Note: Descriptions are shown in the official language in which they were submitted.
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
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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~
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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~
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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~
- -7- 3972
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~
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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
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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~
3972
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
-12- 3972
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~
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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
- -14- 3972
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
-15- 3972
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~
- -16- 3972
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~
-17- 3972
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
-18- 3972
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~
-19- 3972
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~
-20- 3972
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
-21- 3972
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~
-22- 397Z
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~
-Z3- 3972
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~
-24- 3972
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.
-25- 3972
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~
1 0
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~
-28- 3972
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~
-29- 3972
O CH3
H3 CSO2 -S- ( CH2 ) 3 -NH C -CH2CHz O C=O
Fonnul a XI
0~)
