CN1226018C - Oral collection for immunoassay - Google Patents

Abstract

The present invention relates to a method of collecting immunoglobulin and other determinands from an oral cavity for immunity and other tests. An absorbent pad is used for collecting samples of the immunoglobulin with high concentration or other determinands. Basic immune test technologies which are used as a tool for testing patients' diseases can be used on the samples.

Description

The oral collection that is used for immunoassay

The present invention relates to the immunity test field.Specifically, the present invention discloses and a kind ofly is used to analyze immunoglobulin and from the system of other materials of mucosa transudate.

Since the association between the immunoglobulin of blood and saliva, and have the distinctive secretory IgA, sIgA of saliva, thus saliva is carried out the Ag-Ab test, to estimate the value of this class test as the instrument that detects disease.

It is complicated because of following factors to collect saliva from salivary gland: secretory volume is few, the various different anatomic of body of gland distribute and fluid viscosity is higher.Most collection techniques relate to the use capillary tube, suck micropipet, chew on paraffin or inhalation syringe.Yet the limitation of these methods is: the viscosity of saliva makes and is difficult to reclaim no foam material with these technology.The collection method that has proposed other is to eliminate or to reduce bubble quantity and bubble in the sample at least.In these class methods, comprise, by directly collect the saliva in the mouth with sponge or flexible osmotic membranes filler.After absorption, by centrifugal or by extruding absorbing material and from absorbing material, isolate saliva.Yet, absorb generally by Cotton Gossypii, nylon or polyester are realized as absorbing material.The non-conjugated protein specifically of these materials meetings, this causes the response rate of immunoglobulin very low.

The measuring technology of saliva sample is full-scale development not also.Except the problem of collection aspect, the sample of collecting with known method contains the immunoglobulin that the 0.01-0.1% that has an appointment finds usually in serum.Because the decline greatly of immunoglobulin content in the saliva, so when the ill patient of screening, need use more accurate Ag-Ab analytical method.People such as Parry are " whether the screening traveller has the reasonable program (Rational Programme for Screening Travellers for Antibodies to Hepatitis AVirus) of hepatitis A virus (HAV) antibody ", (The Lancet, on June 25th, 1988) in these methods have been discussed, and find that for fear of the glitch in the lower method of other sensitivity preferably IgG catches radioimmunoassay (GACRIA) test more accurately.Certainly, more sensitive test program need increase time and expense usually to obtain result of the test.

The immune system in oral cavity is not only had an effect with the systemic immune system of health, and also has the Ag-Ab response center of himself centralization.Oral cavity perilymph knot and oral cavity endolymph sample aggregation in the oral cavity, have been found.Oral cavity perilymph knot is relevant with the drain of oral mucous membrane, gum and tooth.Yet the function of boil on the nape opposite the mouth intracavity lymphoid tissue is known little.

Oral cavity perilymph knot comprises the meticulous network of lymphatic capillary, and these lymphatic capillarys are positioned at the shallow-layer of mouth, maxillary, cheek, lip, gums and tooth tooth marrow.Lymphatic capillary with bigger, link to each other from the lymphatic vessel of the network depths of lingualis meat and other structures.Antigen can directly enter the oral cavity lymphsystem by capillary tube, is perhaps carried by phagocyte to reach.In case be arranged in network, but just induce immune response of antigen.

What belong to oral cavity endolymph sample tissue generally is 4 kinds of different aggregations of organizing: (a) tonsil, (b) lymph sample aggregation under the dispersive mucosa, (c) salivary gland lymphoid tissue and (d) gums lymphoid tissue.

Tonsil (maxillary with tongue) mainly produces B cell and T cell, and they mainly are arranged in lymphocyte and plasmacytic medicated cap.Antigen typically enters tonsil by different epithelium zone, thereby wherein antigen and T cell and B cells contacting immune stimulatory are replied.Find that the main type of the antibody that forms is IgG, is followed successively by IgA, IgM, IgD and IgE subsequently in tonsil.

