U.S. patent application number 16/776831 was filed with the patent office on 2020-05-28 for method and device for detecting antigen-specific antibodies in a biological fluid sample by using neodymium magnets.
This patent application is currently assigned to The U.S.A., as represented by the Secretary, Department of Health and Human Services. The applicant listed for this patent is The U.S.A. as represented by the Secretary, Department of Health and Human Services. Invention is credited to Peter D. Burbelo.
Application Number | 20200166505 16/776831 |
Document ID | / |
Family ID | 56684314 |
Filed Date | 2020-05-28 |
View All Diagrams
United States Patent
Application |
20200166505 |
Kind Code |
A1 |
Burbelo; Peter D. |
May 28, 2020 |
METHOD AND DEVICE FOR DETECTING ANTIGEN-SPECIFIC ANTIBODIES IN A
BIOLOGICAL FLUID SAMPLE BY USING NEODYMIUM MAGNETS
Abstract
Methods for detecting antigen-specific antibodies in a
biological sample are described. The disclosed methods can be used
for the diagnosis of a variety of autoimmune and infectious
diseases. The methods use a neodymium magnet to efficiently isolate
immune complexes. The disclosed methods are rapid, highly specific
and sensitive, require very small volumes of biological sample, and
do not require the use of radioactivity. With these advantageous
features, the disclosed methods are amendable for point-of-care
testing (POCT), which is currently not available for the detection
of autoantibodies associated with autoimmune disease or for the
detection of many pathogen-specific antibodies.
Inventors: |
Burbelo; Peter D.;
(Bethesda, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The U.S.A. as represented by the Secretary, Department of Health
and Human Services |
Bethesda |
MD |
US |
|
|
Assignee: |
The U.S.A., as represented by the
Secretary, Department of Health and Human Services
Bethesda
MD
|
Family ID: |
56684314 |
Appl. No.: |
16/776831 |
Filed: |
January 30, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15756012 |
Feb 27, 2018 |
10564152 |
|
|
PCT/US2016/046037 |
Aug 8, 2016 |
|
|
|
16776831 |
|
|
|
|
62212973 |
Sep 1, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/566 20130101;
G01N 33/582 20130101; G01N 2469/20 20130101; G01N 33/54326
20130101; G01N 35/0098 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/566 20060101 G01N033/566; G01N 33/58 20060101
G01N033/58; G01N 35/00 20060101 G01N035/00 |
Claims
1. A device for detecting antigen-specific antibodies in a
plurality of biological fluid samples according to a method
comprising: (i) providing a fusion protein comprising an antigen
fused to a light-emitting protein; (ii) contacting the plurality of
biological fluid samples with the fusion protein, thereby forming
immune complexes if antigen-specific antibodies are present in the
plurality of biological fluid samples; (iii) contacting the immune
complexes with magnetic beads coated with an immunoglobulin-binding
protein to form bead-bound immune complexes; (iv) isolating the
bead-bound immune complexes by directly contacting the bead-bound
immune complexes with a neodymium magnet; and (v) detecting
emission of light from the isolated bead-bound immune complexes,
thereby detecting the presence of antigen-specific antibodies in
the plurality of biological fluid samples, wherein the device
comprises a first solid support for housing a plurality of
biological fluid samples; and a second solid support comprising a
plurality of neodymium magnets affixed thereto.
2. The device of claim 1, wherein the first solid support comprises
a multi-well plate.
3. The device of claim 2, wherein the multi-well plate is a 96-well
plate.
4. The device of claim 2, wherein the multi-well plate is a
384-well plate.
5. The device of claim 2, wherein the second solid support
comprises the same number of neodymium magnets as the number of
wells present in the multi-well plate.
6. The device of claim 1, wherein the neodymium magnets are about 1
cm to about 5 cm in length.
7. The device of claim 6, wherein the neodymium magnets are about 2
cm to about 4 cm in length.
8. The device of claim 6, wherein the neodymium magnets are about
2.5 cm to about 3.5 cm in length.
9. The device of claim 1, wherein the neodymium magnets are less
than about 1 mm in diameter.
10. The device of claim 9, wherein the neodymium magnets are about
0.9 mm to about 0.1 mm in diameter.
11. The device of claim 9, wherein the neodymium magnets are about
0.8 mm to about 0.2 mm in diameter.
12. The device of claim 9, wherein the neodymium magnets are about
0.7 mm to about 0.3 mm in diameter.
13. The device of claim 9, wherein the neodymium magnets are about
0.6 mm to about 0.4 mm in diameter.
14. The device of claim 1, which is a hand-held device.
15. The device of claim 1, wherein the total volume of each of the
plurality of biological fluid samples is no more than 10 .mu.L.
16. The device of claim 15, wherein the total volume of each of the
plurality of biological fluid samples is no more than 2 .mu.L.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 15/756,012, filed Feb. 27, 2018, which is the U.S. National
Stage of International Application No. PCT/US2016/046037, filed
Aug. 8, 2016, published in English under PCT Article 21(2), which
claims the benefit of U.S. Provisional Application No. 62/212,973,
filed Sep. 1, 2015. The above-listed applications are herein
incorporated by reference in their entireties.
FIELD
[0002] This disclosure concerns methods for rapidly detecting
antigen-specific antibodies in biological samples using a
fluid-phase immunoassay and neodymium magnets.
BACKGROUND
[0003] Point-of-care testing (POCT) is medical testing at or near
the site of patient care. The driving notion behind POCT is to
bring the test conveniently and immediately to the patient. This
increases the likelihood that the patient, physician, and care team
will receive results quickly, which allows for immediate clinical
management decisions to be made.
[0004] POCT is often accomplished through the use of transportable
and handheld instruments and test kits. Small bench analyzers or
fixed equipment can also be used when a handheld device is not
available. The goal of POCT is to collect the specimen and obtain
the results in a very short period of time at or near the location
of the patient so that the treatment plan can be adjusted as
necessary before the patient leaves. Cheaper, faster, and smarter
POCT devices have increased the use of POCT approaches by making it
cost-effective for many diseases, such as diabetes, carpal tunnel
syndrome and acute coronary syndrome.
[0005] Rapid point-of-care, antibody-based testing is not currently
available for the diagnosis of autoimmune and most infectious
diseases. For detecting autoantibodies associated with most
autoimmune conditions, fluid-phase immunoprecipitation assays are
necessary in order to enable detection of conformation-specific
antibodies; however, these assays usually involve radioactivity,
which is not feasible for point-of-care applications. Therefore,
rapid, POCT diagnostic assays for diagnosis of autoimmune and
infectious diseases would satisfy an important clinical need.
SUMMARY
[0006] An assay for detecting antigen-specific antibodies in
biological samples that is amenable to rapid, POCT diagnostics for
autoimmune and infectious disease is disclosed. The disclosed
methods utilize luciferase immunoprecipitation systems (LIPS) and
neodymium magnets to very rapidly detect antigen-specific
antibodies with high sensitivity and specificity.
[0007] Provided herein is a method for detecting antigen-specific
antibodies in a biological fluid sample. In some embodiments, the
method includes providing a fusion protein comprising an antigen
fused to a light-emitting protein; contacting the biological fluid
sample with the fusion protein, thereby forming immune complexes if
antigen-specific antibodies are present in the biological fluid
sample; contacting the immune complexes with magnetic beads coated
with an immunoglobulin-binding protein to form bead-bound immune
complexes; isolating the bead-bound immune complexes by directly
contacting the bead-bound immune complexes with a neodymium magnet;
and detecting emission of light from the isolated bead-bound immune
complexes.
[0008] In some examples, the biological fluid sample is a serum,
plasma, blood, urine, saliva or bronchoalveolar lavage fluid
sample. In some examples, the light-emitting protein comprises a
fluorescent protein or a luciferase, such as a Renilla luciferase,
a Gaussia luciferase, a modified (optimized) Oplophorus
gracilirostris luciferase (for example, NANOLUC.TM.), a firefly
luciferase or a bacterial luciferase. In some examples, the
immunoglobulin-binding protein is Protein A, Protein G, Protein
A/G, Protein L or a secondary immunoglobulin molecule.
[0009] In some examples, the antibodies are autoantibodies. In
other examples, the antibodies are pathogen-specific
antibodies.
[0010] Also provided herein are methods of diagnosing a subject as
having an autoimmune disease by performing the disclosed methods to
detect autoantibodies in a biological sample from the subject that
are indicative of the autoimmune disease.
[0011] Further provided herein are methods of diagnosing a subject
as infected with a pathogen by performing the disclosed methods to
detect pathogen-specific antibodies in a biological sample from the
subject.
[0012] Also provided herein is a device for detecting
antigen-specific antibodies in a plurality of biological fluid
samples simultaneously, according to the methods disclosed herein.
In some embodiments, the device includes a first solid support for
housing a plurality of biological fluid samples and a second solid
support having a plurality of neodymium magnets affixed
thereto.
[0013] The foregoing and other objects, features, and advantages of
the invention will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a schematic depiction of the LIPSTICKS method
disclosed herein. Only four steps are required for 1 minute
antibody detection by LIPSTICKS: Recombinant luciferase antigen
fusion proteins (Ag-luciferase) are mixed together with diluted
biological sample, such as sera or saliva; paramagnetic protein
A/G-coated beads are added and bind antibody-antigen complexes;
neodymium magnets collect the paramagnetic protein A/G-immune
complexes and are briefly washed; and magnet-bound immune complexes
are placed into tubes containing luciferase substrate and emitted
light is measured with a tube or hand-held luminometer.
[0015] FIGS. 2A-2D are a series of graphs showing the results of
testing various LIPSTICK immunoassay parameters. In each
experiment, sera from a representative control subject (closed
circle) and a representative Sjogren's syndrome patient (closed
square) were tested with Renilla luciferase fused to Ro52
(Ruc-Ro52). The resulting light units (LU) are shown on the Y-axis.
(FIG. 2A) Increasing the volume (.mu.l) of paramagnetic beads
resulted in a better LU signal. (FIG. 2B) A range of sera between
0.001 .mu.l and 4 .mu.l was tested. The results showed that 0.1
.mu.l of sera provided the optimum signal. (FIG. 2C) Increasing the
amount of Ruc-Ro52 extract as input improved the LU signal. (FIG.
2D) Lengthening the time of incubation from 30 seconds to 4 minutes
also increased the Ro52 autoantibody signal for the Sjogren's
syndrome subject sample.
[0016] FIGS. 3A-3B are a pair of graphs showing detection of Ro52
autoantibodies. A sample set of 20 healthy controls (HC) and 28
Sjogren's syndrome (SS) patients was examined for Ro52
autoantibodies. Each dot represents the value for each individual.
(FIG. 3A) The LIPSTICK one minute assay results are shown. (FIG.
3B) For comparison, the data obtained from the standard 2.5 hour
LIPS test performed in 96 well, microtiter and filter plate format
is also presented. The cut-off values (dotted line) for each assay
are based on the mean plus three standard deviations of the HC
group. FIGS. 4A-4B are a pair of graphs showing detection of Ro60
and Ro52 autoantibodies. A sample set of 20 HC and 28 SS patients
was examined by LIPSTICKS for (FIG. 4A) Ro60 autoantibodies or
(FIG. 4B) both Ro60 and Ro52 autoantibodies as a mixture. The
cut-off values used to determine diagnostic performance are
indicated with the dotted line. The mixture assay showed improved
sensitivity compared to either Ro60 or Ro52 alone by LIPSTICK
testing.
[0017] FIGS. 5A-5C are a series of graphs comparing the LIPSTICK
and LIPS assays for detection of La autoantibodies. A sample set of
20 HC and 28 SS patients was examined for La autoantibodies. (FIG.
5A) Results from the LIPSTICKS one minute assay for detection of La
autoantibodies are shown. (FIG. 5B) A prolonged, one hour LIPSTICK
assay for detection of La autoantibodies was also tested. (FIG. 5C)
For comparison, results from the standard 2.5-hour LIPS assay are
shown. The cut-off values used to determine diagnostic performance
are indicated by the dotted line. The one minute LIPSTICK test
yielded 25% sensitivity (100% specificity); the one hour LIPSTICK
test yielded 53% sensitivity (100% specificity); and the standard
2.5 hour LIPS test yielded 61% sensitivity (98% specificity) for
the detection of La autoantibodies. The clinical ELISA test shows
only 45% sensitivity.
