EP0625268A4 - Apparatus and method of saliva collection and verification for dried saliva spot drug and hiv antibody testing. - Google Patents

Apparatus and method of saliva collection and verification for dried saliva spot drug and hiv antibody testing.

Info

Publication number
EP0625268A4
EP0625268A4 EP93900603A EP93900603A EP0625268A4 EP 0625268 A4 EP0625268 A4 EP 0625268A4 EP 93900603 A EP93900603 A EP 93900603A EP 93900603 A EP93900603 A EP 93900603A EP 0625268 A4 EP0625268 A4 EP 0625268A4
Authority
EP
European Patent Office
Prior art keywords
saliva
specimen
zone
absorbent
peroxide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93900603A
Other languages
English (en)
Other versions
EP0625268A1 (fr
Inventor
Jangbir S Sangha
Robert S Shea
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Osborn Group Inc
Original Assignee
Osborn Laboratories Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US07/814,077 external-priority patent/US5334502A/en
Application filed by Osborn Laboratories Inc filed Critical Osborn Laboratories Inc
Publication of EP0625268A1 publication Critical patent/EP0625268A1/fr
Publication of EP0625268A4 publication Critical patent/EP0625268A4/en
Withdrawn legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54391—Immunochromatographic test strips based on vertical flow
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/0045—Devices for taking samples of body liquids
    • A61B10/0051—Devices for taking samples of body liquids for taking saliva or sputum samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5029—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures using swabs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/521—Single-layer analytical elements
    • G01N33/523—Single-layer analytical elements the element being adapted for a specific analyte
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B2010/0009—Testing for drug or alcohol abuse
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/043—Hinged closures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N2001/002—Devices for supplying or distributing samples to an analysing apparatus
    • G01N2001/007—Devices specially adapted for forensic samples, e.g. tamper-proofing, sample tracking

Definitions

  • This invention relates to improvements in an apparatus and method for collecting a saliva sample from human subjects. More particularly, the invention provides indication of the collection of a minimum saliva sample amount as well as providing verification that the sample collected is saliva. The indicator further serves to locate the saliva and to show the sample was properly applied to an absorbent after the saliva has been dried for shipping to a laboratory for HIV antibody testing or drug abuse testing or other viral or bacterial antibody testing, or testing for other analytes of interest.
  • saliva testing is useful as a matter of convenience for the individual seeking insurance.
  • the laboratory Upon receiving an absorbent upon which a saliva sample has dried, the laboratory must be able to locate the saliva thereon (saliva being invisible unlike blood) , determine that a sufficient quantity of the sample has been obtained to perform the tests to be conducted, and verify that the dried sample is, in fact, saliva and not another body fluid or a fake specimen. Once the sample is dried it may be easily transported to a laboratory where a wide range of tests may be performed on the sample.
  • a dried saliva spot on an absorbent may be used for determining drug use such as cocaine and may also be used for analyzing the health status of the potential insured for HIV antibodies or analytes of interest.
  • the utility of saliva as a testing medium is a direct result of its formation in the body.
  • Saliva is formed in the mouth by the salivary glands of the oral cavity and adjacent areas.
  • the salivary glands consist of two parotid, submandibular and sublingual glands in addition to the labial and lingual glands as well as the small mucous glands of the soft and hard pallet.
  • the daily saliva output is between 500 and 1,500 milliliters. This flow may be affected by various factors including age, sex, time of day, time of year, nutritional state and emotional state of the individual.
  • the main constituents of saliva are proteins, sodium, potassium, calcium, magnesium, chloride, bicarbonate, and inorganic phosphate.
  • salivary gland ducts are separated from the human blood circulation by only a layer of epithelial cells many constituents of the blood are passed into the saliva. While most drugs and blood constituents seem to enter the saliva by diffusion across the epithelial cell lipid membrane, it is also possible that there is some active secretion into the saliva of drugs such as penicillin and lithium.
  • samples of saliva for these purposes have been obtained by having the subject expectorate into a container. This is assisted by having or by having the subject suck or chew onto a washed rubber band, a piece of paraffin wax, chewing gum or a strip of citric acid-impregnated filter paper and then having the subject expectorate into a container or allowing the saliva to be absorbed onto an absorbent paper or sponge.
  • saliva collection techniques may be considered less than optimal for use in a business office situation where the form of collection may be obtrusive and out of keeping with the office environment.
  • the sample must then be transported back to the testing laboratory and if the laboratory is some distance from the sample gathering location, the inconvenience and difficulty of shipping a body fluid sample is then incurred.
  • Another object is to provide an apparatus and method for obtaining a saliva sample and determining as the sample is taken that an adequate sample has been obtained.
  • Yet another object of the present invention is to provide a field saliva sample collection apparatus and method which can immediately verify that the sample obtained is in fact saliva.
  • Another object of the present invention is to provide an apparatus and method for saliva sample taking which will at once indicate to the sample collector that a sufficient sample for testing has been collected and that the collected sample is in fact saliva.
  • Yet another object of the present invention is to provide a rapid and convenient apparatus and method for saliva sample collection which can be conveniently and easily stored for law enforcement evidentiary purposes. It is yet another object of the present invention to provide an apparatus and method for saliva sample collection which can be conveniently and easily shipped from one location to another.
  • Yet another object of the invention is to provide an apparatus and method in which the area of application of a clear fluid sample may be observed after the sample is dried.
  • Yet another object of the invention is to provide an apparatus and method that allows determination that the proper sample amount has been applied at the time of sampling as well as after the sample has been allowed to dry.
  • Yet another object of the invention is to provide an apparatus and method which assists in avoiding the need to take repeat samples from subjects due to improper sampling procedures.
  • the apparatus and method of the present invention takes advantage of the ability of the peroxidase enzyme in saliva, in the presence of a peroxide, to oxidize the "leuco" or colorless form of a dye or other indicator to produce a colored form of the dye or indicator.
