United States Patent (19)
`Grow et al.
`
`73) Assignee:
`
`*) Notice:
`
`(54) FECAL SAMPLE IMMUNOASSAY METHOD
`TESTING FOR HEMOGLOBIN
`75 Inventors: Michael A. Grow, San Jose; Wipin D.
`Shah, Saratoga, both of Calif.
`International Immunoassay
`Laboratories, Inc., Santa Clara, Calif.
`The portion of the term of this patent
`subsequent to Mar. 10, 2009 has been
`disclaimed.
`21) Appl. No.: 764,012
`22 Filed:
`Sep. 23, 1991
`Related U.S. Application Data
`Continuation of Ser. No. 329,455, Mar. 28, 1989, Pat.
`No. 5,094,956, which is a continuation-in-part of Ser.
`No. 10,787, Feb. 4, 1987, abandoned.
`51) Int. Cl. ............................................. G01N 33/72
`52 U.S.C. .......................................... 436/66; 436/8;
`436/17; 436/177; 436/815; 436/825
`58) Field of Search ................. 436/66, 8, 17, 63, 177,
`436/815, 825
`
`63
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`3,869,547 3/1975 Mebus et al. .
`4,200,690 4/1980 Root et al. .
`4,424,279 1/1984 Bohn et al. .
`4,533,630 8/1985 Wilkins et al. .
`4,683, 197 7/1987 Gallati.
`FOREIGN PATENT DOCUMENTS
`0030388A2 6/1981 European Pat. Off. .
`0032782A2 7/1981 European Pat. Off. .
`011 1916A1 6/1984 European Pat. Off. .
`0175326A2 3/1986 European Pat. Off. .
`
`||||||||||||||||
`US005198365A
`5,198,365
`11
`Patent Number:
`'Mar. 30, 1993
`(45) Date of Patent:
`
`OTHER PUBLICATIONS
`Julkunen et al, Scand J Infect Dis 17:245-249 (1985).
`Primary Examiner-Sam Rosen
`Attorney, Agent, or Firm-Skjerven, Morrill,
`MacPherson, Franklin & Friel
`(57)
`ABSTRACT
`A method for preparing a fecal sample for immunoassay
`testing comprising the steps of dispersing a sample of
`from 1 to less than 10 wt.% of a stool sample in an
`aqueous fecal test solution formulated with one or more
`preservatives, analyte stabilizing agents and endoge
`nous interference reducing agents. The fecal solids are
`then permitted to settle to form a liquid phase substan
`tially free from fecal solids, and the clarified liquid
`phase is removed to provide a test sample free from
`fecal solids. The fecal test solutions contain suitable
`stool stabilizers such as buffers and antimicrobial agents,
`analyte protecting agents such as proteolytic, reductive
`or oxidative enzyme inhibitors, endogenous assay inter
`fering enzyme inhibitors such as a reducing agent, and
`non-specific binding inhibitors such as animal proteins.
`The stool sample should be fresh or be fresh frozen and
`thawed immediately before dispersion in the buffer
`solution. The sample is suitable for any solid-phase in
`munoassay determination of a fecal sample analyte. A
`method for determining analyte in the stool sample
`comprises conjugating anti-analyte antibody adhered to
`a insoluble support with analyte in the clarified sample,
`and determining the presence and extent of such conju
`gation. For determining human hemoglobin in a sample
`of human stool, the aqueous fecal test solution prefera
`bly contains a proteinase inhibitor, formaldehyde and an
`animal albumin.
`
`4 Claims, 1 Drawing Sheet
`
`Geneoscopy Exhibit 1041, Page 1
`
`

`

`U.S. Patent
`
`Mar. 30, 1993
`
`5,198,365
`
`
`
`
`
`N
`
`aala
`
`N
`
`A/G/
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`Geneoscopy Exhibit 1041, Page 2
`
`

`

`1.
`
`FECAL SAMPLE IMMUNOASSAY METHOD
`TESTING FOR HEMOGLOBIN
`
`5
`
`O
`
`20
`
`CROSS REFERENCE TO RELATED
`APPLICATION
`This application is a continuation of application Ser.
