Substituted phenylacetic acids and salts as TBP blocking agents in iodothyronine immunoassays

An improved immunoassay method, reagent means, and test kit for determining an iodothyronine, e.g., thyroxine (T-4), in a biological fluid, usually serum or plasma, wherein fenclofenac and related phenylacetic acids, or salts thereof, are employed as novel blocking agents for the binding of iodothyronines to thyroxine binding protein (TBP). The present invention is particularly advantageous as applied to homogeneous competitive binding iodothyronine immunoassays wherein a spectrophotometric response is generated in the assay reaction mixture at a wavelength greater than about 300 nm, the blocking agents of the present invention having been found to have no substantial absorbance at wavelengths above 300 nm. Such homogeneous immunoassays include those which employ labels such as fluorescers, enzyme substrates, enzyme prosthetic groups, enzymes, and enzyme inhibitors.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to immunoassays for the determination of 
iodothyronines in biological fluids such as serum or plasma. In 
particular, the present invention relates to competitive binding 
immunoassay methods, reagent means, and test kits for determining 
iodothyronines in unextracted samples of serum or plasma through the use 
of blocking or dissociating agents for the binding of iodothyronines by 
thyroxine binding proteins (TBP) present in such samples. 
The iodothyronines have the general formula: 
##STR1## 
wherein .beta..sup.1 and .beta..sup.2 are, independently, hydrogen or 
iodine. The principal iodothyronines of clinical interest are 
3,5,3',5'-tetraiodothyronine (thyroxine; T-4) wherein .beta..sup.1 and 
.beta..sup.2 are both iodine; 3,5,3'-triiodothyronine (T-3, or simply 
"triiodothyronine") wherein .beta..sup.1 is iodine and .beta..sup.2 is 
hydrogen; 3,3',5'-triiodothyronine ("reverse T-3") wherein .beta..sup.1 is 
hydrogen and .beta..sup.2 is iodine; and 3,3'-diiodothyronine wherein 
.beta..sup.1 and .beta..sup.2 are both hydrogen. The quantitative 
determination of the concentration of the various iodothyronines, 
particularly the hormones T-4 and T-3, in the blood is of important 
significance in the diagnosis of thyroid disorders. 
In the blood, nearly all of the circulating iodothyronines are complexed 
with various carrier proteins including albumin, thyroxine binding 
prealbumin and thyroxine binding globulin (TBG), such carrier proteins 
being generically referred to herein as thyroxine binding protein (TBP). 
In order to measure the concentration of the total amount of an 
iodothyronine in a blood sample, such as serum or plasma, the TBP-bound 
forms must be dissociated to an analytically significant degree and the 
resulting total free iodothyronine determined. The dissociation of 
iodothyronines from TBP, particularly TBG, was originally accomplished by 
an extraction process (U.S. Pat. No. 3,414,383). Under the current 
state-of-the-art, iodothyronines can be determined by immunoassay in 
unextracted samples through the use of compounds found empirically to 
block, and cause dissociation of, TBP binding. In current competitive 
binding iodothyronine immunoassays, a test sample is combined with 
reagents including an antibody to the iodothyronine to be determined, a 
labeled form (e.g., radiolabeled) of such iodothyronine, and one or more 
TBP blocking agents. The iodothyronine in the sample complexed with TBP is 
dissociated therefrom and competes with labeled iodothyronine for binding 
to the antibody. The proportion of labeled iodothyronine that becomes 
antibody-bound compared to that which remains unbound from antibody is 
dependent on the total concentration of the iodothyronine in the sample 
and is measurable in a wide variety of ways depending on the particular 
immunoassay technique employed. 
2. Description of the Prior Art 
Various compounds have been discovered as useful TBP blocking agents, 
including tetrachlorothyronine [Mitsuma et al, J. Clin. Endocrinol. Metab. 
33:365 (1971)], diphenylhydantoin [Lieblich and Utiger, J. Clin. Invest. 
50:60a (1971)], salicylate [Larson, Metab. 20:976 (1971)], and the various 
materials disclosed by Hollander (U.S. Pat. No. 3,928,553) and Chopra 
(U.S. Pat. No. 3,911,096), particularly 8-anilino-1-naphthalenesulfonic 
acid (ANS). The structures and general properties of the known TBP 
blocking agents vary over an extremely wide range. The properties critical 
to operability as a TBP blocking agent in immunoassays, i.e., the ability 
to sufficiently dissociate iodothyronines from TBP at concentration levels 
insufficient to cause significant inhibition of the antibody binding 
reaction, are generally considered unpredictable from purely structural 
comparisons, although some theories of TBG blocking have been propounded 
[Brown and Metheany, J. Pharm. Sci. 63:1214 (1974)]. 
Fenclofenac [2-(2,4-dichlorophenoxy)phenylacetic acid] is a diphenyl ether 
having antirheumatic activity that has been reported to interfere with 
thyroid function tests [Lancet 1:267 (Feb. 2, 1980), Lancet 1:432 (Feb. 
23, 1980), Lancet 1:487 (Mar. 1, 1980), Capper et al, Clin. Chim. Acta 
112:77(1981), and Kurtz et al, Clin. Endocrinol. 15:117(1981)]. Subsequent 
workers have raised the question whether fenclofenac would be suitable as 
a TBG blocking agent in thyroid function radioimmunoassays [Ratcliffe et 
al, Clin. Endocrinol. 13:569(1980)]. Capper et al, supra, also studied the 
effect of diclofenac [2-(2,6-dichlorophenylamino)phenylacetic acid]. 
SUMMARY OF THE INVENTION 
It has now been found that certain phenylacetic acids and salts are 
particularly advantageous TBP blocking agents for use in iodothyronine 
immunoassays. The blocking agent compound is included in the immunoassay 
reaction mixture at a concentration sufficient to release and block the 
binding of an analytically significant percentage of TBP-complexed 
iodothyronine, preferably more than 50% and usually more than 70%, while 
insufficient to interfere significantly with the binding of antibody with 
iodothyronine. While the precise concentrations of the blocking agent 
desired for a particular iodothyronine immunoassay will vary according to 
the iodothyronine under assay and the immunoassay technique followed, as 
well as other factors, the compound is normally used in concentrations in 
the reaction mixture of between about 0.1 millimolar (mM) and about 5 mM, 
particularly where the iodothyronine involved is thyroxine. The blocking 
agents of the present invention are added to the assay reaction mixture as 
the acid or an analytically acceptable salt form thereof, e.g., the 
sodium, potassium, lithium and ammonium salts. 
