Note: Descriptions are shown in the official language in which they were submitted.
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PRET~EATMENT R~AGEN~ AND ~nOD~ U~IN~ THE 8AME
Field of the Invent:Lon
The clinical diagnostic field has seen a broad
expansion in recent years, both as to the variety of
materials (analytes~ that may be readily and accurately
determined, as well as the methods for the determination.
~onvenient and reliable means for detecting the presence
of low concentrations of materials in li~uids is desired.
In clinical chemistry these materials may be present in
body fluids in concentrations below 10-12 molar. The
difficulty of detecting low concentrations of these
materials is enhanced by the relatively small sample
sizes that can be utilized and the fact that some
analytes are associated with other components in the
sample and are difficult to detect accurately.
The monitoring of levels of drugs, such as
therapeutic drugs, in biological fluid samples i5 useful
in the proper treatment of patients receiving such drugs.
It is important to monitor drug levels to avoid toxic
dose levels or dose levels that are therapeutically
ineffective. Many assay techniques have been developed
to determine quantitatively the level of drug in a
patient sample. Such assay techniques generally involve
immunoreactions and include, for example,
radioimmunoassay, enzyme immunoassay, agglutination
immunoassays, fluorescent polarization immunoassays, and
so forth.
In developing an assay there are many
considerations. One consideration is the signal response
to changes in the concentration of an analyte. A second
consideration is the ease with which the protocol for the
assay may be carried out. A third consideration is the
variation in interference from sample to sample. Ease of
preparation and purification of the reagents,
availability of equipment, ease of automation and
interaction of the material of interest with sample
components are some of the additional considerations in
developing a useful assay. For example, some analytes
- SUBSTITUTE SHEET (RULE 26)
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are associated with various other components of a sample
such as proteins, cells and other cellular material, thus
making their detection difficult or impossible.
The body relies upon a complex ; c response
system to distinguish self from non-self. The proper
functioning of the immune system is vital for the long
term health of the body. Deficient immune response can
lead to the body's inability to protect itself from
non-self matter. Excessive ; e response can lead to
the body's over reaction to what would otherwise be
innocuous matter.
At times, the body's immune system must be
controlled in order to either augment a deficient
response or suppress an excessive response. For example,
1~ when organs such as kidney, heart, heart-lung, bone
marrow, and liver are transplanted in humans, the body
will sometimes reject the transplanted tissue by a
process referred to as allograft rejection.
In treating allograft rejection, the immune
system is frequently suppressed in a controlled manner
through drug therapy. Immunosuppressant drugs are
carefully a ; n; ~tered to transplant recipients in order
to help prevent allograft rejection of non-self tissue.
One such drug which finds use as an
immunosuppressant in the United States and other
countries is cyclosporin A (CsA) (U.S. Patent Nos.
4,117,118 (1978~ and 4,396,542 (1983)). CsA may have
other useful properties such as antibiotic,
anti-arthritic and anti-inflammatory activities and may
find use in the treatment of other conditions such as
diabetes, malaria and autoimmune diseases.
~ ven though CsA is a highly effective
immunosuppressant drug, its use must be carefully managed
because the effective dose range is narrow and excessive
3~ dosage can result in serious side effects. Renal
dysfunction, hypertension, cardiovascular cramps,
hirsutism, acne, tremor, convulsions, headache, gum
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hyperplasia, diarrhea, nausea, vomiting, hepatotoxicity,
abdominal discomfort, paresthesia, flushing, leukopenia,
lymphoma, sinusitis and gynecor~ctia have been observed
~ in kidney, heart or liver transplant patients undergoing
CsA treatment. Too little CsA can lead to graft
rejection.
Manageme~t of CsA dosage involves careful
control of the level of the drug present in the patient.
Because the distribution and metabolism of CsA varies
greatly between patients, and because of the wide range
and severity of adverse reactions, accurate monitoring of
drug level is considered essential. CsA associates with
materials present in whole blood samples and, thus, an
accurate assay requires that CsA be extracted from these
other materials.
The methods and compositions of the present
invention relate to reagents for pretreating samples
suspected of containing analytes of interest, in
particular analytes that occur in samples in association
with other materials.
Documents are cited in this disclosure with a
full citation. These documents relate to the state-of-
the-art, and each document is hereby incorporated by
reference.
Brief Description of the Related Art
European patent application No. 283,801 (1988)
(Wang, et al . ) and its priority document U.S. Patent No.
5,239,057 describe a fluorescence polarization assay for
cyclosporin A and ~metabolites and related immunogens and
antibodies.
Quesniaux, et al., Molecular Immunology (1987)
24(11):1159-1168, describe the specificity and cross-
reactivity of monoclonal antibodies to cyclosporin.
Ball, et al ., discuss speci~ic radioimmunoassay
with a monoclonal antibody for monitoring cyclosporine in
blood in Clin. Chem. (1988) 34(2):257-260.
. CA 02230284 1998-02-24
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McBride, et al ., de~cribe the measurement of
cyclosporine in plasma from patients with various
transplants: HPLC and radioimmunoassay with a specific
monoclonal antibody compared in Clin. Chem. (1989)
35(8):1726-1730.
The liquid-chromatographic measurement of
cyclosporin A and its metabolites in blood, bile snd
urine is discussed in Clin. Chem. ~1988) 34(1):34-39.
Bowers, et al ., disclose studies of
cyclosporine blood levels: analysis, clinical utility,
pharmacokinetics, metabolites, and chronopharmacology in
TransPlantation Proceedinqs (1986) XVIII (6):137-143.
U.S. Patent No. 5,135,875 (Meucci, et al.)
discusses a protein precipitation reagent, employing a
glycol, a short-chained aliphatic alcohol and zinc.
European Patent Application No. 0 471 293 A2
(Meucci, et al. ) discloses a solubilization reagent for
biological test samples.
European Patent Application No. 0 473 961 A2
(Morrison, et al . ) discloses immunoassay reagents and
method for determining cyclosporin.
U.S. Patent No. 4,959,303 (Milburn, et al . )
describes an assay for antigens by binding immune
complexes to solid supports free of protein and non-ionic
binders.
Despite the extensive teachings of the prior
art regarding assays for cyclosporin and pretreatment
reagents, the development of a pretreatment formulation
which facilitates the detection of an analyte associated
with other components of a sample, wherein the individual
components of the pretreatment formulation, either alone
or in combination, do not interfere with the assay medium
and subse~uent detection, has not been taught or
suggested by the prior art.
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~U~L~RY OF T~E lNV~JN~llON
Thus, the present invention provides a novel
pretreatment composition for treating samples being
assayed for the presence and/or amount of an associated
analyte, wherein the pretreatment composition facilitates
~ the detection of an analyte associated with other
components of a sample which would otherwise obscure
detection of the analyte under conventional assay
conditions, absent pretreatment. In accordance with the
present invention, a sample is pretreated to render the
associated analyte more readily available to one or more
of the assay reagents. The inventive pretreatment
compositions serve to lyse cells present in the sample,
precipitate proteins which may be present, and solubilize
the analyte of interest, rendering the analyte more
easily detectable.
