ELEVATED TEMPERATURE WASH FOR ELECTROPHORESIS

Abstract

An immunofixation electrophoresis method including separation of proteins (antigens) by electrophoresis, subjecting the separated proteins to various antisera to cause an antibody-antigen reaction and visualizing the results of the antibody-antigen reaction by staining, including a series of blotting and rehydrating steps with an elevated temperature rehydrating solution to remove unbound proteins (antigens), unreacted antisera, and excess stain.

Claims

1. In a method for removing unbound proteins and unreacted antisera from a gel plate having patient samples and antisera, the method including repetitive washing (rehydration) with a washing solution and blotting (drying) the gel plate, the improvement comprising facilitating removal of non-reacted antigens from the patient sample and non-reacted by rehydration with a washing solution having a temperature in the range of about 30° C. to about 50° C. (about 86° F. to about 122° F.).

2. The method of claim 1, wherein the washing solution has a temperature of about 37° C.

3. The method of claim 1, including one or more of: a. denaturing any non-reacted antisera and/or any non-reacted antigen from the patient sample, without damage to the bound antibody-antigen complex formed by contact between the antibody and antigen; b. thermally damaging any non-reacted antibodies and/or any non-reacted antigen from the patient sample, without damage to the bound antibody-antigen complex; and c. expanding the pores in the gel upon which the IFE is performed to facilitate removal of any non-reacted antibodies and/or any non-reacted antigen, without damage to the bound antibody-antigen complex.

4. In a method for removing unbound proteins and unreacted antisera and unbound stain from a gel plate having zones for patient samples and antisera and stain in an IFE system, the method including electrophoresis of patient samples in the zones, contacting the electrophoresed patient samples in the zones with antisera causing reactions between the antisera and antigens in the zones, and application of a stain to the zones to bind to the antisera-antigen reaction, the method further including repetitive washing (rehydration) the gel plate with a washing solution and repetitive blotting (drying) the gel plate, the improvement comprising: facilitating removal of non-reacted antigens from the patient samples on the gel plate and non-reacted antigens and unbound stain on the gel plate by rehydration with a washing solution having a temperature in the range of about 30° C. to about 50° C. (about 86° F. to about 122° F.).

5. The method according to claim 4, wherein the washing solution has a temperature of about 37° C.

6. The method of claim 4, including one or more of: a. denaturing any non-reacted antisera and/or any non-reacted antigen from the patient sample, and/or any unbound stain, without damage to the bound antibody-antigen complex formed by contact between the antibody and antigen; b. thermally damaging any non-reacted antibodies and/or any non-reacted antigen from the patient sample and/or any unbound stain, without damage to the bound antibody-antigen complex; and c. expanding the pores in the gel upon which the IFE is performed to facilitate removal of any non-reacted antibodies and/or any non-reacted antigen and/or any unbound stain without damage to the bound antibody-antigen complex.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In the drawings:

[0015] FIG. 1 is an illustration of a gel plate in which unbound proteins, unreacted antisera, and excess stain, have been removed according to conventional prior art practices; and

[0016] FIG. 2 is an illustration of a gel plate in which unbound proteins, unreacted antisera, and excess stain, have been removed according to the present invention.

DETAILED DESCRIPTION

[0017] FIG. 1 illustrates a typical gel plate used in IFE in which unbound proteins, unreacted antisera, and excess stain, have been removed according to conventional prior art practices. The gel plate, which may be an agarose gel plate, may be used for evaluation of a single patient or for evaluation of multiple patients concurrently.

[0018] A typical gel plate may include areas for multiple patients such as in a 3×3 array such that nine patient samples may be electrophoresed concurrently. The areas for each patient may be generally square in shape and each patient area for an IFE procedure will include columns, referred to as lanes or zones, identified as SP (indicating total serum protein), G, A, M, κ (Kappa) and λ (Lambda).

