U.S. patent application number 16/735604 was filed with the patent office on 2020-07-16 for lateral flow test strip assay for osteoporosis.
The applicant listed for this patent is Optech Ventures, LLC. Invention is credited to Manal Beshay, Morgan Hatch.
Application Number | 20200225220 16/735604 |
Document ID | / |
Family ID | 69410722 |
Filed Date | 2020-07-16 |
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United States Patent
Application |
20200225220 |
Kind Code |
A1 |
Beshay; Manal ; et
al. |
July 16, 2020 |
LATERAL FLOW TEST STRIP ASSAY FOR OSTEOPOROSIS
Abstract
A lateral flow test strip (LFTS) platform measures osteocalcin
(OC) in saliva to identify early indications of bone loss and
minimize bone fracture risk associated with osteoporosis. The OC
assay embodiments are based on the experimentally identified
optimal markers which exhibit selectivity with very low false
positives, and sensitivity relevant to clinical requirements. A
prospective clinical study sampling of 20 patients demonstrated
excellent correlation of OC in saliva with bone mineral density
(BMD). Salivary OC and Dpd levels were validated with a standard
commercial ELISA kit against serum (OC) and urine (Dpd).
Multiplexed LFTS are used to increase specificity of an assay.
Inventors: |
Beshay; Manal; (Rancho Palos
Verdes, CA) ; Hatch; Morgan; (Long Beach,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Optech Ventures, LLC |
Torrance |
CA |
US |
|
|
Family ID: |
69410722 |
Appl. No.: |
16/735604 |
Filed: |
January 6, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15078519 |
Mar 23, 2016 |
10557857 |
|
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16735604 |
|
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62136929 |
Mar 23, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6893 20130101;
G01N 33/558 20130101; G01N 2800/108 20130101; G01N 33/54386
20130101; B01L 3/5023 20130101; G01N 33/5302 20130101; G01N
2021/7786 20130101; B01L 2300/041 20130101; G01N 21/6428 20130101;
G01N 33/549 20130101; B01L 2300/0825 20130101; G01N 2021/7759
20130101; G01N 2333/78 20130101; B01L 2300/069 20130101; A61B 5/00
20130101; G01N 2333/47 20130101; G01N 2333/916 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; B01L 3/00 20060101 B01L003/00; G01N 33/53 20060101
G01N033/53; G01N 33/549 20060101 G01N033/549 |
Goverment Interests
GOVERNMENT LICENSE RIGHTS
[0002] This invention was made with government support under
contract #41R43DE022478-01 awarded by the National Institute of
Health. The government has certain rights in the invention.
Claims
1. A lateral flow test strip for screening for osteoporosis
comprising: a) a sample pad for accepting a subject sample bodily
fluid; b) a migration membrane; c) a plastic backing; d) a
conjugate pad deposited with a first fluorescently labeled detector
antibody; e) a first capture line comprising a first capture
antibody specific to osteocalcin wherein a first antibody pair
comprising the first capture antibody and the first fluorescently
labeled detector antibody are chosen from the group consisting of:
i. 940/939, ii. 11F8/939, iii. 2H9/939, iv. 940/H10, v. 2H9/H10,
vi. 940/3G8, vii. 11F8/3G8.
2. The lateral flow test strip of claim 1 also comprising a second
capture line comprising a second capture antibody reactive specific
to: a) deoxypyridinoline, b) urinary calcium, c) tartare resistant
acid phosphtase, d) bone sialoprotein, e) pryidinoline, f)
N-telopeptide, g) C-telopeptide, h) C-terminal telopeptide of type
I collagen i) bone-specific alkaline phosphatase, j) pro-collagen I
extension peptides, k) amino terminal.
3. The lateral flow test strip of claim 1 wherein the conjugate pad
deposited with the first fluorescently labeled detector antibody is
conjugated to fluorescent microparticles being paired with the
first capture antibody being immobilized on the migration
membrane.
4. The lateral flow test strip of claim 1 wherein the bodily fluid
is whole saliva.
5. The lateral flow test strip of claim 1 wherein the bodily fluid
is urine.
6. The lateral flow test strip of claim 1 also comprising a second
capture line comprising a second capture antibody specific to
osteocalcin wherein a second antibody pair comprising the second
capture antibody and a second fluorescently labeled detector
antibody are chosen from the group consisting of: a) 940/939, b)
11F8/939, c) 2H9/939, d) 940/H10, e) 2H9/H10, f) 940/3G8, g)
11F8/3G8.
7. The lateral flow test strip of claim 6 wherein the first capture
antibody and the second capture antibody are the same.
