CA1084838A - Quencher conjugated antifluorescer in fluorescent immonoassay - Google Patents
Quencher conjugated antifluorescer in fluorescent immonoassayInfo
- Publication number
- CA1084838A CA1084838A CA280,237A CA280237A CA1084838A CA 1084838 A CA1084838 A CA 1084838A CA 280237 A CA280237 A CA 280237A CA 1084838 A CA1084838 A CA 1084838A
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- ligand
- fluorescer
- antifluorescer
- quencher
- antiligand
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Abstract
ABSTRACT OF THE DISCLOSURE
A novel sensitive method for determing quali-tatively and quantitatively the presence of a wide variety of physiologically active organic compounds (ligand) and their receptors is provided. The method employs two novel reagents: the first reagent involves bonding a compound having structural similarity to the compound to be deter-mined (ligand analog) to a fluorescing compound: the second reagent involves bonding of a quencher to a receptor for said fluorescer. The amount of the receptor-quencher conjugate (quencher conjugated antifluorescer) which can bind to the first reagent (ligand analog-fluorescer) will be dependent upon the amount of ligand or ligand receptor (antiligand) present in the medium to be assayed.
A novel sensitive method for determing quali-tatively and quantitatively the presence of a wide variety of physiologically active organic compounds (ligand) and their receptors is provided. The method employs two novel reagents: the first reagent involves bonding a compound having structural similarity to the compound to be deter-mined (ligand analog) to a fluorescing compound: the second reagent involves bonding of a quencher to a receptor for said fluorescer. The amount of the receptor-quencher conjugate (quencher conjugated antifluorescer) which can bind to the first reagent (ligand analog-fluorescer) will be dependent upon the amount of ligand or ligand receptor (antiligand) present in the medium to be assayed.
Description
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This invention relates to a method for detecting a ligand or antiligand in a sample, a reagent kit useful in the method and to a compound useful in the method.
There is a continually expanding need to determine the presence of minute quantities of organic materials, present in concentrations of 10 4 to 10 1 M or lower. In developing systems for measuring the extremely minute quan-tities of organic materials of interest, problems encountered include endogenous interference from materials present in samples and interference introduced into the system from reagents, both of which provide undesired background. For endogenous interference from the sample this can frequently be dealt with by pretreatment of the sample.
Interference introduced with reagents results from the diffculity in obtaining pure materials to work with.
lS For example, in competitive protein binding assays, antibodies are normally employed. The antibodies are obtained as antisera containing a substantial proportion of antibodies which are specific for materials other than the material to be assayed. Therefore, when conjugating the antisera with a label, not only is the antisera of interest labeled but the other antisera as well. Thus, a substantial portion of the label which is introduced as the reagent is involved with antisera uninvolved with the assay.
Where antigen labeling is involved, many materials of interest are available only in extremely small amounts and usually contaminated with other materials of similar properties. Thus, when labeling antigens, frequently a substantial proportion of the label is bound to material which is not involved in the assay. This label acts to qF ~
.
.`' . - , ~,, '.: ' :
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provide the undesired background, which can substantially affect the sensitivity of the assay.
It is therefore desirable to develop assays which can minimize interference from background signal introduced as a result of reagent preparation. It is further desirable to provide assays which are highly sensitive, specific, and allow for simple and rapid protocols for minimizing operator error.
Our U.S. Patent No. 3,998,943 issued December 21, 1976, discloses a double receptor fluorescent immunoassay.
Our U.S. Patent No. 3,996,345, issued December 7, 1976, discloses an assay employing antibodies and fluorescerquen-cher pairs, where the chromophores are bonded either to the ligand or antiligand.
According to the invention reagents are prepared having fluorescer conjugated to ligand and quencher conjugated to antiflourescer. In an assay for ligand, antiligand is included in the assay medium, and ligand competes with the ligand-flourescer conjugate for the antiligand. Antiligand bound to ligand-fluorescer conjugate inhibits the binding of antifluorescer to the fluorescer. In an assay for anti-ligand, any antiligand binds to the ligand-fluorescer con-jugate inhibiting the binding of antifluorescer. Fluoresc-ence is quenched by the presence of the quencher-antifluor-escer conjugate bound to the fluorescer, so that the amount of fluorescence is related to the amount of ligand or antiligand in the medium as the analyte. Any fluorescer present on materials other than ligand is effectively quenched by binding to the quencher-antifluorescer conjugate.
Accordingly the invention is a method for detecting . :
- ~, ~483~3 the presence of a ligand or antiligand in a sample comprising:
combining in an aqueous medium said sample; ligand analog-fluorescer, wherein said ligand analog is specifically recognizable by said antiligand, and the ligand and fluorescer are linked sufficiently close by a linking group so that the simultaneous binding of antiligand and antibody to fluorescer is sterically inhibited; antiligand for the determination of ligand; and quencher conjugated antifluorescer; determining at at least one wavelength the intensity of the fluorescence from said medium as compared to a standard having a known amount of ligand or antiligand.
The subject invention provides a novel sensitive method for determining the presence of a wide variety of organic compounds or compositions, for which a receptor is available or can be prepared, or for the receptor itself.
Fluorescer molecules which are conjugated to materials which ` are not involved in the assay, are effectively quenched, so as to make no substantial contribution to the total fluores-cence observed. In addition, those fluorescer molecules involved in the assay as ligand-fluorescer conjugates are ; also effectively quenched when bound to the antifluorescer-quencher conjugates, so as to minimize the base value of . ~ .
fluorescence, when all fluorescer molecules in the assay medium are bound to quencher-antifluorescer conjugate.
- 25 Definitions :
The following definitions apply throughout this specification:
Analyte - the compound or composition to be measured, which may be mono - or polyepitopic, antigenic or haptenic, a single compound or plurality of compounds which share at .:
.: . .:
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least one common epitopic site, or a receptor.
Ligand - any organic compound for which a receptor naturally exists or can be prepared.
Ligand Analog - a modified ligand which can compete with the analogous ligand for receptor, the modification providing means to join the ligand analog to a fluorescer or other molecule.
Chromophore - flourescer or quencher molecule; in the subject invention, the fluorescer and quencher are interrelated. The flourescer molecule is chromophore which is able to absorb light at one wavelength and emit light at a longer wavelength. The quencher molecule is capable of inhibiting fluorescence, when within a short distance, ; usually less than about lOOA of the fluorescer molecule, by accepting the energy which would otherwise be emitted as fluorescent light.
Ligand Analog-Fluorescer - ligand analog covalently bonded to one or more fluorescent molecules. With small ligands, those below about 10,000 molecular weight, usually below about 2,000 molecular weight, the ligand analog will usually be joined to fewer than 10 fluorescers, usually from one to ten fluorescers, more usually one fluorescer and generally not more than about one fluorescer per 1,000 molecular weight, With a large ligand, at least 2,000 ~ i~4838 1 molecular weight, usually at least about lO,000 molecular
This invention relates to a method for detecting a ligand or antiligand in a sample, a reagent kit useful in the method and to a compound useful in the method.
There is a continually expanding need to determine the presence of minute quantities of organic materials, present in concentrations of 10 4 to 10 1 M or lower. In developing systems for measuring the extremely minute quan-tities of organic materials of interest, problems encountered include endogenous interference from materials present in samples and interference introduced into the system from reagents, both of which provide undesired background. For endogenous interference from the sample this can frequently be dealt with by pretreatment of the sample.
Interference introduced with reagents results from the diffculity in obtaining pure materials to work with.
lS For example, in competitive protein binding assays, antibodies are normally employed. The antibodies are obtained as antisera containing a substantial proportion of antibodies which are specific for materials other than the material to be assayed. Therefore, when conjugating the antisera with a label, not only is the antisera of interest labeled but the other antisera as well. Thus, a substantial portion of the label which is introduced as the reagent is involved with antisera uninvolved with the assay.
Where antigen labeling is involved, many materials of interest are available only in extremely small amounts and usually contaminated with other materials of similar properties. Thus, when labeling antigens, frequently a substantial proportion of the label is bound to material which is not involved in the assay. This label acts to qF ~
.
.`' . - , ~,, '.: ' :
~8~83~
provide the undesired background, which can substantially affect the sensitivity of the assay.
It is therefore desirable to develop assays which can minimize interference from background signal introduced as a result of reagent preparation. It is further desirable to provide assays which are highly sensitive, specific, and allow for simple and rapid protocols for minimizing operator error.
Our U.S. Patent No. 3,998,943 issued December 21, 1976, discloses a double receptor fluorescent immunoassay.
Our U.S. Patent No. 3,996,345, issued December 7, 1976, discloses an assay employing antibodies and fluorescerquen-cher pairs, where the chromophores are bonded either to the ligand or antiligand.
According to the invention reagents are prepared having fluorescer conjugated to ligand and quencher conjugated to antiflourescer. In an assay for ligand, antiligand is included in the assay medium, and ligand competes with the ligand-flourescer conjugate for the antiligand. Antiligand bound to ligand-fluorescer conjugate inhibits the binding of antifluorescer to the fluorescer. In an assay for anti-ligand, any antiligand binds to the ligand-fluorescer con-jugate inhibiting the binding of antifluorescer. Fluoresc-ence is quenched by the presence of the quencher-antifluor-escer conjugate bound to the fluorescer, so that the amount of fluorescence is related to the amount of ligand or antiligand in the medium as the analyte. Any fluorescer present on materials other than ligand is effectively quenched by binding to the quencher-antifluorescer conjugate.
Accordingly the invention is a method for detecting . :
- ~, ~483~3 the presence of a ligand or antiligand in a sample comprising:
combining in an aqueous medium said sample; ligand analog-fluorescer, wherein said ligand analog is specifically recognizable by said antiligand, and the ligand and fluorescer are linked sufficiently close by a linking group so that the simultaneous binding of antiligand and antibody to fluorescer is sterically inhibited; antiligand for the determination of ligand; and quencher conjugated antifluorescer; determining at at least one wavelength the intensity of the fluorescence from said medium as compared to a standard having a known amount of ligand or antiligand.
The subject invention provides a novel sensitive method for determining the presence of a wide variety of organic compounds or compositions, for which a receptor is available or can be prepared, or for the receptor itself.
Fluorescer molecules which are conjugated to materials which ` are not involved in the assay, are effectively quenched, so as to make no substantial contribution to the total fluores-cence observed. In addition, those fluorescer molecules involved in the assay as ligand-fluorescer conjugates are ; also effectively quenched when bound to the antifluorescer-quencher conjugates, so as to minimize the base value of . ~ .
fluorescence, when all fluorescer molecules in the assay medium are bound to quencher-antifluorescer conjugate.
- 25 Definitions :
The following definitions apply throughout this specification:
Analyte - the compound or composition to be measured, which may be mono - or polyepitopic, antigenic or haptenic, a single compound or plurality of compounds which share at .:
.: . .:
~4~8~83~
least one common epitopic site, or a receptor.
Ligand - any organic compound for which a receptor naturally exists or can be prepared.
Ligand Analog - a modified ligand which can compete with the analogous ligand for receptor, the modification providing means to join the ligand analog to a fluorescer or other molecule.
Chromophore - flourescer or quencher molecule; in the subject invention, the fluorescer and quencher are interrelated. The flourescer molecule is chromophore which is able to absorb light at one wavelength and emit light at a longer wavelength. The quencher molecule is capable of inhibiting fluorescence, when within a short distance, ; usually less than about lOOA of the fluorescer molecule, by accepting the energy which would otherwise be emitted as fluorescent light.
Ligand Analog-Fluorescer - ligand analog covalently bonded to one or more fluorescent molecules. With small ligands, those below about 10,000 molecular weight, usually below about 2,000 molecular weight, the ligand analog will usually be joined to fewer than 10 fluorescers, usually from one to ten fluorescers, more usually one fluorescer and generally not more than about one fluorescer per 1,000 molecular weight, With a large ligand, at least 2,000 ~ i~4838 1 molecular weight, usually at least about lO,000 molecular
2 weight, a plurality of fluorescers will be covalently bonded
3 to the ligand analog or ligand. The number of fluorescers
4 present will be limited by the number which may be introduced g without masking too many epitopic sites of the ligand and 6 the need to provide inhibition of antifluorescer binding to 7 fluorescer when antiligand is bound to ligand.
8 Poly(ligand analog)-polyfluorescer - ligand 9 analog and fluorescer are bonded to a high molecular weight (as compared to the ligand analog and fluorescer) water 11 soluble polyfunctionalized hub or nucleus molecule to 12 provide a plurality of ligand analog groups-and fluorescer ;
13 groups spaced on the surface of the molecule, so that when 14 antiligand is bound to ligand analog, antifluorescer will be inhibited from binding to the fluorescer. Convenienily 16 poly(amino acids) or polysaccharides may serve as the hub 17 nucleus.
