Solid support and method of enhancing the recovery of biological material therefrom

Abstract

The present invention relates to solid supports that are used for the storage and further processing of biological materials. The invention is particularly concerned with solid supports which have at least one surface coated with a chemical that enhances the recovery of the biological material from the support. Methods of preparing and using the solid supports are also described.

Claims

1. A method of recovering a biological material from a solid support comprising the steps of: i) providing the solid support having a surface coated with a chemical selected from the group consisting of polyvinyl pyrrolidone (PVP), poly-2-ethyl-2-oxazoline (PEOX), and casein; then ii) applying a sample containing the biological material to be in direct contact with the surface of the solid support and to form a sample spot, wherein the sample is selected from the group consisting of cell, blood, plasma, saliva, and urine; then iii) drying the sample spot to form a dried sample spot on the surface of the solid support; then iv) storing the dried sample spot; and then v) extracting the biological material from the dried sample spot from the surface of the solid support, wherein the solid support is a paper.

2. The method of claim 1, wherein the paper is a cellulose paper.

3. The method of claim 1, wherein step iv) comprises storing the solid support at a temperature in the range of 15 to 40 C.

4. The method of claim 1, wherein the biological material is selected from the group consisting of biomolecule, synthetically-derived biomolecule, cellular component and biopharmaceutical drug.

5. The method of claim 1, wherein the biological material is a biopharmaceutical drug.

6. The method of claim 1, wherein the sample is blood and the dried sample spot is a dried blood spot.

7. The method of claim 1, wherein the providing step further comprises saturation dipping of the solid support in a solution comprising the chemical.

8. A method of recovering a biological material from a solid support comprising the steps of: i) providing the solid support having a surface coated with a chemical selected from the group consisting of polyvinyl pyrrolidone (PVP), poly-2-ethyl-2-oxazoline (PEOX), and casein; then ii) applying a sample containing the biological material to be in direct contact with the surface of the solid support and to form a sample spot, wherein the sample is selected from the group consisting of cell, blood, plasma, saliva, and urine; then iii) drying the sample spot on the surface of the solid support; then iv) storing the dried sample spot; and then v) extracting the biological material from the dried sample spot from the surface of the solid support, wherein the biological material is a biopharmaceutical drug.

9. The method of claim 8, wherein the solid support is selected from the group consisting of paper, glass microfiber and membrane.

10. The method of claim 9, wherein the paper is a cellulose paper.

11. The method of claim 9, wherein the membrane is selected from the group consisting of polyester, polyether sulfone (PES), polyamide (Nylon), polypropylene, polytetrafluoroethylene (PTFE), polycarbonate, cellulose nitrate, cellulose acetate and aluminium oxide.

12. The method of claim 9, wherein step iv) comprises storing the solid support at a temperature in the range of 15 to 40 C.

13. A method of recovering a biological material from a solid support comprising the steps of: i) providing the solid support having a surface coated with a chemical selected from the group consisting of polyvinyl pyrrolidone (PVP), poly-2-ethyl-2-oxazoline (PEOX) and casein; then ii) applying a sample containing the biological material to be in direct contact with the surface of the solid support and to form a sample spot, wherein the sample is selected from the group consisting of cell, blood, plasma, saliva, and urine; then iii) drying the sample spot to form a dried sample spot on the surface of the solid support; then iv) storing the dried sample spot; and then v) extracting the biological material from the dried sample spot from the surface of the solid support, wherein the solid support is selected from the group consisting of paper, glass microfiber and membrane, and wherein the membrane is selected from the group consisting of polyester, polyether sulfone (PES), polypropylene, polytetrafluoroethylene (PTFE), polycarbonate, cellulose nitrate, cellulose acetate and aluminium oxide.

14. The method of claim 13, wherein the biological material is selected from the group consisting of biomolecule, synthetically-derived biomolecule, cellular component and biopharmaceutical drug.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 presents the recovery of exogenously-added IL-2 from dried blood spots applied to various paper matrices.

