CA1241332A - Compounds for linking ligands to enzymes for enzyme- linked immunoassay - Google Patents

Abstract

ABSTRACT
Linker-compounds utllized in performing a novel immunoassay which utilizes an enzyme linked ligand or receptor wherein the enzyme is bacterial luciferase.
The compounds are of general formula: R7-S(O)(O)-S-C(R2)-(R3)-C(R4)(R3)-(CH2)n-R6. The linker-compound-ligand or linker-compound-receptor are of general formula:
R7-S(O)(O)-S-C(R2)(R3)-C(R4)(R5)-(CH2)n-X-Y, wherein n, R2 to R7, X and Y are as defined in the disclosure.

Description

l 3~3~

This is a divisional application of copending applica-tion, Serial No. 444,340, filed December 28, 1983.
FIELD OF INVENTl()N
,_~
The present invention is an enzyme-linked immunoassay and compo-nents utilized in said immunoassay.
DESCRIPTION OF PRIOR ART
-Immunoassays wherein one of the immunotogical components, c either t'ne antigen or the antibody, is labeled with a radioactive iso-tope tracer have an established significant role in medical diagnosis and in the detection of toxins and other substances in industrial enYironments. Gore recently much attention has been focused on assay systems which use labels of a non-radioactive nature, such as chemi-luminescent agents, fluorescent agents and enzymes. Various types of enzyme-labeled or enzyme-linked assays have been described in the literature. For example, U.S. patent 3,654,090 describes an assay wherein one of the two immunological components is covalently linked to an enzyme and the other said component is utilized in an insolubil-ized form, and following incubation with the test sample the enzyme-labeled component in either the solid or liquid phase is a measure of the amount of component in the sample.

2~ In US 3,817,837 there is described a means of detecting sub-stances or ligands for which receptors, e.g., antibodies, can be generated or occur naturally which comprises reacting in an aqueous medium a soluble enzyme-bound ligand, a receptor for the ligand and the substance to be assayed. In this enzyme-linked assay, binding of the receptor to the enz~me-bound ligand results in a substantial reduction in the enzyme activity. Thus, changes in enzyme activity in the assay medium affords a means of measuring the quantity ox ligand in the assay sample. It is important to note that the enzyme in this particular enzyme-bound ligand complex retains its activity whereas once the receptor binds the enzyme-bound ligand complex the enzyme is rendered inactive substantially. A similar enzyme linked assay is described in U.S. 4,039,385.
U.S. 4,171,244 describes an assay for thyroid hormones which uti-lizes an enzyme-bound complex wherein the enzyme loses about 50q~ or more of its aotivity upon forming the complex but which regains a por-tion of this lost activity when bound to a receptor in the assay pro-cedure. Again changes in enzyme activity in the assay medium providP
a means of measuring the quantity of ligand, i.e., thyroid hormone, in ,,~
I

the assay sample.
In U.SO 4,231,999 there is described a modification of the enzyme-linked assay system of U.S. 3,817,837. In the method of the biospecific affinity reaction described in the '999 patent one of the components of said reaction is linked to the label, e.g., an enzyme, by a splittable bond of a covalent nature. Although the enzyme activ-ity is reduced substantially by the biospecific affinity reaction, release of the enzyme by sp1itting the covalent bond optimizes enzyme activity for assay purposes. Although there is an indication that various splittable bonds may be employed, the splittable bond of choice in the '999 patent is the disulfide linkage, i.e., the label, e.g., enzyme and one of the components of the affinity reaction are linked together by a disulfide bridge. The generation of such a disulfide linkage requires the presence of thiol groups in the label and affinity reaction component, and the '999 patent describes means whereby such groupC may be introduced. However, there does not appear to be any convenient or specific means whereby the number of thiol groups introduced into, e.g., an enzyme label may be controlled which factor could ultimately interfere with or decrease the sensitivity of the assay.
Other types of enzyme-linked assay systems are described in U.S.
4,277,560, UoS~ 49281,061, and U.S. 4,233,402. An article by 8.R.
Clark and E. Engvall entitled Enzyme Linked Immunosorbent Assay (ELISA): Theoretical and Practical Aspects in "Enzyme-Immunoassay", 25 E.T. Maggio, ed., CRC Press, Inc., Boca Raton, FL (1980), pp. 167-179, describes the basic aspects of enzyme linked immunoassays.
The present invention provides an enzyme-linked assay system wherein the enzyme is bacterial luciferase.
5acterial luciferase is a flavin-linked monooxygenase (hydroxyl-ase) which catalyzes the bioluminescent oxidation by 2 Of reducedflavin mononucleotide and a long-chain fatty aldehyde depicted below to yield fMN, the corresponding fatty acid, blue-green light and water (M.M. Ziegler and T.O. Baldwin, Current Topics in Bioenergetics, Vol.
12, pp. 65-113 (1981))~
FMNH2 RCHO + 2 luciferase > fMN + RCOOH H20 + blue-green light The luciferase protein is an B dimer with a single active center con-_ 3 3~

fined primarily, if not exclusively, to the a subunit. The precise role of the subunit i9 not clearly understood, but it is required for bioluminescence activity. Detailed chemical modification studies reported in a series of paper during the past ten year show that the lucifera~e of the luminous marine bacterium Vibrio harveyi possesses one particularly reactive sulfhydryl (cysteinyl) group zone of about 15) that is located on the a subunit in or near the active center which when modified with any of a variety of reagent renders the enzyme completely inactive (Ziegler and Baldwin, 1981, ibid.).
This reactive cy~teinyl residue resides in a hydrophobic cleft (M.Z. Nicoli and J.W. Hastings, J. Biol. Chem. 249, 2393-2396 ~1974) and Merritt and Baldwin, Arch. Biochem. Biophys. 202, 499-506 (1980). The apparent second order rate constant for the inactivation of luciferase with a series of N,n-alkyl maleimides shows a marked chaln length effect, apparently due to binding of the hydrophobic alkyl chain in the hydrophobic cleft prior to covalent reaction with the reactive cysteinyl re3idue.
Another class of compounds, the alkylalkanethiol-~ulfonates, has been used to modify the luciferase. Two interesting ob~ervation3 came from these studies. First, modiication o the reactive thiol with a small a group as the -SCH3 rendered the enzyme inactive (M.M~ Ziegler and TØ Baldwin (1981) in "Bioluminescence and Chemilu~inescence:
Basic Chemistry and Analytical Applications" (M.~. DeLuca and rm~

2~3~
- 3a -W.D. McElroy, eds.) Academic Press, New York, pp. 155-160).
Second, the mixed disulfide that results from reaction between luciferase and the thiol~ulfonate was readily reduced by ~-mercaptoethanol, dithiothreitol, or other reducing agent resulting in quantitative recovery of bioluminescence activity (W.R. Welche3 and Tao Baldwin, Biochemistry 20, 512-517 (1981))o These characteristic3 of bacterial luciferase render the enzyme uniquely adaptable for the present invention.

