MICROENCAPSULATED FLUORESCER COMPOSITION,TEST KIT AND SYSTEM FOR IMMUNOASSAY

Abstract: A method, composition, kit and system for carrying out an immunoassay for a particular biological analyte. The method includes: a) contacting a sample containing the analyte with its specific immunochemical binding partner, the binding partner having been labeled with microencapsulated fluorescer material; b) separating the resulting analyte-bound binding partner complex from unbound binding partner; c) disrupting the microcapsules of the complex, thus freeing the fluorescer material to solution; and d) activating the freed fluorescer material and measuring the emitted light.

Description: -1- MICROENCAPSULATED FLUORESCER COMPOSITION, TEST KIT AND SYSTEM FOR IMMUNOASSAY This invention relates to a system for the detection of a biological analyte of interest which comprises a microencapsulated fluorescer which has been conjugated to an immunological specie specific to the biological analyte of interest, a means for disrupting the microcaps-ule to free the fluorescer and an energy source other than electro-magnetic radiation which is capable of activating the fluo¬ rescer. - 2 - Background Of The Invention The clinician is concerned with detecting the presence of, and quantitatively measuring, a variety of substrates via the use of many different analytical techniques. The most commonly used tech- niques employ absorbtiometry, both at visible and ultraviolet wavelengths, however, emission, flame photometry and radioactivity are also commonly used. A novel technique, thus far relatively unexplored in chemistry, is that employing the phenomenon of luminescence.. Analyses based on the measurement of emit¬ ted light offer several distinct advantages over con¬ ventionally employed techniques, including high sensitivity, wide linear range, low cost per test, and relatively simple and inexpensive equipment. It has been predicted that the phenomenon of luminescence, and more particularly chemilumines- cence could have a major 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 oxidas.es and dehydrogenases. In this type of assay the sensitivity of the luir-inees- cence. indicator reaction may be used to quantitate substrates not easily measured by conventional tech¬ niques (e.g. prostaglandins and vitamins ) - The second major clinical application of luminescence must be in the utilization of lumines¬ cent molecules as replacements for radioactive or enzyme labels in immunoassay. In each of these major clinical application - 3 - areas, chemiluminescent reactions can provide a means to achieve a high level of analytical sensitivity. Chemiluminescence may be simply defined. as the chemical production of light. In the litera¬ ture it is often confused with fluorescence. The difference between these two phenomena lies in the source of the energy which promotes molecules to an excited' state. In chemiluminescence this source is the non-radiative energy yielded as the result of a chemical reaction. The subsequent decay of molecules from the excited state back to the ground state is accompanied by emission of light, which is called luminescence. In contrast, in fluorescence, incident radiation is the source of the energy which promotes molecules to an excited state. From an analytical point of view, the types of luminescence that have engendered the most inter¬ est are chemiluminescence and bioluminescence. The latter being the name given to a special form of chemiluminescence found in biological systems, in which a catalytic protein increases the efficiency of the luminescent reaction. Bioluminescent reactions such as the enzymatic firefly process, have been very useful analytically and convert chemical " ." * ;, energy to light with a quantum efficiency of 88%. n contrast to bioluminescence with the longevity and efficiency of the firefly, the history of chemiluminescence (hereinafter referred to as CL) , especially that occurring in the non-aqueous phase, - 4 - is remarkable short. The important aqueous CL substances luminol and lucigenin were discovered in 1928 and 1935, respectively. A series of organic soluble -CL materials were developed in the early 1960's based upon a study of the luminescent re¬ actions of a number of oxalate compounds. A typical organic system useful for CL was disclosed by Bollyky et al. , U.S. Patent No. 3,597,362, and claimed to exhibit a quantum efficiency of about 23% compared with about 3% for the best known available aqueous systems. Chemiluminescence has become increasingly attractive for its potential in the clinical lab- oratory, especially for use in the analysis of a number of biologically associated materials, and its known applicationshave been the subject of thorough reviews, see for example: Whitehead et al. (1979) Analytical Luminescence: Its Potential In The Clinical Laboratory, Clin. Chem. , 25, 9 1531-1546; Gorus et al. (1979) Applications Of Bio- and Chemiluminescence In The Clinical Labora¬ tory, Clin. Chem. , 25, 4 512-519; Isacsson et al. (1974) Chemiluminescence- In Analytical Chemistry, Analytical Chemica Acta, 68, 339-362. With few exceptions, most published CL clinical analytical applications have made use of the less efficient but well known diacylhydrazides, acridinium salts, pyrogallol, or lophine struc¬ tures. It is important to appreciate that due to the nature of the chemical decomposition of the above chemiluminescent structures in the presence - 5 - of hydrogen-peroxide, or generators of H 2 0 2 , as compared to that of the oxidation reaction of diaryloxalate structures, the latter has over 20 times the quantum yield of chemiluminescence, although its requirement for hydrogen peroxide is greater than the former. Hydrogen peroxide, an essential component in many chemiluminescent reactions, has usually been the species selected for use in detecting the analyte of interest. For example, in the deter¬ mination of glucose- uses et al. (1975) , Chemi¬ luminescent Enzyme Method For Glucose. Analytical Chemistry, 47, No. 2, 244-248 employed the oxida- tion of glucose in the presence of glucose oxidase as the source of H-0 2 which, in turn, was-reacted with luminol to produce chemiluminescence in pro¬ portion to the initial glucose concentration. A limit of detection of 8 x 10 -9M peroxide was ob- tained with this system. Williams et al. (1976) , Evaluation Of Peroxyoxalate Chemiluminescence For Determination Of Enayme Generated Peroxide. Anal. Chem. , 48, 7 1003-1006 in a similar reaction concluded the limit of sensitivity of the peroxy- oxalate system is an order of magnitude poorer than that of the luminol system. Therefore, until now the oxalic ester system (oxalate system) was generally thought to have little utility for analytical purposes due to its inefficient conversion of hydrogen peroxide. - 6 In one embodiment the present invention overcomes the deficiency of H-0 2 dependence by making use of the large chemiluminescent reservoir of energy in the oxalate system's chemistry. By 5. using a suitable quantity of hydrogen peroxide and oxalate, a vast amount of energy may be gen¬ erated i a form which is then released as chemi¬ luminescence upon the introduction of a fluorescer. 0 Thus, the oxalate, acting in a fashion which can be visualized 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 5 of a battery to a lamp. This "switch" action causes chemiluminescence and, by incorporating the fluo¬ rescer 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. It is, therefore, an object of the present invention to provide for a system for the detec¬ tion of a biological analyte of interest comprising an encapsulated fluorescer material which has been conjugated to an immunological specie specific to the biological analyte of interest, a means of disrupting the capsule containing the fluorescer and an energy source other than electro-magnetic radiation which is capable of activating the fluorescer. - 7 - A further object of the present invention is to provide for a qualitative method for the detection of a biological analyte of interest comprising: (a) labeling an immunological specie specific to the analyte of interest with an encapsulated fluorescer material which is bio¬ logically compatible with such specie; (b) contacting the encapsulated fluo- rescer labeled specie and the biological of interest to form an encapsulated fluorescer labeled specie/biological complex; (c) separating the fluorescer labeled specie/biological complex; (d) disrupting the capsule containing the fluorescer label thus freeing it to solution; (e) contacting the freed fluorescer with an energy source other than electro-magnetic radiation which is capable of activating the fluorescer label; and (f) determining the presence or absence of chemiluminescent light emitted from the activated fluorescer. A further object of the present invention is to provide for a quantitative method for measur¬ ing the amount of a biological analyte of interest comprising: (a) labeling an immunological specie specific to the analyte of interest with an encap¬ sulated fluorescer material which is biologically compatible with such specie; - 8 - (b) contacting the encapsulated fluo¬ rescer labeled specie and the biological of inter¬ est to form an encapsulated fluorescer labeled specie/biological complex; (c) disrupting the capsule containing the fluorescer label thus freeing it to solution; (d) contacting the freed fluorescer with an energy source other than electro-magnetic radiation which is capable of activating the fluorescer label; and (e) determining the presence or absence of chemiluminescent light emitted from the acti¬ vated fluorescer. A further object of the present intention is to provide for a novel class of micro encap¬ sulated fluorescer materials which may be con¬ jugated to an immunological specie specific to a biological analyte of interest to provide a means for the detection of such biological. A further object of the present invention is to provide for a novel class of conjugated micro- encapsulated fluorescer/biological compositions useful in the detection of various biological analytes of interest. A further object of the present invention is to provide for test kits for the detection of a biological analyte of interest employing the microencapsulated fluorescer materials described herein. - 9 . Summary of the Invention According to the present invention, there is provided a system for the detection of a biological analyte of interest comprising an encapsulated fluorescer material which has been conjugated to