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
  • G01N33/56983—Viruses
• • 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
  • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

• the present invention relates to a method for isolating and/or identifying foreign antigens or nucleic acids associated with leucocytes in fluids derived from a human or animal body, and to kits for carrying this out. More particularly to such a method and kits as specifically adapted to be capable of use 'in the field' in so far as an electric power supply means is not essential to their operation.
• the method and apparatus of the present invention enable antigens to be derived from a human or animal body and identified without the need for complex laboratory bound equipment.
• Such method has application, inter alia, in field testing for infection of humans and animals with various infective agents having known antigenic or nucleic acid characteristics. Such testing may be carried out by a physician or veterinarian in their surgery rather than in a laboratory.
• the processing and therapeutic benefits of using these filters are numerous, but include, inter alia, reduction in platelet refractoriness, prevention of transmission of viruses such as cytomegalovirus, prevention of non-haemolytic febrile transfusion reactions and reduction of transfusion associated thrombocytopenia.
• Direct use to filter a patients blood is helpful in allowing more aggressive anticancer therapy and application to donor tissue fluids and perfusates extends organ preservation time for transplantation. It is known that leucocytes may be analysed to determine the identity of antigens which they have immobilised in the course of their physiological function.
• Fenton et al (1991) described an antigen detection test for diagnosis of Border disease in sheep using leucocytes as a source of viral antigen while Mignon et al (1991) described a similar test for cattle using a monoclonal antibody for capture and signalling of presence of antigen.
• Such methods have focused upon analysis of leucocyte 'buffy coat' derived by methods using flash lysis and centrifugation, buffy coat separation or gradient centrifugation either alone or in combination. It will be immediately recognised that these methods require laboratory equipment that is not available in a surgery or on a farm.
• the present inventors have now provided a novel application for leucocyte retention filters in the form of a method and apparatus for isolating and/or identifying foreign antigens and/or nucleic acids associated with leucocytes in fluids derived from a human or animal body.
• the method and apparatus provides a simple to obtain yet rapid determination of foreign antigens and/or nucleic acids associated with the leucocytes, and thus of the likely presence of organisms of which these antigens are characteristic.
• One significant advantage of this method is that it offers the preparation of a sample outside the laboratory Tjsing a few reagents, a syringe and a filter. Using this method the inventors have assembled a pen-side test that can detect animals persistently infected with BVD virus that takes only 1.5 hours, compared to the 7 days required for a virus isolation test.
• the present invention provides a method for identifying foreign antigens and/or nucleic acids associated with leucocytes in fluids derived from a human or animal body, and/or isolating them in cell free form, comprising
• step (d) using eluant as foreign antigen and/or nucleic acid source may be carried out using standard methods well known to those skilled in the art, as will be exemplified further below.
• Step (a) is conveniently carried out using any of the commercially available leucocyte retaining filter materials or filter units. These are normally comprised of many layers and trap leucocytes by both size exclusion and adsorption with those capable of retaining greater than 99•9997 * of leucocytes being readily available. Suitable examples are available from Bellhouse Biosciences Ltd (Abingdon-UK) , Biotest (UK) Ltd (Shirley, W. Midlands (UK)), Kimal Scientific Products Ltd (Uxbridge-UK) and Pall Biomedical Ltd (Portsmouth-UK) . Some of these are capable of retaining platelets as well as leucocytes but this should not affect the method, particularly the identification step.
• the fluid to be filtered may be any bodily fluid or its derivative.
• filter material or filters eg. such as those sold by Pall Process Filtration Ltd under the trademark 'Leukosorb L '.
• This material is available in sheet form but is particularly conveniently available as 25mm disks in encapsulated form with a syringe-end fitting. The use of such encapsulated form allows facile use of the filter with a Leur syringe whereby use outside a laboratory becomes rapid and convenient.
• an anti-clotting agent is added in an effective amount prior to subjecting the mixture to the filtration step.
• This agent is conveniently that such as EDTA, Na-citrate or heparin, but may be any of the other well known anti-clotting agents that will not significantly damage leucocytes.
• Step (b) is carried out using any agent capable of acting upon whole leucocytes to provide the foreign antigens and/or nucleic acids in an elutable form suitable for the intended end use and is conveniently carried out after the filter has been rinsed through with a rinse liquid, eg. water, saline or buffer, eg. phosphate buffered saline.