Certain unexpected properties of the present blocking agents, particularly 
fenclofenac, make them particularly advantageous for use as TBP blocking 
agents in homogeneous competitive binding immunoassays wherein a 
spectrophotometric response, such as a fluorescence emission or light 
absorption, is generated in the assay reaction mixture at a wavelength 
greater than about 300 nanometers (nm), and usually less than 700 nm, 
which response is a function of the concentration of the iodothyronine in 
the test sample. The present blocking agents have been found to have 
substantially no absorption at wavelengths greater than 300 nm. Thus, 
where the spectrophotometric response is a fluorescence emission, or is 
initiated, although not ultimately expressed, as a fluorescence emission, 
no quenching of such emission is observed when using the present compounds 
as the TBP blocking agent, whereas with prior art agents, particularly 
ANS, a significant quenching can occur resulting in undesirable or 
unacceptable assay performance characteristics, e.g., decreased 
sensitivity, reproducibility, precision, etc. 
Additionally, fenclofenac in particular will exhibit no substantial 
inhibitory effect on the catalytic activity of many enzymes at 
concentrations in which it is an effective TBP blocking agent. Thus, this 
compound is further advantageous as a TBP blocking agent in homogeneous 
competitive binding immunoassays wherein the label employed is a 
participant in an enzymatic reaction, e.g., an enzyme substrate, an enzyme 
inhibitor, a prosthetic group of an enzyme, a coenzyme, or an enzyme 
itself, or a fragment thereof. Prior art TBP blocking agents, particularly 
ANS, can cause significant inhibition of enzyme reactions resulting again 
in decreased assay performance. 
Therefore, the present phenylacetic acids and salts find novel use as TBP 
blocking agents in immunoassays in general, and are particularly 
advantageous when applied to spectrophotometric homogeneous immunoassays, 
especially, in the case of fenclofenac, those in which the label employed 
is a participant in an enzyme-catalyzed reaction. The present invention 
also provides reagent means for performing the novel immunoassays, 
particularly in the form of test kits as commonly used in clinical 
laboratories. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The phenylacetic acids of the present invention are generally of the 
formula: 
##STR2## 
wherein Y is O or NH and one of R.sup.1 and R.sup.2 is chloro and the 
other is hydrogen, and have been found to have unexpected features as TBP 
blocking agents. Fenclofenac (Y=O and R.sup.1 =Cl) has been found to be 
expecially advantageous in this respect, particularly in homogeneous 
iodothyronine immunoassays. Another compound of interest is diclofenac 
(Y=NH and R.sup.2 =Cl). It will, however, be evident to one of ordinary 
skill in the art that various modifications can be made to the basic 
diphenyl structure of the formula above without departing from the present 
inventive concept. Analogs possessing the advantageous TBP blocking agent 
features of the present invention will be considered as equivalents for 
the purposes of the claims hereof. For example, without limitation, the 
oxy or imino linking functionality may be replaced with a suitable 
analogous linker, including such groups as thio, methylene, and keto. 
Further, the dichloro substituents may be replaced by single or multiple 
substituents on either of the phenyl rings, such substituents being 
selected from halo, particularly chloro, bromo, and iodo; alkyl, usually 
lower alkyl (C.sub.1-4), e.g., methyl and ethyl; and alkoxy, usually lower 
alkoxy, e.g., methoxy and ethoxy. Also, the acetate substituent may be 
representative of a series of acid groups, e.g, carboxylic and sulfonic 
acids and their alkyl homologs, particularly the lower alkanoic homologs, 
and such groups may be bonded to a phenyl ring at either the meta or para 
position in addition to the ortho position in the formula. See also U.S. 
Pat. No. 3,766,263. 
The present invention has applicability to iodothyronine immunoassays in 
general. For the purposes hereof, an immunoassay will be understood to 
mean any assay based on antigen-antibody interactions and antibody will be 
understood to mean whole conventional or monoclonal antibody (e.g., of the 
IgG, IgM, IgA, etc., types) or an effective fragment thereof (e.g., Fab, 
F(ab'), etc. fragments of IgG). The most common type of immunoassay to 
which the present invention will be advantageously applied is the 
competitive binding immunoassay. In such an immunoassay for determining an 
iodothyronine, a test sample of a body fluid, usually serum or plasma, is 
combined with an antibody to the iodothyronine under assay, a labeled form 
of the iodothyronine, and a blocking agent for TBP binding. The proportion 
of labeled iodothyronine that becomes bound to the antibody in competition 
with any iodothyronine in the sample compared to that which remains 
unbound is related to the concentration of the iodothyronine in the 
sample. 
Both homogeneous and heterogeneous immunoassay techniques may be followed, 
the former being particularly preferred. In heterogeneous immunoassays, 
the antibody-bound form of the labeled iodothyronine is physically 
separated, as is known in the art, from the unbound form and the label 
measured in one or the other of the separated phases. Various different 
labels are known for use in heterogeneous immunoassays, including 
radioactive isotopes (e.g., U.S. Pat. Nos. 4,111,656 and 3,911,096), 
fluorescers (e.g., U.S. Pat. Nos. 4,201,763; 4,171,311; 4,133,639; and 
3,992,631), enzymes (e.g., U.S. Pat. No. 3,654,090), and so forth. In 
radioimmunoassays for iodothyronines it is particularly advantageous to 
use radioactive iodine as the label, substituting same for one of the 
native iodines in the iodothyronine. 
In homogeneous immunoassays, which are particularly preferred in the 
present invention, the antibody-bound form of the labeled iodothyronine 
expresses a different property from the unbound form and thus the 
separation step required in heterogeneous assays can be avoided. A wide 
variety of homogeneous immunoassay techniques are known in the art. 
Particularly preferred are those wherein the label which is chemically 
conjugated to the iodothyronine is an enzyme, or an enzyme fragment, e.g., 
a prosthetic group, or is a participant in an enzyme-catalyzed reaction, 
e.g., a substrate, a coenzyme, an inhibitor, an activator, or the like. 
The present invention is particularly applicable to homogeneous competitive 
binding immunoassays wherein a spectrophotometric response is generated in 
the assay reaction mixture at a wavelength greater than about 300 nm, and 
usually less than 700 nm, which response is indicative of the 
iodothyronine concentration in the test sample. The present blocking 
agents have been found to have substantially no absorption at such 
wavelengths. By spectrophotometric response is meant an optically 
detectable signal, usually measured at a selected wavelength or 
wavelengths. Exemplary of such signals are light emissions, e.g., 
chemiluminescence (including bioluminescence) and fluorescence, and light 
absorptions or reflections, e.g., color changes or formations, and 
measurable absorbance or reflectance changes in the visible spectrum. The 
following are examples of such assay types: 
1. Fluorescence quenching or enhancement 
The labeled conjugate in this system is composed, in its label portion, of 
a fluorescer whose fluorescence is quenched or enhanced in some measurable 
degree when the labeled iodothyronine conjugate is bound by antibody. The 
fluorescent label is usually measured directly, with its fluorescence 
being the detectable signal. Assay systems of this type are described in 
U.S. Pat. Nos. 4,160,016 and 3,940,475; in U.K. Pat. Spec. 1,583,869; and 
in J. Clin. Path. 30:526 (1977). 
