WO1990004178A1 - Vecteurs immunoreactifs presentant un nouveau revetement intermediaire biocompatible et procede pour leur fabrication - Google Patents

Vecteurs immunoreactifs presentant un nouveau revetement intermediaire biocompatible et procede pour leur fabrication Download PDF

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Publication number
WO1990004178A1
WO1990004178A1 PCT/US1989/004467 US8904467W WO9004178A1 WO 1990004178 A1 WO1990004178 A1 WO 1990004178A1 US 8904467 W US8904467 W US 8904467W WO 9004178 A1 WO9004178 A1 WO 9004178A1
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WIPO (PCT)
Prior art keywords
carrier
immunoreactant
protein gel
coating
polysaccharide
Prior art date
Application number
PCT/US1989/004467
Other languages
English (en)
Inventor
Kenneth H. Kortright
Ravinder K. Gupta
Original Assignee
Coulter Corporation
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 Coulter Corporation filed Critical Coulter Corporation
Priority to AU46259/89A priority Critical patent/AU649397B2/en
Publication of WO1990004178A1 publication Critical patent/WO1990004178A1/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
    • G01N33/53—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
    • G01N33/548—Carbohydrates, e.g. dextran
    • 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
    • G01N33/53—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

Definitions

  • This invention relates generally to immunoreactant carriers, such as, microspheres or beads for use in im- munoassays and more particularly, relates to improve ⁇ ments in immunoreactant carriers having a novel inter ⁇ mediate biocompatible protein or polysaccharide coating to which will be conjugated an antigen or antibody of a single binding pair for immunoassays and the process of making said carriers.
  • immunoreactant carriers such as, microspheres or beads for use in im- munoassays and more particularly, relates to improve ⁇ ments in immunoreactant carriers having a novel inter ⁇ mediate biocompatible protein or polysaccharide coating to which will be conjugated an antigen or antibody of a single binding pair for immunoassays and the process of making said carriers.
  • microspheres or beads as the carrier for one of the members of a single binding pair, such as an antibody or antigen of a single pair of binding mem ⁇ bers.
  • a common immunoassay procedure is to coat the carrier with a specific labelled antibody selected to bind the antigen to be assayed and introduce the coated carrier to a sample of a biological fluid to be tested. The resultant complexing of the antibody coated carrier and bound antigen is detected and measured by known procedures to determine the assay resultant.
  • Another known procedure is to withdraw from a test sample of blood a selected class of blood cells by using magnetic microspheres coated with a monoclonal antibody which binds selectively to the selected class of cells, form the complex of the monoclonal antibody and bound cells and then remove the non-complexed cells from the test sample so as to isolate the bound cells.
  • U.S. Patent No. 4,743,543 describes a procedure for removing red blood cells from a test sample which thereafter enables study of the remaining white blood cells.
  • Cross-linking methods also have been employed to retain a protein on a carrier particle.
  • water-insoluble microcapsules whose circumferential wall contains a protein such as gelatin cross-linked to an antigen or antibody in an acidic solution is dis ⁇ closed in U.S. Patent No. 4,590,170. This method re- quires the use of microcapsules negatively charged at an acidic pH.
  • U.S. Patent No. 4,123,396 describes a procedure for preparing metal containing polymeric microspheres which can be bonded covalently to proteins. Cross- linking of the protein and microspheres is preferred so that the stability and size of the microspheres both in aqueous solution and in organic solvents can be maintained.
  • U.S. Patent No. 4,478,946 teaches carriers such as roughened glass beads to which a film-forming "first layer" protein is cross-linked to the bead and to which a subsequent "second layer” antibody or antigen is covalently coupled.
  • the first layer partially or to ⁇ tally may comprise a protein such as gelatin. Less antibody or antigen is required when the film-forming first layer is composed partially or totally of a protein such as gelatin. Crosslinking methods however are required to retain the first layer on the bead.
