EP0220213A1 - Immunoactive components immobilized in a porous material - Google Patents

Immunoactive components immobilized in a porous material

Info

Publication number
EP0220213A1
EP0220213A1 EP19860902290 EP86902290A EP0220213A1 EP 0220213 A1 EP0220213 A1 EP 0220213A1 EP 19860902290 EP19860902290 EP 19860902290 EP 86902290 A EP86902290 A EP 86902290A EP 0220213 A1 EP0220213 A1 EP 0220213A1
Authority
EP
European Patent Office
Prior art keywords
insoluble
matrix
antibodies
porous
immunosorbent
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP19860902290
Other languages
German (de)
French (fr)
Inventor
Eugeen Bosmans
Henri Martens
Jef Raus
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DR L WILLEMS INSTITUUT
Hasselt Universiteit
Universiteit Maastricht
Original Assignee
DR L WILLEMS INSTITUUT
Hasselt Universiteit
Universiteit Maastricht
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 DR L WILLEMS INSTITUUT, Hasselt Universiteit, Universiteit Maastricht filed Critical DR L WILLEMS INSTITUUT
Publication of EP0220213A1 publication Critical patent/EP0220213A1/en
Withdrawn legal-status Critical Current

Links

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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/549—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic with antigen or antibody entrapped within the carrier

Definitions

  • an "immunoassay” constitutes an analytical technique based on the affinity of an antigen for the sites of association of an antigen on an antibody.
  • Antigens or antibodies are labeled directly or indirectly with an isotope, an enzyme, fluorochrome or another measurable substance.
  • Calibration curves are established on the basis of data obtained after incubation of samples which contain different, but known, concentrations of antigen or antibody.
  • the associated complexes of (antigen-antibody) n are separated from the antigen or from the free antibody. Then, the quantity of complex formed or of antigen or antibody remaining free is measured by means of specific detectors depending on the marker used.
  • Another variant of this technique is based on the addition to the mixture of a second antibody which selectively precipitates the antibody (with the antigen associated therewith), which ultimately leaves only the antigen unaffected. partner in solution.
  • the non-associated antigen is then separated from the antigen-anti ⁇ body complex by centrifugation or by another means.
  • Some researchers have fixed the antibody or antigen on the wall of a plastic container and then a sample containing an antigen or an antibody is introduced into the container. The mixture thus obtained is incubated and then the antigen-antibody complex formed is separated from the antigen or the free antibody by simply emptying the container.
  • the associated antigen or antibody is determined and the quantity of antigen or antibody which was present in the sample is measured using a calibration curve. unknown.
  • the prior techniques essentially have the drawback that the antigens and / or antibodies are generally not covalently linked, that the amounts of these antigens and antibodies attached are not determined with precision and finally that in the case of several antigens and antibodies their respective ratios are not determined with precision.
  • the present invention aims to obtain a method which makes it possible to fix various antigens or antibodies in well defined proportions to an immunosorbent in such a way that the immunosorbent thus prepared can be used in individual or multiple immunological determinations.
  • the invention relates to an immunosorbent for use in immunological determinations.
  • This hydrophilic or hydrophobic sorbent is based on a porous polytetrafluoroethylene (PTFE) matrix to which is attached in a non-covalent manner an organic or inorganic polymer, hydrophilic or hydrophobic, insoluble in l water, such as polyacrylamide, cellulose, silica gel, latex or a similar polymer, with which covalently are associated antigens and / or antibodies in well defined quantities and, if they are various antigens and / or antibodies, in well-defined portions.
  • PTFE polytetrafluoroethylene
  • the porosity is between 20 and 80% by volume.
  • the invention also relates to the process used to produce such an immunosorbent.
  • the antigens or the antibodies can be covalently coupled to a water-insoluble polymer which had already been incorporated beforehand into the base matrix.
  • antigens and / or antibodies can be coupled to a water-insoluble polymer which is subsequently incorporated into the base matrix.
  • This latter method allows the incorporation in various proportions in the basic matrix, insoluble polymers 1 * water to which various antigè- born and / or antibodies were separately coupled.
  • These two operating variants are carried out at a temperature between 4 and 40 ⁇ C, preferably at the temperature room.
  • the incorporation of the insoluble polymer in the base matrix can be carried out dry.
  • biomolecules are copolymerized with other proteins in a matrix which, like that of the present invention is porous.
  • the present invention it is possible to regulate more precisely the quantity of antigens or antibodies by coupling these in advance to the carrier polymer material and then incorporating it into the porous base matrix.
  • the technique of the invention also makes it possible to first couple different antigens or antibodies on different batches of carrier polymer material and then to incorporate in well defined weight proportions these batches thus treated in the porous base matrix.
  • proteins are not used, the chemical and physical nature of which can be the cause of processing difficulties or disturbances, in particular in immunological tests.
  • This technique describes the incorporation of porous silica particles used in small columns and not in a porous matrix as in the present invention.
  • This document describes a "coating" technology comprising the use of a polymer film to which antigens and other biomolecules are coupled. There is no question of applying antigens in predetermined proportions between them.
  • the technique described consists in immobilizing enzymes directly by fixing them physically. non-covalent in a polytetrafluoroethyle ⁇ ne matrix.
  • Materials which can be used as water insoluble carriers are cellulose, polyacrylamide, silica gel, latex, sepharose, agarose ®, etc.
  • the technique used essentially comprises the following stages:
  • the insoluble, activated, non-activated or coupled by the antigen or antibody porous polymer is mixed together with the basic porous matrix in the pulverulent state to form a dry mixture
  • this dry mixture is subjected to an agglomeration treatment to form agglomerates
  • - the agglomerates formed are subjected to a grinding treatment to form crushed agglomerates
  • the tablet is subjected to lamination until a film or sheet is obtained.
  • This film according to the invention can be produced in a substantially continuous manner and then be cut by any suitable means.
  • the technique used makes it possible to work between 4 ° C and 37 ° C approximately, preferably at room temperature (for example 20 ⁇ C approximately) which avoids degradation of the biological components used.
  • a composition consisting of insoluble porous polymers, of identical or different nature, comprising known quantities of biological compounds and in a known ratio of these polymers thus charged.
  • porous insoluble polymers are of different chemical nature, for example hy- drophobes and hydrophilic in order to have activation 'differentiated techniques.
  • This technique makes it possible to detect at the same time human antibodies directed against the cytomegalovirus and human antibodies directed against surface antigens of hepatitis B.
  • the two antigens are associated, by different reactions, with a support in po- lyacrylamide which is then incorporated into a PTFE matrix.
  • the washed gel is then mixed with 35 ml of an aqueous glutaraldehyde solution (25%). This suspension is gently stirred for 5 hours at room temperature. 3. Next, the glutaraldehyde is removed by washing with the aforementioned phosphate buffer. This washing is repeated 5 times. 4. 70 ml of antigen solution (1 mg protein / ml) in phosphate buffer is added.
  • the cytomegalovirus antigen is obtained, inter alia, from an extract of sodium glycine-hydroxy- from a culture of fibroblasts contaminated by the cytomegalovirus having a cytopathic effect of 90%.
  • the hepatitis B surface antigen is obtained in particular from human plasma containing said antigen.
  • the antigen itself is, for example, isolated from the plasma by affinity chromatography with monoclonal antibodies directed against the hepatitis B surface antigen.
  • the two antigens are incubated separately for 1 hour with an activated polyacrylamide gel.
  • the gels are mixed in appropriate proportions.
  • the quantities of antigens are controlled on the basis of the quantities of antibodies which associate with the polyacrylamide gels after incubation.
  • the mixture of gels thus obtained is then incorporated into a PTFE matrix and is transformed into a sheet of 100 micrometers thick. It is possible to produce several other thicknesses.
  • the immunological determination is carried out as follows: a small disc is incubated with 0.5 ml of a 1/200 dilution of patient serum (dilution in a physiological salt solution buffered with phosphate, pH 7.2, which contains 1% bovine albumin and 0.2% TVTEENv 20). The incubation is carried out at room temperature for 1 hour. At the same time as the unknown samples, a positive and a negative control sample (for the hepatitis B surface antigen and the cytomegalovirus) are used.
  • the discs are again washed 5 times with 3 ml of washing buffer (0.15 molar NaCl solution, phosphate buffered, pH ⁇ 7.2, which contains 0.5% of TWEEN ⁇ 20).
  • substrate solution consisting of 10 mg of orthophenylenediamine dissolved in 10 ml of citrate and phosphate buffer (0.1 M, pH ⁇ 6.0) are added to which 10 microliters are added. 30% hydrogen peroxide.
  • the quantity of product formed enzymatically is then measured at 492 nm and compared with the optical density of negative controls. If the optical density of a sample is more than 5 standard deviations above negative control, the sample is considered positive.
  • the 95% limit, determined on a large series of negative sera,. is found in this method around an optical density of 0.200, but this must be determined again experimentally for each application.
  • Example 2 Assay of reactive protein C (C.R.P.) in patient serum using an enzyme immunoassay applied according to a sandwich technique.
  • the gamma globulin fraction of an anti-C.R.P. hyperimmune is covalently associated with a polyacrylamide polymer enclosed in a porous PTFE sheet.
  • Small disks containing this antibody are incubated with patient serum and a series of standards.
  • the associated antigen is then reacted with an antibody labeled with an enzyme.
  • a substrate is added.
  • the kinetic enzymatic reaction measured constitutes a measure of the antigen present.
  • a quantitative determination can be made by comparison with the values obtained for the standards. In an example process, the following operations are carried out.
  • the sheet is dried and small discs 5 mm in diameter are stamped on it.
  • the small disks are transferred into small tubes (for example tubes having a total capacity of 7 ml).
  • a series of standards comprising 100, 500, 1000, 2500 and 5000 micro ⁇ grams of C.R.P./ml. From these dilutions, 500 microliters are transferred into the tubes containing these small disks. The incubation is carried out gently at room temperature for 1 hour.
  • the reaction of the substrate is stopped by means of 1 ml of IN HCl. 14.
  • the optical density of the above-mentioned liquids is measured at 492 nm. With the optical density of the standards, a calibration curve is established on which the concentration of C.R.P. can be read. serum samples.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Inorganic Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Immunosorbant utilisable dans les déterminations immunologiques à base d'une matrice poreuse en polytétrafluoroéthylène où se trouve attaché d'une manière non covalente un polymère organique ou inorganique, hydrophile ou hydrophobe, insoluble dans l'eau auquel sont associés par convalence des antigènes et/ou des anticorps en des quantités bien déterminées et, s'il s'agit de divers antigènes et/ou anticorps, en des proportions bien déterminées entre-eux. Le procédé pour la préparation d'un tel immuno sorbant comporte les étapes suivantes: on mélange ensemble le polymère poreux insoluble, activé, non activé ou couplé par l'antigène ou anticorps avec la matrice poreuse de base à l'état pulvérulant pour former un mélange sec; on soumet ce mélange sec à un traitement d'agglomération pour former des agglomérats; on soumet les agglomérats formés à un traitement de broyage pour former des agglomérats broyés; on presse ensemble les agglomérats broyés pour former un comprimé et on soumet le comprimé à des laminages jusqu'à obtention d'un film ou d'une feuille.Immunosorbent usable in immunological determinations based on a porous polytetrafluoroethylene matrix to which is attached in a non-covalent manner an organic or inorganic polymer, hydrophilic or hydrophobic, insoluble in water with which antigen and / or antibodies in well-defined quantities and, in the case of various antigens and / or antibodies, in well-determined proportions between them. The process for the preparation of such an immunosorbent comprises the following stages: the insoluble porous polymer, activated, not activated or coupled by the antigen or antibody, is mixed together with the basic porous matrix in the pulverulent state to form a dry mix; this dry mixture is subjected to an agglomeration treatment to form agglomerates; the agglomerates formed are subjected to a grinding treatment to form crushed agglomerates; the crushed agglomerates are pressed together to form a tablet and the tablet is subjected to rolling until a film or sheet is obtained.

