U.S. patent number 4,134,792 [Application Number 05/748,005] was granted by the patent office on 1979-01-16 for specific binding assay with an enzyme modulator as a labeling substance.
This patent grant is currently assigned to Miles Laboratories, Inc.. Invention is credited to Robert C. Boguslaski, Robert J. Carrico, James E. Christner.
United States Patent |
4,134,792 |
Boguslaski , et al. |
January 16, 1979 |
Specific binding assay with an enzyme modulator as a labeling
substance
Abstract
A specific binding assay method employing, as a labeling
substance, a reversibly binding enzyme modulator for the detection
of a ligand in a liquid medium. The method follows conventional
specific binding assay techniques of either the homogeneous or
heterogeneous type wherein the liquid medium to be assayed is
combined with reagent means that includes a labeled conjugate to
form a binding reaction system having a bound-species and a
free-species of the conjugate. The amount of conjugate resulting in
the bound-species or the free-species is a function of the amount
of ligand present in the liquid medium assayed. In the present
invention, the labeled conjugate comprises a reversibly binding
enzyme modulator covalently linked to a binding component of the
binding reaction system. The distribution of the conjugate between
the bound-species and the free-species is determined by addition of
an enzyme whose activity is affected, either decreased or
increased, by said modulator and measuring the resulting activity
of the enzyme. The enzyme modulator may be a conventional enzyme
inhibitor, preferably of the coompetitive type, or an allosteric
effector.
Inventors: |
Boguslaski; Robert C. (Elkhart,
IN), Carrico; Robert J. (Elkhart, IN), Christner; James
E. (Ann Arbor, MI) |
Assignee: |
Miles Laboratories, Inc.
(Elkhart, IN)
|
Family
ID: |
25007583 |
Appl.
No.: |
05/748,005 |
Filed: |
December 6, 1976 |
Current U.S.
Class: |
435/4; 435/18;
435/188; 435/20; 435/25; 435/7.5; 435/7.71; 436/537; 436/815;
436/817 |
Current CPC
Class: |
G01N
33/542 (20130101); Y10S 436/817 (20130101); Y10S
436/815 (20130101) |
Current International
Class: |
G01N
33/536 (20060101); G01N 33/542 (20060101); G01N
033/00 (); G01N 031/14 () |
Field of
Search: |
;195/13.5A,99
;424/12 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Miles et al., Nature, vol. 219, (1968), pp. 186-189. .
Landon, "A Suggested Terminology for Non-Isotopic Immunoassays",
Conference on Non-isotopic Immunoassays, (1976)..
|
Primary Examiner: Tanenholtz; Alvin E.
Assistant Examiner: Fan; C. A.
Attorney, Agent or Firm: Klawitter; Andrew L.
Claims
What is claimed is:
1. In a specific binding assay method for determining a ligand in a
liquid medium,
wherein said liquid medium is combined with reagent means including
a conjugate of a labeling substance and a binding component to form
a binding reaction system having a bound-species and a free-species
of said conjugate; and
wherein the amount of ligand present in said liquid medium is
determined as a function of the amount of labeling substance
present in said bound-species or said free-species;
the improvement which comprises employing as said labeling
substance a reversibly binding enzyme inhibitor, the binding
constant for association of said inhibitor and an enzyme whose
activity is inhibited thereby being less than about 10.sup.11
molar.sup.-1 ; adding said enzyme whose activity is affected by
said enzyme inhibitor to said bound-species or said free-species;
and determining the amount of said enzyme inhibitor therein by
measuring the resulting activity of said enzyme.
2. A method as claimed in claim 1 wherein the binding constant for
the association of said reversibly binding enzyme inhibitor and
said enzyme is between about 10.sup.5 and about 10.sup.9
molar.sup.-1.
3. A method as claimed in claim 1 wherein said reversibly binding
enzyme inhibitor is a competitive inhibitor of the enzyme.
4. A method as claimed in claim 3 wherein said reversibly binding
competitive inhibitor and the enzyme inhibited thereby are,
respectively,
(a) acetazolamide, or an effective analog thereof, and carbonic
anhydrase;
(b) sulfanilamide, or an effective analog thereof, and carbonic
anhydrase;
(c) phenyl trimethylammonium ion, or an effective analog thereof,
and acetylcholinesterase;
(d) saccharo-1,4-lactone, or an effective analog thereof, and
.beta.-glucuronidase;
(e) 4-amino-10-methyl-pteroylglutamic acid, or an effective analog
thereof, and dihydrofolate reductase; or
(f) 2,6,8-trichloropurine, or an effective analog thereof, and
uricase.
5. A method as claimed in claim 1 wherein said enzyme inhibitor, as
a component of said conjugate, affects the activity of said enzyme
differently when said conjugate is in said bound-species than when
in said free-species.
6. A method as claimed in claim 5 of the homogeneous type wherein
said enzyme is added to the combined bound-species and
free-species.
7. A method as claimed in claim 1 of the heterogenous type wherein
said bound-species and said free-species are physically separated,
and said enzyme is added to one thereof.
8. A method as claimed in claim 1 wherein said ligand is selected
from the group consisting of antigens and antibodies thereto;
haptens and antibodies thereto; and hormones, vitamins,
metabolites, and pharmacological agents, and their receptors and
binding substances.
9. In a reagent means for use in determining a ligand in a liquid
medium, which means includes a conjugate of a labeling substance
and a binding component, which means and the ligand form a binding
reaction system having a bound-species and a free-species of said
conjugate,
the improvement wherein said labeling substance is a reversibly
binding enzyme inhibitor and said means comprises additionally an
enzyme whose activity is affected by said enzyme inhibitor, the
binding constant for association of said inhibitor and said enzyme
being less than about 10.sup.11 molar.sup.-1.
10. Means as claimed in claim 9 wherein the binding constant for
the association of said reversibly binding enzyme inhibitor and
said enzyme is between about 10.sup.5 and about 10.sup.9
molar.sup.-1.
