Preparation of wax beads containing a reagent for release by heating

• US 5,413,924 A
• Filed: 08/27/1992
• Issued: 05/09/1995
• Est. Priority Date: 02/13/1992
• Status: Expired due to Fees

• Current Assignee: Kenneth M. Kosak, Matthew K. Kosak
• Original Assignee: Kenneth M. Kosak, Matthew K. Kosak
• Inventors: Kosak, Kenneth M., Kosak, Matthew K.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US07/936,357
• Time in Patent Office: 985 Days
• Field of Search: 435/174, 435/177, 435/178, 435/180, 435/182, 435/6, 435/7.1, 436/528, 436/535, 530/812, 530/817
• US Class Current: 435/177
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2547/107 Use of permeable barriers, e.g. waxes
• CPC Class Codes: Y10S 530/812 Peptides or proteins is immobilized on, or in, an organic carrier
• CPC Class Codes: Y10S 530/817 Entrapped within the carrier, e.g. gel, hollow fibre

Abstract

A reagent such as an enzyme is entrapped in a material such as wax or a liposome that releases the reagent when heated. In a preferred embodiment, wax beads containing the reagent are prepared by injecting the reagent into beads of molten wax and cooling to solidify the wax. In another embodiment, droplets of a solution of the reagent are dropped through a layer of molten wax to coat the droplets with the wax and the coated droplets are cooled to solidify the wax. The entrapped reagents have application in nucleic acid hybridizations, polymerase chain reactions (PCR), reverse transcriptase reactions (RTR), nucleic acid sequencing, and product generating reactions such as colorimetic, fluorometric and chemiluminescent enzyme labeled immunoassays.

Claims

1. A method for producing a heat-releasable in-vitro reagent composition comprising the steps of:

(1) injecting an aqueous solution containing a reagent into essentially the center of a bead of molten wax which has a melting temperature greater than 30°

C.; and

(2) cooling said bead of molten wax with injected reagent therein to solidify the wax and form a bead of wax with the reagent entrapped within said bead, such that said bead is essentially water insoluble and water impermeable.

2. The method of claim 1 wherein said reagent is a heat resistant enzyme.

3. The method of claim 1 wherein said reagent is selected from the group consisting of labeled and unlabeled nucleotides, deoxynucleoside triphosphates, dideoxynucleoside triphosphates, ribonucleoside triphosphates, and analogs thereof.

4. The method of claim 1 wherein said reagent is a salt of a metal selected from Mg, Mn, Fe, Co, Cu and Zn.

5. The method of claim 1 wherein said reagent is an enzyme substrate.

6. The method of claim 1, wherein the reagent is an antibody.

7. The method of claim 1, wherein the reagent is an antigen.

8. The method of claim 1, wherein the reagent is a labelling substance.

9. The method of claim 1, wherein the reagent is an enzyme inhibitor.

10. The method of claim 1, wherein the reagent is a chelating agent.

11. A method for producing a heat-releasable in vitro reagent composition comprising the steps of:

(1) dropping droplets of an aqueous solution containing a reagent through a molten layer of wax having a melting temperature greater than 30°

C. whereby the droplets are coated with said wax as the droplets pass through the layer of molten wax; and

(2) passing the wax-coated droplets from step (1) to a cooling zone where the wax is solidified to form beads of wax with said reagent entrapped within said beads, such that said beads are essentially water insoluble and water impermeable.

12. The method of claim 11 wherein said reagent is a heat resistant enzyme.

13. The method of claim 11 wherein said reagent is selected from the group consisting of labeled and unlabeled nucleotides, deoxynucleoside triphosphates, dideoxynucleoside triphosphates, ribonucleoside triphosphates, and analogs thereof.

14. The method of claim 11 wherein said reagent is an enzyme substrate.

15. The method of claim 11, wherein the reagent is an antibody.

16. The method of claim 11, wherein the reagent is an antigen.

17. The method of claim 11, wherein the reagent is a labelling substance.

18. The method of claim 11, wherein the reagent is an enzyme inhibitor.

19. The method of claim 11, wherein the reagent is a chelating agent.