[0018] FIG. 6 is a graph showing detection of human
immunodeficiency virus (HIV) reverse transcriptase (RT) antibodies.
A sample set of 13 healthy uninfected controls (HC) and 13 HIV
patients was examined for antibodies against HIV RT for the
diagnosis of HIV. Compared to the other LIPSTICK assays for Ro52,
Ro60 and La autoantibodies, approximately one tenth the input (20
million total) of Ruc-HIV RT was used. The cut-off values used to
determine diagnostic performance are indicated with the dotted
line. The one minute LIPSTICK RT test showed 100% sensitivity and
100% specificity.
[0019] FIGS. 7A-7D are graphs that provide an overview of the
LIPSTICKS diagnostic performance. (FIG. 7A) Antibody detection by
LIPSTICKS using luciferase-HIV p24 extract with log-dilutions of
sera from HIV negative and HIV positive individuals reveals that
0.1 .mu.l of sera produces the highest signal to noise ratio. (FIG.
7B) Increasing the amount of luciferase-HIV p24 antigen produces a
linear increase in LU for both the HIV negative and HIV positive
samples. (FIG. 7C) HIV reverse transcriptase antibody detection in
a cohort of HIV negative and HIV positive individuals. The
geometric mean in each group is shown by the horizontal bar and the
cut-off value for seropositivity is shown by the dotted line. (FIG.
7D) Epstein-Barr virus (EBV) negative and EBV positive individuals
were tested for antibodies with an EBV Epstein-Barr nuclear antigen
(EBNA) luciferase antigen fusion. The geometric means and the
cut-off value are shown as in FIG. 7C.
[0020] FIGS. 8A-8F are a series of graphs showing LIPSTICKS
autoantibody detection for autoimmune disease diagnosis. (FIG. 8A)
Evaluation of autoantibodies against IFN-.gamma. in the sera of
controls and subjects with disseminated non-tuberculosis infection
demonstrated a diagnostic performance of 95% sensitivity and 100%
specificity. (FIG. 8B) Autoantibodies against BPI fold containing
family B, member 1 (BPIFB1) in the sera of control subjects and
subjects with autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy (APECED). Only a subset of APECED patients had BPIFB1
autoantibodies and LIPSTICKS demonstrated a diagnostic performance
of 95% sensitivity and 100% specificity. (FIG. 8C) Ro60 and (FIG.
8D) Ro52 autoantibodies detected in saliva of healthy controls and
patients with Sjogren's syndrome. (FIG. 8E) Serum autoantibodies
against Ro60 in healthy controls and patients with Sjogren's
syndrome. (FIG. 8F) Comparison of Ro52 serum autoantibodies
detected by tube luminometer vs. a hand held luminometer.
[0021] FIGS. 9A-9B are a pair of graphs showing evaluation of
additional parameters for LIPSTICKS testing. (FIG. 9A) Varying the
amount of protein A/G coated beads produced an increase in LU for
both the HIV negative and HIV positive samples with the
luciferase-HIV p24 antigen. One .mu.l of beads was chosen for
testing. (FIG. 9B) Lengthening the time of incubation from 1 minute
to 10 minutes also modestly increased the LU signal for p24
antibodies of the HIV seropositive sample.
[0022] FIG. 10 is a graph showing the results of LIPSTICKS HIV
reverse transcriptase antibody detection in seven HIV subjects from
before and after 4-5 years of anti-retroviral therapy. A Wilcoxon
matched-pairs signed rank test showed a statistically significant
decrease in RT antibodies with treatment.
[0023] FIG. 11 is a graph showing the results of LIPSTICKS HIV p24
antibody detection in a cohort of HIV negative and HIV positive
individuals. The geometric mean in each group is shown by the
horizontal bar and the cut-off value for seropositivity is shown by
the dotted line.
[0024] FIG. 12 is a graph showing the results of LIPSTICKS
detection of antibodies against equine non-primate hepacivirus
(NPHV) in seronegative (circles) and seropositive (squares) horse
serum samples. The cut-off value for seropositivity is shown by the
dotted line.
[0025] FIG. 13 is a graph showing the results of LIPSTICKS La
autoantibody detection in a cohort of normal volunteers and
Sjogren's syndrome patients. The geometric mean in each group is
shown by the horizontal bar and the cut-off value for
seropositivity is shown by the dotted line.
DETAILED DESCRIPTION
I. Abbreviations
[0026] APECED autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy
[0027] BPIFB1 BPI fold containing family B, member 1
[0028] dNTM disseminated non-tuberculosis mycobacterial
[0029] EBNA Epstein-Barr nuclear antigen
[0030] EBV Epstein-Barr virus
[0031] ELISA enzyme linked immunosorbent assay
[0032] HC healthy control
[0033] HIV human immunodeficiency virus
[0034] LIPS luciferase immunoprecipitation systems
[0035] LU luciferase unit
[0036] NPHV non-primate hepacivirus
[0037] POC point-of-care
[0038] POCT point-of-care testing
[0039] RT reverse transcriptase
[0040] SS Sjogren's syndrome
[0041] VCA viral capsid antigen
II. Terms and Methods
[0042] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0043] In order to facilitate review of the various embodiments of
the disclosure, the following explanations of specific terms are
provided:
[0044] Antibody: A protein (or protein complex) that includes one
or more polypeptides substantially encoded by immunoglobulin genes
or fragments of immunoglobulin genes. The recognized immunoglobulin
genes include the kappa, lambda, alpha, gamma, delta, epsilon, and
mu constant region genes, as well as the myriad of immunoglobulin
variable region genes. Light chains are classified as either kappa
or lambda. Heavy chains are classified as gamma, mu, alpha, delta,
or epsilon, which in turn define the immunoglobulin classes, IgG,
IgM, IgA, IgD and IgE, respectively.
[0045] The basic immunoglobulin (antibody) structural unit is
generally a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kDa) and one "heavy" (about 50-70 kDa) chain. The N-terminus of
each chain defines a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
terms "variable light chain" (V.sub.L) and "variable heavy chain"
(V.sub.H) refer, respectively, to these light and heavy chains.
[0046] As used herein, the term "antibodies" includes intact
immunoglobulins as well as a number of well-characterized
fragments. For instance, Fabs, Fvs, and single-chain Fvs (scFvs)
that bind to target protein (or epitope within a protein or fusion
protein) would also be specific binding agents for that protein (or
epitope). These antibody fragments are defined as follows: (1) Fab,
the fragment which contains a monovalent antigen-binding fragment
of an antibody molecule produced by digestion of whole antibody
with the enzyme papain to yield an intact light chain and a portion
of one heavy chain; (2) Fab', the fragment of an antibody molecule
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain; two Fab' fragments are obtained per antibody molecule;
(3) (Fab').sub.2, the fragment of the antibody obtained by treating
whole antibody with the enzyme pepsin without subsequent reduction;
(4) F(ab').sub.2, a dimer of two Fab' fragments held together by
two disulfide bonds; (5) Fv, a genetically engineered fragment
containing the variable region of the light chain and the variable
region of the heavy chain expressed as two chains; and (6) single
chain antibody, a genetically engineered molecule containing the
variable region of the light chain, the variable region of the
heavy chain, linked by a suitable polypeptide linker as a
genetically fused single chain molecule. Methods of making these
fragments are routine (see, for example, Harlow and Lane, Using
Antibodies: A Laboratory Manual, CSHL, New York, 1999).
[0047] Antibodies for use in the methods of this disclosure can be
monoclonal or polyclonal, and for example specifically bind a
target such as the target antigen. Merely by way of example,
monoclonal antibodies can be prepared from murine hybridomas
according to the classical method of Kohler and Milstein (Nature
256:495-97, 1975) or derivative methods thereof. Detailed
procedures for monoclonal antibody production are described in
Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New
York, 1999.
[0048] Antigen: A compound, composition, or substance that can
stimulate the production of antibodies or a T-cell response in an
animal, including compositions that are injected or absorbed into
an animal. An antigen reacts with the products of specific humoral
or cellular immunity, including those induced by heterologous
immunogens.
[0049] Antigen-specific: As used herein, an "antigen-specific"
antibody is an antibody that was elicited (produced and/or
activated) in response to a particular antigen. An
"antigen-specific" antibody is capable of binding to the antigen,
typically with high affinity.
[0050] Autoantibody: An antibody produced in an organism that is
directed against a constituent of the organism's own tissue (i.e.
an antibody specific for a self-antigen).
[0051] Autoimmune disease: A disease arising from an abnormal
immune response directed against proteins and tissues normally
present in the body. There are currently more than 80 defined types
of autoimmune diseases.
[0052] Contacting: Placement in direct physical association. In the
context of the present disclosure, "directly contacting" bead-bound
immune complexes with a neodymium magnet requires that the
bead-bound immune complexes and neodymium magnet make direct
physical contact without any intervening materials or substances.
Thus, directly contacting the bead-bound immune complexes and
neodymium magnet excludes instances in which a magnet is placed
outside of a tube, container, culture vessel or other structure to
concentrate bead-bound immune complexes within the tube, container,
culture vessel or other structure.
[0053] Fluorescent protein: A protein that emits light of a certain
wavelength when exposed to a particular wavelength of light.
Fluorescent proteins include, but are not limited to, green
fluorescent proteins, blue fluorescent proteins, cyan fluorescent
proteins, yellow fluorescent proteins, orange fluorescent proteins,
red fluorescent proteins and modified versions thereof.
[0054] Fusion protein: A protein containing amino acid sequence
from at least two different (heterologous) proteins or peptides.
Fusion proteins can be generated, for example, by expression of a
nucleic acid sequence engineered from nucleic acid sequences
encoding at least a portion of two different (heterologous)
proteins. To create a fusion protein, the nucleic acid sequences
must be in the same reading frame and contain no internal stop
codons. Fusion proteins, particularly short fusion proteins, can
also be generated by chemical synthesis.
[0055] Helminth: A parasitic worm, such as a fluke, tapeworm or
nematode.
[0056] Heterologous: A heterologous protein or polypeptide refers
to a protein or polypeptide derived from a different source or
species.
[0057] Human immunodeficiency virus (HIV): A retrovirus that causes
immunosuppression in humans (HIV disease), and leads to a disease
complex known as the acquired immunodeficiency syndrome (AIDS).
"HIV disease" refers to a well-recognized constellation of signs
and symptoms (including the development of opportunistic
infections) in persons who are infected by HIV, as determined by
antibody or western blot studies. Laboratory findings associated
with this disease include a progressive decline in T cells. HIV
includes HIV type 1 (HIV-1) and HIV type 2 (HIV-2).
[0058] Immune complex: A protein complex that comprises an antibody
bound to an antigen. In the context of the present disclosure, an
"immune complex" comprises (1) a fusion protein, which is made up
of a selected antigen fused to a light-emitting protein; and (2) an
antibody that specifically binds the antigen. A bead-bound immune
complex is an immune complex that is further bound to at least one
bead (such as a magnetic bead) that is coated with an
immunoglobulin-binding protein. The immunoglobulin-binding proteins
on the surface of the bead bind to the antibody present in the
immune complex.
[0059] Immunoglobulin-binding protein: Any protein that
specifically binds an immunoglobulin molecule. Examples of
immunoglobulin-binding molecules include, but are not limited to,
Protein A, Protein G, Protein A/G, Protein L and secondary
immunoglobulins (for example, anti-IgG, anti-IgM, anti-IgA,
anti-IgE or anti-IgD antibodies).