SUMMARY OF THE INVENTION
The present divisional application claim linker-compound of general formula II, as described below, and a linker-compound-ligand or a linker-compound-receptor derived from the linker-compounds~
The pre3ent inventlon iR a method of assaying for substances using an enzyme-linked assay system wherein the enzyme is bacterial luciferase. Bacterial luciferase can be used as a label or as an analytically indicatable group in assaying for substances by substantially any of the known assay methodologies utilizing reciprocal binding pair members which exhibit bio~pecific affinity for one another The present invention is unique from other known enzyme linked rm/~

3 3~

-4- 3972 immunosorbent assays involving biospeci-Fic affinity reactions ox reciprocal binding pair members in that the enzyme, i.e., bacterial luciferase, i5 linked to one o-F said binding pair members in such a manner that the enzyme is co~lpletely inactivated, and following the various biospecific affinity reactions involved in the assay the enzyme activity is completely restored upon breaking the link enabling one to mPasure said enzyme activity for assay purposes. The bacterial luciferase is linked to one of said reciprocal binding pair members by a splittable bond of a covalent nature.
For purposes of convenience the reciprocal binding pair members are referred to herein as ligand or ligands, i.e., the substance being measured or assayed, and receptor or receptors, i.e., the compound or substance having a binding aFfinity for a liqand. Although the terms ligand and receptor are used herein in the capacity just deFined it is understood that in some instances the substance to be assayed or measured i.e., the ligand, is in fact a receptorO The label, i.e., bacterial luciferase, is reversibly linked to either the ligand or the receptor, forming a luciferase labeled ligand or a luciferase labeled receptor, and in the performance of the assay changes in the number of quanta of light emitted by the enzyme provides a means of measuring levels of substances being assayed. Typically the assay is carried out in any of a variety of well-known manners such as competitive a-Ffinity binding, displacement or disequilibration or using immunomet-ric procedures.
Thus, the present invention provides a method For quantitating the presence of a substance, herein referred to as ligand, in a medium which comprises incubating said medium, a known amount of a luciferase labeled ligand, and a known amount of a receptor capable of binding said ligand and luciferase labeled ligand; separating the receptor-bound material From the unbound material; activating the luciferase in the receptor-bound material and/or unbound material; and measuring the luci-Ferase activity. This assay procedure can be carried out in either the solid or liquid phase, i.e., either one oF the binding pairs may be immobilized by being affixed Jo a solid surface such as beads or a test tube, or all components may remain in solution. When performing the assay in the solid phase the binding pair member which is immobilized will be the unlabeled binding pair member. This competitive type assay may be modified such that the receptor carries l ~33~,

-5- 3972 the label, thus the assay medium comprises the sample or unknown medium being assayed, a known amount of luciferase labeled receptor, and a known amount ox immobilized ligand which is the same ligand as that which is being assayed. Additionally, the competitive type assay S may be performed by incubating the medium containing the sample being assayed; a known amount of immobilized ligand, and a known amount of unlabeled receptor capable of binding said ligand. Following incuba-tion the solid or immobilized phase is separated from the liquid phase and a second receptor carrying the luciferase label is added to the immobilized phaseO The second receptor is capable of binding the first or unlabeled receptor. The medium containing the second recep tor is incubated then the phases are separated, the luciferase acti-vated and enzyme activity measured.
Further, the present invention provides a method for quantitating the presence of a ligand in a medium which comprises combining said medium with an equilibrated composition comprising binding equivalent quantities ox a known amount of luciferase labeled ligand and a known amount of a receptor capable of binding said ligand and said lucifer-ase labeled ligand; incubating the resulting combination; separating the receptor-bound material from the unbound material; activating the luciferase in the receptor-bound material and/or the unbound material;
and measuring the luciferase activity. This displacement type assay may also be carried out in such a manner that the receptor is either immobilized or remains soluble during incubation.
This invention also provides a means of carrying out an immuno-metric assay for determining the presence of a ligand in a medium which assay may be a two-site or a single-site type assay. Thus there is provided a means for determining the presence of a ligand in a medium which comprises incubating a known amount of a luciferase labeled receptor capable of binding said ligand and an equilibrated composition comprising said medium and a known amount of an unlabeled receptor capable ox binding said ligand; separating the bound lucifer-ase labeled receptor material from the unbound luciferase labeled receptor material; activating the luciferase in the bound material or the unbound material; and measuring the luciferase activity. The unlabeled receptor is the same as the receptor contained in the luci-ferase receptor complex. This assay also may be carried out in a solid or liquid phase, i.e., the unlabeled receptor is either immobi-l .3~

-6- 3972 lized or remains soluble in the assay medium.
This two-site immunome~ric type assay may be modified by using a second receptorO Thus there is provided a means for determining the presence of a ligand in a medium which comprises incubating said medium and a known amount of an immobilized first receptor capable of binding said ligand; adding excess of a second receptor capable of binding said ligand said second receptor being from an animal species different from the animal species in which the first receptor is elicited, and incubating the resultant composition; separating the immobilized phase from the liquid phase; adding a known amount of a luciferase labeled receptor capable of binding said second receptor and incubating the resultant composition; separating the immobilized phase from the liquid phase; activating the luciferase in the immobi-lized phase or the liquid phase; and measuring the luciferase activ-ity.
The above-described two-site immunometric assays are employed when assaying for ligands which have more than one antigenic determin-ant The single-site immunometric type assay can be performed in two different manners. Illustratively, the medium containing the ligand to be assayed is incubated with a known amount of an immobilized receptor capable of binding said ligand after which the immobilized phase is separated from the liquid phase. A known amount of a luci-ferase labeled ligand is then added to the immobilized phase and the resultant composition is incubated. Following the second incubation the immobilized phase is separated from the liquid phase, the lucifer-ase is activated in one or the other or both ox these phases, and the enzyme activity is measured. Alternatively, there is incubated a known amount of an immobilized ligand and an equilibrated mixture of said medium containing the ligand to be assayed and a known amount of a luciferase labeled receptor capable of binding said ligand. Follow-ing incubation the immobilized and liquid phases are separated, the luciferase in either the immobilized or liquid phase (or both) is activated, and the enzyme activity is measured.
In the foregoing description of immunoassay procedures in any particular procedure when reference is made to a known amount of a ligand or a luciferase labeled ligand, that phase means the purified form (either unlabeled or luciferase labeled) of the substance, i.e., ~l~F~ L~3~

ligand, being assayed.
This invention also provides a mercantile kit containing reagents useful in performing assays of the present invention which comprises multiple containers wherein one of said containers has therein luci-ferase labeled ligand said ligand of which is a purified form of theligand to be assayed, and another of said containers has therein a receptor capable of binding said ligand which receptor optionally may be immobilized. Also there is provided a mercantile kit useful in performing immunoassays of the present invention which comprises mul-tiple containers wherein one of said containers has therein luciferaselabeled receptor said receptor being capable of binding the ligand to be assayed, and another of said containers has therein either an immo-bilized ligand said immobilized ligand being a purified form of the ligand to be assayed, or has therein unlabeled receptor capable of binding said ligand to be assayed. There is further provided a mer-cantile kit useful in the performance of immunoassays of the present invention which comprises multiple containers wherein one of said con-tainers has therein a quantity of immobilized ligand said immobilized ligand being a purified form of the ligand to be assayed, another of said containers has therein a quantity of unlabeled receptor capable of binding said ligand, and another of said containers has therein a quantity of luciferase labeled receptor said receptor being capable of binding the unlabeled receptor. Additionally there is provided a mer-cantile kit useful in performing assays of the present invention which comprises multiple containers one of said containers having therein a quantity of immobilized receptor capable of binding the ligand to be assayed9 another of said containers has therein a second receptor capable of binding the ligand to be assayed said second receptor being from an animal species different from the animal species in which the immobilized receptor is elicited, and another of said containers has therein a quantity of luciferase labeled receptor said receptor being capable of binding sa;d second receptor.
DETAILED DESCRIPTION OF INVENTION
In practicing the present invention the bacterial lucif~rase is reversibly linked to either the ligand or the receptor to form a luci-ferase labeled ligand or a luciferase labeled receptor. Any reference herein to luciferase means bacterial luciferase. Bacterial luciferase from any species capahle of producing said enzyme may be employed, 3~

