JPH0765996B2 - Method for measuring blood interferon-γ production ability - Google Patents

Description

The present invention relates to a method for measuring blood interferon-γ production ability.

In recent years, clinical chemistry test methods for measuring enzymes or their metabolites present in blood samples have been widely used.

Interferon-γ, which is a component in blood, is also considered to be added to the test items due to its antiviral property, antitumor property, and the like. However, in reality, it has not been realized yet because the amount of interferon-γ contained in blood is very small.

Therefore, the present inventor has diligently studied focusing on measuring the interferon-γ producing ability of the collected blood, rather than directly measuring the interferon-γ contained in the collected blood.

Blood is a liquid in which formed components such as red blood cells, white blood cells, and platelets form a suspension in plasma, which is a liquid component.
In 1 mm ^{3 of} blood, erythrocytes are usually 5.4 × 10 ^{6 in} men
, 4.8 × 10 ^{6 for} women, 7,400 for white blood cells in adults
Contains 300,000 platelets.

Interferon-γ is known to be produced by leukocytes.

Conventionally, leukocytes separated from blood have been used to produce interferon-γ from human blood. For example, Y.
K.YIP et al., Infection and Immunity, Vol.34, No.1,13
1-139 (1981), Tadashi KASAHARA et al., The Journal
As is clear from the description of Immunology, Vol.130, No.4, 1784-1789 (1983), etc., other components are removed from blood, leukocytes are collected, and interferon-γ is produced from these leukocytes. I am letting you.

However, after examining these methods in detail,
Not only is the recovery rate of leukocytes from blood as low as 30 to 50%, but the leukocytes are damaged by the separation operation,
Its survival rate is reduced to 40-60%, and as a result, the effective recovery rate of leukocytes from blood is reduced to about 10-30%, and furthermore, the amount of interferon-γ produced from the collected leukocytes is constant. It has been found that it is extremely difficult to estimate the interferon-γ production ability from blood, for some reason.

The present inventor has used an interferon-
When studies were continued to measure the γ-producing ability, whole blood was contacted with an anticoagulant and mitogen, incubated in a container, and interferon-γ produced in the blood was measured. The present invention has been completed by discovering that Noh can be measured very easily and stably.

After further detailed examination, mitogen was added to whole blood ml.
It has been found that it is preferable to make contact within the range of 10 to 10,000 μg.

In the present invention, whole blood means the collected blood, or the whole formed component obtained by removing the plasma of the liquid component from the blood, is suspended in another liquid such as physiological saline, buffer solution, nutrient medium, etc. Liquid

Any anticoagulant may be used as long as it can prevent coagulation of whole blood and does not adversely affect the production and measurement of interferon-γ. For example, heparin, ACD, CPD and the like are used appropriately.

Further, the mitogen used in the present invention can induce and produce interferon-γ from whole blood, for example, phytohemagglutinin, concanavalin A, pokeweed mitogen, staphylococcus enterotoxin (SE.
A), lipopolysaccharide, endotoxin, β-glucan, polysaccharides such as arabinogalactan, bacteria such as Pseudomonas sp., Corynebacterium sp., Etc. are appropriately used.

Among them, it was found that phytohemagglutinin can induce and produce highly active interferon-γ in a relatively short time, usually about 10 to 30 hours.

The amount of mitogen used is 10 to 10,000μ per ml of whole blood.
A range of g is preferred.

If necessary, it is also possible to increase the amount of interferon-γ produced by coexisting with an interferon-α inducer such as virus or nucleic acid, or to simultaneously produce interferon-α together with interferon-γ.

When whole blood is contacted with an anticoagulant and mitogen and incubated in a container, it is sufficient if the whole blood is contacted with the anticoagulant and mitogen to produce interferon-γ in the container. May be incubated, for example, by adding an appropriate amount of whole blood into a container containing a predetermined anticoagulant and mitogen, or by adding a mixed solution of the anticoagulant and whole blood to the container, You may add mitogen to this and incubate. If necessary, physiological saline, an isotonic buffer, a nutrient medium and the like may be allowed to coexist with the whole blood, anticoagulant, and mitogen upon incubation.

The container to be used can be appropriately selected from flasks, test tubes, ampoules, microplate wells, regardless of its shape and volume.

The incubation conditions may be such that interferon-γ can be produced, for example, 10 to 90 at 30 to 40 ° C.
You only need to keep it for a while.

Incubated in this way, interferon-γ
The whole blood that produced the whole blood, as it is, or after being appropriately diluted with physiological saline, an isotonic buffer solution, etc., if necessary, the supernatant obtained by removing formed components such as blood cells by centrifugation or excess separation operation or The solution is subjected to interferon-γ measurement.

The method for measuring interferon-γ may be any method capable of measuring the amount of interferon-γ produced from the whole blood used, and examples thereof include bioassay methods (Bio Assay), radioimmunoassay methods (Radio Immuno Assay), and enzyme immunoassay. A method such as a measurement method (Enzyme Linked Immuno Sorbent Assay) can be appropriately adopted.

In recent years, an enzyme immunoassay method has been developed as a measurement method that is simple, quick, and highly safe. In the case of the enzyme immunoassay, any method can be used as long as it can measure interferon-γ as an antigen, and for example, a double antibody sandwich method, a modified double antibody sandwich method, etc. can be appropriately selected.

It was found that the interferon-γ producing ability thus measured can be sufficiently applied to clinical chemistry tests of blood-collected individuals.

