Note: Descriptions are shown in the official language in which they were submitted.
W;~ 92/22797 PCI'/US92/03680
2110920
--1--
A88AY FOR TB DETBCT~ON OF 8PECIFIC LIGA~
RELATION TO RELATED APPLICATION
This application is a continuation-in-part of the U.S.
Patent Application, serial no. unknown, filed June 13, 1991,
naming Tzenq, et al., as inventors. Priority of subject
matter in this spplication common with subject matter in that
application is hereby claimed.
~CKGRQUND OF THE INVENTION
Recent technological advances have made it po~sible to
tailor assays for a wide variety of analytes, especially tho~e
molecules exhibiting antigenic characteristics, such as
polypeptides, nucleotides, whole cells, and cellular
fragments, to name but a few. In general, moct assays
currently in use tend to use antibodies to "capture~ antigenic
materials in a liquid-p~a~e or a ~olid-phase format.
Neverthelec~, a~says with an improved sensitivity are
needed, as many conditions and di~ea~e~ do not lend themselves
to early diagno~i~ via antigenic detection. In many
instances, it would be preferable to be able to detect an
increase in a specific population of cells or molecules in an
~ organism, which population is produced in response to an
- "invasion" of the organism by a specific antigenic material.
For example, in the case of certain viral diseases, by
the time a sufficiently detectable titer of ~iral particles i5
present in an organism's blood, the time for effective therapy
may well have passed. In addition, diseases that trigger
highly specific, but virtually undetectable, responses by an
organism's immune system, do not lend themselves to accurate
or easy detection via currently-available a6says. Moreover,
agents which provoke a response by a specific population or
subpopulation of immunoglobulins are often not detectable
until the resultinq disease is full-blown, thus limiting
diagnostic, as well as therapeutic, options. For example, an
assay claiming to facilitate the differential detection of
W092/22797 PCT/USs2/036~D
211092~ ~
--2--
various immunoglob~lin species from each other is described in
U.S. Patent No. 4,292,403 to Duermeyex. Specifically, this
assay purports to detect an antigen-specific immunoglobulin of
a particular immunoglobulin class, which classes include IgM,
IgG, IgA, IgD, or IgE, by using anti-antibodies against the
specific immunoglobulin class. Like many other assays
disclosed in the art which use anti-antibodies (e.g., U.S.
Patent No. 4,818,688 to Adamich, et al., No. 4,828,981 to
Maggio, et al., and No. 4,962,023 to Todd, et al.), the assay
described by Duermeyer is complex and involves a multiplicity
of specific reagents. Furthermore, none of the~e as6ays can
easily distinguish an antigen-specific subpopulation of an
immunoglobulin cla~s from other, non-antigen-specific members
of its class. That is, prior to the advent of the present
invention, it has not been a simple matter to detect
subpopulations of specific IgG or IgM molecules, for example,
which are precisely sensitized to a single antigenic
substance.
An illustrative example of the need for as6ays with
increased sensitivity i8 provided by consideration of
available assays for the detection of antibodies, and in
particular, for the detection of immunoglobulins directed
against specific antigens. Lyme Di~ease provides such an
illustrative example.
Lyme Borreliosis was first identified in Lyme,
Connecticut in 1i75. It has now been reported across several
continents, including North America, Europe, Russia, Asia,
Africa and Australia. The disease is caused by the tick-
transmitted spirochete Bome~a b~gdo~en. This infection can
produce a wide spectrum of clinical symptoms which can be
confusèd with other entities. Therefore, precise d~agnosis is
critical.
~yme disease is the most commonly reported tick-borne
illness in the United States. The disease is most pre~alent
WOs2/22797 PCT/US92/03680
2~ 2 0
in the northeast, upper midwest, and west coast states. The
seasonal onset of the disease is synchronous with the ny~phal
stage of the ticks. Thus, the disease is more common in
summer and early fall.
Antibody titers specific to Bomelia bu~do~en are typically
negative during early illness. Patients with only erythema
migrans also rarely have elevated antibody titers. The
specific IgM levels begin to rise two week~ after the onRet of
the disea~e and peak at three to 6iX week~. The specific IgG
levels tend to lag behind the ~pecific IgM titers by
approximately two weeks, but ~re often positive during the
latter part of the clinical stage of erythema migran~ and
usually remain positive during the second and third clinical
stages with manifestations of carditis, neurological disea~e
or arthriti~.
Generally, the ~natural history" or progre~sion of Ly~e
disease may be divided into three clinical stages. The first
~ta~e is cbaracterized by the development of an eKpanding
annular red rash-like ~kin lesion, erythema migrans, which
occurs at the ~ite of the tick bite and typically la~ts two to
four week~. The erythema migrans may be followed by cardiac,
joint and neurological abnormalities in the second stage,
which occurs one to four months after the disease onset. The
last stage is characterized by arthritiE involving a few large
joint~ which may begin as early as three months after onset.
This stage can last several years or may become chronic.
Early diagnosis permits prompt treatment with appropriate
antibiotics that can halt the progression of the disease.
Since the spirochete is often difficult to culture from
affected skin or body fluids of patients, serological
detection of antibodies is considered the best àvailable
diagnostic means for Lyme diseaeQ. Thè specific IgM against
Bome~a bugdo~en is often not detectable during the first two
weeks, but it usually peaks three to six weeks after the
initial infection, and then persists or declines. The
W092/227s7 PCT/US92/036~0
21 iû92()
~ 4-
response of the specific IgG to the spirochete is frequently
not detectable for four to six weeks, but peaks in the
arthritic stage and often remains elevated for years.
Therefore, in response to an express need for an assay
procedure with diverse applicability, which is capable of
detecting specific ligands, including various polypeptides,
polynucleotides, and immunoglobulins, with great sensitivity,
Applicants disclose the present invention. In addition, the
presently-described assays avoid the agglomeration problems of
other assays, which promotes the goals of improved accuracy
and greater resolution. The present invention, which i~
elegant in its simplicity, is hereby disclosed by Applicants,
including its equivalents thereof.
SUMMARY OF THE INVENTION
In one embodiment of the present invention, Applicants
disclose an analytical device for the detection or
determination of an analyte antibody in a bodily fluid
comprising a layer of a plurality of substantially planar
zones adjacent one another and in absorbent contact with one
another, the layer including a sample application zone, a
conjugate zone containing antigen bound to mobile particles,
and a detection zone containing immobilized antigen, wherein
the antigen is the same in both the conjugate and detection
zones and is an antigen that binds with the analyte antibody,
the liquid 6ample is capable of moving from the 6ample
application zone through the conjugate zone and on to the
detection zone, and if the analyte antibody is present in the
sample it is detected in the detection zone.
