Thus, the luminol acts 9 as a source of CL energy and also as a fluorescer to absorb thè
energy and produce visible light. The luminol system is, thereforc 11 not particularly useful in the context of the present in~entlon 12 since no differentiation between the light emitted upon fluorescer i3 addition and that generated by the luminol itself can be made.

Charts II and III illustrate the fact that for the OX2-late system, hydrogen peroxide does not always produce a species 17 which gives rise to an excited state producing light. Some perox-' 18 ide may be lost in side reactions which are "dark", thus, there 19 lis no predictable stoichiometric relationship ~etween the H2~2 consumption and the quanta of emitted light.

26 /l 28 !/

l/

Chart I
3-Aminopht~halhydrazide Chemiluminescence in Reaction with Pot;assium Persulfate and Hydrogen Per~xide (Luminol) NH2 o NH2 o ~$ + S2022- slow~> ~

l 2 fast N

O decompositio~

~ + N2 O O

_ _ NH2 1l _ ~ fast > ~ + hr 11~;6~33 Chart II
Tentative ~ech~lism for Oxalyl Chloride Chemiluminescence 1~ ClCCCOH + HCl C Cl ~ 00 ClCCOH + HCl 2HCl + 00 + C~2 12 ~ HCCCCOH

decomposition H20 14 + H O
" ____~_ 2 2 13 ~ deco~position 14 ~ æ 2 + H20 + flr' deccmposition flr' > light + f}r , i !

3~

Chart III
Tentative Mechanism for Oxa1ic Ester Chemi1uminescence RooooR + H22 ~ ~OCC-- OR
base ~5H

~ COOH + ROH
15 ~ 16 ~ decomposition ~ ~ + ROH

16 < 17 H + ROH

13 17 + H2O2 17 ~ 2CO2 + flr-deccmposition flr ~ flr + hr f i - 12 -A major difference between the lu~inol system, which has keen used to detect the presence or the quantity of H202, and the oxylate system is the requirement that the oxalates have an addi-tional fluorescer to absorb the chemical energy generated in the reaction and t~en convert that energy to visible light. If the specified fluorescer is absent, the energy generated by the reaction will be dissapated without emitting visible light. The oxalate system is generally employed in an organic solvent and this requirement also has made its use in CL-analytical methods less desirable than other CL materials, which are soluble in an aquPous medium, due to the inccmpatibility of biological anti-analytes to such organic solvents.
; The present invention dramatically differs fram the prior art utilizing CL for analytical purposes in the way the s, !

'I - 12 a -lit;~;l;~3 ,. `` ..
E~LI-l 1 generated CL energy is employed. The present invention makes 2 use of the CL system as a substrate or reselvoir of chemical 3 energy which emits light upon the addition of another compound, 4 i.e. the fluorescer. We have found that by conjugating this fluorescer compound to the anti-analyte of in~erest it is possible to quantify the analyte's concentration in terms of the amount of 7 emitted light. CL as thus applied becomes competitive as a highly' 8 sensitive replacement for radioimmunoassay techniques (RIA).
.9 .' '.
The comparison of Table l-shows various analytical 11 systems employing C~ and illustrates the manner in which components 12 of different reactions may be used to achleve detection. An 13 analyte may be determined using CL by coupling the detector for 14 the analyte to either:
I. A catalyst for generation of the H2O2 CL re~ction, 16 such as glucose-oxidase, or 17 II. A CL compound which generates CL energy and itself 18 emits light, such as luminol, or 19 III. A fluorescer which absorbs chemical energy and e~its' light, such as a perylene derivative.

22 In each case, for the purpose of simplicity in this 23 comparison, the analyte is assumed to be surface antigen to 24 Hepatitis B ~HBSAg) in human serum and is determined by a solid phase "sandwich" technique. This syste is presently widely used ¦
26 with I125, a radioactive isotope, as the la~el or indicator.
27 l/

//

~ 116fi1 33 .,NLI-i 1 e ~ _~ e u e ~ o c~ ~-o u c~ E C~
2 ~ ~ ~ U > ~ o~ - E

. u u - ~ 5 u ~ u - c c ~ Y ~ ù E
. ~ ~ O ~ ,. ~ O ~ N ~ ~ C ~ ~., ~ ~ e ~ ~ ~ 2 u E ~I c~ X ~ ~ ~ C ~ c o 6 ~ ~ _, U ~ o ~ D O ~ ,D ~ ~E u O v c v 3 ~o o o ~ o ~ c~ o c~ ~ ~ ~ o c~ -I
,C C U k o u ~; C ~ u~ U -O U _I C
~ o ~ r ~ V E
8 z .~ ~ ~ S ~ ~ ~ V~ - ~ ~ ~ 03 E ~ ~ c ~ ~ cX X ~ O

1 0 ~ ~ N 1'~ ~J Ul 11 _ C~ ~ r h 12 z 1~ h U ~ u r 13 ~ ~ c 'o4 ~ ~ c 14 e E ~ v u , r ~ ~ ~r--~ .

1 5 H 1~O 1: ~ ~1~ C O C) ~ ~ ~ ~ h 16 ~ c~c o co~ _~, c, E ~ ~ v ~
¢ O ~ ~ v ~ ~
1 7 E~ ~ c~ c cc loo o ~ o 'Q. tJ ~ c _ l . ~ ._ E E~ E ~ ~ o c) c) c o 1 8 ~ o c ~ ~ o _1 N r~ ~
19 I o e E r~~ C ~ ,~ = r ~ v 2 2 Tl ~ c ~:) ~ > u c ~ o u 23 5 o - ~ EX: O U V C ~ r E5 ~ _ ll 3 E O ~ E~ E ~o O~ a 3, 2 6 ~ u~ 3; . .~

23 ~4cu~ O
29 ¦ ~ o , . u~ .
. -14-Fbotnotes to Table 1 1 No solid phase system incorporating the advantages of a separation of CL, enzyme amplification and ~nnunological chemistry has appeared in literature as described here.
2 Williams et al. (1976) EYaluation of Peroxyaxalate, Chemilumi-nescence for Determination of Enzyme Generated Pero~ide, Anal. Chem., 48, 1003-1006.
_ 3 Puget et al. (1977) Light ~mission Techniques For The Micro-estimation of Femtogram Le~rels of Peroxidase.
Anal. Biochem., _ , 447-456.
4 Velan et al. (1978) Chenu`luminescence Dnnunoassay A New Method For Deter~ination of Antigens.
DmmNnochemistry, 15, 331-333.
~kCapra et al. (1977) Assay Method Utilizing Chen~luminescence.
British Patent No. 1,461,877.
6. Hersh et al. (1979) Luminol-Assisted, Ccmpetitive-Bindin~
~mmunoassay of Human ~nnuno-Glokulin G.
Anal. Biochem., _ , 267-271.
7 Pratt et al. (1978) Chemiluminescence-Linked Imnunoassay.
Journal of ~mmunological Methods, 21, 179-184.
8 Simpson et al. (1979) A Stable Chemiluminescent-L`abelled Antibody For Immunological Assays.
Nature, 279, 646-647.
9 Schroeder et al. (1979) Dnnunoassay For Serum Throxine Monitored By Chemiluminescence.
Journal of Immunological Methods, _, 275-282.
10 Olsson et al. (1979) Luninescence Inmunoassay (LI~) A Solid Phase ImmNnoassay Monitored By Chemiluminescence.
Journal of Dmmunological Methods, 25, 127-135.
~

l l In order to detect the antigen-antibody reaction the 2 indicator in all cases illustrated in-the comparison of Table l ¦is taken to be the emission of light from CL. In the '`sandwich 4 Itechnique'', the following steps are taken: ~nti-HBs (Goat) is S ¦coated to controlled pore glass (CPG) particles in tablet fonm I(solid phase). Patien. serum is added to a tube containing a C~G
7 tablet. During incubation the tablet disintegrates. If Hepatitis 8 B Surface Antigen is present in the ser~m tested, it will combine 9 with the antibody on the glass particles. After incubation, the 10 ~!serum is removed and the glass beads rinsed. A label, as dis-ll Icussed below, conjugated to an anti-body specific for HBSAg is 12 !Ithen added. The labeled antibody combines with the antigen bound 13 to the antibody on the glass particles forming the "sandwich".
14 IThe labeled antibody then reacts in a specifled ~anner in the CL
15 ~! system to give light as an indication of antigen presence. This 16 IICL assay is a qualitative test for the presence of Hepatitis B
17 ll Surface Antigen in serum. In general, however, the gre2ter the' 18 1l amount of HBSAg in a s2mple, the greater the intensity of emitted `
19 11 light.
20 ~
21 I The reaction sequence and procedures used in carrying out 22 Ithe Methods' illustrated in Table l were as follows:

24 I Meth'od'I - Enzyme Chemil~ninescent'Immun'o'as'say Label: Antibody to Hepatitis B Surface Antigen conjugated with 26 glucose-oxidase (GLO).