18 Receptor - any compound or composition capable of 19 recognizing a particular spatial and polar organization of a molecule i.e. an epitopic site and frequently polyvalent 21 i.e. having at least two binding sites. Illustrative 22 receptors include naturally occurring receptors, antibodies, 23 Fab fragments, enzymes and the like. For any specific 24 ligand, the receptor will be referred to as antiligand, for example, an antibody for fluorescer will be referred to as 26 antifluorescer. The receptor and its reciprocal ligand form 28 an immunological pair.
29 ` _ 31 .
32~
- ...... ..
`~ 108483~l ~
1 Receptor-quencher - normally, on the average there 2 will be at least one quencher per receptor molecule, and 3 usually more than one quencher molecule, there being not 4 more than about one quencher molecule per l,000 molecular weight, more usually not more than one ~uencher molecule per 6 2,000 molecular weight and preferably not more than about 7 one quencher molecule per 5,000 molecular weight. With 8 antibodies, there will be on the average from about 2 to 25, 9 more usually from about 2 to 20 quencher molecules per antibody molecule, preferably 4 to 16, while with Fab fragments, 11 there will be on the average generally from about l to 12, 12 more usually from about 2 to 8 quencher molecules per Fab 13 fragment.
14 Assay In carrying out the assay, the particular materials 16 employed will depend upon whether a ligand or recepto_ is 17 the analyte. ~ere ligand is the analyte, included in the 18 assay medium will be the unknown sample, antiligand, ligand 19 analog-fluorescer (includes poly(ligand analog)-polyfluorescer) and antifluorescer-quencher. In an assay for antiliqand, 21 included in the assay medium will be the ligand analog-22 fluorescer and antifluorescer-quencher.
23 Various protocols may be employed, the order of 24 addition and combination of reagents being controlled by the 2~ rates at which the receptor binds to the ligand and the 2~ ligand-receptor complex breaks. For example, with antibodies, 27 the rate at which ~he complex breaks is relatively slow. By 28 contrast, with Fab fragments, the rate is relatively rapid.
29 Therefore, depending upon time constraints, one would 32 1 _7_ !
iO8483 1¦ normally not combine ligand analog-fluorescer and antiligand, 2¦ where the antiligand is an antibody, prior to introduction 31 into the assay medium, while one could combine ligand analog-4¦ fluorescer and antiligand prior to introduction into the ~¦ assay medium where the antiligand is a Fab fragment.
61 With antibodies as the antiligand, in an assay for 7 ¦ ligand, one could conveniently combine the unknown, antiligand 8 ¦ and antifluorescer, followed by the addition of ligand 9 ¦ analog-fluorescer; or one could combine the unknown, ligand 10 ~ analog-fluorescer and antiligand, followed by the addition 11 ¦ of antifluorescer; or one could combine substantially 12 ¦ simultaneously all of the reagents.
13 I With a Fab fragment as antiligand and/or antifluorescer, 1~ ~ one could conveniently combine the ligand analog-fluorescer, 15 ¦ antiligand and antifluorescer as a single reagent, which is 16 ¦ added to the unXnown.
17 ¦ After each addition of reagent, an incubation time 18 ~ may be employed. Generally, the incubation times can vary 19¦ from about 0.5min. to 16hr.-or longer, but will usually be 20 ¦ from about 0.5min. to 6hr., and preferably from about 21 O.Smin. to lhr. The temperature for incubation may vary 22 widely, generally being from about 0 to 45C, more usually 231 from about 15 to 40C. The fluorescence value can be 24 ¦ determined either as a rate or equilibrium value. As a rate 25 ¦ value, one can determine the rate of change in fluorescence 2~1 over a predetermined period of time, as measured from the 271 introduction of the final reagent. ~s an equilibrium, one 2~1 can ~ait until there is no significant change in the amount 329~ f fluorescence and read a single value. -2 ! . ~ .
~8~838 In assays for antiligand, the protocol is sub-stantially the same as assay for ligand, except that anti-ligand is not added as a reagent, but is included as the unknown.
The concentrations of ligand which may be assayed for will vary from about 10 4 to 10 14, more usually from about 10 5 to 10 12M. The concentration of ligand to analog-fluorescer will also vary in the same range, usually not differing by more than a factor of 100 from either the ; minimum or maximum concentration of interest. The antili-gand concentrations will generally be from about 0.5-1,000:1 in number of binding sites per equivalent binding site of ligand analog-fluorescer, more usually in the range of about ; 1-10:1 in number of binding sites per equivalent of binding sites of ligand analog-fluorescer. Similar ranges will be employed for antifluorescer. See our U.S. Patent Nos.
3,690,834 issued September 12, 1972, and 3,817,837 issued June 18, 1974, for methods of measuring binding sites. The particular ratios employed will be based on the ratios which optimize the sensitivity of the assay and will generally be affected to a significant degree by the binding constants of the antiligand and antifluorescer.
The medium will normally be aqueous, generally having not more than about 20 volume percent of a polar organic solvent. Various alcohols, ethers and esters may be present in the minor amounts.
The pH of the medium will usually be in the range of about 5 to 10, more usually in the range of about 6 to 9 . :;: : ::, : : : .
1 10~3~83~3 1 ¦and preferably in the range of about 7 to 8.5. Various 2 ¦buffers may be used to achieve the desired pH and maintain 3 ¦the pH during the determination. Illustrative buffers 4 include borate, phosphate, carbonate, tris, barbital and the like. The particular buffer employed is not critical to this invention, but in particular assays, one buffer may be 7 preferred over another.
8 With certain ligands and fluorescers, there may be small but significant amounts of non-specific binding of the ligand or fluorescer to protein. To that extent, it is 11 preferred that the protein concentration resulting from 12 prot~in from the sample in the assay medium ~e less than ~
13 about l weight percent, preferably less than about 0.5 -14 1 weight percent and particularly preferred, less than about 15¦ O.l weight percent. The total protein concentration may be 16 reduced by prior treatment of the unknown sample by ultra-17 filtration, gel filtration, precipitation, dialysis, and the 18 like. Alternately, non-specific binding effects can be 19 mashed by addition of an excess of pure protein, such as albumin.
21 Moderate temperatures are normally employed for 22 carrying out the assay and usually constant temperatures 23 during the period of measurement will be employed. The 24 ¦temperatures normally range from about 15 to 40C, more usually from about 25 to 40C.
26 In carrying out the determination, the assay 271 solution is introduced into the fluorometer cell. The 28 choice of excitation wavelength will depend on the fluorescer.
29 The particular wavelength or band of wavelengths which ~re measured for the emission spectrum will depend on the 31 emission maximum and the amount of interference due to light 32 -lO
, ` ~ ' '~: . ' l - 108483~3 1 scattering. Desirably, an intense source of li~ht of a 2 single wavelength will be used. In this manner, interference 3 from light scattering effects can be minimized. Useful 4 monochromatic light sources that provide greater intensity 3 than conventional sources coupled with a monochromator are 6 low pressure emission lamps and lasers.
7 Analyte 8 As indicated previously, the analyte may be a 9 ligand or a receptor.
The nature of the ligand may vary widely, normally ; 11 having a molecular weight of at least 110, more usually at 12 least 125 with the maximum molecular weight unlimited, 13 although usually not exceeding 10 million. For the most 14 part, the significant factor concerning a ligand is that a receptor can be made to the ligand or is available. Normally, 16 receptors can be made for most organic compounds having a 17 polar functionality. Compounds for which antibodies can be 18 formed by bonding the compound to a compound having antigenic 19 properties are referred to as haptens. Those compounds which elicit antibody formation without chemical modification 21 are referred to as antigens. See Kabat, et al, Experimental 22 Immunochemistry, Charles C. Thomas, Springfield, Illinois, 23 1967.
24 The monoepitopic or haptenic compounds of interest will normally be of from about 125 to 2,000 molecular weight.
26 These compounds involve a wide variety of compounds of 27 varying structure, functionality, and physiological properties.
28 The compounds may be acyclic, alicyclic or heterocyclic, . ~
, . , .`iO8483~3 1¦ both mono- and polycyclic. The heteroatoms involved include 21 oxygen, nitrogen, sulfur, halogen (fluorine, chlorine, 3 ¦ bromine and iodine) boron, phosphorus, metal cations of 4 Groups l~ and 2A of the Periodic Chart and the like.
~ The functionalities include alcohols, ethers, 61 carboxylic acids, esters and amides, amines (primary, 7¦ secondary, tertiary and quaternary) halo, nitrilo, mercapto, 8 ¦ and the like. Normally, the compounds will be composed `
9 ¦ solely of carbon, hydrogen, oxygen, nitrogen, halogen and 10 ¦ phosphorus~r particularIy carbon, hydrogen, oxygen, and 11 ¦ nitrogen and where salts are involved, the appropriate metal 12 ¦ counterion or ammonium counterion. -13 ¦ Heterocyclic rings which are present include 14 ¦ pyrrole, pyridine, piperidine, indole, ~hiazole, piperazine, 15 ¦ pyran, coumarin, pyrimidine, purine, triazine, imidazole, 16 ¦ and the like. -17 ¦ Because of the wide variety of compounds which can 18 ¦ be determined in accordance with the subject assay, the 19 ¦ different groups will be broken down into various, frequently 20 ¦ artificial, categories, either by the presence of a particular 21 ¦ functionality or ring structure, or because of sharing a 22 ¦ particular function or because of being recognized as a 23 1 class. -24 ¦ The first class of compounds of interest are those 25 ¦ having an amino group, either as a heterocyclic me~ber, or 2~ ¦ as a functionality on an aliphatic chain. These compounds 27 ¦ will normally be of from about llO to 800 molecular weight, 28 ¦ more usually of about 125 to 650 molecular weight.
29 ¦ The first group of compounds of interest are the 3~1 alkaloids and the metabolites of those alkaIoids which are 311 . , , .
32 -12- .
108483~ ~
1 ¦ ingested. The first group of important alkaloids are alkaloids 2 ¦ of the morphine group. Included in this group are morphine, 3 1 codeine, heroin, morphine glucuronide and the like.
4 ¦ The next group of alkaloids are the cocaine alkaloids, ~ which include, particularly as metabolites, benzoyl ecgonine 6 and ecgonine.
7 Another group of alkaloids are the cinchona alkaloids 8 which include quinine and quinidine.
9 The isoquinoline group of alkaloids includes mescaline.
The benzylisoquinoline alkaloids includes papaverine.
11 The phthalide isoquinoline alkaloids include narcotine, 12 narceine, and cotarnine.
13 The indolopyridocoline alkaloids include yohimbine 15 ¦ and re rp~le.
32 ~ -13-~ ~084838 :
The ergot alkaloids include ergotamine and lysergic acid.
Other groups of alkaloids include strychnine alkaloids, pyridine alkaloids, piperidine alkaloids, pyrroli-zidine alkaloids and the like.
The alkaloids of primary interest are those whichcome within the category of drugs of abuse, such as morphine, cocaine, mescaline, and lysergic acid, which may be analyzed for the compound or its metabolite, depending on the physio~
logical fluid which is analyzed for its presence.
A number of synthetic drugs mimic the physio-logical properties, in part or in whole, of the naturally occurring drugs of abuse. Included among these drugs are methadone, meperidine, amphetamine, methamphetamine, glut-ethimide, diphenylhydantoin, and drugs which come within thecategory of benzdiazocycloheptanes, phenothiazines and barbiturates.
Drugs of interest because of their physiological properties are those which are referred to as catechol-amines. Among the catecholamines are epinephrine, ephedrine,L-dopa, and norepinephrine.
Another drug of interest is the tranquilizer Meprobamat.
Other compounds of interest are tetrahydroca-nnabinol, cannabinol, and derivatives thereof, primarilycompounds derived from marijuana, synthetic modifications and metabolites thereof.
Another group of compounds of significant interest are the steroids. The steroids include estrogens, gestogens, androgens, adrenocortical hormones, bile acids, cardiotonic ~(~84838 glycoids, algycones, saponins and sapogenins.
Another class of compounds are the vitamins, such as vitamin A, the B group, e.g. vitamin Bl, B6 and sl2, E, K, and the like.
Another class of compounds are the sugars, both the mono-and polysaccharides, particularly di- and higher order polysaccharides.
Another class of compounds is the prostaglandins.
Another group of compounds are the antibiotics such as penicillin, actinomycin, chloromycetin, and the like.
Individual compounds of interest are serotonin, spermin and phenylpyruvic acid.
Another class of compounds of interest are pes-ticides, such as fungicides, insecticides, bactericides, andnematocides.
Another class of compounds are the amino acids, polypeptides and proteins. Polypeptides usually encompass from about 2 to 100 amino acid units (usually less than 20 about 12,000 molecular weight). Larger polypeptides are arbitrarily called proteins and are usually composed of from about l to 20 polypeptide chains. Poly(amino acid) will be used as generic to polypeptides and proteins. Of particular interest among amino acids is thyronines, both the tri- and tetraiodo. The poly(amino acid)s employed in this invention employing two antibodies are reagents will generally range from about 5,000 to 107, usually 104 to 106 molecular weight. Of particular interest among polypeptides and proteins [poly(amino acids)] are hormones, globulins, antigens and compositions found to have specific physio-logical activities.
