Wash for electrophoresis

Abstract

A wash solution for removal of unbound proteins, unreacted antisera, and excess stain from an electrophoresis gel plate and a method of using the wash solution. The wash solution contains one or more of Hexamethylindanopyran, Tetramethyl acetyloctahydronaphthalenes, Hexyl cinnamal, Butylphenyl methylpropional, d-Limonene and Linalool. Alternatively, the wash solution react with lipids and fats in the unbound proteins and/or unreacted antisera, and/or causes pores in the gel to expand yielding a more efficient removal of unbound protein and unbound antisera, and/or contains protease enzymes that digest proteins and an amylase enzyme to hydrolyze starches and sugars.

Claims

1. An improved wash solution for removing unbound proteins and unreacted antisera from a gel plate in an immunofixation (IFE) system, the wash solution including at least one of the components (a) through (f): a) HexamethylindanopyranC.sub.18H.sub.26O also known as 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8,-hexamethyl-cyclopenta[g]benzopyran or HHCB; b) Tetramethyl acetyloctahydronaphthalenesC.sub.16H.sub.26O; c) Hexyl cinnamalC.sub.15H.sub.20O; d) Butylphenyl methylpropionalC.sub.14H.sub.20O; e) d-LimoneneC.sub.10H.sub.16; and f) 3,7-Dimethyl-3-hydroxy-1,6-octadieneC.sub.10H.sub.18O.

2. The improved wash solution according to claim 1 including at least two of the components (a) through (f).

3. A method of removal of unbound proteins and unreacted antisera from a gel plate in an IFE system wherein patient samples are applied to the gel plate in a plurality of lanes, different antisera are applied to at least two of the plurality of lanes to cause an antibody-antisera reaction in each lane, the method including a step of washing the unbound proteins and the unreacted antisera from the gel plate, the improvement of washing the unbound proteins and the unreacted antisera from the gel plate using the wash solution of claim 1.

4. A method of removing unbound proteins and unreacted antisera from an agarose gel plate in an immunofixation (IFE) system wherein patient samples are applied to the agarose gel plate in a plurality of lanes, different antisera are applied to at least two of the plurality of lanes to cause an antibody-antisera reaction in each lane, the improvement of washing the unbound proteins and the unreacted antisera from the agarose gel plate using a wash solution of claim 1 that provides at least one of the following steps: a. reacting with lipids in the unbound proteins to aid in removing the unbound proteins from the agarose gel plate; b. reacting with the lipids in the unreacted antisera to aid in removing the lipids from the agarose gel plate; c. reacting with fats in the unbound proteins to aid in removing the unbound proteins from the agarose gel plate; d. reacting with fats in the unreacted antisera to aid in removing the unreacted antisera from the agarose gel plate; e. causing pores in the agarose gel plate to expand; f. digesting the unreacted proteins on the agarose gel plate; g. hydrolyzing starches on the agarose gel plate; h. hydrolyzing sugars on the agarose gel plate.

5. The method according to claim 3 wherein the step of washing is carried out at a temperature in a range of 12 C. to 32 C.

6. The method according to claim 3 wherein the step of washing is carried out at a temperature in a range of 17 C. to 27 C.

7. The method according to claim 3 wherein the step of washing is carried out at a temperature of approximately 22 C.

8. The method according to claim 4 wherein the step of washing is carried out at a temperature in the range of 12 C. to 32 C.

9. The method according to claim 4 wherein the step of washing is carried out at a temperature in the range of 17 C. to 27 C.

10. The method according to claim 4 wherein the step of washing is carried out at a temperature of approximately 22 C.

11. The improved wash solution according to claim 1 and including three of the components (a) through (f).

12. The improved wash solution according to claim 1 and including four of the components (a) through (f).

13. The improved wash solution according to claim 1 and including five of the components (a) through (f).

14. The improved wash solution according to claim 1 and including all of the components (a) through (f).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a gel plate used in IFE with conventional removal of unbound proteins and unreacted antisera, using a conventional wash such as TBS; and

(2) FIG. 2 illustrates a gel plate used in IFE with removal of unbound proteins and unreacted antisera using the improved wash solution.

