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
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
  • G01N33/586—Liposomes, microcapsules or cells
• • 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
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
• • 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
  • G01N33/54306—Solid-phase reaction mechanisms
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
  • Y10S436/809—Multifield plates or multicontainer arrays
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/819—Multifunctional antigen or antibody
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/825—Pretreatment for removal of interfering factors from sample
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/827—Lectins

Definitions

• the present invention relates to a method for detecting and assaying a biological substance by erythroadsorption.
• the hemagglutination technique for the determination of antibodies consists in mixing red blood cells sensitized by an antigen with a solution containing the antibody to be determined or to be determined. This technique is performed in the liquid phase.
• hemagglutination technique reference may be made to the work cited below for reference: "Handbook of experimental immunology” 3 vol. Ed. D.M. WEIR (1978) Blackwell Scientific.
• compositions with erythrocytes to which antibodies specific for said antigen are coupled, adding a development agent comprising an antibody reactive with said antigen, adding complement and noting the lysis of said erythrocytes as an indication positive of the presence of said antigen.
• the method of the invention makes it possible to avoid the use of erythrocytes sensitized by antibodies or antigens.
• the method according to the invention is a method for detecting and dosing a biological substance by erythroadsorption which consists of:
• the method according to the invention is suitable for serving, as a biological substance, antigens, antibodies, haptens, hormones, immunoglobulins and other substances of biological interest.
• the substance having a fixing affinity with respect to the biological substance to be assayed can be any substance capable of specifically fixing with said biological substance.
• the biological substance to be assayed is an antibody
• the substance having a binding affinity will then be an antigen and vice versa.
• the substance having a binding affinity for the biological substance to be assayed is immobilized on any support by conventional techniques.
• any insoluble support capable of being put in the form of a container can be used.
• constituent substance of such supports one can use cellulose and its derivatives, polyacrylamide, polymethacrylates of alkyl and other polymers of natural or synthetic origin as well as glass.
• microplates are used to immobilize the substance having an affinity for the biological substance to be assayed, for example polystyrene microplates. It has been found that microplates having cavities with a U or V cross section are the most suitable.
• the coupling product used as an assay reagent according to the invention is the coupling product of a specific ligand and of a ligand capable of reacting with erythrocytes. It also constitutes an object of the invention.
• specific ligand in the present description is meant any soluble substance which can react specifically with the substance having an affinity for the biological substance to be assayed or with the biological substance itself.
• soluble substance is meant in the present description any substance soluble in the media commonly used for biological reactions. They may be aqueous media, such as physiological media, or mixtures of aqueous and organic media.
• the specific ligand used must be such that the ligand-specific ligand coupling product capable of reacting with erythrocytes is dissolved in an aqueous medium.
• aqueous media is meant according to the invention the aqueous media, buffered or not, commonly used in the biological field, such as phosphate buffer solutions, buffer solutions containing a detergent, such as "Tween” or gelatin. , bovine serum albumin, bovine lactalbumin and other substances usually used in such fields.
• the specific ligands corresponding to such a definition are in particular antibodies, macromolecular antigens, haptens, hormones and their receptors and similar substances. Among the specific ligands mentioned above, antibodies and antigens are most commonly used.
• the ligand capable of reacting with erythrocytes is a substance which has recognition sites specific determinants of erythrocytes or substances attached to erythrocytes.
• a coupling product between a lectin and a specific ligand has been described and its applications in the biological field, in particular for the determination of biological substances in which the marker can be red blood cells.
• a substance other than a lectin will be used as ligand capable of reacting with normal or modified erythrocytes.
• ligands include anti-red blood cell antibodies, avidin, biotin and the like.
• avidin and biotin are two substances which interact strongly by non-co valent way. This strong interaction has already been used in immunoenzymatic techniques such as the so-called avidin-biotin bridge technique or the technique using an enzyme labeled with avidin. To this end, reference may be made to the article by GUESD0N et al. (The tournal of Histochemistry and Cytochemistry vol. 27, n ° 8, p.1131-1139, 1979).
• erythrocytes denotes both normal erythrocytes and modified erythrocytes, that is to say carrying a specific substance of the ligand used and which is defined generally as a ligand capable of reacting with erythrocytes.
