Note: Descriptions are shown in the official language in which they were submitted.
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Method for the analysis of circulating antibodies
Technical field
The present invention concerns a method for the analysis
circulating antibodies.
Background
Quick, reliable, and cost effective analytical and diagnostic
methods are desirable.
PCT/SE03/00919 (Amic AB) relates to a micro fluidic system
comprising a substrate and provided on said substrate there is
at least one flow path comprising a plurality of micro posts
protruding upwards from said substrate, the spacing between
the micro posts being small enough to induce a capillary
action in a liquid sample applied, so as to force said liquid
to move. There is disclosed that the device can comprise a
denser zone which can act as a sieve preventing for instance
cells to pass. There is also disclosed an embodiment with
microstructures where the shape, size and/or center-to-center
distance forms a gradient so that cells and the like can be
delayed or separated.
PCT/SE2005/000429 (Amic AB) shows a device and method for the
separation of a component in a liquid sample prior to the
detection of an analyte in said sample, wherein a sample is
added to a receiving zone on a substrate, said substrate
further optionally comprising a reaction zone, a transport or
incubation zone connecting the receiving and reaction zone,
respectively, forming a flow path on a substrate, wherein said
substrate is a non-porous substrate, and at least part of said
flow path consists of areas of projections substantially
vertical to the surface of said substrate, and having a
height, diameter and reciprocal spacing such, that lateral
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capillary flow of said liquid sample in said zone is achieved,
and where means for separation are provided adjacent to the
zone for receiving the sample. There is disclosed an
embodiment where red blood cells are removed.
PCT/SE2005/000787 (Amic AB) concerns a device for handling
liquid samples, comprising a flow path with at least one zone
for receiving the sample, and a transport or incubation zone,
said zones connected by or comprising a zone having
projections substantially vertical to its surface, said device
provided with a sink with a capacity of receiving said liquid
sample, said sink comprising a zone having projections
substantially vertical to its surface, and said sink being
adapted to respond to an external influence regulating its
capacity to receive said liquid sample. It is disclosed that
the device can be used when particulate matter such as cells
is to be removed from the bulk of the sample. It is stated
that red blood cells can be separated without significant
rupture of the cells.
PCT/US2003/030965 (The General Hospital Corporation, and GPB
Scientific LLC) discloses methods for separating cells from a
sample. There is disclosed the separation of cells with
different properties. The devices are closed devices with an
input and output channel and a lid. The device comprises
arrays of obstacles that are capable of binding a population
of cells.
US 2007/0059718 Al discloses methods for detecting and
concentrating biohazard analytes such as bacteria, protozoa,
viral pathogens, and toxins.
There is a need for a robust and reliable method for the
analysis of circulating antibodies.
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Summary of the invention
One object of the present invention is to provide an improved
method for the analysis of circulating antibodies.
There is provided a method for the analysis of circulating
antibodies comprising the steps:
a. providing an analysis device comprising a substrate,
and provided on said substrate at least one sample addition
zone, at least one retaining zone, at least one sink, and at
least one flow path connecting the sample addition zone, the
retaining zone and the sink, wherein the flow path is open and
comprises projections substantially vertical to the surface of
said substrate and having a height (H), diameter (D) and
reciprocal spacing (ti, t2) such that lateral capillary flow
of said sample is achieved and such that cells can flow
through the projections, wherein said retaining zone comprises
at least one affinity binding means to which cell structures
are bound,
b. adding at least one liquid sample to a sample
addition zone, and
c. reading a result,
wherein circulating antibodies directed against the bound cell
structures are determined.
In one embodiment the liquid sample is selected from the group
consisting of human or animal blood, urine, lung liquids,
synovial fluid, wound liquids, saliva, tears, and sweat.
In one embodiment the liquid sample is from human blood.
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In one embodiment the cell structures are part of the
haematological antigen system.
In one embodiment the cell structures are part of the antigens
involved in HIV infection or detection.
In one embodiment the liquid sample is from human blood and is
used for the determination of circulating antibodies directed
against bacteria, viruses or small sized single or multi cell
infectious agents.
In one embodiment the liquid sample is from human bone marrow.
