The present invention relates to a method for taking, stabilizing and if necessary storing specimens of body fluids, faeces and secretions for analysing antibodies.

Furthermore the present invention relates to the use of an absorbent material having little or no ability to bind proteins and that is designed for breaking into pieces and is suitable for use in the said method.

Body mucosa have an enormous surface that serves for exchange of gases, taking up nutrients and excreting waste matter.

Therefore they are vulnerable, which is reflected by the fact that these mucous membranes are the point of entry for nearly all infectious agents. Our immune responses against infections are so designed that the reaction to such infections will be to form 1) antibodies and special blood cells that circulate with the blood and reach all parts of the body except for the surface of the mucous membranes and 2) special antibodies that are produced locally in the mucosa and are excreted on the surface. These locally produced antibodies will mix with the mucus on the surface and serve as a protective coating, that is to say, as a barrier against subsequent infection.

Up to the present studies of infections and the effect of vaccines have largely been confined to examining serum, blood plasma and blood cells. However scientists have for some years been working on analysis of antibodies in mucus specimens from various mucous membranes. It has been found that such specimens may be useful in making a diagnosis, for example in the case of infections, and for measuring and characterizing the effect of new types of vaccines that are intended for use direct on the mucosa, either as nasal drops/sprays or as a solution to be taken orally. However progress has been hampered by the lack of practical facilities for taking and storing specimens of mucus so that

they are suitable for later analysis of locally produced antibodies.

The following can be mentioned as examples of mucus specimens for use in the method according to the invention: From respiratory passages specimens of mucus can be taken from the 1) surface of nasal mucosa after administering saline as a nasal spray/douche 2) oral cavity as oral mucus or saliva.

From intestinal surfaces specimens can be taken from 3) faeces.

From female genitals specimens can be taken from 4) cervix uteri (neck of the uterus) 5) vagina From the urinary tract (the renal pelvis, ureters and bladder) specimens can be taken from 6) urine.

It is shown that all of the above mentioned mucus specimens may contain antibodies. Special techniques have previously been developed for taking such mucus specimens, except for faeces, by using a special absorbent material to collect the mucus and freezing the whole. The antibodies can then be extracted by buffered saline with a view to analysing the concentration/quantity and characteristic properties. A condition for this is that this material is capable of binding much fluid, but very little of the proteins that are to be examined.

Antibodies have also been found in faeces that have been frozen. Moreover the present inventor has published studies showing that it is an advantage to extract antibodies from faeces after freeze drying rather than from "moist faeces.

Here reference is made to B. Haneberg, Scandinavian Journal of Immunology, 1974, Vol.3, No.1, pp. 71-76; B.Haneberg, Scandinavian Journal of Immunology, 1974, Vol.3, No.2, pp.

191-197; and K. Hordnes et al., Journal of Reproductive Immunology, 1995, Vol. 28, No.3, pp. 247-262.

WO 90/13819 and HEMO-FEC TEST relate to a device and method for collecting specimens from faeces for determining blood contained therein.

For this invention it is shown that mucus specimens, including faeces and urine, can be placed on absorbent material and dried at ambient temperature without the antibodies being destroyed. They can also be stored at this temperature for a desired period of time, for example several months, without the antibodies being destroyed. Some reduction in the concentration of antibodies in faeces was observed (55% after storage for four months), but this has little material significance since the antibody concen- trations will still be well within the mesurable range.

Similar results are also obtained after drying and storing at normal body temperature (37or). This shows that antibodies of this type break down very little before being collected. This means that it is not critical to take the mucus specimen immediately after the secretion thereof.

This technique will also be useful in countries with high air temperatures, for example in tropical regions where keeping such specimens chilled can be difficult.

The stability of the antibodies under the described con- ditions is explained by the fact that the antibodies are of quite a special type that are found in major concentrations in secretions and that breaking down by for example proteolytic enzymes ceases/is inhibited in the dry state.

However, the finds in connection with faeces were the most surprising, as it was unexpected that the antibodies found in

faeces would be capable of withstanding the large quantities of enzymes therein that are intended to break down all kinds of foodstuffs.

The present invention relates to a method for taking, stabilizing and optionally storing specimens of body fluids, faeces and secretions for analysing antibodies, a method that is characterized by the steps of - placing the specimen on the surface of an absorbent material that has little ability to bind proteins - drying thereof, whereby the stability of antibodies is maintained - isolating by extracting the stabilized antibodies contained therein, and - subsequent analysis of the antibodies.

The present invention further relates to the use of an absorbent material having little or no ability to bind proteins and that is designed for breaking into pieces, in that the material holds a specimen of body fluids, faeces and secretions therein, in a dried state, for isolating by extraction the antibodies in the secretions contained in the specimen.

These specimens are placed on a piece of absorbent material that has a further defined volume which permits collection of a desired amount of the specimen. The specimens can either be dripped on to the absorbent material (for example, nasal secretion, urine) or the absorbent material can be laid on the mucous membrane (for example, for collecting mucus or saliva from the mouth, or mucus from cervix uteri or vagina).

Faeces can be smeared on the surface of the absorbent material using for example a cardboard spatula, so that the moisture penetrates into the material, or loose faeces can be dripped on to or sucked up by the absorbent material.

The absorbent material must have a large surface that permits drying the secretion at ambient temperature for a sufficient

time to maintain stability of the antibodies or other material to be analysed, for example 24 hours.

The requirements imposed for the absorbent material are that it must have a certain mechanical strength, high absorbency and little tendency to bind protein.

After drying the specimens can be stored in the dry state for a long time without refrigeration, for example up to four months at temperatures up to 370C. The period of storage can probably be longer.

The absorbent material must be designed so that all or parts of it are suitable for inserting in tubes with extraction buffer. Since any enzymes will become active in an aqueous solution, protease inhibitors may be added to the solution.

Extraction of antibodies is done by shaking/motion of the tube followed by centrifugating and the extract with the antibodies can then be transferred by e.g. pipette or by straining off into another tube during centrifugating. Like other moist biological material the extracts should be stored in refrigerated state before analysis and characterization by means of conventional immunological techniques.

Examples Saliva was collected from volunteers who had been immunized intranasally with an outer membrane vesicle (OMV) group B meningococcal vaccine, and faeces from other volunteers after immunisation with an oral vaccine against cholera. Portions of saliva and faeces specimens were air-dried and stored for up to six months at room temperature before extraction and analysis by ELISA. The results were compared with those obtained after extraction of specimens stored in the frozen state. After storage for four months at room temperature the concentration of IgA antibodies to OMV in saliva was not reduced, but the concentration of IgA antibodies against cholera toxin (CT) in faeces was reduced by 55%, as compared

with antibody concentrations in corresponding specimens stored in the frozen state.

In a separate test faecal specimens were collected before and after vaccination against cholera by smearing a thin layer on absorbent material ("'MucoSafe", Peterson Sarpsborg AS, Skjeberg, Norway), followed by air-drying and storage at room temperature. The quantity of antibodies to CT in these specimens correlated significantly with anti-CT IgA responses in faeces stored at -700C immediately after defecation (P = 0.0001) and with the number of circulating IgA antibody secreting cells (P = 0.002). Thus air-drying and storage of faeces at room temperature can facilitate the measurement of mucosal immune responses.

Another test showed that antibodies in freeze-dried specimens of faeces correlate with air-dried specimens, with the following results: R = 0.99 and P = 0.0001.