• erythrocytes The modification of erythrocytes is done according to conventional means within the reach of those skilled in the art, that is to say by coupling techniques commonly used in the biological field (see the article by GUESDON et al previously cited).
• biotin can be attached to erythrocytes using biotinyl-N-hydroxysuccinimide according to the procedure described in the article by JASIEWICZ et al
• the specific ligand-ligand coupling capable of reacting with erythrocytes is carried out by means of an appropriate coupling agent, such as for example glutaraldehyde and benzoquinone.
• the coupling is advantageously carried out by covalent bonds. It is carried out according to a process analogous to those used for the coupling of proteins.
• the coupling can be carried out by a process similar to that described for obtaining antibody-enzyme conjugates in Scand. J. Immunol., Vol 8 supp 7 p.7-23 1978.
• Such a coupling process consists in bringing into contact, in one or more stages, a coupling agent with the substances to be coupled.
• Benzoquinone can also be used as a coupling agent.
• the determination of erythroadsorption can be done in several ways. For example, it can be seen visually that the red blood cells are adsorbed on the surface of the support on which the substance having been immobilized has a binding affinity for the biological substance to be assayed.
• the method of the invention makes it possible to identify a particular biological substance in a given biological fluid. Yes, on the contrary, the biological liquid does not contain the particular biological substance, the erythrocytes are not adsorbed and form a pellet at the bottom of the container, for example microplate wells.
• the method of the invention also makes it possible to quantitatively determine a given biological substance. For this purpose, unreacted red blood cells are removed, for example by aspiration with a pipette.
• the adsorbed erythrocytes are lyzed, for example with distilled water, and the substances released by the red blood cells, for example hemoglobin or the substances artificially introduced by the experimenter, are assayed by spectrophotometry at 403 nm, which allows this dosing process to be fully automated.
• Hemoglobin can also be determined by an enzymatic reaction.
• one of the peroxidase substrates such as ortho-dianisidine or ortho-phenylene diamine. Reading is also done by spectrophotometry at 400 nm for orthodianisidine and at 492 nm for ortho-phenylene diamine.
• the quantity of substances released by the red blood cells for example the quantity of hemoglobin released, is proportional to the quantity of the substance to be assayed, which makes it possible to obtain, for example, the assay of an antigen or an antibody present in the sample with reference to a standard range of red blood cell hemolysis established under the same conditions.
• the method according to the invention is particularly suitable for the assay of antibodies and antigens and will be described in more detail below, without however limiting its scope, with reference to the attached single figure, on which the reaction diagram is represented. of the process of the invention.
• (1) represents the first step of the method of the invention during which immobilizes on the support (10) antigens (Ag).
• the support (10) represents a V-shaped well of a microplate.
• the immobilization of the antigens which in this particular case constitute the substance having an affinity for the biological substance to be assayed, namely the antibody, is carried out for example by passive absorption or, if necessary, by covalent bond depending on the nature of the support.
• Step (2) consists of an incubation of the immobilized antigen with the biological fluid containing the antibody (Ac) to be assayed, for example the serum to be titrated.
• Step (3) of the process of the invention consists in incubating the support resulting from step (2) with a specific ligand-ligand coupling product capable of reacting with erythrocytes (P).
• the specific ligand is an antibody directed against the immunoglobulins of the human or animal species of the serum to be titrated.
• erythrocytes (E) are added, which are adsorbed by the coupling product only if the serum to be titrated contains the antibody corresponding to the immobilized antigen, otherwise the erythrocytes are not fixed and fall to the bottom of the container.
• the process of the invention therefore makes it possible to rapidly detect the presence of a given antibody in a patient's serum.
• the quantitative assay that is to say the determination of the amount of antibody contained in the serum, will be easily carried out as described above, that is to say by lysis of the erythrocytes and determination of the amount of substance released by these erythrocytes, for example the amount of hemoglobulin.
• red sheep cells are used for erythroadsorption. Since the sera. show cross-reactions from one species to another and they often contain antibodies against red blood cells, for example antibodies against red blood cells in sheep, care should be taken to absorb the serum with red blood cells. test to avoid false positive reactions. Although such a technique uses means known to those skilled in the art, some of its implementation variants will be indicated below.