; i . : , ~084838 The wide variety of proteins may be considered as to the family of proteins having similar structural features, proteins having particular biological functions, proteins related to specific micro-organisms, particularly disease causing micro-organisms, etc.
The following are classes of proteins related by structure:
protamines histones albumins globulins scleroproteins phosphoproteins mucoproteins chromoproteins lipoproteins nucleoproteins unclassified proteins, e.g. somatotropin, prolactin, insulin, pepsin A number of proteins found in the human plasma are important clinically and includes:
Prealbumin :
Albumin O~l-Lipoprotein al- Acid glycoprotein l 1- Antitryps in ~1- Glycoprotein Transcortin 4.6S-Postalbumin Tryptophan-poor ~ ~1 ~ glycoprotein ~ -16 -: - :" .
iV84838 OL lX- Glycoprotein Thyroxin-binding globulin Inter~- trypsin-inhibitor Gc-Globulin (Gc 1-1) (Gc 2-1) (Gc 2-2) Haptoglobin (Hp 1-1) ' (Hp 2-1) (Hp 2-2) Ceruloplasmin Cholinesterase ~2- Lipoprotein(s) ~2- Macroglobulin a 2- HS-glycoprotein Zn-~2 -glycoprotein ~2- Neuramino-glycoprotein Erythropoietin ~- lipoprotein Transferrin Hemopexin Fibrinogen Plasminogen ~2 glycoprotein I
~2- glycoprotein II
Immunoglobulin G
(IgG) or r G-globulin Mol. formula:
~ -16~-iO8483~3 r2~2 rr2 ~2 Immunoglobulin A (IgA) or rA-globulin Mol. formula:
(~2 ~2)n or (~2 ~2)n Immunoglobulin M
(IgM) or rM-globulin Mol. formula:
(~2 ~2) or (~2 A2) Immunoglobulin D(IgD) or r D-Globulin (r D) Mol. formula:
(~2 ~2) or (~2 ~2) Immunoglobulin E (IgE) or~ E-Globulin (~ E) Mol. formula: .
(~2 ~2) or (2 ~2) Free light chains Complement factors:
C'l C'lq C'lr C ' ls C'2 C'3 ~ A
o~,2D
C'4 C'5 C'6 ~ -16 1~8483~3 C'7 C'8 C ' 9 Important blood clotting factors include:
BLOOD CLOTTING FACTORS
10 International Designation Name I Fibrinogen II Prothrombin IIa Thrombin II~ Tissue thromboplastin V and VI Proaccelerin, accelerator globulin VII Proconvertin VIII Antihemophilic globulin (AHG) IX Christmas factor, plasma thromboplastin component (PTC) X Stuart-Prower factor, autoprothrombin III
XI Plasma thromboplastin antecedent (PTA) XII Hagemann factor XIII Fibrin-stabilizing factor Important protein hormones include:
Peptide and Protein Hormones Parathyroid hormone (parathormone) Thyrocalcitonin Insulin Glucagon Relaxin - , : . - - : . :
.~ ., "~
~84838 Erythropoietin Melanotropin (melanocyte-stimulating hormone; intermedin) Somatotropin (growth hormone) Corticotropin (adrenocorticotropic hormone) Thyrotropin Follicle-stimulating hormone Luteinizing hormone (interstitial cell-stimulating hormone) ~' Luteomammotropic hormone (luteotropin, prolactin) Gonadotropin (chorionic gonadotropin) Tissue Hormones Secretin Gastrin Angiotensin I and II
Bradykinin Human placental lactogen Peptide Hormones from the Neurohypophysis oxytocin Vasopressin Releasing factors (RF) CRF, LRF, TRF, Somatotropin-RF, GRF, FSH-RF, PIF, MIF
~ -16~-~.os4s38 Other polymeric materials of interest are mucop-olysaccharides and polysaccharides.
Illustrative antigenic polysaccharides derlved from micro-organisms are as follows:
SPECIES OF MICRO-ORGANISMS HEMOSENSITIN FOUND IN
Streptococcus pyogenes Polysaccharide Diplococcus pneumoniae Polysaccharide Neisseria meningitidis Polysaccharide Neisseria gonorrhoeae Polysaccharide Corynebacterium diphtheriae Polysaccharide Actinobacillus mallei; Crude extract Actinobacillus whitemori Francisella tularensis Lipopolysaccharide Polysaccharide Pasteurella pestis Polysaccharide Pasteurella pestis 15 Pasteurella multocida Capsular antigen Brucella abortus Crude extract ' Haemophilus influenzae Polysaccharide Haemophilus pertussis Crude Treponema reiteri Polysaccharide Veillonella Lipopolysaccharide Erysipelothrix Polysaccharide Listeria monocytogenes Polysaccharide Chromobacterium Lipopolysaccharide Mycobacterium tuberculosis Saline extract of 90~
phenol extracted mycobacteria and polysaccharide fraction of cells and tuberculin Klebsiella aerogenes Polysaccharide Klebsiella cloacae Polysaccharide Salmonella typhosa Lipopolysaccharide, Polysaccharide Salmonella typhi-murium; Polysaccharide Salmonella derby Salmonella pullorum Shigella dysenteriae Polysaccharide Shigella flexneri Shigella sonnei Crude, polysaccharide Rickettsiae Crude extract 30 Candida albicans Polysaccharide Entamoeba histolytica Crude extract ~ -16~-: . : . .
,:
, :' ~.os4~3s Other than compounds of interest, cells, viruses, and other biological aggregations which are antigenic or to which naturally occurring receptors can be found may also be assayed for.
The micro-organisms which are assayed may be intact, lysed, ground or otherwise fragmented, and the resulting composition or portion, e.g. by extraction, assayed. Micro-organisms of interest include:
!
Corynebacteria .
Corynebacterium diptheriae Pneumococci Diplococcus pneumoniae Streptococci Streptococcus pyogenes Streptococcus salivarus -Staphylococci Staphylococcus aureus Staphylococcus albus `
Neisseriae Neisseria meningitidis Neisseria gonorrheae Enterobacteriaciae Escherichia coli Aerobacter aerogenes ~ The coliform bacteria -16~-- ~ : "- ' . :
11[~8483~3 Klebsiella pneumoniae J
Salmonella typhosa Salmonella choleraesuis The Salmonellae Salmonella typhimurium Shigella dysenteriae Shigella schmitzii Shigella arabinotarda The Shigellae Shigella flexneri Shigella boydii Shigella Sonnei Other enteric bacilli Proteus vulgaris Proteus mirabilis Proteus species Proteus morgani Pseudomonas aeruginosa Alcaligenes faecalis Vibrio cholerae Hemophilus-Bordetella Group Hemophilus influenzae, H. ducreyi H. hemophilus H. aegypticus H. paraifluenzae Bordetella pertussis Pasteurellae Pasteurella pestis Pasteurella tulareusis -l6b- ,' . ~ .
.
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Brucellae Brucella melitensis srucella abortus Brucella suis Aerobic Spore-forming Bacilli Bacillus anthracis Bacillus subtilis Bacillus megaterium Bacillus cereus naerobic Spore-forming Bacilli Clostridium botulinum Clostridium tetani Clostridium perfringens Clostridium novyi ~ :
~ Clostridium septicum - Clostridium histolyticum Clostridium tertium Clostridium bifermentans .
Clostridium sporogenes , , Mycobacteria Mycobacterium tuberculosis hominis Mycobacterium bovis Mycobacterium avium Mycobacterium leprae Mycobacterium paratuberculosis ~ -16~-i~3483~
Actinomycetes (fungus-like bacteria) Actinomyces israelii Actinomyces bovis Actinomyces naeslundii Nocardia asteroides Nocardia brasiliensis The Spirochetes Treponema pallidum Spirillum minus Treponema pertenue Streptobacillus moni-liformis Treponema carateum Borrelia recurrentis Leptospira icterohemorrhagiae Leptospira canicola Mycoplasmas Mycoplasma pneumoniae Other pathogens Listeria monocytogenes Erysipelothrix rhusiopathiae Streptobacillus moniliformis Donvania granulomatis Bartonella bacilliformis Rickettsiae (bacteria-like parasites) Rickettsia prowazekii Rickettsia mooseri Rickettsia rickettsii Rickettsia conori ~ -16j-83~
Rickettsia australis Rickettsia sibiricus Rickettsia akari Rickettsia tsutsugamushi Rickettsia burnetii Rickettsia quintana Chlamydia (unclassifiable parasites bacterial/viral) Chlamydia agents (naming uncertain) Fungi Cryptococcus neoformans Blastomyces dermatidis Histoplasma capsulatum Coccidioides immitis Paracoccidioides brasiliensis Candida albicans Aspergillus fumigatus Mucor corymbifer (Absidia corymbifera) Rhizopus oryzae Rhizopus arrhizus Phycomycetes Rhizopus nigricans Sporotrichum schenkii Fonsecaea pedrosoi Fonsecaea compacta Fonsecaea dermatitidis Cladosporium carrionii Phialophora verrucosa Aspergillus nidulans Madurella mycetomi -16~-. ; : :
.: ..
.
1(~184838 Madurella grisea Allescheria boydii Phialosphora jeanselmei Microsporum gypseum Trichophyton mentagrophytes Keratinomyces ajelloi Microsporum canis Trichophyton rubrum Microsporum andouini Viruses Adenoviruses Herpes Viruses Herpes simplex Varicella (Chicken pox) Herpes Zoster (Shingles) Virus B
Cytomegalovirus Pox Viruses Variola (smallpox) Vaccinia Poxvirus bovis Paravaccinia Molluscum contagiosum Picornaviruses -Poliovirus Coxsackievirus ; 30 Echoviruses ~ -16~-~:, : , !.
~8483~3 Rhinoviruses Myxoviruses Influenza (A, B, and C) Parainfluenza (1-4) Mumps Virus Newcastle Disease Virus Measles Virus Rinderpest Virus Canine Distemper Virus Respiratory Syncytial Virus Rubella Virus :
Arboviruses Eastern Equine Eucephalitis Virus Western Equine Eucephalitis Virus Sindbis Virus Chikungunya Virus Semliki Forest Virus Mayora Virus St. Louis Encephalitis Virus California Encephalitis Virus Colorado Tick Fever Virus Yellow Fever Virus Dengue Virus ;.
Reoviruses Reovirus Types 1-3 -16~-- .
~08483~3 Hepatitis Hepatitis A Virus Hepatitis B Virus Tumor Viruses Rauscher Leukemia Virus Gross Virus Maloney Leukemia Virus Friend Leukemia Virus Mouse Mammary Tumor Virus Avian Leucosis Virus Rous Sarcoma Virus Polyoma Virus Simian Virus 40 Papilloma Virus Preparations of micro-organisms include:
Streptococcus pyogenes, protein Pasteurella pestis, protein toxin Clostridium tetani, toxoid Clostridium perfringens, ~-lecithinase Escherichia coli, filtrates Treponema reiteri, protein extract Corynebacterium diphtheriae, toxin, toxoid Mycobacterium tuberculosis, protein M. tuberculosis, cytoplasm M. tuberculosis, culture filtrate and tuberculin Mycoplasma pneumoniae, "crude" antigen ' ., .. . ~ .
.', . , ' ' .'!
~ ~B483~
l¦ Ligand Analog-Fluorescer 21 The ligand analog will normally differ from the 31 ligand in only minor ways. In most cases, the ligand analog 4 ¦ will replace a hydrogen of the ligand with a bond to a ~ ¦ ligand group. As for example, with morphine, the hydrogen 6 ¦ of the phenolic hydroxyl can be replaced with a bond to the 7 ¦ methylene of an acetyl group. The hydrogen which is replaced 8 by a bond to a linking group may be bonded to carbon, either 9 aliphatic or aromatic, oxygen or nitrogen.
In some instances, an oxocarbonyl may serve as the 11 linking site by modifying the oxocarbonyl to an oxime. In 12 other instances, the hydroxyl of a carboxyl group may be 13 replaced to form a linking group, by forming an ester or 14 amide.
Additional alternatives include introducing 16 functionalities, such as hydroxyl functionalities from which 17 ethers can be formed, amino functionalities from which 18 amides can be formed, amino functionalities, from which 19 diazo groups can be formed, and the like.
The significant factor for the ligand analog is 21 that it has sufficient structural similarity to the ligand .^~
22 so as to be recognized by the antibody for the ligand.
23 Because the manner of addition can be widely varied, the 24 binding constants for the ligand and the ligand analog may be different, but should not differ by more than a factor of 26 103, preferably by not more than a factor of lO2.