Description

COMPOSANTES IMMUNOACTIVES IMMOBILISÉES IMMOBILIZED IMMOBILIZED COMPONENTS
DANS UNE MATIÈRE POREUSEIN A POROUS MATERIAL
Connaissances antérieures • Un "immunoassay" constitue une technique analyti¬ que basée sur l'affinité d'un antigène pour les sites d'association d'un antigène sur un anticorps. Les anti¬ gènes ou les anticorps sont marqués directement ou indi¬ rectement avec un isotope, une enzyme, du fluorochrome ou une autre substance mesurable. On établit des courbes d'étalonnage sur la base de données obtenues après incu¬ bation d'échantillons qui contiennent des concentrations différentes, mais connues, d'antigène ou d'anticorps. Après une incubation, on sépare les complexes associés d' (antigène-anticorps)n de l'antigène ou de l'anticorps libre. Ensuite, on mesure la quantité de complexe formé ou d'antigène ou d'anticorps restant libre au moyen de détecteurs spécifiques dépendant du marqueur utilisé. Ce processus est répété pour un certain nombre d'êchantil- Ions qui contiennent des quantités bien déterminées d'antigènes ou d'anticorps et les résultats obtenus sont présentés sous forme d'un graphique. On y porte la quan¬ tité de marqueur que l'on mesure dans le complexe anti¬ gène-anticorps, ou bien dans l'antigène ou l'anticorps restant, en fonction des diverses concentrations d'anti¬ gène ou d'anticorps. Lorsque cette courbe d'étalonnage a été établie, on peut mesurer la concentration de l'anti¬ gène ou de l'anticorps correspondant dans un échantillon inconnu. Pour pouvoir mesurer la quantité d'antigène ou d'anticorps, il est important que les complexes antigè¬ ne-anticorps soient séparés de l'antigène non associé ou de l'anticorps non associé. Une méthode fréquemment uti¬ lisée à cette fin consiste à ajouter au mélange du dex- trane adsorbé sur du carbone. Le dextrane permet que l'antigène non associé, de bas poids moléculaire, migre à travers lui et s'associe finalement au carbone. Ensui¬ te, le carbone et avec lui l'antigène adsorbé est séparé de l'anticorps ou du complexe antigène-anticorps par centrifugation.Previous knowledge • An "immunoassay" constitutes an analytical technique based on the affinity of an antigen for the sites of association of an antigen on an antibody. Antigens or antibodies are labeled directly or indirectly with an isotope, an enzyme, fluorochrome or another measurable substance. Calibration curves are established on the basis of data obtained after incubation of samples which contain different, but known, concentrations of antigen or antibody. After incubation, the associated complexes of (antigen-antibody) n are separated from the antigen or from the free antibody. Then, the quantity of complex formed or of antigen or antibody remaining free is measured by means of specific detectors depending on the marker used. This process is repeated for a number of samples which contain well-defined amounts of antigens or antibodies and the results obtained are presented in the form of a graph. We carry the quantity of marker which is measured in the anti-gene-antibody complex, or in the remaining antigen or antibody, depending on the various concentrations of anti-gene or antibody. When this calibration curve has been established, it is possible to measure the concentration of the antigen or of the corresponding antibody in an unknown sample. To be able to measure the quantity of antigen or antibody, it is important that the antigen-antibody complexes are separated from the unassociated antigen or from the unassociated antibody. A method frequently used for this purpose consists in adding to the mixture dextrane adsorbed on carbon. Dextran allows the low molecular weight unassociated antigen to migrate through it and ultimately associate with carbon. Next, the carbon and with it the adsorbed antigen is separated of the antibody or antigen-antibody complex by centrifugation.
Une autre variante de cette technique est basée sur l'addition au mélange d'un deuxième anticorps qui fait précipiter sélectivement l'anticorps (avec l'anti¬ gène qui y est associé), ce qui laisse finalement uni¬ quement l'antigène non associé en solution. L'antigène non associé est alors séparé du complexe antigène-anti¬ corps par centrifugation ou par un autre moyen. Certains chercheurs ont fixé l'anticorps ou l'antigène sur la pa¬ roi d'un récipient en plastique et puis on introduit dans le récipient un échantillon contenant un antigène ou un anticorps. Le mélange ainsi obtenu est incubé et puis on sépare le complexe antigène-anticorps formé de l'antigène ou de l'anticorps libre en vidant simplement le récipient.Another variant of this technique is based on the addition to the mixture of a second antibody which selectively precipitates the antibody (with the antigen associated therewith), which ultimately leaves only the antigen unaffected. partner in solution. The non-associated antigen is then separated from the antigen-anti¬ body complex by centrifugation or by another means. Some researchers have fixed the antibody or antigen on the wall of a plastic container and then a sample containing an antigen or an antibody is introduced into the container. The mixture thus obtained is incubated and then the antigen-antibody complex formed is separated from the antigen or the free antibody by simply emptying the container.
Plus récemment, on a cherché' à associer des anti¬ gènes et des anticorps à du dextrane lui-même, à de la cellulose et à des produits similaires. Après incubation le complexe antigène-anticorps est séparé de manière simple des antigènes ou des anticorps non associés.More recently, attempts have been made to combine anti-genes and antibodies with dextran itself, with cellulose and with similar products. After incubation, the antigen-antibody complex is simply separated from the non-associated antigens or antibodies.
Dans .toutes ces méthodes, on détermine l'antigène ou l'anticorps associé et on mesure, à l'aide d'un cour¬ be d'étalonnage, la quantité d'antigène ou d'anticorps qui était présente dans l'échantillon inconnu.In all of these methods, the associated antigen or antibody is determined and the quantity of antigen or antibody which was present in the sample is measured using a calibration curve. unknown.
Inconvénient des techniques antérieuresDisadvantage of prior techniques
Les techniques antérieures présentent essentiel¬ lement 1'inconvénient que les antigènes et/ou anticorps ne sont généralement pas liés par covalence, que les quantités de ces antigènes et anticorps fixés ne sont pas déterminées avec précision et enfin que dans le cas de plusieurs antigènes et anticorps leurs rapports res¬ pectifs ne sont pas déterminés avec précision.The prior techniques essentially have the drawback that the antigens and / or antibodies are generally not covalently linked, that the amounts of these antigens and antibodies attached are not determined with precision and finally that in the case of several antigens and antibodies their respective ratios are not determined with precision.
But de la présente inventionAim of the present invention
La présente invention vise à obtenir un procédé qui permet de fixer divers antigènes ou anticorps en des proportions bien déterminées à un immunosorbant de telle manière que 1'immunosorbant ainsi préparé puisse être employé dans des déterminations immunologiques indivi- duelles ou multiples.The present invention aims to obtain a method which makes it possible to fix various antigens or antibodies in well defined proportions to an immunosorbent in such a way that the immunosorbent thus prepared can be used in individual or multiple immunological determinations.