[0019] Each patient area of the gel plate includes circular depressions or wells. Patient samples are placed in the wells and then subjected to electrophoresis. After electrophoresis, the antisera are applied to the zones for the immunofixation step. The immunofixation step is then followed by the washing and drying (rehydrating) as described above, to remove unbound proteins and unreacted antisera. A stain is applied for visualizing the results, and excess stain is then removed by washing and drying (rehydrating) as described above.

[0020] The nine patient samples on the IFE gel plate for FIG. 1 were processed using a conventional “wash” and “dry” procedure with TBS, that is, conventional blotting and rehydration times and temperatures as describe above with the rehydration occurring at room temperature, i.e., approximately 22° C. (72° F.).

[0021] In FIG. 1, the circular depressions or wells are visible at the bottom, and more particularly at the bottom right portion of the upper-left hand patient area. The reason is that within the depressions one or more of (a) unbound proteins, (b) unreacted antisera, and (c) excess stain are still present. FIG. 1 thus demonstrates the inconsistency among patient sample areas, i.e., some patient sample areas will not be sufficiently washed. Thus, a scanning densitometer (or other equipment) may detect unreacted antisera, unbound proteins and/or excess stain as noise, sometimes referred to as signal noise, leading to a lack of precision in the results.

[0022] Referring next to FIG. 2, another electrophoresis plate is illustrated. The plate also has a 3×3 array of nine patient test areas, each within a generally square border, again with patient test area having zones or lanes SP (indicating total serum protein), G, A, M, κ (Kappa) and λ (Lambda). Each patient area has wells or depressions (not visible) for application of patient samples.

[0023] Using the same blotting and rehydration times and wash solution as described above for FIG. 1, but, contrary to conventional procedures, by elevating the wash or rehydration temperature to a range of about 30° C. to about 50° C. (about 86° F. to about 122° F.) there is a substantial decrease in the “background” in the lanes, i.e., a substantial decrease in unbound proteins, unreacted antisera and unbound stain.

[0024] Thus, the plate of FIG. 2, after the electrophoresis and immunofixation steps, was rehydrated at a temperature of 37° C. (98.6° F.). The various lanes or zones on the plate of FIG. 2 have a greater clarity in the region of the antibody-antigen reaction with more background or signal noise removed, when compared to the patient test areas on the plate of FIG. 1. This may be seen from a detailed lane-by-lane, patient-sample by patient-sample comparison since the patient samples and antisera and stain and the blotting and rehydration (washing) steps and times were identical as between the gel plate of FIG. 1 and the gel plate of FIG. 2 with the sole exception that the rehydration (wash) solution temperature was increased to 37 □C.

[0025] The difference as between the plate of FIG. 1 and the plate of FIG. 2 is more readily apparent when considering that the plate has wells or depressions that are not visible in the plate of FIG. 2. Thus, all the unbound proteins, unreacted antisera, and excess stain have been removed from the plate of FIG. 2 by the washing and elevated temperature rehydration steps. Thereafter, visualization of the results of the reaction will be more accurate and there will be substantially less signal noise.

[0026] The reason for the improved result of rehydration at an elevated temperature is not understood. Various hypotheses have been advanced. One hypothesis is that the elevated temperature rehydration denatures the non-reacted antisera, the non-reacted proteins (the antigen portion) in the patient sample, and the unbound stain, without damage to the bound antibody-antigen complex thus facilitating removal of these undesired components. A second hypothesis is that the elevated temperature rehydration causes thermal damage to the non-reacted antibodies, the non-reacted antigens, and the unbound stain, without damage to the bound antibody-antigen complex thus facilitating removal of these undesired components. A third hypothesis is that the elevated temperature rehydration expands the pores in the agarose gel upon which the IFE is performed to facilitate removal of the non-reacted antibodies, the non-reacted antigen, and the unbound stain, without damage to the bound antibody-antigen complex. Any two or all three hypotheses may be correct.

[0027] The foregoing is a complete description of the present invention for the improved rehydration of IFE gel plates.