• this absorption can be carried out by subjecting the serum to the steps below which consist: a) of decomplementing the serum, that is to say heat it at approximately 56 ° C for approximately 30 minutes, b) adding an excess of sheep red blood cells to said serum, for example to obtain a 10% concentration of red blood cells; c) leaving the serum at room temperature for approximately 1 hour with gentle stirring; d) centrifuge to recover the absorbed serum and remove the pellet of red blood cells.
• the serum will not be decomplemented but it will be absorbed by red blood cells, for example sheep's milk, treated with a tanning agent, preferably glutaraldehyde.
• the method according to the invention is a simple and sensitive method, particularly suitable for the detection or the assay of antibodies and antigens. Its sensitivity is such that it makes it possible to detect an antigen present at a concentration of the order of nanograms per milliliter.
• the coupling product of human anti-immunoglobulin antibodies and sheep anti-red blood cell antibodies obtained by the coupling process was used. single step using glutaraldehyde described in Immunochemistry, 1969, 6, 43-53.
• the antibodies were dialyzed overnight at 4 ° C. against a 0.1 M phosphate buffer, pH 6.8. 30 ⁇ l of a 1% glutaraldehyde solution were added with gentle stirring to 1 ml of a 0.1 M phosphate buffer pH 6.8 containing 2 mg of anti-sheep red blood cell antibodies and 2 mg of anti-human immunoglobulin antibodies. After a 3 hour incubation at room temperature, 50 ⁇ l of a 2 M glycine solution was added and two hours later the mixture was dialyzed for one overnight at 4 ° C against a buffered physiological solution (phosphate buffer). After centrifugation (3000g, 30 minutes) and addition of an equal volume of glycerol, the preparation obtained was stored at - 20 ° C until use.
• phosphate buffer buffered physiological solution
• Polystyrene plates having U or V wells were coated with calf thymus DNA by incubating 0.2 ml of DNA solution (0.5 mg / ral) per well for 2 hours at 37 ° C and 16 hours at 4 ° C.
• 0.2 ml solution of the test serum (containing anti-DNA antibodies) in PBS-TWEEN containing 1% bovine serum albumin was added to each well coated with DNA and allowed to incubate for 16 hours at 4 ° C. .
• the plates were washed 3 times with PBS-TWEEN, and 0.2 ml of the coupling product prepared as above was added to the wells.
• the same assay was carried out using as a coupling product the coupling product of human anti-immunoglobulin antibodies and avidin obtained according to the procedure described above using 30 ⁇ l of a 1% glutaraldehyde solution, and a solution of 1 ml of 0.1 M phosphate buffer pH 6.8 containing 8 mg of avidin and 2 mg of human anti-Ig antibodies.
• the erythrocytes were modified using biotin according to the following procedure, analogous to that described by JASIEWICZ et al. in Exp.Cell. Res. 1976, 100, 213-217 using biotinyl-N-hydroxysuccinimide.
• the erythrocytes were biotinylated by the addition of 7 mg of bio tinyl-N-hydroxysuccinimide dissolved in 80 ⁇ l of dimé thylformamide distilled in 2 ml of a suspension at
• the same assay was carried out using the coupling product co ⁇ canavaline A-anti-human Ig antibody obtained according to the method described in patent application FR 80. 11,470.
• the solution of the anti-human Ig antibody coupling product used in step 3 of the method contained methyl-alpha-D-mannoside to prevent the concanavalin reacting with glycosidic fractions present in the test serum and which would distort erythroadsorption.
• the results obtained according to this procedure are also recorded in Table I.
• the same assay was carried out (anti-DNA antibody) as in Example 1, but the incubation step was replaced tion with the ligand-specific ligand coupling product capable of reacting with erythrocytes by an incubation step with a specific ligand-enzyme coupling product; after incubation and then washing, the enzyme activity was determined.
• glucose oxidase As enzymes, glucose oxidase (Asperqillus niqer glucose oxidase (quality 1) supplied by Boehringer-Mannheim RFA) was used. The procedure for revealing this enzyme is described in detail in the articles below:

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• 1980
  • 1980-07-09 FR FR8015293A patent/FR2486657A1/fr active Granted
• 1981
  • 1981-07-08 EP EP81902035A patent/EP0055751B1/fr not_active Expired
  • 1981-07-08 JP JP56502386A patent/JPS57500997A/ja active Pending
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• 1985
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