Objet de la présente inventionObject of the present invention
L'invention porte sur un immunosorbant utilisable dans les déterminations immunologiques. Ce sorbant (ad- sorbant) hydrophile ou hydrophobe est à base d'une ma¬ trice poreuse en polytétrafluoroéthylène (PTFE) où se trouve attaché d'une manière non covalente un polymère organique ou inorganique, hydrophile ou hydrophobe, in¬ soluble dans l'eau, tel que le polyacrylamide, la cellu- lose, le silicagel, le latex ou un polymère analogue, auquel sont associés par covalence des antigènes et/ou des anticorps en des quantités bien déterminées et, s'il s'agit de divers antigènes et/ou anticorps, en des pro¬ portions bien déterminées entre-eux. De préférence la porosité est comprise entre 20 et 80% en volume.The invention relates to an immunosorbent for use in immunological determinations. This hydrophilic or hydrophobic sorbent (adsorbent) is based on a porous polytetrafluoroethylene (PTFE) matrix to which is attached in a non-covalent manner an organic or inorganic polymer, hydrophilic or hydrophobic, insoluble in l water, such as polyacrylamide, cellulose, silica gel, latex or a similar polymer, with which covalently are associated antigens and / or antibodies in well defined quantities and, if they are various antigens and / or antibodies, in well-defined portions. Preferably the porosity is between 20 and 80% by volume.
L'invention concerne également le procédé mis en oeuvre pour réaliser un tel immunosorbant.The invention also relates to the process used to produce such an immunosorbent.
Eléments essentiels de l'inventionEssential elements of the invention
Selon 1'invention les antigènes ou les anticorps peuvent être couplés par covalence à un polymère insolu¬ ble dans l'eau qui avait déjà été préalablement incorpo¬ ré à la matrice de base. En variante, on peut coupler des antigènes et/ou des anticorps à un polymère insolu¬ ble dans l'eau qui est par la suite incorporé dans la matrice de base. Ce dernier procédé permet d'incorporer en diverses proportions, dans la matrice de base, des polymères insolubles dans 1*eau auxquels divers antigè- nés et/ou anticorps ont été séparément couplés. Ces deux variantes opératoires sont effectuées à une température comprise entre 4 et 40βC, de préférence à la température ambiante.According to the invention, the antigens or the antibodies can be covalently coupled to a water-insoluble polymer which had already been incorporated beforehand into the base matrix. Alternatively, antigens and / or antibodies can be coupled to a water-insoluble polymer which is subsequently incorporated into the base matrix. This latter method allows the incorporation in various proportions in the basic matrix, insoluble polymers 1 * water to which various antigè- born and / or antibodies were separately coupled. These two operating variants are carried out at a temperature between 4 and 40 β C, preferably at the temperature room.
L'incorporation du polymère insoluble dans la ma¬ trice de base peut se faire par voie sèche.The incorporation of the insoluble polymer in the base matrix can be carried out dry.
Avantages de la solution de 1'invention comparée à dif¬ férentes éléments de l'état de la technique US-A-3 951 748 (R.F. DEVLIN)Advantages of the solution of the invention compared to various elements of the state of the art US-A-3,951,748 (R.F. DEVLIN)
La technique décrite dans ce document est sensi- blement différente de celle proposée dans la présente invention pour ce qui a trait à 1'immobilisation des substances immunoactives. Dans la technologie décrite par la référence, des biomolécules sont copolymêrisées avec d'autres protéines dans une matrice qui, comme cel- le de la présente invention est poreuse.The technique described in this document is substantially different from that proposed in the present invention with respect to the immobilization of immunoactive substances. In the technology described by the reference, biomolecules are copolymerized with other proteins in a matrix which, like that of the present invention is porous.
Selon la présente invention il est possible de régler plus précisément la quantité d'antigènes ou d'an¬ ticorps en couplant ceux-ci d'avance au matériau polymè¬ re porteur et I'incorporant ensuite dans la matrice de base poreuse. La technique de l'invention permet aussi de d'abord coupler différentes antigènes ou anticorps sur des lots différents de matériau polymère porteur et d'ensuite d'incorporer dans des proportions pondérales bien déterminée ces lots ainsi traités dans la matrice de base poreuse.According to the present invention it is possible to regulate more precisely the quantity of antigens or antibodies by coupling these in advance to the carrier polymer material and then incorporating it into the porous base matrix. The technique of the invention also makes it possible to first couple different antigens or antibodies on different batches of carrier polymer material and then to incorporate in well defined weight proportions these batches thus treated in the porous base matrix.
Il convient aussi de noter que selon 1'invention on n'utilise pas de protéines dont la nature chimique et physique peut être la cause de difficultés de mise en oeuvre ou de perturbations, en particulier dans des tests immunologiques.It should also be noted that according to the invention, proteins are not used, the chemical and physical nature of which can be the cause of processing difficulties or disturbances, in particular in immunological tests.
La nature de la matière formant la matrice mouil- lable décrite dans le référence US-A-3 951 748 diffère également de la matrice poreuse utilisée selon 1'inven¬ tion qui se présente avantageusement sous forme d'une feuille poreuse de polytetrafluoroethylene dans laquelle le matériau polymère porteur est incorporé, selon l'in¬ vention, à température ordinaire. FR-A-2 531 223 (MOCHIDA PHARMACEUTICAL)(=GB-A-2 125 547)The nature of the material forming the wettable matrix described in the reference US Pat. No. 3,951,748 also differs from the porous matrix used according to the invention, which advantageously takes the form of a porous sheet of polytetrafluoroethylene in which the carrier polymer material is incorporated, according to the invention, at ordinary temperature. FR-A-2 531 223 (MOCHIDA PHARMACEUTICAL) (= GB-A-2 125 547)
La technique décrit dans ce brevet ne permet pas d'incorporer dans des proportions pondérales déterminées un matériau polymère porteur sur lequel ont été préala¬ blement couplé des antigènes ou des anticorps dans une matrice de base poreuse. On n'obtient donc pas les avan¬ tages cités ci-dessus pour l'invention en relation avec la première référence de l'état de la technique.The technique described in this patent does not make it possible to incorporate, in determined weight proportions, a carrier polymer material on which antigens or antibodies have been previously coupled in a porous base matrix. The advantages cited above for the invention are therefore not obtained in connection with the first reference of the state of the art.
US-A-4 166 102 (L.R. JOHNSON)(= FR-A,B-2 222 654)US-A-4 166 102 (L.R. JOHNSON) (= FR-A, B-2 222 654)
