JPS62920B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing protein-fixed plastics. Biologically active proteins, such as enzymes, antibodies, hormones, etc., immobilized on insoluble carriers are useful for utilizing their enzymatic activity, immunoreactive activity, etc. Various methods for manufacture have been published.
For example, a method of immobilizing a protein by covalent bonding to an insoluble polysaccharide carrier activated with cyanogen halide, a method of immobilizing a protein using dialdehydes such as glutaraldehyde as a crosslinking agent, and a method of simply physically immobilizing a protein on the surface of a carrier. There are methods of adsorption. Among these methods, those based on chemical reactions require the use of dangerous reagents such as CNBr, and the reaction operations are complicated and time-consuming. In addition, although the adsorption method is easy to operate, it has the disadvantage that the amount of protein immobilized is not sufficient or cannot be immobilized uniformly, and when the activity of the resulting product is utilized, there is a large variation, making it unreliable and impractical. It was not (Special Publication No. 45-38543, Special Publication No. 28031-1973, Special Publication No. 12712-1971, Japanese Patent Publication No. 53-1973)
―29923, USP3553310, and USP3646346). Therefore, as a result of various studies, the present inventor found that
We have developed a method to produce uniform and stable protein-fixed plastics using simple operations. The present invention may be as simple as an adsorption method in which proteins and plastics are brought into contact in the presence of lauryl sulfate. Plastics refer to synthetic polymer compounds such as those used in materials such as test tubes, such as polyethylene, polystyrene, polypropylene, silicone rubber, and copolymers of these with acrylic acid. Although there are many other examples on page 5 of the above-mentioned Japanese Patent Application Laid-Open No. 53-29923, nylon, polycarbonate, and glass are not suitable. The shape of this insoluble carrier can be appropriately selected depending on the purpose, such as tube, bead, sheet, film, or fiber. Among these, those with removable test tube tips (see drawings in Japanese Patent Application Laid-Open No. 53-29923) are convenient to use. Examples of proteins include various enzymes, antibodies, hormones, etc., and unpurified or purified proteins are used. Due to the nature of the present invention, the type of protein does not need to be particularly limited and can be selected depending on the purpose. It is believed that molecular weight substances are suitable; however, one of ordinary skill in the art will be able to determine suitability through simple preliminary experiments. An example of this is
There are enzymes such as proteolytic enzymes, lipolytic enzymes, starch-degrading enzymes, and other degradative enzymes for the digestive system, as described in column 5 of Publication No. 12712. Various antibodies such as those described in Publication No. 29923, page 6, ie, antibodies against various steroid hormones and peptide hormones, can be used. The antibody may be an antiserum obtained by a conventional method of sensitizing an animal with an antigen and collecting blood, or it may be purified by a conventional method such as salting out with ammonium sulfate or affinity chromatography. For immobilization, lauryl sulfate, such as sodium lauryl sulfate, and protein may be dissolved in a suitable buffer solution and brought into contact with plastics. The appropriate concentration of sodium lauryl sulfate varies depending on the type of protein, degree of purification, and degree of dilution, but is generally 0.001 to 0.05% (W/V), preferably 0.003 to 0.01%. Since we are dealing with proteins, it is not recommended to heat them at room temperature, so it is convenient to do the contact at room temperature, which gives good results, and usually takes several hours to more than 10 hours. . Note that bringing the protein and plastic into contact in the presence of sodium lauryl sulfate means not only adding all three at the same time, but also treating the plastic with sodium lauryl sulfate in advance and washing it or not washing it and then contacting it with the protein. However, as will become clear in later experimental examples, rather than treating the plastic in advance and then contacting the protein, it is better to simultaneously treat the three materials (using a mixture of sodium lauryl sulfate and protein). Better results can be obtained by contacting the plastic (added to plastic). When treating plastics with sodium lauryl sulfate in advance, the concentration of this substance can be used within a fairly wide range, but it is better to use a slightly higher concentration.A value of 0.01% (W/V) or more to 0.1% is appropriate, and 1.0% is also possible. It is. If necessary, the protein-fixed plastic may be stored by soaking it in a stabilizing agent such as a polyvinyl alcohol solution to form a film, or by drying it under vacuum without forming a film. Next, a method for producing a protein-immobilized plastic using an antibody as an example of a protein will be shown, and the results of examining the immobilization state by radioimmunoassay will be shown, but the present invention is not limited to these examples. . The tip of the polyethylene tube was made of a copolymer of 90% ethylene and 10% acrylic acid and had a capacity of 1.1 ml, and after protein fixation, it was used by connecting it to the cylindrical part. The procedure for producing protein-fixed plastics is as follows:
Radioimmunoassay (RIA) was performed according to the Act or B method. Method A a Wash the plastic tube with distilled water (30 minutes) b Contact with antibody and sodium lauryl sulfate solution (control is antibody only) c Wash with distilled water (5 minutes x 4 times) d Contact with 1% polyvinyl alcohol solution ( 30
minutes) e Drying under reduced pressure Method B a Washing the plastic tube with distilled water (30 minutes) b Contacting the sodium lauryl sulfate solution (30 minutes) c Washing with distilled water (5 minutes x 4 times) d Contacting the antibody solution (2 Time) e Washing with distilled water (5 minutes x 4 times) f Contact with 1% polyvinyl alcohol solution (30 minutes) g Drying under reduced pressure (RIA) a Add standard substance (dissolved in buffer solution) to antibody-immobilized plastic tube (5 μl). b Add labeled antigen (dissolved in buffer) (1000μ
l). c Leave to react (2.5 hours at room temperature) d Remove the contents e Wash the inside of the tube with physiological saline (1.5 ml x 1
f) Measuring the amount of radioactivity in the tube using a gamma counter (RIA) (Sand-Germany method) a) Adding the standard material to the antibody-immobilized plastic tube
(dissolved in 0.05M phosphate buffer) and add (100μ
l). b Add 0.05M phosphate buffer (containing 0.5% BSA) (400 μl). c Shaking reaction (room temperature, 4 hours) d Remove the contents e Wash the inside of the tube with physiological saline (1.5 ml x 1
f Labeled antibody (0.05M phosphate buffer (0.5% BSA)
(containing)) (dissolved in) (500 μl). g Shaking reaction (room temperature, 24 hours) h Remove the contents i Wash the inside of the tube with physiological saline (1.5 ml x 1
j) Method for measuring the amount of radioactivity in the tube using a gamma counter (RIA) a) Add the labeled antigen solution to the antibody-immobilized plastic tube (1 ml). b Leave to react (room temperature, 1 day) c Remove the contents d Wash the inside of the tube with physiological saline (1 ml x 1
Example 1: Measurement of the amount of radioactivity in e tubes using a gamma counter Cortisol antibodies were immobilized on polyethylene tubes by Method A, and the effect of the presence of 0.003% sodium lauryl sulfate was investigated. The antibody is cortisol-6α-hexuccinate.
γG salted out with ammonium sulfate from rabbit antiserum against BSA
The fractions were dissolved in 0.001M phosphate buffer (PH6.4) and used. RIA is performed according to the method, and the count (Bo) and total count (T) when no standard substance is added.
The ratio of The labeled antigen is 6α-carboxymethylthiocortisol histamine- ^125I (approximately 30,000
dpm) was used.

