WO1993011434A1

WO1993011434A1 - Apparatus and method of saliva collection and verification for dried saliva spot drug and hiv antibody testing

Info

Publication number: WO1993011434A1
Application number: PCT/US1992/010085
Authority: WO
Inventors: Jangbir S. Sangha; Robert S. Shea
Original assignee: Osborn Laboratories, Inc.
Priority date: 1991-11-27
Filing date: 1992-11-18
Publication date: 1993-06-10
Also published as: EP0625268A4; CA2121361C; EP0625268A1; CA2121361A1

Abstract

A method is provided for rapidly determining during a saliva specimen collection procedure the presence of an amount of saliva, and for verifying that the sample obtained is in fact saliva. Color indication by dye markers and/or enzymatic activation of color indicators (125) provides an indication that at least a predetermined amount of saliva has beed applied to an absorbent (104) and the enzymatic reaction indicates that saliva is contained in the sample collected. Apparatus (100) for collecting a human saliva sample is provided having a dye indicator (125) for signalling the collection of a predetermined sample quantity. Alternatively, the apparatus (100) contains a chromogen capable of undergoing chemical conversion upon contact with saliva to produce a color change thereby verifying the presence of saliva as opposed to another fluid.

Description

APPARATUS AND METHOD OF SALIVA COLLECTION AND

VERIFICATION FOR DRIED SALIVA SPOT

DRUG AND HIV ANTIBODY TESTING

CROSS-REFERENCE Reference is made to the concurrently pending U. S. applications of the present inventor, Jangbir S. Sangha, Serial No. 07/800,649 and Continuation-in-Part Serial No. 07/814,080 and Continuation-in-Part Serial No. 07/954,700 relating to the apparatus and Serial No. 07/800,647 and Continuation-in-Part Serial No. 07/814,077 relating to the associated method for collecting and verifying the quantity and presence of saliva for dried saliva spot drug and HIV antibody testing.

BACKGROUND OF THE INVENTION 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.

In collecting body fluid samples, and in particular human body fluid samples, it is at times necessary to collect samples in the field or outside of a controlled collection environment. Two such cases are immediately evident, that of law enforcement sample collection for evidentiary purposes and the collection of samples by the health insurance industry for health status determinations on prospective insurance purchasers.

In law enforcement sample collection of human body fluid samples, the collection of saliva is potentially useful as a superior indicator of blood alcohol levels in contrast to simple "breathalizer" types of testing. Saliva samples can be saved for future confirmation of the original test results whereas the "breathalizer" tests cannot. For the health and life insurance industry saliva testing is useful as a matter of convenience for the individual seeking insurance. When the sample collection for testing for preexisting conditions can be conducted at their home or within their work site, inconvenience is minimized and there is less resistance to testing.

In the insurance industry it has become common practice to send out a medical on-site examiner and have the fluid sample—saliva—collected at a location most convenient for the subject desiring insurance. Heretofore on-site examiners have collected saliva by having individual expectorate into a container and then adding an anti-bacterial agent to the saliva and shipping it into the laboratory for testing or by collecting a sample of saliva on a swab and then plunging the swab into an anti-bacterial agent for shipment into the laboratory.

Inherent in such "on-site" sample collection situations is that subsequent transport of the sample to a testing laboratory is required. In many cases it is convenient, as well as necessary, to ship the sample to the laboratory by mail or courier in order to minimize the number of laboratory operations. In such cases it is beneficial if the sample can be reduced to a solid phase or at least minimized in volume. As a result it has recently been found advantageous to ship body fluid samples, blood in particular, as a dried spot on an absorbent matrix. In this manner the sample volume is reduced and the sample weight commensurately reduced by evaporation of the fluid component.

Conveyance of these physiological fluid samples to the laboratory often occurs under poor conditions and the use of public and private mail delivery or couriers to transport such samples increases the potential for careless handling. Damaged and leaking fluid samples may alarm or endanger courier or mail system workers who contact the damaged and leaking packages of fluid physiological materials. Thus it is of substantial benefit when the fluid component can be reduced or eliminated in such samples. However, the application of the fluid sample to an absorbent upon which the sample can be dried causes certain problems in the case of saliva. Unlike the on-site examiner, the receiving laboratory does not have the advantage of actual observation of the subject. 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. However, as the 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.

It has been confirmed that intravenous use of cocaine in addition to oral or intra-nasal administrations of cocaine may be determined from saliva. Recently, there has been interest in determining through saliva testing the presence of human immuno-deficiency virus (HIV) antibodies to assist insurance companies in screening out risks to business prior to offering contracts for health and life insurance.

Generally, 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.

These saliva collection techniques, however, 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. In addition, 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.

One avenue around these difficulties has been to allow the subject to place an absorbent swab or paper into the mouth and allow the absorbent material to become saturated with saliva within a set time. However, this presents the problem that it is difficult to determine the degree of saturation of the swab and if a number of saliva samples are being concurrently taken, it may not be possible for the on-site examiner to visually observe each subject and thereby personally witness and ascertain that it is saliva which has been absorbed onto the paper.

More importantly, due to the nature of the absorbent paper, it may be difficult for the on-site examiner to determine that adequate sample has been placed on the absorbent to allow proper testing. In this event it is necessary to return to the subject at a later date to collect an additional sample which is not only inconvenient but adds additional expense to the determination being made and may raise questions as to the competence of the company requesting the test, the competency of the on-site examiner, as well as the health of the subject. Therefore, it is an object of the present invention to provide an apparatus and method for field saliva sampling which will avoid the above debilities of the prior apparatus and methods of saliva sampling.

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 protects technical personnel working with a dried clear fluid sample by making apparent the location of the sample after it is dried. Another object of the invention is to provide an apparatus and method that indicates that the sample was correctly and uniformly applied to the absorbent. Yet another object of the invention is to provide an apparatus and method which can be made and utilized at a low cost.

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.

Other objects and advantages of this invention will become apparent from the following description taken in connection with the accompanying drawings, wherein is set forth by way of illustration and example, an embodiment of this invention.

SUMMARY OF THE INVENTION In broad summary, 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. 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.

Generally, 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.

In this manner, once sufficient saliva has been absorbed so as to allow sufficient saturation of the absorbent to result in saliva contacting and reacting with 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.

To insure against tampering with the absorbent and indicator the portion of the absorbent upon which the indicator is placed may be sandwiched between layers of a clear support stick and the layers of the support stick riveted or sonically welded or joined by adhesive such that if an attempt is made to open the layers and tamper with the absorbent or indicator the attempt will be apparent as the layers cannot be resealed. Other objects and advantages of this invention will become apparent from the following description taken in connection with the accompanying drawings, wherein is set forth by way of illustration and example, an embodiment of this invention. BRIEF DESCRIPTION OF THE DRAWINGS 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; and

soesrⁱTure 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.

DETAI ED DESCRIPTION

The Collection Apparatus In the embodiment of Fig. 1 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. In use, swab 26 is placed into the mouth of a subject and saliva is collected. After collection of saliva on swab 26 probe 22 is removed from the subject's mouth and swab 26 is 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.