2. Fluorescence polarization 
The label in this system is also a fluorescer; however, the affected 
characteristic is polarization of fluorescence due to binding of the 
labeled conjugate by antibody. Assay systems of this type are described in 
J. Exp. Med. 122:1029(1975). 
3. Enzyme substrate-labeled techniques 
In this system, the label is selected so that the labeled conjugate is a 
substrate for an enzyme and the ability of the enzyme to act on the 
substrate-labeled conjugate is affected, either in a positive or negative 
sense, by binding of the labeled conjugate with antibody. Action of the 
enzyme on the substrate-labeled conjugate produces a product that is 
distinguishable in some feature, usually a chemical or physical feature 
such as chemical reactivity in an indicator reaction or such as a 
photometric character, e.g., fluorescence or light absorption (color). 
Assay systems of this type are described in general terms in commonly 
assigned, copending application Ser. No. 894,836, filed Apr. 10, 1978 
(corresponding to U.K. Pat. Spec. No. 1,552,607); and in Anal. Chem. 
48:1933(1976), Anal. Biochem. 77:55(1977) and Clin. Chem. 23:1402(1977). 
In such enzyme substrate-labeled techniques, the labeled conjugate, e.g., 
a substrate-analyte conjugate, will have the property that it can be acted 
upon by an enzyme, by cleavage or modification, to produce a product 
having a detectable property which distinguishes it from the conjugate. 
For example, the conjugate may be nonfluorescent under assay conditions 
but upon reaction with enzyme a fluorescent product is produced. 
Various fluorogenic substrate-labeled conjugates are evident for use in 
such techniques. For example, the labeled conjugate may be of the formula: 
EQU G--D--R--L 
wherein G is a cleavable group such as phosphate, carboxylate, or glycone, 
D is a fluorogenic dye moiety which upon removal of G yields a fluorescent 
product, e.g., D can be umbelliferone, fluorescein, rhodamine, and their 
derivatives, R is a linking group and L is the binding component, usually 
the analyte (e.g., an iodothyronine) or a derivative thereof. Enzymatic 
cleavage (e.g., by phosphatase, carboxylase, glycosidase, etc.) of the 
labeled conjugate is affected by binding, such as by antibody, to the L 
portion of the conjugate. See U.S. Pat. No. 4,279,992. A particularly 
preferred substrate-labeled assay scheme employs a labeled conjugate of 
the type: 
##STR3## 
wherein R is a linking group and L is the binding component, e.g., the 
analyte or analog thereof, whereby the ability of the enzyme 
.beta.-galactosidase to cleave the conjugate yielding a product 
distinguishable by its fluorescence is inhibited by binding of the 
conjugate with antibody. 
Other useful substrate-labeled conjugates are those of the formula: 
EQU D--R--L 
wherein R is an enzyme cleavable linking group, e.g., phosphate, 
carboxylate, and the like, L is the binding component as above, and D is a 
fluorogenic dye moiety as above which upon cleavage of R releases a 
fluorescent indicator. A particularly preferred technique employs a 
labeled conjugate of the type: 
##STR4## 
wherein R.sup.1 is a bond or chain linking the labeled component L to the 
cleavable phosphate group and R.sup.2 is hydrogen or a substituent group 
such as lower alkyl, e.g., methyl and ethyl, N-alkylamido or 
N-(hydroxy-substituted lower alkyl)amido, e.g., --CONH--CH.sub.2).sub.n OH 
wherein n=2-6 (see U.S. Pat. No. 4,273,715). The umbelliferone residue may 
bear other or additional substituents [see Anal. Chem. 40:803(1968)]. 
Cleavage by phosphodiesterase is affected by binding of antibody to the L 
portion of the conjugate. 
4. Energy transfer 
In this system, the label is one member of an energy transfer 
donor-acceptor pair and the antibody is conjugated with the other of such 
pair. Thus, when the labeled conjugate is bound by antibody, the energy 
expression of the donor component of the pair is altered by transfer to 
the acceptor component. Usually, the donor is a fluorescer and the 
acceptor is a quencher therefor, which quencher may or may not be a 
fluorescer as well. In such embodiment, the detectable signal is 
fluorescence, but other detectant systems are possible also. Such assay 
systems are described in U.S. Pat. Nos. 3,996,345; 4,174,384; and 
4,199,559 and in U.K. Pat. Spec. No. 2,018,424. 
5. Chemically-excited fluorescence 
In this system, the label is again a fluorescer, however, the ability of 
the fluorescer label to be chemically excited to an energy state at which 
it fluoresces is affected by binding of the labeled conjugate with 
antibody. Chemical excitation of the label is usually accomplished by 
exposure of the fluorescer label to a high energy compound formed in situ. 
Assay systems of this type are described in commonly owned U.S. Pat. No. 
4,238,195. 
6. Double antibody steric hindrance 
Another assay system is the double antibody immunoassay system described in 
U.S. Pat. Nos. 3,935,074 and 3,998,943. The labeled conjugate comprises 
two epitopes, one of which participates in the immunological reaction with 
the ligand and anti-ligand antibody and the other of which is bindable by 
a second antibody, with the restriction that the two antibodies are 
hindered from binding to the labeled conjugate simultaneously. The second 
epitope can be a fluorescent substance whose fluorescence is quenched by 
the second antibody binding, or may participate in an ancillary 
competitive binding reaction with a labeled form of the second epitope for 
binding to the second antibody. Various detectant systems are possible in 
such a system as described in the aforementioned patents. Related assay 
systems are described in U.S. Pat. Nos. 4,130,462 and 4,161,515 and in 
U.K. Pat. Spec. No. 1,560,852. 
7. Prosthetic group-labeled techniques 
In this system, the label is a prosthetic group of an enzyme, and the 
ability of a catalytically inactive apoenzyme to combine with the 
prosthetic group label to form an active enzyme (holoenzyme) is affected 
by binding of the labeled conjugate with antibody. Resulting holoenzyme 
activity is measurable by conventional detectant systems to yield an 
ultimate detectable signal. Assay systems of this type are described in 
commonly owned U.S. Pat. No. 4,238,565. A particularly preferred 
prosthetic group-labeled assay scheme employs flavin adenine dinucleotide 
(FAD) as the label and apoglucose oxidase as the apoenzyme. Resulting 
glucose oxidase activity is measurable by a colorimetric detectant system 
comprising glucose, peroxidase, and an indicator system which produces a 
color change in response to hydrogen peroxide. Fluorometric detection of 
hydrogen peroxide is also possible using an appropriate fluorogenic 
substrate. 