11. Means as claimed in claim 9 wherein said reversibly binding
enzyme inhibitor is a competitive inhibitor of the enzyme.
12. Means as claimed in claim 11 wherein said reversibly binding
competitive inhibitor and the enzyme inhibited thereby are,
respectively,
(a) acetazolamide, or an effective analog thereof, and carbonic
anhydrase;
(b) sulfanilamide, or an effective analog thereof, and carbonic
anhydrase;
(c) phenyl trimethylammonium ion, or an effective analog thereof,
and acetylcholinesterase;
(d) saccharo-1,4-lactone, or an effective analog thereof, and
.beta.-glucuronidase;
(e) 4-amino-10-methyl-pteroylglutamic acid, or an effective analog
thereof, and dihydrofolate reductase; or
(f) 2,6,8-trichloropurine, or an effective analog thereof, and
uricase.
13. Means as claimed in claim 9 wherein said enzyme inhibitor, as a
component of said conjugate, affects the activity of said enzyme
differently when said conjugate is in said bound-species than when
in said free-species, said means comprising (i) said conjugate
wherein said binding component is said ligand, a specific binding
analog of said ligand, or a specific binding partner of said
ligand, and (ii) if said binding component is said ligand or a
specific binding analog thereof, a specific binding partner of said
ligand.
14. Means as in claim 9 which comprises (i) said conjugate wherein
said binding component is said ligand or a specific binding analog
of said ligand, and (ii) a specific binding partner of said
ligand.
15. Means as in claim 14 wherein one of said conjugate and said
specific binding partner is in a form which is insoluble in said
liquid medium.
16. Means as in claim 9 which comprises (i) said conjugate in a
form which is soluble in said liquid medium and wherein said
binding component is a specific binding partner of said ligand, and
(ii) said ligand or a specific binding analog thereof in a form
which is insoluble in said liquid medium.
17. Means as in claim 9 which comprises (i) said conjugate wherein
said binding component is a specific binding partner of said
ligand, and (ii) a specific binding partner of said ligand.
18. Means as in claim 17 wherein one of said conjugate and said
specific binding partner is in a form which is insoluble in said
liquid medium.
19. Means as in claim 9 wherein said ligand is selected from the
group consisting of antigens and antibodies thereto; haptens and
antibodies thereto; and hormones, vitamins, metabolites, and
pharmacological agents, and their receptors and binding substances.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to assay methods of the homogeneous and
heterogeneous specific binding type for determining qualitatively
or quantitatively a ligand in a liquid medium. In a preferred
embodiment, the present invention relates to competitive binding
immunoassays employing nonisotopic labels.
2. Description of the Prior Art
In conventional specific binding assay techniques, the test sample
is combined with reagent means of various compositions that include
a conjugate of a labeling substance linked to a binding component
which participates with other constituents, if any, of the reagent
means to form a binding reaction system producing two species of
the labeled conjugate, a bound-species and a free-species. The
relative amounts of the labeled conjugate that result in the
bound-species and the free-species are a function of the presence
or amount of the ligand to be detected in the test sample.
As an illustration, a conventional competitive binding assay
technique will now be described. In such a technique the reagent
means would comprise (1) a labeled conjugate in the form of the
ligand to be detected, or of an appropriate analog thereof,
chemically linked to a labeling substance and (2) a specific
binding partner for the ligand, such as an antibody or other
binding protein. Upon combination of the test sample and the
reagent means, the ligand to be detected and the labeled conjugate
would compete in a substantially nondiscriminating manner for
noncovalent binding to the specific binding partner. As a result,
the amount of labeled conjugate that would become bound to the
binding partner, i.e., in the bound-species, or that would remain
free, i.e., unbound to the binding partner, and, thus, in the
free-species, is a function of the amount of competing ligand
present.
Where the labeled conjugate in the bound-species and that in the
free-species are essentially indistinguishable by the means used to
measure the labeling substance, the bound-species and the
free-species must be physically separated in order to complete the
assay. This type of assay is referred to as "heterogeneous." Where
the bound-species and free-species forms of the labeled conjugate
can be distinguished, a "homogeneous" format may be followed and
the separation step avoided.
The first discovered type of specific binding assay was the
radioimmunoassay which employs a radioactive isotope as the
labeling substance. Such an assay necessarily must follow the
heterogeneous format. Because of the hazard and difficulty of
handling radioactive materials, many new assay systems have been
devised using materials other than isotopes as the labeling
substance, including enzymes, fluoroscent molecules,
bacteriophages, coenzymes, and luminescent molecules.
The following describe several different heterogeneous binding
reaction systems in which an enzyme is employed as the labeling
substance: U.S. Pat. Nos. 3,654,909; 3,791,932; 3,839,153;
3,850,752; and 3,879,262; J. Immunol. Methods 1:247 (1972); and J.
Immunol. 109:129(1972). A heterogeneous binding assay utilizing a
non-active precursor of a spectrophotometrically detectable
substance as the labeling substance is suggested in U.S. Pat. No.
3,880,934. Of further background interest pertaining to
heterogeneous assays is Principles of Competitive Protein-Binding
Assays, ed. Odell and Daughaday (J. B. Lippincott Co.,
Philadelphia, 1972). An enzyme-labeled immunoassay of the
homogeneous type is described in U.S. Pat. No. 3,817,834 wherein a
ligand-enzyme conjugate is employed. The enzymatic activity of the
conjugate in the bound-species is measurably less than that in the
free-species, thereby allowing a homogeneous format to be used. The
use of particularly unique materials as labeling substances,
including coenzymes, luminescent molecules, and cleavable
fluorescent substrates, in both homogeneous and heterogeneous
formats, is described in U.S. patent applications Ser. Nos. 667,982
and 667,996, filed on Mar. 18, 1976, and assigned to the instant
assignee.
It is a primary object of the present invention to provide a
specific binding assay method, useable in both the homogeneous and
heterogeneous modes, which employs a unique labeling substance
belonging to a large class of compounds for which there is an
extensive literature background to facilitate selection of specific
labels, approaches to derivatization, and monitoring reactions.