Cette technique décrit 1'incorporation de parti¬ cules de silice poreuse utiisé dans de petites colonnes et non pas dans une matrice poreuse comme dans la pré¬ sente invention.This technique describes the incorporation of porous silica particles used in small columns and not in a porous matrix as in the present invention.
US-A-4 357 142 (R.F.A SCHALL et al.)US-A-4,357,142 (R.F.A SCHALL et al.)
Ce document décrit une technologie de "coating" comportant l'utilisation d'un film polymère auquel des antigènes et d'autres biomolécules sont couplées. Il n'y est pas question d'appliquer des antigènes en propor¬ tions déterminées entre-eux.This document describes a "coating" technology comprising the use of a polymer film to which antigens and other biomolecules are coupled. There is no question of applying antigens in predetermined proportions between them.
0-A-8 200 363 (R.A. HARTE et al.)(= US-A-4 340 564)0-A-8 200 363 (R.A. HARTE et al.) (= US-A-4 340 564)
Il s'agit d'une technologie d'immobilisation dans lequelle une matière poreuse est imprégnée d'une êmul- sion aqueuse de grannules polymères auxquels un biomatê- riau est couplé.It is an immobilization technology in which a porous material is impregnated with an aqueous emulsion of polymeric granules to which a biomaterial is coupled.
CHEMICAL ABSTRACTS, Vol. 99, No. 21, 21 Nov. 1983, p. 308, Abstract 172004 9, Columbus, Ohio, USACHEMICAL ABSTRACTS, Vol. 99, No. 21, 21 Nov. 1983, p. 308, Abstract 172004 9, Columbus, Ohio, USA
La technique décrite consiste à immobiliser di¬ rectement des enzymes en les fixant de manière physique non covalente dans une matrice de polytetrafluoroethyle¬ ne.The technique described consists in immobilizing enzymes directly by fixing them physically. non-covalent in a polytetrafluoroethyle¬ ne matrix.
Description détaillée de 1-'invention La méthode utilisée pour immobiliser des compo¬ santes immunoactives dans une matière poreuse peut être appliquée dans diverses déterminations immunologiques.Detailed description of the invention The method used to immobilize immunoactive components in a porous material can be applied in various immunological determinations.
Les matières qui peuvent être employées comme supports insolubles dans l'eau sont la cellulose, le po- lyacrylamide, le silicagel, un latex, le sépharose, 1'agarose ®, etc.Materials which can be used as water insoluble carriers are cellulose, polyacrylamide, silica gel, latex, sepharose, agarose ®, etc.
La technique utilisée comporte essentiellement les étapes suivantes :The technique used essentially comprises the following stages:
- on mélange ensemble le polymère poreux insoluble, ac- tivé, non activé ou couplé par l'antigène ou anticorps avec la matrice poreuse de base à 1'état pulvérulant pour former un mélange sec,- the insoluble, activated, non-activated or coupled by the antigen or antibody porous polymer is mixed together with the basic porous matrix in the pulverulent state to form a dry mixture,
- on soumet ce mélange sec à un traitement d'aggloméra¬ tion pour former des agglomérats, - on soumet les agglomérats formés à un traitement de broyage pour former des agglomérats broyés,- this dry mixture is subjected to an agglomeration treatment to form agglomerates, - the agglomerates formed are subjected to a grinding treatment to form crushed agglomerates,
- on presse :ensemble les agglomérats broyés pour former un comprimé et- pressing : together the crushed agglomerates to form a tablet and
- on soumet le comprimé à des laminages jusqu'à obten- tion d'un film ou d'une feuille.- the tablet is subjected to lamination until a film or sheet is obtained.
Ce film selon 1'invention peut être produit de manière pratiquement continue pour être ensuite découpé par tous moyens appropriés.This film according to the invention can be produced in a substantially continuous manner and then be cut by any suitable means.
La technique utilisée permet de travailler entre 4°C et 37°C environ, de préférence à la température ambiante (par exemple 20βC environ) ce qui évite une dégradation des composants biologiques mis en oeuvre.The technique used makes it possible to work between 4 ° C and 37 ° C approximately, preferably at room temperature (for example 20 β C approximately) which avoids degradation of the biological components used.
Dans le cas ou l'on désire que plusieurs compo- santés immunoactives soient immobilisées sur une matrice poreuse, on peut soumettre à l'opération de mélange spé¬ cifié dans la première étape ci-dessus, une composition constituée de polymères poreux insolubles, de nature identique ou différentes, comportant des quantités con¬ nues de composés biologiques et dans un rapport connu de ces polymères ainsi chargés. Dans certains cas d'application, il peut être in¬ téressant que lesdites polymères poreux insolubles soient de nature chimiques différents, par exemple hy- drophobes et hydrophyles afin de disposer de techniques d'activation' différenciées.In the case where it is desired that several immunoactive components are immobilized on a porous matrix, one can submit to the mixing operation specified in the first step above, a composition consisting of insoluble porous polymers, of identical or different nature, comprising known quantities of biological compounds and in a known ratio of these polymers thus charged. In certain applications, it may be in¬ téressant that said porous insoluble polymers are of different chemical nature, for example hy- drophobes and hydrophilic in order to have activation 'differentiated techniques.
Exemples d'exécution de l'inventionExamples of Execution of the Invention
L'invention sera décrite plus en détail en réfé¬ rence aux exemples d'exécution de l'invention, donnés uniquement à titre d'illustration sans caractère limita- tif.The invention will be described in more detail with reference to the exemplary embodiments of the invention, given solely by way of illustration without limitation.
Exemple 1Example 1
Cette technique permet de dépister en même temps des anticorps humains dirigés contre le cytomêgalovirus et des anticorps humains dirigés contre des antigènes de surface de l'hépatite B. Les deux antigènes sont asso¬ ciés, par des réactions différentes, à un support en po- lyacrylamide qui est ensuite incorporé dans une matrice en PTFE. 1. On hydrate 10 g de polyacrylamide (dimensions des granules 40 micromètres, limite d'exclusion 6000 daltons) au moyen de 100 ml de tampon au phosphate (0,1 M, pH = 7,2). On lave ensuite le gel deux fois avec le même tampon, puis on sépare le tampon par aspiration.This technique makes it possible to detect at the same time human antibodies directed against the cytomegalovirus and human antibodies directed against surface antigens of hepatitis B. The two antigens are associated, by different reactions, with a support in po- lyacrylamide which is then incorporated into a PTFE matrix. 1. 10 g of polyacrylamide (granule dimensions 40 micrometers, exclusion limit 6000 daltons) are hydrated using 100 ml of phosphate buffer (0.1 M, pH = 7.2). The gel is then washed twice with the same buffer, then the buffer is separated by suction.