8. Coenzyme-labeled techniques 
The labeled conjugate in this system is composed, in its label portion, of 
a coenzyme-active functionality, and the ability of such coenzyme label to 
participate in an enzymatic reaction is affected by binding of the labeled 
conjugate with antibody. The rate of the resulting enzymatic reaction is 
measurable by conventional detectant systems to yield an ultimately detect 
able signal. Assay systems of this type are described in commonly 
assigned, copending application Ser. No. 894,836, filed Apr. 10, 1978 
(corresponding to U.K. Pat. Spec. No. 1,552,607); and in Anal. Biochem. 
72:271(1976), Anal. Biochem. 72:283(1976) and Anal. Biochem. 76:95 (1976). 
9. Enzyme modulator-labeled techniques 
The labeled conjugate in this system is composed, in its label portion, of 
an enzyme modulating functionality such as an enzyme inhibitor or 
stimulator, and the ability of such modulator label to modulate the 
activity of an enzyme is affected by binding of the labeled conjugate with 
antibody. The rate of the resulting enzymatic reaction is measurable by 
conventional detectant systems to yield an ultimately detectable signal. 
Assay systems of this type are described in U.S. Pat. Nos. 4,134,792 and 
4,273,866. Particularly preferred is the use of methotrexate as the label 
with dihydrofolate reductase as the modulated enzyme. 
10. Enzyme-labeled techniques 
In this system, the label is an enzyme and the activity of the enzyme label 
is affected by binding of the labeled conjugate with antibody. Resulting 
enzyme activity is measurable by conventional detectant systems to yield 
an ultimately detectable signal, e.g., absorption or fluorescence. Assay 
systems of this type are described in U.S. Pat. Nos. 3,817,837 and 
4,043,872. 
Other homogeneous competitive binding immunoassay techniques can be 
followed without departing from the present inventive concept. 
Since fenclofenac in particular also will have insubstantial inhibitory 
effect on the catalytic activity of many enzymes at concentrations in 
which it is effective as a TBP blocking agent, the present invention is 
further advantageous in homogeneous immunoassays involving enzymatic 
reactions. Such assays include the enzyme substrate-labeled, prosthetic 
group-labeled, coenzyme-labeled, enzyme modulator-labeled, and 
enzyme-labeled techniques described above. By insubstantial inhibitory 
effect on enzymatic activity is meant that the rate of catalysis is not 
decreased more than about 70%, more usually less than 50%, and preferably 
less than 30%. 
The biological fluid to be tested may be any in which the iodothyronine(s) 
of interest may be undesirably associated with binding proteins. In the 
usual situation, the biological fluid is a blood sample such as whole 
blood, serum or plasma. 
The reagent means of the present invention comprises all of the essential 
chemical elements required to conduct a desired iodothyronine immunoassay 
method encompassed by the present invention. The reagent means is 
presented in a commercially packaged form, as a composition or admixture 
where the compatibility of the reagents will allow, in a test device 
configuration, or as a test kit, i.e., a packaged combination of one or 
more containers holding the necessary reagents. Included in the reagent 
means are the reagents appropriate for the binding reaction system desired 
and having a compound of the present invention, e.g., fenclofenac, as a 
TBP blocking agent. Such binding reaction reagents usually include, in 
addition to the blocking agent, a labeled iodothyronine conjugate, 
antibody to the iodothyronine under assay, and possibly other TBP blocking 
agents as may be desired. Of course, the reagent means can include other 
materials as are known in the art and which may be desirable from a 
commercial and user standpoint, such as buffers, diluents, standards, and 
so forth. Particularly preferred is a test kit for the homogeneous 
competitive binding immunoassay of the present invention comprising (a) an 
antibody to the iodothyronine to be determined, (b) a labeled 
iodothyronine conjugate which has a detectable property which is altered 
when bound with the antibody and (c) a compound of the present invention 
as a TBP blocking agent. The specific label used will depend on the 
technique followed, as described hereinabove. Also preferred is a test 
device comprising a reagent composition including an iodothyronine 
antibody, a labeled iodothyronine conjugate which has a detectable 
property which is altered when bound with the antibody, and a compound of 
the present invention as a TBP blocking agent, and a solid carrier member 
incorporated with the reagent composition. The various forms of such test 
device are described in U.S. patent application Ser. No. 202,378, filed 
Oct. 30, 1980, which is incorporated herein by reference.

The present invention will now be illustrated, but is not intended to be 
limited, by the following examples. 
EXAMPLES 
I. Dissociation of Thyroxine from Human Serum with Fenclofenac and 
Diclofenac 
Approximately 3 milliliters (ml) of human serum was allowed to equilibrate 
with radioactive iodine-labeled thyroxine (.sup.125 I-thyroxine obtained 
from Amersham-Searle, Arlington Heights, Ill., USA) for about 8 hours. 
Then 100 microliter (.mu.l) aliquots of this serum were combined with 300 
.mu.l aliquots of 0.1 molar (M) sodium phosphate buffer, pH 6.5, 
containing various concentrations of fenclofenac (British Pat. No. 
1,308,327; example 6). Then 180 .mu.l aliquots of each of these mixtures 
were applied to 2 ml columns of Sephadex LH-20 (Pharmacia Fine Chemicals, 
Piscataway, N.J. USA) equilibrated with 0.1M sodium phosphate buffer, pH 
6.5. The radioactivity on the columns was measured and the columns were 
washed with 5 ml of the buffer. The radioactivity of each column was 
measured again and the results (shown below in Table 1) used as estimates 
of thyroxine dissociated from serum proteins. 
TABLE 1 
______________________________________ 
Fenclofenac Percent Thyroxine 
(mM) Dissociated 
______________________________________ 
0 19 
0.25 50 
0.50 72 
1.0 79 
2.0 98 
5.0 100 
______________________________________ 
A second experiment was run to compare the dissociation characteristics of 
fenclofenac and diclofenac. Radioactive iodine labeled thyroxine (.sup.125 
I-thyroxine obtained from Amersham-Searle, Arlington Heights, IL, USA) was 
equilibrated with 5 ml of normal human serum for 48 hours at 4.degree. C. 