28 Reaction: ~2 Glass.ab.ag + ab.GLO + glucose ~ H2O2 29 1 C22 Luminol ~ NaOH + H22 Cfrom reaction l) ~ light l -16-133- ~

1 Procedure: After incubation of the oxidase label to f~rm the 2 "sandwich" as described abov2, the complex is washed to remove excess label. The washed complex is then incubated for a fi~ed 4 time with a standard glucose solution to allow the glucose sub-strate to form H2O2, the quantity of which is proportional to the 6 original GLO present in the sandwich. An aliquot of the solution ¦
7 is then added to a standard catalyzed alkaline l~minol solution 8 with the light emission proportional to the HBSAg in the ori~inal 9 sample.

11 Method II - Chemiluminescent-labeled Immunoassay .
12 Label: Antibody to ~epatitis B Surface Antigen labeled with lu~inol ¦

15 ~ ~Reaction: ) Glass: ab.ag.ab.1um1no1 + H2O2+ hemin + lioht 16 Procedure: After incubation of the luminol label to form the 17 "sandwich" as described above, ~he complex is washed to remove 18 excess label. To the washed complex is added a standard hydr~gen 19 peroxide alkaline hemin reagent: The light emission is p~opor-tional to the HBSAg in the original sample. It is noteworthy that 21 HeIsh et al. (1979 Luminol-~ssisted, Competitive-Binding ~mmuno- i 22 Assay Of Human Immuno-Globulin, G. Anal. Biochem., 93 267-271, end !
23 their paper describing a similar use of luminol with the foIlowing 24 summary:
"The luminol-based chemiluminescent label can be 26 employed as a su~stitute for radiolabels in immunoassay 27 for serum components at concentrations greater than 10-9 28 mol/liter. The main factor limiting the sensitivity of 29 the method is the relatively low overall chemilu~inescent efficiency (CE~ of the luminol tag. The CE Of underiva-31 tized luminol is reported to be 1.5% (5~. Our luminol-ll~bl;13 FI~LI-l 1 possible that a more efficient means of coupling luminol, I
2 if found, would increase sensitivity by a maximum of 600%. The most efficient chemiluminescent system 4 reported to date (not involving enzymes) is the hydrogen S peroxide-oxalate ester reaction C6~. This reaction has 6 an overall chemiluminescence efficiency of 23%. The 7 use of the oxalate ester as a chemiluminescent label 8 would provide the more substantial gain of 1500% compared 9 to the luminol system."
11 Thus, while earlier investigators recognized ihe quantum.
12 efficiency of the oxalate system for CL, they, like others, never 13 appreciated the most efficient way to use this oxalate as a source 14 of energy, would be by controlling the "switch" and not the "source" of the energy.

17 Me hod III - Chem_luminescent Labeled Light Amplification System 18 (The method of the present invention~ cLAsslc`l2 lq Label: Antibody to Hepatitis B Surface Antigen conjugated to a perylene derivative fluorescer~

22 Reaction: Cl2 Glass.ab.ag.ab. perylene-+ TCPO + H22 ~ light 24 Procedure: After incubation of the perylene label to form the "sandwich" as described above, the comple~ is washed to remo~e 26 excess label. The "sandwich" is t~en washed with tertiary butanol 27 to remove excess buffer salts. Then an excess of bistrichloro- j 28 phenyl oxalate and hydrogen peroxide in dimethylphthalate are 29 added to cause t~e fluorescer conjugate to e~mit light. The light emission is proportional to the HBSAg in the original sample.

-18- `
." . .

lii33~
.` . . ' ENLI-l 1 The light intensity may be measured qualitatively by eye, or 2 quantitatively by using a photodlode in the sa~e manner that a I photomultiplier in proximity to a sodium iodide crystal responds 4 to the photons released by the gamma rays from the I125 label.
S`
DISCUSSION OF ~THODS I, II AND III
8 The use of an oxidizer conjugated to an antibody 9 (Method I) is in reality an adaptation of the well-known enzyme-10 1 immunoassay systems of S~Ja Corporation CU.S. Patent ~o. 3,817,837?
11 ¦ and Organon Co. (U.S. Patent No. 3,654,090) but here using CL as 12 ~ a light indicator instead of a dye color change. We are not aware 13 of an analogous system lncorporating all the solid phase sequences 14 suggested herein. Nonetheless, the detection limit of this 15 1 method is governed by the ability of the oxidase enzyme conjugate 16 to liberate sufficient H22 as in the above enzyme i~munoassays.
17 Some increase in detection level may be 2chieved by using CL
18 because of the better sensitivity o~ CL vs. dye color ch~nge, 19 ¦ this sensitivity does not however ~?proach tne detection le~el of the fluorescer conjugate of Method III.

22 In Method II a number of analysts have suggested labeling 23 the analyte detector with a CL compound or derivative. This 24 method is inferior to Methods I or III in that the amount of light emitted can never be more than the tot~l energy content of the 26 amount of CL compound conjugated - i.e., luminol or oxalate. A
27 further disadvantage in coupling the CL compound directly to the 28 antibody, for example, ls the loss in CL capacity of the conjugate 29 and the continued loss of li~ht as the compound is consumed in the reaction. Finally, the entire loss of the consu~ed C~ compounds Il -19-~ 3 `

c~ l 1 before test completion prevents the analyst from repeating or 2 rechecking the sample's CL.
4 Method III, alternatively referred to as "CLASSIC", the I
method of the present invention, o~ercomes the inherent disadvan- I
6 tages of Methods I and II. With "CLASSIC" it is possible to 7 achieve the highest order of activity and specificity of the B analyte detector because one can carefully select the preferred 9 attachment site on the biological to be labeled. It is also possible to design the linkage of an eficient and durable 11 fluorPscer to conjugate with the biological effectively at this 12 site without damaging the biological. Damage in specificity and 13 activity of biologicals from I125 labeling, and damage to enzymes 14 by conjugation is well kno~ and an accepted act in the prepa-ration of immunodiagnostic reagents. A fluorescent label of 16 preferred utility in CL, by its ~ery structure, must be stable to 17 ~ the oxidizing conditions of the oxalate reaction. This inertness 18 augers well in making fluorescers a particularly efficient form 19 of 12bel for immunochemical analyses.
.
21 The various levels of sensitivity and variations in 22 different types of amplification is e~aluated in a 1976 review 23 by G. Wisdom, Enzym,e-immunoassay, Clin. Chem., 22 1234-1255.
24 T~ese systems provide the amplification for enzyme labels since enzyme catalytic properties allow them to act as amplifiers, and 26 many enzy~e molecules can catalyze the formation of more than 105 1 27 product molecules per minutes.

