WO1993010452A1 - Reactif de diagnostic, ses applications dans un procede pour la determination d'un analyte et dispositif pour la mise en ×uvre dudit procede - Google Patents

Reactif de diagnostic, ses applications dans un procede pour la determination d'un analyte et dispositif pour la mise en ×uvre dudit procede Download PDF

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Publication number
WO1993010452A1
WO1993010452A1 PCT/FR1992/001049 FR9201049W WO9310452A1 WO 1993010452 A1 WO1993010452 A1 WO 1993010452A1 FR 9201049 W FR9201049 W FR 9201049W WO 9310452 A1 WO9310452 A1 WO 9310452A1
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WIPO (PCT)
Prior art keywords
ligand
dye
reaction
binding substance
coupling
Prior art date
Application number
PCT/FR1992/001049
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English (en)
French (fr)
Inventor
Hamidou Samake
Philippe Goumard
Gérard Somme
Original Assignee
Clonatec
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 Clonatec filed Critical Clonatec
Priority to JP5509027A priority Critical patent/JPH06504625A/ja
Publication of WO1993010452A1 publication Critical patent/WO1993010452A1/fr

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    • 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
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

  • the present invention relates to a diagnostic reagent and to its applications in a method for the rapid qualitative and quantitative determination of a ligand or analyte in a fluid.
  • the present invention also relates to a device for implementing said method.
  • the invention applies more particularly, but not limitatively, to the detection of the presence of antibodies or antigens in a biological fluid, to the detection of toxic substances, viruses or bacteria in any fluid.
  • Patents or Patent Applications provide a vast literature on the different techniques for the production of conjugates or discernible signals in immunoassays.
  • IMMUNOMEDICS INC. Describes antibodies modified by a synthetic poly type polymer (amide / urea / thiourea)
  • the antibody described in this Request is present a structure of the Ac- (lr-PAUT) n type , in which Ac is the antibody, lr is a binding element between the antibody and the poly (amide / urea / thiourea), such as an ester, an amide , urea, carbamate or thiourea.
  • KODAK Corp. describes a composition also based on leucoimidazole, comprising hydroxy-4 'acetanilide, at a concentration up to 2.5 mmol.
  • composition can be used in an immunological test in which a peroxidase-labeled conjugate is also used.
  • the labeled complex formed in essei is then detected by adding the dye-forming composition, which will color in the presence of the peroxidase and its substrate such as H 2 O 2 .
  • composition has the advantage of making it possible to increase the concentration of conjugate labeled with the enzyme, to detect low concentrations of antigens and to decrease the duration of the test, without however modifying the sensitivity or increase background noise.
  • the present invention has therefore set itself the aim of providing a ready-to-use colored reagent which is stable and which makes it possible to avoid the stage of development with a chromogen, the reading of the result being in effect direct.
  • Such a reagent can be used in particular in all reactions of the ligand-receptor type, in which the ligand is defined as any substance whose detection may be of interest and the receptor is defined as any substance capable of specifically binding to a ligand.
  • receptor of any molecule of polymeric, protein, polypeptide, carbohydrate, lipid nature, the physicochemical or biological properties of which are of interest in the field of diagnosis, immunology, molecular biology. , pathology, immunohistochemistry or biochemistry.
  • the subject of the present invention is a colored reagent, characterized in that it corresponds to formula I below:
  • L represents a binding substance capable of specifically binding either to a ligand to be detected in a fluid, or to a receptor of said ligand;
  • r is a reactive group providing the link between the
  • Col dye and substance L which group r is capable of reacting by addition, substitution or any other reaction with substance L;
  • s constitutes the support of the reactive group and is of the type (XH) x - (Y) y - (Z) z , in which
  • X represents a nitrogen atom
  • Y represents a CO group or an SO 2 group
  • Z represents a CH 2 group
  • x and y represent 0 or 1
  • z 0, 1 or 2;
  • Col is a chemical molecule whose structure
  • S represents one or more solubilizing groups of
  • L is an antibody, an antigen, a nucleic acid or any other molecule capable of binding to the desired ligand, or to a receptor for the latter, of formula of the type R'-NH 2 or R'-OH, R 'constituting the residue of an amino acid, peptide, protein, nucleic acid or any other suitable molecule.
  • . r is a reactive group capable of reacting with substances L (antibodies, antigens, nucleic acid, etc.) and is in particular chosen from:
  • aromatic groups such as halogenitrobenzenes, benzoylazides, sulfofluorides, or * heterocyclic groups such as cyanuryl chloride derivatives (chlorotriazine, in particular), tetrachloropyrimidine, trichloropyrimidine, trifluorochloropyrimidine, dichloroquinoxichlorinichloride dichloroquinoxichloridine, dichloroquinoxinichloride