In another embodiment, the mobile particles are colored
plastic particles or a metal 801- Another variation di6closes
a device wherein the antigen will bind to antibodies which in
turn bind to epitopes of the Bome~abu~do~en microorganism. In
another variation, the mobile particles are colored
3S polystyrene microparticles. In yet another embodiment, the
layer is made from nitrocellulose.
W~92/22797 2 11 0 9 2 D PC~r/US92/03680
The present invention also discloses a method employing
the disclosed device~, which compri~es adding sample su~pected
of containing the analyte antibody to the ~ample application
zone and waiting for sufficient time for the sample to
traverse the layer through the detection zone, and reading
the recults in the detection zone. In one variation, the
mobile particles are colored plastic particleE; or al-etal 601.
In another variation, the antigen used will bind to antibod1es
which in turn bind to epitopes of the ~on~ba b~do~on
microorganism. Yet another aspect discloses that the mobile
particles are colored poly~tyrene microparticles. In another
embodiment, the layer is made from nitrocellulo~e.
The present invention further discloses a proce~s for the
determination of the presence or concentration of an analyte
antibody in a sample fluid which comprises contacting a sample
of the fluid with a fir~t antigen for the analyte antibody,
wherein the first antigen is labelled, in order to form a
soluble complex between the fir~t antigen and the analyte
antibody; contacting the ~oluble complex with a 6econd
antigen, wherein the second antigen is bound to a solid phase
insoluble in the fluid, in order to form an in~oluble complex
of thæ fir6t antigen, the analyte antibody, and the ~econd
antigen; ~eparating the solid phase from the fluid ~ample and
the unreacted, first antigen; measuring either the fir~t,
labelled antigen associated with the solid pha~e of the
unreacted amount of the first, labelled antigen; relating the
amount of first, labelled antigen measured for a control
sample prepared in accordance with the first four steps, the
control sample being free of the analyte antibody, to
determine the presence of the analyte antibody in the fluid
sample, or relating the amount of first, labelled antigen
measured for the fluid sample with the amount of làbelled
antigen measured for samples containing known amounts of
analyte antibody prepared in accordance with the first four
steps in order to determine the concentration of the analyte
antibody in the fluid sample; wherein both the fir6t and
w092/22797 PCT/US92/036~0
2~ g20,
second antigen are the same before they are labelled or
attached to the solid phase; respectively. Those ~killed in
the art will realize that, in the case where the label is a
visible particle, such as a gold sol or colored or colorable
microparticle, the complex fo~med by the labelled antigen and
the analyte antibody may be insoluble.
In yet another embodiment, the label is an enzyme or a
radioisotope. In another alternative embodiment, the antigen
reacts with antibodies that in turn react with epitopes of
lo Bomel~ bu~do~en. Other variations disclose that the label is
alkaline phosphatase and the solid phase i8 a bead, the inner
walls of a test tube or the wells of a microtitre plate;
alternatively, the label is alkaline phosphatase and the solid
phase is a non-chromatographic device.
~5 The present invention further discloses an a ~ay kit
comprising a first antigen bound to a solid phase insoluble in
the fluid to be tested and a reagent containing a second
antigen bound to a label, wherein the ~olid phase and the
reagent are present in sufficient amount to perform at lea~t
one assay for analyte antibody in the fluid, and wherein the
first and second antigen are the same before they are bound to
the solid phase or labelled, respectively. In another
variation, the label is an enzyme or a radioisotope. In yet
another embodiment, the antigen is an antigen that bind~ to
antibodies which in turn bind to epitopes of Bome~a bu~do~on.
Another variation discloses that the solid pha~e is~a bead,
the inner walls of a test tube or a non-chromatographic
apparatus, and the label is alkaline phosphata6e. In another
embodiment, the label is alkaline pho~phata~e and the solid
phase is a non-chromatographic device.
In another embodiment, an a88ay kit i8 disclosed, which
comprises a first antigen bound to a solid phase insoluble in
the fluid to be tested and a reagent containing a second
antigen bound to a label, wherein the solid phase and the
reagent are present in sufficient amounts to perform at least
` W O 92/22797 PC~r/US92/03680
~li332~ ;
one assay for analyte antibody in the fluid, and wherein the
first and second antigens are the Fame before they are bound
to the solid phase or are labelled, re~pectively.
In another embodiment, a process is disclosed, whereby
S the process is for the determination of the pre~ence or
concentration of an analyte antibody in a fluid comprising the
following steps: a) simultaneously contacting a sample of the
fluid with a first and second antigen, wherein the fir~t
antigen is bound to a solid phase insoluble in the fluid and
the second antigen is labelled and provided in a measured in
order to form a insoluble complex between the fir~t and ~econd
antigens and the analyte antibody; b) separating the ~olid
carrier from the fluid sample containing unreacted ~econd,
labelled antigen; c) measuring the amount of the second,
labelled antigen associated with the solid phase or the amount
of unreacted second, labelled antigen; d) relating the amount
of second labelled antigen with the amount of labelied antigen
measured for a control sample prepared in accordance with
steps (a) through (c), the control sample known to be free of
analyte antibody, to determine the prQsence of analyte
antibody in the fluid sample, or relating the amount of
labelled antigen mea6ured for the fluid sample with the amount
~f labelled antigen measured for 6amples containing known
amount of analyte antibody prepared in accordance with ~teps
a) through c) to determine the concentration of the an~lyte
antibody in the fluid sample; wherein the first antigen and
second antigen are the same before they are bound to the solid
phase or labelled, respectively.
In alternative embodiments, the label is an enzyme or
raaioisotope; further, the first and second antigen may bind
to antibodies that in turn bind to epitope~ of Bo~el~b~do~en.
In another variation, the label i8 alkaline phosphatase and
the solid phase is a plastic bead, the inner walls of a test
tube, the wells of a microtitre plate. In ye~ another
embodiment, the label is alkaline phosphata6e and the solid
phase is a non-chromatographic device.
w092/22797 211~ PCT/US92/036gO
, . ` . .