Lymphoid cell under the dispersive mucosa also there is not broad research.It is similar that these cell masses and tonsil are organized on the histology.

Find that main salivary gland (parotid gland, inframandibular and the Sublingual) and time salivary gland both are contained lymphocyte and plasma cell.Most plasma cell secretion IgA and some IgG or IgM.Synthetic IgA has dimeric structure in salivary gland.Such IgA is called as secretory IgA, sIgA (sIgA), and it is an immunoglobulin component main in the saliva.

T cell and B cell in the gums lymphoid tissue, have been found.In the normal individuality of gingival tissues, the T cell is in the majority clinically.In infection period,, find that the B cell is in the majority as when suffering from gingivitis.

Also in the gums lymphoid tissue, find plasma cell.These cell clusters are general near blood vessel and mainly produce IgG.Also make less I gA and IgM.The more important thing is, people such as Brandtzaeg show in " human saliva: clinical chemistry and microbiology (Human Saliva:Clinical Chemistry andMicrobiology) " (Jorma O.Tenovuo edits), and are directly related from the immunoglobulin of finding in the immunoglobulin of gingival tissues secretion place and the blood.

In order to eliminate or to significantly reduce inherent problem in the Ag-Ab of saliva is analyzed, we have proposed a kind of method previously, and it is collecting immunoglobulin with hyperosmotic solution under as the assistance of gargling washing liquid from the oral cavity.Referring to EP 0418 739A1.We are also former to have proposed, and uses in the oral cavity and collects pad (handling with hyperosmotic solution) absorption immunoglobulin for immunoassay.Referring to WO 91/13355.

We find that a kind of undressed pad can be used for collecting the oral immunisation globulin that is used for immunity test of capacity.The output of using the immunoglobulin that this pad forms is greater than required, and it can combine as the disease detection instrument with basic antibody-antigen test technology.We find that also the present invention can be used for collecting the material of the NIg that is used to test.In fact, successfully collected the material that molecular weight is about 176 (but ferrum is peaceful)-950,000 (IgM) with the present invention.Unrestricted when using the present invention to collect to bulk of molecule.If molecular energy passes capillary wall and other oral cavity tissue, it just can be collected by the present invention.This pad can contact with oral mucosa without just chewing.

Fig. 1 is a longitudinal sectional drawing that stores the pad container instance;

Fig. 2 is the longitudinal sectional drawing that shows Fig. 1 example of pad and support;

Fig. 3 is that another is applicable to axonometric chart of twisting the example of the container that removes lid of the present invention;

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

Fig. 5 is the longitdinal cross-section diagram of Fig. 4 container along Fig. 4 line 5-5;

Fig. 6 is the axonometric chart that is used for the lid of container shown in Figure 3;

Fig. 7 is the vertical view of Fig. 6 lid;

Fig. 8 is the cross-sectional view of lid along Fig. 7 line 8-8.

The present invention relates to collect the oral immunisation globulin is used for immunity test and collects other material being used for test. Use a kind of pad to collect the sample with high concentration immunoglobulin (Ig) or other material. High level oral immunisation globulin is considered to the concentration that every milliliter of total Ig surpasses 50 μ g. Can use basic experimental technique to sample, this technology can be used as patient disease's testing tool or is used as the testing tool of some allosome material.

The representative molecule that the application of the invention has successfully been collected has:

Measured object Molecular weight

But ferrum peaceful 176

Glucose 180

Theophylline 180

Cocaine 303

B2M 11,818

Hbs antigen 24,000

β-human chorionic gonadotropin 37,900

IgG--people's antibody 150,000

Total IgG (unreceipted antigen)

HIV-1

Hepatitis A

Hepatitis B

Measles

The non-helical isoantigen of syphilis

IgA--people's antibody 160,000

Total IgA (unreceipted antigen)

IgM-people's antibody 950,000

Total IgM (unreceipted antigen)

