JPH0122903B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying somatostatin. In recent years, with advances in medicine, it has become possible to treat various complex diseases, and one of the achievements of this is the improvement of diagnostic techniques, that is, the analysis of various physiologically active components. Diagnosing diseases by measuring various physiologically active components requires rapid, accurate, and reproducible quantification of trace amounts of physiologically active components present in blood. For example, by quantifying somatostatin present in the blood, central disease, various gastrointestinal diseases,
It is possible to diagnose diseases such as adrenal gland tumors, and somatostatin can be used, for example, by radioimmunoassay (RIA).
It is quantified by an immunoassay method that utilizes antigen-antibody reactions, such as (method). An example of the principle of the RIA method is as follows. That is, ¹²⁵ I-labeled Tyr-somatostatin (labeled antigen) and unlabeled somatostatin (unlabeled antigen) are competitively reacted with the antibody, and then the unbound labeled antigen (F) and the labeled antigen-antibody are reacted competitively with the antibody. The bound substance (B) is separated and its radioactivity is measured to determine the B/B+F value. At this time, an inverse correlation is established between the B/B+F value and the amount of unlabeled antigen. Therefore, when measuring the unlabeled antigen contained in a sample, add the labeled antigen and its antibody to a solution containing appropriate amounts of unlabeled antigen in advance and perform the same procedure as above to measure the known amount. Calculate the B/B+F (B/B ₀ ) value for the unlabeled antigen and create a standard curve. Next, add the labeled antigen and its antibody to a sample containing an unknown amount of unlabeled antigen, and perform the above procedure. The content of unlabeled antigen in the sample can be determined by applying the B/B+F value obtained by the same operation to the above standard curve. Somatostatin contained in plasma has also been measured by the above method, but recent research has shown that when Tyr-somatostatin labeled with ¹²⁵ I (hereinafter referred to as "labeled somatostatin") is mixed with plasma, It has been reported that somatostatin is degraded into In particular, in the RIA method, the immune reaction (antigen-antibody reaction) usually requires several reactions, but during this time labeled somatostatin is considerably degraded, making it impossible to accurately quantify somatostatin contained in plasma. . From the above viewpoint, various attempts have been made to accurately quantify somatostatin contained in plasma by suppressing the decomposition of labeled somatostatin. One of the representative attempts is to stabilize labeled somatostatin by first extracting somatostatin from plasma, removing substances that inhibit the stability of labeled somatostatin, and then using the somatostatin-containing extract in an immunoassay. There is a so-called indirect method that attempts to quantify somatostatin by subjecting it to . However, this indirect method requires an additional extraction operation, which does not meet the demands of the medical community that a large amount of samples must be processed quickly, and it is difficult to completely extract somatostatin from plasma. There is a problem with this. In addition, from a completely different perspective from this indirect method, without extracting somatostatin from plasma, various inhibitors are present in the reaction system during the immune reaction to stabilize labeled somatostatin, and somatostatin in plasma is stabilized. A so-called direct method has also been attempted to quantify the For example, Clinica Chimica Acta.
Vol. 87, pp. 275-283 (1978) and DIABETES, Vol. 27, No. 5, pp. 523-529 (1978), disclose that aprotinin (trasylol) and sodium ethylenediaminetetraacetate are added to plasma. A method has been reported in which the plasma is then subjected to the RIA method to quantify somatostatin. However, in this direct method, the inhibitor is not effective in stabilizing labeled somatostatin, and labeled somatostatin is still unstable in plasma, and it is inevitable that labeled somatostatin will be gradually degraded over time. It is not possible to accurately quantify somatostatin in In view of the current situation, the present inventors have conducted extensive research in order to develop a new method that can accurately quantify somatostatin in plasma by a direct method. As a result, it was discovered that by adjusting the pH to 4 to 5.5 in an immune reaction system containing plasma, labeled somatostatin was stabilized and no decomposition of labeled somatostatin was observed over a long period of time. The present invention was completed based on this knowledge. That is, the present invention is characterized in that when somatostatin present in blood is quantified by an immunoassay using ¹²⁵ I-labeled Tyr-somatostatin as a labeled antigen, the immunoassay is carried out within a pH range of 4 to 5.5. Relates to a method for quantifying somatostatin. Tyr-somatostatin in the present invention has the following structure. According to the method of the present invention, labeled somatostatin can be stabilized for a long time during an immune reaction, that is, when labeled somatostatin is mixed with plasma, and therefore somatostatin in plasma can be accurately and reproducibly determined. Can be quantified. Furthermore, the method of the present invention does not require the operation of extracting somatostatin from plasma as in the conventional indirect method, and is therefore suitable for rapidly processing a large amount of samples. In the present invention, when quantifying somatostatin in plasma, labeled somatostatin, plasma containing somatostatin, and an immune reaction system containing these antibodies are used.