27 For the most part, the ligand analog will have the 28 same or substantially the same structure and charge distribution 29 (spatial and polar organization) as the ligand for a significantr 32 ~ -17-~ 8~838 l if not major, portion of the molecular volume. Since frequently 2 the linking site for a hapten will be the same in prepariny 3 the antigen for production of antibodies as used for linking 4 to the fluorescer, the same portion of the ligand molecule which provides the template for the antibody will be exposed ~ by the ligand analog when bound to fluorescer.
71 Because of the steric inhibition of the presence 81 of one antibody preventing the binding of another antikody 9¦ to the ligand analog-fluorescer, the linking group will .
10¦ normally be relatively short. Usually, the linking group ll ~ will be substantially less than 25 A, more usually less than 121 20 A, and preferably less than 15 A. Normally, the linking ~
13¦ group will be from about 1.5-lOA. -14 ¦ With large molecules or macromolecules as ligands, ~¦ such as polypeptides and proteins, there will be a number of 16¦ different epitopes available on the surface of the molecule, 17¦ each of which will have a complementary antibody. When the 18¦ macromolecule is conjugated with fluorescer, normally there 19¦ will be a plurality of fluorescer molecules bonded to the 20¦ macromolecule. Depending on the spatial relationship of the 21¦ fluorescer molecule to an epitope, there may or may not be 221 steric inhibition to the simultaneous binding of antibody to 231 the ligand epitope and antibody to the fuorescer. ~1owever, 241 there will normally be a plurality of pairs of epitope siies 25 I and fluorescer molecules, where to various degrees, steric 26 1 inhibition between the two different antibodies will exist.
271 In referring to ligand analog-fluorescer molecules, it is 28 intend d to include mo1ecu1es where there is at least one ~ l - ~
1~34838 1 epitope-fluorescer pair, which is in appropriate juxtaposition 2 for steric interaction. The statement concerning the steric 3 inhibition for simpler molecules having one epitope and one 4 fluorescer will be normally appropriate to the epitope-fluorescer pairs present in macromolecules.
6 In choosing the fluorescer, a wide variety of 7 ¦ considerations will come into play. As already indicated, 8 the choice of fluorescer will, to a degree, be governed by 9¦ the ligand. Therefore, one consideration is that the 10¦ fluorescer have absorption at higher wavelengths than a 11 ¦ fluorescent ligand or ligand bound to antibody.
12¦ In addition to the considerations which relate to 13¦ the particular ligand being determined, there will be a 14 ¦ number of other considerations which limit the particular 15¦ choice of fluorescer. As a practical matter, since one is 16¦ concerned with a change in the em ssion spectrum as a result 17¦ of being bound or unbound to an antifluorescer, one would lB ¦ desire a large environmental effect on the emission intensity 19¦ at a particular wavelength. This can be a result of a 20¦ substantial changê in quantum yield or a change in the 21 ¦ emission or absorption spectrum in going from the bound to 22¦ unbound fluorescer.
231 Since proteins absorb at a wavelength of about 241 280, the fluorescer should have an absorption maximum about 251 300, usually about 350 and preferably above 400nm. The 2~1 extinction coefficient should be greatly in excess of 10, 271 preferably in excess of 103, and particularly preferred, in 28¦ excess of 104M lcm 1.
3~
: :, 1~84838 .
1 In addition, it is desirable that the fluorescer 2 have a large Stokes shift. That is, it is preferred that 3 there be a substantial spread or difference in wavelengths 4 for the fluorescer between its absorption maximum and
8 Poly(ligand analog)-polyfluorescer - ligand 9 analog and fluorescer are bonded to a high molecular weight (as compared to the ligand analog and fluorescer) water 11 soluble polyfunctionalized hub or nucleus molecule to 12 provide a plurality of ligand analog groups-and fluorescer ;
13 groups spaced on the surface of the molecule, so that when 14 antiligand is bound to ligand analog, antifluorescer will be inhibited from binding to the fluorescer. Convenienily 16 poly(amino acids) or polysaccharides may serve as the hub 17 nucleus.
18 Receptor - any compound or composition capable of 19 recognizing a particular spatial and polar organization of a molecule i.e. an epitopic site and frequently polyvalent 21 i.e. having at least two binding sites. Illustrative 22 receptors include naturally occurring receptors, antibodies, 23 Fab fragments, enzymes and the like. For any specific 24 ligand, the receptor will be referred to as antiligand, for example, an antibody for fluorescer will be referred to as 26 antifluorescer. The receptor and its reciprocal ligand form 28 an immunological pair.
29 ` _ 31 .
32~
- ...... ..
`~ 108483~l ~
1 Receptor-quencher - normally, on the average there 2 will be at least one quencher per receptor molecule, and 3 usually more than one quencher molecule, there being not 4 more than about one quencher molecule per l,000 molecular weight, more usually not more than one ~uencher molecule per 6 2,000 molecular weight and preferably not more than about 7 one quencher molecule per 5,000 molecular weight. With 8 antibodies, there will be on the average from about 2 to 25, 9 more usually from about 2 to 20 quencher molecules per antibody molecule, preferably 4 to 16, while with Fab fragments, 11 there will be on the average generally from about l to 12, 12 more usually from about 2 to 8 quencher molecules per Fab 13 fragment.
14 Assay In carrying out the assay, the particular materials 16 employed will depend upon whether a ligand or recepto_ is 17 the analyte. ~ere ligand is the analyte, included in the 18 assay medium will be the unknown sample, antiligand, ligand 19 analog-fluorescer (includes poly(ligand analog)-polyfluorescer) and antifluorescer-quencher. In an assay for antiliqand, 21 included in the assay medium will be the ligand analog-22 fluorescer and antifluorescer-quencher.
23 Various protocols may be employed, the order of 24 addition and combination of reagents being controlled by the 2~ rates at which the receptor binds to the ligand and the 2~ ligand-receptor complex breaks. For example, with antibodies, 27 the rate at which ~he complex breaks is relatively slow. By 28 contrast, with Fab fragments, the rate is relatively rapid.
29 Therefore, depending upon time constraints, one would 32 1 _7_ !
iO8483 1¦ normally not combine ligand analog-fluorescer and antiligand, 2¦ where the antiligand is an antibody, prior to introduction 31 into the assay medium, while one could combine ligand analog-4¦ fluorescer and antiligand prior to introduction into the ~¦ assay medium where the antiligand is a Fab fragment.
61 With antibodies as the antiligand, in an assay for 7 ¦ ligand, one could conveniently combine the unknown, antiligand 8 ¦ and antifluorescer, followed by the addition of ligand 9 ¦ analog-fluorescer; or one could combine the unknown, ligand 10 ~ analog-fluorescer and antiligand, followed by the addition 11 ¦ of antifluorescer; or one could combine substantially 12 ¦ simultaneously all of the reagents.
13 I With a Fab fragment as antiligand and/or antifluorescer, 1~ ~ one could conveniently combine the ligand analog-fluorescer, 15 ¦ antiligand and antifluorescer as a single reagent, which is 16 ¦ added to the unXnown.
17 ¦ After each addition of reagent, an incubation time 18 ~ may be employed. Generally, the incubation times can vary 19¦ from about 0.5min. to 16hr.-or longer, but will usually be 20 ¦ from about 0.5min. to 6hr., and preferably from about 21 O.Smin. to lhr. The temperature for incubation may vary 22 widely, generally being from about 0 to 45C, more usually 231 from about 15 to 40C. The fluorescence value can be 24 ¦ determined either as a rate or equilibrium value. As a rate 25 ¦ value, one can determine the rate of change in fluorescence 2~1 over a predetermined period of time, as measured from the 271 introduction of the final reagent. ~s an equilibrium, one 2~1 can ~ait until there is no significant change in the amount 329~ f fluorescence and read a single value. -2 ! . ~ .
~8~838 In assays for antiligand, the protocol is sub-stantially the same as assay for ligand, except that anti-ligand is not added as a reagent, but is included as the unknown.
The concentrations of ligand which may be assayed for will vary from about 10 4 to 10 14, more usually from about 10 5 to 10 12M. The concentration of ligand to analog-fluorescer will also vary in the same range, usually not differing by more than a factor of 100 from either the ; minimum or maximum concentration of interest. The antili-gand concentrations will generally be from about 0.5-1,000:1 in number of binding sites per equivalent binding site of ligand analog-fluorescer, more usually in the range of about ; 1-10:1 in number of binding sites per equivalent of binding sites of ligand analog-fluorescer. Similar ranges will be employed for antifluorescer. See our U.S. Patent Nos.
3,690,834 issued September 12, 1972, and 3,817,837 issued June 18, 1974, for methods of measuring binding sites. The particular ratios employed will be based on the ratios which optimize the sensitivity of the assay and will generally be affected to a significant degree by the binding constants of the antiligand and antifluorescer.
The medium will normally be aqueous, generally having not more than about 20 volume percent of a polar organic solvent. Various alcohols, ethers and esters may be present in the minor amounts.
The pH of the medium will usually be in the range of about 5 to 10, more usually in the range of about 6 to 9 . :;: : ::, : : : .
1 10~3~83~3 1 ¦and preferably in the range of about 7 to 8.5. Various 2 ¦buffers may be used to achieve the desired pH and maintain 3 ¦the pH during the determination. Illustrative buffers 4 include borate, phosphate, carbonate, tris, barbital and the like. The particular buffer employed is not critical to this invention, but in particular assays, one buffer may be 7 preferred over another.
8 With certain ligands and fluorescers, there may be small but significant amounts of non-specific binding of the ligand or fluorescer to protein. To that extent, it is 11 preferred that the protein concentration resulting from 12 prot~in from the sample in the assay medium ~e less than ~
13 about l weight percent, preferably less than about 0.5 -14 1 weight percent and particularly preferred, less than about 15¦ O.l weight percent. The total protein concentration may be 16 reduced by prior treatment of the unknown sample by ultra-17 filtration, gel filtration, precipitation, dialysis, and the 18 like. Alternately, non-specific binding effects can be 19 mashed by addition of an excess of pure protein, such as albumin.
21 Moderate temperatures are normally employed for 22 carrying out the assay and usually constant temperatures 23 during the period of measurement will be employed. The 24 ¦temperatures normally range from about 15 to 40C, more usually from about 25 to 40C.
26 In carrying out the determination, the assay 271 solution is introduced into the fluorometer cell. The 28 choice of excitation wavelength will depend on the fluorescer.
29 The particular wavelength or band of wavelengths which ~re measured for the emission spectrum will depend on the 31 emission maximum and the amount of interference due to light 32 -lO
, ` ~ ' '~: . ' l - 108483~3 1 scattering. Desirably, an intense source of li~ht of a 2 single wavelength will be used. In this manner, interference 3 from light scattering effects can be minimized. Useful 4 monochromatic light sources that provide greater intensity 3 than conventional sources coupled with a monochromator are 6 low pressure emission lamps and lasers.
7 Analyte 8 As indicated previously, the analyte may be a 9 ligand or a receptor.
The nature of the ligand may vary widely, normally ; 11 having a molecular weight of at least 110, more usually at 12 least 125 with the maximum molecular weight unlimited, 13 although usually not exceeding 10 million. For the most 14 part, the significant factor concerning a ligand is that a receptor can be made to the ligand or is available. Normally, 16 receptors can be made for most organic compounds having a 17 polar functionality. Compounds for which antibodies can be 18 formed by bonding the compound to a compound having antigenic 19 properties are referred to as haptens. Those compounds which elicit antibody formation without chemical modification 21 are referred to as antigens. See Kabat, et al, Experimental 22 Immunochemistry, Charles C. Thomas, Springfield, Illinois, 23 1967.
24 The monoepitopic or haptenic compounds of interest will normally be of from about 125 to 2,000 molecular weight.
26 These compounds involve a wide variety of compounds of 27 varying structure, functionality, and physiological properties.
28 The compounds may be acyclic, alicyclic or heterocyclic, . ~
, . , .`iO8483~3 1¦ both mono- and polycyclic. The heteroatoms involved include 21 oxygen, nitrogen, sulfur, halogen (fluorine, chlorine, 3 ¦ bromine and iodine) boron, phosphorus, metal cations of 4 Groups l~ and 2A of the Periodic Chart and the like.
~ The functionalities include alcohols, ethers, 61 carboxylic acids, esters and amides, amines (primary, 7¦ secondary, tertiary and quaternary) halo, nitrilo, mercapto, 8 ¦ and the like. Normally, the compounds will be composed `
9 ¦ solely of carbon, hydrogen, oxygen, nitrogen, halogen and 10 ¦ phosphorus~r particularIy carbon, hydrogen, oxygen, and 11 ¦ nitrogen and where salts are involved, the appropriate metal 12 ¦ counterion or ammonium counterion. -13 ¦ Heterocyclic rings which are present include 14 ¦ pyrrole, pyridine, piperidine, indole, ~hiazole, piperazine, 15 ¦ pyran, coumarin, pyrimidine, purine, triazine, imidazole, 16 ¦ and the like. -17 ¦ Because of the wide variety of compounds which can 18 ¦ be determined in accordance with the subject assay, the 19 ¦ different groups will be broken down into various, frequently 20 ¦ artificial, categories, either by the presence of a particular 21 ¦ functionality or ring structure, or because of sharing a 22 ¦ particular function or because of being recognized as a 23 1 class. -24 ¦ The first class of compounds of interest are those 25 ¦ having an amino group, either as a heterocyclic me~ber, or 2~ ¦ as a functionality on an aliphatic chain. These compounds 27 ¦ will normally be of from about llO to 800 molecular weight, 28 ¦ more usually of about 125 to 650 molecular weight.
