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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to novel chemiluminescent-labeled conjugates for use
in specific binding assays for a ligand, such as an antigen, hapten or
antibody, in a liquid medium such as a body fluid. The invention further
relates to intermediate compounds produced in the synthesis of the novel
labeled conjugates.
2. Brief Description of the Prior Art
Specific binding assay methods have undergone a technological evolution
from the original competitive binding radioimmunoassay (RIA) in which a
radioisotope-labeled antigen is made to compete with antigen from a test
sample for binding to specific antibody. In the RIA technique, sample
antigen is quantitated by measuring the proportion of radioactivity which
becomes associated with the antibody by binding of the radiolabeled
antigen (the bound-species of the labeled antigen) to the radioactivity
that remains unassociated from antibody (the free-species) and then
comparing that proportion to a standard curve. A comprehensive review of
the RIA technique is provided by Skelly et al, Clin. Chem. 19:146(1973).
While by definition RIA is based on the binding of specific antibody with
an antigen or hapten, radiolabeled binding assays have been developed
based on other specific binding interactions, such as between hormones and
their binding proteins.
From the radiolabeled binding assays have evolved nonradioisotopic binding
assays employing labeling substances such as enzymes as described in U.S.
Pat. Nos. 3,654,090 and 3,817,837. Recently further improved
nonradioisotopic binding assays have been developed as described in German
Offenlegungschriften Nos. 2,618,419 and 2,618,511 based on U.S. Ser. Nos.
667,982 and 667,996, filed on Mar. 18, 1976 and assigned to the present
assignee, both now abandoned, employing particularly unique labeling
substances, including coenzymes, cyclic reactants, cleavable fluorescent
enzyme substrates, and chemiluminescent molecules. The chemiluminescent
labels consist of an organic molecule which undergoes a change in chemical
structure with the production of light.
Specific examples of substances useful as chemiluminescent labels mentioned
in German OLS No. 2,618,511 are luminol, isoluminol, pyrogallol and
luciferin. In particular, an example is provided in the OLS [and in Anal.
Chem. 48:1933(1976) based on the same work] of an isoluminol-labeled
conjugate wherein isoluminol is coupled through its amino function by a
2-hydroxypropylene bridge to the ligand biotin. The isoluminol-labeled
conjugate is monitored in the binding assay by measuring the production of
light in the presence of either hydrogen peroxide and peroxidase or
potassium superoxide. The chemiluminescent phthalhydrazide-labeled
conjugates wherein an amino-phthalhydrazide is coupled through its amino
function by a 2-hydroxyalkylene bridge to a ligand are described in
pending U.S. patent application Ser. No. 927,622, filed on even date
herewith and assigned to the present assignee.
The efficiency of the amino-phthalhydrazides as chemiluminescent labels has
been improved by coupling through the amino function with a straight chain
lower alkylene bridge as described in pending U.S. patent application Ser.
No. 927,621, filed on even date herewith and assigned to the present
assignee. The use of more efficient labels enables more sensitive
detection of ligands.
SUMMARY OF THE INVENTION
Labeled conjugates comprising even more efficient chemiluminescent labels
have now been devised having the formula:
##STR2##
wherein R is hydrogen or straight chain alkyl containing 1-4 carbon atoms,
perferably ethyl, n.times.2-6, preferably 4, and L(CO-- is a specifically
bindable ligand, or a binding analog thereof, bound through an amide bond.
The subject chemiluminescent naphthalene-1,2-dicarboxylic acid
hydrazide-labeled conjugates are used in specific binding assays for
detecting the ligand or a binding partner thereof. The labeled conjugates
are monitored in the performance of a binding assay by oxidizing the
labeled conjugates and measuring the light produced either as total light
produced or peak light intensity. For instance, a specific binding assay
for determining a hapten in a liquid medium might be carried out by
incubating a sample of the liquid medium with an antibody for such hapten
and with a labeled conjugate of the present invention wherein such hapten
or a binding analog is labeled with the subject chemiluminescent moiety.
