Note: Descriptions are shown in the official language in which they were submitted.
~0~38
B~CKGROUND OF T~iE INVENTION
The present invention is directed to medical and
laboratory fluid specimen collecting and testing apparatus,
and more specifically to an apparatus for testing for the
presence of specific antigens in biological fluids.
The family of immulloassay works upon the simple
principle that is the specific recognition of an antigen by
an antibody. Thus specific antigen detection and
quantifica-tion requires an antibody which recognizes the
uniqueness of an an-tigen. The antigen binding site of
antibodies recognizes about six amino acids or their
equivalent in mass. One unigue binding site serves as an
identifying marker for that protein.
When a definitive antibody for a given antigen is
available i-t is used to identify the antigen in t~As sample
mi.xture. Once the antibody combines with the antigen a
means is needed to recognize the complex. There presently
exists a need to concentrate antigens from volumes o fluid
when the antigen is not present .tn measurable quanti.ties in
specific fluid volul11es.
The present invention ls directed toward a method
which can use imn1unoassay in sample treatment apparatus for
diagnostic and testing purposes of specific urine antigen by
concentrat:i.ng the specific urine an-tigen by capturing it
with an immobilized an-tibody on a bead in a small volume
area, washing the an-tigen-antibody complex, engaging the
antigen-antibody complex with a solution of prelabelled
primary antibody so that the captured an-tigen bonds with the
prelabelled primary antibody as a sandwich complex and
mixing the antigen-antibody sandwich compl~x with a coloring
reagent to obtain a test color.
I~t is generally necessary in diagnosing and
testing for many diseases to collec-t biological fluids from
a patient, e.g., sputum, blood, pleural and peritoneal
cavity f]uids, urine, etc. for analysis. It is important
during the collection handling o~ biological fluid specimens
that the poten-tial of specimen contamina-tion and the spread
of any infection from the specimen be minimi~ed. While
urine is con1111only collected in lO0 ml containers the actual
urirle testing :is commonly conducted with relatively small
amounts of sample around .2 - .5 ml in volume. Thus because
of the small tes-t quantity, cancer producing antigen can
only be ascertained after the cancer is in an advanced or
late tumor stage. The rest of the urine sample is used for
further testing or is thrown away. Additional problems
occur in shipment when dealing with urine because of the
relatively large volume of fluid invo:Lved in the collection
specimer1 sample. 'rh~re :Ls cllS0 tll~ rls1c of sample
deterioratic)1l because of -the relativel.y short sample shelf
life of urine unless kept in specific temperat1lre
conditions. In addition there is also the potential for
specln1er1 damage or spillage during the collection and/or
shipment process as well as the potential for destruction of
certain rnolecular components of the specimen such as
antigens contained therein, because the packaging does not
Q ~ ~
protec-t the u:rine or causes chemical changes of different
- fluid componen-ts which will negate the test results or
resu~.t in false data being obtained when the specimen is
-tested.
'I'here currently exists a need to provide a tsst
procedure to -test for molecular components of biological
fluids for the presence of cancer at an early stage in -the
development of the cancer.
A typical specimen collecting apparatus is shown
by U.S. Patent 4,741,346. This apparatus includes a base
stand which supports the specimen vial in an upright
position. ~ I.unne:l. is inserted in the open end of the
specimen v.ial arld surrounds and encloses the upper portion
of the via].. The base stand has an upwardly extending
tubular wall which at least partially surrounds the vial in
connection with -the cap and allows the user -to remove -the
vial without touching the surface or coming in contact with
the specimen. Examples of various types of li~uid
containers for collecting and -transporting urine are shown
by U.S. Patents 3,777,739; 3,881,465; A,042,337; 4,084,937;
4,244,920; 4,~92,258 and 4,700,'7:L4.
Ano-ther specimen collec-tion device shown by U.S.
Patent 4,040,791 discloses a collection receptacle haviny a
nipple upon whi.ch is mounted a specimen container which
receives a prede-terlllined amount of the specimen in a sealed
condi.ti.on. The specimen contairler is provided with an
inteyally formed cap which is placed over the openiny :in
which the collector nipple is inserted. U.S. Patent
~,557,274 c~i.scloses a midstream urine collector having a
funnel which transmits urine into a cup member which is
covered by a membrane cover.
