U.S. patent application number 15/440628 was filed with the patent office on 2017-06-08 for methods and reagents for determining isomeric analytes.
This patent application is currently assigned to Siemens Healthcare Diagnostics Inc.. The applicant listed for this patent is Siemens Healthcare Diagnostics Inc.. Invention is credited to Izak Bahar, Tie Q. Wei.
Application Number | 20170160295 15/440628 |
Document ID | / |
Family ID | 51388523 |
Filed Date | 2017-06-08 |
United States Patent
Application |
20170160295 |
Kind Code |
A1 |
Wei; Tie Q. ; et
al. |
June 8, 2017 |
METHODS AND REAGENTS FOR DETERMINING ISOMERIC ANALYTES
Abstract
Methods include determining in a sample an amount of a first
isomeric analyte and a second isomeric analyte. A first measurement
value and a second measurement value are determined. The first
measurement value represents a total amount of the first isomeric
analyte and the second isomeric analyte. The second measurement
value represents an amount of the second isomeric analyte only. The
second measurement value is subtracted from the first measurement
value to obtain a resulting value and the resulting value is
equated to an amount of the first isomeric analyte in the
sample.
Inventors: |
Wei; Tie Q.; (Wilmington,
DE) ; Bahar; Izak; (Hockessin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Siemens Healthcare Diagnostics Inc. |
Tarrytown |
NY |
US |
|
|
Assignee: |
Siemens Healthcare Diagnostics
Inc.
Tarrytown
NY
|
Family ID: |
51388523 |
Appl. No.: |
15/440628 |
Filed: |
February 23, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13780305 |
Feb 28, 2013 |
9618523 |
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15440628 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/585 20130101;
G01N 33/5308 20130101; G01N 33/82 20130101 |
International
Class: |
G01N 33/82 20060101
G01N033/82; G01N 33/58 20060101 G01N033/58 |
Claims
1-20. (canceled)
21. A method of determining in a sample an amount of
non-epi-25-hydroxy vitamin D.sub.3 and 3-epi 25-hydroxy vitamin
D.sub.3, the method comprising: (a) conducting an assay on a first
portion of the sample using an assay protocol wherein assay
reagents utilized in the assay protocol of this step (a) comprise
vitamin D.sub.3 conjugated to a label and a first antibody having a
binding affinity for each of non-epi-25-hydroxy vitamin D.sub.3 and
3-epi 25-hydroxy vitamin D.sub.3 of 10.sup.8 to 10.sup.14
liters/mole to form a first complex comprising the first antibody
and non-epi-25-hydroxy vitamin D.sub.3 and a second complex
comprising the first antibody and 3-epi 25-hydroxy vitamin D.sub.3
wherein the first complex and the second complex include vitamin
D.sub.3 conjugated to a label wherein an amount of signal from the
first complex and the second complex is related to a total amount
non-epi-25-hydroxy vitamin D.sub.3 and 3-epi 25-hydroxy vitamin
D.sub.3 in the sample to obtain a first measurement value; and (b)
conducting the assay on a second portion of the sample using the
same assay protocol as in step (a) wherein assay reagents utilized
in the assay protocol in this step (b) comprise the vitamin D.sub.3
conjugated to a label and the first antibody, wherein a second
antibody having a binding affinity for the non-epi-25-hydroxy
vitamin D.sub.3 of 10.sup.6 to 10.sup.8 liters/mole and a binding
affinity for the 3-epi 25-hydroxy vitamin D.sub.3 of less than
about 10.sup.4 liters/mole is employed in an amount of about 5 to
about 200 times the amount of the first antibody, wherein the
binding affinity of the second antibody for non-epi-25-hydroxy
vitamin D.sub.3 is less than the binding affinity of the first
antibody for non-epi-25-hydroxy vitamin D.sub.3 by a factor of at
least about 10, wherein the second antibody binds the
non-epi-25-hydroxy vitamin D.sub.3 such that the non-epi-25-hydroxy
vitamin D.sub.3 does not bind to the first antibody, and wherein a
complex is formed comprising the first antibody and the
epi-25-hydroxy vitamin D.sub.3, wherein the complex includes the
vitamin D.sub.3 conjugated to a label wherein an amount of signal
from the complex is related to an amount of the 3-epi 25-hydroxy
vitamin D.sub.3 in the sample to obtain a second measurement value;
wherein an amount of the non-epi-25-hydroxy vitamin D.sub.3 in the
sample is determined by subtracting the second measurement value
from the first measurement value.
22. The method according to claim 21, wherein the assay protocol is
a competitive homogeneous assay protocol.
23. The method according to claim 21, wherein the assay protocol
employs reagents that comprise a particle.
24. The method according to claim 21, wherein the assay protocol
employs reagents that comprise a photosensitizer reagent and a
chemiluminescent particle.
25. The method according to claim 24, wherein the photosensitizer
reagent comprises a particle.
Description
BACKGROUND
[0001] This invention relates to compositions, methods and kits for
determining the presence and/or amount of each of two or more
isomeric analytes in a sample suspected of containing the isomeric
analytes.
[0002] Many small molecule compounds or haptens such as, for
example, drugs and vitamins, exist in isomeric forms, of which only
one form is active. In order to obtain an accurate measurement of
the active form of an analyte, the presence of the non-active
isomer of the analyte must be addressed. Measurements of both
isomeric forms of an analyte, that is, active and non-active forms,
can lead to inaccuracies that may be detrimental to an individual
depending on the function of the active form of the analyte.
Accurately assessing the level of each of a pair of isomeric
analytes in biological samples is important especially where only
one of the isomers is active and measurements that include the
amount of the non-active isomer distort the level of the analyte in
a sample. For example, measuring vitamin D levels in biological
samples is important since vitamin D deficiency is related to a
number of disorders in mammals. In infants, for example, vitamin D
measurements that include amounts of 3-epi isomers can lead to
inaccurate assessment of vitamin D levels in the infant, which in
turn can lead to a lack of proper supplementation. It is important
to measure the active form of vitamin D so that an infant can
receive proper vitamin D therapy, if necessary.
[0003] The term "vitamin D" refers to a group of fat-soluble
secosteroids. In humans, vitamin D is unique because it can be
ingested as cholecalciferol (vitamin D.sub.3) or ergocalciferol
(vitamin D.sub.2) and because the body can also synthesize it (from
cholesterol) when sun exposure is adequate. Because of this latter
property, vitamin D is considered by some to be a non-essential
dietary vitamin although most consider it an essential nutrient.
Vitamin D has an important physiological role in the positive
regulation of calcium ion homeostasis. Vitamin D.sub.3 is the form
of the vitamin synthesized by animals. It is also a common
supplement added to milk products and certain food products as is
vitamin D.sub.2.
[0004] Both dietary and intrinsically synthesized vitamin D.sub.3
must undergo metabolic activation to generate bioactive
metabolites. In humans, the initial step of vitamin D.sub.3
activation occurs primarily in the liver and involves hydroxylation
to form the intermediate metabolite 25-hydroxycholecalciferol (also
referred to as calcidiol, calcifediol, 25-hydroxycholecalciferol,
or 25-hydroxyvitamin D.sub.3. Calcidiol is the major form of
Vitamin D.sub.3 in the circulatory system. Vitamin D.sub.2 also
undergoes similar metabolic activation to 25-hydroxyvitamin
D.sub.2. Collectively these compounds are called 25-hydroxyvitamin
D (abbreviated 25(OH)D) and they are the major metabolites that are
measured in serum to determine vitamin D status; 25(OH)D and its
epimers are both pre-hormones that need to be converted into
1,25(OH)D to exert biological functions. The comparison of
bioactivity of 1,25(OH)D versus that of 3-epi-1,25(OH)D is
complex.
[0005] The vitamin D compounds 25-hydroxyvitamin D.sub.3 and
25-hydroxyvitamin D.sub.2 are epimeric at the 3-position with the
epimers being designated 25-hydroxyvitamin D.sub.3 and
3-epi-25-hydroxyvitamin D.sub.3 and 25-hydroxyvitamin D.sub.2 and
3-epi-25-hydroxyvitamin D.sub.2, respectively. Only one of the
epimers of each of these epimeric compounds, namely,
25-hydroxyvitamin D.sub.3 and 25-hydroxyvitamin D.sub.2,
respectively, are active. The structures for the epimers of
25-hydroxyvitamin D.sub.3 and 25-hydroxyvitamin D.sub.2 are set
forth in FIG. 1.
[0006] There is a need for reagents and methods for accurate and
sensitive determinations of concentrations of isomeric analytes in
samples suspected of containing such analytes. For example, there
is a need for reagents and methods for accurate and sensitive
determinations of concentrations of epimeric forms of vitamin
D.
SUMMARY
[0007] Some examples in accordance with the principles described
herein are directed to methods of determining in a sample an amount
of a first isomeric analyte and a second isomeric analyte. In the
method a first measurement value and a second measurement value are
determined. For determination of the first measurement value, a
total amount of the first isomeric analyte and the second isomeric
analyte is measured by conducting an assay on a portion of the
sample using a first antibody that exhibits sufficient assay
binding affinity for each of the first isomeric analyte and the
second isomeric analyte. For determination of the second
measurement value, an amount of the second isomeric analyte is
measured by conducting the assay on a portion of the sample using
the first antibody, wherein a second antibody that binds to the
first isomeric analyte but exhibits insufficient assay binding
affinity for the first isomeric analyte and substantially no assay
binding affinity for the second isomeric analyte is employed in
excess to block binding of the first isomeric analyte to the first
antibody. The second measurement value is equated to an amount of
the second isomeric analyte in the sample. The second measurement
value is subtracted from the first measurement value to obtain a
resulting value and the resulting value is equated to an amount of
the first isomeric analyte in the sample.
[0008] Some examples in accordance with the principles described
herein are directed to methods of determining in a sample an amount
of a first isomeric analyte and a second isomeric analyte. In the
method a first measurement value and a second measurement value are
determined. For determination of the first measurement value, a
total amount of the first isomeric analyte and the second isomeric
analyte is measured by conducting an assay on a portion of the
sample using a first antibody having a binding affinity for each of
the first isomeric analyte and the second isomeric analyte of at
least about 10.sup.7 liters/mole. For determination of the second
measurement value, an amount of the second isomeric analyte is
measured by conducting the assay on a portion of the sample using
the first antibody to obtain a second measurement value, wherein a
second antibody that has a binding affinity for the first isomeric
analyte of about 10.sup.6 to about 10.sup.12 liters/mole and a
binding affinity for the second isomeric analyte of less than about
10.sup.4 liters/mole is employed in excess to block binding of the
first isomeric analyte to the first antibody. The second
measurement value is equated to an amount of the second isomeric
analyte in the sample and the second measurement value is
subtracted from the first measurement value to obtain a resulting
value, which is equated to an amount of the first isomeric analyte
in the sample.
BRIEF DESCRIPTION OF DRAWINGS
[0009] FIG. 1 is a depiction of the chemical formulas for the
epimeric forms of 25-hydroxyvitamin D.sub.3 and 25-hydroxyvitamin
D.sub.2.
[0010] FIG. 2 is a graph depicting vitamin D measurements of
3-epi-25-hydroxyvitamin D.sub.3 with and without the addition of a
second antibody in accordance with examples in accordance with the
principles described herein.