--8--
Another embodiment discloses a proces~ for the
determination of the presence or amount of an analyte antibody
in a fluid sample comprising the steps of: a) contacting a
sample of the fluid with a first antigen, wherein the first
antigen is bound to a solid phase insoluble in the fluid, in
order to form an insoluble complex between the first antigen
and the analyte antibody; b) separating the fluid 6ample
containing the unreacted analyte antibody from the insoluble
complex of the analyte antibody and first antigen; c) reacting
a measured amount of a second, labelled antigen with the
insoluble complex of the first antigen and the analyte
antibody to form an insoluble complex composed of the first
and second antigens and the analyte antibody; d) separating
the solid phase from the unreactQd second, labelled antigen;
e) mea~uring either the amount of second, labelled antigen
a6sociated with the solid phase or the amount of unreacted
~econd, labelled antigen;
f) relating the amount of ~econd, labelled antigen`measured
with the amount of labelled antigen mea6urQd for a control
sample prepar~d in accordance with 6teps a) through e) being
known to be free of the analyte antibody to determine the
presence of analyte antibody, or relating the amount of
labelled ~ntigen measured in the fluid sample with the amount
of labelled antigen measured for 6amples containing known
amounts of analyte antibody prepared in accordance with stQps
a) through e) to determine the concentration of analyte
antibody in the fluid sample; wherein the first and second
antigens, before they are bound to the solid phase or
labelled, respectively, are the same.
In various alternative ~mbodiment6, the label is an
enzyme, a radioisotope, or alkaline phosphata~e. In another
variation, the first and second antigens are both antigen6
that complex with epitopes of the Bon~l~ b~do~en
microorganism. Yet another embodiment discloses a process
W ~ 92/22797 2 1 ~ O 9 2 0 PC~r/US92/03680
wherein the solid phase is a bead, the inner walls of a test
tube, or the wells of a microtitre plate; in another
variation, the solid phase is a non-chromatographic device.
~E~rAILED DESC~RIPTION
The present invention is directed to devices and methodR
for simply and rapidly performing assays that are capable of
differentiating specific molecules, such as antibodie6. While
any convenient solid material may be u6ed as a solid support,
it is preferable to use any one (or more) of several bibulous
or absorbent solid materials, which may be employed to allow
for capillary transport of a liquid away from the interface
Detween the sir and liquid. Various bibulous material~
include paper, cellulose particles, silica gel, cellulosic
beads, glass fiber, filter paper, and the like. The surface
of the solid support should be relatively smooth, 80 ~8 to
allow for the formstion of a concentrated particle 6ite, for
example, in the form of a sharp band or point. The Qize and
shape of the bibulous material may be varied widely depending
upon the purpose of the as~ay. For example, the bibulou~
material may be ~haped as a narrow strip of from about one to
about five millimeters in width, or it may be in any other
convenient geometric or non-gesmetric shape. In each ca~e,
the bibulous material will usually have a support which
provides structural strength. The non-bibulous material may be
a water impermeable layer or coating.
One embodiment of the present invention has the antigens
as capture antigens bound to a porous membrane. A porous
membrane may be comprised of a flexible or rigid matrix made
from any of a variety of filtration or chromatographic
materials including glass fibers, micro-fibers, and natural or
synthetic materials. Fluids should be able to flow into and
pass easily through the porous membrane. The membrane should
also preferably have pore sizes of at least 0.1~ and
preferably no more than 20~. The porous membrane can be used
alone or as part of a more elaborate device. Such devices
W O 92/22797 2 1 ~ O 9 2 0 PC~r/US92~0368~ .
. ~, , ., ~ ~ .
--10--
include the non-chromatographic ICON0 and like devices
described in Valkirs, et al., U.S. Pat. Nos. 4,632,901 ~nd
4,727,01~, issued December 20, 1986 and February 23, 1988,
respectively, herein incorporated by reference. ICON~ is a
s trademark of Hybritech Incorporated (San Diego, CA) for the
devices described in the Valkirs, et al. patents listed above.
More specifically, Valkirs, et al. describe an apparatus
for the detection of a target antigen in a liquid sample,
comprising: (a) a first member which is a porous membrane or
filter and to which is bound an antibody against the target
antigen, which member has upper and lower surfaces, the sample
being applied to the upper surface, and wherein the antibody
is bound within an area smaller than the area of the me~ber to
which the sample is applied; and (b) a ~econd member, which is
a body of absorbent material having a ~urface over which the
first member is placed and having capillaries therethrough in
a direction generally transverse to the surface over which the
first member is placed, which capillaries are in communication
with the pores on the lower surface of the fir6t ~1~ ber BO aB
to draw liquid added to the upper ~urface which has perme~ted
the first member into the capillaries of the me~ber, the
capillary communication between said first and ~econd ~e~ber~
having been established prior to, and maintained during, the
addition of liquids to the apparatus in the immunoassay
process.
Another such device is the TestPack~ device of Abbott
Laboratories (North Chicago, IL), described in European Patent
Application No. 217,403, published April 8, 1987. Still other
devices containing porous membranes useful in the present
invention include the devices of Bauer, et al., U.S. Pat. No.
3,811,8`40, issuèd May 21, 1974; Brown, III, et al., U.S. Pat.
No. 4,916,056, issued April 10, 1990; Cole, et al., U.S. Pat.
No. 4,407,943, issued Oct. 4, 1983; Cole, eg al., U.S. Pat.
No. 4,246,339, is~ued Jan. 20, 1981; Intengan, U.S. Pat. No.
4,440,301, issued April 3, 1984; ~olley, U.S. Pat. No.
4,704,255, issued Nov. 3, 1987; Katz, et al., U.S. Pat. No.
4,496,654, issued Jan. 29, 1985; and Tom, et al., U.S. Pat.
W~s2/22797 PCT/US92/03680
21i~920
--11--
No. 4,366,241, issued Dec. 28, 1982, all of which are
incorporated herein by reference.
The present methods also may be accomplished by
chromatoqraphic methods such as, for example, those de6cribed
in Weng, et al., U.S. Pat. No. 4,740,468, issued April 26,
1988, incorporated herein by reference, and published European
Application No. 186,100 to Yue, et al., published July 2,
1986.
The porous membrane of the present invention may also be
used in chromatographic assays de6cribed, for exaJple, in U.S.
Pat. No. 4,861,711, issued August 29, 1989 to Friesen, et al.;
U.S. Pat. No. 4,855,453, issued August 8, 1989 to Rosenstein,
et al.; U.S. Pat. No. 4,857,453, i~sued August 15, 1989 to
Ullman, et al., all of which are incorporated herein by
reference, and May, et al., EP0 Publication No. 291,194,
published Nov. 17, 1988; Ching, et al., EP0 Publication No.