Hepatitis A

Hepatitis B

For the degree that will minimize in the degraded in the sample of collecting, the antiseptic of can in the container of the storage pad after using hyperosmotic solution of the present invention, packing into.This antiseptic can suppress to destroy the activity of the proteolytic enzyme of antibody molecule.Chemical compound as antiseptic comprises antibacterial agent, antifungal, bacterial inhibitor, fungistat and enzyme inhibitor.Use benzoic acid at one preferably in the example, sorbic acid or their salt are as antifungal.With regard to bacterial inhibitor, the salt of high concentration is preferably with the chemical compound that hyperosmotic solution can be remained on low pH value.This salt comprises thiomersalate (or thimerosal), phenylmercuric acetate, phenylmercuric nitrate and sodium azide.Other preferably antiseptic comprise be usually used in medicine and mouth-wash in antiseptic.Its example comprises ethanol and gluconic acid hibitane.Another kind of antimicrobial preferably is the cleanser that can be used as the local bactericidal medicine or be used for mouth-wash.Its example is a benzalkonium chloride.The better consumption of this antiseptic is about 0.01-0.2 weight %.

In the present invention, use a pad from the oral cavity, to adsorb mucosa secretions.This pad is made by the adsorbing material that can insert effectively in the oral cavity.Can use a kind of plastics or carbohydrate materials (as cellulose) as adsorbing material, but be preferably a kind of thicker Cotton Gossypii paper that absorbability is arranged.A kind of example of the thicker Cotton Gossypii paper that absorbability is arranged is by being positioned at Keene, the #300 product that the Schleicher and Schuell of New Hampshire produces.The preferably non-foam shape of this pad or spongiform is although foam or sponge also can be used.

Because the non-specificity of the material of many preparation pads meeting ground is conjugated protein.Therefore, some immunoglobulin can be combined on the pad with not needing, needs to use the blocker blocks protein to be combined on the pad.Because blood itself contains blocker (being the human serum albumin), so when collecting blood sample, there is not the bonded problem of non-specificity usually.

Bonding in order in the collection of oral cavity sample, to reduce non-specificity, can in sneaking into the hyperosmotic solution of pad, add blocker.Normally a kind of soluble protein of blocker is used to prevent that the non-specificity of other albumen is bonded to the surface of solids.The chemical compound that can be used as the blocker interpolation comprises albumin and gelatin, but any water miscible avirulence albumen can both be used as blocker, as long as the non-confrontational body molecule of this albumen has a negative impact.Be preferably the use Bos taurus domesticus Gmelin.In general, blocker can be made into the solution that concentration is about 0.01-0.2 weight %.By dipping or spraying this solution is infiltrated pad subsequently, then drying.

For collecting oral substance, can pad be put into the oral cavity by means of support.Pad and support are as shown in drawings.Pad support 1 can be the hollow plastic stick that one one end has groove 2.Pad 3 inserts groove, utilizes support pad can be put into the oral cavity, preferably is put between gingiva bottom and the buccal.Pad is put in helps between buccal bottom and the gingiva bottom to adsorb from the secretions of gums lymphoid tissue and from the secretions of tela submucosa lymphoid tissue and salivary gland lymphoid tissue.Pad is rubbed between gingiva and buccal about 10 seconds back and forth, subsequently the position of pad between gingiva-buccal placed about 2 minutes kinds and collect sample.Should not chew, can stimulate unwanted salivation because chew to pad.

After collecting sample, pad is stored in the container until carrying out immunity test.A kind of container is illustrated in figures 1 and 2.Require container to have a centrifuge tube as the container exterior portion, the inside of container partly has to manage in one inserts in the centrifuge tube.Pad places interior pipe, and the article in the interior pipe are fixing by the pipe lid.