e.g., luninous bacteria or any mutant thereof.
In forming the l uciferase labeled ligand or the l uciferase labeled receptor, the ligand or receptor is reacted with a bi~unc~
tional linker compound to give a linker compound-ligand intermediate 5 or a linker comFound-receptor intennediate. The thus formed intermed-iates are brought together and bound to bacterial l uciferase by a reversible or cleavable covalent linkage. Th;s covalent binding of the intermediates and bacterial luciferase renders the enzyme inactive while the ligand or receptor is available to bind its counterpart.
10 Upon cleaving the covalent linkage the luciferase activity is restored and the concentration of ligand bei ng assayed can be determined by measuring the luminescence.
The substances to be measured or detected in the performance of the assay, i.e., the ligand, includes those substances which are anti-15 genic or can be rendered antigenic, i.e., haptens, or which have natu-rally occurring receptors. Reference is made to U.S. patent 3,817,837 wherein the foregoing categories of ligands are defined and exempli-vied. The ligands to be assayed can be substantially any of the sub-stances which are recognized in the art as being detectable by assay 20 procedures known heretofore or for which a receptor naturally exists or can be prepared. Thus, the ligands to be assayed by the methods of the present i nvention incl ude, for example, those substances described in U.S. patents, 3,817,837 (columns 6-26); 4,039,385 columns 1~3);
4,108,975 (column 5); 4,191,613 (columns 4-8); 4,235,960 (columns 25 3-4); and 4,233,40Z (col umns 10-16). It is necessary, however, that the ligand be capable of, or can be modified to render it capable of, reacting with the bifunctional linker compounds. More specifically, the ligands to be assayed include steroids, such as dihydrotestoster-one, aldosterone, estradiol, estrone, estriol, dehydroepiandroster-30 one-S (DHEA-S), cortisol, corticosterone, deoxycortisol, deoxycortico-sterone, progesterone, pregnanediol, male testosterone, female test-osterone, androstenedione, and 17-hydroxyprogesterone; cardiac glyco-sides, such as, digitoxin, digoxin and gitalin; cannabinoids, such as, tetrahydrocannabinol s; opi2tes incl udi ng morphine and thebaine; pe p-35 tide hormones, such as, leutinizing hormone, follicle stimulating hor-mone, thyroid stimulating hormone, human growth hormone, human growth factor ACTH, glucagon, insulin, human placental lactogen, prolactin, human chorionic gonadotropin, gastrins, C-peptide of insulin, folate, l 33~
_ 3972 intact parathyroid stimulating hormone or the C~terminal thereon, and N-cholylglycine, prostaglandins and related substances, such as, PGA1, PGA2, PGD2, PGE1, PGE2, PGF1, PGF2, thromboxane B2~ 6-keto PGF1al the 6,15-diketo-dinor derivative of PGF1, PGF2, PGE2 and PGE1, the 13,14-dihydro-15-keto derivative of PGF1, PGF2, PGE1 and PGE2; and bicyclic forms of the 13,14-dihydro derivative ox 15-keto-PGE2 and 15-keto-PGF2a; vitamins, such as, vitamin B-12, folic acid, and vitamin A;
neurotransmitters or bioactiYe amines, such as, norepinephrine, dopa-mine, and epinephrine; nuoleic acids; tumor markers, such as, alpha fetoprotein, carcinoembryonic antigen (CEA), and prostatic acid phos-phatase; drugs, such as, acetomenophen, N-acetylprocainamide, amika-cin, acetazolamide, amobarbitol, butabarbitol, chloramphenicol, car-isoprodol, carbamazepine, chlorazepate, disopyramide, diazepam, diox-epin, ethosuximide, ethclorvynol, gentamicin, glutethimide, kanamycin, lidocaine, librium, meprobamate, methaqualone, methpyrlon, mepheny-toin, norpropoxyphene, phenobarbital, phenytoin, procainamide, primid-one, pentobarbitol, quinidine, secobarbitol, theophylline, tobramycin, thoridazine, valproic acid, vetilmicin, imipramine, amitriptyline, desipramine, nortriptyline, propranolol, thorazine, fluorazepam, clon-azepam, alprazolam, valium and propoxyphene; proteins, such as,thyroid binding globulin, ferritin, myoglobin, and thyroglobulin, IgG, IgA, IgM, IgE, antitrypsin, rheumatoid factor, factor YIII, myelin basic protein, cross reactive protein, complement factors C3, CL~ and activated complement components C3a, C4a and Csa, enzymes, such as, renin, angiotensin I, malic dehydrogenase, pyruvic kinase, glucose 6-phosphate dehydrogenase, lactic dehydrogenase, creatine phosphokin-ase, and pepsinogen; nonsteroidal hormones, such as, thyroxine-4 (T4) and 3,5,3-triiodothyronine; and chemical mediators such as, cyclic AMP; viral antigens resulting in, e.g., herpes simplex, hepatitis B, rubella, and rabies as well as antibodies to such antigens; antibac-terial antibodies, such as, an~igonococcus; parasite antigens result-ing in toxoplasmosis, malaria, schistosomiasis, trypanosomiasis and syphilis and antibodies thereto.
The term receptors as used herein includes solution receptors found or generated in the plasma or cytoplasm such as antibodies, which may be naturally occurring or induced by well known procedures, cytosol, testosterone binding globulin (TEBG), trans cortin, or enzymes. Also the term receptors includes cell-bound receptors such g y S"!~
l 33 as those for acetylcholine, catecholamines, insulin, estrogen, proges-terone, testosterone and T-cell and B cell markers. The fo~nation and isolation of the receptors is well known in the art, e.g., see E.V.
Jensen, et alp, Receptors for Reproductive Hormones (B.W. O'Malley, A.R. Means, eds.), p. 60, Plenum Press, New York, London, 1973;
W.I.P. Mainwarning, et al., ibid., p. 197; G.E. Block, et al., Ann.
Surg. 18?, 342, 1975, F. Suzuki, et al., Endocrinology, , 1220 (1972); R. E. Cone, "The Search for the T Cell Antigen Receptor,"
Progress in Immunology III, Australian Acad. of Sci., pp. 47-57, 1977.
The linker compounds employed in forming the luciferase labeled ligand or the luciferase labeled receptor can be any bifunctional ccmFound which contains as one of the functional moieties a reactive group such as carboxyl or a reactive derivative thereof or an amino group or other reactive moiety designed to react with the ligand or receptor and contains as the other functional moiety a sulfide or sulfoxide group which will react with the reactive sul~hydryl of luciferase. The linker compound is designed to deliver the ligand or receptor to the active sulfhydryl of luciferase, and hold the ligand or receptor in position to achieve recognition and binding by its counterpart then upon appropriate treatment release the luciferase.
Particularly useful as linker compounds are the compounds of Formulas I and II depicted in the Formula Chart.
The linker compounds are of two classes represented by Formulas I
and II. In Formulas I and II Rl is any group which will render the sulfur to which it is attached electron deficlent, i.e., R1 is an electron withdrawing group such as 2-benzothiazolyl, 2-pyridyl, 4-. .
pyridyl, ~-nitro-2-pyridyl, 2-pyridyl-N-oxide, or a carbonate, i.e., o -c-O-R8 wherein R8 is any ester forming group such as lower alkyl or benzyl or phenethyl.
R7 can be substantially any group which will not interfere with the reaction of the sulfoxide compound with luciferase; typically R7 is a lower C1-6 alkyl group, ego methyl or ethyl or an aromatic group such as phenyl, substituted phenyl such as fluorophenyl or p-nitrophenyl or lower C1-4 alkyl substituted phenyl 2~ or 4-pyridyl;
n is zero to 15, preferably zero to 4; R6 is COOH, COOsuccinimide;
-COCl; -COBr; Cl; Br; SCN; NH2;

3~S~

NH2 Cl- NH2 Cl -C-OCH3; or C-OCH3.