[0060] Isolated: An "isolated" biological component (such as a
nucleic acid molecule, protein, or cell) has been substantially
separated or purified away from other biological components in the
cell, blood or tissue of the organism, or the organism itself, in
which the component naturally occurs, such as other chromosomal and
extra-chromosomal DNA and RNA, proteins and cells. Nucleic acid
molecules and proteins that have been "isolated" include those
purified by standard purification methods. The term also embraces
nucleic acid molecules and proteins prepared by recombinant
expression in a host cell as well as chemically synthesized nucleic
acid molecules and proteins.
[0061] Light-emitting protein: Any protein that is capable of
emitting light or inducing the emission of light by acting on a
particular substrate. Light-emitting proteins include, for example,
fluorescent proteins and bioluminescent proteins. Fluorescent
proteins include, for example, green fluorescent proteins and
variants thereof (including blue, cyan, yellow, orange and red
fluorescent proteins) and phycobiliproteins, such as
B-phycoerythrin (B-PE), R-phycoerythrin (R-PE) and allophycocyanin
(APC). Bioluminescent proteins include, for example, aequorin and
luciferase (which acts on the substrate luciferin to emit
light).
[0062] Linker: One or more nucleotides or amino acids that serve as
a spacer between two molecules, such as between two nucleic acid
molecules or two peptides (such as in a fusion protein).
[0063] Luciferin: A light-emitting compound found in organisms that
generate bioluminescence. Luciferins are small molecule substrates
that undergo an enzyme-catalyzed oxidation and the resulting
excited state intermediate emits lights upon decaying to its ground
state.
[0064] Luciferase: An oxidative enzyme that generates light by
reacting with luciferin. Commonly used luciferase proteins include
Renilla luciferase, Gaussia luciferase, Oplophorus gracilirostris
(deep sea shrimp) luciferase (a modified version with increased
stability, NANOLUC.TM., is commercially available), firefly
luciferase and bacterial luciferase. Exemplary luciferases and the
substrates they act upon are shown in the table below.
TABLE-US-00001 Organism Luciferase Substrate Photinus pyralis North
American firefly luciferase D-luciferin Luciola cruciata Japanese
firefly (Genji-botaru) D-luciferin luciferase Luciola italica
Italian firefly Luciferase D-luciferin Luciola lateralis Japanese
firefly (Heike) luciferase D-luciferin Luciola mingrelica East
European firefly luciferase D-luciferin Photuris pennsyl vanica
Pennsylvania firefly luciferase D-luciferin Pyrophorus
plagiophthalamus Click beetle luciferase D-luciferin Phrixothrix
hirtus Railroad worm luciferase D-luciferin Renilla reniformis
Renilla luciferase Coelenterazine Rluc8 (mutant of Renilla
luciferase) Coelenterazine Green Renilla luciferase Coelenterazine
Gaussia princeps Gaussia luciferase Coelenterazine Gaussia-Dura
luciferase Coelenterazine Cypridina noctiluca Cypridina luciferase
Vargulin/Cypridina luciferin Cypridina hilgendorfii Cypridina
(Vargula) luciferase Vargulin/Cypridina luciferin Metridia longa
Meiridia luciferase Coelenterazine Oplophorus gracilorostris
NANOLUC .TM. (optimized) Furimazine (Nano- Glo .TM. assay
substrate)
[0065] Neodymium magnet: A type of rare-earth magnet made from an
alloy of neodymium, iron and boron. Neodymium magnets are the
strongest type of permanent magnet that is commercially
available.
[0066] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein-coding regions, in the same reading frame.
[0067] Pathogen: A biological agent that causes disease or illness
to its host. Pathogens include, for example, bacteria, viruses,
fungi, helminths, protozoa and other parasites. Pathogens can also
be referred to as infectious agents.
[0068] Plurality: Any number that is more than one. In some
embodiments herein, a "plurality" means at least 6, at least 12, at
least 24, at least 48, at least 96, or at least 384.
[0069] Protein A: An immunoglobulin-binding protein. Protein A is a
42 kDa surface protein originally found in the cell wall of the
bacterium Staphylococcus aureus. The presence of five
immunoglobulin-binding domains allows Protein A to bind
immunoglobulin molecules. Protein A binds with high affinity to the
Fc portion of human IgG, IgG.sub.1, IgG2, and IgG4, and also binds
with lesser affinity to IgG.sub.3, IgM, IgE and IgA (including
IgA.sub.1 and IgA.sub.2). Protein A also binds to immunoglobulin
molecules from a variety of different species.
[0070] Protein G: An immunoglobulin-binding protein expressed in
group C and G Streptococcal bacteria. Protein G can binds strongly
to human IgG, IgG.sub.1, IgG.sub.2, IgG.sub.3, and IgG.sub.4, as
well as IgG molecules from a variety of different species. When
used for purification of antibodies, a recombinant form of Protein
G lacking the albumin binding domain is used.
[0071] Protein A/G: A recombinant fusion protein that combines
immunoglobulin binding domains of Protein A and Protein G. Protein
A/G contains four Fc binding domains from Protein A and two Fc
binding domains from Protein G. Protein A/G binds with high
affinity to all subclasses of human IgG, and also binds to human
IgA, IgE and IgM (and to a lesser extent IgD), as well
immunoglobulin molecules from a variety of different species.
[0072] Protein L: An immunoglobulin-binding protein first isolated
from the surface of the bacterial species Peptostreptococcus
magnus. Protein L binds to the kappa light chain of immunoglobulin
molecules. Protein L binds strongly to all subclasses of human IgG,
IgM, IgA, IgE, IgD, as well as scFv and Fab fragments. Protein L
can also bind to immunoglobulin molecules of several non-human
species.
[0073] Protozoa: Unicellular eukaryotic organisms. Some protozoa
are parasites that cause disease in humans, for example, malaria
(Plasmodium species), amoebiasis (Entamoeba species), giardiasis
(Giardia lamblia), toxoplasmosis (Toxoplasma gondii),
cryptosporidiosis (Cryptosporidium species). trichomoniasis
(Trichomonas vaginalis), Chagas disease (Trypanosoma cruzi),
Leishmaniasis (Leishmania species), sleeping sickness (Trypanosoma
brucei), amoebic dysentery (Entamoeba histolytica), acanthamoeba
eeratitis (Acanthamoeba species), and primary amoebic
meningoencephalitis (Naegleria fowleri).
[0074] Recombinant: A recombinant nucleic acid molecule or protein
is one that has a sequence that is not naturally occurring or has a
sequence that is made by an artificial combination of two otherwise
separated segments of sequence. This artificial combination can be
accomplished by chemical synthesis or by the artificial
manipulation of isolated segments of nucleic acid molecules, such
as by genetic engineering techniques. The term "recombinant" also
includes nucleic acids and proteins that have been altered solely
by addition, substitution, or deletion of a portion of the natural
nucleic acid molecule or protein.
[0075] Sample: Refers to any biological or environmental sample. A
biological sample is a sample obtained from a subject (such as a
human or veterinary subject). In particular examples, the
biological sample is a biological fluid sample. Biological fluid
samples from a subject include, but are not limited to, serum,
blood, plasma, urine, saliva, cerebral spinal fluid (CSF),
bronchoalveolar lavage fluid or other bodily fluid.
[0076] Sjogren's syndrome: An autoimmune disorder characterized by
immune cells that attack and destroy the glands that produce tears
and saliva. Sjogren's syndrome is not life-threatening or
life-shortening, but can significantly reduce quality of life. The
hallmark symptoms of the disorder are dry mouth and dry eyes.
Sjogren's syndrome may also cause skin, nose and vaginal dryness,
and can affect other organs of the body including the kidneys,
blood vessels, lungs, liver, pancreas and brain. Sjogren's syndrome
affects 1-4 million people in the United States, with women being
nine times more likely to develop the disease. The majority of
Sjogren's sufferers are at least 40 years old at the time of
diagnosis. Sjogren's syndrome can occur as a primary condition or
as a secondary disorder in association with a connective tissue
disease, such as systemic lupus erythematosus ("lupus"), rheumatoid
arthritis or scleroderma.
[0077] Subject: Living multi-cellular vertebrate organisms, a
category that includes human and non-human mammals.
[0078] Synthetic: Produced by artificial means in a laboratory, for
example a synthetic nucleic acid can be chemically synthesized in a
laboratory.
[0079] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. "Comprising A or B"
means including A, or B, or A and B. It is further to be understood
that all base sizes or amino acid sizes, and all molecular weight
or molecular mass values, given for nucleic acids or polypeptides
are approximate, and are provided for description. Although methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the present disclosure,
suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
explanations of terms, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
III. Overview of Several Embodiments
[0080] There is great interest in point-of-care clinical
immunoassays for the diagnosis of infectious and autoimmune
diseases. Lateral flow immunoassays have been utilized as one
approach to produce rapid qualitative, positive/negative diagnostic
results for detecting antigens and for antibody-based detection of
selected infectious agents including HIV and hepatitis C virus
(Smith et al., Antivir Ther 17(7 Pt B):1409-1413, 2012; Yager et
al., Annu Rev Biomed Eng 10:107-144, 2008). Other technologies,
such as miniaturized ELISAs (mChip) have also been employed for the
rapid, serological diagnosis of HIV and syphilis infections (Chin
et al., Nat Med 17(8):1015-1019, 2011; Laksanasopin et al., Sci
Transl Med 7(273):273re1, 2015). However, only a few rapid
immunoassays, such as for celiac disease (Bienvenu et al., BMC
Gastroenterol 14:186, 2014) and vasculitis (Offermann et al., J
Immunol Methods 403(1-2):1-6, 2014) have been reported for the
detection of autoantibodies associated with autoimmune
diseases.
[0081] Fluid-phase immunoprecipitation assays show the highest
sensitivity for the diagnosis of autoimmune diseases due to their
ability to effectively detect conformational autoantibodies
(Burbelo et al., Transl Res 165(2):325-335, 2015; Liu and
Eisenbarth, Clin Immunol 125(2):120-126, 2007). However, these
assays are often not feasible for point-of-care applications due to
the usual requirement of radioactivity. One alternative fluid phase
immunoassay, luciferase immunoprecipitation systems (LIPS), employs
light-emitting luciferase antigen fusions for detecting
antigen-specific antibodies (Burbelo et al., Transl Res
165(2):325-335, 2015). In LIPS, if antibodies are present, they
bind to light-emitting antigens and the antigen-antibody complexes
are then captured by protein A/G beads, washed and luciferase
activity is measured. The amount of light produced is proportional
to the amount of antibody present. Several key advantages of LIPS
are the high signal to noise detection, the ability to efficiently
detect conformational epitopes and the ability to use antigen
mixtures. Although a number of formats including tube (Burbelo et
al., BMC Biotechnol 5:22, 2005), plate (Burbelo et al., J Vis Exp
32:1549, 2009) and microfluidic (Zubair et al., Biomed Microdevices
13(6):1053-1062, 2011) formats exist for LIPS, the development of a
rapid format that requires limited assay manipulation and liquid
handling is highly desirable.
[0082] Disclosed herein is the development of an immunoassay
("LIPSTICK") for measuring antibodies by employing neodymium
magnetic sticks in combination with LIPS (FIG. 1). In this method,
cell extracts of light-emitting protein-antigen fusions and a
biological fluid sample, such as a serum sample, are incubated
together, which is then followed by the addition of paramagnetic
beads coated with an immunoglobulin-binding protein, such as
protein A/G. Next axially, magnetized neodymium magnets are used to
directly capture bead-bound, antibody-antigen complexes. The
magnets are washed twice in buffer, and the antigen-specific
antibody present in the sample is quantified. For example, if a
Renilla luciferase-antigen fusion protein is used as the
light-emitting protein, the antibody is quantified by placing the
magnet in a tube of coelenterazine and the luciferase activity can
be measured, for example, in a tube luminometer.