  • the reaction of the saliva peroxidase with the indicator may be used to determine that the sample is, in fact, saliva and also that sufficient sample has been placed on the absorbent.
  • a second enzymatic method for verification of saliva presence and sample sufficiency utilizes the amylase enzyme of saliva to effect a color change, from colorless to colored, in a substrate compound.
  • the compound a polysaccharide or oligosaccharide having a chromogenic moiety or molecule attached thereto, is applied to the absorbent at a location distant from the zone of application.
  • the substrate Upon being contacted by the amylase of saliva the substrate reacts with the amylase to release the chromogen from the polysaccharide or oligosaccharide substrate to thereby provide a free chromogen which is colored and visible to an observer.
  • An alternate apparatus and method relies on the application of a simple food dye placed at a particular predetermined distance from the zone of saliva application.
  • the mixing effect of the saliva with the dye indicates that at least a predetermined amount of saliva has been placed on the absorbent.
  • the determination of adequate saliva absorption is accomplished by first determining the absorptive capacity of the selected absorbent. Once this is determined the proper position on the absorbent for application of the pre- positioned indicator may be selected. The proper position is that distance from the zone of saliva application which will require application and absorption of sufficient sample quantity for testing by the laboratory prior to the saliva contacting the indicator.
  • the on-site examiner can easily observe the color change of the indicator or the diffusing or migration of the indicator in the saliva and be assured that sufficient sample has been placed on the absorbent.
  • Fig. 1 is a plan view of the preferred embodiment showing an applicator transferring a saliva sample to the sample application zones of an absorbent layer. Each zone is shown surrounded by an indicator component, three of the indicators are shown in the migrated state after having been contacted by the applied saliva;
  • Fig. 2 is a plan view of an ' alternate embodiment showing an absorbent material bisected by perforations and having tandem indicator components one which surrounds the sample area and one isolated indicator for saliva verification;
  • Fig. 3 is a side elevational view of the embodiment of Fig. 2;
  • Fig. 4 is a front elevational view of an alternative embodiment of the apparatus with an absorbent material affixed to a handle and having an indicator component as a ring of dots around the upper most portion of the absorbent material;
  • Fig. 5 is a front elevational view of another embodiment of the invention showing a portion of the absorbent partially detached to allow separate testing with the indicator component;
  • Fig. 6 is a side elevational view of the absorbent swab of Fig. 5 with the detachable portion intact and the line of detachment shown in phantom lines;
  • Fig. 7 is a side elevational view of yet another embodiment showing a sampling device within an outer protective tube
  • Fig. 8 shows the sampling device of Fig. 7 withdrawn from the outer protective tube and showing the absorbent material extending from the base of the capillary tube connecting the absorbent to the cap;
  • Fig. 9 is a perspective view of the capillary tube showing the internal channel for insertion of the absorbent
  • Fig. 10 is a plan view of the indicator component taken along line 10-10 of Fig. 8 and showing the indicator component on a one-way barrier on top of the absorbent material;
  • soesr i Ture SHEET Fig. 11 is a perspective view of the absorbent material which is inserted into the capillary tube of the collection apparatus shown in Fig. 8 and showing on one end the one-way barrier and the indicator component.
  • sample probe 22 is utilized to obtain a saliva sample from a subject and then to apply the sample to an absorbent sheet or layer 20.
  • Sample probe 22 is comprised of support stick 24 having swab 26 for saliva collection thereon.
  • swab 26 is placed into the mouth of a subject and saliva is collected.
  • saliva application zones 28a-f on absorbent sheet or layer 20 are touched to saliva application zones 28a-f on absorbent sheet or layer 20. In this manner saliva is transferred from swab 26 onto absorbent layer 20.
  • swab 26 is first touched to application zone 28a where saliva begins to be absorbed upon absorbent layer 20. As the saliva passes from swab 26 to absorbent layer 20 it diffuses through absorbent layer 20 and reaches indicator component 29. Indicator 29 is spaced a distance from the center of application zone 28a-f. The spacing is such that as saliva is absorbed onto absorbent layer 20 and migrates across absorbent layer 20 to contact indicator 29, it may be concluded that the proper quantity of saliva has been applied to absorbent layer 20 when indicator 29 reacts to the presence of the saliva. The on-site examiner can then proceed to each of the next zones until the complete sampling has been accomplished.
  • Indicator 29 of Fig. 1 may be either a vegetable dye or a colorless chemical substrate which acts as a chromogen in the presence of saliva.
  • indicator 29 is a vegetable dye the contact of saliva with the vegetable dye at indicator 29 will cause some of the vegetable dye to begin to diffuse. This migration of the vegetable dye can be observed by the on-site examiner who is then assured that sufficient sample quantity has been collected to allow the testing laboratory to complete analysis of the saliva.
  • indicator 29 may be one of several compounds which becomes a colored chromogen in the presence of saliva. In this case, the saliva absorbs through layer 20 to
  • SUBSTIT contact indicator 29 Upon contact with the chromogen substrate in indicator 29 a color change in the indicator occurs. This color change shows the on-site examiner that the material on absorbent layer 20 is, in fact, saliva.
  • the use of the chromogen at indicator 29 will simultaneously serve to indicate that sufficient saliva has been absorbed onto absorbent layer 20 at zone 28a-f to permit the laboratory to complete testing of the saliva sample.
  • the chemical substrate enzyme reaction is more particularly discussed infra.
  • the cost of shipping the sample is substantially lowered as both weight and volume of the sample are diminished through evaporation of the excess fluid.
  • the dried sample also offers added protection to laboratory personnel and shipping personnel as they do not have to deal with fluid samples which can spill or deal with leaking packages.
  • the marker dye or chromogen substrate allows the laboratory technician to ascertain where the proper saliva sample was located so he or she can avoid contaminating the sample area and avoid the chance of infection.
  • the sample obtained from the area within the marker will be uniform, therefore presenting a more accurate sample for analysis.