  • azo and phenylazopyrazolonics for example, if one wishes to obtain a yellow coloring, one will rather choose azo and phenylazopyrazolonics; to obtain an orange or red coloration, the monoazoics derived from J acid and H acid; for obtaining a blue coloring, the derivatives of 1-amino anilino-4 anthraquinone; to obtain a coloring blue, turquoise or green, copper and nickel phthalocyanines; and to obtain a violet, blue, brown, gray and black coloration, the copper, chromium and cobalt complexes of the azo series.
  • Procion ® Procion H ® , Procilan ® (HERE); Cibacron ® , Lanasol ® (CIBA); Remazol ® (HOECHST); Levafix ® , Verofix ® (BAYER); Reacton ® , Reactofil ® (GEIGY); Drimaren ® , Drimalan F ® (SANDOZ); Primazin ® (BASF); Solidazol ® (CASSELLA); Reatex ® (FRANCOLOR).
  • binding substance-dye reagents when implemented in a diagnostic method, have the advantage of increasing the sensitivity and reactivity of said method significantly compared to a method in which the dye is fixed on particles (EP 165 633) as well as making it possible to carry out said detection in a single step; they can also be further advantageously coupled to one half of an affinity pair.
  • An antibody-dye reagent for example, can be used to reveal an antigen-antibody immunological reaction, in the context of:
  • bacterial or viral antigens in the detection of polyspecific hormones, tumor markers or cell differentiation, cytokines;
  • the reagent according to the invention allows, in all cases, the direct reading of the result on the support used.
  • ELISA type tests direct or indirect competition, sandwich, immunocapture
  • bil-type supports of polystyrene, tubes, microtitration plates, fins, etc. and in which the reagent conforms to the the invention, for example an antibody-dye reagent is used in place of the enzymatic, fluorescent or (chemi) luminescent conjugate; . in agglutination tests in which the particles are sensitized with a reagent according to the invention;
  • the present invention also relates to a process for preparing the colored reagent according to the invention, characterized in that it comprises the coupling of a dye of formula rs-Col- (S) n , in which r, s. Col, S and n have the same meaning as above with a binding substance L, which coupling is carried out by reaction between r and L.
  • the reactive groups r can react directly with the binding substance L or indirectly, after chemical modification (activation) or by means of bifunctional reagents.
  • said coupling is carried out by direct reaction by a substitution process with a ⁇ - or a ⁇ -elimination.
  • coupling by direct reaction by substitution can be carried out by using any of the following reactions:
  • R-NH 2 or R-OH type can also be coupled to a binding substance of the R-NH 2 or R-OH type by a direct substitution reaction.
  • said direct reaction coupling comprises an elimination reaction followed by an addition reaction.
  • coupling by direct reaction by elimination followed by addition can be carried out as follows:
  • said coupling is carried out by indirect reaction, by activating the dye and / or the binding substance, prior to the reaction between r and L.
  • the subject of the present invention is also a method for rapid qualitative and quantitative detection of the presence of a ligand in a fluid, of the type comprising bringing a sample possibly containing the ligand to be detected with at least one binding substance specific, at least one of which is a ligand receptor, characterized in that one of said specific binding substances (which binds either to the ligand or to a ligand receptor) is coupled to a colorant formula rs-Col- (S) n to form a reagent according to the invention.
  • At least one of the specific binding substances is fixed on an appropriate solid support.
  • solid support means both beads (polyacrylamide, glass, polystyrene, etc.) as well as particles, tubes, discs, membranes, strips or microplates.
  • all of the reagents are fixed on an appropriate solid support; such support is said reagent.
  • At least one of the reagents is able to migrate on said support.
  • At least one of the reagents is in a fixed position on said support.
  • said support is a reactive membrane with low protein retention.
  • the method comprises at least:
  • the ligand present in the sample reacts with the receptive reagent coupled to a half affinity pair and / or a dye of formula rs-Col - (S) n and in particular makes it possible to obtain a ligand concentration effect; then the labeled ligand / receptor complex reacts with the reagent of step (2), fixed on the support, which stops the migration of said ligand and allows direct reading of the result (presence of a coloration in the reading window ).
  • affinity pairs of the biotin-avidin or streptavidin pairs, a heavy metal derivative-thio group, various homopolynucleotides such as poly dG-poly dC, poly dA-poly dT and poly dA-poly dU, hapten-antibody anti-hapten, enzyme-substrate, enzyme-inhibitor, lectin-glycan.
  • the number of reagents used in the process according to the invention essentially depends on the method used (sandwich, direct, competition, capture in particular).
