A METHOD FOR THE COLLECTION OF ANTIBODIES AND THE DETECTION OF A SPECIFIC ANTIBODY SPECIES IN A FLUID SAMPLE AND A DEVICE FOR CARRYING OUT SAID METHOD AS WELL AS A KIT COMPRISING SAID DEVICE

Abstract: A method for the collection of antibodies and the detection of a specific antibody species in a fluid sample. Said fluid sample is brought into contact with a solid phase matrix comprising a porous membrane and latex particles bound with recombinant protein A or G, resulting in a specific binding of antibodies from the sample. An antigen that is specific for the antibody that is to be detected is subsequently brought into contact with the matrix and forms a detectable complex. The invention is also directed at a device and a kit for carrying out immunodiagnostic assays.

Description: A method for the collection of antibodies and the detection of a specific antibody species in a fluid sample and a device for carrying out said method as well as a kit comprising said device. This invention concerns a method for the collection of antibodies and the detection of a specific antibody species in a fluid sample and a device for carrying out said method as well as a kit comprising said device. European patent application 0200381 describes a solid phase system for use in a ligand-receptor assay for the detection of a selected analyte in a fluid sample comprising a porous matrix, wherein microspheres are entrapped, wherein said microspheres are bound with a receptor capable of capturing a specific target ligand. Upon use of different receptors either different target ligands can be bound or one species of target ligand can be bound to different places for example upon use of different antibodies that bind the antigen at different non-interfering epitopes. The solid phase according to EP-A-0200381 can consist of a membrane or filter made of glass fiber, nylon or ceramic material, wherein microspheres are entrapped, whereby said microspheres are made of a polymeric material such as for example latex that can bind to the receptor substance. Preferably a system is used here consisting of membrane-microspheres-immunological pair, whereby the pair consists of antibody-antigen or antigen-antibody. The method that is described in this EP-A comprises the application of a fluid sample on the solid phase system, whereby the receptor that is bound to the microspheres specifically binds the selected target ligand, as the fluid through passes the solid phase system. Subsequently a solution of a receptor conjugate is adc d, whereby said conjugate is capable of specifically binding and labelling the target ligand in order for the bound target ligand to be detected on the solid phase. The term "receptor conjugate" is directed at a complex comprising a receptor and a label that can be detected. If the target ligand is an antigen the receptor conjugate can be a labelled antibody preferably a monoclonal antibody. On the other hand if the target ligand is an antibody labelled antigen can be used as receptor conjugate. Non-bound receptor conjugate can subsequently be removed by rinsing. The presence of bound receptor conjugate on the solid phase system implies the presence of the analyte in the sample. The EP-A also describes a device for carrying out such a method, whereby said device comprises the solid phase system as described, whereby said device is provided with means permitting the flow of a fluid sample through the solid phase system. It is known that protein A generally binds immunoglobulin and that it binds to Fc, it's constant region. (J.J. Langone, Advances in Immunology, Vol. 32, page 179-211). Natural protein A is derived from Staphylococcus aureus. a pathogen, and is therefore not free of Staphylococcus toxins. American patent 4,189.466 describes the detection of the presence of rheumatoid factor (RF) in an animal sample by mixing the sample with a suspension of microbial cells containing protein A that have been sensitised with free Fc fragments of IgG for which RF is specific. If the mixing results in agglutination this indicates a positive result. Further protein A is coupled to for example agarose beads via chemically stable amide bonds and this complex is used in affinity chromatography for antibodies such as for example in American patent 4,879,211. DuPont has come on to the market with a recombinant protein A coupled to Sepharose, whereby said protein is expressed in E.coli, a non-pathogen rather than in the pathogen Staphylococcus aureus from which the natural protein A is obtained, which implies that the ligand is free of Staphylococcus toxins. DuPont uses recombinant protein A in the perflex 35RS affinity column. The molecule contains 5 homologous binding regions which are highly specific for the Fc portion of immunoglobulins. Immunoglobulin G (IgG) is the major immunoglobulin which binds to recombinant protein A. Other immunoglobulins will also be adsorbed, however the class and sub-class specificity varies from species to species. The protein A can therefore be used for purifying IgG or for selectively removing IgG for analysis of the IG classes or