299,428, published Jan. 18, 1989, and Devereaux, et al., EP0
publication No. 323,605, published July 12, 1989.
The capture antigens may be directly or indirectly bound
to the membrane. The direct binding may be covalent or non-
covalent and may be accompli6hed by any method known in the
art 6uch as, for example, the use of glutaraldehyde and
aminosilanes as well a~ other methods described in
~Immobilized Enzymes", Ichiro Chibata, Halstead Pre~s, NY
(1978); Cuatreca6as, J. Bio. Chem. 245: 3059 (1970); and
March, et al., Anal. Biochem. 60: 149, et seq. (1974). The
non-covalent binding takes advantage of the natural adhe~ion
of antibodies to the non-synthetic and especially the
6ynthetic fiber~. Thus, appropriately buffered 601ution6 can
be mixed with the membrane then evaporated, leaving a coating
of the desired ligand on the membrane.
The non-direct method f or applying the ligand to the
membrane employs microparticles that may be bound to or
entrapped by the membrane, such that the microparticles are
within the matrix of the membrane, on the ~urface of the
membrane, or bound to other particles which are in turn bound
to the membrane. The microparticles may be any 6hape,
W092/22797 PCT/US92/03680
~110~2~ -12-
preferably spherical. The size of the particles should be
such that they do not migrate through the membrane to any
significant degree. The size of the particles may vary, but
in general, they may be slightly larger than the minimum pore
size of the membrane and smaller-than the Daximum pore size,
and in addition or in the alternative, may be larger than the
maximum pore size. Thus, the particle may be bound within the
matrix of the membrane, on the surface of the membrane, or to
other particles which are in turn bound to the membrane. The
particles may be made of a variety of naturally occurring or
synthetic materials. Exemplary of such partic~es are those
made from polyethylene, polyacrylates, polyacrylamide, and
preferably poly~tyrene or naturally occurring materials such
as cross-linked polysaccharides like agaro~e, dextran,
cellulose, starch, or the like. The primary requirement is
that the particles do not contribute a signal, usually light
absorption, that would cause the zone in which the p rticles
were located to have a different gignal than the re~t of the
membrane.
The ligand may be covalently or non-covalently bound to
the microparticle. The binding of ligand to the particle may
be by methods similar to tho~e discus~ed above for binding the
ligand directly to the membrane or other ~ethod~ known to
those skilled in the art. The preferred method for coating
ligands to the microparticles i~ de6cribed in the Ex~mple~
herein.
The particles may be applied (or "spotted"~ to the
membrane in a zone within the surface area of the membrane.
Thus, spotting localizes the antigen-coated microparticles to
a discrete area on the membrane to localize the antigen-coated
microparticles on or within the membrane. Any of the methods
known in the art may be employed. One such method employs
various mechanical means such as, for example, the Sandy
Springs spotting Machine (Germantown, MD) to apply a
suspension, frequently aqueous (nlatexn), to the membrane.
The ~ethods of preparing and using such microparticles
for the instant invention are further discussed in Weng, et
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211032~ :
-13-
al., U.S. Pat. No. 4,740,468, is~ued April 26, 1988 (~ee
especially columns 13-15) ineorporated herein by referenee;
Brown, III, et al., European Patent App. No. 217,403,
published April 8, 1987, ; and A.S. Rubenstein, European
Patent App. No. 200,381, published Nov. 5, 1986.
The separation steps for the various assay formats
(e.g.,, forward, simultaneou~, and reverse) may be performed
by any of the methods known in the art. For membranes and
. .
filters, additional washing with buffer may often be
suffieient, preferably drawing the liquid through the membrane
or filter by applying vacuum to the opposite side of the
membrane or filter or eontacting the oppo~ite side of the
filter or membrane with a liguid absorbing member that draw6
the liquid through, for example, via eap~llary aetion. The
ICON deviee (Hybritech, Incorporated, San Diego, CA), which
i~ preferred for use in one e~bodi~ent of the pre~ent
invention, uses th~ latter method.
In addition, non-ehromatographie devices and assay~ are
appropriate for use aceording to the present invention. Bead
as~ay~, for example, provide an alternative to ehromatographie
as~ays. See, e.g., Geigel, et al., U.S. Pat. No. 4,517,288,
issued May 14, 1985, whieh is ineorporated herein by
reference.
Moderate temperatùres are normally employed for earrying
out the assay. Constant temperature~ during the period of the
mea~urement are generally required only if the assay is
performed without eomparison with a eontrol sample. The
temperatures for the determination will generally range from
about 15--45-C.
The term "labeled antigen" refers to any antigen baving
speeifie reaetivity with the partieular antibody of intere~t.
Sueh an antigen may be labeled by eonventional methods to form
all or part of a signal generating sy~tem. For example, the
antigen may be labeled with radioaetive isotopes, enzymes,
biotin, avidin, ehromogenie or fluorogenie ~ubstances,
W092/22797 PCT/USg2/036~0
2 1 1 ~ 9 2 0 -14-
chemiluminescent labels, colloidal metal particles, colored
microparticles, colorable particles, and colorable latex
particles.
Thus, the labelled antigen may be covalently bound to
s radioisotopes such as tritium, carbon 14, phocphorus 32,
iodine 125, and iodine 131 by methods well known in the art.
For example, Il~ can be introduced by procedures such as the
chloramine-T procedure, enzymatically via the lactoperoxidase
procedure, or by the prelabeled Bolton-Hunter technique.
These techniques plus others are discus~ed in h. Van Vunakis
and J.J. Langone, eds., Methods in Enzymolo~y 70: Part A
(1980). See also U.S. Pat. No~. 3,646,346, issued Feb. 29,
1972, and 4,062,733, issued Dec. 13, 1979, incorporated herein
by reference, for further examples of radioactive
labels. Chromogenic labels are those compounds that absorb
light in the visible ultraviolet wavelengths. Such compound~
are usually dyestuffs and include quinoline dye~,
triarylmethane dyes, phthalein~, insect dyQs, azo dyes,
anthraquimoid dyes, cyanine dyes, and phenazoxonium dyes.