[0011] FIG. 3 is a graph depicting vitamin D measurements of
3-epi-25-hydroxyvitamin D.sub.3 with and without the addition of a
second antibody in accordance with examples in accordance with the
principles described herein.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
General Discussion
[0012] Methods in accordance with the principles described herein
minimize cross-reactivity of, and measure an amount of, one of two
isomers of an analyte where one of the isomers or its metabolic
product is potent and the other or its metabolic product is not.
The term "potent" refers to the degree of an activity of an analyte
with respect to a particular function, which may be, for example, a
biological function such as, e.g., bone metabolism. For example,
biological activity of a substance relates to the ability of the
substance to enhance or suppress a biological function such as, for
example, maintaining appropriate levels of minerals and salts in a
subject, cellular function. Vitamin D, by way of illustration and
not limitation, maintains appropriate levels of calcium and
phosphate in a subject, which relate to calcium homeostasis and
bone metabolism.
[0013] Methods include determining in a sample an amount of a first
isomeric analyte and a second isomeric analyte. A first measurement
value and a second measurement value are determined. The first
measurement value represents a total amount of the first isomeric
analyte and the second isomeric analyte. The second measurement
value represents an amount of the second isomeric analyte only. The
second measurement value is subtracted from the first measurement
value to obtain a resulting value and the resulting value is
equated to an amount of the first isomeric analyte in the
sample.
[0014] In methods in accordance with the principles described
herein, at least two portions of a sample to be analyzed are
utilized. An assay is carried out on a first portion of the sample
suspected of containing at least two isomeric forms of an analyte
using an antibody (first antibody) that binds to both isomeric
forms of the analyte. The first antibody exhibits sufficient assay
binding affinity for each of the first isomeric analyte and the
second isomeric analyte.
[0015] The phrase "assay binding affinity" refers to the strength
with which an antibody binds to a corresponding analyte to produce
a complex of antibody bound to analyte.
[0016] The phrase "sufficient assay binding affinity" means that
the binding affinity of an antibody for an analyte is that which
produces a detectable complex in an amount sufficient to obtain an
assay signal that results in an accurate and sensitive
determination of the analyte. The binding affinity of the first
antibody is strong enough to form detectable complexes of the first
antibody and the each of the first isomeric analyte and the second
isomeric analyte where the detectable complexes accurately
represent the amount of the first isomeric analyte and the second
isomeric analyte in the sample once the assay system and instrument
have been subjected to suitable calibration and any correction
factors for antibody recognition of one or both of the isomeric
analytes have been applied. This assay on the first portion of the
sample measures the amount or concentration of both isomeric forms
of an analyte in a sample.
[0017] The same assay is conducted on a second portion of the same
sample using both the first antibody that binds to both isomeric
forms of the analyte and a second antibody that binds to one of the
isomeric forms (first isomeric form) but exhibits substantially no
binding affinity for the other isomeric form (second isomeric form)
leaving the second isomeric form free for detection by the first
antibody in the assay conducted on the second portion. The second
antibody that binds to the first isomeric form but not to the
second isomeric form exhibits insufficient assay binding affinity
for the first isomeric analyte. In some examples, the second
antibody that binds to one of the isomeric forms but not to the
other isomeric form binds to the active isomeric form but not to
the non-active isomeric form. This assay on the second portion of
the sample measures the concentration of only one of the isomeric
forms of the analyte, namely, the second isomeric analyte in the
above description. The signal values obtained may be used to
determine the total analyte concentration and the concentration of
each of the two isomeric forms of the analyte.
[0018] The phrase "insufficient assay binding affinity" means that
the binding affinity of the second antibody for an isomeric analyte
is less than the binding affinity of the first antibody for the
isomeric analyte. In some examples, the phrase "insufficient assay
binding affinity" means that the binding affinity of the second
antibody for an isomeric analyte is not great enough to form
detectable complexes between the second antibody and the first
isomeric analyte and thus any detectable complexes do not
accurately represent the amount of the first isomeric analyte. The
second antibody, when used in excess, exhibits sufficient binding
affinity for the first isomeric analyte to block binding of the
first antibody to the first isomeric analyte in the assay on the
second portion of the sample. The binding affinity of the second
antibody for the first isomeric form of the analyte is too low to
generate complexes of second antibody and first isomeric analyte so
that sufficient signal for accurate detection of the first isomeric
analyte is produced in the assay employed.
[0019] The phrase "exhibits substantially no binding affinity" for
the second isomeric form means that substantially no detectable
complexes are formed between the second antibody and the second
isomeric form of the analyte.
[0020] It should be noted that, if the result from the assay on the
second portion of the sample is substantially equivalent to zero,
then the result obtained from the assay on the first portion of the
sample represents the concentration of only one of the two isomeric
forms of the analyte since that the other isomeric form of the
analyte is not detected in the assay on the second portion of the
sample. In such a circumstance, it would not be necessary to
conduct assays on two portions of the sample in question since the
results from the methods in accordance with the principles
described herein indicate that only one of the isomeric forms of
the analyte is contributing to signal obtained in the assay on the
first portion of the sample. On the other hand, if the result from
the assay on the second portion of the sample is not substantially
equivalent to zero, then the result obtained from the assay on the
first portion of the sample represents the concentration of both of
the two isomeric forms of the analyte since the other isomeric form
of the analyte is detected in the assay on the second portion of
the sample. In such a circumstance, it would be necessary to
conduct assays on two portions of the sample in question since the
results from the methods in accordance with the principles
described herein indicate that both of the isomeric forms of the
analyte are contributing to signal obtained in the assay on the
first portion of the sample.
Preparation of Antibodies
[0021] Examples of methods of preparing antibodies in accordance
with the principles described herein are described by way of
illustration and not limitation. At least two different antibodies
are required, which have the properties in accordance with the
principles described herein. One antibody exhibits sufficient assay
binding affinity for both the first isomeric analyte and the second
isomeric analyte. The other antibody binds to the first isomeric
analyte but exhibits insufficient assay binding affinity for the
first isomeric analyte and exhibits substantially no assay binding
affinity for the second isomeric analyte.
[0022] In some examples in accordance with the principles described
herein, sufficient assay binding affinity is at least about
10.sup.7 liters/mole, or at least about 10.sup.8 liters/mole, or at
least about 10.sup.9 liters/mole, or at least about 10.sup.10
liters/mole, or at least about 10.sup.11 liters/mole, or at least
about 10.sup.12 liters/mole, or at least about 10.sup.13
liters/mole, or at least about 10.sup.14 liters/mole, for example,
and the amount of detectable complex is sufficient to obtain an
assay signal that results in an accurate and sensitive
determination of the analyte. In some examples in accordance with
the principles described herein, sufficient assay binding affinity
is about 10.sup.7 to about 10.sup.14 liters/mole, or about 10.sup.7
to about 10.sup.11 liters/mole, or about 10.sup.7 to about
10.sup.12 liters/mole, or about 10.sup.8 to about 10.sup.14
liters/mole, or about 10.sup.8 to about 10.sup.11 liters/mole, or
about 10.sup.8 to about 10.sup.12 liters/mole, for example,
[0023] In some examples, insufficient assay binding affinity means
that the binding affinity of the second antibody for a first
isomeric analyte is less than the binding affinity of the first
antibody for the first isomeric analyte. In some examples,
depending on the binding affinity of the first antibody for the
first isomeric analyte, the binding affinity of the second antibody
for a first isomeric analyte is less than the binding affinity of
the first antibody for the first isomeric analyte by a factor, for
example, of about 10, or about 10.sup.2, or about 10.sup.3, or
about 10.sup.4, or about 10.sup.5. For example, if the binding
affinity of the first antibody for the first isomeric analyte is
about 10.sup.9 liters/mole, the binding affinity of the second
antibody may be less that about 10.sup.7 liters/mole, or less than
about 10.sup.6 liters/mole. In some examples in accordance with the
principles described herein, insufficient assay binding affinity
means that the binding affinity of an antibody for an analyte is
about 10.sup.6 to about 10.sup.8 liters/mole, or about 10.sup.6 to
about 10.sup.7 liters/mole, for example, depending on the nature of
the antibody and the nature of the analyte.
[0024] In some examples, substantially no binding affinity means
that an antibody has a binding affinity for an isomeric analyte of
less than about 10.sup.4 liters/mole, or less than about 10.sup.3
liters/mole, or less than about 10.sup.2 liters/mole, or less than
about 10 liters/mole, for example.
[0025] In the above discussion, binding affinity is specific
binding affinity, which involves the specific recognition of one of
two different molecules for the other compared to substantially
less recognition of other molecules. On the other hand,
non-specific binding involves 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.
[0026] The antibody may be monoclonal or polyclonal. 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.
[0027] Monoclonal antibodies can be prepared by techniques that are
well known in the art such as preparing continuous hybrid cell
lines and collecting the secreted protein (somatic cell
hybridization techniques). 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).
[0028] 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. This approach involves
cloning and expressing nucleotide sequences or mutagenized versions
thereof coding at least for the amino acid sequences required for
specific binding of natural antibodies.
[0029] In one approach for the production of monoclonal antibodies,
a first step includes immunization of an antibody-producing animal
such as a mouse, a rat, a goat, a sheep, or a cow with the antigen,
for example, with an immunogen. Immunization can be performed with
or without an adjuvant such as complete Freund's adjuvant or other
adjuvants such as monophosphoryl lipid A and synthetic trehalose
dicorynomycolate adjuvant. A next step includes isolating spleen
cells from the antibody-producing animal and fusing the
antibody-producing spleen cells with an appropriate fusion partner,
typically a myeloma cell, such as by the use of polyethylene glycol
or other techniques. Typically, the myeloma cells used are those
that grow normally in hypoxanthine-thymidine (HT) medium but cannot
grow in hypoxanthine-aminopterin-thymidine (HAT) medium, used for
selection of the fused cells. A next step includes selection of the
fused cells, typically by selection in HAT medium. A next step
includes screening the cloned hybrids for appropriate antibody
production using immunoassays such as enzyme-linked immunosorbent
assay (ELISA) or other immunoassays appropriate for screening.
[0030] The term "immunogenic carrier" means a group or moiety
which, when conjugated to a hapten and injected into a mammal or
otherwise employed as an immunogen, induces an immune response and
elicits production of antibodies that bind to the hapten.
Immunogenic carriers are also sometimes referred to as antigenic
carriers. In some examples in accordance with the principles
described herein, immunogens comprising immunogenic carriers,
including poly(amino acid) and non-poly(amino acid) immunogenic
carriers, linked to an immunosuppressant compound at a particular
position are synthesized and used to prepare antibodies. Haptens
are compounds capable of binding specifically to corresponding
antibodies, but do not themselves act as immunogens (or antigens)
for preparation of the antibodies. Consequently, a hapten is linked
to an immunogenic carrier, which is employed to raise
antibodies.
[0031] The molecular weight range (in Daltons) for poly(amino
acids) that are immunogenic carriers is about 5,000 to about
10,000,000, or about 20,000 to about 600,000, or about 25,000 to
about 250,000 molecular weight, for example. Poly(amino acid)
immunogenic carriers include proteins such as, for example,
albumins, serum proteins, e.g., globulins, ocular lens proteins and
lipoproteins. Illustrative proteins include, but are not limited
to, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH),
egg ovalbumin, and bovine gamma-globulin (BGG), for example.