`No. 07/329,455, filed Mar. 28, 1989, now U.S. Pat. No.
`5,094,956 which is a continuation-in-part of application
`Ser. No. 07/010,787 filed Feb. 4, 1987, now abandoned
`and incorporated herein by reference in its entirety.
`. FIELD OF THE INVENTION
`This invention relates to an improved method for
`analyzing fecal samples. In particular, this invention
`15
`relates to a method for preparing fecal samples for im
`munoassays, the prepared fecal sample compositions
`and to solid-phase immunoassays for determining the
`presence and quantity of analytes in the samples.
`BACKGROUND OF THE INVENTION
`Stool or fecal samples are routinely tested for the
`presence of parasites, fat, occult blood, viruses, bacteria
`and other organisms and chemicals in the diagnosis for
`various diseases. The stool is usually collected, placed
`in a clean container and processed for testing.
`Stool collection is usually non-invasive and theoreti
`cally ideal for testing pediatric or geriatric patients, for
`testing away from a clinical site, for frequently repeated
`tests and for determining the presence of analytes which
`are likely to be found in the digestive tract. Stool can
`also be collected with a swab or finger cot during exam
`ination and applied directly to a test surface. For micro
`scopic examination or occult blood testing, the sample
`can be spread directly on a test surface. For other tests,
`such as testing for fat, the stool may be suspended in a
`liquid medium such as water.
`Traditional sample examinations have used complex
`chemical or microbiological procedures. These proce
`dures are being rapidly replaced with immunoassay
`methods. Immunoassay techniques are highly sensitive
`and require only a small sample. Solid-phase techniques
`such as latex agglutination and enzyme immunoassays
`have been developed to such a stage of simplicity that
`they can be performed at home, at the doctor's office or
`45
`other test sites without the need for highly trained labo
`ratory technicians or expensive instruments. Applica
`tion of solid-phase immunoassay procedures to the anal
`ysis of stool samples is thus highly desirable.
`Application of immunoassay techniques to fecal anal
`50
`ysis has proven to be difficult for several reasons. Stool
`handling is disagreeable and biohazardous, and sanitary
`and inoffensive procedures for processing stool have
`proven to be awkward and frequently complex. Ana
`lytes in stool samples are frequently unstable. Weighing,
`55
`extracting and centrifuging, and storing samples are
`difficult except in a clinical laboratory equipped with
`suitable apparatus and skilled technicians.
`Constituents of stool are known to interfere with
`solid-phase immunoassays. Immunoreactants immobi
`lized on solid-phase are desorbed by stool constituents.
`Non-specific reactions occur.
`To increase the commercial use of immunoassay
`techniques for measuring analytes in stool, a number of
`problems must be solved. Instability of the analyte in
`65
`the stool, interference from stool constituents, needs for
`extensive handling of the stool, equipment contamina
`tion, and instrumentation needs must be minimized.
`
`5, 198,365
`2
`Simple preparation steps avoiding the use of expensive
`equipment and instruments are required to extend the
`use of immunoassay testing procedures to sites outside
`hospital and clinical laboratory environments.
`DESCRIPTION OF THE PRIOR ART
`Assay procedures including the preparation of con
`centrated suspensions of 10 and 25 wt.% stool in water
`or buffer solution have been described by Vellacott et
`al, The Lancet p 18 (Jan. 3, 1981) and Jilkunen et al,
`Scand.J. Infec.D. 17:245 (1985). These were centrifuged
`and sterile-filtered to provide a sample for testing.
`Desorption of immunoreactants has been reduced by
`either heat treatment of the sample or by mixing 50 vol.
`% fetal calf serum or acid-protein buffer containing 5
`vol. 9% bovine serum albumin (BSA) with the test sam
`ple.
`Non-specificity problems have been overcome by
`heat-treating samples in the presence of a reducing
`agent.
`Current stool handling procedures include storing
`and transporting stool samples in clean containers and
`reducing deterioration of analyte by maintaining the
`sample at low temperatures. Problems of non-uniform
`ity are resolved by forming a suspension of an entire
`sample or by assaying several samples; the suspension is
`then treated by centrifugation, filtration, extraction and
`sterilization.