In making the transfer of saliva, 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, as well as the other embodiments, may be either a vegetable dye or a colorless chemical substrate which acts as a chromogen in the presence of saliva. Where 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. Alternatively, 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. In addition, 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. Once the sample has been applied to absorbent layer 20 the saliva sample spots are allowed to dry for shipping to the laboratory. By allowing the saliva spot to dry on the absorbent layer the saliva sample becomes more stable and can better withstand elevated temperatures. In addition, 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.

At the laboratory, portions of the dried saliva sample are punched from the absorbent and tested for HIV virus antibodies or drugs of abuse such as cocaine. Alternatively, 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.

In using the simple dye marker method with the embodiment of Fig._.1, 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.

Alternatively, where 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. As 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. Once the applied saliva contacts 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.

In addition to indicating that the sample is saliva by the color change of the chromogen substrate, application of the dye or the chromogen in a pattern can provide additional information to the laboratory regarding the sample quality. It is intended that the on-site examiner apply the saliva to the center of the application zone. When this is the case 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. As to application accuracy, if the on-site examiner applies the sample in the center of zone 28a-f, 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.

As workers, with the aid of the inventive indicator, can now see where the sample has been applied, they may avoid touching that part of the absorbent conraining the sample. In the prior art absorbents, the laboratory technician was unable to observe the location of the colorless dried saliva and therefore could contact those areas containing sample which not only leads to erroneous results from manual contamination, but leads to infection of personnel.

The second attribute—observation of the amount of sample applied to the zone—is provided by noting the distance of outward migration of indicator 29. If the on-site examiner has applied the proper quantity of sample to application zone 28a-f a particular distance of migration of the indicator will result and can be observed by the on-site examiner at the time of sampling and by the laboratory personnel upon receipt. If the on-site examiner has applied an improper quantity of sample to the application zone the distance of migration by the indicator will be less than expected and a correction by the on- site examiner can be made by resampling or that zone can be avoided by the laboratory analyst at testing.

Referring now to Figs. 2 and 3, another embodiment of the present invention is shown. In the embodiment of Fig. 2, absorbent sheet or layer 30 is bisected by perforations 32 into absorbent portions 34, 36. 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. Referring to 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. 2 is presented to a 10 subject who is instructed to expectorate onto absorbent layer 30 in the vicinity of perforations 32. After several saliva samples are placed onto absorbent layer 30, the saliva will spread into both absorbent layer halves 34, 36. As saturation of absorbent layer 30 is completed the saliva will pass through 15 one-way barrier 38 and contact indicator 39. Upon contact of indicator 39 with saliva the indicator will show the on-site examiner that saliva has approached that point on absorbent layer 30 and that sampling may be terminated.

Alternatively, the indicator in the embodiment of Fig. '20 2 may be placed at indicator 39 as well as perimeter indicator 37. In this situation 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.

Referring now to Fig. 4, another embodiment of the invention is shown. 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. Again, 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.

Referring now to Figs. 5 and 6, an alternate apparatus similar to that of Fig. 4 will be discussed. In the embodiment of Fig. 5 the entirety of absorbent 54 may be inserted into the mouth of a subject by support stick 52 for collection of saliva thereon. Thus, the entirety of absorbent 54 is the zone of application. After the collection of saliva, 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.

Referring to 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. Referring now to 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. It is desirable that 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.

In Fig. 8, 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.

Referring now to 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.

Referring now to 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. 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.

Again indicator component 84 can interact with the presence of saliva in two ways. First, 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. In this manner 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. In Fig. 11, a perspective view of absorbent 80 is shown having barrier 82 and indicator 84 in place on an end of absorbent 80.

The Method of Indication of Sample Sufficiency As outlined in the Summary of the Invention, the inclusion of a simple marker or dye which is at least partially soluble in saliva or which can migrate along the absorbent layer with the moving saliva front as it is wicked up the absorbent can provide a means for determining that a sufficient amount of saliva sample has been applied to the absorbent. In this method, and referring now to Figure 4, a simple dye or marker is placed on the absorbent layer as shown in Figure 4 at reference numeral 48. This colored marker or dye need only be observable against the background of the absorbent 48. As the saliva saturates absorbent 44 in zone of application 45, the saliva will move along the absorbent and approach marker or dye 48 as the amount of saliva applied to absorbent 44 increases. This migration of a saliva front along absorbent 44 will eventually reach the point on the absorbent where marker or dye 48 has been applied. Upon contact of the saliva with marker or dye 48 an interaction between the saliva and the marker or dye will occur.

This interaction between the dye or marker and the saliva may be of various types. In the case where 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.

Alternatively, if 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. 2) at a predetermined position on the absorbent sufficiently distant from zone of application 28a-f, 35, 45 that the minimum quantity of saliva required for proper testing of the sample must be applied to zone of application 28a-f, 35, 45 prior to the saliva having sufficiently wetted absorbent 44, 20, 30, to come into contact with marker or dye 48, 29, 37. Thus, at the moment the dye or marker diffuses into the saliva or begins to move with the saliva front along absorbent 44, 20, 30 the on-site examiner is made immediately aware that sufficient quantity has been collected and sampling can cease.

In an alternate embodiment, not illustrated, 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.

In this embodiment, any of a number of suitable vegetable or food dyes may be used as the marker. Examples of 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.

In using the inventive apparatus it is necessary to first select the particular absorbent for collecting the saliva sample. Suitable examples of absorbents include an absorbent paper, S&S^® 903™, manufactured by Schleicher & Schuell, Inc. or equivalent absorbents from other vendors such as Whatman, Inc. of Clifton, N.J. Also 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. As regards 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.

By way of example and not limitation, it was determined that 300 microliters of saliva sample (wet volume) were required for conducting a particular series of laboratory tests on the saliva sample. The selected absorbent material was capable of absorbing 50 microliters of saliva per square centimeter of absorbent material. This resulted in a required total saturated absorbent area of four cubic centimeters to accomplish proper testing. 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. Thus, if 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.

Should the on-site examiner misapply the sample in place it outside the application zone this too will be noted. Such an application outside the zone would result in the saliva migrating inwardly—toward the center of the application zone as shown in Fig.- 1 reference numeral 28e. In this case the on-site examiner would immediately note the improper application and a second application could be made. This therefore avoids the premature releasing the subject and the need to request that they return again for additional testing.

In the embodiment of Fig. 1, 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.

It will be apparent to those skilled in the art that a number of variations on this methodology and apparatus can be made. It is to be appreciated that such modifications of the embodiments shown are contemplated in the invention.

Method of Verification of Saliva Presence

As previously discussed, during the course of obtaining saliva samples either by law enforcement officers or by an on-site examiner for an insurance company or other interested party, it may be necessary to provide verification that the sample was in fact saliva. For law enforcement officers it may be necessary for evidentiary purposes to be able to swear that the sample obtained was observed to be saliva at the time of sampling. For on-site examiners of insurance companies it may be necessary for the on-site examiner to sample multiple subjects at one time resulting in diversion of the on- site examiner's attention from any single subject. The opportunity presented by non-observation could permit persons so disposed to substitute a water sample or some other clear fluid which would provide negative test results at the laboratory. The present method of verifying saliva presence avoids such attempts at substitution by reacting with components present in saliva to produce a reaction with an indicator which has been previously placed on the absorbent or which may be added to a portion of the absorbent after the sample has been collected.