Non-poly(amino acid) immunogenic carriers include polysaccharides,
nucleic acids and particles (biologic and synthetic materials). A
wide variety of immunogenic carriers are disclosed in Davalian, et
al., U.S. Pat. No. 5,089,390, column 4, line 57 to column 5, line
5, which is incorporated herein by reference.
[0032] As mentioned above, the immunogenic carrier may be a
polysaccharide, which is a high molecular weight polymer of
monosaccharides that may be prepared naturally or synthetically and
usually involves repeated condensations of monosaccharides.
Examples of polysaccharides are starches, glycogen, cellulose,
carbohydrate gums, such as gum arabic, agar, and so forth. The
polysaccharide can also contain poly(amino acid) residues and/or
lipid residues.
[0033] As mentioned above, in some examples in accordance with the
principles described herein, the immunogenic carrier may be linked
to an analyte analog at a predetermined position on the analyte by
means of a linking group. In some examples, the linking group may
comprise about 2 to about 50 atoms, or 4 to about 30 atoms, not
counting hydrogen and may comprise a chain of from 2 to about 30
atoms, or 3 to about 20 atoms, each independently selected from the
group normally consisting of carbon, oxygen, sulfur, nitrogen, and
phosphorous. Part or all of the linking group may be a portion of
the molecule being linked to the immunosuppressant compound such
as, but not limited to, an amino acid residue on a poly(amino
acid), for example. In some examples, the linking group comprises
an oxime functionality.
[0034] The number of heteroatoms in the linking group may be in the
range from 0 to about 20, or 1 to about 15, or about 2 to about 10.
The linking group may be aliphatic or aromatic. When heteroatoms
are present, oxygen is normally present as oxo or oxy, bonded to
carbon, sulfur, nitrogen or phosphorous, nitrogen is normally
present as nitro, nitroso or amino, normally bonded to carbon,
oxygen, sulfur or phosphorous; sulfur is analogous to oxygen; while
phosphorous is bonded to carbon, sulfur, oxygen or nitrogen,
usually as phosphonate and phosphate mono- or diester. Common
functionalities in forming a covalent bond between the linking
group and the molecule to be conjugated are alkylamine, amidine,
thioamide, ether, urea, thiourea, guanidine, azo, thioether and
carboxylate, sulfonate, and phosphate esters, amides and
thioesters. One specific embodiment of a linking group comprising
heteroatoms is an oxime functionality as mentioned above.
[0035] For the most part, when a linking group has a linking
functionality (functionality for reaction with a moiety) such as,
for example, a non-oxocarbonyl group including nitrogen and sulfur
analogs, a phosphate group, an amino group, alkylating agent such
as halo or tosylalkyl, oxy (hydroxyl or the sulfur analog,
mercapto) oxocarbonyl (e.g., aldehyde or ketone), or active olefin
such as a vinyl sulfone or .alpha.-, .beta.-unsaturated ester,
these functionalities are linked to amine groups, carboxyl groups,
active olefins, alkylating agents, e.g., bromoacetyl. Where an
amine and carboxylic acid or its nitrogen derivative or phosphoric
acid are linked, amides, amidines and phosphoramides are formed.
Where mercaptan and activated olefin are linked, thioethers are
formed. Where a mercaptan and an alkylating agent are linked,
thioethers are formed. Where aldehyde and an amine are linked under
reducing conditions, an alkylamine is formed. Where a ketone or
aldehyde and a hydroxylamine (including derivatives thereof where a
substituent is in place of the hydrogen of the hydroxyl group) are
linked, an oxime functionality (.dbd.N--O--) is formed. Where a
carboxylic acid or phosphate acid and an alcohol are linked, esters
are formed. Various linking groups are well known in the art; see,
for example, Cautrecasas, J. Biol. Chem. (1970) 245:3059.
[0036] Each different antibody is selected for its binding affinity
to one or both of two isomeric analytes as described above.
Accordingly, a first antibody is prepared and selected by means of
an appropriate screening method such that the first antibody
exhibits sufficient assay binding affinity for each of the first
isomeric analyte and the second isomeric analyte. A second antibody
is prepared and selected by means of an appropriate screening
method such that the second antibody binds to the first isomeric
analyte but exhibits insufficient assay binding affinity for the
first isomeric analyte and further exhibits substantially no assay
binding affinity for the second isomeric analyte. An antibody with
the requisite binding affinity for an analyte as set forth above
may be selected by well-known screening methodologies, which
include, by way of illustration and not limitation, ELISA, dot
blots, Western analysis, and Surface Plasmon Resonance, for
example.
General Description of Assays
[0037] The following discussion is by way of illustration and not
limitation. Any appropriate assay that utilizes an antibody may be
employed on portions of the sample in the determinations involved
in accordance with the principles described herein. The assays can
be performed either without separation (homogeneous) or with
separation (heterogeneous) of any of the assay components or
products. Heterogeneous assays usually involve one or more
separation steps and can be competitive or non-competitive. The
assays may be manual or automated.
[0038] The sample to be analyzed is one that is suspected of
containing an analyte. The samples may be biological samples or
non-biological samples. Biological samples may be from a mammalian
subject or a non-mammalian subject. Mammalian subjects may be,
e.g., humans or other animal species. Biological samples include
biological fluids such as whole blood, serum, plasma, sputum,
lymphatic fluid, semen, vaginal mucus, feces, urine, spinal fluid,
saliva, stool, cerebral spinal fluid, tears, mucus, and the like;
biological tissue such as hair, skin, sections or excised tissues
from organs or other body parts; and so forth. In many instances,
the sample is whole blood, plasma or serum. Non-biological samples
including, but not limited to, waste streams, for example, may also
be analyzed using compounds in accordance with the principles
described herein.
[0039] The sample can be prepared in any convenient medium, which
may be, for example, an assay medium, which is discussed more fully
hereinbelow. In some instances a pretreatment may be applied to the
sample such as, for example, to lyse blood cells. In some examples,
such pretreatment is performed in a medium that does not interfere
subsequently with an assay.
[0040] In many embodiments immunoassays involve labeled reagents.
Immunoassays that involve labeled reagents include
chemiluminescence immunoassays, enzyme immunoassays, fluorescence
polarization immunoassays, radioimmunoassays, inhibition assay,
induced luminescence assays, and fluorescent oxygen channeling
assays, for example.
[0041] One general group of immunoassays includes immunoassays
using a limited concentration of one of the assay reagents. Another
group of immunoassays involves the use of an excess of one or more
of the principal reagents. Another group of immunoassays are
separation-free homogeneous assays in which labeled reagents
modulate the label signal upon binding of one of the antibodies in
accordance with the principles described herein to one or both of
two isomeric analytes in the sample.
[0042] As mentioned above, the assays 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 (Siemens
Healthcare Diagnostics Inc., Deerfield, Ill.) disclosed in
Rubenstein, et al., U.S. Pat. No. 3,817,837, column 3, line 6 to
column 6, line 64; the induced luminescence immunoassay ("LOCI.RTM.
technology") disclosed in U.S. Pat. No. 5,340,716 (Ullman, et al.);
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 immunoassays ("ECIA") such as those
disclosed in Maggio, et al., U.S. Pat. No. 4,233,402, column 6,
line 25 to column 9, line 63; the fluorescence polarization
immunoassay ("FPIA") as disclosed, for example, in, among others,
U.S. Pat. No. 5,354,693; enzyme immunoassays such as the enzyme
linked immunosorbant assay ("ELISA"). 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.
[0043] Other enzyme immunoassays are the enzyme modulate mediated
immunoassay ("EMMIA") discussed by Ngo and Lenhoff, FEBS Lett.
(1980) 116:285-288; the substrate labeled fluorescence immunoassay
("SLFIA") disclosed by Oellerich, J. Clin. Chem. Clin. Biochem.
(1984) 22:895-904; the combined enzyme donor immunoassays ("CEDIA")
disclosed by Khanna, et al., Clin. Chem. Acta (1989) 185:231-240;
homogeneous particle labeled immunoassays such as particle enhanced
turbidimetric inhibition immunoassays ("PETINIA"), and particle
enhanced turbidimetric immunoassay ("PETIA"), etc.; for
example.
[0044] Other assays include the sol particle immunoassay ("SPIA"),
the disperse dye immunoassay ("DIA"); the metalloimmunoassay
("MIA"); the enzyme membrane immunoassays ("EMIA");
luminoimmunoassays ("LTA"); and so forth. Other types of assays
include immunosensor assays involving the monitoring of the changes
in the optical, acoustic and electrical properties of a reagent
upon the binding of an analyte. Such assays include, for example,
optical immunosensor assays, acoustic immunosensor assays,
semiconductor immunosensor assays, electrochemical transducer
immunosensor assays, potentiometric immunosensor assays, and
amperometric electrode assays.
[0045] Heterogeneous assays usually involve one or more separation
steps and can be competitive or non-competitive. A variety of
competitive and non-competitive heterogeneous 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
an example of a competitive heterogeneous assay, a support having
an antibody for analyte bound thereto is contacted with a medium
containing the sample suspected of containing the analyte and a an
analyte analog that comprises a label. Analyte in the sample
competes, for binding to the analyte antibody, with the labeled
analyte analog. After separating the support and the medium, the
label activity of the support or the medium is determined by
conventional techniques and is related to the amount of analyte in
the sample. In a variation of the above competitive heterogeneous
assay, the support comprises an analyte analog, which competes with
analyte of the sample for binding to an antibody reagent in
accordance with the principles described herein.
[0046] In some examples, the sample to be analyzed is subjected to
a pretreatment to release analyte from endogenous binding
substances such as, for example, plasma or serum proteins that bind
the analyte. The release of the analyte from endogenous binding
substances may be carried out, for example, by addition of a
digestion agent or a releasing agent or a combination of a
digestion agent and a releasing agent used sequentially. The
digestion agent is one that breaks down the endogenous binding
substances so that they can no longer bind the analyte. Such agents
include, but are not limited to, proteinase K and proteinase K and
protein denaturing agents such as, e.g., detergents (sodium dodecyl
sulfate, for example). Releasing agents for releasing the analyte
from endogenous binding substances include, by way of illustration
and not limitation, acidic denaturing agents such as, for example,
salicylic acid, warfarin, sulfonic acids, toluene sulfonic acids,
naphthalene sulfonic acid, anilinonaphthalene sulfonic acids (ANS)
(including, e.g., 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS)
and 8-anilinonapthalene-1-sulfonic acid (8-ANS)), salicylic acids
and derivatives of the above.
[0047] The conditions such as, for example, duration, temperature,
pH and concentration of the releasing agent in the medium for
carrying out the digestion or releasing actions are dependent on
the nature of the analyte, the nature of the endogenous binding
substances, the nature of the sample, and the nature of the
releasing agent, for example. In general, the conditions are
sufficient to achieve the desired effect or function. In some
examples in accordance with the principles described herein, an
effective concentration of releasing agent is about 0.01 to about
20 mg/mL, or about 0.01 to about 10 mg/mL, or about 0.01 to about 5
mg/mL, or about 0.1 to about 20 mg/mL, or about 0.1 to about 10
mg/mL, or about 0.1 to about 5 mg/mL, or about 0.1 to about 1
mg/mL. The pretreatment of the sample to release the analyte from
endogenous binding substances may be carried out as a separate step
prior to conducting an assay or as a first step in an assay. In
either case, one or more reagents may be required to stop the
action of the digestion agent and/or the releasing agent.