• the agent is preferably a detergent and most preferably is a detergent which is capable of providing the foreign antigens associated with the leucocytes in a form whereby they are provided on the surface of micelles in the eluant.
• any detergent capable of liberating the required elutable forms from the leucocytes, and preferably acting upon the cell membranes to solubilise them, may be used but preferably non-ionic detergents will be selected.
• detergents which are known to interfere with step (d) they may be removed prior to such step using adsorbents such as Bio-beads (available from Bio-Rad) .
• a suitable range of detergents are available from suppliers such as Sigma and include the glucopyranoside range of non-ionic detergents and Nonidet P-4 ⁇ .
• Use of an optimal micelle forming amount of Nonidet takes about 1 hour contact with the filter to achieve a suitable solubilising effect.
• Use of preferred detergent eg. n-octyl- ⁇ -D-glucopyranoside, achieves suitable solubilisation in about 15 minutes.
• the concentration of the detergent needs to be optimised if the desired optimal micellular formation is to be achieved.
• the optimal concentration is about a 2% solution, eg. in water or commercial preparation, but other suitable concentrations for this and other detergents will be derivable by those skilled in the art by simple bench experimentation using the optimised system as a measure of what can be achieved.
• the step (b) is conveniently carried out by adding the detergent to the filter, eg. by passing it onto it and through its thickness, and leaving the two in contact for a period suitable for transformation of the foreign antigens and/or leucocytes to elutable form, preferably by solubilisation of the leucocytes.
• an encapsulated filter such as the Leukosorb ⁇ Leur syringe-end fitting filter
• the syringe is conveniently that used for the taking up and applying the sample blood and rinse solution.
• Step (c) is carried out after the selected time for step (b) has passed whereby the detergent solution and its contents are conveniently expelled with a rinse solution or air, preferably with air, eg. by operation of the empty syringe, application of an air-line or' by manual blowing.
• the expelled solution is preferably collected in a container for further processing.
• Step (d) will be common to many other methods of detecting antigens or nucleic acids, or methods for further isolating these, and will be carried out after step (c) with or without an optional detergent removal step as required.
• native antigens may be removed by adsorption with antibodies directed at them. Nucleic acid removal will be carried out before or after this step by methods utilising the very different properties of the materials.
• the eluted material may be passed down an affinity column on which are immobilised antibodies which will specifically bind the material. Techniques for isolating nucleic acids are known to those skilled in the art, eg. SDS gel chromatography against known standards.
• step (d) may be carried out a variety of methods, some of which are particularly adapted for use in the field.
• nucleic acid hybridization probes of labelled nature these being used on their own or in combination with polymerase chain reaction primers which can be used to amplify a target sequence prior to specific probing.
• Such method usually involves use of radioactively labelled probes which renders it dependent upon laboratory equipment for detection of positive results.
• use of biologically labelled probes makes it possible to produce a colour forming end point on successful probing by removal of any non-hybridized material before activating the label reaction.
• step (d) are again various, including radio immuno-assay (RIA) and enzyme linked immuno-assay (ELISA) techniques and their derivatives. More suitable for field assays however are simple slide agglutination methods utilising antibodies targeted at the antigen of interest or use of specific antibodies in immunostick 'dipstick' immobilised form.
• test kits of the present invention are those comprising components specifically associated with the method of the invention and thus comprise
• kits will also be any one or more of the following components;
• affinity reagents for immobilising target antigens and/or nucleic acids and optionally liberating them from their immobilised state once the fluid from which they have been separated has been removed,
• the reagents (a) to (g) will be those designated as preferable in the description of the method as outlined above.
• the reagents (d) are most conveniently those of a colour forming immunoassay targeted at a specific antigen associated with an organism for which infection with the test kit is intended to detect.
• the reagents (e) are those of a dipstick assay suitable for obtaining rapid detection of a target antigen in the field with a colour forming end-point.
• Figure 1 shows a graph plotting Optical Density (0D) v Reciprocal dilution for colour assay of antigen prepared by the method of the invention ( ) as compared to flash lysed and centrifuged derived antigen ( ).
• Virus titre at neat is 10*' 18 TCID 0 /ml. Assay was carried out as detailed in Example 2.
