CN1226018C - Oral collection for immunoassay - Google Patents

Abstract

A method of collecting immunoglobulins and other analytes from the oral cavity for use in immunization and other tests. Use an absorbent pad to collect samples with high concentrations of immunoglobulin or other analytes. Basic immunoassay techniques as a tool for detection of disease in patients can be used on this sample.

Description

Oral collection for immunoassays

The present invention relates to the field of immunoassay. In particular, the present invention discloses a system for the analysis of immunoglobulins and other substances from mucosal exudates.

Because of the correlation between blood and saliva immunoglobulins, and the presence of saliva-specific secretory IgA, antigen-antibody assays have been performed on saliva to evaluate the value of such assays as tools for detecting disease.

Collection of saliva from salivary glands is complicated by low secretion volume, various anatomical distribution of the glands, and high fluid viscosity. The vast majority of collection techniques involve the use of capillaries, aspiration micropipettes, chewing on paraffin, or aspiration into syringes. However, a limitation of these methods is that the viscosity of saliva makes recovery of non-foaming material difficult with these techniques. Other collection methods have been proposed to eliminate or at least reduce the number of air bubbles in the sample. Included in such methods is the collection of saliva in the mouth by direct absorption with sponges or flexible, permeable membrane pads. After absorption, the saliva is separated from the absorbent material by centrifugation or by squeezing the absorbent material. However, absorption is generally achieved by using cotton, nylon or polyester as the absorbent material. These materials bind proteins non-specifically, which results in poor recovery of immunoglobulins.

Testing techniques for saliva samples have not been fully developed. In addition to collection problems, samples collected by known methods typically contain about 0.01-0.1% of the immunoglobulins found in serum. Because of the greatly reduced immunoglobulin content in saliva, more accurate antigen-antibody assays are required in screening patients for disease. Parry et al. discuss these methods in "Rational Program for Screening Travelers for Antibodies to Hepatitis AVirus", (The Lancet, 25 June 1988) and find that for To avoid false signals in other, less sensitive methods, the more accurate IgG capture radioimmunoassay (GACRIA) assay is preferred. Of course, more sensitive testing procedures generally require additional time and expense to obtain test results.

The oral immune system not only interacts with the body's systemic immune system, but also has its own centralized antigen-antibody response center. Extraoral lymph nodes and intraoral lymphoid aggregates were found in the oral cavity. Extraoral lymph nodes are involved in the drainage of the oral mucosa, gingiva, and teeth. However, little is known about the function of lymphoid tissues in the oral cavity.

Extraoral lymph nodes include a fine network of lymphatic capillaries in the superficial layers of the mouth, palate, cheeks, lips, gingiva, and dental pulp. Lymphatic capillaries connect to larger lymphatic vessels that originate deep in the network of tongue muscles and other structures. Antigens can enter the oral lymphatic system directly through capillaries, or they can be transported by phagocytes. Once in the network, the antigen can induce an immune response.

Lymphoid tissue in the oral cavity is generally classified as 4 different tissue aggregates: (a) tonsils, (b) scattered submucosal lymphoid aggregates, (c) salivary gland lymphoid tissue, and (d) gingival lymphoid organize.

The tonsils (palatal and lingual) primarily produce B and T cells, which are located primarily in the caps of lymphocytes and plasma cells. Antigens typically enter the tonsils through different epithelial regions where the antigens come into contact with T and B cells to stimulate an immune response. It has been found that the major class of antibodies formed in the tonsils is IgG, followed by IgA, IgM, IgD and IgE.

Dispersed submucosal lymphoid cells have not been extensively studied. These cell clusters are histologically similar to tonsil tissue.

Both the primary salivary glands (parotid, submandibular and sublingual) and secondary salivary glands have been found to contain lymphocytes and plasma cells. Most plasma cells secrete IgA and some IgG or IgM. IgA synthesized in salivary glands has a dimeric structure. This type of IgA is called secretory IgA (sIgA), and it is the major immunoglobulin component in saliva.

T cells and B cells were found in the gingival lymphoid tissue. In individuals with clinically normal gingival tissue, T cells predominated. During the infectious period, such as when suffering from gingivitis, B cells are mostly found.

Plasma cells are also found in the gingival lymphoid tissue. These cell clusters are generally close to blood vessels and mainly produce IgG. Less IgA and IgM are also produced. More importantly, Brandtzaeg et al. in "Human Saliva: Clinical Chemistry and Microbiology" (eds. Jorma O. Tenovuo) showed that immunoglobulins from the secretion site of gingival tissue were similar to those found in blood. Immunoglobulins are directly related.