It was also found that the method of the present invention showed that the blood collected from a cancer patient had an extremely low interferon-γ-producing ability as compared with that of a healthy person.

The present invention will be described below by experiments.

Experiment 1 Effect of Presence or Absence of Blood Treatment on Interferon-γ Production Ability The effect of presence or absence of blood treatment on interferon-γ production was examined. As the blood used, heparinized fresh blood collected from three healthy persons was used.

The treated blood is a plasma-removed suspension obtained by suspending all the formed components obtained by centrifuging blood to remove liquid component plasma in RPMI1640 medium to the same concentration as blood, and 0.75% ammonium chloride according to the conventional method. Tris-HCl buffer solution (pH 7.
The erythrocytes were treated with 2), the erythrocytes were hemolyzed, and the formed erythrocyte-free components collected by centrifugation were suspended in RPMI1640 medium at the same concentration as that of the blood.
Take 1 ml of blood or treated blood in a plastic test tube and add phytohemagglutinin-P to it at 0, 50, 500 μ.
0.1 ml of physiological saline containing g was added and the mixture was incubated at 37 ° C. for 24 hours. Then, using the supernatant obtained by centrifugation, the interferon-γ activity per 1 ml of blood was measured and used as the blood's ability to produce interferon-γ.

The activity of interferon-γ was measured using a human interferon-γ radioimmunoassay kit (product name GAMMA INTERFERON IRMA KIT, manufactured by Celtec, UK).

The results are shown in Table 1.

As is clear from the results in Table 1, in the case of blood or a plasma-cleared suspension containing all the formed components in blood, the interferon-γ production ability is high and the value is stable. It is suitable as a sample for measuring γ productivity. It was found that the interferon-γ production ability was low in the case of the ammonium chloride-treated suspension in which red blood cells were hemolyzed and the value thereof was not constant.

Experiment 2 Effect of mitogen amount on interferon-γ producing ability The effect of mitogen amount on interferon-γ producing ability was examined. As the blood used, heparinized fresh blood collected from 3 healthy subjects and cancer patients (1 each for liver cancer and gastric cancer patients) was used. According to the method of Experiment 1, 1 ml of blood was placed in a test tube and phytohemagglutinin-P was used as a mitogen.
Saline containing 0, 1, 10, 100, 1,000, 10,000 μg
After adding 0.1 ml of each and incubating, the interferon-γ activity per 1 ml of blood was measured and used as the blood's ability to produce interferon-γ.

In addition, phytohemagglutinin-P 100,000μ per 1 ml of blood
An attempt was made to add g, but it was difficult to dissolve to satisfy this, and it was difficult to carry out.

The results are shown in Table 2.

As is clear from the results in Table 2, the amount of mitogen is blood.
10 to 10,000 μg per ml is preferred.

Interferon-γ in blood collected from cancer patients
The productivity was found to be extremely low compared to that of healthy people. Further, blood samples were taken from 20 healthy individuals and 20 cancer patients, respectively, and the interferon-γ-producing ability was similarly measured using 100 μg of mitogen per ml of blood. It showed ± 10 units.
This can be used for early detection of cancer by measuring the interferon-γ production ability of the collected blood.

Hereinafter, a few examples will be described.

Example 1. Heparinized fresh blood 1 ml collected from a healthy person (male, 28 years old)
Was placed in a plastic test tube, phytohemagglutinin-P (250 µg) was added thereto, incubated at 37 ° C for 24 hours, and then centrifuged to obtain the interferon-γ activity in the supernatant obtained in the same manner as in Experiment 1 It was measured at.

The interferon-γ producing ability per 1 ml of blood was about 420 units.

Example 2. Heparinized fresh blood 1 ml collected from a healthy person (female, 33 years old)
Concanavalin A (500 μg) was added thereto and incubated at 37 ° C. for 64 hours, and then the interferon-γ production ability was measured in the same manner as in Example 1. Productivity is about per ml of blood
It was 380 units.

Example 3 Heparinized fresh blood collected from a healthy person (male, 61 years old) was centrifuged to remove plasma, and the obtained formed components were centrifugally washed with physiological saline. A suspension was formed in RPMI1640 medium so that the formed components had the same concentration as blood.

Take 1 ml of this suspension in a plastic test tube, add 300 μg of porkweed mitogen to it, and incubate at 37 ° C for 48 hours.
The γ activity was measured by the enzyme immunoassay method using the two-antibody sandwich method.

The interferon-γ production ability per ml of blood was about 200 units. The value measured by the enzyme immunoassay was in good agreement with that of the radioimmunoassay.

Example 4. Interferon-treated with heparinized fresh blood collected from a lung cancer patient (male, 68 years old) in the same manner as in Example 1.
When the γ-producing ability was measured, it was about 20 units.

Example 5. Using the heparinized fresh blood collected from a uterine cancer patient (female, 55 years old), the same treatment as in Example 1 was carried out to measure the interferon-γ production ability, which was about 10 units.

Claims (3)

[Claims] 1. A method for measuring interferon-γ production ability of blood, which comprises contacting whole blood with an anticoagulant and mitogen, incubating in a container, and measuring interferon-γ produced. 2. The method for measuring interferon-γ production ability of blood according to claim (1), wherein the mitogen is in the range of 10 to 10,000 μg per ml of whole blood. 3. The method for measuring the interferon-γ production ability of blood according to claim (1) or (2), wherein the mitogen is phytohemagglutinin.