Fluorogenic compounds include tho~e which emit light in
the ultraviolet or visible wavelength sub~equent to
irradiation by light. The fluorogens can be employed by
themselves or with quencher molecules. The primary fluorogens
are those of the rhodamine, fluorescein and umbelliferone
families. The method of conjugation and use for these and
other fluorogens can be found in the art. See, for example,
Langone and Van Vunakis, Methods in Enzymolooy 74: apart C
~1981), especially at pages 3-105. For a representative
listing of other suitable fluorogens, see Tom, et al., U.S.
Pat. No. 4,366,241, issued Dec. 28, 1982, especially at
columns 28 and 29; and U.S. Pat. No. 3,996,345, both of which
are incorporated herein by reference.
Chemiluminescent labels may also be used in t~e present
invention. For example, the labels listed in Maier, et al.,
U.S. Pat. No. 4,104,029, issued August 1, 1978, herein
incorporated by reference, may be used as detection signals in
the present invention.
W O 92/22797 2 1 1 0 ~ 2 0 PC~r/US92/03680 ~
Alternate methods of detection include the u~e of colored
microparticles, colorable particle~, including colorable latex
particles and colloidal metal particles. Colored
microparticle~ and their use in assays known in the art
include, for example,- those described in U.S. Pat. No.
4,703,017, issued Oct. 27, 1987 to Campbell, et al.,
incorporated herein by reference. The use of colloidal metal
particles in assay6 is also well known in the art. See, for
example, U.S. Pat. No. 4,313,734, i~sued Feb. 2, 1982 to
Leuvering; U.S. Pat. No. 4,775,636, issued Oct. 4, 1988 to
Moerman~, et al.; both of which are incorporated herein by
reference, and by Yost, et al., EP0 Pub. No. 298,368,
published Jan. 11, 1989. Colorable particles and colorable
latex particles are also known in the art to be useful as
marker~ and are described, for example, in U.S. Pat. No.
4,373,932, is6ued Feb. 15, 1983 to Gribnau, et al., and U.S.
Pat. No. 4,837,168, issued June 6, 1989 to deJaeger, et al.,
re~pectively, both of which are incorpor~ted herein by
reference.
The6e non-enzymatic signal sy6tems are adequate for the
pre~ent invention. However, tho6e skilled in the art will
recognize that an enzyme-catalyzed ~ignal systQm i~ in general
more ~ensitive than a non-enzymatic sy~tem and is, therefore,
preferred. Catalytic label~ are well known in the art and
include ~ingle and dual (nchanneledn) enzymes ~uch as alkaline
phosphatase, horseradi6~ peroxidase, lucifera~e, ~-
galactosidase, glucose oxidase, lysozyme, malate
dehydrogenase, glucose-6-phosphate dehydrogenase, and the
like. Dual catalytic systems include, for example, alkaline
phosphatase and glucose oxidase using gluco~e-6-pho~phate as
the initial substrate. A ~econd example of a dual catalytic
sy6tem is illustrated by the oxidation of glucose to hydrogen
peroxide by glucose oxidase, which hydrogen peroxide would
react with a leuco dye to produce a signal generator. A more
detailed discussion of catalytic systems can be found, for
example, in U.S. Pat. No. 4,366,241 to Tom, et al.,
W O 92/22797 P ~ /US92/0368~
2ll0.,s~n,.,
-16-
particularly columns 27-40; U.S. Pat. No. 4,843,000, issued
June 27, 1989 to Litman, et al., and U.S. Pat. No. 4,849,338,
issued July 18, 1989 to Litman, et al., all of which are
incorporated herein by reference. A1BO~ ~;ee Weng, et al.,
U.S. Pat. No. 4,740,468, which is also incorporated herein by
reference, especially at columns 2 and 6-8.
The procedures for coupling enzymes to the antigen~ are
well known in the art and are describ~d, for example, in
Kennedy, et al., Clin. Chim. ACTA 70: 1 (1976). Reagents that
may be u6ed for this procedure include, for example,
glutaraldehyde, p-toluene diisocyanate, various carbodiimide
reagents, p-benzoquinone m-periodate, N,N'-o-
phenylenedimaleimide and the like.
- The substrates for the catalytic systems include simple
chromogens and fluorogens such a8 para-nitrophenyl phosphate
(PNPP), ~-D-glucose (plus pos~ibly a suitable redox dye),
homovanillic acid, o-dianisidine, bxomocre601 purpie powder,
4-alkyl-umbelliferone, luminol, para-dimethylaminolophine,
paramethyloxylophine, and the like, with indoxyl phosphate
being the preferred substrate.
Depending on the nature of the label and catalytic signal
producing system, a signal can be detected by irradiating with
light and observing the level of fluorescence; by providing
for a catalyst 6ystem to produce a dye, fluorescence, or
chemiluminescence, where the dye can be observed visually or
in a spectrophotometer and the fluorescence could be observed
visually or in a fluorometer; or in the case of
chemiluminescence or a radio~ctive label, by employing a
radiation counter. Where the appropriate equipment is not
available, it will normally be desirable to have a chromophore
produced which results in a visible color. Where
sophisticated equipment is involved, any of the technigues is
applicable. For example, when the preferred combination of
alkaline phosphatase is used as the enzyme and indoxyl
phosphate ~s the substrate, a color change may be detected
visually for a qualitative positive reaction. For a
W092/22797 PCT/US92/03680
2110921)
-17-
quantitative analysis, the ICON~ reader and accompanying
software (Hybritech Incorporated, San Diego, CA) may be used
according to the manufacturer's in6truction~ and are preferred
for use in the present invention.
While latex particles tend to be preferred for use in
conjunction with the presently-di~closed invention, it 6hould
be appreciated that other particles may be used aucceFsfully.
For example, the particles which are involved in the assay may
be present in the sample, may be added a~ reagents or formed
~ s~u. The n~ture of the particle may vary widely, being
naturally occurring or synthetic, being a single material, a
few materials, or a combination of a wide variety of
materials. Naturally-occurring particles include nuclei,
mycoplasma, plasmids, pl~stids, mammalian cells, unicellular
microorganisms (e.g., bacteria). Synthetic particles may be
prepared from synthetic or natur~lly occurring materials, such
as metal colloids or latex particles made from polystyrene
polyacrylates or naturally-occurring material~, such as
polysaccharides, e.g., agarose, or the like. Non-naturally-
occurring particles may be varied depending upon the
particular as~ay, the protocol for the assay, or other
considerations. (See, e.g., Gould, et al., U.S. Pat. No.