8. A system for immunochromatographic bone loss marker assay
comprising: a) a lateral flow test strip comprising: i) a sample
pad for accepting a subject sample bodily fluid; ii) a migration
membrane; iii) a plastic backing; iv) a conjugate pad deposited
with a first fluorescently labeled detector antibody; v) a first
capture line comprising a first capture antibody specific to
osteocalcin wherein the first capture antibody and the first
fluorescently labeled detector antibody are chosen from the group
consisting of: (1) 940/939, (2) 11F8/939, (3) 2H9/939, (4) 940/H10,
(5) 2H9/H10, (6) 940/3G8, (7) 11F8/3G8. b) a calibrated florescence
test strip reader.
9. The system of claim 8 wherein the conjugate pad deposited with
the first fluorescently labeled detector antibody is conjugated to
fluorescent microparticles being paired with the first capture
antibody being immobilized on the migration membrane.
10. The system of claim 8 wherein the lateral flow test strip also
comprises a second capture line comprising a second antibody
capture antibody reactive specific to: a) deoxypyridinoline, b)
urinary calcium, c) tartare resistant acid phosphtase, d) bone
sialoprotein, e) pryidinoline, f) N-telopeptide, g) C-telopeptide,
h) C-terminal telopeptide of type I collagen i) bone-specific
alkaline phosphatase, j) pro-collagen I extension peptides, k)
amino terminal.
11. The system of claim 8 wherein the bodily fluid is whole
saliva.
12. The system of claim 8 wherein the bodily fluid is urine.
13. The system of claim 8 wherein the immunochromatographic assay
also comprises a diagnostically correlated evaluation for reader
output measurements of reagent lines following the application of a
serum specimen to the lateral flow test strip.
14. A multi-plexed lateral flow test strip for screening for
osteoporosis comprising: a) a sample pad for accepting a subject
sample bodily fluid; b) a migration membrane; c) a plastic backing;
d) a conjugate pad deposited with a first fluorescently labeled
detector antibody; e) a first capture line comprising a first
capture antibody specific to osteocalcin wherein a first antibody
pair comprising the first capture antibody and the first
fluorescently labeled detector antibody are chosen from the group
consisting of: f) a second capture line comprising a second capture
antibody specific to secondary marker for osteocalcin wherein a
second antibody pair comprise a second capture antibody and a
second fluorescently labeled detector antibody. are chosen from the
group consisting of i. osteocalcin, ii. deoxypyridinoline, iii.
urinary calcium, iv. tartare resistant acid phosphtase, v. bone
sialoprotein, vi. pryidinoline, vii. N-telopeptide, viii.
C-telopeptide, ix. C-terminal telopeptide of type I collagen x.
bone-specific alkaline phosphatase, xi. pro-collagen I extension
peptides, xii. amino terminal.
Description
RELATED APPLICATIONS
[0001] This application for patent claims the benefit of and is a
continuation of U.S. non-provisional patent application Ser. No.
15/078,519, filed on Mar. 23, 2016 and claims the benefit of
provisional application 62/136,929 filed on Mar. 23, 2015. Both
applications are incorporated herein in their entirety.
FIELD OF THE INVENTION
[0003] This field of this disclosure relates generally to the assay
of biological markers for bone loss.
BACKGROUND
[0004] Bone health is regulated in a tightly coupled metabolic
process between bone formation (by osteoblasts) and bone resorption
(by osteoclasts). In healthy bone these processes are in balance;
however, these rates may become uncoupled due to diseases affecting
this regulation (Paget's disease, metastatic bone cancer), or
hormonal changes, as in postmenopausal women. When bone resorption
occurs more than bone formation, a net loss in bone mineral density
(BMD) results, which can lead to diseases such as osteoporosis. The
traditional approach to measuring BMD is dual energy X-ray
absorption (DEXA), reported as a T-score (standard deviation from
mean BMD) or Z-score (standard deviation from age-matched BMD).
However, DEXA is an expensive procedure, and is not readily
available for general population screening. The development of a
viable screening diagnostic exam for early detection of BMD loss is
a long-standing problem.
SUMMARY
[0005] Disclosed is a system for screening is the detection of
biomarkers of bone formation and degradation, which can be assayed
in human serum or urine via concentrations of osteocalcin (OC) and
deoxypyridinoline (Dpd), respectively. Various embodiments of
diagnostic systems disclosed reliably identify the concentrations
of these biomarkers in human saliva which provides clinicians an
opportunity to improve the diagnosis and prevention of
osteoporosis, as well as provide a noninvasive method for
convenient population screening.