By providing a substrate comprising projections in combination
with a retaining zone where particles and/or cells are
retained by attractive forces, several advantages are
obtained.
,
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The projections give a large surface for the substrate and the
large surface of the retaining zone is an advantage because
cells are bound more efficiently. The kinetics of the analysis
device is improved with the combination of projections and
affinity binding.
The projections in combination with the affinity binding mean
provide a possibility to create a suitable flow of sample
liquid in the device. This allows problems such as unwanted
clogging to be avoided.
A further advantage of the present invention is that the
reading of a result is easier in an open system according to
the present invention. Moreover there are no problems with
entrapped gases in an open system.
Another advantage of using substantially vertical projections
to analyse cells is that this allows design of the projections
so that the cells are handled carefully.
Definitions
Before the present device and method is described, it is to be
understood that this invention is not limited to the
particular configurations, method steps, and materials
disclosed herein as such configurations, steps and materials
may vary somewhat. It is also to be understood that the
terminology employed herein is used for the purpose of
describing particular embodiments only and is not intended to
be limiting since the scope of the present invention will be
limited only by the appended claims and equivalents thereof.
It must also be noted that, as used in this specification and
the appended claims, the singular forms "a", "an", and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to a reaction mixture
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containing "an antibody" includes a mixture of two or more
antibodies.
The term "about" when used in the context of numeric values
denotes an interval of accuracy, familiar and acceptable to a
person skilled in the art. Said interval is + 10 96.
In describing and claiming the device and method, the
following terminology will be used in accordance with the
definitions set out herein.
As used throughout the claims and the description the wording
"affinity binding means to which cells are bound" denotes an
element which binds to cells by attractive forces between the
binding means and the cells.
As used throughout the claims and the description the term
"analysis" means the process in which at least one analyte is
determined.
As used throughout the claims and the description the term
"analysis device" means a device by the aid of which an
analysis can be performed.
As used throughout the claims and the description the term
"analyte" means a substance or chemical or biological
constituent of which one or more properties are determined in
an analytical procedure. An analyte or a component itself can
often not not be measured, but a measurable property of the
analyte can. For instance, it is possible to measure the
concentration of an analyte.
As used throughout the claims and the description the term
"capillary flow" means flow induced mainly by capillary force.
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As used throughout the claims and the description the term
"casing" means an element enclosing a part of or the entire
device.
As used throughout the claims and the description the term
"circulating antibody" means an antibody in solution.
As used throughout the claims and the description the term
"detectable group" means any arrangement of molecules or atoms
that can be detected when present on a substrate.
As used throughout the claims and the description the term
"flow path" means an area on the device where flow of liquid
can occur between different zones.
As used throughout the claims and the description the term
"fluid connection" means a connection in which a fluid can be
transported.
As used throughout the claims and the description the term
"lid" means an element covering a part of or the entire
device.
As used throughout the claims and the description the term
"open" used in connection with capillary flow means that the
system is open i.e. the system is not enclosed. Examples of an
open system include a system without at lid in capillary
contact with the sample liquid. In an open system a lid shall
not be in capillary contact with the sample liquid, i.e. a lid
shall not take part in creating the capillary force.
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As used throughout the claims and the description the term
"reciprocal spacing" means the distance between adjacent
projections.
As used throughout the claims and the description the term
"retaining zone" means a zone where at least some part of a
sample is retained.
As used throughout the claims and the description the term
"sample" means a mixture or a solution to be analysed.
As used throughout the claims and the description the term
"sample addition zone" means a zone where a sample is added.
As used throughout the claims and the description the term
"sink" means an area with the capacity of receiving liquid
sample.
As used throughout the claims and the description the term
"substance" means any pure chemical or biological entity or
any mixture or solution comprising at least one chemical or
biological entity.
