U.S. patent application number 17/354023 was filed with the patent office on 2022-04-21 for device for detection of vitamin d metabolites.
The applicant listed for this patent is Affimedix, Inc.. Invention is credited to Kevin C. Wang.
Application Number | 20220120767 17/354023 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-21 |
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United States Patent
Application |
20220120767 |
Kind Code |
A1 |
Wang; Kevin C. |
April 21, 2022 |
DEVICE FOR DETECTION OF VITAMIN D METABOLITES
Abstract
The present invention provides methods, devices, and
compositions to rapidly detect analytes, including small analytes,
using a lateral flow device. Described herein is such a lateral
flow device that can detect and quantify vitamin D in a whole
blood, serum, or plasma sample by employing a sandwich-based
immunoassay.
Inventors: |
Wang; Kevin C.; (San
Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Affimedix, Inc. |
Hayward |
CA |
US |
|
|
Appl. No.: |
17/354023 |
Filed: |
June 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16041582 |
Jul 20, 2018 |
11073524 |
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17354023 |
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PCT/US2017/014620 |
Jan 23, 2017 |
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16041582 |
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62286297 |
Jan 22, 2016 |
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International
Class: |
G01N 33/82 20060101
G01N033/82; G01N 33/543 20060101 G01N033/543; G01N 33/558 20060101
G01N033/558; C07K 16/44 20060101 C07K016/44 |
Claims
1.-39. (canceled)
40. A method for quantifying one or more vitamin D levels in a
biological sample using a test device configured to perform a
binding assay, said method comprising: (a) contacting said
biological sample to said test device; (b) subjecting said
biological sample to said binding assay that utilizes a reaction
mixture comprising a complex formed by: (i) a vitamin D binding
agent; and (ii) one or more vitamin D molecules; (c) further
exposing said complex to a detection agent that binds to an epitope
that is formed by complexing said vitamin D binding agent with said
one or more vitamin D binding molecules, wherein said binding assay
has a sensitivity of detection in said biological sample that is
comparable to that of liquid chromatography-tandem mass
spectrometry, thereby quantifying said one or more vitamin D levels
in said biological sample.
41. The method of claim 40, wherein said quantifying comprises
classifying said biological sample as having a sufficient, an
insufficient, or a deficient level of said one or more vitamin D
molecules.
42. The method of claim 41, wherein said sufficient level is at
least about 30 ng/mL.
43. The method of claim 41, wherein said insufficient level is at
least about 10 ng/mL and less than about 30 ng/mL.
44. The method of claim 41, wherein said deficient level is less
than about 10 ng/mL.
45. The method of claim 40, wherein said biological sample is
selected from the group consisting of: whole blood, serum, plasma,
urine, saliva, ocular fluid, spinal fluid, and perspiration.
46. The method of claim 40, wherein said detection agent exhibits a
higher binding affinity to said complex than said vitamin D binding
agent.
47. The method of claim 40, wherein said detection agent comprises
a scFv, VH, Fab, or (Fab)2 binding unit.
48. The method of claim 40, wherein said one or more vitamin D
molecules are selected from the group consisting of:
25-hydroxyvitamin D.sub.2, 25-hydroxyvitamin D.sub.3,
25-hydroxyvitamin D.sub.4, 25-hydroxyvitamin D.sub.5,
1,25-hydroxyvitamin D.sub.3, and any combination thereof.
49. The method of claim 40, wherein said one or more vitamin D
molecules are selected from the group consisting of
25-hydroxyvitamin D.sub.2 and 25-hydroxyvitamin D.sub.3.
50. The method of claim 40, wherein said one or more vitamin D
molecules are 25-hydroxyvitamin D.sub.2 and 25-hydroxyvitamin
D.sub.3.
51. The method of claim 40, wherein said one or more vitamin D
molecules consist of 25-hydroxyvitamin D.sub.2.
52. The method of claim 40, wherein said one or more vitamin D
molecules consist of 25-hydroxyvitamin D.sub.3.
53. The method of claim 40, wherein said test device is a lateral
flow test device.
54. The method of claim 40, wherein said quantifying comprises
quantifying a concentration of about 3 ng/mL to about 105 ng/mL of
said one or more vitamin D molecules in said biological sample.
55. The method of claim 40, further comprising detecting levels of
said one or more vitamin D molecules in said biological sample by
visualizing a signal appearing in said test device.
56. The method of claim 55, wherein said quantifying comprises
using an imaging device to provide an image of a detection membrane
on said test device.
57. The method of claim 56, wherein said quantifying further
comprises using software on a programmed computer configured to
quantify said one or more vitamin D molecules in said biological
sample based on said image of said detection membrane.
58. The method of claim 40, wherein said vitamin D binding agent is
conjugated to a detection reagent.
59. The method of claim 58, wherein said detection reagent is
selected from the group consisting of: a gold particle, a latex
particle, a carbon nanoparticle, a selenium nanoparticle, a silver
nanoparticle, a quantum dot, a fluorescent compound, a dye, an
enzyme, and a liposome.
Description
CROSS-REFERENCE
[0001] This application is a continuation application of U.S.
patent application Ser. No. 16/041,582, filed Jul. 20, 2018, which
is a continuation application of International Patent Application
No. PCT/US2017/014620, filed Jan. 23, 2017, which claims the
benefit of U.S. Provisional Application No. 62/286,297, filed Jan.
22, 2016, each of which is incorporated by reference in its
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy is named
49821-701.301.txt and is 22,877 bytes in size.
BACKGROUND OF THE INVENTION
[0003] Vitamin D refers to group of steroid hormones responsible
for enhancing intestinal absorption of calcium and the regulation
of its homeostasis. Two common forms of vitamin D are vitamin
D.sub.2 and vitamin D.sub.3. Vitamin D.sub.3 is naturally produced
in the human skin through the exposure to ultraviolet light,
whereas Vitamin D.sub.2 is mainly obtained from foods and
supplements. Vitamin D.sub.2 and vitamin D.sub.3 are biologically
inactive; activation requires the transport of vitamin D.sub.2 and
vitamin D.sub.3 to the liver where they can be metabolized by
hydroxylation to an active form, referred to herein as 25-hydroxy
vitamin D ("25-(OH)D"). Vitamin D binding protein DBP is the
predominant serum transport protein for all vitamin D metabolites.
DBP transports 95-99% of the total 25-OHD with only 1-5% carried by
albumin and lipoproteins. Vitamin D deficiency has been linked to
many diseases including osteoporosis, osteopenia, rickets, cancers,
autoimmune diseases, cardiovascular diseases, and infectious
diseases, and it is also associated with increased risk of
mortality.
[0004] Unfortunately, vitamin D deficiency is a significant
worldwide health concern and has become a global epidemic. An
estimated 1 billion people globally do not have adequate vitamin D
levels. Furthermore, it is estimated that 64% of Americans do not
have enough vitamin D sub-optimal vitamin D levels. The major cause
of vitamin D deficiency is the lack of moderate sun exposure, which
is the major source of vitamin D for most humans.
[0005] A 25-(OH)D blood test can be used to determine the
circulating vitamin D concentration in a subject. The blood
concentration of 25-(OH)D, including 25-OH (D.sub.2) and
25-OH(D.sub.3), is considered the best indicator of vitamin D
status. The past decade has seen a world-wide increase in demand
for the analysis of 25-(OH)D levels in patient populations.
[0006] Vitamin D is a challenging analyte to measure accurately,
due to its highly lipophilic nature and high-affinity binding to
vitamin D binding protein DBP. Current measurements are largely
performed in specialist laboratories employing time-consuming
methods, such as liquid chromatography-tandem mass spectrometry
(LC-MS/MS) assay, radioimmunoassay (RIAs), enzyme-linked
competitive immunosorbent assays (ELISAs), and competitive
protein-binding assays (CPBA).
[0007] There exists a need for improved methods to detect and
quantify vitamin D metabolites. Provided herein are methods,
compositions, and devices to detect and quantify vitamin D
metabolites by a simplified, rapid and accurate assay.
SUMMARY OF THE INVENTION
[0008] There exists a considerable need for alternative devices,
methods, and kits for detecting and measuring one or more vitamin D
molecules. The present invention addresses this need and provides
additional advantages. In one aspect, the present invention
provides for a test device for detecting one or more vitamin D
molecules comprising: (a) a housing, contained therein: (i) a
sample application pad configured to absorb a biological sample and
transport the biological sample to a conjugate pad; (ii) the
conjugate pad comprising a vitamin D binding agent that
specifically binds to one or more vitamin D molecules; and (iii) a
detection zone comprising a first region immobilized therein a
detection antibody that specifically binds an epitope that is
generated by complexing the vitamin D binding agent with the one or
more vitamin D molecules.
[0009] In some embodiments provided herein, the one or more vitamin
D molecules are selected from the group consisting of
25-hydroxyvitamin D.sub.2, 25-hydroxyvitamin D.sub.3,
25-hydroxyvitamin D.sub.4, 25-hydroxyvitamin D.sub.5, and
1,25-hydroxyvitamin D.sub.3. In some embodiments provided herein,
the one or more vitamin D molecules are selected from the group
consisting of 25-hydroxyvitamin D.sub.2 and 25-hydroxyvitamin
D.sub.3. In some embodiments provided herein, the one or more
vitamin D molecules are 25-hydroxyvitamin D.sub.2 and
25-hydroxyvitamin D.sub.3. In some embodiments provided herein, the
one or more vitamin D molecules consist of 25-hydroxyvitamin
D.sub.2. In some embodiments provided herein, the one or more
vitamin D molecules consist of 25-hydroxyvitamin D.sub.3.
[0010] In some embodiments provided herein, the detection zone
further comprises a second region immobilized therein a third
antibody that is capable of binding to the vitamin D binding agent
whether or not the vitamin D binding agent is bound to the one or
more vitamin D molecules. In some embodiments provided herein, the
vitamin D binding agent is conjugated to a detection reagent. In
some embodiments provided herein, the detection reagent is selected
from the group consisting of gold particle, latex particle, carbon
nanoparticle, selenium nanoparticle, silver nanoparticle, quantum
dot, fluorescent compound, dye, enzyme and liposome. In some
embodiments provided herein, the detection reagent is the gold
particle. In some embodiments provided herein, the detection
reagent is the latex particle.
[0011] In some embodiments provided herein, the sample application
pad, the conjugate pad, and the detection zone are aligned from
upstream to downstream along a fluid path through which the
biological sample travels. In some embodiments provided herein, the
test device further comprises a filtering component between the
sample application pad and the conjugate pad configured to separate
particulate portion of the biological sample from aqueous portion
of the biological sample.
[0012] In one aspect, the present invention provides a method,
comprising: applying a biological sample to the sample application
pad of any one of the test devices disclosed herein; applying a
chase buffer to the sample application pad; and detecting the one
or more vitamin D molecules. In some embodiments provided herein,
the method further comprises quantifying the one or more vitamin D
molecules in the sample. In some embodiments provided herein, the
quantifying classifies the blood sample as having a sufficient, an
insufficient, or a deficient level of the one or more vitamin D
molecules. In some embodiments provided herein, the sufficient
level is at least 30 ng/mL. In some embodiments provided herein,
the insufficient level is at least 10 ng/mL and less than 30 ng/mL.
In some embodiments provided herein, the deficient level is less
than 10 ng/mL. In some embodiments provided herein, the quantifying
further comprises using an imaging device to provide an image of
the detection membrane and software on a programmed computer
configured to quantify the one or more vitamin D molecules in the
sample based on the image of the detection membrane. In some
embodiments provided herein, the imaging device and the programmed
computer are a single device.
[0013] In some embodiments provided herein, the chase buffer
comprises reagents to dissociate the one or more vitamin D
molecules from vitamin D binding protein. In some embodiments
provided herein, the biological sample is selected from the group
consisting of whole blood, serum, plasma, urine, saliva, ocular
fluid, spinal fluid, and perspiration.
[0014] In some embodiments, the detection antibody comprises a
light chain and a heavy chain, wherein the light chain comprises a
sequence selected from the group consisting of SEQ ID NOs 1-5 and
16-20; and wherein the heavy chain comprises a sequence selected
from the group consisting of SEQ ID NOs 5-15. In some embodiments,
the detection antibody exhibits a higher binding affinity to the
immunocomplex than the vitamin D binding agent. In some
embodiments, the detection antibody comprises a scFv, VH, Fab, or
(Fab)2 binding unit.
