Patent Application
20080009079
The present invention will become more fully understood from the detailed description given herein below for illustration only, and which thus is not limitative of the present invention, and wherein:
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In a third embodiment of the present invention, the flushed-away immunoadsorbents after formation of the stacking gel layer are further recovered. And then the recovered immunoadsorbents can be used to form another immunoadsorption wall.
The invention will now be described with reference to the following specific Examples which are illustrative, and not limiting, of the invention.
Acrylamide-bisacrylamide (29:1) solution (Sigma, St. Louis, Mo., U.S.A.) is prepared by dissolving 29 g of acrylamide and 1.0 g of bisacrylamide in a total volume of 100 mL of water. Ammonium persulfate (10%, w/v) (Sigma) serving as the initiator of polymerization is made fresh just before use. N,N,N′,N′-tetramethylethylenediamine (TEMED) (Bio-Rad, Hercules, Calif., U.S.A.) is added as accelerator of the polymerization process without pretreatment. The immunoadsorbents is prepared by coupling Rabbit CNBr-activated Sepharose 4B (Amersham Biosciences, Piscataway, N.J., U.S.A.) with anti-β2-microglobulin (β-2M) antibodies (Dako, Glostrup, Denmark). The immunoadsorbent thus prepared is stored at 4° C. in 10 mM sodium phosphate buffer with 0.15 M NaCl (pH 7.4) (PBS) (Sigma) containing 0.02% NaN3 (Merck, Whitehouse Station, N.J., U.S.A.). Human β-2M solution is fractionated from urine of hemodialysis patients as reported (Vallar L, et al., 1995). The determination of β-2 M level is made by enzyme immunoassay kit (BioCheck, Foster City, Calif., U.S.A.). Regeneration buffer is 0.3 M (pH 2.8) glycine-HCl (Sigma).
In the first embodiment, a stationary phase comprising a supporting gel layer (total concentration of monomer, T %=15%) and a stacking gel layer (T %=7.5%) with immobilized immunoadsorbents is formed in a thick-wall 25-mm diameter glass tube. The present invention is a two-stage polymerization method. The first stage is polymerization of higher content acrylamide to form a more restrictive supporting gel layer. The supporting gel layer is formed by polymerization of 5 mL acrylamide-bisacrylamide (29:1) polymer solution (acrylamide-bisacrylamide=15%) containing a catalyst system of 20 μL ammonium persulfate (w/v=10%) and 5 μL N,N,N′,N′-tetramethylenediamine (TEMED). At the beginning of the second stage, the temperature of the reaction system is lowered and maintained at 0° C. by an ice-water bath. Then, lower acrylamide content polymer solution (the stacking solution) containing 2 mL of immunoadsorbents is added on top of the supporting gel layer. The stacking solution includes 5 mL acrylamide-bisacrylamide (29:1) polymer solution (acrylamide-bisacrylamide=7.5%), 20 μL ammonium persulfate (w/v=10%) and 5 μL N,N,N′,N′-tetramethylenediamine (TEMED). The immunoadsorbents of the first embodiment are the complexes made by coupling CNBr-activated Sepharose 4B with rabbit anti-β-2M antibodies. Because the temperature of the reaction system is maintained at 0° C., the polymerization of acrylamide-bisacrylamide polymer solution could be inhibited. Therefore, the stacking solution remains in an unpolymerization state and the sedimentation of the immunoadsorbents could be completed. Then, part of the supernatant of the stacking solution is removed and the surface of the stacking solution is rinsed with PBS buffer for dilution. Subsequently, the reaction system returned to room temperature (c. 30° C.) and the polymerization reaction of the stacking solution initiated spontaneously. Accordingly, antihuman β-2M immunoadsorbents are copolymerized with acrylamide to form a stacking gel layer. Actually, the upper portion of the remaining stacking solution did not polymerize due to previous dilution and the unpolymerized portion is washed away by a flush of regeneration buffer once the inner portion is thoroughly polymerized. As a result, a stationary phase superficially embedded with the immunoadsorbents is formed. To prevent dehydration and potential microbial contamination, the surface of the stationary phase is supplied with PBS containing 0.02% NaN3.
The prepared stationary phase is then tested for its adsorption capacity by adding 3 mL of β-2M in pH 7.4 phosphate buffer with concentration level of approximately 180 μg/mL at 37° C. Samples (20 μL) are collected from the supernatant at 0, 1, 2, 3, 4, and 5 h, respectively (n=3). Further, the binding activity of the stationary phase is regenerated by washing three times with glycine-HCl solution. Three adsorption-desorption cycles are made. All immunoadsorption cycles and quantitative assays are carried out in the same operating conditions.
Referring to
In the second embodiment, the formation of a stationary phase is still the two-stage polymerization method. But Rabbit anti-β2-microglobulin (β-2M) antibodies are not coupled with CNBr-activated Sepharose 4B at the start. Rather, CNBr-activated Sepharose 4B are alone embedded in the finished stationary phase, and then β-2M antibodies are coupled with CNBr-activated Sepharose 4B settled on the surface of the finished stationary phase.