SUMMARY OF THE INVENTION
The present invention provides a specific binding assay method
employing, as a labeling substance, a reversibly binding enzyme
modulator. The inventive labeling substance may be used in both
homogeneous and heterogeneous binding assay formats wherein the
liquid medium to be assayed for a ligand is combined with reagent
means which includes a labeled conjugate to form a binding reaction
system having a bound-species and a free-species of the conjugate.
The amount of conjugate resulting in the bound-species or the
free-species is a function of the presence or amount of ligand
present in the liquid medium assayed. In the present invention, the
labeled conjugate comprises a reversibly binding enzyme modulator
covalently linked to a binding component of the binding reaction
system. The distribution of the conjugate between the bound-species
and the free-species is determined by addition of an enzyme whose
activity is affected, either in an inhibitory or stimulatory
manner, by said modulator and measuring the resulting activity of
the enzyme following the desired homogeneous or heterogeneous assay
scheme.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a graphic representation of the inhibitory effect of
various concentrations of biotin-modulator conjugate on the
esterase activity of carbonic anhydrase measured by hydrolysis
rate; and
FIG. 2 is a graphic representation of the effect of various
concentrations of avidin on the inhibitory effect of
biotin-modulator conjugate on the esterase activity of carbonic
anhydrase measured by hydrolysis rate.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the context of this disclosure, the following terms shall be
defined as follows: "ligand" is the substance, or group of
substances, whose presence or the amount thereof in a liquid medium
is to be determined; "specific binding partner of the ligand" is
any substance, or group of substances, which has a specific binding
affinity for the ligand to the exclusion of other substances;
"specific binding analog of the ligand" is any substance, or group
of substances, which behaves essentially the same as the ligand
with respect to the binding affinity of the specific binding
partner for the ligand; and "monitoring reaction" is a reaction
catalyzed by an enzyme whose activity is affected (i.e., decreased
or increased) by the enzyme modulator of the present invention.
The novel labeling substance of the present invention is any
chemical substance which binds specifically or nonspecifically with
an enzyme in a reversible, non-covalent manner and thereby
measurably affects, either in an inhibitory or stimulatory manner,
the catalytic activity of the enzyme relative to a predetermined
reaction, i.e., the monitoring reaction for the binding reaction
system formed in the present assay method. By "reversibly binding"
is meant that the binding constant for the association of the
enzyme modulator and the enzyme that catalyzes the monitoring
reaction is less than about 10.sup.11 molar.sup.-1. Preferably, the
binding constant is between about 10.sup.5 and about 10.sup.9
molar.sup.-1.
One class of substances which encompasses usable enzyme modulators
is the group of reversibly binding enzyme inhibitors, whether of
the competitive, uncompetitive, or noncompetitive type. Competitive
inhibitors combine with the free enzyme in such a way that they
compete with the normal substrate for binding at the active site of
the enzyme, however, the inhibitor molecule is not chemically
transformed by the enzyme. In contrast, uncompetitive inhibitors do
not combine with the free enzyme or affect its association with the
normal substrate; rather, they combine with the formed
enzyme-substrate complex to yield an inactive adduct that is
incapable of releasing the normal product. Noncompetitive
inhibitors, on the other hand, can combine with either the free
enzyme or the enzyme-substrate complex to interfere with normal
catalysis, usually by binding at a site on the enzyme other than
the active site to alter the sensitive configuration of the active
enzyme.
Of the conventional enzyme inhibitors, those of the competitive
type are preferred. Following is a table of some useful competitive
inhibitors, the enzymes whose activity is inhibited thereby, and an
approximation of the inhibitor constants (K.sub.i) for the
dissociation of the respective inhibitors from the enzymes.
______________________________________ Inhibitor Enzyme (K.sub.i
(molar) ______________________________________ acetazolamine
carbonic anhydrase 6.times.10.sup.-7 sulfanilamide carbonic
anhydrase 10.sup.-9 phenyl trimethyl- acetylcholin- 10.sup.-6
ammonium ion esterase saccharo-1,4- .beta.-glucuronidase 10.sup.-6
lactone 4-amino-10- dihydrofolate 10.sup.-9 methyl- reductase
pteroylglutamic acid 2,6,8-trichloro- uricase 10.sup.-6 purine
______________________________________
Effective analogs, i.e., derivatives, homologs, and so forth, of
the above-listed inhibitors can also be used. For example,
acetylcholinesterase is also inhibited by p-aminophenyl
trimethylammonium ion and also by the N-methyl-N-(p-aminophenyl)
carbamate ester of m-(trimethylamino) phenol.
Another class of substances from which a useful enzyme modulator
can be selected is that of allosteric effectors. Such substances
act in biological processes as regulators of enzymatic metabolism.
Their action is accomplished by binding specifically to a site on
the regulated enzyme other than the substrate active site and
thereby to inhibit or stimulate, such as by a conformational
change, the activity of the enzyme. Following is a table of some
allosteric effectors contemplated to be useful as the enzyme
modulator of the present invention. In the table, inhibitory or
negative effectors are indicated by (-) and stimulatory or positive
effectors are indicated by (+). The source of the affected enzyme
is also given in parentheses.