`Current techniques for measuring hemoglobin in
`stool exemplify the problems. A widely used semi-quan
`titative procedure for measuring hemoglobin uses gua
`iac resin paper on which the stool is reacted with hydro
`gen peroxide. The reaction of hemoglobin with guaiac
`resin forms a blue color, the intensity of which is a
`function of the quantity of hemoglobin in the sample.
`This method does not distinguish hemoglobin derived
`from animal blood in food from human hemoglobin.
`Because this method is subject to variables derived from
`chemical and biochemical interference with the hemo
`globin-guaiac resin reaction and variations in water
`content of the paper and stool, it is not truly quantita
`tive.
`A quantitative method for measuring hemoglobin in
`stool described in U.S. Pat, No. 4,378,971 involves heat
`ing a small amount of stool in a reducing acid milieu.
`Porphyrin, free from other contaminating fluorescent
`compounds, is extracted from the mixture. This proce
`dure provides a very sensitive, quantitative measure
`ment of hemoglobin in stool. However, it requires ex
`tensive handling, does not differentiate human and ani
`mal hemoglobins, and cannot be carried out rapidly.
`The radialimmunodiffusion (RID) procedure de
`scribed by Barrows, G.H. et al, Ann.J. Clin. Path.
`69:342-346 (1977) uses antibodies to human hemoglobin
`in conjunction with calibrators of known hemoglobin
`concentrations. A disk is punched out from filter paper,
`stool sample is applied to the disk, and the disk is placed
`on a RID plate. There it is allowed to react for 24 hours
`with a disk impregnated with the calibrators. This test
`has a detection limit of 0.3 mg of hemoglobin in 8 mg of
`stool. It requires overnight incubation. Use of filter
`paper limits sensitivity since all hemoglobin placed on
`the paper is not made available for the antigen-antibody
`reaction. Irreversible protein absorption may permit the
`release of as little as 5 to 10 percent of the hemoglobin
`placed on the paper.
`
`25
`
`30
`
`35
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`Geneoscopy Exhibit 1041, Page 3
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`

`

`5
`
`20
`
`25
`
`5, 198,365
`3
`4.
`fecal solids. The clarified liquid phase is removed to
`U.S. Pat. No. 4,582,811 describes a procedure includ
`ing binding hemoglobin in a sample with antibody in
`provide a test sample free from fecal solids.
`pregnated in paper, and then reacting the product with
`The preferred stool stabilizing agents include buffer
`a substrate to measure pseudoperoxidase activity of the
`ing agents, and antimicrobial agents such as antibacte
`hemoglobin.
`rial and/or antimycotic agents. The preferred analyte
`protecting agents include inhibitors of proteolytic, re
`U.S. Pat. No. 4,427,769 describes an immunological
`method involving extraction of hemoglobin applied to a
`ductive and/or oxidative enzymes. For immunoassays
`using alkaline phosphatase enzyme labels, the preferred
`guaiac resin coated paper, and measuring it with a sand
`endogenous enzyme inhibitor inhibits the activity of
`wich enzyme immunoassay technique. Kim et al, Clin.-
`endogenous alkaline phosphatase, such as formaldehyde
`Chimacta. 152:175 (1985) describes a still further ap
`10
`or equally effective enzyme inhibitors.
`proach wherein a stool sample applied to a glass fiber
`The stool sample is preferably freshly collected or
`filter is placed on a gel and allowed to incubate for 2-4
`hours. Hemoglobin is quantitatively determined down
`has been chilled to a temperature below -20°C. imme
`diately upon collection. Frozen stool is preferably then
`to a limit of 0.2 mg of hemoglobin per gram of stool
`raised to a temperature within the range of from 2 to 6'
`based on the presence or absence of a visible band.
`C. immediately before being dispersed in the aqueous
`The procedures describe above require the direct
`transfer of a stool specimen to the test system. Transfer
`fecal test solution.
