U.S. patent application number 11/108229 was filed with the patent office on 2005-09-01 for reagents for assays for ligands.
Invention is credited to Haley, Carolyn J., Parrish, Richard F., Staples, Mark A., Zmolek, Wesley W..
Application Number | 20050191762 11/108229 |
Document ID | / |
Family ID | 21807986 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050191762 |
Kind Code |
A1 |
Staples, Mark A. ; et
al. |
September 1, 2005 |
Reagents for assays for ligands
Abstract
One aspect of the present invention relates to a method for
releasing a ligand from a complex thereof. The method comprises
contacting a medium suspected of containing such complex with an
effective amount of a compound effective in releasing the ligand.
Another aspect of the present invention is an improvement in a
method for the determination of an analyte that is a member of a
specific binding pair in a sample suspected of containing such
analyte. The method comprises the steps of (a) providing in an
assay medium the sample and a binding partner for the analyte and
(b) detecting the binding of the binding partner to the analyte.
The improvement comprises including in the assay medium a compound
of the invention in an amount sufficient to enhance the accuracy of
the determination. The invention has particular application to a
method for releasing mycophenolic acid from a complex thereof. The
method provides an improvement in a method for the determination of
mycophenolic acid in a sample suspected of containing mycophenolic
acid. The present invention also provides assay reagents as well as
packaged kits useful for performing the methods of the
invention.
Inventors: |
Staples, Mark A.; (San Jose,
CA) ; Haley, Carolyn J.; (Morgan Hill, CA) ;
Parrish, Richard F.; (San Jose, CA) ; Zmolek, Wesley
W.; (Freemont, CA) |
Correspondence
Address: |
DADE BEHRING INC.
LEGAL DEPARTMENT
1717 DEERFIELD ROAD
DEERFIELD
IL
60015
US
|
Family ID: |
21807986 |
Appl. No.: |
11/108229 |
Filed: |
April 18, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11108229 |
Apr 18, 2005 |
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09542445 |
Apr 4, 2000 |
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6887669 |
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09542445 |
Apr 4, 2000 |
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08896244 |
Jul 17, 1997 |
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6171801 |
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60022133 |
Jul 18, 1996 |
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Current U.S.
Class: |
436/518 |
Current CPC
Class: |
Y10S 435/961 20130101;
G01N 33/94 20130101; Y10S 435/962 20130101; G01N 33/9493 20130101;
G01N 33/5306 20130101; Y10S 436/825 20130101; Y10T 436/25 20150115;
G01N 33/53 20130101; Y10T 436/25375 20150115 |
Class at
Publication: |
436/518 |
International
Class: |
G01N 033/543 |
Claims
1-28. (canceled)
29. A kit for conducting an assay for the determination of an
analyte, said kit comprising in packaged combination: (a) a binding
partner for said analyte and (b) a compound of the formula:
6wherein R.sup.1 is alkyl; R.sup.2 is hydrogen or alkyl; X is O, S
or N; n is 1 when X is O or S and n is 2 when X is N; and m is 1 or
2.
30. The kit of claim 29 which further comprises a compound
comprising said analyte bound to a detectable label.
31. The kit of claim 29 wherein said compound is methoxybenzoic
acid.
32. The kit of claim 29 wherein said binding partner is an
antibody.
33. A kit for conducting an assay for the determination of
mycophenolic acid, said kit comprising in packaged combination: (a)
an antibody capable of binding to mycophenloic acid, (b) a compound
comprising mycophenolic acid bound to a detectable label, and (c) a
compound of the formula: 7wherein R.sup.1 is alkyl; R.sup.2 is
hydrogen or alkyl; X is O, S or N; n is 1 when X is O or S and n is
2 when X is N; and m is 1 or 2.
34. The kit of claim 33 wherein said compound is methoxybenzoic
acid.
35. The kit of claim 33 wherein said compound is o-methoxybenzoic
acid.
36. The kit of claim 33 wherein said antibody is a monoclonal
antibody.
37-39. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to ligand-receptor binding assay
techniques. The determination of the presence or concentration of a
ligand analyte that is a member of a specific binding pair ("sbp
member") consisting of ligand and its complementary receptor, in
serum or other body fluids relies increasingly upon specific
binding assay techniques. These techniques are based on formation
of a complex between sbp members in which one or the other of the
complex may be labeled with a moiety that produces a signal either
directly or indirectly. In the case of competitive specific binding
assay techniques, analyte in a sample of fluid being tested for its
presence competes with a known quantity of labeled analyte in
binding to a limited amount of a complementary sbp member. Thus,
the amount of labeled analyte bound to the sbp member varies
inversely with the amount of analyte in the sample. In immunometric
assays, the analyte is usually a ligand and the assay employs a
complementary sbp member and a second labeled receptor, usually an
antibody. In such an assay, the amount of labeled receptor
associated with the complex is directly related to the amount of
analyte substance in the fluid sample. Numerous variations of the
above are also used in the detection of analytes such as the use of
a receptor for a receptor for the analyte or other binding pairs
such as avidin-biotin and the like.
[0003] The presence in the sample of one or more interfering
substances such as proteins, e.g., albumin, that bind
non-specifically to the analyte in question or to a reagent being
employed in an assay for such analyte can be a serious factor in
compromising the quantitative character of a ligand-receptor assay.
The analyte is usually present in very small amounts. An
interfering substance may be present in greater amounts and can
bind to a significant number of analyte molecules and, thus, reduce
assay sensitivity. In many situations, the amount of interfering
substance will vary from sample to sample thereby preventing
accurate reference to a standard or calibrator normally employed to
provide for translating the observed signal into the concentration
of the analyte. In order to enhance the accuracy of an assay, it is
desirable to diminish or to completely remove the effect of the
interfering substance on the observed signal.
[0004] Mycophenolic acid ("MPA") is produced by the fermentation of
several penicillium species. It has a broad spectrum of activities,
specific mode of action, and is tolerable in large doses with
minimal side effects, Epinette, et al., Journal of the American
Academy of Dermatololgy 17(6):962-71 (1987). MPA has been shown to
have antitumor, antiviral, antipsoriatic, immunosuppressive,
anti-inflammatory activities, Lee, et al., Pharmaceutical Research
7(2):161-166 (1990), along with antibacterial and antifungal
activities, Nelson, et al., Journal of Medicinal Chemistry
33(2):833-838 (1990). It inhibits inosine monophosphate
dehydrogenase, an enzyme in the de novo synthesis of purine
nucleotides (Wu, Perspectives in Drug Discovery and Design (1994)
2:185-204). Since T and B lymphocytes depend largely upon this de
novo synthesis, MPA is able to inhibit lymphocyte proliferation,
which is a major factor of the immune response.
[0005] The morpholinoethyl ester of MPA, morpholinoethyl
(E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)--
4-methyl-4-hexenoate ("MPA-M") is rapidly hydrolyzed in vivo to
MPA. Administration of MPA in the form of this ester, greatly
improves MPA's bioavailability.
[0006] Because MPA is a potent biologically active material, an
effective immunoassay could be useful in monitoring its
bioavailability. In addition, it may be important to monitor
therapeutic drug levels, i.e., optimal drug levels necessary for
adequate immunosuppression. Since MPA-M is rapidly hydrolyzed to
MPA, an assay for MPA would allow a means of regulating and
optimizing MPA-M dosages. It is known that MPA is highly
protein-bound in plasma (83->98%) and any factors that alter
plasma protein concentrations in patients could affect the accuracy
of an MPA assay (Shaw, et al., Therapeutic Drug Monitoring
(1995)17:690-699).
[0007] Patients under treatment with MPA and cyclosporin or
tacrolimus may be co-administered numerous drugs including, but not
limited to, azathioprine, prednisone, methylprednisolone,
antivirals, antibiotics, antifungals, cardiovascular agents,
diabetic agents and diuretic agents. Many of these drugs have
profound effects on metabolism and result in changes in
concentrations of various serum/plasma components. There exists,
therefore, a potential for interference by these components, either
directly or indirectly, in the determination of MPA in the target
patient population.
[0008] Serum assays in general are limited by the difficulty of
variations in plasma protein concentrations in patient populations.
Furthermore, variations in sample matrix components that alter free
and bound fractions of MPA, such as albumin concentration, can lead
to inaccurate immunoassay results without releasing MPA from its
bound fraction. In particular, the apparent concentration of MPA
would be higher or lower depending on the protein concentration of
the sample. For example, in the early post transplant period,
albumin concentrations are low relative to a calibrator with normal
plasma protein concentrations so that MPA quantitation of these
patient samples could be high. A number of factors including time
post transplant an metabolic differences due to co-administered
drugs or disease states of the patient can result in abnormal
protein concentrations. These abnormal protein concentrations may
alter the free-to-bound ratio of MPA and, therefore, affect the
accuracy of the immunoassay results. Variable recovery as a
function of protein concentration of the samples prevents selection
of one average protein concentration for calibrators that
represents all samples.
