Biodegradable gelatin-aminodextran particle coatings of and processes for making same
First Claim
1. Colloidal particles having a plurality of pendent functional groups on an exterior coating of aminodextran in which each particle comprises a solid metallic core coated with a first gelatin layer of type B, alkali cured gelatin of Bloom in the range 60 to 225 and a second layer of an aminodextran, said layers having been either (a) crosslinked by the action of a chemical crosslinking agent or (b) joined by a condensation reaction between said gelatin and said aminodextran, such that said so layered particles can be stored as predominantly discrete colloidal particles having pendent functional groups.
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Accused Products
Abstract
The invention relates generally to colloidal particle having a core material and a gelatin/aminodextran coating with pendent functional groups attached thereto. Biological substances or molecules, especially monoclonal antibodies, may be attached to said particles. The monoclonal antibody containing particles are useful in a variety of positive and negative biological assays.
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Citations
41 Claims
- 1. Colloidal particles having a plurality of pendent functional groups on an exterior coating of aminodextran in which each particle comprises a solid metallic core coated with a first gelatin layer of type B, alkali cured gelatin of Bloom in the range 60 to 225 and a second layer of an aminodextran, said layers having been either (a) crosslinked by the action of a chemical crosslinking agent or (b) joined by a condensation reaction between said gelatin and said aminodextran, such that said so layered particles can be stored as predominantly discrete colloidal particles having pendent functional groups.
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16. A process for preparing discrete colloidal particles having a plurality of pendent functional groups on an exterior coating of aminodextran in which each particle comprises a solid metallic core coated either with biodegrable, crosslinked or condensed layers of type B, alkali cured gelatin of Bloom 60 to 225 and an aminodextran, said process comprising;
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(a) (i) (1) preparing metallic core particles in said gelatin or (2) adsorbing as a first layer said gelatin onto said metallic core particles and adsorbing as a second layer an aminodextran onto the surface of the gelatin coated particles; (ii) crosslinking the coating of step (a) (i) by reaction with a chemical crosslinking agent; and (iii) blocking free, unreacted crosslinking agent functional groups present on the surface of the product of step (a) (ii) by reaction of said groups with a sufficient amount of a polyamine such that one of the amine --NH2 groups reacts with said unreacted crosslinking agent functional group and the other NH2 group or groups remain unreacted;
or(b) (1) preparing metallic core particles in said gelatin or (2) adsorbing as a first layer said gelatin onto said metallic core particles and joining to said gelatin by a condensation reaction an aminodextran as a second layer; and (c) separating the coated particles of steps (a) and (b), washing the same and, if desired, derivatizing said particles by reaction with a bifunctional bridging reagent to obtain colloidal particles having additional pendent functional groups. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. Particles with a polyclonal and/or monoclonal antibody covalently bonded thereto, each of said particles comprising:
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(a) a colloidal sized solid metallic core material; (b) (i) a first gelatin coating and a second aminodextran coating on the surface of said solid core and crosslinked thereon by a chemical crosslinking agent, or (ii) a first gelatin coating adsorbed onto the surface of said solid core and a second aminodextran coating joined to said gelatin coating by a condensation reaction, wherein said gelatin coating consists of a type B, alkali cured gelatin of Bloom in the range 60 to 225; (c) an antibody; and (d) a bridging group having an end covalently bonded to said aminodextran and another end covalently bonded to said antibody. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33)
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34. A process for preparing particles with a polyclonal and/or monoclonal antibody bound thereto, said process comprising:
