JPS6276435A - Method for measuring bactericidal action power of leucocyte by chemiluminescence method - Google Patents

Abstract

PURPOSE:To measure the bactericidal action power of leucocyte by acting a non-fluorescent material to H2O2 formed when the leucocyte develops the bactericidal action in the presence of an oxidation catalyst to convert the same to a fluorescent material, then bringing the fluorescent material into reaction with diesters of oxalic acid and separately added H2O2 under the condition to inhibit the oxidation catalyst and measuring the emission intensity thereof. CONSTITUTION:If opsonized zymosan is applied to the leucocyte, the phagositosis thereof is stimulated and is conducted to the state in which the macrophage and neutiocyte of leucocyte develop the bactericidal action. An NADPH oxidase system is activated and releases O2<-> as the bactericidal action in this case and the O2<-> is released in the form of H2O2 by the dismutase action into the system. If the non-fluorescent material is acted to the H2O2 in the presence of the oxidation catalyst, the material is converted to the fluorescent material and if the diesters of oxalic acid and H2O2 are acted thereto, the formed dioxycetanedion provides energy dislocation to the fluorescent material and the fluorescent material emits light.

Description

Detailed Description of the Invention Field of Application of Ash Suhi The present invention relates to a method for measuring the bactericidal activity of leukocytes by chemiluminescence, and more specifically, a method for measuring the bactericidal activity of leukocytes by chemiluminescence, and more specifically, a method for measuring the bactericidal activity of leukocytes by chemiluminescence. This invention relates to a method for quantifying hydrogen oxide by chemiluminescence and thereby measuring the bactericidal activity of leukocytes.

It is well known that when foreign substances such as bacteria invade a living body, white blood cells take them into the cells through phagocytosis and exert a bactericidal effect. Then, macrophages and neutrophils in white blood cells phagocytose them, and at that time, active enzymes (superoxide 02-1 hydrogen peroxide H202, hydroquine radical 0II-, songlet oxidation +02) are used as bactericidal substances.
Of these, O,'- is disproportioned by superoxide nosmutase in the cell and promptly becomes 820
It is known that it can be converted into 2.

Therefore, by measuring H t Ot in such a leukocyte phagocytic expression system, the bactericidal activity of leukocytes can be measured. However, since the amount of H2O2 produced is extremely small, the 202 detection system is required to have extremely high sensitivity. Such a highly sensitive method for measuring H3O2 is the chemiluminescence method using luminol-peroxidase [Lawrence et al.
ce, R, et al, J.

Immun, ), 129.1589-1593 (19
82); Descombouracha et al., An Immuno (Dc
scomps-Latscha, B, et at,
Anntmmun, ), 133.349-364 (1
982); De Sole, P. et al., Ad.
v, Exp, Med.

Biol, ), A±, 591-Gol (1982
).

Kunihiko Suwa & Shigeru Tanaka, History of Medicine, Volume 131.

ioo ~ p. 104 (1984)'], Chemiluminescence method using lucigenin-peroxidase [Minkelherg and Felber, J.I.
Kenberg, T., & Perber,
E, J.

Immunol, Methods), 71, 6
1-67 (1984), and a fluorescence method using homovanillic acid-peroxidase [Ruch, W., et al., J.
, Immunol.

Methods), 63, 347-357 (1983)
] etc. are known.

However, chemiluminescence using luminol or lucigenin has the drawback that the luminescence is significantly attenuated by cells or cell-derived proteins. In addition, in the detection system using fluorescence method,
In order to measure the fluorescence intensity relative to the standard liquid, a standard curve must be used for each measurement, which is extremely cumbersome.
It is E. However, since this is a photometric method based on light excitation, measurement sensitivity may decrease due to light scattering by existing cells. Furthermore, the sensitivity of the fluorescence method is 1 to 2 times higher than that of the luminescence method.
It has the disadvantage of being low in order.

Purpose of the Invention After the above-mentioned circumstances, the inventor of the present invention has conducted various studies to find a more stable and highly sensitive method for measuring the bactericidal activity of leukocytes, and as a result, it has been found that leukocytes exhibit bactericidal activity. In the presence of an oxidation catalyst, a non-fluorescent substance is applied to the H, O, produced during the reaction to convert it into a fluorescent substance, and then, under conditions of inhibition of the oxidation catalyst, the fluorescent substance is added to oxalic acid diesters and separately. H, 0
The present inventors have discovered that 2 can be quantified, and thereby the bactericidal activity of leukocytes can be measured, leading to the completion of the present invention.

When opsonized zymogen is given to the leukocytes constituting the invention, their phagocytosis is stimulated, and the leukocytes' macrophages and neutrophils are brought into a state where they exhibit bactericidal activity. In this case, the NADPH oxidase system is activated as a bactericidal action, and 0
2- is released, which is converted into HtOt by dismutase action.
and is released into the system.