[0006] Disclosed are embodiments of a lateral flow test strip
(LFTS) platform which measure osteocalcin (OC) and
deoxypyridinoline (Dpd) in saliva to identify early indications of
bone loss and minimize bone fracture risk associated with
osteoporosis. The OC assay embodiments are based on the
experimentally identified optimal markers which exhibit selectivity
with very low false positives, and sensitivity relevant to clinical
requirements. A prospective clinical study sampling of 20 patients
demonstrated excellent correlation of OC in saliva with bone
mineral density (BMD). Salivary OC and Dpd levels were validated
with a standard commercial enzyme-linked immunosorbent assay
(ELISA) kit against serum (OC) and urine (Dpd).
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 shows a single biomarker lateral flow test strip.
[0008] FIG. 2A shows a photograph of sample quantified lateral flow
assay (LFA) test strips with measured levels of Dpd.
[0009] FIG. 2B shows a photograph of sample quantified LFA test
strips with measured levels of OC.
[0010] FIG. 3 shows a multiplex biomarker lateral flow test strip
and test strip reader apparatus.
[0011] FIG. 4 shows a chart of clinical patient results for testing
salivary OC.
[0012] FIG. 5A shows a chart of measured salivary OC compared
against measured OC from serum.
[0013] FIG. 5B shows a chart of measured salivary Dpd compared
against measured Dpd from urine.
[0014] FIG. 6 shows a correlation computation for the embodiment
measured OC against the laboratory ELISA measured OC.
[0015] FIG. 7A shows a chart of LFTS measured OC compared with BMD
levels in subjects.
[0016] FIG. 7B shows a detailed graph comparing levels of subject
BMD and measured OC from the developed embodiment LFTS.
[0017] FIG. 8 is a chart of tested and identified optimal
combinations of reagents for detecting biomarkers.
[0018] FIG. 9 is a chart of identified biomarkers suitable for
various embodiments of the disclosed LFTS platform.
DETAILED DESCRIPTION
[0019] The disclosed LFTS platform is a rapid immunochromatographic
assay comprised of a test strip with several membranes that house
all the reagents necessary for the test. The analyte of interest is
applied in the sample medium (OC or Dpd in saliva), wherein it is
captured in the test in a sandwich-antibody immunocomplex coupled
to fluorescent detection. Monoclonal antibodies specific for OC or
Dpd are conjugated to fluorescently labeled microparticles and
deposited on the conjugate pad. Upon adding the sample to the
sample pad, the saliva resolubilizes the dried antibody conjugates
and forms an analyte-antibody conjugate complex, which is captured
by another monoclonal antibody specific for OC or Dpd immobilized
to the nitrocellulose membrane. Excitation of captured fluorescent
particles generates signal response proportional to the
concentration of reagent in the sample.
[0020] Shown in FIG. 1 is an exemplar LFTS 101 for a single
biomarker configuration. The LFTS platform includes sample pad 102,
conjugate pad 103, nitrocellulose membrane 104 and wicking pad 110.
Detector antibodies (OC or Dpd) 105 are carried on the conjugate
pad and capture antibodies (OC or Dpd) 108 are carried by the
capture line 106. A control line 109 is also located on the
nitrocellulose membrane. Flow of the sample 107 is mediated by a
combination of capillary action through the membranous components
from the sample pad 102 to the absorbent pad 110, and by pressure
from the liquid in the sample pad pushing toward the absorbent
pad.
[0021] Experimental results for determining optimal configuration
of the LFTS system were conducted by the following procedure.
Separate, finalized test strips for OC and Dpd were placed in
plastic cassettes for testing. The cassettes have an open window to
view the test results, and a sample port where the sample is
applied. In this embodiment the widely-used Qiagen ESE test strip
fluorescent reader was adapted and calibrated with customized
optical settings of emission and excitation wavelengths to read the
selected fluorescent label. Other readout devices can be used for
the reported platform measurements; such as LRE/SOFIA by Quidel,
Cell-phone readout by Holomics, and RDS-1500 PRO by Detekt
Biomedical. Saliva samples from 20 donor patients were obtained.
Collected saliva samples were kept frozen at -80.degree. C. until
tested. Frozen samples were thawed, centrifuged to remove large
particulates, and diluted 1 to 1 with our running buffer. Tests
were performed in triplicate with each saliva sample. After adding
100 uL of sample volume to each test, the tests flowed for 10
minutes before the results were read with the ESE reader.
[0022] Shown in FIG. 2A and FIG. 2B are exemplar samples. Shown are
samples with Dpd concentrations of 30 nmol/L 201A, 15 nmol/L 202A,
7.5 nmol/L 203A, 3.7 nmol/L 204A, 1.8 nmol/L 205A and 0 nmol/L
206A. Formation of sandwich antibody detection with decreasing
concentrations of Dpd. Fluorescence was observed under an
ultra-violet (UV) light source. Shown in FIG. 2B are exemplar OC
samples with intensity observed from spiking synthetic OC in
saliva. Shown tests were run in cassettes. Fluorescence was
observed under UV light source for the samples 0 ng/mL 201B, 5
ng/mL 202B, 10 ng/mL 203B, 15 ng/mL 204B and 30 ng/mL 205B
[0023] Shown in FIG. 3 is the fluorescent reader apparatus 301
utilized for single and multiplex test strips. Shown in the inset
is an embodiment for a multiplex LFTS 302 with multiple detection
lines (multiplex) 303 and 304 and control line 305.