29 ¦ The first group of compounds of interest are the 3~1 alkaloids and the metabolites of those alkaIoids which are 311 . , , .
32 -12- .
108483~ ~
1 ¦ ingested. The first group of important alkaloids are alkaloids 2 ¦ of the morphine group. Included in this group are morphine, 3 1 codeine, heroin, morphine glucuronide and the like.
4 ¦ The next group of alkaloids are the cocaine alkaloids, ~ which include, particularly as metabolites, benzoyl ecgonine 6 and ecgonine.
7 Another group of alkaloids are the cinchona alkaloids 8 which include quinine and quinidine.
9 The isoquinoline group of alkaloids includes mescaline.
The benzylisoquinoline alkaloids includes papaverine.
11 The phthalide isoquinoline alkaloids include narcotine, 12 narceine, and cotarnine.
13 The indolopyridocoline alkaloids include yohimbine 15 ¦ and re rp~le.
32 ~ -13-~ ~084838 :
The ergot alkaloids include ergotamine and lysergic acid.
Other groups of alkaloids include strychnine alkaloids, pyridine alkaloids, piperidine alkaloids, pyrroli-zidine alkaloids and the like.
The alkaloids of primary interest are those whichcome within the category of drugs of abuse, such as morphine, cocaine, mescaline, and lysergic acid, which may be analyzed for the compound or its metabolite, depending on the physio~
logical fluid which is analyzed for its presence.
A number of synthetic drugs mimic the physio-logical properties, in part or in whole, of the naturally occurring drugs of abuse. Included among these drugs are methadone, meperidine, amphetamine, methamphetamine, glut-ethimide, diphenylhydantoin, and drugs which come within thecategory of benzdiazocycloheptanes, phenothiazines and barbiturates.
Drugs of interest because of their physiological properties are those which are referred to as catechol-amines. Among the catecholamines are epinephrine, ephedrine,L-dopa, and norepinephrine.
Another drug of interest is the tranquilizer Meprobamat.
Other compounds of interest are tetrahydroca-nnabinol, cannabinol, and derivatives thereof, primarilycompounds derived from marijuana, synthetic modifications and metabolites thereof.
Another group of compounds of significant interest are the steroids. The steroids include estrogens, gestogens, androgens, adrenocortical hormones, bile acids, cardiotonic ~(~84838 glycoids, algycones, saponins and sapogenins.
Another class of compounds are the vitamins, such as vitamin A, the B group, e.g. vitamin Bl, B6 and sl2, E, K, and the like.
Another class of compounds are the sugars, both the mono-and polysaccharides, particularly di- and higher order polysaccharides.
Another class of compounds is the prostaglandins.
Another group of compounds are the antibiotics such as penicillin, actinomycin, chloromycetin, and the like.
Individual compounds of interest are serotonin, spermin and phenylpyruvic acid.
Another class of compounds of interest are pes-ticides, such as fungicides, insecticides, bactericides, andnematocides.
Another class of compounds are the amino acids, polypeptides and proteins. Polypeptides usually encompass from about 2 to 100 amino acid units (usually less than 20 about 12,000 molecular weight). Larger polypeptides are arbitrarily called proteins and are usually composed of from about l to 20 polypeptide chains. Poly(amino acid) will be used as generic to polypeptides and proteins. Of particular interest among amino acids is thyronines, both the tri- and tetraiodo. The poly(amino acid)s employed in this invention employing two antibodies are reagents will generally range from about 5,000 to 107, usually 104 to 106 molecular weight. Of particular interest among polypeptides and proteins [poly(amino acids)] are hormones, globulins, antigens and compositions found to have specific physio-logical activities.
; i . : , ~084838 The wide variety of proteins may be considered as to the family of proteins having similar structural features, proteins having particular biological functions, proteins related to specific micro-organisms, particularly disease causing micro-organisms, etc.
The following are classes of proteins related by structure:
protamines histones albumins globulins scleroproteins phosphoproteins mucoproteins chromoproteins lipoproteins nucleoproteins unclassified proteins, e.g. somatotropin, prolactin, insulin, pepsin A number of proteins found in the human plasma are important clinically and includes:
Prealbumin :
Albumin O~l-Lipoprotein al- Acid glycoprotein l 1- Antitryps in ~1- Glycoprotein Transcortin 4.6S-Postalbumin Tryptophan-poor ~ ~1 ~ glycoprotein ~ -16 -: - :" .
iV84838 OL lX- Glycoprotein Thyroxin-binding globulin Inter~- trypsin-inhibitor Gc-Globulin (Gc 1-1) (Gc 2-1) (Gc 2-2) Haptoglobin (Hp 1-1) ' (Hp 2-1) (Hp 2-2) Ceruloplasmin Cholinesterase ~2- Lipoprotein(s) ~2- Macroglobulin a 2- HS-glycoprotein Zn-~2 -glycoprotein ~2- Neuramino-glycoprotein Erythropoietin ~- lipoprotein Transferrin Hemopexin Fibrinogen Plasminogen ~2 glycoprotein I
~2- glycoprotein II
Immunoglobulin G
(IgG) or r G-globulin Mol. formula:
~ -16~-iO8483~3 r2~2 rr2 ~2 Immunoglobulin A (IgA) or rA-globulin Mol. formula:
(~2 ~2)n or (~2 ~2)n Immunoglobulin M
(IgM) or rM-globulin Mol. formula:
(~2 ~2) or (~2 A2) Immunoglobulin D(IgD) or r D-Globulin (r D) Mol. formula:
(~2 ~2) or (~2 ~2) Immunoglobulin E (IgE) or~ E-Globulin (~ E) Mol. formula: .
(~2 ~2) or (2 ~2) Free light chains Complement factors:
C'l C'lq C'lr C ' ls C'2 C'3 ~ A
o~,2D
C'4 C'5 C'6 ~ -16 1~8483~3 C'7 C'8 C ' 9 Important blood clotting factors include:
BLOOD CLOTTING FACTORS
10 International Designation Name I Fibrinogen II Prothrombin IIa Thrombin II~ Tissue thromboplastin V and VI Proaccelerin, accelerator globulin VII Proconvertin VIII Antihemophilic globulin (AHG) IX Christmas factor, plasma thromboplastin component (PTC) X Stuart-Prower factor, autoprothrombin III
XI Plasma thromboplastin antecedent (PTA) XII Hagemann factor XIII Fibrin-stabilizing factor Important protein hormones include:
Peptide and Protein Hormones Parathyroid hormone (parathormone) Thyrocalcitonin Insulin Glucagon Relaxin - , : . - - : . :
.~ ., "~
~84838 Erythropoietin Melanotropin (melanocyte-stimulating hormone; intermedin) Somatotropin (growth hormone) Corticotropin (adrenocorticotropic hormone) Thyrotropin Follicle-stimulating hormone Luteinizing hormone (interstitial cell-stimulating hormone) ~' Luteomammotropic hormone (luteotropin, prolactin) Gonadotropin (chorionic gonadotropin) Tissue Hormones Secretin Gastrin Angiotensin I and II
Bradykinin Human placental lactogen Peptide Hormones from the Neurohypophysis oxytocin Vasopressin Releasing factors (RF) CRF, LRF, TRF, Somatotropin-RF, GRF, FSH-RF, PIF, MIF
~ -16~-~.os4s38 Other polymeric materials of interest are mucop-olysaccharides and polysaccharides.
Illustrative antigenic polysaccharides derlved from micro-organisms are as follows:
SPECIES OF MICRO-ORGANISMS HEMOSENSITIN FOUND IN
Streptococcus pyogenes Polysaccharide Diplococcus pneumoniae Polysaccharide Neisseria meningitidis Polysaccharide Neisseria gonorrhoeae Polysaccharide Corynebacterium diphtheriae Polysaccharide Actinobacillus mallei; Crude extract Actinobacillus whitemori Francisella tularensis Lipopolysaccharide Polysaccharide Pasteurella pestis Polysaccharide Pasteurella pestis 15 Pasteurella multocida Capsular antigen Brucella abortus Crude extract ' Haemophilus influenzae Polysaccharide Haemophilus pertussis Crude Treponema reiteri Polysaccharide Veillonella Lipopolysaccharide Erysipelothrix Polysaccharide Listeria monocytogenes Polysaccharide Chromobacterium Lipopolysaccharide Mycobacterium tuberculosis Saline extract of 90~
phenol extracted mycobacteria and polysaccharide fraction of cells and tuberculin Klebsiella aerogenes Polysaccharide Klebsiella cloacae Polysaccharide Salmonella typhosa Lipopolysaccharide, Polysaccharide Salmonella typhi-murium; Polysaccharide Salmonella derby Salmonella pullorum Shigella dysenteriae Polysaccharide Shigella flexneri Shigella sonnei Crude, polysaccharide Rickettsiae Crude extract 30 Candida albicans Polysaccharide Entamoeba histolytica Crude extract ~ -16~-: . : . .
,:
, :' ~.os4~3s Other than compounds of interest, cells, viruses, and other biological aggregations which are antigenic or to which naturally occurring receptors can be found may also be assayed for.
The micro-organisms which are assayed may be intact, lysed, ground or otherwise fragmented, and the resulting composition or portion, e.g. by extraction, assayed. Micro-organisms of interest include:
!
Corynebacteria .
Corynebacterium diptheriae Pneumococci Diplococcus pneumoniae Streptococci Streptococcus pyogenes Streptococcus salivarus -Staphylococci Staphylococcus aureus Staphylococcus albus `
Neisseriae Neisseria meningitidis Neisseria gonorrheae Enterobacteriaciae Escherichia coli Aerobacter aerogenes ~ The coliform bacteria -16~-- ~ : "- ' . :
11[~8483~3 Klebsiella pneumoniae J
Salmonella typhosa Salmonella choleraesuis The Salmonellae Salmonella typhimurium Shigella dysenteriae Shigella schmitzii Shigella arabinotarda The Shigellae Shigella flexneri Shigella boydii Shigella Sonnei Other enteric bacilli Proteus vulgaris Proteus mirabilis Proteus species Proteus morgani Pseudomonas aeruginosa Alcaligenes faecalis Vibrio cholerae Hemophilus-Bordetella Group Hemophilus influenzae, H. ducreyi H. hemophilus H. aegypticus H. paraifluenzae Bordetella pertussis Pasteurellae Pasteurella pestis Pasteurella tulareusis -l6b- ,' . ~ .
.
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Brucellae Brucella melitensis srucella abortus Brucella suis Aerobic Spore-forming Bacilli Bacillus anthracis Bacillus subtilis Bacillus megaterium Bacillus cereus naerobic Spore-forming Bacilli Clostridium botulinum Clostridium tetani Clostridium perfringens Clostridium novyi ~ :
~ Clostridium septicum - Clostridium histolyticum Clostridium tertium Clostridium bifermentans .
Clostridium sporogenes , , Mycobacteria Mycobacterium tuberculosis hominis Mycobacterium bovis Mycobacterium avium Mycobacterium leprae Mycobacterium paratuberculosis ~ -16~-i~3483~
Actinomycetes (fungus-like bacteria) Actinomyces israelii Actinomyces bovis Actinomyces naeslundii Nocardia asteroides Nocardia brasiliensis The Spirochetes Treponema pallidum Spirillum minus Treponema pertenue Streptobacillus moni-liformis Treponema carateum Borrelia recurrentis Leptospira icterohemorrhagiae Leptospira canicola Mycoplasmas Mycoplasma pneumoniae Other pathogens Listeria monocytogenes Erysipelothrix rhusiopathiae Streptobacillus moniliformis Donvania granulomatis Bartonella bacilliformis Rickettsiae (bacteria-like parasites) Rickettsia prowazekii Rickettsia mooseri Rickettsia rickettsii Rickettsia conori ~ -16j-83~
Rickettsia australis Rickettsia sibiricus Rickettsia akari Rickettsia tsutsugamushi Rickettsia burnetii Rickettsia quintana Chlamydia (unclassifiable parasites bacterial/viral) Chlamydia agents (naming uncertain) Fungi Cryptococcus neoformans Blastomyces dermatidis Histoplasma capsulatum Coccidioides immitis Paracoccidioides brasiliensis Candida albicans Aspergillus fumigatus Mucor corymbifer (Absidia corymbifera) Rhizopus oryzae Rhizopus arrhizus Phycomycetes Rhizopus nigricans Sporotrichum schenkii Fonsecaea pedrosoi Fonsecaea compacta Fonsecaea dermatitidis Cladosporium carrionii Phialophora verrucosa Aspergillus nidulans Madurella mycetomi -16~-. ; : :
.: ..