  • One desirable advantage in preparing such an im ⁇ munoreactant microsphere is to achieve a desirable high ratio of bound antibody to bead surface.
  • Another desirable advantage is to be able to apply an intermediate biocompatible coating to the micro- spheres directly, which coating will enable direct con ⁇ jugation thereto of the antibody or antigen.
  • Yet another desirable advantage is the ability to use reduced quantities of the antibody or antigen to be coated to the microsphere or bead yet achieve the desired immunoassay determinations by means of the herein invention. All of the desired advantages enumerated and others which will become apparent are derived from the methods embodying the invention as herein disclosed. An ancillary advantage is to produce the immunoreactant coated microspheres prepared by said methods of the in- vention for use in immunoassay kits and/or procedures.
  • the invention provides an immunoreactant carrier comprising a biocompatible coating medium retained directly on the surface of the carrier body without an intermediate binding agent and an immunoreactant covalently bound to said coating medium by means of a bifunctional reagent, and the method of producing the carrier.
  • One method of the invention comprises coating the peripheral surface of the carrier body with a biocompatible protein gel by hydrophobic interaction. After coating the carrier, an immunoreactant member of a single binding pair such as an antigen or antibody, is covalently bound to the coated carrier to form the immunoreactant carrier.
  • Another method of the invention provides coating the carriers with a biocompatible protein gel or a polysaccharide by covalent binding.
  • the carrier coated with the biocompatible medium then is covalently bound to an immunoreactant member of a single binding pair to form the immunoreactant carrier.
  • biocompatible as used herein to de ⁇ scribe the intermediate coating medium is intended to mean a medium which does not interact nonspecifically and does not interfere with the immunological reaction or other components of the biological fluid test sample, and does not detrimentally influence the im- munoreactants involved in the immunological reaction.
  • immunological reactant as used herein is in ⁇ tended to mean a substance capable of being one of the reaction members of a single binding pair, as in an antigen or antibody complex formation.
  • Gelatin capsules available from Eli Lilly & Co.: lOmg/ml dissolved in phosphate buffered saline (PBS), pH 7.3, containing 0.1% sodium azide
  • Gelatin capsules available from Eli Lilly & Co.: 20 mg/ml dissolved in PBS, pH 7.3, containing 0.1 sodium azide
  • PBS Phosphate Buffered Saline
  • Bifunctional reagent such as Sulfo-SMCC (2 mg/ml sulfosuccinimidyl-4-[N- maleimidomethyl] cyclohexane-1-carboxylate in PBS), available from Pierce Chemical Co.
  • 2-iminothiolane hydrochloride (2 mg/ml in
  • Dextran T-110 molecular weight 110,000 daltons
  • Dextran T-500 molecular weight 500,000 daltons
  • Dextran T-2M molecular weight 2,000,000 daltons
  • Fluka, Pharmacea or Ficoll (molecular weight 70,000 daltons), available from Sigma Chemical Co. 5g
  • the aminopolysaccharide coated beads may be con ⁇ jugated to immunoreactants by using a bifunctional reagent.
  • bifunctional reagents are as follows:
  • the biocompatible intermediate coating medium can be a protein or a polysaccharide.
  • the intermediate coating medium is a protein, such as gelatin.
  • the biocompatible intermediate coating medium can also be a polysaccharide.
  • Polysaccharides tested in ⁇ cluded Dextran T-40 (molecular weight 40,000 daltons), Dextran T-110 (molecular weight 110,000 daltons), Dex ⁇ tran T-500 (molecular weight 500,000 daltons), Dextran T-2M (molecular weight 2,000,000 daltons), Fluka, Pharmacea, and Ficoll (molecular weight 70,000 daltons), Sigma Chemical Co.
  • Non-magnetic microspheres Commercially available microspheres tested in- eluded divinyl-benzene/polystyrene magnetic micro- spheres (0.7 micron, 20% magnetic; 0.7 micron, 42% mag ⁇ netic; and 1.5 micron, 13% magnetic), Seragen Corpora ⁇ tion; Non-magnetic microspheres also may be employed. Although the diameter size of the microspheres tested ranged from 0.7 to 1.5 microns, diameter sizes larger or smaller than those tested may be employed.
