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
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
  • G01N33/56922—Campylobacter

Definitions

• This invention relates to a method of treating saliva.
• it relates to a method of treating saliva for use in a diagnostic test for the presence of antigens to H. pylori .
• Saliva is a complex mixture of secretions originating from various glands within the oral cavity. Among its many constituents are various enzymes, immunoglobulins and mucins, together with cellular debris and a host of bacterial flora.
• the mucins are responsible for the viscoelastic properties of saliva, and act as lubricants with protective and antibacterial properties.
• the viscoelastic properties of these glycoproteins depend on the protein and carbohydrate composition acquired during synthesi's, but are also influenced by the extent of their interaction with other components of saliva. For example, lipids can also contribute significantly to the viscoelastic properties of saliva.
• Saliva which contains immunoglobulins can be used for the diagnosis of various diseases.
• the detection of IgG specific to antigens produced by pathogens is indicative of infection or recent contact.
• One example of such an infection is that caused by H. pylori .
• the diagnosis of H. pylori infection can be made by microscopy, microbiological culture or urease detection in gastric mucosal biopsies, urea breath test or by the presence of specific antibodies in serum detected by ELISAs.
• H. pylori infects the gastric mucosa and would be expected to elicit an IgA antibody response that could be detected in mucous in gastric secretions.
• the predominant H is the predominant H.
• pylori -specific antibody found in mucous secretions is IgG class and not IgA.
• AU-A-9067676 describes a method for the detection of IgG in mucous secretion specific to H. pylori antigen, thereby providing a means of monitoring infection by that microorganism in mammals.
• the corresponding academic publication is Witt et al , Frontiers in Mucosal Immunology 1 693-696 (1991) .
• WO-A-9322682 discloses a convenient and reliable in vi tro test for H. pylori . This test utilises an antigen preparation that reacts with. IgG antibody in a mucous secretion from a mammal being tested.
• Particulates which are present in saliva can bind to the membrane, by simple deposition on the surface and reduce flow times.
• the mucins have viscoelastic properties and this can result in a gel-like matrix being deposited on the membrane which will reduce liquid flow.
• the flow- through time of the saliva must be a balance between the need for accurate binding of IgG to antigen, which means that the flow-through time must not be too fast, and the need for the test to be practical, i.e. the flow-through time must not be too slow.
• the binding of particulates and mucins to the surface of any membrane or pad to which the antigen is bound can lead to greatly reduced flow- through time.
• a further problem associated with deposition of mucins and particulates on the membrane is related to signal development.
• One preferred method of signal development utilises IgG-Colloidal Gold conjugates which can be trapped by the deposits. This, can lead to false positive results, and in a worst case, where no conjugate can pass freely, the whole membrane can bind conjugate.
• saliva can be processed using a system known as Omnisal ® in which saliva is collected onto a cotton pad then extracted in a buffer and filtered through a porous plastic filter.
• Omnisal ® a system known as Omnisal ® in which saliva is collected onto a cotton pad then extracted in a buffer and filtered through a porous plastic filter.
• the liquid so produced can still have poor flow characteristics.
• the present invention provides a method of improving the flow characteristics of saliva, which comprises the step of altering the properties of the saliva sample.
• the invention relates to altering the viscoelastic properties of the saliva.
• the properties of the saliva are altered by passing the saliva through a filtration medium which achieves this effect.
• Preferred filtration media include charcoal, DEAE-sepharose and diatomaceous earth, e.g. Celite ® , with diatomaceous earth being particularly preferred.
• the filtration medium removes particulates. Mechanical shear, removal of high molecular weight aggregates and lipids may disrupt the polymeric structure of mucins, leading to improved flow characteristics.
• Mucins present are not removed by the method, however, their polymeric structure may be altered, thus improving flow characteristics.
• the properties of the saliva sample can be manipulated by the addition of detergents and adjusting the ionic strength and/or pH of the saliva sample. This can be achieved by passing the saliva sample into a suitable buffer solution.
• a suitable buffer solution e.g., a suitable buffer solution
• the saliva sample is not only passed through a filtration medium which alters the properties of the saliva, but also has its pH and/or ionic strength adjusted and detergent added by means of passing into a suitable buffer solution.
• the present invention finds particular application in rapid diagnostic tests for specific antibodies found in saliva. Therefore, in a further aspect, the present invention provides a method for the detection of an antibody in a sample of saliva which comprises the step of altering the properties, e.g. the viscoelastic properties, of the saliva to improve flow characteristics. As described herein, this can be achieved by means of filtration and/or buffer manipulation.
• the test is for the detection of H. pylori specific antibody, usually IgG.
• the present invention provides a kit for the detection of an antibody in a sample of saliva which comprises a means for altering the properties, e.g. the viscoelastic properties, of the saliva sample, those means being provided in the form of a filtration material and/or a buffer solution, either or both of which have the ability to alter the flow properties of the saliva sample before application to the test area.
• a means for altering the properties e.g. the viscoelastic properties
• Fi ⁇ ure 1 shows saliva flow times with or without a Celite ® filtration step
• Fi ⁇ ure 2 shows IgG Gold conjugate flow times with or without a Celite ® filtration step.
• Saliva samples were collected from a number of individuals using the Omnisal ® (Saliva Diagnostic Systems, Inc., 11719 NE 95th Street, Vancouver, WA 98682, USA) saliva collection and processing system. However, the saliva was extracted into the following buffer: 20 mM Phosphate pH 7.0-8.0 containing 0.5% v/v Tween 60, 125 mM NaCl, 10 mM Sodium azide and 0.1% w/w BSA.
• the saliva was either extracted into the buffer by filtering through the Omnisal ® filter or through Celite ® . 1 ml of the resulting solutions were then placed onto a Helisal ® rapid test card, which contains membrane spotted with antigen, and the flow through times recorded. After saliva, 1 ml of IgG-gold conjugate was added and the flow through time also recorded. The results are shown below.
• FIGS. 1 and 2 are a representation of the above data graphically. The following points are clear:
• Samples processed as described above also show a significant reduction in background colour due to trapping of IgG-Gold, and the number of false positives is greatly reduced.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Immunology (AREA)
• Engineering & Computer Science (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Biochemistry (AREA)
• Cell Biology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Biotechnology (AREA)
• Food Science & Technology (AREA)
• Virology (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Microbiology (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Peptides Or Proteins (AREA)
• Sampling And Sample Adjustment (AREA)
• Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)