29 To be suitable as-a label, an enzyme must meet the several criteria set forth by Wisdom (1976~ Csupra~ which are . - . I
~ -2Q-~

1 as follows:
- (1) Available cheaply in hig~ purity.
C2~ High specific activity.
4 (3~ Stable under assay and storage conditions.
(4~ Soluble.
6 C5) Assay method that is simple, sensitive, rapid, 7 and cheap.
8 ` (:6~ Absent from biological fluids.
9 (7) Substrate, inhibitors, and disturbing factors, 2bsent from biological fluids.
11 (8) Capable of retaining activity while undergoing 12 app~opriate linkage re2ctions.
'3 (92 Capable of inhibition or rèactivation when anti-14 body binds to the enzyme-hapten conjugate.
' Clo) Assay conditions compati~le with hapten-antibody 16 ~inding.

18 These specifications are easily met by fluorescent 19 Qrgànic compounds ~hich may be readily coupled 2s labels capable of absorbing the ch~mical energy from the oxalate "substrate".
21 In addition, as has been shown ~y Rau~ut, certain selected 22 1uorescer structures ar~ capable of catalyzing the peroxyoxalate 2~ reaction products, thus pro~iding the type of amplification 24 a~ailable with enzymes. Whether such ampli~ication does in fact take place has been quesLioned by Hastings et al. C1976) P~otochem 26 Phot'ob'i'ol., 23, 461.

28 The CL system OI the present invention; '`CLASSIC`', ~l~o 29 has certain advantages over fluorescent antibody techniques which ma~e use of the ability of a fluorescent tag to emit light of a 11ti~133 LI-l 1 particular wave length when excited by radiant energy of a lower wave length. A number of clinical analyses which utilize fluore-scent "prohes" or tags have been described in a recent revie~-4 by Soini (179) Fluoroimmunoassay: Present Status And Key Problems.
Clin. Chemistry, 25, 353-361. In general, the detection level, or 6 sensitivity, bf fluoroimmunoassay techniques is greater than 7 en~yme immunoassay techniques and approaches the capability of 8 radioimmunoassay systems.
'. .9 The use of fluore~scent probes to replace radioactive 11 iso.opes is hindered by the decreased sensit~vity ob~ained with 12 fluorescence. This is due, to a great extent, to the sample's 13 or serum's own fluorescence. The intensity of this background 14 ¦¦ is affected by many fluorescing compounds, such as protein whlch 15 llmay be present in the sample, and whlch also increase scattering 16 ll caused by the specimen.
17 I'!
18 1I Fluorescence methods are now extensively applied in 19 ~! immunology, mainly in fluorescence microscopy, for studying 20 1l various types of tissues, cells, bacteria, viruses and so on~
21 ll A number of ~luorescent materials and procedures for coupling t~.em 22 ¦ to the above biologicals and haptens ls well developed~
23 I .
24 I To take advantage of the full scope of th~s invention, 25 I special high intensity fluorescent molecules are required. These 26 i¦ must be capable of biological coupling with protein, poly-27 saccharide and hapten substances, especially immunoglobulins -28 1l i. e., IgG - and antigens without disturbing the specificity or 29 ¦, activity of these biological materials.

Il -22- I
l!

~-~LI~ llin (1968) Phot~physical and Photochemical Effects 2of Dye Binding. Photochem. and Photobiol., 8 383-342 and Porro _ et al. Cl963 and 1965) Fluorescence And Absorption Spectra of Biological Stains Stain Technolo~y, Vol. 38, and Fluorescence And Absorption Spectra Of Biological Dyes (II). Stain Technology,¦
6 Vol. 40, No. 3, 173-175, respectively, have shown that there is a 7 reduction in efficiency in the light output of fluorescers as 8 a result of bonding or conjugation to proteins as compared to the ¦
9 output of these fluorescers in free solution. Our work has shown , i0 a slmilar loss in output, however, the energy efficiency of the 11 oxalate system compensates for this loss. While this loss in 12 light output effects all other known applications of conjugated 13 fluorescers, the analytical method of the present invention 14 requires a conjugate only during the biological antibody/antigen formation phase of the analysis. Procedures are well known for 16 preparing a conjugate of a fluorescer in a manner which permits 17 the conjugate to be subsequently separated at will by changing the 18 ¦pH, or other parameter, of the conjugate so]ution. It should also 19 be noted that the i~munochemical reaction of CLASSIC, ~fethod III, Imay be carried out in the environment best suited fcr the optimum !
21 detection of the analyte of interest. After the label has been 2~ identified with the analyte one may then separate the label, the 23 fluorescer, from the conjugate which allows the fluorescer to 24 enter the solvent phase of the CL system to yield the maximum light efficiency.

27 In general, it is desirable to provide the high quantum ¦
28 efficiency of fluorescing aromatic and substituted hydrocarbons, 29 heterocyclic compounds, dyestuffs, and metal chelates with the ease of conjugation to the biological now available for microscopy~

_23-.

reagents. We have found that we can couple the fluorescer using kncwn procedures currently accepted for use ~ith the fluorescent a~njugates such as set forth in Soini (1979) supra.
m e followqng Tables 2 and 3 frcm Soini (1979) supra, set forth data on various fluorescent materials which can be advanta-geously employed as labels in the environment of the present inventlon.

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*(Of = quantum yield of free fluorochrome, b = quantum yield of fluorochrome bound to protein).

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E~LI-l l In addition to the organic fluorescers listed above, a 2 number of metal organic materials have been suggested for laser fluorescent assay systems: Ruthenium (II)-tri(bipyridyl) complex 4 has been identified by Curtis et al. (1977), Chemiluminescence;
A New Method For Detecting Fluorescent Compounds Separated 3y Thin 6 Layer ~hromatography, J. Chromato~raPhy, 134/ 343-350 for CL appli 7 cations; Metal Complexes by Sherman (1978), Analytical Application 8 Of Peroxyoxalate Chemiluminescence, Analytical Chim. Acta, 97, 9 21-27, and Soini (1979) supra. Weider U.S. Patent No. 4,058,732 disclosed and sugges.ed their immunofluorescent application. It i ll also well known, Van Uitert (1960), Factors Influencing The Lumi- ¦
12 nescent Emission States Of The Rare Earths. J. Electrochem. Soc., ~3 107, 803, that smaIl additions of the rare earth and/or transition 14 metals function as promotors, activators or coactivators in inor-ganiC and organic phosphors. Thus, it is not unexpected that 16 trace impurities will behave in a similar manner in other organic 17 and metallo-organic systems and have a profound effect on the 18 quzntum éfficiency of the fluorescer.

The discussion has thus far centered around the novel 21 2nalytical use o~ a fluorescer-biological conjugate activated by 22 the chemical energy from a peroxyoxalate CL system. The preferred 23 peroxyoxalate system is advantageous for CL because of its quantum 24 efficiency and because there is no background light in the absence of a fluorescer conjugate. This system is particularly "noise 26 free'; when certain intensity control additives are eliminated, 27 such as are disclosed by Bollyky C1972) Chemiluminescent Additives 28 U.S. Patent No. 3,704,231. A system for analytical purposes need 29 only provide light of high intensity for a short period, that is, for example, under about 30 minutes.