2. On mélange ensuite le gel lavé avec 35 ml d'une solution aqueuse de glutaraldêhyde (25%). Cette suspension est agitée doucement pendant 5 heures, à la température ambiante. 3. Ensuite, le glutaraldêhyde est éliminé par la¬ vage avec le tampon au phosphate susmentionné. Ce lavage est répété 5 fois. 4. On ajoute 70 ml de solution d'antigène (1 mg de protéine/ml) dans du tampon au phosphate.2. The washed gel is then mixed with 35 ml of an aqueous glutaraldehyde solution (25%). This suspension is gently stirred for 5 hours at room temperature. 3. Next, the glutaraldehyde is removed by washing with the aforementioned phosphate buffer. This washing is repeated 5 times. 4. 70 ml of antigen solution (1 mg protein / ml) in phosphate buffer is added.
L'antigène du cytomegalovirus est obtenu, entre autres, à partir d'un extrait de glycine-hydroxy- de de sodium d'une culture de fibroblastes conta¬ minée par le cytomegalovirus ayant un effet cyto- pathogèhe de 90%.The cytomegalovirus antigen is obtained, inter alia, from an extract of sodium glycine-hydroxy- from a culture of fibroblasts contaminated by the cytomegalovirus having a cytopathic effect of 90%.
L'antigène de surface de l'hépatite B est ob¬ tenu notamment à partir de plasma humain conte- nant ledit antigène. L'antigène même est, par ex¬ emple, isolé du plasma par chromatographie d'af¬ finité avec des anticorps monoclonaux dirigés contre l'antigène de surface de l'hépatite B.The hepatitis B surface antigen is obtained in particular from human plasma containing said antigen. The antigen itself is, for example, isolated from the plasma by affinity chromatography with monoclonal antibodies directed against the hepatitis B surface antigen.
Les deux antigènes sont incubés séparément pendant 1 heure avec un gel de polyacrylamide ac¬ tivé.The two antigens are incubated separately for 1 hour with an activated polyacrylamide gel.
5. Après l'incubation, ces gels sont lavés 5 fois au moyen de 100 ml de tampon au phosphate (cf. ci-dessus) . 6. Les groupes aldéhydiques libres du gel activé sont bloqués par une incubation de 4 heures au moyen : d'éthanolamine (éthanolamine 0,1M, pH =7,6).5. After the incubation, these gels are washed 5 times with 100 ml of phosphate buffer (see above). 6. The free aldehyde groups of the activated gel are blocked by a 4 hour incubation using : ethanolamine (0.1M ethanolamine, pH = 7.6).
7. Ensuite, les gels sont lavés 3 fois au moyen de 100 ml de tampon au phosphate (0,02M, pH =7. Then, the gels are washed 3 times using 100 ml of phosphate buffer (0.02M, pH =
7,2), après quoi le tampon est éliminé et les gels sont séchés.7,2), after which the buffer is removed and the gels are dried.
8. Après un contrôle séparé de la quantité d'an¬ tigènes qui ont été associés par covalence, les gels sont mélangés en des proportions appro¬ priées. Le contrôle des quantités d'antigènes s'effectue sur la base des quantités d'anticorps qui s'associent aux gels de polyacrylamide après l'incubation. Le mélange de gels ainsi obtenu est ensuite incorporé dans une matrice en PTFE et est transformé en une feuille de 100 micromètres d'é¬ paisseur. Il est possible de produire plusieurs autres épaisseurs.8. After a separate control of the quantity of antigens which have been covalently combined, the gels are mixed in appropriate proportions. The quantities of antigens are controlled on the basis of the quantities of antibodies which associate with the polyacrylamide gels after incubation. The mixture of gels thus obtained is then incorporated into a PTFE matrix and is transformed into a sheet of 100 micrometers thick. It is possible to produce several other thicknesses.
9. A partir de cette feuille on estampe de petits disques de 5 mm de diamètre. Ces disques sont examinés par détermination immunologique. 10. La détermination immunologique est effectuée comme suit : un petit disque est incubé avec 0,5 ml d'une dilution au 1/200 de sérum de patients (dilution dans une solution de sel physiologique tamponnée au phosphate, pH 7,2, qui contient 1% d'albumine bovine et 0,2% de TVTEENv 20) . L'incu¬ bation s'effectue à la température ambiante pen¬ dant 1 heure. En même temps que les échantillons inconnus, on met en oeuvre un échantillon de con¬ trôle positif et un négatif (pour l'antigène de surface de l'hépatite B et le cytomegalovirus).9. From this sheet, small discs 5 mm in diameter are stamped. These discs are examined by immunological determination. 10. The immunological determination is carried out as follows: a small disc is incubated with 0.5 ml of a 1/200 dilution of patient serum (dilution in a physiological salt solution buffered with phosphate, pH 7.2, which contains 1% bovine albumin and 0.2% TVTEENv 20). The incubation is carried out at room temperature for 1 hour. At the same time as the unknown samples, a positive and a negative control sample (for the hepatitis B surface antigen and the cytomegalovirus) are used.
11. Après l'incubation, les disques sont lavés 5 fois avec 3 ml de tampon de lavage (solution de NaCl 0,15 molaire tamponnée au _phosphate, pH = 7,2, qui contient 0,5% de TWEEN ^< '-0) • 12. 200 microlitres d'une solution d'anticorps antihumains marqués à la peroxydase sont incubés avec les petits disques pendant 1 heure.11. After the incubation, the discs are washed 5 times with 3 ml of washing buffer (0.15 molar NaCl solution buffered with phosphate, pH = 7.2, which contains 0.5% of TWEEN ^ <'- 0) • 12. 200 microliters of a solution of anti-human antibodies labeled with peroxidase are incubated with the small disks for 1 hour.
13. Les disques sont de nouveau lavés 5 fois avec 3 ml de tampon de lavage (solution de NaCl 0,15 molaire tamponnée au phosphate, pH ≈ 7,2, qui contient 0,5% de TWEEN ^20).13. The discs are again washed 5 times with 3 ml of washing buffer (0.15 molar NaCl solution, phosphate buffered, pH ≈ 7.2, which contains 0.5% of TWEEN ^ 20).
14. On ajoute 500 microlitres de solution de sub¬ strat, consistant en 10 mg d'orthophénylènediami- ne dissous dans 10 ml de tampon au citrate et au phosphate (0,1M, pH ≈ 6,0) auquel on a ajouté 10 microlitres de peroxyde d'hydrogène à 30%.14. 500 microliters of substrate solution, consisting of 10 mg of orthophenylenediamine dissolved in 10 ml of citrate and phosphate buffer (0.1 M, pH ≈ 6.0) are added to which 10 microliters are added. 30% hydrogen peroxide.
15. L'incubation du substrat est arrêtée après 30 min au moyen de 1 ml d'HCl IN.15. The incubation of the substrate is stopped after 30 min using 1 ml of IN HCl.
16. La quantité de produit formé enzymatiquement est ensuite mesurée à 492 nm et comparée à la densité optique de contrôles négatifs. Si la den¬ sité optique d'un échantillon se trouve à plus de 5 déviations standard au-dessus du contrôle néga¬ tif, l'échantillon est considéré comme positif. La limite de 95%, déterminée sur une grande série de sêrums négatifs, . se trouve dans cette méthode aux alentours d'une densité optique de 0,200, mais celle-ci doit être de nouveau déterminée ex¬ périmentalement pour chaque application.16. The quantity of product formed enzymatically is then measured at 492 nm and compared with the optical density of negative controls. If the optical density of a sample is more than 5 standard deviations above negative control, the sample is considered positive. The 95% limit, determined on a large series of negative sera,. is found in this method around an optical density of 0.200, but this must be determined again experimentally for each application.