`of hemoglobin from the sample to the test system is only
`The solid-phase immunoassay method of this inven
`tion for determining analyte in a fecal sample comprises
`partial. Undesirable reactions caused by stool constitu
`contacting the clarified liquid phase with a solid support
`ents are difficult to control with reagents due to their
`uniform distribution throughout the sample. Most of the
`to which an anti-analyte is adhered for a time sufficient
`to permit antibody conjugation with analyte; and deter
`procedures require a well equipped laboratory and
`mining hemoglobin adhering to the insoluble support.
`trained technicians.
`For determining human hemoglobin in a sample of
`Adams and Layman, Ann. Clin. Labs. Sci 4:343 (1974)
`describe a latex agglutination test involving blending 1
`human stool according to this method, the aqueous
`fecal solution preferably contains an inhibiting amount
`gm of stool in 100 ml of buffer solution and filtering the
`of a proteinase inhibitor, from 0.02 to 0.5 wt.% of
`suspension. This test can detect hemoglobin down to a
`formaldehyde and from 0.01 to 10 wt.% of a non
`level of 10 ml of blood per gram of stool.
`immune animal protein.
`Vellacott, et al, The Lancet. 1:18 (1981) have de
`It is an object of this invention to provide an im
`scribed a fluorescent immunoassay method in which a
`proved procedure for preparing stool samples for solid
`20% suspension of stool in water is used as a test sample.
`phase immunoassay which is simple, quick and can be
`This sample is sonicated and centrifuged prior to test
`carried out in the home or other non-laboratory site by
`1ng.
`a technically unskilled person with simple, inexpensive
`Japanese Patent Application 60173471 (Dialog Der
`resources. It is a further object of this invention to pro
`went World Patent Acc. No. 85-259806/42) describes
`35
`vide a procedure for preparing a stool sample composi
`applying stool containing an analyte to a porous nate
`tion which provides reduced interference with solid
`rial. The porous material contains a carrier to which an
`immunoassay procedures. A still further object of this
`antibody which binds with the analyte is attached. The
`invention is an improved immunoassay method for fecal
`sample is washed and contacted with further reagents to
`provide a change in spectroscopic characteristics.
`occult blood testing of stool samples.
`European Patent Application 70366 (Dialog Derwent
`BRIEF DESCRIPTION OF THE DRAWINGS
`World Patent Acc. No. 83-12612K/06) describes an
`FIG. 1 is a view of a sample preparation vial suitable
`immunoperoxidase sandwich test method for determin
`ing hemoglobin, albumin or globulin in stool samples
`for use in the method of this invention
`using beads upon which antibodies for the analyte are
`FIG. 2 is a cross-sectional view of the sample prepa
`45
`immobilized and a peroxidase labeled secondary anti
`ration vial of FIG. 1.
`body.
`DETAILED DESCRIPTION OF THE
`INVENTION
`This invention is a method for preparing a fecal or
`stool sample for analysis using immunological tech
`niques. This procedure is described hereinafter in con
`junction with an immunoassay procedure for determin
`ing human hemoglobin in a stool sample, by way of
`example, not by way of limitation. The sample prepara
`tion procedure described hereinbelow is equally suit
`able for preparing stool samples for determining other
`stool analytes by immunoassay methods, and the use of
`this procedure for all such applications is intended to be
`included within the scope of this invention.