4,837,168, which describes the use of a variety of particles.)
Uniform latex particles (nULPs~) are, in general,
extremely uniform sphere~ of 6mall diameter. Typical
diameters range from le6s than about 0.1~m to about 100 ~.
Particles smaller than 5 ~m are usually prepared by emulsion
polymerization. The result of this process is a ~eries o$
particles with extremely uniform size distribution~.
The principal use for ULPs is in the medical diagnostic
area, wherein the particles are utilized for latex
agglutination test6. Other varieties of particles, 6uch as
amide-modified latex ( HAML~ ) and carboxylate-modified latex
(nCML~) have amide and carboxylic acid groups, respectively,
W O 92/22797 PC~r/US92/03680
~ 1~0920
-18-
on their surfaces. These functional groups permit covalent
bindin~ of ligands -- for example, antigens or antibodies --
to the surface of the ULPs for improved agglutination te~t~.
For example, if one is attempting to measure a particular
antibody ("~b"), an appropriate antigen (nAgN) is coated onto
the latex particles. Since the Ab is divalent, it may bind to
identical sites on two adjacent particle6 and link them
together. Thus, if Ab present in an individual's sample is
mixed with the Ag-coated particles, it will cause
lo agglutination or coagulation of the particles; the~e
aggregates are generally visible to the naked eye. This
phenomenon is, essentially, the basis for latex agglutination
te8ts ( nL~.Tn )
one major difficulty with mobile particles is the fact
that the coated particles tend to spontaneously agglutinate.
Latex suspensions, in particular, are colloidal suspen~ions of
- hydrophobic particles. The stability of the ~uspension is
dependent upon the surface active charges; addition of small
amounts of protein (approximately 10 ~g per mg of latex) can
cause agglomeration, whereas continued addition of larger
amount~ of protein tends to increase particle stability. This
type of agglutination is also a problem in the chromat~graphic
assays using colored or visible particles.
Various methods of addressing this problem and the
related problem of nonspecific agglutination have been
suggested, including the u~e of linkers and spacer~. However,
few of these suggestions prove entirely satisfactory, as they
tend to interfere with the assay, many doing 80 in a manner
that inhibits agglutination. Thi6 is, of course, a completely
unacceptable result for most assays.
The means for detecting a detectable ~ignal at or away
from the concentration site may or may not be an intrinsic
property of the particles. The particleF may be labeled with
a wide variety of materials which allow for detection, such as
radionuclides, dyes, fluorescer~, enzymes, or other convenient
label providing for a detectable signal, either vi~ually
~ .
W092/22797 2 1 1 0 9 ~ O PCT/US92/03680
--19-- ,
observable or detectable by instrumentation. The variou6
labels would normally be covaiently bonded to the particle,
using linking arms as appropriate. The labels may be bound to
the surface or, when feasible, extend throughout the particle.
The size of the particles may vary widely, generally
ranging from about 0.05 to 100 micrometers (~m), more u6ually
from about 0.1 to 75~m. The particles may be charged, either
positively or negatively, may be amphoteric ~r lack any
charge, being neutral. The presence or absence of charge may
affect other parameters involved in the a6say.
A large number of patents have been issued which de6cribe
a wide variety of labels which have found u~e in diagno6tic
assays. Various protocols can be developed where the~e label6
may be used with advantage. Illustrative of 6uch patent6 are
U.S. Pat. Nos. 3,850,752; 4,255,329; 4,233,402; and 4,;208,479.
For performing the a~ay, kit6 can be provided where the
various reagent6 are combinod in predetermined amount6 in
combination with various ancillary materials for co~bination
with the 6ample or for other u~es in the a66ay. In view of
- 20 the wide spectrum of protocols and reagents, a wide variety of
kits may be prepared. In general, where the method involve6
the addition of particles, the kits will include particle~
which have a ligand bound to the particle, either covalently
or non-covalently. Also, there may be a label bound to the
surface of the particle or disper~ed therein, particularly a
dye, which may be colored in the visible range. The following
references describe various labeling methods:
chemiluminescence (Maier, et al., U.S. Patent No. 4,104,029);
colored particles (Campbell, et al., U.S. Pat. No. 4,703,017);
colorable particles (deJaeger, et al., U.5. Pat. No.
4,837,168); or fluorescence (Langone and Van Vunakis, eds,
Methods in EnzYmolooy 74; Part C (1981)). Radioisotopes may
also be u~ed to label the particle6. (See, e.g., Langone and
Van Vunakis, eds, Methods in EnzymoloqY 70; Part A (1980),
which describes radioisotopic labeling methods.) In other
instances, the particle may also be labelled with an enzyme.
WO 92/22797 PCI`/US92/036&0
21~ 2n~`
-20-
Where particles are not to be included, the reagents will
normally involve labelled receptors or ligands, where the
labels provide for a detectable signal and may provide for the
inhibition of migration of the particles present in the as~ay
medium. In addition to the labelled reagents, there may be
ancillary reagents ~uch as buffers, stabilizers, detergent~,
and as appropriate substrates for enzymes, bulking agents, and
the like. Also likely to be included in such kits would be
the wicking material, prepared in strip form or some other
lo convenient shape. In preferred embodiment~ of the invention,
the as~ay kit contain~ desiccant material; more preferably,
the desiccant material is contained within the ~say device or
housing.
The invention can be better understood by way of the
following examples which are representative of the preferred
embodiments thereof, but which are not to be construed as
limiting the ~cope of the invention. `
EXANPLE I
~yme Antibody As~ay
Extracted Lyme antigens are prepared from ~n~abu~d~on
strain B-31 purchased from the American Type Culture
Collection. The microorganism was grown in modified BSK II
Nedium as described below. It was incubated at 33-C and
harvested by centrifugation. The harvested cells were washed
three times with phosphate buffered saline (pH 7.4) containing
S mM MgCl2, then extracted with 2% 6arcosyl in 10 mM tri6
buffered ~olution (pH 8.2) containing 1 mM EDTA. Thi6
extraction procedure was carried out until all the sarco~yl
soluble material was extracted. The incoluble material was
further blended with an Omni mixer. The blended material is
hereinafter referred to as the "Lyme antigensn. It should
further be noted that the same Lyme antigens or Lyme antigens
from the same sourCe may be used to detect Lyme-specific
antibodies in a variety of mammals, including cattle, dogs,
cats and humans.
w~s2/22797PCT/US92/03680
~11û920
-21-
EXANPLE II
PreDaration of Coniuaates
While latex particles are used in the presently-described
experiments, it should be appreciated that other particle6 may
s be used successfully. In addition, while microparticles that
are not conjugated with ligands are added to ligand-particle
conjugates in various examples, it should be under~tood that
there are various embodiments of the pre~ent invention in
which such admixing is not necessary to the practice the
present invention.