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(I) (a) (1) preparing metallic core particles in type B, alkali cured gelatin of Bloom in the range 60 to 225, or (2) coating a preformed solid metallic core material with gelatin by mixing said core material with a 1% w/v aqueous solution of said gelatin, and (3) isolating and washing said particles of (1) or (2) with a solution of an aminodextran solution; (b) storing the washed particles of step (a) in suspension in an aqueous aminodextran solution until used in step (c), a time in the range of up to about six months, or immediately using the particles of step (a) in step (c); (c) suspending the particles of step (a) or the stored and subsequently separated particles of step (b) in an aminodextran coating solution; (d) mixing the suspension of step (c) with a solution of glutaraldehyde for a time in the range of about 1 hour, thereby crosslinking the surface adsorbed gelatin/aminodextran; (e) adding ethylenediamine to the suspension of step (d) and mixing the new suspension for a time in the range of 1 to 4 hours; (f) adding NaBH4 to the suspension step of (e) and mixing the new suspension; (g) separating the particles of step (f) from the suspending solution and washing the particles with 0.2M aqueous NaCl; (h) reacting, with mixing, the resultant particles of step (f) or (g) with ethylenediamine in 0.2M NaCl aqueous solution containing 1-ethyl-3-(3-diamethylaminopropyl)-carbodiimide at ambient temperature; (i) separating the particles of step (h) from the reaction solution and washing them with phosphate buffered saline solution; (j) reacting the particles of step (i) with a bifunctional bridging reagent in phosphate buffered saline solution at ambient temperature for a time in the range of approximately 0.50 to 1.5 hours to prepare particles having reactive terminal maleimidyl or sulfhydryl groups bound to the particles'"'"' surface; and (k) separating the particles of step (j) and washing them with phosphate buffered saline solution; (II) separately preparing an antibody for conjugation to the particles of step (I)(k) by generating reactive substituents consisting of sulfhydryl groups or maleimidyl groups on said antibody; (III) reacting the particles of step (I)(k) and the antibody of step (II), with mixing, for a time in the range of about 1-3 hours, whereby said reactive substituents of said antibody are coupled to the particles'"'"' reactive groups, separating the resulting antibody containing particles from the reaction medium and washing them with buffered saline solution; (IV) blocking unreacted groups present on the product of step (III); and (V) separating and washing the antibody containing particles of step (IV) with about 1% bovine serum albumin in 0.1% NaN3 in phosphate buffered saline solution, sorting the washed particles in said solution at about 4°
C. for a period in the range of 8 to 16 hours, separating the antibody containing particles, again washing the particles with bovine serum albumin buffer solution, and storing the resulting antibody containing particles in about 1% bovine serum albumin, 0.1% NaN3 in phosphate buffered saline solution until required for use. - View Dependent Claims (35, 36, 37)
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38. A process for preparing particles with a polyclonal and/or monoclonal antibody bound thereto, said process comprising:
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(I) (a) (1) preparing metallic core particles in type B, alkali cured gelatin of Bloom in the range 60 to 225, or (2) coating a preformed solid metallic core material with gelatin by mixing said core material with a 1% w/v aqueous solution said gelatin; (b) coating the particles of step (a) with an aminodextran through a condensation reaction between gelatin carboxylate groups and aminodextran amine groups; (c) separating the particles of step (b) from the reaction solution and washing them with phosphate buffered saline solution; (d) reacting the particles of step (c) with a bifunctional bridging reagent in phosphate buffered saline solution at ambient temperature for a time in the range of approximately 0.50 to 1.5 hours to prepare particles having reactive terminal maleimidyl or sulfhydryl groups bound to the particles'"'"' surface; and (II) separately preparing an antibody for conjunction to the particles of step (I)(d) by generating reactive substituents consisting of sulfhydryl groups or maleimidyl groups on said antibody; (III) reacting the particles of step (I)(d) and the antibody of step (II), with mixing, for a time in the range of about 1-3 hours, whereby said reactive substituents of said antibody are coupled to the particles'"'"' reactive groups separating the resulting antibody containing particles from the reaction medium and washing them with buffered saline solution; (IV) blocking unreacted groups present on the product of step (III); and (V) separating and washing the antibody containing particles of step (IV) with about 1% bovine serum albumin in 0.1% NaN3 in phosphate buffered saline solution, storing the washed particles in said solution at about 4°
C. for a period in the range of 8 to 16 hours, separating the antibody containing particles, again washing the particles with bovine serum albumin buffer solution, and storing the resulting antibody containing particles in about 1% bovine serum albumin, 0.1% NaN3 in phosphate buffered saline solution until required for use. - View Dependent Claims (39, 40, 41)
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Specification