[0009] Salicylate is known to increase MPA free fraction in normal
human plasma when present at concentrations that may be observed in
chronic administration of aspirin (Nowak, infra). However, we have
found that the use of salicylate as a releasing agent can result in
a 25-50% decrease in the total dose-response curve in an MPA enzyme
immunoassay. It is also known that 8-anilino-1-naphthalenesulfonic
acid (ANS) functions as a releasing agent in immunoassays (Nerli,
et al., Arch Int Physiol Biochim Biophys (1994) 102(1):5-8 and
Seth, et al., Clin Chem (1975) 21(10):1406-1413. However, ANS has
disadvantages, because of its background absorbance at 340 nm and
its susceptibility to light degradation. The background absorbance
is particularly disadvantageous in enzyme immunoassays. However,
ANS has been used in certain enzyme assays under conditions where
its disadvantages can be tolerated.
[0010] The present invention avoids the deficiencies of the above
known compounds used as releasing agents in assays for ligands.
[0011] 2. Description of the Related Art
[0012] Nowak, et al., Clin. Chem. (1995) 41(7): 1011-1017 discusses
mycophenolic acid binding to human serum albumin: characterization
and relation to pharmacodynamics.
[0013] Langman, et al., Therapeutic Drug Monitoring (1994)
16:802-807 discusses blood distribution of mycophenolic acid.
[0014] European Patent 0 218 309 B1 discloses a method for
measuring free ligands in biological fluids. Sodium salicylate and
2,4-dinitrophenol were employed to prevent labeled analogs of
triiodothyronine and tetraiodothyronine from binding to albumin and
thyroid binding pre-albumin.
[0015] European Patent Application 0 392 332 A2 discloses a
fluorescent polarization immunoassay and reagents therefor. Various
compounds were disclosed for converting a marijuana metabolite,
which was bound to serum albumin and other proteins in urine, to
free form. These compounds included, among others, ANS, salicylic
acid and 5-methoxysalicylic acid.
SUMMARY OF THE INVENTION
[0016] One aspect of the present invention relates to a method for
releasing a ligand from a complex thereof. The method comprises
contacting a medium suspected of containing such complex with an
effective amount of a compound of the formula: 1
[0017] wherein R.sup.1 is alkyl; R.sup.2 is hydrogen or alkyl; X is
O, S or N; n is 1 when X is O or S and n is 2 when X is N; and m is
1 or 2 (Compound I).
[0018] Another aspect of the present invention is an improvement in
a method for the determination of an analyte that is a member of a
specific binding pair in a sample suspected of containing such
analyte. The method comprises the steps of (a) providing in an
assay medium the sample and a binding partner for the analyte and
(b) detecting the binding of the binding partner to the analyte.
The improvement comprises including in the assay medium Compound I
in an amount sufficient to enhance the accuracy of the
determination.
[0019] Another embodiment of the present invention relates to a
method for releasing mycophenolic acid from a complex thereof. The
method comprises contacting a medium suspected of containing the
complex with an effective amount of Compound I.
[0020] Another embodiment of the present invention is an
improvement in a method for the determination of mycophenolic acid
in a sample suspected of containing mycophenolic acid. The method
comprises (a) providing in an assay medium the sample and a binding
partner for mycophenolic acid and (b) detecting the binding of the
binding partner to mycophenolic acid. The improvement comprises
including in the assay medium Compound I in an amount sufficient to
enhance the accuracy of the determination.
[0021] A further embodiment of the present invention is an
improvement in a method for measuring the amount of mycophenolic
acid in a sample suspected of containing mycophenolic acid and
endogenous proteins that bind to the mycophenolic acid. The method
comprises (a) combining in an aqueous medium the sample,
mycophenolic acid conjugated to a detectable label, and an antibody
capable of binding to mycophenolic acid, and (b) determining the
effect of the sample on the activity of the label. The improvement
comprises including in the medium a compound of the formula: 2
[0022] wherein R.sup.1 is alkyl and R.sup.2 is hydrogen or alkyl
(Compound II) in an amount effective in releasing the mycophenolic
acid from the endogenous proteins.
[0023] The present invention further includes kits for conducting
an assay for the determination of an analyte. The kit comprises in
packaged combination a binding partner for the analyte and Compound
I.
[0024] A kit for conducting an assay for the determination of
mycophenolic acid comprises in packaged combination an antibody
capable of binding to mycophenolic acid, a compound comprising
mycophenolic acid bound to a detectable label, and Compound I.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0025] Before proceeding with the description of the specific
embodiments of the invention, a number of terms will be
defined.
[0026] Analyte--the compound or composition to be detected. The
analyte can be comprised of a member of a specific binding pair
(sbp) and may be a ligand, which is usually monovalent
(monoepitopic), usually haptenic, and is a single compound or
plurality of compounds which share at least one common epitopic or
determinant site.
[0027] The monoepitopic ligand analytes will generally be from
about 100 to 2,000 molecular weight, more usually from 125 to 1,000
molecular weight. The analytes include drugs, metabolites,
pesticides, pollutants, and the like. Representative analytes, by
way of example and not limitation, include (I) alkaloids such as
morphine alkaloids, which includes morphine, codeine, heroin,
dextromethorphan, their derivatives and metabolites; cocaine
alkaloids, which include cocaine and benzyl ecgonine, their
derivatives and metabolites; ergot alkaloids, which include the
diethylamide of lysergic acid; steroid alkaloids; iminazoyl
alkaloids; quinazoline alkaloids; isoquinoline alkaloids; quinoline
alkaloids, which include quinine and quinidine; diterpene
alkaloids, their derivatives and metabolites; (ii) steroids, which
include the estrogens, androgens, andreocortical steroids, bile
acids, cardiotonic glycosides and aglycones, which includes digoxin
and digoxigenin, saponins and sapogenins, their derivatives and
metabolites; steroid mimetic substances, such as
diethylstilbestrol; (iii) lactams having from 5 to 6 annular
members, which include the barbituates, e.g. phenobarbital and
secobarbital, diphenylhydantoin, primidone, ethosuximide, and their
metabolites; (iv) aminoalkylbenzenes, with alkyl of from 2 to 3
carbon atoms, which include the amphetamines; catecholamines, which
includes ephedrine, L-dopa, epinephrine; narceine; papaverine; and
metabolites of the above; (v) benzheterocyclics which include
oxazepam, chlorpromazine, tegretol, their derivatives and
metabolites, the heterocyclic rings being azepines, diazepines and
phenothiazines; (vi) purines, which includes theophylline,
caffeine, their metabolites and derivatives; (vii) drugs derived
from marijuana, which includes cannabinol and tetrahydrocannabinol;
(viii) hormones such as thyroxine, cortisol, triiodothyronine,
testosterone, estradiol, estrone, progestrone, polypeptides such as
angiotensin, LHRH, and immunosuppressants such as cyclosporin,
tacrolimus, mycophenolic acid (MPA), and so forth; (ix) vitamins
such as A, B, e.g. B12, C, D, E and K, folic acid, thiamine; (x)
prostaglandins, which differ by the degree and sites of
hydroxylation and unsaturation; (xi) tricyclic antidepressants,
which include imipramine, dismethylimipramine, amitriptyline,
nortriptyline, protriptyline, trimipramine, chlomipramine,
doxepine, and desmethyldoxepin; (xii) anti-neoplastics, which
include methotrexate; (xiii) antibiotics, which include penicillin,
chloromycetin, actinomycetin, tetracycline, terramycin, the
metabolites and derivatives; (xiv) nucleosides and nucleotides,
which include ATP, NAD, FMN, adenosine, guanosine, thymidine, and
cytidine with their appropriate sugar and phosphate substituents;
(xv) miscellaneous individual drugs which include methadone,
meprobamate, serotonin, meperidine, lidocaine, procainamide,
acetylprocainamide, propranolol, griseofulvin, valproic acid,
butyrophenones, antihistamines, chloramphenicol, anticholinergic
drugs, such as atropine, their metabolites and derivatives; (xvi)
metabolites related to diseased states include spermine, galactose,
phenylpyruvic acid, and porphyrin Type 1; (xvii) aminoglycosides,
such as gentamicin, kanamicin, tobramycin, and amikacin; and
(xviii) pesticides such as polyhalogenated biphenyls, phosphate
esters, thiophosphates, carbamates, polyhalogenated sulfenamides,
their metabolites and derivatives.
[0028] The present invention may be applied to polyvalent ligand
analytes having a site subject to the same non-specific binding as
a monovalent analyte. Such polyvalent analytes are normally
poly(amino acids) such as polypeptides and proteins,
polysaccharides, nucleic acids, and combinations thereof. Such
combinations include components of bacteria, viruses, chromosomes,
genes, mitochondria, nuclei, cell membranes and the like.
[0029] The term analyte can further include oligonucleotide and
polynucleotide analytes such as m-RNA, r-RNA, t-RNA, DNA, DNA-RNA
duplexes, etc.
[0030] The analyte may be a molecule found directly in a sample
such as biological tissue, including body fluids, from a host. The
sample can be examined directly or may be pretreated to render the
analyte more readily detectable by removing unwanted materials. The
sample may be pretreated to separate or lyse cells; precipitate,
hydrolyze or denature proteins; hydrolyze lipids; solubilize the
analyte; or the like. Such pretreatment may include, without
limitation: centrifugation; treatment of the sample with an organic
solvent, for example, an alcohol, such as methanol; and treatment
with detergents. The sample can be prepared in any convenient
medium which does not interfere with the assay. An aqueous medium
is preferred.
[0031] Furthermore, the analyte of interest may be determined by
detecting an agent probative of the analyte of interest such as a
specific binding pair member complementary to the analyte of
interest, whose presence will be detected only when the analyte of
interest is present in a sample. Thus, the agent probative of the
analyte becomes the analyte that is detected in an assay.