One aspect of the present invention relates to
a composition comprising a lower alkyl alcohol in an
amount of about 30% to about 40% weight/volume (w/v), a
glycol in an amount of about 20% to about 40% w/v, and at
least about 30% w/v of an aqueous component comprising
about 20 mM to about 30 mM metal salt.
Another embodiment of the present invention is
a composition comprising methanol in an amount of about
30% to about 40% w/v, propylene glycol or ethylene glycol
in an amount of about 20% to about 40% w/v, and about 30%
to about 40% w/v of an aqueous component comprising about
20 mM to about 30 mM copper salt, about 0.5 mM to about
20 mM of a buffer, and about 0.005~ to about 0.1% w/v of
a non-ionic detergent, such that the aqueous component
has a pH of about 3.0 to about 4.6.
Another aspect of the present invention is an
improvement in an assay for the determination of an
associated analyte in a sample suspected of containing
the associated analyte. The assay comprises contacting a
sample suspected of containing the associated analyte
with a pretreatment reagent, contacting the sample with
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assay reagents for detection of the analyte. The
improvement comprises employing as the pretreatment
reagent one of the above compositions.
Another aspect of the present invention is a
kit comprising in packaged combination (a) one or more
reagents for conducting a determination of an associated
analyte and (b) one of the above compositions.
Another aspect of the present invention is an
i _Lovement in an assay for the determination of an
immunosuppressant drug in a sample suspected of
containing the drug. The assay comprises contacting the
sample with a specific binding member for the drug and
detecting binding of the specific binding ~ h~ to the
drug. The present improvement comprises contacting the
sample with one of the above compositions either prior to
or in conjunction with the contacting step.
Another embodiment of the present invention is
an improvement in an assay for the determination of an
immunosuppressant drug in a sample suspected of
containing the drug. The assay comprises contacting the
sample with antibodies for the drug and a conjugate of a
label and a compound recognized by the antibodies, and
detecting immune complexes of the label conjugate and the
antibodies. The improvement comprises contacting the
~5 sample with the one of the above compositions either
prior to or in conjunction with the first step of the
assay.
These and other objects and embodiments are
disclosed or will be obvious from the following Detailed
Description.
BRIEF ~ESCRIPTION OF THE FIGURE8
Fig. 1 is a graphical representation of the
results of an assay for tacrolimus (FK506) with the use
of the inventive pretreatment composition versus with
methanol alone.
CA 02230284 1998-02-24
W 0~8/OOC96 PCTAUS97112420
DETAILED DESCRIP~IO~N
The present invention provides compositions and
kits for pretreating samples, to facilitate the
determination of the presence and/or the amount of an
associated analyte. In accordance with the present
invention, a sample is pretreated to make the associated
analyte more readily available to one or more of the
assay reagents. Samples to be analyzed are pretreated to
lyse cells precipitate proteins, and to solubilize
cyclosporin, each of which may be present in the sample
to be analyzed. The pretreatment compositions are
characterized in that they exhibit lower volatility than
some of the known pretreatment materials. This permits
the pretreated sample to be assayed by analyzers using
sample cups open to the environment with greater sample
stability and with reduced risk to the operator.
Before proceeding with a description of the
specific embodiment:s, a number of terms will be defined.
A sample is a biological material, usually a
biological fluid, which can include human, animal or
man-made samples. Typically, the sample is a natural
fluid, such as, for example, urine, whole blood, serum,
plasma, semen, spinal fluid, saliva, and so forth or an
aqueous solution or extract thereof.
An associated analyte is an analyte that is
present in a sample in association with, e.g., complexed
to, other components of the sample such as, for example,
cellular material, phospholipids, proteins and the like.
Such analytes include therapeutic drugs, such as, for
example, immunosuppressant drugs, e.g., cyclosporin,
mycophenolic acid, FK-506, rapamycin, azathioprine and
steroids.
- Cyclosporin is a natural or synthetic cyclic
undecapeptide, used as an immunosuppressive drug to
suppress unwanted immune response leading to organ
rejection after transplantation. The exact structure of
a particular cyclosporin may vary in minor ways from one
CA 02230284 1998-02-24
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to the next. Cyclosporins include cyclosporin A,
cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin
E, cyclosporin F, cyclosporin G, cyclosporin H,
cyclosporin I, atiocyclosporin, and so forth. Also
included in the term cyclosporin are metabolites of
cyclosporin which retain the undecapeptide ring such as,
for example, major metabolites AM9 (Ml), AM19 (M8), AMl
(M17) and AM4N (M21), and other metabolites such as M9,
MlO, M16 and M18.
Lower alkyl alcohols are branched or straight
chain alkyl alcohols containing from 1 to 5 carbon atoms,
such as, e.g., methanol, ethanol, propanol, butanol,
isopropanol, isobutanol, pentanol, isopentanol, etc., and
mixtures thereof, preferably methanol.
The term aliphatic relates to an acyclic
hydrocarbon; an aliphatic hydrocarbon is a compound of
carbon and hydrogen having an open chain.
Polyhydroxy aliphatic compounds are aliphatic
compounds contA;~;ng two or more hydroxyl groups such as,
for example, alkylene polyols, e.g., glycols, glycerol
and the like, and mixtures thereof, preferably, a glycol,
more preferably, propylene glycol.
Glycols are aliphatic compounds containing two
hydroxyl groups such as, for example, ethanediol
(ethylene glycol), propanediol (propylene glycol),
polyethylene glycol, polypropylene glycol, and the like.
Glycerol (glycerin) is an alkylene polyol,
namely, 1,2,3-propanetriol.
A metal salt is a compound containing a metal
ion and a non-metal counterion. The metal ion is one that
provides for precipitation of hemoglobin (heme), plasma
proteins, and other interfering substances in a sample to
be analyzed. Preferred metal ions include ions of
copper, iron, cobalt and molybdenum, more preferably,
copper ion. The particular metal ion used is determined
in part so as to avoid interference in the wavelength of
the measurement of a signal obtained in an assay for the
CA 02230284 1998-02-24
W O 9~J'~ C PCTrUS97/12420
associated analyte. The non-metal anion can be, by way of
illustration and not limitation, a sulfate, a halide such
as chloride, bromide, etc., an acetate, and so forth.
A buffer is a buffering agent generally having
a pKa of about 4.5 to about 8.5. The buffer is chosen so
that a stable pH is obtained within the desired range of
pH values for the aqueous component, and thus a
composition, in accordance with the present invention.
The buffer should have minimal effect on the stability of
the components of the present compositions and should not
have a deleterious effect on the reagents used in an
assay for an associated analyte or on the assay
measurement or results in general. Suitable buffers are
homopiperazine-N,N'-bis-2-ethanesulfonic acid, acetate,
citrate, tris-HCl, and the like.
The term detergent, in the context of the
present disclosure, refers to a surfactant that is
capable of rupturing red blood cells. A non-ionic
detergent refers to a non-ionic surfactant such as, for
example, a nonionic polyglycol detergent, for example,
polyoxypropylene (available as Pluronic 25R2 from BASF
Chemicals, Mount Olive, NJ), saponins, digitonin,
polyoxyethylene alcohols (e.g., Brij series and Lubrol),
polyoxyethylene p tert octylphenols (e.g., Triton X-100,
Triton X-114 and Nonidet P-40), fatty acid esters of
polyoxyethylene sorbitan (e.g., Tween-20), B-D-
octylglucoside, ~-D-dodecylmaltoside and alkyl-N-
methylglucamides (all available from Sigma Chemical
Company, St. Louis, M0, or Fluka Chemie AG, Buchs,
Switzerland or Ronkonkoma, NY, or Aldrich Chemical
Company, Milwaukeer WI), and so forth. Preferably, the
non-ionic detergent is a polyoxypropylene or
polyoxyethylene.