Aliquots of this serum (100 .mu.l) were added to 300 .mu.l of 0.1M sodium 
phosphate, pH 7.0, containing various concentrations of fenclofenac or 
diclofenac (see Example XI) to give the final concentrations given in 
Table 1A. After 5 minutes of incubation at room temperature, a 165 .mu.l 
aliquot was applied to a Sephadex.RTM. column from a Seralute.RTM. 
thyroxine assay kit (Miles Laboratories, Inc., Ames Division, Elkhart, IN, 
USA), which had been equilibrated with 0.1M sodium phosphate, pH 7.0. The 
total radioactivity applied to the columns was measured and the 
undissociated material was washed through the column with 3 ml of buffer. 
The columns were counted to determine the percentage of thyroxine 
dissociated from the serum proteins. 
TABLE 1A 
______________________________________ 
Dissociating Agent 
Percent Dissociated 
Percent Dissociated 
Concentration (mM) 
(Fenclofenac) (Diclofenac) 
______________________________________ 
0 9 9 
0.25 40 48 
0.50 61 61 
1.00 71 73 
2.00 75 77 
4.00 79 80 
8.00 81 82 
______________________________________ 
Therefore, 0.25 mM fenclofenac in an iodothyronine immunoassay reaction 
mixture can be expected to release and block the binding of about 40-50% 
of the protein-bound iodothyronine, and 0.50 mM about 60-70%. The second 
study showed that both dissociating agents are equally effective in 
dissociating iodothyronine from serum proteins in an immunoassay reaction. 
II. Effect of Fenclofenac on the Binding of Thyroxine with Antibody 
A series of antibody binding reactions were set up in 0.1M sodium phosphate 
buffer, pH 6.5, to give final volumes of 0.6 ml containing various 
concentrations of fenclofenac. Each reaction contained .sup.125 
I-thyroxine, 20 .mu.l of normal rabbit immunoglobulin, and 2 .mu.l of 
antibody to thyroxine. The mixtures were incubated at room temperature for 
about 3 hours and then 400 .mu.l of 50% (w/v) polyethyleneglycol was 
added. The precipitated proteins were collected by centrifugation and the 
radioactivity in each precipitate was measured. The results are shown in 
Table 2 as percent of the radioactivity in the precipitate without 
fenclofenac. 
TABLE 2 
______________________________________ 
Fenclofenac Percent .sup.125 I-Thyroxine 
(mM) in Precipitate 
______________________________________ 
0 100 
0.2 97 
0.5 80 
1.0 69 
2.5 61 
5.0 43 
______________________________________ 
The data indicate that concentrations of fenclofenac below about 5 mM 
inhibit the antibody binding reaction only about 50% and below about 2.5 
mM only about 40%. Based on this data and that of Example I, preferred 
fenclofenac concentrations in a thyroxine immunoassay reaction mixture 
would be in the range of 0.25-1.0 mM. 
III. Radioimmunoassay for Thyroxine 
A radioimmunoassay for thyroxine in serum was conducted using fenclofenac 
as the TBP blocking agent. Serum standards in 100 .mu.l aliquots 
containing known concentrations of thyroxine were combined with 290 .mu.l 
of 0.1M sodium phosphate buffer, pH 6.5, an amount of fenclofenac to give 
a concentration of 0.67 mM in the final assay mixture, 2 .mu.l of rabbit 
antibody to thyroxine, and a fixed amount of .sup.125 I-thyroxine 
(approximately 34,000 counts per minute per 100 .mu.l in the final 
volume). After incubation at room temperature for 2 hours, 400 .mu.l of 
50% (w/v) polyethyleneglycol was added and the resulting precipitates 
collected by centrifugation. The radioactivity of each precipitate was 
then measured. The results are shown in Table 3 (.mu.g/dl is micrograms 
per deciliter). 
TABLE 3 
______________________________________ 
Thyroxine Counts per Minute 
(.mu.g /dl) in Precipitate 
______________________________________ 
0 23,700 
1.0 21,800 
2.5 17,300 
5.0 14,400 
10.0 10,000 
20.0 7,500 
______________________________________ 
The data indicate that as the thyroxine level in the serum sample 
increased, the amount of labeled thyroxine bound to antibody decreased. It 
was accordingly demonstrated that fenclofenac can be used effectively in 
competitive binding immunoassays for the iodothyronine thyroxine in serum. 
IV. Optical Absorption Spectra of Fenclofenac and Diclofenac 
A 50 mM solution of fenclofenac in dilute sodium hydroxide solution was 
prepared and observed to have no visible color. The optical absorption 
spectrum of a 0.5 mM solution in 0.1M sodium phosphate buffer, pH 6.5, was 
recorded and showed no significant absorption above 300 nanometers (nm). 
Accordingly, fenclofenac could have no significant effect on 
spectrophotometric signals generated above such wavelength. 
In contrast, the conventionally used blocking agent ANS has a significant 
absorption above 300 nm. A 50 .mu.M solution of ANS in 0.1M phosphate 
buffer, pH 7.0, showed a broad absorption band from 300-400 nm with a peak 
of 0.5 at about 350 nm. Thus, ANS gives significant absorption at very low 
concentrations, concentrations far below those at which ANS is normally 
used as a blocking agent (around 1 mM). 
In a second study, solutions of fenclofenac and diclofenac were prepared by 
dissolving them in 0.1M sodium hydroxide and the spectra were made with a 
Bausch & Lomb Spectronic 2000 dual beam scanning spectrophotometer. 
TABLE 3A 
______________________________________ 
Fenclofenac* 
Diclofenac* 
______________________________________ 
.lambda.max 275 275 
269 
NaOH 
.epsilon. 2.06 .times. 10.sup.3 
11.8 .times. 10.sup.3 
max 2.05 .times. 10.sup.3 
.lambda.min 257 248 
NaOH 
.epsilon. 1.52 .times. 10.sup.3 
6.4 .times. 10.sup.3 
min 
______________________________________ 
*Wavelength in nanometers and extinction coefficient is M.sup.-1 
.multidot. cm.sup.-1. 
While both compounds have a near-UV absorbance spectrum, neither of them 
have any absorbance above 320 nm. Neither compound would contribute any 
interference with spectrophotometric generated signals above this 
wavelength. 