[0032] The biological tissue includes excised tissue from an organ
or other body part of a host and body fluids, for example, urine,
whole blood, plasma, serum, saliva, semen, stool, sputum, cerebral
spinal fluid, tears, mucus, and the like. Preferably, the sample is
plasma or serum.
[0033] Mycophenolate ester--includes, but is not limited to, esters
of MPA at the carboxylic acid group of the side chain attached at
the 1'-position of the MPA isobenzofuranyl ring system such as
MPA-M.
[0034] MPA metabolite--a product of the metabolism of MPA,
preferably a product containing the isobenzofuranyl ring system,
more preferably products also containing a portion of the side
chain such as the acyl or phenolic glucuronide of MPA.
[0035] Measuring the amount of an analyte--quantitative,
semiquantitative, and qualitative methods as well as all other
methods for determining an analyte are considered to be methods of
measuring the amount of an analyte. For example, a method which
merely detects the presence or absence of an analyte in a sample
suspected of containing the analyte is considered to be included
within the scope of the present invention. The terms "detecting"
and "determining", as well as other common synonyms for measuring,
are contemplated within the scope of the present invention.
[0036] Capable of distinguishing between--the ability of a receptor
or antibody to bind preferentially to a first ligand relative to a
second ligand. Usually at least 5-fold more of the first ligand
than the second ligand will be bound when the antibody is combined
with a sample containing the ligands. Preferably, at least 10-fold
more and, more preferably, at least 20-fold more of the first
ligand will be bound. Although the relative binding of each ligand
will depend on the relative concentrations in the sample, usually
these conditions are met when the binding constant of the antibody
to the first ligand is at least equal to the binding constant to
the second ligand, and preferably, is at least 10-fold, more
preferably, at least 50-fold the binding constant to the second
ligand.
[0037] Conjugate--a molecule comprised of two or more molecules
bound together, optionally through a linking group, to form a
single structure. The binding can be made either by a direct
connection (e.g. a chemical bond) between the molecules or by use
of a linking group. For example, an analyte analog conjugated to an
enzyme is an analyte analog-enzyme conjugate.
[0038] Member of a specific binding pair ("sbp" member)--one of two
different molecules having an area on the surface or in a cavity
that specifically binds to and is therefore defined as
complementary with a particular spatial and polar organization of
the other molecule. The members of the sbp can be referred to as
ligand and receptor such as members of an immunological pair, e.g.,
antigen-antibody. Complementary sbp members bind to one another, as
for example, a ligand and its complementary receptor. With respect
to two complementary sbp members, one may be referred to as the
"binding partner" for the other. Sbp members can be immunological
pairs such as antigen and antibody, or non-immunological pairs such
as avidin and biotin. Sbp members can also be small molecules or
residues of small molecules and their receptors. Small molecules
have a molecular weight of from 100-2000; preferably 150-1000, and
a receptor for the small molecule either exists or can be prepared.
Examples of small molecules include derivatives of biotin, lysergic
acid, fluorescein or a fluorescein derivative, and vitamin
B.sub.12, with the corresponding receptors being avidin or
streptavidin, anti-lysergic acid, anti-fluorescein and intrinsic
factor, respectively. Small molecules are often covalently bound to
other sbp members to form a conjugate having at least one, and
frequently 2-20, small molecules. Bonding of the small molecule to
the sbp member may be accomplished by chemical reactions which
result in replacing a hydrogen atom of the small molecule with a
bond to the sbp member or by a linking group between the small
molecule and the sbp member of any size but preferably no larger
than necessary to permit binding to the conjugate of both a
receptor for the small molecule and the sbp member.
[0039] Ligand--any organic compound for which a receptor naturally
exists or can be prepared.
[0040] Ligand analog (or analyte analog)--a modified ligand, an
organic radical or analyte analog, usually of a molecular weight
greater than 100, which can compete with the analogous ligand for a
receptor, the modification providing means to join a ligand analog
to another molecule. The ligand analog will usually differ from the
ligand by more than replacement of a hydrogen with a bond which
links the ligand analog to a hub or label, but need not. The ligand
analog can bind to the receptor in a manner similar to the ligand.
The analog could be, for example, an antibody directed against the
idiotype of an antibody to the ligand.
[0041] Receptor ("antiligand")--any compound or composition capable
of recognizing a particular spatial and polar organization of a
molecule, e.g., epitopic or determinant site. Illustrative
receptors include naturally occurring receptors, e.g., thyroxine
binding globulin, antibodies, enzymes, Fab fragments, lectins,
nucleic acids, protein A, complement component C1 q, and the
like.
[0042] Specific binding--the specific recognition of one of two
different molecules for the other compared to substantially less
recognition of other molecules. Generally, the molecules have areas
on their surfaces or in cavities giving rise to specific
recognition between the two molecules. Exemplary of specific
binding are antibody-antigen interactions, enzyme--substrate
interactions, polynucleotide interactions, and so forth.
[0043] Non-specific binding--non-covalent binding between molecules
that is relatively independent of specific surface structures.
Non-specific binding may result from several factors including
hydrophobic interactions between molecules.
[0044] Non-specific complex of a ligand--a ligand bound
non-specifically to another substance, usually, endogenous
substances present in a sample to be analyzed. The endogenous
substances generally are endogenous proteins such as plasma
proteins, e.g., albumin, globulins, glycoproteins, lipoproteins,
and the like.
[0045] Antibody--an immunoglobulin which specifically binds to and
is thereby defined as complementary with a particular spatial and
polar organization of another molecule. The antibody can be
monoclonal or polyclonal and can be prepared by techniques that are
well known in the art such as immunization of a host and collection
of sera (polyclonal) or by preparing continuous hybrid cell lines
and collecting the secreted protein (monoclonal), or by cloning and
expressing nucleotide sequences or mutagenized versions thereof
coding at least for the amino acid sequences required for specific
binding of natural antibodies. Antibodies may include a complete
immunoglobulin or fragment thereof, which immunoglobulins include
the various classes and isotypes, such as IgA, IgD, IgE, IgG1,
IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab,
Fv and F(ab').sub.2, Fab', and the like. In addition, aggregates,
polymers, and conjugates of immunoglobulins or their fragments can
be used where appropriate so long as binding affinity for a
particular molecule is maintained.
[0046] Antiserum containing antibodies (polyclonal) is obtained by
well-established techniques involving immunization of an animal,
such as a rabbit, guinea pig, or goat, with an appropriate
immunogen and obtaining antisera from the blood of the immunized
animal after an appropriate waiting period. State-of-the-art
reviews are provided by Parker, Radioimmunoassay of Biologically
Active Compounds, Prentice-Hall (Englewood Cliffs, N.J., U.S.,
1976), Butler, J. Immunol. Meth. 7: 1-24 (1975); Broughton and
Strong, Clin. Chem. 22: 726-732 (1976); and Playfair, et al., Br.
Med. Bull. 30: 24-31 (1974).
[0047] Antibodies can also be obtained by somatic cell
hybridization techniques, such antibodies being commonly referred
to as monoclonal antibodies. Monoclonal antibodies may be produced
according to the standard techniques of Kohler and Milstein, Nature
265:495-497, 1975. Reviews of monoclonal antibody techniques are
found in Lymphocyte Hybridomas, ed. Melchers, et al.
Springer-Verlag (New York 1978), Nature 266: 495 (1977), Science
208: 692 (1980), and Methods of Enzymology 73 (Part B): 3-46
(1981). Samples of an appropriate immunogen preparation are
injected into an animal such as a mouse and, after a sufficient
time, the animal is sacrificed and spleen cells obtained.
Alternatively, the spleen cells of a non-immunized animal can be
sensitized to the immunogen in vitro. The spleen cell chromosomes
encoding the base sequences for the desired immunoglobins can be
compressed by fusing the spleen cells, generally in the presence of
a non-ionic detergent, for example, polyethylene glycol, with a
myeloma cell line. The resulting cells, which include fused
hybridomas, are allowed to grow in a selective medium, such as
HAT-medium, and the surviving immortalized cells are grown in such
medium using limiting dilution conditions. The cells are grown in a
suitable container, e.g., microtiter wells, and the supernatant is
screened for monoclonal antibodies having the desired
specificity.
[0048] Various techniques exist for enhancing yields of monoclonal
antibodies, such as injection of the hybridoma cells into the
peritoneal cavity of a mammalian host, which accepts the cells, and
harvesting the ascites fluid. Where an insufficient amount of the
monoclonal antibody collects in the ascites fluid, the antibody is
harvested from the blood of the host. Alternatively, the cell
producing the desired antibody can be grown in a hollow fiber cell
culture device or a spinner flask device, both of which are well
known in the art. Various conventional ways exist for isolation and
purification of the monoclonal antibodies from other proteins and
other contaminants (see Kohler and Milstein, supra).
[0049] In another approach for the preparation of antibodies the
sequence coding for antibody binding sites can be excised from the
chromosome DNA and inserted into a cloning vector which can be
expressed in bacteria to produce recombinant proteins having the
corresponding antibody binding sites.
[0050] In general, antibodies can be purified by known techniques
such as chromatography, e.g., DEAE chromatography, ABx
chromatography, and the like, filtration, and so forth.