One method of accomplishing the verification of saliva presence is through use of the salivary peroxidase (Myeloperoxidase) enzyme and/or catalase in saliva. 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.

It should be noted that the function of 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:

Peroxidase + H₂0₂ → Compound I Compound I + AH_{2(oxidizable substrate) (colorless)} ^→ Compound II + AH° Compound II + AH₂ → Peroxidase + AH"

2 AH → Oxidized product_(colored)

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.

Alternatively, 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.

In the present 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.

In 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. A wide variety of such compounds exist and are well known in the art. In the present methodology 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. Historically 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. Once the textile has been saturated with the leuco form of the dye the textile is removed from the vat and air oxidized into the colored form of the compound.

It is this ability of these compounds to become colored upon oxidation which has been utilized in the inventive method to visibly indicate the presence of saliva. This is accomplished by relying on the reduction-oxidation (redox) reaction of the leuco-dyes to change from their colorless- reduced-('leuco') form into their colored-oxidized-form. The catalyst for performing this reaction is saliva peroxidase or saliva catalase which in the presence of an oxygen donor molecule such as a hydroperoxide can accept two hydrogen atoms from the leuco compound to thereby oxidize the leuco form enzyme substrate into its colored form. This reaction is generally presented in the following equation:

leuco form (colorless) Oxidized form (colored)

n = 1: indigo, benzo-, naphtho- and anthraquinone n = 2: anthanthrone, dibenzpyrenequinone, etc. n = 4: pyranthrone, dibenzanthrone, dipyrazolanthrone, etc.

An alternate form of the leuco compounds is present in the aromatic amines and in particular derivatives of benzidine:

R₁=R₂=H benzidine

R,=(___- and R^H o-tolidine R^OCH-; R₂=H o-dianisidine R.^{= =}__i— tetramethylbenzidine (TMB)

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. In the case of amine compounds a hydrogen is extracted from each amine group to result in the tertiary amine. In the case of diols the hydrogen is removed from the two alcohol moieties to form the colored oxidized form of the compound and a molecule of water. Where 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.

As previously described for the simple dye or marker method, 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. Thus is indicated 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. By way of example, a solution of tetramethylbenzidine

(TMB) dissolved in dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) to give a final concentration of 660 μgrams/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. The TMB solution is then allowed to dry and a solution of cumene hydroperoxide in DMF (0.03 percent v/v) is then applied to the same area as was the TMB. After the peroxide has dried the absorbent layer 80 is protected from light. Alternatively, 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). When 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. In this manner 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. In a second example, 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. The 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. In a third example, 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.

In like manner this procedure can be accomplished with solutions of the below listed compounds at a concentration in a range of between 100 μgrams 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) . At this concentration 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. It should also be appreciated that the buffers that can be utilized to maintain the pH of the peroxidase reaction (pH 4-7) may be citrate, succinate, phthalate or phosphate. It will be appreciated by those familiar with the art of redox reactions that a number of additional compounds will be effective with the inventive method. It is to be understood that the substitution of such compounds for those particularly specified is contemplated within the present invention and will produce a method equivalent to that described herein.

As an alternative to reliance upon the peroxidase of saliva, 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 α- 1,4 glycoside bonds in oligosaccharides (from trisaccharides upwards) and polysaccharides such as starch, glycogen and dextrins. Amylase acts upon the α-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. It should be appreciated that in the present description the term 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.

In the inventive method, 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.

The reactions by which amylase, in combination with other enzymes, cleaves the substrate molecule to release the chromogen is:

amylase chromo-(glucose)- chro o-(glucose)₄₃₂ + (glucose)- ₄₃

(colorless) (colorless)

glucoamylase; α-glucosidase chromo-(glucose)₄₃₂ chromo + chromo(glucose)₄ + x-glucose

(colored)

In the above reaction sequence the chromogen containing oligosaccharide (e.g. silyl-blocked-4- n trophenylmaltoheptaoside) is first cleaved at the α-1,4 linkages to produce the chromogen linked to oligosaccharides of 2, 3 and 4 glucose units and oligosaccharides of 3, 4 and 5 glucose units. In the second step of the reaction glucoamylase and α-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 α-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 α- glucosidase.

Examples of polysaccharide or oligosaccharide molecules having chromogens incorporated include: α-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-α-D-maltoheptaoside. Also, 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.

In use within the inventive method a solution is composed of the chromogen-containing amylase substrate selected from the above compounds or their equivalents, α-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 following examples of this inventive method are presented by way of illustration and not limitation. 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.

In a first example, the solution applied to the substrate was composed as follows:

Actual Amount Alternative Used Per Approximate 100 μl Working Ranges Per 100ml

Silyl-blocked substrate 0.08 nM 0.008-0.8 nM (4-nitrophenylmalto- heptaoside) α-Glucosidase 3.0 units 0.2 - 30 Units

Glucoamylase 3.0 units 0.1 - 30 Units

Sodium Chloride 5.0 nM 0.5 Nm - 25 nM Calcium Gluconate 0.5 nM 0.05 Nm - 5 nM

PIPES buf er 13.5 nM 3.9 nM - 40.5 nM Approximately 100 microliters of this solution was applied to the absorbent in the area of the saliva zone of application. The absorbent was allowed to dry prior to application of saliva thereto. Saliva was then applied to the absorbent and upon the saliva contacting the area to which the solution was applied, a yellow color was observed.

In a second example the following concentrations were used in formulating the substrate solution for application to the absorbent:

Actual Amount Alternative Used Per Approximate 100 μl Working Ranges Per 100ml

Amylase substrate 0.05 nM 0.005 nM - 0.5 nM (4-nitrophenylmalto- heptaoside PNPG-7)

Sodium Chloride 5.0 nM 0.5 nM - 25 nM

Calcium Chloride 0.5 nM 0.05 nM - 5 nM α-Glucosidase 2.5 Units 0.25 - 25 Units Glucoamylase 1.0 Units 0.1 - 25 Units

PIPES buffer 13.5 nM 3.9 nM - 40.5 nM

Approximately 100 microliters of this solution are applied to the absorbent in the area of the saliva zone of application. The absorbent may be then allowed to dry prior to the application of saliva thereto. Saliva is then applied to the absorbent and upon the saliva contacting the area to which the solution was applied a yellow color will be observed.

Though the foregoing specific examples set forth the working concentrations of each ingredient, the concentrations may be varied according to the speed of the visual color reaction required.

When an on-site examiner is ready to utilize the absorbent containing the chromogen linked substrate, saliva is applied to the absorbent by any convenient method. Upon the amylase of saliva contacting the chromogen-linked substrate, the chromogen will be released from the remaining glucose units and become colored and visible to the examiner.

Referring to Fig. 12, an embodiment of the present invention is shown which further comprises the addition of tamper resistant features to sample probe 100 and features to notify testing personnel of such tampering attempts. 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. In 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. In Fig. 15 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.