(2) FIG. 2 presents the recovery of exogenously-added IL-2 from dried blood spots applied to 903 Neonatal STD papers coated with various chemicals.

(3) FIG. 3 presents the recovery of exogenously-added IL-2 from dried blood spots applied to DMPK-C papers coated with various chemicals.

DETAILED DESCRIPTION OF THE INVENTION

(4) Recombinant IL-2carrier (R & D Systems; Cat. 202-IL-CF-10 g; lot AE4309112 and Cat. 202-IL-10g; lot AE4309081 respectively) was dissolved in either Dulbecco's PBS without calcium and magnesium (PAA; Cat. H15-002, lot H00208-0673), EDTA-anti-coagulated human, rabbit or horse blood (TCS Biosciences) at 50 pg or 100 pg/l.

(5) Aliquots (1 l containing 0, 50 or 100 pg of IL-2) were applied to the following GE Healthcare filter papers; 903 Neonatal STD card, Cat. 10538069, lot 6833909 W082; DMPK-A card, Cat. WB129241, lot FT6847509; DMPK-B card, Cat. WB129242, Lot FE6847609 and DMPK-C card, Cat. WB129243, Lot FE6847009. Samples were allowed to dry overnight at ambient temperature and humidity.

(6) Punches (3 mm diameter) were extracted from each paper type using the appropriately sized Harris Uni-core punch (Sigma, Cat.Z708860-25ea, lot 3110). Single punches were placed into individual wells of the IL-2 microplate derived from the Human IL-2 Quantikine ELISA (R & D Systems, Cat. D0250, lot 273275). These plates are coated with a mouse monoclonal antibody against IL-2. The IL-2 protein was eluted from the paper punch using the assay buffer (100 l) supplied with the Quantikine kit. All subsequent steps were performed according to the instructions supplied with the Quantikine kit using a paper in method (paper punches are placed directly into the assay buffer and the analyte eluted directly in situ). On completion of the assay the optical density of the microplate was monitored at 450 nm using a Thermo Electron Corporation, Multiskan Ascent. The recovery of IL-2 was determined by comparing values to a standard curve of known IL-2 concentrations. A fresh IL-2 standard curve was prepared for each individual experiment.

(7) Additional experiments involved the addition of IL-2-spiked blood to the 903 Neonatal STD and DMPK-C cards after the cards had been saturation dipped in several chemical solutions (as described below).

Chemicals Used

(8) A list of the chemicals and their sources is given below. Poly-ethyl-enemine, 50% in water (Fluka; Cat. P3143, lot 29k1492). Poly-vinyl-pyrolodine, 1% in water (Sigma; Cat.PVP40-100 mg, lot 11 pk0097). Inulin, 1% in water (Sigma; Cat. 12255-100 g, lot 079F7110). Poly-2-ethyl-2-oxazoline, 1% in water (Aldrich Cat. 372846, lot 30498PJ). Albumin, 1% in water (Sigma, Cat A2153-10 g, lot 049k1586). Caesin from bovine milk, 1% in water (Sigma, Cat. C5890-500 g, lot 089k0179). Poly-ethylene glycol 1000, 1% in water (Biochemika, Cat. 81189, lot 1198969). Poly-ethylene glycol 200, 1% in water (Fluka, Cat. 81150, lot 1384550).

Experimental Results

(9) When IL-2 was dissolved in EDTA-anti-coagulated blood, the 903 and DMPK-C cards facilitated the recovery of 45-55% of the cytokine, while only 2-3% was recovered from the DMPK-A and B cards (see Table 1 and FIG. 1). The 903 and DMPK-C cards are the basic base papers and have not been dipped or coated with any chemical, whilst the DMPK-A and B cards are coated with a proprietary mixture of chemicals that facilitate the denaturation and inactivation of proteins, micro-organisms and cells respectively. These cards have been designed to facilitate the transportation and prolonged storage of nucleic acids. Therefore the low IL-2 recovery levels observed when using the DMPK-A and B cards may actually be a reflection of the presence of these denaturing reagents and the ELISA-based antibody detection system used. The ELISA detection system requires the eluted IL-2 to exhibit an intact native structure.