[0083] Provided herein is a method for detecting antigen-specific
antibodies in a biological fluid sample. The method includes
providing a fusion protein comprising an antigen fused to a
light-emitting protein; contacting the biological fluid sample with
the fusion protein, thereby forming immune complexes if
antigen-specific antibodies are present in the biological fluid
sample; contacting the immune complexes with magnetic beads coated
with an immunoglobulin-binding protein to form bead-bound immune
complexes; isolating the bead-bound immune complexes by directly
contacting the bead-bound immune complexes with a neodymium magnet;
and detecting emission of light from the isolated bead-bound immune
complexes. A key feature of this assay is the direct contact of the
neodymium magnet with bead-bound immune complexes in the biological
fluid sample. This differs from other immunoassays involving the
use of magnetic beads in which magnets are used on the exterior of
a tube, culture plate or other vessel as a means to concentrate
bead-bound complexes within the vessel, while the remaining fluid
within the vessel is removed. Directly contacting the bead-bound
immune complexes with the neodymium magnet significantly increases
the rapidity of the assay, in which only a simple and short wash
step is required.
[0084] Although antibodies present in the biological sample that
are not specific for the antigen of interest may also bind the
magnetic beads coated with the immunoglobulin-binding protein, this
will not interfere with the readout of the assay since these
antibodies will not bind the fusion protein and therefore will not
emit a detectable signal.
[0085] The biological fluid sample can be any biological fluid in
which antibodies can be present. In some embodiments, the
biological fluid sample is a serum, plasma, blood, urine, saliva or
bronchoalveolar lavage fluid sample. The disclosed methods are
capable of detecting antigen-specific antibodies in very small
sample volumes. In some examples, the total volume of the
biological fluid sample is no more than 10, no more than 8, no more
than 6, no more than 4, no more than 2, no more than 1, no more
than 0.5, no more than 0.25, no more than 0.1, no more than 0.05,
no more than 0.025, or no more than 0.01 .mu.L. The biological
fluid sample can be diluted in an appropriate buffer as needed to
carry out the assay. In some instances, the biological sample is
diluted prior to use in the LIPSTICKS assay, such as diluted 1:2,
1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In other instances, the
biological sample is used undiluted. In one non-limiting
embodiment, the biological sample is an undiluted saliva sample and
the total volume of the saliva sample is no more than 10 .mu.L.
[0086] In some embodiments, the light-emitting protein comprises a
luciferase. In some examples, the luciferase is Renilla luciferase,
Gaussia luciferase, firefly luciferase or a bacterial luciferase.
When a luciferase is used as the light-emitting protein, luciferase
activity is used as a measure of the quantity of antigen-specific
antibody present in the sample. Luciferase activity is measured by
contacting the magnet bound to bead-bound immune complexes with an
appropriate luciferase substrate (such as luciferin or
coelenterazine) and measuring the emission of light, for example
with a luminometer.
[0087] In other embodiments, the light-emitting protein comprises a
fluorescent protein, such as a green fluorescent protein, a blue
fluorescent protein, a cyan fluorescent protein, a yellow
fluorescent protein, an orange fluorescent protein, a red
fluorescent protein, or a modified version thereof, or a
phycobiliprotein, such as B-phycoerythrin (B-PE), R- phycoerythrin
(R-PE) or allophycocyanin (APC). When a fluorescent protein is used
as the light-emitting protein, fluorescence intensity is used as a
measure of the quantity of antigen-specific antibody present in the
sample. Fluorescence intensity is measured by exposing the magnet
bound to bead-bound immune complexes with an appropriate wavelength
of light and measuring light emission.
[0088] In some embodiments, the immunoglobulin-binding protein
comprises Protein A, Protein G, Protein A/G, or Protein L. In
particular examples, the immunoglobulin-binding protein comprises
Protein A/G. In other embodiments, the immunoglobulin-binding
protein comprises a secondary antibody, such as anti-IgG antibody,
anti-IgM antibody, anti-IgA antibody, anti-IgE antibody, anti-IgD
antibody, or any combination or two or more thereof. In particular
examples, the secondary antibody comprises anti-IgG antibody. One
of skill in the art can select an appropriate
immunoglobulin-binding protein based, for example, on the
particular immunoglobulin binding properties of each
protein/antibody.
[0089] The neodymium magnet used in the disclosed methods can be
any size or shape suitable for binding immune complexes within a
tube, multi-well plate, culture vessel or other container.
Generally, the magnets are rod-shaped and narrow in diameter, such
as less than about 1/4 inch in diameter (less than about 6.35 mm in
diameter). In some embodiments, the magnet is rod-shaped and has a
diameter of about 1/32 inch to about 1/4 inch (about 0.79 mm to
about 6.35 mm), such as about 1/16 inch to about 3/16 inch (about
1.59 mm to about 4.76 mm). In particular non-limiting examples, the
magnet is rod-shaped and has a diameter of about 1/32 inch (0.79
mm), about 1/16 inch (1.59 mm), about 1/8 inch (3.18 mm), about
3/16 inch (4.76 mm), or about 1/4 inch (6.35 mm). In particular
non-limiting examples, the magnet is rod-shaped and has a diameter
of about 1/8 inch (3.18 mm). In other examples, particularly when
the method is carried out using a high-throughput device (for
example, using a multi-well plate), the magnet is very thin, such
as less than about 1 mm in diameter. In non-limiting examples, the
magnet is about 0.9 mm, about 0.8 mm, about 0.7 mm, about 0.6 mm,
about 0.5 mm, about 0.4 mm, about 0.3 mm, about 0.2 mm or about 0.1
mm in diameter. An important advantage of the disclosed methods
compared to prior art methods for detecting antigen-specific
antibodies in a biological sample is the rapid nature of the assay,
with each step requiring little time to achieve high sensitivity
and specificity. Thus, in some embodiments, the step of contacting
the biological fluid sample with the fusion protein (to form immune
complexes if antigen-specific antibodies are present in the
biological fluid sample) is performed for a maximum of 4 minutes; a
maximum of 3 minutes; a maximum of 2 minutes; a maximum of 1
minute; a minimum of 10 seconds to a maximum of 4 minutes; a
minimum of 20 seconds to a maximum of 3 minutes; or a minimum of 30
seconds to a maximum of 2 minutes. In some instances, the step of
contacting the biological fluid sample with the fusion protein (to
form immune complexes if antigen-specific antibodies are present in
the biological fluid sample) is performed for a slightly longer
period as needed (depending upon, for example, the affinity of the
antibodies to be detected), such as for a maximum of about 3 hours,
2 hours, 1 hour, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10
minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes or 5
minutes.
[0090] In some embodiments, the step of contacting the immune
complexes with magnetic beads coated with an immunoglobulin-binding
protein to form bead-bound immune complexes is performed for a
maximum of 6 minutes; a maximum of 5 minutes; a maximum of 4
minutes; a maximum of 3 minutes; a maximum of 2 minutes; a minimum
of 10 seconds to a maximum of 6 minutes; a minimum of 20 seconds to
a maximum of 5 minutes; or a minimum of 30 seconds to a maximum of
4 minutes.
[0091] In some instances, the step of contacting the immune
complexes with magnetic beads coated with an immunoglobulin-binding
protein to form bead-bound immune complexes is performed for a
slightly longer period as needed, such as for a maximum of about 3
hours, 2 hours, 1 hour, 30 minutes, 25 minutes, 20 minutes, 15
minutes, 10 minutes, 9 minutes, 8 minutes or 7 minutes.
[0092] In some embodiments, the step of isolating the bead-bound
immune complexes by directly contacting the bead-bound immune
complexes with a neodymium magnet is performed for a maximum of 5
seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 45
seconds, 1 minute or two minutes.
[0093] In some embodiments, the steps of contacting the biological
fluid sample with the fusion protein, thereby forming immune
complexes if antigen-specific antibodies are present in the
biological fluid sample; contacting the immune complexes with
magnetic beads coated with an immunoglobulin-binding protein to
form bead-bound immune complexes; isolating the bead-bound immune
complexes by directly contacting the bead-bound immune complexes
with a neodymium magnet; and detecting emission of light from the
isolated bead-bound immune complexes are carried out in a total of
less than 10 minutes, less than 9 minutes, less than 8 minutes,
less than 7 minutes, less than 6 minutes, less than 5 minutes, less
than 4 minutes, or less than 3 minutes.
[0094] The emission of light can be detected using any means known
in the art. In some embodiments, the emission of light is detected
using a luminometer, such as a hand-held luminometer.
[0095] The disclosed methods can be used to detect, for example,
autoantibodies or pathogen-specific antibodies.
[0096] In some embodiments, the autoantibodies are indicative of
any one of a number of autoimmune diseases, such as but not limited
to, Sjogren's syndrome, type 1 diabetes, rheumatoid arthritis,
systemic lupus erythematosus, celiac disease, myasthenia gravis,
Hashimoto's thyroiditis, Graves' disease, autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),
disseminated non-tuberculosis mycobacterial (dNTM) infection, or
any other autoimmune disease listed in section IV or known in the
art. In some examples, the autoantibodies associated with patients
with Sjogren's syndrome and several other rheumatologic diseases
include antibodies against Ro52, Ro60 or La. In some examples, the
autoantibodies are indicative of dNTM infection, such as
autoantibodies specific for interferon-y. In some examples, the
autoantibodies are associated with APECED including autoantibodies
against BPI fold containing family B, member 1 (BPIFB1). Autoimmune
diseases as well as autoantibodies that are indicative of
particular autoimmune diseases are discussed further in section
IV.
[0097] In some embodiments in which the antibodies are
pathogen-specific antibodies, the pathogen is a viral pathogen, a
bacterial pathogen, a fungal pathogen, a parasitic helminth, or a
parasitic protozoan. In some examples, the viral pathogen is HIV,
hepatitis C virus (HCV), Epstein-Barr virus (EBV), human
T-lymphotropic virus 1 (HTLV-1), Kaposi's sarcoma herpesvirus
(KSHV), equine non-primate hepacivirus (NPHV), or Ebola virus. In
some examples, the bacterial pathogen is Helicobacter pylori,
Borrelia burgdorferi (Lyme disease), Escherichia coli, Mycobacteria
tuberculosis, Staphylococcus aureus, Neisseria gonorrhoeae,
Streptococcus pneumoniae, Corynebacterium diphtheria, or Vibrio
cholera. In some examples, the fugal pathogen is Candida albicans.
In some examples, the protozoan parasite is Plasmodium falciparum,
Trypanosoma cruzi, Giardia lamblia, Toxoplasma gondii, Trichomonas
vaginalis, or Entamoeba histolytica. In some examples, the helminth
is Strongyloides stercoralis, Onchocerca volvulus, Loa loa, or
Wuchereria bancrofti.
[0098] Any pathogen listed in section V below or known in the art
can be detected using the disclosed methods.
[0099] In one non-limiting embodiment. the method for detecting
antigen-specific antibodies in a serum sample comprises providing a
fusion protein comprising an antigen fused to a luciferase;
contacting a serum sample having a volume of less than 2 .mu.l with
the fusion protein, thereby forming immune complexes if
antigen-specific antibodies are present in the sample; contacting
the immune complexes with magnetic beads coated with protein A/G;
isolating the bead-bound immune complexes by directly contacting
the bead-bound immune complexes with a neodymium magnet; and
detecting emission of light from the isolated bead-bound immune
complexes, thereby detecting the presence of antigen-specific
antibodies in the biological sample, wherein all steps of the assay
are completed in less than five minutes.
[0100] Also provided herein is a device for detecting
antigen-specific antibodies according to the methods disclosed
herein, wherein the device is capable of processing a plurality of
samples simultaneously. For example, the device may be used to
process multiple samples from a single subject, with each sample
being used to detect antibodies specific for a different antigen.
