CA2040088A1 - Method for testing urine antigen - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A method for testing for biological molecular indicators in urine. Urine is transported through a sample container under pressure to flow through the sample container so that antigens in the urine, are collected and bound on antibodies immobilized on the beads to form antigen-antibody complex. The beads are washed to remove cell debris and charged molecules and a specific prelabelled antibody solution is passed through the sample container with the prelabelled antibodies attaching to a receptor site on the captured antigen to form are antibody-antigen-prelabelled antibody sandwich complex. This sandwich complex is washed to remove cell debris and charged molecules and mixed with a color reagent solution which reacts with the prelabelled antibody to produce an color indicating the presence of a specific cancer antigen.

Description

~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.

....
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 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:

Claims (3)

1. Claim 1. A method of testing for the presence of cancer antigen comprising the steps of:
   a. collecting urine into an apparatus for collecting biological fluids;
   b. passing the urine through a urine treatment container holding an immobilized antibody bead means to capture antigen from the urine;
   c. washing the bead means antibody-antigen complex formed in step b to remove non bound materials, d. mixing the bead means antibody complex with a prelabelled primary antibody solution to form a sandwich complex of antibody antigen prelabelled primary antibody;
   e. washing the sandwich complex bead means to remove non bound materials; and f. immersing the washed sandwich bead means in a coloring reagent which reacts with the prelabelled primary antibody to produce a color which indicates cancer presence.
2. Claim 2. A method as claimed in claim 1 including repeating steps b and d a plurality of times.
3. Claim 3. A method of testing for predetermined antigen in urine comprising the steps of:
   a. placing an antigen collecting apparatus in fluid communication with urine and causing the urine to pass through the antigen collecting apparatus so that antigens are captured by antibody bead means located in said collecting apparatus forming an antigen-antibody complex bead means;
   b. washing the antigen-antibody complex bead means to remove non bound debris, c. causing a prelabelled primary antibody solution to contact the antigen-antibody complex bead means to form an antigen-antibody sandwich complex with the prelabelled primary antibody being bound to the antigen of the antigen-antibody complex;
   d. washing the sandwich complex to remove non bound debris; and e. mixing the sandwich complex in a reactant solution to form an indicator indicating the presence of a specific cancer.
   Claim 4. The method as claimed in claim 3 including repeating steps a and c at least one time.
   Claim 5. The method as claimed in claim 3 including repeating steps a and c three times.
   Claim 6. The method as claimed in claim 3 including repeating steps a and c a plurality of times.
   Claim 7. The method as claimed in claim 3 including the step of mixing said urine with a buffering solution before causing urine flow through the antibody bead means.
   Claim 8. A method of collecting antigen from urine for testing for cancer comprising the steps of:
   a. mixing a buffer solution in urine;
   
   b. withdrawing the buffered urine into a syringe means containing a filter and beads with immobilized antibodies to obtain a concentration of antigen on said beads with immobilized antibodies;
   c. washing the complexed antigen-antibody beads to remove the stray ligands and debris;
   d. mixing the antigen-antibody complex beads with a prelabelled primary antibody solution to form a sandwich complex with the prelabelled primary antibody fastening on to the captured antigen receptor sites;
   e. washing the sandwich antigen-antibody complex to remove stray ligands and debris; and f. treating the washed sandwich complex with a coloring reagent to obtain a colored solution indicating the presence of a specific antigen cancer marker.
   Claim 9. The method as claimed in claim 8 including the step of adding another solution to said reactant solution.
   Claim 10. The method as claimed in claim 8 wherein said preblabelled primary antibody as labelled with HRP.
   Claim 11. The method as claimed in claim wherein said reactant solution is ABTS.
   Claim 12. The method as claimed in claim 8 wherein said reactant solution is TMB.
   Claim 13. The method as claimed in claim 8 wherein said reactant solution is OPD.
   
   Claim 14. The method as claimed in claim 9 wherein said another solution added to said reactant solution is hydrogen peroxide.