An anionic detergent is an anionic surfactant,
such as, for example, alkyl-, alkyletherdialkyl esters-,
alkylaryl-, and alpha olefin- sulfates, sulfonic acids,
sulfonates, sulfosuccimates, and sulfosuccinic acids,
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polymerized alkyl naphthalene sulfonates, phosphate
esters, free acid of complex organic phosphate esters,
aliphatic hydroxylated phosphate esters, sulfated fatty
acid esters, sulfated oils such as castor, sperm, soya
bean, glycerol trioleate, neatsfoot, tallow and oleic
acid, n-fatty acid acyl glutamates such as n-lauroyl, n-
cocoyl, n-hydrogenated tallowyl, n-mixed fatty acid acyl,
carboxylated polyelectrolytes, disproportionated resins,
and so forth. Examples of common anionic detergents
include sodium dodecylsulfate and sodium dodecyl-~-
sarcosinate.
The term alkylene refers to an organic radical
derived from an unsaturated aliphatic hydrocarbon, e.g.
ethylene.
An antibody is 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
t~c-hn;ques that are well known in the art such as
;~m~ln;zation of a host and collection of sera from which
the immunoglobulin can be separated by known techniques
(polyclonal), 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')2,
Fab', and the like.
Antiserum containing antibodies (polyclonal) is
obtained by well-established techniques involving
~ n;zation of an animal, such as a rabbit, guinea pig,
or goat, with an appropriate immunogen and obtaining
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11
antisera from the blood of the immunized ~n; ~l after an
appropriate waiting period. State-of-the-art reviews are
provided by Parker, Radioimmunoassay of Biologically
Active Compounds, Prentice-~all (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).
Antibodies can also be obtained by somatic cell
hybridization t~ch~;ques, such antibodies being commonly
referred to as monoclonal antibodies. Monoclonal
antibodies may be produced according to the st~n~rd
techniques of Antiserum containing antibodies
(polyclonal) is obtained by well-established t~ hrl; ques
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 ;m~nn;zed 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)-
Antibodies can also be obtained by somatic cell
hybridization techni~ues, such antibodies being commonlyreferred to as monoclonal anti~odies. Monoclonal
antibodies may be produced according to the st~ndA~d
~echn;ques of Kohler and Milstein, Na~ure ~65:495-497,
1975. Reviews of monoclonal antibody t~-hn;ques 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 ~n; ~- is
sacrificed and spleen cells obtained. Alternatively, the
spleen cells of a non-;~nn;zed animal can be sensitized
CA 02230284 1998-02-24
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to the immunogen n 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.
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 ~lask
device, both of which are well known in the art. Various
conventional ways exist for isolation and puri~ication of
the monoclonal antibodies from other proteins and other
cont~ ;n~nts (see Kohler and Milstein, supra).
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.
Reviews of monoclonal antibody t~chn;ques are
found in Lymphocyte Hybridomas, ed. Melchers, et al.
Springer-Verlag (New York 1978~, Nature 266: 495 (1977),
35 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
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13
a mouse and, after a sufficient time, the An~l is
sacrificed and spleen cells obtained. Alternatively, the
spleen cells of a non~ n;zed animal can be sensitized
to the immunogen n vi~ro. The spleen cell chromosomes
S 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, w~ich 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.
Various techniques exist for enhancing yields
of monoclonal antihodies, 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 ;n 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
cont~;nAnts (see Rohler and Milstein, supra).
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 wllich can be expressed in bacteria to
produce recombinant proteins having the corresponding
antibody binding sites.
In general, antibodies can be purified by known
techniques such as chromatography, e.g., DEAE
~ CA 02230284 1998-02-24
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14
chromatography, ABx chromatography, and the like,
filtration, and so forth.
Reagents for conducting the determination of an
associated analyte are otherwise referred to herein as
reagents used in an assay for the associated analyte.
Such reagents depend on the nature of the assay, e.g.,
whether the assay is heterogeneous or homogeneous; on the
nature of the binding reactions and the signal producing
system utilized, e.g., enzyme immunoassay, fluorescent
immunoassay, chemiluminescent immunoassay, agglutination
assay, and so forth. In general, such reagents are those
that permit an accurate determination to be conducted for
the presence and/or amount of an associated analyte.
For example, for an immunoassay such reagents
may include a conjugate of a label and a specific binding
pair member, e.g., antibody or hapten, other members of a
signal producing system of which the label is a part,
other specific binding pair members, ancillary materials
and the like.
A label is any molecule which produces or can
be induced to produce a signal. The label may be
conjugated to a ~h~r of a specific binding pair such as
an analyte or an antibody, or to another molecule such as
a receptor or a molecule that can bind to a receptor such
as a ligand, particularly a hapten. The label can be a
member of the signal producing system, as defined below,
that includes a signal producing means. The
label may be isotopic or nonisotopic, preferably
nonisotopic. By way of example and not limitation, the
label can be a part of a catalytic reaction system such
as enzymes, enzyme fragments, enzyme substrates, enzy~e
inhibitors, coenzymes, or catalysts; part of a chromogen
system such as fluorophores, dyes, chemiluminescers,
luminescers, or sensitizers; a dispersible particle that
can be non-magnetic or magnetic, a solid support, a
liposome, a ligand, a receptor, a hapten, and so forth.
A wide variety of non-enzymatic catalysts which may be
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employed are found in U.S. Patent No. 4,160,645 ~1979),
the appropriate portions of which are incorporated herein
by reference. A large number of enzymes and coenzymes
for providing such products are indicated in U.S. Patent
No. 4,275,149 columns 19 to 23, and U.S. Patent No.
4,318,980, columns 10 to 14, which disclosures are
incorporated herein by reference. A number of enzyme
combinations are set forth in U.S. Patent no. 4,275,149,
columns 23 to 28, which combinations can find use in the
subject invention. This disclosure is incorporated
herein by reference.
Illustrative enzymes include dehydrogenases
such as malate dehydrogenase, glucose-6-phosphate
dehydrogenase, and lactate dehydrogenase. Of the
~5 oxidases, glucose oxidase is exemplary. Of the
peroxidases, horse radish peroxidase is illustrative. Of
the hydrolases, alkaline phosphatase, b-glucosidase and
lysozyme are illustrative.
A conjugate is a molecule comprised of two or
more subunits bound together, optionally through a
linking group, to form a single structure. The binding
is a chemical interaction which can be made either by a
direct connection ~e.g. a chemical bond) between the
subunits or by use of a linking group. Conjugation is
any process wherein two subunits are linked together to
form a conjugate. The conjugation process can be
comprised of any number of steps.