.
1(~184838 Madurella grisea Allescheria boydii Phialosphora jeanselmei Microsporum gypseum Trichophyton mentagrophytes Keratinomyces ajelloi Microsporum canis Trichophyton rubrum Microsporum andouini Viruses Adenoviruses Herpes Viruses Herpes simplex Varicella (Chicken pox) Herpes Zoster (Shingles) Virus B
Cytomegalovirus Pox Viruses Variola (smallpox) Vaccinia Poxvirus bovis Paravaccinia Molluscum contagiosum Picornaviruses -Poliovirus Coxsackievirus ; 30 Echoviruses ~ -16~-~:, : , !.
~8483~3 Rhinoviruses Myxoviruses Influenza (A, B, and C) Parainfluenza (1-4) Mumps Virus Newcastle Disease Virus Measles Virus Rinderpest Virus Canine Distemper Virus Respiratory Syncytial Virus Rubella Virus :
Arboviruses Eastern Equine Eucephalitis Virus Western Equine Eucephalitis Virus Sindbis Virus Chikungunya Virus Semliki Forest Virus Mayora Virus St. Louis Encephalitis Virus California Encephalitis Virus Colorado Tick Fever Virus Yellow Fever Virus Dengue Virus ;.
Reoviruses Reovirus Types 1-3 -16~-- .
~08483~3 Hepatitis Hepatitis A Virus Hepatitis B Virus Tumor Viruses Rauscher Leukemia Virus Gross Virus Maloney Leukemia Virus Friend Leukemia Virus Mouse Mammary Tumor Virus Avian Leucosis Virus Rous Sarcoma Virus Polyoma Virus Simian Virus 40 Papilloma Virus Preparations of micro-organisms include:
Streptococcus pyogenes, protein Pasteurella pestis, protein toxin Clostridium tetani, toxoid Clostridium perfringens, ~-lecithinase Escherichia coli, filtrates Treponema reiteri, protein extract Corynebacterium diphtheriae, toxin, toxoid Mycobacterium tuberculosis, protein M. tuberculosis, cytoplasm M. tuberculosis, culture filtrate and tuberculin Mycoplasma pneumoniae, "crude" antigen ' ., .. . ~ .
.', . , ' ' .'!
~ ~B483~
l¦ Ligand Analog-Fluorescer 21 The ligand analog will normally differ from the 31 ligand in only minor ways. In most cases, the ligand analog 4 ¦ will replace a hydrogen of the ligand with a bond to a ~ ¦ ligand group. As for example, with morphine, the hydrogen 6 ¦ of the phenolic hydroxyl can be replaced with a bond to the 7 ¦ methylene of an acetyl group. The hydrogen which is replaced 8 by a bond to a linking group may be bonded to carbon, either 9 aliphatic or aromatic, oxygen or nitrogen.
In some instances, an oxocarbonyl may serve as the 11 linking site by modifying the oxocarbonyl to an oxime. In 12 other instances, the hydroxyl of a carboxyl group may be 13 replaced to form a linking group, by forming an ester or 14 amide.
Additional alternatives include introducing 16 functionalities, such as hydroxyl functionalities from which 17 ethers can be formed, amino functionalities from which 18 amides can be formed, amino functionalities, from which 19 diazo groups can be formed, and the like.
The significant factor for the ligand analog is 21 that it has sufficient structural similarity to the ligand .^~
22 so as to be recognized by the antibody for the ligand.
23 Because the manner of addition can be widely varied, the 24 binding constants for the ligand and the ligand analog may be different, but should not differ by more than a factor of 26 103, preferably by not more than a factor of lO2.
27 For the most part, the ligand analog will have the 28 same or substantially the same structure and charge distribution 29 (spatial and polar organization) as the ligand for a significantr 32 ~ -17-~ 8~838 l if not major, portion of the molecular volume. Since frequently 2 the linking site for a hapten will be the same in prepariny 3 the antigen for production of antibodies as used for linking 4 to the fluorescer, the same portion of the ligand molecule which provides the template for the antibody will be exposed ~ by the ligand analog when bound to fluorescer.
71 Because of the steric inhibition of the presence 81 of one antibody preventing the binding of another antikody 9¦ to the ligand analog-fluorescer, the linking group will .
10¦ normally be relatively short. Usually, the linking group ll ~ will be substantially less than 25 A, more usually less than 121 20 A, and preferably less than 15 A. Normally, the linking ~
13¦ group will be from about 1.5-lOA. -14 ¦ With large molecules or macromolecules as ligands, ~¦ such as polypeptides and proteins, there will be a number of 16¦ different epitopes available on the surface of the molecule, 17¦ each of which will have a complementary antibody. When the 18¦ macromolecule is conjugated with fluorescer, normally there 19¦ will be a plurality of fluorescer molecules bonded to the 20¦ macromolecule. Depending on the spatial relationship of the 21¦ fluorescer molecule to an epitope, there may or may not be 221 steric inhibition to the simultaneous binding of antibody to 231 the ligand epitope and antibody to the fuorescer. ~1owever, 241 there will normally be a plurality of pairs of epitope siies 25 I and fluorescer molecules, where to various degrees, steric 26 1 inhibition between the two different antibodies will exist.
271 In referring to ligand analog-fluorescer molecules, it is 28 intend d to include mo1ecu1es where there is at least one ~ l - ~
1~34838 1 epitope-fluorescer pair, which is in appropriate juxtaposition 2 for steric interaction. The statement concerning the steric 3 inhibition for simpler molecules having one epitope and one 4 fluorescer will be normally appropriate to the epitope-fluorescer pairs present in macromolecules.
6 In choosing the fluorescer, a wide variety of 7 ¦ considerations will come into play. As already indicated, 8 the choice of fluorescer will, to a degree, be governed by 9¦ the ligand. Therefore, one consideration is that the 10¦ fluorescer have absorption at higher wavelengths than a 11 ¦ fluorescent ligand or ligand bound to antibody.
12¦ In addition to the considerations which relate to 13¦ the particular ligand being determined, there will be a 14 ¦ number of other considerations which limit the particular 15¦ choice of fluorescer. As a practical matter, since one is 16¦ concerned with a change in the em ssion spectrum as a result 17¦ of being bound or unbound to an antifluorescer, one would lB ¦ desire a large environmental effect on the emission intensity 19¦ at a particular wavelength. This can be a result of a 20¦ substantial changê in quantum yield or a change in the 21 ¦ emission or absorption spectrum in going from the bound to 22¦ unbound fluorescer.
231 Since proteins absorb at a wavelength of about 241 280, the fluorescer should have an absorption maximum about 251 300, usually about 350 and preferably above 400nm. The 2~1 extinction coefficient should be greatly in excess of 10, 271 preferably in excess of 103, and particularly preferred, in 28¦ excess of 104M lcm 1.
3~
: :, 1~84838 .
1 In addition, it is desirable that the fluorescer 2 have a large Stokes shift. That is, it is preferred that 3 there be a substantial spread or difference in wavelengths 4 for the fluorescer between its absorption maximum and
5 emission maximum.
6 Another consideration where physiological fluids
7 are concerned is non-specific binding of the fluorescer to
8 protein. Preferred fluorescers will have minimal non-
9 specific binding, so that the primary or sole effect seen is the binding of the fluorescer to its antibody.
11 A number of different fluorescers are described in 12 Brand and Zohlke, Annual Review of Biochemlstry, 41 843-868 -13 (1972) and Stryer, Science, 162 526 (1968).
14 One group of fluorescers having a number of the desirable properties described previously are the xanthene 16 dyes, ~-hich include the fluoresceins derived from 3,6-I7 dihydroxy-9-phenyl-xanthhydrol and rosamines and rhodamines, 18 derived from 3,6-diamino-9-phenylxanthhydrol. The rhodamines 19 and fluoresceins have a 9-o-carboxyphenyl group, and are derivatives of 9-o-carboxy-phenylxanthhydrol.
21 These compounds are commercially available with 22 substituents on the phenyl group which can be used as the 23 site for bonding or as the bonding functionality. For 2~ example, amino and isothiocyanate substituted fluorescein compounds are available.
26 Another group of fluorescent compounds are the 27 naphthylamines, having an amino group in the alpha or beta 28 position, usually alpha position. Included among the : , ., -lQ8483~
1~ na~h hy.amino compoundS re l-dimethylaminonaphth 2 sulfonate, 1-anilino-8-naphthalene sulfona~e and 2-p-3 toluidinyl-6-naphthalene sulfonate. The naphthalene compounds 4 are found to have some non-specific binding to protein, so that their use requires their employ in an assay medium where 6 the amount of protein is minimized.
71 As already indicated, the linking group may be 81 derived from a func-tionality which is present on the fluorescer 9¦ or a functionality which is present on the ligand analog.
11 A number of different fluorescers are described in 12 Brand and Zohlke, Annual Review of Biochemlstry, 41 843-868 -13 (1972) and Stryer, Science, 162 526 (1968).
14 One group of fluorescers having a number of the desirable properties described previously are the xanthene 16 dyes, ~-hich include the fluoresceins derived from 3,6-I7 dihydroxy-9-phenyl-xanthhydrol and rosamines and rhodamines, 18 derived from 3,6-diamino-9-phenylxanthhydrol. The rhodamines 19 and fluoresceins have a 9-o-carboxyphenyl group, and are derivatives of 9-o-carboxy-phenylxanthhydrol.
21 These compounds are commercially available with 22 substituents on the phenyl group which can be used as the 23 site for bonding or as the bonding functionality. For 2~ example, amino and isothiocyanate substituted fluorescein compounds are available.
26 Another group of fluorescent compounds are the 27 naphthylamines, having an amino group in the alpha or beta 28 position, usually alpha position. Included among the : , ., -lQ8483~
1~ na~h hy.amino compoundS re l-dimethylaminonaphth 2 sulfonate, 1-anilino-8-naphthalene sulfona~e and 2-p-3 toluidinyl-6-naphthalene sulfonate. The naphthalene compounds 4 are found to have some non-specific binding to protein, so that their use requires their employ in an assay medium where 6 the amount of protein is minimized.
71 As already indicated, the linking group may be 81 derived from a func-tionality which is present on the fluorescer 9¦ or a functionality which is present on the ligand analog.
10¦ Either the fluorescer or ligand analog may be modified in
11 ¦ order to provide the necessary linkage between the two
12¦ compounds.
13¦ Both the ligand and the fluorescer may be conjugated
14 ¦ to hub nucleii to provide poly(ligand analog)-polyfluorescer.
15¦ The hub nucleus will normally be a polyfunctionalized molecule, 161 generally having from about 5,000 to one million, more 17¦ usually from about 30,000 to 600,000 molecular weight.
18¦ Generally, there will not be more than one fluorescer or one 19¦ ligand per 1,000 molecular weight, more usually not more 20¦ than one fluorescer and/or one ligand per 5,000 molecular 21 ¦ weight, and usually at least one ligand or one fluorescer 22¦ per S0,000 molecular weight, more usually at least one 23¦ ligand or one fluorescer per 30,000 molecular weight. The 241 hub nucleus may be a poly(amino acid), either naturally 2~ occurring or synthetic e.g. proteins or polypeptides, 26¦ polyhydroxylic compounds, such as cellulose or dextran, or 271 the like. The functionalities which may be employed for 281 linking the ligand to the fluorescer, may also be employed 29l ~or linking the ligand and fluorescer to the hub nucleus.
3~1 ~
32 -~17 ~ l 1~84838 ¦ ntifluorescer-Quencher 2 The antifluorescers will normally be antibodies 3 prepared by introducing fluorescer conjugated to an antigen 4 into a vertebrate to produce antisera specific for the 51 fluorescer. The antisera may then be harvested and purified 61 by conventional techniques, such as fractionation, precipitation, 71 chromatography, and the like. The quencher may then be 81 conjugated to the antisera by any convenient means. The 9 ¦same methods employed for conjugating ligand analog to 10 ¦fluorescer can be employed for conjugating quencher to 11 jantifluorescer, recognizing the fact that the most convenient 12 ¦functionality present in the antifluorescer is the amino 13 ¦group. Convenient methods for conjugation employ carboxylic -14 ¦acid derivatives to form amides, imidates to form amidines, 15 ¦and carbonyl groups, particularly aldehydes, to form imines 1~¦ which may be reduced to amines.