11~i61;~ ~

E~ While peroxyoxalates which are "noise free", or non-fluorescent are preferred, other naturally self-fluorescent oxalate esters or CL compounds are also useful with the proper selection o~
4 a barrier filter and use of a conjugate fluorescer of longer wavelength. Such esters include 2-napthol-3,6,8-trisulfonic acid, 6 2~carboxyphenyl, 2-carboxy-6-hydroxyphenol, 1,4-dihydroxy-9, 10 diphenylanthracene, 2-napthol, as well as aqueous C~ materials 8 such as luminol, lophine, pyrogallol, luciferin, and related l compounds.
! -Other systems besides those mentioned are also c2~able 11 of activating a CL fluorescer-conjugate.
12 These include~ zone, which has been shown by 13 Randhawa (1967), OZonesonde For Rocket Flight, l~ature, 213, .53 to 14 activate Rhodamine-B. (2) Keszthelyi et al. (196g), Electrogene-rated Chemiluminescence: Determination Of Absolute Luminescence 16 Efficiency, etc., A. Chem., 47, 249-256, has demonstrated electro-¦
17 ll generated CL in 9,10-diphenylanthr2cene, thianthrene, and rubrene,l 18 with some systems. Thus, Ozone or electro-generated CL in the p.re-19 sence of the fluorescer-conjugate can provide other useful ener~y sources for the CL fluorescer systems of the present inven,ion. In 21 addition, other known energy sources such as have been ound uselu~
22 i~ applications involving the distortion of various pol~ers by 23 mechanical energy and other similar systems which yield free radi-24 cals are also useful in the present invention.
It should be understood that many analytical system 26 variations are possible, but all have in common the use of a 27 labelled i~munological specie specific to the anal~te. The analyst 28 has the latitude in selecting a procedure which provides the detec 29 tion level required from a minimum amount of sample and ~hich uses~
the least expensive and most reliable instrument. The detection - 31 level required is a function of the antigen, antibody or hapten 32 concentration in the analyte and its clinical significance~

_~T-l 1 For clinically signi~icant dosage testing - i.e. Digoxin standard curves are obtained from known samples analyzed together with the unknown and run under carefully controlled duplicate 4 analyses on highly calibrated instruments. While a presumative test for an immunoglobin requires a much lower level of sophis-6 tication, it is highly advantageoùs for a single analytical system 8 to be able to cover this analytical spectrum.
9 The sophisticated analytical requirements may be met by ;
using a Centrifugal Fast Analyzer such as that made by Electro- I
11 Nucleonics, Inc. Burtis et al. C1975). Development Of a 12 Multipurpose Optical System For Use With A Centrifugal Fast 13 Analyzer. Clinical Chemistry, 21 1225-1232. For the Nth nations 14 lacking the ability or need for such sophistication, or for presumptive testing at the physician's office or clinic, no instru 16 ment is required. The "CLASSIC" system of the present invention 17 delivers sufficient intensity to the labeled biological to enable 18 the clinician to make a simple go-no-go determination by "eye- '~
19 balling".
.20 21 The clinician may also modify the role of t~e labeled 22 specie used in carrying out the analyses. While solid phase 23 techniques have been used as examples to illustrate t~e advantages¦
24 of the present invention, it should be recognized that homogeneous and heterogeneous assays also will benefit from the use of the 26 "CLASSIC" system. Acceptable alternative variations in test 27 procedure include:
28 (1) Competitive binding of labeled antigen.
29 ~2) Competitive binding of labeled antibody.
(3) Quenching analyses.
..

i 1~ 3 1 (42 I~munoprecipitant reactions.
2 ¦ (5) Ion exchange ~ethods.
3 (6) Ion exclusion methods.

DESCRIPTION OF lXE PREFFERE~ EMBODIME~TS

7 The major components for the preferred "light-switch"
8 or "light indicator" of the present invention are similar to those 9 disclosed in V.S. Pa~ent No. 3,597,362. They include an oxalic ester, a hydroperoxide, a fluorescer (or fluorescent compound2 11 and a diluent. Furthermore, in order to generate max~mu~ inten-12 sity of light, the employment of an additional catalytic accelera-13 tor is sometimes necessary. The choice and the concentration and 14 other parameters of a suitable catalytic accelerator is also described in U.S. Patent No. 3,704,231.

17 The present invention differs fro~ ~he teaching of 18 U.S. Patent No. 3,597,362 in that the fluorescent compound Cor 19 fluorescer) employed in this invention is covalently bonded to a biological material, such as inmunoglobulin, enzymes, proteins, 21 bacteria, and so oni or to an organic material, such as haptens 22 or polvmers; or to zn inorganic materialj such as glass, silica, 23 ceramic, or the like. The organic and inorganic materials to 24 w~lch suitable fluorescer may be bonded can be in the form of particles, crystals, tubes, rods, plates, blocks and the like, or 26 in solution. The fluorescent compound, or fluorescer, bonded to 27 the above mentioned substances can then be utilized as a label in 28 place of radioactive materials or as an indicator in place of 29 color dye, for use in various well-known assays. -//

I -44- ' I ' ` `, 3;~
Especially suitable fluorescent ccmpounds, or fluorescers, for use in the present invention are those which have a spectral emission falling between 260 millimicrons and 1,000 millimicrons.
m e structure of the fluorescent compounds or fluorescers useful in the present invention must possess one or more functional groups capable of reacting with those materials to be coupled to it. Examples of preferred functional groups are: alkylamino-, arylamino-, isocyano-, cyano-, isothiocyano-, thiocyano-, carboxy-, hydrcxy-, mercapto-, phenol-, imidiazole-, aldehyde-, epoxy-, thionyl~, halide-, sulfonyl halide-, nitrobenzoyl halide-, carbonyl halide-, triazo-, succinimido-~, anhydride-, haloacetate-, hydrazino-, dihalo triazinyl-.
Typical examples of suitable fluorescer derivatives are: 3, 4, 9, 10 perylene tetracarboxylic dianhydride, amino-chrysene, fluorescein isothiocyanate, teteramethylrhodamine isothiocyanate, amino-pyrene, amino-anthracene, and similar ccmpounds as will be familiar to those skilled in the art.
It has been observed that on binding a fluorescent ccmpound, fluorescer, to a solid material, the wavelength of emission of the bonded fluorescer shifts to either a longer or a shorter wavelength depending on the specific fluorescer employed.
We have also found that the length of "space arm", the ligand between the fluorescent compound and the material bonded to it, effects the emission wavelength of the bonded fluorescer.
m e exact concentration of fluorescer derivative employed for bin~ing is not critical providing that the immuno-logical or enzymatic active conjugates produced therefrom 1 i~Ç133 l have the desired activity, and that the intensity of ligh~ thus 2 produced is visible, with or without the help of instruments, and may be differentiated from the background.
The intensity of the light generated by the coupled 6 fluorescer depends upon the structure of the fluorescer, the type 7 of linkage between the fluorescer and the bonded materials, ~nd 8 the available functional groups of the anchored substance. In 9 general, the intensity of the light produced by a fluorescer is not 2S ~reat after coupling as it is when in free solution. It 11 is also important that the flllorescer conjugate be stable in the 12 presence of the chemiluminescent reaction.

14 The following ex~nples are given to illustrate the various ways the fluorescer may be attached to another moiety by 16 covalently bonding using an inorganic su?port for convenience, i7 which is in no way intended to limit the scope of th~ invention `~
18 ¦ described herein.
191 . I
20 ¦ EXA~LES I-V
21 In each of the Examples I-V the linkage a tached to 22 a controlled pore glass surface was synthesized to imitate the 23 representative chemically active sites on a typical protein or 24 biological conjugate. For example, amino-, carboxyl-, merca?to-, ¦
or hydroxyl- groups are representa~ive of attac~ment sites.