A receptor is any compound or composition
capable of recognizing a particular spatial and polar
organization of a molecule. These organized areas of a
molecule are referred to as epitopic or determ;n~nt
sites. Illustrati~e naturally occurring receptors
~ include antibodies r enzymes, FAb fragments, poly(nucleic
acids), complement component, i.e. thyroxine binding
- 35 globulin, lectins, protein A, and the like. Receptors
are also referred to as antiligands. Natural receptors
exists that binds specifically to cyclosporin.
CA 02230284 1998-02-24
W098~9C PCT~S971~20
16
A ligand is any organic molecule for which a
receptor naturally exists or can be prepared.
A member of a specific binding pair (s~p
member) is one of two different molecules, having an area
on the surface or in a cavity which specifically bi~ds to
and is thereby defined as complementary with a particular
spatial and polar organization of the other molecule.
The members of the specific binding pair are referred to
as ligand and receptor (antiligand). These will usually
be members of an immunological pair such as
antigen-antibody, although other specific binding pairs,
such as biotin-avidin, hormones-hormone receptors,
nucleic acid duplexes, IgG-protein A, DNA-DNA, DNA-RNA,
and the like, are not immunological pairs but are
specific binding pairs.
A support or surface is a porous or non-porous
water insoluble material. The support can be hydrophilic
or capable of being rendered hydrophilic and includes
inorganic powders such as silica, magnesium sulfate, and
alumina; natural polymeric materials, particularly
cellulosic materials and materials derived from
cellulose, such as fiber containing papers, e.g., filter
paper, chromatographic paper, etc.; synthetic or modified
naturally occurring polymers, such as nitrocellulose,
cellulose acetate, poly (vinyl chloride), polyacrylamide,
cross linked dextran, agarose, polyacrylate,
polyethylene, polypropylene, poly(4-methylbutene),
polystyrene, polymethacrylate, poly(ethylene
terephthalate), nylon, poly(vinyl butyrate), etc.; either
used by themselves or in conjunction with other
materials; glass available as Bioglass, ceramics, metals,
and the like. Natural or synthetic assemblies such as
liposomes, phospholipid vesicles, and cells can also be
employed. Other materials which can be employed are
described above in the definition of immunogenic carrier
particles and below in the definition of a signal
producing system.
CA 02230284 1998-02-24
_.
W0~8~ 9~ PCT~S97/12420
17
Binding of sbp members to the support or
surface may be accomplished by well-known t~chniques,
commonly available in the literature, and described above
- in the definition of immunogenic carrier particles. The
surface can have any one of a number of shapes, such as
strip, rod, particle, including bead, and the like.
The function of the signal producing system is
to produce a product which provides a detectable signal
related to the amount of bound and/or unbound label. The
signal producing sy~tem may have one or more components,
at least one component being a label. The signal
producing system includes all of the reagents required to
produce a measurable signal including signal producing
means capable of interacting with the label to produce a
signal.
The signal producing system provides a signal
detectable by external means, normally by measurement of
electromagnetic radiation, desirably by visual
~;~tion. In one approach, the signal producing
system includes a chromophoric substrate and enzyme,
where chromophoric substrates are enzymatically converted
to dyes which absorb light in the ultraviolet or visible
region, phosphors or fluorescers.
The signal producing means is capable of
2S interacting ~ith the label to produce a detectable
signal. Such means include, for example, electromagnetic
radiation, heat, chemical reagents, and the like. Where
chemical reagents are employed, some of the chemical
reagents can be included as part of a developer solution.
The chemical reagents can include substrates, coenzymes,
enhancers, second enzymes, activators, cofactors,
inhibitors, scavengers, metal ions, specific binding
~ substances required for binding of signal generating
substances, and the like. Some of the chemical reagents
such as coenzymes, substances that react with enzymic
products, other enzymes and catalysts, and the like can
be bound to other molecules or to a support.
. CA 02230284 1998-02-24
WO ~81C~C PCTAUS97/12420
18
The signal producing system including the label
can include one or more particles, which are insoluble
particles of at least about 50 nm and not more than about
50 microns, usually at least about 100 nm and less than
about 25 microns, preferably from about 0.2 to 5 microns,
diameter. The particle may be organic or inorganic,
porous or non-porous, preferably of a density
approximating water, generally from about 0.7 to about
1.5 g/mL, and composed of material that can be
transparent, partially transparent, or opa~ue.
Further, ancillary materials will frequently be
employed in an assay in accordance with the present
invention. For example, buffers will frequently be
present in the assay medium, as well as stabilizers,
preservatives and/or antimicrobial agents ~or the assay
medium and the assay components.
QuantitatiVe, semiquantitative, and qualitative
methods for deter~in;ng an associated analyte are
considered to be methods of measuring the amount of such
analyte. For example, a method which detects the
presence or absence of such analyte in a sample suspected
of containing the analyte is considered to be included
within the scope of the above phrase. Synonymous with
the recitation of the phrase dete~ in;ng the amount of an
associated analyte are the following, non-limiting
recitations, including: detecting or measuring an
associated analyte; detecting or measuring the presence
of an associated analyte; and detecting or measuring the
amount of such analyte.
One aspect of the present invention relates to
compositions comprising a lower alkyl alcohol, a
polyhydroxy aliphatic compound, and an aqueous component
containing a metal salt. The nature and amount of alcohol
in the present composition is governed by a number of
considerations. The alcohol should have ~;n;~l effect
on the binding of specific binding reagents used in an
assay for the associated analyte. Also, the alcohol
CA 02230284 1998-02-24
WO 9~J~C9C PCTrUS97/1242
19
should have minimal effect on the activity of other
reagents for conducting an assay such as, for example,
labels, e.g., enzymes, and so forth. The amount of
alcohol is at least sufficient to provide for substantial
extraction of the associated analyte from other sample
components and also to ~in; ;ze the amount of hemoglobin
and other interfering substances in the extracted sample.
The amount of alcohol should be low enough so that a
substantial portion, preferably all, of the metal salt
r~ ~; n~ in solution in the composition. The amount of
alcohol is not so great as to result in an unacceptable
evaporation rate for the present composition when used to
pretreat a sample in accordance with the present
invention or to result in an unacceptable integrity for
the insolubilized interfering substances that form during
the present pretreatment. Generally, the insolubilized
interfering substances form a pellet during the course of
the present pretreatment. The integrity of the pellet
should be such as to not permit any significant amount of
the interfering substances to be removed when the sample
solution is separated from the pellet. In general, the
amount of alcohol in the composition is about 30% to
about 40%, preferably about 33% to about 37%, more
preferably, about 35%, w/v. In a preferred embodiment,
the alcohol is methanol, and it is present in an amount
of about 33% to 35% w/v.
The polyhydroxy aliphatic compound should be
present in an amount that provides for rapid ~;x;ng of
the present composition with the sample so that minimal
overall handling time is realized. Desirably, the amount
of the polyhydroxy aliphatic compound is sufficient when
the viscosity of the present composition is about that of
the sample to be pretreated. Where the sample is whole
blood, the viscosity of the composition of the invention
should ideally approximate that of whole blood. In this
way, the sample and the composition are rapidly and
easily mixed together. The amount of polyhydroxy
CA 02230284 1998-02-24
WO ~8/00~96 PCT~US97tl2420
aliphatic compound should not be great enough to result
in the extraction of hemoglobin or other interfering
substances together with the extracted analyte. Usually,
the polyhydroxy aliphatic compound is present in an
amount of about 20% to about 40%, preferably, about 27%
to about 33.3~, more preferably, about 30~ to 33.3%, w/v.