an immunological specie specific to the biological analyte of interest, a means for disrupting the capsule containing the fluor¬ escer and an energy source other than electro¬ magnetic radiation which is capable of activating the fluorescer. There is also provided a method for the qualitative and/or quantitative detection of a biological of interest comprising: (a) labeling an immunological specie specific to the analyte of interest with an en¬ capsulated fluorescer material which is biologi¬ cally compatible with such specie; (b) contacting the encapsulated fluorescer labeled specie and the biological of interest to form an encapsulated fluorescer labeled specie/biological complex; (c) separating the encapsulated fluorescer labeled specie/biological complex; (d) disrupting the capsule containing the fluorescer label thus freeing it to solu¬ tion; - (e) contacting the freed fluorescer with an energy source other than electro-magnetic radiation which is capable of activating the fluorescer label; and - 10 - (f) detecting the presence of and/or measuring the quantum of chemilumin scent light emitted. Additionally, there is provided novel microencapsulated fluorescer and conjugated microencapsulated fluorescer/immunological specie compositions useful in the detection of various biological analytes of interest. Further there is provided novel test kits for the detection of a biological analyte of interest employing the microencapsulated fluorescer materials described. With respect to Charts I, II, and III, Rauhut et al. (1969) , Chemiluminescence From Concerted Peroxide Decomposition Reactions, Accounts of Chemical Research, Vol. 2_, 80-87, it can be seen that one mole of H_0 2 is necessary to convert one mole of luminol into one mole of the energized or excited molecule. This excited molecule then reverts to its ground state and emits light. Of interest is the fact that the CL com- pound, in Chart I, luminol or its derivatives, is also capable of converting the chemical energy of the system to light. Thus, the luminol acts as a source of CL energy and also as a fluorescer to absorb the energy and produce visible light. The luminol system is, therefore, not particularly useful in the context of the present invention since no differentiation between the light - 11 - emitted upon fluorescer addition and that generated by the luminol itself can be made. Charts II and III illustrate the fact that for the oxalate system, hydrogen peroxide does not always produce a species which gives rise to an excited state producing light. Some peroxide may be lost in side reactions which are "dark", thus, ' there is no predictable stoichiometric relationship between the H 2 0- consumption and the quanta of emitted light. - 12 - Chart I -Aminophihalydrazide Chemiluminescence in Reaction with Potassium Persulfate and Hydrogen Peroxide (Luminol) - 13 - Chart II Tentative Mechanism for Oxalyl Chloride Chemiluminescen • oo w.o, Bϊ 00 CICCDOH HCI ft! 12 ciccα 00 HP αc ScFoH- uσ— * . 2HC1+ CO ÷ CO, C3 Chart III Tentative Mechanism for Oxalic Ester Chemiluminescence IS OO ROCCOOH _•ROH 33 13 17 -f HA n fir * - 15 - A major difference between the luminol system, which has been used to detect the pres¬ ence or the quantity of ∑-^O^ and the oxalate system is the requirement that the oxalates have an additional fluorescer to accept the chemical energy generated in the reaction and then con¬ vert that energy to visible light. If the speci¬ fied fluorescer is absent, the energy generated by the reaction will be dissipated without emit¬ ting useful signal. 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 aqueous medium, due to the imcompatibility of biologi¬ cal anti-analytes to such organic solvents. The present invention dramatically differs from the prior art utilizing CL for analytical purposes in the way the generated CL energy is ' employed. The present invention makes use of the CL system as a substrate or reservoir of chemical energy which emits light upon the addition of another compound, i.e. the fluorescer. We have found that by con¬ jugating this fluorescer compound to the anti- analyte of interest it is possible to quantify the analyte 1 s concentration in terms of the amount of emitted light. CL as thus applied becomes competitive as a highly sensitive replacement for radioimmunoassay techniques (RIA) . - 16 - The comparison of Table 1 shows various analytical systems employing CL and illustrates the manner in which components of different reactions may be used, to achieve detection. An analyte may be determined using CL by coupling the detector for the analyte to eithers I. A catalyst for generation of the H-0- CL reaction, such as glucose- oxidase, or II. A CL compound which generates CL energy and itself emits light, such as luminol, or III. A fluorescer which accepts chemical - energy and emits light, such as a perylene derivative. In each case, for the purpose of simpli¬ city in this comparison, the analyte is assumed to be surface antigen to Hepatitis B (HBo C Ag_) in human serum and is determined by a heteroσeneous "sandwich" technique. This system is presently widely used wwiitthh II 1 1 2 2 5 5, , a radioactive isotope, as the label or* indicator. t t H * H 1 O O O n- TABLE I COMPARISON OF METHODS FOR USING COHPONEHTS OF CL. REACTION 1 Me thod I Method II Method III Con jugat ion of OxldUer Conluf.ation of Luminol Conluxation of Fluorescer Object of ot Detection H 2 0j luminol Fluorescer conjugate Label example glucαaa oxidase lumlnol and derivatives 3,1,9, 10-perylen* tetracarboxyllc di.anhydride Anslonous UlUlama 2 (1976). ruget 1 (1977) llersh 6 fl979), Pratt 7 (1978) Nona Systems . Valan (1978), HcCapra' (1977) Simpson* (1979), Schroedar" (1979) Olsson 10 (1979) Advantages 1) Eniyme catalyst amplifica¬ 1) Iromunonologtcal reaction separata and tion system for hydrogen Nona distinct from CL production. peroxide. 2) Highest level of light Intensity, 211. I 2) System can provide • number 3) Sample may be reteated with of readings before deatructlon, additional oxalic eater. -J 3) Highest sensitivity for A) Label la stable for coupling and detecting t^O j . storage. I 5) Host desirable functional group may be used for attachment to biological material, minimizing destruction of label, specificity snd activity. 6) Fluorescer inexpensive compared with - 18 - Footnotes to Table 1 No solid phase system incorporating the ad¬ 6 Hersh et al. (1979) Luminol-Assisted, Competitive- Binding Immunoassay Of Human Immuno-Globulin G. Schroeder et al. (1979) Immunoassay F or Serum T hroxine Monitored By Chemiluminescence. Journal of Immunological Methods, 25, 275-28 2, O lsson et al. (1979) Luminescence Immunoassay ( LI A) A Solid Phase * Immunoassay Monitore d By C hemiluminescence. Journal of Immunological M ethods, 25_, 127-135. - 20 - In order to detect the antigen-anti¬ body reaction the indicator in all cases illus¬ trated in the comparison of Table 1 is taken to be the emission of light from CL- In the "sandwich" technique, the following steps are taken: anti-HB g (Goat) is coated to controlled pore glass (CPG) particles in tablet form (solid phase) . Patient serum is added to a tube containing a CPG tablet. During incubation the tablet disintegrates. If Hepatitis B Surface Antigen is present in the ■ serum tested, it will combine with the antibody on the glass particles. After incubation, the serum is removed and the glass beads rinsed. A label, as discussed below, conjugated to an anti- body specific for HB-A is then added. The labeled antibody combines with the antigen bound to the antibody on the glass particles forming the "sandwich". The labeled antibody then reacts in a specified manner in the CL system to give light as an indi- cation of antigen presence. This CL assay is a qualitative test for the presence of Hepatitis B Surface Antigen in serum. In general, however, the greater the amount of HBo C Ag in a sample, the greater the intensity of* emitted light. The reaction sequence and procedures used in carrying out the Methods illustrated in Table 1 were as follows: Method 1 - Enzyme Che iluminescent Immunoaasay Label: Antibody to Hepatitis B Surface Antigen conju¬ gated with glucose-oxidase (GLO) Reaction': (1) Glass.ab.ag + ab.GLO + glucose -→ H2- V 2 (2) Luminol + NaOH + H 2 0 2 (from reaction 1) -→ light Procedure: After incubation of the oxidase label to form the "sandwich" as described above, the complex is washed to remove excess label. The washed complex is then incubated for a fixed 10 time with a standard glucose solution to allow the glucose substrate to form H 2 G 2 , the quantity of which is proportional to the original GLO pres¬ ent in the sandwich. An aliquot of the solution is then added to a standard catalyzed alkaline - j c luminol solution with the light emission pro¬ portional to the HB g in the original sample. Method II - Chemiluminescent-labeled Immunoassay Label: Antibody to Hepatitis B Surface Antigen 0 labeled with luminol. Reaction: (1) Glass.ab.ag.ab.luminol + H-0 2 -→ light 5 Procedure: After incubation of the luminol label to form the "sandwich" as described above, the complex is washed to remove excess label. To the washed complex is added a standard hydrogen pero¬ xide alkaline he in reagent. The light emission 0 is proportional to the HB g in the original sample. It is noteworthy that Hersh et al. (1979 Luminol- Assisted, Competitive-Binding Immuno-Assay of Human I muno-Globulin, G. Anal. Biochem. , 93, 267-271, 5 - 22- end their paper describing a similar use of luminol with the following summary: "The luminol-based chemiluminescent label can be employed as a substitute for radiolabels in immunoassay for serum components at concen- -9 trations greater than 10 ol/liter. The main factor limiting the sensitivity of the method is the relatively low overall chemilum- inescent efficiency (CΞ) of the luminol tag. The CE of underivatized luminol is reported to be 1.5% (5) . Our luminol-IgG label had a final efficiency of- about 0.3%...It is possible that a more efficient means of coupling luminol, if found, would increase sensitivity by a maximum of 600%. The most efficient chemiluminescent system reported to date (not involving enzymes) is the hydrogen peroxide-oxal te ester reaction (6) . This reaction has an overall chemilumin¬ escence efficiency of 23%. The use of the oxalate ester as a chemiluminescent label would provide the more substantial gain of 1500% compared to the luminol system." Thus, while earlier investigators recogni¬ zed the quantum efficiency of the oxalate system for CL, they, like