During the incubation, any hapten present in the liquid medium competes
with the labeled conjugate for binding with the antibody. Thereafter, the
amount of labeled conjugate resulting in the bound-species compared to the
free-species (which amount is an inverse function of the amount to hapten
in the liquid medium assayed) is determined (i.e., monitored) either in a
homogeneous fashion, if the chemiluminescent character of the labeled
conjugate is different when in the bound-species than when in the
free-species, or in a heterogeneous fashion, if such character is
essentially the same in both species. In the homogeneous assay, the
unseparated reaction mixture containing both species of the labeled
conjugate is combined with an appropriate oxidation system for the
chemiluminescent label and the light produced is measured. In the
heterogeneous assay, the bound- and free-species are separated by any
conventional technique, the oxidation system combined with one thereof,
and the light produced is measured.
The monitorable chemiluminescent reaction may be illustrated as follows:
##STR3##
wherein h.nu. represents electromagnetic radiation emitted. Useful
oxidation systems include hydrogen peroxide combined with any of the
following catalysts, peroxidase (particularly microperoxidase), catalase,
deuterohemin, hematin or ferricyanide ions; hypochlorite ions combined
with cobalt ions; persulfate ions; potassium superoxide; periodate ions;
hypoxanthine combined with xanthine oxidase; or potassium t-butoxide.
The chemiluminescent-labeled conjugates may be employed in any conventional
homogeneous or heterogeneous binding assay method, including competitive
binding methods, sequential saturation methods, direct binding methods,
and "sandwich" binding methods. Further details concerning the state of
the art for binding assay techniques may be found in the aforementioned
German OLS Nos. 2,618,419 and 2,618,511.
In the context of this disclosure, the following terms shall be defined as
follows unless otherwise indicated: "specifically bindable ligand" is an
organic substance of analytical interest for which there is specific
binding partner; "specific binding partner of the ligand" is the substance
which has a noncovalent binding affinity for the ligand to the exclusion
of other substances; and "binding analog of the ligand" is an organic
substance which is different in chemical structure from the ligand but
which behaves essentially the same as the ligand with respect to the
binding affinity of the specific binding partner of the ligand.
The specifically bindable ligand or analog thereof in the present labeled
conjugates, in terms of its chemical nature, usually is a protein,
polypeptide, peptide, carbohydrate, glycoprotein, steroid, or other
organic molecule for which a specific binding partner is obtainable. In
functional terms, the ligand will usually be an antigen or an antibody
thereto; a hapten or an antibody thereto; or a hormone, vitamin, or drug,
or a receptor or binding substance therefor. Most commonly, the ligand is
an immunologically-active polypeptide or protein of molecular weight
between 1,000 and 4,000,000 such as an antigenic polypeptide or protein or
an antibody; or is a hapten of molecular weight between 100 and 1,500.
The present labeled conjugates are prepared usually by forming a peptide or
amide couple between (1) an amino derivative of a chemiluminescent
naphthalene-1,2-dicarboxylic acid hydrazide and (2) either the ligand,
where such contains a carboxylic acid function, or a binding analog of the
ligand (e.g., a derivative of the ligand) which analog contains the
desired carboxylic acid function. Such condensation reactions can be
accomplished by reacting the amino derivative of the label directly with
the carboxylic acid-containing ligand or ligand analog using conventional
peptide condensation reactions such as the carbodiimide reaction [Science
144:1344 (1964)], the mixted anhydride reaction [Erlanger et al, Methods
in Immunology and Immunochemistry, ed. Williams and Chase, Academic Press
(New York 1967)p. 149], and the acid azide and active ester reactions
[Kopple, Peptides and Amino Acids, W.A. Benjamin, Inc. (New York 1966)].
See also for a general review Clin. Chem. 22:726(1976).