In a preferred embodiment, the polyhydroxy aliphatic
compound is propylene glycol, and it is present in an
amount of about 30% to 33.3~ w/v.
The amount of the aqueous component is
generally determined by default based on the amount of
the lower alkyl alcohol and the polyhydroxy aliphatic
compound. The amount of the aqueous component is at
least that necessary to i n; mi ze precipitation of the
metal salt in the present composition. Normally, the
agueous component is present in an amount of at least
30%, preferably, at about 30% to about 40%, more
preferably about 33~ to about 38%, more preferably, about
33.3% to 36% w/v. The amount of metal salt present in
the aqueous component is at least sufficient to
precipitate a substantial portion of the hemoglobin and
other interfering substAnc-e~ in the sample to be
analyzed. However, the amount of the metal ion should
not be great enough to cause a significant effect on the
activity of assay reagents used in the analysis. In
particular, the metal ion concentration should not be so
great as to have a detrimental effect on the activity of
a label, e.g., an enzyme, used as part of a signal
producing system. Generally, the copper salt is at a
concentration of about 20 mM to about 30 mM, preferably,
about 23 mM to about 27 mM, more preferably, about
25 mM.
The aqueous component may contain one or more
other reagents such as a buffer and a non-ionic or
anionic detergent. The concentration of a buffer is not
critical. In general, the lowest concentration is chosen
that achieves a desired pH for the final composition of
CA 02230284 1998-02-24
W O 98/00696 PCTrUS97/1242
21
the invention. The concentration of the buffer should
not be so high as to offset the required pH for other
reagents used in an assay for the associated analyte when
the extracted sample is mixed with such reagents to
conduct the assay. The amount of the buffer is chosen so
that a stable pH is obtained within the desired range of
pH values for the aqueous component, and thus a
composition, in accordance with the present invention.
The concentration of the buffer should have minimal
effect on the stability of the components of the present
compositions and should not have a deleterious effect on
the reagents used in an assay for a associated analyte or
on the assay measurement or results in general. The
buffer is usually present at a concentration of about 0.5
mM to about 20 mM, preferably, about 0.8 to about 19 mM,
more preferably, about 1 to 18.5 mM.
The detergent concentration should be
sufficient to result in a substantial extraction of the
associated analyte and to bring about substantial
rupturing of cells present in the sample to be analyzed.
Furthermore, the nature of the detergent as well as its
concentration should provide for consistency in the
results obtained in an assay for the associated analyte.
Usually, the detergent is present in a concentration of
about 0.005% to about 0.2%, preferably, about 0.01% to
about 0.1%, more preferably, about 0.01%, w/v.
The final pH of the composition should not be
so low that it would have a deleterious effect on
reagents used in an assay for the associated analyte, nor
should the pH be great enough to cause any significant
precipitation of the metal ion. The pH of the
composition is dependent primarily on the pH of the
~ aqueous component. Usually, the pH of the aqueous
component is about 2.0 to about 4.6, preferably, about
3.0 to 4.6, more preferably, about 4.0 to about 4.4, more
preferably, about 4.3. The amount of the buffer and its
pKa generally determines the final pH, but other
CA 02230284 1998-02-24
W09~ 96 PCT~S97/1~20
22
components of the composition should be taken into
consideration in this regard. Upon combining the aqueous
component with the lower alkyl alcohol and the
polyhydroxy aliphatic compound, there will be a slight
change in the overall pH of the combination. Generally,
the pH of the combination will be lowered approximately
O.l to 0.3, usually, O.l to 0.2, pH units with respect to
the pH of the aqueous component.
While the addition of preservatives,
antimicrobials or chelating agents to the present
compositions is not necessary, such agents may be
included in the pretreatment composition without
interfering with the function of the inventive
compositions. Examples of such ancillary agents which
may be included in the inventive compositions, include,
but are not limited to, sodium azide, EDTA and
streptamycin.
The composition of the present invention is
usually packaged in a single container for convenience of
use. However, components of the composition may ~e
provided in separate containers. In that regard the
lower alkyl alcohol, the polyhydroxy aliphatic compound,
and the aqueous component, respectively, may be packaged
in separate containers or two of these components may be
combined and packaged separately from the remaining
component. Further, the compositions of the instant
invention may be packaged, in one or more containers,
acc~ ~anied by directions for suitable admixture and use.
A preferred embodiment of the present invention
is a composition comprising methanol in an amount of
about 30% to about 40% w/v, propylene glycol or ethylene
glycol in an amount of about 20% to about 40% w/v, and
about 30% to about 40% w/v of an a~ueous component
comprising about 20 mM to about 30 mM copper salt, about
0.5 mM to about 20 mM of a buffer, about 0.005% to about
O.l~ w/v of a non-ionic detergent and a pH of about 3.0
to about 4.6.
CA 02230284 l998-02-24
W 0~8~'~t~g6 PCTAUS97/12420
23
Another aspect of the present invention is an
improvement in an a~say for the determination of an
associated analyte such as an immunosuppressant drug,
e.g., cyclosporin, in a sample suspected of containing
the associated analyte. The assay comprises contacting a
- sample suspected of cont~; n; ~g the associated analyte
with a pretreatment reagent, contacting the sample with
reagents for conducting a determination of the analyte
and analyzing for the results of the determination. The
improvement comprises employing as the pretreatment
reagent one of the above compositions. The inventive
compositions are particularly compatable with the EMIT~
assay format, due to the use of copper ion as the metal
in the pretreatment composition. It has been
surprisingly found that the use of copper is particularly
compatable with the EMIT~ assay system, whereas other
metal ions tend to interfere with the detection of
analyte by the EMIT~ assay. The EMIT~ assay system may
be used for the detection of a variety of analytes,
including, but not limited to, cyclosporin. The EMIT~
assay format or Enzyme Multiplied Immunoassay Technique,
is a homogeneous competitive enzyme immunoassay
introduced by Rubenstein, K.E., Schneider, R.S., Ullman,
E.F. "Homogeneous" enzyme immunoassay. A new
immunochemical t~c~n;que. Biochem. Biophys, Res. Commun.
47: 846, 1972. This techique avoids the necessity to
separate labeled antigen bound to antibody from that
which is unbound. It depends upon a change in the
specific enzyme activity when antibody is bound to enzyme
labeled antigen or conjugate. The activity of the
unseparatd assay mixture is proportional to the amount of
conjugate to which antibody is bound.
~ One broad category of techniques for conducting
an assay involves the use of a receptor for the analyte.
The observed effect of binding by the receptor will
depend upon a label used. In some instances the binding
of the receptor merely provides for a differentiation in
CA 02230284 l998-02-24
W O 98~ S96 PCT~US97/12420
24
molecular weight between bound and unbound labeled
ligand. In other instances the binding of the receptor
will facilitate separation of bound labeled ligand from
free labeled ligand or it may affect the nature of the
signal obtained from the label so that the signal varies
with the amount of receptor bound to labeled ligand. A
further variation is that the receptor is labeled and the
ligand unlabeled. Alternatively, both the receptor and
ligand are labeled or different receptors are labeled
with two dif~erent labels, whereupon the labels interact
when in close proximity and the amount of ligand present
affects the degree to which the labels of the receptor
may interact. The analysis for the results of the
determination is then based on the nature of the label.