[0051] Hapten--a compound capable of binding specifically to
corresponding antibodies, but do not themselves act as immunogens
(or antigens) for preparation of the antibodies. Antibodies which
recognize a hapten can be prepared against compounds comprised of
the hapten linked to an immunogenic (or antigenic) carrier. Haptens
are a subset of ligands.
[0052] MPA analog--modified MPA. The modification provides means to
join this analog to another molecule. The analog will usually
differ from MPA by more than replacement of a hydrogen with a bond
which links the analog to a hub or label.
[0053] Immunogenic carrier--a group which, when conjugated to a
hapten and injected into a mammal, will induce an immune response
and elicit the production of antibodies that bind to the hapten, in
this case MPA. Immunogenic carriers are also referred to as
antigenic carriers. Typical immunogenic carriers include, without
limitation, poly(amino acids), polysaccharides, nucleic acids and
particles (biologic and synthetic materials). A wide variety of
such carriers are disclosed in Davalian, et al., U.S. Pat. No.
5,089,390, column 4, line 57 to column 5, line 5, incorporated
herein by reference. Other suitable immunogenic carriers include
albumins, serum proteins, e.g., globulins, ocular lens proteins and
lipoproteins. Illustrative proteins include bovine serum albumin,
keyhole limpet hemocyanin ("KLH"), ovalbumin and bovine
gamma-globulin.
[0054] Support or surface--a solid phase, typically a support or
surface, which is a porous or non-porous water insoluble material
that can have any one of a number of shapes, such as strip, rod,
plate, well, particle and bead. A wide variety of suitable supports
are disclosed in Ullman, et al. U.S. Pat. No. 5,185,243, columns
10-11, Kurn, et al., U.S. Pat. No. 4,868,104, column 6, lines 21-42
and Milburn, et al., U.S. Pat. No. 4,959,303, column 6, lines
14-31, which are incorporated herein by reference. Binding of sbp
members to a support or surface may be accomplished by well-known
techniques, commonly available in the literature. See, for example,
"Immobilized Enzymes," Ichiro Chibata, Halsted Press, New York
(1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970). As used
herein, the term "capable of being bound to a support" means, for
example, that a reagent, such as the anti-analyte antibody, is
bound to a first sbp member or a small molecule and a complementary
second sbp member or receptor for the small molecule, is in turn
bound a support. Alternately, a receptor for the anti-analyte
antibody, such as an anti-mouse antibody, is bound to a support and
used to capture the anti-analyte antibody. Therefore, the
anti-analyte antibody is not actually bound to a support, but will
become bound, when a complementary sbp member or receptor is
added.
[0055] Signal producing system ("sps")--one or more components, at
least one component being a detectable label, which generate a
detectable signal that relates to the amount of bound and/or
unbound label, i.e. the amount of label bound or not bound to the
compound being detected. The label is any molecule that produces or
can be induced to produce a signal, and preferably is a fluorescer,
radiolabel, enzyme, chemiluminescer or photosensitizer. Thus, the
signal is detected and/or measured by detecting fluorescence or
luminescence, radioactivity, enzyme activity or light
absorbance.
[0056] Suitable labels include, by way of illustration and not
limitation, enzymes such as alkaline phosphatase,
glucose-6-phosphate dehydrogenase ("G6PDH") and horseradish
peroxidase; ribozyme; a substrate for a replicase such as QB
replicase; promoters; dyes; fluorescers, such as fluorescein,
rhodamine compounds, phycoerythrin, phycocyanin, allophycocyanin,
o-phthaldehyde, and fluorescamine; chemiluminescers such as
isoluminol; sensitizers; coenzymes; enzyme substrates; radiolabels
such as .sup.125I, .sup.131I, .sup.14C; .sup.3H, .sup.57Co and
.sup.75Se; particles such as latex or carbon particles; metal sol;
crystallite; liposomes; cells, etc., which may be further labeled
with a dye, catalyst or other detectable group. Suitable enzymes
and coenzymes are disclosed in Litman, et al., U.S. Pat. No.
4,275,149, columns 19-28, and Boguslaski, et al., U.S. Pat. No.
4,318,980, columns 10-14; suitable fluorescers and chemiluminescers
are disclosed in Litman, et al., U.S. Pat. No. 4,275,149, at
columns 30 and 31; which are incorporated herein by reference.
[0057] There are numerous methods by which the label can produce a
signal detectable by external means, for example, by visual
examination, electromagnetic radiation, heat, and chemical
reagents. The label or other sps members can also be bound to an
sbp member, another molecule or to a support.
[0058] The label can directly produce a signal, and therefore,
additional components are not required to produce a signal.
Numerous organic molecules, for example fluorescers, are able to
absorb ultraviolet and visible light, where the light absorption
transfers energy to these molecules and elevates them to an excited
energy state. This absorbed energy is then dissipated by emission
of light at a second wavelength. Other labels that directly produce
a signal include radioactive isotopes and dyes.
[0059] Alternately, the label may need other components to produce
a signal, and the signal producing system would then include all
the components required to produce a measurable signal, which may
include substrates, coenzymes, enhancers, additional enzymes,
substances that react with enzymic products, catalysts, activators,
cofactors, inhibitors, scavengers, metal ions, and a specific
binding substance required for binding of signal generating
substances. A detailed discussion of suitable signal producing
systems can be found in Ullman, et al. U.S. Pat. No. 5,185,243,
columns 11-13, incorporated herein by reference.
[0060] The label can be bound covalently to numerous sbp members:
an antibody; a receptor for an antibody; a receptor that is capable
of binding to a small molecule conjugated to an antibody; or a
ligand analog. Bonding of the label to the sbp member may be
accomplished by chemical reactions which result in replacing a
hydrogen atom of the label with a bond to the sbp member or may
include a linking group between the label and the sbp member. Other
sps members may also be bound covalently to sbp members. For
example, two sps members such as a fluorescer and quencher can each
be bound to a different antibody that forms a specific complex with
the analyte. Formation of the complex brings the fluorescer and
quencher in close proximity, thus permitting the quencher to
interact with the fluorescer to produce a signal. Methods of
conjugation are well known in the art. See for example, Rubenstein,
et al., U.S. Pat. No. 3,817,837, incorporated herein by reference.
This invention also contemplates having an antibody bound to a
first sps member and a detectable label as the second sps member.
For example, when the detectable label is bound to a ligand analog,
the extent of binding of the antibody to the analog can be measured
by detecting the signal produced by the interaction of the sps
members.
[0061] Ancillary Materials--Various ancillary materials will
frequently be employed in an assay in accordance with the present
invention. For example, buffers will normally be present in the
assay medium, as well as stabilizers for the assay medium and the
assay components. Frequently, in addition to these additives,
additional proteins may be included, such as albumins, or
surfactants, particularly non-ionic surfactants, binding enhancers,
e.g., polyalkylene glycols, preservatives, antimicrobials, or the
like.
[0062] Linking Group--a portion of a structure which connects 2 or
more substructures. The linking group can be a bond or it can have
at least 1 uninterrupted chain of atoms other than hydrogen (or
other monovalent atoms) extending between the substructures. The
number of atoms in the chain will be at least one and is determined
by counting the number of atoms other than hydrogen along the
shortest route between the substructures being connected, and is
typically 1-30, usually 2-10, preferably 3-8, atoms each
independently selected from the group consisting of carbon, oxygen,
nitrogen, sulfur and phosphorous. The number of total atoms in the
linking group is determined by counting the total carbon, oxygen,
nitrogen, sulfur and phosphorous atoms, i.e. the atoms other than
hydrogen. Typically, the linking group has a total of less than 30
atoms, preferably less than 20 atoms, more preferably less than 10
atoms. As a general rule, the length of a particular linking group
can be selected arbitrarily to provide for convenience of synthesis
and the incorporation of any desired group. The linking groups may
be aliphatic or aromatic, although with diazo groups, aromatic
groups will usually be involved. Oxygen will normally be present as
oxo or oxy, bonded to carbon, sulfur, nitrogen or phosphorous;
nitrogen will normally be present as nitro, nitroso or amino,
normally bonded to carbon, oxygen, sulfur or phosphorous; sulfur
would be analogous to oxygen; while phosphorous will be bonded to
carbon, sulfur, oxygen or nitrogen, usually as phosphonate and
phosphate mono- or diester.
[0063] Common functionalities in forming a covalent bond between
the linking group and the molecule to be conjugated are alkylamine,
amidine, thioamide, dithiol, ether, urea, thiourea, guanidine, azo,
thioether and carboxylate, sulfonate, and phosphate esters, amides
and thioesters.
[0064] Alkyl--a monovalent branched or unbranched radical derived
from an aliphatic hydrocarbon by removal of one H atom; includes
both lower alkyl and upper alkyl.
[0065] Lower alkyl--alkyl containing from 1 to 5 carbon atoms such
as, e.g., methyl, ethyl, propyl, butyl, isopropyl, isobutyl,
pentyl, isopentyl, etc.
[0066] Upper alkyl--alkyl containing more than 6 carbon atoms,
usually 6 to 20 carbon atoms, such as, e.g., hexyl, heptyl, octyl,
etc.
[0067] One aspect of the present invention relates to a method for
releasing a ligand from a non-specific complex thereof such as a
complex wherein the ligand is bound non-specifically to another
substance such as endogenous sample proteins and other non-specific
substances. The method comprises contacting a medium suspected of
containing such complex with an effective amount of Compound I.