Exemple 2 Dosage de protéine C réactive (C.R.P.) dans du sérum de patients au moyen d'un immunoassay enzymatique appliqué suivant une technique en sandwich. Dans cette application, la fraction de gammaglobuline d'un sérum anti-C.R.P. hyperimmunisé est associée par covalence à un polymère de polyacrylamide enfermé dans une feuille de PTFE poreuse. De petits disques contenant cet anti¬ corps sont incubés avec du sérum de patients et une sé¬ rie d'étalons. On fait ensuite réagir l'antigène associé avec un anticorps marqué par une enzyme. Après élimina- tion par lavage de la fraction conjuguée non associée, on ajoute un substrat. La réaction enzymatique cinétique mesurée constitue une mesure de l'antigène présent. On peut effectuer une détermination quantitative par compa¬ raison avec les valeurs obtenues pour les étalons. Dans un processus servant d'exemple on réalise les opérations suivantes.Example 2 Assay of reactive protein C (C.R.P.) in patient serum using an enzyme immunoassay applied according to a sandwich technique. In this application, the gamma globulin fraction of an anti-C.R.P. hyperimmune is covalently associated with a polyacrylamide polymer enclosed in a porous PTFE sheet. Small disks containing this antibody are incubated with patient serum and a series of standards. The associated antigen is then reacted with an antibody labeled with an enzyme. After washing off the unassociated conjugate fraction, a substrate is added. The kinetic enzymatic reaction measured constitutes a measure of the antigen present. A quantitative determination can be made by comparison with the values obtained for the standards. In an example process, the following operations are carried out.
1. Une feuille de 100 cm^ ayant 100 micromètres d'épaisseur et comprenant du polyacrylamide (grosseur des granules = 40 micromètres, limite d'exclusion 6000 daltons) est lavée 3 fois au moyen de 10 ml d'une solution de NaCl 0,15 molai¬ re tamponnée au phosphate, pH = 7,2.1. A sheet of 100 cm 3 having a thickness of 100 micrometers and comprising polyacrylamide (size of the granules = 40 micrometers, exclusion limit 6000 daltons) is washed 3 times with 10 ml of a solution of NaCl 0, 15 phosphate buffered molai¬ re, pH = 7.2.
2. La feuille est ensuite transférée dans 60 ml d'une solution de NaCl 0,15 molaire tamponnée au phosphate, pH = 7,2 mélangée avec une solution de 30 ml de glutaraldêhyde (25% de glutaraldêhyde dans de l'eau) et incubée pendant 15 heures à la température ambiante.2. The sheet is then transferred to 60 ml of a 0.15 molar phosphate buffered NaCl solution, pH = 7.2 mixed with a solution of 30 ml of glutaraldehyde (25% glutaraldehyde in water) and incubated for 15 hours at the ambient temperature.
3. Le glutaraldêhyde est ensuite éliminé par la¬ vage en 5 opérations de lavage au moyen de 100 ml de tampon au phosphate 0,1M, pH = 7,2. 4. La feuille activée est transférée dans 50 ml d'une solution d'anticorps anti-C.R.P., en une concentration adaptée, dans du tampon au phospha¬ te (pH = 7,2). Cette concentration varie entre 1 mg et 0,001 mg/ml suivant la qualité de l'antisé- rum.3. The glutaraldehyde is then removed by washing in 5 washing operations using 100 ml of 0.1M phosphate buffer, pH = 7.2. 4. The activated leaf is transferred into 50 ml of a solution of anti-C.R.P. antibodies, in a suitable concentration, in phosphate buffer (pH = 7.2). This concentration varies between 1 mg and 0.001 mg / ml depending on the quality of the antiserum.
5. Après une incubation de 1 heure à 4 degrés Celsius la protéine non associée est éliminée par lavage en 5 opérations de lavage au moyen de 100 ml de tampon. 6. Les groupes aldéhydiques libres du gel activé sont bloqués par une incubation de 4 heures au moyen d'éthanolamine (éthanolamine 0,1M, pH5. After an incubation of 1 hour at 4 degrees Celsius, the unassociated protein is eliminated by washing in 5 washing operations using 100 ml of buffer. 6. The free aldehyde groups of the activated gel are blocked by a 4 hour incubation using ethanolamine (0.1M ethanolamine, pH
=7,6).= 7.6).
_ .7. On sèche la feuille et on y estampe des petits disques de 5 mm de diamètre._ .7. The sheet is dried and small discs 5 mm in diameter are stamped on it.
8. Pour la détermination quantitative, on trans¬ fère 'les petits disques dans des petits tubes (par exemple des tubes ayant une capacité totale de 7ml) . 9. Les sérums à tester sont dilués 500 fois dans un tampon de dilution (solution de NaCl 0,15 mo¬ laire tamponnée au phosphate, pH = 7,2, 1% d'al¬ bumine bovine et 0,5% de TWEEN^ θ). On dilue 500 fois, de la même manière, une série d'étalons comprenant 100, 500, 1000, 2500 et 5000 micro¬ grammes de C.R.P./ml. De ces dilutions, on trans¬ fère 500 microlitres dans les tubes contenant ces petites disques. L'incubation s'effectue douce¬ ment en rotation à température ambiante, pendant 1 heure.8. For the quantitative determination, the small disks are transferred into small tubes (for example tubes having a total capacity of 7 ml). 9. The test sera are diluted 500 times in a dilution buffer (0.15 mol NaCl solution, phosphate buffered, pH = 7.2, 1% bovine al¬ bumine and 0.5% TWEEN ^ θ). One dilutes 500 times, in the same way, a series of standards comprising 100, 500, 1000, 2500 and 5000 micro¬ grams of C.R.P./ml. From these dilutions, 500 microliters are transferred into the tubes containing these small disks. The incubation is carried out gently at room temperature for 1 hour.
10. Les disques sont ensuite lavés 5 fois avec 3 ml d'un tampon de lavage (solution de NaCl 0,15 molaire tamponnée au phosphate, pH =*7,2, 0,5% de10. The discs are then washed 5 times with 3 ml of washing buffer (0.15 NaCl solution phosphate buffered molar, pH = * 7.2, 0.5% of
TWEEN 5)20) .TWEEN 5) 20).
11. On ajoute ensuite aux disques, en 1 heure, un anticorps anti-C.R.P., conjugué avec de la per- oxydase, porté à une dilution appropriée dans une solution de NaCl 0,15 molaire tamponnée au phos¬ phate, pH = 7,2, contenant 1% d'albumine de boeuf et 0,5% de TWEEN0 .11. Then added to the discs, in 1 hour, an anti-CRP antibody, conjugated with peroxidase, brought to an appropriate dilution in a 0.15 molar NaCl solution buffered with phosphorus, pH = 7, 2, containing 1% beef albumin and 0.5% TWEEN0.
12. Après 5 opérations de lavage au moyen de 3 ml de tampon de lavage (solution de NaCl 0,15 molai¬ re tamponnée au phosphate, pH = 7,2, 0,5% de ajoute 500 microlitres de substrat (10 g d'orthophénylènediamine, 10 ml de tampon au citrate et au phosphate, pH = 6,0, 0,1M et 10 microlitres de peroxyde d'hydrogène à 30%) et on incube pendant 30 minutes à 22 degrés Celsius en l'absence de lumière.12. After 5 washing operations using 3 ml of washing buffer (0.15 molar NaCl solution, phosphate buffered, pH = 7.2, 0.5% of add 500 microliters of substrate (10 g of orthophenylenediamine, 10 ml of citrate and phosphate buffer, pH = 6.0, 0.1M and 10 microliters of 30% hydrogen peroxide) and incubate for 30 minutes at 22 degrees Celsius in the absence of light.
13. On arrête la réaction du substrat au moyen de 1 ml d-HCl IN. 14. On mesure la densité optique des liquides susmentionnés à 492 nm. Avec la densité optique des étalons, on établit une courbe d'étalonnage sur laquelle on peut lire la concentration en C.R.P. des échantillons de sérum. 13. The reaction of the substrate is stopped by means of 1 ml of IN HCl. 14. The optical density of the above-mentioned liquids is measured at 492 nm. With the optical density of the standards, a calibration curve is established on which the concentration of C.R.P. can be read. serum samples.