  • portions of the dried saliva sample are punched from the absorbent and tested for HIV virus antibodies or drugs of abuse such as cocaine.
  • the sample may be tested to identify tobacco smoking subjects through testing for nicotine and its metabolites or adducts in the saliva, or the dried saliva spot sample may be tested for other analytes of interest such as auto-antibodies, hormones, proteins, or drugs.
  • swab 26 contacts application zone 28b and in the fashion previously described saliva from swab 26 is transferred to application zone 28b. After a period of saliva transfer the saliva will contact indicator 29. Upon contact of the saliva with indicator 29 the dye indicator itself will begin to migrate in the absorbent layer 20. This is shown in application zone 28a of Fig. 1 where saliva, previously applied, has diffused partially into the absorbent layer. It is this migration or diffusion of indicator 29 which alerts the on-site examiner that sufficient saliva has been applied to absorbent layer 20.
  • indicator 29 is composed of a colorless chromogen substrate which changes color upon contact by saliva
  • the colorless chromogen is incorporated into the apparatus at the location of indicator 29.
  • the substrate is colorless until it contacts saliva or saliva components, it is useful to mark the center of application zone 28a-f so the on- site examiner can apply the sample to the center of the zone 28a-f.
  • the indicator a chemical reaction with the enzymes present in saliva is initiated and the chromogen substrate is transformed into a visible colored compound.
  • the reaction which produces the color change in the chromogen is specific to the presence of saliva as opposed to water or other colorless fluids such as soft drinks. Thus it serves to indicate that the subject has properly saturated absorbent 20 with saliva and not with some other convenient fluid such as toilet water which would cause the simple dye marker indicator to change as described above.
  • the on-site examiner apply the saliva to the center of the application zone.
  • the contacting of indicator 29 by the saliva will cause the dye, or the now colored chromogen, to migrate outwardly as shown in Fig. 1 at reference numeral 28a.
  • the direction and degree of dye or colored chromogen migration will be a result of two factors: first, the accuracy with which the on-site examiner applied the sample to the center of the application zone; and second, the amount of sample applied to the zone.
  • indicator 29 will be evenly contacted by saliva and the indicator will evenly migrate outwardly from the center of the application zone. Such an even outward migration may be observed at 28a or 28d. If the on-site examiner were to apply the sample outside of the indicator, the sample would migrate on absorbent 20 such that the indicator would flow inwardly toward the center of the zone of application as shown at application zone 28e. In the manner the laboratory can, by simple inspection determine if the sample has been properly applied. This opportunity to infer the point of sample application also imparts added safety to the work of the laboratory workers who must analyze the samples.
  • FIG. 2 absorbent sheet or layer 30 is bisected by perforations 32 into absorbent portions 34, 36.
  • indicator 39 On one of absorbent portions 34, 36 is provided indicator 39 for determination of saliva presence.
  • Indicator 39 is situated atop one-way barrier 38.
  • One-way barrier 38 allows saliva to move upwardly from absorbent sheet or layer 30 to contact indicator 39, but does not allow the reversed migration of saliva which has become mixed with 5 indicator 39 to then mingle with the saliva on absorbent layer 30.
  • Fig. 3 a front side elevational view of the embodiment of Fig. 2 is illustrated having perforation 32 bisecting absorbent layer 30 into absorbent portions 34, 36. In use, the embodiment of Fig.
  • the indicator in the embodiment of Fig. '20 2 may be placed at indicator 39 as well as perimeter indicator 37.
  • indicator 37 it may be useful to utilize the simple vegetable or food dye marker for indicator 37 to indicate to the on-site examiner that there is a sufficiency of sample quantity while using the chemical substrate at indicator 29 as a means of 25 indicating that the sample is in fact saliva.
  • a sample probe 40 comprises a support stick 42 having an absorbent 44 attached to one end of support 42.
  • a on-site examiner can, by grasping support 42, insert zone of 30 application 45 into the mouth of a subject. Saliva from the subject is then wicked upwardly on absorbent 44 and diffuses through absorbent 44 toward support 42. Upon saturation of substantially all of absorbent 44, saliva will approach indicator 48.
  • indicator 48 may be a simple vegetable dye 35 or- it may be a chemical indicator of the type which will undergo a color change upon contact with saliva or components of saliva. In this manner the on-site examiner can observe when sufficient sample has been absorbed and/or verify that the sample is saliva by observation of the change in indicator 48.
  • Figs. 5 and 6 an alternate apparatus similar to that of Fig. 4 will be discussed.
  • the entirety of absorbent 54 may be inserted into the mouth of a subject by support stick 52 for collection of saliva thereon.
  • the entirety of absorbent 54 is the zone of application.
  • sample probe 50 is removed from the subject's mouth and testing of the sample can be accomplished to determine if the sample truly is saliva.
  • the indicator component of this embodiment is separate from the absorbent 54.
  • Indicator test portion 58 of absorbent 54 is detached from sample portion 57 along perforations 56. After separation of test portion 58 from absorbent 54 the indicator component for saliva presence may be applied to test portion 58. If the test demonstrates that the sample on absorbent 54 is in fact saliva, sample portion 57 of absorbent 54, attached to support 52, may be allowed to dry and sent to the laboratory for testing for HIV antibodies or abuse of drugs such as cocaine or for testing of other analytes of interest such as islet cell antigen antibodies.
  • FIG. 6 a side elevational view of the embodiment of Fig. 5 is illustrated showing perforations 56 on absorbent 54 between test portion 58 and sample portion 57.
  • FIG. 7 another embodiment, collection device 70, is shown assembled for shipment.
  • Sample probe 72 is contained within tube 74 to protect the sample probe during shipment.
  • Probe 72 is secured within tube 74 by cap 76 which contacts tube 74 by a frictional fit or by registrable threads on tube 74 and cap 76.
  • tube 74 be constructed from a light excluding material to protect the sample, the indicator and other components of collection device 70 from degradation by light exposure.