V. Effect of Fenclofenac on the Activation of Apoglucose Oxidase by 
FAD-labeled Conjugates 
A series of apoenzyme reactivation measurements were set up with different 
concentrations of fenclofenac. The assays were performed at 37.degree. C. 
and the final reagent concentrations in 0.1M phosphate buffer, pH 7.0, 
were 1.0 nanomolar (nM) FAD-labeled conjugate (an FAD-theophylline 
conjugate as described in U.S. Pat. No. 4,238,565), 50 nM apoglucose 
oxidase (U.S. Pat. No. 4,268,631), 2.5 .mu.l/ml anti(glucose oxidase) 
antiserum, 2 mM sodium dichlorohydroxybenzene sulfonate (DHSA), 0.2 mM 
4-aminoantipyrine, 0.1M glucose, 20 .mu.g/ml peroxidase, and 0.1% (w/v) 
bovine serum albumin. The apoenzyme and anti(glucose oxidase) were 
preincubated and the reaction then started by simultaneously mixing in the 
other reagents. The reaction mixtures were incubated for 5 minutes and 
then absorbance at 520 nm read. The results shown in Table 4 relate 
fenclofenac concentration to the generation of active glucose oxidase. 
TABLE 4 
______________________________________ 
Fenclofenac Percent Apoglucose 
(mM) Oxidase Activity 
______________________________________ 
0 100 
0.5 81 
1.0 66 
1.5 61 
2.0 64 
2.5 47 
______________________________________ 
The data indicate that fenclofenac concentrations below 2.5 mM permit the 
recombination of apoglucose oxidase and FAD-labeled conjugates to proceed 
at a rate sufficient for use of a prosthetic group-labeled immunoassay 
(U.S. Pat. No. 4,238,565). 
For the purposes of comparison, a series of apoenzyme reactivation 
measurements were set up with different concentrations of the 
conventionally used blocking agent ANS. The following reagents were 
prepared: 
Reagent A 
0.105M potassium phosphate buffer, pH 7.0 
0.105M Glucose 
2.2 mM DHSA 
21 .mu.g/ml peroxidase 
1.1% (w:v) bovine serum albumin 
5.26 nM FAD-labeled conjugate, supra 
Reagent B 
4 .mu.M apoglucose oxidase, supra 
30% (w:v) glycerol 
50 mM phosphate buffer, pH 7.0 
8 mM 4-aminoantipyrine 
ANS was dissolved directly into separate aliquots of Reagent A in the 
concentrations shown in Table 5. Apoenzyme activity was determined by 
placing 50 .mu.l of Reagent B in a cuvette and starting the reaction by 
addition of 1.90 ml of Reagent A. The assay reactions were incubated for 
10 minutes at room temperature and the absorbances at 520 nm recorded. The 
results are shown in Table 5. 
TABLE 5 
______________________________________ 
ANS Percent Apoglucose 
(mM) Oxidase Activity 
______________________________________ 
0 100 
0.1 64 
0.2 46 
0.5 13 
1.0 0 
______________________________________ 
The data show that ANS concentrations above about 1.0 mM totally inhibit 
the recombination reaction. Since ANS concentrations around this 
concentration are required for blocking agent purposes, ANS could not be 
used in the immunoassay. 
In a second study, the effect of fenclofenac on the activation of 
apoglucose oxidase by an FAD-iodothyronine (T-4) conjugate was 
investigated. The activation of apoglucose oxidase was set up with 
different concentrations of fenclofenac and performed at 37.degree. C. To 
two sets of assays containing 96 mM sodium phosphate, pH 7.0, 95 mM 
glucose, 1.0 mM sodium dichlorohydroxybenzene sulfonate (DHSA), 19 
.mu.g/ml peroxidase and various concentrations of sodium fenclofenac were 
added apoglucose oxidase, 4-aminoantipyrine, and anti(glucose oxidase) at 
final concentrations of 100 nM, 100 .mu.M, 5 .mu.l/ml respectively or 2.1 
nM final concentration of an FAD-thyroxine conjugate. After preincubating 
the assay media for 5 min at 37.degree. C. the FAD-thyroxine conjugate or 
the apoglucose oxidase reagent was added to the appropriate assay set. The 
absorbance at 520 nm was recorded after a 330 second incubation. The data 
are presented as a percentage of the absorbance recorded when no 
fenclofenac is present. 
TABLE 4A 
______________________________________ 
Started with Started with 
Fenclofenac (mM) 
Apoenzyme Addition 
Conjugate Addition 
______________________________________ 
0 100 100 
0.16 100 97 
0.31 98 92 
0.63 97 94 
1.25 93 95 
2.5 90 85 
5.0 78 74 
10.0 53 54 
______________________________________ 
The colorimetric response was diminished by only 8-10% at about 2 mM 
fenclofenac when an FAD-thyroxine conjugate was used to activate the 
apoglucose oxidase under the conditions employed for an actual 
iodothyronine immunoassay. 
VI. Studies on Fluorescence Quenching by Fenclofenac 
Fluorescence measurements were conducted in 50 mM Bicine buffer 
(N,N-bis-(2-hydroxyethyl)glycine, Calbiochem-Behring, LaJolla, Calif., 
USA), pH 8.3, using an Aminco Bowman Fluorometer (American Instruments, 
Silver Springs, Md., USA), excitation set at 400 nm and emission at 450 
nm, which are the fluorescence conditions for the 
.beta.-galactosyl-umbelliferone enzyme substrate-labeled fluorescent 
immunoassay (SLFIA) described in U.S. Pat. No. 4,279,992. Under these 
conditions, 10 mM fenclofenac did not exhibit any fluorescence. 
Quenching studies were performed by measuring the fluorescence of a 1.3 
.mu.M solution of 2-[7-hydroxy-3-carboxamidocoumarin]ethanol (U.S. Pat. 
No. 4,273,715) in the presence of various levels of fenclofenac. The 
ratios of observed fluorescence (F) to fluorescence in the absence of 
fenclofenac (Fo) versus fenclofenac concentration were calculated and are 
presented in Table 6. 
TABLE 6 
______________________________________ 
Fenclofenac 
(mM) F/Fo 
______________________________________ 
0 1.00 
0.2 1.00 
1.0 1.01 
2.0 1.00 
5.0 1.00 
10. 1.02 
______________________________________ 
The data indicate that fenclofenac exhibits essentially no quenching of the 
umbelliferone fluorescer used in the SLFIA technique and accordingly is 
well suited for use as a TBP blocking agent in such homogeneous 
immunoassay technique (U.S. Pat. No. 4,279,992). 
VII. Effect of Fenclofenac on the Activity of the Enzyme Dihydrofolate 
Reductase 
Assay mixtures were prepared to contain various concentrations of 
fenclofenac and, in a 1 ml final volume at 37.degree. C., 0.3 mM thiazoyl 
blue, 0.115M dihydrofolate, 0.5 mM NADPH, and 0.012 Units/ml of 
dihydrofolate reductase. The absorbance of each reaction mixture at 560 nm 
was read over a 10 minute incubation period at 37.degree. C. The results 
are given in Table 7. 