What Fig. 1 showed is the assembly that uses for the present invention through improving.Container 2 comprises the centrifuge tube 4 with conical lower end or bottom 5, and bottom 5 has downward conical deep hole 6, gathers the solid matter after centrifugal in this deep hole; Also comprise the upper pipes or container 7 and pipe close or the stopper 11 that have radially outwards outstanding ring-shaped edge 8 and be positioned at the cylindrical upper part 9 of its upper end.Cylindrical part 9 is identical with the upper part of centrifuge tube 4 with pipe close 11 acquisition size and dimensions, make it to align with the outer surface of centrifuge tube and make assembly have well-balanced outward appearance, but these features is not important to the present invention's practice.Be hole 17 in the end 13 of container 7 bottoms, when whole assembly being carried out when centrifugal, liquid flows to centrifuge tube 4 by this hole from container 7.Container 7 is made by any suitable material (as polyethylene, glass etc.).Equally, pipe close 11 can be made by any suitable material, as known in the art polyethylene.

A movably stopple 17 is arranged in hole 17.Stopple can be made by any suitable material (as paraffin, plastics etc.).In container 7, be placed with an amount of listerine 21.

As long as pad is put into pipe, just in pipe, add listerine 21.This listerine plays inhibitory enzyme activity (this kind of enzyme can destroy antibody molecule) effect or as antimicrobial.

The chemical compound that is used for interior pipe as antiseptic comprises antibacterial agent, antifungal, bacterial inhibitor, fungistat and enzyme inhibitor.With regard to antibacterial agent, preferably use gluconic acid hibitane or thimerosal.

The listerine that uses in interior pipe can contain one or more antiseptic.In general, the concentration of preservatives that is contained should be able to restriction micro-organisms be polluted and the absorption of immunoglobulin in the pad is not had a negative impact.

The listerine that uses in interior pipe also can contain a kind of cleanser, and it can improve the separation of antibody from the pad in centrifugal process.Tween20 (polyoxyethylene sorbitan monoleate) is a cleanser preferably, because it can also prevent the non-specificity combination of antibody at the surface of solids.Be preferably and use the mixture that comprises about 0.01-0.2% gluconic acid hibitane and 0.2-0.7%Tween 20.Preferably use the mixture that comprises about 0.1% gluconic acid hibitane and 0.5%Tween 20.

Behind pipe in the listerine adding, will manage lid and insert container with the sealing inclusions.Pad can be stored several days until beginning to carry out immunity test in this way.

In this example, use pad 3 on support 1 as mentioned above.After pad 3 taken out from the user oral cavity, from container 7, remove pipe close 11 and pad is put into container 7.In the outside of container 7 openings support 1 is fractureed and to make it to protrude upward container.Put back to pipe close 11 subsequently.Because pipe close 11 is hollow, it can make the fracture end of support 1 stretch into pipe close and sealed container 7 securely.Preferably the suitable position of support 1 being carried out indentation makes it to fracture easily.When pad 3 was inserted container 7, it is absorbed portion listerine 21 at least.

Pad 3 is stored in the container 7 until beginning tests.In laboratory, the container 7 that has pipe close 11 securely is inverted, remove the stopple of making by paraffin or other suitable material 19, and container 7 is put into centrifuge tube 4.Centrifugal subsequently a whole set of assembly 2 passes through hole 17 suction centrifuge tubes 4 with all liq (comprising listerine, saliva etc.).Then test with known technology.

Use the pad collection system to collect and analysis oral cavity sample for simplifying, can use a kind of kit.This kit comprises pad and is used to collect and prepares the compositions of analyzing with the kit of oral cavity sample.A better example of kit comprises treated pad 3 and pad support 1; Have the container 7 of pipe close 11 and store antiseptic 21.Also can randomly comprise centrifuge tube 4.

The container that another store collected material of the present invention is used for testing subsequently comprises that one is suitable for removable pipe close sealed opening upper end and one and has the bottom that makes the hole that is interlinked to the outside in the container, this hole is chosen as fragile papillae and seals when stored substance, and perhaps blow-by is used for testing subsequently to take out the material of collecting.Comfortable and safe for user, fragile papilliferous end is unfolded, and is preferably globular.The upper end of opening the most handy threaded " stopple sealing " lid sealing.In addition, the silk screen that the container bottom that tilts to medium pore has many settings is to prevent front No.641, and the described pad of 739 patent applications is shelved on the bottom of container and prevents the obstruction of base apertures.