  • said method comprises:
  • the present invention also relates to a ready-to-use diagnostic kit for implementing the method for detecting a ligand according to the invention, characterized in that it comprises at least:
  • the present invention further relates to a device for the rapid qualitative and quantitative determination of the presence of a ligand, in a fluid, of the type comprising a reaction zone containing a reagent intended to react with the desired ligand and a development zone, which device is characterized in that it comprises:
  • reaction zone which comprises an activated membrane intended to receive a sample of fluid to be tested and intended to be associated with at least one specific binding substance of the ligand to be detected (receptor) suitably coupled to a half of pair 'affinity, and / or a dye of formula rs-Col- (S) n , able to migrate;
  • a revelation zone associated with a substance attached to said support chosen from the group which comprises the other half of the affinity pair, a ligand-specific binding substance and a receptor-specific binding substance and in which the complex possibly formed in zone (a) is fixed.
  • a zone for checking the correct functioning of the test is provided and comprises a suitable solid phase containing a reference reagent.
  • the reference reagent, unlabeled is capable of recognizing a reagent of the reaction zone (a) and can in particular be identical to the ligand that it is sought to detect.
  • the binding substance coupled to one half of an affinity pair and / or to a dye of formula I is a receptor for the desired ligand, in particular an antigen, an antibody or a nucleic acid.
  • said device comprises:
  • a first reaction zone which includes an activated membrane intended to receive a sample of fluid to be tested and intended to be associated with at least one reagent suitably coupled to one half of an affinity pair, capable of recognizing a desired ligand and of migrating;
  • a second reaction zone which comprises an activated membrane which is associated with a colored reagent according to the invention, capable of binding to the complex formed in the first reaction zone, and
  • a zone for checking the correct functioning of the test is provided and comprises a suitable solid phase containing a reference reagent.
  • the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention.
  • EXAMPLE 1 Coupling of polyclonal antibodies (Ab) and an activated dye type ⁇ -sulfatoethyl sulfone (EFR Royal Blue ® Bayer).
  • the conjugate obtained is purified, by gel filtration chromatography on a GF05 IBF column equilibrated beforehand in 0.1 M carbonate bicarbonate buffer pH 9.5 (2 mg of gel / 1 mg of conjugate).
  • the characterization of the conjugate is done first by HPLC chromatography on a TSK column on SW3000 balanced in PBS.
  • the measurement of the optical densities at 280 nm and 600 nm makes it possible to calculate the concentrations of antibodies and dye.
  • the dye concentration (C) is given by the following relationship:
  • the antibody concentration is given by the following relationship:
  • the conjugate thus obtained is stored at + 4oC while waiting for its use.
  • EXAMPLE 2 Coupling comprising activation of the dye (indirect coupling).
  • a solution of 100 mM NaI ⁇ 4 is added in an amount sufficient to obtain a molarity of 10 mM and incubation is carried out for 2 hours at room temperature.
  • the dye is put on hold while waiting for coupling at 4oC in the dark.
  • the final volume must be ⁇ 1, 3 ml.
  • a volume of 0.5 M DTT (77.1 mg / ml) in PBS pH 7.4 50 mM phosphate, 150 mM NaCl equal to 1/20 of the volume of antibody is added to the Ig-SPDP solution obtained above. above. Incubate 18-20 min at room temperature with stirring, then filter through Millex ® 0.8 ⁇ m.
  • Coupling of 0.15 mg of dye per mg of antibody is carried out overnight at + 4oC in the dark on a slow speed roller shaker.
  • reaction is stopped with 25% by volume of 0.12 M N-ethylamine (12.51 mg / ml) in 0.1 M phosphate buffer pH 6, 5 mM EDTA, for 30 minutes at room temperature on rollers.
  • the conjugate is purified by chromatography on GF05 in 0.1 M carbonate bicarbonate buffer pH 9.5.
  • CC (mg / ml) OD 600 ⁇ 0.12.
  • the antibody concentration is given by the following relationship: ,
  • the immunofilter consists of '' a plastic cylinder on which a reactive membrane is fixed. All reagents filtered through the membrane are trapped inside the immunofilter cylinder.
  • the principle is based on the capture of specific antibodies by two homologous synthetic peptides, derived from the transmembrane glycoproteins of the HIV1 and HIV2 viruses (peptides identical to those described in the CLONATEC RAPID HIV1 / HIV2 Ab ® kit).
  • the reagents provided in the kit allow 30 tests to be carried out:
  • R1 ready-to-use immunofilters
  • R4 colored tracer (anti-IgG-Royal Blue EFR conjugate), R5: R4 reconstitution buffer,
  • R6 to R8 control sera (negative, weak positive and strong positive).
  • the samples to be tested are diluted 1/10 in the diluent meant for that purpose.