Referring to Fig. 16, 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. Once absorbent 104 is inserted into 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

110 to support stick 102, the area of support stick 102 near holder 106 is placed on a solid surface and struck from above with a hammer or other blunt instrument. This impact causes the tip of crimp rivet 114, extending through receptacle 116, to become expanded into flattened rivet 130 on bottom surface 132 of support stick 102 (Fig. 13) . Flattened rivet 130 (Fig. 13) prevents any reopening of compression lid 110 and withdrawal of absorbent 104 from holder 106. The only method of opening compression lid 110 is by resort to prying tools which will cause deformation of probe 100 structure and tearing of breakaway connectors 112.

Once compression lid 110 is affixed within holder 106 and rivet 114 compressed into flattened rivet 130, the apparatus appears as shown in Figs. 12-14. In Fig. 14 flattened crimp rivet 130 is shown on bottom surface 132 of 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. In this manner 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. It will be appreciated from the foregoing discussion that 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) . As 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. In this manner 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. As stated earlier once 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. As substantial pressure is required to be applied by a blade-type wedge instrument to remove compression lid 110 from holder 106 and to force crimp rivet 114 from receptacle 116 the sudden release of compression lid 110 from holder 106 will be strong and rapid and uncontrolled and will cause the rupture of at least one of breakaway connectors 112. As these cannot be reattached it will be obvious to the testing laboratory that sample probe 100 has been the subject of tampering. Such separated breakaway connectors 127 are illustrated in Fig. 15.

An additional tamper resistant feature is provided by 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

129 and blocking the insertion of a bogus absorbent into support stick 102.

An alternative means of providing secure closure of compression lid 110 within holder 106 is through the use of an adhesive applied to holder 106 as shown in Fig. 15 by adhesive 134. 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.

The embodiment of 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. In Fig. 12 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. In actual use, 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. The activation of indicator 125 demonstrates to the on- site examiner that a saliva sample of sufficient quantity has been obtained. Sample probe 100 is then sent to the laboratory for analysis of the saliva sample. In the laboratory, the identification information of sample probe 100, as recorded 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.

In such a retest situation, it also will be clear to the retest- investigator that the sample probe 100 has not been tampered with during the intervening period between initial testing and repeat testing as all security indicia remain in tact. During the course of original testing there is no occasion to damage break-away connectors 112, or to make any change to indicator 125, or to open compression lid 110. Therefore, the integrity and security of the original sample is maintained by archive area 122 and assurance is derived that no substitution of absorbent 104 of archive area 122 has occurred during the intervening time period between original testing and the retest of archive area 122.

As stated previously indicator 125 may be composed of a simple vegetable dye or composed of any of the biochemically reactive indicators discussed herein. When a simple vegetable dye is utilized indicator 125 indicates only that sufficient saliva quantity has been applied to absorbent 104. When the biologically reactive indicators are utilized 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.

It is to be understood that while certain forms of this invention have been illustrated and described, it is not limited thereto, except insofar as limitations are included in the following claims.

Claims

C AIMS Having thus described the invention, what is claimed as new and desired to be secured by Letters Patent is as follows: 1. A method of collecting and identifying saliva for analysis, said method comprising the steps of:

(a) applying a fluid specimen to an absorbent element;

(b) providing a testing component that upon contact with said specimen gives an indication of the presence of saliva in said specimen; and

(c) following said step (a) , contacting said element with said component whereby the presence of saliva in the specimen results in said indication.

2. The method as claimed in claim 1, further comprising the steps of:

(d) allowing said fluid specimen, containing saliva therein, to dry on said element; and

(e) utilizing the dried saliva to test for the presence of drugs of use, drugs of abuse, nicotine and/or its metabolites and adducts, auto antibodies, hormones or islet cell antigen antibodies, anti-bodies to infectious agents or other salivary analytes of interest.

3. The method as claimed in claim 1, wherein said testing component comprises: a peroxide; and a leuco dye for oxidation by the interaction of said peroxide and the saliva such that a colored compound is produced to provide said indication.

4. The method as claimed in claim 3, wherein said peroxide is selected from the group consisting of hydrogen peroxide, methyl hydroperoxide, ethyl hydroperoxide, isopropyl hydroperoxide, butyl hydroperoxide, benzoyl hydroperoxide and cumene hydroperoxide.

5. The method as claimed in claim 3, wherein said peroxide is the product of a reaction of glucose oxidase with glucose.

6. The method as claimed in claim 3, wherein said leuco dye is selected from the group consisting of o-phenylenediamine, o-dianisidine, o-tolidine, guaiacol, 4-aminoantipyrine, pyrogallol, benzidine, tetramethyl benzidine, 4-chloro-l-naphthol and 2,2 '-azino-di (3-ethylbenzthiazoline sulphonic acid-6) .

7. A method of collecting saliva for analysis, said method comprising the steps of:

(a) applying a fluid specimen of saliva to an absorbent element at a zone of application thereon;

(b) providing a component that upon contact with said specimen gives an indication of the presence thereof; and

(c) positioning said component on said element in sufficient spaced relationship to said zone to require application of at least a predetermined amount of said specimen to said element before the presence of the specimen is indicated.

8. The method as claimed in claim 7, wherein said element comprises a sheet of material having a portion presenting said zone and upon which the applied specimen is dried, and wherein said step (c) includes locating said component on said sheet in said spaced relationship to said zone.

9. The method as claimed in claim 7, wherein said element comprises a sheet of material having a portion presenting said zone and upon which the applied specimen is dried, and wherein said step (c) includes locating said component on said sheet in circumscribing relationship to said zone radially therefrom in said spaced relationship thereto.

10. The method as claimed in claim 7, wherein said element comprises a wick having a portion presenting said zone, and wherein said step (c) includes locating said component on said wick in said spaced relationship to said zone.

11. The method as claimed in claim 7, wherein said element comprises an elongated wick having an end portion presenting said zone, and wherein said step (c) includes locating said component on an opposite end portion of said wick in said spaced relationship to said zone.

12. The method as claimed in claim 7, wherein said component comprises a dye at least partially soluble in saliva such that contact therewith solubilizes the dye to indicate the presence of at least said predetermined amount of the specimen on said absorbent element.

13. The method as claimed in claim 12, wherein said dye is a vegetable dye.

14. The method as claimed in claim 12, wherein said dye is non-toxic to humans.

15. The method as claimed in claim 7, wherein said component comprises saliva-insoluble, dyed or fluorescent micro particles which migrate with the specimen as it diffuses on said element to indicate the presence of at least said predetermined amount of the specimen.

16. A method of collecting and identifying saliva for analysis, said method comprising the steps of:

(a) applying a fluid specimen to an absorbent element;

(b) providing a testing component that upon contact with said specimen gives an indication of the presence of saliva in said specimen; and (c) positioning said component on said element for contact by the applied specimen, whereby the presence of saliva therein results in said indication.

17. The method as claimed in claim 16, wherein said step (a) includes applying the specimen to said element at a zone of application thereon, and wherein said step (c) includes positioning said component in sufficient spaced relationship to said zone to require application of at least a predetermined amount of saliva-containing specimen before the presence of saliva is indicated.