Alternatively or in addition, the device may be used to process
biological samples from a multitude of different subjects for the
detection of a single type of antibody or for the detection of
multiple different antigen-specific antibodies. The device includes
a first solid support for housing a plurality of biological fluid
samples and a second solid support comprising a plurality of
neodymium magnets affixed thereto. In some embodiments, the first
solid support is a multi-well plate, such as a 6-well plate, a
12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, a
384-well plate or a 1536-well plate. A number of different
multi-well plates are commercially available from a variety of
sources; a suitable plate can be selected by one of skill in the
art. In some embodiments, the second solid support is matched to
the first solid support such that the second solid support includes
the same number of neodymium magnets as the number of wells present
in the multi-well plate, and the neodymium magnets are spaced on
the second solid support such that each well is only contacted by a
single neodymium magnet.
[0101] The length and diameter of the neodymium magnets for the
device can be selected based on the corresponding size of the wells
of the multi-well plate and volume contained within each well. In
some embodiments, the neodymium magnets are about 1 cm to about 5
cm in length, such as about 2 cm to about 4 cm, or about 2.5 cm to
about 3.5 cm in length. In some embodiments, the diameter of the
neodymium magnet is less than about 1 mm in diameter. In
non-limiting examples, the magnet is about 0.9 mm, about 0.8 mm,
about 0.7 mm, about 0.6 mm, about 0.5 mm, about 0.4 mm, about 0.3
mm, about 0.2 mm or about 0.1 mm in diameter.
IV. Diagnosis of Autoimmune Disorders
[0102] Also provided by the present disclosure are methods of
diagnosing a subject as having an autoimmune disease by performing
the disclosed methods to detect autoantibodies in a biological
sample from the subject that are indicative of the autoimmune
disease. In some embodiments, the method includes providing a
fusion protein comprising an antigen (such as an autoantigen) fused
to a light-emitting protein; contacting a biological fluid sample
(from a subject suspected of having an autoimmune disease) with the
fusion protein, thereby forming immune complexes if autoantibodies
are present in the biological fluid sample; contacting the immune
complexes with magnetic beads coated with an immunoglobulin-binding
protein to form bead-bound immune complexes; isolating the
bead-bound immune complexes by directly contacting the bead-bound
immune complexes with a neodymium magnet; and detecting emission of
light from the isolated bead-bound immune complexes, thereby
diagnosing the subject as having an autoimmune disease.
[0103] In some embodiments, the autoimmune diseases is selected
from 21-hydroxylase deficiency, acute anterior uveitis, acute
disseminated encephalomyelitis (ADEM), acute necrotizing
hemorrhagic leukoencephalitis, Addison's disease,
gammaglobulinemia, alopecia areata, amyloidosis, ankylosing
spondylitis, anti-GBM/Anti-TBM nephritis, antiphospholipid syndrome
(APS), autoimmune angioedema, autoimmune aplastic anemia,
autoimmune dysautonomia, autoimmune hepatitis, autoimmune
hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear
disease (AIED), autoimmune myocarditis, autoimmune oophoritis,
autoimmune pancreatitis, autoimmune retinopathy, autoimmune
thrombocytopenic purpura (ATP), autoimmune thyroid disease,
autoimmune urticarial, axonal and neuronal neuropathies, Balo
disease, Behcet's disease, bullous pemphigoid, cardiomyopathy,
Castleman disease, celiac disease, Chagas disease, chronic
inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent
multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial
pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans
syndrome, cold agglutinin disease, congenital heart block,
coxsackie myocarditis, CREST disease, cryoglobulinemia,
demyelinating neuropathies, dermatitis herpetiformis,
dermatomyositis, Devic's disease (neuromyelitis optica), discoid
lupus, Dressler's syndrome, endometriosis, eosinophilic
esophagitis, eosinophilic fasciitis, erythema nodosum, experimental
allergic encephalomyelitis, Evans syndrome, fibrosing alveolitis,
giant cell arteritis (temporal arteritis), giant cell myocarditis,
glomerulonephritis, Goodpasture's syndrome, granulomatosis with
polyangiitis (GPA), Graves' disease, Guillain-Barre syndrome,
Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic
anemia, Henoch-Schonlein purpura, herpes gestationis,
hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP),
IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory
lipoproteins, inclusion body myositis, inflammatory bowel disease,
interstitial cystitis, juvenile arthritis, juvenile diabetes (type
1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton
syndrome, leukocytoclastic vasculitis, lichen planus, lichen
sclerosus, ligneous conjunctivitis, linear IgA disease (LAD),
membranous nephropathy, Meniere's disease, microscopic
polyangiitis, mixed connective tissue disease (MCTD), Mooren's
ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia
gravis, myositis, narcolepsy, neutropenia, ocular cicatricial
pemphigoid, optic neuritis, palindromic rheumatism, pediatric
autoimmune neuropsychiatric disorders associated with streptococcus
(PANDAS), paraneoplastic cerebellar degeneration, paroxysmal
nocturnal hemoglobinuria (PNH), Parry Romberg syndrome,
Parsonnage-Turner syndrome, pars planitis (peripheral uveitis),
pemphigus, peripheral neuropathy, perivenous encephalomyelitis,
pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I,
II, & III autoimmune polyglandular syndromes, polymyalgia
rheumatic, polymyositis, postmyocardial infarction syndrome,
postpericardiotomy syndrome, progesterone dermatitis, primary
biliary cirrhosis, primary sclerosing cholangitis, psoriasis,
psoriatic arthritis, pulmonary fibrosis (idiopathic), pyoderma
gangrenosum, pure red cell aplasia, Raynauds phenomenon, reactive
arthritis, reflex sympathetic dystrophy, Reiter's syndrome,
relapsing polychondritis, restless legs syndrome, retroperitoneal
fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis,
Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm
and testicular autoimmunity, stiff person syndrome, subacute
bacterial endocarditis (SBE), Susac's syndrome, sympathetic
ophthalmia, systemic lupus erythematosus (SLE), Takayasu's
arteritis, temporal arteritis/Giant cell arteritis,
thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse
myelitis, type 1 diabetes, ulcerative colitis, undifferentiated
connective tissue disease (UCTD), uveitis, vasculitis,
vesiculobullous dermatosis, and vitiligo.
[0104] In particular non-limiting embodiments, the autoimmune
disease is selected from Sjogren's syndrome, type 1 diabetes,
rheumatoid arthritis, systemic lupus erythematosus, celiac disease,
myasthenia gravis, Hashimoto's thyroiditis and Graves' disease.
[0105] In some examples, the autoimmune disease is selected from
one of the autoimmune diseases listed in the table below and the
method detects autoantibodies against the listed target.
TABLE-US-00002 Disease Autoantibody Target Acute motor axonal
neuropathy Ganglioside GD3 (AMAN) Antiphospholipid syndrome
Phospholipid APECED BPIFB1 Celiac disease tTG Chronic autoimmune
hepatitis Smooth muscle CREST syndrome Centromere Dermatitis
herpetiformis eTG dNTM infection interferon-.gamma. Graves' disease
TSH receptors Hashimoto's thyroiditis Thyroid peroxidase,
thyroglobulin, TSH receptors Immunodysregulation, Harmonin, villin
polyendrocrinopathy, enteropathy X-linked syndrome (IPEX)
Inflammatory myopathy Jo1 Lambert-Eaton myasthenic syndrome
Voltage-gated calcium channel (P/Q type) Limbic encephalitis
Voltage-gated potassium channel (VGKC) Miller-Fisher syndrome
Ganglioside GQ1B Mixed connective tissue disease Ribonucleoprotein
(RNP) Multifocal motor neuropathy with Ganglioside GM1 conduction
block (MMN) Myasthenia gravis Nicotinic acetylcholine receptor,
muscle- specific kinase (MUSK) Neuromyelitis optica (Devic's
disease) Aquaporin-4 (AQP4) Polymyositis Signal recognition peptide
(SRP) Primary biliary cirrhosis Nucleoporin 62 (p62), sp100 nuclear
antigen, nucleoporin 210 kDa, mitochondria Rheumatoid arthritis
IgG, cyclic citrullinated peptide, ADAM33, honerin, HCN3 Sjogren's
syndrome SSA (Ro52 and Ro60), SSB (La) Sporadic inclusion body
myositis Cytosolic 5-nucleosidase 1A (cN1A) Stiff person syndrome
Glutamic decarboxylase-65 (GAD65), amphiphysin, glutamate
decarboxylase Systemic lupus erythematosus (SLE) Sm proteins,
U1-RNP-A1, U1-70K RNP, small nuclear RNA (snRNA), dsDNA, histones,
thrombin, Ro52, R06O, La Systemic sclerosis Topoisomerase, P0LR3A
Type 1 diabetes Insulinoma-associated protein-2 (IA2), IA2- .beta.,
insulin, GAD65, glutamate decarboxylase, zinc transporter-8
(Znt8)
[0106] In one non-limiting example, the method diagnoses a subject
as having Sjogren's syndrome by detecting autoantibodies specific
for Ro52, Ro60 and/or La.
V. Diagnosis of Infectious Diseases
[0107] The present disclosure also provides methods of diagnosing a
subject as infected with a pathogen by performing the disclosed
methods to detect pathogen-specific antibodies in a biological
sample from the subject. In some embodiments, the method includes
providing a fusion protein comprising a pathogen-specific antigen
fused to a light-emitting protein; contacting a biological fluid
sample (from a subject suspected of having an infectious disease)
with the fusion protein, thereby forming immune complexes if
pathogen-specific antibodies are present in the biological fluid
sample; contacting the immune complexes with magnetic beads coated
with an immunoglobulin-binding protein to form bead-bound immune
complexes; isolating the bead-bound immune complexes by directly
contacting the bead-bound immune complexes with a neodymium magnet;
and detecting emission of light from the isolated bead-bound immune
complexes, thereby diagnosing the subject as infected with a
pathogen.
[0108] In some embodiments, the pathogen is a viral pathogen, a
bacterial pathogen, a fungal pathogen, a parasitic helminth, or a
parasitic protozoan.
[0109] Examples of viruses include, but are not limited to those in
the following virus families: Retroviridae (for example, human
immunodeficiency virus (HIV), human T-cell leukemia viruses;
Picornaviridae (for example, poliovirus, hepatitis A virus,
enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses,
foot-and-mouth disease virus); Caliciviridae (such as strains that
cause gastroenteritis, including Norwalk virus); Togaviridae (for
example, alphaviruses (including chikungunya virus, equine
encephalitis viruses, Simliki Forest virus, Sindbis virus, Ross
River virus, rubella viruses); Flaviridae (for example, hepatitis C
virus, equine non-primate hepacivirus (NPHV), dengue viruses,
yellow fever viruses, West Nile virus, Zika virus, St. Louis
encephalitis virus, Japanese encephalitis virus, Powassan virus and
other encephalitis viruses); Coronaviridae (for example,
coronaviruses, severe acute respiratory syndrome (SARS) virus,
Middle East respiratory syndrome (MERS) virus; Rhabdoviridae (for
example, vesicular stomatitis viruses, rabies viruses); Filoviridae
(for example, Ebola virus, Marburg virus); Paramyxoviridae (for
example, parainfluenza viruses, mumps virus, measles virus,
respiratory syncytial virus); Orthomyxoviridae (for example,
influenza viruses); Bunyaviridae (for example, Hantaan viruses, Sin
Nombre virus, Rift Valley fever virus, bunya viruses, phleboviruses
and Nairo viruses); Arenaviridae (such as Lassa fever virus and
other hemorrhagic fever viruses, Machupo virus, Junin virus);
Reoviridae (e.g., reoviruses, orbiviurses, rotaviruses);
Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae
(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses,
BK-virus); Adenoviridae (adenoviruses); Herpesviridae (herpes
simplex virus (HSV)-1 and HSV-2; cytomegalovirus; Epstein-Barr
virus; varicella zoster virus; Kaposi's sarcoma herpesvirus (KSHV);
and other herpes viruses, including HSV-6); Poxviridae (variola
viruses, vaccinia viruses, pox viruses); and Iridoviridae (such as
African swine fever virus); Astroviridae; and unclassified viruses
(for example, the etiological agents of spongiform
encephalopathies, the agent of delta hepatitis (thought to be a
defective satellite of hepatitis B virus). In some examples, the
viral pathogen is HIV, HCV, EBV, HTLV-1, KSHV, or Ebola virus.