  • a result is positive if at least one blue spot appears on the membrane. Depending on its position, it indicates the type of antibody detected:
  • HIV1 or HIV2 will be given by the most intense spot.
  • Table I collates the results obtained with some sera tested using the kit described above and according to the procedure described.
  • the test consists of a strip on which an activated membrane is fixed.
  • the principle is based on the capture of specific antibodies by a synthetic peptide fixed on the membrane.
  • the biological sample is brought into contact with the reactive membrane.
  • the specific antibodies bind to the synthetic peptide and are detected by molecules conjugated to a dye (reagent according to the invention) according to FIG. 1, or to latex particles (control reagent). If the sample contains anti-HIV antibodies a colored band appears at the end of the reaction.
  • Table II a colored band of intensity "+" appears with the 10 positive HIV sera tested, without significant background noise on the membrane. No signal or background noise on the membrane is observed with the 10 HIV negative sera tested.
  • the results show that the use of a colored tracer allows both better specificity and satisfactory sensitivity to be obtained, compared with the use of latex particles.
  • the use of latex particles also has the disadvantage of a longer technique for sensitizing latex particles, compared to that of colored molecules.
  • the use of the reagent according to the invention allows a faster result to be obtained and has the advantage of being of reduced cost.
  • EXAMPLE 7 Determination of HCG with the process according to the invention.
  • a reagent according to the invention is prepared according to the protocols specified above:
  • the reagent according to the invention is an anti-HCG antibody conjugated to a dye as defined above; migration occurs, according to Figure 2a, on a nitrocellulose membrane attached to a Mylar ® sheet.
  • the serum to be tested is deposited in (1); in the presence of HCG in said serum tested; an anti-HCG dye HCG antibody 1 complex (complex 1) forms in (2) and migrates to the level of the anti-HCG antibody 2 fixed in (3), where it is stopped by the formation of a complex 2 between the HCG of -complex 1 and the anti-HCG antibody 2 fixed; part of the anti-HCG-dye antibody 1 fixes at the level of the operating indicator (4), which is HCG; the positive result is indicated by a colored "+" sign.
  • the anti-HCG-dye antibody 1 (2) migrates and becomes fixed at the level of the operating indicator (4) (HCG fixed); this negative result is indicated by a "
  • the reagent according to the invention is an anti-HCG-dye-biotin antibody; the migration takes place in accordance with FIG. 2b, on the same type of membrane as above.
  • the serum to be tested is deposited in (1); in the presence of HCG in said serum tested, an anti-HCG-dye-biotin HCG complex (complex 1) forms in (2) and migrates; this complex 1 saturated with HCG, does not bind to the level of fixed HCG (3) and migrates to the level of streptavidin (4) where it is stopped by the formation of a biotin-streptavidin complex; in (2), due to the presence of both an anti-HCG-dye-biotin and mouse-dye antibodies, the mouse-dye antibodies bind to the corresponding operating control (5) anti-mouse antibodies; the positive result is then materialized by a colored "+" sign.
  • mice-dye antibodies present in (2) migrate and bind to the anti-mouse function control in (5); this negative result is indicated by a "
  • the dye is represented by the sign "X" in Figures 2a and 2b.
  • EXAMPLE 8 dosage of thyroid hormones.
  • a reagent according to the invention is prepared according to the protocols specified above:
  • the reagent according to the invention is an anti-thyroid hormone antibody (T3 or T4) conjugated to a dye as defined above; migration occurs, in accordance with Figure 3a, on a nitrocellulose membrane mounted on a Mylar ® sheet.
  • T3 or T4 anti-thyroid hormone antibody
  • the serum to be tested is deposited in (1); in the presence of thyroid hormones (HT), an HT anti-HT-dye-biotin complex is formed in (2) and migrates to (4) where a biotin-streptavidin complex is formed; the positive result is indicated by a colored "-" sign.
  • HT thyroid hormones
  • the reagent according to the invention anti HT-dye-biotin present in (2) migrates and forms a complex with the HT fixed in (3); the negative result is materialized by the absence of sign in (4).
  • the reagent according to the invention is an anti-HT-dye-biotin antibody; the migration takes place in accordance with FIG. 3b, on the same type of membrane as above.
  • the serum to be tested is deposited in (1); in the presence of HT in said serum, the latter binds in (3) to the anti-HT-dye conjugate and is in competition with the HT-biotin conjugate deposited in (2); a result is positive in the absence of coloring because the HT-biotin conjugate, which migrates to (4) and results in the formation of a biotin-streptavidin complex is not colored.
  • the negative result is materialized by the presence of a color in (4) because the HT-biotin (2) conjugate when migrating has formed a complex with the anti HT-dye (3) before stopping in (4).
  • the dye is represented by the sign "X" in Figures 3a and 3b.