A combined strip testing device and collection
apparatus i~s shown by U. S . Patent 4,473,530 and is directed
to an apparatus which integrates testing and collection by
having chemical reagent test strips present within the tube
together with specific gravity reading means allowing
immediate testi.ng of the urina. U.S. Patent 4,573,983 is
directed towards a liquid collection system having an
antiseptic member on the discharge section which uses a
filter of a.tr an(l bacteria impervious material to filter the
urine.
It is therefore desirable to provide an easy to
hand].e method for allowing more sensitive cancer detection
from the sample while also providing that the test specirnen
can be compactly stored for a period of time in concentrated
form allowing cancer testing to be performed quickly and
accurately by distal testing facilities with minimum elapse
of -time.
~ tl-,F SUMM~Y OE~ TIIE INVENTION
The inven-tion is directed toward a urlne antigen
testing process. This method uses a removable stackable
sealed uri.ne al-ltigen sample container having an interior
chamber wi.tll primary antibody covalently bound to beads.
The uri.ne is pumped through the corltainer where it engages
and passes through a filter which allows passage of filtered
urine fluid and antigen through an antibody bead bed. The
beads ill the bead bed have specific antibodies covalently
bound thereto -to capture specific antigen carried by the
urine fluid. ~'he resultant antigen-antibody complex and
associated ~ beads are washed and then recombined with
prelabelled primary an-tibodies in solution to form a
sandwich complex. The sandwich complex is washed and
combined with coloring reayents to give a color indication
if a cancer marking antigen is present in the urine.
I-t is thus an object of the invention,
particularly where ligarlds such as antigens are being
removecl from the body flu:lds for testing to provide a easy
visual color test to determine the presence of specific
antigens in the body fluid samples. Previously such testing
has been accomplished by a series of tests involving a
number of different containers and expensive laboratory
equlpment of a limited sensitivity.
In the accompanying drawings, there is shown an
illustrative elllbodiment of the invention from which these
and other o~ o~Jectives, novel features and aclvalltages w.ill
be readily apparent.
E3RIEF DESCRIPTION OF THE DRAWINGS
....
Figure 1 is an exploded cross sectional schematic
view of the inventive uri.ne testing device;
Figure 2 is a cross sectional schematic view of
the device of figure 1 showing the urine test container
immersed in urine with urine entering the syringe in the
direction of movement shown by arrow A for antigen capture,
Figure 3 is a cross sectional schematic view of
the device of figure 1 showing sequential movement of the
syringe plunger from that shown in figure 2 with urine being
discharged from the syringe with direction of movement shown
by arrow B~and the immobilized antibody beads and captured
antigen positioned in the test container;
Figure 4 is cross sectional schematic view of the
syringe plunger shown in Figure 3 after the urine has been
fully discharged from the syringe, washing the beads to
separate cells, unbound ligands and debris from the antigen-
antibody on t}-e beads;
~ igure 5 is cross sectional schematic view of the
container af-ter wash1llg as shown in Figure 4 mixing the
antigen-an-tlbody complex with prelabelled primary antibody
solution to obtain a sandwich complex bead;
Figure 6 is cross sectional schematic view
showing mixing of the sandwich complex beads after washing
as shown in Figure 4 with coloring reagents which react to
the prelabelled primary antibody on the sandwich complex to
present a readable test color; and
Figures 7(a)-(d) are a pe~spective schematic
/ showing the Einal results of the test which can be read by
the naked eye or using a reader.