Unifor~ latex particles (nULPs~) are, in general,
extremely uniform ~pheres of small diameter. Typical
diameters range from less than about O.l~m to about 100 ~m.
Particles smaller than 5 ~m are usually prepared by emulsion
polymerization.
a. Pre~r~ion of Liaand-Particle Con~uqa~e~
The basic process of ligand-particle conjugation, for
example, via simple adsorption or covalent binding, is well
known in the art, as i~ the use of colored latex particle~,
which increase the resolution and readability of assays.
Various procedures are described, in general ter~, in Bangs,
L.B., "Uniform Latex Particles,~ presented at a work~hop at
the 41st National ~eeting, Amer. A~soc. Clin. Che~., 1989, and
available in printed form from Seragen Diagnostic~ Inc.,
Indianapolis, IN; or Galloway, R.J., "Development of
microparticle tests and immunoassays,~ Seradyn, Inc~,
Indianapolis, IN. These articles, and references cited to
therein, are hereby incorporated by reference.
One method of preparinq coated latex particles, for
example, is the adsorption method. In general terms, one
should: 1) utilize pure reagents; 2) clean the particles
prior to coating; and 3) determine the quantitative surface
covèrage of the particle and the ligand chemistry.
~or example, ligand-latex conjugates ("L-latexN) ~ay be
prepared according to the following method: in the simplest
ca e, the appropriate ligand is dissolved in a buffer
W092/22797 PCT/US~2/0368~
2110~ 22-
solution, added to a latex suspension, and stirred for times
ranging from a few minutes to more than 24 hours. After
equilibration, the latex is centrifuged and the supernatant
containing any unadsorbed ligand is discarded. The latex is
re-suspended in fresh buffer and centrifuged; the supernatant
is again discarded. These steps are to be repeated until the
latex is determined to be wached free of any residual un-
adsorbed ligand. At this juncture, the latex coating process
may be complete and the latex ready to use in latex
agglutination assays.
Covalent coupling involves the permanent or covalent
binding of a ligand or othér material to the latex particle
surface. If covalent coupling is the method of choice, one
must first couple the ligand to the latex particle6, then
maintain the stability of the latex particle suspension,
followed by preventing the protein from becoming denatur~d.
(For a general di~cu~ion of covalent coupling technique6, and
citation to more detailed references, see Bangs, L.B.,
"Uniform L~tex Particles," which has been incorporated herein
by reference.)
While the foregoing di6cus~ion is in the context of latex
particles, it will be appreciated that other particle~,
including, without limitation, naturally-occurring (e.g.
plasmids) or synthetic particles (e.g. polymer6), and metal
colloids or particles (e.g., gold sol particles), may be used.
These particles and their methods of extraction or preparation
are well known in the art.
b. PreDaration of BSA-Latex Coniuaates
Preparation of bovine serum albumin - latex conjugates
("BSA-latex") is similar to ligand-latex (L-latex)
preparàtion, a6 described above, except that no ligand is used
in the preparation, and BSA i6 u~ed instead. Alternatively,
other proteins may be u6ed in place of the BSA, such a~ other
albumins (including lactalbumin), ca6ein, globulin, non-
~pecific immunoglobulin (which does not participate in theantigen-antibody reaction), and the like that can prevent
nonspecific binding.
. .
W092/22797 PCT/US92/03680
21~3~-~
-23-
c. Mixture of Liqand-latex and BSA-latex
L-latex and BSA-latex are mi~ed together in varying
ratios, depending upon the test to be performed. For example,
in preparing mixtures for use as set out in some of the
Examples, L-latex and BSA-latex were mixed in approximately a
2:1 ratio to a 5:1 ratio, volume to volume, for u~e in the
assays. Depending upon the nature of the as~ay, the ratio~
ean vary substantially, with areater amounts of protein-
labeled latex resulting in greater reduetion of non~pecific
binding. The amount of latex (or other partiele) that does
not have ligand attached can be any amount that ia effective
to appreeiably decrease nonspecifie binding, or false
positives. Sueh amounts are readily determined by obvious
empirieal methods.
d. Mixture of Liaand-latex and ~Nak~d" latex
L-latex and naked latex are mixed together in varying
ratios, depending upon the test to be performed, a~ noted
above in regard to Ligand-latex/BSA-latex ~ixtures. As noted
above, the ratios of L-latex to naked latex ean vary
substantially, with greater a~ounts of protein-labeled latex
resulting in greater reduetion of non~peeifie binding. The
amount of latex (or other partiele) that does ~ot have ligand
attaehed ean be any amount that i~ effeetive to appreeiably
deerease nonspeeifie binding, or false positives. Sueh
amounts are readily determined by obvious empirieal methods.
EXAMPLE III
Pre~aration of Bome~a Con~uaate
10 ml of blue earboxylated latex (preferably about 0.4
in diameter) having 50 mg/ml solid, obtained from Magsphere,
is diluted to 0.5 mg/ml in 10% ~uerose and 20mM MES buffer, pH
5.5. Add Sulfo-NHS (Pieree) and EDAC (Sigma, St. Louis, M0)
to a final eoneentration of 1.087 mg/ml and 2 mg/ml,
respeetively. Mix by end-to-end rotation for about 30
minutes. The aetivated latex i~ washed five times with 10%
sucrose, 0.02% SDS in 20mM MES, pH 6.5, in an amieon
W O 92~22797 PC~r/US92/03680
2 ~
-24-
concentrator with 0.2~m nylon membrane. Re-suspend to
approximately 50 mg/ml with 10% 6ucrose, 2mM MES (pH 6.5), and
sonicate four times for 15 seconds each time.
About 0.5 mg of extracted Bomel~ b~do~en antigenB are
mixed in 0.01% SDS, 2mM ME~ buffer (pH 6.Sj, containing 10%
sucro~e and the 500 mg of activated latex ~uspen~ion. The
latex and extracted Bon~a b~do~en are allowed to react for
two hours at 22-C.