______________________________________ Allosteric Effector Enzyme
______________________________________ L-isoleucine (-)
biosynthetic L-threonine deaminase L-valine (+) (E. coli K12) and
(yeast) cytosine triphosphate aspartate transcarbamylase (E. coli)
(CTP) (-) adenosine triphosphate (ATP) (+) deoxythymidine tri-
deoxycytidylate aminohydrolase phosphate (dTTP) (-) (ass spleen)
deoxycytosine triphosphate (dCTP) (+) ATP(-) phosphofructokinase
(guinea pig 3',5'-adenosine heart) monophosphate (AMP) (+) dTTP(-)
deoxythymidine kinase (E. coli) deoxycytosine diphos- phate dCDP(+)
.alpha.-ketoglutarate(-) nicotinamide adenine dinucleotide citrate
(+) (NAD)-isocitric dehydrogenase (N. crassa) 5'-AMP(+)
NAD-isocitric dehydrogenase (yeast) L-threonine(-) homoserine
dehydrogenase (R. rubrum) L-isoleucine(+) L-methionine(+)
addenosine diphosphate L-threonine deaminase (ADP) (+) (C.
tetanomorphum) L-valine(+) acetolactate synthetase (E. coli)
L-threonine(-) threonine aspartokinase (E. coli) uridine
diphosphate L-glutamine-D-fructose-6-phosphate (UDP)-N-acetyl-
transaminase (rat liver) glucosamine(-) glucose-6-phosphate(+)
glycogen synthetase (yeast) and (lamb muscle) ATP(-) glutamate
dehydrogenase (beef liver) guanosine triphosphate (GTP) (-) reduced
NAD(-) estrogens(-) thyroxine(-) ADP(+) leucine(+) methionine(+)
ATP(-) phosphorylase b (rabbit muscle) 5'-AMP(+) cytosine
monophosphate UDP-N-acetyl-glucosamine-2-epimerase (CMP)-N-acetyl-
(rat liver) neuraminic acid(-) L-threonine(-) homoserine
dehydrogenase (E. coli) L-lysine(-) lysine aspartokinase (E. coli)
5'-AMP(-) fructose-1,6-diphosphatase (frog muscle) and (rat liver)
______________________________________
Further details may be found in J. Mol. Biol. 12:88(1965).
To form the labeled conjugate which participates in the binding
reaction system, the enzyme modulator is covalently linked to an
appropriate binding component of such reaction system. Such binding
component is the ligand to be detected, a specific binding analog
of the ligand, or a specific binding partner of the ligand,
depending upon the binding reaction scheme selected. Some of the
various available binding reaction schemes will be described in
detail hereinafter. The method used to covalently link the enzyme
modulator and the binding component is not critical so long as the
resulting enzyme modulator moiety retains a measurable amount of
modulating activity and, similarly, the resulting binding component
moiety retains a useful amount of activity with respect to the
binding reaction system. In general, the enzyme modulator and the
binding component are linked directly or by a chain bridge group
having active coupling groups at opposite ends to covalently bind
to the respective moieties to be linked. The bridge group usually
comprises between 1 and 50, and preferably between 1 and 10, carbon
atoms or heteroatoms such as nitrogen, oxygen, sulfur, phosphorus,
and so forth. Examples of a bridge group comprising a single atom
would be a methylene group (one carbon atom in the chain) and an
amino group (one heteroatom in the chain). The bridge group usually
has a molecular weight not exceeding 1000 and preferably less than
200. The bridge group, if present, comprises a chain of carbon
atoms and/or heteroatoms and is joined to the enzyme modulator and
the binding component, respectively, by connecting groups, usually
selected from ester, amide, ether, thioester, acetal, methylene,
and amino groups.
Following are structural formulae of contemplated labeled
conjugates including the competitive inhibitors previously listed
as the labeling substance and a general method for their
preparation.
A. Acetazolamide - binding component conjugates ##STR1##
Example 1, Parts B and C provide details of preparations of these
types of conjugates.
B. Phenyl trimethylammonium ion - binding component conjugates
##STR2##
The binding component of interest is covalently linked to the amino
function of an o-aminoalkyl carboxylic acid through a reaction such
as nucleophilic displacement. The resulting product is then coupled
to p-amino-N,N-dimethylaniline via an activating agent, such as
carbodiimide. The resulting product is then treated with methyl
iodide to give the desired conjugate. ##STR3## The binding
component of interest is coupled through any appropriate means
(activation of the carboxyl group, nucleophilic displacement) to
one amino group of 3,3'-diaminodipropylamine. The resulting product
is then treated with succinic anhydride. The resulting material is
then coupled to the free amino function of the
N-methyl-N-(p-aminophenyl) carbamate ester of
m-(triethylamino)-phenol via a carboxylic acid activating agent.
Alternatively, the inhibitor could be extended further from the
ligand by treatment of the above succinylated derivative with
3,3'-diaminodipropylamine and carbodiimide, followed by succinic
anhydride and coupling to the inhibitor (n=2).
C. Saccharo-1,4-lactone - binding component conjugate ##STR4##
Saccharo-1,4-lactone is coupled to an .alpha.,.omega.-diaminoalkyl
derivative through its free carboxyl group using a carboxylic acid
activating agent. The resulting product is then coupled to the
binding component of interest using any appropriate reaction, e.g.,
nucleophilic displacement, carboxylic acid activation, etc.
D. 4-Amino-10-methylpteroylglutamic acid - binding component
conjugate ##STR5## The binding component of interest is coupled to
amino function of an .alpha.,.omega.-diaminoalkyl derivative
through an appropriate reaction sequence. The product from this
reaction is then coupled to one of the carboxylic acid functions of
4-amino-10-methyl pteroyl glutamic acid via an appropriate
carboxylic acid activating agent, e.g., carbodiimide.
E. 2,6,8-Trichloropurine - binding component conjugate ##STR6##
2,6,8-Trichloropurine is treated with dihydropyran and a catalytic
amount of acid to produce a 9-substituted tetrahydropyranyl ether.
This product is then reacted with an .alpha.,.omega.-diaminoalkane
to produce a C-8 substituted aminoalkylamine derivative. The
tetrahydropyranyl blocking group is then removed under acidic
conditions to give a 2,6-dichloro-substituted purine. The binding
component of interest is then coupled to the free alkyl amino group
through any appropriate reaction sequence.