I7 ¦ Normally, there will be at least one quencher 18¦ molecule per 100,000 molecular weight of the antifluorescer, l9 ¦more usually one quencher molecule per 75,000 molecular 201 weight of the antifluorescer, and usually not more than ~ 21 ¦ about one quencher molecule per l,000 molecular weight of 22¦ the antifluorescer, more usually not more than one quencher 231 molecule per 2,000 molecular weight of the antifluorescer.
24 ¦Primarily, the consideration is one of solubility, since 25 ¦many dyes have low aqueous solubility and may desolubilize 26 ¦ the receptor. For the most part, the number of quencher 27 ¦ molecules will be in the range of about ? to 25, more usually 28¦ 2 to 20, and preferably about ~ to 16, more preferably about 291 6 to 16 where antibodies, y-globulin, are involved.
32 ~2~-~ 1~8483~
1 ¦ The quenchers are dyes which are chosen so as to 21 have an absorption band which overlaps the emission band of 31 the fluorescer. In referring to the absorption and emission 41 bands of fluorescer and quencher, what is intended is the B¦ bands as observed in the medium of the assay, as influenced 61 by the assay medium and conjugation to protein and not bands ~¦ as reported in a different environment.
81 Molecules which have been reported as fluorescers 91 previously, may also be used as quenchers. For example, 10 ¦ rhodamine may be used as a quencher of fluorescein. Other 11 ¦ combinations may also be found in the literature.
12 I By employing antifluorescer-quencher in the 13 ¦ subject assay, numerous advantages accrue. In the preparation 14 ¦ of ligand analog-fluorescer, particularly with high molecular 15¦ weight naturally occurring materials, there is usually a
18¦ Generally, there will not be more than one fluorescer or one 19¦ ligand per 1,000 molecular weight, more usually not more 20¦ than one fluorescer and/or one ligand per 5,000 molecular 21 ¦ weight, and usually at least one ligand or one fluorescer 22¦ per S0,000 molecular weight, more usually at least one 23¦ ligand or one fluorescer per 30,000 molecular weight. The 241 hub nucleus may be a poly(amino acid), either naturally 2~ occurring or synthetic e.g. proteins or polypeptides, 26¦ polyhydroxylic compounds, such as cellulose or dextran, or 271 the like. The functionalities which may be employed for 281 linking the ligand to the fluorescer, may also be employed 29l ~or linking the ligand and fluorescer to the hub nucleus.
3~1 ~
32 -~17 ~ l 1~84838 ¦ ntifluorescer-Quencher 2 The antifluorescers will normally be antibodies 3 prepared by introducing fluorescer conjugated to an antigen 4 into a vertebrate to produce antisera specific for the 51 fluorescer. The antisera may then be harvested and purified 61 by conventional techniques, such as fractionation, precipitation, 71 chromatography, and the like. The quencher may then be 81 conjugated to the antisera by any convenient means. The 9 ¦same methods employed for conjugating ligand analog to 10 ¦fluorescer can be employed for conjugating quencher to 11 jantifluorescer, recognizing the fact that the most convenient 12 ¦functionality present in the antifluorescer is the amino 13 ¦group. Convenient methods for conjugation employ carboxylic -14 ¦acid derivatives to form amides, imidates to form amidines, 15 ¦and carbonyl groups, particularly aldehydes, to form imines 1~¦ which may be reduced to amines.
I7 ¦ Normally, there will be at least one quencher 18¦ molecule per 100,000 molecular weight of the antifluorescer, l9 ¦more usually one quencher molecule per 75,000 molecular 201 weight of the antifluorescer, and usually not more than ~ 21 ¦ about one quencher molecule per l,000 molecular weight of 22¦ the antifluorescer, more usually not more than one quencher 231 molecule per 2,000 molecular weight of the antifluorescer.
24 ¦Primarily, the consideration is one of solubility, since 25 ¦many dyes have low aqueous solubility and may desolubilize 26 ¦ the receptor. For the most part, the number of quencher 27 ¦ molecules will be in the range of about ? to 25, more usually 28¦ 2 to 20, and preferably about ~ to 16, more preferably about 291 6 to 16 where antibodies, y-globulin, are involved.
32 ~2~-~ 1~8483~
1 ¦ The quenchers are dyes which are chosen so as to 21 have an absorption band which overlaps the emission band of 31 the fluorescer. In referring to the absorption and emission 41 bands of fluorescer and quencher, what is intended is the B¦ bands as observed in the medium of the assay, as influenced 61 by the assay medium and conjugation to protein and not bands ~¦ as reported in a different environment.
81 Molecules which have been reported as fluorescers 91 previously, may also be used as quenchers. For example, 10 ¦ rhodamine may be used as a quencher of fluorescein. Other 11 ¦ combinations may also be found in the literature.
12 I By employing antifluorescer-quencher in the 13 ¦ subject assay, numerous advantages accrue. In the preparation 14 ¦ of ligand analog-fluorescer, particularly with high molecular 15¦ weight naturally occurring materials, there is usually a
16¦ substantial amount of contamination. When conjugating
17¦ fluorescer to the analyte of interest, the contaminants will
18¦ also frequently be conjugated. Thus, when introducing the ~9¦ ligand analog-fluorescer lnto the assay medium, a substantial 20¦ amount of fluorescer will also be introduced with the 21 ¦ contaminants. While antifluorescer can significantly 22¦ change the amount of fluorescence of a fluorescer when bound 231 to fluorescer, either an increase or decrease can occur, and 241 even with a decrease a substantial background of fluorescence 251 will be present in the assay. In addition, the fluorescence 26¦ of the fluorescer bound to ligand analog is also incompletely 271 inhibited. Thus, even w th all the fluorescer present in 28~ the ssay m~dium bound by antif1uorescer, there will still ¦
332 l ` -23-~ ;
-' :' 1~848;~8 1 be a substantial amount of fluorescence in the medium. ~
2 Thus, in carrying out the assay, one would be substracting 3 two relatively large numbers to obtain a small number. The 4 smaller the differences between the two large numbers, the less sensitive is the assay to small variations in concentration 6 of the analyte. By employing quencher conjugated to the 7 antifluorescer, 95% or better of the fluorescence may be 8 quenched. Therefore, the fluorescers adventitiously introduced 9 by conjugation of fluorescer with contaminants present with the ligand will only introduce an extremely small amount of 11 background fluorescence to the assay. Furthermore, the 12 fluorescer conjugated to ligand analog wil r also be substantially 13 quenched when bound to antifluorescer conjugated to quencher.
14 Thus, the background is greatly diminished, so that only a ;
15 small background number need be substrated from the observed ;~
1~ result.
17 The following examples are offered by way of 18 illustration and not by way of limitation.
332 l ` -23-~ ;
-' :' 1~848;~8 1 be a substantial amount of fluorescence in the medium. ~
2 Thus, in carrying out the assay, one would be substracting 3 two relatively large numbers to obtain a small number. The 4 smaller the differences between the two large numbers, the less sensitive is the assay to small variations in concentration 6 of the analyte. By employing quencher conjugated to the 7 antifluorescer, 95% or better of the fluorescence may be 8 quenched. Therefore, the fluorescers adventitiously introduced 9 by conjugation of fluorescer with contaminants present with the ligand will only introduce an extremely small amount of 11 background fluorescence to the assay. Furthermore, the 12 fluorescer conjugated to ligand analog wil r also be substantially 13 quenched when bound to antifluorescer conjugated to quencher.
14 Thus, the background is greatly diminished, so that only a ;
15 small background number need be substrated from the observed ;~
1~ result.
17 The following examples are offered by way of 18 illustration and not by way of limitation.
19 EXPERI~lENTAL
All temperatures not otherwise indicated are in 21 centigrade. All percents not otherwise indicated are by 22 weight, except for mixtures of liquids, which are by volume.
231 The following abbreviations are used 24 BSA-bovine serum e1bumin D~-dimethyl'ormamide.
29~ _~
31 .
- , -: , , : : :
.. . .
: :: . . , . :
1~84838 Example I. Preparation of Fluorescein-BSA Conjugate:
Into a scintillation vial was placed 180 mgs of BSA (2.6 X 10 6 Moles) (Pentex (Trade Mark) crystallized) dissolved in 6 mls H20 containing 180 mgs K2CO3. To this was added 18.3 mgs fluorescein isothiocyanate (FITC) t4.85 X
10 5 Moles) and the mixture was stoppered, covered with aluminum foil then placed in the center hole of a vortex mixer and shaken gently overnight at room temperature.
The following morning the reaction mixture was acidified to pH 4 with lN HCl theavY precipitate), then made basic to pH
8 with 0.1N NaOH, applied to a 2.5 X 30cm column of Sephadex G-10 tTrade Mark) and eluted with 0.05M PO4, pH 8.0 at 5.4 mls/hr. 0.9 ml/fraction. Fractions 43-72 were pooled.
Hapten number was calculated from the W absorption of the conjugate at 493mu and the extinction coefficient for protein-bound fluorescein of 7.2 X 104. Hapten number was thus calculated to be 14.5. The above conjugate was employed to prepare antifluorescein antisera according to known procedures.
.. : . .
.: . : , , .
1Example II. Conjugation of rhodamine to anti(f~ rescein) 2 I ; , 3A lml aliquot of antisera to fluorescein was 4precipitated against 50% saturated ammonium sulfate, and the 51 precipitate dissolved in lml of O.lM K2HPOa and dialyzed 61 against the same solution to yield a solution having a 71 concentration of 9mg/ml. Into a reaction vessel was intro- r 81 duced 0.4ml (3.6mg) of the anti(fluorescein) antisera and ¦ 0.17ml of glycerol, the pH brought to 9.5 and 0.8mg of 10 ¦ tetramethylrhodamine isothiocyanate in 100~1 D.~F added with 11 ¦ stirring at room temperature. After continuing the reaction 12 ¦ for 3hrs., the solution was poured onto a-Sephadex LH-20 j I
13 ¦ column (0.9xi5cm) and the anti(fluorescein) recovered in ' 14 ¦ lml.
1~ I . ' ' . -~ ~
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31 .
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~ ~134838 I . .;,-l EXA~IPLE III.Conjugation of Human Gamma-Clobulin 2 (hIgG) to Fluorescein (FhIgG) r 3 ¦ One mg of HIgG ~Human IgG) dissolved in 0.4ml of .
4 ¦ 0.l~ phosphate buffer pH 7.5, was brought to pH 9.5 with 5 1 crystalline Na2C03. A solution (l0~l) of FITC (70~g) in 6 ¦ acetone was added with stirring and mixed for 3 hrs at room 1 temperature. The resulting solution was separated two times 8 ¦ on Sephadex G-25(M) column (lxl5cm) equilibrated ~Jith 0.05~1 I phosphate buffer pH 8Ø The eluted FITC-hIgG coniugate ~' 10 ¦ solution was 0.58mg/ml in concentration and had D/P ~ 5.5 12 t~l/M) s det~rmiDed by the We11a nomograph.
4 ~ .
16 I ` . ;
18 . . .
19 I . ~,
All temperatures not otherwise indicated are in 21 centigrade. All percents not otherwise indicated are by 22 weight, except for mixtures of liquids, which are by volume.
231 The following abbreviations are used 24 BSA-bovine serum e1bumin D~-dimethyl'ormamide.
29~ _~
31 .
- , -: , , : : :
.. . .
: :: . . , . :
1~84838 Example I. Preparation of Fluorescein-BSA Conjugate:
Into a scintillation vial was placed 180 mgs of BSA (2.6 X 10 6 Moles) (Pentex (Trade Mark) crystallized) dissolved in 6 mls H20 containing 180 mgs K2CO3. To this was added 18.3 mgs fluorescein isothiocyanate (FITC) t4.85 X
10 5 Moles) and the mixture was stoppered, covered with aluminum foil then placed in the center hole of a vortex mixer and shaken gently overnight at room temperature.
The following morning the reaction mixture was acidified to pH 4 with lN HCl theavY precipitate), then made basic to pH
8 with 0.1N NaOH, applied to a 2.5 X 30cm column of Sephadex G-10 tTrade Mark) and eluted with 0.05M PO4, pH 8.0 at 5.4 mls/hr. 0.9 ml/fraction. Fractions 43-72 were pooled.
Hapten number was calculated from the W absorption of the conjugate at 493mu and the extinction coefficient for protein-bound fluorescein of 7.2 X 104. Hapten number was thus calculated to be 14.5. The above conjugate was employed to prepare antifluorescein antisera according to known procedures.
.. : . .
.: . : , , .
1Example II. Conjugation of rhodamine to anti(f~ rescein) 2 I ; , 3A lml aliquot of antisera to fluorescein was 4precipitated against 50% saturated ammonium sulfate, and the 51 precipitate dissolved in lml of O.lM K2HPOa and dialyzed 61 against the same solution to yield a solution having a 71 concentration of 9mg/ml. Into a reaction vessel was intro- r 81 duced 0.4ml (3.6mg) of the anti(fluorescein) antisera and ¦ 0.17ml of glycerol, the pH brought to 9.5 and 0.8mg of 10 ¦ tetramethylrhodamine isothiocyanate in 100~1 D.~F added with 11 ¦ stirring at room temperature. After continuing the reaction 12 ¦ for 3hrs., the solution was poured onto a-Sephadex LH-20 j I
13 ¦ column (0.9xi5cm) and the anti(fluorescein) recovered in ' 14 ¦ lml.