TABLE 7 
______________________________________ 
Fenclofenac Absorbance Change 
(mM) Over 10 Minutes 
______________________________________ 
0 0.360 
0.1 0.359 
0.2 0.365 
0.5 0.355 
1.0 0.332 
2.5 0.273 
5.0 0.118 
______________________________________ 
The data indicate that fenclofenac did not give substantial inhibition of 
enzyme activity at concentrations below 1.0 mM which are effective in 
dissociating thyroxine from serum proteins and accordingly is well suited 
for use as a TBP blocking agent in enzyme modulator-labeled homogeneous 
immunoassays (U.S. Pat. No. 4,134,792). 
VIII. Enzyme Inhibitor-Labeled Immunoassay for Thyroxine 
An enzyme inhibitor-labeled immunoassay (U.S. Pat. No. 4,134,792) for 
thyroxine in serum was conducted using fenclofenac as the TBP blocking 
agent. Serum standards in 40 .mu.l aliquots containing known 
concentrations of thyroxine were combined with 0.2 ml of an antibody 
reagent consisting of 21 .mu.l rabbit antiserum to thyroxine in 1 ml of 
0.1M sodium phosphate buffer, pH 6.5, containing 0.3M potassium chloride, 
0.05% sodium azide, and 0.5 mM fencolfenac. After a 30 second incubation, 
to each mixture was added 0.2 ml of a conjugate reagent consisting of 0.63 
mg/ml NADPH (0.65 mM), 0.0175 .mu.M of a methotrexate-thyroxine conjugate 
[prepared as described in the U.S. patent application filed on even date 
herewith, assigned to the present assignee, entitled "Methotrexate-Labeled 
Iodothyronine Conjugates Ser. No. 318,028, which application is 
incorporated herein by reference] in 10 mM sodium carbonate buffer, pH 
9.5. After another 30 second incubation, to each mixture was added 0.2 ml 
of an enzyme reagent consisting of dihydrofolate reductase at a 
concentration of 27.5 nM methotrexate binding sites in 0.1M Tris-HCl 
buffer (tris-(hydroxymethyl)aminomethane hydrochloric salt, 
Calbiochem-Behring, La Jolla, Calif., USA), pH 8.5, containing 0.5% (w:v) 
gelatin and 0.005% (w:v) chlorhexidine (Sigma Chemical, St. Louis, Mo., 
USA). After a further 5 minute incubation, to each mixture was added 50 
.mu.l of 2.5 mM dihydrofolate in 0.1M Tris.HCl, pH 8.5. Forty-five (45) 
seconds later, the absorbance of each solution at 340 nm was read over a 1 
minute period. The results are given in Table 8. 
TABLE 8 
______________________________________ 
Thyroxine Absorbance Change 
(.mu.g/dL) per Minute 
______________________________________ 
0 0.1682 
1.0 0.1659 
2.5 0.1648 
10. 0.1571 
20. 0.1498 
______________________________________ 
Accordingly, as the thyroxine level in the serum sample increased, the 
amount of enzyme inhibition by the inhibitor-thyroxine conjugate 
increased. Fenclofenac did not substantially interfere with either the 
spectrophotometric response at 340 nm or the enzymatic reaction. 
VIII. Enzyme Substrate-Labeled Immunoassay for Thyroxine 
A. Synthesis of labeled conjugate-5-(thyroxinamido) pentyl, 
4-methylumbelliferone, hydrogen phosphate 
##STR5## 
A solution of 8.73 g (10 mmol) of N-trifluoroacetyl L-thyroxine, 1.133 g 
(11 mmol) of 5-amino-1-pentanol, and 2.7 g (20 mmol) of 
1-hydroxybenzotriazole in 125 ml of dry dimethylformamide (DMF) was cooled 
to -5.degree. C. while stirring under argon. To this was added 2.28 g (11 
mmol) of dicyclohexylcarbodiimide. The cooling bath was removed and the 
reaction allowed to come to room temperature and stir for 3 hours. The 
solvent was removed in vacuo. The residue was taken up in 250 ml of ethyl 
acetate, filtered, and washed with 150 ml of saturated aqueous sodium 
bicarbonate solution and 5% aqueous citric acid solution. This residue was 
purified by preparative liquid chromatography in a slica gel column 
eluting with 5:1 (v/v) methylene chloride:acetone. This gave 6.5 g (67% 
yield) of the N-(5-hydroxypentyl)amide of N-trifluoroacetylthyroxine as a 
white solid, mp 202.degree.-203.degree. C. 
Analysis: Calculated for C.sub.22 H.sub.21 F.sub.3 I.sub.4 N.sub.2 O.sub.5 
: C, 27.58; H, 2.21; N, 2.92. Found: C, 27.89; H, 2.18; N, 3.36. 
The N-(5-hydroxypentyl amide (5.75 g, 6 mmol) and 2.01 g (6 mmol) of the 
pyridinine salt of 4-methylumbelliferone-monophosphate were suspended in 
75 ml of dry DMF. The mixture was concentrated to about 25 ml in volume on 
a rotary evaporator attached to a vacuum pump. An additional 20 ml of dry 
DMF was added followed by 30 ml of dry pyridine. Solid 
dicyclohexylcarbodiimide (2.48 g, 12 mmol) was then added and the reaction 
stirred under argon at room temperature for 24 hours. Solvent was removed 
under vacuum and the residue stirred with 300 ml of 0.1M aqueous 
triethylammonium bicarbonate for 1 hour. The white precipitate was 
filtered and then stirred for 30 minutes in 300 ml of ether and filtered. 
This left 8 g of the desired product contaminated with dicyclohexyl urea. 
This residue (1.6 g) was taken up in methanol (some insoluble material was 
removed by filtration) and chromatographed on Sephadex LH-20 (60 cm 
.times.5 cm) eluting with methanol. The flow rate was 0.5 ml/minute and 10 
ml fractions were collected. 
Fractions 69 through 76 were pooled and evaporated to give, as a white 
glassy solid, 390 mg of 5-[N-(trifluoroacetamido) thyroxinamido]pentyl, 
4-methyl hydrogen phosphate, triethylammonium salt. 
Analysis: Calculated for C.sub.38 H.sub.43 F.sub.3 I.sub.4 PN.sub.3 
O.sub.10 : C, 35.18; H, 3.34; N, 3.24. Found: C, 37.01; H, 3.63; N, 3.36. 
A 200 mg portion of the N-trifluoroacetamido-protected phosphodiester was 
dissolved in 50% aqueous methanol, pH 12, for 3 hours. The reaction was 
quenched with 0.5 ml of acetic acid and then concentrated to dryness under 
vacuum. The residue was taken up in methanol containing a little ammonium 
hydroxide, 2.5 g of silica gel added, and solvent removed. The impregnated 
adsorbent was placed atop a column of 25 g of silica gel made up in 10:5:1 
(v/v/v) chloroform:methanol:concentrated ammonium hydroxide. The column 
was eluted with this solvent and 9 ml fractions were collected. 