[0015] In some embodiments, the detection antibody comprises a
light chain and a heavy chain, wherein the light chain a sequence
sharing at least 80% sequence homology to a sequence selected from
the group consisting of SEQ ID NOs 1-5, and 16-20, and wherein the
heavy chain comprises a sequence having least 80% sequence homology
to a sequence selected from the group consisting of SEQ ID NOs
6-15.
[0016] In yet another aspect, the present disclosure provides for a
method for detecting one or more vitamin D levels in a biological
sample with a test device configured to perform a binding assay,
comprising: (a) contacting the biological sample to the test
device; (b) subjecting the biological sample to the binding assay
that utilizes a reaction mixture comprising a complex formed by (1)
a vitamin D binding agent and (2) one or more vitamin D molecules;
(c) further exposing the complex to a detection agent that binds to
an epitope that is formed by complexing the vitamin D binding agent
with the one or more vitamin D binding molecules, wherein the
binding assay has a sensitivity of detection in the biological
sample that is comparable to that of liquid chromatography-tandem
mass spectrometry. In some embodiments, the biological sample is
selected from the group consisting of whole blood, serum, plasma,
urine, saliva, ocular fluid, spinal fluid, and perspiration.
[0017] In some embodiments the detection agent comprises a light
chain and a heavy chain, wherein the light chain comprises a
sequence selected from the group consisting of SEQ ID NOs 1-5 and
16-20, and wherein the heavy chain comprises a sequence selected
from the group consisting of SEQ ID NOs 6-15. In some embodiments,
the detection agent exhibits a higher binding affinity to the
complex than the vitamin D binding agent. In some embodiments, the
detection agent comprises a scFv, VH, Fab, or (Fab)2 binding
unit.
[0018] In some embodiments, the detection agent comprises a light
chain and a heavy chain, wherein the light chain comprises a
sequence sharing at least 80% sequence homology to a sequence
selected from the group consisting of SEQ ID NOs 1-5, and 16-20,
and wherein the heavy chain comprises a sequence having least 80%
sequence homology to a sequence selected from the group consisting
of SEQ ID NOs 6-15.
[0019] In some embodiments, the biological sample is selected from
the group consisting of whole blood, serum, plasma, urine, saliva,
ocular fluid, spinal fluid, and perspiration.
[0020] In one aspect, the present invention provides a kit,
comprising: any one of the test device disclosed herein; and
written instructions for use of the kit. In some embodiments
provided herein, the kit further comprises one or more components
selected from the group consisting of a sterilization agent; a
device to puncture skin; gauze; chase buffer; and a micropipette.
In some embodiments provided herein, the sterilization agent is an
alcohol wipe. In some embodiments provided herein, the device to
puncture skin is selected from the group consisting of a lancet, a
needle, and a syringe.
[0021] In some embodiments provided herein, the chase buffer is
configured to dissociate one or more vitamin D molecules from
vitamin D binding protein in a blood sample. In some embodiments
provided herein, the kit further comprises a color chart relating
signal strength to a quantity of the one or more vitamin D
molecules in the sample.
INCORPORATION BY REFERENCE
[0022] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0024] FIGS. 1A-1B provide an exemplary lateral flow device for
detecting vitamin D metabolite levels. FIG. 1A displays band
densities related to the level of vitamin D metabolites in the
subject. FIG. 1B displays a color chart relating the band density
to a quantitative range of vitamin metabolite concentration in the
blood sample. FIG. 1C provides a schematic of an exemplary lateral
flow device for detecting vitamin D.
[0025] FIG. 2 illustrates the results of an enzyme-linked
immunosorbent assay (ELISA) to test the binding of mouse antibody
clones to the 25-(OH)D.sub.3:AF10 antibody immunocomplex.
[0026] FIG. 3 illustrates the results of an ELISA testing the
binding of synthetic antibody clones to the immunocomplex of
25-(OH)D.sub.3:AF10 antibody.
[0027] FIG. 4. illustrates a linear relationship between serum
vitamin D levels and the signal of a sandwich ELISA using an
antibody that specifically binds the immunocomplex of 25-(OH)D:AF10
antibody.
[0028] FIG. 5 displays the results of using an exemplary lateral
flow device to test 25-(OH)D levels in subject blood and serum,
showing a linear correlation between signal strength and 25-(OH)D
levels ranging from 10 ng/ml to 80 ng/ml.
[0029] FIG. 6 illustrates the results of a comparison between
vitamin D levels in as measured using (i) an exemplary lateral flow
device to measure vitamin D levels of capillary blood as measured
by a smartphone-based reader and (ii) an ELISA-based serum and or
blood test.
[0030] FIG. 7 shows an exemplary computer system that can be used
for the analysis of images of the detection zone of the test device
disclosed herein.
[0031] FIG. 8 shows an exemplary vitamin D measurement procedure
using a portable lateral flow assay reader.
[0032] FIG. 9 shows a correlation between test results generated
from a lateral flow assay and a liquid chromatography-tandem mass
spectrometry assay.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The systems and methods of this disclosure as described
herein may employ, unless otherwise indicated, conventional
techniques and descriptions of molecular biology (including
recombinant techniques), cell biology, biochemistry, microarray and
sequencing technology, which are within the skill of those who
practice in the art. Such conventional techniques include polymer
array synthesis, hybridization and ligation of oligonucleotides,
sequencing of oligonucleotides, and detection of hybridization
using a label. Specific illustrations of suitable techniques can be
had by reference to the examples herein. However, equivalent
conventional procedures can, of course, also be used. Such
conventional techniques and descriptions can be found in standard
laboratory manuals such as Green, et al., Eds., Genome Analysis: A
Laboratory Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds.,
Genetic Variation: A Laboratory Manual (2007); Dieffenbach,
Dveksler, Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and
Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003);
Mount, Bioinformatics: Sequence and Genome Analysis (2004);
Sambrook and Russell, Condensed Protocols from Molecular Cloning: A
Laboratory Manual (2006); and Sambrook and Russell, Molecular
Cloning: A Laboratory Manual (2002) (all from Cold Spring Harbor
Laboratory Press); Stryer, L., Biochemistry (4th Ed.) W.H. Freeman,
N.Y. (1995); Gait, "Oligonucleotide Synthesis: A Practical
Approach" IRL Press, London (1984); Nelson and Cox, Lehninger,
Principles of Biochemistry, 3rd Ed., W.H. Freeman Pub., New York
(2000); and Berg et al., Biochemistry, 5th Ed., W.H. Freeman Pub.,
New York (2002), all of which are herein incorporated by reference
in their entirety for all purposes.
[0034] It is to be understood that this disclosure is not limited
to the specific systems and methods, compositions, targets and uses
described, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular aspects only and is not intended to limit the
scope of the present disclosure, which will be limited only by
appended claims.
[0035] The term "about" or "approximately" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviation, per the practice in the art.
Alternatively, "about" can mean a range of up to 20%, up to 10%, up
to 5%, or up to 1% of a given value. Alternatively, particularly
with respect to biological systems or processes, the term can mean
within an order of magnitude, preferably within 5-fold, and more
preferably within 2-fold, of a value. Where particular values are
described in the application and claims, unless otherwise stated
the term "about" meaning within an acceptable error range for the
particular value should be assumed.
[0036] The terms "polynucleotide," "nucleic acid," and
"oligonucleotide" are used interchangeably. As used herein, they
generally refer to a polymeric form of nucleotides of any length,
either deoxyribonucleotides or ribonucleotides, or analogs thereof.
Polynucleotides may have any three dimensional structure, and may
perform any function, known or unknown. Non-limiting examples of
polynucleotides are coding or non-coding regions of a gene or gene
fragment, intergenic DNA, loci (locus) defined from linkage
analysis, exons, introns, messenger RNA (mRNA), transfer RNA,
ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA
(shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, cDNA,
recombinant polynucleotides, branched polynucleotides, plasmids,
vectors, isolated DNA of any sequence, isolated RNA of any
sequence, nucleic acid probes, adapters, and primers. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and nucleotide analogs. If present,
modifications to the nucleotide structure may be imparted before or
after assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component.
[0037] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non amino acids.
The terms also encompass an amino acid polymer that has been
modified; for example, disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other
manipulation, such as conjugation with a labeling component. As
used herein the term "amino acid" includes natural and/or unnatural
or synthetic amino acids, including glycine and both the D or L
optical isomers, and amino acid analogs and peptidomimetics.
[0038] A "control" is an alternative subject or sample used in an
experiment for comparison purpose.
[0039] The terms "subject," "individual," and "patient" are used
interchangeably herein to refer to a vertebrate, preferably a
mammal, more preferably a human. Mammals include, but are not
limited to, murines, simians, humans, farm animals, sport animals,
and pets. Tissues, cells, and their progeny of a biological entity
obtained in vivo or cultured in vitro are also encompassed.
[0040] The terms "determining", "measuring", "evaluating",
"assessing," "assaying," "detecting", and "analyzing" can be used
interchangeably herein to refer to any form of measurement, and
include determining if an element is present or not (for example,
detection). These terms can include both quantitative and/or
qualitative determinations. Assessing may be relative or absolute.
"Detecting the presence of" can include determining the amount of
something present, as well as determining whether it is present or
absent.
[0041] "Antibody" is an immunoglobulin, or derivative or fragment
or active fragment thereof, having an area on the surface or in a
cavity which specifically binds to and is thereby defined as
complementary with a particular spatial and polar organization of
another molecule. The antibody can be monoclonal or polyclonal and
can be prepared by techniques that are well known in the art such
as, for example, immunization of a host and collection of sera or
hybrid cell line technology.
Test Devices
[0042] In one aspect, the present disclosure provides for a test
device for detecting one or more vitamin D molecules, comprising
(a) a housing, contained therein: (i) a sample application unit
configured to absorb a biological sample and transport said
biological sample to a conjugate pad; (ii) said conjugate pad
comprising a first antibody that specifically binds to one or more
vitamin D molecules; (iii) a detection zone comprising a first
region immobilized therein a second antibody that specifically
binds to an immunocomplex, said immunocomplex comprising (a) said
first antibody and (b) said one or more vitamin D molecules, or an
epitope that is generated by complexing said first antibody and
said one or more vitamin D molecules.
[0043] "One or more vitamin D molecules" refers to members of the
group of fat-soluble secosteriods responsible for enhancing
intestinal absorption of calcium, iron, magnesium, phosphate, and
zinc. Exemplary members of this group are vitamin D.sub.1, vitamin
D.sub.2, vitamin D.sub.3, vitamin D.sub.4, and hydroxylated
versions thereof. Exemplary hydroxylated vitamin D molecules are
25-hydroxyvitamin D.sub.2 (25-OHD.sub.2 or 25-(OH)D.sub.2) and
25-hydroxyvitamin D.sub.3 (25-OHD.sub.3 or 25-(OH)D.sub.3). One or
more vitamin D molecules can include vitamin D compounds with an
additional hydroxy group attached at the 1-.alpha. position, such
as 1,25-hydroxyvitamin D.sub.3 (1,25-OHD.sub.3, or
1,25-(OH)D.sub.3).
[0044] The test device can be a lateral flow test device. A lateral
flow device can comprise a housing, enclosed therein a test strip.
A test strip can comprise a sample application unit, a conjugate
pad, and/or a detection unit. A test strip can include one or more
materials. If a test strip comprises more than one material, the
one or more materials are preferably in fluid communication. One
material of a test strip may be overlaid on another material of the
test strip, such as for example, filter paper overlaid on
nitrocellulose. Alternatively or in addition, a test strip may
include a region comprising one or more materials followed by a
region comprising one or more different materials. In this case,
the regions are in fluid communication and may or may not partially
overlap one another. Suitable materials for test strips include,
but are not limited to, materials derived from cellulose, such as
filter paper, chromatographic paper, nitrocellulose, and cellulose
acetate, as well as materials made of glass fibers, nylon,
polyethylene terephthalate, polyvinyl chloride, polyacrylamide,
cross-linked dextran, agarose, polyacrylate, ceramic materials, and
the like. The material or materials of the test strip may
optionally be treated to modify their capillary flow
characteristics or the characteristics of the applied sample. For
example, the sample application region of the test strip may be
treated with buffers to correct the pH or specific gravity of an
applied urine sample, to ensure optimal test conditions.
[0045] The test strip material or materials can be a single
structure such as a sheet cut into strips or it can be several
strips or particulate material bound to a support or solid surface
such as found, for example, in thin-layer chromatography and may
have an absorbent pad either as an integral part or in liquid
contact. The material can also be a sheet having lanes thereon,
capable of spotting to induce lane formation, wherein a separate
assay can be conducted in each lane. The material can have a
rectangular, circular, oval, triagonal or other shape provided that
there is at least one direction of traversal of a test solution by
capillary migration. Other directions of traversal may occur such
as in an oval or circular piece contacted in the center with the
test solution. However, the main consideration is that there be at
least one direction of flow to a predetermined site. In the
following discussion strips will be described by way of
illustration and not limitation.