Accordingly, for enzyme labels, enzyme activity is
measured; for fluorescent labels, the amount of
fluorescence is measured, and so forth.
One particular embodiment of an assay involving
a receptor is an immunoassay where the receptor is an
immunoreactant such as an antibody and immune complexes
are formed in relation to the presence or amount of
analyte present in the sample. The analysis for the
results of the determination generally involves the
detection of the immune complexes. The immune complexes
are detected directly, ~or example, where an immune
reagent such as an antibody employed is conjugated to a
label. The immune complex is detected indirectly by
~ m;ning 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 ; ~ reagent used in the assay.
The assay of the invention has application to
all immunoassays for associated analytes. The assay can
be performed either without separation (homogeneous) or
with separation (heterogeneous) of any of the assay
components or products. Exemplary of heterogeneous
assays are enzyme linked immllnoaSSays such as the enzyme
CA 02230284 l998-02-24
W O ~8/00696 PCT~US97/1242
linked immunosorbant assay ~ELISA), see
"Enzyme-Immunoassay'l by Edward T. Maggio, CRC Press
Incorporated, Boca Raton, Florida, 1980. Homogeneous
immunoassays are exemplified by enzyme multiplied
immunoassay techniques (e.g. see U.S. Patent No.
- 3,817,837), immunofluorescence methods such as those
disclosed in U.S. Patent No. 3,993,345, enzyme channeling
t~chn i ques such as those disclosed in U.S. Patent No.
4,233,402, and other enzyme immunoassays as discussed in
Maggio, suPra. The disclosures of the above patents are
incorporated herein by reference in their entirety as to
the description of the particular assay methods
mentioned.
The sample to be analyzed is treated with the
present composition. Accordingly, the sample is combined
with a composition in accordance with the present
invention. Usually, the sample is combined with an excess
amount of the present composition, normally, about 100 to
500 ,UL, preferably, about 200 to 400 ~lL, more preferably,
about 250 to 350 ,uL, of the composition per about 100 ,~L
of the sample. The pretreatment is conducted at a
temperature of about 15 to 30~C, preferably, about 20 to
25~C, more preferably, at ambient t~ _?r-ature. The
sample and the present composition are incubated for a
period of about 10 seconds to about 5 minutes,
preferably, about 0.5 to 3 minutes, more preferably,
about 1 to 2 minutes. Generally, the pretreatment is
carried out prior to the addition of reagents for
conducting an assay. However, there may be circumstances
where one or more of the reagents for conducting an assay
are present with the pretreatment composition where the
nature of the reagent or an assay permits. Next, the
~ treated sample is subjected to centrifugation, usually at
ambient temperature for a period sufficient and force to
form any solids in the medium into a mass, usually for a
period of about 2 to 6 minutes at about 10,000 to 25,000
CA 02230284 1998-02-24
W O 98J'~gG PCTAUS97/12420 26
relative centrifugal force. The medium is then separated
from the solid mass.
After, or coincident with subjecting the sample
to pretreatment in accordance with the present invention,
an assay for the associated analyte is conducted. One or
more reagents are added to an aqueous medium con~in;ng
the pretreated sample. The assay for the analyte will
normally be carried out in an aqueous buffered medium at
a moderate pH, generally that which provides optimum
assay sensitivity. The aqueous assay medium may be
solely water or may include from 0 to 40 volume percent
of a cosolvent. The pH for the medium will usually be in
the range of about 4 to 11, more usually in the range of
about 5 to 10, and preferably in the range of about 6.5
to 9.5. The pH will usually be a compromise between
optimum binding of the binding members of any specific
binding pairs and the pH optimum for other reagents of
the assay such as members of the 6 ignal producing system.
Various buffers may be used to achieve the
desired pH and maintain the pH of the assay medium during
the determination. Illustrative buffers include borate,
phosphate, carbonate, tris, barbital and the like. The
particular buffer employed is not critical to this
invention, but in an individual assay one or another
buffer may be preferred.
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 about 5~ to 45~C, more usually from
about 15~ to 40~C. Temperatures during measurements will
generally range from about 10~ to 50~C, more usually from
about 15~ to 40~C.
The concentration of analyte that may be
assayed will generally vary from about 10-5 to 10-l3 M,
more usually from about 10 6 to 10-8 M. Considerations,
such as whether the assay is gualitative,
CA 02230284 1998-02-24
. _
WOg8l00C96 PCT~S97tl2420
27
semiquantitative or quantitative (relative to the amount
of cyclosporin present in the sample), the particular
detection technique and the concentration of the analyte
will normally determine the concentrations of the various
reagents. Further, the kit may optionally contain
directions for suitable admixture and use.
While the concentrations of the various
reagents in the assay medium will generally be determined
by the concentration range of interest of the analyte,
the final concentrat:ion 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.
While the order of addition of the reagents for
conducting an assay 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 assay
reagents simultaneously and to analyze for the results by
determining 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 about 30 seconds to 6
hours, more usually from about l minute to 1 hour.
Another aspect of the present invention is an
il..~o~ement in an assay for the determination of an
immunosuppressant drug in a sample suspected of
containing the drug. The assay comprises contacting the
sample with a specific binding member for the drug and
detecting the binding of the specific binding member to
~ the drug. The present improvement comprises contacting
the sample with one of the above compositions either
prior to or in conjunction with the contacting step.
Another embodiment of the present invention is
an improvement in an assay for the determination of an
CA 02230284 1998-02-24
W098~ 96 PCT~S97/12420
28
immunosuppressant drug in a sample suspected of
con~A;ning such drug. The assay comprises contacting the
sample with antibodies for the drug and a conjugate of a
label and a compound recognized by the antibodies and
detecting immune complexes of the label conjugate and the
antibodies. The improvement comprises contacting the
sample with a c-~mpo~ition in accordance with the present
invention.
Another aspect of the present invention is a
kit comprising in packaged combination (a) one or more
reagents for conducting a determination of a associated
analyte and (b) a composition in accordance with the
present invention. To enhance the versatility of the
subject invention, the reagents can be provided in
packaged combination, in the same or separate containers,
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.
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.
CA 02230284 l998-02-24
W O 98/00696 PCTrUS97/12420
29
EXi~MPLBS
The invention is demonstrated further by the
following illustrative examples. Parts and percentages
- are by weight/volume (w/v) unless indicated otherwise.
Temperatures are in degrees centigrade (~C) unless
otherwise indicated.
Reagents
Methanol & Ethylene glycol - Mallinckrodt Chemical, Inc.,
St. Louis, MO
Propylene glycol - Aldrich Chemical Co., Milwaukee, WI
Homopipes~ buffer - homopiperazine-N,N'-bis-2-
ethanesulfonic acid from Research Organics, Inc.,
Cleveland, OH
Cupric sulfate pentahydrate & Tris-HCl - Fluka Chemical
Corporation, Ronkonkoma, NY
Pluronic 25R2~ - BASF Chemicals, Mount Olive, NJ
ExamPle 1
AssaY for CyclosPori~e
Z0 Assays for cyclosporine were conducted using
the EMIT~ Cyclosporine Specific Assay kit manufactured by
Behring Diagnostics Inc., San Jose, California, Part No.