The present assay method may be applied to the detection of any
ligand for which there is a specific binding partner. The ligand
usually is a peptide, protein, carbohydrate, glycoprotein, steroid,
or other organic molecule for which a specific binding partner
exists in biological systems or can be synthesized. The ligand, in
functional terms, is usually selected from the group consisting of
antigens and antibodies thereto; haptens and antibodies thereto;
and hormones, vitamins, metabolites and pharmacological agents, and
their receptors and binding substances. Specific examples of
ligands which may be detected using the present invention are
hormones such as insulin, chorionic gonadotropin, thyroxine,
liothyronine, and estriol; antigens and haptens such as ferritin,
bradykinin, prostaglandins, and tumor specific antigens; vitamins
such as biotin, vitamin B.sub.12, folic acid, vitamin E, and
ascorbic acid; metabolites such as 3',5' adenosine monophosphate
and 3',5' guanosine monophosphate; pharmacological agents such as
dilantin, digoxin, morphine, digitoxin, and barbiturates;
antibodies such as microsomal antibody and antibodies to hepatitis
and allergens; and specific binding receptors such as thyroxine
binding globulin, avidin, intrinsic factor, and transcobalamin.
As stated previously, the present assay method may follow, in
appropriate circumstances, either a homogeneous or a heterogeneous
scheme.
HOMOGENEOUS SCHEMES
A homogeneous scheme, i.e., one which does not require a physical
separation of the bound-species and the free-species, is available
where reaction between the binding component of the labeled
conjugate and a corresponding binding partner causes a measurable
change, either in a positive or a negative sense, in the ability of
the conjugated enzyme modulator to affect the activity of the
enzyme that catalyzes the monitoring reaction. In such a case, the
distribution of the enzyme modulator between the bound-species and
the free-species can be determined by adding the enzyme directly to
the combined species and measuring therein the activity of the
enzyme. Several manipulative schemes are available for carrying out
a homogeneous assay with the preferred being the direct binding and
the competitive binding techniques.
In the direct binding technique, a liquid medium suspected of
containing the ligand to be detected is contacted with a conjugate
comprising the enzyme modulator coupled to a specific binding
partner of the ligand, and any change in the activity of the enzyme
modulator is assessed. In the competitive binding technique, the
liquid medium is contacted with a specific binding partner of the
ligand and with a conjugate comprising the enzyme modulator coupled
to one or both of the ligand or a specific binding analog thereof,
and thereafter any change in the activity of the enzyme modulator
is assessed. In both techniques, the activity of the enzyme
modulator is determined by contacting the liquid medium with at
least one reagent which forms, with the enzyme modulator, the
predetermined monitoring reaction. Qualitative determination of the
ligand in the liquid medium involves comparing a characteristic,
usually the rate, of the resulting reaction to that of the
monitoring reaction in a liquid medium devoid of the ligand, any
difference therebetween being an indication of a change in activity
of the enzyme modulator. Quantitative determination of the ligand
in the liquid medium involves comparing a characteristic of the
resulting reaction to that of the monitoring reaction in liquid
media containing known amounts of the ligand.
In general, when following the homogeneous assay scheme, the
components of the specific binding reaction, i.e., the liquid
medium suspected of containing the ligand, the conjugate, and/or a
specific binding partner of the ligand, may be combined in any
amount, manner, and sequence, provided that the activity of the
enzyme modulator in the conjugate is measurably altered when the
liquid medium contains the ligand in an amount or concentration of
significance to the purposes of the assay. Preferably, all of the
components of the specific binding reaction are soluble in the
liquid medium.
Where a direct binding homogeneous technique is used, the
components of the binding reaction are the liquid medium suspected
of containing the ligand and a quantity of a conjugate comprising
the enzyme modulator coupled to a specific binding partner of the
ligand. The activity of the conjugated modulator on contact with
the liquid medium varies inversely with the extent of binding
between the ligand in the liquid medium and the specific binding
partner in the conjugate. Thus, as the amount of ligand in the
liquid medium increases, the activity of the conjugated modulator
decreases.
Where a competitive binding homogeneous technique is used, the
components of the binding reaction are the liquid medium suspected
of containing the ligand, a quantity of a conjugate comprising the
enzyme modulator coupled to the ligand or a specific binding analog
of the ligand, and a quantity of a specific binding partner of the
ligand. The specific binding partner is contacted substantially
simultaneously with both the conjugate and the liquid medium. Since
any ligand in the liquid medium competes with the ligand or
specific binding analog thereof in the conjugate for binding with
the specific binding partner, the activity of the conjugated
modulator on contact with the liquid medium varies directly with
the extent of binding between the ligand in the liquid medium and
the specific binding partner. Thus, as the amount of the ligand in
the liquid medium increases, the activity of the conjugated
modulator increases.
A variation of the competitive binding homogeneous technique is the
displacment binding homogeneous technique wherein the conjugate is
contacted first with the specific binding partner of the ligand and
thereafter with the liquid medium. Competition for the specific
binding partner then occurs. In such a method, the amount of the
conjugate contacted with the specific binding partner is usually
that which comprises the ligand or analog thereof in excess of that
capable of binding with the amount of the specific binding partner
present during the time that the conjugate and the specific binding
partner are in contact prior to contact with the liquid medium
suspected of containing the ligand. This order of contact may be
accomplished in either of two convenient ways. In one method, the
conjugate is contacted with the specific binding partner in a
liquid environment prior to contact with the liquid medium
suspected of containing the ligand. In the second method, the
liquid medium suspected of containing the ligand is contacted with
a complex comprising the conjugate and the specific binding
partner, the specific binding substance in the conjugate and the
specific binding partner being reversibly bound to each other. The
amount of the conjugate that becomes bound to the specific binding
partner in the first method or which is in complexed form in the
second method is usually in excess of that capable of being
displaced by all of the ligand in the liquid medium in the time
that the specific binding partner, or complex, and the medium are
in contact prior to the completion of the assessment of any change
in the activity of the conjugated modulator.
Another variation of the competitive binding homogeneous technique
is the sequential saturation homogeneous technique wherein the
components of the specific binding reaction are the same as those
used in the competitive binding technique, but the order of
addition or combination of the components and the relative amounts
thereof used are different. Following a sequential saturation
technique, the specific binding partner of the ligand is contacted
with the liquid medium suspected of containing the ligand for a
period of time prior to the contact of said liquid medium with the
conjugate. The amount of the specific binding partner contacted
with the liquid medium is usually in excess of that capable of
binding with all of the ligand thought to be present in the liquid
medium in the time that the specific binding partner and the liquid
medium are in contact prior to the time that the liquid medium is
contacted with the conjugate. Further, the amount of the ligand or
ligand analog in conjugated form is usually in excess of that
capable of binding with the remaining unbound amount of the
specific binding partner during the time that the liquid medium and
the conjugate are in contact prior to the completion of the
assessment of any change in activity of the conjugated
modulator.