others, never appreciated the most efficient way to use this oxalate as a source of energy, would be by controlling the "switch" and not the "source" of the energy. Method III - Chemiluminescent Labeled Light Amplification System (The method of the present invention - "Classic") - 23 - Label: Antibody to Hepatitis B Surface Antigen conjugated to a perylene derivative fluorescer. Reaction: (1) Glass.ab.ag.ab. perylene + TCPO + H 2 0 2 -→ light Procedure: After incubation of the perylene label to form the "sandwich" as described above, the complex is washed to remove excess label. The "sandwich" is then washed with tertiary butanol to remove excess buffer salts. Then an excess of bistricholorphenyl oxalate and hydrogen peroxide in dimethyIphthalate are added to cause the fluor¬ escer conjugate to emit light. The light emission is proportional to the HB g A in the original sample. The light intensity may be measured qualitatively by eye, or quantitatively by using a photodiode in the same manner that a photomultiplier in proximity to a sodium iodide crystal responds to the photons released by the gamma rays from the I 125 label. Discussion of Methods I, II and III The use of an oxidizer conjugated to an antibody (Method I) is in reality an adaptation of the well-known enzyme-i munoassay systems of Syva Corporation (U. S. Patent No. 3,817,837) and Organon Co. (U. S. Patent No. 3,654,090) but here using CL as a light indicator instead of a dye color change. We are not aware of an analogous system incorporating all the solid phase sequences suggested herein. Nonetheless, the detection limit - 24 - of this method is governed by the ability of the oxidase enzyme conjugate to liberate sufficient H 2 0 2 as in the above enzyme immunoassays. Some increase in detection level may be achieved by using CL because of the better sensitivity of CL vs. dye color change, this sensitivity does not however approach the detection level'of the fluorescer conjugate of Method III. In Method II a number of analysts have suggested labeling the analyte detector with a CL compound or derivative. This method is inferior to Methods I or III in that the amount of light emitted can never be more than the total energy content of the amount of CL compound conjugated - i.e., luminol or oxalate. A further disadvantage in coupling the CL compound directly to the anti¬ body, for example, is the loss in CL capacity of the conjugate and the continued loss of light as the compound is consumed in the reaction. Finally, the entire loss of the consumed CL compounds before test completion prevents the analyst from repeating or rechecking the sample's CL. Method III, alternatively referred to as "CLASSIC", the method of the present invention, overcomes the inherent disadvantages of Methods I and II. With "CLASSIC" it is possible to achieve the highest order of activity and specificity of the analyte detector because one can carefully select the preferred attachment site on the biolo¬ gical to be labeled. It is also possible to design the linkage of an efficient and durable fluorescer - 25 - to conjugate with the biological effectively at this site without damaging the biological. Damage in specificity and activity of biologicals from I 125 labeling, and damage to enzymes by conjuga¬ tion is well known and an accepted fact in the pre¬ paration of immunodiagnostic reagents. A fluorescent label of preferred utility in CL, by its very structure, must be stable to the oxidizing condi¬ tions of the oxalate reaction. This inertness augers well in making fluorescers a particularly efficient form of label for immunochemical analyses. The various levels of sensitivity and variations in different types of amplification is evaluated in a 1976 review by G. Wisdom, Ξnzyme- immunoassay, Clin. Chem. , 22 1234-1255. • These systems * provide the amplification for enzyme labels since enzyme catalytic properties allow them to act as amplifiers, and many enzyme molecules can 5 ccaattaallyyzzee tthhee ffoorrmmaattiioonn of more than 10 product molecules per minutes. To be suitable as a label, an enzyme must meet the several criteria set forth by Wisdom .(1976) (supra) which are as follows: (1) Available cheaply in high purity. (2) High specific activity. (3) Stable under assay and storage conditions. (4) Soluble. (5) Assay method that is simple, sensitive, rapid, and cheap. (6) Absent from biological fluids. -26 - (7) Substrate, inhibitors, and disturbing factors, absent from biological fluids. (8) Capable of retaining activity while undergoing appropriate linkage reactions. (9) Capable of inhibition or reactivation when antibody binds to the enzyme- hapten conjugate. (10) Assay conditions compatible with hapten-antibody binding. ' These specifications are easily met by fluorescers which may be readily incorporated as labels capable of accepting the chemical energy from the oxalate "substrate". In addition, as has been shown by Rauhut, certain selected fluorescer structures are capable of catalyzing the peroxyoxalate reaction products, thus providing the type of ampli¬ fication available with enzymes. A rationale for this catalysis has been advanced by Schuster (1979) Accts. of Chem. Res. , 12, 366. The CL system of the present invention, "CLASSIC", also has certain advantages over fluores¬ cent antibody techniques which make use of the ability of a fluorescent tag to emit light of a particular wave length when excited by radiant energy of a lower wave length. A number of clinical analyses which utilize fluorescent "probes" or tags have been described in a recent review by Soini (1979) Fluoroimmunoassay: Present Status And Key Problems, Clin. Chemistry, 25, 353-361. In general, the detection level, or sensitivity, of fluoro- OMP - 27 - immunoassay techniques is greater than enzyme immuno¬ assay techniques and approaches the capability of radioimmunoassay systems. The use of fluorescent probes to replace radioactive isotopes is hindered by the decreased sensitivity obtained with fluorescence. This is due, to a great extent, to the sample's or serum's own fluorescence. The intensity of this background is affected by many fluorescing compounds, such as protein which may be present in the sample, and which also increase scattering caused by the specimen. Fluorescence methods are now extensively applied in immunology, mainly in fluorescence microscopy, for studying various types of tissues, cells, bacteria, viruses and so on. A number of fluorescent materials and procedures for coupling them to the above biologicals and haptens is well developed. To take advantage of the full scope of this invention, special high intensity fluorescent molecules are required. These must be capable of biological coupling with protein, polysaccharide and hapten substances, especially immunoglobulins. — i.e., I G - and antigens without disturbing the specificity or activity of these biological materials, Bellin (1968) Photophysical and Photo¬ chemical Effects of Dye Binding. Photocheπ. and Photobiol., 8 383-342 and Porro et al. (1963 and ■ 28 * and 1965) Fluorescence And Absorption Spectra of Biological Stains. Stain Technology, Vol. 3_8_, and Fluorescence And Absorption Spectra Of Biological Dyes (II). Stain Technology, Vol. 4 , No. 3, 173- 175, respectively, have shown that there is a reduction in efficiency in the light output of fluorescers as a result of bonding or conjugation to proteins as compared to the output of these fluorescers in free solution. Our work has shown a similar loss in output, however, the energy efficiency of the oxalate system compensates for this loss. While this loss in light output effects all other known applications of conjugated fluorescers, the analytical method of the present invention re¬ quires a conjugate only during the biological anti¬ body/antigen formation phase of the analysis. Pro¬ cedures are well known for preparing a conjugate of a fluorescer in a manner which permits the con¬ jugate to be subsequently separated at will by changing the pH, or other parameter, of the conjugate solution. It should * also be noted that the immuno¬ chemical reaction of CLASSIC, Method III, may be carried out in the environment best suited for the optimum detection of the analyte of interest. After the label has been identified with the analyte one may then separate the label, the fluorescer, from the conjugate which allows the fluorescer to enter the solvent phase of the CL system to yield the maximum light efficiency. In general, it is desirable to provide the high quantum efficiency of fluorescing aromatic and substituted hydrocarbons, heterocyclic compounds. - 29 - dyestuffs, and metal chelates with the ease of conjugation to the biological now available for microscopy reagents. We have found that we can couple the fluorescer using known procedures cur¬ rently accepted for use with the fluorescent con¬ jugates such as set forth in Soini (1979) supra, the teachings of which are incorporated herein by reference. The following Tables 2 and 3 from Soini (1979) supra, set forth data on various fluorescent materials some of which can be advantageously employed as labels. U> O O O Cn Table' 2 Published Dnln on Iho Propoiilos of Fluorochromos Used for Various Purposes, Including Dccny Tlmos (r), Qunnlum Yields (0), Excllnllon and Emission Wnvolonolho, ΛbsorbnncoB (e), and the . • Sotislllvltles ol Fluorescence to Polarity Changes In Iho Environment * • Stntll lly », E-c. n__. _*m ( ,πι*>«_. Mot. at lluo<»*«*»rκ-« r x>τ cli_-o<n« n* ( o» r-m nm dbtoi-p. lo ChΛrnjoi l.*l*ftnc*(«) FΓTC -4,5 0,05 492 510 72000 slαblo • 60,51,52.53 Tnrrc 510- 40000 stable 55-60 ' πorrc 3 0.7 550 505 12300 stable 65, 57.50 no 200SC 1 • 0.04 530, 505 595,710 slablo 40,49.55 DNS-CI 1-4 0,2 0,3 .3-10 520-100 3400 sensitive 31.32,42.47 flixxam 7 0,00 0.1 39-1 475 6300 sensitive 46,65 1 • MDPF 0 0,1 390 400 G400 not ( J1-G4 o NDD-CI 466 530 12900 senslllvo 44,45 • ΛNS (ΛN ) 16 0,0 0,9 305 471 sensitive 41.29 NPM (PDΛ) 100 0,0 3-10 392.375 sonsllivo 37.30,39,40 DΛCM 5 0,0 0,5 303-390 400-402 24200 sonslllvo 29,33.34,35 DIPM 1 0,0 0.5-0, fl 310-315 3G0 20000 sonsllivo 29.33 nnllvaccoo-fTC 29 0,6 357 4G0 3040 nol 4ft FAM 20 0,2 362 462 29,36 -3 t t I- * l_n O O t_n O l_π Tab le 3 Fluorescence Maxima and Detection Llmils ol Some Probe Conjugates Based on Measuremenls by Ihe Authors * D*l»ctl«<ι IVnrt P*ob-» D D --Kκjfllrrv>gg ll**rrpp Eicl tlon Emliilc-n t*-* •rrwn-* ( iπo) π»m»-ffc» ΠTC-BSA covalenl 491 517 30 ng/mL 100C ng/mL Inlcrlcrcncc by scattering covalcnt 491 517 40 ng/nnL 1400 ng/rnL Interference by scattering -thyroxlno covalenl 490 515 1 pmol/mL 35 pM/mL Inlorlerencθ by scattering RBfTC-BSA covalenl 552 572 100 ng/mL 430 ng/mL Interference by scattering . DNS-BSA covalenl 360 514 100 ng/mL 7000 ng/mL Intcrlerence by I scrum fluorescence t -hlgG covalenl 360 514 -thyroxine covalenl 330 460 30 nmol/rnL? 2100 nmol/mL Interference by serum lluorescenco -cys-digoxin covalenl 356 533 140 pmol/mL 98 nmol/mL Interference by serum fluorescence Fl oraπvBSA covalenl 393 465 1500 ng/mL 32000 ng/mL Interference by sorum fluorescence -hlgG covalenl 393 465 -thyroxlno covalenl 395 400 25 pmol/mL 535 pmol/mL Interference by scattering llυo-fcscencβ '. NP -BSA ' covalont 335 392 100 ng/mL 1500 ng/mL Interference by scattering and fluorescence •IgG covalenl 340 392 2000 ng/mL ANS-BSA . noncovalonl 305 470 ANSC-B..A covalenl 3B0 470 10000 ng/mL TNS-BSA noncovalenl 322, 360 429 NBD-BSA covalenl . 