  • sample probe 72 is shown removed from tube 74. It may now be appreciated that in this embodiment cap 76 is joined to sample probe 72 and is utilized for holding sample probe 72 during sample collection. Attached to cap 76 of sample probe 72 is capillary tube 78. Capillary tube 78 extends from cap 76 approximately four to six inches. Absorbent or wick 80 is inserted into the end of capillary tube 78 opposite cap 76. In use sample probe 72 is inserted into the mouth of a subject and allowed to remain until such time as absorbent or wick 80 becomes saturated with saliva. The absorption of saliva onto absorbent 80 is assisted by capillary action provided by capillary tube 78.
  • FIG. 9 a perspective view of capillary tube 78 is shown to illustrate hollow channel 79 which extends through tube 78 and into which absorbent 80 is inserted.
  • FIG. 10 a cross-sectional view of sample probe 72 taken along line 10-10 of Fig. 8 is presented.
  • Capillary tube 78 surrounds absorbent 80.
  • On top of absorbent 80 is one-way barrier 82 and on one-way barrier 82 is indicator component 84.
  • indicator component 84 As saliva migrates or is wicked along absorbent 80 it approaches barrier 82 which is situated atop absorbent 80 as shown in Fig. 8. The saliva will pass through one-way barrier 82 to interact with indicator component 84. However, once the saliva has passed upwardly through barrier 82 to contact indicator 84 the saliva is unable to migrate back through barrier 82. Thus contact between the subject and the saliva that has interacted with indicator 84 is avoided.
  • indicator component 84 can interact with the presence of saliva in two ways.
  • indicator 84 may be a simple dye marker which, when contacted by saliva, will become partially dissolved and then diffuse in a bleeding fashion. Such a change in the dye would indicate that saliva had entirely coated absorbent 30 and contacted indicator 84.
  • the change in the dye marker indicator 84 serves to show the on-site examiner when a sufficient saliva sample has saturated absorbent 80 to allow testing to be properly accomplished at the laboratory.
  • a second function of indicator 84 is accomplished when a chemical chromogen substrate is incorporated into indicator 84.
  • the apparatus provides rapid indication, at the time of taking a saliva sample from a subject, that not only has sufficient sample been applied to absorbent 80, but that the sample applied to absorbent 80 is, in fact, saliva.
  • Fig. 11 a perspective view of absorbent 80 is shown having barrier 82 and indicator 84 in place on an end of absorbent 80.
  • This interaction between the dye or marker and the saliva may be of various types.
  • the dye or marker is soluble or partially soluble in saliva, the dye or marker will begin to diffuse into the saliva and the bleeding effect of the diffusing dye will be noted by the on-site examiner as illustrated in Figure 1 at zones 28a, 28d.
  • the dye or marker is insoluble in saliva but exhibits mobility with respect to its position on absorbent 44, 20, 30 such as colored fluorescent plastic micro particles, it is possible to accomplish the migration of the marker or dye along the absorbent with the migration of the saliva front along absorbent 44, 20, 30.
  • This manner of utilizing a simple dye marker is suitable for indication to the on-site examiner that a sufficient level of saliva has been acquired on absorbent 44, 20, 37. This signaling that sufficient saliva has been applied is accomplished by positioning marker or dye 48 (Fig. 4) or 29 (Fig. 1) or 37 (Fig.
  • a dye suitable for human consumption or external contact could be placed at a position on absorbent 44 (Fig. 4) below the known position at which sufficient sample is indicated. The dye is then allowed to migrate along absorbent 44 with the moving front of absorbed saliva. In this manner when the moving marker or dye front reaches the known point of sample sufficiency along absorbent 44 the on-site examiner would be aware that sufficient saliva sample had been collected.
  • any of a number of suitable vegetable or food dyes may be used as the marker.
  • suitable vegetable or food dye markers are the FDA approved dyes for use as food, drug or cosmetic dyes such as red No. 1 or No. 40 or yellow No. 5 or Blue No. 1 or a combination thereof or any other colorant which is non-toxic to humans.
  • the dye or marker need only be easily visible against the absorbent selected and be safe for human consumption or human external contact.
  • absorbents include an absorbent paper, S&S ® 903TM, manufactured by Schleicher & Schuell, Inc. or equivalent absorbents from other vendors such as Whatman, Inc. of Clifton, N.J.
  • porous plastic absorbent supports formed from polyethylene, polypropylene, polyvinylidene fluoride, ethylene-vinyl acetate, styrene-acrylonitrile, or polytetrafluoroethylene having known absorption characteristics may be used in place of cellulose or sponge material.
  • the absorbent it is principally necessary that the material selected absorb a known reproducible quantity of saliva when it is provided with sufficient sample to saturate the material.
  • the amount of saliva required for accurate laboratory testing of the sample is then calculated.
  • the amount of required sample in milliliters is divided by the number of milliliters of saliva which can be absorbed per square centimeter of absorbent material.
  • the dye or marker is then imprinted upon the absorbent material at a location sufficiently distant from the area of sample application so as to require that at least the minimum necessary sample volume will be applied before the saliva interacts with the indicator. In this manner the desired degree of accuracy is assured.
  • the design of the saliva sample apparatus is therefore a card of absorbent material provided with six saliva application zones 28a-f having a diameter of 1.25 cm and circumscribed by an imprinted dashed indicator line which encloses 1.23 square centimeters of absorbent area.
  • each zone is properly filled it will contain 61.36 microliters of saliva and the card will contain approximately 368 microliters of total saliva.
  • the on-site examiner is to apply the saliva to center of the application zone. As the saliva absorbs on the absorbent layer 20 (Fig. 1) it moves outwardly from the application zone and after application of slightly more than 50 microliters of saliva the saliva will contact the indicator. The contact of the saliva will cause the indicator to migrate outwardly with the moving saliva and will present the diffusion pattern shown in Fig. 1 at reference numeral 28a. If less than the required amount of saliva is applied to the card the saliva will only partially contact the indicator. In this case only a slight migration of the indicator will be noted and a diffusion pattern such as shown in Fig. 1 at reference, numeral 28d will be observed.