18. The method as claimed in claim 16, wherein said testing component comprises: a peroxide; and a leuco dye for oxidation by the interaction of said peroxide and the saliva such that a colored compound is produced to provide said indication.

19. The method as claimed in claim 18, wherein said peroxide is selected from the group consisting of hydrogen peroxide, methyl hydroperoxide, ethyl hydroperoxide, isopropyl hydroperoxide, butyl hydroperoxide, benzoyl hydroperoxide and cumene hydroperoxide.

20. The method as claimed in claim 18, wherein said peroxide is the product of a reaction of glucose oxidase with glucose.

21. The method as claimed in claim 18, wherein said leuco dye is selected from the group consisting of o-phenylenediamine, o-dianisidine, o-tolidine, guaiacol, 4-aminoantipyrine, pyrogallol, benzidine, tetramethyl benzidine, 4-chloro-l-naphthol and 2,2 '-azino-di (3-ethylbenzthiazoline sulphonic acid-6) .

22. A method of collecting and identifying saliva for analysis, said method comprising the steps of:

(a) applying a fluid specimen to an absorbent element having a sample portion and a separable test portion; (b) providing a testing component that upon contact with said specimen gives an indication of the presence of saliva in said specimen; (c) following said step (a) , removing said test portion from said absorbent element; and (d) applying said component to said test portion to contact the applied specimen, whereby the presence of saliva therein results in said indication and analysis may proceed utilizing said sample portion.

23. The method as claimed in claim 22, wherein said testing component comprises: a peroxide; and a leuco dye for oxidation by the interaction of said peroxide and the saliva such that a colored compound is produced to provide said indication.

24. The method as claimed in claim 23, wherein said peroxide is selected from the group consisting of hydrogen peroxide, methyl hydroperoxide, ethyl hydroperoxide, isopropyl hydroperoxide, butyl hydroperoxide, benzoyl hydroperoxide and cumene hydroperoxide.

25. The method as claimed in claim 23, wherein said peroxide is the product of a reaction of glucose oxidase with glucose.

26. The method as claimed in claim 23, wherein said leuco dye is selected from the group consisting of o-phenylenediamine, o-dianisidine, o-tolidine, guaiacol,

4-aminoantipyrine, pyrogallol, benzidine, tetramethyl benzidine, 4-chloro-l-naphthol and 2,2 '-azino-di (3-ethylbenzthiazoline sulphonic acid-6) .

27. The method as claimed in claim 16, wherein said testing component comprises: a substrate reactive with an amylase enzyme of said saliva, said substrate having a portion thereof which upon reaction with said amylase presents visible color.

28. The method as claimed in claim 27, wherein said substrate is a polysaccharide having a chromogen attached thereto.

29. The method as claimed in claim 28, wherein said chromogen-containing polysaccharide is a p-nitrophenyl- containing polysaccharide.

30. The method as claimed in claim 28, wherein said chromogen-containing polysaccharide is selected from the group consisting of α-nitrophenylmaltoheptaoside, β-4- nitrophenylmaltoheptaoside, 4,6, ethylidine-blocked-4- nitrophenylmaltoheptaoside, silyl-blocked-4- nitrophenylmaltoheptaoside, and 3-ketobutylidene-beta-2 chloro- 4-nitrophenylmaltopentaoside.

31. The method as claimed in claim 28, wherein said chromogen-containing polysaccharide is an indole-containing polysaccharide.

32. The method as claimed in claim 28, wherein said chromogen-containing polysaccharide is selected from the group consisting of indoly-α-maltoheptaoside; indolyl-|_-maltoheptaoside; 4,6, ethylidine-blocked-indolyl maltoheptaoside; silyl-blocked-indolyl maltoheptaoside; 3- ketobutylidene-beta-2-chloro-indolyl maltopentaoside; and indolyl-α-D-maltoheptaoside.

33. Apparatus for collecting a saliva specimen comprising: an absorbent element for receiving a fluid saliva specimen at a zone of application thereon; and means on said element responsive to contact by the received specimen for indicating the presence thereof on the element and positioned in sufficient spaced relationship to said zone to require application of at least a predetermined amount of the specimen to said element for response by said indicating means.

34. The apparatus as claimed in claim 33, wherein said absorbent element comprises cellulose fibers.

35. The apparatus as claimed in claim 33, wherein said absorbent element comprises a sponge material.

36. The apparatus as claimed in claim 33, wherein said absorbent element comprises a porous plastic material.

37. The apparatus as claimed in claim 33, wherein said indicating means comprises a dye at least partially soluble in saliva such that contact therewith solubilizes the dye to indicate the presence of at least said predetermined amount of the specimen on said absorbent element.

38. The apparatus as claimed in claim 37, wherein said dye is a vegetable dye.

39. The apparatus as claimed in claim 37, wherein said dye is non-toxic to humans.

40. The apparatus as claimed in claim 33, wherein said indicating means comprises saliva-insoluble, dyed or fluorescent micro particles which migrate with the specimen as it diffuses on said ^element to indicate the presence of at least said predetermined amount of the specimen.

41. The apparatus as claimed in claim 33, wherein said element comprises a sheet of material having a portion thereon presenting said zone, said indicating means being applied to said sheet.

42. The apparatus as claimed in claim 33, wherein said element comprises a sheet of material having a portion thereon presenting said zone, and wherein said indicating means is applied to said sheet in circumscribing relationship to said zone radially therefrom in said spaced relationship thereto.

43. The apparatus as claimed in claim 33, wherein said element comprises a wick having a portion presenting said zone, said indicating means being disposed on said wick in said spaced relationship to said zone.

44. The apparatus as claimed in claim 33, wherein said element comprises a wick having opposed end portions, one of said end portions presenting said zone, said indicating means being disposed on the opposite end portion in said spaced relationship to said zone.

45. The apparatus as claimed in claim 43, further comprising a sample probe having an elongated, tubular member provided with a distal end receiving and holding said opposite end portion of the wick with said indicating means disposed within said member, and an elongated container for housing said probe upon longitudinal insertion of the probe thereunto.

46. The apparatus as claimed in claim 33, wherein said indicating means is further characterized by the property that upon contact with said specimen, an indication is given of the presence of saliva therein to thereby verify that the collected specimen contains saliva.

47. The apparatus as claimed in claim 45, wherein said indicating means comprises: a peroxide; and a leuco dye for oxidation by the interaction of said peroxide and the saliva such that a colored compound is produced to provide said saliva verification.

48. The apparatus as claimed in claim 46, wherein said peroxide is selected from the group consisting of hydrogen peroxide, methyl hydroperoxide, ethyl hydroperoxide, isopropyl hydroperoxide, butyl hydroperoxide, benzoyl hydroperoxide and cumene hydroperoxide.

49. The apparatus as claimed in claim 46, wherein said peroxide is the product of a reaction of glucose oxidase with glucose.

50. The apparatus as claimed in claim 46, wherein said leuco dye is selected from the group consisting of o-phenylenediamine, o-dianisidine, o-tolidine, guaiacol, 4-aminoantipyrine, pyrogallol, benzidine, tetramethyl benzidine, 4-chloro-l-naphthol and 2,2'-azino-di (3-ethylbenzthiazoline sulphonic acid-6) .