27 - A glass support is used so that the activity and speci- ¦
28 ficity of the functional group is easily controlled, and to 29 ;~mobilize the fluorescer so that it may be readily separated from the fr e or unbound fluorescent compound in order`that the -46- `

i33 fluorescent spectra may easily be recognized as distinct from the o~llate CL reagent.
The results of visual observation as to the color of the fluorescent glass, and color and intensity of emitted light for l-aminopyrene covalently bonded to porous glass (CPG) (500A pore size) fluorescer with various different linkages are set forth in the attached Table 7.
m e methodology employed for preparing each fluorescer/
glass sample was as follows:
E~AMPLE 1 Ten grams of porous glass of 500 (A) (angstrom pore size) was treated with 100 ml 15~ gamma-aminopropyltrimethoxysilane in toluene and refluxed for at least 16 hours, then removed. The unbound silane was thoroughly washed with methanol filtered and the glass air dried before use. Approximately 25 milligrams l-amino-pyrene was dissolved in dioxane (20 millimeter). To this solution about 153 milligrams of succinic anhydride was added. After tw~ hours, 10 millimeter of 5 m mole N,N~dicyclohexyl-carbodiimide dioxane solution was added. 500 mg of this gamma-aminopoply-trimethoxy-silane treated glass (from here on, aminopropyl-glass) as prepared akove was added to dioxane solution. m e slurry was then stirred for one hour and let stand overnight at room temperature. Continuous stirring is preferable. The excess pyrene-dioxane solution was decanted and the glass washed exhaustively with dioxane, methanol and acetone (15 ~1 of each wash and three times for each solvent). m e wet pyrene coupled glass was filtered and allowed to air dry.

_XLI-l 1 EXAMPLE II
2 500 ~R of the aminopropyl-glass prepared as stat~d in 3 Example I was added to 25 ml of lOqo thiophosgene in chloroform 4 and the slurry was refluxed for 4 hours. The chloroform was decanted and then washed with chloroform, methanol, acetone (25 6 ml of each wash and three times for each solvent~. The slurry 7 was filtered and air dried. 30 milligrams of l-aminopyre~e was 8 dissolved in 15 ml dioxane. To this solution, the dry isocyanato-~9 gl2ss was added and stirred for one hour and ~hen allowed to stand at room temperature overnig~t. A~ter the reaction was 11 compiete, aminopyrene dioxane solution was decanted and the 12 pyrene coupled glass was washed in the same manner as sta~ed in 13 Example I.

EXAMPLE III
16 500 mg of aminopropyl-glass, prepared as stated in 17 Example I, was added to 10 ml of dioxane dissolved with 50 mg 18 succi~ic anhydride. The slurry was allowed to stand o~rernight 19 at room temperature preferably~with continous stirring. After the reaction was complete, the aminopropyl-glass, being converted I
21 to carboxy-glass, was washed in the same manner as stated in 22 Example I. Approximately 23 mg l-aminopyrene was dissolved in 23 1 ml of dioxane. To this solution, 58 mg of N-acetyhomocystein 24 was dissolved. The solutlon was then kept 4 hours at room tempe-rature. 5Q mg of N,N-dicyclohexyl-carbodiimide was then added 26 ~o it. At the same time,-the-prepared and dried carboxyl-glass 27 was added to the solution---for-co~pling. The reaction was allowed 28 to stand at room t~mperature for 24 hours. Pyrene coupled glass 29 was then washed and dried in the 5ame manner as stated before.
~/
. ,~

ENLI-l 1 EXAMPLE IV
2 - 4 grams of aminopropyl-glass prepared from Example I
was added to 10% p-nitro~enzoyl chloride with 1 ml of triethyl-4 amine in SO ml chloroform solution. The slurry was stirred and refluxed for at least 8 hours. The resulting acylated glass was 6 thoroughly washed with chloroform and let air dry. O~lM of 7 sodiu~ dithionite C30 ml2 was prepared and the acylated glass was 8 added. The temperature was then raised to 40C. The reaction 9 was completed in one hour. The glass was washed thoroughly with warm water. The arylamino-glass thus prepared was ready to 11 diazotize. 1 gm of arylamino-glass was added to 20 ml a~,ueous 12 solution of 350 mg sodium nitrite and 0.2 ml lN hydrochloric acid., 13 The temperature was brought down to 4C using an ice bath. The 14 reaction.was allowed to continue for one hour. The acid solution I
was then decanted, the glass was thoroughly washed and the pH was ' 16 adjusted to above 8Ø The filtered glass was then added to lO ml~
17 of 20 mg aminopyrene dioxane solution. Reaction was complete in 18 8 hours at room temperature. The pyrene coupled glass was then 19 washed in the same manner as in Example I.
`~
21 EX~MPLE-V
22 One gram of 500 (A) pore size porous glass treated with 23 lO ml 15% gamma-glycidoxypropyltrimethoxysilane in toluene and 24 refluxed for at least 16 hours, then washed the glass with acetone thoroughly and air dried. To 30 ml aqueous solution containing 26 1.5 mg/ml of m-sodium periodate,the silane treated glass ~epoxy- I
27 glass~ was adde-d.--The reaction w~s allo~7ed to go on for 2 hours. ¦
28 Then the glass was washed with water thoroughly. 25 mg of l-amino 29 pyrene was dissolved in 30 ml dioxane. To this solution, the filtered wet cake glass was added. The slurry was stirred .

-49- ~ ' "' ' ` ' , . ` ' ` 1, I

1 1:~6`1;~3 ENLI-l 1 for one hour and then let stand overnigh.t at room temperature.
Then pyrene coupled glass was washed in the sa~e manner as stated 4 ¦ n Examp I.
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li~bi:~3 ~NLI-1 1 EX~MPLE VI
2 Different lengths of "space arm" for binding of the 3 fluorescer were used to study the effect on the chemiluminescence 4 quality of the resultant bonded fluorescer.
S . O
6 A long "space arm'` of about 20 ~A~ in length stretching 7 out from a controlled glass pore surface was prepared as follows:
a 500 ~g of carboxy-glass prepared as stated in Example III was 9 ¦ activated by adding a 20 ~1 dioxane solution containing 200 mg of N,~-dicyclohexyl carbodiimide. The glass was stirred Ior 24 ll hours and then w~shed with dioxane and methanol. 20 ml of 2~0 mg , 12 ihe~amethylene diamine a~ueous solution was prepared and cooled 13 Ibeforehand. The activated carboxy-glass was added to the cooled 14 !Isolution and stirred for five hours, then allowed to stand for 24 lS ;ihours at 4~C. The glass was then washed hDroughly wi~h water, 16 !Imethanol and dioxane. 20 ml dioxane containing 50 mg succinic 17 l~anhydride was then added to the glass. This reaction was completed 18 lin 24 hours. The glass was subsequently washed thoroughly with l9 llmethànol. 25 mg l-a~inopyrene was dissolved in 30 ml dio~ane.
20` ¦I To this- solution 5 m mole N,N-dicylohexylcarbodiimide was added 21 iiand dissolved prior to adding the prepared glass. The slurry 22 Iwas stirred for one hour and then let stand o~ernight at room 23 ¦temperature. After 24 hours reaction, pyrene coated glass w2s 24 ¦then washed in the same manner as in Example I.
26 Pyrene coated glass with a short "space arm" of about 27 1 lQ (A) in length was prepared as stated in Ex~mple I, as the 28 i control. The results of these two glasses is set forth in 29 attached Ta~le 8.
//
.. `' 1.

3 3 ~

l 1TABLE 8 - EXAMPLE VI
2Effect of "Space Arm'l Length Between Glass Surface -3and the Fluorescer on Chemiluminescent Characteristics ' Approximate Length of "Space'Arm" _ Color of L'ight Intensity Observed*'¦
6 .
7 (,Example I~ Control lO(A~ bluish-green M
8 Example V~ 20 (A~ green W-M

12 * M = medium; W = weak '' '13 , . '' 14 ,, 15 , ~ S'~}' 16 Porous glass having various pore sizes were coated with 17 l-a~inopyrene to show t~e effect of pore size on the chemilumi-18 nescence. Three different porous glasses having 170 (A~
19 .~angstrom2, 500 (A~ and 3000 (A) pore size, respectively, ~7ere coated with l-aminopyrene in the same manner as stated in 21 Example I. T~e effect on the chemiluminescence is set forth in 22 attached Table 9.
,23 ll ', 24 l/
/l 26 /l ''.
27 1/ ' 28 l/' 29 l/ , ' ' ' : , ' ' /, ' -53- `' ' ' , 1~ 116~i133 I
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~ 3 ENLI_l 1 EX~PLES X-XV`
2 Several different fluorescers were coated on porous 3 glass to study the effect of structure on color emission. l-amino 4 pyrene and 2-amino-anthracene were coated on the porous glass S (500 A) in the same manner as described in Example I.
7 20 mg of 3,4,9,10-perylenetetracarboxylic dianhydride 8 was added to 25 ml of dioxane, to this solution 25 mg of amino-9 propyl-glass was added and stirred for one hour before allowing to stand for another 6 hours at room temperature. The glass was 11 then washed thoroughly with methanol or acetone, then filtered 12 and air ~ried.