  • the application of the saliva to the absorbent card could be accomplished by having the subject expectorate into a container and then applying the saliva with a pipette to the absorbent sheet 20.
  • the peroxidase of saliva is typical of most peroxidases in that it catalyzes the oxidation by hydrogen peroxide of a relatively narrow number of classes of organic compounds. These classes of compounds include phenols; aromatic, primary, secondary, and tertiary amines; the leuco-dyes; certain heterocyclic compounds such as ascorbic acid and indole; as well as certain inorganic ions such as the iodide ion.
  • peroxidase is not restricted to hydrogen peroxide in particular, but that any number of compounds which are of the hydroperoxide type may act as the oxygen provider according to the following reaction scheme:
  • Some of the hydrogen peroxide compounds which may be utilized in this reaction are hydrogen peroxide, cumene hydroperoxide, benzoyl hydroperoxide, methyl hydrogen peroxide, ethyl hydrogen peroxide, and butyl hydrogen peroxide.
  • the peroxide necessary for the reaction with peroxidase or catalase may be generated in situ on the absorbent utilizing glucose oxidase on the absorbent to react with the glucose of the saliva sample to produce hydrogen peroxide.
  • the generated hydrogen peroxide could then be utilized in reaction with the catalase or peroxidase of saliva to react with the colorless substrate compound to provide the inventive indicating system of the invention.
  • the peroxidase and/or catalase of saliva is used to catalyze a reaction upon the absorbent which visibly indicates to the on-site examiner that the fluid sample obtained contains peroxidase and/or catalase and may be concluded to be saliva. Also, it may be indicated to the on-site examiner that sufficient sample has been collected for laboratory testing. The latter attribute is determined by the specific pre-positioning of the peroxidase/catalase substrate and reactants at a particular location on the paper as previously described for the simple marker methodology in determining sample sufficiency.
  • the inventive method utilizing saliva peroxidase/catalase to verify the presence of saliva in a fluid sample, it is useful to employ an enzyme substrate which reacts with peroxidase/catalase to undergo an easily observed color change.
  • an enzyme substrate which reacts with peroxidase/catalase to undergo an easily observed color change.
  • a wide variety of such compounds exist and are well known in the art.
  • the preferred class of enzyme substrate compounds for verification of saliva presence is known generally as the leuco-dyes or leuco compounds.
  • Leuco-dyes may be thought of as chromogens in that they will, in at least one of their molecular forms, become colored compounds easily observable in the visible spectrum.
  • the classification "leuco” is due to the fact that these compounds present one molecular form which is white or colorless therefore exacting the name "leuco” dye (from the Greek leukos meaning clear) from observers.
  • leuco-dyes have received much attention as vat-dyes for textiles. In this application the leuco form of the dye is soluble in alkalinic aqueous solutions whereas the colored or oxidized form of the compound is essentially insoluble.
  • n 1: indigo, benzo-, naphtho- and anthraquinone
  • n 2: anthanthrone, dibenzpyrenequinone, etc.
  • n 4: pyranthrone, dibenzanthrone, dipyrazolanthrone, etc.
  • R, (___- and R ⁇ H o-tolidine R ⁇ OCH-;
  • R 2 H o-dianisidine
  • the above reaction between the peroxidase of saliva and a hydroperoxide in the presence of a leuco dye proceeds generally as follows.
  • Peroxidase joins with the hydroperoxide to provide an oxygen atom to the reactants which then acts as a hydrogen acceptor.
  • the peroxidase-hydroperoxide complex then extracts two hydrogens from the leuco form of the molecule to oxidize the molecule to its colored form.
  • amine compounds a hydrogen is extracted from each amine group to result in the tertiary amine.
  • diols the hydrogen is removed from the two alcohol moieties to form the colored oxidized form of the compound and a molecule of water.
  • hydrogen peroxide is used, two molecules of water are formed from the destruction of hydrogen peroxide.
  • Inventive use of this enzyme catalyzed reaction of leuco form enzyme substrate compounds into their oxidized colored form is accomplished by the application of the leuco form of the substrate compound in solution and application of a suitable hydroperoxide to a preselected location on an absorbent.
  • the proper position for the leuco compound on the absorbent is determined by calculating the amount of saliva sample required for laboratory testing and then calculating the particular distance from a zone of saliva application for placement of the leuco compound.
  • the proper distance is that which would require placement of sufficient saliva sample on the absorbent to saturate the absorption to the preselected point to accomplish contact between the saliva and the leuco reactants.
  • the leuco substrate compound and the hydroperoxide compound are then applied to the absorbent at that location so that when saliva is wicked from the zone of application on the absorbent to the point of application of the leuco enzyme substrate and the hydroperoxide the peroxidase or catalase enzyme presence in saliva will contact the peroxide and the leuco compound substrate to oxidize the leuco substrate to its colored form.
  • the presence of saliva to the on-site examiner as well as indicatinq that a sufficiency of saliva sample had been applied to the absorbent.
  • TMB dimethyl formamide
  • DMSO dimethyl sulfoxide
  • cumene hydroperoxide may be replaced by a 0.03% aqueous hydrogen peroxide solution in an acetate buffer (0.05 mol/1) containing 1 mmol/ml EDTA at a pH of 4.7 and dried immediately at elevated temperatures (50°C ⁇ 5°C).
  • an on-site examiner is prepared to take a sample the absorbent layer 20 (Fig. 1) is removed from the light protective cover and a sample probe 22 containing saliva from the subject is applied to the center of zone 28a Fig. 1. The swab is left in contact with absorbent layer 20 until the on- site examiner observes a color change as represented in Fig. 1 at indicator 28a.
  • the swab 26 of probe 22 then may be removed from zone 28a and moved to zone 28b for application of saliva to that zone.