51. Apparatus for collecting and identifying a saliva specimen comprising: an absorbent element for receiving a fluid specimen; and means on said element responsive to contact by the received specimen for indicating the presence of saliva in said specimen.

52. The apparatus as claimed in claim 51, wherein said indicating means comprises: a peroxide; and a leuco dye for oxidation by the interaction of said peroxide and the saliva such that a colored compound is produced to provide said saliva verification.

53. The apparatus as claimed in claim 51, wherein said peroxide is selected from the group consisting of hydrogen peroxide, methyl hydroperoxide, ethyl hydroperoxide, isopropyl hydroperoxide, butyl hydroperoxide, benzoyl hydroperoxide and cumene hydroperoxide.

54. The apparatus as claimed in claim 51, wherein said peroxide is the product of a reaction of glucose oxidase with glucose.

55. The apparatus as claimed in claim 51, wherein said leuco dye is selected from the group consisting of o-phenylenediamine, o-dianisidine, o-tolidine, guaiacol, 4-aminoantipyrine, pyrogallol, benzidine, tetramethyl benzidine, 4-chloro-l-naphthol and 2,2 '-azino-di (3-ethylbenzthiazoline sulphonic acid-6) .

56. The apparatus as claimed in claim 51, wherein said indicating means comprises: a substrate reactive with an amylase enzyme of said saliva, said substrate having a portion thereof which upon reaction with said amylase presents visible color.

57. The apparatus as claimed in claim 56, wherein said substrate is a polysaccharide having a chromogen attached thereto.

58. The apparatus as claimed in claim 57, wherein said chromogen-containing polysaccharide is a p-nitrophenyl- containing polysaccharide.

59. The apparatus as claimed in claim 57, wherein said chromogen-containing polysaccharide is selected from the group consisting of α-4-nitrophenyl maltoheptaoside, 3-4-nitrophenylmaltoheptaoside, 4,6, ethylidine-blocked-4- nitrophenylmaltoheptaoside, silyl-blocked-4- nitrophenylmaltoheptaoside, and 3-ketobutylidene-beta-2 chloro- 4-nitrophenylmaltopentaoside.

60. The apparatus as claimed in claim 57, wherein said chromogen-containing polysaccharide is an indole-containing polysaccharide.

61. The apparatus as claimed in claim 57, wherein said chromogen-containing polysaccharide is selected from the group consisting of indoly-α-maltoheptaoside; indolyl-,3-maltoheptaoside; 4,6, ethylidine-blocked-indolyl maltoheptaoside; silyl-blocked-indolyl maltoheptaoside; 3-ketobutylidene-beta-2-chloro-indolyl maltopentaoside; and indolyl-α-D-maltoheptaoside.

62. The apparatus as claimed in claim 33 further comprising: a support for said absorbent element, and means on said support for capture of a portion of said absorbent element such that attempts at removal of said absorbent from capture in said holder are observable.

63. The apparatus as claimed in claim 62, wherein said means for capture comprises: a holder for receiving a portion of said absorbent element, and a lid for registration with said holder, said lid upon registration with said holder capturing said portion and said lid then being permanently attached to said holder.

64. The apparatus as claimed in claim 63 further comprising means attached to said holder and said lid for evidencing tampering.

65. The apparatus as claimed in claim 64, wherein said means for evidencing tampering comprises a breakable connector linking said lid to said holder. 66. An apparatus for collection of a saliva specimen comprising: a support, means for tamper resistance on said support, an absorbent element for receiving a fluid saliva specimen at a zone of application thereon, said absorbent element comprising: a first saliva application zone, and a second saliva application zone in contact with said first zone, and means on said support for capture of a portion of said second saliva application zone such that said first zone is detachable from said second zone without damaging said means for tamper resistance such that said apparatus provides in said second zone a permanent tamper resistant archive test portion.

67. The apparatus as claimed in claim 66, wherein said means for capture comprises: a holder for receiving a portion of said absorbent element, and a lid for registration with said holder, said lid upon registration with said holder being capable of capturing said portion within said holder and said lid then being permanently attached to said holder.

68. The apparatus as claimed in claim 66, wherein said means for tamper resistance comprises a rivet for permanently closing said means for capture.

69. The apparatus as claimed in claim 68, wherein said means for tamper resistance further comprises a breakable connector linking said lid to said holder. 70. The apparatus as claimed in claim 66 further comprising means on said absorbent element responsive to contact by the received specimen for indicating the presence thereof on the element and positioned in sufficient spaced relationship to said zone to require application of at least a predetermined amount of the specimen to said element for response by said indicating means.

71. The apparatus as claimed in claim 70, wherein said indicating means comprises a dye at least partially soluble in saliva such that contact therewith solubilizes the dye to indicate the presence of at least said predetermined amount of the specimen on said absorbent element.

72. The apparatus as claimed in claim 71, wherein said dye is a vegetable dye.

73. The apparatus as claimed in claim 70, wherein said indicating means is further characterized by the property that upon contact with said specimen, an indication is given of the presence of saliva therein to thereby verify that the collected specimen contains saliva.

74. The apparatus as claimed in claim 73, wherein said indicating means comprises: a peroxide; and a leuco dye for oxidation by the interaction of said peroxide and the saliva such that a colored compound is produced to provide said saliva verification.

75. The apparatus as claimed in claim 67, wherein said means for tamper resistance is sonic welding of said holder to said lid.

76. The apparatus as claimed in claim 67, wherein said means for tamper resistance is an adhesive for sealing said lid to said holder. _£ AMENDED C AIMS

[received by the International Bureau on 26 February 1993 (26.02.93); original claims 1,2,5,7-9,11,16,17,20,22,25,33,41-54 amended; new claims 77-86 added; other claims unchanged (16 pages)]

Having thus described the invention, what is claimed a new and desired to be secured by Letters Patent is as follows:

1. A method of collecting and identifying saliva for analysis, said method comprising the steps of:

(a) applying a fluid specimen to an absorbent element;

(b) providing a testing component that upon contact with said specimen gives an indication of the presence o saliva in said specimen; and (c) providing an absorbent path along said element uncontaminated by said component for migration of t applied specimen to said component, whereby an uncontaminated sample of the specimen is collected said element and a portion of the specimen is contacted with said component.

2. The method as claimed in claim 1, further comprising the steps of:

(d) allowing said fluid specimen, containing saliva therei to dry on said element; and (e) utilizing said dried saliva to test for the presence of foreign agents such as drugs, drugs of abuse, nicotine and/or its metabolites and adducts, auto antibodies, hormones or islet cell antigen antibodies, antibodies to infectious agents or othe salivary analytes of interest.

3. The method as claimed in claim 1, wherein said testing component comprises: a peroxide; and a leuco dye for oxidation by the interaction of said peroxid and the saliva such that a colored compound is produced to provide said indication.

4. The^"method as claimed in claim 3, wherein said peroxide is selected from the group consisting of hydrogen peroxide, methyl hydroperoxide, ethyl hydroperoxide, isopropyl hydroperoxide, butyl hydroperoxide, benzoyl hydroperoxide and 5. The method as claimed in claim 3, wherein said peroxide is produced by a reaction of glucose oxidase with glucose.