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Citations (6)

• 1994
  • 1994-11-15 GB GB9422991A patent/GB9422991D0/en active Pending
• 1995
  • 1995-11-14 AU AU38529/95A patent/AU3852995A/en not_active Abandoned
  • 1995-11-14 MX MX9703563A patent/MX9703563A/es unknown
  • 1995-11-14 CA CA002205088A patent/CA2205088A1/en not_active Abandoned
  • 1995-11-14 WO PCT/GB1995/002668 patent/WO1996015455A1/en not_active Application Discontinuation
  • 1995-11-14 EP EP95936683A patent/EP0792459A1/en not_active Withdrawn
  • 1995-11-14 JP JP8511169A patent/JPH10508689A/ja active Pending
  • 1995-11-14 BR BR9509677A patent/BR9509677A/pt not_active Application Discontinuation
  • 1995-11-15 ZA ZA959731A patent/ZA959731B/xx unknown
  • 1995-11-15 IL IL11600995A patent/IL116009A0/xx unknown
  • 1995-12-06 TW TW084112976A patent/TW340183B/zh active
• 1997
  • 1997-05-14 FI FI972055A patent/FI972055A0/sv not_active Application Discontinuation
  • 1997-05-14 NO NO972221A patent/NO972221L/no unknown

Patent Citations (6)

Non-Patent Citations (2)

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