WO1990007121A1 - Methode pour detecter et quantifier l'acide hyaluronique - Google Patents

Methode pour detecter et quantifier l'acide hyaluronique Download PDF

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Publication number
WO1990007121A1
WO1990007121A1 PCT/AU1989/000549 AU8900549W WO9007121A1 WO 1990007121 A1 WO1990007121 A1 WO 1990007121A1 AU 8900549 W AU8900549 W AU 8900549W WO 9007121 A1 WO9007121 A1 WO 9007121A1
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WO
WIPO (PCT)
Prior art keywords
diagnostic kit
label
kit according
habp
labelled
Prior art date
Application number
PCT/AU1989/000549
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English (en)
Inventor
Peter Ghosh
Prachya Kongtawelert
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The University Of Sydney
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Publication date
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Publication of WO1990007121A1 publication Critical patent/WO1990007121A1/fr

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose

Definitions

  • the present invention relates to an improved method of detecting hyaluronan in biological fluids and to a diagnostic kit for use in that method.
  • Hyaluronic acid or hyaluronan is a linear unbranched polysaccharide consisting of repeating
  • HA disaccharide units of ß-(1,4)-D-glucuronic acid-ß-(1,3)-D-N-acetylglucosamine.
  • cartilage In cartilage, it has been shown to be an important component for the macromolecular
  • HA provides the backbone for multiple site interactions with proteoglycan monomers, the interaction being electrostatic in nature and stabilized by link protein (Hascall et al., 1974).
  • hexuronate containing compounds and neutral sugars also generate colour in this assay.
  • Some selectivity may be achieved using specific hyaluronidases (e.g. from Streotomvces hyalurolyticus) to generate reducing end groups which can then be quantified using
  • HA-binding proteins isolated from bovine cartilage.
  • This method has enabled submicrogram quantities of HA to be detected and quantified in biological samples, but since 125 I-HABP has a relatively short half-life, the assay must be performed with freshly prepared material.
  • An ELISA method has been devised by Delpech and co-workers (Delpech et al., 1985), using the HA binding protein, hyaluronectin. However the method requires pre-coating of the plates with another HA binding protein and this has presented some difficulties in the routine application of this approach.
  • the assay is conducted at a pH within the range of 5.8 to 7.3 and at a temperature in the range of 0°C to 27°C.
  • HABP would not assume native conformation in that pH range and that consequently a more alkaline pH is desirable to ensure that disulphide bridges present in the native form of HABP are maintained. This is supported in the literature [Plaas et al, 1986]
  • European Patent Application No. 87850360.6 describes the direct binding of HA to a solid support. This has also been described by other workers (Delpech et al, 1985).
  • the present inventors have found that in their hands HA does not bind well to solid support materials.
  • the present inventors have employed a polycationic material interposed between the solid support material and the HA.
  • the present inventors have found that the inclusion of a polycationic coating material provides a highly sensitive assay for HA.
  • hyaluronan is intended to encompass hyaluronan and hyaluronic acid.
  • the present invention provides an improved method of detecting and/or quantifying hyaluronan in biological fluids and tissues which involves the interaction of hyaluronan in a sample with an excess of a labelled hyaluronan binding protein (HABP).
  • HABP labelled hyaluronan binding protein
  • HA is an anionic macromolecule which should not bind directly to polyvinyl chloride or
  • a method for the detection and/or quantification of hyaluronan in a sample containing HA which method comprises:
  • the method comprises:
  • the method comprises; providing a solid support coated with the
  • the polycationic coating substance is selected from poly-L-lysine, polyarginine, glutaraldehyde and a cationic protein, Awhich does not produce non-specific interactions.
  • the polycationic coating substance is poly-L-lysine.
  • the cationic protein is secretory
  • the method is carried out at a pH between 7.5 and 9.0.