6. The method as claimed in claim 3, wherein said leuco dye is selected from the group consisting of o- phenylenediamine, o-dianisidine, o-tolidine, guaiacol, 4- aminoantipyrine, pyrogallol, benzidine, tetramethyl benzidine, 4-chloro-l-naphthol and 2,2 '-azino-di (3-ethylbenzthiazoline sulphonic acid-6) .

(c) positioning said component on said element in sufficient spaced relationship to said zone to require application of at least a predetermined amount of said specimen to said element before the presence of the specimen is indicated, said spaced relationship providing for uncontaminated collection of said predetermined amount of specimen by said component.

8. The method as claimed in claim 7, wherein said element comprises a sheet of material having a portion presenting said zone and upon which the applied specimen is dried, and wherein said step (c) includes locating said component on said sheet in said spaced relationship to said zone prior to the application of said specimen to said absorbent element. o

9. The method as claimed in claim 7, wherein said element comprises a sheet of material having a portion presenting said zone and upon which the applied specimen is dried, and wherein said step (c) includes locating said component on said sheet in circumscribing relationship relative to said zone and in said spaced relationship thereto.

11. The method as claimed in claim 7, wherein said element comprises an elongated wick having an end portion presenting said zone, and wherein said step (c) includes locating said component on an opposite end portion of said wick from said zone and in said spaced relationship to said zone.

13. The method as claimed in claim 12, wherein said dye is a vegetable dye.

14. The method as claimed in claim 12, wherein said dye is non-toxic to humans.

15. The method as claimed in claim 7, wherein said component comprises saliva-insoluble, dyed or fluorescent micro particles which migrate with the specimen as it diffuses on said element to indicate the presence of at least said predetermined amount of the specimen. 16. A method of collecting and identifying saliva for analysis, said method comprising the steps of:

(a) applying a fluid specimen to an absorbent element at a zone of application; (b) providing a testing component on said element that upon contact with said specimen gives an indication of the presence of saliva in said specimen; and (c) positioning said component on said element in sufficient spaced relationship to said zone of application to indicate the presence of saliva in said applied specimen upon contact thereby and to provide specimen uncontaminated by said component.

17. The method as claimed in claim 16, wherein said step (c) includes positioning said component in sufficient spaced relationship to said zone to require application of at least a predetermined amount of saliva-containing specimen before the presence of saliva is indicated.

20. The method as claimed in claim 18, wherein said peroxide is produced by a reaction of glucose oxidase with glucose. 21. The method as claimed in claim 18, wherein said leuco dye is selected from the group consisting of o-phenylenediamine, o-dianisidine, o-tolidine, guaiacol, 4-aminoantipyrine, pyrogallol, benzidine, tetramethyl benzidine, 4-chloro-l-naphthol and 2,2 '-azino-di (3-ethylbenzthiazoline sulphonic acid-6) .

(a) applying a fluid specimen to an absorbent element having a sample portion and a separable test portion;

(b) providing a testing component that upon contact with said specimen gives an indication of the presence of saliva in said specimen;

(c) following said step (a), removing said test portion from said sample portion; and

(d) applying said component to said test portion to contact the applied specimen, whereby the presence of saliva therein results in said indication and analysis may proceed utilizing said sample portion.

25. The method as claimed in claim 23, wherein said peroxide is produced by a reaction of glucose oxidase with glucose. 26. The method as claimed in claim 23, wherein said leuco dye is selected from the group consisting of o-phenylenediamine, o-dianisidine, o-tolidine, guaiacol, 4-aminoantipyrine, pyrogallol, benzidine, tetramethyl benzidine, 4-chloro-l-naphthol and 2,2'-azino-di (3-ethylbenzthiazoline sulphonic acid-6) .

30. The method as claimed in claim 28, wherein said chromogen-containing polysaccharide is selected from the group consisting of α-nitrophenylmaltoheptaoside, ,9-4- nitrophenylmaltoheptaoside, 4,6, ethylidine-blocked-4- nitrophenylmaltoheptaoside, silyl-blocked-4- nitrophenylmaltoheptaoside, and 3-ketobutylidene-beta-2 chloro- 4-nitrophenylmaltopentaoside.

31. The method as claimed in claim 28, wherein said chromogen-containing polysaccharide is an indole-containing polysaccharide. 32. The method as claimed in claim 28, wherein said chromogen-containing polysaccharide is selected from the group consisting of indolyl-α-maltoheptaoside; indolyl-?- maltoheptaoside; 4,6, ethylidine-blocked-indolyl maltoheptaoside; silyl-blocked-indolyl maltoheptaoside; 3- ketobutylidene-beta-2-chloro-indolyl maltoheptaoside; and indolyl-α-D-maltoheptaoside.

33. Apparatus for collecting a saliva specimen comprising: an absorbent element for receiving a fluid saliva specimen at a zone of application thereon; means for indicating the presence of the specimen upon contact therewith; and means for migration of the specimen defining a path of a predetermined distance uncontaminated by said indicating means said migration means communicating the specimen with said indicating means such that said distance requires application of at least a predetermined amount of the specimen to said element for response by said indicating means.

38. The apparatus as claimed in claim 37, wherein said dye is a vegetable dye. 39. The apparatus as claimed in claim 37, wherein said dye is non-toxic to humans.

40. The apparatus as claimed in claim 33, wherein said indicating means comprises saliva-insoluble, dyed or fluorescent micro particles which migrate with the specimen as it diffuses on said element to indicate the presence of at least said predetermined amount of the specimen.

41. The apparatus as claimed in claim 33, wherein said element comprises a sheet of material having a portion thereon presenting said zone, said indicating means being applied to said sheet and said means for migration positioned between said zone and said indicating means to communicate a portion of said specimen therebetween.

42. The apparatus as claimed in claim 33, wherein said element comprises a sheet of material having a portion thereon presenting said zone, and wherein said indicating means is applied to said sheet in circumscribing relationship to said zone radially therefrom in said spaced relationship thereto and said means for migration positioned between said zone and said indicating means to communicate the applied specimen from said zone to said indicating means.

43. The apparatus as claimed in claim 33, wherein said element comprises a wick having a portion presenting said zone, said indicating means being disposed on said wick in said spaced relationship to said zone and said means for migration being juxtaposed between said zone and said indicating means.

44. The apparatus as claimed in claim 33, wherein said element comprises a wick having opposed end portions, one of said end portions presenting said zone, said indicating means being disposed on the opposite end portion in said spaced relationship to said zone and said migration means being positioned between said zone and said indicating means to comsunciate the specimen therebetween. 45. The apparatus as claimed in claim 44, further comprising a sample probe having an elongated, tubular member provided with a distal end receiving and holding said opposite end portion of the wick with said indicating means disposed within said member, and an elongated container for housing said probe upon longitudinal insertion of the probe thereunto.

46. The apparatus as claimed in claim 33, wherein said indicating means chemically reactive with saliva such that upon contact with said specimen, an indication is given of the presence of saliva therein to thereby verify that the collected specimen contains saliva.

