WO1996004850A1 - Oral collection for immunoassay - Google Patents
Oral collection for immunoassay Download PDFInfo
- Publication number
- WO1996004850A1 WO1996004850A1 PCT/US1994/009096 US9409096W WO9604850A1 WO 1996004850 A1 WO1996004850 A1 WO 1996004850A1 US 9409096 W US9409096 W US 9409096W WO 9604850 A1 WO9604850 A1 WO 9604850A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- pad
- container
- immunoglobulins
- testing
- collected
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
- A61B10/0045—Devices for taking samples of body liquids
- A61B10/0051—Devices for taking samples of body liquids for taking saliva or sputum samples
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
- A61B2010/0009—Testing for drug or alcohol abuse
Definitions
- the present invention relates to the field of immunological testing.
- a system for analyzing immunoglobulins and other substances derived from mucosal transudate is dis ⁇ closed.
- Collection of saliva from the salivary glands is complicated by the low volumes secreted, the diverse anatomic dispersion of the glands, and the relatively high viscosity of the fluid.
- Most techniques for collection involve the use of capillary tubes, suction into micropipettes, chewing on paraffin or aspiration into syringes. These methods, however, are limited in that viscosity of the saliva makes the recovery of bubble-free material by these techniques difficult.
- Other methods of collection have been suggested to eliminate or at least reduce the quantity of bubbles in the sample. Among such methods include collecting saliva in the mouth by direct absorption with a sponge or flexible wad of osmotic membrane.
- the saliva can be separated from the absorptive material by centrifugation or by compressing the absorptive material.
- absorption is generally accomplished by using cotton, nylon, or polyester as the absorptive material. These materials can non-specifically bind proteins which can result in an undesirably low recovery of immunoglobulins.
- the immune system of the mouth not only interacts with the general immune system of the body, but also has its own central ⁇ ized center for antigen-antibody response.
- extraoral lymph nodes and intraoral lymphoid aggregations are involved in the drainage of the oral mucosa, gum and teeth.
- the function of the intraoral lymphoid tissue is little understood.
- the extraoral lymph nodes include a fine network of lymph capillaries which are superficially located in the mouth, palate, cheeks, lips, gingiva, and pulp of the teeth.
- the capillaries join to larger lymph vessels which originate from a network deep in the muscle of the tongue and other structures.
- An antigen can gain entry into the oral lymphatic system directly through the capillaries or be transported there by phagocytes. Once inside the network, the antigen can induce an immune response.
- tissue aggregations include (a) the tonsils, (b) scattered submucosal lymphoid aggregations, (c) salivary gland lymphoid tissue, and (d) gingival lymphoid tissue.
- the tonsils (palatine and lingual) primarily produce B-cells and T-cells which are generally contained within a cap of lymphocytes and plasma cells. Antigen typically gains entry into the tonsils through a distinct epithelial region wherein the antigen can come into contact with the T- and B-cells to stimulate an immune response.
- the predominant type of antibody formed in the tonsils is found to be IgG followed, in order, by IgA, IgM, IgD and IgE.
- Scattered submucosal lymphoid cells have not been extensive ⁇ ly studied. These cell masses are histologically similar to tonsillar tissue.
- T-cells and B-cells are found in the gingival lymphoid tissue. In subjects having clinically normal gingival tissue, T-cells predominate. During an infectionary period, such as during the development of gingivitis, B-cells have been found to predominate.
- Plasma cells are also found in the gingival lymphoid tissue. Clusters of these cells are generally located near the blood vessels and predominantly produce IgG. To a lesser extent, IgA and IgM are also manufactured. More importantly, Brandtzaeg et al. in, Human Saliva: Clinical Chemistry and Microbiology edited by Jorma O. Tenovuo, have shown that the immunoglobulins from the secretions from the gingival tissue area are directly related to the immunoglobulins found in the blood.
- an untreated pad can be used to collect a sufficient quantity of oral immunoglobulin for immunological testing.
- the use of the pad results in a yield of immunoglobulins greater than would be expected and can incorpo ⁇ rate basic antigen-antibody testing techniques as a screening tool for diseases.
- the present invention can be used to collect substances other than immunoglobulins for testing.