  • the method is carried out at a pH of 8.6.
  • the method is carried out at a temperature between 18°C and 37°C.
  • the method is carried out at 37°C.
  • the solid support is selected from polystyrene, PVC, Sepharose and agarose.
  • the support is PVC.
  • the support may be formed as a microtitre plate, tube or bead.
  • the HA coating source is derived from synovial fluid, cocks comb, umbilical cord, serum, plasma dermis, cartilage, intervertebral disc or a microorganism.
  • the HA coating substance is derived from umbilical cord.
  • the HABP is derived from a proteoglycan monomer.
  • proteoglycan monomer is derived from animal hyaline cartilage.
  • the animal hyaline cartilage is articular cartilage from a human, bovine, porcine or lapine.
  • the HABP may be labelled with an enzyme, a
  • radioisotope a fluorescent label or biotin.
  • the label is: an enzyme selected from alkaline phosphatase, horse radish peroxidase,
  • ⁇ -galactosidase and urease a radioisotope selected from 125 I and 131 I; a fluorescent label selected from a
  • fluorochrome fluorochrome, FITC and TRITC or biotin.
  • the label is biotin.
  • biotinylated HABP is detected using a labelled avidin or streptavidin.
  • the labelled avidin or streptavidin is enzyme labelled.
  • the avidin or streptavidin is labelled with an enzyme selected from alkaline phosphatase, horse radish peroxidase, ⁇ -galactosidase and urease.
  • the avidin or streptavidin is labelled with alkaline phosphatase.
  • enzymes are used for labelling either a single enzyme, an oligomeric form of the enzyme, or an
  • enzyme/antienzyme complex may be used.
  • the enzyme may be coupled to an alternative detection system, such as an amplification system.
  • the signal from alkaline phosphatase can be amplified 100-5000 fold using a redox cycle based on the cycling of NAD.
  • the sample is a synovial fluid sample, vitreous aqueous humor, dermis, cartilage, serum, plasma or intervertebral disc.
  • a diagnostic kit for use in the detection and/or quantification of hyaluronan in a sample containing hyaluronan comprising:
  • the kit is provided with the solid support coated with the polycationic material and the HA coating coated on the coated solid support.
  • the solid support is provided coated with the polycationic coating material and the HA coating is provided to be applied to the coated support.
  • the solid support is PVC, polystyrene, agarose or Sepharose.
  • the solid support is PVC.
  • the support may be formed as a microtitre plate, tube or bead.
  • the polycationic coating material is selected from poly-L-lysine, polyarginine, glutaraldehyde and a cationic protein, which does not produce non-specific interactions.
  • the cationic protein is secretory proteinase leukocyte inhibitor.
  • the polycationic material is poly-L-lysine.
  • the HA coating source is derived from synovial fluid, cocks comb, umbilical cord, serum, plasma, .dermis, cartilage, invertebral disc, or a microorganism.
  • the HA coating substance is derived from umbilical cord.
  • the HABP is derived from a proteoglycan monomer.
  • proteoglycan monomer is derived from animal hyaline cartilage.
  • the animal hyaline cartilage is articular cartilage from a human, bovine, porcine or lapine.
  • the HABP is labelled with an enzyme, a radioisotope, a fluorescent label or biotin.
  • the enzyme is selected from alkaline phosphatase, horse radish peroxidase, ⁇ -galactosidase and urease.
  • the radioisotope is selected from 125 I and
  • the fluorescent label is a fluorochrome
  • the label is biotin.
  • the biotinylated HABP is detected using a labelled avidin or streptavidin.
  • the avidin or streptavidin is enzyme labelled.
  • the label is selected from alkaline phosphatase, horse radish peroxidase, ⁇ -galactosidase and urease.
  • the label is alkaline phosphatase.
  • the HA standard is selected from Healon and human umbilical cord HA.
  • enzymes are used for labelling either a single enzyme, an oligomeric form of the enzyme, or an
  • enzyme/antienzyme complex may be used.
  • the enzyme may be coupled to an alternative detection system, such as an amplification system.
  • Figure 1 is a diagrammatic representation of the principle used for the quantification of HA by the labelled avidin - biotin technique.
  • Figure 2 shows a typical standard inhibition curve obtained when quantifiying HA by the labelled avidin-biotin technique using Healon in 6% BSA as a standard reagent.