In the second embodiment, the supporting gel layer is formed by polymerization of 5 mL acrylamide-bisacrylamide (29:1) polymer solution (acrylamide-bisacrylamide=15%) containing a catalyst system of 20 μL ammonium persulfate (w/v=10%) and 5 μL N,N,N′,N′-tetramethylenediamine (TEMED). Subsequently, the temperature of the reaction system is lowered and maintained at 0° C. by an ice-water bath. Then, the stacking solution containing 0.3 g CNBr-activated Sepharose 4B is added on top of the supporting gel layer. The stacking solution included 5 mL acrylamide-bisacrylamide (29:1) polymer solution (acrylamide-bisacrylamide=7.5%), 20 μL ammonium persulfate (w/v=10%) and 5 μL N,N,N′,N′-tetramethylenediamine (TEMED). The stacking solution remains in an unpolymerization state (0° C.), and the sedimentation of CNBr-activated Sepharose 4B are completed. Then, part of the supernatant of the stacking solution is removed and the surface of the stacking solution is rinsed with PBS buffer for dilution. Subsequently, the reaction system returned to room temperature, which brought the stacking solution to the polymerization state. As a result, a stationary phase superficially embedded with CNBr-activated Sepharose 4B is formed. Then P-2M antibodies are loaded onto the surface of the stationary phase and coupled with CNBr-activated Sepharose 4B. Then, 0.1M Tris-HCl solution (pH 8.0) is added to block the coupling reaction. Finally, the surface of the stationary phase is alternately washed with 0.1M acetate buffer (pH 4.0) and 0.1M Tris-HCl solution (pH 8.0) for at least three cycles. The post-coupling process of the formation of a stationary phase is completed. In addition, the activation of Sepharose 4B can be done after finishing the stationary phase embedded with Sepharose 4B.
The prepared stationary phase is then tested for its adsorption capacity by adding 3 mL of P-2M in pH 7.4 phosphate buffer with concentration level of approximately 180 μg/mL at 37° C. Samples (20 μL) are collected from the supernatant at 0, 1, 2, 3, 4, and 5 h, respectively (n=3). Further, the binding activity of the stationary phase is regenerated by washing three times with glycine-HCl solution. Three adsorption-desorption cycles are made. All immunoadsorption cycles and quantitative assays are carried out in the same operating conditions.
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In the third embodiment, in order to enhance the utilization efficiency of immunoadsorbents, the flushed-away immunoadsorbents are further recovered, and the copolymerization is conducted in series to produce three consecutive immunoadsorption walls.
In the third embodiment, the supporting gel layer is formed by polymerization of 5 mL acrylamide-bisacrylamide (29:1) polymer solution (acrylamide-bisacrylamide=15%) containing a catalyst system of 20 μL ammonium persulfate (w/v=10%) and 5 μL N,N,N′,N′-tetramethylenediamine (TEMED). Then, the temperature of the reaction system is lowered and maintained at 0° C. by an ice-water bath and lower acrylamide content polymer solution (the stacking solution) containing 2 mL of immunoadsorbents is added on top of the supporting gel layer. The stacking solution includes 5 mL acrylamide-bisacrylamide (29:1) polymer solution (acrylamide-bisacrylamide=7.5%), 20 μL ammonium persulfate (w/v=10%) and 5 μL N,N,N′,N′-tetramethylenediamine (TEMED). Further, remove the supernatant of the stacking solution and rinse the surface of the remained stacking solution with buffer for dilution, and let the temperature of the reaction system return to room temperature (e.g. 30° C.) so that the polymerization reaction initiates spontaneously to form a more porous stacking gel layer. Once the inner portion is thoroughly polymerized, flush away the unpolymerized portion of the stacking gel with neutral phosphate buffer (The above-mentioned steps can refer to the first embodiment). The flushed-away immunoadsorbents are extensively washed with three cycles of high and low pH buffers, and then kept at 4° C. for subsequent recycled use. Finally, make up the required amount of immunoadsorbents with an intact one and repeat the above steps to manufacture additional immunoadsorption walls. In the third embodiment, three consecutive immunoadsorption walls using antihuman β-2M immunoadsorbents are made and labeled Nos. 1-3 in serial order. The immunoadsorption walls thus prepared is supplied with PBS buffer for prevention of dehydration and kept at 4° C.
In addition, the uncoupling antibodies in the second embodiment also can be recovered and applied to form another immunoadsorption wall.
The prepared immunoadsorption walls are then tested for their immunoadsorption efficacy by adding 1 mL of P-2M in pH 7.4 phosphate buffer with concentration levels of approximately 30 μg/mL at 37° C. Samples (20 μL) are collected from the supernatant at 0, 0.16, 0.33, 0.5, 1 h, respectively. The time course change of the β-2M levels is assayed by the enzyme-linked immunoassay. Referring to
The method for formation of a stationary phase in an immunoadsorption wall of the present invention is mainly a two-stage polymerization reaction. Further, in order to avoid shielding immunoadsorbents from toxins by polyacrylamide, the most important key step is the intentional dilution of the surface of the stacking solution settled with the immunoadsorbents in the second stage. When the surface of the stacking solution settled with the immunoadsorbents is rinsed with buffer, a non-uniform concentration distribution of acrylamide would be created. Thus, copolymerization of acrylamide with immunoadsorbent in the upper portion will not begin under relatively low concentration of monomers while the copolymerization in the inner portion proceeds. Relying on the stationary phase provided by the present invention, the toxin can immediately contact with the surface of the stationary phase and is bound to the exposed ligands of the immunoadsorbents with little or no hindrance until saturation. Moreover, the discontinuous porous structure of the stationary phase could produce a considerable molecular sieving effect, thereby preventing leakage of some essential components. On the other hand, the procedure for coupling CNBr-activated support matrices with antibody could be done after finishing the stationary phase embedded with the CNBr-activated carriers (referring to the second embodiment). Moreover, the washed-away immunoadsorbents or antibodies during preparation could be pooled for recycling (referring to the third embodiment).
While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.