1-+ thus generated 20. When a non-fluorescent substance is applied in the presence of an oxidation catalyst, the non-fluorescent substance is converted to a fluorescent substance, and oxalic acid diesters and 1-ttot (added separately) are applied to this under conditions that inhibit the oxidation catalyst. When this is done, the generated dioxycetanedione causes energy transfer to the fluorescent substance, causing the fluorescent substance to emit light. Therefore, I-12 released by the expression of bactericidal action of leukocytes
0 and 0th- are quantified by measuring the peak value in luminescence, and further, the bactericidal activity of leukocytes is calculated based on the H20 and quantified value.

The reaction formula for the above reaction is as follows.

Leukocyte bactericidal action +0. →-HzOt(Ot-) (
1) Ht Ot + non-fluorescent substance - 1111 = fluorescent substance + 2
1-120 (2) Fluorescent substance + oxalic acid diester + HzOz-hv + 2CCL
+Product (3) The non-fluorescent substance in reaction formula (2) is oxidized by reacting with Ht 2 Ot in the presence of an oxidation catalyst, or converted into an oxidized (condensed) substance into a fluorescent substance.

Such a non-fluorescent substance is required to be a substance that, after being converted into a fluorescent substance, emits light with high sensitivity even in a trace amount due to the action of oxalic acid diesters, I-I 202. Examples of such non-fluorescent substances include 2'', 7°-dichlorofluorescin diacetate, p-cresol, etc. The amount of this non-fluorescent substance used is usually 0001 to 5.
It is mM.

Examples of the oxidation catalyst include enzymes such as peroxidase, microperoxidase, myeloperoxidase, and catalase, and chemical oxidizing agents such as ferrin ampotassium. The amount used is usually 1-100 units for enzymes, and usually 1-100 mM for chemical oxidants.

As the oxalic acid noester used in reaction formula (3), H
It is preferable to use a compound that forms a stable dioquinosetanedione upon reaction with tOt, and a suitable example thereof is bis(2,4,
Examples include 6-)lichlorophenyl)oxalic acid, bis(2,4-dinitrophenyl)oxalic acid, and the amount used is usually 01 to 50mM.

L added separately (202 is usually 0.1 to 50 mtvi
Used within the range of

Note that the reaction of reaction formula (3) needs to be carried out under conditions where the oxidation catalyst is inhibited. If there is an oxidation catalyst that is not deactivated, the separately added HzOt and the remaining non-fluorescent substance will react in the reaction of reaction formula (3), producing an extra fluorescent substance and causing an error in the measured value. Undesirable. Such oxidation catalyst inhibition conditions include adding inhibitors such as potassium cyanide, sodium cyanide, sodium azide, sulfide, fluoride, ferrocyanide, hydroxyphenylhydranone, and 2,3-dimercaptopropanol, or adding inhibitors such as acetone, ethyl acetate, This is carried out using an organic solvent such as acetonitrile. In particular, when an organic solvent is used, it not only inhibits the oxidation catalyst but also inhibits the reaction equation (
This is preferable because the luminescence reaction of 3) can be carried out more efficiently.

In the reaction of reaction formula (1), leukocytes, especially neutrophils, collected from blood are suspended in an appropriate buffer, such as a phosphate buffer (p+45 to 9) containing salt, and opsonized zymogen or phorbol is added to the suspension. Myristate acetate is added to stimulate leukocyte phagocytes. This additive amount is not particularly limited, but is usually 11g-100mg/
This is the range of m children.

The reaction of reaction formula (2) is also generally carried out in a suitable aqueous buffer at about 20-40°C and 1) H5-9. By adding 10-5 to 10-'M cyclodextrin to this reaction system, it is possible to stabilize the non-fluorescent substance and increase the luminescence of the produced fluorescent substance. In addition, to prevent oxidation of non-fluorescent substances, zinc sulfate heptahydrate was added to
It is preferable to add 04 mg/m, M.

The reaction of reaction formula (3) is preferably carried out in an organic solvent as described above (water is used as a carrier), and the organic solvents may be used alone or in combination of two or more. This reaction usually takes about 2
It is carried out for about 30 seconds at 0 to 406C.

The luminescence activity generated as described above can be measured using the usual method, for example, by measuring the peak luminescence amount using a Lumiphotometer TD400.
It can be measured by counting at 0 (manufactured by Labo Science). Since the luminescence value is always expressed per reaction time, it also indicates the reaction rate. Therefore, by sampling the reaction system of reaction formula (3) over time and measuring the luminescence intensity, it is possible to know the initial rate of H2O2 release based on the increase in fluorescent substance, and also to determine the peak value of luminescence. Due to the height of the released I-1,0
.

m of (0,) can be measured, and as a result, the bactericidal activity of leukocytes can be calculated. In addition, when measuring the amount of luminescence, treat the standard H202 solution in the same way and measure the amount of luminescence, prepare a 14202 calibration curve for the standard solution, and compare it with the actual measurement 1-110 and calculate the amount. Ru.