- the invention has been successfully used to collect substances having molecular weights ranging from about 176 (cotinine) to about 950,000 (IgM).
- IgM 950,000
- Fig. 1 is a longitudinal section of one embodiment of a container for storing the pad
- Fig. 2 is a longitudinal section of the embodiment of Fig. 1 with the pad and holder shown;
- Fig. 3 is an elevational view of another embodiment of a container useful in the present invention with the cap removed;
- Fig. 4 is a top plan view of the container of Fig. 3 ;
- Fig. 5 is a longitudinal cross-sectional view of the container of Fig. 4 taken along the line 5—5 of Fig. 4;
- Fig. 6 is an elevational view of a cap for use with the container shown in Fig. 3;
- Fig. 7 is a top plan view of the cap of Fig. 6; and Fig. 8 is a cross-sectional view of the cap taken along the line 8—8 of Fig. 7.
- the present invention is concerned with collecting oral immunoglobulins for immunological testing and other substances for testing.
- a pad is used to collect a specimen having a high concentration of immunoglobulins or the other substances.
- High levels of immunoglobulins from the oral cavity are considered to be concentrations in excess of 50 ⁇ g total Ig per ml.
- the specimen can be subjected to a basic testing technique which can be used as a tool for screening a patient for diseases or for the presence of certain foreign substances.
- HIV-1 Total IgG (antigen not specified) HIV-1
- IgA human antibody 160,000
- container in which the pad is stored after use can include a preservative.
- a preservative can act to inhibit proteolytic enzymatic activity which can be responsible for the destruction of antibody molecules.
- Compounds contemplated as a preservative include anti-bacterial agents, anti-fungal agents, bacteriostatic agents, fungistatic agents, and enzyme inhibitors.
- benzoic acid, sorbic acid or the salts thereof are used as anti-fungal agents.
- bacteriostatic agents salts in high concentration and compounds capable of maintaining the hypertonic solution at low pH are contemplated.
- Such salts include thi erosal (or merthiolate) , phenyl mercuric acetate, phenyl mercuric nitrate and sodium azide.
- Other preferred preservatives include preservatives which are typically used in medicines and outhwashes. Examples include ethyl alcohol and chlorhexidine gluconate.
- Another class of preferred anti ⁇ microbial agents are detergents which can be used as topical germicides or in mouthwashes. An example is benzalkonium chloride. It is preferred to use these preservatives in a range of about 0.01% to about 0.2% by weight.
- a pad is used to absorb mucosal secretions from the oral cavity.
- the pad is made of an absorbent material which can be effectively placed into the oral cavity.
- a plastic or carbohydrate material such as cellulose can be used as the absorbent material, but a thick, absorbent cotton paper is preferred.
- An example of a thick, absorbent cotton paper is product #300 manufactured by Schleicher and Schuell in Keene, New Hampshire.
- the pad is preferably not in the form of a foam or sponge, although foam or sponge could be used.
- a blocking agent can be added to the hypertonic solution to be incorporated into the pad.
- a blocking agent is generally a soluble protein which is used to prevent non-specific binding of another protein to a solid surface.
- Compounds which can be added as blocking agents include albumin and gelatin, but any water soluble, non-toxic protein can be used as a blocking agent as long as the protein does not adversely affect antibody molecules. It is preferred to use bovine gelatin.
- blocking agents can be made into a solution at a concentration of between about 0.01% and 0.2% by weight. The solution is then incorporated into the pad by soaking or spraying followed by drying.
- the pad can be placed into the mouth with the aid of a holder.
- the pad and holder are shown in the drawing.
- the pad holder 1 can be a hollow, plastic stick having a groove 2 at one end.
- the pad 3 is inserted into the groove and the holder can be manipulated to place the pad into the oral cavity, preferably between the lower gums and cheek. Placement of the pad between the lower cheek and gums facilitates absorption of secretions originating from gingival lymphoid tissue as well as secretions from submucosal lymphoid tissue and salivary gland lymphoid tissue.
- the specimen be collected by rubbing the pad back and forth between the gums and cheek for about ten seconds and then holding the pad in position for about two minutes.
- the pad should not be chewed since chewing stimulates salivation which is undesirable.