It will be recognized, of course, that other well known methods are
available for coupling the ligand or a derivative thereof to the
amino-derivative of the label. In particular, conventional bifunctional
coupling agents may be employed for coupling a ligand, or its derivative,
containing a carboxylic acid or amino group to the amino-derivative of the
label. For example, amine-amine coupling agents such as bis-isocyanates,
bis-imidoesters and glutaraldehyde [Immunochem. 6:53(1969)] may be used to
couple a ligand or derivative containing an amino group to the
amino-derivative of the label. Also, appropriate coupling reactions are
well known for inserting a bridge group in coupling an amine (e.g., the
amino-derivative of the label) to a carboxylic acid (e.g., the ligand or a
derivative thereof). Coupling reactions of this type are thoroughly
discussed in the literature, for instance in the above-mentioned Kopple
monograph and in Lowe & Dean, Affinity Chromatography, John Wiley & Sons
(New York 1974).
Such coupling techniques will be considered equivalents to the previously
discussed peptide condensation reactions in preparing useful labeled
conjugates. The choice of coupling technique will depend on the
functionalities available in the ligand or analog thereof for coupling to
the label derivative and on the length of bridging group desired. In all
cases, for purposes of this disclosure, the resulting labeled conjugate
will comprise the label derivative bound to the remaining portion of the
conjugate through an amide bond. Such remaining portion of the conjugate
will be considered as a residue of a binding analog of the ligand, unless
the ligand itself is directly coupled to the label derivative. Thus, in
this description and in the claims to follow, the abbreviation L(CO--
represents the ligand or a binding analog thereof coupled through an amide
bond, wherein such analog may be a derivative of the ligand coupled by
peptide condensation to the label derivative or may be the ligand or
derivative thereof coupled through a bridging group inserted by coupling
of the ligand or derivative to the label derivative with a bifunctional
coupling agent.
Preparation of the present chemiluminesent-labeled conjugates proceeds
according to the following general synthetic sequence:
##STR4##
Reaction of an N-(.omega.-bromoalkyl)phthalimide (I) [available from
Aldrich Chemical Co., Milwaukee, Wisconsin USA, or see Derscherl and
Weingarten, Justus Liebig's Annalen der Chemie 574:131(1951)]with dimethyl
7-aminonaphthalene-1,2-dicarboxylate [Gundermann et al, Justus Liebig's
Annalen der Chemie 684:127(1965)]produces the intermediate dimethyl
7-[.omega.-N-(phthalimido)alkyl] aminonaphthalene-1,2-dicarboxylate (II).
##STR5##
Alkylation of the amine group in the intermediate dicarboxylate (II) is
obtained by reaction with a dialkyl sulfate (III) [Rodd, Chemistry of
Carbon Compounds, vol. 1, Elsevier Publ. Co. (New York 1951) p. 337]
##STR6##
to yield the alkylated derivative (IV)
##STR7##
wherein R' is straight chain alkyl containing 1-4 carbon atoms.
Treatment of the intermediate dicarboxylate (II) or the alkylated
derivative (IV) with hydrazine produces the amino-hydrazide (V)
##STR8##
wherein R is hydrogen or straight chain alkyl containing 1-4 carbon atoms.
Condensation of the amino-hydrazide (V) with (a) the ligand to be labeled,
where such contains a carboxylic acid function, (b) a binding analog of
the ligand, such analog being a carboxylic acid derivative of the ligand,
or (c) the ligand or an appropriate derivative of the ligand in the
presence of a bifunctional coupling agent, produces the
chemiluminescent-labeled conjugate (VI)
##STR9##
wherein R is the same as defined above and L(CO-- represents the
specifically bindable ligand, or a binding analog thereof (formed by
derivation of the ligand and/or insertion of a bridge by a bifunctional
coupling agent), bound through an amide bond.
Other variations of labeled conjugates based on the above-described
synthetic scheme are clearly evident. In particular, various
ring-substituted dimethyl 7-aminonaphthalene-1,2-dicarboxylates may be
used as starting material to produce ring-substituted labeled conjugates
possessing substantially the same qualitative properties as the conjugates
prepared according to the above-described scheme. Such conjugates will be
recognized as equivalents and are exemplified by the addition of one, two
or more simple substituents to an available aromatic ring site, such
substituents including without limitation, alkyl, e.g., methyl, ethyl and
butyl; halo, e.g., chloro and bromo; nitro; hydroxyl; alkoxy, e.g.,
methoxy and ethoxy, and so forth.