  • the immunoreactant may be labelled or unlabelled, depending upon the immunoassay system to be utilized. Suitable labels include enzymes, radioactive elements or dyes.
  • the carriers are washed in Step I.
  • the washing reagent and procedure will depend upon both the biocompatible medium and the coating method to be used to apply the biocompatible medium on the carrier.
  • biocompatible medium is prepared in Step II. Again, reagents and methods will vary depending upon which coating medium is used and which coating method is employed.
  • Step III the solution of the biocompatible me ⁇ dium is coated on the carrier and excess coating medium is removed.
  • the coating method may be by either hydrophobic interaction or covalent binding if gelatin is the biocompatible coating medium used.
  • the coating method is by covalent binding if a polysaccharide is used.
  • the carrier coated with the biocompatible coating medium can be sterilized by known irradiation techni- ques prior to Step IV.
  • Step IV the immunoreactant is covalently bound to the carrier which has been coated with the biocompatible medium by use of a bifunctional reagent.
  • Step I 1 milliliter ' (ml) of a 10% suspension comprised of 0.7 micron, 42% magnetic microspheres is diluted to 4 ml with PBS and 0.1% azide and washed three (3) times using 4 ml of Phosphate Buffered Saline (PBS) containing a bacteriostatic agent such as 0.1% sodium azide and recovered in the conventional manner.
  • PBS Phosphate Buffered Saline
  • Step II the gelatin solution is prepared. Gelatin powder or capsules is weighed and then combined with PBS to obtain a concentration of 10 mg/ml. The solution is heated gently to approximately 50°C on a magnetic stir plate until the gelatin solution is clear. The gelatin solution is cooled to room tempera ⁇ ture by stirring before further use.
  • Step III the gelatin solution is coated onto the microspheres by hydrophobic interaction. 4 ml of the gelatin solution is combined with the washed mi ⁇ crospheres, sonicated in a water bath at room tempera ⁇ ture for 1 minute and roller-mixed for 3 to 16 hours.
  • 2 ml of a 2.5% suspension of microspheres coated with gelatin by hydrophobic interaction are resuspended to 2 ml in PBS.
  • 27 microliter ( /l) of sulfosuccinimid ⁇ l-4-[N-maleimido-meth ⁇ l] cyclohexane-1- carboxylate (sulfo-SMCC) is added to the microsphere suspension and this mixture is roller-mixed for 1 hour at room temperature. Then, the mixture is washed four (4) times with 2 ml of PBS each wash in the conven ⁇ tional manner, resuspended to a volume of 2 ml in PBS and stored at 4°C until used.
  • the* immunoreactant is thiolated using 2- imino-thiolane hydrochloride according to the method of R. Jue et al., Biochemistry 17:5399 (1978).
  • immunoreactant (2mg) at >10 mg/ml concentration is reacted with 13 ⁇ l of 2-iminothiolane hydrochloride for 1 hour at 22°C.
  • the thiolated immunoreactant is separated by gel filtration and the protein concentra ⁇ tion determined by absorbance at 280 nanometers (nm) in a spectrophotometer.
  • 4 ml of a 2.5% suspension of coated microspheres is treated with 40 ⁇ l of 2-iminothiolane hydrochloride at room temperature for 1 hour, washed 4 times in the conventional manner with 4 ml of PBS each wash, resuspended to 3.8 ml in PBS and stored at 4°C until used. Then, 1 mg of immunoreactant is treated with 40 ⁇ l sulfo- SMCC at 10 mg/ml protein concentra- tion in PBS for 1 hour at room temperature. This treated immunoreactant then is passed through a Sephadex G-50 column in PBS, the protein peak collected and the protein concentration calculated from the A230 value.
  • the modified immunoreactant is added to 4 ml of the thiolated microspheres.
  • the suspension is roller-mixed at room temperature for 2 hours.
  • the reaction is quenched by adding 120 ⁇ l of cysteine per ml of reaction volume to the suspension for 15 minutes.
  • the free sulphydral groups are capped by adding 120 ⁇ l each of Iodoacetamide and 1M Borate per ml of reaction volume to the suspension, and roller-mixing for 30 minutes at room temperature.
  • the microspheres then are blocked with 1% BSA for 1 hour at room temperature, washed 4 times with 4 ml of 1% BSA each wash, resuspended to 4 ml in 1% BSA and stored at 4°C until used.