- the pad is stored in a container until immunological testing can be performed.
- One type of container is shown in Figs. 1 and 2. It is desired that the container have a centrifuge tube as an outer portion of the container, and that an inner portion of the container have an inner tube which mounts into the centrifuge tube. The pad is to be placed into the inner tube, and the contents therein are secured by a tube cap.
- the container 2 comprises a centrifuge tube 4 having a tapered lower end or base 5 with a downwardly tapering recess
- an upper tube or container 7 having a radially outwardly projecting annular flange 8 and a cylindrical upper portion 9 at its upper end; and a plug or stopper 11.
- the cylindrical portion 9 and stopper 11 are of the same size and shape as the upper part of centrifuge tube 4 so they are flush with the outer surface of the centrifuge tube and the assembly presents a uniform appearance, although this feature is not important to the practice of the instant invention.
- the container 7 is a bore 17 to allow liquid to flow from container 7 to centrifuge tube 4 when the complete assembly is centrifuged.
- the container 7 is made of any suitable material such as polyethyl ⁇ ene, glass, etc.
- the stopper 11 is made of any suitable material such as polyethylene as is well-known in the art.
- the plug could be made of any suitable material such as wax, a plastic, etc.
- a suitable quantity of a preservative solution 21 is placed in the container 7.
- a preservative solution 21 is added.
- a preservative solution can act to inhibit enzymatic activity which can be responsible for the destruction of antibody molecules or can function as an anti ⁇ microbial agent.
- Compounds contemplated for use in the inner tube as a preservative include anti-bacterial agents, anti-fungal agents, bacteriostatic agents, fungistatic agents, and enzyme inhibitors.
- an antibacterial agent it is preferred to use chlorhexidine gluconate or thimerosal.
- the preservative solution to be used in the inner tube can contain one or a combination of the preservatives.
- the preservatives are included in a concentration which limits microbial contamination and does not adversely effect the immunoglobulins absorbed into the pad.
- the preservative solution to be used in the inner tube can also contain a detergent which improves removal of antibody from the pad during centrifugation.
- Tween 20 polyoxyethylene sorbitan monooleate
- a combination comprising about 0.1% chlorhexidine gluconate and 0.5% Tween 20 is most preferred.
- the tube cap is inserted into the container to seal in the contents.
- the pad can be stored in this manner for several days until immunological testing can be initiated.
- the pad 3 on holder 1 is used as already described. After the pad 3 is removed from the user's mouth, stopper 11 is removed from container 7 and the pad is placed within the container 7. The holder 1 is broken off at a point outside the mouth of container 7 so it will project upwardly from the container. Then the stopper 11 is replaced. Since stopper 11 is hollow, it will securely seal the container 7 with the broken end of holder 1 extending into it. Holder 1 is preferably scored at a suitable location to provide for easy breaking. When the pad 3 is inserted in container 7, it will absorb at least a part of preservative solution 21.
- the pad 3 is stored in container 7 until testing can be initiated.
- the container 7, with the stopper 11 securely in place is inverted, the seal 19 of wax or other suitable substance is removed, and the container 7 is placed in a centrifuge tube 4.
- the complete assembly 2 is then centrifuged whereby all the liquid, including preservative solution, saliva, etc., is drawn down through bore 17 into the centrifuge tube 4. Testing is then performed using known techniques.
- kits can be provided.
- the kit can include a combination of the pad and implements used to collect and prepare the oral specimen for analysis.
- One preferred embodiment of the kit includes the treated pad 3 and pad holder 1; the container 7 having the stopper 11 and the storage preservative 21.
- a centrifuge tube 4 could be included.
- Another container for storing collected substances for subsequent testing comprises an open upper end adapted to be sealed with a removable stopper and a lower end having an opening communicating the interior of the container with the outside, the opening being selectively sealed by a frangible nipple during storage of the substances and unsealed for removal of the collected substances for subsequent testing.
- the distal end of the frangible nipple is enlarged, preferably in the form of a ball.
- the open upper end is preferably sealed with a screw-on "plug seal" cap.
- the floor of the container which slopes towards the opening in the center thereof, has a plurality of upstanding webs to prevent the pad described in the aforemen ⁇ tioned application serial No. 641,739 from resting on the bottom of the container and blocking the opening at the bottom.