[Table] As seen in this table, more labeled antigen was bound to the tube that was in contact with the antibody in the presence of sodium lauryl sulfate, which means that more antibodies were also immobilized on the tube. do. Moreover, the variation is small and the effect of the present invention is clear. Example 2 A TSH antibody was immobilized on a polyethylene tube using method A, and the effect of sodium lauryl sulfate was examined using RIA using method A. The antibody used was rabbit anti-human TSH serum, and the contact time required for fixation was 2 hours. The results are as shown _in the drawing.
Bo is the count measured without the standard substance (non-specific binding amount), and T is the count measured without ^the standard substance (non-specific binding amount). This is a count of ¹²⁵ I-labeled chloramine T purified by affinity chromatography using chlorinated Sepharose 4B. As seen in this figure, there are some differences in the optimal concentration of sodium lauryl sulfate depending on the degree of dilution of the antiserum. However, when the concentration reaches 0.1%, no effect is seen. Example 3 Sodium lauryl sulfate and antiserum were added separately under the same conditions as in Example 2, and the difference in effects depending on the order was examined.

[Table] Although there is no noticeable difference between these, there is a slight variation in the cases where antiserum is added last. Example 4 A method in which plastic is pretreated with a sodium lauryl sulfate solution, washed, and then contacted with antiserum (B
Method) and Method A were compared. The tube is made of polyethylene, the antibody is a 0.001M phosphate buffer solution of TSH antiserum, RIA is performed by the method, and the labeled antigen is
¹²⁵ I-labeled human TSH was used (5 cases in each group)

[Table] From this result, method B using pretreatment is inferior to method A, but is recognized to be effective. Example 6 Using rabbit anti-human TSH serum as an antibody, it was immobilized on a polyethylene tube by method A, and RIA was performed by method to examine the effect of sodium lauryl sulfate. 5 cases in each group.

[Table] Due to the presence of sodium lauryl sulfate, the bound percentage is high and the variation is small. Example 7 A TSH antibody was immobilized on polystyrene tubes, polypropylene tubes, and silicone pieces using Method A, and the effects were examined using the method (the conditions were the same as in Example 2). In all cases, it was observed that several times more antibodies were immobilized, and the variation was reduced to about 1/3. It is thought that it is possible to immobilize a large amount of protein in these tubes by modifying their inner surfaces to make them suitable for adsorption. Example 8 The following surfactants (concentration: 0.01%) were investigated by immobilizing the TSH antibody using Method A in the same manner as in Example 7 and confirming their effects using the method, but no effect was observed with any of them. Sodium oleate (anionic) Dotesyl trimethyl ammonium chloride (cationic) Dimethylalkyl betaine (Anon BF) (zwitterionic) Polyoxyethylene sorbitol monooleate (Tween80) (nonionic)

[Brief explanation of the drawing]

The figure illustrates the relationship between the concentration of sodium lauryl sulfate and B/T.

Claims (1)

[Claims] 1. A method for producing protein-fixed plastics, which comprises bringing proteins into contact with plastics in the presence of lauryl sulfate.