  • the on-site examiner can quickly and conveniently deliver the required amount of saliva to the absorbent and also verify that, in fact, it is saliva which is being applied to the absorbent.
  • the saliva which has been applied to zone 28a migrates until it contacts the peroxide and the TMB.
  • the peroxidase enzyme in the migrating saliva contacts the peroxide and TMB and oxidizes the TMB into its colored form whereupon the on-site examiner can observe the color change due to the reaction and determine that the sample is saliva and that the sample quantity was sufficient.
  • a solution of o-phenylenediamine dissolved in DMSO to give a final concentration of 1 mg/ml in an acetate buffer (0.05 mol/1) (pH 4.7 to 4.8) and containing 1 mmol/ml EDTA is applied to a preselected position (indicator 29) on absorbent layer 20 in Fig. 1.
  • o-phenylenediamine solution is then allowed to dry and a solution of hydrogen peroxide in DMF (0.03 percent v/v) is then applied to the same area as was the o-phenylenediamine. After the peroxide has dried the absorbent layer 80 is protected from light. This is then utilized in the manner previously described.
  • a solution of guaiacol dissolved in DMSO to give a final concentration of 50% v/v is applied to a preselected position (indicator 29) on absorbent layer 20 in Fig. 1.
  • the guaiacol solution is then allowed to dry and a solution of hydrogen peroxide in acetate buffer, 0.05 mol/1, pH 4.7, 1 mmol/ml EDTA (0.03 percent v/v) is then applied to the same area as was the guaiacol. After the hydrogen peroxide has dried the absorbent layer 80 is protected from light. This is then utilized in the manner previously described.
  • this procedure can be accomplished with solutions of the below listed compounds at a concentration in a range of between 100 ⁇ grams to 1 milligram per milliliter dissolved in a suitable solvent such as an acetate buffer and organic solvent mixture (e.g. DMSO, DMF or alcohol) .
  • a suitable solvent such as an acetate buffer and organic solvent mixture (e.g. DMSO, DMF or alcohol) .
  • DMSO acetate buffer and organic solvent mixture
  • the reaction of the leuco compound with peroxidase and the selected hydroperoxide will arrive at a sufficient end point (color change observation by the on-site examiner) within a period of 40 seconds to approximately 1/2 minutes.
  • Suitable leuco enzyme substrate and indicator compounds which can react in accordance with this method are benzidine; o-tolidine; o-dianisidine; o-phenylenediamine; 2,2 '-azino-di(3- ethylbenzthiazoline sulphonic acid-6) (ABTS) ; 4-amino antipyrine; guaiacol; pyrogallol; 4-chloro-l-naphthol; and nitrotetrazolium blue chloride.
  • the buffers that can be utilized to maintain the pH of the peroxidase reaction may be citrate, succinate, phthalate or phosphate.
  • the amylase enzyme of saliva may be utilized to produce a visible color change in a substrate compound. This color change is observable to the on-site examiner and indicates that the substance applied to the absorbent is saliva. Also, by placing the amylase-reactive compound at a specified distance from the zone of application, the on-site examiner can determine that sufficient sample has been applied to the absorbent to permit proper testing at the laboratory. Amylase is present in all living organisms and in mammals is present in high concentration in saliva.
  • Amylase is a member of the group of hydrolase enzymes which cleave the ⁇ - 1,4 glycoside bonds in oligosaccharides (from trisaccharides upwards) and polysaccharides such as starch, glycogen and dextrins. Amylase acts upon the ⁇ -l,4-glucan links in large linear polysaccharides having three or more __-l,4-linked D- glucose units.
  • the high amylase activity in saliva can be used to drive a reaction which releases a portion of a polysaccharide substrate. If the released portion of the polysaccharide contains sufficient conjugated double bonds within its structure it will absorb light and be visible to an observer as a colored compound on the absorbent paper.
  • polysaccharide embraces oligosaccharide and the two are used interchangeably when generally referencing an amylase substrate to which is attached a chromogen. Therefore the chromogen derivative reactive to amylase could be a tetraoside, pentaoside, hexaoside, heptaoside, or other polysaccharide.
  • a colorless form of an amylase substrate (an oligosaccharide or a polysaccharide attached to a chromogen) in a proper combination of enzymes and buffer system is applied to an absorbent and dried.
  • Saliva is then applied to the absorbent by an on-site examiner.
  • the substrate is then cleaved by the saliva amylase and enzymes present in the applied substrate solution to release the chromogen compound from the oligosaccharide. This results in a colored area on the absorbent paper to which saliva has been applied. This colored area is then visible to an on-site examiner or other observer.
  • amylase in combination with other enzymes, cleaves the substrate molecule to release the chromogen is:
  • glucoamylase ⁇ -glucosidase chromo-(glucose) 432 chromo + chromo(glucose) 4 + x-glucose
  • the chromogen containing oligosaccharide e.g. silyl-blocked-4- n trophenylmaltoheptaoside
  • oligosaccharide e.g. silyl-blocked-4- n trophenylmaltoheptaoside
  • glucoamylase and ⁇ -glucosidase further act on the chromogen-linked oligosaccharides of 2, 3 and 4 glucose units to hydrolase the chromogen from the 2 and 3 glucose unit oligosaccharides.
  • a few of the 4 glucose unit oligosaccharides are also hydrolysed, but at a much slower rate. This hydrolysis results in release of a sufficient quantity of the chromogen to permit visual observation of a color presence on the absorbent by the on-site examiner. Thus the examiner is able to quickly and easily verify the presence of saliva on the absorbent. If the chromogen- containing oligosaccharide is placed at a particular distance from the zone of application the examiner may also determine that sufficient sample quantity has been placed on the absorbent to permit analysis at the laboratory. In this manner the amylase present in saliva may be utilized within the inventive method and apparatus to determine both sufficiency of saliva quantity as applied to the absorbent and also verify that the substance applied to the absorbent is in fact saliva.
