EP0438534A4 - Immunoreactant carriers having a novel biocompatible intermediate coating and process of making same - Google Patents
Immunoreactant carriers having a novel biocompatible intermediate coating and process of making sameInfo
- Publication number
- EP0438534A4 EP0438534A4 EP19900900465 EP90900465A EP0438534A4 EP 0438534 A4 EP0438534 A4 EP 0438534A4 EP 19900900465 EP19900900465 EP 19900900465 EP 90900465 A EP90900465 A EP 90900465A EP 0438534 A4 EP0438534 A4 EP 0438534A4
- Authority
- EP
- European Patent Office
- Prior art keywords
- carrier
- immunoreactant
- protein gel
- coating
- polysaccharide
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
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)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Materials For Medical Uses (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US25574388A | 1988-10-11 | 1988-10-11 | |
US255743 | 1988-10-11 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0438534A1 EP0438534A1 (en) | 1991-07-31 |
EP0438534A4 true EP0438534A4 (en) | 1991-09-11 |
Family
ID=22969662
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19900900465 Withdrawn EP0438534A4 (en) | 1988-10-11 | 1989-10-05 | Immunoreactant carriers having a novel biocompatible intermediate coating and process of making same |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP0438534A4 (en) |
JP (1) | JPH04501313A (en) |
CN (1) | CN1041825A (en) |
AU (1) | AU649397B2 (en) |
ES (1) | ES2017153A6 (en) |
WO (1) | WO1990004178A1 (en) |
ZA (1) | ZA897708B (en) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3842700A1 (en) * | 1988-12-19 | 1990-06-21 | Boehringer Mannheim Gmbh | METHOD FOR PROTEIN IMMOBILIZATION ON A SOLID PHASE, PROTEIN-CARRYING SOLID PHASE PRODUCED THEREOF AND THE USE THEREOF |
WO1992003732A2 (en) * | 1990-08-28 | 1992-03-05 | Bioprobe International, Inc. | Compositions and methods for enhanced binding in biological assays |
WO1995006254A1 (en) * | 1993-08-24 | 1995-03-02 | Applied Immune Sciences, Inc. | Site-specific covalent attachment of conglutinin to solid phase materials and related methods |
US7179660B1 (en) | 2000-03-06 | 2007-02-20 | Dade Behring Marburg Gmbh | Carriers coated with polysaccharides, their preparation and use |
EP2839286B1 (en) * | 2012-04-18 | 2018-02-28 | Siemens Healthcare Diagnostics Inc. | Compounds and methods for preparation of conjugate reagents |
CN109270064B (en) * | 2018-11-12 | 2020-12-01 | 新乡医学院 | Immunoprecipitation reagent based on dextran microspheres and preparation method and application thereof |
CN109444401A (en) * | 2018-12-12 | 2019-03-08 | 郑州安图生物工程股份有限公司 | A kind of preparation method of magnetic microparticle chemiluminescence product |
Family Cites Families (6)
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 |
GB2013688B (en) * | 1978-01-26 | 1982-06-30 | Technicon Instr | Insolubilised proteins and immunoassays utilising them |
US4452773A (en) * | 1982-04-05 | 1984-06-05 | Canadian Patents And Development Limited | Magnetic iron-dextran microspheres |
DE3524451A1 (en) * | 1985-07-09 | 1987-03-12 | Behringwerke Ag | Article for use in immunoassay coated with polymer |
SE8701962D0 (en) * | 1987-05-13 | 1987-05-13 | Bo Hakan Nygren | SET TO INSULATE AND / OR DETERMINE THE CONTENT OF AN ORGANIC SUBSTANCE THROUGH COVALENT COUPLING OF A FOR-SPECIFICALLY SPECIFIC MOTOR REACTANT TO SURFACE PREPARED, HYDROPHOBATED WATER SOLUBLE POLYMER |
-
1989
- 1989-10-05 AU AU46259/89A patent/AU649397B2/en not_active Ceased
- 1989-10-05 EP EP19900900465 patent/EP0438534A4/en not_active Withdrawn
- 1989-10-05 WO PCT/US1989/004467 patent/WO1990004178A1/en not_active Application Discontinuation
- 1989-10-05 JP JP2500309A patent/JPH04501313A/en active Pending
- 1989-10-10 CN CN89107707A patent/CN1041825A/en active Pending
- 1989-10-10 ES ES8903417A patent/ES2017153A6/en not_active Expired - Lifetime
- 1989-10-11 ZA ZA897708A patent/ZA897708B/en unknown
Non-Patent Citations (2)
Title |
---|
No further relevant documents have been disclosed. * |
See also references of WO9004178A1 * |
Also Published As
Publication number | Publication date |
---|---|
AU4625989A (en) | 1990-05-01 |
ZA897708B (en) | 1991-06-26 |
JPH04501313A (en) | 1992-03-05 |
AU649397B2 (en) | 1994-05-26 |
WO1990004178A1 (en) | 1990-04-19 |
ES2017153A6 (en) | 1991-01-01 |
CN1041825A (en) | 1990-05-02 |
EP0438534A1 (en) | 1991-07-31 |
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