for absorbing immunocomplexes for the purification of antigens. The method according to the invention is directed at a method for the collection of antibodies and the detection of a specific antibody species in a fluid sample comprising the steps of a) contacting said fluid sample with a solid phase matrix, said matrix comprising porous membrane, latex particles distributed in said porous membrane, recombinant protein A or G being covalently bound to said latex particles resulting in an aspecific binding of antibodies present in the fluid sample to form a complex [antibodies-protein A/G- latex-membranej, b) optionally removing excess fluid sample from said solid phase matrix, c) adding an antigen to said complex [antibodies-protein A/G- latex-membrane], said antigen being specific for the antibody species to be detected to allow formation of a complex containing said specific antibody species: [antigen-antibody-protein A/G-latex-membrane], d) optionally removing excess antigen from said porous matrix, e) using a detection method specific for the antigen of step c) . The invention is directed at a method whereby all antibodies in a fluid sample are entrapped on a solid phase matrix and subsequently one or optionally more antibodies can specifically be detected. The antigens and antibodies that can be used with this method can be associated with bacteria, fungi, parasites or virus. One can consider a retrovirus such as for example a HIV-1 virus. This invention is directed at a new method for the collection of immunoglobulins from for example a body fluid for example for a diagnostic test. The invention is directed at the collection and detection of antibodies from body fluids of a mammal (including a human being) such as sweat, tears, blood, urine or preferably saliva. It is therefore possible to use body fluids in which the amounts of antibodies are low as fluid sample. The substance that can bind immunoglobulin is a recombinant protein G or recombinant protein A derived from E.coli or recombinant protein A and G and this protein is covalently bound to latex particles. These latex particles are surface modified for example by carboxylate, amide and/or hydroxy to enable them to bind protein (page 30, Uniform Latex Particles L.R. Bangs). The latex particles bound with protein G, A or G/A are entrapped in a porous membrane and form the solid phase matrix. The porous membrane can be made for example from cellulose, ceramic material, glass fiber, nylon or plastic. Polystyrene latex microparticles possess an intrinsic large surface which allows a large contact area for the immunoglobulin binding substance, recombinant protein A/G, and also for the sample fluid, resulting in a large amount of immunoglobulins that can be entrapped on the matrix, whereby the substrate for a very sensitive immunodiagnostic test is created. The high affinity of proteins G and A for the Fc region of immunoglobulin permits a fast collection of immunoglobulin G's (IgG) and in connection with the large surface area that is offered by latex microparticles this combination of protein A/G-latex microparticles provides an ideal system for the collection and detection of immunoglobulins in fluids, particularly in body fluids such as saliva which contain a smaller amount of antibodies than serum. Simultaneously the antigen position of the captured antibodies remains free as protein G and A bind to the Fc region of the antibodies so that these captured antibodies can react with their free specific regions with the corresponding specific antigens. The matrix consisting of membrane-latex-protein A/G plays the part of collector of immunoglobulins and also serves as solid phase carrier for the identification of antibodies in an immunodiagnostic test. It is therefore only necessary to use one specifically binding antigen for the detection, in contrast to the test described in EP-A-0200381, whereby at least two specifically binding receptors of the analyte are necessary. Furthermore a sample that is analysed according to the method of the invention can be examined for the presence of various specific antibodies by use of different solutions with specific antigens. This was also possible with the method according to EP-A-0200381 however only by filling various areas of the matrix with microspheres bound to various specific receptors, whereby the detection possibilities of different antibodies with such a matrix are restricted to a few antibodies determined at the preparation of the matrix. This contrast with the matrix used in the method according to the invention in which, in accordance with the antibody that is to be detected, a specific corresponding antigen is added after binding the antibodies and whereby the matrix is suited for the binding of every antibody. In this method every possible antibody species that is present in the sample is bound and therefore every antibody can be detected by use of a specific antigen. The" specificity of this detection method depends on the antigen that is selected as means of detection. In the method according to the invention one can select the