As stated hereinabove, the ligand which is comprised in the labeled
conjugage or whose binding analog is comprised in the labeled conjugate is
in most circumstances an immunologically-active polypeptide or protein of
molecular weight between 1,000 and 4,000,000 such as an antigenic
polypeptide or protein or an antibody; or is a hapten of molecular weight
between 100 and 1,500. Following will now be presented various methods for
coupling such ligands or analogs thereof to the amino-derivative (V) of
the label through an amide bond.
Polypeptides and Proteins
Representative of specifically bindable protein ligands are antibodies in
general, particularly those of the IgG, IgE, IgM and IgA classes, for
example hepatitis B antibodies; and antigenic proteins such as insulin,
chorionic gonadotropin (e.g., HCG), carcinoembryonic antigen (CEA),
myoglobin, hemoglobin, follicle stimulating hormone, human growth hormone,
thyroid stimulating hormone (TSH), human placental lactogen, thyroxine
binding globulin (TBG), instrinsic factor, transcobalamin, enzymes such as
alkaine phosphatase and lactic dehydrogenase, and hepatitis-associated
antigens such as hepatitis B surface antigen (HB.sub.s Ag), hepatitis e
antigen (HB.sub.e Ag) and hepatitis core antigen (HB.sub.c Ag).
Representative of polypeptide ligands are angiotensin I and II, C-peptide,
oxytocin, vasopressin, neurophysin, gastrin, secretin, and glucagon.
Since, as peptides, ligands of this general category comprise numerous
available carboxylic acid and amino groups, coupling the amino-derivative
of the chemiluminescent label can proceed according to conventional
peptide condensation reactions such as carbodiimide reaction, the mixed
anhydride reaction, and so forth as described hereinabove, or by the use
of conventional bifunctional reagents capable of coupling carboxylic acid
or amino functions to the amino group in the label derivative as likewise
described above. General references concerning the coupling of proteins to
primary amines or carboxylic acids are mentioned in detail above.
Haptens
Haptens, as a class, off a wide variety of organic substances which evoke
an immunochemical response in a host animal only when injected in the form
of an immunogen conjugate comprising the hapten coupled to a carrier
molecule, almost always a protein such as albumin. The coupling reactions
for forming the immunogen conjugates are well developed in the art and in
general comprise the coupling of a carboxylic acid ligand or a carboxylic
acid derivative of the ligand to available amino groups on the protein
carrier by formation of an amide bond. Such well known coupling reactions
are directly analogous to the present formation of labeled conjugates by
coupling carboxylic acid ligands or binding analogs to the
amino-derivative of the chemiluminescent label.
Hapten ligands which themselves contain carboxylic acid functions, and
which thereby can be coupled directly to the amino-derivative of the
label, include the iodothyronine hormones such as thyroxine and
liothyronine, as well as other materials such as biotin, valproic acid,
folic acid and certain prostaglandins. Following are representative
synthetic routes for preparing carboxylic acid binding analogs of hapten
ligands which themselves do not contain an available carboxylic acid
function whereby such analogs can be coupled to the amino-derivative of
the label by the aforementioned peptide condensation reactions or
bifunctional coupling agent reactions (in the structural formulae below, n
represents an integer, usually from 1 through 6).
Carbamazepine
Dibenz[b,f]azepine is treated sequentially with phosgene, an
.omega.-aminoalkanol, and Jones reagent (chromium trioxide in sulfuric
acid) according to the method of Singh, U.S. Pat. No. 4,058,511 to yield
the following series of carboxylic acids:
##STR10##
Quinidine
Following the method of Cook et al, Pharmacologist 17:219(1975), quinidine
is demethylated and treated with 5-bromovalerate followed by acid
hydrolysis to yield a suitable carboxylic acid derivative.
Digoxin and Digitoxin
The aglycone of the cardiac glycoside is treated with succinic anhydride
and pyridine according to the method of Oliver et al, J. Clin. Invest.
47:1035(1968) to yield the following:
##STR11##
Theophylline
Following the method of Cook et al, Res. Comm. Chem. Path. Pharm.