Fractions 10 through 21 were combined and evaporated to give 100 mg of 
5-(thyroxinamide)pentyl, 4-methylumbelliferone, hydrogen phosphate, as a 
white microcrystalline solid, mp 191.degree.-192.degree. C. (darkens from 
188.degree. C.). 
Analysis: Calculated for C.sub.30 H.sub.29 I.sub.4 PH.sub.2 O.sub.9.H.sub.2 
O: C, 32.22; H, 2.79; N, 2.50. Found: C, 32.37; H, 2.71; N, 2.46. 
B. Assay method 
The following reagents were assembled: 
Reagent A: 50 mM Bicine buffer with 0.1% sodium azide, pH 8.5 
Reagent B: 60 mM fenclofenac in 0.5N sodium hydroxide 
Reagent C: thyroxine standards with concentrations of 0, 2, 4, 6, 8, 10, 
12, 16 and 20 .mu.g/dl prepared by adding thyroxine (Sigma) to thyroxine 
free serum. 
Reagent D: antibody/enzyme reagent containing 50 .mu.l antiserum per ml and 
0.12 units per ml of phosphodiesterase (Sigma, Type VII) in Bicine buffer. 
Reagent E: 1.24 .mu.M of the labeled conjugate (Part A above) in 5 mM 
formate, 0.1% sodium azide buffer, pH 3.5. 
The assay protocol involved adding 75 .mu.l of Reagent B and 0.5 ml of 
Reagent A to a cuvette, followed by 75 .mu.l of an appropriate Reagent C 
and another 0.5 ml of Reagent A, and finally 75 .mu.l of Reagent D and a 
third 0.5 ml of Reagent A. After a 1 hour incubation at room temperature, 
the assay reaction was started by adding 75 .mu.l of Reagent E and 0.5 ml 
of Reagent A to the cuvette. The fluorescence (excitation=360 nm, 
emission=450 nm) was measured 20 minutes after addition of Reagent E. 
C. Results 
The assay was run for each Reagent C and the results were as shown in Table 
9. 
TABLE 9 
______________________________________ 
thyroxine fluorescence 
(.mu.g /dl) units 
______________________________________ 
0 24.9 
2 25.5 
4 26.4 
6 27.2 
8 26.7 
10 27.1 
12 30.0 
16 30.1 
20 30.8 
______________________________________ 
As thyroxine concentration increased, the fluorescence emission increased. 
Thus, an assay was established for thyroxine. 
IX. The Use of Fenclofenac or Diclofenac in an Apoenzyme Reactivation 
Immunoassay System for Serum Thyroxine 
Standard curves for serum thyroxine were generated using a semiautomated 
assay protocol on the Gilford Clinical Chemistry Analyzer System 203-S 
(Gilford Instrument Laboratories, Oberline, OH, USA). The buffer comprised 
of 96 mM sodium phosphate, pH 7.0, 2.1 mM dichlorohydroxybenzene sulfonate 
(DHSA), 21 .mu.g/ml peroxidase, 105 mM glucose and 2 mM sodium fenclofenac 
or sodium diclofenac was preheated to 37.degree. C. before 0.8 ml were 
added to the reaction cup with 0.05 ml of 200 .mu.l/ml thyroxine standards 
in T.sub.4, T.sub.3 free human serum (AMF Biological and Diagnostic 
Products, Sequin, TX, USA), 100 .mu.l/ml anti(glucose oxidase) antiserum, 
15 .mu.l/ml anti(thyroxine) antiserum, 0.03M sodium phosphate, pH 7.0. The 
FAD-thyroxine conjugate (40 nM in 0.1M sodium phosphate, pH 7.0, 0.01% 
Triton X-100) as a 0.05 ml aliquot was added to the reaction cup and 
allowed to equilibrate for 30 seconds. The reaction was initiated by 
addition of 0.10 ml 1.0 .mu.M apoglucose oxidase, 2 mM 4-aminoantipyrine, 
12% glycerol, 80 mM sodium phosphate, pH 7.0. The absorbance at 520 nm was 
recorded after an 8 minute incubation. 
TABLE 10 
______________________________________ 
Thyroxine 
Standard Absorbance Absorbance 
(.mu.g/L) (with Fenclofenac) 
(with Diclofenac) 
______________________________________ 
0 0.797 0.619 
25 0.823 0.620 
75 0.893 0.647 
125 0.965 0.678 
200 1.078 0.719 
______________________________________ 
With fenclofenac or diclofenac as the iodothyronine dissociating agent, a 
correlation between the absorbance readout and the concentration of 
thyroxine in serum can be observed using a homogeneous apoenzyme 
reactivation immunoassay system. 
X. Effect of pH on the Dissociation of Iodothyronine from Serum Proteins 
Using the column procedure described in the second study in Example 1, the 
pH of the 0.1M sodium phosphate was varied and the concentration of the 
dissociating agent held constant at 2.0 mM. The columns were equilibrated 
with buffer containing the dissociating agent at the given pH and 165 
.mu.l of the serum diluted in buffer at the given pH (as described above) 
was applied to the column. The total counts were measured and then the 
columns were washed with buffer at the given pH. The counts remaining on 
the column represent the percentage of iodothyronine dissociated. 
TABLE 11 
______________________________________ 
Percent Dissociated 
Percent Dissociated 
pH by Fenclofenac 
by Diclofenac 
______________________________________ 
6.0 84 86 
6.5 84 86 
7.0 86 86 
7.5 85 85 
8.0 83 85 
______________________________________ 
The dissociation of iodothyronine from serum proteins by fenclofenac or 
diclofenac is independent of pH (over the range 6.0-8.0) at which the 
incubation of the immunoassay is conducted. 
XI. Preparation of Diclofenac 
Following the method of Japanese Kokai patent document No. 80-79,352 (Chem. 
Abs. 94:121132u), 2-iodobenzoic acid was treated with thionyl chloride and 
dimethylamine to give N,N-dimethyl-2-iodophenylacetamide. Reaction upon 
heating with 2,6-dichloroaniline in the presence of potassium carbonate 
following the method of Japanese Kokai patent document No. 80-87,748 
(Chem. Abs. 94:30378q) gave N,N-dimethyl-o-(2,6-dichlorophenylamino) 
phenylacetamide. Hydrolysis with 15% potassium hydroxide (British Pat. 
Appln. No. 2,027,028) gave diclofenac 
[o-(2,6-dichlorophenylamino)phenylacetic acid].