`In general, the method of this invention provides a
`stabilized stool sample solution which yields an im
`proved result in immunoassays. The stool is suspended
`in an aqueous fecal test solution in a concentration of
`less than 10 wt.%. The fecal test solution contains
`agents which stabilize the stool and protect the analyte
`from deterioration. The solution also preferably con
`tains agents which reduce endogenous sources of immu
`
`SUMMARY AND OBJECTS OF THE
`INVENTION
`The method of this invention for preparing a fecal
`sample composition for immunoassay testing comprises
`forming a dispersion of 1 up to 10 wt.% and preferably
`from 1 to 5 wt.% of a stool sample in an aqueous fecal
`test solution. The aqueous fecal test solution can contain
`preservatives and endogenous interference reducing
`agents to protect the test sample components against
`assay related deterioration and the assay from interfer
`ence. The aqueous fecal test solution of this invention
`contains at least one stool stabilizing agent or at least
`one analyte stabilizing agent. The aqueous fecal test
`solution also preferably contains agents which facilitate
`the immunoassay such as at least one inhibitor of endog
`enous enzymes which may interfere with the immuno
`assay and one or more non-immune animal proteins or
`65
`polyamino acid polymers to reduce non-specific bind
`ing. Fecal solids in the dispersion are permitted to settle
`to form a clarified liquid phase substantially free from
`
`50
`
`55
`
`Geneoscopy Exhibit 1041, Page 4
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`

`

`10
`
`15
`
`5, 198,365
`6
`5
`lung described by Kraut, H. et al, Z. Physiol Chem.
`noassay interference and proteins to reduce non-specific
`198:97-101 (1930). The proteolytic activity inhibitor is
`binding. The clarified liquid from the stool suspension
`contains sufficient analyte for a highly accurate immu
`present in a concentration sufficient to inhibit a major
`proportion of the proteolytic activity. Aprotinin can be
`noassay determination. We have discovered that be
`present in concentrations of from 10,000 to 30,000 kalli
`cause the stool and analyte are diluted to low levels, the
`protective and inhibiting functions can be achieved
`krein inactivator units per liter of fecal test solution.
`with such low concentrations of reagents that the rea
`The aqueous fecal test solution also preferably con
`gents do not significantly interfere with the immunoas
`tains reagents which inhibit or deactivate enzymatic
`activity which may interfere with the particular immu
`say. Prior to this invention, effective stool stabilization
`and analyte protection with chemical or biochemical
`noassay procedure used. Particularly when the immu
`reagents was incompatible with immunoassay methods.
`noassay uses alkaline phosphatase labeled reagents, en
`The high reagent concentrations required greatly inter
`dogenous alkaline phosphatase naturally present in the
`fered with the immunoassays. Accordingly, expensive,
`sample presents substantial interference. If alkaline
`time-consuming non-reagent purification and stabiliza
`phosphatase levels in the sample are not adequately
`tion techniques were found necessary to prepare fecal
`suppressed by other reagents, the broadly active en
`samples for immunoassay analysis.
`zyme inhibitors or deactivators are useful. The pre
`The method of this invention for preparing a fecal
`ferred test system uses formaldehyde as an inhibitor of
`sample composition for immunoassay testing involves a
`alkaline phosphatase in the sample. Other enzyme inhib
`first step of forming a dispersion of from 1 up to less
`itors include metal chelating agents, heavy metal ions,
`than 10 wt.% and preferably from 1 to 5 wt.% of a
`20
`certain amino acids such as tyrosine and phenylalanine,
`stool sample in an aqueous fecal test solution. The aque
`and high concentrations of zinc or inorganic phos
`ous fecal test solution of this invention contains at least
`phates. Any conventional enzyme inhibitor can be used
`one stool stabilizing agent and/or at least one analyte
`to prepare the aqueous fecal test solution, if it does not
`stabilizing agent. The aqueous fecal test solution also
`interfere with the later assay procedure and particularly
`preferably contains agents which facilitate the immuno
`25
`any enzyme reactions used in the immunoassay proce
`assay such as at least one inhibitor of endogenous en
`dure. The level of enzyme inhibitor or deactivator is
`zymes which may interfere with the immunoassay and
`selected to be sufficient to achieve the level of inhibition
`one or more non-immune animal protein or polyamino
`required for the sample. For the general stool samples,
`acid polymer to reduce non-specific binding.
`alkaline phosphatase inhibitors such as formaldehyde
`For stabilizing the stool, the aqueous fecal test solu
`30
`can be used in concentrations of from 0.01 to 0.5 wt.%
`tion contains buffering agents and/or antimicrobial
`and preferably in concentrations of from 0.01 to 0.2 wt.
`agents. The aqueous fecal test solution can be buffered
`%.
`to a pH selected to increase the stability of the stool.