In order to eliminate or greatly reduce the problems inherent in the antigen-antibody analysis of saliva, we previously proposed a method for collecting immunoglobulins from the oral cavity with the assistance of a hypertonic solution as a rinse. See EP 0418 739A1. We have also previously proposed the use of collection pads (treated with a hypertonic solution) in the oral cavity to absorb immunoglobulin for immunological testing. See WO 91/13355.

We have found that an untreated spacer can be used to collect sufficient quantities of oral immunoglobulin for immunoassays. Yields of immunoglobulin produced using this spacer are greater than required, and it can be combined with basic antibody-antigen assay techniques as a disease detection tool. We have also found that the present invention can be used to collect non-immunoglobulin material for testing. In fact, the present invention has been used successfully to collect species with molecular weights ranging from about 176 (cotinine) to 950,000 (IgM). There is no limit to the size of the molecules when collected using the present invention. If a molecule can pass through capillary walls and other oral tissues, it can be collected by the present invention. The spacer comes into contact with the oral mucosa without chewing.

Fig. 1 is a longitudinal sectional view of an example of a storage spacer container;

Figure 2 is a longitudinal sectional view of the example of Figure 1 showing the spacer and bracket;

Figure 3 is another perspective view of an example of a container suitable for unscrewing the lid of the present invention;

Fig. 4 is the top view of Fig. 3 container;

Fig. 5 is a longitudinal sectional view of Fig. 4 container along Fig. 4 line 5-5;

Figure 6 is a perspective view of a lid for the container shown in Figure 3;

Fig. 7 is the top view of Fig. 6 cover;

FIG. 8 is a cross-sectional view of the cover taken along line 8-8 of FIG. 7. FIG.

The present invention relates to the collection of oral immunoglobulins for immunoassays and other substances for assays. A spacer is used to collect samples with high concentrations of immunoglobulin or other substances. High levels of oral immunoglobulins are considered to be concentrations exceeding 50 μg per milliliter of total Ig. Basic assay techniques can be applied to samples that can be used as a detection tool for disease in a patient or as a detection tool for certain foreign substances.

Representative molecules that have been successfully collected using the present invention are:

Molecular weight of the analyte

cotinine 176

Glucose 180

Theophylline 180

Cocaine 303

β2-microglobulin 11,818

Hepatitis B surface antigen 24,000

β-human chorionic gonadotropin 37,900

IgG--human antibody 150,000

Total IgG (unspecified antigen)

HIV-1

Hepatitis A

Hepatitis B

measles

syphilitic nontreponemal antigen

IgA--human antibody 160,000

Total IgA (unspecified antigen)

IgM-human antibody 950,000

Total IgM (unspecified antigen)

Hepatitis A

Hepatitis B

In order to minimize degradation in the collected samples, the container for the storage pad after application of the hypertonic solution of the present invention can be filled with a preservative. This preservative inhibits the activity of proteolytic enzymes that destroy antibody molecules. Compounds that act as preservatives include antibacterial agents, antifungal agents, bacteriostatic agents, fungal inhibitors, and enzyme inhibitors. In a preferred embodiment, benzoic acid, sorbic acid or their salts are used as the antifungal agent. As far as bacterial inhibitors are concerned, high concentrations of salts and compounds that maintain hypertonic solutions at low pH are preferred. Such salts include sodium ethylmercury thiosalicylate (or thimerosal), phenylmercuric acetate, phenylmercuric nitrate, and sodium azide. Other preferred preservatives include those commonly used in medicines and mouthwashes. Examples thereof include ethanol and chlorhexidine gluconate. Another class of preferred antimicrobial agents are purging agents which can be used as topical antiseptics or in mouthwashes. An example of this is benzalkonium chloride. Such preservatives are preferably used in an amount of about 0.01-0.2% by weight.

In the present invention, a pad is used to absorb mucosal secretions from the oral cavity. The pad is made of absorbent material that sits effectively in the mouth. A plastic or carbohydrate material such as cellulose can be used as the absorbent material, but a thicker absorbent cotton paper is preferred. An example of a thicker absorbent cotton paper is product #300 produced by Schleicher and Schuell in Keene, New Hampshire. The spacer is preferably non-foam or sponge, although foam or sponge can also be used.

Because many materials from which spacers are made will bind proteins non-specifically. As a result, certain immunoglobulins will undesirably bind to the spacer, requiring the use of blocking agents to block protein binding to the spacer. Since the blood itself contains blocking agents (ie, human serum albumin), there is usually no problem of non-specific binding when collecting blood samples.