Claims

REVENDICATIONS
1. Immunosorbant utilisable dans les détermina¬ tions immunologiques, caractérisé en ce qu'il est à base d'une matrice poreuse en polytetrafluoroethylene où se trouve attaché d'une manière non covalente un polymère organique ou inorganique, hydrophile ou hydrophobe, in¬ soluble dans l'eau, auquel sont associés par covalence des antigènes et/ou des anticorps en des quantités bien déterminées et, s'il s'agit de divers antigènes et/ou anticorps, en des proportions bien déterminées entre- eux.1. An immunosorbent which can be used in immunological determinations, characterized in that it is based on a porous polytetrafluoroethylene matrix to which is attached in a non-covalent manner an organic or inorganic, hydrophilic or hydrophobic, in¬ soluble polymer in water, to which are covalently associated antigens and / or antibodies in well-defined quantities and, in the case of various antigens and / or antibodies, in well-determined proportions between them.
2. Immunosorbant selon la revendication 1 carac¬ térisé en ce que la porosité de matrice est comprise en¬ tre 20 et 80% en volume. 2. Immunosorbent according to claim 1 charac¬ terized in that the matrix porosity is between en¬ be 20 and 80% by volume.
3. Immunosorbant selon la revendication 1 ou 2 caractérisé en ce que les antigènes ou les anticorps sont couplés par covalence à un polymère insoluble dans l'eau qui avait déjà été préalablement incorporé à la matrice de >ase. 3. Immunosorbent according to claim 1 or 2 characterized in that the antigens or antibodies are covalently coupled to a polymer insoluble in water which had already been previously incorporated into the matrix of> ase.
4. Immunosorbant selon la revendication 1 ou 2 caractérisé en ce que les antigènes et/ou ces anticorps sont couplés à un polymère insoluble dans 1'eau qui est par la suite incorporé dans la matrice de base.4. An immunosorbent according to claim 1 or 2 characterized in that the antigens and / or these antibodies are coupled to a water-insoluble polymer which is subsequently incorporated into the basic matrix.
5. Immunosorbant selon la revendication 4 carac- térisé en ce que les polymères insolubles dans l'eau auxquels divers antigènes et/ou anticorps ont été sépa¬ rément couplés sont incorporés en proportions définies, dans la matrice de base.5. An immunosorbent according to claim 4, characterized in that the water-insoluble polymers to which various antigens and / or antibodies have been separately coupled are incorporated in defined proportions in the basic matrix.
6. Immunosorbant selon l'une quelconque des re- vendications 3 à 6 caractérisé en ce que les opérations de préparation sont effectuées à une température compri¬ se entre 4 et 40°C, de préférence à la température am¬ biante.6. Immunosorbent according to any one of claims 3 to 6 characterized in that the preparation operations are carried out at a temperature compri¬ se between 4 and 40 ° C, preferably at the am¬ biante temperature.
7. Immunosorbant selon 1'une quelconque des re- vendications 3 à 6 caractérisé en ce que 1'incorporation du polymère insoluble dans la matrice de base est effec¬ tuée par voie sèche. 7. An immunosorbent according to any one of claims 3 to 6, characterized in that the incorporation of the insoluble polymer into the base matrix is carried out dry.
8. Immunosorbant selon l'une quelconque des re¬ vendications 1 à 7 caractérisé en ce que la matière em¬ ployée comme polymère insoluble dans 1'eau est une cel¬ lulose, un polyacrylamide* le silicagel, un latex, le sêpharose, ou l'agarose «'.8. Immunosorbent according to any one of the re¬ vendications 1 to 7 characterized in that the material em¬ employed as a polymer insoluble in water is a celulose, a polyacrylamide * silica gel, a latex, sepharose, or agarose "'.
9. Procédé pour la préparation d'un immunosorbant selon l'une quelconque des revendications 1 à 8 caracté¬ risé en ce qu'il comporte les étapes suivantes :9. Process for the preparation of an immunosorbent according to any one of claims 1 to 8, characterized in that it comprises the following steps:
- on mélange ensemble le polymère poreux insoluble, ac- tivé, non activé ou couplé par l'antigène ou anticorps avec la matrice poreuse de base à l'état pulvérulant pour former un mélange sec,the insoluble, activated, non-activated or coupled by the antigen or antibody porous polymer is mixed together with the basic porous matrix in the pulverulent state to form a dry mixture,
- on soumet ce mélange sec à un traitement d'aggloméra¬ tion pour former des agglomérats, - on soumet les agglomérats formés à un traitement de broyage pour former des agglomérats broyés,- this dry mixture is subjected to an agglomeration treatment to form agglomerates, - the agglomerates formed are subjected to a grinding treatment to form crushed agglomerates,
- on presse ensemble les agglomérats broyés pour former un comprimé et- the crushed agglomerates are pressed together to form a tablet and
- on soumet- le comprimé à des laminages jusqu'à obten- tion d'un film ou d-'une feuille.- The tablet is subjected to lamination until a film or sheet is obtained.
10. Procédé selon la revendication 9 caractérisé en ce que ce film selon l'invention est produit de ma¬ nière pratiquement continue pour être ensuite découpée par tous moyens appropriés. 10. Method according to claim 9 characterized in that this film according to the invention is produced in a substantially continuous manner in order to be then cut by any suitable means.
11. Procédé selon la revendication 9 ou 10 carac¬ térisé en ce qu'on travaille à une température comprise entre 4°C et 37°C environ, de préférence à la températu¬ re ambiante (par exemple 20"C environ) pour éviter une dégradation des composants biologiques mis en oeuvre. 11. The method of claim 9 or 10 charac¬ terized in that one works at a temperature between 4 ° C and 37 ° C, preferably at room temperature (for example 20 "C) to avoid degradation of the biological components used.
12. Procédé selon l'une quelconque des revendica¬ tions précédentes caractérisé en ce que, dans le cas où l'on désire que plusieurs composantes immunoactives soient immobilisées sur une matrice poreuse, on soumet à l'opération de mélange spécifié dans la première étape ci-dessus, une composition constituée de polymères po¬ reux insolubles, de nature identique ou différentes, comportant des quantités connues de composés biologiques et dans un rapport connu de ces polymères ainsi chargés.12. Method according to any one of the preceding claims, characterized in that, in the case where it is desired that several immunoactive components are immobilized on a porous matrix, the mixing operation specified in the first step is subjected above, a composition consisting of insoluble porous polymers, of identical or different nature, comprising known amounts of biological compounds and in a known report of these polymers thus charged.
13. Procédé selon la revendication 12 caractérisé en ce que lesdites polymères poreux insolubles sont de nature chimiques différents, notamment hydrophobes et hydrophyles afin de disposer de techniques d'activation différenciées. 13. The method of claim 12 characterized in that said porous insoluble polymers are of different chemical nature, in particular hydrophobic and hydrophylic in order to have differentiated activation techniques.
EP19860902290 1985-04-25 1986-04-24 Immunoactive components immobilized in a porous material Withdrawn EP0220213A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
LU85868A LU85868A1 (en) 1985-04-25 1985-04-25 IMMUNOACTIVE COMPONENTS IMMOBILIZED IN A POROUS MATERIAL
LU85868 1985-04-25