[0048] The conditions for conducting an assay on a portion of a
sample in accordance with the principles described herein include
carrying out the assay 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 from 0.1 to about
40 volume percent of a cosolvent. The pH for the medium will be in
the range of about 4 to about 11, or in the range of about 5 to
about 10, or in the range of about 6.5 to about 9.5, for example.
The pH will usually be a compromise between optimum binding of the
binding members of any specific binding pairs, the pH optimum for
other reagents of the assay such as members of the signal producing
system, and so forth. Various buffers may be used to achieve the
desired pH and maintain the pH during the assay. Illustrative
buffers include, by way of illustration and not limitation, borate,
phosphate, carbonate, TRIS, barbital, PIPES, HEPES, MES, ACES,
MOPS, and BICINE, for example. The particular buffer employed is
not critical, but in an individual assay one or another buffer may
be preferred.
[0049] Various ancillary materials may be employed in the assay
methods. For example, in addition to buffers the medium may
comprise stabilizers for the medium and for the reagents employed.
In some embodiments, in addition to these additives, proteins may
be included, such as, for example, albumins; organic solvents such
as, for example, formamide; quaternary ammonium salts; polyanions
such as, for example, dextran sulfate; binding enhancers, for
example, polyalkylene glycols; polysaccharides such as, for
example, dextran or trehalose. The medium may also comprise agents
for preventing the formation of blood clots. Such agents are well
known in the art and include, but are not limited to, EDTA, EGTA,
citrate, heparin, for example. The medium may also comprise one or
more preservatives such as, but not limited to, sodium azide,
neomycin sulfate, PROCLIN.RTM. 300, Streptomycin, for example. The
medium may additionally comprise one or more surfactants. Any of
the above materials, if employed, is present in a concentration or
amount sufficient to achieve the desired effect or function.
[0050] One or more incubation periods may be applied to the medium
at one or more intervals including any intervals between additions
of various reagents employed in an assay including those mentioned
above. The medium is usually incubated at a temperature and for a
time sufficient for binding of various components of the reagents
and binding of the analyte in the sample to occur. Moderate
temperatures are normally employed for carrying out the method and
usually constant temperature, preferably, room temperature, during
the period of the measurement. In some examples, incubation
temperatures range from about 5.degree. to about 99.degree. C., or
from about 15.degree. C. to about 70.degree. C., or from about
20.degree. C. to about 45.degree. C., for example. The time period
for the incubation, in some examples, is about 0.2 seconds to about
24 hours, or about 1 second to about 6 hours, or about 2 seconds to
about 1 hour, or about 1 minute to about 15 minutes, for example.
The time period depends on the temperature of the medium and the
rate of binding of the various reagents, which is determined by the
association rate constant, the concentration, the binding constant
and dissociation rate constant.
[0051] Many assays discussed herein use a signal producing system,
which may have one or more components, at least one component being
a label. The signal producing system generates a signal that
relates to the presence of an analyte in a sample. The signal
producing system includes all of the reagents required to produce a
measurable signal. Other components of the signal producing system
may be included in a developer solution and can include, but are
not limited to, substrates, enhancers, activators, chemiluminescent
compounds, cofactors, inhibitors, scavengers, metal ions, and
specific binding substances required for binding of signal
generating substances, for example. Other components of the signal
producing system may be coenzymes, substances that react with
enzymic products, other enzymes and catalysts, for example. The
signal producing system provides a signal detectable by external
means, by use of electromagnetic radiation, desirably by visual
examination. Exemplary signal-producing systems are described in
U.S. Pat. No. 5,508,178, the relevant disclosure of which is
incorporated herein by reference.
[0052] The term "label" includes poly(amino acid) labels and
non-poly(amino acid) labels. The term "poly(amino acid) label
moieties" includes labels that are proteins such as, but not
limited to, enzymes, antibodies, peptides, and immunogens, for
example. With label proteins such as, for example, enzymes, the
molecular weight range will be from about 10,000 to about 600,000,
or from about 10,000 to about 300,000 molecular weight. There is
usually at least one compound in accordance with the principles
described herein (analog group) per about 200,000 molecular weight,
or at least about 1 per about 150,000 molecular weight, or at least
about 1 per about 100,000 molecular weight, or at least about 1 per
about 50,000 molecular weight, for example, of the protein. In the
case of enzymes, the number of analog groups is usually from 1 to
about 20, about 2 to about 15, about 3 to about 12, or about 6 to
about 10.
[0053] Enzymes include, by way of illustration and not limitation,
redox enzymes such as, for example, dehydrogenases, e.g.,
glucose-6-phosphate dehydrogenase and lactate dehydrogenase;
enzymes that involve the production of hydrogen peroxide and the
use of the hydrogen peroxide to oxidize a dye precursor to a dye
such as, for example, horseradish peroxidase, lactoperoxidase and
microperoxidase; hydrolases such as, for example, alkaline
phosphatase and .beta.-galactosidase; luciferases such as, for
example firefly luciferase, and bacterial luciferase; transferases;
combinations of enzymes such as, but not limited to, saccharide
oxidases, e.g., glucose and galactose oxidase, or heterocyclic
oxidases, such as uricase and xanthine oxidase, coupled with an
enzyme that employs hydrogen peroxide to oxidize a dye precursor,
that is, a peroxidase such as horseradish peroxidase,
lactoperoxidase or microperoxidase, for example.
[0054] The term "non-poly(amino acid) labels" includes those labels
that are not proteins. The non-poly(amino acid) label is capable of
being detected directly or is detectable through a reaction that
produces a detectable signal. The non-poly(amino acid) label can be
isotopic or non-isotopic and can be, by way of illustration and not
limitation, a radioisotope, a luminescent compound (which includes,
but is not limited to fluorescent compounds and chemiluminescent
compounds, for example), a polynucleotide coding for a catalyst, a
promoter, a dye, a coenzyme, an enzyme substrate, a radioactive
group, and an amplifiable polynucleotide sequence, for example.
[0055] In some examples one member of the signal producing system
is a small organic molecule refers to a molecule of molecular
weight of about 200 to about 2,000, or about 200 to about 1,500, or
about 200 to about 1,000, or about 200 to about 500. Such small
organic molecules include, but are not limited to, biotin,
fluorescent molecules (such as fluorescein and rhodamine, for
example), chemiluminescent molecules and dinitrophenol, for
example. A binding partner for a small organic molecule is a
molecule that specifically recognizes and binds to the small
molecule. Binding partners for a small molecule are defined by the
nature of the small molecule and include, but are not limited to,
avidin, streptavidin, antibody for the small organic molecule
(which include, but are not limited to, antibody for a fluorescent
molecule (such as antibody for fluorescein and antibody for
rhodamine, for example), antibody for a chemiluminescent molecule,
antibody for dinitrophenol, for example.
[0056] In some examples of assays, a support is utilized. The
support may be comprised of an organic or inorganic, solid or
fluid, water insoluble material and which may be transparent or
partially transparent. The support can have any of a number of
shapes, such as, but not limited to, a particle (particulate
support) including bead, a film, a membrane, a tube, a well, a
strip, a rod, a fiber, or a planar surface such as, e.g., a plate
or paper, for example. The support may or may not be suspendable in
the medium in which it is employed. Examples of suspendable
supports are polymeric materials such as latex, lipid bilayers or
liposomes, oil droplets, cells and hydrogels, and magnetic
particles, for example. Other support compositions include
polymers, such as, by way of illustration and not limitation,
nitrocellulose, cellulose acetate, poly (vinyl chloride),
polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4
methylbutene), polystyrene, polymethacrylate, poly(ethylene
terephthalate), nylon, poly(vinyl butyrate), for example, either
used by themselves or in conjunction with other materials. The
support may or may not be further labeled with a dye, catalyst or
other detectable group, for example.
[0057] In some examples, the support may be a particle. The
particles have an average diameter of at least about 0.02 microns
and not more than about 100 microns. In some examples, the
particles have an average diameter from about 0.05 microns to about
20 microns, or from about 0.3 microns to about 10 microns. The
particle may be organic or inorganic, swellable or non-swellable,
porous or non-porous, preferably of a density approximating water,
generally from about 0.7 g/mL to about 1.5 g/mL, and composed of
material that can be transparent, partially transparent, or opaque.
The particles can be biological materials such as cells and
microorganisms, e.g., erythrocytes, leukocytes, lymphocytes,
hybridomas, streptococcus, Staphylococcus aureus, and E. coli,
viruses, for example. The particles can also be particles comprised
of organic and inorganic polymers, liposomes, latex particles,
magnetic or non-magnetic particles, phospholipid vesicles,
chylomicrons, lipoproteins, and the like. In some examples, the
particles are chromium dioxide (chrome) particles or latex
particles.
[0058] Chemiluminescent particles are particles that have
associated therewith a chemiluminescent compound. The phrase
"associated therewith" as used herein means that a compound such
as, for example, a chemiluminescent compound and a particle may be
associated by direct or indirect bonding, adsorption, absorption,
incorporation, or solution, for example. Examples of
chemiluminescent compounds that may be utilized are those set forth
in U.S. Pat. Nos. 5,340,716 and 6,251,581, the relevant disclosures
of which are incorporated herein by reference. In some examples in
accordance with the principles described herein, the
chemiluminescent compound is a photoactivatable substance that
undergoes a chemical reaction upon direct or sensitized excitation
by light or upon reaction with singlet oxygen to form a metastable
reaction product that is capable of decomposition with the
simultaneous or subsequent emission of light, usually within the
wavelength range of 250 to 1200 nm. The term "photoactivatable"
includes "photochemically activatable". In some examples, the
chemiluminescent compounds are those that react with singlet oxygen
to form dioxetanes or dioxetanones. The latter are usually electron
rich olefins. Exemplary of such electron rich olefins are enol
ethers, enamines, 9-alkylidene-N-alkylacridans, arylvinylethers,
dioxenes, arylimidazoles, 9-alkylidene-xanthanes and lucigenin.
Other compounds include luminol and other phthalhydrazides and
chemiluminescent compounds that are protected from undergoing a
chemiluminescent reaction by virtue of their being protected by a
photochemically labile protecting group, such compounds including,
for example, firefly luciferin, aquaphorin, and luminol. Examples
of such chemiluminescent compounds that may be utilized are those
set forth in U.S. Pat. No. 5,709,994, the relevant disclosure of
which is incorporated herein by reference.
[0059] Sensitizer particles are particles that have associated
therewith a sensitizer compound, which includes, but is not limited
to, a photosensitizer compound. Examples of sensitizer compounds
that may be utilized are those set forth in U.S. Pat. Nos.
5,340,716 and 6,251,581, the relevant disclosures of which are
incorporated herein by reference.