[0068] Representative examples of compounds having the above
formula are found in Table 1, by way of illustration and not
limitation.
1TABLE 1 Compound X R.sup.1 R.sup.2 n m Ring position* I-A O alkyl
H 1 2 ortho I-B O alkyl H 1 2 meta I-C O alkyl alkyl 1 2 ortho I-D
O alkyl alkyl 1 2 meta I-E S alkyl H 1 2 ortho I-F S alkyl H 1 2
meta I-G S alkyl alkyl 1 2 ortho I-H S alkyl alkyl 1 2 meta I-I N
alkyl H 2 2 ortho I-J N alkyl H 2 2 meta I-K N alkyl alkyl 2 2
ortho I-L N alkyl alkyl 2 2 meta I-M O alkyl H 1 1 ortho I-N O
alkyl H 1 1 meta I-O O alkyl H 1 1 para I-P O alkyl alkyl 1 1 ortho
I-Q O alkyl alkyl 1 1 meta I-R O alkyl alkyl 1 1 para I-S S alkyl H
1 1 ortho I-T S alkyl H 1 1 meta I-U S alkyl H 1 1 para I-V S alkyl
alkyl 1 1 ortho I-W S alkyl alkyl 1 1 meta I-X S alkyl alkyl 1 1
para I-Y N alkyl H 2 1 ortho I-Z N alkyl H 2 1 meta I-AA N alkyl H
2 1 para I-BB N alkyl alkyl 2 1 ortho I-CC N alkyl alkyl 2 1 meta
I-DD N alkyl alkyl 2 1 para *Ring position refers to the position
on the benzene ring of the -- XR.sup.1 group in relation to the
carboxy group.
[0069] Preferably, compounds for use in the methods of the present
invention have an ortho relationship between the carboxy group and
the substituent--XR.sup.1 and have the formula (designated
Compounds I'): 3
[0070] wherein R.sup.1 is alkyl; R.sup.2 is hydrogen or alkyl; X is
O, S or N. Representative compounds in this category, by way of
example and not limitation, are Compounds I-M, I-P, I-S, I-V, I-Y
and I-BB above.
[0071] Preferred compounds of formula I above are those of the
formula of Compound II. Representative compounds in this group, by
way of illustration and not limitation, are Compounds I-M, I-N,
I-O, I-P, I-Q and I-R above.
[0072] More preferably, compounds useful in the present invention
are those of the following formula (Compound III): 4
[0073] wherein R.sup.1 is alkyl and R.sup.2 is hydrogen or alkyl.
Representative compounds in this group, by way of illustration and
not limitation, are Compounds I-M and I-P above. Particularly
preferred compounds with this category are those wherein R.sup.1 is
lower alkyl, more preferably, methyl.
[0074] More preferred are compounds of the following formula
(Compounds IV): 5
[0075] wherein R.sup.3 is lower alkyl. Particularly preferred is
o-methoxybenzoic acid also known as o-anisic acid.
[0076] It is important in the present invention that the particular
Compound I employed not bind to any significant degree to an sbp
member or binding partner for an analyte used in an assay for the
analyte. What constitutes a significant degree is dependent on the
sensitivity necessary for the assay; the higher the sensitivity
required for the assay, the less tolerable is the amount of binding
between an sbp member or binding partner for the analyte. By the
term the "significant degree" is meant the particular Compound I
not bind to an sbp member or binding partner to an extent that
would affect the accuracy or the quantitative or qualitative nature
of the assay result. Accordingly, any binding between a particular
Compound I and an sbp member or a binding partner should be,
preferably, less than 1%, more preferably, less than 0.01%, most
preferably 0%. Such percentage is determined by measuring the
apparent analyte quantitation of the given amount of releasing
agent and expressing the result as a fraction of the actual
concentration. Furthermore, the particular Compound I selected must
have minimal, if any, interference with the binding of sbp members
to one another or with the ability of the signal producing system
to produce a signal in relation to the presence or amount of
analyte in a sample.
[0077] By the term "effective amount" is meant an amount sufficient
to bring about the release of the ligand from such complex so that
preferably at least about 90%, more preferably, at least 99% and
most preferably 100% of the ligand is in a form free of such
complex. The effective amount of Compound I to be used in a
particular assay will depend on the nature of the ligand and of the
assay and reagents employed therein. Preferably, the effective
amount is determined empirically based on the suspected
concentration range of analyte in the sample. In general, an
effective amount of Compound I is an excess amount over the
suspected amount of the analyte. Considering an assay for MPA, by
way of illustration and not limitation, where the expected level of
drug in a sample is about 1.5 to 45 .mu.M, the effective amount of
Compound I in the assay medium is about 0.1 to about 100 mM,
preferably, about 1 to about 25 mM, more preferably, about 2 to 12
mM.
[0078] Many of the compounds useful in the present invention are
commercially available and/or their synthesis is known in the
literature. The compounds useful in the methods of the present
invention may be prepared by known procedures from starting
materials that are readily available such as benzoic acid or
hydroxybenzoic acid. Such procedures involve formation of one or
more alkoxy groups on the benzene ring and esterification of the
resulting compound where an ester is desired. Both of the above may
be accomplished by well-known procedures that will not be repeated
here. See, for example, Heathcock, et al., "Introduction to Organic
Chemistry," MacMillan Publishing Company, New York, N.Y., Third
edition, 1985, pages 814 and 493; Carey, "Organic Chemistry,"
McGraw Hill Inc., New York, N.Y., Second edition, 1987, pages
994-995 and 609.
[0079] The method of the present invention may be applied to most
assays for the determination of an analyte that is an sbp member,
particularly those for the determination of free analyte, e.g.,
free hapten. In general, a sample suspected of containing an
analyte is combined in an assay medium with a binding partner for
the analyte and other reagents depending on the particular assay
performed. The binding of the binding partner to the analyte, if
present, is detected. An effective amount of Compound I is included
in the assay medium. The assay can be performed either without
separation (homogeneous) or with separation (heterogeneous) of any
of the assay components or products. Homogeneous immunoassays are
exemplified by the EMIT.RTM. assay products (Behring Diagnostics
Inc., formerly Syva Company, San Jose, Calif.) disclosed in
Rubenstein, et al., U.S. Pat. No. 3,817,837, column 3, line 6 to
column 6, line 64; immunofluorescence methods such as those
disclosed in Ullman, et al., U.S. Pat. No. 3,996,345, column 17,
line 59 to column 23, line 25; enzyme channeling techniques such as
those disclosed in Maggio, et al., U.S. Pat. No. 4,233,402, column
6, line 25 to column 9, line 63; and other enzyme immunoassays such
as the enzyme linked immunosorbant assay ("ELISA") are discussed in
Maggio, E. T. supra. Exemplary of heterogeneous assays are the
radioimmunoassay, disclosed in Yalow, et al., J. Clin. Invest.
39:1157 (1960). The above disclosures are all incorporated herein
by reference.
[0080] A typical non-competitive sandwich assay is an assay
disclosed in David, et al., U.S. Pat. No. 4,486,530, column 8, line
6 to column 15, line 63, incorporated herein by reference. In this
method, an immune sandwich complex is formed in an assay medium
containing an effective amount of Compound I. The complex comprises
the analyte, a first antibody (monoclonal or polyclonal) that binds
to the analyte and a second antibody that binds to the analyte or a
complex of the analyte and the first antibody. Subsequently, the
immune sandwich complex is detected and is related to the amount of
analyte in the sample. The immune sandwich complex is detected by
virtue of the presence in the complex of a label wherein either or
both the first antibody and the second antibody contain labels or
substituents capable of combining with labels, such as, for
example, providing the antibody linked to biotin and providing
avidin bound to a label.
[0081] Another method that is useful in carrying out the assay of
this invention is disclosed in Ullman, et al., U.S. Pat. No.
4,857,453, column 11, line 21 to column 14, line 42, and column 18,
line 21 to column 21, line 55, incorporated herein by
reference.
[0082] The assay is normally carried out in an aqueous buffered
medium at a moderate pH, generally that which provides optimum
assay sensitivity. The aqueous medium may be solely water or may
include some percentage of a cosolvent, for example, from 0-40
volume percent of a cosolvent. The pH for the medium will usually
be in the range of 4-11, more usually in the range of 5-10, and
preferably in the range of 6.5-9.5. The pH will usually be a
compromise between optimum binding of the binding members of any
specific binding pairs, optimum release of the analyte from a
non-specific complex thereof in accordance with the present
invention, and the pH optimum for other reagents of the assay such
as members of the signal producing system.
[0083] Various buffers may be used to achieve the desired pH and
maintain the pH during the determination. Illustrative buffers
include borate, phosphate, carbonate, tris and barbital. The
particular buffer employed is not critical to this invention, but
in an individual assay one or another buffer may be preferred.
[0084] Moderate temperatures are normally employed for carrying out
the assay and usually constant temperatures during the period of
the measurement, particularly for rate determinations. Incubation
temperatures will normally range from 5-45.degree. C., more usually
from 15-40.degree. C. Temperatures during measurements will
generally range from 10-50.degree. C., more usually from
15-40.degree. C.
[0085] The concentration of analyte that may be assayed will
generally vary from 10.sup.-4 to 10.sup.-13 M, more usually from
10.sup.-5 to 10.sup.-7 M. Considerations, such as whether the assay
is qualitative, semiquantitative or quantitative (relative to the
amount of analyte present in the sample), the particular detection
technique and the concentration of the analyte will normally
determine the concentrations of the various reagents.