R2 and R3 are the same and are hydrogen3 methyl, or ethyl;
R4 and Rs are the same and are hydrogen, methyl, or ethyl, or R2, R3, R4 and Rs taken together with the carbon atoms to which they are attached represent a cycloalkyl group having from 4 to 6 carbon atoms, or represent 1,4-phenylene.
The compounds of general Formula I are known in the art or are prepared by procedures well known in the art as set forth, for exam-ple, in U.S. patents 4~149~003; 4,232,119; 4~175~073; 4~258~193; and 4~187~345 as well as S.J. Brois, et al., J. Am Chem. Soc. 92, 7629-7631 (1970~; J.E. Dunbar and J.H. Rogers, J. Org. Chem. 31, 2842-2846 (1966); and L. Field and P.M. Giles, J. Org. Chem., 309-313 (1971)o The compounds of Formula II wherein R7 is methyl or p-methyl phenyl, n is zero each of R2, R3, I, and R5 is hydrogen R6 is NH2 are known in the art. The compounds of Formula II other than the two aforedescribed compounds are a part of the present invention.
The compounds of Formula II are prepared by procedures generally 20 known in the art. Illustratively, a derivative of the formula R,SO2SK
prepared as generally described by Boldyner and Zakharchuk, Dolk.
Akad. Naak. SSR 95, 877 (1954) is reacted with a compound of Formula III wherein n, R2, R3, and R5 have the meanings defined in Formula II and R~ is -COOH, Cl, Br, SCN, NH2, CN or 4-cyanophenyl, by the general procedure described by Johnston and Gallagher, J. Org. Chem.
26, 37~0 (1961). The nitrile derivatives obtained by the foregoing are used to prepare the carboxy imidates of Formula II by the general procedure described in U.S. patent 4,237,267. The nitrile intermedi-ates are also a part of the present invention. The compounds of For-mulas I and II wherein R6 is -COO-succinimido are prepared by treating the corresponding carboxylic acid with N-hydroxysuccinimide as gener-ally described, for example, in U.S. 4,237,267. Compounds of Formulas I and II wherein R6 is -COCl or -COBr are prepared by, e.g., treatment of the corresponding carboxyl derivative with thionyl chloride or thionyl bromide by procedures known in the art.
The compounds of Formula III are known in the art or are prepared by procedures generally known in the art.
The compounds of Formulas I and II wherein R6 is -COO or NH2 are preferred for use in preparing the luciferase labeled ligand and luci ferase labeled receptor of the present inventionr The bacterial luciferase employed in the present invention is isolated and purified by means known in the art, e.g., as described by Holanan and Baldwin, Biophysical Journal 33, 255 (1981).
In preparing the luciferase labeled ligand and the luciferase labeled receptor the ligand or the receptor is brought together with a linker compound of Formula I or II to effect a reaction between the group designated R6 of said compound and the ligand or receptor. The linker compound chosen depends on the nature of the reactive function present on the ligand or receptor. If the ligand or receptor does not have present thereon a suitable function for reaction with the R6 group of the com unds of Formula I or II then such function is intro-duced by various procedures known in the art. Also if the ligand or receptor to be assayed or to be labeled with bacterial luciferase contains any reactive sulfhydryl groups it is important that they be blocked prior to reaction with a compound of Formula I or II, or assaying, by treatment, for example, with iodoacetamide or N-ethyl-maleimide.
It is apparent that the compounds of Formulas I and II will react with a variety of functional groups commonly present on, or which can be introduced into, the ligands or receptors to be labeled. Thus 9 compounds of Formula I or II wherein R6 is a carboxyl, a carboxysuc-cinimide, or an acyl chloride or acyl bromide group will react with primary amine groups forming an amide linkage. See, J.C. Sheehan and G.P. Hess, J7 Am. Chem. Soc. 77, 1067 (1955~; N.F. Albertson, Organic Reactions 12, 205 (1962); R. Paul and G.W. Anderson, J. Org. Chem. 27, 2094-2099 (1962); J.C. Sheehan amd PA Cruickshank, J. Orgy Chem. 26, 2525 (1961); J.C. Sheehan, et al., J. em. Chem. Soc. 87, 2492 (196~).
Compounds of Formulas I and II wherein R6 is Br or Cl will react with primary or secondary amines present on the ligand or receptor to be labeled forming an alkyl amine linkage. Also compounds of Formulas I
and II wherein R6 is Br or Cl will react with carboxyl groups present on the ligand or receptor to form an ester linkage. Also a compound wherein R6 is -COOH will react with a chlorine or bromine moiety which may be present on a ligand to form an ester linkage. Compounds of Formulas I and II wherein R6 is SCN are useful in reacting with amine groups which may be present on the substance to be labeled forming a l .3 3~

thiocarbonate linkage. Compounds of Formulas I and II wherein R6 is NH2 are useful in reacting with carboxyl groups present on the ligand or receptor forming an amide bond. Also, compounds of Formulas I and II wherein R6 is NH2 Jill react with aldehydes present on the ligand or receptor undergoing a Schiff base formation. Compounds of Formulas I and II wherein R6 is a carboxymethoximc or a phenylcarboxymethoxime group are useful in reacting with amine groups present on the ligand or receptor to be labeled forming an amînoimidate linkage. Compounds of Formulas I and II wherein R6 is NH2 and each of R2, R3, and Rs is hydrogen tend to decompose after about three days at room tempera-ture. Therefore, these compounds should be used preferably within a day or two following preparation.
Many ligands or receptors to be labeled will contain a suitable functional group capable of reacting with the moiety of the com-pounds of Formulas I and II. For example, protein and peptide typeligands or receptors will contain amine and/or carboxy groups suitable for reaction with compounds of Formulas I and II. Prostaglandins generally will contain a carboxy group suitable for reaction or can be derivatized to render said compound suitable for reaction by means known in the art. Either the carboxyl or amine present on thyroxine and triodothyronine are suitable for reacting with compounds of Formu-las I and II. Many drugs, e.g., N-acetylprocainamide, amibacin, car-isoprodol, carbamazepine, gentamicin, elipten, kanamycine, meprobam-ate, desipramine, valproic acid, chlorazepate, ethosoximide, propran~
olol, etc., will contain amine, carboxyl or other groups which can be utilized in producing the linker compound-ligand intermediate. Other ligands will require some modification. For example steroids and car-diac glycosides containing hydroxyl groups can be treated with phos gene to give a chlorocarbonate which can be reacted with an amine of Formula I or II; or the hydroxyl can be derivatized using succinic anhydride to give an acid moiety suitable for reaction. Hydroxymethyl groups present in, e.g., corticosteroids, can be used to form hemisuc-cinates suitable for reaction. Also carbonyl moieties present at var-ious ring positions can be derivatized using O-(carboxymethyl)hydrox-ylamine as described by B. Erlanger, et al., J. Biol. Chem. 228, 713(1957). Hydroxyl groups present in cannaboids and various drugs, e.g., chloramphenicol, can be utilized in a manner similar to that described for steroids to give a suitably derivatized ligandO Hydr-g ox~l gruups can be introduced into aliphatic chains (see Chinn, "Sel-ection of Oxidants in Synthesis," pp. 7-11, Marcel Cekker, Inc., New ~ork~ 1971 and Let, in Augustine9 "Qxidation," vol. 1, pp. 2-6, Marcel Dekker, Inc., New York, 1969) then derivatized as described above which provides a means of derivatizing~ e.g., barbiturates such as pentobarbital and secobarbital, and retaining the immunogenicity of the ligand. Conpounds such as diazepam, methaqualone, mephenytoin, reticillin, norpropoxyphene, phenobarbital 9 and pyrimidone can be derivatized by introducing a nitro group into an aromatic ring thereof ~0 then either reducing the nitro group to an amine or oxidizing the nitro group to a carboxyl group via the nitrile by means generally known in the art. Of course compounds already containing nitro groups, e.g9, nitroazepam, could be reduced to amines to provide a suitable coupling moiety. Ligands containing carbohydrate moieties can be treated with periodate oxidizing the ring hydroxyls to alde-hydes which can be reacted with amines of Formulas I and II by Schiff base condensation. Also, treatment with epichlorohydrin Jill give an epoxide derivative suitable for reacting with a diamine. See R. oxen, et al., J. Acta Chem. Scand., B-29, 471 (1975). The thus formed primary amine function can be utilized to link the ligand to a suitable compound of Formula I or II. Various other means suitable for introducing suitable reactive functions into ligands will be apparent to those skilled in the art.
In reacting the ligand or receptor with the linker compound the quantity of reactants employed will vary depending on the nature of the receptor or ligand and the number of reactive functions present thereon. It is apparent that in some instances more than one linker compound will bind to the ligand or receptor. A sufficient quantity of linker compound is employed to bind all or substantially all of the reactive functions ox the ligand or receptor. For protein or other ligand or receptor for which structure is unknown one can titrate for sulfhydryl groups to determine the optimum ligand (receptor)/linker compound ratio. Specific examples set forth below illustrate further the coupling of linker compounds of Fonnulas I and II with ligands or receptors to form the appropriate intermediates represented by For-mulas IV and V suitable for reaction with bacterial luciferaseO
In the compounds of Formulas IV(a) and IV(b), R1, R2, R3, R~, R5, R7 and n have the meanings defined in Formulas I and II; m is an lnteger equivalent to the number of reactive functions present on the ligand or receptor capable of reacting with the linker compound and preferably is an integer of from 1 to 40, Y represents the ligand or receptor to be labeled or assayed absent the functional group which reacted with the linker compound; and X represents the functional linkage between the linker compound and the ligand or receptor and is o -CNH-; -NR- wherein R is hydrogen or any group which may be present on the ligand or receptor functional secondary amine, 10 o o s o o NH2 Il 11 11 11 11 11 -OC-, -CO-; -NCNH-; -NHC-; ~C---NH~; or -O-NH-. The intermediates of Fonmula IV(b) are a part of the present invention.
Once the linker compound-ligand and the linker ccmpound-receptor intermediates are formed said intenmediates are brought together with bacterial luciferase to effect thiolalkylation of the reactive sulf-hydryl group of luciferase and the reactive R1S-S- or the R~S(02)S-moieties of the linker compounds of Formulas I and II respectively.
The quantity of reactants employed varies with the number of sulfide or sulfoxide reactive groups available on intermediates and is con-trolled such that each such group binds a luciferase molecule. As the luciferase reacts with the linker compound-ligand or linker compound-receptor intermediate the luciferase is inactivated. Therefore it is convenient to add to an excess of luciferase in a buffer, e.g., 0.2M
phosphate buffer, pH 7.5, the intermediate of Formula IV(a) or IV(b) portionwise until there is a disappearance of enzyme activity. We have found that the addition ot a nonionic surfactant or-detergent such as polysorbate 80 or polyethylene glycol p-isoacetylenephenyl ether to the reaction medium may improve the assay sensitivity. This is believed to be related to the hydrophobicity o-f the region of the luciferase molecule wherein the reactive sulfhydryl group resides. As the carbon chain length of the linker compound increases the need to add a surfactant generally decreases, however, the presence of a non-ionic surfactant in the reaction medium wherein any of the intermedi-ates of Formula IV(a) or IV(b) is not detrimental. A final concentra-tion of about 0.05~ to 0.5%, preferably 0.05% to 0.1%, of surfactant in the reaction mixture is suitable. The luciferase labeled ligand or luciferase labeled receptor as depicted by Formula V may be separated 3~