[0110] Examples of bacterial pathogens include, but are not limited
to: Helicobacter pylori, Escherichia coli, Vibrio cholerae, Borelia
burgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M.
tuberculosis, M. avium, M. intracellulare, M. kansaii, M.
gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria
meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group
A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans group), Streptococcus faecalis,
Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus
pneumoniae, pathogenic Campylobacter sp., Enterococcus sp.,
Haemophilus influenzae, Bacillus anthracis, corynebacterium
diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,
Clostridium perfringers, Clostridium tetani, Enterobacter
aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides
sp., Fusobacterium nucleatum, Streptobacillus moniliformis,
Treponema pallidium, Treponema pertenue, Leptospira, Bordetella
pertussis, Shigella flexnerii, Shigella dysenteriae and Actinomyces
israelli.
[0111] Examples of fungal pathogens include, but are not limited
to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides
immitis, Blastomyces dermatitidis, Chlamydia trachomatis and
Candida albicans.
[0112] Other pathogens (such as parasitic pathogens) include, but
are not limited to: Plasmodium falciparum, Plasmodium vivax,
Trypanosoma cruzi and Toxoplasma gondii. (Plasmodium species),
amoebiasis (Entamoeba species), giardiasis (Giardia lamblia),
toxoplasmosis (Toxoplasma gondii), cryptosporidiosis
(Cryptosporidium species), trichomoniasis (Trichomonas vaginalis),
Chagas disease (Trypanosoma cruzi). Leishmaniasis (Leishmania
species), sleeping sickness (Trypanosoma brucei), amoebic dysentery
(Entamoeba histolytica), acanthamoeba eeratitis (Acanthamoeba
species), and primary amoebic meningoencephalitis (Naegleria
fowleri)
[0113] Examples of helminth pathogens include Strongyloides
stercoralis (causes strongyloidiasis); Onchocerca volvulus (causes
river blindness/Robles disease); Loa loa (filarial nematode that
causes Loa boa filariasis); and Wuchereria bancrofti (roundworm
that causes lymphatic filariasis).
[0114] In one non-limiting example, the method diagnoses a subject
as having HIV by detecting antibodies specific for reverse
transcriptase.
VI. Luciferase Immunoprecipitation Systems (LIPS)
[0115] LIPS has been previously described in, for example, Burbelo
et al., J Vis Exp 32:1549, 2009; Burbelo et al., Expert Rev
Proteomics 8(3): 309-316, 2011; Burbelo and O'Hanlon, Curr Opin
Rheumatol 26:717-723, 2014; and Burbelo et al., Transl Res
165(2):325-335, 2015. LIPS is a fluid-phase immunoassay that
employs light-emitting tagged antigens (e.g., Renilla luciferase
(Ruc)-tagged antigens) to detect antibodies specific for target
antigens, such as autoantigens or pathogen-specific antigens.
Chimeric nucleic acid molecules encoding antigens fused to a
light-emitting protein, such as Renilla luciferase, are expressed
in mammalian cells, and crude extracts are prepared and used in
immunoprecipitations assays to yield quantitative antibody
profiles. LIPS has been shown to be capable of detecting both
linear and conform-specific epitopes.
[0116] In many cases, the gene encoding the 30-kDa Renilla
luciferase (from the soft coral Renilla reniformis) is used as the
reporter in LIPS because it has a highly linear output spanning
over seven orders of magnitude. Mammalian expression vectors
encoding Ruc-antigen fusions are constructed using standard
molecular biological techniques. For example, the pREN2 vector can
be used to fuse antigens in frame with Ruc. Any one of a number of
protein antigen targets can be used with this system, including
full-length proteins, protein variants, protein fragments and short
peptides.
[0117] To perform LIPS, plasmids encoding light-emitting antigen
fusions are transfected into mammalian cells, such as Cosl cells.
Since the antigen is directly tagged with the light-emitting
protein, crude extracts can be used without the need for protein
purification following expression. Crude extracts of the
light-emitting protein-antigen fusions can be stored frozen for
later use. Defined amounts (based on light units) of the
light-emitting protein-antigen fusion is incubated with the serum
sample. If antigen-specific antibodies are present in the serum,
they bind to the fusion protein. The reaction mixture is then
transferred to a filter plate containing antibody capturing
reagents, such as Protein A/G beads or secondary
immunoglbulin-immobilized beads. Unbound luciferase-tagged antigen
is removed from the microtiter filter plate by multiple washing
steps. The relative amount of antibody bound to the
luciferase-tagged antigen can be determined by measuring the light
produced in the presence of luciferase substrate.
[0118] Although LIPS alone is more rapid than performing a standard
ELISA or Western blot, the assayusually takes about 2.5 hours and
requires extensive washing steps usually on a vaccum manifold.
However, the methods disclosed herein, which combine LIPS with a
neodymium magnet, greatly improve the speed of the assay such that
antigen-specific antibodies can be detected, with high specificity
and sensitivity, within one minute. The neodymium magnet is capable
of rapidly and efficiently collecting suspended paramagnetic beads
bound to immune complexes. Furthermore, there is very low
background binding to the neodymium magnetic sticks.
[0119] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the disclosure to the particular features or
embodiments described.
EXAMPLES
Example 1
Rapid Antibody Detection for the Diagnosis of Autoimmune and
Infectious Diseases
[0120] Disclosed herein is the development of "LIPSTICK" for
measuring antibodies by employing magnetic sticks in
immunoprecipitation (FIG. 1). In this method, cell extracts of
luciferase-antigen fusions and sera are incubated together, which
is then followed by the addition of paramagnetic protein A/G beads.
Next axially, magnetized neodymium magnets (2''.times.0.75'') are
used to capture protein A/G-antibody-antigen complexes. The magnets
are washed twice in buffer and the antigen-specific antibody is
quantified by placing the magnet in a tube of coelenterazine and
the luciferase activity is measured in a tube luminometer. In
addition to the single, simple washing step for LIPSTICK, one major
difference compared to the previous LIPS formats is the deployment
of paramagnetic protein A/G beads, which have a much lower
IgG-binding capacity (0.5 .mu.gG/ml) than the previously employed
ULTRALINK.TM. protein A/G beads (vs. 25 .mu.g/ml). However, unlike
the protein A/G ULTRALINK.TM. beads which settle out quickly during
the incubation period, the paramagnetic protein A/G beads remain in
suspension (FIG. 1).
Results
[0121] Detection of autoantibodies against three different
autoantigens, Ro52, Ro60 and La) is useful for the diagnosis of the
autoimmune disease, Sjogren's syndrome (SS) (Burbelo et al.,
Autoimmunity 42(6):515-524, 2009). To examine the characteristics
of the LIPSTICK assay, a previously described Renilla luciferase
(Ruc)-Ro52 fusion protein (Burbelo et al., Am J Transl Res
2(2):145-155, 2010) was utilized with representative serum samples
from a seronegative, control subject and a seropositive patient
with Sjogren's syndrome (SS). The assay conditions for initial
testing consisted of 0.1 .mu.l of sera, 200 million light units
(LU) of Ruc-Ro52 as input, which were tested by a short 25 second
incubation step for formation of the antigen-antibody complex,
followed by adding the paramagnetic protein A/G beads for 25
seconds, and then a single wash and LU read (10 seconds). As shown
in FIG. 2A, this one minute assay demonstrated that increasing the
amount of paramagnetic protein A/G beads over a range of 2-20 .mu.l
yielded LU that were proportional to the amount of paramagnetic
protein A/G beads employed. Over the different protein A/G
concentrations, there were over 20 times more light units with the
SS sample than the control samples.
[0122] Using 10 .mu.l of the Ruc-Ro52 extract as input, but varying
the amount of serum from 0.001 .mu.l to 4 .mu.l in the one minute
assay, demonstrated that the highest signal was obtained with 0.1
.mu.l of serum (FIG. 2B), which likely reflected the maximum amount
of immunoglobulin captured by this volume of paramagnetic beads.
The LU signal in the seropositive SS sample also increased roughly
linearly with the addition of increasing amounts of the Ruc-Ro52
extract (FIG. 2C).
[0123] Lastly, extending the assay time from 30 seconds to 4
minutes produced a 5-fold increase in the signal to noise ratio,
yielding an overall difference between the seronegative and
seropositive samples of 50-fold (FIG. 2D). Additional experiments
revealed that the formation of the antibody-antigen complex during
the first incubation step was rate-limiting and that the second
incubation step involving binding of the immunoglobulin-complex to
the paramagnetic beads occurred essentially instantaneously.
Overall, these experiments highlight the linearity and robustness
of the signal Obtained over a wide range of LIPSTICK conditions and
suggests that only a single, rapid and simple wash step of the
neodymium magnetic sticks is sufficient to produce useful
serological data.
[0124] Based on a one-minute LIPSTICK format (0.1 .mu.l of sera,
200 million Ro52 input and 10 .mu.l of paramagnetic protein A/G
beads), a cohort of control (n=20) and SS (n=28) serum samples were
evaluated and the results were compared with the standard 2.5 hour
LIPS format. For all further mention of diagnostic performance, the
mean plus three standard deviations of the controls was used to
calculate sensitivity and specificity. As shown in FIG. 3, LIPSTICK
demonstrated a dynamic range of 4,330 to 856,347 LU within the
cohort of controls and SS samples. Evaluation of the sensitivity
and specificity of a LIPSTICKS test for detecting SS revealed 54%
sensitivity (15/28) and 100% specificity and produced results with
a lower sensitivity (75% sensitivity and 100% specificity) than the
2.5 hour LIPS test. Several of the SS samples detected in the
standard LIPS assay were not detected by LIPSTICKS, which were
likely due to the presence of low affinity and/or low titer
antibodies in these samples.
[0125] To determine the performance of another autoantigen in the
LIPSTICKS format for the diagnosis of SS, the Ro60 autoantigen was
tested (Ching et al., PLoS ONE 7(2):e32001, 2012). The input for
the Ruc-Ro60 LIPSTICK test was 200 million. The Ro60 LIPSTICK test
demonstrated 57% sensitivity (16/28) and 100% specificity and
produced diagnostic results again slightly lower than the 2.5 hour
Ro60 LIPS test (FIG. 4A). Based on the ability of combining
antigens in LIPS (Burbelo et al., Transl Res 165(2):325-335, 2015;
Burbelo et al., PLoS Negl Trop Dis 3(5):e438, 2009; Burbelo et al.,
Clin Vaccine Imunol 16(5):621-627, 2009), a mixture of Ro52 and
Ro60 (200 million of each extract) was used in LIPSTICK in the one
minute assay. As shown in FIG. 4B, there was an additive response
employing Ro52 and Ro60 together as a mixture in LIPSTICK testing,
which showed improved sensitivity of 64% sensitivity and 100%
specificity than using either by itself. These results with the one
minute LIPSTICK test are also as good as a clinical ELISA that
takes 3-4 hours to perform. These findings highlight how mixtures
of antigens can be analyzed simultaneously in LIPSTICK to improve
diagnostic performance.
[0126] A third autoantigen, La/SSB (Burbelo et al., Autoimmunity
42(6):515-524, 2009), was also evaluated with the cohort. The input
for each assay was approximately 250 million. In contrast to Ro52
and Ro60 LIPSTICK testing, only a few of the samples were
seropositive for La compared to the standard assay (compare FIG. 5A
and FIG. 5C). To determine if longer incubation might improve the
detection of the SS samples containing low affinity and/or low
titer autoantibodies, the assay was extended to 59 minutes and
protein A/G beads were added for only one minute and processed. As
shown in FIG. 5B, lengthening the time improved the signal and
sensitivity. The one minute La LIPSTICK test yielded 25%
sensitivity (7/28), the one hour La LIPSTICK test yielded 53%
sensitivity (15/28) and the standard 2.5 hour LIPS test yielded 61%
sensitivity (17/28). The clinical ELISA test only has 45%
sensitivity.