[0086] The concentrations of the various reagents in the assay
medium will generally be determined by the concentration range of
interest of the analyte. However, the final concentration of each
of the reagents will normally be determined empirically to optimize
the sensitivity of the assay over the range. That is, a variation
in concentration of analyte which is of significance should provide
an accurately measurable signal difference.
[0087] While the order of addition may be varied widely, there will
be certain preferences depending on the nature of the assay. The
simplest order of addition is to add all the materials
simultaneously and determine the effect that the assay medium has
on the signal as in a homogeneous assay. Alternatively, the
reagents can be combined sequentially. Optionally, an incubation
step may be involved subsequent to each addition, generally ranging
from 30 seconds to 6 hours, more usually from 1 minute to 1
hour.
[0088] The following examples further describe the specific
embodiments of the invention, and are intended to describe and not
to limit the scope of the invention. MPA assays are referred to by
way of example and not limitation.
[0089] In a homogeneous assay after all of the reagents have been
combined, the signal is determined and related to the amount of MPA
in the sample. For example, in an EMIT assay for MPA, a sample
suspected of containing MPA is combined in an aqueous medium either
simultaneously or sequentially with an MPA-enzyme conjugate and
antibody capable of recognizing MPA and the conjugate. The medium
also contains an effective amount of Compound I. Generally, a
substrate for the enzyme is added which results in the formation of
a chromogenic or fluorogenic product upon enzyme catalyzed
reaction. Preferred enzymes are glucose-6-phosphate dehydrogenase
and alkaline phosphatase. Compound I acts to release MPA from any
non-specific complex thereof that may be present in the sample. The
MPA and the MPA-enzyme conjugate compete for binding sites on the
antibody. The enzyme activity in the medium is then determined,
usually by spectrophotometric means, and is compared to the enzyme
activity determined when calibrators or reference samples are
tested in which a known amount of MPA is present. Typically, the
calibrators are tested in a manner similar to the testing of the
sample suspected of containing MPA. The calibrators will typically
contain differing, but known, concentrations of the MPA analyte to
be determined. Preferably, the concentration ranges present in the
calibrators will span the range of suspected MPA concentrations in
the unknown samples.
[0090] Heterogeneous assays usually involve one or more separation
steps and can be competitive or non-competitive. A variety of
competitive and non-competitive assay formats are disclosed in
Davalian, et al., U.S. Pat. No. 5,089,390, column 14, line 25 to
column 15, line 9, incorporated herein by reference. In a typical
competitive assay a support having an antibody for MPA bound
thereto is contacted with a medium containing the sample and MPA
conjugated to a detectable label such as an enzyme. The medium also
contains an effective amount of Compound I. MPA in the sample
competes with the conjugate for binding to the antibody. After
separating the support and the medium, the label activity of the
support or the medium is determined by conventional techniques and
related to the amount of MPA in the sample.
[0091] As mentioned above, the present invention provides
advantages over known compounds for releasing analytes from
non-specific binding substances present in samples for analysis.
With the present invention up to 100% of the analyte can be
released from endogenous non-specific binding substances such as
plasma proteins. Thus, variable recovery of the analyte as a
function of protein concentration relative to a calibrator matrix
is substantially reduced or eliminated. Furthermore, variable
recovery of analyte as a function of the presence of
co-administered drugs is substantially reduced or eliminated
because competition for binding sites on endogenous non-specific
binding substances between an analyte and other drugs that bind to
such substances is reduced or eliminated. High background
absorbance associated with some of the known agents is avoided in
the present invention. Accordingly, the present methods provide
particular advantages for assays wherein a signal producing system
is used that produces signal in the range of about 300 to about 700
nm. This is an important advantage particularly for
spectrophotometric based immunoassays. Yet another advantage of the
use of compounds of the present invention is the avoidance of
deleterious effects on the total dose response curves otherwise
obtained in assays not utilizing the present compounds. Still
another advantage of the present invention is that the present
releasing agent does not exhibit light sensitive degradation.
[0092] In one MPA assay in accordance with the present invention,
antibodies are employed that are capable of binding to MPA and to
its esters and metabolites. In another MPA assay in accordance with
the present invention, antibodies are used that are capable of
distinguishing between MPA and mycophenolate esters, such as MPA-M.
In another embodiment of an MPA assay in accordance with the
invention, the antibodies employed are able to distinguish between
MPA and MPA metabolites, such as MPA-G.
[0093] The binding of the antibody to MPA can be detected in
numerous ways that are well known in the art. Binding of the
antibody and MPA forms an immune complex that can be detected
directly or indirectly. The immune complexes are detected directly,
for example, when the antibodies employed are conjugated to a
label. The immune complex is detected indirectly by examining for
the effect of immune complex formation in an assay medium on a
signal producing system or by employing a labeled receptor that
specifically binds to an antibody of the invention.
[0094] Another aspect of the present invention relates to kits
useful for conveniently performing an assay for the determination
of an analyte. A kit in accordance with the present invention
comprises in packaged combination a binding partner for the analyte
and Compound I. The kit may further comprise a conjugate of the
analyte bound to a detectable label. A kit for the determination of
a MPA comprises in packaged combination an antibody capable of
binding to MPA, a conjugate of MPA and a label, and Compound I.
[0095] To enhance the versatility of the subject invention, the kit
reagents can be provided in packaged combination, in the same or
separate containers, in liquid or lyophilized form so that the
ratio of the reagents provides for substantial optimization of the
method and assay. The reagents may each be in separate containers
or various reagents can be combined in one or more containers
depending on the cross-reactivity and stability of the reagents.
For example, an aqueous solution of a Compound I can be provided in
a separate container. Alternatively, a Compound I can be included
in one of the reagents for conducting an assay. For example,
Compound I can be included in an aqueous medium containing an
antibody reagent; such aqueous medium can be packaged in a separate
container.
[0096] The kit can further include other separately packaged
reagents for conducting an assay such as additional sbp members,
ancillary reagents such as an ancillary enzyme substrate, and so
forth. The relative amounts of the various reagents in the kits can
be varied widely to provide for concentrations of the reagents
which substantially optimize the reactions that need to occur
during the present method and to further substantially optimize the
sensitivity of the assay. Under appropriate circumstances one or
more of the reagents in the kit can be provided as a dry powder,
usually lyophilized, including excipients, which on dissolution
will provide for a reagent solution having the appropriate
concentrations for performing a method or assay in accordance with
the present invention. The kit can further include a written
description of a method in accordance with the present invention as
described above.
EXAMPLES
[0097] The invention is demonstrated further by the following
illustrative examples. Parts and percentages herein are by weight
unless otherwise indicated. Temperatures are in degrees Centigrade
(.degree. C.).
Example 1
Ortho-Anisic Acid as a Releasing Agent in an Assay for MPA
[0098] The following reagents were prepared:
2 REAGENT A Comp. by wt./vol. % Comp. (g/L) COMPONENT # COMPONENT
(by weight) (at 20.degree. C.) SOURCE 1 NAD 2.346 23.88 Boehringer
Mannheim 2 G6P 0.615 6.26 Calzyme 3 sodium chloride 0.491 5.00
Mallinckrodt 4 MIT 0.098 1.00 Boehringer Mannheim 5 Na2 EDTA 0.036
0.37 Sigma 6 Pluronic .RTM. 25R2 0.010 0.1028 BASF Chemicals 7
o-anisic acid 0.149 1.52 Sigma 8 BSA 0.098 1.00 Miles Diagnostics 9
sodium azide 0.092 0.94 Amersham USB 10 Antibody to MPA 0.001
0.0075 (1) 11 water 96.062 977.82 Millipore deionized 100.00 1017.9
pH 5.6 .+-. 0.1
[0099]
3 REAGENT B Comp. by wt./vol. % Comp. (g/L) COMPONENT # COMPONENT
(by weight) (at 20.degree. C.) SOURCE 12 Tris Base 2.120 21.54
Sigma 13 Tris HCl 3.447 35.02 Sigma 14 BLG 0.098 1.00 International
Enzymes 15 Na2 EDTA 0.036 0.37 Sigma 16 MIT 0.098 1.00 Boehringer
Mannheim 17 sodium azide 0.093 0.94 Amersham USB 18 Pluronic 25R2
0.030 0.3084 BASF Chemicals 19 MPA-G6PDH 0.00005 0.0005 (2) 20
Stabilizing 0.00007 0.00075 (1) antibody 21 water 94.077 955.92
Millipore deionized 100.000 1016.1 pH 8.15 .+-. 0.15
[0100] (1) Prepared in a manner similar to that described by
Galfre, et al., (1981) Preparation of monoclonal antibodies:
strategies and procedures,
[0101] Methods Enzymol. 73: 3-46
[0102] (2) Prepared in a manner similar to that described by
Grabarek, et al. (1990) Zero-length crossliking procedure with the
use of active esters.
[0103] Anal. Biochem. 185: 131-135.
[0104] (3) Pluronic is a registered trademark of BASF
Corporation.