466 526 4000 ng/mL 240 /ig/mL Footnotes to Table 3 a Measurement results have been obtained with some commonly used probes as conjugates of bovine serum albumin, IgG, thyroxine , and digαxin. Conjugation was by common methods described in the literature (47 , 37 , 46 , 44) . No attempt was made to optimize measurement in any way, " this was done directly at emission maxima for bandwidths of 10 nm. • No cut-off filters were used. It would probably have been possible to reduce the detection limits of some probes cons iderably by altering the slit- values and by adjus ting the measurement wavelengths , and by using suitable cut-off filters . (The emission of fluorescein for example is usually measured at 540 nm, although the emiss ion maximum occurs at 515 nm. ) The fluores cence and detection limits for different probe- conjugates were measured with, a Perkin-Elmer " fluorescence spectrometer , Model MPF-2A. The detection limits were measured in the regions of excitation and emission maxima, and the values compared with the back¬ ground fluorescence values of diluted serum at the same ■ t. wavelengths and with the same instrument sensitivity, No reaction in IgG, SH-groups . Serum background, may bind to different proteins . Interference by protein fluorescence. __ Interference by serum fluorescence, own fluorescence weak. BSA, bovine serum albumin; hlgG, human i munoglobulin G. - 3 3- Typical of fluorescers which provide derivatives to which the biological may be coupled are those set forth by Pringsheim (1946) Luminescence Of Liquids And Solids And Its Practical Applications. Interscience Publishers / Inc., New York, NY. In addition to the organic fluorescers listed by Pringsheim, a number of metal organic materials have been suggested for laser fluorescent assay systems: Ruthenium (II-tri-(bibyridyl) complex has been identified by Curtis et al. (1977) , Chemi¬ luminescence; A New Method For Detecting Fluorescent Compounds Separated By Thin Layer Chro atography, J. Chromatography , 134, 343-350 for CP applications; Metal Complexes by Sherman (1978) , Analytical Appli¬ cations Of Peroxyoxalate Chemiluminescence, Analy¬ tical Chem. Acta, 97, 21-27 and Soini (1979) supra. eider U. S. Patent No. 4,058,732 disclosed and suggested their jjnmunofluorescent application. It is also well known. Van Uitert (1960) , Factors In¬ fluencing The Luminescent Emission States Of The Rare Earths. J. Electrochem. Soc, 107, 803, that small additions of the rare earth and/or transition metals function as promotors, activators or co- activators in inorganic and organic phosphors. Thus, it is not unexpected that trace impurities will behave in a similar manner in other organic and metallo-organic systems and have a profound effect on the quantum efficiency of the fluorescer. The discussion has thus far centered around the novel analytical use of a fluoresce -biological conjugate activated by the chemical energy from a - 34 " peroxyoxalate CL system. The preferred peroxyoxalate system is advantageous for CL because of its quantum efficiency and because there is minimal background light in the absence of a fluorescer conjugate. This system is particularly "noise free" when certain intensity control additives are eliminated, sucn as t are disclosed by Bollyky (1972) Chemiluminescent Additives ϋ. S. Patent No. 3,704,231. A system for analytical purposes need only provide light of high intensity for a short period, that is, for example, under about 30 minutes. 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 wavelength. Such esters include 2-napthol-3,6,8-trisulfonic acid, 2-carboxyphenyl, 2-carboxy-6-hydroxyphenol, l,4-dihydroxy-9, 10 diphenylanthracene, 2-napthol, as well as aqueous CL materials such as luminol, lophine, pyrogallol, luciferin, and related compounds. Other systems besides those mentioned are also capable of activating a CL fluorescer-conjugate. These include: (1) Ozone, which has been shown by Randhawa (1967) , Ozonesonde For Rocket Flight, Nature , 213, 53 to activate Rhodamine-B. (2) Keszthelyi et al. (1969) , Electrogenerated Chemiluminescence: Determination Of Absolute Lum¬ inescence Efficiency, etc., Analytical Chem. , 47, 249-256, has demonstrated electro-generated CL in 9,10-diphenylanthracene, thianthrene, and rubrene, - 3 5 - with some systems. Thus, ozone or electro-generated CL in the presence of the fluorescer-conjugate can provide other useful energy sources for the CL fluorescer systems of the present invention. In addition, other known energy sources such as have been found useful in applications involving the distortion of various polymers by mechanical energy and other similar systems which yield free -radicals are also useful in the present invention. It should be understood that many analytica system variations are possible, but all have in common the use of a labelled immunological specie specific to the analyte. The analyst has the lati¬ tude in selecting a procedure which provides the detection level required from a minimum amount of samples and which uses the lease expensive and most reliable instrument. The detection level required is a function of the antigen, antibody or hapten concentration in the analyte and its clinical signi¬ ficance. For clinically significant dosage testing - i. e. Digoxin, standard curves are obtained from known samples analyzed ' together with the unknown and run under carefully controlled duplicate analyses on highly calibrated instruments. While a presumptive test for an im unoglobin requires a much lower level of sophistication, it is highly advantageous for a single analytical system to be able to cover this analytical spectrum. - 36- The sophisticated analytical requirements may be met by using a Centrifugal Fast Analyzer such as that made by Electro-Nucleonics, Inc. Burtis et al. (1975) , Development Of a Multipurpose Optical System For Use With A centrifugal Fast Analyzer. Clinical Chemistry, 21 1225-1232. For the N th nations lacking the ability or need for such sophis¬ tication, or for presumptive testing at the physician' office or clinic, no instrument is required. The "CLASSIC" system of the present invention delivers sufficient intensity to the labeled biological to enable the clinician to make a simple go-no-go determination by "eye-balling" * . The clinician may also modify the role of the labeled specie used in carrying out the analyses. While solid phase techniques have been used as examples to illustrate the advantages of the pre¬ sent invention, it should be recognized that homo¬ geneous and heterogeneous assays also will benefit from the use of the "CLASSIC" system. Acceptable alternative variations in test procedure include: (1) Competitive binding of labeled antigen. (2) Competitive binding of labeled antibody. (3) Quenching analyses. (4) Immunoprecipitant reactions. (5) Ion exchange methods. (6) Ion exclusion methods. - 3 7 - DESCRIPTION OF THE INVENTION The major components for the preferred "light-switch" or "light indicator" of the present invention are similar to those disclosed in ϋ; S. Patent No. 3,597,362. They include an oxalic ester, a hydroperoxide, a fluorescer (or fluorescent com¬ pound) and a diluent. Furthermore, in order to generate maximum intensity of light, the employment of an additional catalytic accelerattor is sometimes necessary. The choice and the concentration and other parameters of a suitable catalytic accelerator is also described in ϋ. S. Patent No. 3,704,231. The present invention differs from the teaching of U. S. Patent No. 3,597,362 in that the fluorescent compound (or fluorescer) employed in this invention is covalently bonded to a biological material, such as immunoglobulin, enzymes, proteins, bacteria, and so on; or to an organic material, such as haptens or polymers; or to an inorganic material, such as glass, silica, ceramic, or the like. The organic and inorganic materials with which a suitable fluorescer may be incorporated can be in the form of particles, crystals, tubes, rods, plates, blocks and the like, or in solution. The fluorescent com¬ pound, or fluorescer, incorporated with the above mentioned substances can then be utilized as a label in place of radioactive materials or as an indicator in place of color dye, for use in various well-known assays. 3 8 " Espeσially suitable fluorescent compounds, or fluorescers" for use in the present invention are those which have a spectral emission falling between 350 millimicrons and 1,000 millimicrons. The struc¬ ture of the fluorescent compounds or fluorescers use¬ ful 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-, thionyl halide-, sulfonyl halide-, nitrobenzoyl halide-, carbonyl halide-, triazo-, succinimido-, anyhdride-, haloacetate-, hydrazino-, dihalo trizinyl-. Typical examples of suitable fluorescer derivatives are: 3,4,9,10 perylene tetracarboxylic dianhydride, . amino- chrysene, fluorescein isothiocyanate, tetera- methylrhodamine iosthiocyanate, amino-pyrene, amino- anthracene, and similar compounds as will be -familiar to those skilled in the art. It has been observed that on binding a fluorescent compound, or fluorescer, to a solid material, the wavelength of emissio of the incor¬ porated fluorescer shifts to either a longer or a shorter wavelength depending on the specific fluorescer icroenvironment employed. We have also found that the length of "space arm" the ligand between the fluorescers and the material bonded to it, effects the emission wave¬ length of the bonded fluorescer. 