HETEROGENEOUS SCHEMES
The use of an enzyme modulator as labeling substance can also be
applied to the conventional heterogeneous type assay schemes
wherein the bound- and free-species of the labeled constituent are
separated and the quantity of labeling substance in one and/or the
other is determined. The reagent means for performing such a
heterogeneous assay may take on many different forms. In general,
such means comprises three basic constituents, which are (1) the
ligand to be detected, (2) a specific binding partner of the
ligand, and (3) a labeled constituent which is normally a labeled
form of (a) the ligand, (b) a specific binding analog of the
ligand, or (c) the specific binding partner. The binding reaction
constituents are combined simultaneously or in a series of
additions, and with an appropriate incubation period or periods,
the labeled constituent becomes bound to its corresponding
competing binding partners such that the extent of binding, i.e.,
the ratio of the amount of labeled constituent bound to a binding
partner to that unbound, is a function of the amount of ligand
present. To follow is a brief description of some of the different
heterogeneous binding reaction schemes that may be used in carrying
out the method of the present invention.
For the diagrams which are set out hereinafter, the following
legend shall apply:
______________________________________ Symbol Definition
______________________________________ L ligand to be detected in
sample .circle.L ligand or specific binding analog thereof B
binding partner for the ligand * labeling substance, i.e., enzyme
modulator ##STR7## insoluble phase ##STR8## incubation period (sep)
appropriate separation of the bound- and free-species (lim)
limited; present in an amount less than that capable of being bound
to the total available binding sites under the selected reaction
conditions during the selected incubation period; i.e., the con-
stituent for which the other con- stituents compete for binding
(exc) excess; present in an amount greater than that capable of
being bound by the total available binding sites under the selected
reaction condi- tions during the selected incubation period
______________________________________
1. Competitive binding heterogeneous formats ##STR9## This is the
classical competitive binding approach. Examples of insolubilizing
agents are specific precipitating antibodies, specific
insolubilized antibodies, and, where B or L * is a proteinaceous
material, protein precipitators such as ammonium sulfate, or where
B or L * is a small adsorbable molecule, dextran-coated charcoal.
Description of parallel systems may be found in Biochem. J. 88:137
(1963) and U.S. Pat. No. 3,839,153. ##STR10## This approach is
commonly referred to as the solid-phase technique. Descriptions of
parallel radioimmunoassay and enzyme immunoassay techniques may be
found in U.S. Pat. Nos. 3,505,019; 3,555,143; 3,646,346; and
3,654,090. ##STR11## Reference: U.S. Pat. No. 3,654,090. ##STR12##
Reference: U.S. Pat. No. 3,850,752.
2. Sequential saturation heterogeneous formats ##STR13## In the
sequential saturation technique, some or all the binding sites on B
remaining after the first incubation period are bound by the
labeled constituent. ##STR14## Descriptions of parallel
radioimmunoassay and enzyme immunoassay techniques may be found in
U.S. Pat. No. 3,720,760 and J. Immunol. 209:129(1972).
##STR15##
3. "Sandwich" heterogeneous format ##STR16## In the sandwich
technique, some or all of the ligand molecules bound to the
insolubilized binding partners are bound by the labeled
constituent.
Reference: U.S. Pat. No. 3,720,760.
4. Solid-phase ligand adsorbent format ##STR17## In this technique,
the ligand and the labeled constituent are bound to a non-specific
binder and thereafter proportional amounts are dissociated
therefrom by binding with a binding partner having a greater
affinity than the binder for the ligand and the labeled
constituent. The most useful form of this technique employs a
column of the non-specific binder as described in U.S. Pat. No.
3,659,104. Such a technique is useful where the ligand is bound to
endogenous binding substances in the sample which unless removed
would interfer with the competitive binding reaction. Upon being
bound to the non-specific binder, the endogenous binding substances
may be removed by appropriate washes.
For further discussion of the parameters involved in conventional
heterogeneous assay systems, such as more detailed descriptions of
assay formats and alternative separation techniques, reference may
be had to Principles of Competitive Protein-Binding Assays, ed.
Odell and Daughaday (J. B. Lippincott Co., Philadelphia, 1972).
It is contemplated that manipulative schemes involving other orders
of addition and other binding reaction formats may be devised for
carrying out homogeneous and heterogeneous specific binding assays
without departing from the inventive concept embodied herein.
The liquid medium to be tested may be a naturally occurring or
artificially formed liquid suspected of containing the ligand, and
usually is a biological fluid or a liquid resulting from a dilution
or other treatment thereof. Biological fluids which may be assayed
following the present method include serum, plasma, urine, and
amniotic, cerebral, and spinal fluids. Other materials such as
solid matter, for example tissue, or gases may be assayed by
reducing them to a liquid form such as by dissolution of the solid
or gas in a liquid or by liquid extraction of the solid.
The present invention will now be illustrated, but is not intended
to be limited, by the following examples.
EXAMPLE 1
Preparation of Materials
A. Carbonic anhydrase
The enzyme was isolated from human red blood cells by the procedure
of Armstrong et al, J. Biol. Chem. 241:5137-5149 (1966), except
that the dialysis was omitted prior to the precipitation with
ammonium sulfate. The precipitate was dissolved in 0.05 M
tris-(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl) buffer,
pH 8.7, and chromatographed on a 2.5.times.55 cm column of
DEAE-cellulose equilibrated with the same buffer. The enzyme was
eluted as one peak with the Tris-HCl buffer.
B.