[0060] A photosensitizer is a sensitizer for generation of singlet
oxygen usually by excitation with light. In some examples, the
photosensitizer absorbs at a longer wavelength than the
chemiluminescent compound and has a lower energy triplet than the
chemiluminescent compound. The photosensitizer can be
photoactivatable (e.g., dyes and aromatic compounds). The
photosensitizer is usually a compound comprised of covalently
bonded atoms, usually with multiple conjugated double or triple
bonds. The compound should absorb light in the wavelength range of
200-1100 nm, usually 300-1000 nm, preferably 450-950 nm. Typical
photosensitizers include, but are not limited to, acetone,
benzophenone, 9-thioxanthone, eosin, 9,10-dibromoanthracene,
methylene blue, metallo-porphyrins (e.g., hematoporphyrin),
phthalocyanines, chlorophylls, rose bengal, buckminsterfullerene,
for example, and derivatives of these compounds. Examples of other
photosensitizers are enumerated in N.J. Turro, "Molecular
Photochemistry", page 132, W. A. Benjamin Inc., N.Y. 1965. The
photosensitizer assists photoactivation where activation is by
singlet oxygen. Usually, the photosensitizer absorbs light and the
thus formed excited photosensitizer activates oxygen to produce
singlet oxygen, which reacts with the chemiluminescent compound to
give a metastable luminescent intermediate.
[0061] Some known assays utilize a signal producing system (sps)
that employs first and second sps members. The sps members may be
related in that activation of one member of the sps produces a
product such as, for example, light or an activated product, which
results in activation of another member of the sps.
[0062] In an example of such an assay, the sps members comprise a
sensitizer such as, for example, a photosensitizer, and a
chemiluminescent composition that includes a chemiluminescent
compound where activation of the sensitizer results in a product
that activates the chemiluminescent composition. The second sps
member usually generates a detectable signal that relates to the
amount of bound and/or unbound sps member, i.e., the amount of sps
member bound or not bound to the analyte being detected. In some
examples in accordance with the principles described herein, one of
either the sensitizer reagent or the chemiluminescent reagent
comprises an antibody reagent in accordance with the principles
described herein.
Examples of Methods in Accordance with the Principles Described
Herein
[0063] As discussed above, methods in accordance with the
principles described herein are directed to determining in a sample
an amount of a first isomeric analyte and a second isomeric
analyte. In the method a first measurement value and a second
measurement value are determined. For determination of the first
measurement value, a total amount of the first isomeric analyte and
the second isomeric analyte is measured by conducting an assay on a
first portion of the sample using a first antibody that exhibits
sufficient assay binding affinity for each of the first isomeric
analyte and the second isomeric analyte. In this example, the first
antibody is a monoclonal antibody prepared by one of the procedures
described above.
[0064] The sample portion can be prepared in any convenient medium
that does not interfere with an assay; an aqueous medium generally
is employed. The size of the sample portion is dependent on one or
more of the nature of the isomeric analytes, the nature of the
assay, the nature of the various reagents for conducting the assay,
and the nature of the complex comprising the analyte, for example.
The size of the sample portion should be essentially the same for
both measurements involved in the determination. In some examples,
the volume of the sample portion is about 1 .mu.L to about 100
.mu.L, or about 2 .mu.L to about 100 .mu.L, or about 5 .mu.L to
about 100 .mu.L, or about 10 .mu.L to about 100 .mu.L, or about 1
.mu.L to about 80 .mu.L, or about 1 .mu.L to about 60 .mu.L, or
about 1 .mu.L to about 40 .mu.L, or about 1 .mu.L to about 20
.mu.L, or about 5 .mu.L to about 50 .mu.L, or about 10 .mu.L to
about 50 .mu.L, for example.
[0065] The assay selected for the determination of the first
measurement value is performed on the first sample portion, which
may be pretreated as discussed above to release the analyte from
endogenous binding substances. An amount of a complex comprising
the first antibody for the analyte and the first and second
isomeric analytes is measured by measuring a level of signal
generated by the complex. Signal observed is related to a total
amount of combined first isomeric analyte and second isomeric
analyte in the sample.
[0066] For determination of the second measurement value, an amount
of the second isomeric analyte is measured by conducting the assay
on a second portion of the sample using the first antibody and the
assay medium further comprises a second antibody that binds to the
first isomeric analyte but exhibits insufficient assay binding
affinity for the first isomeric analyte and substantially no assay
binding affinity for the second isomeric. The second sample portion
may be pretreated as discussed above to release the analyte from
endogenous binding substances. Alternatively, the sample may be
pretreated prior to taking portions to be employed in the methods
in accordance with the principles described herein. In this
example, the second antibody is a monoclonal antibody prepared by
one of the procedures described above. The second antibody is
employed in excess relative to the first antibody in the assay
medium comprising the second portion of the sample to block binding
of the first isomeric analyte to the first antibody. The excess
amount is an amount greater than that of the first antibody
required to bind a majority of the first isomeric analyte that
might be present in a sample. The amount of the second antibody
employed depends on the nature of the second antibody, the nature
of the first antibody, the nature of the isomeric analytes, the
nature of the assay medium, and the nature of the assay, for
example. In some examples in accordance with the principles
described herein an excess amount of the second antibody is about 5
to about 200 times, or about 5 to about 150 times, or about 5 to
about 100 times, or about 5 to about 50 times, or about 10 to about
200 times, or about 10 to about 150 times, or about 10 to about 100
times, or about 10 to about 50 times, or about 20 to about 200
times, or about 20 to about 150 times, or about 20 to about 100
times, or about 20 to about 50 times that of the first antibody,
for example. An amount of a complex comprising the first antibody
for the analyte and the second isomeric analyte is measured by
measuring a level of signal generated by the complex. Signal
observed is related to an amount of second isomeric analyte in the
sample. The second measurement value is subtracted from the first
measurement value to obtain a resulting value and the resulting
value is equated to an amount of the first isomeric analyte in the
sample.
[0067] As discussed more fully above, any suitable assay may be
employed. The assay comprises adding reagents for determining the
concentration of an analyte in the sample. The reagents include at
least the first antibody and the second antibody and, thus, the
assay is an immunoassay. The assays conducted on the sample
portions may be carried out sequentially or concomitantly in
separate reaction vessels or sequentially in the same reaction
vessel for each sample portion. The term "complex" refers to a
complex wherein antibody for the analyte is bound to analyte in the
sample.
[0068] As mentioned above, measurements of the isomeric analytes
may be carried out on samples that have been treated with a
releasing agent. The amount of releasing agent that is added to the
sample is that which is sufficient to displace substantially all of
the isomeric analytes from the endogenous binding substances. The
phrase "displace substantially all of the isomeric analytes that
are bound by endogenous binding substances" means that the isomeric
analytes are at least 80%, or at least 90%, or at least 95%, or at
least 99%, or at least 99.5%, or at least 99.9% or is 100%
displaced from endogenous binding substances and available for
detection during an assay.
[0069] After addition of a releasing agent, the sample is incubated
for a period of time under conditions to displace substantially all
of the isomeric analytes from endogenous binding substances. The
length and conditions of the incubation are dependent on one or
more of the nature of the releasing agent, the nature of the
analyte, and the suspected concentration of the analyte, for
example. In some embodiments incubation temperatures for this step
may be about 5.degree. C. to about 99.degree. C., or about
15.degree. C. to about 70.degree. C., or about 20.degree. C. to
about 45.degree. C., for example. The time period for the
incubation is about 0.2 seconds to about 24 hours, or about 1
second to about 6 hours, or about 2 seconds to about 1 hour, or
about 1 to about 15 minutes, for example. The incubation may be
carried out in a medium that, for convenience, may be an assay
medium as discussed herein, but need not be.
[0070] One particular example in accordance with the principles
described herein is directed to a method that employs the following
assay reagents on the first and second portions of the sample
suspected of containing the analyte: (i) an antibody reagent in
accordance with the principles described herein, (ii) a
chemiluminescent particle reagent comprising an analyte analog, and
(iii) a photosensitizer particle reagent comprising a small
molecule-binding moiety or a binding partner for the small
molecule.
[0071] In the following particular examples, the isomeric analytes
are the non-epi and epi forms of vitamin D by way of illustration
and not limitation. An induced luminescence immunoassay may be
employed. The induced luminescence immunoassay is referred to in
U.S. Pat. No. 5,340,716 (Ullman), which disclosure is incorporated
herein by reference. In one approach, the assay uses a particle
having associated therewith a photosensitizer where a vitamin D
analog is bound to the particle (particle-analog reagent). For the
assay on the first portion of a sample suspected of containing both
the non-epi and the epi forms of vitamin D analyte, the
chemiluminescent reagent comprises a first antibody that exhibits
sufficient assay binding affinity for each of the non-epi and epi
forms of the vitamin D analyte. For the assay on the second portion
of a sample, the chemiluminescent reagent comprising the first
antibody is employed along with a second antibody that binds to the
non-epi form of the vitamin D analyte but exhibits insufficient
assay binding affinity for the non-epi form of the vitamin D
analyte and substantially no assay binding affinity for the epi
form of the vitamin D analyte. In the above example, the first
antibody is linked to a small molecule, which is bound to a binding
partner for the small molecule on a chemiluminescent particle. This
chemiluminescent reagent may be pre-formed or formed in situ. The
vitamin D analyte (non-epi and epi forms) competes with the
particle-analog reagent for binding to the antibody for vitamin D
in accordance with the principles described herein. If the vitamin
D analyte is present, the fewer is the number of molecules of
particle-analog reagent that come into close proximity with the
chemiluminescent reagent. Therefore, there will be a decrease in
the assay signal. The photosensitizer generates singlet oxygen and
activates the chemiluminescent reagent when the two labels are in
close proximity. The activated chemiluminescent reagent
subsequently produces light, where a decrease in signal is observed
in the presence of the analyte. The amount of light produced is
related to the amount of the complex formed, which in turn for the
assay on the first sample portion is related to the amount of both
the non-epi and epi forms of the vitamin D analyte present in the
sample (first measurement value) and for the assay on the second
samples portion is related to the amount of the epi form of the
vitamin D analyte present in the sample (second measurement value).
Subtraction of the second measurement value from the first
measurement value gives the amount of the non-epi form of the
vitamin D analyte in the sample.