13:497(1976), 4,5-diamino-1,3-dimethylpyrimidine-2,6-dione is heated with
glutaric anhydride to yield the following:
##STR12##
Phenobarbital and Primidone
Sodium phenobarbital is heated with methyl 5-bromovalerate and the product
hydrolyzed to the corresponding acid derivative of phenobarbital [Cook et
al, Quantitative Analytic Studies in Epilepsy, ed. Kelleway and Peterson,
Raven Press (New York 1976) pp. 39-58]:
##STR13##
To obtain the acid derivative of primidone following the same Cook et al
reference method, 2-thiophenobarbital is alkylated, hydrolyzed, and the
product treated with Raney nickel to yield:
##STR14##
Diphenylhydantoin
Following the method of Cook et al, Res. Comm. Chem. Path. Pharm.
5:767(1973), sodium diphenylhydantoin is reacted with methyl
5-bromovalerate followed by acid hydrolysis to yield the following:
##STR15##
Morphine
Morphine free base is treated with sodium .beta.-chloroacetate according to
the method of Spector et al, Science 168:1347 (1970) to yield a suitable
carboxylic acid derivative.
Nicotine
According to the method of Langone et al, Biochem. 12(24):5025(1973),
trans-hydroxymethylnicotine and succinic anhydride are reacted to yield
the following:
##STR16##
Androgens
Suitable carboxylic acid derivatives of testosterone and androstenedione
linked through either the 1- or 7-position on the steroid nucleus are
prepared according to the method of Bauminger et al, J. Steroid Biochem.
5:739(1974). Following are representative testosterone derivatives:
##STR17##
Estrogens
Suitable carboxylic acid derivatives of estrogens, e.g., estrone, estradiol
and estriol, are prepared according to the method of Bauminger et al,
supra, as represented by the following estrone derivative:
##STR18##
Progesterones
Suitable carboxylic acid derivatives of progesterone and its metabolites
linked through any of the 3-, 6- or 7-positions on the steroid nucleus are
prepared according to the method of Bauminger et al, supra, as represented
by the following progesterone derivatives:
##STR19##
The methods described above are but examples of the many known techniques
for forming suitable carboxylic acid derivatives of haptens of analytical
interest. The principal derivation techniques are discussed in Clin. Chem.
22:726(1976) and include esterification of a primary alcohol with succinic
anhydride [Abraham and Grover, Principles of Competitive Protein-Binding
Assays, ed. Odell and Daughaday, J. B. Lippincott Co. (Philadelphia 1971)
pp. 140-157], formation of an oxime from reaction of a ketone group with
carboxylmethyl hydroxylamine [J. Biol. Chem. 234:1090(1959)], introduction
of a carboxyl group into a phenolic residue using chloroacetate [Science
168:1347(1970)], and coupling to diazotized p-aminobenzoic acid in the
manner described in J. Biol. Chem. 235:1051(1960).
The hereinbefore-described general synthetic sequence for preparing the
present chemiluminescent-labeled conjugates is specifically exemplified by
the following description of the preparation of the labeled thyroxine
conjugate
7-[N-ethyl-N-(4-thyroxinylamido)butyl]aminonaphthalene-1,2-dicarboxylic
acid hydrazide. The reaction sequence for this synthesis is outlined in
Table 1 which follows.
A. PREPARATION OF THE LABELED CONJUGATE
Dimethyl 7-[4-N-(Phthalimido)butyl]aminonaphthalene-1,2-dicarboxylate (3)
A solution of 14.1 grams (g) (0.05 mol) of N-(4-bromobutyl)phthalimide (1)
(Aldrich Chemical Co., Milwaukee, Wis. USA) and 25.0 g (0.1 mol) of
dimethyl 7-aminonaphthalene-1,2-dicarboxylate (2) [Gundermann et al,
Justus Liebig's Annalen der Chemie 684:127(1965)] in 100 milliliters (ml)
of 2,2,2-trifluoroethanol was refluxed under argon for 16 hours.