14 500 mg of aminopropyl-glass was added to 30 ml dioxane containing 50 mg succinic anhydride and stirred for one hour 16 before being allowed to stand overnight at room te~perature. The 17 glass was~ then washed thoroughly with acetone, filtered and air 18 dried. One part of 250 mg of such glass (-carboxyl-glass2 was 19 added to 25 ml 0.01M potassium~phosphate of pH-7.6 solution containing 20 mg of isothiocyanate fluorescein. Another part of .
21 250 mg of carboxyl-glass was added to acetone/diox2ne (:50/50 by 22 volume) solution containing 20 mg of 3-amino-phthalhydrazide. The 23 two glass slurries were stirred for one hour and then allowed 24 to stand at room temperature overnight. After the reaction was completed, the glass was washed with deionized water and acetone, 26 respecti~ely. Finally, they both were washed with acetone, then 27 filtered and air dried.
28 .
29 300 mg of aminopropyl-~lass prepared as sho~m Exampie I was added to 50 ml 0.01M potassium phosphate of p~-7.6 .,' ' . .' `'.

E~LI- 1 l solution containing 25 mg of Q-phthalicdicar~oxaldehyde. The 2 glass slurry was stirred for one hour, then allowed to stand at 3 room temperature for another 24 hours. The glass was then washed 4 thoroughly with deionized water, acetone, then filtered and air drled.

7 The attached Table lb sets forth the observed chemilumi-8 nescence c~aracteristics of different fluorescers bonded to 9 porous glass in an oxalate esterlperoxide system.
10 /1 , ., , 11 // '' //

19 ~/
20 // `

25 // 1, 27 /~

I ,--''; ' , ' ' ' 1~

11~

El~
2 .
V bO O ~
O ~ ~d ~ C ~ aJ ~S
3 ~ ~ b~
~ . ~ a~
4 .~ ~ ~ o ~ ~
6 ~ a~~o I "
Fs~ N~ L.
~
~n ~ o ~ ~ o c~
o O ~r~ ~ ~ ~~ ~ 0 5~ ~ O
8 . " o ~ ~DrD ~ s~ bO ,D ~
9 ~P~
~o~
~o~ ~
~ U~
11 ~ ~ ~ ~
~ o . ~ u~ . 3 ~ ,. ~ ~ ~
L~ ~ ~ o o . .
~3 x ~
X ~C~
14 u~ o ~
~ Q~ Oa) ~ :: i l C
:~ 0 Q) bO h ;~ a) p,~ o , u~
¢ t~ O I u~ ,C J~
16 x q o ~ ~ ~ o J ~ ,~
~ O ~ O ~ O
1 7 l ~,1 ~ ~~~ ~--( h ., l E~ Q~ o ,~ o~ ~ ~> o bD ,D b~) ~ j I o . ~ c~

19 1.¢ .~¢ . ~ ~
~r. ~ ~ C~ a~
~v o ~, o ~ ~ ~o ~ 3 ~ `~
~ a) ~ ~ ~q .~ bO O ~D
21 o~ o o u~ Q~
1 ~ O
~ ~ o 22 ~ . C)~ ~ ~ o ~ a) 24 ~
2 5 ~ z ~ H H ~ X

X a "- I o~v I I ~ ~ r~
QJ
s~
2 ~ . a v, ~ 5 . u~
" ~ a~ p" ~ o h ~
7 ' ~-~ O O r~ O 0 -~ 0 . O S~ h S ~ S X
. 1:~ ~ 0 ,~ ~ C)~') ~J :~~ ~ ~,.C`-- 'O U

'l i ~ 3 ~NLI-l 1 EXA~PLE XVI
2 Aminopyrene conju~_e with antibody to Hep`atitis B Surface . I
3 Anti~en coated on porous glass.

30 mg commercially availàble antibody to Hepatitis B
6 Sùrface Antigen coated porous glass was added to 5 ml of 0.01 m 7 potassium phosphate of pH=7.6. 24 mg of l-aminopyrene was dis-8 ¦solved in 2 ml dioxane. To this solution 45 mg of succinic anhy-9 ¦dride was added and mixed for two hours. Approximately 95`~g of 10 ¦ N,N-dicyclohexyl-carbodiimide was dissolved in 1 ml of dioxane.
11 ¦ T'ne latter two solutions were mixed together and stirred for 30 12 ¦ minutes. Then 250 lamda of pyrene solution was transferred 13 to the glass slurry solution. The slurry was stirred for two 14 1 hours at room temperature and then allowed to stand at 4C
15 ¦1 overnight. The glass was washed four times with 10 ml phosphate 16 1l! buffer CpH=7.6) each wash, and was given two additional t-butanol 17 washes with 10 ml phosphate bufer each time before testing. If 18 ll:necessary, the slurry was washed until no light could be detected ~ from the supernate of the slurry. Then the l-~..inopyrene-antibody`
20 I conjugate coated on the porous glass was tested by reacting with 21 ¦l oxalate and peroxide. It was found th2t only the glass particle 22 ! glowed in faint blue color.

24 ! "
26 ~l // .

28 1 /1 . ,i 29 // : `
30 /!

5 8 -1 I - EXAMPLE XVIl 2 Fluorescein isothiocyanate anti-human gamma-globulin 3 conjugate was prepared as follows '4 mg of fluorescein'isothio-4 cyanate thoroughly mixed in 10 ml 0.1 M potassium phosphate buffer of pH=9Ø 4 ml of anti-human gamma-globulin (protein

6 concentration of 20 mg/ml~ was then added to the fluorescein

7 phosphate solution. The mixture was continuously stirred for one

8 hour at 4C and allowed to stand at the same temperature for

9 another 24 hours. Excess fluorescein was removed by extensive dialysis against 0.1 M potassium phosphate buffer of pH=7.2.
ll During dialysis~100 ml of buffer each time was used, and the 12 buffer was changed every 2 hours for 5 times.

14 Gamma-globulin coated porous glass was prepared as Ifollows: 50 mg of epoxy-glass (3000 (A) pore size) was prepared 16 ¦in the same way as described in Example IV. 2.5 mg m-sodium 17 ~,periodate was dissolved in 5 ml of deionized water. Gl2ss ~,7as 18 Ithen added to this solution and stirred at room temperature for ', 19 12 hours. The glass was washed thoroughly with deionized water and then with lC ml Q.l M potassium phosphate pH=9.Q bufer and 21 kept for one hour. The glass was then filtered and was ready for , 22 coupling. 5 ml human gamma-globulin (:protein concentration of 23 30 mg!ml) was diluted with 5 ml of 0.1 M, pX=9.0 phosphate buffer.
24 The activated glass was then added to this solution and was stirred at 4C for 2 hours before being allowed to stand overnight¦
26 'at the same te~perature. After reaction was completed, the glass 27 Iwas washed extensively with 0.1 M potassium phosphate buffer of 28 ¦pH=7.2 and then fil'tered for immediate use.
29 /l , , 3~ //

' " -59-.. .. .

