JPH0513271B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing clear serum that does not become cloudy even after long-term storage. [Prior Art] Conventionally, in order to measure components in biological fluids, standard serum for calculating measured values, pooled serum for quality control, control serum for accuracy management, etc. have been used as control serum. Recently, in clinical testing, various methods such as SRID (single immunodiffusion) and laser nephelometry are used to measure trace amounts of proteins, complements, hormones, etc. in biological fluids.
It is becoming possible to quantitatively measure drugs, etc., and it is becoming widely used as a routine test. In particular, a method (immunoturbidimetry) has been developed in which an antigen and a specific antiserum are reacted in a solution and the amount of the generated antigen-antibody complex is quantified by absorbance measurement. It became possible to quantify. The principle of this optical measurement method utilizes the fact that the antigen-antibody reaction produces reactants depending on the amount of antigen in the antibody excess region. Therefore, the turbidity produced by reacting the specific antiserum with the antigen in the specimen is measured optically, and the antigen concentration in the specimen is determined from a standard curve created by reacting the standard serum in the same manner. I can do it. The standard serum used in this measurement method is preferably free of turbidity, so the drug manufacturer degreases it, partially purifies it, dilutes it to an appropriate concentration, and supplies it in a ready-to-use form. I am using it as is. Furthermore, there is currently no clear serum for immunoassays containing major serum proteins for controlling the precision and accuracy of machines and reagents. [Problems to be Solved by the Invention] Conventionally, in order to clarify serum, a method of extracting it with an organic solvent such as Freon or ether has been known. However, Freon treatment has insufficient degreasing effect, and when diluted with physiological saline,
Another problem is that turbidity occurs when dissolved after freeze-drying, Freon itself is expensive, and treated Freon cannot be destroyed by combustion. In addition, in the case of ether treatment, although the degreasing effect is good, the ether extraction must be carried out at a low temperature of -20°C, which causes problems such as complicated and dangerous operations. Both processes are difficult to handle in large quantities. [Means for Solving the Problems] Under these circumstances, the present inventor conducted various studies in order to produce a stable serum that does not become cloudy even when stored for a long period of time using simple operations. The present inventor first added a highly concentrated mixture of sodium phosphotungstate and magnesium chloride to serum to precipitate lipoproteins such as low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) in the serum. When the serum was removed, clear serum was obtained. However, the serum thus obtained became cloudy when left for one day, and the above objective could not be achieved. In addition, sodium phosphotungstate and magnesium chloride that had not taken part in the reaction remained, which sometimes affected the measurements. Therefore, as a result of further intensive research, the inventors of the present invention discovered that LDL and VLDL were extracted from serum as described above.
The present inventors have discovered that a clear serum that is stable for a long period of time can be obtained by desalting the supernatant obtained by precipitating and removing lipoproteins such as the above by ultrafiltration, and have completed the present invention. That is, the present invention provides serum with sodium phosphotungstate (0.2-0.3% final concentration) and magnesium chloride (approximately 0.025M final concentration).
The present invention provides a method for producing clear serum, which is characterized by adding a liquid mixture containing the following: removing the resulting precipitate mainly consisting of lipoproteins, and then ultrafiltrating the supernatant. To carry out the method of the present invention, first, a mixed solution containing sodium phosphotungstate in an amount to give a final concentration of 0.2 to 0.3% and magnesium chloride in an amount to give a final concentration of about 0.025M is added to serum. For example, the mixed liquid contains 0.4 to 0.6% sodium phosphotungstate and approximately 0.05M magnesium chloride.
It is preferable to dissolve it in a buffer solution with a pH of 5 to 7 at a concentration such that The amount of the mixed liquid is preferably approximately equal to the amount of serum. After adding the mixed solution to the serum, stir gently at room temperature for about 1 hour, and leave in a cool place overnight to complete precipitation. The resulting precipitate is removed by centrifugation or the like. The supernatant thus obtained is subjected to ultrafiltration. Any type of ultrafiltration device can be used, but one that uses a molecular sieve membrane (for example, the one manufactured by Amicon)
8400 type), the pressure is 3 to 5 Kg/cm ² . In the case of a type using hollow fibers (for example, DC4 manufactured by Amicon), it is preferable to carry out at 0.8 to 1.2 Kg/cm ² . In this way, the serum supernatant is desalted and concentrated. This product can be diluted to an appropriate concentration by adding physiological saline and used as it is, but it can also be lyophilized and stored. [Effects of the Invention] The serum and lyophilized product prepared according to the present invention dissolved in purified water do not become turbid even when stored for a long period of time. Alternatively, it can be extremely advantageously used as a standard serum, control serum, or pooled serum for biochemistry. [Example] Next, an example will be given and explained. Example 1 (i) A 0.1M Tris-HCl buffer (PH6.7 to 6.8, room temperature) containing 0.6% sodium phosphotungstate 18 hydrate and 0.05M magnesium chloride 6H ₂ O was prepared. Mix equal amounts of the above treatment solution and human serum,
This was slowly stirred at room temperature for 1 hour. Thereafter, the mixture was left in a cool place overnight and centrifuged at 3000 xg for 30 minutes to remove the precipitate and obtain a supernatant. This supernatant
Pass through a 0.45 μm filter and desalt at 1.0 Kg/cm ² using an ultrafilter (manufactured by Amicon, DC4 type).
Concentrated. In this case, physiological saline was added to avoid reducing the amount below the amount of serum before treatment. next,
Add NaN ₃ to 0.1% and again
The mixture was passed through a 0.2 μm filter, dispensed in 2 ml portions into 10 ml tea bottles, and freeze-dried. The lyophilized product was stored in a cool place and used by dissolving it in purified water when necessary. (ii) Turbidity (blank value) test Dissolve 200 mg of the freeze-dried product obtained in (i) in 2 ml of purified water, and measure the turbidity immediately after dissolution and when stored at room temperature for one week as absorbance A ₃₄₀ , A ₆₀₀ , A Measured at ₆₆₀ . The results are shown in Table 1, and no turbidity occurred even after storage for one week after dissolution.