6RO19UL, containing Reagents A and B. Calibrators used
were manufactured by Behring Diagnostics Inc., San Jose,
California, Part NoO 6R119UL. Manufacturer's
instructions were followed. Assays were performed on the
COBAS MIRA analyzer~
Prior to conducting the assays, samples were
pretreated in accordance with the present invention.
Accordingly, one hundred microliters (100~L) of a whole
blood sample and 6 calibrators were each separately
treated with 300~L of the following pretreatment
composition: methanol (35%), propylene glycol (29%), an
aqueous component (36%) cont~;n;ng 1 mM Homopipes~
biological buffer (pKa=4.6), 25.0 mM cupric sulfate
(pentahydrate) and 0.01% Pluronic 25R2~ nonionic
detergent; pH=4.3.
CA 02230284 1998-02-24
W O98~LCg~ PCT~US97/12420
Each sample or ~A 1; hrator together with the
pretreatment composition were treated ~y vortex at 500 to
1000 rpm at ambient temperature for a period of greater
than or equal to 10 seconds. After the above treatment,
the mixture was allowed to incubate for greater than or
equal to 2 minutes at ambient temperature and then the
mixture was centrifuged for greater than or equal to 2
minutes at 20,800 relative centrifugal force (rcf). On
the analyzer, 27.5~L of the resulting pretreated sample
was incubated for 75 seconds with 155~L of Reagent A of
the above EMIT assay kit. Subsequently, 75 ~L of Reagent
B of the EMIT assay kit was added. After a 175 second
incubation, enzyme activity, (a function of drug
concentration) was monitored by following the production
of NADH spectrophotometrically at 340 nm for 100 seconds.
A total of 60 samples and six calibrators were
pretreated and assayed as described above using the EMIT
assay kits.
AssaY parameters on the COBAS MIRA analyzer
20 Assay Temperature 37~C
Wavelength 340 nm
Volume of Pretreated Sample 27.5~L
Diluent Volume (water) 47.5~L
Reagent A Volume 155 ~L
25 Incubation Time (sample + Reagent A) 75 sec
Reagent B Volume 75 ~L
Delay Time (sample + Reagent A + Reagent B) 175 sec
Read Time 100 sec
The assay standard curve range extended to 500
ng/mL. Analytical recovery within the curve range varied
from 90 to 103%. Within run precision with trilevel
controls ranged from 3.2 to 3.9% CV.
The results are summarized in the following
table:
CA 02230284 1998-02-24
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31
Table 1
CYCLOS~N r llu~ltXllJ~_Il I~TE t~
(n~/ml) INVf NnDNr MErHANoL
0 89.5 89.5
5 50 98.4 92.1
100 98.9 95.2
200 106.3 102.1
350 113.4 108.8
500 117.7 114.0
*Net aL,so-L,ance adjusted; e~tla~lion rate reflects the chan~e in abso.Lance which is related
to the amount of analyte extracted from a spe~,i..-en.
Table 2
CYCLOSr~N ~ llr~nnuu~noN RATE~ (n~/ml) % _~
(n~/ml)
~ O
44.8 89.6
lOO 95.4 95.4
200 205.0 102.5
350 348.8 99 7
500 474.3 94.9
25 ~P.t~t.edl---.:.. l in acco.dance with the present invention, quantitated
from .,.~ll.anol ~ la~tiOn curve; t~,clla~,lion rate is the quantity of analyte t:~t-a~d.
As a control, the above assay method was
repeated using methanol pretreatment. One hundred
microliters of a whole blood sample and 6 calibrators
were each separately vortexed with 200 mL methanol.
Incubation and centrifugation were as described above.
The supernatant was used as the pretreated sample for
CA 02230284 l998-02-24
WO ~ 59~ PCT~US97/12420
32
conducting the r~in~er of the assay. The assay
stAn~d curve range extended to 500 ng/mL. Analytical
recovery within the curve range varied from 89 to 102.5%.
Within run precision with trilevel controls ranged from
5 2.8 to 7.5% CV. Between run precision with the same
controls ranged from 5. 3 to 15.8% CV.
Exam~le 2
Assay for Tacrolimus tFR506)
Assays for tacrolimus were conducted using a
method similar to that described above in Example 1 for
cyclosporine. Assays were performed on the COBAS MIRA
analyzer.
Tacrolimus samples were prepared at 25 to 400
ng/mL ~lood hemolysate spiked with a stock solution of
tacrolimus in methanol. After m; ~; ng of each sample,
aliquots were taken and subjected to extraction. In
addition, tacrolimus calibrators were prepared at 6.25 to
100 ng/mL (concentrations same as above plus the
appropriate dilution factor) using methanol (no
hemolysate), spiked directly with a stock a solution of
tacrolimus in methanol.
Prior to conducting the assays, tacrolimus
spiked samples were pretreated in accordance with the
present invention. Accordingly, one hundred microliters
(100 ~L) of the tacrolimus spiked hemolysate and 6
calibrators were each separately treated with 300~L of
the following pretreatment composition: methanol (35%),
propylene glycol ~29~), an aqueous component (36%)
cont~;n;ng 1 mM Homopipes~ biological buffer (pKa=4.6),
25.0 mM cupric sulfate ~pentahydrate) and 0.01% Pluronic
25~2~ nonionic detergent; pH=4.3. In addition, the
reference extraction was performed using methanol.
Accordingly, one hundred microliters (100 ~L) of the
tacrolimus spiked hemolysate were each separately treated
with 300 ~L of methanol. The remainder of the assay was
as described above in Example 1. Enzyme conjugates were
-
CA 02230284 1998-02-24
. _
WO98i~C~9G PCT~S97/12420
33
~ prepared in a ~nne1~ similar to that described in U.S.
Patent No. 4,727,022, the relevant disclosure of which is
incorporated herein by reference. Antibodies used were
monoclonal antibodies for tacrolimus prepared by st~n~d
hybrid cell t~ohnology (see, for example, Kohler and
Milstein, Nature, suPra). The results are summarized in
the following tables:
Table 3
-OONCENlhAI_ ~ng/ml) _ ~ RATE EXTRI~CTI011 I~TE
I 1. . I RETIWIOL
0 0 178 . 7 178 . 7
6.25 185.2186.6
12.5 190.7190.0
196.8197.2
207.2208.2
100 217.1216.4
*Net absorbance adiusted; extraction rate reflects
the change in absorbance which i5 related to the amount
of analyte extracted from a specimen.
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WO ~8~'C C 69C PCT~US97/12420
34
Table 4
1~: ! -T~ n~/ml~ RATE~ (ng/ml) X RECOVEllr ~n~l)
~ O
6.25 5.8 93.4
12.5 12.6 100.8
89.9
49. 4 98.7
100 102.7 102.7
10 *Pretreatment in accordance with the present
invention, quantitated from methanol extraction curve.
The results of the assay for tacrolimus are
shown graphically in Fig. 1.