detection method of step e) from labelling with an enzyme, with a radionuclide with fluorescent agents, phosphorous agents, polymers containing colouring agents or chemiluminescent portions, whereby preferably a detection procedure is used that will generate a colour change which can be detected by instruments or preferably by sight. When labelling with an enzyme it is possible to subsequently apply a substrate that brings about a colour change. For example 4-chloro-l-naphthol or TMB (tetramethylbenzidin) can be used as substrates. This invention is also directed at the use of a non-toxic, non-infectious complex of [recombinant protein A or G-latex-membrane] for immunological tests as well as a device for carrying out immunological tests according to the method of the invention. This device comprises a carrier containing the complex [protein A/G-latex- membrane]. The carrier comprises an absorbent means incorporated in an elongated container with an opening at one end, whereby said end contains said complex and said opening enables the entrance of fluid via the complex absorbent means. The absorbent means is associated with the solid phase system complex in such a manner that the absorbent means draws fluid sample, that comes into contact with the solid phase system, through the complex. Said absorbent means can for example contain capillaries in a direction perpendicular to the surface over which the solid phase system is placed so that the capillaries are in direct communication with the pores of the solid phase system. An embodiment of the device according to the invention is schematically illustrated in cross section in the drawing. In said drawing 1 represents an elongated container of synthetic material provided with an opening at one end in which a membrane permeable for fluid 2 has been placed. Porous carrier 3 containing the complex [protein A or G-latex] is present under membrane 2 in the container 1. Carrier 3 is in contact with a fluid absorbing material 4 that can completely or partially fill the container 1. Various materials can be used as absorbent means such as cellulose acetate fibers, cotton, paper, plastic or polyester. The device can further for example be made of plastic with the following dimensions, a length of approximately 4- 14 cm, a depth of 1-5 mm and a width of 0,5-5 cm with an opening with a diameter of approximately 1-4 cm at one end. The absorbent means can have a length of 3-13 cm, a depth of 0,5-4,5 mm and a width of 0,3-4,5 cm. The open end of the device can be covered with a membrane. A preferred embodiment of the device is a device whereby the open end of the device containing the substance that -is capable of binding immunoglobulin can be inserted into the mouth of a test person. In particular for this application the importance of the use of recombinant protein A without toxins that are present upon use of natural protein is an advantage. This is a new and extremely simple fast and handy method for collecting immunoglobulins G and the further use of said immunoglobulins in an immunodiagnostic test on a solid phase matrix. It is only necessary to keep the device in the mouth for the duration of 1 to 3 minutes. Saliva that comes into contact with the matrix passes through the latex particles bound with protein A, whereby the immunoglobulins that are present will bind quickly to protein A. The size and shape of the device make it simple to use and the discomfort of collecting blood or urine for an immunodiagnostic test is prevented. Furthermore this same device in which the immunoglobulins are collected serves as carrier to be used in the identification of desired specific antibodies associated with a certain disease. Once the antibodies have been bound to the protein A-latex particles membrane an antigen of choice can be used to detect the specific antibodies from the collected saliva in an enzyme immunotest. An example of such a specific experiment in which saliva of a patient was collected is described below. A patient known to be HIV-1-antibody positive kept the device according to the invention under the tongue for 1 minute. After collecting the saliva 0.5 ml of a buffer containing PBS/0, 5% Tween- 20/2# BSA/0.01# Thimerosal with pH 7.8 was pipetted on to the membrane to rinse the excess proteins. After the buffer had been completely absorbed 0.2 ml of a HIV-1-solution containing 1 microgram of biotinilated protein recombinant envelope and core HIV-1 in PBS/0, 5% Tween-20/0.1% gelatine was added and also absorbed. The membrane was subsequently washed with 0.5 ml PBS/0.5% Tween-20/2% BSA/0.01J. Thimerosal pH 7»8 and 1.5 microgram of HRP labelled avidin was pipetted on to the membrane. It is also possible to bind biotin to avidin in advance so that this step can be omitted during the test. The membrane was rinsed again with 1 ml of PBS/0.5% Tveen-20/2% BSA/0.012 Thimerosal with pH 7-8. The buffer was absorbed again. Then 100 microliter of a substrate, 4-chloro-l-naphthol, was pipetted on to the membrane and within 1 minute a positive blue colour developed, characterizing an antibody positive