  • Figure 3 shows an elution profile of HABP on
  • Figure 4 shows a saturation curve obtained for the optimal concentration of human umbilical cord HA in PBS, pH 7.4 for absorption on poly-L-lysine (50 ⁇ /ml; 100 ⁇ g/well) pre-coated polyvinyl chloride immunoassay plates.
  • Figure 5 shows the relationship between dilution of HABP and alkaline phosphatase conjugated streptavidin at dilutions of 1:1000, 1:2000 and 1:4000 on an HA coated plate.
  • Figure 6 shows a comparison of the sensitivity of the assay of the invention with the Pharmacia HA test and the ELISIA assay.
  • Figure 7 shows a comparison of amounts of poly-L-lysine for precoating plates.
  • the samples in which the HA is to be detected are prepared in accordance with standard techniques of sample preparation.
  • coated plates, tubes or beads may be carried out in accordance with standard techniques.
  • the HABP for use in the method of the invention may be derived from a variety of sources and is prepared in accordance with standard techniques.
  • the HA coating and standard materials may be derived from a variety of sources and are isolated and formulated for use in accordance with standard techniques.
  • Standard curves for the estimation of HA in unknown samples was in accordance with standard techniques for the .preparation of standard curves.
  • the preparation of the diagnostic kits of the invention is also in accordance with standard techniques.
  • Example 1 Detection and guantification of HA by measuring excess bjotinylated-HAPP
  • Proteoglycans were extracted from human articular cartilage with buffered 4M guanidine hydrochloride, pH7.4 containing protease inhibitors and purified under
  • the keratan sulphate core protein fraction was further partially digested with trypsin.
  • the HABP can be obtained by
  • HABP Biotinylation of HABP was performed by the method of Rappuoli et al, (1981).
  • N-hydroxysuccinimidobiotin (34.1mg/ml of dimethylsulfoxide) at room temperature for 18 hours.
  • the mixture was applied to a Sephadex G-25 column and eluted with 0.1M Tris-0.05M NaOAc pH 8.6.
  • the excluded protein peak was collected, aliguoted and stored at -20°C as stock solution.
  • Activated polyvinyl chloride immuno-assay plates were pre-coated with poly-L-lysine hydrochloride by the addition of 100 ⁇ l/well of a 50 ⁇ g/ml solution in distilled water.
  • Alkaline phosphatase substrate [1mg/ml of p-nitrophenyl phosphate in
  • absorbance at 405/690nm was determined using a microtiter plate reader (Titertek Twinreader, Flow Laboratories,
  • a standard inhibition curve for HA was constructed using log/linear co-ordinates and the HA levels in the test samples were determined by comparing their capacity to inhibit colour development at OD 405/690nm relative to this standard curve.
  • Figure 2 shows a typical standard inhibition curve for HA using the standard conditions described. It was found using this method that it was possible to reproducibly quantify between 10-200pg/ml of HA in biological samples.
  • Figure 6 illustrates the improvement in sensitivity by this method compared with ELISIA assay and the Pharmacia HA test.
  • HA-test The mean value of HA in diluted normal sera was 24 ⁇ 12ng/ml by our labelled-avidin biotin technique and 22 ⁇ 16ng/ml using a commercial radioassay kit (HA-test,
  • HA-affinity column chromatography of the trypsin digested PG-monomers. As is evident, HABP was eluted from the column by 4M GuHCl. In order to determine capacity of the
  • poly-L-lysine pre-coated plates for HA they were incubated with various concentrations of human umbilical cord HA.
  • the amount of HA adhering t ⁇ the poly-L-lysine pre-coated plates was determined by adding B-HABP, enzyme-conjugated
  • the major advantage of the present method against other published HA assays is its ability to allow HA to be
  • the level of HA is reported to be elevated in
  • the present invention is of use in the detection of hyaluronan in biological fluids and tissues.
  • the detection of hyaluronan is a useful adjunct to the diagnosis of diseases such as rheumatoid arthritis, neoplastic diseases, scleroderma and liver disease.
  • hyaluronate in rheumatoid arthritis relationship to inflammatory activity and the effect of corticosteroid therapy. Ann. Rheum. Dis., 44: 83-88, 1985.
  • synthesized proteoglycan for hyaluronic acid can be enhanced by exposure to mild alkali. Biochem. J. 234: 221-223, 1986.