To reduce non-specific binding during buccal sample collection, a blocking agent can be added to the hypertonic solution mixed into the pad. A blocking agent is usually a soluble protein used to prevent non-specific binding of other proteins to the solid surface. Compounds that 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 the antibody molecule. Preferably bovine gelatin is used. Generally, the blocking agent can be made into a solution at a concentration of about 0.01-0.2% by weight. The solution is then impregnated into the gasket by dipping or spraying, followed by drying.

To collect oral material, the spacer can be placed in the oral cavity with the aid of a stand. Spacers and brackets are shown in the accompanying drawings. The spacer support 1 can be a hollow plastic rod with a groove 2 at one end. The spacer 3 is inserted into the groove, and the spacer can be placed in the oral cavity by using the bracket, preferably between the bottom of the gum and the cheek. Placing the spacer between the bottom of the cheek and the bottom of the gum helps to absorb secretions from the gingival lymphoid tissue as well as secretions from the submucosa and salivary gland lymphoid tissue. Preferably, the pad is rubbed back and forth between the gum and cheek for about 10 seconds, and then the pad is placed in the gum-cheek position for about 2 minutes to collect the sample. Spacers should not be chewed, as chewing stimulates unwanted salivation.

After specimen collection, the pads are stored in containers until immunoassays are performed. A container is shown in Figures 1 and 2. The container is required to have a centrifuge tube as the outer part of the container and the inner part of the container to have an inner tube inserted into the centrifuge tube. The spacer is placed in the inner tube, and the contents of the inner tube are secured by the tube cap.

Figure 1 shows an assembly modified for use with the present invention. The container 2 comprises a centrifuge tube 4 having a conical lower end or bottom 5 with a downwardly conical deep hole 6 in which centrifuged solid matter accumulates; a ring having a radially outwardly protruding An upper end tube or container 7 and a plug or plug 11 with a cylindrical edge 8 and a cylindrical upper portion 9 at its upper end. Cylindrical portion 9 and tube plug 11 obtain the same size and shape as the upper portion of centrifuge tube 4 to align with the outer surface of the centrifuge tube and give the assembly a symmetrical appearance, although these features are not critical to the practice of the invention. In the bottom 13 at the bottom of the container 7 is a hole 17 through which liquid flows from the container 7 to the centrifuge tube 4 when the whole assembly is centrifuged. Container 7 is made of any suitable material (eg polyethylene, glass, etc.). Likewise, the plug 11 may be made of any suitable material, such as polyethylene as is well known in the art.

In the hole 17 there is a removable plug 17 . The hole plug can be made of any suitable material (eg paraffin, plastic, etc.). An appropriate amount of antiseptic solution 21 is placed in the container 7 .

As soon as the gasket is placed in the inner tube, the anti-corrosion solution 21 is added to the tube. This preservative solution acts to inhibit the activity of enzymes that destroy antibody molecules or acts as an antimicrobial agent.

Compounds used in the inner tube as preservatives include antibacterial agents, antifungal agents, bacteriostatic agents, fungal inhibitors and enzyme inhibitors. As far as antibacterial agents are concerned, chlorhexidine gluconate or thimerosal are preferred.

The preservative solution used in the inner tube may contain one or more preservatives. In general, preservatives should be included in concentrations that limit microbial contamination and do not adversely affect the adsorption of immunoglobulins to the gasket.

The preservative solution used in the inner tube may also contain a decontamination agent that improves the separation of antibodies from the spacer during centrifugation. Tween 20 (polyoxyethylene sorbitan monooleate) is a preferred scavenger because it also prevents non-specific binding of antibodies to solid surfaces. Preferably a mixture comprising about 0.01-0.2% chlorhexidine gluconate and 0.2-0.7% Tween 20 is used. Preferably a mixture comprising about 0.1% chlorhexidine gluconate and 0.5% Tween 20 is used.

After the embalming solution is added to the inner tube, the cap is inserted into the container to seal the contents. In this way the shims can be stored for several days until immunoassays are started.

In this example, the spacer 3 on the bracket 1 was used as described above. After the spacer 3 is removed from the user's mouth, the plug 11 is removed from the container 7 and the spacer is placed in the container 7 . The support 1 is broken off outside the opening of the container 7 so that it stretches out of the container upwards. Then replace the plug 11. Since the plug 11 is hollow, it enables the broken end of the bracket 1 to protrude into the plug and seal the container 7 securely. It is preferable to score the appropriate part of the bracket 1 to make it easy to break. When the gasket 3 is inserted into the container 7 it will absorb at least part of the embalming solution 21 .