`A non-specific binding inhibitor is preferably present
`For hemoglobin immunoassays, a buffersolution having
`in the fecal test solution. Suitable non-specific binding
`a pH of from 7.0 to 8.0 is desirable. Any conventional
`inhibitors are non-immune water-soluble animal prote
`buffering agents can be used to prepare the test buffer
`ins and polyamino acids which would not interfere with
`solution, if they do not interfere with the later assay
`the later assay procedure and particularly any protein
`procedure and assay reagents. One example of a suitable
`measurements in the immunoassay procedure. Suitable
`buffering solution is standard phosphate buffered saline,
`animal proteins are include bovine (BSA), human
`pH 7.2 to 7.6 and preferably about 7.4.
`(HSA), rabbit (RSA), goat (GSA), sheep (SHA), and
`The stool stabilizing agent can include any biocidal or
`horse (HOSA) serum albumins, for example; serum
`biostatic agents which will inhibit microbial growth in
`gamma globulin, of the previously described animals
`the sample but will not interfere with the immunoassay.
`and other animal proteins such as ovalbumin, fibrino
`Any conventional biocidal or biostatic agent can be
`gen, thrombin, transferin, glycoproteins, etc. Suitable
`used which will not interfere with the later assay proce
`45
`water-soluble amino acid polymers include polylysine,
`dure and assay reagents. An example of suitable biocidal
`polyglutamic acid, polyalanine, polyhistidine, poly
`agents are antibiotics such as penicillin or streptomycin,
`methionine, polyproline, and the like. For assays where
`and antimycotic agents such as fungizone. A commer
`non-specific binding presents a problem, the concentra
`cial buffer solution containing these antimicrobial
`tion of the non-specific binding inhibitor can be from 0.1
`agents is available from Gibco Co., New York, N.Y.
`to 1.0 wt.% and is preferably from 1 to 5 wt.%.
`The concentration of the antibiotic and antimycotic
`The preserved aqueous fecal test solution can also
`agents is adjusted based on the activity of the reagent
`contain other protective agents including proteins, car
`selected. In general, the level is sufficient to suppress
`bohydrates, salts and the like which provide a protec
`the reproduction of the microbes and preferably is suffi
`tive function.
`cient to kill a majority of them.
`Microbial and chemical changes in the sample should
`Particularly for proteinaceous analytes, the analyte
`be inhibited and preferably completely arrested immedi
`stabilizing or protecting agent can be an inhibitor of
`ately after the sample is obtained. The stool is prefera
`enzyme activity which would affect the analyte. Inhibi
`bly dispersed in the aqueous fecal test solution immedi
`tors of proteolytic, reductive and oxidative enzymes are
`ately after being obtained, where the preservatives and
`useful. Any conventional inhibitors of proteolytic, re
`other reagents in the buffer solution will stabilize the
`ductive or oxidative enzyme activity can be used to
`sample. If the stool is to be stored or shipped before
`prepare the aqueous fecal test solution, if they do not
`testing, it should be quickly frozen to a temperature
`interfere with the later assay procedure and assay rea
`below -20 C. immediately after being obtained to
`gents. Examples of suitable proteolytic activity inhibi
`prevent chemical and microbial changes, and the sam
`tors include phenylmethylsulfonylfluoride (PMSF),
`65
`ple should be maintained at this temperature until the
`pepstatin A, Bestatin, and chymostatin (Sigma Chemi
`sample is to be dispersed in the aqueous fecal test solu
`cal Co.). A suitable commercial product is the proteo
`tion. Immediately before being dispersed, the frozen
`lytic activity inhibitor aprotinin, a derivative of bovine
`
`50
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`35
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`5
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`O
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`5
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`5, 198,365
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`7
`wich immunoassays can be carried out with a sample
`sample temperature is raised to a temperature within the
`prepared according to this invention. These procedures
`range of from 2 to 6 C.
`can use antibodies or reagent analytes labeled with a
`The fecal solids in the dispersion are then permitted
`wide variety of physically detectable labels or with
`to settle to form a liquid phase substantially free from
`active labels such as enzymes or enzyme substrates
`fecal solids and the clarified liquid phase is removed,
`which upon a further reaction yield physically detect
`providing a test sample substantially free from fecal
`able labels.