[Table] (iii) Stability test 200 mg of the lyophilized product obtained in (i) was dissolved in 2 ml of purified water, and the serum components were analyzed using the SRID method and immunoturbidimetry (TIA) immediately after dissolution and after being stored at room temperature for one week. method). The results are shown in Tables 2 and 3, and it was stable even one week after dissolution.

【table】

[Table] The abbreviations in the table are as follows. C ₃ : Complement C ₃ C ₄ : Complement C ₄ T _f : Transferrin α ₁ AG: α ₁ acidic protein C _p : Ceruloplasmin H _p : Haptoglobin α ₁ AT: α ₁ antitrypsin H _x : Hemopexin AT: Antithrombin (iv) 200 mg of the lyophilized product obtained in standard curve (i) was dissolved in 1 ml of purified water, and three types of solutions were diluted to 1/2 and 1/4. Figure), IgA (Figure 2), IgM (Figure 3)
), C ₃ (Figure 4) and C ₄ (Figure 5) were prepared. Example 2 The concentrated serum obtained by ultrafiltration in (i) of Example 1 was diluted 2 times with physiological saline, and the product of the present invention and the treatment solution were added to remove the precipitate. A turbidity test was conducted in the same manner as in (ii) of Example 1 for the sample (comparative product). The results are shown in Table 4, and those that did not pass the ultraviolet rays became cloudy within one day. Furthermore, in Example 1(i), the serum obtained by performing only ultrafiltration without treating the serum with the mixture of sodium phosphotungstate and magnesium chloride became cloudy over time.

[Table] Example 3 200 mg of the lyophilized product obtained in (i) of Example 1 was dissolved in 2 ml of purified water, and the stability of the biochemical items of the clear serum was investigated immediately after dissolution and when stored at room temperature for one week. Ta. The results are shown in Table 5, and it was stable even one week after dissolution.

[Table] Example 4 Sodium phosphotungstate at various concentrations.
0.1M Tris-HCl buffer containing 18 hydrate and 44 mM magnesium chloride hexahydrate (PH6.8, room temperature)
was prepared. Equal amounts of the above mixed liquid and pooled mixed serum whose total cholesterol amount had been measured in advance were mixed, and the mixture was slowly stirred at room temperature for 1 hour. Thereafter, the mixture was left in a cool place overnight and centrifuged at 3000 xg for 30 minutes to remove the precipitate and obtain a supernatant. The total amount of cholesterol in the obtained supernatant was measured, and the lipid sedimentation rate due to the above mixed solution treatment was calculated.
The results are shown in Table 6 and Figure 6. From the above results, it can be seen that the final concentration of sodium phosphotungstate is required to be 0.2% or more in order to sufficiently precipitate lipids and obtain clear serum for a long period of time through the subsequent ultrafiltration operation.

【table】 [Brief explanation of drawings]

Figure 1 shows the standard curve of IgG of the standard serum of the present invention, Figure 2 shows
The figure shows the calibration curve for the same IgA, and Figure 3 shows the calibration curve for the same IgM.
Figure 4 is the calibration curve for C ₃ , Figure 5 is the calibration curve for C ₄ ,
FIG. 6 is a diagram showing the relationship between sodium phosphotungstate concentration and lipid sedimentation rate.

Claims (1)

[Claims] 1. Add a mixture of sodium phosphotungstate (0.2-0.3% final concentration) and magnesium chloride (approximately 0.025M final concentration) to serum, remove the resulting precipitate mainly consisting of lipoproteins, and then remove the resulting supernatant. A method for producing clear serum characterized by ultrafiltration of the liquid.