  • the substrate molecules utilized are generally composed of a polysaccharide or oligosaccharide chain having a chromogenic moiety or molecule attached to one end of the chain.
  • the other end of the chain is either open or has attached a molecule which inhibits the enzymatic action of glucoamylase or ⁇ -glucosidase and therefore amylase.
  • the purpose of such a blocking agent is to promote the action of amylase on the substrate molecule over the action of glucoamylase or ⁇ - glucosidase.
  • polysaccharide or oligosaccharide molecules having chromogens incorporated include: ⁇ -4-nitrophenylmaltoheptaoside, 0-4-nitrophenylmaltoheptaoside, 4,6, ethylidine-blocked-4-nitrophenylmaltoheptaoside, silyl- blocked-4-nitrophenylmaltoheptaoside, 3-ketobutylidene-beta-2 chloro-4-nitrophenylmaltopentaoside, and derivatives of the above wherein Indole moiety is substituted for the p-nitrophenyl moiety such as Indolyl- ⁇ -D-maltoheptaoside.
  • saccharogenic substrates such as thiazine-labeled starches, amylopectin-azure and amylochro e dissolved in a suitable buffer may be used. However, if these substrates are used the eluate from the absorbent will require centrifugation to remove the substrate material.
  • a solution is composed of the chromogen-containing amylase substrate selected from the above compounds or their equivalents, ⁇ -glucosidase, glucoamylase, sodium chloride, calcium gluconate or calcium chloride dissolved in a suitable buffer.
  • Suitable buffers include: ACES ⁇ N-2-acetamido-2-aminoethanesulfonic acid ⁇ , ADA ⁇ N-2-acetamidoiminodiacetic acid ⁇ , BIS-Tris-Propane ⁇ 1 3- bis[ins(hydroxymethyl)methylamino]propane ⁇ , HEPES ⁇ N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid ⁇ , HEPPS ⁇ N-2- hydroxyethylpiperazine-N'-3'propanesulfonic acid ⁇ , MES [2- ⁇ N- morpholino ⁇ ethanesulfonic acid], MOPS [3- ⁇ N- morpholino ⁇ propanesulfonic acid], PIPES [piperazine-N,N'-bis(2- ethanesul onic acid)], TES [2 ⁇ [(tris- hydroxymethyl)methyl]amino ⁇ ethanesul onic acid] .
  • the method first involves formulation of a solution containing the chro ogen-linked substrate and secondary enzymes for release of the chromogen after action on the substrate molecule by amylase.
  • the solution applied to the substrate was composed as follows:
  • Glucoamylase 3.0 units 0.1 - 30 Units
  • Amylase substrate 0.05 nM 0.005 nM - 0.5 nM (4-nitrophenylmalto- heptaoside PNPG-7)
  • the concentrations may be varied according to the speed of the visual color reaction required.
  • saliva is applied to the absorbent by any convenient method.
  • the chromogen will be released from the remaining glucose units and become colored and visible to the examiner.
  • Sample probe 100 is composed of support stick 102 having absorbent 104 attached to an end thereof.
  • Absorbent 104 is affixed within two registrable portions of support stick 102.
  • the specifics of the capture of absorbent 104 within support stick 102 are described herein. However, the purpose of such capture is to prevent substitution of a false absorbent layer into sample probe 100 and to allow easy and sure identification of any sample probes subjected to tampering attempts.
  • Fig. 13 the capture of absorbent 104 within the portions of support stick 102 is shown.
  • Absorbent 104 is sandwiched between two registrable portions of support stick 102.
  • An absorbent holder portion 106 on support stick 102 is provided to receive absorbent 104.
  • a compression lid which fits into or in registration with holder 106 may then be fitted in place within holder 106 to securely fasten absorbent 104 within sample probe 100.
  • absorbent 104 is shown already having been captured in or sandwiched between holder 106 by compression lid 110 and the lid reopened. This, as Fig. 15 reveals, results in absorbent 104 being attached to spikes 108 of compression lid 110. The capture of absorbent 104 between holder 106 and compression lid 110 is accomplished in the following manner.
  • unassembled support stick 102 is illustrated with compression lid 110 opened.
  • Breakaway connectors 112 are attached between compression lid 110 and holder 106 to secure lid 110 to support stick 102.
  • Breakaway connectors 112 serve as a hinge-like structure allowing compression lid 110 to be folded over onto holder 106 and brought; into registration for capture of absorbent 104. It will be appreciated that both compression lid 110 and absorbent 104 are sized to closely register with or fit within holder 106. Still referring to Fig. 16, absorbent 104 has been shown in phantom lines to indicate its positioning under the opened compression lid 110 and placement in holder 106.
  • compression lid 110 is then pivoted on breakaway connectors 112 and aligned with holder 106. A slight pressure on compression lid 110 will press spikes 108 through absorbent 104 and into spike receptacles 129. Crimp rivet 114 then enters rivet receptacle 116 on support stick 102. When it is desired to securely fasten compression lid
  • sample probe 100 After preparation of sample probe 100 as described above, it may then be used for obtaining samples of saliva from a subject without the need for the on-site examiner to observe the subject at all times during the sampling procedure. This is of particular importance in relieving the on-site examiner from the burden of personally verifying the collection of each sample through observation. This also permits the on-site examiner to simultaneously make collections from multiple subjects in different areas. As it requires a few moments for sufficient saliva to saturate absorbent 104, the on-site examiner is able to attend to other duties and testing of other personnel as it is no longer necessary for the entire saliva collection from each subject to be actually observed by the on-site examiner. In use, absorbent 104 is placed within the mouth of the subject and saliva is allowed to saturate absorbent 104.
  • the entirety of support stick 102 is exterior of the mouth.
  • the application of saliva to absorbent 104 will saturate a first saliva application zone or test area 120 and a second saliva application zone or archive area 122 prior to contacting indicator 125.
  • indicator 125 is not contacted by saliva until saliva has saturated test area 120 and archive area 122. After such saturation saliva is wicked onto covered area 126 (Fig. 15) .