Detailed description
In a first aspect there is provided a method for the analysis
of circulating antibodies comprising the steps:
a) providing an analysis device comprising a substrate, and
provided on said substrate at least one sample addition zone,
at least one retaining zone, at least one sink, and at least
one flow path connecting the sample addition zone, the
retaining zone and the sink, wherein the flow path is open and
comprises projections substantially vertical to the surface of
said substrate and having a height (H), diameter (D) and
reciprocal spacing (ti, t2) such that lateral capillary flow
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of said sample is achieved and such that cells can flow
through the projections, wherein said retaining zone comprises
at least one affinity binding means to which cell structures
are bound,
b) adding at least one sample to a sample addition zone, and
c) reading a result,
wherein circulating antibodies directed against cell
structures are determined.
Reciprocal spacing (ti, t2) denotes the reciprocal spacing in
x and y direction in an orthogonal coordinate system. In one
embodiment all projections have the same spacing in x-
direction and/or y-direction. In an alternative embodiment the
projections have different spacings in the x-direction. In one
embodiment the distance of the different projections in x-
direction are t11, t12, t13... In a further embodiment the
projections have different spacings in the y-direction. In one
embodiment the distance of the different projections in y-
direction are t21, t22,
The device comprises a substrate. In one embodiment the
substrate is partly or entirely enclosed by a casing or a lid.
If a casing or a lid is used, the distance between the
substrate is such that the casing or lid does not contribute
to the capillary force acting on the sample liquid.
There is at least one sample addition zone to which sample
liquid is added. There is a flow path in fluid connection with
the sample addition zone and the retaining zone and the sink.
In one embodiment the sample flows in a flow path from a
sample addition zone via a retaining zone to a sink.
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In one embodiment the retaining zone is placed across the
entire path/paths where the sample fluid flows so that no
sample liquid is able to pass by the retaining zone. In an
alternative embodiment the retaining zone is placed so that a
part of the sample liquid passes the retaining zone without
any essential interaction with the retaining zone.
In one embodiment at least one of a) the sample addition zone,
b) the retaining zone and c) the sink, comprises projections
substantially vertical to the surface of said substrate and
having a height (H), diameter (D) and reciprocal spacing (ti,
t2) such that lateral capillary flow of said sample is
achieved and such that cells can flow through the projections.
In one embodiment the height, diameter and reciprocal spacing
of the a) flow path, b) the sample addition zone, c) the
retaining zone, and d) the sink are the same. In an
alternative embodiment the height, diameter and reciprocal
spacing of at least one of a) flow path, b) the sample
addition zone, c) the retaining zone, and d) the sink are
different.
In one embodiment the affinity binding means is selected from
an antibody, an aptamer, a receptor, a ligand, a single chain
antibody, a fragmented antibody, and a lectin.
In one embodiment the micro posts are arranged with micro post
distances of 5-200 pm. In another embodiment the micro post
distances is 20-100 pm.
In one embodiment the micro posts are arranged with micro post
heights of 1-1000 pm. In another embodiment the micro posts
height is 10-100 pm.
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In one embodiment the liquid sample is selected from the group
consisting of human or animal blood, urine, lung liquids,
synovial fluid, wound liquids, saliva, tears, and sweat.
In one embodiment the liquid sample is from human blood.
In one embodiment the cell structures are part of the
haematological antigen system.
In one embodiment the cell structures are part of the antigens
involved in HIV infection or detection.
In one embodiment the liquid sample is from human blood and is
used for the determination of circulating antibodies directed
against bacteria, viruses or small sized single or multi cell
infectious agents.
In one embodiment the liquid sample is from human bone marrow.
Other features and uses of the invention and their associated
advantages will be evident to a person skilled in the art upon
reading the description and the examples.
It is to be understood that this invention is not limited to
the particular embodiments shown here. The following examples
are provided for illustrative purposes and are not intended to
limit the scope of the invention since the scope of the
present invention is limited only by the appended claims and
equivalents thereof.
Examples
Example 1
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Adherence of cells to the an analyse device according to the
invention.
The projections of the chip had different centre to centre
spacing with the largest spacing in the flow direction. The
projections were narrowing towards the top. The height of the
projections was 65 pm. The diameter of the projections at the
bottom was 70 pm and the diameter at the top was 50 pm. The
spacing between the projections were tl = t2 = 31.77 pm at the
bottom of the projections and tl.t2. 51.77 pm at the top of
the projections.