1~ I . ' ' . -~ ~
~ ~ ~
222,21, ~ ~
31 .
I . ''". .
. . - - ~ . .
~ ~134838 I . .;,-l EXA~IPLE III.Conjugation of Human Gamma-Clobulin 2 (hIgG) to Fluorescein (FhIgG) r 3 ¦ One mg of HIgG ~Human IgG) dissolved in 0.4ml of .
4 ¦ 0.l~ phosphate buffer pH 7.5, was brought to pH 9.5 with 5 1 crystalline Na2C03. A solution (l0~l) of FITC (70~g) in 6 ¦ acetone was added with stirring and mixed for 3 hrs at room 1 temperature. The resulting solution was separated two times 8 ¦ on Sephadex G-25(M) column (lxl5cm) equilibrated ~Jith 0.05~1 I phosphate buffer pH 8Ø The eluted FITC-hIgG coniugate ~' 10 ¦ solution was 0.58mg/ml in concentration and had D/P ~ 5.5 12 t~l/M) s det~rmiDed by the We11a nomograph.
4 ~ .
16 I ` . ;
18 . . .
19 I . ~,
20;
23 ~
. -.
26 I . .
271 . .
28~l 32 ~ -27-I
~8483~
In order to establish the utility of the subject compounds, the following assays were carried out.
In performing the assay, 200,ul of buffer (O.lM
K2HPO4, pH 7.8, 0.05% NaN3 is combined with 30 ,ul of fluo-rescein conjugated hIgG (1.6 X 10 9M) having a fluorescein/
; hIgG ratio of 14:1, and the prescribed amount of anti (hIgG) and the mixture incubated at room temperature for 0.5 hour.
At the end of this time, 2.75 ml of buffer is added and 50,ul of the rhodamine conjugated anti(fluorescein) at a 1:10 dilution. Readings are then taken of the fluorescence ofthe sample. The following table indicates the amount of anti (hIgG) added at a concentration of 2.4 mg/ml.
~ .
TABLE II
Anti (hIgG) ~ul Fluorescence .
1 17.5, 18.5 2 21.5, 21.5 22.5, 24.5 29.5, 30.5 31.5, 33.5 It is evident from the above results that the rhodamine conjugated anti(fluorescein) is an effective quencher of fluorescence. Furthermore, increasing amounts of anti(hIgG) provide increasing protection of the fluore-scence from quenching by the anti(fluorescein).
.~
1~8~831~
In the next assay, varying concentrations of hIgG were employed to complete with the fluorescein con-jugated hIgG for a limited amount of anti(hIgG). The protocol employed combining 100 ,ul of the fluorescein conjugated hIgG (1.6 X 10 M), 100~1 of hIgG at various concentrations and 20,ul of anti(hIgG) at a concentration of about 0.4 mg/ml. The mixture was incubated for 0.5 hour at room temperature, followed by the addition of 2.75 ml buffer (see above) and 50,ul of the rhodamine conjugated anti(fluor-escein) diluted 1:10 and the mixture incubated for an additional 0.5 hour. Fluorescence readings were then taken after 0.5 hour. The following table indicates the results.
TABLE III
hIgG
M Fluorescence _ 85, 84 1 X 10 10, 9 1 X 10 8 10, 11 1 X 10 9 59, 63 1 X 10 10 76, 80 1 X 10 11 82, 87 The presence of the hIgG has the effect of binding of the anti(hIgG) and reducing the available anti(hIgG) for binding to the fluorescein conjugated hIgG. Thus, at high hlgG eoneentrations, the fluoreseein is not protected by the presence of anti(hIgG) and is quenched by the rhodamine eonjugated anti(fluoreseein).
~ ~8483~ r It is evident from the prior results, that by employing antifluorescer conjugate with quencher, fluor-escence of the fluorescer is substantially eliminted when the antifluorescer is bound to fluorescer, thus background values are greatly diminished.
In addition, ligands which are relatively impure can be employed in the preparation of ligand analog-fluorescer, avoiding costly purification procedures. The subject invention also finds advantage with haptenic ligands in substantially eliminating fluorescence from ligand analog-fluorescer bound to antifluorescer-quencher. By virtue of the diminution of background fluorescence more sensitive and accurate assays are provided.
23 ~
. -.
26 I . .
271 . .
28~l 32 ~ -27-I
~8483~
In order to establish the utility of the subject compounds, the following assays were carried out.
In performing the assay, 200,ul of buffer (O.lM
K2HPO4, pH 7.8, 0.05% NaN3 is combined with 30 ,ul of fluo-rescein conjugated hIgG (1.6 X 10 9M) having a fluorescein/
; hIgG ratio of 14:1, and the prescribed amount of anti (hIgG) and the mixture incubated at room temperature for 0.5 hour.
At the end of this time, 2.75 ml of buffer is added and 50,ul of the rhodamine conjugated anti(fluorescein) at a 1:10 dilution. Readings are then taken of the fluorescence ofthe sample. The following table indicates the amount of anti (hIgG) added at a concentration of 2.4 mg/ml.
~ .
TABLE II
Anti (hIgG) ~ul Fluorescence .
1 17.5, 18.5 2 21.5, 21.5 22.5, 24.5 29.5, 30.5 31.5, 33.5 It is evident from the above results that the rhodamine conjugated anti(fluorescein) is an effective quencher of fluorescence. Furthermore, increasing amounts of anti(hIgG) provide increasing protection of the fluore-scence from quenching by the anti(fluorescein).
.~
1~8~831~
In the next assay, varying concentrations of hIgG were employed to complete with the fluorescein con-jugated hIgG for a limited amount of anti(hIgG). The protocol employed combining 100 ,ul of the fluorescein conjugated hIgG (1.6 X 10 M), 100~1 of hIgG at various concentrations and 20,ul of anti(hIgG) at a concentration of about 0.4 mg/ml. The mixture was incubated for 0.5 hour at room temperature, followed by the addition of 2.75 ml buffer (see above) and 50,ul of the rhodamine conjugated anti(fluor-escein) diluted 1:10 and the mixture incubated for an additional 0.5 hour. Fluorescence readings were then taken after 0.5 hour. The following table indicates the results.
TABLE III
hIgG
M Fluorescence _ 85, 84 1 X 10 10, 9 1 X 10 8 10, 11 1 X 10 9 59, 63 1 X 10 10 76, 80 1 X 10 11 82, 87 The presence of the hIgG has the effect of binding of the anti(hIgG) and reducing the available anti(hIgG) for binding to the fluorescein conjugated hIgG. Thus, at high hlgG eoneentrations, the fluoreseein is not protected by the presence of anti(hIgG) and is quenched by the rhodamine eonjugated anti(fluoreseein).
~ ~8483~ r It is evident from the prior results, that by employing antifluorescer conjugate with quencher, fluor-escence of the fluorescer is substantially eliminted when the antifluorescer is bound to fluorescer, thus background values are greatly diminished.
In addition, ligands which are relatively impure can be employed in the preparation of ligand analog-fluorescer, avoiding costly purification procedures. The subject invention also finds advantage with haptenic ligands in substantially eliminating fluorescence from ligand analog-fluorescer bound to antifluorescer-quencher. By virtue of the diminution of background fluorescence more sensitive and accurate assays are provided.
Claims (10)
1. A method for detecting the presence of a ligand or antiligand in a sample comprising:
combining in an aqueous medium:
said sample;
ligand analog-fluorescer, wherein said ligand analog has sufficient structural similarity to the ligand so as to be recognized by antiligand, said ligand analog-fluorescer has at least one ligand epitope-fluorescer pair for steric inhibition of simultaneous binding of antiligand and antifluorescer, so that binding of antiligand to ligand analog inhibits the binding of antifluorescer to fluorescer;
antiligand for determination of ligand; and quencher conjugated antifluorescer, which sub-stantially inhibits the fluorescence of said fluorescer when bound to said fluorescer;
wherein the amount of quencher conjugated anti-fluorescer, which binds to said fluorescer, is related to the amount of ligand or antiligand in said sample, so that the fluorescence observed from said medium when said fluor-escer is excited, is related to the amount of ligand or antiligand in said medium; and determining at about the wavelength of the emis-sion maximum of said fluorescer the intensity of the fluore-scence from said medium as compared to a standard having a known amount of ligand or antiligand.
combining in an aqueous medium:
said sample;
ligand analog-fluorescer, wherein said ligand analog has sufficient structural similarity to the ligand so as to be recognized by antiligand, said ligand analog-fluorescer has at least one ligand epitope-fluorescer pair for steric inhibition of simultaneous binding of antiligand and antifluorescer, so that binding of antiligand to ligand analog inhibits the binding of antifluorescer to fluorescer;
antiligand for determination of ligand; and quencher conjugated antifluorescer, which sub-stantially inhibits the fluorescence of said fluorescer when bound to said fluorescer;
wherein the amount of quencher conjugated anti-fluorescer, which binds to said fluorescer, is related to the amount of ligand or antiligand in said sample, so that the fluorescence observed from said medium when said fluor-escer is excited, is related to the amount of ligand or antiligand in said medium; and determining at about the wavelength of the emis-sion maximum of said fluorescer the intensity of the fluore-scence from said medium as compared to a standard having a known amount of ligand or antiligand.
2. A method according to claim 1, wherein the pH
of said aqueous medium is in the range of about 6 to 9, the concentration of ligand analog-fluorescer is in the range of about 10-4 X 10-12M and the temperature of said medium is in the range of about 15 to 40°C.
of said aqueous medium is in the range of about 6 to 9, the concentration of ligand analog-fluorescer is in the range of about 10-4 X 10-12M and the temperature of said medium is in the range of about 15 to 40°C.
3. A method according to claim 2, wherein said quencher conjugated antifluorescer has a ratio of quencher to antifluorescer on the average of from about 2 to 25.
4. A method according to claim 2, wherein said ligand is a poly(amino acid).
5. A method according to claim 2, wherein said ligand is an alkaloid.
6. A method according to claim 2, wherein said ligand is a steroid.
7. A method according to claim 2, wherein said ligand is a synthetic drug.
8. A method according to claim 2, wherein said fluorescer is fluorescein and said quencher is rhodamine.
9. A method according to claim 1 wherein said fluorescence is from said fluorescer.
10. Quencher conjugated antifluorescer having at least one quencher molecule per 100,000 molecular weight of the antifluorescer up to about one quencher molecule per 1,000 molecular weight of antifluorescer, said quencher being a dye having an absorption band that overlaps the emission band of the fluorescer to which said antifluorescer speci-fically binds, said fluorescer having an absorption maximum in excess of about 350nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA280,237A CA1084838A (en) | 1977-06-10 | 1977-06-10 | Quencher conjugated antifluorescer in fluorescent immonoassay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA280,237A CA1084838A (en) | 1977-06-10 | 1977-06-10 | Quencher conjugated antifluorescer in fluorescent immonoassay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1084838A true CA1084838A (en) | 1980-09-02 |
Family
ID=4108858
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA280,237A Expired CA1084838A (en) | 1977-06-10 | 1977-06-10 | Quencher conjugated antifluorescer in fluorescent immonoassay |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1084838A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0343346A1 (en) * | 1988-03-29 | 1989-11-29 | Matsushita Electric Industrial Co., Ltd. | Fluorescence immunoassay method utilizing pseudo-antigens combined with fluorescent quenchers |
| EP0349520A2 (en) * | 1988-06-28 | 1990-01-03 | Ernst Dr. Koller | Fluorescence immunoassay based on fluorescent quenching |
| US5229302A (en) * | 1988-03-29 | 1993-07-20 | Matsushita Electric Industrial Co., Ltd. | Fluorescence immunoassay method utilizing pseudo-antigens combined with fluorescent quenchers |
-
1977
- 1977-06-10 CA CA280,237A patent/CA1084838A/en not_active Expired
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0343346A1 (en) * | 1988-03-29 | 1989-11-29 | Matsushita Electric Industrial Co., Ltd. | Fluorescence immunoassay method utilizing pseudo-antigens combined with fluorescent quenchers |
| US5229302A (en) * | 1988-03-29 | 1993-07-20 | Matsushita Electric Industrial Co., Ltd. | Fluorescence immunoassay method utilizing pseudo-antigens combined with fluorescent quenchers |
| EP0349520A2 (en) * | 1988-06-28 | 1990-01-03 | Ernst Dr. Koller | Fluorescence immunoassay based on fluorescent quenching |
| EP0349520A3 (en) * | 1988-06-28 | 1990-12-19 | Ernst Dr. Koller | Fluorescence immunoassay based on fluorescent quenching |
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