  • Step I of this procedure 1 ml of a 10% suspension of 0.7 micron, 42% magnetic microspheres is diluted with 3 ml of 0.2M Sodium Chloride (NaCl), washed once in the conventional manner with 4 ml of 0.2M NaCl and suspended to a volume of 4 ml with 0.2M NaCl. The microspheres then are treated with 15 ⁇ l of l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDAC) (2 mg/ml) for 15 minutes.
  • EDAC l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride
  • the gelatin solution is prepared (Step II). Gelatin powder or capsules is weighed and then combined with PBS to obtain a con- centration of 20 mg/ml. The solution is heated gently to approximately 50°C on a magnetic stir plate until the gelatin solution is clear. The gelatin solution is cooled to room temperature by stirring before further use. Then, Step III, the microspheres are bath- sonicated for 30 seconds, treated with 500 ⁇ l of the gelatin solution, bath-sonicated again for 30 seconds, and roller-mixed overnight at room temperature. The reaction is stopped by adding 100 ⁇ l of gly ⁇ ine to the treated microspheres for 30 minutes.
  • microspheres then are washed in the conventional manner four (4) times with 4 ml of water each wash, resuspended to a volume of 4 ml in water containing 0.1% sodium azide to achieve a concentration of 2.5% and stored at 4°C until used.
  • Step IV these coated microspheres are covalently bound with an immunoreactant by Conjugation Procedure A.
  • the carrier also may be coated with a polysac- charide.
  • Step I of this coating procedure 18 ml of a 10% suspension of 0.7 micron, 42% magnetic micro- spheres is diluted to 72 ml with 200 mM NaCl and washed once with 200 ml of 200 mM NaCl. Then, Step II, the polysaccharide solution is prepared.
  • the method of R. S. Molday and L. L. Molday (FEBS Letters 170, No.2:232 [1984]) for T-40 was adapted as herein described.
  • Aminoderivatives of the following polysaccharides were prepared in this manner: Dextran T-40, Dextran T- 110, Dextran T-500, Dextran-2M and Ficoll.
  • Step III the aminoderivatized polysac ⁇ charide is covalently bound to the microspheres by use of a carbodiimide reagent.
  • the washed microspheres are resuspended to 72 ml in 200 mM NaCl containing the aminoderivatized polysaccharide at 3.75 mg/ml.
  • 450 ⁇ l of EDAC then is added to the suspension and the suspen ⁇ sion roller-mixed overnight at room temperature.
  • the microspheres are washed 3 times with 72 ml of water and resuspended to 72 ml in water containing 0.1% sodium azide.
  • microspheres coated with a polysaccharide by this method included 1.5 micron, 13% magnetic micro- spheres and 0.7 micron, 20% magnetic microspheres.
  • Step IV the polysaccharide coated microspheres are covalently bound to an immunoreactant by either Conjugation Procedure ' A or Conjugation Procedure B.
  • a preferred method is by using thiolated immunoreactant and SMCC-treated microspheres as previously described in Conjugation Procedure A.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
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  • Food Science & Technology (AREA)
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  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Materials For Medical Uses (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Est décrit un vecteur immunoréactif renfermant un élément antigène ou anticorps d'une seule paire de liaison liée par covalence à un milieu biocompatible contenant un gel protéinique ou un polysaccharide enrobant la surface d'un corps vecteur. Est également décrit un procédé de production de ce vecteur immunoréactif. Dans les cas où le milieu biocompatible est un gel protéinique, il est enrobé sur la surface du corps vecteur par interaction hydrophobe ou liaison covalente. Dans le cas où le milieu biocompatible est un polysaccharide, il est enrobé sur la surface du vecteur par liaison covalente. Le vecteur immunoréactif s'utilise dans un dosage mettant en ÷uvre un élément d'une seule paire de liaison, tel qu'un antigène ou un anticorps d'une seule paire de liaison dans un échantillon biologique.
PCT/US1989/004467 1988-10-11 1989-10-05 Vecteurs immunoreactifs presentant un nouveau revetement intermediaire biocompatible et procede pour leur fabrication WO1990004178A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU46259/89A AU649397B2 (en) 1988-10-11 1989-10-05 Immunoreactant carriers having a novel biocompatible intermediate coating and process of making same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US25574388A 1988-10-11 1988-10-11
US255,743 1988-10-11