DETAILED DESCRIPTION

(3) FIG. 1 illustrates a conventional gel plate after IFE, staining, and conventional removal of unbound proteins and unreacted antisera. The gel plate in FIG. 1 accommodates nine patient samples in a 33 array. The first horizontal row illustrates the results for patients 1, 2, and 3, the second horizontal row illustrates the IFE results for patients 4, 5 and 6, and the third horizontal row illustrates the IFE results for patients 7, 8 and 9.

(4) For each patient there are six vertical columns, referred to as lanes or zones, identified as SP (indicating total serum protein), G, A, M, (Kappa) and (Lambda)

(5) The nine patient samples on the IFE gel plate for FIG. 1 were processed using a conventional wash and dry procedure with conventional blotting and rehydration times and temperatures as describe above with the rehydration occurring at room temperature, i.e., approximately 22 C. (72 F.).

(6) FIG. 2 illustrates a conventional gel plate after IFE, staining, and removal of unbound proteins and unreacted antisera using the improved wash solution. The gel plate in FIG. 2 accommodates nine patient samples in a 33 array. The first horizontal row illustrates the results for patients 1, 2, and 3, the second horizontal row illustrates the IFE results for patients 4, 5 and 6, and the third horizontal row illustrates the IFE results for patients 7, 8 and 9.

(7) For each patient there are six vertical columns, referred to as lanes or zones, identified as SP (indicating total serum protein), G, A, M, (Kappa) and (Lambda)

(8) The nine patient samples on the IFE gel plate for FIG. 2 were processed using the same patient samples or specimens and the same wash and dry procedure with conventional blotting and rehydration times and temperatures as describe above with the rehydration occurring at room temperature, i.e., approximately 22 C. (72 F.) but using the improved wash.

(9) A comparison of the actual gel plates indicates greater clarity in the lanes of the of the FIG. 2 gel plate, less background and thus more reliable results. While the Figures cannot provide the total degree of clarity as would be apparent on inspection of the actual gel plates, the Figures indicate, for example in the Lambda () lane, that there has been a substantial decrease in background, i.e., a substantial decrease in unbound proteins and unreacted antisera.

(10) It is believed, from a theoretical perspective, that the wash solution may react with lipids and fats in the unbound proteins and/or unreacted antisera to aid in removing unbound proteins and/or unreacted antisera from the agarose plate. Alternatively, or additionally, it is believed from a theoretical perspective, that the wash solution may cause the agarose pores in the gel to expand yielding a more efficient removal of unbound protein and unbound antisera.

(11) The enhanced removal of unbound proteins and/or unreacted antisera reduces unwanted background staining.

(12) A suitable wash solution includes: (1) Water; and (2) One or more of: a. HexamethylindanopyranC.sub.18H.sub.26O also known as Galaxolide; b. Tetramethyl acetyloctahydronaphthalenesC.sub.16H.sub.26O; c. Hexyl cinnamalC.sub.15H.sub.20O; d. Butylphenyl methylpropionalalso known as LilialC.sub.14H.sub.20O, e. d-LimoneneC.sub.10H.sub.16; and f. LinaloolC.sub.10H.sub.18O.

(13) It is believed, subject to further investigation, that one or more of the ingredients 2(a) through 2(f) contains protease enzymes that digest proteins (including lipoproteins) and an amylase enzyme to hydrolyze starches and sugars. These enzymes seem to aid in clearing the background from the gel.

(14) The temperature for the blotting step is in the range of 40 C. to 60 C., preferably 45 C. to 55 C. and most preferably approximately 50 C. The temperature for the rehydration step is in the range of 12 C. to 32 C., preferably 17 C. to 27 C. and most preferably approximately 22 C.