from any unreacted intermediate by various known techniques, e.g., molecular seive solumn chromatography or ultrafiltration. In Formula V, R2, R3, R~, R5, n, m, X and Y are as defined in Formulas IV(a) and (b) and E is as depicted below in Formula VII.
In performing the assay the incubation medium containing the sam-ple being assayed must be free of any reducing agent or must be deter-mined and corrected for by using, e.g., methyl methanethiolsulfonate labeled luci~erase in a control sample. If the free sulfhydryl leYels in the sample to be tested are very high and producing a high back-ground it may be advantageous to add a reducing agent scavenger to the test sample prior to assaying.
The assay incubation medium is buffered to a pH of about 6 to 9.5, and the buffer ideally contains a high concentration of anions such as phosphate, arsenate, citrate, sulfate, pyrophosphate. Usually the incubation medium will contain a high concentration of protein but if not protein, such as, bovine serum albumen (BSA)) s-hould be added. Generally 0.1 mg of BSA per ml of incubation medium is ade-quate. The temperature of the incubation medium can vary from about 0 to 40C but preferably is about room temperature, i.e., 25-30C.
The incubation period varies with the ligand being assayed but is usually less than one hour. Of course, for prolonged incubation periods antibacterial agents such as EDTA could be added to the mediumO Addition of a nonionic surfactant, e.g., as identified hereinabove, in the incubation medium may also be useful in improving the sensitivity of the assay for the reasons indicated hereinabove.
Following incubation and in those assay systems wherein either the ligand or the receptor is immobilized the incubation medium is decanted and the immobilized material is washed with a buffer solution of the type used in the incubation medium. In those assay systems wherein the ligand and receptor remain soluble the receptor-bound material is separated frcm the unbound material by various means commonly known in the art. For example, this separation can be achieved by treatment with polyethylene glycol [B. Desbuquois and G.D.
Aurback, J. Clin. Endocrinol. Metab. 33, 732 (1971)] or IgG Sorb or by contacting the incubate with a second antibody. The second antibody9 which is prepared by standard procedures, e.g., as described by Doughaday, et al., "Principles of Competitive Protein Binding Assay,"
J.B. Lippincott, Philadelphia (1971), is particularly preferred.O

l 3~

In those assay systems wherein more than one receptor is employed the conditions of the incubation medium and the separation techniques are substantially the same as described above with the additional receptors being added sequentially and at time intervals Jo permit binding of the various components involved.
Once the bound material is separated it is combined with a buffer of the type used in the incubation medium along with a sulfide reduc ing agent, such as, 0.1M ~-mercaptoethanol, 0.01 to 0.05M dithiothrei-tol, 0.01 to 0.05M dithioerythritol or sodium dithionate, and protein (no protease) such as BSA. The time required for reduction and recovery of active luciferase can be standardized for each ligand, and generally will be from one to 60 minutes. The luciferase is then activated by any one of the various known techniques, ego the dithionate method; or the flavin squirt or injection technique (J.W.
Hastings, et al., Methods Enzymol. 57, 135-152 (1978); or the coupled assaying flavin reductase method (E. Jablonki and M. Deluca, Methods Enzymolr 57, 202-214 (1978); and P.E. Stanley, Methods Enzymol. 57, 215-222 (1978~.
The light emitted by the luciferase can be measured by using a luminometer, a photomultiplier photometer, or a liquid scintillation counter, and by comparison to standard curves for known quantities of ligand or receptor the concentration of substance being measured in the sample is determined. Standard curves are generated by the foregoing procedure using known quantities of ligand or receptor.
The following represent preferred representative procedures for performing the immunoassay of the present invention.
Procedure I: Double antibody method A. Immunoassay: A prepared sample (0.1 ml) is incubated with 0.1 ml of a first antibody or receptor and luciferase-labeled ligand (0.1 ml) for a period of time ranging from 5 to 20 minutes depending on the affinity oF the receptor. Following incubation, 0.1 ml of a second antibody or receptor is added and the medium is incubated for an additional 30 minutes after which 2 ml of saline solution is added, and the mixture is centrifuged at 3000 G for 5 minutes. The pellet is resuspended in 0.94 ml dit~iothreitol (20 mM) containing assay buffer (0.02 M phosphate buffer, pH 7.0, with 0.2% bovine serum albumin and 20 mM dithiothreitol) and incubated for 30 minutes.
B. Detection of the bound ligand: Any one of the three methods to detect the luciferase activity as described hereinabove can be used, for example, using the methods described by J.W. Hastings, et al., Methods Enzymol. 57, 135-152 ~1978). The unknowns are estimated against a standard curve which is established using the identical assay.
Procedure II: Solid phase method The receptor can be attached onto a solid matrix, such as, Staph-~lococci aureus, microbeads, polyethylene/polystyrene tubes, by vari-ous known methods. See, Clark and Engvall, ibid.; Sheehan and Cruick-shank, ibid.; and E. O'Keefe and R. Vann, J. Biol. Chem. 255, 561-5fi~
(1980). The immunoassay procedures are designed according to the nature of solid matrix-receptor complex.
Ao Receptor-coated beads or S. aureus 1. Immunoassay: 0.1 ml of prepared sample, 0.1 ml of luci-ferase labeled ligand, and 0.1 ml of the receptor-coated beads are incubated for a period of time as described above. At the end of the incubation, 2.0 ml of saline solution is added and then centrifuged at 3000 G for 15 minutes, The pellet is resuspended in the buffer and incubated as described above. Then the resuspension is centrifuged at 3000 G for 10 minutes and 0.5 ml of the supernatant is transferred to a new assay tube for detection.
20 Detection of bound lisand: 0.45 ml of the assay buffer is added to the 0.5 ml supernatant and the activity is detected as described above in Procedure I.
8. Receptor-coated tubes 1. Immunoassay: 0.1 ml of prepared sample, 0.1 ml of luci-ferase labeled ligand, and 0.3 ml of phosphate buffered saline ~solu-tion are incubated for a period of time as described above. At the end of the incubation, 2.8 ml of saline solution is addedg and the whole mixture is decanted, after which the assay buffer as described above is added and incubated. detection of bound antigen is carried out as described above.
The following specific examples further illustrate the invention.