- the container is dimensioned to permit its insertion into a standard 15 ml. conical centrifuge tube. This allows centrifu ⁇ gation after the nipple is broken off, thereby transferring the vial contents into the centrifuge tube.
- the container is intended to hold a volume of preservative liquid (from about 0.5 ml. to about 2.0 ml.). When the pad is placed in the container, the preservative is absorbed into the pad. Since the container with the preservative will be stored for up to a year without significant loss of liquid volume, the water transmission rate must be low.
- the container is fabricated of any suitable plastic such as polyethylene, polycarbonate, polystyrene, PET (polyethylene terephthalate) , polypropylene, EPC (ethylene propylene copolymer) , and the like. Polycarbonate is the least desirable due to excessive "breathing," that is, allowing moisture to escape. The preferred material is poly ⁇ propylene.
- the container of this embodi ⁇ ment generally designated by the numeral 10, comprises an elongated body portion 12.
- Body portion 12 is generally cylindrical although, it is preferably slightly tapered from the upper end to the floor 15.
- the upper end 16 is open and is provided with a screw thread 18 to accommodate the cap.
- a nipple 20 extends downwardly from the floor 15.
- the floor 15 preferably has a slight slope at an angle ⁇ from the outside to the center. Angle is preferably about 20°.
- a depression 22 At the center of the inside of the floor 15 is a depression 22, preferably "V" shaped and preferably forming an angle of about 88.50°.
- the depression 22 causes the base end of nipple 20 to be weakened, thereby allowing the same to break off when sufficient pressure is applied.
- a bulb 24 is formed at the distal end of nipple 20 to make it easier and safer to break off the frangible nipple 20.
- a plurality of upstanding webs 26 are provided on floor 15. Preferably, there are four webs 26.
- a cap generally designated by the numeral 28 for sealing the open upper end 16 of container 10.
- Cap 28 is of the type commonly known as a "plug seal" cap.
- Cap 28 has internal screw threads 30 to cooperate with threads 18 on the container.
- Annular depression 32 creates an annular space 34 bounded by annular depression 32 and the inside of outer wall 36. The wall of the container thus fits into annular space 34 and creates a perfect seal.
- the outside surface of cap 28 is knurled as shown at 38 to facilitate removal and replacement of the cap.
- the container 10 is used in the same manner as the contain ⁇ ers described above with respect to Figs. 1 and 2. The following examples show the effectiveness of the pad of the present invention.
- Oral samples were collected from ten subjects.
- the subjects collected saliva by chewing on the "absorbent body" sold as part of the Sarstedt SalivetteTM kit with a cotton plug as an absor ⁇ bent body until the cotton plug became saturated with saliva.
- the subject then collected mucosal transudate by collecting oral fluid using the treated pad described in WO 91/13355. After the saliva or transudate was collected, the liquid was removed from the cotton plug and treated pad by centrifugation.
- the oral specimens were analyzed for the presence of IgG antibody using an enzyme immunoassay.
- Table 1 shows the levels of IgG collected by the cotton plug versus the treated pad.
- Saliva samples collected by chewing on an "absorbent body" of the Sarstedt SalivetteTM kit contain approximately 25 times less IgG antibody than samples collected by the treated pad of WO 91/13355.
- a total of four pads were used for oral specimen collection from each voluntary participant, two of the pads being treated and two untreated. Each collection (two like pads collected simultaneously) lasted two minutes. The participant rinsed his/her mouth with tap water after the initial collection and waited at least one-half hour before the second collection. Participants in the study were divided into two groups. Half of the participants initially collected an oral sample using two untreated pads, followed later by two treated pads. The other participants collected with two treated pads first, and two untreated pads later.
- the participant After collection the participant inserted the pad into an empty vial which has been labeled with the specimen number and the type of pad used for collection (treated, or untreated) . The stick was snapped-off and the vial capped tightly. The specimen remained at 4°C before and during centrifugation. Once centri- fuged, the oral specimens collected by the two like pads from each participant were pooled. Specimens were stored at 4°C.
- Each specimen pool was assayed using Epitope, Inc.'s EIA for Total IgG to quantitate the IgG present in each specimen.