  • saliva migrates along covered area 126 of absorbent 104 and toward indicator 125 it moves between compression lid 110 and holder 106.
  • the migrating saliva then contacts indicator 125 causing either migration of indicator 125 or a color change in the indicator or both.
  • the on-site examiner can observe that sufficient saliva quantity has been applied to the absorbent and/or that the substance applied to the absorbent is in fact saliva.
  • the type of analysis enabled will depend upon the type of substance used as indicator 125.
  • the apparatus provides test security both through the type of indicator 125 which is utilized and also through the structural features of compression lid 110 and holder 106 which evidence attempts to tamper with sample probe 100.
  • compression lid 110 has been affixed within holder 106 through the flattening crimp rivet 114 strong pressures are required to reopen compression lid 110. In such an event, crimp rivet 114 will be pulled back through receptacle 116, but crimp rivet 114 will not be recompressed. Therefore, it is not possible to insert a second time crimp rivet 114 through receptacle 116.
  • a second tamper resistant feature is provided by breakaway connectors 112.
  • spikes 108 which protrude from compression lid 110 and upon closure of compression lid 110 within holder 106 pierce absorbent 104 and extend through spike receptacles 129. As spikes 108 pierce absorbent 104 and secure the absorbent within support stick 102 it is not possible to slide absorbent 104 out of support stick 102 when compression lid 110 is closed and secured within holder 106. If an attempt is made to withdraw absorbent 104 when compression lid 110 is secured absorbent 104 will be shredded by the attempt. Additionally, it will not be possible to insert a second absorbent sheet into the closed holder as spikes 108 are projecting through spike receptacles
  • Adhesive 134 can serve to seal compression lid 110 within holder 106 and also serve as evidence of tampering. Once adhesive 134 dries it is in contact with both absorbent holder 106, compression lid 110, and absorbent 104. An attempt to open compression lid 110 will result in shredding and release of absorbent 104 which will be clearly obvious upon examination of sample probe 100.
  • Figs. 12-16 also provides the laboratory with a means for both testing the saliva sample upon its receipt in the laboratory and a method of archiving a portion of the same exact sample for future confirmation of test results. This is accomplished through the use of a two-part absorbent 104.
  • absorbent 104 is shown composed of test area 120 and archive area 122. These two areas are separated on a line between tear points 136. Tear points 136 permit test area 120 to be separated from archive area 122 so the desired tests may be performed on test area 120.
  • a sample probe 100 is inserted into a subject's mouth and absorbent 104 is saturated with saliva. The saliva migrates into covered area 126 and activates indicator 125.
  • sample probe 100 is then sent to the laboratory for analysis of the saliva sample.
  • identification information of sample probe 100 is noted on label 138, by writing or by bar code, is noted.
  • Test area 120 is then separated from sample probe 100 by tearing along tear point 136. The desired tests are conducted on the saliva present in test area 120.
  • the remainder of sample probe 100, inclusive of archive absorbent area 122, is then stored under proper conditions for as long as the laboratory believes necessary. This provides the laboratory with a convenient reference sample composed of a specimen in archive area 122 which is identical to the specimen in test area 120 upon which the initial testing was conducted. If a question arises regarding the results of the analysis on test area 120, the laboratory can be contacted, the archive specimen contained in archive area 122 retrieved, and testing repeated.
  • indicator 125 may be composed of a simple vegetable dye or composed of any of the biochemically reactive indicators discussed herein.
  • indicator 125 indicates only that sufficient saliva quantity has been applied to absorbent 104.
  • indicator 125 will demonstrate both that sufficient quantity of saliva has been applied and that the substance applied to absorbent 104 is in fact saliva.

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Abstract

Procédé destiné à déterminer rapidement pendant une procédure de collecte d'un échantillon de salive la présence d'une quantité de salive et à vérifier que l'échantillon obtenu est en fait de la salive. Une indication de couleur par des marqueurs colorants et/ou l'activation enzymatique d'indicateurs de couleur (125) indique qu'au moins une quantité prédéterminée de salive a été appliquée sur un absorbant (104) et la réaction enzymatique indique que l'échantillon collecté contient de la salive. Un appareil (100) destiné à collecter un échantillon de salive humaine est décrit. Il comporte un indicateur de couleur (125) pour signaler la collecte d'une quantité prédéterminée d'un échantillon. Alternativement, ledit appareil (100) contient un chromogène capable de subir une conversion chimique au contact de salive pour produire un changement de couleur, ce qui permet ainsi de vérifier la présence de salive par opposition à un autre liquide.
EP93900603A 1991-11-27 1992-11-18 Apparatus and method of saliva collection and verification for dried saliva spot drug and hiv antibody testing. Withdrawn EP0625268A4 (en)

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
US80064991A 1991-11-27 1991-11-27
US80064791A 1991-11-27 1991-11-27
US800647 1991-11-27
US81408091A 1991-12-24 1991-12-24
US07/814,077 US5334502A (en) 1991-11-27 1991-12-24 Method of collecting, identifying, and quantifying saliva
US814080 1991-12-24
US814077 1991-12-24
US95470092A 1992-09-30 1992-09-30
US954700 1992-09-30
PCT/US1992/010085 WO1993011434A1 (fr) 1991-11-27 1992-11-18 Appareil et procede de collecte et de verification de salive destines a detecter la toxicomanie et les anticorps hiv a partir d'une tache de salive sechee
US800649 1997-02-04

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EP0625268A1 EP0625268A1 (fr) 1994-11-23
EP0625268A4 true EP0625268A4 (en) 1996-02-28

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EP93900603A Withdrawn EP0625268A4 (en) 1991-11-27 1992-11-18 Apparatus and method of saliva collection and verification for dried saliva spot drug and hiv antibody testing.

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CA (1) CA2121361C (fr)
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CA2121361C (fr) 1997-03-04
EP0625268A1 (fr) 1994-11-23

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