~2~3q~

Example 1 Potassium methyl sulfonate Hydrogen sulfide gas was passed through a stirred solution of 10 y (150 mm) of potassium hydroxide in 80 ml H20, cooled in an iee bath until the solution was saturated. While cooling in ice bath, there was added very 510wly (syringe pump) over one hour 5.8 ml methane sulfonyl chloride (75 mm). Stirring was continued for another hour, then the mixture was filtered and evaporated to dryness under reduced pressure9 To the resultant residue was added 25 ml dimethyl formamide and the mixture was warmed to 60 C with stirring for about 45 minutes under a nitrogen atmosphere. The mixture was then filtered, washed with DMF and dried under reduced pressure to give the title compound which was recrystallized from isopropyl alcohol.
Exame~e 2 3-(Methylsulfonylthio)propionic acid One 9 (6.6 em) of homopropionic acid in 10 ml DMF was treated with 2.0 9 (13 mm) of CH3SO2SK with stirring under No in a 60C oil bath for 4 hours. Upon cooling the mixture W2S diluted with H20, acidified in 2N KHS04, extracted with ethyl acetate, washed sequentially with ice cold KHS04, H20, then brine, dried over Na2SO4, and evaporated to give the title compound.
Example 3 3-(Methylsulfonylthio)propylamine HBr 1.74 9 of 3-homopropylamine-HBr and 1.35 9 of CH3~02SK in 5 ml DMF was stirred under N2 at 60C for 3 hours, then filtered through Celite, washed in more DMF and evaporated using a viscous oil. The oil was combined with 7 ml ox 1:1 acetonitrile-ether and stirred for one hour. The resulting precipitate was washed with ether, dried, dissolved in hot acetonitrile, filtered and cooled. Upon cooling a precipitate formed which was recrystallized from methanol-ethylacetate to give the title compoundO
Example 4 5-(Methylsulfonylthio)pentanoic acid A mixture of 19 of 5-bromovaleric acid, 1 9 of CH3SO2SK and 10 ml DMF was stirred under N2 at 60C for 3 hours. The mixture was then filtered, washed with DMF, and evaporated under reduced pressure to dry. The resulting residue was chromatographed on 100 9 Cc4 packed in 50% EtOAc-Skellysolve B eluting with 1 l 100% EtOAc-Skellysolve B to give the title compound. M.P. 69-71C.
When in the above procedure 6-bromohexanoic acid or 4-bromobutan-oic acid is substituted for 5-bromovaleric acid one obtains 6-(methyl-sulfonylthio)hexanoic acid, M.P. 71-76C, and 4-(methylsulfonylthio)-3~

butanoic acid respectively.
Ex?mple 5 2-(Methylsulfonylthio)ethylamine-HBr A mixture of 1.63 9 of 2 bromoethylamine hydrobrcmide and 1.35 9 of CH3SO2SK in 5 ml DMF was stirred under Nz at 60C for 3 hours. The 5 mixture was then filtered through Celite* washed with DMF, and evapor-ated under reduced pressure to give an oil. The oil was combined with about 7 ml of 1:1 acetonitrile-ether solution and stirred for one hour then chromatographed on 300 9 CC4 packed in 10X MeOH-EtOAc, and eluted with 3 l 10-50/5 MeOH-EtOAc to give the title compound. M. P.
lO 109-114O. When in the foregoing procedure one substitutes 3-bromo-propylamine-HBr for 2-bromoethylamine HBr, the product obtained is 2-(methyl sulfonylthio) propylamine-HBr.
Example 6 (a) Estriol-6- (O)-carboxymethyl oxime (E3-CMO) is prepared from 15 estriol (1,3,5-estratrien-3,16~,17~-triol) and carboxymethyloxime by known procedures. See F. Knhen, et al., "Preparation of Antigenic Steroid-Protein Conjugates" in Steroid Immunoassay, E.D.H. Cameron, S.CO Hiller, and K. Griffiths, eds., Alpha Omega Publishing Ltd., Cardiff (1975), pp. 11-32.
A solution of 5 mg of E3-CMO in 0.2 ml of tetrahydrofuran is reacted with 2.5 mg of carbonyldiimidazole for 30 minutes at about 25C after which 3.85 mg of 5-(methylsulfonylthio)pentylamine is added. The pH of the reaction mixture is adjusted to 8.0 by the addition of 0.01 ml of 1.0 M aqueous sodium hydroxide. The reaction is pennitted to proceed for 18-19 hours at about 25C, and the product is isolated by thin layer chromatography using silica gel G and a 6:4 mixture of chloroform:methanol. The UV absorbing spots were scraped from the plate, eluted with chloroform:methanol (6:4) followed by THF :ethanol (5:5), then dried under nitrogen to give estriol-6-0[5-30 (methylsulfonylthio)pentylaminocarbonyl]methoxime,, having Formula VI.
The compound was identified by NMR and quanti~ated by UV at 262 nm.
(b) A mixture of 1.0 ml of luciferase from Vibrio harveyi (2.2 x 10 6 M) having a weight extinction coefficiency f ~280%nmlo mm of 0.94, in phosphate buffer 0.1 M, pH 7.5, containing 0.8~ sodium chloride and 0.05% Tween*80 was reacted with 0.06 ml or 3.5 x 10-5 M
from 6(a) having a molar extinction coefficiency of ~621nmmm of 1.18 x 10-4 [O.D. 262 nm = 0.49 for 3.5 x 10-3 M]. The reaction was carried out at about 25C for 30 minutes after which the reaction mix-* trade mark ture was ultrafiltered. The filtrate was diluted with 4.5 ml of phos-phate buffer 0.02 M, pH 7.0~ to yield 7.7 x 10 6 M of the product of Formula VII wherein E represents luciferase absent the reactive sulf-hydryl group. When 0.1 m1 of the product of Formula VII was incubated with 1.0 ml of buffer containing 0.02 M phosphate buffer, pH 7.0, 0.2g BSA and 20 mM dithiothreitol, the recovery of light as compared with the equivalent quantity of untreated luciferase was over 90%.
Example 7 la) Triiodothyronine methyl ester 325 mg of triodothyronine was dissolved in 40 ml of dry methanol saturated with hydrogen chloride gas at room temperature. After com-plete dissolutian, the mixture was allowed to stand overnight. The triiodothyronine methyl ester HCl was precipitated by distillation under vacuum. The precipitate was filtered off, washed with alcohol and ether, then dried. The ester hydrochloride was dissolved in 5 ml of 80% ethanol and treated with 2N NaOH to neutral. Recrystallization of the ester was completed by addition of 5 to 10 ml H20 and standing at 4C. The crystals were then collected on a sintered glass funnel and dried in a dessicator under reduced pressure at 4C. The recovery was about 80%. The resulting product of the reaction was separated by thin layer chromatography on silica gel G plate (methanol:triethyl-amine = 90:10). UV absorbing material of Rf = 0.56 was collected and used as the product (b) Coupling of triiodothyronine (T3)-methyl ester to 4-(methylsulfonylthio)butanoic acid 150 mg T3-methy1 ester was neutralized with 4.0 ml of 0.133 N
NaOH containing 0.5 ml of THF:DMF mixture (1:1) (37.5 mg/m1 final concentration). A 0.03 ml (1.125 mg) portion of this mixture was introduced into a mixture containing 4-(methylsulfonylthio)butanoic acid (1.0 mg) and 1-ethyl-3,3-dimethylaminophenylcarbodiimide EDAC
(loO mg) at pH 6.0 in 0.01 M phosphate buffer. The reaction was allowed to proceed at room temperature overnight. The resulting product was used as described in part (c) below without further purification.
(c) Conjugation of product of Formula VIII with luciferase Inhibition of luciferase forming the product of Formula IX
wherein E has the meaning defined above was carried out mixing the product of Formula YIII with luciferase under the follawing condi-~L2 3~ 7~