The gasket 3 was stored in the container 7 until the start of the test. In the laboratory, the container 7 securely with the plug 11 is inverted, the plug 19 made of paraffin or other suitable material is removed, and the container 7 is placed in the centrifuge tube 4 . The whole assembly 2 is then centrifuged and all liquid (including embalming solution, saliva, etc.) is drawn through the hole 17 into the centrifuge tube 4 . Tests are then carried out using known techniques.

To simplify the collection and analysis of buccal samples using the pad collection system, a kit is available. The kit includes a combination of pads and a complete set of tools for collecting and preparing oral samples for analysis. A preferred example of a kit includes treated gasket 3 and gasket holder 1 ; container 7 with pipe plug 11 and storage preservative 21 . A centrifuge tube 4 may also optionally be included.

Another container of the present invention for storing collected substances for subsequent testing comprises an open upper end adapted to be sealed by a removable stopper and a bottom end with a hole communicating the inside and outside of the container, which hole is selected as a fragile nipple when storing the substance Sealed by the shaped protrusion, or left unsealed to withdraw collected material for subsequent testing. The ends of the frangible papillae are flared, preferably bulbous, for the comfort and safety of the user. The open upper end is preferably sealed with a threaded "hole plug seal" cap. In addition, the bottom of the container sloping toward the central hole has a number of upstanding screens to prevent the gasket described in the aforementioned Patent Application No. 641,739 from resting on the bottom of the container and preventing blockage of the bottom hole.

The size of the container is chosen so that it can fit into a standard 15ml conical centrifuge tube. This enables the contents of the vial to be centrifuged into a centrifuge tube after the papillae have snapped off.

The container will contain a certain volume (about 0.5-2.0ml) of preservative liquid. When the gasket is placed in the container, the embalming fluid is drawn into the gasket. Since the container with embalming fluid will be stored for up to one year without appreciable reduction in the volume of embalming fluid, the water transmission rate must be low. The container is made of any suitable plastic such as polyethylene, polycarbonate, polystyrene, PET (polyethylene terephthalate), polypropylene, EPC (ethylene-propylene copolymer), etc.). Polycarbonate is the worst since it "breathes" excessively, even if moisture escapes. A preferred material is polypropylene.

Referring to FIGS. 3 and 4 , the container of the present example (designated by numeral 10 ) includes an elongated body portion 12 . The body portion 12 is generally cylindrical, although it is preferably slightly tapered from the upper end to the bottom 15. The upper end 16 is open and has threads 18 to fit the cap.

The papillae 20 protrude downwards from the base 15 . The bottom 15 is preferably slightly inclined at an angle α from the outside towards the center. The angle α is preferably about 20°. Centrally on the inside of base 15 is a depression 22 which is preferably "V" shaped and preferably forms an angle of about 88.5°. The depression 22 makes the root of the papillae 20 brittle so that it breaks off easily when sufficient pressure is applied. A ball 24 is formed at the end of the papillae 20 for easier and safer breaking of the fragile papillae 20 . In order to prevent gaskets inserted into the container 10 from resting in the holes of the bottom 15 created by the nipples 20 , a number of upstanding wire meshes 26 are placed on the bottom 15 . Preferably 4 layers of screen 26 are placed.

Let us now turn to Figures 5, 6 and 7, which show a lid (marked by numeral 28) for sealing the opening at the upper end 16 of the container 10. The shape of the cap 28 is often referred to as a "plug seal" cap. The cap 28 has internal threads 30 that mate with the threads 18 on the container. The ring recess 32 creates a ring region 34 bounded by the inner side of the ring recess 32 mixing outer wall 36 . The container wall is thus adapted to the ring region 34 and produces a better seal. On the outside surface of the cover 28 is a ridge shown at 38 to facilitate removal and replacement of the cover.

The method of use of the container 10 is the same as that of the container described above with reference to FIGS. 1 and 2 . The following examples demonstrate the effectiveness of the inventive gasket.

Example 1

Comparison of IgG in Saliva and Mucosal Exudates

The purpose of this study was to compare IgG levels in saliva and mucosal exudates.

Oral samples were collected from 10 subjects. Saliva was collected from the subjects by chewing on the "adsorbent" (part of the Sarstedt Salivette( ^TM) assembly, with a cotton swab as the adsorbent) until the cotton swab was saturated with saliva. After about 4 hours thereafter, mucosal exudates were collected from the subjects by collecting oral fluid with a treatment pad as described in WO 91/13355. After collecting saliva or exudate, centrifuge the fluid off the cotton swab and dispose of the pad. Oral samples were analyzed for the presence of IgG antibodies by enzyme immunoassay.

Table 1 lists the IgG collection levels of cotton swabs and treated pads.