Publications (1)

Publication Number Publication Date
EP0220213A1 true EP0220213A1 (en) 1987-05-06

Family

ID=19730450

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19860902290 Withdrawn EP0220213A1 (en) 1985-04-25 1986-04-24 Immunoactive components immobilized in a porous material

Country Status (3)

Country Link
EP (1) EP0220213A1 (en)
LU (1) LU85868A1 (en)
WO (1) WO1986006491A1 (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4829010A (en) * 1987-03-13 1989-05-09 Tanox Biosystems, Inc. Immunoassay device enclosing matrixes of antibody spots for cell determinations
US5100777A (en) * 1987-04-27 1992-03-31 Tanox Biosystems, Inc. Antibody matrix device and method for evaluating immune status
DE3814370A1 (en) * 1988-04-28 1989-11-09 Boehringer Mannheim Gmbh TEST PROVIDER FOR THE ANALYSIS OF A SAMPLING FLUID, METHOD FOR CARRYING OUT SUCH ANALYSIS AND METHOD OF PRODUCTION
DK269889A (en) * 1988-06-06 1989-12-07 Photest Diagnostics Inc HETEROGEN IMMUNOASSAY
WO1994015216A1 (en) * 1992-12-23 1994-07-07 Niyazmatov Agzamdzhan Akhtamov Process for obtaining a diagnostic reagent for detecting antigens and antibodies of infectious and other illnesses

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1150138A (en) * 1956-05-02 1958-01-08 Caoutchouc Et Matieres Plastiq Polyurethane reglomerated material
US3951748A (en) * 1974-11-11 1976-04-20 Medical Products, Inc. Sensitized matrix for detection of disease
US4166102A (en) * 1975-04-07 1979-08-28 Becton, Dickinson And Company Immobilized immunoadsorbent
US4357142A (en) * 1980-07-18 1982-11-02 Akzona Incorporated Glass support coated with synthetic polymer for bioprocess
US4340564A (en) * 1980-07-21 1982-07-20 Daryl Laboratories, Inc. Immunoadsorptive surface coating for solid-phase immunosubstrate and solid-phase immunosubstrate
GB2125547B (en) * 1982-07-31 1986-04-23 Mochida Pharm Co Ltd Simultaneous immunoassay of two or more substances

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8606491A1 *

Also Published As

Publication number Publication date
WO1986006491A1 (en) 1986-11-06
LU85868A1 (en) 1986-11-05

Similar Documents

Publication Publication Date Title
Hucknall et al. Simple fabrication of antibody microarrays on nonfouling polymer brushes with femtomolar sensitivity for protein analytes in serum and blood
US4332783A (en) Process for immunologic determination tests
AU753093B2 (en) Antigens embedded in thermoplastic
US5310885A (en) Process for immobilizing a protein containing substance on a solid phase
US4415700A (en) Hydrophilic latex particles and use thereof
US5061640A (en) Process for preparing a carrier useful in immunoassays by deposition of a complex of a specifically binding substance with hydrophobic protein, and the resulting carrier
US4347311A (en) Enzyme immunoassay for determining antigen specific antibodies and test kit for carrying out this assay
EP0106769B1 (en) Surface-grafted particulate support, its preparation and adsorbents for affinity-chromatography comprising this support, and its use, especially in biology
FR2490826A1 (en) PROCESS FOR MEASURING FREE THYROXIN OR FREE 3,5,3&#39;-TRIIODOTHYRONIN IN A LIQUID SAMPLE
US4317810A (en) Waffle-like matrix for immunoassay and preparation thereof
JPH0614050B2 (en) Preparation of test strips for immunological analysis
WO1994003530A1 (en) A method of chemical coupling on solid phases
CA1170180A (en) Process for the enzyme immuno determination in the heterogenous phase
EP0220213A1 (en) Immunoactive components immobilized in a porous material
JPH028271B2 (en)
FR2598811A1 (en) ENZYMATIC IMMUNOASSAY.
FR2543972A1 (en) METHOD FOR FIXING BIOLOGICAL MACROMOLECULES ON SUPPORTS
FR2547057A1 (en) METHOD FOR THE STABLE FIXING OF ANTIGENS AND ALLERGENS ON SOLID MEDIA, AND MEDIA FOR SUCH USE
EP2660603B1 (en) Immunological assay method
JPH01209370A (en) Method and apparatus for measuring immunologically active substance
AU621935B2 (en) Biological diagnostic assay system
FR2534031A1 (en) MEDIA FOR IMMUNOCHEMICAL ASSAY AND ASSAY REAGENTS USING SUCH MEDIA
JPS59182804A (en) Reactive fine particle and production thereof
JPS62231171A (en) Labeling complex
CA1203741A (en) Process for the detection and assay of a biological substance by erythroadsorption

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19870130

RIN1 Information on inventor provided before grant (corrected)

Inventor name: RAUS, JEF

Inventor name: BOSMANS, EUGEEN

Inventor name: MARTENS, HENRI