tions. (Product of Formula VIII 3.5 x 10 5 M:luciferase 2.2 x 10 6 M
in 0.1 M, phosphate buffer, pH 7.5, with 0.8% NaCl and 0.05g Tween 80.) The reaction mixture after 30 minutes was diluted with 0.02 M
phosphate buffer at pH 7.0 to 4~5 ml.
Example 8 Immune reaction 0.5 ml rabbit anti-T3 antiserum was mixed with 10 ml of 10%
suspension of IgGsorb (S. aureus Cowan I inactivated particles in 0.9%
_ NaCl). The mixture after 2 hours at room temperature was washed with 0.g~ NaCl extensively, and reconstituted to the original volume (10 ml)O A 002 ml portion of this suspension was used as solid phase antibody suspension, to which 0.1 ml of the solution from Example 7(c) (product of Formula IX) was added and incubated at room temperature for 2 hours. The reaction mixture was centrifuged and washed with 1 ml of phosphate bl~fered saline (pH 7.5`0.01 M phosphate), and 20 mM
(final) of dithiothreitol (DTT) was added. Control assay was carried out with S. aureus beads absorbed with norma1 rabbit serum.
After reaction with dithiothreitol, the reaction mixture was centrifuged 3000 G for 5 minutes. The supernatant was assayed for the presence of luciferase as an indicator for triiodothyronine. There was a significant difference in light emission between antibody-C.
aureus particles and normal rabbit serum-S. aureus-particle. This indicates that anti-T3 recognized the product of Formula VIII.
Reaction with T3 luciferase Light emission Ra-anti T3-S. aureus 28 mN x 10 multiplication factor Normal-Ra-serun~S. aureus 7 mN x 10 multiplication.
Example 9 Luminescence-enzyme-immunoassay (a)~~0.1 ml of the product of Formula VII (specific activity:4~6 mV/pg, 1~1.5 V/0.1 ml) obtained in Example 6(b) and 0.9 ml of phos-phate buffer 0.1 M, pH 7.0, containing 0.88% NaCl (PBS) are incubated in tubes coated with anti-estriol antibodies at room temperature for 1.0 hour after which 2 ml of saline is added and the whole liquid is decanted. Then 1.0 ml of the assay buffer (0.02 M phosphate buffer, pH 7.0, with 0.2~ BSA and 20 mM DTT) is added and incubated at room temperature for 15 minutes. The luciferase activity is measured using FMNH-injection method. A comparable control is performed by the identical method except using a blank tube not coated with antibody.
(b) 0.1 ml of the product of Formula IX (1.5-2.0 ~N/pg, 200-400 mV/0.1 ml) 9 0~1 ml of P6S and 0.1 ml of anti-T3 antibodies immobilized l L~3 3~

on S. aureus at room temperature for two hours. At the end of the incubation, the reac-tion mixture is washed twice with 2.0 ml saline by resuspension and centrifugationO Then 1.0 ml of the assay buffer as described above is added and incubated a room temperature for 15 min-utes. The So aureus are spun down and the supernatant is transferredto an assay tube for the measurement of luciferase activity by the FMNH-injection method.
Example 10 T4-Thyroxine-C5 acid-luciferase conjugate To a solution of 20 mg (8.62 x 10 2 mmole) of 6-~methylsulfonyl-thio)hexanoic acid in 5.0 ml ethanol, 5.0 ml 0~20 M phosphate, pH 4.5,and 2.0 ml water was added 17 mg (8.62 x 10 2 mmole) of EDAG. The mixture was stirred for 30 minutes during which time the pH maintained at 4~5 after which 68 mg (8.62 x 10 2 mmole) of thyroxine methyl ester was addedq The pH of the reaction mixture was raised to 8.5 using 1.0 N aqueous NaOH and maintained for three hours. The resulting product was extracted with ethyl acetate (10 ml, 3X), and the extracts were dried on a rotovap. The residue was resuspended in 3 ml of ethanol and stored at -10C for 48 hours whereupon a precipitate formed giving the product of Formula X.
Example 11 T4-assay An inactivation of mixture of 100 l of luciferase (at 8.0 x 10 7 M) and 3 l of the product of Formula X in dimethylformamide (DMF) was incubated for 12 minutes. A control mixture of 100 l of luciferase (at 8.0 x 10 7 M) and 3 l of DMF was also incubated. The inactiva-tion mixture and the control mixture were added to separate plastictubes coated with anti-thyroxine (anti-T~) antibody and containing 1.0 ml of 20 mM phosphate and 0.2% BSA, pH 7.0) to give a final volume of 1.1 ml. The mixtures were incubated for one hour at 37~C with occa-sional stirring after which each was washed with two 1.0 ml volumes of 10 mM phosphate pH 7Ø Following the washing step 1.0 ml of 10 mM
phosphate and 20 l of 10 mM ~-mercaptoethanol ME was added to each of the inactivation sample and the control sample. Also 20 l of B-ME was added to each of the washes. Each sample and the washes were incubated for 60 minutes at about 22C after which each was assayed for luciferase activity. There was no observed enzyme activity in any of the washes or the control sample. There was an observed 0.0182 light units (LU) in the inactivation sample or an efficiency of 2.2 x 0 3% based on the following assumptions: (1) The known activity of l Lo 3~3~

luciferase is ~1.25 x 106 LU/~mole of enzyme and 1 LU 1 x 101 quanta/sec.; (2) The amount of anti-T4 antibody per tube was about 100 ~g or about 6.67 x 10 4 mole; (3) There was a 1:1 stoichometry of antibody to antigen; and (4) The maximum detectable LU was 16.67 x 10-4 ~mole)(1.25 x 106 LU/~mole) = 834.
Example 12 Progesterone-C3amine-intermediate To a solution of 35.4 mg (0.08 mmole) of 11~- progesterone hem;-succinate in 8 ml of dimethylformamide, 5 ml waxer, and 0.30 ml 1.0 M
phosphate, pH 7.0, was added 15.~ mg (0.08 mmole) of EDAC. The pH of the mixture was adjusted to l with 1.0 NHCl. The mixture was stirred at room temperature for 1/2 hour maintaining the pH at 5.1 after which 20 mg (0.08 mole) of 3-(methylsulfonylthio)propylamine-HBr was added. Maintaining a pH of 8.0 the reaction was permitted to pro-ceed for 90 minutes at 22C, then the mixture was evaporated to dry-ness on a rotovap. The resulting residue was taken up in 3 ml ofethanol and stored at ^10C. The product, as depicted by Formula XI, was purified on a C18 reverse phase HPLC column. The ethanol soluble material was applied to the column in 20% ethanol/80% water. Lucifer-ase inactivating activity was eluted to the end o-F a solvent gradient to 100% ethanol. All of the luciferase activity was recovered upon addition of ME
Example 13 Progesterone assay An inactivation mixture of 100 Al of luciferase (at 8.0 x 10 7 M
in 10 mM phosphate, pH 7.0, 22C) and 2 Al of purified product of For-mula XI was incubated for about three minutes after which 1.0 Al ofanti-progesterone antibody was added and incubation was continued for three minutes. Following the second incubation 2 mg of protein A-sepharose was added and the medium was incubated another three minutes with moderate agitation. The medium was then centrifuged. The pellet and supernatant were separated. The supernatant was retained for enzyme content analysis. The pellet was washed with three 100 Al volumes of 10 mM phosphate buffer, pH 7Ø Following the wash step 2 Al of ME was added to each of the washes, the supernatant, and the pellet then each was assayed for luciferase activity. Most of the enzyme activity (98.4%) was recovered from the supernatant. From the first wash about 0.4~ enzyme activity was recovered; no activity was found in the second wash; and 1% of the enzyme activity was recovered from the pellet.