[0046] The support for the test strip, where a support is desired
or necessary, will generally be water insoluble, frequently
non-porous and rigid but may be elastic, usually hydrophobic, and
porous and usually will be of the same length and width as the
strip but may be larger or smaller. The support material can be
transparent, and, when a test device of the present invention is
assembled, a transparent support material can be on the side of the
test strip that can be viewed by the user, such that the
transparent support material forms a protective layer over the test
strip where it may be exposed to the external environment, such as
by an aperture in the front of a test device. A wide variety of
non-mobilizable and non-mobilizable materials, both natural and
synthetic, and combinations thereof, may be employed provided only
that the support does not interfere with the capillary action of
the material or materials, or non-specifically bind assay
components, or interfere with the signal producing system.
Illustrative polymers include polyethylene, polypropylene,
poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene
terephthalate), nylon, poly(vinyl butyrate), glass, ceramics,
metals, and the like. Elastic supports may be made of polyurethane,
neoprene, latex, silicone rubber and the like.
[0047] The test device can comprise a sample application aperture
leading to the sample application unit. In some cases, "sample
application aperture" can refer to the portion of a test device or
test strip where where an opening in the test device provides
access to the sample application unit of the test strip. In one
embodiment of the present invention, a sample application aperture
is created by an open-ended channel at the proximal end of the test
device. Preferably, a test strip is engaged in the open-ended
channel such that sample contacted with the sample application
aperture is thereby applied to the test strip. In an alternate
embodiment, a sample application aperture is formed by an opening
in the front of a test device, such that the sample application
unit of the test strip is in fluid communication with the exterior
of the test device.
[0048] "Sample application unit" can be the portion of a test strip
where sample can be applied. The sample application zone of a test
strip of the present invention preferably occurs at the sample
application aperture of a test device of the present invention, and
is in fluid communication with the sample application aperture. In
some cases, the sample application unit comprises a filtering
component configured to remove particulate portions of a biological
fluid, leaving only the aqueous component. Components for filtering
out blood cells, for example, are described in WO2003014726 and
WO2009069017, which are hereby incorporated by reference in their
entirety.
[0049] "Conjugate pad" refers to a region of a test strip where
reagent is provided, which can be referred to as a reagent zone.
The conjugate pad can be a separate segment of bibulous or
non-bibulous material included on the test strip, or it can be a
region of a bibulous or non-bibulous material of a test strip that
also includes other zones, such as an analyte detection zone. The
reagent zone can carry a detection reagent, which may be a direct
or indirect label. Preferably the detection reagent is provided in
a form that is immobile in the dry state and mobile in the moist
state. A reagent can be a specific binding member (e.g., an
antibody), an analyte or analyte analog, an enzyme, a substrate,
indicators, components of a signal producing system, chemicals or
compounds such as buffering agents, reducing agents, chelators,
surfactants, etc., that contribute to the function of the test
strip assay.
[0050] In some cases, a label may be any molecule attached to a
specific binding member that can produce a detectable signal. In
the present invention, the label may be inert and provide a signal
by concentrating in the detection zone, or it may serve solely as a
binding site for a member of the signal producing system, or it may
spontaneously produce a detectable signal or may produce a
detectable signal in conjunction with a signal producing system. A
label can be selected from the group consisting of gold particle,
latex particle, carbon nanoparticles, selenium nanoparticles,
silver nanoparticles, quantum dots, fluorescent compound, textile
dyes, enzymes, and liposomes. The label can be gold particle. The
label can be latex particle.
[0051] "Specific binding member" is one of two different molecules
having an area on the surface or in a cavity which specifically
binds to and is thereby defined as complementary with a particular
spatial and polar organization of the other molecule. Specific
binding members can be members of an immunological pair such as
antigen-antibody, although other specific binding pairs such as
ligand-carrier protein, biotin-avidin, hormone-hormone receptor,
nucleic acid duplexes, IgG-protein A, DNA-DNA, DNA-RNA, and the
like are not immunological pairs but are included in the
definition. A specific binding member can be a binding agent.
[0052] A binding agent can be a molecule that complementarily binds
one or more molecules. A binding agent can be a protein, a nucleic
acid, a ligand, a receptor, or the like.
[0053] Exemplary proteins that are binding agents can include
hemagglutinins, small molecule binding proteins, active or inactive
enzymes or fragmented antibodies. Hemagglutinins can comprise
antibodies or lectins. Antibodies can be of one or more classes of
immunoglobulins (Ig). For instance, an antibody can be an IgA, IgD,
IgE, IgG1, IgG2, IgG3, IgG4, IgM, IgW, or a modified variation
thereof. Antibodies can be monoclonal or polyclonal. An antibody
can be a humanized antibody, a chimeric antibody, or a bispecific
antibody. Antibodies can comprise various types of binding regions.
For instance, an antibody can have scFv, VH, Fab, or (Fab)2 binding
regions. An antibody can comprise various modifications. For
example, an antibody can be modified with one or more detection
reagents. An antibody may be conjugated to another binding agent,
oligonucleotide or protein.
[0054] A binding agent can be a fusion protein that incorporates
various combinations of protein subunits. The combinations of
protein subunits can be non-naturally occurring or naturally
occurring. As a non-limiting example, a fusion protein can
incorporate aspects of an antibody (e.g., an Fc region) and one or
more receptor domains. A fusion protein can comprise two or more
subunits. For example, a fusion protein can be a dimer, trimer,
tetramer, or pentamer.
[0055] In one aspect of the present invention, one or more binding
agents can be used to detect one or more vitamin D molecules. In an
embodiment, a vitamin D binding agent specifically binds to one or
more vitamin D molecules. A vitamin D binding agent can be a
specific binding member. A vitamin D binding agent can be a
specific binding member that is capable of binding to one or more
vitamin D molecules. A vitamin D binding agent can be a first
antibody, as referred to herein.
[0056] A detection agent can be used and may bind to a complex of
the vitamin D binding agent and one or more vitamin D molecules. A
detection agent can be used to detect one or more vitamin D
molecules complexed with a vitamin D binding agent or a first
antibody. The detection agent can be a detection antibody that
specifically binds to a first complex of vitamin D binding agent
and a vitamin D molecule. A detection agent can be a second
antibody or a detection antibody, as referred to herein.
[0057] A third binding agent can be used that is capable of binding
to a vitamin D binding agent whether or not the vitamin D binding
agent is bound to the one or more vitamin D molecules.
[0058] A binding agent may comprise any of the amino acid sequences
listed in Table 1 (SEQ ID Nos 1-20), or a combination thereof.
[0059] In some embodiments, a conjugate pad comprises a detection
reagent, wherein the detection reagent is a first antibody that
specifically binds to one or more vitamin D molecules. Vitamin D
antibodies are known in the art. An exemplary vitamin D antibody,
AF10, was produced as described in Example 1. Other antibodies
capable of binding one or more vitamin D molecules are known in the
art, such as those described in US20130059825, which is hereby
incorporated by reference in its entirety.
[0060] A "detection zone" is a region of the test strip in which a
dye as described above can be observed to shift location, appear,
change color, or optionally to disappear. Detection or observation
of the detection zone can be performed by any convenient means,
depending upon the choice of detectable label. For example,
detection or observation can be performed visually, fluorescently,
by reflectance, radiographically, or any other means known the one
of ordinary skill in the art.
[0061] A detection zone can comprise a first region, immobilized
therein a detection agent or second antibody that specifically
binds to an immunocomplex comprising (a) the first antibody (e.g.,
AF10) that specifically binds one or more vitamin D molecules and
(b) one or more vitamin D molecules. In some cases, the detection
agent or second antibody binds an epitope created when the complex
of the vitamin D binding agent or first antibody and one or more
vitamin D molecules is formed. For example, a second antibody can
bind the junction between the one or more vitamin D molecules and
the first antibody. In some cases, the binding of a vitamin D
binding agent or first antibody to the one or more vitamin D
molecules can cause the formation of an epitope on the vitamin D
binding agent or the first antibody that is recognized by the
detection agent or second antibody. In some cases the epitope
formed upon the binding of one or more vitamin D molecules is not
the binding site of the one or more vitamin D molecules, but an
epitope at another site of the first antibody. In some cases, the
second antibody can recognize the one or more vitamin D molecules
when bound to the first antibody. The first region of the detection
zone can provide a detectable signal indicating the presence of the
analyte. The first region of the detection zone can include an
immobilized binding reagent specific for an analyte ("specific
binding member"), and/or an enzyme that reacts with the analyte.
Other substances that may allow or enhance detection of the
analyte, such as substrates, buffers, salts, may also be provided
in the detection zone. One or more members of a signal producing
system may be bound directly or indirectly to the detection zone. A
detection zone can optionally include a second region comprising
one or more control zones that provide indication that the test has
been performed properly.
[0062] In some instances, the detection zone can further comprise a
second region. This region can be a control region, immobilized
therein a specific binding member that binds the first antibody
whether or not it is bound to one or more vitamin D molecules. For
example, the specific binding member can be a third antibody can
bind the constant region of the first antibody. The third antibody
can bind an epitope on the first antibody that is unchanged whether
the one or more vitamin D molecules are bound or not. In some
cases, the second region can be a control region, and the specific
binding member immobilized therein can be protein A. In some cases,
the conjugate pad comprises an additional antibody derived from a
different species than the first antibody, wherein the additional
antibody does not bind the first antibody, vitamin D, or vitamin D
binding protein. In some cases, the second region is a control
region, and the third antibody recognizes the additional antibody,
but does not recognize the first antibody or second antibody. For
example, if the first and second antibody are derived from mouse
and rat, respectively, the additional antibody can be derived from
chicken, and the third antibody can be goat anti-chicken, rat
anti-chicken, etc.
[0063] Signal appearing in the second region can be used to
indicate that the first antibody can be detected and that the test
strip is functioning properly. The second region can be a region of
a test strip in which a detectable label can be observed to shift
location, appear, change color, or optionally to disappear.
Detection or observation of the second region may be done by any
convenient means, depending upon the particular choice of dye,
especially, for example but not limited to, visually,
fluorescently, by reflectance, radiographically, by a lateral flow
reader and the like.
[0064] A biological sample is any material to be tested for the
presence or amount of an analyte. Preferably, a biological sample
is a fluid sample, preferably a liquid sample. Examples of liquid
samples that may be tested using a test device of the present
invention include bodily fluids including blood, serum, plasma,
saliva, urine, ocular fluid, semen, perspiration, and spinal fluid.
The biological sample can be blood or plasma. Viscous liquid,
semi-solid, or solid specimens may be used to create liquid
solutions, eluates, suspensions, or extracts that can be samples.
For example, throat or genital swabs may be suspended in a liquid
solution to make a sample.
[0065] An exemplary lateral flow assay is provided in FIG. 1C. The
exemplary lateral flow assay comprises a sample application pad,
conjugate pad, and said detection zone aligned from upstream to
downstream along a fluid path along which said biological sample
travels. A sample may be applied to the sample application pad or
sample pad. Application of a sample may be followed by a chase
buffer. From the blood sample, vitamin D (e.g., 25-(OH)D) may flow
to a conjugate pad comprising a first antibody (Mab-1) that
specifically binds to one or more vitamin D molecules to generate
an immunocomplex. A first antibody may be conjugated with a
detection reagent, such as colloidal gold. Immunocomplexed and
uncomplexed antibody may flow to the detection zone. A detection
zone may comprise a control line and a test line. The test line may
comprise a second antibody (Mab-2) that specifically binds to an
epitope generated by complexing the Mab-1 with one or more 25-(OH)D
molecules. Immunocomplexed and non-complexed Mab-1 may laterally
flow to a control line comprising a third antibody that is capable
of binding to Mab-1 whether or not Mab-1 is bound to one or more
vitamin D molecules (Anti-IgG antibody). The uncomplexed and/or
immunocomplexed Mab-1 antibody can be detected at the control line.
The Mab-2 bound immunocomplexed Mab-1 antibody can be detected at
the test line. A level of detection at the test line can be
indicative of a level of vitamin D.
Methods
[0066] In one aspect, the present disclosure provides methods
capable of detecting levels of one or more vitamin D molecules
using test devices disclosed herein. The methods can comprise
applying a biological sample to the sample application unit of a
test device disclosed herein; applying a chase buffer to the sample
application unit, and detecting levels of the one or more vitamin D
molecules. Such detection can occur by visualizing signal appearing
in the second region of the test device, such as by concentration
of a labeled first antibody in a detection zone by a specific
binding agent.
