WO1998038512A1 - Collection, stabilization and optional storage of samples of body fluids, faeces and secretions for analyses of antibodies - Google Patents

Collection, stabilization and optional storage of samples of body fluids, faeces and secretions for analyses of antibodies Download PDF

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Publication number
WO1998038512A1
WO1998038512A1 PCT/NO1998/000061 NO9800061W WO9838512A1 WO 1998038512 A1 WO1998038512 A1 WO 1998038512A1 NO 9800061 W NO9800061 W NO 9800061W WO 9838512 A1 WO9838512 A1 WO 9838512A1
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WO
WIPO (PCT)
Prior art keywords
antibodies
faeces
absorbent material
specimen
secretions
Prior art date
Application number
PCT/NO1998/000061
Other languages
French (fr)
Norwegian (no)
Inventor
Björn HANEBERG
Original Assignee
M. Peterson & Søn As
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by M. Peterson & Søn As filed Critical M. Peterson & Søn As
Priority to AU61249/98A priority Critical patent/AU6124998A/en
Publication of WO1998038512A1 publication Critical patent/WO1998038512A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L99/00—Subject matter not provided for in other groups of this subclass
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • A61B10/0096—Casings for storing test samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/10—Devices for withdrawing samples in the liquid or fluent state
    • G01N1/14—Suction devices, e.g. pumps; Ejector devices
    • G01N2001/1472—Devices not actuated by pressure difference
    • G01N2001/149—Capillaries; Sponges
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G01N2001/2826—Collecting by adsorption or absorption

Definitions

  • the present invention relates to a method for taking, stabilizing and if necessary storing specimens of body fluids, faeces and secretions for analysing antibodies.
  • 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.
  • 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.
  • WO 90/13819 and HEMO-FEC TEST relate to a device and method for collecting specimens from faeces for determining blood contained therein.
  • mucus specimens including faeces and urine
  • absorbent material 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.
  • 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 conditions 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.
  • 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.
  • OMV outer membrane vesicle
  • 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.
  • CT cholera toxin

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Urology & Nephrology (AREA)
  • Clinical Laboratory Science (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention relates to a method of taking, stabilizing and possibly storing of samples of body fluids, faeces and secretions, for analysis of antibodies, said method comprising the steps of placing the sample on the surface of an absorbent material having little ability to bind protein, drying thereof, whereby the stability of antibodies is maintained, isolation by extraction of stabilized antibody contained therein, and subsequent analysis of antibodies. Moreover, the invention relates to the use of an absorbent material suitable for usage in the above method.

Description

Collection, stabilization and optional storage of samples of body fluids, faeces and secretions for analyses of antibodies .
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 (37oc) . 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 conditions 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 37°C. 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 -70°C 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.

Claims

1. A method for taking, stabilizing and optionally storing specimens of body fluids, faeces and secretions for analysing antibodies, c h a r a c t e r i z e d 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.
2. The method of claim 1, c h a r a c t e r i z e d i n that the absorbent material is selected from materials having mechanical strength and high absorbency .
3. The method of claims 1 or 2 , c h a r a c t e r i z e d i n that a certain volume of the specimen placed on the surface of the absorbent material, is absorbed thereby.
4. The method of any of claims 1 to 3 , c h a r a c t e r i z e d i n that the drying of the material takes place at ambient temperature.
5. The method of any of claims 1 to 4 , c h a r a c t e r i z e d i n that, after the drying stage the absorbent material and the specimen contained therein are stored at ambient temperature for a desired period of time, for example up to several months .
6. The method of any of claims 1 to 5, c h a r a c t e r i z e d i n that, after being isolated, the antibodies are analysed by conventional immunological techniques .
7. The method of any of claims 1 to 6, c h a r a c t e r i z e d i n that the specimens are selected from the group of saliva, mucus such as oral mucus, faeces and urine.
8. 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 contains an inserted specimen of body fluids, faeces and secretions, in dried form, for isolation, by extraction, of antibodies in secretions contained in the specimen.
9. The use of claim 8, wherein the absorbent material is selected from materials having mechanical strength and high absorbency.
10. The use of claims 8 or 9 , wherein the specimen is selected from the group of saliva, mucus such as oral mucus, faeces and urine.
PCT/NO1998/000061 1997-02-26 1998-02-26 Collection, stabilization and optional storage of samples of body fluids, faeces and secretions for analyses of antibodies WO1998038512A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU61249/98A AU6124998A (en) 1997-02-26 1998-02-26 Collection, stabilization and optional storage of samples of body fluids, faecesand secretions for analyses of antibodies

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NO970858A NO970858D0 (en) 1997-02-26 1997-02-26 Procedure for taking and storing samples from body fluids / secretions, faeces and the like
NO970858 1997-02-26

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988010272A1 (en) * 1987-06-26 1988-12-29 Garth Alexander Nicholson Method of preserving a body fluid sample, prior to assay for hiv (aids) infection
WO1990013819A1 (en) * 1989-05-09 1990-11-15 Beckman Instruments, Inc. Device and method for collecting fecal occult blood specimens
EP0418739A1 (en) * 1989-09-21 1991-03-27 Epitope, Inc. Oral immunoglobulin collection for immunoassay
EP0750185A2 (en) * 1995-06-24 1996-12-27 Roche Diagnostics GmbH Element and system for collecting, transporting and saving analytical samples

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988010272A1 (en) * 1987-06-26 1988-12-29 Garth Alexander Nicholson Method of preserving a body fluid sample, prior to assay for hiv (aids) infection
WO1990013819A1 (en) * 1989-05-09 1990-11-15 Beckman Instruments, Inc. Device and method for collecting fecal occult blood specimens
EP0418739A1 (en) * 1989-09-21 1991-03-27 Epitope, Inc. Oral immunoglobulin collection for immunoassay
EP0750185A2 (en) * 1995-06-24 1996-12-27 Roche Diagnostics GmbH Element and system for collecting, transporting and saving analytical samples

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
C. HAMBLIN et al., "Blood Dried on Filter or Blotting Paper for the Detection of Antibody Against Swine Vesicular Disease Virus by Enzyme-Linked Immunosorbent Assay", VETERINARY RECORD, Volume 111, November 1982, pages 460-461. *
S. TSAO et al., "Two-Site Enzyme Immunoassay for Alpha-Fetoprotein in Dried-Blood Samples Collected on Filter Paper", pages 2079-2082. *

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AU6124998A (en) 1998-09-18
NO970858D0 (en) 1997-02-26

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