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PCT/FR1992/001049 1991-11-15 1992-11-12 Reactif de diagnostic, ses applications dans un procede pour la determination d'un analyte et dispositif pour la mise en ×uvre dudit procede WO1993010452A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP5509027A JPH06504625A (ja) 1991-11-15 1992-11-12 診断用試薬、それの試験液中における分析物の迅速な定性及び定量的測定法への適用、及びその方法を実施する装置

Applications Claiming Priority (2)

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FR9114117A FR2683911B1 (fr) 1991-11-15 1991-11-15 Reactif de diagnostic, ses applications dans un procede pour la determination qualitative et quantitative rapide d'un analyte dans un fluide a tester et dispositif pour la mise en óoeuvre dudit procede.
FR91/14117 1991-11-15

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WO1993010452A1 true WO1993010452A1 (fr) 1993-05-27

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EP (1) EP0542627A1 (US20100012521A1-20100121-C00001.png)
JP (1) JPH06504625A (US20100012521A1-20100121-C00001.png)
DE (1) DE542627T1 (US20100012521A1-20100121-C00001.png)
ES (1) ES2049702T1 (US20100012521A1-20100121-C00001.png)
FR (1) FR2683911B1 (US20100012521A1-20100121-C00001.png)
GR (1) GR930300109T1 (US20100012521A1-20100121-C00001.png)
WO (1) WO1993010452A1 (US20100012521A1-20100121-C00001.png)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0015519A1 (en) * 1979-03-01 1980-09-17 Fuji Photo Film Co., Ltd. Method for immunological analysis
GB2081257A (en) * 1980-07-30 1982-02-17 Abbott Lab Biologically Interesting Compounds Labeled with Chlorotriazinyl-aminofluorescein
EP0064833A2 (en) * 1981-04-27 1982-11-17 The Public Health Laboratory Service Board High pressure liquid affinity chromatography
EP0201633A2 (en) * 1984-12-20 1986-11-20 Abbott Laboratories Substituted anilide compounds and their use
US4743551A (en) * 1984-11-05 1988-05-10 Miles Inc. Purification of microbial rennet from Mucor miehei
WO1990007950A1 (en) * 1989-01-12 1990-07-26 Eaton John W Biocompatible materials comprising albumin-binding dyes

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0015519A1 (en) * 1979-03-01 1980-09-17 Fuji Photo Film Co., Ltd. Method for immunological analysis
GB2081257A (en) * 1980-07-30 1982-02-17 Abbott Lab Biologically Interesting Compounds Labeled with Chlorotriazinyl-aminofluorescein
EP0064833A2 (en) * 1981-04-27 1982-11-17 The Public Health Laboratory Service Board High pressure liquid affinity chromatography
US4743551A (en) * 1984-11-05 1988-05-10 Miles Inc. Purification of microbial rennet from Mucor miehei
EP0201633A2 (en) * 1984-12-20 1986-11-20 Abbott Laboratories Substituted anilide compounds and their use
WO1990007950A1 (en) * 1989-01-12 1990-07-26 Eaton John W Biocompatible materials comprising albumin-binding dyes

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GR930300109T1 (US20100012521A1-20100121-C00001.png) 1993-10-29
ES2049702T1 (es) 1994-05-01
DE542627T1 (de) 1993-11-04
FR2683911B1 (fr) 1995-02-03
EP0542627A1 (fr) 1993-05-19
JPH06504625A (ja) 1994-05-26
FR2683911A1 (fr) 1993-05-21

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