[0072] In another particular example of an induced luminescence
immunoassay using vitamin D as an example, by way of illustration
and not limitation, the assay uses a particle having associated
therewith a chemiluminescent compound where a vitamin D analog is
bound to the particle (particle-analog reagent). For the first
sample portion, a photosensitizer reagent comprises a first
antibody that exhibits sufficient assay binding affinity for each
of the non-epi and epi forms of the vitamin D analyte, which is
linked to a small molecule that is in turn bound to a binding
partner for the small molecule on a chemiluminescent particle. For
the second sample portion, the photosensitizer reagent and a second
antibody are employed. The second antibody binds to the non-epi
form of the vitamin D analyte but exhibits insufficient assay
binding affinity for the non-epi form of the vitamin D analyte and
substantially no assay binding affinity for the epi form of the
vitamin D analyte. For the first sample portion, the both the
non-epi form and the epi form of the vitamin D analyte compete with
the particle-analog reagent for binding to the first antibody for
vitamin D. If the vitamin D analyte is present, the fewer is the
number of molecules of particle-analog reagent that come into close
proximity with the photosensitizer reagent. Therefore, there will
be a decrease in the assay signal. For the second sample portion,
the epi form of the vitamin D analyte competes with the
particle-analog reagent for binding to the first antibody for
vitamin D because the non-epi form of the vitamin D analyte is
bound by the second antibody. If the epi form of the vitamin D
analyte is present, the fewer is the number of molecules of
particle-analog reagent that come into close proximity with the
photosensitizer reagent. Therefore, there will be a decrease in the
assay signal. The photosensitizer generates singlet oxygen and
activates the chemiluminescent compound of the particle-analog
reagent when the two labels are in close proximity. The activated
chemiluminescent compound subsequently produces light, where a
decrease in signal is observed in the presence of the analyte. The
amount of light produced is related to the amount of the complex
formed, which in turn for the assay on the first sample portion is
related to the amount of both the non-epi and epi forms of the
vitamin D analyte present in the sample (first measurement value)
and for the assay on the second samples portion is related to the
amount of the epi form of the vitamin D analyte present in the
sample (second measurement value). Subtraction of the second
measurement value from the first measurement value gives the amount
of the non-epi form of the vitamin D analyte in the sample.
[0073] In another particular example of an induced luminescence
assay using vitamin D by way of illustration and not limitation, a
photosensitizer particle is employed that is conjugated to a
binding partner for a small molecule such as, for example, avidin
or streptavidin (which are binding partners for biotin). An
antibody reagent in accordance with the principles described herein
that comprises biotin linked to a first antibody that binds to both
the non-epimeric and epimeric forms of the vitamin D analyte is
employed. A chemiluminescent reagent is employed as part of the
detection system. The reaction medium for the first sample portion
or the second sample portion, as the case may be, is incubated to
allow the avidin or streptavidin of the photosensitizer particles
to bind to the biotin of the antibody reagent by virtue of the
binding between avidin and biotin and to also allow the specific
binding between the first antibody of the antibody reagent in
accordance with the principles described herein, which is now
attached to the photosensitizer particles, to bind to the analyte
of the sample and to the analyte that is part of the
chemiluminescent reagent. Then, the medium is irradiated with light
to excite the photosensitizer, which is capable in its excited
state of activating oxygen to a singlet state. Because less of the
chemiluminescent reagent is now in close proximity to the
photosensitizer because of the presence of the analyte, there is
less activation of the chemiluminescent reagent by the singlet
oxygen and less luminescence. The medium is then examined for the
presence and/or the amount of luminescence or light emitted, the
presence thereof being related to the presence and/or amount of the
analyte where a decrease in signal is observed in the presence of
the analyte. The amount of light produced is related to the amount
of the complex formed, which in turn for the assay on the first
sample portion is related to the amount of both the non-epi and epi
forms of the vitamin D analyte present in the sample (first
measurement value) and for the assay on the second samples portion
is related to the amount of the epi form of the vitamin D analyte
present in the sample (second measurement value). Subtraction of
the second measurement value from the first measurement value gives
the amount of the non-epi form of the vitamin D analyte in the
sample.
[0074] Another example of an assay format for detection of vitamin
D, by way of illustration and not limitation, in a sample is the
ACMIA assay format. For the ACMIA assay format, chrome particles,
which are coated with vitamin D or a vitamin D analog (chrome
particle reagent), are employed as a first component. A second
component is an antibody reagent that comprises an antibody for
vitamin D in accordance with the principles described herein. In
the antibody reagent, the antibody is linked by means of a linking
group to a reporter enzyme (for example, .beta.-galactosidase) to
form an antibody-enzyme conjugate. The antibody reagent is added to
a reaction vessel in an excess amount, i.e., an amount greater than
that required to bind all of the vitamin D analyte that might be
present in a sample. A first portion of a sample, which is
previously subjected to treatment with a releasing agent, is
treated with a first antibody reagent as described above, which
comprises an antibody that exhibits sufficient assay binding
affinity for each of the non-epi and epi forms of the vitamin D
analyte; the antibody binds to vitamin D in the sample. The
antibody-enzyme conjugate is mixed with sample in the medium to
allow the vitamin D analyte to bind to the antibody. Next, the
chrome particle reagent is added to bind up any excess
antibody-enzyme conjugate. Then, a magnet is applied, which pulls
all of the chrome particles and excess antibody-enzyme out of the
suspension, and the supernatant is transferred to a final reaction
container. The substrate of the reporter enzyme is added to the
final reaction container, and the enzyme activity is measured
spectrophotometrically as a change in absorbance over time. The
amount of this signal is related to the amount of both the non-epi
and epi forms of the vitamin D in the sample. A second portion of a
sample, which is previously subjected to treatment with a releasing
agent, is treated with the first antibody reagent and a second
antibody as described above, which comprises a second antibody,
which binds to the non-epi form of the vitamin D analyte but
exhibits insufficient assay binding affinity for the non-epi form
of the vitamin D analyte and substantially no assay binding
affinity for the epi form of the vitamin D. The antibody-enzyme
conjugate is mixed with sample in the medium to allow the vitamin D
analyte to bind to the antibody. Next, the chrome particle reagent
is added to bind up any excess antibody-enzyme conjugate. Then, a
magnet is applied, which pulls all of the chrome particles and
excess antibody-enzyme out of the suspension, and the supernatant
is transferred to a final reaction container. The substrate of the
reporter enzyme is added to the final reaction container, and the
enzyme activity is measured spectrophotometrically as a change in
absorbance over time. The amount of this signal is related to the
amount of both the non-epi and epi forms of the vitamin D in the
sample.
[0075] Another example of an assay for isomeric forms of vitamin D
(by way of illustration and not limitation) in a sample is an
acridinium ester label immunoassay using paramagnetic particles as
a solid phase (ADVIA immunoassay). The detection system employed
for this example of a vitamin D assay includes a small
molecule-labeled vitamin D (capture moiety) as the small molecule
conjugate or capture conjugate, binding partner for the small
molecule-coated paramagnetic latex particles as a solid phase (SP),
and an acridinium ester labeled antibody for vitamin D (detection
antibody) in accordance with the principles described herein. The
small molecule may be, for example, biotin or fluorescein and the
respective binding partner may be streptavidin or antibody for
fluorescein. The vitamin D may be linked to the small molecule
directly or through a linking group such as, for example, a
protein, e.g., bovine serum albumin (BSA). Vitamin D in a patient
sample competes with vitamin D of the capture moiety for binding to
the acridinium ester labeled detection anti-vitamin D antibody. The
sample suspected of containing vitamin D is subjected to a
pretreatment with 1,8-ANS. The assay may be carried out on first
and second sample portions using respective antibodies in
accordance with the principles described herein and a CENTAUR.RTM.,
CENTAUR.RTM. XP or CENTAUR.RTM. CP apparatus (Siemens Healthcare
Diagnostics Inc., Newark Del.) in accordance with the
manufacturer's directions supplied therewith.
[0076] Another example of an assay for an analyte in accordance
with the principles described herein is an acridinium ester label
immunoassay using paramagnetic particles as a solid phase (ADVIA
immunoassay). The detection system employed for this example of an
assay for isomeric analytes includes an antibody reagents in
accordance with the principles described herein, in which a small
molecule is linked to the antibody for the analyte (capture
antibody) as the capture conjugate, paramagnetic latex particles as
a solid phase (SP) coated with a binding partner for the small
molecule of the antibody reagent, and an acridinium ester labeled
analyte analog (detection hapten). The acridinium ester label may
be directly bound to the analyte to form the detection hapten or a
linking group may be employed including, for example, a protein
such as, e.g., BSA. The analyte of a sample competes with the
acridinium ester labeled detection hapten for binding with
anti-analyte antibody. The sample suspected of containing the
analyte may be subjected to a pretreatment with one or more of a
releasing agent and a digestion agent. The assay may be carried out
on the first and second sample portions using a CENTAUR.RTM.,
CENTAUR.RTM. XP or CENTAUR.RTM. CP apparatus (Siemens Healthcare
Diagnostics Inc., Newark Del.) in accordance with the
manufacturer's directions supplied therewith. In variations of the
above acridinium ester assays, the small molecule may be, for
example, biotin or fluorescein and the binding partners for the
small molecule may be, for example, avidin or streptavidin or
antibody for fluorescein, respectively.
[0077] The concentration of the isomeric analytes in a sample that
may be assayed generally varies from about 10.sup.-5 to about
10.sup.-17 M, or from about 10.sup.-6 to about 10.sup.-14 M, for
example. Considerations such as whether the assay is qualitative,
semi-quantitative or quantitative (relative to the amount of the
analyte present in the sample), the particular detection technique
and the expected concentration of the analyte normally determine
the concentrations of the various reagents.
[0078] The concentrations of the various reagents in the assay
medium will generally be determined by the concentration range of
interest of the analyte, the nature of the assay, and the like.
However, the final concentration of each of the reagents is
normally determined empirically to optimize the sensitivity of the
assay over the range of interest. That is, a variation in
concentration of analyte that is of significance should provide an
accurately measurable signal difference. Considerations such as the
nature of the signal producing system and the nature of the
analytes normally determine the concentrations of the various
reagents.
[0079] As mentioned above, the sample and reagents are provided in
combination in the medium. While the order of addition to the
medium may be varied, there will be certain preferences for some
embodiments of the assay formats described herein. The simplest
order of addition, of course, 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, each of the
reagents, or groups of reagents, can be combined sequentially. In
some embodiments, an incubation step may be involved subsequent to
each addition as discussed above. In heterogeneous assays, washing
steps may also be employed after one or more incubation steps.
[0080] Some examples in accordance with the principles described
herein are directed to methods of determining one or both of the
presence and the amount of one or both of two isomeric forms of
vitamin D in a sample suspected of containing vitamin D and may be
referred to herein as "assays for vitamin D." As used herein in
reference to assays, the term "vitamin D" refers to one or more of
the non-epi and epi forms of one or more of
25-hydroxycholecalciferol (also referred to as calcidiol,
calcifediol, 25-hydroxycholecalciferol, or 25-hydroxyvitamin D
(abbreviated 25(OH)D); calcidiol; 1,25-dihydroxyvitamin D.sub.3
(calcitriol; 1,25(OH).sub.2D.sub.3); 1,25-dihydroxy vitamin
D.sub.4; 1,25-dihydroxy vitamin D.sub.5; and 1,25-dihydroxy vitamin
D.sub.6; including metabolites of all of the above.
Examination Step
[0081] In one step of an assay method, the medium is examined for
the presence of a complex comprising one or more isomeric forms of
the analyte and antibody for an analyte in accordance with the
principles described herein. The presence and/or amount of one or
both of the complexes indicates the presence and/or amount of one
or more of the isomeric forms of the analyte in the sample.
[0082] The phrase "measuring the amount of analyte" refers to the
quantitative, semiquantitative and qualitative determination of one
or more of the isomeric forms of an analyte. Methods that are
quantitative, semiquantitative and qualitative, as well as all
other methods for determining the analyte, are considered to be
methods of measuring the amount of the analyte. For example, a
method, which merely detects the presence or absence of the 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.