~ 3 ENLI-l 1 I
30 mg of human gamma-globulin coated porous glass was 3 added to 0.5 ~1 of fluorescein-antihuman gam~a-globulin conjugate.
The slurry was incubated on 24 cycles of agitationlsettling .. ~60/90 seconds ratio2. Excess antibody solution was decanted and ¦
the glass was washed with 0.01 M potassium phosphate buffer of pH=7.2 until no light was detected by testing the decanted buffer 7 in oxalate/peroxide system.
8 . .

The glass was then washed with 5 ml t-butanol and excess butanol was withdrawn. Green color light was observed on glass . particles.upon addition of oxalate and peroxide.

13 ~lthough.the above examples illustrate various modif~-cations of the present invention, other variations will suggestlS themselves to those skilled in the art in the li~ht of the above 16 disclosure. It is to be understood, therefore, that changes may 17 ' be made .in the particular embodiments described above which are 13 ¦ within the full intended scope of the inventions as defined in appended claims.
20 /~ `.

22 l/
23 .//
24 l/
- 25 l/
26 l/

28 !/
29 l/
30 ll .

l -60-

Claims (61)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A system for the detection of a biological analyte of interest which comprises a fluorescer-catalyst which has been conjugated to an immunological species specific to the biological analyte of interest, in the presence of an energy source other than electromagnetic radiation which is capable of activating the fluorescer-catalyst.

2. A system for the detection of a biological analyte of interest which comprises a fluorescer-catalyst which has been conjugated to an immunological species specific to the biological analyte of interest, in the presence of an excess of an energy source other than electromagnetic radiation which is capable of activating the fluorescer-catalyst.

3. A method for the qualitative detection of a biological analyte of interest comprising:
(a) labeling an immunological species specific to the analyte of interest with a fluorescer-catalyst material which is biologically compatible with such species;
(b) contacting the fluorescer-catalyst labeled species and the biological of interest, (c) separating the fluorescer-catalyst labeled species/biological complex, (d) contacting the separated fluorescer-catalyst labeled species/biological complex of (c) with an energy source other than electromagnetic radiation which is capable of activating the fluorescer label, and (e) determining the presence or absence of light emitted from the activated fluorescer-catalyst.

4. A quantitative method for measuring the amount of a biological analyte of interest comprising:
(a) labeling an immunological species specific to the analyte of interest with a fluorescer-catalyst mate-rial which is biologically compatible with such species;
(b) contacting the fluorescer-catalyst labeled species and the biological of interest;
(c) separating the fluorescer-catalyst labeled species/biological complex:
(d) contacting the separated fluorescer-catalyst labeled species/biological complex of (c) with an energy source other than electromagnetic radiation which is capable of activating the fluorescer label, and (e) determining the amount of light emitted from the activated fluorescer-catalyst.

5. The method of claim 3, wherein the fluorescer-catalyst of (a) is chemically conjugated to the immuno-logical species specific to the biological of interest.

6. The method of claim 5, wherein the chemical con-jugation of the fluorescer-catalyst material to the immuno-logical species specific to the biological of interest is carried out in such a way as to prevent substantial bio-logical damage to the attached species.

7. The method of claim 3, wherein the fluorescer-catalyst material utilized has a spectral emission of from about 260 millimicrons to about 1000 millimicrons.

8. The method of claim 3, wherein the fluorescer-catalyst material utilized has a spectral emission above the light absorption wavelength of either the immunological species specific to the biological of interest or the biologi-cal of interest and below the light absorption wavelength of any solvent system utilized.

9. The method of claim 3, wherein the fluorescer-catalyst material utilized has a structure which possesses one or more functional groups capable of reacting with the immunological species specific to the biological of interest without adversely affecting such species.

10. The method of claim 3, wherein the fluorescer-catalyst material utilized has a structure which possesses one or more functional groups selected from the group consisting of alkylamino-, arylamino-, isocyano-, cyano-, isothiocyano-, thiocyano-, carboxy-, hydroxy-, mercapto-, phenol-, imidiazole-, aldehyde-, epoxy-, thionyl halide-, sulfonyl halide-, nitrobenzoyl halide-, carbonyl halide-, triazo-, succinimido-, anhydride-, haloacetate-, hydrazino-and dihalo triazinyl-.

11. The method of claim 3, wherein the fluorescer-catalyst material utilized is selected from the group com-prising 3,4,9,10 perylene tetracarboxylic dianhydride, amino-chrysene, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, amino-pyrene, and aminoanthracene.

12. The method of claim 3, wherein the energy source of (d) which is contacted with the separated fluorescer-catalyst labeled species/biological complex is present in excess of the amount required to activate all of the fluorescer-catalyst labeled species.

13. The method of claim 3, wherein the energy source of (d) which is contacted with the separated fluorescer-catalyst labeled species/biological complex is any source other than electromagnetic radiation which is capable of activating the particular fluorescer-catalyst selected to be compatible with the labeled species.

14. The method of claim 3, wherein the energy source of (d) which is contacted with the separated fluorescer-catalyst labeled species/biological complex is the peroxy-oxylate reaction.

15. A method according to claim 13, wherein the energy source is a chemical reaction selected from the group consisting of 2-napthol-3,6,8-trisulfonic acid, 2-carboxy-phenyl, 2-carboxy-6-hydroxyphenol, 1,4-dihydroxy-9, 10-diphenylanthracene, 2-napthol, luminol, lophine, pyrogallol and luciferin reactions.

16. A method according to claim 13, wherein the energy source is derived from a chemical reaction, ozone, an electric current, an electrochemical reaction or a mechanically generated species.

17. A method according to claim 3, which is carried out utilizing solid phase analytical techniques.

18. A method according to claim 3, which is carried out utilizing a sandwich technique.

19. A method according to claim 3, which is carried out utilizing heterogeneous analytical techniques.

20. A method according to claim 3, which is carried out utilizing heterogeneous competitive binding techniques.

21. A method according to claim 3, which is carried out without separating the fluorescer-catalyst labeled species/biological complex utilizing quenching analyses techniques.

22. A method according to claim 3, which is carried out utilizing immuno-precipitant reaction techniques.

23. A method according to claim 3, which is carried out utilizing ion exchange techniques.

24. A method according to claim 3, which is carried out utilizing ion exclusion techniques.

25. A method according to claim 3, which is carried out utilizing masking techniques.

26. A method of claim 4, wherein the fluorescer-catalyst of (a) is chemically conjugated to the immuno-logical species specific to the biological of interest.

27. The method of claim 4, wherein the chemical conjugation of the fluorescer-catalyst material to the immunological species specific to the biological of interest is carried out in such a way as to prevent substantial bio-logical damage to the attached species.

28. The method of claim 4, wherein the fluorescer-catalyst material utilized has a spectral emission of from about 260 millimicrons to about 1000 millimicrons.

29. The method of claim 4, wherein the fluorescer-catalyst material utilized has a spectral emission above the light absorption wavelength of either the immunological species specific to the biological of interest or the biological of interest and below the light absorption wave-length of any solvent system utilized.

30. The method of claim 4, wherein the fluorescer-catalyst material utilized has a structure which possesses one or more functional groups capable of reacting with the immunological species specific to the biological of interest without adversely affecting such species.

31. The method of claim 4, wherein the fluorescer-catalyst material utilized has a structure which possesses one or more functional groups selected from the group con-sisting of alkylamino-, arylamino-, isocyano-, cyano-, isothiocyano-, thiocyano-, carboxy-, hydroxy-, mercapto-, phenol-, imidiazole, aldehyde-, epoxy-, thionyl halide-, sulfonyl halide-, nitrobenzoyl halide-, carbonyl halide-, triazo-, succinimido-, anhydride-, haloacetate-, hydra-zino-, and dihalotriazinyl-.

32. The method of claim 4, wherein the fluroescer-catalyst material utilized is selected from the group consisting of 3,4,9,10 perylene tetracarboxylic dianhydride, amino-chrysene, fluorescein isothiocyanate, tetramethyl-rhodamine isothiocyanate, amino-pyrene, and amino-anthracene.