reaction. The total time required for the test was less than 5 minutes. A similar experiment was carried out on a patient known to be HIV-1-antibody negative and no colouring reaction of the device was observed, which is characteristic of an antibody negative reaction. These examples show the utility of the device for collecting antibodies from body fluid with a lower concentration of antibodies than in serum. The antibodies that are collected in this manner are bound to a matrix in a concentration that is suited for a specific immunodiagnostic test. Furthermore the specificity of the test has been demonstrated upon use of a specific antigen for detection of a certain antibody. The protein A/polystyrene microparticles were coupled covalently to each other in the following manner: latex particles were used that had been carboxylate modified (CML) with a diameter of 0.3*95 mm and are commercially available from Serydin, Inc., Indianapolis. The COOH-groups on the surface enable a fast and simple formation of amide bonds via activation of water soluble carbodiamide. 1 ml of 10 solid latex particles with a diameter of 0.3-95 mm was rinsed with 10 ml of deionised water via centrifugation during 10 minutes at 10-12,000 rpm. The water was carefully removed by suction without absorbing the latex beads. The beads were resuspended in 10 ml deionised H 2 0 and sonified for a short period. The beads were mixed by using a vortex and centrifuged during 10 minutes at 10-12,000 rpm. This step was repeated twice. During this rinsing procedure the 0.07 molar carbodiamide solution was prepared: 67 mg of EDC (ethyl-3-(3-dimethylamino-propylcarbodiamide-HCl) (Pearce Rockford II) with ml of deionised H 2 0 with 1 N NaOH with pH 10 and 40 mg of sulfo-NHS (N-hydroxysulfosuccinamide) were added and this was quickly dissolved. After the last rinsing step of the beads the water was poured off and 3 ml of the above-mentioned EDC/sulfo-NHS-solution were added. The beads were mixed vigorously, sonified for a short period of time and incubated at room temperature under gentle agitation at room temperature during 2 hours. After 2 hours of incubation the beads were rinsed another three times with the deionised water as described before. During this rinsing step the £-amino-N-caproic acid solution was made by dissolving 1 g £-amino-N-caproic acid in 0.5 molar potassium phosphate with pH 7«4. The beads were resuspended in 3 ml of this solution. This was powerfully shaken on a vortex and incubated at room temperature in a mixer for 8 hours. After said 8 hours of incubation 0.3 ml of 1 molar ethanolamine was added to stop the reaction and the solution was incubated at room temperature for 1 hour: the beads were washed anoter three times in 10 ml of deionised water and centrifuged during ten minutes at 12,000 rpm. During the rinsing steps a new carbodiamide solution was prepared as described earlier. The carbodiamide activation was repeated and at the end of the 2 hours of incubation the beads were rinsed again in the deionised water as is described previously. The beads were resuspended in 1 ml of 0.5 KPOή with pH 7-4 and 1 mg of recombinant protein A was added that had dissolved in 2 ml of 0.5 KPO4 of pH 7-4 and was incubated at room temperature over night. To stop the coupling reaction 0.3 ml of 1 molar ethanolamine was added and was incubated for 1 hour at room temperature. The latex beads coupled to protein were rinsed again as described earlier with deionised water. The latex beads were resuspended in 2 ml of PBS and mixed vigorously. The mixture was sonified for 10 seconds and vortexed again. This procedure was repeated one more time. It is known that latex microparticles bind strongly to microporous glass fiber. The selection of glass fiber membrane should be made so that the latex particles can be incorporated in the pores of the membrane. In this example a Schleier and Schuell glass fiber membrane No. 3^ was used. The freshly prepared microparticles bound with protein A were dissolved 1:20 in PBS. 0.2 ml of diluted latex microparticles/protein A was pipetted in the shape of a cross onto the glass fiber membrane and allowed to dry at room temperature. To prevent the non-specific binding of other proteins that could disturb the immunoenzyme test the glass fiber membrane that contained latex-protein A-complex was blocked with a blocking buffer containing PBS/2 BSA/0.1 gelatine/0.5% Tween-20. In order to complete the blockage the membrane was placed on an absorbent cushion/filter paper and 2 ml of the blocking buffer were added to the membrane in order for the buffer to be absorbed by the membrane so that an in depth blockage of the glass fiber matrix was accomplished. The matrix was allowed to dry at room temperature. The blocked matrix was stored before use in a plastic bag with a desiccator. These glass fiber-protein bound latex particles serve as solid matrix for collecting immunoglobulin in an increased concentration, which is obtained by a) the large surface area that is offered by the latex microparticles and b) the known high affinity of protein A for immunoglobulin G.