Table 1

Subject ID Saliva IgG (μg/ml) Exudate IgG (μg/ml) 1 1.6 38.3 2 3.2 60.3 3 0.7 21.0 4 1.8 48.5 5 2.4 135 6 4.9 86.7 7 1.1 72.3 8 1.3 31.1 9 3.0 63.1 10 4.1 37.5 Average IgG 2.4 59.4

The IgG antibody content of the saliva sample collected by chewing the "adsorbent" of the Sarstedt Salivette ^™ assembly was about 25 times lower than that of the sample collected by the treatment pad of WO 91/13355.

Example 2

A comparison test was carried out between the treated gasket of WO 91/13355 and the untreated gasket of the present invention.

Oral samples were collected using 4 pads per volunteer subject, two of which were treated and two were untreated. Each collection (two identical pads collected simultaneously) lasted 2 minutes. After the first collection, subjects' mouths were rinsed with tap water and the second collection was performed at least 1.5 hours apart. The subjects in this study were divided into two groups. Half of the subjects used two untreated pads first, followed by two treated pads to collect oral samples, and the remaining subjects used two treated pads first, followed by untreated pads for collection.

After collection, subjects inserted the spacers into empty vials labeled with the sample number and the type of spacer used for collection (treated or untreated). Break off the standoff rod and cap the vial tightly. Samples were maintained at 4°C before or during centrifugation. After centrifugation, buccal samples collected from each subject using two identical spacers were pooled. Samples were stored at 4°C.

The total amount of IgG in each pooled sample was analyzed using an EIA manufactured by Epitope, Inc. to determine the amount of IgG in each sample.

Table 2

Subject number IgG (μg/ml) untreated gasket Processing Gasket 1 ^* 6.7 9.1 2 ^* 4.9 7.8 3 4.6 11.2 4 12.4 23.9 5 ^* 10.2 26.4 6 ^* 9.2 43.9 7 5.3 45.4

^* : first collected with untreated shims

Samples collected from treated pads contained higher immunoglobulin G (IgG) levels than samples collected from untreated pads, as analyzed by in-house IgG-specific enzyme immunoassay (EIA).

From the analysis of the WO 91/13355 pad collection and the Sarstedt "adsorbate" collection, the pad collects IgG from mucosal exudates but not saliva and the difference is evident.

From the analysis of the treated pad collection of WO 91/13355 and the untreated pad collection of the present invention, although the IgG collected by the untreated pad is less than that of the treated pad, the IgG collected by the untreated pad is still significantly more Based on the amount expected to be collected from saliva (5.3 μg/ml vs. 2.4 μg/ml).

Claims (16)

1. A method for preferentially collecting mucous membrane exudates for determination from the oral cavity, comprising the steps of: (a) inserting the adsorption pad into the oral cavity, wherein the adsorption pad is not immersed in the hypertonic saline solution; (b) bringing the spacer into contact with the oral mucosa without tearing said spacer; (c) Remove the spacer from the mouth. 2. The method of claim 1, wherein the pad is retained in a container after the pad is removed from the oral cavity. 3. The method of claim 2, wherein the container contains a preservative solution. 4. The method of claim 3, wherein the preservative solution comprises chlorhexidine gluconate or thimerosal. 5. The method of claim 4, wherein the preservative solution comprises chlorhexidine gluconate. 6. The method of claim 2, wherein the container includes an open upper end sealed by a removable lid, and a lower end having a hole connecting the inside and outside of the container, the hole being selectively sealed during storage of said gasket, And when taking out the collected mucosal exudate for subsequent determination, choose to open the seal. 7. The method of claim 6, wherein the hole is selectively sealed by frangible papillae. 8. The method of claim 1, wherein said mucosal exudate contains analytes with a molecular weight of 176-950,000. 9. The method according to claim 8, wherein said measured substance is selected from the group consisting of cotinine, glucose, theophylline, cocaine, β2-microglobulin, hepatitis B surface antigen, β-human chorion stimulating Gonadal hormones, immunoglobulins, and mixtures thereof. 10. The method of claim 9, wherein the analyte is an immunoglobulin. 11. The method according to claim 10, characterized in that said immunoglobulin is selected from the group consisting of IgG, IgA and IgM. 12. The method according to claim 10, wherein the immunoglobulin is at least one antibody among HIV-1, hepatitis A, hepatitis B, measles and non-treponemal pallidum antigens. 13. The method of claim 1, wherein said analysis is an immunoassay. 14. The method of claim 13, wherein the immunoassay is an ELISA assay. 15. The method of claim 1 wherein said shim is made of carbohydrate material. 16. The method of claim 1, further comprising (d) storing the pad in a hypertonic solution for subsequent removal of the collected mucosal exudate from the pad for use in Determination.

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