[0083] In many embodiments the examination of the medium involves
detection of a signal from the medium. The presence and/or amount
of the signal is related to the presence and/or amount of one or
more of the isomeric forms of an analyte in the sample. The
particular mode of detection depends on the nature of the signal
producing system. As discussed above, there are numerous methods by
which a label of a signal producing signal can produce a signal
detectable by external means. Activation of a signal producing
system depends on the nature of the signal producing system
members.
[0084] Temperatures during measurements generally range from about
10.degree. C. to about 70.degree. C. or from about 20.degree. C. to
about 45.degree. C., or about 20.degree. C. to about 25.degree. C.,
for example. In one approach standard curves are formed using known
concentrations of vitamin D analyte. Calibrators and other controls
may also be used.
[0085] Luminescence or light produced from any label can be
measured visually, photographically, actinometrically,
spectrophotometrically, such as by using a photomultiplier or a
photodiode, or by any other convenient means to determine the
amount thereof, which is related to the amount of analyte in the
medium. The examination for presence and/or amount of the signal
also includes the detection of the signal, which is generally
merely a step in which the signal is read. The signal is normally
read using an instrument, the nature of which depends on the nature
of the signal. The instrument may be, but is not limited to, a
spectrophotometer, fluorometer, absorption spectrometer,
luminometer, and chemiluminometer, for example.
Kits Comprising Reagents for Conducting Assays
[0086] Kits for conducting assays on portions of a sample suspected
of containing isomeric forms of an analyte may be prepared. The
kits comprise antibody reagents for assays to be carried out on
respective portions of the sample. Accordingly, one antibody
reagent comprises an antibody that exhibits sufficient assay
binding affinity for each of a first isomeric analyte and a second
isomeric analyte. A second antibody is included that binds to the
first isomeric analyte but exhibits insufficient assay binding
affinity for the first isomeric analyte and substantially no assay
binding affinity for the second isomeric analyte. The kit may
further include other reagents for performing the assay, the nature
of which depend upon the particular assay format.
[0087] 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 kit can further
include other separately packaged reagents for conducting an assay
such as additional specific binding pair members, signal producing
system members, and ancillary reagents, for example.
[0088] The relative amounts of the various reagents in the kits can
be varied widely to provide for concentrations of the reagents that
substantially optimize the reactions that need to occur during the
present methods and further to optimize substantially the
sensitivity of an 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 using a compound
reagent in accordance with the principles described herein. The kit
can further include a written description of a method utilizing
reagents that include a compound reagent in accordance with the
principles described herein.
[0089] The designation "first" and "second" as used herein is
completely arbitrary and is not meant to suggest any order or
ranking among moieties referred to or any order of addition of
moieties in the present methods.
[0090] The phrase "at least" as used herein means that the number
of specified items may be equal to or greater than the number
recited. The phrase "about" as used herein means that the number
recited may differ by plus or minus 10%; for example, "about 5"
means a range of 4.5 to 5.5.
[0091] The following discussion is directed to specific examples in
accordance with the principles described herein by way of
illustration and not limitation; the specific examples are not
intended to limit the scope of the present disclosure and the
appended claims. Numerous modifications and alternative
compositions, methods, and systems may be devised without departing
from the spirit and scope of the present disclosure.
Examples
[0092] Unless otherwise indicated, materials in the experiments
below may be purchased from the Sigma-Aldrich Chemical Corporation
(St. Louis Mo.) or Fluka Chemical Corporation (Milwaukee Wis.).
Parts and percentages disclosed herein are by weight to volume
unless otherwise indicated.
DEFINITIONS
[0093] mg=milligram
[0094] g=gram(s)
[0095] ng=nanogram(s)
[0096] mL=milliliter(s)
[0097] .mu.L=microliter(s)
[0098] .mu.mol=micromolar
[0099] .degree. C.=degrees Centigrade
[0100] min=minute(s)
[0101] sec=second(s)
[0102] hr=hour(s)
[0103] w/v=weight to volume
[0104] v/v=volume to volume
[0105] TLC=thin layer chromatography
[0106] HPLC=high performance liquid chromatography
[0107] EDTA=ethylenediaminetetraacetate
[0108] PEG=polyethylene glycol
[0109] EtOAc=ethyl acetate
[0110] DMF=dimethylformamide
[0111] DMSO=dimethylsulfoxide
[0112] MeOP=1-methoxy-2-propanol
[0113] MES=2-(N-morpholino)ethanesulfonic acid
[0114] DI=distilled
[0115] UPA=Ultra Particle Analyzer
[0116] LOCI=luminescent oxygen channeling immunoassay
[0117] Ab=antibody
Preparation of Biotinylated First Antibody that Exhibits Sufficient
Assay Binding Affinity for Both Non-Epi Vitamin D and Epi-Vitamin
D
[0118] A solution (0.8 mL at 2.63 mg/mL) of vitamin D antibody 5H10
(sheep monoclonal from Bioventix, Farnham, Surrey, UK) in 10 mM
PO.sub.4, 300 mM NaCl, pH 7.0 was mixed with 43.2 .mu.L of an
aqueous solution (2.0 mg/mL) of NHS-dPEG.RTM. 4-biotin (Quanta
Biodesign Ltd., Powell Ohio, part number 10200). The amount of
biotinylation reagent added represents a 10-fold molar challenge of
the biotinylating agent with the antibody. The reaction mixture was
incubated at room temperature for 3 hr and then the reaction was
quenched by addition of 80 .mu.L of 0.5 M TRIS. The reaction
mixture was subjected to buffer exchange with 10 mM PO.sub.4, 300
mM NaCl, pH 7.0 in an AMICON.RTM. (YM10) device until absorption at
260 nm of the effluent was .ltoreq.0.03. The antibody solution
(1.04 mL at 2.1 mg/mL protein) was mixed with 10 .mu.L of
PROCLIN.RTM. 300 and 10 .mu.L of an aqueous solution of neomycin
sulfate (10 mg/mL) filtered using a 0.2 um ACRODISC.RTM. syringe
filter (Pall Corporation) and was stored at 2-8.degree. C.
Preparation of EPRM-EDA Beads
[0119] EPRM beads (2000 mg, 20.0 mL) are added to a 40-mL vial. The
EPRM beads are prepared by a procedure similar to that described in
U.S. Pat. No. 7,179,660 and the chemiluminescent compound is
2-(4-(N,N, di-tetradecyl)-anilino-3-phenyl thioxene with europium
chelate. EDA (800 mg, 890 .mu.L) is combined with 10 mL MES pH 6
buffer (the "Buffer") and about 4.2 mL 6N HCl. The pH of the
mixture is, or is adjusted to be, about 6.9. The EDA solution is
added to the EPRM beads with vortexing and the mixture is rocked at
room temperature for 15 minutes. Sodium cyanoborohydride (400 mg)
is combined in a 15 mL vial with 10 mL DI water and the combination
is added to the bead mixture from above. The mixture is shaken at
37.degree. C. for 18-20 hours. The beads are transferred to six 40
mL centrifuge tubes. MES buffer is added to bring the volume to 35
mL and the mixture is centrifuged at 19,000 rpm for 30 min. The
supernatant is decanted and the beads are re-suspended in 2 mL of
the Buffer with a stir-rod and additional Buffer is added to 35 mL.
The mixture is sonicated at 18 Watts power for 30 sec, using ice to
keep the mixture cold. The wash/sonication step is performed 4
times to remove all activation chemical. After the last MES Buffer
centrifugation, 2 mL of the Buffer containing 5% MeOP and 0.1%
Tween.RTM. 20 (the "second Buffer") is added to the tubes for the
re-suspension step. Additional second buffer is added to 35 mL
before sonication. The bead suspension is centrifuged at 19,000 rpm
for 30 min. The supernatant is discarded. The final sonication used
12 mL of the second Buffer in each tube to give a 25 mg/mL
dilution. Particle size is 277 nm as determined on a UPA
instrument.
[0120] The EPRM chemibead is prepared in a manner similar to the
method described in U.S. Pat. No. 6,153,442 and U.S. Patent
Application Publication No. 20050118727A, the relevant disclosures
of which are incorporated herein by reference. The EPRM chemibead
comprises an aminodextran inner layer and a dextran aldehyde outer
layer having free aldehyde functionalities. See, for example, U.S.
Pat. Nos. 5,929,049, 7,179,660 and 7,172,906, the relevant
disclosures of which are incorporated herein by reference. The
reaction is carried out at a temperature of about 0 to about
40.degree. C. for a period of about 16 to about 64 hours at a pH of
about 5.5 to about 7.0, or about 6, in a buffered aqueous medium
employing a suitable buffer such as, for example, MES. The reaction
is quenched by addition of a suitable quenching agent such as, for
example, carboxymethoxyamine hemihydrochloride (CMO), and
subsequent washing of the particles.
[0121] Aldehyde groups on the outer dextran aldehyde layer are
reacted with ethylene diamine under reductive amination conditions
to form reagent EPRM-EDA having pendant moieties comprising an
ethylene chain and a terminal amine group. The reductive amination
conditions include the use of a reducing agent such as, for
example, a metal hydride. The reaction is carried out in an aqueous
medium at a temperature during the reaction of about 20.degree. C.
to about 100.degree. C. for a period of about 1 hour to about 48
hours.
Synthesis of 25-OH Vitamin D.sub.3 3-Carbamate (25-OH Vitamin
D.sub.2 3-Carbamate)
[0122] A mixture of 22 mg (55 .mu.mol) 25-OH VD.sub.3 purchased
from ChemReagents.com, Sugarland Tex., 100 mg (420 .mu.mol)
disuccinimidyl carbonate (DSC), 100 .mu.L triethylamine in 1 mL
anhydrous acetonitrile in a 5-ml flask (covered with foil) was
stirred at room temperature for 18 hr under nitrogen to prepare
activated 25-OH VD.sub.3. TLC (EtOAc:Hexane=2:1) showed no starting
material left. A suspension was prepared by adding 150 mg of
carboxymethoxylamine hemihydrochloride (CMO), 0.3 ml triethylamine
and 1 ml DMF to a 10 ml flask. A solution containing activated
25-OH VD.sub.3 was added dropwise to the CMO suspension with
stirring, which was continued for another 18 hr. Vacuum was applied
to remove the solvents as much as possible (the heating bath
temperature should not be over 50.degree. C.). EtOAc (25 ml) was
added to the residue, which was washed three times with 2 ml brine.
The organic phase was dried with anhydrous Na.sub.2SO.sub.4 and was
filtered; solvent was removed using rotavap. Crude product (42 mg)
was obtained after drying and was purified by HPLC. Pure product
(24 mg) was obtained after being dried under high vacuum. The
product was dissolved into 1.2 ml anhydrous DMSO. Aliquots were
transferred into vials, which were kept at -70.degree. C.
Coupling of EPRM-EDA and 25-OH Vitamin D.sub.3 3-Carbamate to Give
Chemibead Reagent
[0123] 25-OH Vitamin D.sub.3 3-Carbamate (10 .mu.L of aliquot in
DMSO prepared as described above) (0.2 mg) was added to a 2-mL
vial. EDAC (6.8 mg) and SNHS (9.4 mg) plus 2.27 mL dry DMSO (3
mg/mL) were added to a 5-mL vial. The EDAC/SNHS solution (190
.mu.L) was combined with the contents of the 2-mL vial from above
(1 mg/mL) to prepare activated 25-OH vitamin D.sub.3 3-carbamate.