Evaporation gave a residue that was partitioned between 400 ml of ether
and 300 ml of water (H.sub.2 O). The ether phase was separated, dried, and
evaporated. The resulting dark red residue was chromatographed on a column
of 1200 g of silica gel (E. Merck, Darmstadt, West Germany) eluting with a
19:1 volume to volume (v:v) mixture of benzene and methanol. After the
first 700 ml of eluent was discarded, 20 ml fractions were collected.
Fractions numbered 219 to 251 were combined and evaporated to give 9 g of
the substituted carboxylate (3) as a clear red oil.
Analysis:
NMR Spectrum (CDCl.sub.3): .delta. 1.7 (m, 4H), 3.9 (s, 3H), 4.0 (s, 3H)
Infrared Spectrum (CDCl.sub.3): 1720 cm.sup.-1 (carbonyl)
Mass Spectrum (70 eV) m/e: 461 [MH.sup.+ ], 460 [M.sup.+ ], 429 [M.sup.+
minus OCH.sub.3 ]
TABLE 1
__________________________________________________________________________
##STR20##
##STR21##
##STR22##
##STR23##
##STR24##
##STR25##
##STR26##
##STR27##
##STR28##
##STR29##
##STR30##
__________________________________________________________________________
Dimethyl
7-{N-Ethyl-N-[4-(N-phthalimido)butyl]amino}naphthalene-1,2-dicarboxylate
(4)
A mixture of 9 g (0.02 mol) of the substituted carboxylate (3) and 20 ml of
diethyl sulfate was heated at 130.degree. C. for two hours under argon.
The dark solution was poured into a beaker of crushed ice containing 200
ml of saturated aqueous sodium bicarbonate solution. When all the ice had
melted, the mixture was extracted with three 250 ml volumes of ether. The
ether extracts were combined, dried, and evaporated to give a red oil. The
oil was chromatographed on a column of 600 g of silica gel eluting with a
19:1 (v:v) mixture of benzene and methanol and the light yellow fractions
combined and evaporated. Excess diethyl sulfate was removed by evaporative
sublimation at 50.degree. C. and reduced pressure of 0.01 millimeters (mm)
mercury leaving a residue of 4 g of the N-ethyl substituted carboxylate
(4) as a light red oil.
Analysis:
NMR Spectrum (CDCl.sub.3): .delta. 3.9 (s, 3H), 4.1 (s, 3H)
Mass Spectrum (70 eV) m/e: 489 [MH.sup.+ ], 488 [M.sup.+ ], 457 [M.sup.+
minus OCH.sub.3 ]
7-[N-(4-Aminobutyl)-N-ethyl]aminonaphthalene-1,2-dicarboxylic Acid
Hydrazide (5)
A mixture of 4 g (0.008 mol) of the N-ethyl substituted carboxylate (4), 15
ml of 85% hydrazine, and 50 ml of methanol was refluxed for three hours.
When cool, the mixture was evaporated to dryness on a rotary evaporator
and the crystalline residue scraped out and dried overnight at 80.degree.
C. under high vacuum. The resulting dark solid was chromatographed on a
column of 200 g of silica gel (E. Merck, Darmstadt, West Germany), eluting
with a 7:3 (v:v) mixture of ethanol and 1 molar (M) triethylammonium
bicarbonate and collecting 20 ml fractions. Fractions numbered 25 to 75
were combined and evaporated to give a yellow solid. After two
recrystallizations from pyridine, there was collected 1.1 g of the
amino-hydrazide (5) as five yellow crystals, melting point (m.p.)
246.degree.-247.degree. C.
Analysis:
Calculated for C.sub.18 H.sub.22 N.sub.4 O.sub.2 : C, 66.24; H, 6.80; N,
17.17. Found: C, 66.05; H, 6.69; N, 17.65
NMR Spectrum (d.sub.6 -DMSO): .delta. 1.1 (m, 3H), 1.5 (m, 4H)
Infrared Spectrum (KCl): 1615 cm.sup.-1 (carbonyl)
The efficiency of the amino-derivative (5) of the label in a
chemiluminescent reaction and the detection limit of such derivative were
determined as follows.