39 The exact concentration of fluorescer derivative incorporated is not critical providing that the immunological or enzymatically active fluorescent incorporated product has the desired activity, and that the intensity of light thus 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 fluorescer depends upon the structure of the fluorescer, the type of linkage between the fluorescer and the bonded materials, and the avail¬ able functional groups of the anchored substance. In general, the intensity of the light produced by a fluorescer is not as great after coupling as it is when in free solution. It is also important that the fluorescer conjugate be stable in the pre¬ sence of the chemiluminescent reaction. united States Patent No. 4,238,195, which issued on December 9, 1980 describes an assay method employing a fluorescer as a label and claims the improvement comprising measuring said fluorescent label by chemically exciting said label to cause the same to emit light and then measuring the light emitted. This reference sets out a number of assay methods in which the claimed invention may be employed. The teachings of said United States Patent No. 4,238,195 are hereby incorporated by reference in the present application. - 40- The present invention provides for an improved method for carrying out an assay employing an encapsulated fluorescer or quencher/poison label wherein the encapsulated fluorescer label * is to be excited either chemically or by other non-radiant energy means. It has been found that higher quantum efficiency of fluorescer and light output may be obtained by encapsulating fluorescers. The micro- capsule is then conjugated as a label to the biologi¬ cal. After completion of the biological analysis which carries the microcapsule containing the fluo¬ rescer label through the aquous phases, the micro- capsule is disrupted releasing the fluorescer in an unbound state. This improvement offers the following advantages over previous art: (1) More fluorescer label is available without effecting the biologibal activity of the conjugate. (2) The fluorescer in solution is more efficient than bound fluorescer. (3) There is no requirement for any reactive group on the fluorescer. (4) Excess fluorescer is more readily separated from the biological after incorporation. (5) The ability to select either hyro- phillic or hydrophobic fluorescer. (6) Allows one to select the most efficient fluorescer catalyst/or poison for the high energy intermediate. (7) Allows one to minimize the ncn- - 41 - specific absorption of the fluorescer probe to other proteins which increases the overall sensitivity of the analysis. While the use of fluorescer labels has been previously described and the advantages of the use of excited or non-radiative energy to enhance the fluorescence signal from these fluore¬ scers when conjugated to a biological for use ' as iinmunological assay's probe has been noted, the advantage of the chemiluminescence assay's sensiti¬ vity is often limited by non-specific binding re¬ actions and the inherently limited choice in selecting the quantity of the most advantageous fluorescer molecule for the specific assay to be undertaken. It has now been discovered that by utili¬ zing recently developed encapsulation techniques we can eliminate the restructions imposed by direct conjugation of the fluorescer label to the biological. Encapsulation means any method whereby a carrier such as a liposome or polymer for a fluorescer or quencher/poison incorporates such fluorescer or quencher/poison in such a manner that it may be attached to a biological specie specific to an analyte of interest and later released free into solution. Many encapsulation processes are known to the art, which will allow one to prepare suitable encapsulated fluorescers as the mechanism as now - 4 2 " under stood. In order to function in any efficient immu¬ noassay the encapsulating system should possess the following properties: (1) Colloidal size sufficient small to permit free suspension and not inhibit the movement and immunological binding of the labelled species, uniform size is essential. (2) Internal volume of the microcapsule 10 should be as large as possible, i.-.e.. that is to say the microcapsule should have a minimal wall thick¬ ness for containment. Alternatively the microcapsule should have a maximum wall thickness for embedding the fluorescer, in the event such a technique is 15 employed. (3) The microcapsule should have one or. more reactive groups to enable its conjugation with the biological. (4) The microcapsule should be stable in 20 the environment of the biological assay. (5) The membrane of the microcapsule should be easily disrupted to allow the fluorescer to escape to solution, but with minimal detriment to the chemiluminescent signal to be generated by 25 the fluorescer. (6) The enclosed fluorescer should be contained as a liquid or solid with minimal leakage from the microcapsule. (7) The microcapsule should be stable 30 and provide long shelf life with minimal storage and environmental requirements. (8) The microcapsule may possess positive, 35 - 43 " negative or neutral charge on its surface. (9) The microcapsule should be easily and economically produced. The improved method of the present inventio allows for the utilization in any conventional assay, such as those previously described, or as set forth in the United States Patent No. 4,238,195, referred to above, an encapsulated fluorescer as the label, so long as the encapsulating technique used to form the microcapsule or sac containing the fluorescer material permits the inclusion of a large concen¬ tration of fluorescer, has appropriate binding sites on the surface of the microcapsule or sac to allowing for conjugation to a immunological specie specific to the analyte of interest and the encapsulating material is readily disruptable to free the contained fluorescer which may subsequently be excited to emit light. Liposomes as small as 250A° have been pre¬ pared by techniques described by Papamadjopoulos (1978), Korn (1973), Huang (1969) and Bangham (1976). The incorporation of fluorescers as markers for cell surface studies when excited by u.v. light has been demonstrated by Weinstein (1977) . Torchilin (1979) and Heath (1980) have successfully incorporated a conjugatable binding site to the liposome bilayer. - 44- This work provides us with the ability to place bound and unbound fluorescent probes in micro¬ capsules or sacs. The sacs may be filled with far more of the fluorescer of choice than could hereto¬ fore effectively be conjugated directly to the biolo¬ gical to be used for the assay probe. The fluorescer to be incorporated into the microcapsule or sac of the present invention may also be selected to optimize the catalysis of the high energy intermediate, and also to optimize - the emission spectra from the probe. As a result one can achieve improved signal/noise output of the system by at least 25 times and vastly broaden the category of practical fluorescers, which may be utilized in carrying out any fluorescer ' labelled assay. The preferred fluorescers to be incor¬ porated into the microcapsule of the present inven¬ tion include 5,12-dihydro quinoxalo (2,3,6) phena- zine magnesium and zinc metalo porphyrins, neutral red, magdalla red, acridine red, acridine orange, dianiasyl ethynlyl tetracene phenizine, rhodamine, 3,4,9,10 perylene tetracarboxylic dianhydride, and derivatives thereof or similar materials which meet the requirements stated above. The following examples are given to illus¬ trate the various ways the fluorescer may be attached to another moiety by covalently bonding using an inorganic support for convenience, which is in no way intended to limit the scope of the invention " 45 " described herein. EXAMPLES I-V In each of the Examples I-V the linkage attached to a controlled pore glass surface was synthesized to imitate the representative chemically active sites on a typical protein or biological con¬ jugate. For example, amino-, carboxyl-, mercapto-, or hydroxyl- groups are representative of attach¬ ment sites. A glass support is used so that the activity and specificity of the functional group is easily controlled, and to immobilize the fluorescer so that it may be readily separated from the free or unbound fluorescent compound in order that the fluorescent spectra may easily be recognized as distinct from the oxalate CL reagent. The results of visual observation as to the color of the fluorescent glass, and color and intensity of emitted light for 1-aminopyrene coval- ently bonded to porous glass (CPG) (500A° pore size) fluorescer with various different linkages are set forth in the attached Table 4. The methodology employed for preparing each fluorescer/glass sample was as follows: EXAMPLE I o Ten grams of porous glass of 500 (A) (ang¬ strom pore size) was treated with 100 ml 15% gamma- aminopropyltrimethoxysilane in toluene and refluxed -4 6 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 1-aminoρyrene was dissolved in dioxane (20 millimeter) . To this solution about 153 milligrams of succinic anhydride was added. After two hours, 10 millimeter of 5 m mole N,N- dicyclohexyl-carbodiimide dioxane solution was added. 500 mg of this gamma-aminopoplytrimethoxy- silane treated glass (from here on, aminopropyl- 10 glass) as prepared above was added to dioxane solu¬ tion. . The 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 exhaustivel -'"- ■ ' with dioxane, methanol and acetone (15 ml of each wash and three times for each solvent) . The wet pyrene coupled glass was filtered and allowed to air dry. 0 EXAMPLE II 500 mg of the aminopropyl-glass prepared as stated in Example I was added to 25 ml of 10% thiophosgene in chloroform and the slurry was refluxed for 4 hours. 'The chloroform was decanted 5 and then washed with chloroform, methanol, acetone (25 ml of each wash and three times for each solvent) , The slurry was filtered and air dried. 30 milligrams of 1-aminopyrene was dissolved in 15 ml dioxane. To this solution, the dry isocyanato-glass was 0 added and stirred for one hour and then allowed to stand at room temperature overnight. After the reaction was complete, aminopyrene dioxane solution 5 -47 - was decanted and the pyrene coupled glass was washed in the same manner as stated in Example I. EXAMPLE III 500 g of aminopropyl-glass, prepared as stated in Example I, was added to 10 ml of dioxane dissolved with 50 mg s ' uccinic anhydride. The slurry was allowed to stand overnight at room temperature preferably with continuous stirring. After the reaction was complete, the aminopropyl-glass, being converted to carboxy-glass, was washed in the same manner as stated in Example I. Approximately 23 mg 1-aminoρyrene was dissolved in 1 ml of dioxane. To this solution, 58 mg of N-acetyhomoc stein was dissolved. The solution was then kept 4 hours at room temperature. 