Claims: C L A I M S 1. A method for the collection of antibodies and the detection of a specific antibody species In a fluid sample comprising the steps of a) contacting said fluid sample with a solid phase matrix, said matrix comprising a porous membrane, latex particles distributed in said porous membrane, - recombinant protein A or G being covalently bound to said latex particles resulting in an aspecific binding of antibodies present in sa d fluid sample to form a complex [antibodies-protein A/G-latex-membrane], b) optionally removing excess fluid from said solid phase matrix c) adding an antigen to said complex [antibodies-protein A/G-latex-membrane} said antigen being specific for the antibody species to be detected to allow formation of a complex containing said specific antibody species: [antigen-antibody-protein A/G-latex-membrane], d) optionally removing excess antigen from said porous matrix, e) using a detection method specific for the antigen of step c) . 2. The method according to claim 1, wherein said antigens and antibodies are associated with a bacterium, fungus, parasite or virus. 3- The method according to claim 2, wherein said virus is HIV-1. 4. The method according to any of claims 1-3 . wherein said latex is surface-modified, e.g. modified by carboxylate, amide and/or hydroxy. 5. The method according to any of claims 1-4, wherein said fluid sample is a body fluid of a mammal including a human being, such as sweat, tears, blood, urine or preferably saliva. 6. The method according to any of claims 1-5 . wherein said porous matrix is made of ceramic material, nylon, synthetic material or paper, preferably glass fiber. 7« The method according to any of claims 1-6, wherein the antibody to be detected is associated with the HIV virus, steps b) and d) comprise rinsing with a buffer having a pH-value in the range of 7- 8.5, step c) comprises the addition of HIV-1 solution containing biotin labelled recombinant envelope and core HIV-l * and step e) rinsing with a buffer having a pH-value in the range 7-8.5 and addition of a suitable labelled enzyme solution and addition of a non-mutagenic, non- carcinogenic substrate for detection. 8. Method according to claim 7. wherein said enzyme solution co - prises avidin labelled with HRP (horseradish peroxidase). 9. Method according to claim 7 or 8, wherein the substrate of step e is chosen from .-chloro-1-naphthol or TMB (tetramethylbenzidin ). 10. Method according to any of claims 1-9. wherein the detection ■ethod of step e) is selected from labelling with an enzyme, with a radionuclide with fluorescent agents, phosphorous agents, polymers containing dyes or chemilumlnescent moieties, preferably a labelling which will generate a colour change which nay be detected by Instrumental means or preferably visually by the human eye. 11. Use of a non-toxic, non-infectious complex [recombinant protein A or G-latex-membrane] for the collection of antibody from body fluids such as saliva. 12. Use of the complex according to claim 11 for immunological assays. 13. Device comprising a carrier which contains the complex [protein A/G-latex-membrane] suitable for the collection of antibodies from the mouth and/or carrying out immunological tests according to a method of any of the claims 1-10. 14. Device according to claim 13, wherein the carrier comprises an absorbent means in an elongated container with an opening at one end whereby said opening contains the complex according to claim 11 which is connected to the absorbent means in such a way that the absorbent means draws fluid that comes into contact with the complex through the complex. whereby the complex is also covered with a membrane layer which lets fluid through. 1 * Device according to claim 14, wherein the absorbent means is chosen from an absorbent material such as cellulose, acetate fibers, porous synthetic material, cotton or wood. 16. Kit for carrying out immunodiagnostic assays comprising at least the following components: 1) at least one device according to the claims 13-15. 2) at least the components required for making buffers necessary for carrying out a method according to any of the claims 1-10, optionally in the shape of one or more solutions, 3) at least a solution containing antigen for a specific antibody which is to be detected optionally already containing a label. 17. Kit according to claim 16 comprising at least a solution for making the antigen solution mentioned in point 3 of claims 16 detectable. 18. Kit according to claim 16 or 17 for detecting a HIV-1 antibody, characterized in that point 2 of claim 16 contains components for making a PBS/0. % Tween-20/2 % BSA/0.01 % Thimerosaie buffer with pH 7 * 8 and the solution of antigen mentioned under point 3 of claim 16 contains a HIV-1 solution which contains 1 microgram of biotinilated recombinant envelope and core HIV-1 in PBS/0.5 % Tween-20/0.1 % gelatine, that the detection mentioned in point 4 consists of addition of a solution of avidin-labelled with HRP and the addition of a solution of 4-chloro-l-naphthol substrate.

Inventors: GAVOJDEA STEFAN, GAVOJDEA, STEFAN
Applicants: TENTA PROPERTIES NV, TENTA PROPERTIES N.V.
Application year: 1991
Main classification: G01N33
IPC codes: A61B10, G01N33, G01N37