Select the size of container to make it to insert 15ml standard taper centrifuge tube.Thereby can the inclusion in the bottle is centrifugal to centrifuge tube after papillae fractures.

The anticorrosion liquid of certain volume (about 0.5-2.0ml) will be housed in the container.When pad is put into container, impregnating fluid is inhaled into this pad.The water transmitance because will storing the volume of the impregnating fluid up to a year, significantly do not reduce by the container of band impregnating fluid, so must be low.Container is made by any suitable plastic (as polyethylene, Merlon, polystyrene, PET (polyethylene terephthalate), polypropylene, EPC (ethylene-propylene copolymer) etc.).Since Merlon over-drastic " breathing ", though the moisture escape, so it is the poorest.Material is a polypropylene preferably.

Referring to Fig. 3 and 4, the container of this example (being designated as numeral 10) comprises elongated body part 12.Although body part 12 15 is preferably had a taper slightly from the upper end to the bottom, it is normally cylindrical.Upper end 16 be open wide and to have a screw thread 18 adaptive with lid.

Papillae 20 stretches out bottom 15 downwards.Bottom 15 is preferably tilted with angle [alpha] a little by export-oriented central authorities.Preferably about 20 ° of angle [alpha].15 inboard central authorities are depressions 22 in the bottom, it preferably " V " shape and be preferably formed as about 88.5 ° angle.Depression 22 becomes fragile the root of papillae 20, thereby it is fractureed easily when applying enough pressure.End at papillae 20 forms ball 24, so that the easier and fragile papillae 20 that more safely fractures.Shelve in the hole that bottom 15 produces by papillae 20 in order to prevent to insert pad in the container 10, the silk screen 26 of many settings is placed on the bottom 15.Be preferably and place 4 layers of silk screen 26.

Present let us Fig. 5,6 and 7, shown is the lid (with digital 28 labellings) that is used for sealed container 10 upper ends 16 openings.The shape of lid 28 often is called " stopple sealing " lid.The screw thread 18 that lid 28 has on female thread 30 and the container is complementary.It is the ring zone 34 on boundary that ring depression 32 produces the inboard of mixing outer wall 36 with ring depression 32.Thereby make chamber wall and ring zone 34 adaptive and generation better seal.Be that protuberance shown in 38 is so that the removing and replace of lid on lid 28 outer surface.

The using method of container 10 is same as top using method with reference to the described container of Fig. 1 and 2.Following embodiment shows the effectiveness of pad of the present invention.

Embodiment 1

The comparison of IgG in saliva and the mucosa transudate

The purpose of this research is the IgG level in comparison saliva and the mucosa transudate.

From 10 experimental subjecies, collect the oral cavity sample.By chewing " adsorbate " (Sarstedt Salivette ^TMThe part of molectron, with Cotton swab as adsorbate) collect saliva by saliva is saturated from experimental subject until Cotton swab.After about subsequently 4 hours, collect the mucosa transudate from experimental subject by collecting mouth cavity liquid with WO 91/13355 described processing pad.After collecting saliva or transudate, centrifugal is gone out Cotton swab and handles pad.Analyze the existing IgG antibody of oral cavity sample with enzyme immunoassay (EIA).

Table 1 has been listed Cotton swab and has been handled the IgG collection level of pad.

Table 1

The experimental subject numbering	Saliva IgG (μ g/ml)	Transudate IgG (μ g/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

By chewing Sarstedt Salivette ^TMThe IgG antibody content of the saliva sample that " adsorbate " of molectron collected hangs down about 25 times than the IgG antibody content of the sample that the processing pad of WO 91/13355 is collected.

Embodiment 2

Carry out the be untreated comparative test of pad of the processing pad of WO 91/13355 and the present invention.