[0105] Abbreviations
[0106] NAD: nicotinamide adenine dinucleotide
[0107] G6P: D-glucose-6-phosphate, monosodium salt
[0108] o-anisic acid: o-methoxybenzoic acid
[0109] BSA: bovine serum albumin
[0110] Na2 EDTA: ethylenediaminetetraacetic acid, disodium salt,
dihydrate
[0111] MIT: N-methylisothiazolone, hydrochloride
[0112] MPA: mycophenolic acid
[0113] Tris Base: tris(hydroxymethyl)aminomethane
[0114] Tris HCl: Tris hydrochloride
[0115] BLG: .beta.-lactoglobulin
[0116] MPA-G6PDH: mycophenolic acid conjugated to
glucose-6-phosphate dehydrogenase
[0117] Reagents A and B were prepared as follows.
[0118] A 1.028% weight per weight solution of Pluronic 25R2 was
prepared at 2 to 25.degree. C. for use in both reagents.
[0119] Reagent A was prepared by first making an anisic acid
solution. Anisic acid was dissolved in 1N NaOH in an amount equal
to 25 mL per 1.52 grams of anisic acid. In a separate container was
weighed 70% of the final weight of deionized water, to which was
added components 1 through 5 inclusive and also component 6 using
the prepared solution of Pluronic 25R2 at 10 mL thereof per 1017.9
g (or 1.0 liter) of final solution. This solution was stirred and
the anisic acid solution was added. If necessary, the pH of the
preparation was adjusted within the range of 5.50 to 5.70 with 6 N
NaOH. Next, components 8 and 9 were added and the solution was
stirred. If necessary, the pH was adjusted to the above range. The
solution was then brought to the final weight with deionized water
and was filtered through a 0.2 micron filter. Final pH was 5.50 to
5.70. The resulting solution was designated the A diluent. Reagent
A was completed by adding antibody, i.e., component 10, to the A
diluent to a final antibody concentration of 7.5 mg/L (or 7.5
.mu.g/mL).
[0120] It is noted that Reagent A contained BSA, which like human
serum albumin binds MPA. However, the BSA was found to be a
preferred stabilizer of Reagent A over certain other proteins that
were evaluated, and thus was included in Reagent A for this reason.
Any releasing agent for MPA, therefore, would be formulated to
overcome this effect of BSA as well as any binding from the sample
being analyzed. The above formulation had greater than a 500-molar
excess of o-anisic acid to BSA in Reagent A. This concentration of
o-anisic acid was found to be more than sufficient to release all
MPA in the system and keep it displaced.
[0121] For Reagent B deionized water was weighed in an amount equal
to 80% of the final weight. To this water was added with stirring
components 12 through 17 inclusive as well as component 18, using
30 mL of Pluronic 25R2 solution per 1016.1 g (or 1.0 liter) of
final solution. The solution was brought to the final weight with
deionized water, pH in the range of 8.0 to 8.3, and was filtered
through 0.2 micron filter. The solution at this point was
designated the B diluent, which was used to make Reagent B by
addition of relatively negligible volumes or weights of components
19 and 20. For example 0.37 mL of stabilizing antibody at 20.6
mg/mL and 6.1 mL of conjugate at 0.8 mg/mL were added to 10 L (or
10.18 kg) of Reagent B. The stabilizing antibody, component 20, was
added to a final concentration of 0.75 mg/L (or 0.75 .mu.g/mL). The
conjugate, component 19, was added to achieve a rate of 300.+-.10
mA/min; rate is defined as the change in absorbance at 340 nm per
minute of reaction time and is usually expressed as mA/min.
[0122] Rates were determined on a Cobas Mira Plus.RTM. instrument
(Roche Diagnostics Systems, Inc., Branchburg, N.J.). The
temperature was kept at 37.degree. C. for the entire assay. Timings
were carried out in cycles with each cycle being 25 seconds. In
cycle 1, the first cycle, 75 .mu.L of water and 3 .mu.L of a sample
were mixed with 155 .mu.L of A diluent in a 0.6 cm path length
cuvette. This mixture was incubated until the addition of Reagent B
in cycle 7. To establish the conjugate rate, the sample used did
not contain any MPA. In cycle 7, seventy-five .mu.L of Reagent B
followed by 20 .mu.L of water was then added to the cuvette, mixed,
and incubated until the end of cycle 25 at which time the assay was
finished. During the assay, absorbance readings at 340 nm were made
at the end of every cycle. A best linear fit was then made using
only the 12 consecutive absorbance readings of cycles 14 through 25
versus time in minutes. The slope of this line was the rate.
[0123] Reagent A preparations were then made by adding antibody at
different levels to the A diluent. These titration levels were then
run with Reagent B and calibrators with different levels of MPA
(e.g., 0, 0.5, 2.0, 5.0, 10.0, and 15.0 .mu.g/mL). The level of
antibody giving the maximum rate separations between the two low
end calibrators and between the two high end calibrators was then
used to formulate the final Reagent A.
[0124] Once Reagent A and B were prepared they were used along with
calibrators to determine unknown concentrations of MPA.
[0125] To determine an unknown MPA concentration, calibrators were
run with Reagents A and B, and rates were determined for each as
previously described, except that Reagent A containing antibody was
substituted for the A diluent. Duplicate rates were typically
determined for each calibrator and averaged. The calibration curve
parameters were calculated using the MPA concentrations, average
calibrator rates and an appropriate mathematical model such as a
logit/log 4 model fit. The fit can be made on line by the analyzer
or by appropriate computer programs which optimize the parameters
Ro, Kc, a and b in the following equation: 1 R = Ro + Kc 1 + exp [
- ( a + b ln C ) ]
[0126] where
[0127] Ro, Kc, a, b are curve parameters
[0128] C is the MPA concentration
[0129] R is the rate observed with the MPA concentration
[0130] Solving this equation for C allowed the unknown MPA
concentration to be determined from its rate, R, and the curve
parameters as:
C=exp[[a+ln[Kc/(R-Ro)-1.0]]/-b]
[0131] The results are summarized as follows:
[0132] The effectiveness of the formulation with o-anisic acid was
evaluated by measuring the agreement between the quantitation of a
10 .mu.g/mL MPA spike in a normal human plasma pool (NHP) and the
quantitation of a similar spike into Dulbecco's phosphate buffered
saline (PBS), purchased from BioWhittaker, Walkersville, Md. For
both NHP and PBS, two separate spikes were made. PBS contained 0.2
g/L KCl, 0.2 g/L KH.sub.2PO.sub.4, 2.16 g/L
Na.sub.2HPO.sub.4.7H.sub.2O, and 8.0 g/L NaCl at a final pH 6.4 to
7.6. The PBS had no protein and thus no protein binding of MPA can
occur. Calibrators were NHP with 7 levels of MPA at 0, 0.3, 0.5,
2.5, 5, 10, and 20 .mu.g/mL. The NHP and PBS spikes quantitated
nearly the same, giving respective averages of 10.2 and 10.4
.mu.g/mL MPA (pooled standard deviation (sd)=0.4 .mu.g/mL). These
results indicate that the method was measuring total MPA and was
not affected by normal serum albumin binding of MPA.
Example 2
Comparison of Anisic Acid Isomers and ANS as Releasing Agents in an
MPA Assay
[0133] In this example four agents, o-anisic acid (o-AA), its meta
(m-AA) and para (p-AA) isomers, and the known releasing agent
8-anilino-1-naphthalene sulfonic acid (ANS) were compared. All the
anisic acids were purchased from Sigma Chemical Company, St. Louis,
Mo.; the ANS was obtained from Calbiochem, La Jolla, Calif.
[0134] In these experiments, the diluent for Reagent A (Rgt A) was
made somewhat differently than that in Example 1. However, the
composition was the same, except for component 7 (releasing agent,
o-anisic acid) and component 10 (concentration of MPA antibody).
Five diluents were prepared, four with one of each of the above
agents and one control with no agent. First, a 2.times. solution
was made in the manner described in Example 1 but which had only
components 1 through 5 and 9 at twice the amounts listed. Next, for
three of the diluents, each of the three anisic acid isomers was
weighed to achieve a final molarity of 12.5 mM and predissolved in
one quarter the final volume of water and a minimum amount of 6 N
NaOH (approximately 6 drops per 0.2 grams anisic acid). For the
fourth diluent, the agent, ANS, was weighed to give a 0.25 mM final
concentration and added to one quarter the final volume of water.
The fifth diluent, the control, had no releasing agent. To make
each of these five diluents, the appropriate amounts of the
2.times. solution, the 1.times. amount of BSA, component 8, and the
1.times. amount of the Pluronic 25R solution, component 6, were
combined. Four of these then received the appropriate agent. All
were mixed well. Where necessary, pH adjustments were made as
described previously and each preparation was brought to the final
volume with deionized water to achieve 1.times. concentrations of
components. Each of these A diluents was then filtered through a
0.2 micron filter. Final pH measurements on each were all between
5.5 to 5.7. For each of these five A diluents, a corresponding
Reagent A was made by adding antibody to MPA for a final
concentration of 6.5 .mu.g/mL. Reagent B (Rgt B) was prepared as
described in Example 1 except that component 20 was omitted and
0.1% BSA (Miles Diagnostics, Kankakee, Ill.) was substituted for
BLG.
[0135] It should be noted that the concentration of ANS in Reagent
A was limited due to its contribution to background absorbance.
Higher concentrations of ANS created an offscale absorbance
reading, preventing the collection of rate data. It should be
further noted that all of the Reagent A preparations were visually
colorless except for the Reagent A with ANS which had a tannish
yellow color.