- IgG immunoglobulin G
- EIA enzyme immunoassay
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Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU76313/94A AU7631394A (en) | 1994-08-09 | 1994-08-09 | Oral collection for immunoassay |
KR1019970700829A KR970704384A (en) | 1994-08-09 | 1994-08-09 | Oral collection for immunological analysis (ORAL COLLECTION FOR IMMUNOASSAY) |
BR9408607A BR9408607A (en) | 1994-08-09 | 1994-08-09 | Preferential collection process of mucosal transudate from an oral cavity for testing |
PCT/US1994/009096 WO1996004850A1 (en) | 1994-08-09 | 1994-08-09 | Oral collection for immunoassay |
CA002180025A CA2180025C (en) | 1994-08-09 | 1994-08-09 | Oral collection for immunoassay |
JP8507254A JPH09509495A (en) | 1994-08-09 | 1994-08-09 | Oral sampling for immunoassay |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BR9408607A BR9408607A (en) | 1994-08-09 | 1994-08-09 | Preferential collection process of mucosal transudate from an oral cavity for testing |
PCT/US1994/009096 WO1996004850A1 (en) | 1994-08-09 | 1994-08-09 | Oral collection for immunoassay |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1996004850A1 true WO1996004850A1 (en) | 1996-02-22 |
Family
ID=29402841
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1994/009096 WO1996004850A1 (en) | 1994-08-09 | 1994-08-09 | Oral collection for immunoassay |
Country Status (4)
Country | Link |
---|---|
JP (1) | JPH09509495A (en) |
AU (1) | AU7631394A (en) |
BR (1) | BR9408607A (en) |
WO (1) | WO1996004850A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002031105A1 (en) * | 2000-10-12 | 2002-04-18 | Sapporo Immuno Diagnostic Laboratory | Method and instrument for collecting fluid in the oral cavity |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020106891A1 (en) | 2018-11-20 | 2020-05-28 | Spectrum Solutions, Llc | Sample collection system including sealing cap and valve |
US11701094B2 (en) | 2019-06-20 | 2023-07-18 | Spectrum Solutions L.L.C. | Sample collection system including valve and plug assemblies |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4635488A (en) * | 1984-12-03 | 1987-01-13 | Schleicher & Schuell, Inc. | Nonintrusive body fluid samplers and methods of using same |
EP0418739A1 (en) * | 1989-09-21 | 1991-03-27 | Epitope, Inc. | Oral immunoglobulin collection for immunoassay |
WO1991013355A2 (en) * | 1990-02-28 | 1991-09-05 | Epitope, Inc. | Oral collection for immunoassay |
WO1994004078A1 (en) * | 1992-08-25 | 1994-03-03 | Epitope, Inc. | Oral collection device and kit |
-
1994
- 1994-08-09 JP JP8507254A patent/JPH09509495A/en active Pending
- 1994-08-09 BR BR9408607A patent/BR9408607A/en not_active Application Discontinuation
- 1994-08-09 AU AU76313/94A patent/AU7631394A/en not_active Abandoned
- 1994-08-09 WO PCT/US1994/009096 patent/WO1996004850A1/en not_active Application Discontinuation
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4635488A (en) * | 1984-12-03 | 1987-01-13 | Schleicher & Schuell, Inc. | Nonintrusive body fluid samplers and methods of using same |
EP0418739A1 (en) * | 1989-09-21 | 1991-03-27 | Epitope, Inc. | Oral immunoglobulin collection for immunoassay |
WO1991013355A2 (en) * | 1990-02-28 | 1991-09-05 | Epitope, Inc. | Oral collection for immunoassay |
WO1994004078A1 (en) * | 1992-08-25 | 1994-03-03 | Epitope, Inc. | Oral collection device and kit |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002031105A1 (en) * | 2000-10-12 | 2002-04-18 | Sapporo Immuno Diagnostic Laboratory | Method and instrument for collecting fluid in the oral cavity |
Also Published As
Publication number | Publication date |
---|---|
BR9408607A (en) | 1997-12-23 |
JPH09509495A (en) | 1997-09-22 |
AU7631394A (en) | 1996-03-07 |
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