[0067] In some cases, methods further comprise quantifying the one
or more vitamin D molecules in the sample. Quantification can be
performed by determining a relationship between signal strength in
the first region of the detection zone and the amount of analyte
present in biological sample. It will be appreciated that the
relationship between signal strength and the amount of analyte
present in the biological sample may be determined for each batch
or each test strip by the application of standards containing known
amounts of one or more vitamin D molecules.
[0068] In some instances, the quantification will be
semi-quantitative, such as by classifying a subject as having
sufficient levels, insufficient levels, or deficient levels of one
or more vitamin D molecules in the biological sample. Sufficient
levels of vitamin D can be at least 10 ng/mL, at least 15 ng/mL, at
least 20 ng/mL, at least 25 ng/mL, at least 30 ng/mL, at least 35
ng/mL, at least 40 ng/mL, or at least 50 ng/mL. Insufficient levels
of vitamin D can be at least 5 ng/mL and at most 45 ng/mL, at least
5 ng/mL and at most 40 ng/mL, at least 5 ng/mL and at most 35
ng/mL, at least 5 ng/mL and at most 30 ng/mL, at least 5 ng/mL and
at most 25 ng/mL, at least 5 ng/mL and at most 20 ng/mL, at least 5
ng/mL and at most 15 ng/mL, at least 5 ng/mL and at most 10 ng/mL,
at least 10 ng/mL and at most 45 ng/mL, at least 10 ng/mL and at
most 40 ng/mL, at least 10 ng/mL and at most 35 ng/mL, at least 10
ng/mL and at most 30 ng/mL, at least 10 ng/mL and at most 25 ng/mL,
at least 10 ng/mL and at most 20 ng/mL, at least 10 ng/mL and at
most 15 ng/mL, at least 15 ng/mL and at most 45 ng/mL, at least 15
ng/mL and at most 40 ng/mL, at least 15 ng/mL and at most 35 ng/mL,
at least 15 ng/mL and at most 30 ng/mL, at least 15 ng/mL and at
most 25 ng/mL, at least 15 ng/mL and at most 20 ng/mL, at least 20
ng/mL and at most 45 ng/mL, at least 20 ng/mL and at most 40 ng/mL,
at least 20 ng/mL and at most 35 ng/mL, at least 20 ng/mL and at
most 30 ng/mL, at least 20 ng/mL and at most 25 ng/mL, at least 25
ng/mL and at most 45 ng/mL, at least 25 ng/mL and at most 40 ng/mL,
at least 25 ng/mL and at most 35 ng/mL, at least 25 ng/mL and at
most 30 ng/mL, at least 30 ng/mL and at most 45 ng/mL, at least 30
ng/mL and at most 40 ng/mL, at least 30 ng/mL and at most 35 ng/mL,
at least 35 ng/mL and at most 45 ng/mL, at least 35 ng/mL and at
most 40 ng/mL, at least 40 ng/mL and at most 45 ng/mL. Deficient
levels of vitamin D can be at most 10 ng/mL, at most 15 ng/mL, at
most 20 ng/mL, at least 25 ng/mL, at most 30 ng/mL, at most 35
ng/mL, at most 40 ng/mL, or at most 50 ng/mL. For example, the
subject may have greater than 30 ng/mL of one or more vitamin D
molecules and be classified as having sufficient levels of one or
more vitamin D molecules. For example, the subject may have between
10-30 ng/mL of one or more vitamin D molecules and be classified as
having insufficient levels of one or more vitamin D molecules. For
example, the subject may have less than 10 ng/mL of one or more
vitamin D molecules and be classified as having deficient levels of
one or more vitamin D molecules.
[0069] In some instances, quantifying further comprised using an
imaging device to produce an image of the detection zone and
software on a programmed computer configured to quantify the one or
more vitamin D molecules in the biological sample based on the
image of the detection zone. The image can be analyzed for signal
strength. In some cases, signal strength is compared against the
signal strength of the control region to normalize the image for
variables such as light intensity, light quality, and variation
between imaging devices.
[0070] FIG. 7 shows a computer system 701 that is programmed or
otherwise configured to implement methods of the present
disclosure. The computer system 701 can be integral to implementing
methods provided herein, which would be otherwise extremely
difficult to perform in the absence of the computer system 701. The
computer system 701 can regulate various aspects of methods of the
present disclosure, such as, for example, methods that quantify one
or more vitamin D molecules in a biological samples based on an
image of a detection zone. The computer system 701 can be an
electronic device of a user or a computer system that is remotely
located with respect to the electronic device. The electronic
device can be a mobile electronic device. As an alternative, the
computer system 701 can be a computer server.
[0071] The computer system 701 includes a central processing unit
(CPU, also "processor" and "computer processor" herein) 705, which
can be a single core or multi core processor, or a plurality of
processors for parallel processing. The computer system 701 also
includes memory or memory location 710 (e.g., random-access memory,
read-only memory, flash memory), electronic storage unit 715 (e.g.,
hard disk), communication interface 720 (e.g., network adapter) for
communicating with one or more other systems, and peripheral
devices 725, such as cache, other memory, data storage and/or
electronic display adapters. The memory 710, storage unit 715,
interface 720 and peripheral devices 725 are in communication with
the CPU 705 through a communication bus (solid lines), such as a
motherboard. The storage unit 715 can be a data storage unit (or
data repository) for storing data. The computer system 701 can be
operatively coupled to a computer network ("network") 730 with the
aid of the communication interface 720. The network 730 can be the
Internet, an internet and/or extranet, or an intranet and/or
extranet that is in communication with the Internet. The network
730 in some cases is a telecommunication and/or data network. The
network 730 can include one or more computer servers, which can
enable distributed computing, such as cloud computing. The network
730, in some cases with the aid of the computer system 701, can
implement a peer-to-peer network, which may enable devices coupled
to the computer system 701 to behave as a client or a server.
[0072] The CPU 705 can execute a sequence of machine-readable
instructions, which can be embodied in a program or software. The
instructions may be stored in a memory location, such as the memory
710. The instructions can be directed to the CPU 705, which can
subsequently program or otherwise configure the CPU 705 to
implement methods of the present disclosure. Examples of operations
performed by the CPU 705 can include fetch, decode, execute, and
writeback.
[0073] The CPU 705 can be part of a circuit, such as an integrated
circuit. One or more other components of the system 701 can be
included in the circuit. In some cases, the circuit is an
application specific integrated circuit (ASIC).
[0074] The storage unit 715 can store files, such as drivers,
libraries and saved programs. The storage unit 715 can store user
data, e.g., user preferences and user programs. The computer system
701 in some cases can include one or more additional data storage
units that are external to the computer system 701, such as located
on a remote server that is in communication with the computer
system 701 through an intranet or the Internet.
[0075] The computer system 701 can communicate with one or more
remote computer systems through the network 730. For instance, the
computer system 701 can communicate with a remote computer system
of a user (e.g., patient, healthcare provider, or service
provider). Examples of remote computer systems include personal
computers (e.g., portable PC), slate or tablet PC's (e.g.,
Apple.RTM. iPad, Samsung.RTM. Galaxy Tab), telephones, Smart phones
(e.g., Apple.RTM. iPhone, Android-enabled device, Blackberry.RTM.),
or personal digital assistants. The user can access the computer
system 701 via the network 730.
[0076] Methods as described herein can be implemented by way of
machine (e.g., computer processor) executable code stored on an
electronic storage location of the computer system 701, such as,
for example, on the memory 710 or electronic storage unit 715. The
memory 710 can be part of a database. The machine executable or
machine readable code can be provided in the form of software.
During use, the code can be executed by the processor 705. In some
cases, the code can be retrieved from the storage unit 715 and
stored on the memory 710 for ready access by the processor 705. In
some situations, the electronic storage unit 715 can be precluded,
and machine-executable instructions are stored on memory 710.
[0077] The code can be pre-compiled and configured for use with a
machine having a processer adapted to execute the code, or can be
compiled during runtime. The code can be supplied in a programming
language that can be selected to enable the code to execute in a
pre-compiled or as-compiled fashion.
[0078] Aspects of the systems and methods provided herein, such as
the computer system 701, can be embodied in programming. Various
aspects of the technology may be thought of as "products" or
"articles of manufacture" typically in the form of machine (or
processor) executable code and/or associated data that is carried
on or embodied in a type of machine readable medium.
Machine-executable code can be stored on an electronic storage
unit, such as memory (e.g., read-only memory, random-access memory,
flash memory) or a hard disk. "Storage" type media can include any
or all of the tangible memory of the computers, processors or the
like, or associated modules thereof, such as various semiconductor
memories, tape drives, disk drives and the like, which may provide
non-transitory storage at any time for the software programming.
All or portions of the software may at times be communicated
through the Internet or various other telecommunication networks.
Such communications, for example, may enable loading of the
software from one computer or processor into another, for example,
from a management server or host computer into the computer
platform of an application server. Thus, another type of media that
may bear the software elements includes optical, electrical and
electromagnetic waves, such as used across physical interfaces
between local devices, through wired and optical landline networks
and over various air-links. The physical elements that carry such
waves, such as wired or wireless links, optical links or the like,
also may be considered as media bearing the software. As used
herein, unless restricted to non-transitory, tangible "storage"
media, terms such as computer or machine "readable medium" refer to
any medium that participates in providing instructions to a
processor for execution.
[0079] Hence, a machine readable medium, such as
computer-executable code, may take many forms, including but not
limited to, a tangible storage medium, a carrier wave medium or
physical transmission medium. Non-volatile storage media include,
for example, optical or magnetic disks, such as any of the storage
devices in any computer(s) or the like, such as may be used to
implement the databases, etc. shown in the drawings. Volatile
storage media include dynamic memory, such as main memory of such a
computer platform. Tangible transmission media include coaxial
cables; copper wire and fiber optics, including the wires that
comprise a bus within a computer system. Carrier-wave transmission
media may take the form of electric or electromagnetic signals, or
acoustic or light waves such as those generated during radio
frequency (RF) and infrared (IR) data communications. Common forms
of computer-readable media therefore include for example: a floppy
disk, a flexible disk, hard disk, magnetic tape, any other magnetic
medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch
cards paper tape, any other physical storage medium with patterns
of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other
memory chip or cartridge, a carrier wave transporting data or
instructions, cables or links transporting such a carrier wave, or
any other medium from which a computer may read programming code
and/or data. Many of these forms of computer readable media may be
involved in carrying one or more sequences of one or more
instructions to a processor for execution.
[0080] The computer system 701 can include or be in communication
with an electronic display 735 that comprises a user interface (UI)
740 for providing, for example, genetic information, such as an
identification of disease-causing alleles in single individuals or
groups of individuals. Examples of UI's include, without
limitation, a graphical user interface (GUI) and web-based user
interface (or web interface).
[0081] A chase buffer may be used to detect one or more vitamin D
molecules. The chase buffer can be configured comprise reagents to
dissociate the one or more vitamin D molecules from vitamin D
binding protein and/or albumin. Non-limiting examples of reagents
to dissociate the one or more vitamin D molecules are acidic
solution, alkaline solution, 8-anilino-1-napthalenesulfonic acid,
3-(acetonylbenzyl)-4-hydroxycoumarin, alkyl amino fluoro
surfactants, perfluorhexanoic acid, perfluoroctanoic acid,
proteinase K, urea, and guanidine hydrochloride. For example, the
chase buffer can be an acidic solution or an alkaline solution, as
described in WO2004063704, which is hereby incorporated by
reference in its entirety. In another, the chase buffer can rely on
the competitive displacement of Vitamin D from endogenous binding
proteins using 8-anilino-1-napthalenesulfonic acid and/or
3-(acetonylbenzyl)-4-hydroxycoumarin, as described in U.S. Pat. No.
7,482,162, which is hereby incorporated by reference in its
entirety.
[0082] Chase buffer can, for example, be a reagent with a pH from
3.8 to 4.8 and 5-30% DMSO, a liquid organic amide and optionally
0.5-5% of a short chain alcohol, such as described in WO2007039194,
which is hereby incorporated by reference in its entirety. Chase
buffer can comprise stabilizing agents and capture ligands,
including alkyl amino fluoro surfactants as described in
WO2008039266, which is hereby incorporated by reference in its
entirety.
Kits
[0083] In one aspect, the present disclosure provides kits capable
of detecting levels of one or more vitamin D molecules comprising
test devices disclosed herein and written instructions for the use
of the kit. In some instances, kits can further comprise one or
more components selected from the group consisting of a
sterilization agent, a device to puncture skin, gauze, chase
buffer, and a micropipette.