(10) TABLE-US-00001 TABLE 1 The Recovery of exogenously-added IL-2 from dried blood spots applied to various paper types. The p-value compares carrier for each paper type. The presence of the carrier had no significant effect on the recovery of IL-2 (p-value > 0.05). Paper type IL-2 recovery (%) p-value 903; minus carrier 46.9 13.3 >0.05 903; plus carrier 50.7 5.8 DMPK A; minus carrier 2.0 0.0 >0.05 DMPK A; plus carrier 2.0 0.0 DMPK B; minus carrier 2.0 0.0 >0.05 DMPK B; plus carrier 2.0 0.0 DMPK C; minus carrier 53.9 4.8 >0.05 DMPK C; plus carrier 45.2 5.4

(11) No IL-2 recovery was observed when the cytokine was dissolved in PBS irrespective of the paper type used (data not shown). The IL-2 recovery levels observed in the absence of added IL-2 were essentially equivalent to background levels indicating that the EDTA-anti-coagulated blood contain negligible amounts of endogenous IL-2 (data not shown).

(12) Several chemicals were used to saturation dip the 903 Neonatal STD and DMPK-C cards, some of which appeared to facilitate the recovery of elevated IL-2 levels compared to non-dipped papers (p-value<0.05). For both the 903 Neonatal STD and DMPK-C cards (Tables 2 and 3; FIGS. 2 and 3), chemicals such as poly-vinyl-pyrolodine, poly-2-ethyl-2-oxazoline, albumin and casein facilitated a significant increase in IL-2 recovery levels (mean>55% compared to 45% observed for the corresponding un-dipped paper).

(13) TABLE-US-00002 TABLE 2 The Recovery of exogenously-added IL-2 from dried blood spots applied to 903 Neonatal STD papers coated with various chemicals. The table is derived from 2 independent experiments (n = 6). The p-value compares the values derived from the dipped papers to those derived from the Un-dipped 903 paper. Chemical IL-2 recovery (%) p-value Un-dipped 44.9 6.5 n/a Poly-ethyl-enemine (PEI) 41.8 6.0 >0.05 Poly-vinyl-pyrolodine (PVP) 62.0 10.7 <0.05 Inulin 50.4 7.6 >0.05 Poly-2-ethyl-2-oxazoline (PeOX) 66.1 12.6 <0.05 Albumin 73.8 13.6 <0.05 Caesin 55.0 7.8 <0.05 Poly-ethylene glycol 1000 (PEG 1000) 42.5 9.1 >0.05 Poly-ethylene glycol 200 (PEG 200) 43.3 11.0 >0.05

(14) TABLE-US-00003 TABLE 3 The Recovery of exogenously-added IL-2 from dried blood spots applied to DMPK-C coated with various chemicals (n = 3). The p-value compares the values derived from the dipped papers to those derived from the Un-dipped DMPK-C paper. Albumin* n = 1. Chemical IL-2 recovery (%) p-value Un-dipped 49.0 2.1 n/a Poly-ethyl-enemine (PEI) 55.8 12.2 >0.05 Poly-vinyl-pyrolodine (PVP) 74.7 7.8 <0.05 Inulin 33.6 15.4 >0.05 Poly-2-ethyl-2-oxazoline (PeOX) 62.2 2.0 <0.05 Albumin* 63.7 increase Caesin 57.7 1.5 <0.05 Poly-ethylene glycol 1000 (PEG 1000) 31.0 2.8 >0.05 Poly-ethylene glycol 200 (PEG 200) 33.5 15.7 >0.05

(15) While preferred illustrative embodiments of the present invention are described, one skilled in the art will appreciate that the present invention can be practised by other than the described embodiments, which are presented for the purposes of illustration only and not by way of limitation. The present invention is limited only by the claims that follow.