Example 3
ASSaY for Cyclo~porine
Assays for cyclosporine were conducted using
the EMIT0 Cyclosporine Specific Assay kit, described
hereinabove
Prior to conducting the assays, samples were
pretreated in accordance with the present invention.
Accordingly, one hundred microliters (lOO~L) of a whole
blood sample and 6 calibrators were each separately
treated with 300~L of the following pretreatment
composition: methanol (33.3%), ethylene glycol (33.3%),
an aqueous component (33.3%) containing 18.3 mM Tris-HCl
(pKa=7.8), 25.0 mM cupric sulfate (pentahydrate), 0.01%
Pluronic 25R2~ nonionic detergent; 0.33 mM sodium EDTA,
sodium azide (0.033%) and streptomycin (0.0017~); pH=4.5.
Each sample or calibrator together with the
pretreatment composition were treated by vortex at 500 to
1000 rpm at ambient temperature for a period of greater
CA 02230284 l998-02-24
W 098/00696 PCTAUS97tl2420
than or equal to 10 seconds. After the above treatment,
the mixture was allowed to incubate for greater than or
equal to 2 minutes at ambient temperature and then the
mixture was centrifuged for greater than or equal to 2
minutes at 20,800 relative centrifugal force (rcf). On
the analyzer, 27.5,uL of the resulting pretreated sample
was incubated for 75 seconds with 155,1LL of Reagent A of
the above EMIT assay kit. Subsequently, 7 5 ,uL of Reagent
B of the EMIT assay kit was added. After a 175 second
incubation, enzyme activity, (a function of drug
concentration~ was monitored by following the production
of NADH spectrophotometrically at 340 nm for 100 seconds.
A total of 10 curves each derived from six
calibrators were generated with invention and methanol as
reference. These samples were pretreated and assayed as
described above using the EMIT assay.
AssaY parameters on the COBAS MIRA analyzer
Assay Temperature 37~C
Wavelength 340 nm
20 Volume of Pretreated Sample 27.5,uL
Diluent Volume (water) 47. 5,uL
Reagent A Volume 155 ,uL
Incubation Time (sample + Reagent A~ 75 sec
Reagent B Volume 75 ,uL
25 Delay Time (sample + Reagent A + Reagent B) 175 sec
Read Time 100 sec
The results are summarized in the following
table:
Table 5
~n~/ml) INVB~llO~ MEllWlOL
0 93.5 93.5
97.8 96.6
35100 102.5 100.7
CA 02230284 1998-02-24
WO98,~~~9~ PCT~S97112420
3 6
2~ 110 4 1~1
~o 1179 1160
5~ 121 8 1206
~N~ ~s~ adju~ed; extraction rate reflects the change
in absorbance which is related to the amount of analyte
extracted from the specimen
Table 6
0 CYCLOSPORIN f I 1~. EX~N R~TE~ % pFf~
(n~ ) (n~/mi)
0 0.4
~1 1282
1~ 1192 119 2
lS 2~ 2~7 1143
~o 398 8 1139
5~ 5~9 1 12 8
dll,~ in a~o,~lc."~ with th~ present invention, quantitated
2 0 from " ,e,ll ,a,~ Al,~ n ~urve; ~AI,~.,Iion rate is the quantity of analyte ~AII
As a control, the above assay method was
repeated using methanol pretreatment. one hundred
microliters of a whole blood sample and 6 calibrators
2 5 were each separately vortexed with 2 00 mL methanol.
Incubation and centrifugation were as described above.
The supernatant was used as the pretreated sample for
conducting the r~ ~ of the assay. The assay st~n~A~d
curve range extended to 500 ng/mL. Analytical recovery
3 0 within the curve range varied from 1 12 to 128%. Within
run precision with trilevel controls ranged from 2. 8 to
7.5% CV. Between run precision with the same controls
ranged from 5.3 to 15.8% CV.
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WO ~8~ C PCTAUS97/12420
37
ExamPle 4
AssaY~ for Tacrolimus (F~506)
Assays for tacrolimus were conducted using a
method similar to that described above in Example 3 for
cyclosporine. Assays were performed on the COBAS MIRA
analyzer.
Tacrolimus samples were prepared at 25 to 400
mg/mL using blood hemolysate spiked with a stock solution
of tacrolimus in methanol. After mixing of each sample,
ali~uots were taken and subjected to extraction. In
addition, tacrolimus calibrators were prepared at 6.25 to
100 ng/mL (concentrations same as above plus the
appropriate dilution factor) using methanol (no
hemolysate), spiked directly with a stock a solution of
tacrolimus in methanol.
Prior to conducting the assays, tacrolimus
spiked samples were pretreated in accordance with the
present invention. Accordingly, one hundred microliters
(100 ~L) of the tac~olimus spiked hemolysate and 6
calibrators were each separately treated with 300~L of
the following pretreatment composition: methanol
(33.3%~, ethylene glycol (33.3%), an a~ueous component
(33.3%) containing 18.6 mM Tris-HC~ (pKa=7.8), 2~.0 mM
cupric sulfate (pentahydrate), 0.01% Pluronic 25R2~
nonionic detergent, 0.33 mM sodium EDTA, sodium azide
(0.033%) and streptomycin (0.0017%; pH=4.5. In addition,
the reference extraction was performed using methanol.
Accordingly, one hundred microliters (lOO~L) of the
tacrolimus spiked hemolysate were each separately treated
with 300~1 of methanol. The remainder of the assay was
as described above in Example 3. Enzyme conjugates were
prepared in a manner similar to that described in U.S.
Patent No. 4,727,022, the relevant disclosure of which is
incorporated herein by reference. Antibodies used were
monoclonal antibodies for tacrolimus prepared by standard
hybrid cell technology (see, for example, Kohler and
CA 02230284 l998-02-24
W O98~ 9G PCT~US97/1242
38
Milstein, Nature, suPra). The results are ~- ~ized in
the following tables:
Table 7
T~CROLIMUS CONCENTR~TION (n~/ml~ L~ r~E ~. - r~TE
,, METHWN OL
0 182.7 ~82.7
6.25 185.1 186.4
12.5 189.0 190.8
195.7 195.3
207.6 210.0
0 100 220.4 221.4
~Net dL,so,L,ancO adjusted; OAIId-:lion rate reflects the change in d~so,l,a"ce which is relatsd
to the amount of analytO OxtractOd from the s,ve~
~k~
T~ (n~/ml) E~cmA~N w~TE~ (n~/m~ "~
~ O
6.25 4.7 75.2
2 0 12.5 11.4 90.8
22.4 89.6
46.7 83.4
100 92.8 92.8
2 5 ~ trodtl l l~ in acco, ~ance with the prssent invention, quantitatsd
from methanol OAIId~.lion curve; eAt~d~tion rate is the quantity of analyte eAlld~tOd.
While the present invention has been described
with reference to the specific embodiments thereof, it
will be understood by and obvious to those skilled in the
art that various changes may be made and equivalents may
be substituted without departing from the true spirit and
CA 02230284 1998-02-24
W098/00696 PCT~S97/12420
39
scope of the inventYon. In addition, many modifications
may be made to adapt a particular situation, material,
composition of matter, process, process step or steps, to
~ the o~jective, spirit and scope of the present invention.
All such modifications are intended to be within the
scope of the claims appended hereto.