Publications (1)

Publication Number Publication Date
WO1990004178A1 true WO1990004178A1 (fr) 1990-04-19

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PCT/US1989/004467 WO1990004178A1 (fr) 1988-10-11 1989-10-05 Vecteurs immunoreactifs presentant un nouveau revetement intermediaire biocompatible et procede pour leur fabrication

Country Status (7)

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EP (1) EP0438534A4 (fr)
JP (1) JPH04501313A (fr)
CN (1) CN1041825A (fr)
AU (1) AU649397B2 (fr)
ES (1) ES2017153A6 (fr)
WO (1) WO1990004178A1 (fr)
ZA (1) ZA897708B (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992003732A2 (fr) * 1990-08-28 1992-03-05 Bioprobe International, Inc. Compositions et procedes pour obtenir une liaison amelioree dans des analyses biologiques
WO1995006254A1 (fr) * 1993-08-24 1995-03-02 Applied Immune Sciences, Inc. Fixation covalente specifique au site de conglutinine a des matieres en phase solide et procedes afferents
US7179660B1 (en) 2000-03-06 2007-02-20 Dade Behring Marburg Gmbh Carriers coated with polysaccharides, their preparation and use

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3842700A1 (de) * 1988-12-19 1990-06-21 Boehringer Mannheim Gmbh Verfahren zur proteinimmobilisierung an einer festphase, so hergestellte protein tragende festphase sowie deren verwendung
CN104303058A (zh) * 2012-04-18 2015-01-21 西门子医疗保健诊断公司 用于制备缀合物试剂的化合物和方法
CN109270064B (zh) * 2018-11-12 2020-12-01 新乡医学院 一种基于葡聚糖微球的免疫沉淀试剂及其制备方法与应用
CN109444401A (zh) * 2018-12-12 2019-03-08 郑州安图生物工程股份有限公司 一种磁微粒化学发光产品的制备方法

Citations (4)

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US3639558A (en) * 1968-02-19 1972-02-01 Louis Csizmas Immunological reagent particles having proteinaceous materials covalently bonded thereto
US4210418A (en) * 1976-08-30 1980-07-01 Mallinckrodt, Inc. Container for immunochemical and enzymatical determinations or procedures
US4253844A (en) * 1978-01-26 1981-03-03 Technicon Instruments Corporation Insolubilized proteins and immunoassays utilizing them
US4452773A (en) * 1982-04-05 1984-06-05 Canadian Patents And Development Limited Magnetic iron-dextran microspheres

Family Cites Families (2)

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Publication number Priority date Publication date Assignee Title
DE3524451A1 (de) * 1985-07-09 1987-03-12 Behringwerke Ag Mit ueberzuegen versehene formkoerper zur bindung von bioaffinen substanzen, verfahren zu ihrer herstellung sowie ihre verwendung
SE8701962D0 (sv) * 1987-05-13 1987-05-13 Bo Hakan Nygren Sett att isolera och/eller bestemma halten av ett organiskt emne genom kovalent koppling av en for emnet specifik motreaktant till ytadsorberad, hydrofoberad vattenloslig polymer

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3639558A (en) * 1968-02-19 1972-02-01 Louis Csizmas Immunological reagent particles having proteinaceous materials covalently bonded thereto
US4210418A (en) * 1976-08-30 1980-07-01 Mallinckrodt, Inc. Container for immunochemical and enzymatical determinations or procedures
US4253844A (en) * 1978-01-26 1981-03-03 Technicon Instruments Corporation Insolubilized proteins and immunoassays utilizing them
US4452773A (en) * 1982-04-05 1984-06-05 Canadian Patents And Development Limited Magnetic iron-dextran microspheres

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0438534A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992003732A2 (fr) * 1990-08-28 1992-03-05 Bioprobe International, Inc. Compositions et procedes pour obtenir une liaison amelioree dans des analyses biologiques
WO1992003732A3 (fr) * 1990-08-28 1992-04-16 Bioprobe Int Inc Compositions et procedes pour obtenir une liaison amelioree dans des analyses biologiques
WO1995006254A1 (fr) * 1993-08-24 1995-03-02 Applied Immune Sciences, Inc. Fixation covalente specifique au site de conglutinine a des matieres en phase solide et procedes afferents
US7179660B1 (en) 2000-03-06 2007-02-20 Dade Behring Marburg Gmbh Carriers coated with polysaccharides, their preparation and use

Also Published As

Publication number Publication date
AU649397B2 (en) 1994-05-26
EP0438534A4 (en) 1991-09-11
ES2017153A6 (es) 1991-01-01
CN1041825A (zh) 1990-05-02
EP0438534A1 (fr) 1991-07-31
AU4625989A (en) 1990-05-01
JPH04501313A (ja) 1992-03-05
ZA897708B (en) 1991-06-26

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