• Figure 2 shows a Leur syringe (1) fitted with an encapsulated Leukosorb filter (2) in a configuration for passing blood (3) from its barrel (4) onto the filter material (5) •
• EXAMPLE 1 Test kit for detection of Bovine Viral Diarrhoea (BVD) in cattle whole blood.
• BVD Bovine Viral Diarrhoea
• a field adapted test kit consisting of: 1 x Leur syringe plus sampling needle
• Heparin 143 USP Unit 10 ml tubes both tubes by Becton Dickinson.
• the basic kit according to the invention was augmented by a separate set of reagents making up a dip-stick assay for BVD; such reagents optionally being provided within the same packaging as the reagents above.
• reagents consisted of:
• mAbs non-competitive monoclonal antibodies having specificity for the p ⁇ O/125 protein of BVD-prepared by conventional mAb raising techniques as ascites fluid in Balb C mice.
• test system was first configured using polypropylene plates and then adapted to use with Nunc Maxisorb immunosticks; mAb was bound to the dipstick in carbonate buffer for 18 hours minimum. Suitable test tubes and measuring/dispensing pipettes were also provided as optional extras.
• Samples of blood were taken from cattle and treated with an amount of EDTA suitable for prevention of blood clotting. Aliquots of 5mls of the EDTA treated blood were passed through the filter using the syringe, the syringe was used to pass a similar amount of phosphate buffered saline through the filter for rinsing purposes. The syringe was then used to pass 0.5ml of the 2% n-octyl- ⁇ -D-glucopyranoside solution onto the filter where it was left for 15 minutes before being expelled into a test tube by air. The air was provided by operation of the empty syringe.
• the treated sample could be used for further isolation steps directly from the test tube but in the present example the test tube was that already containing the monoclonal antibody WB103 in biotinylated form.
• the dipstick from the kit was rinsed in tap water and placed into the mixture of the sample and mAb solution, as is conventional in the art, and after 30 minutes removed, again rinsed with tap water and then placed into the solution of streptavidin-biotin-horseradish peroxidase and left for a further 30 minutes. Finally the dipstick was washed again and placed in a solution of tetra-methyl benzidine wherein the colour reaction was allowed to develop for 15 minutes after which the tube was examined. Presence of a blue colour indicated the presence of the BVD characteristic antigen.
• EDTA or heparin was added to blood by use of Vacutainer tubes as described in the alternatives for the kit in Example 1.
• Vacutainer tubes as described in the alternatives for the kit in Example 1.
• lOmls of blood was added to such tube direct from the sampling syringe whereby it contacts EDTA or heparin that has been coated onto the tube wall.
• the EDTA or heparin anti-clotting agent is provided in such an amount as to produce 0.34M EDTA or 143 USP Units per lOmls, and the resultant treated blood was then taken up into the sampling syringe and passed onto and through the filter.
• Results were obtained showing that the filter method of sample preparation successfully provided a detectable amount of antigen in blood derived from infected animals. See Figure 1 where detectability of antigen in blood derived by flash lysed and centrifugation is compared with that derived from the method of the present invention.
• dipstick reagent Further investigation into the storability of the dipstick reagent was carried out whereby it was shown that the test worked with dipsticks prepared at weekly intervals for nine weeks and kept at 4°C and room temperature in the dark. At the end of the period the performance of tests using them was assessed and found to be unimpared in all cases.

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• 1992
  • 1992-10-02 GB GB929220773A patent/GB9220773D0/en active Pending
• 1993
  • 1993-10-01 GB GB9410166A patent/GB2276005B/en not_active Expired - Fee Related
  • 1993-10-01 EP EP93921042A patent/EP0619883A1/en not_active Ceased
  • 1993-10-01 WO PCT/GB1993/002047 patent/WO1994008239A1/en not_active Application Discontinuation
  • 1993-10-01 CA CA 2124585 patent/CA2124585A1/en not_active Abandoned
  • 1993-10-01 NZ NZ25611793A patent/NZ256117A/en unknown
  • 1993-10-01 AU AU48318/93A patent/AU671299B2/en not_active Ceased
  • 1993-10-01 JP JP6508854A patent/JPH07502123A/ja active Pending

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