5-(Cis-hexahydro-2-oxo-1H-thieno[3,4-d]imidazole)-N-(5-sulfonamido-1,3,4-t
hiadiazo-2-yl)valeramide ##STR18##
A solution of anhydrous biotin (300 mg; 1.2 mmol) in dry dimethyl
formamide (19.5 ml) was stirred at -10.degree. C. under dry
nitrogen and dry triethylamine (0.17 ml; 1.2 mmol) was added
[Knappe et al, Biochem Z.338:559(1963)]. A solution of freshly
distilled ethyl chloroformate (0.141 ml in 3 ml of dry
diethylether) was added dropwise and a white precipitate formed.
The mixture as stirred at -10.degree. C. for 30 min and then
filtered under an atmosphere of dry nitrogen. The filtrate was
cooled immediately to -10.degree. C. and a solution of
2-amino-1,3,4-thiadiazole-5-sulfonamide (438 mg; 2.43 mmol) [Roblin
et al, J. Am. Chem. Soc. 72:4890-4892(1950)] in 3 ml of dry
pyridine was added. The resulting solution was stirred at
-10.degree. C. for 15 min and then at room temperature for 30 min.
The solvents were evaporated under vacuum at 40.degree. C., leaving
an oily residue. The oil was stirred ast 0.degree. C. with 50 ml
0.3 N hydrochloric acid. The resulting white solid (400 mg) was
collected by filtration and dissolved in 10% (by weight) sodium
bicarbonate. This solution was cooled in an ice bath and adjusted
to pH 6.5 with concentrated hydrochloric acid. The resulting beige
precipitate was collected by filtration, giving 130 mg (m.p.
254.degree. C. with decomposition). Crystallization from methanol
gave 60 mg of a white product (m.p. 262.degree.-263.degree. C. with
decomposition).
Analysis calculated for C.sub.12 H.sub.18 N.sub.6 O.sub.4 S.sub.3
:C, 35.46; H, 4.46; N, 20.67. Found: C, 35.62; H, 4.47; N,
20.55.
C. 6-(2,4-Dinitroanilino)-N-(5-sulfonamide-1,3,4-thiadiazo-2-yl)
caproamide ##STR19##
A solution of anhydrous 6-(2,4-dinitroanilino) caproic acid (357
mg; 1.2 mmol), [Garkusha et al, Obshchei Khim 29: 1554-1558(1959)]
was converted to the mixed anhydride following the stepwise
addition of triethylamine and ethylchloroformate as described in
Part B above. The filtrate was cooled immediately to -10.degree. C.
and the product was mixed at -10.degree. C. with a solution of
2-amino-1,3,4-thiadiazole-5-sulfonamide (438 mg; 2.43 mmol)
(obtained in the same manner as described in part B above) in 3 ml
of dry pyridine. The reaction was stirred at 0.degree. C. for 20
hrs and then the solvents were evaporated at 30.degree. C. under
vacuum. The residue was stirred for 1 hr at 0.degree. C. with 100
ml 1.5 N hydrochloric acid and the resulting yellow precipitate was
collected by filtration and stirred for 1 hr at 0.degree. C. with
100 ml 10% (by weight) sodium hydroxide. The yellow solid was
collected by filtration and recrystallized from aqueous methanol to
give 50 mg of yellow solid (9% yield, m.p. 229.degree.-233.degree.
C.).
Analysis calculated for C.sub.14 H.sub.17 N.sub.7 O.sub.7 S.sub.2 :
C, 36.60; H. 3.73; N, 21.34. Found: C, 37.07; H, 3.96; N,
21.19.
D. Reagent Solutions
(1) Acetazolamide Reagent -
2-Acetylamino-1,3,4-thiadiazole-5-sulfonamide (1.6 mg) was
dissolved in several drops of dimethylsulfoxide and diluted to 50
ml with water. Further dilutions were with water.
(2) Biotin- and DNP-Modulator Reagents - The biotin -modulator
conjugate (2.0 mg) and the DNP-modulator conjugate (1.7 mg) were
each dissolved in 10 ml 0.1 M sodium carbonate buffer, pH 10.5.
Further dilutions were with water.
(3) Avidin Reagent - Lyophilized avidin (Sigma Chemical Co., St.
Louis, Mo.) was dissolved in 10 mM Tris-HCl buffer, pH 7.4, at a
level of 10.5 activity units per ml (one unit = amount capable of
binding 1 .mu.g of biotin).
(4) Antibody to Dinitrophenyl (DNP) Reagent - Antisera raised
against DNP was chromatographed at 4.degree. C. on a 5 .times. 70
cm column of Sephadex G-200 (Pharmacia AB, Uppsala, Sweden) with
0.1 M Tris-HCl buffer, pH 8.2, containing 1 M sodium chloride. The
second eluted peak determined by optical density monitored at 280
nm contained the antibody activity to DNP.
(5) p-Nitrophenyl Acetate Reagent - 5 mg in 0.3 ml acetone adjusted
to 10 ml with distilled water with stirring.
EXAMPLE 2
Esterase Activities of Carbonic Anhydrase as a Function of
Acetazolamide Concentration
Carbonic anhydrase, 0.013 International Units, was incubated for 5
min at room temperature in a series of 0.66 ml aqueous solutions
containing, respectively, 100 .mu.l 0.1 M diethylmalonic buffer, pH
7.4, and the concentrations of acetazolamide and avidin given in
Table 1 (concentrations of acetazolamide and avidin based on 1 ml
final volume). p-Nitrophenyl Acetate Reagent (0.33 ml) was added to
each solution to give a total volume of 1 ml. The hydrolysis rate
for p-nitrophenyl acetate was determined by monitoring absorbance
at 348 nm.
The results, expressed as the average of duplicate measurements,
are presented in Table 1.
Table 1 ______________________________________ Acetazolamide Avidin
Hydrolysis Rate Concentration (.mu.M) (units/ml) (.DELTA.OD.sub.348
/min/ml) ______________________________________ 0.000 -- 0.136
0.144 -- 0.088 0.288 -- 0.047 0.144 0.105 0.089 0.000 -- 0.111
0.000 0.105 0.106 ______________________________________
These data indicate that acetazolamide inhibited the enzyme
carbonic anhydrase and that the presence of avidin had no
significant effect on such inhibition.