[0024] A standard commercial enzyme-linked immunosorbent assay
(ELISA) test kit was used to validate the embodiment LFTS platform
with patient saliva samples. A correlation value of 0.85 was
obtained with OC.
[0025] In various embodiments salivary OC and Dpd concentrations
are correlated with serum (OC) and urinary (Dpd) levels from the
same patient using ELISA measurements. Samples are normalized by
protein concentration to adjust for salivary specific gravity. The
resulting high correlation confirms the reliability of salivary
markers for the disclosed embodiment.
[0026] Shown in FIG. 4 are results for patient samples categorized
into three main groups--normal, osteopenic, and
osteoporotic--showed high amounts of OC in normal patients, and low
OC in osteopenic/osteoporotic patients.
[0027] Note that for purposes of testing various embodiments, after
adding 100 uL of sample volume to each test, the tests flowed for
10 minutes before the results were read with the ESE reader.
[0028] To determine the optimum LFA components, incremental assay
optimization steps were carried out for both OC and DPD assays,
including capture antibody concentration, assay running buffers,
purification of the antibody reagents, addition of surfactants,
selection of LFA membranes, conjugate pad selection, and
fabrication/drying protocols.
[0029] FIG. 8 shows a chart of tested capture and detector reagents
along with those combinations which proved to be effective for the
disclosed embodiment. To identify the antibody pair with the best
affinity to osteocalcin (OC), OC reagents were screened from
Fitzgerald, Novus Biologicals, Thermo Scientific, and Hytest that
includes mouse anti-OC monoclonal and rabbit polyclonal antibodies
for sandwich pairing and antigen testing. The following pairs were
the only combinations that bound synthetic osteocalcin as
membrane/detector: 940/939, 11F8/939, 2H9/939, 940/H10, 2H9/H10,
940/3G8, 11F8/3G8, 2H9/3G8. Two pairs bound strongly with native
osteocalcin as membrane/detector: 940/H10, and 2H9/H10, and were
used for further assay optimization and testing. Mouse monoclonal
antibodies against DPD were acquired from Quidel and screened along
with an antibody from a human DPD ELISA kit (MyBioSource).
[0030] Salivary OC and Dpd concentrations were correlated with
serum (OC) and urinary (Dpd) levels from the same patient using
ELISA measurements. Samples were normalized by protein
concentration to adjust for salivary specific gravity. The
resulting high correlation suggested the reliability of salivary
markers. Shown in FIG. 5A are results for salivary experimental
measurements vs. the serum OC validation marker. Shown in FIG. 5B
are results for salivary experimental measurements vs. the urine
Dpd validation marker.
[0031] A standard commercial ELISA test kit was used to validate
the disclosed LFTS platform with patient saliva samples. A
correlation value of 0.85 was obtained with OC. FIG. 6 shows the
correlation of OC measurements.
[0032] In various embodiments, the disclosed quantifiable LFTS may
contain multiplexed biomarkers for both OC and Dpd. The LFTS shown
in FIG. 3 illustrates a multiplexed LFA.
[0033] In various embodiments, additional biomarkers are utilized
in the disclosed LFTS in either the disclosed single or multiplexed
quantifiable methodology. Bone turnover biomarkers are represented
in compounds such as collagen precursors, enzymes, and by-products,
or degradation products involved with the bone formation
(osteoblast) and bone resorption (osteoclast) processes. In various
embodiments, one or more of these biomarkers are utilized in the
disclosed LFTS for optimal or additional accuracy for screening
examination. FIG. 9 provides a chart of the biomarkers identified
for use in these embodiments.
[0034] The disclosed sensitive lateral flow assay-based technique
are capable of the detecting bone formation marker osteocalcin in
saliva at clinically relevant levels which has been demonstrated
and correlated to BMD, utilizing the disclosed readout system which
as disclosed is easily integrated in a single platform for
point-of-care (POC) applications.
[0035] Other embodiments of the invention utilize equivalent
monoclonal antibody capture and detector reagents and reagent pairs
for the assay.
[0036] What has been described herein is considered merely
illustrative of the principles of this invention. Accordingly, it
is well within the purview of one skilled in the art to provide
other and different embodiments within the spirit and scope of the
invention.
* * * * *