Adjust the pH to 4-5.5. A wide variety of conventionally known buffers can be used to adjust the pH, and such buffers may be used in an appropriate amount so that the pH of the immune reaction system is 4 to 5.5. Specifically, the buffering agent used is
Phthalate-sodium hydroxide buffer such as 0.2M potassium hydrogen phthalate-0.2M sodium hydroxide (PH4.8), 0.2M glycine-0.2M hydrochloric acid (PH4.8), etc.
Glycine-hydrochloric acid buffer such as 5.0), 0.2M acetic acid-
Acetic acid buffer such as 0.2M sodium acetate (PH4-5.2), 0.1M citric acid - 0.2M disodium hydrogen phosphate (PH5.4), 0.1M citric acid - 0.1M sodium citrate (PH4.4-5) ), 0.1M secondary sodium citrate - 0.1M hydrochloric acid (PH4-5), 0.1M primary potassium citrate - 0.1M sodium hydroxide (PH4-5)
Citric acid buffer such as 5.5), 0.2M succinic acid
Examples include succinic acid buffers such as 0.2M sodium hydroxide (PH4.4). In the present invention, amino acids commonly used in the immune response system, such as histidine, phenylalanine, arginine,
It is also possible to add tyrosine and the like. By adding amino acids, labeled somatostatin can be stabilized for a longer period of time, and somatostatin can be quantified with higher accuracy. The amount of amino acids to be added varies depending on the type of amino acid, etc., but it cannot be stated unconditionally, but it can be adjusted as appropriate within the range in which proteins such as somatostatin contained in plasma do not precipitate under the immunoassay conditions described below, and the amino acids precipitate appropriately. It is better to choose. Furthermore, in the present invention, gelatin, bovine serum albumin, sodium azide, sodium chloride, etc. may be appropriately selected for the immune reaction system. Taking the RIA method as an example, the procedure for quantifying somatostatin in plasma will be described in detail below. First, plasma is collected using a conventional method, and the plasma is separated using a centrifuge. Blood collection and plasma separation are usually preferably carried out in an atmosphere of about 4 to 30°C. A predetermined amount of labeled somatostatin, antiserum (antibody), a buffer, and components such as amino acids, if necessary, are added to the separated plasma, and the mixture is incubated usually at 4 to 37°C for about 1 to 100 hours. Next, the labeled somatostatin-antibody conjugate (B) and unbound somatostatin (F) are separated using conventional methods such as dextran charcoal method, dual antibody method, solid phase method, polyethylene glycol method, talc method, ammonium sulfate method, and ethanol method. etc., and their radioactivity is γ
- The B/B+F value is obtained by measuring using a counter, and the content of somatostatin in plasma is determined from a standard curve prepared in advance. Note that the creation of the standard curve is no different from the conventional method. Examples are given below to further clarify the present invention. Example 1 Production of Tyr-somatostatin labeled with ¹²⁵ I Added 2 μg of Tyr-somatostatin (Protein Promotion Foundation) to 50 μg of 500 mM phosphate buffer (PH = 7.2) with stirring (in 0.2 M acetic acid and 0.2 M NaCl). 20 μl as a solution), then Na ¹²⁵ I1 milliquiry (200 m
20μ as a solution in M phosphate buffer (PH=7.2))
Add 20μg of chloramine T (20μg as a solution in 50mM phosphate buffer (PH = 7.2) and incubate for 45 seconds. D,L-tyrosine 100μg (200μg)
100μ as a solution in mM phosphate buffer (PH=7.2)
) In addition, unreacted Na ¹²⁵ I is consumed to stop the reaction. This reaction mixture was applied to a CM-Sephadex C-25 column [eluent: 0.13M-NaCl, 0.1w/
v%BSA (bovine serum albumin) in 50mM phosphate buffer (PH=6.8)] to obtain ^125I -Tyr-somatostatin-labeled antigen. Example 2 Prepare a 50mM citric acid solution in 150mM NaCl, 0.1 w/v% BSA. Similarly, 150mM NaCl, 0.1
Prepare a 100 mM disodium phosphate solution of % BSA. Mix the two while measuring the pH with a PH meter to obtain a citric acid/phosphate buffer solution of 150mM NaCl and 0.1% BSA with a pH of 4.0. Similarly, obtain a citric acid/phosphate buffer solution of 150 mM NaCl and 0.1% BSA at pH 4.5. 0.1ml of normal human plasma in 0.5ml of the above PH4.0 buffer solution,
¹²⁵ I-Tyr-somatostatin obtained in Example 1 above
0.1 ml of the above buffer (PH4.0) at 10,000 cpm and 0.1 ml of the above buffer (×70,000 final) of somatostatin antiserum (OAL272; see JP-A-16-16415)
and incubate at 4°C for 20 hours. Dextran charcoal powder (0.5% NoritA, 0.05%
Add 1 ml of the above PH4.0 buffer solution of Dextrane T-70 and incubate at 4°C for 1 hour at 8000 rpm15.