[0136] In a total of two runs the effects of each of the four
agents relative to a control of no agent was examined with respect
to (1) background absorbance at 340 nm, (2) the rate of a negative
MPA sample, (3) the rate span between 0 and 10 .mu.g/mL MPA, and
(4) the closeness of rate matching of a 10 .mu.g/mL MPA spike in
NHP and in buffer. Both runs included the control Reagent A. The
first run evaluated Reagent A with ANS while the second run
evaluated Reagent A preparations containing the structural isomers
of anisic acid.
[0137] MPA was spiked into NHP and buffer (Buff) to achieve a final
concentration of 10 .mu.g/mL MPA. Buffer in this example was 50 mM
MES (2-[N-morpholino]-ethanesulfonic acid, obtained from Sigma
Chemical Company) with 0.1% (weight/volume) sodium azide, pH 7.1.
As with PBS, this buffer has no protein and thus no protein binding
of MPA can occur.
[0138] In both runs, the background absorbance at 340 nm for each
Reagent A was measured in duplicate on a 230-.mu.L combination of 3
.mu.L negative NHP, 72 .mu.L of deionized water, and 155 .mu.L
Reagent A. The cell path length was 0.6 cm. The average A.sub.340
(A=absorbance) values are found in Column C of Table 1. Duplicate
rates were determined on the spiked and unspiked NHP and buffer
similar to Example 1. The only changes from Example 1 for rate
determinations were in the protocol timings. These changes are
noted as follows. Reagent B and water were added in cycle 4. The
analysis was finished at the end of cycle 15. Rates were determined
using the 5 absorbance readings of cycles 11 through 15.
[0139] Averages of these rates are summarized in Table 2 in columns
E through H.
4TABLE 2 E F J NHP NHP G H I (H - F) with with Buff with Buff with
(H - G) Buff- B 0 .mu.g/ml 10 .mu.g/ml 0 .mu.g/ml 10 .mu.g/ml Buff
NHP CONC. D MPA MPA MPA MPA Rate Rate A in Rgt A C .DELTA. A =
agent - control Rate Rate Rate Rate Span Diff. RUN # Agent mM A 340
nm .DELTA. A 340 nm mA/min mA/min mA/min mA/min mA/min mA/min 1
NONE 0 0.457 -- 161.0 240.5 159.4 250.8 91.4 10.3 (control) ANS
0.25 0.779 0.322 158.2 274.2 156.0 276.2 120.2 2.0 2 NONE 0 0.486
-- 159.1 238.7 159.5 250.6 91.1 11.9 (control) o-AA 12.5 0.490
0.004 169.5 276.9 168.2 277.5 109.3 0.6 m-AA 12.5 0.494 0.008 188.1
280.3 189.4 279.7 90.3 -0.6 p-AA 12.5 0.494 0.008 182.7 280.4 182.3
278.3 96.0 -2.1
[0140] The effectiveness of the releasing agent was determined by
subtracting the spiked NHP rate from the spiked buffer rate (column
H-column F). The closer this value was to zero, the better the
releasing effect. Based on this criterion, all three of the anisic
acid agents showed better MPA release when compared to the control,
as seen by the results in column J. Thus, all three of the anisic
acid agents diminished or eliminated the effect of NHP on MPA
measurement, indicating total MPA was being measured.
[0141] Furthermore, it is seen that, although ANS can function as a
releasing agent, ANS contributed detrimentally to background
absorbance (column C and D) while the structural isomers of anisic
acid have little or no effect on background absorbance. Also,
Reagent A with ANS was found to become visibly discolored when
exposed to light. This did not occur with the control or anisic
acid reagents.
[0142] All four agents showed some non-detrimental or tolerable
effects on either the negative rate and/or rate span as compared to
control values. This indicated that the agents were having effects
other than reducing the matrix effect of the NHP. ANS had the
largest increase on rate span. The anisic acids had a marked
increase in negative rate over the control whereas ANS had little
or no effect. Although there were these effects on the negative
rate relative to reagents without anisic acid, there did not appear
to be an impact on assay performance because the negative rates for
buffer and NHP were the same (see columns E and G). Very good assay
sensitivity was achieved with o-anisic acid.
[0143] In summary, the results of the study showed that all three
of the anisic acids performed well as releasing agents with
o-anisic acid showing somewhat better results than the meta and
para isomers based on the smaller effect on the negative rate. The
results showed that ANS had several effects on performance that
were not desirable such as increased absorbance and discoloration
of reagent when exposed to light. ANS has a significant
contribution to background absorbance even at the relatively low
level of 0.25 mM in Reagent A, thus limiting its use in assays,
particularly those having an absorbance maximum.
[0144] The anisic acid isomers did not demonstrate these effects on
absorbance or coloration with light exposure and all were effective
in the release of MPA from NHP as seen by the equivalent or near
equivalent rates of MPA in a non-protein matrix (buffer) and NHP.
Without these agents, the MPA in buffer gave a higher rate (more
apparent MPA) than the same concentration of MPA in NHP. The ortho
isomer had the least effect on the negative rate. Furthermore,
o-anisic acid was shown to quantitate MPA equivalently in a
protein-free matrix and NHP.
Example 3
[0145] Salicylic Acid as a Releasing Agent
[0146] In a separate study another agent, salicylic acid
(2-hydroxybenzoic acid), was formulated into Reagent A at 12.5 and
25 mM. This agent reduced the Buffer rate span by 47% and increased
the negative rate by 38% relative to the control without releasing
agent. Rates were determined in a manner similar to that described
in Example 2. Additionally, the 25 mM salicylic acid contributed
0.311 absorbance units above the control background at 340 nm. It
is also noted that the Reagent A preparation of salicylic acid had
a slight pink tinge. This visual color and the background
absorbance at 340 nm may be due to the interaction of salicylic
acid with the merest levels of ferric salts as reported in the
Merck Index, eleventh edition, Merck & Co., Inc., Rahway, N.J.,
page 8300. Salicylic acid was eliminated from more extensive
comparative studies due to these experimental results.
Example 4
Effect of o-Anisic Acid on MPA Rates in the Presence of
Co-Administered Drugs and Salicylic Acid
[0147] In a separate study, the ability of o-anisic acid to
eliminate the effect of salicylate on rates of MPA plasma spikes
was compared. Two sets of antibody reagents (Reagent A) were
prepared to the basic formulation described in Example 2 above. In
one reagent, no releasing agent was added; in another reagent,
o-anisic acid was added at 12.5 mM. A common Reagent B was prepared
as described in Example 2.
[0148] Rates for four levels of MPA spikes into plasma and a plasma
control were measured with each reagent set. A similar set of
plasma spikes was prepared with plasma containing non-interfering
co-administered drugs, and another set of plasma spikes was
prepared containing non-interfering drugs plus salicylate.
Concentrations of the non-interfering drugs in .mu.g/mL were as
follows: ampicillin, 36; cefaclor, 26; chloramphenicol, 64;
trimethoprim, 2; albuterol, 7; isoproterenol, 18; metoprolol, 77;
diltiazem, 28; nifedipine, 22; verapamil 12; fenoprofen, 16;
indomethacin, 36; ketoconazole, 75; miconazole, 106; isoniazid,
120; 5-fluorouracil, 23; griseofulvin, 10; methotrexate, 2;
diphenhydramine, 93; dl-ephedrine, 103; phenylephrine, 76;
disopyramide, 52; procainamide, 64; metoclopramide, 5; niacin, 25;
niacinamide, 47; acetaminophen, 87; and lidocaine, 43. Salicylate
was present at 659 .mu.g/mL.
[0149] Rates were determined as described in Example 2 above with
the exception of the absorbance read window, which was extended to
cycle 20 where the assay was ended. Rates were determined using the
10 consecutive absorbance readings of cycles 11 through 20. The
results are summarized in Table 3.
5 TABLE 3 Antibody Reagent Antibody Reagent + o- Only Anisic Acid
MPA + MeOH* 0 .mu.g/mL 149 161 0.5 .mu.g/mL 155 171 2.5 .mu.g/mL
181 210 5 .mu.g/mL 201 234 15 .mu.g/mL 240 264 MPA + NI** 0
.mu.g/mL 149 161 0.5 .mu.g/mL 158 173 2.5 .mu.g/mL 183 211 5
.mu.g/mL 204 234 15 .mu.g/mL 242 263 MPA + NI + Salicylate** 0
.mu.g/mL 149 161 0.5 .mu.g/mL 157 171 2.5 .mu.g/mL 190 211 5
.mu.g/mL 210 234 15 .mu.g/mL 250 263 *MeOH = plasma spiked with
methanol as control for NI spike. **NI = plasma contains spikes of
non-interfering co-administered drugs in addition to MPA.
[0150] In summary, salicylate increased rates of MPA in normal
human plasma in the absence of releasing agents, which could result
in inaccurate quantitation of MPA in an assay. The presence of
o-anisic acid as a releasing agent in the antibody reagent
eliminated this potential problem in an assay for MPA.
[0151] The above discussion includes certain theories as to
mechanisms involved in the present invention. These theories should
not be construed to limit the present invention in any way, since
it has been demonstrated that the present invention achieves the
results described.
[0152] The above description and examples fully disclose the
invention including preferred embodiments thereof. Modifications of
the methods described that are obvious to those of ordinary skill
in the art are intended to be within the scope of the following
claims.
* * * * *