Effects of the Invention According to the method of the present invention, the luminescence of proteins present in a biological sample is not hindered even by particles, and the luminescence intensity does not decrease even in a turbid sample, making it possible to measure with extremely high sensitivity. . Or, since the amount of luminescence is measured after converting to a fluorescent substance, even a very small amount of produced H,02 can be quantified with high sensitivity without being affected by white blood cells, so the bactericidal activity of white blood cells can be measured accurately and with high sensitivity. . Furthermore, since the luminescent reaction reaches its peak value within 5 seconds, the peak value can be measured in an extremely short time. Thus,
The method of the present invention determines whether leukocytes in a living body exhibit sufficient phagocytosis.

Next, the method of the present invention will be explained in more detail with reference to Examples.

Example (+) Preparation of a white blood cell (neutrophil) bactericidal solution Neutrophils are separated from the venous blood of a healthy person collected using heparin (5 IU/mjL) using dextran and Barcol High Bark. The neutrophils were collected at 0.9mMcac47.0.
5 mM Mgc, and glucose (Ig/l in saline phosphate buffer (p+-r7.OX or less, P B
(abbreviated as S/Ca/Mg/glucose) (Neutrophil count: 2XIO5CeL/mR,). To 1 ml of this suspension, 1 ml of an aqueous solution of Dimosan (100 μg/J) opsonized with fresh human serum was added, and the mixture was allowed to react at 37°C for 1 hour, and then 1 ml of EDTA-glinone buffer was added to stop the reaction. , 2000
After centrifuging at Xg for 10 minutes, neutralize with weak hydrochloric acid to prepare a test leukocyte bactericidal solution.

(2) Preparation of detected amount of H202 A predetermined amount of H2O2 is dissolved in the PBS/Ca/M4 glucose solution, and a HzOt calibration curve is prepared for each 1 mal as follows.

(i) Preparation of fluorescent reagent: Dissolve 5 mg of leucodiacetyl-2゜7-dichlorofluorescein in 10 ml of ethanol. 01 Add NNa0I-140J to deacetylate activated 2゛,7゜-dichlorofluorenone noacetate, and add 25mM phosphorus buffer (pH 7) containing 2% β-cyclodextrin to this 75
Add m children and stir.

This was added with 25mM phosphate buffer e, (pi-17) 75m
Further add l- to make the total amount 200J.

(ii) Preparation of luminescence reagent: 0.5mM His (2.4°6
-Dolichlorophenyl) oxalic acid and 2 mMi-(20t) are mixed and diluted 5 times with acetonitrile to prepare a luminescent reagent.

(iii) Preparation of oxidizing enzyme solution: 5 U of peroxodase
/mfl 25mM phosphate buffer (pH 7)
dissolve in

(iV) Preparation of I(102 calibration curve) Add 5 U/mfl peroxidase itmJ2. to 3 mal of the luminescence reagent prepared above, and add H2 at each concentration to the mixture.
Add 1 m each of 2O and liquid and react at 30°C for 1 hour.
The sample 0. Take 5 mfl, add 0.5 mjJ of luminescence reagent to it, and measure its peak value using Lumiphotometer TD4000.

Draw a graph based on this measurement and calculate 10"-"M-1
A calibration curve showing a straight line at 0'MH, 0, is obtained.

(3) Neutrophil 1420. Measurement of release ability 1 m of the leukocyte bactericidal solution prepared as described above (+) was added to the above (2X
Mix with 3 mfl of the fluorescent reagent prepared in i) and (ii), oxidase IJ, and 1 g of polystyrene beads, and subject to a fluorescence reaction at 30°C for 1 hour. Take 0.5 ml of this reaction solution, add 0.5 mfl of the luminescence reagent prepared in (2) (iii) above, and measure the peak value using a Lumiphotometer TD4000.

Claims (1)

[Claims] Hydrogen peroxide produced during phagocytosis of leukocytes is converted into a fluorescent substance by the action of a non-fluorescent substance in the presence of an oxidation catalyst, and then the fluorescent substance is converted into an oxalic acid diester under conditions where the oxidation catalyst is inhibited. The amount of hydrogen peroxide is measured by measuring the luminescence intensity produced by reacting with hydrogen peroxide, and the bactericidal ability of leukocytes is calculated by the chemiluminescence method. Measuring method.