[0127] To determine the effectiveness of LIPSTICKS for the
diagnosis of an infectious agents, a Ruc-HIV reverse transcriptase
antigen (Burbelo et al., J Infect Dis 209(10):1613-1617, 2014;
Burbelo et al., Biochem Biophys Res Commun 352(4):889-895, 2007)
was evaluated for HIV testing in the one minute rapid LIPSTICK
format. In this assay, only 20 million of total input LU was added
per sample. As shown in FIG. 6, using only 20 million LU of input
showed a low background in all the healthy uninfected controls.
However, seropositive signals were detected in all 13 HIV samples.
These g results represent the fastest diagnostic test for HIV that
has ever been developed.
Diagnostic Applications
[0128] Described herein is a simple and rapid immunoassay format
based on performing an immunoprecipitation reaction in solution and
capturing the immune complexes on the end of a magnet. It is
believed that LIPSTICKS can be used to obtain a diagnostic result
in less than five minutes for any one of a number of infectious and
autoimmune diseases. One of the key features of the assay is the
magnets that show low background binding. Although magnets have
been used previously used in various scientific applications, a key
feature of LIPSTICKS is to directly use the magnet in the reaction
and not on the outside of the vessel. For manual testing, the
magnets are recyclable and can be used over and over again. The
magnets can also be used with hand-held luminometers (which are
commercially available) to further enable the assay for POCT. The
use of the magnetic sticks provides a simple format for processing
the reaction and eliminates extensive washing and liquid handling
steps that are needed in other immunoprecipitation formats. In
addition to the simple wash step needed for LIPSTICK, one major
difference compared to the previous LIPS format is the deployment
of paramagnetic protein A/G beads, which have a much lower
IgG-binding capacity (0.5 .mu.g IgG/ml) than the previously
employed ULTRALINK.TM. protein A/G beads (vs. 25 .mu.g/ml).
However, unlike the protein A/G ULTRALINK.TM. beads which settle
out quickly during the incubation period, the paramagnetic protein
A/G beads remain in suspension (FIG. 1).
Example 2
Ultrarapid Measurement of Diagnostic Antibodies by Magnetic Capture
of Immune Complexes
[0129] Rapid and inexpensive antibody quantitation is needed for
clinical diagnostics. This example describes LIPSTICKS, a simple
and robust fluid-phase immunocapture method utilizing neodymium
magnetic sticks to capture protein A/G coated paramagnetic beads
bound to antibody/luciferase-labeled antigen complexes. The data in
this example demonstrates that this system effectively measures
specific antibody levels in serum samples from subjects with a
variety of different infectious or autoimmune disorders. In the
case of Sjogren's syndrome, antibody levels are measured directly
from saliva, requiring only about one minute per assay.
Background
[0130] There is great interest in developing point-of-care (POC)
clinical immunoassays to detect antibodies for the rapid diagnosis
of infectious and autoimmune diseases. Solid-phase POC formats such
as lateral flow immunoassays and miniaturized ELISAs show great
promise, however, the number of available tests are few and they
require 10 minutes to several hours for completion, often not
yielding quantitative results (Chin et al., Nat Med
17(8):1015-1019, 2011; Laksanasopin et al., Sci Transl Med
7(273):273re1, 2015).
[0131] Fluid-phase immunoprecipitation assays efficiently detect
linear and conformational epitopes and typically show the highest
sensitivity for assessing the presence and the level of serum
antibodies in various autoimmune and infectious diseases (Liu and
Eisenbarth, Clin Immunol 125(2):120-126, 2007; Burbelo Pet al.,
Transl Res 165(2):325-335, 2015). One fluid-phase immunoassay
technology, the Luciferase Immunoprecipitation Systems (LIPS),
utilizes light-emitting luciferase-antigen fusion proteins added to
the sample and recaptured in the presence of specific antibody
reacting with it to detect antibodies in clinical samples (Burbelo
Petal., Transl Res 165(2):325-335, 2015). LIPS has demonstrated
high sensitivity and specificity in the detection of antibodies in
many different infectious and autoimmune diseases (Burbelo Petal.,
Transl Res 165(2):325-335, 2015). LIPS has several advantages over
other methods, including a high signal-to-noise ratio, modular
format and the ability to multiplex. Described herein is an
alternative high-speed, streamlined modification of the LIPS assay,
termed LIPSTICKS.
Methods
Serum and Saliva Samples
[0132] Control and patient serum and/or saliva samples were
obtained from human subjects. Five cohorts of human samples along
with corresponding controls were used: HIV-infected subjects,
Epstein-Barr Virus (EBV)-infected, disseminated non-tuberculosis
mycobacterial (dNTM) infection, autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and
Sjogren's syndrome (SS) samples. Additional samples for animal
health monitoring included uninfected and HNPV-infected serum
samples from horses.
[0133] HIV cohort: Serum samples from HIV uninfected (n=24) and
HIV-infected subjects (n=25) were used. The samples from untreated
HIV-infected subjects had a median viral load of 29,494 copies/ml
(interquartile range of 8,814-79,989). Additional testing from
eight HIV-infected subjects from before and after long term
anti-retroviral treated were also used and have been previously
described (Burbelo et al., J Infect Dis 209(10):1613-1617,
2014).
[0134] EBV cohort: Human samples were serologically evaluated for
EBV infection with an EBV viral capsid antigen (VCA) ELISA (Trinity
Biotech). However, following LIPSTICKS analysis, two samples
detected as positive by LIPSTICKS but negative by ELISA were
further studied in detail and were confirmed to be seropositive for
two additional EBV antigens (p18 and p24) consistent with published
studies. In total, 39 samples (15 EBV uninfected and 24
EBV-infected sera) were studied.
[0135] dNTM: Disease controls and subjects having dNTM with
interferon-.gamma. autoantibodies have been previously described
(Browne et al., N Engl J Med 367(8):725-734, 2012). For LIPSTICKS,
a random set of controls (n=17) and dNTM patients (n=13) was
studied and compared to previous testing.
[0136] APECED: Serum samples from healthy controls (n=17) and
APECED subjects (n=23) were used. Since no available ELISA data was
available for BPIFB1 autoantibodies, the 2.5 hour LIPS assay with a
BPIFB1-Gaussia luciferase detector was used to assess
seropositivity.
[0137] Sjogren's syndrome: Healthy controls and SS patients were
studied, in which the diagnosis of SS fulfilled the revised
European consensus criteria (Vitali et al., Ann Rheum Dis
61(6):554-558, 2002). Autoantibodies against SSA and SSB were
determined by ELISA. Saliva samples obtained from the parotid gland
were obtained from SS (n=17) and healthy volunteers (n=18). Another
set of serum samples from SS (n=29) and healthy volunteers (n=19)
were also studied.
[0138] Equine nonprimate hepacivirus virus (NPHV) infection: A
cohort of horses with and without NPHV infection has been
previously described (Burbelo et al., J Virol 86(11):6171-6178,
2012). A subset of horse samples with (n=4) and without (n=7) NPHV
infection were used in LIPSTICKS.
Plasmids and Recombinant Antigens
[0139] Table 1 provides details about the ten different luciferase
antigen fusion constructs used in this study. Mammalian expression
plasmids expressing Renilla luciferase light-emitting antigen
fusions for HIV p24 (Burbelo et al., J Infect Dis
209(10):1613-1617, 2014), HIV reverse transcriptase (Burbelo et
al., J Infect Dis 209(10):1613-1617, 2014), EBV EBNA1 (Bu et al.,
Clin Vaccine Immunol 23(4):363-369, 2016), NHPV helicase (Burbelo
et al., J Virol 86(11):6171-6178, 2012), human Interferon-.gamma.
(Browne et al., N Engl J Med 367(8):725-734, 2012), human Ro52
(Burbelo et al., Autoimmunity 42(6):515-24, 2009), human Ro60
(Ching et al., J Dent Res 90(4):445-449, 2011) and human La
(Burbelo et al., Autoimmunity 42(6):515-24, 2009), have been
previously described. Two new mammalian vectors expressing Gaussia
and NANOLUC.TM. luciferases for BPIFB1 and Ro52, respectively, were
also generated.
TABLE-US-00003 TABLE 1 Description of Luciferase-Antigen Fusions
Type of Plasmid Disease/Infection Antigen Luciferase Antigen Fusion
PREN-p24 HIV P24 HIV capsid Renilla Luciferase C-terminal pREN2-RT
HIV P24 HIV reverse Renilla Luciferase C-terminal transcriptase
pREN2-EBNA1 EBV EBNA1 Renilla Luciferase C-terminal pREN2-CHV NHPV
Helicase Renilla Luciferase C-terminal pREN2-IFN-.gamma. dNTM
Interferon-.gamma. Renilla Luciferase C-terminal pGaus3-BPIFB1
APECED BPIFB1 Gaussia Luciferase N-terminal pREN2-Ro52 SS Ro52
Renilla Luciferase C-terminal pREN2-Ro60 SS R06O Renilla Luciferase
C-terminal pREN2-La SS La Renilla Luciferase C-terminal pNano-Ro52
SS Ro52 NANOLUC .TM. N-terminal
[0140] To prepare recombinant light emitting proteins, Cos-1 cells
were grown in DMEM supplemented with 10% FBS, 1% glutamine and
penicillin-streptomycin. Cells were seeded in 100 mm dishes the day
before transfection. Transfections (2 .mu.g) were performed with
Fugene-6 (Promega) per the manufacturer's instructions. Forty-eight
hours after transfection, the plates were washed once with PBS,
scraped in 0.2 ml Buffer A (20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM
MgCl.sub.2, 1% Triton X-100), the cells were collected and
centrifuged twice at 13,000.times.g for 4 minutes, then the
supernatants were collected and used immediately. Alternatively,
the extracts are harvested in Buffer A containing protease
inhibitors and 50% glycerol and then stored frozen at -80.degree.
C. Total luciferase activity in 1 .mu.l of each crude extract was
measured by adding it directly to 100 .mu.l of assay buffer and
substrate mixture (Renilla Luciferase Reagent Kit, Promega) in a
clear 1.5 ml microfuge tube, vortexing and immediately measuring in
a luminometer (Turner design 20/20, Promega) for 1 second.
Magnets and Paramagnetic Beads
[0141] Two sizes of neodymium magnetic cylinders ("sticks"; K&G
Magnets) were initially tested: 1.59 mm diameter.times.25.4 mm
thick (Cat #D2X0; 1/8'' diameter X 1'' thick) and 1/16''
diameter.times.25.4 mm thick (Cat #D1X0; 1/16'' diameter X 1''
thick). The 1/16'' magnets produced a better detectable light
signal, which was likely due to less physical quench of the light.
To simplify handling of the magnets during the assay, two different
size magnets were combined, whereby the 1/16'' diameter magnet was
stuck together with the 1/8 diameter such that 1/16'' diameter
magnet side was utilized to capture the paramagnetic beads.
Following the assay, the neodymium magnets were reused after
decontamination and stripping of the bound beads. This was easily
accomplished by first placing in 0.1% bleach disinfectant,
physically removing the paramagnetic beads from the magnets with a
paper towel, and then rinsing the cleaned magnets with water.
[0142] Three different sizes of paramagnetic protein A/G-coated
beads were tested including 1-2.5 .mu. diameter (Product #88803;
Thermo Scientific/Pierce protein A/G magnetic beads), 1 .mu.
diameter Hi-Sur Mag Protein A/G (Ocean Nanotech), and 500 nm
diameter Supermag protein A/G beads (Ocean Nanotech). The two
smaller beads of 1 .mu. and 500 nm diameters needed more time,
approximately 15 and 45 seconds, respectively, to be captured by
the magnets from the 100 .mu.L reaction volumes. Thus, the Thermo
Scientific/Pierce protein A/G magnetic beads were used in
LIPSTICKS. Additional experiments titrating these beads revealed
that 5 .mu.L of beads (diluted 1:5 in water from the stock)
provided an adequate signal to noise output for the LIPSTICKS
assay.