50 mg of N,N-dicyclohexyl-carbo- diimide was then added to it. At the same time, the prepared and dried carboxyl-glass was added to the solution for coupling. The reaction was allowed to stand at room temperature for 24 hours. Pyrene coupled glass was then washed and dried in the same manner as stated before. EXAMPLE IV 4 grams of aminopropyl-glass prepared from Example I was added to 10% p-nitrobenzoyl chloride with 1 ml of triethylamine in 50 ml chloro¬ form solution. The slurry was stirred and refluxed for at least 8 hours. The resulting acylated glass was thoroughly washed with chloroform and let air dry. 0.1M of sodium dithionite (30 ml) was pre¬ pared and the acylated glass was added. The temp¬ erature was then raised to 40°C. The reaction was ^^GRE - 4 8 - completed in one hour. The glass was washed thoro¬ ughly with warm water. The arylamino-glass thus prepared was ready to diazotize. 1 gm of arylamino- glass was added to 20 ml aqueous solution of 350 mg sodium nitrite and 0.2 ml IN hydrochloric acid. The temperature was brought down to 4°C using an ice bath. The reaction was allowed to continue for one hour. The acid solution was then decanted, the glass was thoroughly washed and the pH was adjusted to above 8.0. The filtered glass was then added to 10 ml of 20 mg aminopyrene dioxane solution. Reaction was complete in 8 hours at room temperature. The pyrene coupled glass was then washed in the same manner as in Example I. EXAMPLE V One gram of 500 (A) pore size porous glass treated with 10 ml 15% gamma-glycidoxypropyltrimethoxy- silane in toluene and refluxed for at least 16 hours, then washed the glass with acetone thoroughly and air dried. To 30 ml aqueous solution containing 1.5 mg/ml of m-sodium periodate, the silane treated glass (epoxy-glass) was added. The reaction was allowed to go on for 2 hours. Then the glass was washed with water thoroughly. 25 mg of 1-aminopyrene was dissolved in 30 ml dioxane. To this solution, the filtered wet cake glass was added. The slurry was stirred,for one hour and then let stand overnight at room temperature. Then pyrene coupled glass was washed in the same manner as stated in Example I. LO NJ O o J- * •_, Oi TABLE - EXAMPLES I-V Effect of Different LlnkβRββ Color of Color the Light** Type of Fluorescent Emits on Glass Intenβlty* Example No. Linkaβe Structure Glass Particle Observed I Λmldo 11 0 blue-green bluish green M " 1 II Flr-N-C--(CPC) II Thlourea 11 S 11 blue-green bluish green M Flr-N-C-N—(CPG) m Thlo-eβter • 0 grayish green bluish green W-M Flr-S-C—(CPG) IV D azo Flr-N=N--(CPG red-brown red-brown VW Amide 11 H as CPG blue M Flr-N-C--(CPG) * H - medlumi W - weak; VU - very weak ** After addition of oκnlntc enter nnd hydrogen poroxl.de - 50- EXA PLE VI Different lengths of "space arm" for binding of the fluorescer were used to study the effect on the chemiluminescence quality of the resultant bonded fluorescer. o A long "space arm" of about 20 (A) in length stretching out from a controlled glass pore surface was prepared as follows: 500 mg of carboxy-glass prepared as stated in Example III was activated by adding a 20 ml dioxane solution containing 200 mg of N / N-dicyclohexyl carbodiimide. The glass was stirred for 24 hours and then washed with dioxane and methanol. 20 ml of 200 mg hexamethylene diamine aqueous solution was prepared and cooled beforehand. The activated carboxy-glass was added to the cooled solution and stirred for five hours, then allowed to stand for 24 hours at 4°C. The glass was then washed thoroughly with water, methanol and dioxane. 20 ml dioxane containing 50 mg succinic anhydride was then added to the glass. This reaction was completed in 24 hours. The glass was subsequently washed thoroughly with methanol. 25 mg l-aminopyrene was dissolved in 30 ml dioxane. To this solution 5 mole N,N-dicylohexylcarbodiimide was added and dissolved prior to adding the prepared glass. The slurry was stirred for one hour and then let stand overnight at room temperature. After 24 hours reaction, pyrene coated glass was then washed in the same manner as in Example I. Pyrene coated glass with a short "space OMPI arm" of a b out 10 (A) in length was prepare d as stated in Example I, as the control. The results of these two glasses is set forth in attached Table 5. J _S.__ι. OMPI 52 TABLE 5 - EXAMPLE VI Effect of "Space Arm" Length Between Glass Surface and the Fluorescer on Chemiluminescent Characteristics Approximate Length Intensity of "Space Arm" Color of Light Observed* (Example I) Control 10(A) bluish-green M o Example VI 20 (A) green - * M = medium; W = weak EXAMPLES VI - X Porous glass having various pore sizes were coated with 1-aminopyrene to show the effect of pore size on the chemiluminescence. Three o different porous glasses having 170 (A) (angstrom) , 500 (A) and 3000 (A) pore size, respectively, were coated with 1-aminoρyrene in the same manner as stated in Example I. The effect on the chemilum¬ inescence is set forth in attached Table 6. N3 t o Ln Cn TABLE 6 - EXAMPLES VII-IX Ef fect Of Pore S ize On Chemllumlneacence Color of Fluorescent Gli IBS Pore Size Surface Area Before After Color of Intensity Example No. (In nnftntrom) (In M2/p.m) CootlnR Con inς Llp.ht Emitted Observed VII 170 110 clear green yellowlβh green M VIII 500 50 It. blue It. blue bluish green W-H tn IX 3000 10 white white blue W I - 5 4 - EXAMPLES X-XV Several different fluorescers were coated on porous glass to study the effect of structure on color emission. 1-aminoρyrene and 2-amino-anthracene o were coated on the porous glass (500 A) in the same •5 manner as described in Example I. 20 mg of 3,4,9,10-perylenetetracarboxylic dianhydride was added to 25 ml of dioxane, to this solution 25 mg of aminopropyl-glass was added and 10 stirred for one hour before allowing to stand for another 6 hours at room temperature. The glass was then washed thoroughly with methanol or acetone, then filtered and air dried. i-5 500 mg of aminopropyl-glass was added to 30 ml dioxane containing 50 mg succinic anhydride and stirred for one hour before being allowed to stand overnight at room temperature. The glass was then washed thoroughly with acetone, filtered 20 and air dried. One part of 250 mg of such glass (carboxyl-glass) was added to 25 ml 0.01M potassium . phosphate of pH-7.6 solution containing 20 mg of isothiocyanate fluorescein. Another part of 250 of carboxyl-glass was added to acetone-dioxane ^ ■ - > (50/50 by volume) solution containing 20 mg of 3- amino-phthalhydrazide. The two glass slurries were stirred for one hour and then allowed to stand at room temperature overnight. After the reaction was completed, the glass was washed with deionized 30 water and acetone, respectively. Finally, they both were washed with acetone, then filtered and air dried. 35 - 55 " 300 mg of aminopropyl-glass prepared as shown Example I was added to 50 ml 0.01M potassium phosphate of pH»7.6 solution containing 25 mg of O-phthalicdicarboxaldehyde. The glass slurry was stirred for one hour, then allowed to stand at room temperature for another 24 hours. The glass was then washed thoroughly with deionized water, acetone, then filtered and air dried. The attached Table 7 sets forth the ob¬ served chemiluminescence characteristics of different fluorescers bonded to porous glass in an oxalate ester/peroxide system. 3 LO l- NJ O «_n l O TABLE 7 - EXAMPLES X-XV Comparison of Chemiluminescence of Different Fluorescers Free nnd Covnlently Attached to Porous (. aaa of 500(A) Pore Size Color of Color of Color of Light - Intensity Light - Fluorescence Example Fluorescent Uσndcd of Light Free U.V. Fluorescer No. Glass Fluorescer Observed Fluorescer Light 1-amlnopyrene blue green bluish green M green negative 2-nmlno-nntrα- XI It. brown violet M blue- negative cene violet 3,4,9, 10-pecyl- XII orange red orange-red M none orange ene Cctra-carboκ- I yllc dlαnhydride Fluorcaceln XIII yellow green M green yellow lsothlocyanαte 3-nmtno-phthal XIV It. blue bright blue M blue blue hydrazide (luminol) 0-phthalicdi- XV rellowiβh- greenlβh- W-M none negative carboxoldehyde >rown yellow - 57 - EXAMPLE XVI Aminopyrene conjugate with antibody to Hepatitis B Surface Antigen coated on porous glass. 30 mg commercially available antibody to Hepatitis B Surface Antigen coated porous glass was added to 5 ml of 0.01 m potassium phosphate of pH=7-6. 24 mg of 1-aminopyrene was dissolved in 2ml dioxane. To this solution 45 mg of succinic anhydride was added and mixed for two hours. Approximately 95 mg of N,N-dicyclohexyl-carbodiimide was dissolved in 1 ml of dioxane. The latter two solutions were mixed together and stirred for 30 minutes. Then 250 lamda of pyrene solution was transferred to the glass slurry solution. The slurry was stirred for two hours at room tempera¬ ture and then allowed to stand at 4°C overnight. The glass was washed four times with 10 ml phosphate buffer (pH=7.β) each wash, and was given two additional t-butanol washes with 10 ml phosphate buffer each time before testing. If necessary, the slurry was washed until no light could be detected from the supernate of the slurry. Then the 1-amino¬ pyrene-antibody conjugate coated on the porous glass was tested by reacting with oxalate and peroxide. It was found that only the glass particle glowed in faint blue color. EXAMPLE XVII Fluorescein isothiocyanate anti-human gamma-globulin conjugate was prepared as follows: 4 mg of fluorescein isothiocyanate thoroughly mixed in 10 ml 0.1 M potassium phosphate buffer of pH=9.0 , 5 4 ml of anti-human gamma-globulin (protein concen¬ tration of 20 mg/ml) was then added to the fluores¬ cein phosphate solution. The mixture was continu¬ ously stirred for one hour at 4°C and allowed to stand at the same temperature for another 24 hours. Excess fluorescein was removed by extensive dialysis against 0.1 potassium phosphate buffer of H=7.2. During dialysis, 100 ml of buffer each time was used, and the buffer was changed every 2 hours for 5 times. Gamma-globulin coated porous glass was prepared as follows: 50 mg of epoxy-glass (3000 o (A) pore size) was prepared in the same way as described in Example IV. 2.5 mg m-sodium periodate was dissolved in 5 ml of deionized water. Glass was then added to this solution and stirred at room temperature for 2 hours. The . glass was washed thoroughly with deionized water and then with. 10 ml 0.1 M potassium phosphate pE=9.0 buffer and kept for one hour. The glass was then filtered a___d was ready for coupling. 