DETAILED DESCRIPTION OF TIIE INVE'NTION
The preferred embodiment and best mode of the
invention is seen in Figures 1 through 6 The initial
collection of the urine is normally housed in a graduated
lOO ml container 10. Such a container is currently
manufactured by Bec-ton Dickerson Labware under the
designation 4013 specimen container. This collection
container holds 4.5 oz. (approx. 133 ml) and is graduated
with a pol~ethylene snap lid. The invention utili7Aes a
urine sample con-tainer ll with treatment filter 12 mounted
therein. The filter 12 preferably has a filter particle
size of 5 microns but can range from l-5 rnicrons or any si~e
which is suitable to allow fluid flow with antigens to pass
therethrough but also prevent the passage of beads 50. The
urine sample con-tainer ll can be a disposable sterile single
use filter asselTIbly manufactured by Gelman Sciences under
the trademarlc AC~ODISC with a 5 VM filter. However, any
suitable fil-ter can be used such as the acIueous glass
microfiber filter manufactured by Xydex, a subsidiary of
Genex Corporation or a membrane member manufactured by
Millipore Corporation. One end l~ of the container is fitted
with a threaded projection which is adapted to fit onto the
luer lock of a 30 cc syringe 18, manufactured by Becton
Dickinson & C:o. It should be noted t}lat any pUIllp t~pe
device could be used Ln place of the syrlnge as Eor exalllple
an au-tovlal spunglass f:Llter manufac-tured by Genex
Corporation. The syringe 18 has a barrel 20, piston 22 and
piston head 24. While the invention can be used for any
body fluid it is pr:imarily designed for use in collecting
concentrated urine antigen samples for use in -testing for
the presence of various kinds of cancer in the body to
determine the presence and stage of the cancer.
2 ~
As shown in Figures 1 through 7 a urine sample
container 11 is constructed of polystyrene. The container
hous~ng has walls which define an urine entrance port 36 and
exit port 38. The chamber 40 of the urine sample container
con-tains a'filter 12 with a filter size ranging from .5 to
5 microns mounted at one end and a bed of beads 50 with
immobilized an-tibodies positioned on the syringe side of the
filter.
The beads 50 may be visible so that their flow
into the syringe and bac)c to the container can be visually
observed to malce sure of maximum bead contact with the
urine. ~ntibodies are irnmobilized (covalently bound) on
beads 50 and are designed to have binding sites which have
a high affinity for the epitopes of the cancer rnarking
antigens carried in the urine. It should be noted that the
volume of beads 50 is important and the beads
should not be greater then volume of the contain0r chamber
~0 so that the syringe neck will not become jammed.
The principle of affinity chromatography requires
that a successful separation of a biospecifio llgand is
available and that it can be cheltlioall~ lmmobilized to a
chromatographic bed material, the matrix. Numbers of
methods well known in the art have been used to couple or
immobilize antibodies to a variety of activated resins.
Examples of immobilization techniques which exhibit variable
linkage are those formed by the reaction of -the reactive
groups on the support with amino, thiol, hydroxyl, and
carboxyl groups on the protein ligand. The selection of the
h ~ $
ligand is influenced by two fac-tors. First, the ligand
should exhibit specific and reversible binding affinity for
the substance to be purified and secondly it should have
chemically modifiable groups which allow it to be attached
to the ma'trix without destroying its binding activity.
(Examples of such are Protein G Sepharose manufactured by
Pllarmacia, llydrazide AvidGel Ax manufactured by BioProbe
International, and Actigel-ALD manufactured by Sterogene
Bioseparation Inc.)
An advantage to the use of Actigel-ALD is that
it does not cross link proteins therefore allowing proteins
to re-tain hlgh bioactivity after their immobilization.
~ctigel-ALO SUPER FLOW also available from S-terogene
Bioseparation Inc. permits a linear flow rate of up to 3000
cm/h which would fit nicely with the flow rates in the
apparatus (approx 10-100 cm/min).
The resin beads 50 with ma-trix and primary ligand
(in this case imrnobilized antibody) having had flow contact
with the filtered urine in buffered form from the addition
of 200ml of M Tris buffer, pll 7.8 man~lfactured by Pharmacia,
captures through antigen-antibody reaction or immune
reaction the specific ligand component carried by the urine
namely, the non complexed antigen.
The buffer solution can be added to the cvllection
container 10 by directly adding it from the syringe 18 prior
to withdrawing the urine into the syringe or simply adding
it from another container. When the specific antigen is
present in the urine testing sample 100 the antigen reacts
with the antibody to form antigen-antibody complexes. The
complexed antigen-antibody carried by beads 50 remains in
the housing chamber 40. If there is an absence of the
antigen in the specimen sample 100 the antibody will remain
unoccupied.'