47. The apparatus as claimed in claim 46, wherein said indicating means comprises: a peroxide; and a leuco dye for oxidation by the interaction of said peroxide and the saliva such that a colored compound is produced to provide said saliva verification.

48. The apparatus as claimed in claim 47, wherein said peroxide is selected from the group consisting of hydrogen peroxide, methyl hydroperoxide, ethyl hydroperoxide, isopropyl hydroperoxide, butyl hydroperoxide, benzoyl hydroperoxide and cumene hydroperoxide.

49. The apparatus as claimed in claim 47, wherein said peroxide is the product of a reaction of glucose oxidase with glucose.

50. The apparatus as claimed in claim 47, wherein said leuco dye is selected from the group consisting of o-phenylenediamine, o-dianisidine, o-tolidine, guaiacol, 4-aminoantipyrine, pyrogallol, benzidine, tetramethyl benzidine, 4-chloro-l-naphthol and 2,2 '-azino-di (3-ethylbenzthiazoline sulphonic acid-6) . 51. Apparatus for collecting and identifying a- saliva specimen comprising: an absorbent element for receiving a fluid specimen at a zone of application thereon; means for indicating the presence of the specimen upon contact therewith; means for migration of the specimen defining a path of a predetermined distance uncontaminated by said indicating means said migration means communicating the specimen with said indicating means such that said distance requires application of at least a predetermined amount of the specimen to said element for response by said indicating means; and a barrier between said migration means and said absorbent to prevent contamination of said absorbent by said indicator means, said barrier permitting migration of said specimen to said indicator means.

53. The apparatus as claimed in claim 52, wherein said peroxide is selected from the group consisting of hydrogen peroxide, methyl hydroperoxide, ethyl hydroperoxide, isopropyl hydroperoxide, butyl hydroperoxide, benzoyl hydroperoxide and cumene hydroperoxide.

54. The apparatus as claimed in claim 52, wherein said peroxide is the product of a reaction of glucose oxidase with glucose. 55. The apparatus as claimed in claim 51, wherein said leuco dye is selected from the group consisting of o-phenylenediamine, o-dianisidine, o-tolidine, guaiacol, 4-aminoantipyrine, pyrogallol, benzidine, tetramethyl benzidine, 4-chloro-l-naphthol and 2,2 '-azino-di (3-ethylbenzthiazoline sulphonic acid-6) .

59. The apparatus as claimed in claim 57, wherein said chromogen-containing polysaccharide is selected from the group consisting of α-4-nitrophenyl maltoheptaoside, 3-4- nitrophenylmaltoheptaoside, 4,6, ethylidine-blocked-4- nitrophenylmaltoheptaoside, silyl-blocked-4- nitrophenylmaltoheptaoside, and 3-ketobutylidene-beta-2 chloro- 4-nitrophenylmaltopentaoside.

60. The apparatus as claimed in claim 57, wherein said chromogen-containing polysaccharide is an indole-containing polysaccharide. 61. The apparatus as claimed in claim 57, wherein said chromogen-containing polysaccharide is selected from the group consisting of indolyl-α-maltoheptaoside; indolyl-/3- maltoheptaoside; 4,6, ethylidine-blocked-indolyl maltoheptaoside; silyl-blocked-indolyl maltoheptaoside; 3- ketobutylidene-beta-2-chloro-indolyl maltoheptaoside; and indolyl-α-D-maltoheptaoside.

65. The apparatus as claimed in claim 64, wherein said means for evidencing tampering comprises a breakable connector linking said lid to said holder.

66. An apparatus for collection of a saliva specimen comprising: a support, means for tamper resistance on said support, an absorbent element for receiving a fluid saliva specimen at a zone of application thereon, said absorbent element comprising: a first saliva application zone, and a second saliva application zone in contact with said first zone, and means on said support for capture of a portion of said second saliva application zone such that said first zone is detachable from said second zone without damaging said means for tamper resistance such that said apparatus provides in said second zone a permanent tamper resistant archive test portion.

69. The apparatus as claimed in claim 68, wherein said means for tamper resistance further comprises a breakable connector linking said lid to said holder.

70. The apparatus as claimed in claim 66 further comprising means on said absorbent element responsive to contact by the received specimen for indicating the presence thereof on the element and positioned in sufficient spaced relationship to said zone to require application of at least a predetermined amount of the specimen to said element for response by said indicating means.

72. The apparatus as claimed in claim 71, wherein said dye is a vegetable dye.

73. The apparatus as claimed in claim 70,. wherein said indicating means is further characterized by the property that upon contact with said specimen, an indication is given of the presence of saliva therein to thereby verify that the collected specimen contains saliva.

76. The apparatus as claimed in claim 67, wherein said means for tamper resistance is an adhesive for sealing said lid to said holder.

77. The apparatus as claimed in claim 33, wherein said means for migration comprises a portion of said absorbent for communicating the migration of said specimen between said zone of application and said indicating means.

78. The apparatus as claimed in claim 33 further comprising a one-way barrier between said migration means and said indicating means to prevent migration of a specimen-diluted indicating means in a reverse direction along said means for migration and toward said zone of application.

79. A method of detecting the collection of a predetermined quantity of a fluid specimen comprising: providing an absorbent of predetermined fluid absorption capacity, said absorbent having a zone of application thereon, positioning a means for indicating specimen presence at a selected distance from said zone of application said distance being sufficient to require application of at least a predetermined amount of specimen to said absorbent for response by said indicating means, juxtaposing a means for migration of said specimen between said zone and said indicating means said migration means being uncontaminated by said indicating means and permitting communication of said specimen from said zone to said indicating means, applying sufficient specimen to saturate said zone and said absorbent to result in contact of said means for indicating by said specimen, and observing said means for indicating for specimen contact therewith.

80. The method as claimed in 79, wherein said indicating means comprises a dye at least partially soluble in saliva such that contact therewith solubilizes the dye to indicate the presence of at least said predetermined amount of the specimen on said absorbent element.

81. The method as claimed in claim 33, wherein said indicating means is chemically reactive with saliva such that upon contact with said specimen, an indication is given of the presence of saliva therein to thereby verify that the collected specimen contains saliva.

82. The method as claimed in claim 79, wherein said indicating means comprises: a peroxide; and a leuco dye for oxidation by the interaction of said peroxide and the saliva such that a colored compound is produced to provide said saliva verification.

83. The method as claimed in claim 79, wherein said peroxide is selected from the group consisting of hydrogen peroxide, methyl hydroperoxide, ethyl hydroperoxide, isopropyl hydroperoxide, butyl hydroperoxide, benzoyl hydroperoxide and cumene hydroperoxide.

84. The method as claimed in claim 79, wherein said peroxide is the product of a reaction of glucose oxidase with glucose.

85. The method as claimed in claim 79, wherein said leuco dye is selected from the group consisting of o-phenylenediamine, o-dianisidine, o-tolidine, guaiacol, 4-aminoantipyrine, pyrogallol, benzidine, tetramethyl benzidine, 4-chloro-l-naphthol and 2,2'-azino-di (3-ethylbenzthiazoline sulphonic acid-6) .

86. The method as claimed in claim 1 further comprising the step of establishing the length of said path such that application of at least a predetermined amount of the specimen is required for an indication from said component.