5 ml human gamma-globulin (pro¬ tein concentration of 30 mg/ml) was diluted with 5 ml of 0.1 M, pH=9.0 phosphate buffer. The acti¬ vated glass was then added to this solution and was stirred at 4°C for 2 hours before being allowed to stand overnight at the same temperature. After reaction was completed, the glass was washed exten¬ sively with 0.1 M potassium phosphate buffer of ρH=7.2 and then filtered for immediate use. 30 mg of human gamma-globulin coated porous - 59- glass was added to 0.5 ml of fluorescein-antihuman gamma-globulin conjugate. The slurry was incubated on 24 cycles of agitation/settling (60/90 seconds ratio) . Excess antibody solution was decanted and the glass was washed with 0.01 potassium phosphate buffer of ρH=7.2 until no light was detected by testing the decanted buffer in oxalate/ peroxide system. 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. EXAMPLES XVIII-XXI USE OF ENCAPSULATED FLUORESCER AS A LABEL IN CHEMILUMINESCENT IMMUNOASSAYS The following Examples are given to illus¬ trate the preparation and use of encapsulated fluorescers as labels, and are in no way intended to limit the scope of the invention described herein. EXAMPLE XVIII Chemiluminescence and entrapment of hydrophillic fluorescer (Hhodamine B) in lipso e A chloroform solution of phosphatidyl- choline (egg) cholesterol and phosphatidylethanoamine in molar ratio of 6:2:2 (total concentration about -60 - 26 mg) was placed into a 50 ml round bottom flask and the solvent evaporated on a rotary vacuum evaporator at room temperature. The lipid film was then purged with N-. 2.0ml of 0.01M borate buffer, pH 8.5 containing Rhoda ine B was added to the lipid film, the film was dislodged from the walls of the flask by vigorous shaking. The resulting emulsion liposome suspension was removed and was sonically treated for 5 minutes in ice bath employing an ultrasound generator. The liposome solution was passed through a sepharose 6B column to separate the uniform single compartment liposomes from the multilameller ones. The uniform single compartment liposome fraction was again passed through G-75 sephadex column to remove free Rhodamine B. 25 ul 0.0144M of TCPO (2,4,5-Trichloro- phenyloxalate) in Glyme .and 25ul of 1.23M H_0 2 were pipetted into 6 X 50 mm test tube. The liposome enclosed Rhodamine B solution was first treated with Triton X-100 (to release the Rhodamine 3) , 50ul of this solution was then injected to the H_0 2 /oxalate mixture, light was detected by Pico- Lite luminometer with a red filter. EXAMPLE XIX Chemiluminescence and entrapment/embedding of hydrophobic fluorescer DAΞT (Dianisylethynyltetracene) in liposome - 61 - A chloroform solution containing ganglio- sides, phosphatidylcholine, cholesterol (10:45:45 mole ratio) and 200mM DAET was evaporated in rotary vacuum evaporator at room temperature. The lipid film was purged with N~ and flooded with 2.0ml of 0.010M borate buffer pH8.5. The lipid emulsion solution was then sonicated for five (5) minutes in an ice bath. The liposome solution was first passed through Sepharose 6B column and again G-75 Sephadex column to separate undispersed lipids, multilameller liposomes and free fluorescer. The liposome solution was treated with Triton X-100 to ensure the rupture of the lipid membrane. 25ul of 0.0144M TCPO and 25 ul of 1.2 3M H-0- were pipetted into a 6 X 50 mm- test tube and the tube was loaded into the analyzer of Pico- Lite luminator. A 50 ul of the Triton X-100 treated liposome solution was injected into the test tube and the emission of light was. detected by Pico-Lite luminometer with a red filter. EXAMPLE XX Chemiluminescence and entrapment of fluorescer (Hydrophillic or hydrophobic) bound onto silica sol 0.5 ml silica sol of 5nm size was diluted with 0.5 ml of 0.01M borate buffer pH12.5. 25ul of δ -a inopropyltriethoxysilane was then added to the OMP -62 - sol solution. The mixture was then vigorously shaken to disperse the sole particles. The sol solution was then dialyzed extensively against de¬ ionized water pH=9.0. LRSC (lissamine Rhodamine sulfonyl chloride) 5 mg in 1.0ml 0.01M borate pH=12.5 was then added to the sol solution and incubated overnight. The Rh-B/sol was then separated from free fluorescer by either dialysis or gel filtration. The fluorescent sol was then dispersed in borate buffer and enclosed by liposome as indicated in Example XIX. The entrapped Rh-3/sol was treated with Triton X-100, and the solution was then injected to a mixture of TCPO/H-0-. Light was detected from Pico-Lite lumino eter with a red filter. EXAMPLE XXI Chemiluminescent immunoassay utilizing liposome entrapped fluorescer as a label 20ul of 25% glutaraldehyde was added to 2ml liposome containing Rhodamine B as prepared in Example XVIII and incubated at 20°C for 10 minutes. Excess glutaraldehyde was dialyzed against 1 liter of 0.145M NaCl for 1 hour and again 1 liter of borate buffer for another hour at room temperature. The activated liposome was then incubated with Anti-Hb Ag solution at 4°C overnight. After incubation, the liposome conjugated with antibody was purified by passing through a separose 4B column. 63 " The immunoassay procedure was carried out by using the reagents of RIAUSURE kit with the lipo¬ some conjugated antibody replacing the I labelled antibody. Triton X-100 solution was added to the test tube after the biological assay and before the addition of TCPO/H-0- solution. Light ' was generated by positive sample and detected by Pico-Lite luminator with a red filter. The preferred energy source for carrying out the method of the present invention is that generated by the reaction of a peroxide and an oxalate/oximide selected from the group comprising: bis (2,4,6 - trichloroρhenyl oxalate, bis (3-trifluoro methyl-4-nitrophenyl)- oxalate, bis (2-formyl-4-nitrophenyl)- oxalate, bis (2,6-dichloro-4nitrophenyl)- oxalate, N,N'-bis (2,4,5-trichlorophenyl)- N,N'- bis (trifluoromethyl sulfonyl)- oximide, N,N'-bis (2,4 - dichlorophenyl)- N,N'- bis (trifluoromethyl sulfonyl- oximide, N,N'-bis (2-methoxy phenyl -N,N'- bis (trifluoromethyl sulfonyl)- oximide, and N,N*-bis (2-nitro ethyl)- N,N'- bis (trifluorό methyl sulfonyl)- oximide. Although the above examples illustrate various modifications of the present invention, other variations 'will suggest themselves to those skilled in the art in the light of the above dis¬ closure. It is to be understood, therefore, that changes may be made in the particular embodiments described above which are within the full intended scope of the inventions as defined in appended claims. OMPI

Claims: - 64- CLAIMS 1. A system for the detection of a bio¬ logical analyte of interest which comprises an encapsulated fluorescer material which has been conjugated to an immunological specie specific to the biological analyte of interest, a means of dis¬ rupting the capsule containing the fluorescer and an energy source other _than electro-magnetic radia¬ tion which is capable of activating the fluorescer. 10 2. A system for the detection of a biolo¬ gical analyte of interest which comprises an encap¬ sulated fluorescer which has been conjugated to an immunological specie specific to the biological 15 analyte of interest, a means of disrupting the capsule containing the fluorescer and an excess of an energy source other than electro-magnetic radiation which is capable of activating the fluorescer. 0 3. A method for the qualitative detection of a biological analyte of interest comprising: (a) labeling an immunological specie specific to the analyte of interest with an encapsu¬ lated fluorescer material which is biologically 2 compatible with such specie; (b) contacting the encapsulated fluorescer labeled specie and the biological of interest to form an encapsulated fluorescer labeled specie/biological complex; n JU (c) separating the encapsulated fluorescer labeled specie/biological complex; (d) disrupting the capsule containing the 35 -6 5 - - ■ fluorescer label thus freeing it to solution; (e) contacting the freed fluorescer with an energy source other than electro-magnetic radia¬ tion which is capable of activating the fluorescer label; and (f) determining the presence of chemilumi¬ nescent light emitted. 4. A quantitative method for measuring the amount of a biological analyte of interest comprising: (a) labeling an immunological specie specific to the analyte of interest with an encapsu¬ lated fluorescer material which is biologically compatible with such specie; (b) contacting the encapsulated fluorescer labeled specie and the biological of interest to form an encapsulated fluorescer labeled specie/ biological complex; (c) separating the encapsulated fluorescer labeled specie/biological complex; (d) disrupting the capsule containing the fluorescer label thus freeing it to solution; (e) contacting the freed fluorescer witn an energy source other than electro-magnetic radia¬ tion which is capable of activating the fluorescer label; and (f) measuring the quantum of chemilumi¬ nescent light emitted. 5. The method of claim 3 wherein the encapsulated fluorescer material of (a) is chemically conjugated to the immunological specie specific to - 6 6- the biological of interest. 6. The method of claim 5 wherein the chemical conjugation of the encapsulated fluorescer material to the immunological specie specific to the biological of interest is carried out using known techniques in such a way as to prevent sub¬ stantial biological damage to the attached specie. 7. The method of claim 3 wherein the encapsulated fluorescer material utilized has a spectral emission of from about 350 millimicrons to about 1000 millimicrons. 8. The method of claim 3 wherein the encapsulated fluorescer material utilized has a spectral emission above the emission wavelength of the immunological specie specific to the biolo¬ gical of interest, the energy source, or any solvent system utilized. 9. The method of claim 3 wherein the encapsulated fluorescer material utilized has been formed via known encapsulation techniques to pro¬ duce a microcapsule having a high concentration of fluorescer material. 10. The method of claim 3 wherein the encapsulated fluorescer material utilized has a microcapsule structure having one or more reactive groups to enable the microcapsule's conjugation with, an immunological specie specific to the analyte of interest. -6 7 " 11. The method of claim 3 wherein the encapsulated fluorescer material utilized possesses a microcapsule of uniform colloidal size whose structure has a membrane which is easily disrupted to free the fluorescer material. 