As a control to demonstrate the viability of the enzyme integrity 100 Al of luciferase (8.0 x 10 7 M, 10 mM phosphate pH 7,0) and 2 Al of the product of Formula XI in ethanol (6.4 x 10-7 M) were combined whereupon about 80% of the enzyme activity was lost within 10 minutes. 5 Addition of 2 Al of ME resulted in recovery of 90~ of the initial activity within two minutes.
Also to demonstrate the stability of luciferase in the presence of antibody and protein A-sepharose 100 Al of luciferase (8.0 x 10 7 M) (10 mM phosphate, pH 7.0) and 5 mg of lyophilized protein A-sepha rose were combined and there was no observed effect on enzyme activ-ity. Similarly 100 Al of luciferase (8.0 x 10 7 M) (10 mM phosphate, pH 7.~) and 1 Al of anti-progesterone antibody were combined and no effect on luciferase activity was observed after 50 minutes.
Example 14 Insulin-C3amine-luciferase conjugate (a) A mixture of 20 Al (1.75 x 10-4 mmoles) of porcine insulin (having about 5 moles of carboxyl groups per mole of protein), 10 Al EDAC (1.4 x 10 3 mmoles) and 7.74 ml of 50 mM phosphate buffer, pH
4.7, was reacted for about one hour at 22C after which 2.0 ml of 3-(methylsulfonylthio)propylamine-HBr (1.6 x 10 3 mmoles) in ethanol was added. The pH of the mixture was adjusted to 8.2 and the reaction - was allowed to proceed for 6 hours at 22C whereupon the mixture was transferred to a spectrapore 6 dialysis tubing (1000 mOw. cut off) and dialyzed against 500 ml of 50 mM phosphate buffer, pH 7.1 at 4 C for 16 hours to give the insulin C3amine intermediateO
A 100-fold excess of the above-obtained intermediate (88 EM) and luciferase in 10 mM phosphate buffer, pH 7.0, were incubated at 22C
for about 8 minutes to give the insulin-C3amine-luciferase conjugate.
Example 15 The conjugate obtained in Example 14 is used to determine the concentration of cell surface insulin receptors in rat hepatoma cells as follows The cell lines, buffers and reagents are preparPd as described by procedures known in the art. See, J.F. Ballard, et al., J. Cell Physio. 105, 335-336 (1980) and J.M. Gunn, et al., J. Cell Physio., In Press.
The insulin C3-amine luciferase conjugate is added to a monolayer of RH35 hepatcma cells in 100 EM TES, pH 7.85, with Earl's Balanced Salts for a final concentration of 0.1-10 nM. A control mixture is prepared in the identical manner except that unlabeled insulin for a 33~
26= 3972 final concentration of 0.1-1 EM is employed. Each of the mixtures is washed 3 times with volunes of PBS equivalent to that of each mixture after which ~-mercaptoethanol is added to a final concentration of 0.05-0.2 M to each of the wash volumes and the final experimental and control monolayer mixtures. After 60 minutes at-about 22C each wash or mixture is assayed for luciferase activity using the flavin i ection method to determine the concentration of receptors.
Example 16 Estriol (E3) standard curve 0.1 ml of estriol-luciferase product of Formula ~II in 0.1 M
phosphate buffer, pH 7.0, with 0.05% Tween 80 plus 0.1 ml phosphate buffer, pH 7.0, containing 0.05% Tween and 0.85~ NaCl plus 0.1 ml of E3 standard plus 0.1 ml primary antibody solution containing 1/300 diluted sheep anti-E3, 1/60 diluted normal sheep serum in 0.1 M PBS, pH 7~0, with 0.05% Tween 80. The mixture was incubated at room tem-perature for 60 minutes, then 0.1 ml of secondary antibody solution(4.8% polyethylene glycol and 1/2.5 diluted donkey-anti-sheep antibody in the PUS buffer described above). The mixture was incubated for an additional 30 minutes and then cenkrifuged at 2500 x G for 15 minutes.
The supernate was decanted and the pellet was resuspended in 0.2 ml dithiothreitol solution (10 mM) and incubated at room temperature for another 15 minutes. Then 0.72 ml of the assay buffer (as described above) was added and the luciferase activity was determined by the FMNH2-injection method. The standard curve then was constructed with B/Bo of 59%, 42%, and 36% for 50, 250 and 500 pg estriol/tube. A
linear standard curve was obtained over the range stated on a log logit scale.

L3 ~33~
-27- 3g72 FORI~LA CHART

R~ R~
Rl ~S-S-C--C- ( CHz ) n-R6 - -R3 R5 Formul a I

O R~ R~

R7 -S -S -C--C- ( OH ) "- R~
O R~ Rs Forml~l a I I

Rz R4 Br-CI--Ci- OH n-Rg R~ R5 Formula lII

Rz R4 I i nY
R3 Rs --m Formula IV(a) r 1l R~ R~ 1 ~~~
R7 -S -S - C C - ( OH ) no X Y
O R3 Rs _ m Formul a IV ( b) r R~ R4 E-S-S -C--C- ( OH ) n-X Y
R3 R5 m Formul a V

393~

0~1 Formula Vl N-O-CH2-C-NH- (CH2 ~5-S-SOzCH3 OH
f I- -OH
~~
HO l~J Formula VII
N-O-CH2-C-NH ( CH2 )5-S -S -E

O
Ho~30~3CH2-CH2-NH-C-(CH2)3-S-502CH3 C=O
OCH3Formul a VI I I

O
HO O CH2-CH2-NH-C- ( CH2 )3 -S-S -E

C=O
OCH3Formul a IX

- HO o~3CH2-CH2-NH-C- (CH2 )5-S-SO2-CH3 C=O

OCH3 Fonnul a X

3q3~

H3 CSO2 -S- ( CH2 ) 3 -NH C -CH2CHz O C=O
Fonnul a XI

0~)

Claims (2)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A compound of the formula wherein R7 is a lower alkyl group having from 1 to 6 carbon atoms;
phenyl; phenyl substituted with fluoro, nitro or a lower alkyl group having from 1 to 4 carbon atoms; 2-pyridyl; or 4-pyridyl; n is zero to 15; R6 is -COOH; -COOsuccinimide; -COCl; -COBr; Cl; Br; SCN; NH2; CN;
4-cyanophenyl; or ;
R2 and R3 are the same and are hydrogen, methyl or ethyl; R4 and R5 are the same and are hydrogen, methyl or ethyl; or R2, R3, R4 and R5 taken together with the carbon atoms to which they are attached repre-sent a cycloalkyl group having from 4 to 6 carbon atoms, or represent 1,4-phenylene with the proviso that when R7 is a lower alkyl group having one carbon atom or phenyl substituted with a lower alkyl group having one carbon atom and n is zero and each of R2, R3, R4 and R5 is hydrogen, R6 is other than -NH2.

2. A linker-compound-ligand or a linker-compound-receptor of the formula wherein R7 is a lower alkyl group having from 1 to 6 carbon atoms;
phenyl; phenyl substituted with fluoro, nitro or a lower alkyl group having from 1 to 4 carbon atoms; 2-pyridyl; or 4-pyridyl;
n is zero to 15;
R2 and R3 are the same and are hydrogen, methyl or ethyl;
R4 and R5 are the same and are hydrogen, methyl or ethyl; or R2, R3, R4 and R5 taken together with the carbon atoms to which they are attached represent a cycloalkyl group having from 4 to 6 carbon atoms, or represent 1,4-phenylene;
m is an integer equivalent to the number of reactive functions present on the ligand or receptor capable of reacting with the linker compound;
X represents the functional linkage between the linker compound and the ligand or receptor and is ; -NR- wherein R is hydrogen or any group which may be present on the ligand or receptor functional secondary amine;

; and Y represents the ligand or receptor to be labeled or assayed absent the functional group which reacted with the linker compound.