EXAMPLE 3
Esterase Activities of Carbonic Anhydrase as a Function of
Biotin-Modulator Conjugate Concentration
Carbonic anhydrase, 0.027 International Units, was incubated for 5
min at room temperature in a series of 0.66 ml aqueous solutions
containing 100 .mu.l 0.1M diethylmalonic buffer, pH 7.4, and the
concentrations of the biotin-modulator conjugate (prepared
according to Example 1, Part B) indicated in Table 2 (concentration
of conjugate based on 1 ml final volume). Esterase activity, as
measured by hydrolysis rate, was determined for each reaction
mixture as in Example 2. The results are presented in Table 2 and
in graphical form in FIG. 1 of the drawing.
Table 2 ______________________________________ Biotin-Modulator
Conjugate Hydrolysis Rate Concentration (.mu.M) (.DELTA.OD.sub.348
/min/ml) ______________________________________ 0.00 0.136 0.05
0.120 0.10 0.109 0.20 0.103 0.30 0.071 0.40 0.057 0.50 0.040
______________________________________
These data indicate that the biotin-modulator conjugate possesses
useful inhibitory properties relative to carbonic anhydrase and is
slightly less efficient than the underivatized inhibitor,
acetazolamide.
EXAMPLE 4
Direct Binding Assay for Avidin; Competitive Binding Assay for
Biotin; Use of Derivatized Acetazolamide as Labeling Substance
A series of binding reaction mixtures were prepared, each to a
volume of 0.66 ml and containing 100 .mu.l 0.1M diethylmalonic
buffer, pH 7.4, and the concentrations of biotin-modulator
conjugate (prepared as in Example 1, Part B), biotin, and avidin
indicated in Table 3 (indicated concentrations based on 1 ml final
volume). After a 5 min incubation at room temperature, 0.027
International Units carbonic anhydrase was added to each reaction
mixture followed by an additional 5 min incubation. p-Nitrophenyl
Acetate Reagent (0.33 ml) was then added to each and the rate of
hydrolysis measured by monitoring absorbance at 348 nm.
The results, expressed as the average of duplicate measurements,
are presented in Table 3. The results of reaction mixtures 2
through 9 are presented in graphical form in FIG. 2 of the
drawing.
The results of reaction mixtures 2 through 9 demonstrate the effect
of various levels of avidin on the inhibitory activity of
biotin-modulator conjugate on esterase activities of carbonic
anhydrase. For a constant level of biotin-modulator conjugate, the
rate of enzymatic hydrolysis is directly related to the amount of
avidin present. The results of reaction mixtures 8, 10, and 11 show
that when biotin is present, the rate of hydrolysis is inversely
related to the amount present.
Table 3 ______________________________________ Reaction
Biotin-Modulator Biotin Auidin Rate of Mixture Conjugate (.mu.M)
(.mu.M) (units/ml) Hydrolysis
______________________________________ 1 -- -- -- 0.128 2 0.50 --
0.011 0.049 3 0.50 -- 0.026 0.056 4 0.50 -- 0.040 0.066 5 0.50 --
0.053 0.075 6 0.50 -- 0.065 0.079 7 0.50 -- 0.079 0.090 8 0.50 --
0.105 0.129 9 0.50 -- 0.126 0.136 10 0.50 0.10 0.105 0.104 11 0.50
0.60 0.105 0.080 ______________________________________ *Increase
in Absorbance per min at 348 nm
EXAMPLE 5
Competitive Binding Assay for Dinitrophenyl (DNP) Derivatives; Use
of Derivatized Acetazolamide as Labeling Substance
A series of binding reaction mixtures were prepared, each to a
volume of 0.66 ml and containing 100 .mu.l 0.1M diethylmalonic
buffer, pH 7.4, and the concentrations of acetazolamide,
DNP-modulator (prepared as in Example 1, Part C), N-DNP-6
-aminocaproate [Biochem. J. 42:287(1948)], and antibody to DNP
indicated in Table 4 (indicated concentrations based on 1 ml final
volume). After a 5 min incubation at room temperature, 0.03
International Units carbonic anhydrase was added to each reaction
mixture followed by an additional 5 min incubation. p-Nitrophenyl
Acetate Reagent (0.33 ml) was then added and the rate of hydrolysis
measured by monitoring absorbance at 348 nm.
The results, expressed as the average of duplicate measurements,
are presented in Table 4. A comparison of the results of reaction
mixtures 1, 3, and 5 shows that acetazolamide inhibited the
carbonic anhydrase reaction and was substantially unaffected by the
presence of antibody to DNP. Comparing the results of reaction
mixtures 1, 2, and 4 reveals that the DNP-modulator conjugate also
inhibited the carbonic anhydrase reaction, with the degree of
inhibition being decreased by the presence of antibody to DNP. The
result of reaction mixture 6 indicates that the presence of a DNP
derivative had no substantial effect on the carbonic anhydrase
reaction. A comparison of the results of reaction mixtures 2 and 7
shows that the presence of the DNP derivative could be determined
by observing the increased inhibition of the carbonic anhydrase
reaction by the DNP-modulator conjugate.
Table 4
__________________________________________________________________________
Reaction Acetazolamide DNP-Modulator N-DNP-6-amino Antibody Rate of
Mixture (.mu.M) Conjugate (.mu.M) caproate (.mu.M) to DNP
(.infin.l/ml) Hydrolysis*
__________________________________________________________________________
1 -- -- -- 100 0.153 2 -- 0.278 -- 100 0.108 3 0.216 -- -- 100
0.091 4 -- 0.278 -- -- 0.074 5 0.216 -- -- -- 0.088 6 -- -- 1.8 --
0.154 7 -- 0.278 1.8 100 0.098
__________________________________________________________________________
*Increase in Absorbance per min at 348 nm
* * * * *