[0084] A sterilization agent can be, for example, an alcohol wipe
capable of disinfecting human skin.
[0085] A device to puncture the skin can be a small, sharp object
capable of penetrating the skin of the subject to produce a small
volume of blood. The device to puncture the skin can be selected
from the group consisting of a lancet, a needle, and a syringe. The
lancet can, for example, be spring-powered.
[0086] Chase buffer can comprise reagents for dissociating one or
more vitamin D molecules from vitamin D binding protein, as
described previously.
[0087] A micropipette can provide a known volume of biological
sample to the device.
[0088] The kit can further comprise a color chart relating signal
strength to a quantity of said one or more vitamin D molecules in a
sample. Due to potential batch-to-batch variability, the color
chart can be generated empirically for each batch of test devices
by running standards containing known amounts of one or more
vitamin D molecules and determining the colors corresponding to
different ranges of amounts of one or more vitamin D molecules in
the biological sample.
EXAMPLES
Example 1: Generation of Monoclonal Antibody to 25-(OH)D
[0089] Monoclonal antibodies against 25-(OH)D were prepared by a
modified method of Kohler and Milstein (G. Kohler and C. Milstein
Nature, 1975, 256, 495). Mice were immunized by subcutaneous
injection of 25-(OH)D conjugated at its 3-position to carrier
protein KHL. The complete Freund's adjuvant was injected with the
antigen. The incomplete Freund's adjuvant was used for antigen
boosts. The immune response was monitored by ELISA against
25-(OH)D.sub.3. After four to five antigen boosts, the spleen cells
were harvested and fused with myeloma cells in the presence of
polyethylene glycol (PEG). The fused cells were seeded in 96-well
plates and grown in the presence of selective hypoxanthine,
aminopterin and thymidine (HAT) medium. The supernatants from the
fused cells were tested by ELISA for binding activity to
25-(OH)D.sub.3 and 25-(OH)D.sub.2. The clone AF10, which has a high
affinity for binding both 25-(OH)D.sub.3 and 25-(OH)D.sub.2, was
selected for large scale of antibody production and
purification.
Example 2: Generation of a Monoclonal Antibody Against an
Immunocomplex of 25-(OH)D and mAb from Immunized Mice
[0090] Mice were immunized by subcutaneous injection of 25-(OH)D
conjugated at its 26-position to carrier protein keyhole limpet
hemocyanin (KLH). The complete Freund's adjuvant was injected with
antigen. KLH conjugate and the incomplete Freund's adjuvant was
used for the first and third boosts, and the immunocomplex of
25-(OH)D:antibody AF10 was used for the second and fourth boosts.
The immune response was monitored by ELISA assay to the
immunocomplex of 25-(OH)D:antibody AF10. After 4 boosts, the spleen
cells were harvested. The RNA from the spleen cells was then
isolated and variable gene amplification was performed.
[0091] The mRNA was isolated by using Dynabeads.RTM. mRNA
Purification Kit (ThermoFisher) according to the manufacturer's
protocol. Subsequently, first strand cDNA was generated using
Superscript II reverse transcriptase (Invitrogen, Carlsbad,
Calif.). From the cDNA, heavy and light chain variable genes were
amplified separately by PCR using primer sets for mouse VH, VK and
VX, as described according to Phage Display: A Laboratory Manual
(Cold Spring Harbor Laboratory Press). Heavy and light chain
variable region genes were further amplified by PCR to add partial
sequence of (G53).sub.4 linker to the 3' VH and 5' VL. In the third
PCR reaction, an equal amount of second round amplified VH and VL
DNA was mixed for single-chain variable fragment (scFv) assembly.
The final scFv DNA was purified with Qiagen PCR purification column
(QIAGEN Inc., Germany). The purified scFv DNA and PCANTAB5 vector
DNA were digested with restriction enzymes NcoI and Not1 (New
England Lab) and then size-selected by 1% agarose gel. Excised
bands were purified with the QIAquick Gel Extraction Kit. Digested
scFv, vector DNA and T4 ligase (New England Lab) were mixed for
overnight ligation at 16.degree. C. 20 .mu.g of ligated DNA was
purified and transformed into E coli TG1 competent cells by
electroporation. After transformation, TG1 cells were suspended
into SOC medium and incubated for 1 h at 37.degree. C. and 250 rpm
shaking. The TG1 cells were plated on 2YT-agar plates containing
100 .mu.g/ml of carbenicillin and 2% glucose. Following overnight
30.degree. C. incubation, the TG1 transformants were harvested into
2YT containing 15% glycerol, 100 .mu.g/ml of carbenicillin and 2%
glucose, and the library aliquots were kept at -80.degree. C. for
storage. This procedure resulted in a library size of
1.2.times.10.sup.9 colonies.
[0092] Phage displayed library was prepared as follows. 200 mL 2YT
medium (with 100 .mu.g/ml of carbenicillin and 2% glucose) was
inoculated with library TG1 cells at starting OD 0.1. After 3-4
hours of incubation at 37.degree. C. with 250 rpm shaking, KO7
helper phage was added at a 1:10 ratio of cells to phage, and the
culture was incubated at 37.degree. C. for 1 hour without sharking.
The TG1 cells were centrifuged and the cell pellet was re-suspended
into 2 L of 2YT with 100 .mu.g/ml carbenicillin and 35 .mu.g/ml
Kanamycin, followed by overnight incubation at 30.degree. C. with
250 rpm sharking. TG1 cells were then centrifuged for 30 minutes at
6000.times.g at 4.degree. C. The library phage particles were
purified by PEG-precipitation from culture supernatant, resuspended
into phosphate-buffered saline (PBS) and titered by OD268
measurement. The phage display antibody library was stored at
-80.degree. C. in PBS with 20% glycerol.
[0093] Immunocomplex-specific antibodies were selected from the
above mouse scFv phage display library by the following selection
procedure. The microtiter well was coated with 1 .mu.g of mAb AF10
overnight at 4.degree. C. After 2 hour blocking with 1.times.
chemiblocker (EMD Millipore), 1 .mu.g of 25-(OH)D.sub.3 was added
to each well for overnight incubation at 4.degree. C. The well was
washed 3 times with PBS and blocked with 1.times. chemiblocker for
1 hour at room temperature. Prior to performing affinity selection
against the immunocomplex of 25-(OH)D:antibody AF10, 400 .mu.l of
the phage library solution (a total of 1,012 phage particles) was
pre-incubated in a well containing antibody AF10 without vitamin D
for 2 hours at room temperature, after which the unbound phages
were transferred into 4 wells with the immunocomplex of
25-(OH)D:antibody AF10 for 2 hour incubation at room temperature.
The unbound phages were removed and the well was washed 10 times
with PBS-Tween. Bound phages were eluted with 100 .mu.l of 100 mM
HCl for 10 minutes, and eluted phages were harvested and
neutralized with 10% 1 M Tris-HCl. The eluted phages were then
added to 10 mL of fresh TG1 cells (OD600.apprxeq.0.8) for 1 hour
incubation at 37.degree. C. Infected TG1 cells were plated on two
2YT plates with carbenicillin and glucose for overnight growth, and
overnight cells were harvested for first round phage preparation as
described above. The affinity selection procedure was performed on
these second round phages as just described. A total of three
rounds of affinity selection were performed.
[0094] The infected TG1 colonies from the third round of affinity
selection were picked for expression of scFv-p3 fusion and
confirmation of binding activity. Briefly, isolated TG colonies
were picked into 96 well plates (5 plates and a total of 480
colonies) with 100 .mu.l of 2YT/carbenicillin and glucose, and
incubated overnight at 30.degree. C. The second day, 10 .mu.l of
culture per well was transferred into a corresponding well in a
96-well deep plate containing 500 .mu.l per well 2YT medium
containing carbenicillin and 0.1% Glucose. The deep-well plates
were incubated in a 37.degree. C. shaker incubator shaking at 250
rpm until the cultures reach OD600 of 0.8-1. 100 .mu.l of 2YT
containing 6 mM IPTG was then added to each well and the expression
plate was incubated overnight at 30.degree. C. shaking at 250 rpm.
160 .mu.L of lysis buffer containing 2.5 mg/mL lysozyme and 5 mM
EDTA were then added to each well of the expression plate, and the
cultures were shaken for 1 h at room temperature. The culture
supernatant was mixed with 140 .mu.l 2.times. ChemiBlocker per well
and incubated for an additional 30 min while shaking at 250 rpm.
The culture supernatants were centrifuged and prepared for binding
assay as below.
[0095] To screen for the clones capable of binding the
immunocomplex of 25-(OH)D:antibody AF10, two 96-well assay plates
were prepared for each expression plate, one plate coated with AF10
antibody only, the other coated with the immunocomplex of
25-(OH)D:antibody AF10. The assay plates were blocked with 1.times.
ChemiBlocker for 2 hours at room temperature. The culture
supernatants from the expression plate were transferred to the
corresponding well of the assay plate. After 1 hour incubation at
room temperature and three PBS-Tween washes, 100 .mu.L of mouse
anti-M13 p3 antibody (New England Lab) was added to each well for 1
hour incubation at room temperature. The samples were washed three
times with PBS-Tween, and 100 .mu.L of horse radish peroxidase
(HRP)-conjugated goat anti-mouse IgG was added to each well for 1
hour incubation. The samples were washed three times with
PBS-Tween, and 100 .mu.L of substrate
3,3',5,5'-Tetramethylbenzidine (TMB) was added to each well. The
OD630 was measured after 10-30 minutes development. The clones that
bound to the immunocomplex of 25-(OH)D:antibody AF10, but not AF10
antibody alone, were selected for confirmation. The positive
culture supernatants were serially diluted in a 1:3 ratio and were
assayed again following the above procedure. FIG. 2 shows six
clones with binding activity to the immunocomplex of
25-(OH)D.sub.3:antibody AF10. The unique sequences of the heavy
chain variable regions (SEQ ID NOs 6-15) and light chain variable
regions (SEQ ID NOs 1-5 and 16-20) are listed in TABLE 1.
[0096] A light chain can comprise a sequence selected from the
group consisting of SEQ ID NOs 1-5, and 16-20. A heavy chain can
comprise a sequence selected from the group consisting of SEQ ID
NOs 6-15. In some instances, the light chain can comprise a
sequence sharing at least 80% sequence homology to a sequence
selected from the group consisting of SEQ ID NOs 1-5, and 16-20. In
some instances, the heavy chain can comprise a sequence having
least 80% sequence homology to a sequence selected from the group
consisting of SEQ ID NOs 6-15.
[0097] An antibody may comprise light chain CDR and said heavy
chain CDR with any of the following amino acid sequences: a heavy
chain of SEQ ID NO 6 and a light chain of SEQ ID NO 1, a heavy
chain of SEQ ID NO 6 and a light chain of SEQ ID NO 2, a heavy
chain of SEQ ID NO 6 and a light chain of SEQ ID NO 3, a heavy
chain of SEQ ID NO 6 and a light chain of SEQ ID NO 4, a heavy
chain of SEQ ID NO 6 and a light chain of SEQ ID NO 5, a heavy
chain of SEQ ID NO 6 and a light chain of SEQ ID NO 16, a heavy
chain of SEQ ID NO 6 and a light chain of SEQ ID NO 17, a heavy
chain of SEQ ID NO 6 and a light chain of SEQ ID NO 18, a heavy
chain of SEQ ID NO 6 and a light chain of SEQ ID NO 19, a heavy
chain of SEQ ID NO 6 and a light chain of SEQ ID NO 20.
[0098] An antibody may comprise light chain CDR and said heavy
chain CDR with any of the following amino acid sequences a heavy
chain of SEQ ID NO 7 and a light chain of SEQ ID NO 1, a heavy
chain of SEQ ID NO 7 and a light chain of SEQ ID NO 2, a heavy
chain of SEQ ID NO 7 and a light chain of SEQ ID NO 3, a heavy
chain of SEQ ID NO 7 and a light chain of SEQ ID NO 4, a heavy
chain of SEQ ID NO 7 and a light chain of SEQ ID NO 5, a heavy
chain of SEQ ID NO 7 and a light chain of SEQ ID NO 16, a heavy
chain of SEQ ID NO 7 and a light chain of SEQ ID NO 17, a heavy
chain of SEQ ID NO 7 and a light chain of SEQ ID NO 18, a heavy
chain of SEQ ID NO 7 and a light chain of SEQ ID NO 19, a heavy
chain of SEQ ID NO 7 and a light chain of SEQ ID NO 20.