`solids.
`By way of example, the clarified liquid can be used
`FIG. 1 shows an open vial and spatula combination
`for determining human hemoglobin in the sample using
`which is suitable for treating the stool samples accord
`an anti-(human hemoglobin) antibody. For this proce
`ing to this invention, and FIG. 2 is a cross-sectional
`dure the fecal test solution preferably contains as stool
`representation thereof. The container 2 has a body 4
`stabilizing agents, buffers and antimicrobial agents; and
`made of transparent organic polymer or glass with level
`as an analyte protecting agent, a proteolytic enzyme
`or volume graduation lines 6 on the outer surface
`inhibitor. For immunoassays using a phosphatase la
`thereof. The bottom of the body is formed into a conical
`beled reagent, the aqueous fecal test solution preferably
`solids receptor 8, enclosed within a cylindrical stand 10
`which maintains the body 4 in an axially upright posi
`also contains a reducing agent and a non-specific bind
`ing inhibitor. A. aqueous fecal test solution for this assay
`tion. The cap 12 is threaded to form a sealed, threaded
`engagement with the body threads 14. A spatula-stirrer
`preferably is buffered to a pH of from 7.0 to 8.0, and
`16 having a flattened end 18 is mounted in the cap 12,
`contains a biocidal amount of an antimicrobial agent, an
`extending downward into the dispersion and settling
`inhibiting amount of a proteinase inhibitor, from 0.02 to
`cavity 20 formed by the body 4.
`0.5 wt.% and optimally from 0.02 to 0.1 wt.% of form
`The spatula or sample spoon 16 is dimensioned to
`aldehyde, and from 1 to 10 wt.% of an animal albumin.
`take a selected amount of stool and disperse it in a se
`This invention is further illustrated by the following
`lected volume of liquid 22 in the vial 2. The suspension
`specific, but non-limiting examples. Temperatures are
`given in degrees Centigrade and percents as weight
`is permitted to settle, and the solids are collected in the
`25
`conical bottom 8, leaving a clarified liquid in the upper
`percents unless otherwise specified. Examples which
`portion of the container. This container is suitable for
`are constructively reduced to practice herein are pres
`preparing dilutions of stool for qualitative immunoas
`ented in the present tense, and examples representing
`laboratory experiments previously reduced to practice
`says.
`A further dilution can be made by transferring clari
`are presented in the past tense.
`fied liquid from the vial to another container of fecal
`EXAMPLE 1
`test solution or another suitable aqueous fecal test dilu
`tion medium. For competition immunoassays, the fecal
`Fecal Occult Blood Immunoassay
`test solution or dilution medium can be used for obtain
`Goats are immunized by standard procedures with
`ing the desired dilutions. For sandwich immunoassays,
`purified human hemoglobin Variance A (Isolab PH 100,
`the fecal test solution or dilution medium can contain
`Akron, O.H.). Bleeds from the goats are tested against
`suitable reducing agents as required for the analyte
`human hemoglobin until the presence of antibodies to
`determinations.
`hemoglobin are confirmed. Polystyrene macrobeads
`Containers of the type shown in FIG. 1 and FIG. 2
`(6-8 mm) are suspended in the anti-serum diluted in
`are also suitable for storage and shipment of stool sam
`glycine-saline buffer, pH 8.2 to provide a coating of the
`ples dispersed in the fecal test solution. With dilution in
`goat anti-(human hemoglobin) antibody.
`an inhibitory buffer solution, the odor is minimized and
`Dialyzed calf-intestine alkaline phosphatase is mixed
`the appearance of the sample made less objectionable.
`with hemoglobin in the presence of gluteraldehyde to
`However, in the preferred method, the fresh stool is
`form a hemoglobin-alkaline phosphatase conjugate. The
`transferred into liquid medium in the container for clari
`reaction product is purified on a SEPHACYL S-300
`fication and testing.