As shown by figures 1-3 the container 11 is placed
on the luer lock of syringe 18 with ports 36 and 38
opened by the removal of caps 37. The container 11 is
screwed into a 30 cc syringe and immersed in urine 100
contained in cup 10. The syringe plunger is pulled
withdrawing 25 cc of the urine sample through the container.
The syringe i.s erllptied tl-rough the container and the steps
of withdraw and discharge are repeated until a total volume
of 100 cc of the urine sample has come in contact with -the
antibody beads 50. The antibody beads are carried by the
urine into the barrel of the syringe each time the urine is
withdrawn from cup 10.
After the urine sample has been processed as
described to concentrate the antigen on the antibody beads,
the beads are washed by withdraw:Lnt3 25 cc of washing
solution 200 throuyh the corrtaint-~r 11 and with(~rawing and
discharying the syringe three times. This washing carrries
off cells, debris, and non bonded ligands which may have
been attached to the bead matrix or antigen-antibody complex
by lodging or charge attraction.
After washing, a solution 300 of lO ml of
prelabelled primary antibody of a 1:500 dilution is
prepared. The total amount or 10 ml of the antibody
solu-tion 300 is withdrawn and passed -through the container
11 to discharge and empty the syringe three times. When
the primary labelled antibody solution has been processed
so that the primary labelled antibody is bound to the
antigen of'the an-tigen-antibody complex to form a sandwich
complex, the container is washed as previously described.
The container 11 is placed in a coloring solution reagent
400 and 5 cc of coloring reagent is withdrawn into the
syringe. The syringe is emptied and the color read in the
container.
The color solution or reactant 400 is preferably a
substrate manufactured by Kirkegaard & Perry Labs under one
of several acronyms namely: ABI'S (2,2'-azino-di-[3-
ethylbenzthiazoline sulfonate (6)]; OPD (ortho-phenylene
diamine); or TMB (tetramethylkbenzidine). In choosing the
substrate, the sensitivity of the immunoassay is de-termined
by the discrimination of the antibody rea~ents. When this
occurs, the use of a more sensitive substrate serves only to
proportionately increase the signal and the background. The
result is more color but the same .signal-to-noise ratio.
Should the more .sensitive substrate pusll the absorbance over
the cut-off ol-~ the reader, the faster substrate may in fact
reduce the signal-to-noise ratio.
The preferred color solution as used in the
embodiment of the present invention is ABTS. The
preferred ABTS substrate is a one-component substrate. The
HRP label Oll the primary labelled antibody added after the
initial antigen-antibody complex turns ABTS to a blue-green
color and -tl-ere is no change in color or absorbance when the
reac-tion is stopped with SDS (sodium dodecyl sulfate). If
the assay optimi.zation indicates the sensitivity of the
immunoasssay is limited by the color generated by the HRP
(horseradish peroxidase) then the more sensitive TMB
substrate would give more color d0velopmen-t without a
corresponding increase in the background. Another advantage
of the TMB substrate is that it often lowers the arnount of
antibody and anti.gen reagents required for the immunoassay.
TMB substrate is a two component liquid substrate and
requires hydrogen peroxide. Thus when using this substiate
the additionaJ. step of imrnersing in hydrogen peroxide would
be requirecl. I-IRP converts TMB to a blue product. When the
reaction is stopped by acidification, the TMB product
becomes yellow. ODP is generally provided as a table-t that
is d.issolved in buffer at the time of use. HRP converts OPD
to a yellow product which continues to oxidize into a brown
pr0cipi.tate. Upon acidification the OPD product becomes
orange.
As can be seen this antigen-antibody sandwich
complex metl-od allows for the first time for the deteot:Lon
of the early stages of cancer as compared with present day
testing which can only identify the later tumor stage. Thus
the physician now has available the capability of quick
testi.ng for cancer patients after surgery or for the testing
of patients with suspected cancer.
In the foregoing description, the invention has
been described with reference to a particular preferred
12
embodiment, althouc3h it is to be understood that specific
details shown are merely illustrative, and the invention may
be carried out in other ways without cleparting from the true
spirit and scope of the following clai.ms:
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