[0099] An antibody may comprise light chain CDR and said heavy
chain CDR with any of the following amino acid sequences: a heavy
chain of SEQ ID NO 8 and a light chain of SEQ ID NO 1, a heavy
chain of SEQ ID NO 8 and a light chain of SEQ ID NO 2, a heavy
chain of SEQ ID NO 8 and a light chain of SEQ ID NO 3, a heavy
chain of SEQ ID NO 8 and a light chain of SEQ ID NO 4, a heavy
chain of SEQ ID NO 8 and a light chain of SEQ ID NO 5, a heavy
chain of SEQ ID NO 8 and a light chain of SEQ ID NO 16, a heavy
chain of SEQ ID NO 8 and a light chain of SEQ ID NO 17, a heavy
chain of SEQ ID NO 8 and a light chain of SEQ ID NO 18, a heavy
chain of SEQ ID NO 8 and a light chain of SEQ ID NO 19, a heavy
chain of SEQ ID NO 8 and a light chain of SEQ ID NO 20.
[0100] An antibody may comprise light chain CDR and said heavy
chain CDR with any of the following amino acid sequences: a heavy
chain of SEQ ID NO 9 and a light chain of SEQ ID NO 1, a heavy
chain of SEQ ID NO 9 and a light chain of SEQ ID NO 2, a heavy
chain of SEQ ID NO 9 and a light chain of SEQ ID NO 3, a heavy
chain of SEQ ID NO 9 and a light chain of SEQ ID NO 4, a heavy
chain of SEQ ID NO 9 and a light chain of SEQ ID NO 5, a heavy
chain of SEQ ID NO 9 and a light chain of SEQ ID NO 16, a heavy
chain of SEQ ID NO 9 and a light chain of SEQ ID NO 17, a heavy
chain of SEQ ID NO 9 and a light chain of SEQ ID NO 18, a heavy
chain of SEQ ID NO 9 and a light chain of SEQ ID NO 19, a heavy
chain of SEQ ID NO 9 and a light chain of SEQ ID NO 20.
[0101] An antibody may comprise light chain CDR and said heavy
chain CDR with any of the following amino acid sequences: a heavy
chain of SEQ ID NO 10 and a light chain of SEQ ID NO 1, a heavy
chain of SEQ ID NO 10 and a light chain of SEQ ID NO 2, a heavy
chain of SEQ ID NO 10 and a light chain of SEQ ID NO 3, a heavy
chain of SEQ ID NO 10 and a light chain of SEQ ID NO 4, a heavy
chain of SEQ ID NO 10 and a light chain of SEQ ID NO 5, a heavy
chain of SEQ ID NO 10 and a light chain of SEQ ID NO 16, a heavy
chain of SEQ ID NO 10 and a light chain of SEQ ID NO 17, a heavy
chain of SEQ ID NO 10 and a light chain of SEQ ID NO 18, a heavy
chain of SEQ ID NO 10 and a light chain of SEQ ID NO 19, a heavy
chain of SEQ ID NO 10 and a light chain of SEQ ID NO 20.
[0102] An antibody may comprise light chain CDR and said heavy
chain CDR with any of the following amino acid sequences: a heavy
chain of SEQ ID NO 11 and a light chain of SEQ ID NO 1, a heavy
chain of SEQ ID NO 11 and a light chain of SEQ ID NO 2, a heavy
chain of SEQ ID NO 11 and a light chain of SEQ ID NO 3, a heavy
chain of SEQ ID NO 11 and a light chain of SEQ ID NO 4, a heavy
chain of SEQ ID NO 11 and a light chain of SEQ ID NO 5, a heavy
chain of SEQ ID NO 11 and a light chain of SEQ ID NO 16, a heavy
chain of SEQ ID NO 11 and a light chain of SEQ ID NO 17, a heavy
chain of SEQ ID NO 11 and a light chain of SEQ ID NO 18, a heavy
chain of SEQ ID NO 11 and a light chain of SEQ ID NO 19, a heavy
chain of SEQ ID NO 11 and a light chain of SEQ ID NO 20.
[0103] An antibody may comprise light chain CDR and said heavy
chain CDR with any of the following amino acid sequences: a heavy
chain of SEQ ID NO 12 and a light chain of SEQ ID NO 1, a heavy
chain of SEQ ID NO 12 and a light chain of SEQ ID NO 2, a heavy
chain of SEQ ID NO 12 and a light chain of SEQ ID NO 3, a heavy
chain of SEQ ID NO 12 and a light chain of SEQ ID NO 4, a heavy
chain of SEQ ID NO 12 and a light chain of SEQ ID NO 5, a heavy
chain of SEQ ID NO 12 and a light chain of SEQ ID NO 16, a heavy
chain of SEQ ID NO 12 and a light chain of SEQ ID NO 17, a heavy
chain of SEQ ID NO 12 and a light chain of SEQ ID NO 18, a heavy
chain of SEQ ID NO 12 and a light chain of SEQ ID NO 19, a heavy
chain of SEQ ID NO 12 and a light chain of SEQ ID NO 20.
[0104] An antibody may comprise light chain CDR and said heavy
chain CDR with any of the following amino acid sequences: a heavy
chain of SEQ ID NO 13 and a light chain of SEQ ID NO 1, a heavy
chain of SEQ ID NO 13 and a light chain of SEQ ID NO 2, a heavy
chain of SEQ ID NO 13 and a light chain of SEQ ID NO 3, a heavy
chain of SEQ ID NO 13 and a light chain of SEQ ID NO 4, a heavy
chain of SEQ ID NO 13 and a light chain of SEQ ID NO 5, a heavy
chain of SEQ ID NO 13 and a light chain of SEQ ID NO 16, a heavy
chain of SEQ ID NO 13 and a light chain of SEQ ID NO 17, a heavy
chain of SEQ ID NO 13 and a light chain of SEQ ID NO 18, a heavy
chain of SEQ ID NO 13 and a light chain of SEQ ID NO 19, a heavy
chain of SEQ ID NO 13 and a light chain of SEQ ID NO 20.
[0105] An antibody may comprise light chain CDR and said heavy
chain CDR with any of the following amino acid sequences: a heavy
chain of SEQ ID NO 14 and a light chain of SEQ ID NO 1, a heavy
chain of SEQ ID NO 14 and a light chain of SEQ ID NO 2, a heavy
chain of SEQ ID NO 14 and a light chain of SEQ ID NO 3, a heavy
chain of SEQ ID NO 14 and a light chain of SEQ ID NO 4, a heavy
chain of SEQ ID NO 14 and a light chain of SEQ ID NO 5, a heavy
chain of SEQ ID NO 14 and a light chain of SEQ ID NO 16, a heavy
chain of SEQ ID NO 14 and a light chain of SEQ ID NO 17, a heavy
chain of SEQ ID NO 14 and a light chain of SEQ ID NO 18, a heavy
chain of SEQ ID NO 14 and a light chain of SEQ ID NO 19, a heavy
chain of SEQ ID NO 14 and a light chain of SEQ ID NO 20.
[0106] An antibody may comprise light chain CDR and said heavy
chain CDR with any of the following amino acid sequences: a heavy
chain of SEQ ID NO 15 and a light chain of SEQ ID NO 1, a heavy
chain of SEQ ID NO 15 and a light chain of SEQ ID NO 2, a heavy
chain of SEQ ID NO 15 and a light chain of SEQ ID NO 3, a heavy
chain of SEQ ID NO 15 and a light chain of SEQ ID NO 4, a heavy
chain of SEQ ID NO 15 and a light chain of SEQ ID NO 5, a heavy
chain of SEQ ID NO 15 and a light chain of SEQ ID NO 16, a heavy
chain of SEQ ID NO 15 and a light chain of SEQ ID NO 17, a heavy
chain of SEQ ID NO 15 and a light chain of SEQ ID NO 18, a heavy
chain of SEQ ID NO 15 and a light chain of SEQ ID NO 19, a heavy
chain of SEQ ID NO 15 and a light chain of SEQ ID NO 20.
TABLE-US-00001 TABLE 1 Exemplary heavy and light chain variable
regions Sequence No. Amino acid sequences 1
DIQMTQSPAIMSASPGEKVTITCSASSSVSYMHWYQQKSGTSPK
LWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQ RSSYPYTFGGGTKLEIK 2
DIVLTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQS
PKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC QQHYSTPYTFGGGTKLEIK 3
DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKP
GQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAAT
YYCQQSNEDPFTFGSGTKLELK 4
DIVLTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTIKR
LIYATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCQQHG ESPLTFGAGTKLEIK 5
DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQS
PKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFC QQYASSPYTFGGGTKLEIK 6
EVQLQQSGAELAKPGASVKMSCKASGYTFTSYWMHWVKQRPG
QGLEWIGYINPSTGYTEYNQKFKDKATLTADKSSSTAYMQLSSL
TSEDSVVYYCARQDGYYVGYFDYWGQGTTLTVSS 7
EVELQESGAELVRPGASVKLSCKASGYSFTNYWMNWVRQRPG
QGFEWIGEINPSNGDTFYNQKFKGKATLIVDKSSSTAYMELLSLT
SEDSAVYYCARIGGYYFDYWGQGTTLTVSS 8
EVNVVESGAELVKPGASVRLSCTTSGFNIEDSYIHWVKQRPEQG
LEWIGRIDPANGNIKSDPKFQGKATISADTSSNTAYLQLSSLTSED
TAVYYCLYYYDSSDYWGQGTTLTVSS 9
EVKLVESGPELEKPGASVKISCKASGYSFTGYNMNWVKQSNGK
SLEWIGNIDPYYGGTSYNQKFKGKATLTVDKSSSTAYMQLKSLT
SEDSAVYYCARWSYYGNYVYWYFDVWGAGTTLTVSS 10
EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQ
GLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTS
EDTAVYYCARSYYFDYWGQGTTLTVSS 11
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTVNWVRQAPGK
GLEWLSVISGDGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKALNAGWGFDYWGQGTLVTVSS 12
QVQLLESGGGLVKPGGSLRLSCAASGFTFSGSAMHWVRQAPGK
GMEWVSAISGSGGSTYYADSMKGRFTISRDNSKNTLYLQMNSL
RAEDTAVYYCARNGYTDGYGMDYWGQGTLVTVSS 13
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDSSVNWVRQAPGK
GLEWLAVISGDGGSTYYADSVKGRVTISRDNSKNTLYLQMNSL
RAEDTAVYYCARAIFPSYMDVWGQGTLVTVSS 14
EVQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVDWVRQARGQ
RLEWIGWIVVGSGNTSYAQKFQERVTITRDMSTSTAYMELSSLR
SEDTAVYYCAKSVTYYYMDHWGQGTLVTVSS 15
QVQLVQSGAEVKKPGSSVKVSCKASGGTFNSYAISWVRQAPGQ
GLEWMGVIIGIFGTATYAQSVQGRVTITADESTSTAYMELSSLRS
EDTAVYYCARSGRYSRSFDVWGQGTLVTVSS 16
EIVLTQSPGTLSLSPGERATLSCRANRLVSSSMLAWYQQKPGQA
PRLLIGASKRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCAQ YDGSSYTFGQGTKLEIKR
17 QSVLTQPPSVSGSPGQRVTISCTGNNLSGYYVSWYQQLPGTAPK
LLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAMDEADYYCA AYYSGTYVFGQGTKLTVLGQ
18 SYELTQPLSVSVSLGQTARITCWDNVGGYNVHWYQQKPGQSPV
LVIYRDSERPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCSSY YQSTVLFGGGTKLTVLGQ
19 DIQMTQSPSSLSASVGDRVTITCQASNVGGNYLNWYQQKPGKA
PKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCS AYHQSTYTFGQGTKVEIKR
20 DIQMTQSPSSLSASVGDRVTITCRASKVGSSYVNWYQQKPGKAP
KLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAS
YYSTSTFGGGTKVEIKR
Example 3: Generation of a Monoclonal Antibody to the Immunocomplex
of 25-(OH)D:mAB from a Synthetic Library
[0107] The synthetic antibody library was designed based on the
natural VH/VL pairing frequency (described in Protein Engineering,
Design & Selection pp. 1-7, 2012 doi:10.1093/protein/gzs043),
the frameworks of frequently paired human VH1, VH3, VK1, VK3,
V.lamda.1, V.lamda.2 and V.lamda.3 were selected as master genes
for gene synthesis. The selected master genes were synthesized by
PCR assembly of overlapping oligos. The length of oligos was
between 60 and 80 bases, and the oligos for all CDRs were
degenerate oligos using the nucleotides NNS and/or MVS to randomize
amino acid residues. After the first round of PCR assembly, 5 light
chain master genes were assembled with a light chain constant
region by PCR. The resulting full-length light chain genes were
digested with restriction enzymes HindIIII and NcoI (New England
Lab) and size-selected on a 1% agarose gel. The size-selected
fragments were purified and extracted with the QIAquick Gel
Extraction Kit. Digested light chain genes, pAXD20 vector DNA, and
T4 ligase (New England Lab) were mixed and ligation was allowed to
proceed overnight at 16.degree. C. For each light chain gene
construct, 20 .mu.g of ligated DNA was purified and transformed
into E coli TOP10 competent cells by electroporation. Transformants
were then grown on 100 2YT-agar plates containing 100 .mu.g/ml of
carbenicillin and 2% glucose. After overnight 30.degree. C.
incubation, library transformants were harvested and suspended in
2YT with 15% glycerol. Half of the stock was stored as library
aliquots at -80.degree. C., and the rest was processed by Qiagen
Maxiprep kit. The resulted library size is listed in Table 2. The
DNA of AFK1 and AFk3 were combined, and AFL1, AFL2 and AFL3 were
also combined for the next step of heavy chain cloning.