The mixture was allowed to rotate at room temperature for 18 hr. A
0.4 mL aliquot of a 16% GAFAC.RTM. surfactant solution (GAF
Corporation, Wayne N.J.) (0.15%) was diluted to 1.6% with 3.6 mL DI
water.
[0124] Vitamin D.sub.3 (8.5 mg) and 850 .mu.L DMSO (10 mg/mL) were
combined. To a 10-mL round bottom flask (labeled 3323-064B)
equipped with a stir-bar was added 2.0 mL (200 MG) EPRM-EDA
followed by 400 .mu.L (4 mg) of the Vitamin D.sub.3 solution from
above. The mixture stirred overnight at room temperature.
[0125] To a 10-mL round bottom flask equipped with a stir-bar was
added 2.0 mL (200 mg) EPRM-EDA (prepared as described above)
followed by 260 .mu.L 1.6% GAFAC.RTM. surfactant solution (0.15%)
with moderate stirring. To a small test tube was added 504 .mu.L
anhydrous DMSO followed by 60 .mu.L (0.06 mg) activated Vitamin
D.sub.3-3-carbamate prepared as described above; and the mixture
was added to the EPRM-EDA bead mixture. The total DMSO content of
the bead suspension was 20%. The reaction vessel was allowed to
stir overnight at room temperature. Then, the beads were washed by
means of diafiltration.
[0126] Each bead lot was taken up to 20 mL working volume with 10%
MeOP/1% GAFAC.RTM./MES pH6 buffer. The mixture was diafiltered with
5 volumes of the buffer and then sonicated with a probe sonicator
at 18-21 Watts using ice to keep the mixture cold. The
diafiltration/sonication continued through 50 volumes with effluent
samples being taken at 35, 40, 45 and 50 volumes. The buffer was
changed to LOCI Hapten Wash Buffer (50 mM HEPES, 300 mM NaCl, 1 mM
EDTA, 0.01% neomycin sulfate, 0.1% TRITON.RTM. 405X and 0.15%
PROCLIN.RTM. 300, pH 7.2) with 10 volumes being used. The mixture
was reduced to about 7 mL and a UPA performed. Particle sizes were
3323-064A=289 nm and 3323-064B=298 nm. Percent solids were
determined and the bead lot was brought up to 10 mg/mL with LOCI
Hapten Wash Buffer pH7.2. Yield was 160.4 mg.
Assay for Non-Epi-Vitamin D and Epi-Vitamin D
[0127] Assays were carried out on a DIMENSION.RTM. VISTA.RTM.
analyzer (Siemens Healthcare Diagnostics Inc., Deerfield, Ill.)
following the protocol for a LOCI assay and using calibrator
solutions containing varying amounts of non-epi-25-hydroxyvitamin
D.sub.3 and/or 3-epi-25-hydroxyvitamin D.sub.3. In this example,
the assay uses, as a chemiluminescent reagent, the chemibead
reagent prepared as described above. Sample portions are reacted
with either (i) the first biotinylated antibody reagent (first
sample portion) prepared as described above or (ii) the first
biotinylated antibody and a second antibody (second sample portion)
and then with the chemibead reagent. For the second sample portion,
the second antibody is a solution (0.8 mL at 2.63 mg/mL) of vitamin
D antibody 10H9 (mouse monoclonal found in CENTAUR.RTM. vitamin D
assay, Siemens Healthcare Diagnostics Inc., Newark Del.); the
second antibody was present in excess amount (75 .mu.g/mL or 100
times that of the 5H10 antibody). The chemibeads bind to the
fraction of the monoclonal antibody binding sites that is not
occupied by analyte from the sample. Subsequently, streptavidin
coupled sensitizer beads are added to the reaction mixture. This
leads to the formation of chemibead/sensibead pairs whose
concentration is inversely related to a concentration of either
both forms of the vitamin D (first sample portion) or the epi form
of vitamin D (second sample portion). Upon illumination at 680 nm,
the sensitizer beads generate singlet oxygen which diffuses into
the chemibeads which are paired with sensibeads, reacts with the
olefinic dye and triggers a chemiluminescent signal at
approximately 612 nm which is inversely related to the analyte
concentration.
[0128] The streptavidin-sensitizer bead ("sensibead(s)") is
prepared using a method analogous to that described in U.S. Pat.
Nos. 6,153,442, 7,022,529, 7,229,842 and U.S. Patent Application
Publication No. 20050118727A. The photosensitizer was
bis-(trihexyl)-silicon-t-butyl-phthalocyanine. The concentration of
sensibead reagent was 200 .mu.g/mL in HEPES buffer, pH 8.0
containing 150 mM NaCl. The EPRM-EDA-25-OH Vitamin D.sub.3 particle
reagent prepared as described above was employed as the "chemibead
reagent" at a concentration of 200 .mu.g/mL in HEPES buffer, pH
7.2, containing 150 mM NaCl and 0.1% detergent.
[0129] For a respective sample portion, at time t=zero sec, 20
.mu.L biotinylated antibody reagent and 20 .mu.L water were added
to a reaction vessel. Sample, 12 .mu.L, was added 21.6 seconds
later, followed by 8 .mu.L water. At t=414.0 seconds, 40 .mu.L
chemibead reagent was added followed by 20 mL of water. Sensibead
reagent was then dispensed at 457.2 seconds. Measurements were
taken 601.2 seconds after initiation of the reaction sequence. A
first measurement value representing an amount of both the epi and
non-epi forms of vitamin D and a second measurement value
representing an amount of only the epi form of vitamin D were
obtained.
[0130] Using the above assay format, assays were carried out on
serum samples that were spiked with varying amounts of
non-epi-25-hydroxyvitamin D.sub.3 (non-epi-VD) but not with
3-epi-25-hydroxyvitamin D.sub.3 (3-epi-VD). This set of assays was
performed to calibrate the instrument and the samples that
contained or did not contain 10H9 second antibody. The results are
summarized in Table 1 below and are plotted in a graph depicted in
FIG. 3.
TABLE-US-00001 TABLE 1 10H9 Ab absent 10H9 Ab present Non-epi-VD
(ng/mL) Non-epi-VD (ng/mL) 0.0 0.0 9.2 7.5 19.6 10.8 71.6 19.7 167
29.1
The results show that the assay is still detecting some non-epi-VD
even with an excess amount of the 10H9 second antibody present.
Therefore, results obtained in other assays employing the 10H9
second antibody on this instrument system will have to be adjusted
to account for the results of this calibration.
[0131] Using the above assay format, assays were carried out on
serum samples that were spiked with varying amounts of non-epi-VD
and with 3-epi-VD. The assays were carried out both with (+10H9)
and without (-10H9) the 10H9 second antibody. The "Predicted 1" and
"Predicted 2" values are obtained with reference to the graphs in
FIGS. 2 and 3. Values are ng/mL; Diff=difference between +10H9
value and Predicted 1 value. "Amount spiked" is the amount of
3-epi-VD that was spiked into the samples. The results are
summarized in Table 2 below.
TABLE-US-00002 TABLE 2 Predicted 3-epi-VD Predicted Amount -10H9
+10H9 1 Diff present 2 spiked 53 48.5 18 30.6 Yes 170 167 25.2 24.1
12 12.1 Yes 67 70 15.3 14.5 9 5.7 Yes 32 30 10.8 10.6 7 7 Yes 20 10
9.2 7.5 6 1.2 No 0 0 19.6 10.8 10 0.5 No 0 0 71.6 19.7 21 -0.9 No 0
0
[0132] Using the above data in Table 2, a corrected amount of
3-epi-VD is calculated as follows; the predicted amount of 3-epimer
is listed in the second to the last column. Explanations for each
column of Table 2 above are as follows: [0133] Column 1: -10H9 is
ng/mL 25(OH)D measured in the absence of 10H9 Ab. This represents
the total amount of 25(OH)D ng/mL
(D.sub.2+D.sub.3+3epi.times.cross-reactivity) [0134] Column 2:
+10H9 is ng/mL 25(OH)D measured in the presence of 10H9 Ab. This
represents suppressed total 25(OH)D ng/mL (partial D2+partial
D3+3epi.times.cross-reactivity) [0135] Column 3: Predicted 1 is the
amount of ng/mL 25(OH)D in the presence of 10H9 if there were no
3-epimer present in sample (partial D.sub.2+partial D.sub.3) [0136]
Column 4: Diff is column 2 minus column
3=3epi.times.cross-reactivity [0137] Column 5: Predicted 2 is
column 4 divided by cross-reactivity or
(3epi.times.cross-reactivity)/cross-reactivity=3epi ng/mL) [0138]
Column 6: Amount spiked is how much 3-epimer is spiked in sample.
This column should be compared to column 5 to show how close the
results are in column 5 and column 6. The results are summarized in
Table 3. Corrected means 25(OH)D ng/mL calculated after the
measured 3-epimer ng/mL is subtracted from the total ng/mL 25(OH)D
measured in the absence of 10H9 Ab. CXR means 3-epimer
cross-reactivity of the 2 reaction vessel assay after 3-epimer
interference is removed. This is not the same as the
cross-reactivity referred to above. The cross-reactivity in Table 2
is the cross-reactivity of 5H10 Ab with the 3-epimer. The
cross-reactivity in Table 3 is the cross-reactivity of the assay
after the concentration of 3-epimer is subtracted from the final
ng/mL 25(OH)D concentration.
TABLE-US-00003 [0138] TABLE 3 Amount 3-epi-VD 3-epi-VD -10H9 +10H9
Spiked Diff present Corrected CXR 53 48.5 167 30.6 Yes 14 3% 25.2
24.1 70 12.1 Yes 10 2% 15.3 14.5 30 5.7 Yes 10 3% 10.8 10.6 10 7
Yes 9 2% 9.2 7.5 0 1.2 No 0 0 19.6 10.8 0 0.5 No 0 0 71.6 19.7 0
-0.9 No 0 0
In Table 3, the columns 1, 2, 3, 4 and 5 correspond to columns 1,
2, 7, 4 and 5 in Table 2, respectively. Column 6 is corrected,
which is ng/mL 25(OH)D without 3-epimer. Basically, ng/mL 25(OH)D
values for calibrators L2-L5 was plotted against the differences in
ng/mL between 25(OH)D values in the presence and absence of 10H9
antibody. The coefficients generated from this plot were used to
predict non-3-epimer 25(OH)D values by the differences. This
results because the difference is the suppressed signal that
represents non-3-epimer signal since 10H9 antibody only binds to
non-epimers.
[0139] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0140] It should be understood that the above-described examples
are merely illustrative of some of the many specific examples that
represent the principles described herein. Clearly, those skilled
in the art can readily devise numerous other arrangements without
departing from the scope as defined by the following claims.
[0141] Unless otherwise indicated, materials in the experiments
below may be purchased from the Sigma-Aldrich Chemical Corporation
(St. Louis Mo.) or Fluka Chemical Corporation (Milwaukee Wis.).
Parts and percentages disclosed herein are by weight to volume
unless otherwise indicated.
* * * * *