LIPSTICKS Tube Assay
[0143] The LIPSTICKS tube assay involves several steps and can be
performed in approximately 45 seconds, in part due to the tube
luminometer requiring only a 1 second integration time to read the
sample (FIG. 1). To initiate the assay, 5 .mu.l of the diluted
serum sample (1:10 in buffer A) or 10 .mu.l of undiluted saliva was
added to 5 .mu.l of the luciferase-tagged antigen in a 1.5 ml
microfuge tube. For the different antigen fusions used in the
assays, the total input activity was approximately 50-100 million
LU/.mu.l. Next, 100 .mu.l buffer A was added and the reaction
mixture was immediately vortexed for 2 seconds. Then, 5 .mu.l
diluted paramagnetic beads (1:5 in water) was pipetted into the
reaction mix and the tube was tapped two times to evenly disperse
the beads. The magnetic stick was then immersed into the tube
containing the beads for 5 seconds. The magnet was removed and
dipped twice in wash buffer. Lastly, the magnetic stick was placed
in a tube, preloaded in the luminometer (Turner design 20/20)
containing 100 .mu.l coelenterazine substrate, and then read for an
integration time of 1 second.
Handheld LIPSTICKS
[0144] The hand-held, battery operated, portable, EnSURE
luminometer (Hygiena) was also employed for testing. In order to
measure luciferase activity from the beads bound to the magnet, the
Ultrasnap cap tubes supplied by the manufacturer (Hygiena) were
first emptied of their contents and then rinsed three times with
distilled water. For LIPSTICKS testing, the cleaned, empty tubes
were refilled with 100 .mu.l Nanoglow substrate (Promega). The
magnet was then simply dropped into the Ultrasnap tube, recapped
and placed in the EnSURE luminometer. An integration time of 15
seconds was used to read the light emitted by the antigen/antibody
complex. Unlike Renilla luciferase antigen fusions, the
Ro52-NANOLUC.TM. produced a high output with a stable glow with its
substrate and is highly detectable during the long integration time
with the handheld luminometer. Due to additional integration time
of 15 seconds, these tests required 1 minute for completion. For
additional comparison, LIPSTICK testing of the Ro52-NANOLUC.TM. was
also performed with the tube luminometer.
Results
[0145] LIPSTICKS is based on the magnetic capture of
luciferase-tagged immune complexes for the ultrafast measurement of
antibodies in clinical samples (FIG. 1). In LIPSTICKS, extracts
from cells producing recombinant luciferase-antigen fusion proteins
are incubated with serum or saliva samples in a microfuge tube,
followed by the addition of buffer and then paramagnetic protein
A/G-coated beads. A unique feature of the assay is the use of
axially neodymium magnetized sticks that are placed directly into
the reaction mixture, capturing in seconds the immune complexes.
Non-specifically associated labeled antigen is removed by simply
dipping the sticks in wash buffer. The antigen-specific antibody is
measured by placing the sticks into tubes containing coelenterazine
substrate preloaded in a luminometer and the luciferase activity
present is quantified in light units (LU).
[0146] It was found that the detection of antibodies by LIPTICKS
was affected by several parameters. With the aid of a tube
luminometer, the assay was optimized using a Renilla luciferase-HIV
p24 antigen fusion protein (Burbelo et al., J Infect Dis
209(10):1613-1617, 2014) and serum samples from HIV-infected and
uninfected subjects. It was determined that 1.59 mm diameter
neodymium magnetic sticks bound with beads produced a larger LU
signal than 3.18 mm diameter sticks because they quenched less
luciferase-generated light. Additional comparisons of various bead
diameters revealed similar immunoglobulin-binding capacities, but
the 1.mu.m diameter protein A/G beads were chosen because they
required only a short time (<5 sec) to be captured (Table
1).
[0147] Varying the amount of serum from 0.001 to 4 .mu.L in the one
minute assay produced 24-200 times more LU from the HIV-positive
compared to the uninfected sample (FIG. 7A). The highest
signal-to-noise ratio for detecting antibodies was obtained with
0.1 .mu.L, serum, yielding 21,740 LU for the HIV-negative sample
vs. 4,545,000 LU for the HIV-positive sample yielding acceptable
positive and negative signals given their magnitude. Using more
than 0.1 .mu.L of serum decreased the signal due to uncomplexed
immunoglobulins competing and displacing the binding of the
luciferase-tagged antigen-antibody complexes to the protein A/G
beads. The addition of larger amounts of the luciferase-p24 extract
demonstrated a roughly parallel increase in the light signal in
both the seronegative and seropositive samples (FIG. 7B).
Similarly, varying the amount of beads over a range of 0.1 to 5
.mu.L also yielded linearity in the LU signal in both the negative
and positive samples, but 1 .mu.L beads yielded a sufficiently high
signal-to-noise ratio (FIG. 9A). Lastly, extending the incubation
time from one minute to five minutes generally produced a 2-fold
increase in the signal-to-noise ratio (FIG. 9B).
[0148] To evaluate the performance of LIPSTICKS for sensitivity and
specificity, a luciferase-HIV reverse transcriptase (RT) antigen
(Burbelo et al., J Infect Dis 209(10):1613-1617, 2014) was used in
the assay with a cohort of uninfected (n=24) and HIV-infected
patient serum samples (n=24). As shown in FIG. 7C, the geometric
mean level of antibodies in the HIV-infected samples was 291,000 LU
(95% CI 180,100-470,000), 78-fold higher than the uninfected
controls, where the value was 3,742 LU (95% CI 3417-4099). This
rapid testing was highly reproducible with a coefficient of
variation 16%.+-.3%. The mean of the controls plus three standard
deviations was used as a cutoff value, which revealed 100% (28/28)
sensitivity and 100% specificity for HIV diagnosis. Additional
rapid testing of serum samples from before and after long term
anti-retroviral treated HIV patients showed a statistically
significant drop in anti-RT antibody levels (FIG. 10), which
previously was not observed using the standard LIPS format (Burbelo
et al., J Infect Dis 209(10):1613-1617, 2014). These results
suggest that the rapid, non-equilibrium conditions of LIPSTICKS are
useful for monitoring antibody changes associated with HIV
treatment. Lastly, analysis with a luciferase-HIV p24 capsid fusion
protein as probe, an antigen known to have lower diagnostic
sensitivity than HIV RT, demonstrated 96% sensitivity and 100%
specificity (FIG. 11).
[0149] Disease-related antibodies were evaluated by LIPSTICKS for
two additional infectious diseases. First, a luciferase-EBNA fusion
protein (Bu et al., Clin Vaccine Immunol 23(4):363-369, 2016) was
utilized in a cohort of seronegative and seropositive EBV human
serum samples. Compared to a VCA EBV ELISA, two positive samples
were not detected by LIPSTICKS (FIG. 7D). However, two other ELISA
negative samples were positive by LIPSTICKS and additional testing
confirmed them as true EBV positives (FIG. 7D). Thus, the one
minute assay produced results with 92% sensitivity and 100%
specificity and showed identical diagnostic performance to an
ELISA, which requires several hours for completion. Additionally,
LIPSTICKS was used for a potential veterinary application, the
serological detection of equine nonprimate hepacivirus virus (NPHV)
infection (Burbelo et al., J Virol 86(11):6171-6178, 2012). As
shown in FIG. 12, a luciferase-NHPV helicase antigen fusion
detected seropositive horse serum samples and produced results
similar to the standard LIPS assay.
[0150] The efficacy of LIPSTICKS for the detection of
autoantibodies in several autoimmune diseases was also assessed.
First, anti-interferon-.gamma. autoantibodies were examined, which
are associated with an acquired immune condition often
characterized by non-tuberculosis mycobacterial infection (Browne
et al., N Engl J Med 367(8):725-734, 2012). Similar to previous
results with the standard LIPS assay, ultrarapid testing with
neodymium magnets showed 95% sensitivity and 100% specificity (FIG.
8A). Next, autoantibodies against BPIFB1, a secreted protein, which
have been reported to be associated with interstitial lung disease
in patients with autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) were
evaluated (Shum et al., Sci Transl Med 5(206):206ra139, 2013). For
these studies, a Gaussia luciferase reporter, which expresses
better for secreted proteins, was fused to the C-terminus of
BPIFB1. Testing by LIPSTICKS with the BPIFB1-Gaussia luciferase
fusion protein showed 95% sensitivity and 100% specificity in the
cohort compared to the standard 2.5 hour LIPS assay (FIG. 8B).
[0151] Sjogren's syndrome (SS) involves chronic inflammation and
autoimmune attack on the salivary and lacrimal glands resulting in
the loss of saliva and tear production, respectively (Fox, Lancet
366(9482):321-331, 2005). Autoantibodies against SSA, composed of
the Ro52 and Ro60 proteins, and SSB (La protein), are present in a
subset of patients with of SS (Burbelo et al., Autoimmunity
42(6):515-24, 2009). It has been previously shown that saliva can
be used in LIPS for the assessment of autoantibody levels in SS
(Ching et al., J Dent Res 90(4):445-449, 2011). Due to the
simplicity of collecting saliva directly, a cohort of saliva
samples (10 .mu.L) from healthy volunteers (n=18) and SS patients
(n=17) was tested. The results were compared serum testing of these
same subjects based on clinical diagnosis and SSA ELISA data
measuring both Ro52 and Ro60 together. As shown in FIG. 8C, the
Renilla luciferase-Ro60 saliva LIPSTICKS test had 70% sensitivity
(100% specificity) for SS diagnosis and showed identical results
with the serum ELISA test. The Renilla luciferase-Ro52 saliva
LIPSTICKS test on the same samples was less informative with 53%
sensitivity (100% specificity) for SS diagnosis (FIG. 8D). These
results demonstrate that saliva is a practical clinical sample for
use in a one minute Ro60 autoantibody test for the diagnosis of
Sjogren's syndrome.
[0152] LIPSTICKS was also performed with a different cohort of
serum samples from healthy controls and SS patients using Renilla
luciferase-Ro60 and Renilla luciferase-La autoantigen extracts and
the results were compared with clinical ELISA data. As shown in
FIG. 8E, much higher LU values for Ro60 autoantibodies were
observed in the SS cohort compared to the healthy controls,
yielding 59% (17/29) sensitivity and 100% specificity and produced
identical results with the conventional ELISA. The SSB/La, known to
have lower diagnostic sensitivity (Burbelo et al., Autoimmunity
42(6):515-24, 2009), also showed promising results by LIPSTICKS
with 52% sensitivity and 100% specificity and picked up two
additional positives missed by the ELISA (FIG. 13).
[0153] To extend the LIPSTICKS technique into portable antibody
testing, a handheld, battery operated luminometer designed for
ATP-based assaying of bacterial contamination was utilized
(Omidbakhsh et al., PLoS One 9(6):e99951, 2014). Initial
calibration experiments with the handheld luminometer revealed that
the light output generated by the Renilla luciferase-antigen flash
with coelenterazine substrate or even a glow Renilla substrate was
insufficiently detected by the photodiode detector. To enhance the
luciferase signal, a different luciferase (NANOLUC.TM.) was used,
which has a sustained glow and higher specific activity (Hall et
al., ACS Chem Biol 7(11):1848-1857, 2012). Ro52-NANOLUC.TM. extract
in the LIPSTICKS assay with healthy volunteer and SS patient sera
produced values by the handheld luminometer that tracked those
obtained with the tube luminometer and produced results that
matched the ELISA (FIG. 8F). These findings indicate that LIPSTICKS
is capable of being used with a portable device.
[0154] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only preferred
examples of the invention and should not be taken as limiting the
scope of the invention. Rather, the scope of the invention is
defined by the following claims. We therefore claim as our
invention all that comes within the scope and spirit of these
claims.
* * * * *