TABLE-US-00002 TABLE 2 Characteristics of light chain libraries
Library Library size AFK1 1.1 .times. 10.sup.9 AFK3 12 .times.
10.sup.9 AFL1 8.5 .times. 10.sup.8 AFL2 7.6 .times. 10.sup.8 AFL3 8
.times. 10.sup.8
[0108] PCR assembled VH1 and VH3 master genes were further
assembled with CH1 constant region by PCR, the resulting Fd genes
were digested with restriction enzymes NcoI and SalI (New England
BioLab) and size-selected by 1% agarose gel. The size-selected
fragment was purified and extracted with the QIAquick Gel
Extraction Kit. Digested Fd genes, light chain library DNA and T4
ligase (New England Lab) were mixed and ligation was allowed to
proceed overnight at 16.degree. C. For each Fd chain gene library,
40 .mu.g of ligated DNA was purified and transformed into E. coli
TG1 competent cells by electroporation. Transformants were then
grown on 400 2YT-agar plates containing 100 .mu.g/ml of
carbenicillin and 2% glucose. After overnight 30.degree. C.
incubation, library transformants were harvested and suspended in
2YT with 15% glycerol. Library aliquots were prepared and stored at
-80.degree. C. The resulting library sizes are listed in Table
3.
TABLE-US-00003 TABLE 3 Characteristics of phage display libraries
Library Library size AFH1K 3.6 .times. 10.sup.9 AFH3K 3.3 .times.
10.sup.9 AFH1L 3.5 .times. 10.sup.9 AFH3L 3.2 .times. 10.sup.9
Total 1.4 .times. 10.sup.10
[0109] Phage display libraries were prepared in the following
manner. A 500 2YT (with 100 .mu.g/ml of carbenicillin and 2%
glucose) culture was inoculated with library TG1 cells at a
starting OD of 0.1. After 3-4 hours incubation at 37.degree. C.
with 250 rpm shaking, KO7 helper phage was added in a 1:10 ratio.
The culture was then incubated at 37.degree. C. for 1 hour without
shaking. The TG1 cells were centrifuged and the cell pellet was
re-suspended into 2 L of 2YT containing 100 .mu.g/ml carbenicillin
and 35 .mu.g/ml Kanamycin for overnight incubation at 30.degree. C.
with 250 rpm shaking. TG1 cells were then centrifuged for 30 min at
6000.times.g at 4.degree. C. The library phage particles were
purified by PEG-precipitation from culture supernatants,
resuspended into PBS and titered by OD268 measurement. The phage
display antibody library was stored at -80.degree. C. in PBS with
20% glycerol. Each library was prepared separately and kept
separately.
[0110] The procedure to select immunocomplex-specific antibodies is
similar to the description in the Example 2. Briefly, two kinds of
microtiter wells were prepared, one coated with AF10 antibody only,
and the second with the immunocomplex of 25-(OH)D.sub.3:AF10.
Before affinity selection, the individual library was pre-incubated
in AF10-only wells for to deplete for phages that bind AF10 alone.
The supernatant is then transferred to the microtiter wells coated
with the immunocomplex of 25-(OH)D.sub.3:AF10. In the first round
affinity selection, a total of eight wells were used, and each
library phage was added to two wells. In the second round of
affinity selection, the phages from first round were mixed and 4
wells were used for incubation. A total of three rounds of affinity
selection were performed.
[0111] The colonies from the third round of affinity selection were
picked for expression of p3 fusion and confirmation of binding
activity. The procedure is similar to that described in Example 2.
Briefly, five 96-well plates and a total of 480 colonies were
picked to make master plates. During the second day, 10 .mu.l of
culture per well was transferred into a corresponding well in a
96-well deep-well expression plate. The deep-well plates were
incubated in a 37.degree. C. shaker incubator with shaking at 250
rpm until the cultures reach OD600 nm of 0.8-1. 100 .mu.l of 2YT
containing 6 mM IPTG was added to each well, and the expression
plate was incubated overnight at 30.degree. C. while shaking at 250
rpm. 160 .mu.l of lysis buffer containing 2.5 mg/ml lysozyme and 5
mM EDTA was added to each well of the expression plate, and the
plate was incubated with shaking for 1 hour at room temperature.
The culture supernatant was mixed with 140 .mu.l 2.times.
ChemiBlocker per well and incubated for an additional 30 min at 250
rpm. The culture supernatants were centrifuged and prepared for
binding assay as below.
[0112] The ELISA screening procedure was similar to that described
in Example 2. Briefly, for each expression plate, two 96-well assay
plates were prepared, one plate coated with AF10 antibody only, and
the second with the immunocomplex of 25-(OH)D.sub.3:AF10. The
culture supernatants from the expression plate were transferred to
the corresponding well of the assay plate. After 1 hour incubation
at room temperature, the samples were washed three times with
PBS-Tween. 100 .mu.l of HRP-conjugated anti-Fab antibody (Jackson
ImmunoReaearch Lab, USA) were added to each well for 1 hour
incubation at room temperature. After 3 times wash with PBS-Tween,
100 .mu.l of substrate TMB was added to each well for 10-30 min
development, and 50 .mu.l of stop solution was added. OD450 was
then measured. The clones that bound to the immunocomplex of
25-(OH)D.sub.3:AF10 but not AF10 antibody alone were selected for
confirmation. 1:3 serial dilutions of the positive culture
supernatants were assayed following above procedure. FIG. 3 shows
the binding activity of five clones to the immunocomplex of
25-(OH)D.sub.3:AF10, with variable region sequences listed in Table
1.
Example 4: Recombinant Antibody Expression and Purification
[0113] For recombinant Fab expression, VH and VL gens were cloned
into E. coli expression vector pEx. For each individual clone, 20
ml overnight cultures were prepared in 2YT with 2% glucose and 100
.mu.g/ml Carbenicillin and incubated in a 37.degree. C. shaker
incubator. The next day, 700 mL of 2YT containing 0.1% glucose and
100 .mu.g/mL Carbenicillin was inoculated in a 2 L Ultra yield
flask (Thomson Instrument Company) by transferring 7 ml of the
overnight culture. The cultures were grown at 35.degree. C. with
280 rpm shaking for approximately 2-3 hours until OD600 was
.about.0.8. Then 350 .mu.l of 1M IPTG was added to the culture for
overnight induction at 22.degree. C. with 280 rpm shaking. The
cells pellets were collected by centrifugation at 7500.times.g for
20 minutes, and resuspended into lysis buffer containing 1 mg/ml
lysozyme and 5 mM EDTA for 1 hour incubation at room temperature.
The supernatant with soluble Fab was collected by centrifuging
twice at 15,000 rpm 25000.times.g at 4.degree. C. The supernatant
was then loaded onto a 5 ml protein-G column (GE healthcare) for
Fab binding and washed wash with 50 ml PBS. Fab protein was then
eluted with 0.3M acetic acid, pH3 buffer. The eluted fractions were
collected, and neutralized with 0.5 volumes of 1M Tris-HCl, pH8.5
buffer. The Fab samples were buffer-exchanged to PBS, and
concentrated to a concentration of 3-5 mg/ml. The Fab samples were
stored at -80.degree. C.
[0114] For IgG expression, the heavy chain variable domains and
light chain variable domains were cloned into a cytomegalovirus
promoter-based mammalian expression vectors PMX30 and pMX31,
respectively. The heavy chain and light chain vector DNA was mixed
in a ratio of 1:3 and transiently transfected into a 30 ml
suspension HEK293 cells in serum-free medium. After 20 hours, cells
were sampled to determine viability and viable cell count, and
titer was measured (Octet QKe, ForteBio). The cultures were
harvested at day 5, and an additional sample for each was measured
to determine cell density, viability, and titer. The conditioned
media containing IgG was harvested and clarified from the transient
transfection production by centrifugation and filtration. The
supernatant was run over a Protein A column and eluted with a low
pH buffer (pH=3). Filtration using a 0.2 .mu.m membrane filter was
performed before aliquoting. After purification and filtration, the
protein concentration was calculated based on the OD280 and the
extinction coefficient. Generally, 1-5 mg of IgG was generated from
this procedure.
Example 5: Sandwich ELISA Assay for Serum 25-(OH)D Measurement
[0115] A monoclonal antibody to the immunocomplex of 25-(OH)D:AF10
were used to prepare a sandwich based ELISA assay. Free
25-(OH)D.sub.3 was used for the assay. Briefly, 100 .mu.L/well of
AF10 antibody (3 .mu.g/ml in PBS) was used to coat the microtiter
plate overnight at 4.degree. C.
[0116] The assay plates were washed with PBS and blocked with
1.times. ChemiBlocker for 2 hours at room temperature. 5 .mu.L/well
of 25-(OH)D.sub.3 at concentration of 0.5-50 ng/ml was added to the
plate, and 200 .mu.L of anti-imunocomplex antibody at 3 .mu.g/ml
was added thereafter. After 1 hour incubation at room temperature,
the plate was washed 3 times with PBS-Tween, and 100 .mu.l of
HRP-conjugated goat anti-mouse antibody (Jackson ImmunoReaearch
Lab, USA) was added to each well for a 30-minutes incubation at
room temperature. After washing three times with PBS-Tween, 100
.mu.l of substrate TMB was added to each well for a 30-minute
development. 50 .mu.L of stop solution was added, and OD450 was
measured. This data showed a dose-dependent response and served as
proof-of-concept for a sandwich ELISA for 25-(OH)D detection. To
further confirm the utility of the assay for clinical sample
detection, patient serum samples with 25-(OH)D values (as
determined by ADVIA Centaur VitaminD Total assay, Siemens) were
measured using the sandwich ELISA assay. The assay was performed as
for the free 25-(OH)D.sub.3, except that to each well 200 .mu.L of
CalBiotech release buffer (Calbiotech, Calif., USA) containing 3
.mu.g/ml of the monoclonal antibody to the immunocomplex of
25-(OH)D:AF10 was added, followed by 5 .mu.l of the serum sample.
After mixing, the plate was incubated at room temperature for 1.5
hours. FIG. 4 shows a successful sandwich ELISA detection of serum
25-(OH)D, demonstrating a linear relationship between the two
methods of measuring vitamin D concentration.
Example 6: Lateral Flow Assay for 25-(OH)D Measurement
[0117] An exemplary lateral flow device comprises a sample
application unit (e.g., from Ahlstrom or Millipore) for sample
transportation, a conjugate pad comprising labeled antibody and a
detection zone comprising immobilized capture reagents and an
absorbent pad (e.g., from Ahlstrom or Millipore).
[0118] Dry conjugate pad was prepared as follows. A 0.04% colloidal
solution was prepared by reducing gold chloride with sodium citrate
at boiling temperature. 25 .mu.g of anti-25-(OH)D antibody was
added to 1 ml of 0.04% colloidal solution and incubated at room
temperature for 30 minutes. The conjugate was then centrifuged for
20 min at 10000 rpm. The supernatant was then decanted, and the
pellet suspended in 1 mL of 15 mM phosphate buffer, pH 7.4. A
similar procedure was performed to prepare the conjugate of
colloidal gold-anti-chicken IgG antibody used for the positive
control. The anti-25-(OH)D conjugate and the anti-chicken IgG
conjugate were mixed with conjugate pad base in a 1:10 ratio. 10 ml
of the conjugate pad base mixture was applied to a piece of 8
cm.times.8 cm fiberglass sheet, and vacuum dried at room
temperature for 4 hours. The pad was stored with desiccant in a dry
room.