To neutralize Furface carboxyl groups not bound to the
extracted Bome~a b~do~on antigen~, a solution of Tris buffer
(pH 8.5), ie added to 20mM, followed by a solution of case~n
(Sigma) and Zwittergent 3-12 (Calbiochem, San Diego, CA) of
0.5% and 0.1%, respectively. The Eon~a b~do~on-latex is
diluted to a desired concentration containing 20% ~ucro~e,
20mM Tris (pH 8.5), 0.1% Zwittergent 3-12, 0.5% casein, and
0.3% BSA-latex, and i~ ready for printing onto the solid
support or matrix.
EXANPLE IV
Reagent Preparation
1. Preparation of BSK-II Medium
To 1 liter of fortified RPMI-1640 without glutamine add
the following and mix well: 5 g Neopeptone (Difco) and 50 g
BSA (Sigma A-4503; St. Louis, M0). Dissolve the neopeptone
and BSA completely and then add the following: 2g TC
Yeastolate (Difco); 6g HEPES (Sigma); 0.7g sodium citrate; 5g
glucose; 0.8g sodium pyruvate; 0.4g N-acetylgluco~amine
(Sigma); and 2.2g sodium bicarbonate. Adjust the pH to 7.6-
7.8.
Modified BSK-II medium was prepared according to the
method set forth by Barbour, A.G., in Yale J. B. Med. 57: 521-
525 (1984), which is incorporated herein by reference. The
modified medium was prepared from fortified RPMI-1640 without
glutamine ~nd 7% gelatin.
Wos2/227s~ 0 9 2 0 PCT/USs2/03680
-25-
2. Inoculation and Harvesting of Bo~e~a Cultures
The equipment used included a 2.2 L Erlenmeyer fla6k; a
vacuum equipped with a trap; 0.2 micron 1 liter filter unit6
(Nalgene 90mm diameter); and a laminar flow hood. The medium
s was inoculated with Bome~a according to appropriate, safe
laboratory procedures. The media flask~ are incub~ted at 33-
35-C overnight as a precaution, to check for contamination.
Signs of contamination include the appearance of turbidity
and/or an acidic pH shift.
EXAMPLE V
Assay PreDaration and Procedures
1. Preparation of solid pha~e
one example of the use of the pre6ent invention in a
solid phase assay format may proceed es~entially as follows.
The illustrated reaction device consists of a 601id --
typically plastic -- housing containing a solid ~upport for
the a6say. Generally, a ~e~brane strip such as nitrocellulo~e
is used; preferably, an i~unochromatographic ~trip i6 u~ed.
For example, the right end of the membrane provides contact
with the sample well. The ~ample well contain6 an absorbent
pad which provides an even flow of the sample fluid from right
to left along the membrane. ~obile particles are applied to
a first zone of the membrane -- for example, latex beads --
which particles are conjugated with the appropriate ligand or
antiligand. For example, antigens extracted from Bon~Ma
b~db~en were used in one experiment described herein.
Various methods of isolating and preparing viral or bacterial
antigens are knGwn in the art. For example, and without
limitation, Hepatitis A antigen may be prepared according to
the method of Markus, et al., as described in U.S. Pat. No.
4,301,249, which is incorporated herein by reference.
In a second zone on the membrane, ligand or antiligand i8
immobilized. A third reagent may be used as well; for
ex2mple, such an agent may be immobilized in a zone on the
solid support. This agent may be capable of binding particle~
~ that migrate from the first zone after sample is added. This
: .
W O 92/22797 PC~r/US92/0368~ .
9 ~ ()
-26-
third agent can act as a procedural control and may serve to
indicate that the assay i~ complete, or that it has been
properly performed, if, for example, a detectable response
occurs in the zone in which the third agent is immobilized.
In a ~typical" chromatographic acsay test procedure, a
liquid specimen is applied to the solid support proximal to
the first zone. As the fluid moves via capillary action to
the first zone of the me~brane, it mobilizes the mobile
particles. The fluid continues to move the particles acro~s
the membrane to the next zone or zones. If analyte iB pre6ent
in the sample, a "sandwich" of solid phase-
antigen/analyte/antigen-conjugated particles is for~ed and a
detectable re6ult occurs. A6 the fluid continues to move the
particles across the membrane, the fluid/particles come into
contact with the third reagent, which is immobilized on the
support. A detectable response should then occur in that
zone, indicating that the test is valid, or that the te~t is
complete.
2. Test Procedure
Place the solid phase assay (or membrane alone) on a
well-lighted, level surface. Place one drop (about 50~L) of
liquid sample in/on the 6ample application area proximal to
the first zone. The application area may also contain a
buffer solution to further promote assay perfor~ance. One
example of such a buffer comprises 2% ca~ein and 0.5%
Zwittergent in O.lM Tris (pH 8.0). Alternatively, the 6ample
may be mixed with another substance -- e.g., saline ~olution -
- prior to administration of the sample or sample mixture to
the sample application area. Immediately thereafter, add
several (preferably about 6) drops of developer solution to
the sample application area. A useful developer solution
contains the following, for example, in aqueous solution: 1%
Zwittergent, 1% Triton x-100, 0.02% SDS, 0.2% NaN3, and 50mM
Tris buffer (pH 8.5).
Allow the test to run for about 10 minutes and then read
t~e results. Although a signal may appear in the third zone
W~92/22797 PCT/US92/03680
_~LL~920
before the lo minutes elapse, the results are likely to be
read with greater accuracy if one wait~ the full 10 minute6.
After the appropriate amount of time elapRe~, provided the
appropriate signal is detected in the approprate zone or
s zones, then the assay results may be read. For example, if a
symbol appears in the third zone, then the test re~ult~ may be
interpreted as valid and negative. If a 6ymbol appears in the
second and third zones, then the re~ult~ may be interpreted as
valid and positive.
Control solutions may further be provided for the purpose
of comparison with as~ay re~ults or to te~t reagent viability.
For example, a positive control solution may comprise about
0.5mg/ml purified rabbit anti-Lyme antibody in 2mg/ml o*
rabbit gamma globulin and 50mM Tris (pH 8.0). A negative
control may comprise 2mglml of rabbit gamma globulin in 50rM
Tris (pH 8.0). These solution~ may be provided in a kit for~
with any or all of the reagentC and as~ay component~ di~clo~ed
herein.
Although the invention has been de~cribed in the context
of particular embodiment~, it is intended that the scope of
coverage of the patent not be limited to those particular
embodiment~, but be determined by reference to the following
claims.