12. The method of claim 3 wherein the encapsulated fluorescer material utilized is selected from the group consisting of 5,12 -dihydro quinoxalo (2,3,6) phenazine, magnesium and zinc etalo por- phyrins, neutral red, magdalla red, acridine red, acridine orange, dianiasyl ethynlyl tetracene, phenizine, rhodamine, 3,4,9,10 perylene tetracarbo- xylic dianhydride, and derivatives thereof. 13. The method of claim 3 wherein the energy source of (e) which is contacted with the freed fluorescer material is present in excess of the amount required to activate all of the freed fluorescer material. 14. The method of claim 3 wherein the energy source of (e) is any source other than electro-magnetic radiation which is capable of activating the particular fluorescer selected. 15. The method of claim 3 wherein tne energy source of (e) is the reaction product of a peroxide and an oxalate/oximide selected from the group comprising: - 68 - Q bis (2,4,6 - trichlorophenyl)- oxalate, bis (3-trifluoro methyl-4-nitrophenyl)- oxalat bis (2-formyl-4-nitroρhenyl)> oxalate, bis (2,6-dichloro-4nitrophenyl)- oxalate, N,N*-bis (2,4,5 - trichlorophenyl)- N,N'- bis (trifluoromethyl sulfonyl)- oximide, N,N'-bis (2,4 - dichlorophenylf N,N*- bis (trifluoromethyl sulfonyl)- oximide N,N'-bis (2-methoxyl phenyl)- N,N'- bis (trifluoromethyl sulfonyl)- oximide, and N,N'-bis (2-nitro ethyl)- N,N'- bis (trifluoro methyl sulfonyl)- oximide. 16. A method according to claim 14 wherein the energy source is a chemical reaction selected from the group consisting of 2-naρthol- 3,6,8-tri- . sulfonic acid, 2-carboxyphenyl, 2-carboxy-6-hydroxy- phenol, 1,4-dihydroxy-9, 10-diphenylanthracene, 2-naρthol, luminol, lophine, pyrogallol, luciferin, dioxetanes, dioxetaneones, and other peroxide reactions 17. A method according to claim 14 wherein the energy source is derived from a chemical reaction, ozone, an electrical current, an electro-chemical reaction, or a mechanically generated species. 18. The method according to claim 3 which is carried out utilizing a known assay technique. 19. The method according to claim 3 which is carried out utilizing a heterogeneous sandwich assay technique. 20. The method according to claim 3 which is carried out utilizing a heterogeneous competitive assay technique. - 6 9- 21. A system for the detection of a biolo¬ gical analyte of interest which comprises an encap¬ sulated quenching/poisoning material which has been conjugated to an immunological specie specific to the biological analyte of interest, a chemiluminescent reaction which generates an energy signal, and a means of disrupting the capsule containing the quencher/poison thereby liberating the quencher/ poison to free solution and vitiating or diminishing the chemiluminescent reaction generated energy signal. 22. The method of claim 4 wherein the encapsulated fluorescer material of (a) is chemically conjugated to the immunological specie specific to the biological of interest. 23. The method of claim 22 wherein the chemical conjugation of the encapsulated fluorescer material to the immunological specie specific to the biological of interest is carried out using known techniques in such a way as to prevent sub¬ stantial biological damage to the attached specie. 24. The method of claim 4 wherein the encapsulated material utilized has a spectral emis¬ sion of from about 350 millimicrons to about 1000 millimicrons. 25. The method of claim 4 wherein the encapsulated fluorescer material utilized has a spectral emission above the emission wavelength of - 70- i the immunological specie specific to the biological ϊ of interest, the energy source, or any solvent system utilized. i 26. The method of claim 4 wherein the ' 5 encapsulated fluorescer material utilized has been formed via known encapsulation techniques to pro¬ duce a microcapsule having a high concentration of fluorescer material-. 10 27. The method of claim 4 wherein the encapsulated fluorescer material utilized has a microcapsule structure having one or more reactive groups to enable the microcapsule's conjugation with an immunological specie specific to the 15 analyte of interest. 1 28. The method of claim 4 wherein the I encapsulated fluorescer material utilized possesses j a microcapsule of uniform colloidal size whose _ 20 structure has a membrane which is easily disrupted to free the fluorescer material, j i 29. The method of claim 4 wherein the encapsulated fluorescer material utilized is selected 25 from the group consisting of 5,12 - dihydro quinoxalo (2,3/6) phenazine, magnesium and zinc etalo por- phyrins, neutral red, magdalla red, acridine red, acridine orange, dianiasyl ethynlyl tetracene, phenizine, rhodamine, 3,4,9,10 perylene tetracar- 30 boxylic dianhydride, and derivatives thereof. 35 - 71 - 30. The method of claim 4 wherein the energy source of (e) which is contacted with the freed fluorescer material is present in excess of the amount required to activate all of the freed fluorescer material. 31. The method of claim 4 wherein the energy source of (e) is any source other than electro-magnetic radiation which is capable of activating the particular fluorescer selected. 32. The method of claim 4 wherein the energy source of- (e) is the reaction product of a peroxide and an oxalate/oximide selected from the group comprising: bis (2,4,6 -trichlorophenyl)- oxalate, bis (3-trifluoro methyl-4-nitroρhenyl)- oxalat bis (2-formyl-4-nitrophenyl)- oxalate, bis (2,6-dichloro-4nitrophenyl)- oxalate, N,N*-bis (2,4,5 - trichlorophenyl)- N, * N*-=_*. , bis (trifluoromethyl sulfonyl)- oximide, N,N'-bis (2,4 - dichlorophenyl)- N,N'- bis (trifluoromethyl sulfonyl)- oximide, N,N'-bis (2-methoxy phenyl)- N,N*- bis (trifluoromethyl sulfonyl)- oximide, and N,N'-bis (2-nitro ethyl)- N,N*- bis (trifluoro methyl sulfonyl)- oximide. 33. A method according to claim 31 wherein the energy source is a chemical reaction selected from the grou consisting of 2-napthol- 3,6,8-tri- • sulfonic acid, 2-carboxyphenyl, 2-carboxy- 6 hydroxy- phenol, 1,4-dihydroxy-9, 10-diphenylanthracen , 2-naρthol, luminol, lophine, pyrogallol, luciferin, dioxetanes, dioxetaneones, and other peroxide reactions. - 7 2- 34. A method according to claim 31 wherein the energy source is derived from a chemical reaction, ozone, an electrical current, an electro-chemical reaction, or a mechanically generated species. 35. The method according to claim 4 which is carried out utilizing a known assay technique. 36. The method according to claim 4 which is carried out utilizing a heterogeneous sandwich assay technique. 37. The method according to claim 4 which is carried out utilizing a heterogeneous competitive assay technique. 38. An encapsulated fluorescer composition which has been formed utilizing known encapsulation techniques to produce a microcapsule having a high concentration of fluorescer material. 39. An encapsulated fluorescer composition possessing a microcapsule structure having one or more reactive groups to enable the microcapsule's conjugation with an immunological specie specific to the analyte of interest. 40. An encapsulated fluorescer composition possessing a microcapsule of uniform colloidal size whose structure has a membrane which is easily dis¬ rupted to free the fluorescer material. -73 - 41. An encapsulated fluorescer composition useful in the labeling of an immunological specie specific to and for the detection of a biological of interest, wherein the fluorescer selected from the group consisting of: 5,12 -dihydro quinoxalo (2,3,6) phenazine, magnesium and zinc metalo porphyrins, neutral red, magdalla red, acridine red, acridine orange, dianiasyl ethynlyl tetracene, phenizine, rhodamine, 3,4,9,10 perylene tetracarboxylic dianhydride, and derivatives thereof. 42. A conjugated encapsulated fluorescer/ immunological specie composition useful in the detec¬ tion of a biological of interest wnich has been formed via reacting an immunological specie with an encap¬ sulated possessing a microcapsule structure having one or more reactive groups to enable the micro- capsule's conjugation with an immunological specie specific to the analyte of interest. 43. A conjugated encapsulated fluorescer/ immunological specie composition useful in the detection of a biological of interest which has been formed by reacting an immunological specie with an encapsulated fluorescer possessing a microcapsule of uniform colloidal size whose structure has a membrane which is easily disrupted to free the fluorescer material. 44. A conjugated encapsulated fluorescer/ immunological specie composition useful in the detec¬ tion of a biological of interest which has been - 7 4- formed by reacting an immunological specie with an encapsulated fluorescer selected from the group consisting of 5,12 - dihydro quinoxalo (2,3,6) phena-. zine, magnesium and zinc metalo porphyrins, neutral red, magdalla red, acridine red, acridine orange, dianiasyl ethynlyl tetracene, phenizine, rhodamine, 3,4,9,10 perylene tetracarboxylic dianhydrid , and derivatives thereof. 45. A test kit for use in the detection of a biological of interest by means of a conventional assay technique comprising, in a packaged combination, one or more containers holding (1) a microencapsulated fluorescer material conjugated to an immunolo¬ gical specie specific to the biolo¬ gical of interest, (2) a means for disrupting the membrane structure of the microencapsulated fluorescer, and (3) chemical reagents capable of reacting to produce a high energy intermediate which will excite the freed fluorescer material to cause same to emit light. 46. The kit of claim 45 wherein the chemical reagents comprise (i) hydrogen peroxide or a chemical system for generating hydrogen peroxide and (ii) an oxamide or a bis-oxalate ester. 47. A test kit for use in the detection of a biological of interest by means of a conventional assay technique comprising, in a packaged combination. one or more containers holding (1) a micro encapsulated quenching/ poisoning material conjugated to an immunological specie specific to the biological of interest, (2) chemical reagents capable of reacting to produce a chemiluminescent reaction generating an energy signal, and (3) a means for disrupting the membrane structure of the microencapsulated quencher/poisoning material to liberate the quencher/poison to free solution.

Inventors: MANDLE RICHARD M, WONG YUAN N
Applicants: ELECTRO NUCLEONICS, ELECTRO NUCLEONICS INC
Application year: 1981
Main classification: G01N33
IPC codes: G01N33