`column (Pharmacia), eluting with tris buffer containing
`Surprisingly, the sample preparation procedure of
`MgCl2.
`this invention, when carried out with suitable reagent
`The buffered aqueous fecal test solution is 0.02 M
`inhibitors in the fecal test solution, reduces interfering
`phosphate buffered saline, pH 7.4, containing 0.1 wt.%
`activities far more efficiently than the prior art methods
`sodium azide, wt.% bovine serum albumin, 10,000
`and without their complicated, lengthy and elaborate
`u/L aprotinin (TRASYLOL, Mobay Chemical, New
`procedures. The dispersion of sample in the buffer solu
`York, N.Y.) and 694 microliters/liter of formaldehyde.
`tion allows a uniform and rapid inhibitory and protec
`A stool sample (1 gm) is dispersed in 30 ml of the fecal
`tive action by the reagents. The arrest of the potential
`test solution.
`harmful effects of the stool constituents in the subse
`55
`Hemoglobin calibrator controls are made by dis
`quent immunoassay is more efficient and complete than
`solving human hemoglobin in desired concentrations in
`with the prior art procedures.
`the testing buffer solution.
`A solid-phase immunoassay for determining an ana
`The enzyme substrate p-nitrophenyl phosphate
`lyte in a human stool sample according to this invention
`(SIGMA 104 phosphatase substrate tablets, Sigma
`comprises the steps of contacting the clarified liquid test
`Chemical Company, St. Louis, M.o.) is dissolved in a
`sample with a solid support to which an anti-analyte
`buffer which contains 0.05 M. NaHCO3, 0.05 M. NaCl
`antibody is adhered for a time sufficient to permit anti
`and 0.1 mM MgCl2. The tablet must be completely
`body conjugation with analyte, and determining analyte
`adhering to the insoluble support. The choice of aque
`dissolved.
`ous buffer solution inhibitors and reagents is selected to
`65
`facilitate the immunoassay procedure to be carried out.
`A wide variety of solid-phase immunoassay procedures
`including latex agglutination, competition and sand
`
`Assay Procedure
`1. Allow all assay reagents and specimens to equilibrate
`to room temperature.
`
`20
`
`30
`
`35
`
`45
`
`50
`
`Geneoscopy Exhibit 1041, Page 6
`
`

`

`10
`TABLE A-continued
`Concentration
`Calibrators (microgm/ml)
`Control
`
`ng hemoglobin/gm stool = micro
`
`Average
`O.D. (405 nm)
`500
`
`O.D. (405 nm)
`1,422
`In hemoglobin x 30
`stool wt., (ng)
`
`5
`
`10
`
`5
`
`20
`
`5, 198,365
`2. Label test tubes in duplicate for each calibrator, con
`trol and sample.
`3. Pipette 50 microliters each of calibrator, control and
`clarified stool sample solution into the bottom of each
`tube. The volume can be selected from the range of
`from 10 to 100 microliters.
`4. Pipette 1 ml of the buffer solution into each tube,
`shaking the tubes to mix.
`5. Place the required number of beads onto absorbent
`paper and drop one bead into each tube. Shake to mix
`the bead and liquid.
`6. Incubate all tubes at rm temp for 30 min. During each
`incubation, shake tubes 3 to 4 times at regular inter
`vals with sufficient force to lift the bead off the tube
`bottom.
`7. Decant or aspirate liquid from all tubes. Wash each
`bead with 5 ml of distilled water at rm temp. Decant
`or aspirate liquid from all tubes.
`8. Repeat washing step (7) two more times, and after the
`last wash, remove all residual water with an aspirator
`or invert the tubes over absorbent paper.
`9. Add 200 microliters of prepared hemoglobin-alkaline
`phosphatase conjugate solution to all tubes, and incu
`bate all tubes at rm temp for 30 min. Shake tubes 3 to
`25
`4 times during incubation. The incubation times for
`each tube must be the same. This can be accom
`plished by pipetting all reagents into all tubes in the
`same order without interruptions and in the same
`elapsed time interval.
`30
`10. Wash each bead with 5 ml of distilled water at rm
`temp. Deca