[0119] To prepare the detection membrane, the monoclonal antibody
to the immunocomplex of 25-(OH)D:AF10 was prepared in 15 mM
phosphate buffer at pH 7.4 at the range of concentration of 0.2-1.5
mg/ml as the test line solution. The test line solution was
dispensed on to nitrocellulose membrane (Millipore 125 or GE AE99)
at an amount between 0.6-1.0 .mu.L/cm. Similarly, the control line
solution with anti-chicken IgG was dispensed onto the
nitrocellulose membrane at the control line position. The membrane
was then allowed to air-dry in an environment with less than 30%
humidity for one hour, and then stored with desiccant in a dry
room.
[0120] In order to construct the test device, the detection
membrane, conjugate pad, and sample wicking materials were aligned
and the assembled strip was placed into plastic housing.
[0121] The device was evaluated by using serum or blood samples
with known 25-(OH)D concentrations. 5 .mu.L of serum or 10 .mu.L of
capillary blood was applied to the sample pad, followed by 3 drops
(100 .mu.L) of sample chase buffer. The test line and control line
were read after 15 min. The results, shown in FIG. 5, indicate that
the density of signal on the test line increased in samples with
higher serum vitamin D concentrations. The test device indicates
that a subject has sufficient, insufficient, or deficient vitamin D
(FIG. 1A). Furthermore, the test device demonstrated a linear
relationship between signal intensity and 25-(OH)D levels in
samples ranging from 10 ng/ml to 80 ng/ml. Additional samples were
tested for vitamin D levels as measured using a semi-quantitative
color chart (FIG. 1B). The data were compared with the vitamin D
levels determined by ADVIA Centaur VitD Assay. As shown in TABLE 4,
the results of the lateral flow test device were consistent with
the test from ADVIA Centaur VitD assay. The tests were reproduced
with the devices from different production lot.
TABLE-US-00004 TABLE 4 Comparison of measurements using lateral
flow test device and ADVIA Centaur VitD Assay ADVIA Lateral flow
test Centaur VitD results (Semi- Assay Quantitative) Patient
25-(OH)D 25-(OH)D VitD # Patient ID ng/mL ng/mL Sufficiency 1
03928913 4.2 <10 Deficient 2 03928920 4.2 <10 Deficient 3
1023110935 9 .ltoreq.10 Deficient 4 1023110936 10 10 Deficient 5
1023110968 10 10 Deficient 6 1023110755 10 10 Deficient 7
1023110768 10 .ltoreq.10 Deficient 8 1022110849 11 10 Deficient 9
1023110943 11 10 Deficient 10 1023110949 11 10 Deficient 11
1023110957 19.4 .ltoreq.20 Insufficient 12 1023110763 20 20
Insufficient 13 1023110788 20 .gtoreq.20 Insufficient 14 1023110831
20 20 Insufficient 15 1023110866 20 .ltoreq.20 Insufficient 16
1023110876 20 20 Insufficient 17 1023110958 20 20 Insufficient 18
1023110743 21 20 Insufficient 19 1022110782 29 30 Sufficient 20
1022110783 29 30 Sufficient 21 1022110784 30 .gtoreq.30 Sufficient
22 1022110863 30 30 Sufficient 23 1023110910 30 30 Sufficient 24
1023110813 29.1 30 Sufficient 25 10411110254 32.7 30 Sufficient 26
10411110267 33.5 .gtoreq.30 Sufficient 27 10052015WK 42 >30
Sufficient 28 10052015JA 41 >30 Sufficient 29 1120834 67
>>30 Sufficient 30 1124761 80 >>30 Sufficient 31
1123647 97 >>30 Sufficient
[0122] Furthermore, a smartphone-based reader was used for
quantitative detection of vitamin D levels in capillary blood
samples. 10 .mu.l of capillary blood was applied to the sample pad,
followed by 3 drops (100 .mu.l) of sample chase buffer. The test
lines were read after 15 minutes. The serum samples of
corresponding individuals were collected and subjected to an ELISA
test. FIG. 6 shows the comparison between the capillary blood test
using the lateral flow test device and an ELISA-based serum test.
The data shows a positive linear correlation with a correlation
coefficient, R, of 0.91.
Example 7: Lateral Flow Assay for 25-(OH)D Measurement Using
Cube-Reader
[0123] The Cube-Reader (from opTricon GmbH, Germany) was used for
fully quantitative measurement of 25-OH vitamin D. Cube-Reader is a
portable lateral flow assay reader, with a cube-shape at an edge
length of approximate 41 mm and weigh of 40 g. The measurement
procedure is shown in FIG. 8.
[0124] Total 20 human serum samples with 25-OH Vitamin D values
determined by Liquid Chromatography Mass Spectrometry (LC-MS/MS)
assay were applied to lateral flow test. The measurement procedure
is described in the Example 6. The results listed in Table 5 show
that the 25-OH vitamin D values from lateral flow test are very
close to the true values generated from LC-MS/MS assay (a "gold"
standard method for 25-OH vitamin D measurement).
TABLE-US-00005 TABLE 5 Test Results from Lateral Flow Test and
Liquid Chromatography Mass Spectrometry Test 25-OH Vitamin 25-OH
Vitamin Sample D (ng/ml) D (ng/ml) ID LC-MS/MS test Lateral flow
test S-1 14.3 14.4 S-2 14.0 16.0 S-3 18.9 19.9 S-4 19.8 19.1 S-5
20.0 22.4 S-6 26.4 22.1 S-7 29.3 31.0 S-8 40.1 37.2 S-9 40.3 47.4
S-10 45.9 51.8 S-11 46.4 39.2 S-12 49.0 42.6 S-13 56.3 57.4 S-14
60.3 72.7 S-15 63.1 67.8 S-16 75.5 71.2 S-17 87.4 85.9 S-18 92.3
97.9 S-19 92.4 94.4 S-20 103.9 93.1 Average 49.8 50.2
The comparison of 25-OH vitamin D values between lateral flow test
and LC-MS/MS assay yield a linear regression Y=0.98X+1.49, with
correlation coefficient of 0.98. The comparison result is
illustrated in the FIG. 9.
[0125] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 1
1
211106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Asp Ile Gln Met Thr Gln Ser Pro Ala Ile Met
Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Ser
Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Ser Gly Thr
Ser Pro Lys Leu Trp Ile Tyr 35 40 45Ser Thr Ser Asn Leu Ala Ser Gly
Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser
Leu Thr Ile Ser Ser Met Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Tyr Thr 85 90 95Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 1052107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Asp Ile Val Leu Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly1 5
10 15Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr
Asn 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala
Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg
Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln
His Tyr Ser Thr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 1053111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 3Asp Ile Val Leu Thr Gln Ser Pro Ala
Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg
Ala Ser Lys Ser Val Ser Thr Ser 20 25 30Gly Tyr Ser Tyr Met His Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu
Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Glu
Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp
Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys 100 105
1104107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 4Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Leu Gly1 5 10 15Glu Arg Val Ser Leu Thr Cys Arg Ala Ser
Gln Asp Ile Gly Ser Ser 20 25 30Leu Asn Trp Leu Gln Gln Glu Pro Asp
Gly Thr Ile Lys Arg Leu Ile 35 40 45Tyr Ala Thr Ser Ser Leu Asp Ser
Gly Val Pro Lys Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Ser Asp Tyr
Ser Leu Thr Ile Ser Ser Leu Glu Ser65 70 75 80Glu Asp Phe Val Asp
Tyr Tyr Cys Gln Gln His Gly Glu Ser Pro Leu 85 90 95Thr Phe Gly Ala
Gly Thr Lys Leu Glu Ile Lys 100 1055107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
5Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5
10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu
Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg
Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
Tyr Ala Ser Ser Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 1056120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 6Glu Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Ala Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Ser
Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Thr
Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu
Ser Ser Leu Thr Ser Glu Asp Ser Val Val Tyr Tyr Cys 85 90 95Ala Arg
Gln Asp Gly Tyr Tyr Val Gly Tyr Phe Asp Tyr Trp Gly Gln 100 105
110Gly Thr Thr Leu Thr Val Ser Ser 115 1207117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
7Glu Val Glu Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala1 5
10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn
Tyr 20 25 30Trp Met Asn Trp Val Arg Gln Arg Pro Gly Gln Gly Phe Glu
Trp Ile 35 40 45Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Phe Tyr Asn
Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Ile Val Asp Lys Ser Ser
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Leu Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ile Gly Gly Tyr Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr Thr 100 105 110Leu Thr Val Ser Ser
1158116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Glu Val Asn Val Val Glu Ser Gly Ala Glu Leu
Val Lys Pro Gly Ala1 5 10 15Ser Val Arg Leu Ser Cys Thr Thr Ser Gly
Phe Asn Ile Glu Asp Ser 20 25 30Tyr Ile His Trp Val Lys Gln Arg Pro
Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly
Asn Ile Lys Ser Asp Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Ile Ser
Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Ser Ser
Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Tyr Tyr Tyr
Asp Ser Ser Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110Thr Val
Ser Ser 1159123PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 9Glu Val Lys Leu Val Glu Ser Gly Pro
Glu Leu Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30Asn Met Asn Trp Val Lys Gln
Ser Asn Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Asn Ile Asp Pro Tyr
Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr
Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu
Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Trp Ser Tyr Tyr Gly Asn Tyr Val Tyr Trp Tyr Phe Asp Val 100 105
110Trp Gly Ala Gly Thr Thr Leu Thr Val Ser Ser 115
12010115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 10Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Ser Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly
Phe Asn Ile Lys Asp Tyr 20 25 30Tyr Met His Trp Val Lys Gln Arg Pro
Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly
Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Ile Thr
Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Ser Ser
Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Tyr
Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110Val Ser
Ser 11511119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 11Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Thr Val Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Ser Val Ile Ser Gly
Asp Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Lys Ala Leu Asn Ala Gly Trp Gly Phe Asp Tyr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser 11512120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
12Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly
Ser 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Met Glu
Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Met 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Gly Tyr Thr Asp Gly Tyr
Gly Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 12013118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 13Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Ser Val Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Ala Val Ile Ser Gly
Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Val
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Ala Ile Phe Pro Ser Tyr Met Asp Val Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 11514118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
14Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser
Ser 20 25 30Ala Val Asp Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu
Trp Ile 35 40 45Gly Trp Ile Val Val Gly Ser Gly Asn Thr Ser Tyr Ala
Gln Lys Phe 50 55 60Gln Glu Arg Val Thr Ile Thr Arg Asp Met Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Val Thr Tyr Tyr Tyr Met
Asp His Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
11515119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 15Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Asn Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Val Ile Ile Gly Ile Phe Gly
Thr Ala Thr Tyr Ala Gln Ser Val 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Gly
Arg Tyr Ser Arg Ser Phe Asp Val Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 11516108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 16Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Asn Arg Leu Val Ser Ser Ser 20 25 30Met Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Gly Ala
Ser Lys Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Ala Gln Tyr Asp Gly Ser Ser Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
10517108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ser Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Asn Asn
Leu Ser Gly Tyr Tyr Val 20 25 30Ser Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu Leu Ile Tyr 35 40 45Gly Asn Ser Asn Arg Pro Ser Gly
Val Pro Asp Arg Phe Ser Gly Ser 50 55 60Lys Ser Gly Thr Ser Ala Ser
Leu Ala Ile Thr Gly Leu Gln Ala Met65 70 75 80Asp Glu Ala Asp Tyr
Tyr Cys Ala Ala Tyr Tyr Ser Gly Thr Tyr Val 85 90 95Phe Gly Gln Gly
Thr Lys Leu Thr Val Leu Gly Gln 100 10518107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
18Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ser Leu Gly Gln1
5 10 15Thr Ala Arg Ile Thr Cys Trp Asp Asn Val Gly Gly Tyr Asn Val
His 20 25 30Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile
Tyr Arg 35 40 45Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser
Gly Ser Asn 50 55 60Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala
Gln Ala Gly Asp65 70 75 80Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Tyr
Gln Ser Thr Val Leu Phe 85 90 95Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly Gln 100 10519108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 19Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Gln Ala Ser Asn Val Gly Gly Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn
Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Ile Ala Thr Tyr Tyr Cys Ser Ala Tyr His Gln Ser Thr Tyr 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
10520107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 20Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Lys Val Gly Ser Ser Tyr 20 25 30Val Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Ser Tyr Tyr Ser Thr Ser
Thr 85 90 95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100
1052116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Gly Ser Ser Ser Gly Ser Ser Ser Gly Ser Ser Ser
Gly Ser Ser Ser1 5 10 15
* * * * *