In determining efficiency, the label derivative and luminol
(5-amino-2,3-dihydrophthalazine-1,4-dione) were oxidized individually at
several levels in the picomolar range and related to the peak light
intensities by a graph plot. Linear portions of the resulting curves
allowed calculation of change in peak light intensity per unit
concentration for the label derivative and for luminol. Efficiency of the
label derivative was expressed as a percentage of the slope produced with
luminol.
Reaction mixtures (150 .mu.l) of the following composition were assembled
in 6.times.50 mm test tubes mounted in a Dupont 760 Luminescence Biometer
(E. I. duPont de Nemours and Co., Wilmington, Delaware USA) with a
sensitivity setting of 820: 50 mM sodium hydroxide, 57.5 mM barbital
adjusted to pH 8.6, 0.27 .mu.M microperoxidase (Sigma Chemical Co., St.
Louis, Missouri USA) and either the amino-derivative of the label or
luminol at varying concentrations in the picomolar (pM) range (diluted
with H.sub.2 O from a stock solution at 1 mM in 0.1 M sodium carbonate, pH
10.5). The final pH of the reaction mixture was 12.6. Each mixture was
incubated 10 minutes at room temperature and 10 .mu.l of 90 mM hydrogen
peroxide in 10 mM Tris-HCl buffer [tris-(hydroxymethyl)-aminomethane
hydrochloride], pH 7.4 was added to initiate the chemiluminescent
reaction. Peak light intensity values were recorded from the instrument
readings. All reactions were performed in triplicate and averaged. The
efficiency of the label derivative (5 ) was found to be 420%.
Detection limit was defined as the concentration of the label derivative
that produced a peak light intensity one and a half times the background
chemiluminescence in the reaction mixture. The detection limit for the
label derivative (5) was found to be 0.1 pM.
N-Trifluoroacetylthyroxine (6)
A solution of 20 g [25.6 millimoles (mmol)] of L-thyroxine (Sigma Chemical
Co., St. Louis, Missouri USA) in 240 ml of dry ethyl acetate containing 46
ml of trifluoroacetic acid and 7.6 ml of trifluoroacetic anhydride was
stirred at 0.degree. C. for one hour. Upon warming to room temperature and
adding 200 ml of H.sub.2 O, a suspension formed which was then saturated
with sodium chloride. The organic phase of the mixture was separated,
washed with saturated aqueous sodium chloride solution, dried over
anhydrous magnesium sulfate, filtered and evaporated to give 21.3 g of the
N-protected thyroxine derivative (6). A sample was recrystallized from
ether-pentane to give fine crystals, m.p. 233.degree.-235.degree. C.
(decomposed).
Analysis:
Calculated for C.sub.17 H.sub.10 F.sub.3 I.sub.4 NO.sub.5 : C, 23.39; H,
1.15; N, 1.60. Found: C, 23.23; H, 1.12; N, 1.56
Infrared Spectrum (KCl): 1700 cm.sup.-1 (carbonyl)
Optical Rotation [.alpha.].sub.D.sup.25 =-14.97.degree. (c 1.0,
dimethylsulfoxide)
7-[N-Ethyl-N-(4-thyroxinylamido)butyl]aminonaphthalene-1,2-dicarboxylic
Acid Hydrazide (7)
A solution of 1.746 g (2 mmol) of N-trifluoroacetylthyroxine (6) in 20 ml
of dry pyridine was cooled to -10.degree. C. with stirring under argon. To
this solution was added 450 milligrams (mg) (2.2 mmol) of dicyclohexyl
carbodiimide, followed 45 minutes later by 980 mg (3 mmol) of the
amino-hydrazide (5). After stirring for 3 hours at -10.degree. C., the
reaction was allowed to warm to room temperature overnight. The reaction
mixture was diluted with 10 ml of pyridine, 10 g of silica gel was added,
and the solvent removed under vacuum. The resulting impregnated silica gel
was placed atop a 200 g column of silica gel made up in a 7:3 (v:v)
mixture of ethanol and 1 M triethylammonium bicarbonate, eluting with the
same solvent mixture. After the first 400 ml of eluent was discarded, 20
ml fractions were collected. Fractions numbered 19 to 30 were combined and
evaporated to give a yellow crystalline residue. This product was refluxed
for 3 hours in 300 ml of 1 M triethylammonium bicarbonate to complete
removal of the trifluoroacetyl protecting group. The solution was then
filtered while hot. When cool, 15 g of silica gel was added and the
solvent evaporated. The impregnated silica gel was placed atop a 200 g
column of silica gel and eluted with a 7:3 (v:v) mixture of ethanol and 1
M triethylammonium bicarbonate, collecting 20 ml fractions. Fractions
numbered 30 to 50 were combined and evaporated to give 910 mg of the
labeled thyroxine conjugate (7) as a yellow solid. A 110 mg sample was
chromatographed on a 45.times.3.2 centimeter (cm) column of Sephadex LH-20
(Pharmacia Fine Chemicals, Uppsala, Sweden), eluting with methanol.