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Abstract

Méthode de détermination et/ou de quantification de l'acide hyaluronique (ou hyaluronane ou HA). Il s'agit d'une méthode d'inhibition utilisant une protéine liant l'acide hyaluronique et de l'acide hyaluronique lié à un support solide qui a été préalablement revêtu d'une substance de revêtement polycationique. L'invention porte en outre sur une trousse de diagnostic pour la mise en ÷uvre de ce procédé.
PCT/AU1989/000549 1988-12-19 1989-12-19 Methode pour detecter et quantifier l'acide hyaluronique WO1990007121A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0643305A3 (fr) * 1993-09-13 1996-01-24 Ciba Corning Diagnostics Corp Sous-revêtement de surfaces de phases solides et méthode de revêtement direct.
WO1998037222A1 (fr) * 1997-02-22 1998-08-27 Uhlenküken, Jochen Procede d'immobilisation reversible d'oligo et/ou de polysaccharides
CN114829929A (zh) * 2019-12-02 2022-07-29 尤碧护理有限责任公司 测定水凝胶中透明质酸钠含量的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0271461A1 (fr) * 1986-12-08 1988-06-15 Kabi Pharmacia AB Procédé de détermination d'acide hyaluronique
EP0283779A1 (fr) * 1987-03-03 1988-09-28 Chugai Seiyaku Kabushiki Kaisha Procédé pour déterminer un acide hyaluronique au poids moléculaire élevé

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0271461A1 (fr) * 1986-12-08 1988-06-15 Kabi Pharmacia AB Procédé de détermination d'acide hyaluronique
EP0283779A1 (fr) * 1987-03-03 1988-09-28 Chugai Seiyaku Kabushiki Kaisha Procédé pour déterminer un acide hyaluronique au poids moléculaire élevé

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANALYTICAL BIOCHEMISTRY, Vol. 160, 1987 (KEISER H.D.), "A Solid-Phase Immunoassay for the Binding of Cartilage Proteoglycan to Hyaluronic Acid", pages 462-467. *
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, Vol. 237, No. 1, 15 February 1985 (TURNER RAYMOND E. et al.): "Cationic Dye Binding by Hyaluronate Fragments: Dependence on Hyaluronate Chain Length", pages 253-260. *
INT. J. BIOCHEM, Vol. 17, No. 1, 1985, (Great Britain), (KALPAXIS D.L. et al.): "Immobilisation of Hyaluronate or Cellulose Fibres and Its Use for the Isolation of Cartilage Components", pages 61-66. *
JOURNAL OF CHROMATOGRAPHY, Vol. 350, 1985, (Amsterdam), (KALPAXIS, D.L.): "Comparative Study of Affinity Chromatography of Components of the Hyaluronate - Proteoglycan Complex to Immobilised Hyaluronate", pages 227-236. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0643305A3 (fr) * 1993-09-13 1996-01-24 Ciba Corning Diagnostics Corp Sous-revêtement de surfaces de phases solides et méthode de revêtement direct.
WO1998037222A1 (fr) * 1997-02-22 1998-08-27 Uhlenküken, Jochen Procede d'immobilisation reversible d'oligo et/ou de polysaccharides
EP0861903A1 (fr) * 1997-02-22 1998-09-02 Lansing, Manfred Procédé pour immobiliser de façon reversible des oligo- et polysaccharides
CN114829929A (zh) * 2019-12-02 2022-07-29 尤碧护理有限责任公司 测定水凝胶中透明质酸钠含量的方法

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