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.

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• Health & Medical Sciences (AREA)
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• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

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Cited By (5)

Citations (6)

• 1985
  • 1985-04-25 LU LU85868A patent/LU85868A1/fr unknown
• 1986
  • 1986-04-24 WO PCT/BE1986/000013 patent/WO1986006491A1/fr unknown
  • 1986-04-24 EP EP19860902290 patent/EP0220213A1/de not_active Withdrawn

Patent Citations (6)

Non-Patent Citations (1)

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