Fractions of 7 ml volume were collected and those numbered 64 to 76 were
combined and evaporated to give 60 mg of the labeled conjugate (7) as a
yellow solid, m.p. 218.degree. C. (decomposed).
Analysis:
Calculated for C.sub.33 H.sub.31 I.sub.4 N.sub.5 O.sub.5 : C, 36.52; H,
2.88; I, 46.77; N, 6.45. Found: C, 35.61; H, 3.02; I, 44.69; N, 6.25.
B. BINDING ASSAY FOR THYROXINE
Competitive binding reaction mixtures (200 .mu.l) were assembled in
triplicate by combining the following reagents: 50 .mu.l of 10 nM labeled
conjugate (7) in 75 mM barbital buffer (pH 8.6), 50 .mu.l of a preparation
of antibody to thyroxine in the same buffer, varying volumes of 54.8 nM
thyroxine in the same buffer, and a sufficient volume of the buffer to
make a final volume of 200 .mu.l. After a 10 minute incubation at room
temperature, the free- and bound-species of the labeled conjugate were
separated for each reaction mixture by applying a 150 .mu.l aliquot to
small Sephadex G-25 (Pharmacia Fine Chemicals, Uppsala, Sweden) columns.
The columns had a bed volume of 1.5 ml and were pre-washed with successive
3 ml volumes of 7% acetic acid (3 times), H.sub.2 O 0.1 M sodium hydroxide
(3 times), and 75 mM of the barbital buffer. The bound-species of the
labeled conjugate was eluted from the column with 1.5 ml of the barbital
buffer leaving the free-species in the column.
An aliquot (95 .mu.l) of each column effluent was added to 55 .mu.l of a
solution of 134 mM sodium hydroxide, 0.73 .mu.M microperoxidase, and 27 mM
barbital in a 6.times.50 mm test tube. After a 10 minute incubation at
room temperature, each tube was placed in the Dupont 760 Biometer and 10
.mu.l of 90 mM hydrogen peroxide in 10 mM Tris-HCl buffer (pH 7.4) was
added. The resulting peak intensity of the light produced in the
chemiluminescent reaction was recorded from the instrument reading. Each
binding mixture was monitored in triplicate to give a total of 9
individual peak light intensity values which were averaged for each
different volume of thyroxine added to the initial reaction mixture.
The peak intensity values were also related as a percentage of total
labeled conjugate in the bound-species by ratioing such values to the peak
light intensity measured in the chemiluminescent reaction using 95 .mu.l
of 0.25 nM labeled conjugate in place of the column effluent. Background
chemiluminescence in the monitoring reaction was found to be 0.3 peak
intensity units.
The relationships of the amount of thyroxine in the binding reaction to
peak light intensity and percent of labeled conjugate in the bound-species
are shown in Table 2 below.
TABLE 2
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volume of thyroxine
peak light percent in
solution added (.mu.l)
intensity bound-species
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0 26.5 63.1
5 25.3 59.5
10 23.3 55.5
20 23.7 56.4
40 19.1 45.4
60 14.7 35.0
80 13.7 32.6
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The results demonstrate that the labeled conjugate of the present invention
is useful in binding assays for determining a ligand in a liquid medium.
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