Use 4 pads to collect the oral cavity sample to each trial volunteer, wherein handle for two, two is untreated.Each collection (two same pads are collected simultaneously) continues 2 minutes.Collect the back first also carried out collecting the second time with the oral cavity that tap water cleans the experimenter at least at interval in 1.5 hours.Experimenter in this research is divided into two groups.The half experimenter uses two untreated pads first, uses two to handle pad collection oral cavity sample subsequently, and other experimenter uses two to handle pads earlier, uses untreated pad to collect subsequently.

After the collection, the experimenter inserts pad in the empty bottle, and this bottle indicated specimen coding and collected the type (processing or untreated) of using pad.The carriage bar that fractures is also covered tight bottle.Make sample remain on 4 ℃ before centrifugal or in the centrifugal process.After centrifugal, merge the oral cavity sample of from each experimenter, collecting with two identical pads.Sample storage is at 4 ℃.

Use Epitope, the EIA that Inc. produces analyzes each IgG total amount that merges sample to determine the amount of IgG in each sample.

Table 2

The study subject numbering	IgG(μg/ml)
				Pad is untreated	Handle pad
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

^*: at first collect with untreated pad

By the analysis of inner (in-house) IgG characteristic enzyme immunoassay (EIA) (EIA) method as can be known, contain higher immunoglobulin G (IgG) level by the sample of handling the pad collection than the sample of collecting by the pad that is untreated.

By the analysis of WO 91/13355 pad gleanings and Sarstedt " adsorbate " gleanings as can be known, pad is from the mucosa transudate but not collect IgG the saliva, and difference wherein is tangible.

By the processing pad gleanings of WO 91/13355 and the present invention be untreated the pad gleanings analysis as can be known, the IgG that pad is collected although be untreated is less than the pad of processing, the amount (5.3 μ g/ml are to 2.4 μ g/ml) that the IgG that the pad that is untreated is collected still obviously collects from saliva more than hope.

Claims (16)

1. a method of collecting the mucosa transudate that is used to measure from the oral cavity according to qualifications comprises the steps:

(a) adsorption gasket is inserted the oral cavity, wherein, described adsorption gasket does not immerse in the hypertonic salt solution;

(b) pad is contacted with oral mucosa and do not tear described pad;

(c) pad is shifted out from the oral cavity.

2. the method for claim 1 is characterized in that after pad takes out from the oral cavity this pad being kept in the container.

3. method as claimed in claim 2 is characterized in that containing in the container listerine.

4. method as claimed in claim 3 is characterized in that listerine comprises gluconic acid hibitane or thimerosal.

5. method as claimed in claim 4 is characterized in that listerine comprises the gluconic acid hibitane.

6. method as claimed in claim 2, it is characterized in that container comprises the open upper end by removable sealing of lid, and lower end with the inside and outside hole of connection container, sealing is selected in this hole in preserving described pad process, and selects Kaifeng when taking out described collection mucosa transudate and being used for measuring subsequently.

7. method as claimed in claim 6 is characterized in that this hole is sealed by the papillae selectivity of fragility.

8. the method for claim 1 is characterized in that it is 176-950 that described mucosa transudate contains molecular weight, 000 measured object.

9. method as claimed in claim 8, but it is characterized in that described measured object is selected from that ferrum is peaceful, glucose, theophylline, cocaine, B2M, hbs antigen, β-human chorionic gonadotropin, immunoglobulin and composition thereof.

10. method as claimed in claim 9 is characterized in that described measured object is an immunoglobulin.

11. method as claimed in claim 10 is characterized in that described immunoglobulin is selected from IgG, IgA and IgM.

12. method as claimed in claim 10 is characterized in that described immunoglobulin is at least a antibody in the non-helical isoantigen of HIV-1, hepatitis A, hepatitis B, measles and syphilis.

13. the method for claim 1 is characterized in that described analysis is immunoassay.

14. method as claimed in claim 13 is characterized in that described immunoassay are that ELISA measures.

15. the method for claim 1 is characterized in that described pad made by carbohydrate materials.

16. the method for claim 1 is characterized in that, described method comprises that also (d) is kept at described pad in the hyperosmotic solution, is used for measuring so that take out the mucosa transudate of collecting subsequently from pad.

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