33. The method of claim 4, wherein the energy source of (d) which is contacted with the separated fluorescer-catalyst labeled species/biological complex is present in excess of the amount required to activate all of the fluorescer-catalyst labeled species.

34. The method of claim 4, wherein the energy source of (d) which is contacted with the separated fluorescer-catalyst labeled species/biological complex is any source other than electromagnetic radiation which is capable of activating the particular fluorescer-catalyst selected to be compatible with the labeled species.

35. The method of claim 4, wherein the energy source of (d) which is contacted with the separated fluorescer-catalyst labeled species/biological complex is the peroxy-oxylate reaction.

36. A method according to claim 35, wherein the energy source is a chemical reaction selected from the group consisting of 2-napthol-3,6,8-trisulfonic acid, 2-carboxyphenyl, 2-carboxy-6-hydroxyphenol, 1,4-dihydroxy-9,10-diphenylanthracene, 2-napthol, luminol, lophine, pyro-gallol, luciferin reactions.

37. A method according to claim 35, wherein the energy source is derived from a chemical reaction, ozone, an electrical current, an electrochemical reaction or a mechanically generated species.

38. A method according to claim 4, which is carried out utilizing solid phase analytical techniques.

39. A method according to claim 4, which is carried out utilizing heterogeneous analytical assay techniques.

40. A method according to claim 4, which is carried out utilizing heterogeneous competitive binding techniques.

41. A method according to claim 4, which is carried out without separating the fluorescer-catalyst labeled species/
biological complex utilizing quenching analyses techniques.

42. A method according to claim 4, which is carried out utilizing immuno-precipitant reaction techniques.

43. A method according to claim 4, which is carried out utilizing ion exchange techniques.

44. A method according to claim 4, which is carried out utilizing ion exclusion techniques.

45. A method according to claim 4, which is carried out utilizing masking techniques.

46. A fluorescer-catalyst composition useful in the labeling of an immunological species specific to and for the detection of a biological of interest, such fluorescer-catalyst having a chemical structure which possesses one or more functional groups capable of chemical reaction with the immunological species without adverse effect on the specificity of such species to the biological of interest.

47. A fluorescer-catalyst composition useful in the labeling of an immunological species specific to and for the detection of a biological of interest, such fluorescer having a chemical structure which possesses one or more functional groups selected from the group consisting of alkylamino-, arylamino-, isocyano-, cyano-, isothiocyano-, thiocyano-, carboxy-, hydroxy-, mercapto-, phenol-, imidia-zole-, aldehyde-, epoxy-, thionyl halide-, sulfonyl halide-, nitrobenzoylhalide-, carbonyl halide-, triazo-, succinimido-, anhydride-, haloacetate-, hydrazino- and dihalo triazinyl-.

48. A fluorescer-catalyst composition useful in the labeling of an immunological species specific to and for the detection of a biological of interest, such fluorescer-catalyst selected from the group consisting of 3,4,9,10-perylene tetracarboxylic dianhydride, amino-chrysene, fluorescein isothiocyanate, tetramethylrhodamine isothio-cyanate, aminopyrene and amino-anthracene.

49. A conjugated fluorescer-catalyst/immunological species composition useful in the detection of a biological of interest which has been formed via reacting an immuno-logical species with a fluorescer-catalyst having a chemical structure which possesses one or more functional groups capable of chemical reaction with the immunological species without adverse effect on the specificity of such species to the biological of interest.

50. A conjugated fluorescer-catalyst/immunological species composition useful in the detection of a biological of interest which has been formed via reacting an immuno-logical species with a fluorescer-catalyst having a chemical structure which possesses one or more functional groups selected from the group comprising alkyl-amino, arylamino-, isocyano-, cyano-, isothiocyano-, thiocyano-, carboxy-, hydroxy-, mercapto-, phenol-, imidiazole-, aldehyde-, epoxy-, thionyl halide, sulfonyl halide-, nitrobenzoyl halide-, carbonyl halide-, triazo-, succinimido-, anhydride, halo-acetate-, hydrazino- and dihalo triazinyl-.

51. A conjugated fluorescer-catalyst/immunological species composition useful in the detection of a biological of interest which has been formed via reacting an immuno-logical species with a fluorescer-catalyst selected from the group consisting of 3,4,9,10 perylene tetracarboxylic dianhydride, amino-chrysene, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, amino-pyrene and amino-anthracene.

52. A method for the qualitative detection of a bio-logical analyte of interest according to claim 3, wherein the fluorescer-catalyst labeled species/biological complex is not separated prior to activating the fluorescer-catalyst label.

53. A method for the quantitative measurement of an amount of abiological analyte of interest according to claim 4, wherein the fluorescer-catalyst labeled species/-biological complex is not separated prior to activating the fluorescer-catalyst label.

54. A system for the detection of a biological analyte of interest which comprises a fluorescer-catalyst which has been conjugated to an immunological species specific to the biological analyte of interest, said fluorescer-catalyst being biologically compatible with said species and conjugated to said species in an amount effective to provide a determinable light emission for the fluorescer-catalyst, said fluorescer-catalyst being activatable by an energy source other than electromagnetic radiation.

55. A method for the qualitative detection of a biological analyte of interest comprising:
(a) labeling an immunological species specific to the analyte of interest with a fluorescer-catalyst material which is biologically compatible with such species;
(b) contacting the fluorescer-catalyst labeled species and the biological of interest to form a fluorescer catalyst labeled species/biological complex;
(c) contacting the separated fluorescer-catalyst labeled species/biological complex of (b) with an energy source other than electromagnetic radiation which is capable of activating the fluorescer label; and (d) determining the presence or absence of light emitted from the activated fluorescer-catalyst.

56. A method for the qualitative detection of a biological analyte of interest according to claim 55, wherein the fluorescer-catalyst labeled species/bio-logical complex is separated prior to activating the fluorescer-catalyst label.

57. A quantitative method for measuring the amount of a biological analyte of interest comprising:
(a) labeling an immunological species specific to the analyte of interest with a fluorescer-catalyst material which is biologically compatible with such species, (b) contacting the fluorescer-catalyst labeled species and the biological of interest to form a fluorescer-catalyst labeled species/biological complex, (c) contacting the separated fluorescer-catalyst labeled species/biological complex of (b) with an energy source other than electromagnetic radiation which is capable of activating the fluorescer label, and (d) determining the amount of light emitted from the activated fluorescer-catalyst.

58. A method for the quantitative measurement of an amount of a biological analyte of interest according to claim 57, wherein the fluorescer-catalyst labeled species/biological complex is separated prior to activating the fluorescer-catalyst label.

59. In a method for the detection of a biological analyte of interest in which an immunological species specific to and for the detection of a biological of interest is labeled, the improvement wherein said species is labeled with a fluorescer-catalyst having a chemical structure which possesses one or more functional groups capable of chemical reaction with the immunological species without adverse effect on the specificity of such species to the biological of interest.

60. A method according to claim 59, wherein said fluorescer-catalyst has a chemical structure which possesses one or more functional groups selected from the group consisting of alkylamino-, arylamino-, iso-cyano-, cyano-, isothiocyano-, thiocyano-, carboxy-, hydroxy-, mercapto-, phenol-, imidiazole-, aldehyde-, epoxy-, thionyl halide-, sulfonyl halide-, nitrobenzoyl-halide-, carbonyl halide-, triazo-, succinimido-, anhydride-, haloacetate-, hydrazino- and dihalo triazinyl-.

61. A method according to claim 59, wherein said fluorescer-catalyst is selected from the group consisting of 3,4,9,10-perylene tetracarboxylic dianhydride, amino-chrysene, fluorescein isothiocyanate, tetramethylrhod-amine isothiocyanate, aminopyrene and amino-anthracene.