Centrifuge for minutes at 4°C. The supernatant and sediment were separated, radioactivity was measured using a γ-counter, and B/
Find the B+F (%) value. Let this be the value of day 0. Similarly, 0.1ml of normal human plasma in 0.5ml of pH4.0 buffer.
and ¹²⁵ I-Tyr-somatostatin 10,000 cpm buffer solution (PH4.0) 0.1 ml was added and incubated at 4°C for 1 day.
Samples were incubated for 2 days, 3 days, 4 days, and 5 days, respectively, and frozen at -20°C. After thawing them in the same manner as above, somatostatin antiserum was added and incubated at 4°C for 20 hours.
BF is separated by the dextran charcoal method, radioactivity is measured, and each B/B+F value is determined. These values are taken as values after 1 day, 2 days, 3 days, 4 days, and 5 days. In the PH4.5 buffer solution, measurements were taken on day 0, day 1, day 2, and day 3 in the same manner as in the case of the buffer solution with pH 4.0.
The B/B+F values are determined after 1 day, 4 days, and 5 days. The results are shown in Figure 1. It can be seen from FIG. 1 that ¹²⁵ I-Tyr-somatostatin is stable at PH4.0 and PH4.5. Comparative Example 1 150m at PH6.5 in the same manner as in Example 2
Obtain a citrate-phosphate buffer of MNaCl and 0.1% BSA. 0 in the same manner as in Example 2 using the buffer solution.
Find the B/B+F value after 5 days. The results are also shown in FIG. 1 above. Example 8 150mM NaCl, 100mM histidine, 0.1% BSA
Prepare a 50mM citric acid solution. 150mM NaCl, 100mM histidine, 0.1% BSA
Prepare a 100 mM disodium phosphate solution.
Mix the two while measuring the PH with a PH meter, and check the PH
5.5 150mM NaCl, 100mM histidine, 0.1%
Obtain BSA citrate/phosphate buffer. Using the buffer solution, the same procedure as in Example 2 was carried out on day 0, day 2, and day 4.
Find the B/B+F value after 1 day. The results are shown in Figure 2. Example 4 A 200mM citric acid solution of 100mM histidine, 0.1 gelatin, and 0.02% sodium azide was prepared,
Prepare a buffer solution by adjusting the pH to 5.0 with an aqueous sodium hydroxide solution while measuring the pH with a pH meter. 0.2ml of the above buffer with antiserum in 0.2ml of the above buffer
(OAL272 x 70,000 final), 0.2 ml of the above buffer containing 10,000 cpm of ¹²⁵ -Tyr-somatostatin and a known amount of somatostatin (Protein Promotion Association)
Add 0.1 ml of the above buffer and incubate at 4°C for 72 hours. BF separation is performed in the same manner as in Example 2 using the dextran charcoal method to determine B/B ₀ . Note that B ₀ indicates the B value when the somatostatin concentration is 0. The standard curve shown in FIG. 8 is thus obtained. Example 5 In the same manner as in Example 4, a 200 mM citrate buffer with pH 5 of 100 mM histidine, 0.1% gelatin, and 0.02% sodium azide is prepared. Heparin (0.01
Collect 5 ml of blood (mg/ml). Separate plasma from each
Store at -20℃. In the same manner as in Example 4 above, 0.1 ml of the above plasma (specimen) is added instead of the known amount of somatostatin, immune reaction and BF separation are performed, and B/B ₀ is calculated. The somatostatin concentration of the sample is determined from the standard curve of Example 4. The results are shown in Table 1 below. 【table】

[Brief explanation of drawings]

FIGS. 1 and 2 are graphs showing the relationship between the number of days of incubation and B/B+F (%).
FIG. 3 is a graph showing the relationship between somatostatin concentration and B/B0 (%).

Claims (1)

[Claims] 1 When quantifying somatostatin present in blood by immunoassay using ¹²⁵ I-labeled Tyr-somatostatin as a labeled antigen, PH4-
A method for quantifying somatostatin, characterized by performing immunoassay within a range of 5.5.