The principle of adherence of cells to the device surface is
exemplified by firm binding of red blood cells (RBC). RBCs was
firmly attached during free flow to a defined area of the chip
surface by means of different principles including RBC
agglutinins, charge and antibodies directed to surface
antigens.
Small amounts (0.1 pl) of lectins 1 mg/ml in 50 mM Na-
phosphate buffer, pH 7.5 were applied in a single lane on the
chip where after 20 pl RBCs 0.8 96- in suspension were applied
and let to flow through the detection zone containing the
lectins. The results showed a variable RBC binding to
different lectins (PHA-E, PHA-M, WGA, Jacalin) with WGA as the
most efficient one. The bound RBCs that are clearly visible by
eye remained attached after washing with 50 pl of buffers
containing e.g. 0.1 96 of the detergent Tween 20. Bound RBCs
was determined quantitatively by adding 10 pl of rabbit anti-
human RBC in combination with 10 pl of Cy5 goat anti-rabbit
IgG.
A firm binding of RBC to the chip surface was also obtained
using antibodies against RBC surface antigens such as
glycophorin, a major surface protein of the human RBC.
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Polylysine of high molecular weight which generally bind cells
firmly in cell cultures was also able to bind RBC in numbers
comparable to wGA.
Attachment of RBC to the 4castchip was also possible using
biotin labelled RBC in combination with deposited
streptavidin. Streptavidin (0.13 pl of 2 mg streptavidin/ml)
in PBS pH 7,5 was applied in a single lane on the chip. 20 pl
of RBCs 1.6 % labelled with Biotin using Sulfo-NHS-biotin was
let to flow through the detections zone containing the
streptavidin. The results showed a clearly visible firm
binding of RBC to the streptavidin and remained attached after
washing with 80 pl of buffers containing e.g. 0.1 % of the
detergent Tween 20.
Example 2
Detection of soluble human antibodies directed to RBC surface
antigens (indirect antiglobin test, IAT).
The principle of antibody detection is exemplified with
detection of anti-D antibodies present in low titre in human
serum. The assay principle involves firm adherence or binding
of viable RBCs on the chip surface by means of deposited
catching e.g. antibodies against RBC surface antigens. The
same device as in example 1 was used. Thus, RBCs that are
transported by free flow through the micro pillars of the chip
are captured by chip bound antibodies located in the detection
zone. A small volume (10 pl)of a human serum samples diluted
1:100 in LISS buffer containing 0.5 % gelatine containing
anti-D antibodies of different titres was applied to sensitize
the RBCs. Following washing (30 pl) the presence of IgG on the
RBC surface was detected using 10 pl of an anti-human globulin
antibody (AHG) conjugated with transfluosphere. The results
showed a dose dependent binding of AHG conjugate to RBCs with
respect to the anti-D antibody titre. Optimal sensitization
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was obtained in the absence of detergent and using low -ionic-
strength saline (LISS) washing buffer containing 0,5%
gelatine.
In the high sensitivity IAT assay the detection with the AHG-
conjugate is done with fluorescent dye combinations with
extremely large stoke shift (the separation between excitation
and emission maxima) like in the Transfluosphere and europium
conjugates.
Example 3
ABO blood group antigen testing
The ABO blood group antigens on RBCs are determined with high
specificity. RBCs from donor blood samples were prepared by
washing twice in LISS buffer and then re-suspended to a 0.8%
RBC solution. The washed donor RBCs (4 %, 20 pl) in LISS
buffer were attached to the device using deposited anti-
glycophorin (1 mg/ml, 0.13 p1/chip). The A- and B-antigens
respectively were detected using 10 pl of monoclonal anti-
RBC-A and RBC-B antibodies followed by 10 pl of an anti-mouse
IgM antibody conjugated with Cy5. The chip was finally washed
with 60 pl of an assaybuffer (20 mM Tris, 0.135 M NaC1, 10 mM
EDTA, 0.1% Tween 20, 1% BSA, pH 7.4). The fluorescence signal
read at 635 nm was clearly positive for A positive RBCs and
negative (equal to background signal) for B positive RBCs
using anti- RBC-A antibodies. The same high specificity was
obtained in experiments with B positive RBCs.
