JPH07280808A - Immuno-staining for organic sample - Google Patents

Abstract

PURPOSE:To realize highly accurate detection by inactivating an intrinsic peroxidase before a sample is subjected to immuno-staining using a labeled peroxidase antibody thereby protecting the antigen against damage. CONSTITUTION:When an organic sample is immersed into a methanol containing both agents of sodium azide and hydrogen peroxide and brought into contact therewith for a predetermined time, an intrinsic peroxidase present in cytoplasm is inactivated but an antigenic protein present in the nucleus is not damaged. Upon finishing the inactivation, it is cleaned with water to produce an organic sample which is then subjected to immunoreaction with a peroxidase labeled antibody or a fragment of antibody having antigen recognition characteristics. Upon finishing the immunoreaction, the organic sample is cleaned and added with a solution containing a substrate for peroxidase in order to develop a color. It is then cleaned and a counterstain solution is placed on the organic sample to cause counterstain. The stained organic sample is cleaned and encapsulated with glycerol or polyvinyl alcohol and the stained state is observed by means of an optical microscope.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a method for immunostaining a biological sample,
In particular, it relates to a method for immunostaining a biological sample containing human leukocytes.

[0002]

2. Description of the Related Art Methods for immunostaining biological samples, particularly for biological samples containing human leukocytes are known. A method for collecting a biological sample containing white blood cells from a patient infected with or suspected of being infected with a virus that infects human white blood cells, and detecting an antigenic protein associated with the virus to be analyzed using an antibody Is also known.

In such a case, a method is generally practiced in which an antibody is labeled with peroxidase and the presence of an antigenic protein, and thus the presence of a virus, is detected by color development by the peroxidase. However, in a biological sample containing white blood cells, other blood cell components such as eosinophils, neutrophils and red blood cells are mixed, and the activity of endogenous peroxidase derived from them is the activity of peroxidase labeled on the antibody. It will be overlaid and interfere with the original detection.

As a means for inactivating the activity of such an endogenous peroxidase, treatment with a hydrogen peroxide-added methanol solution, a periodate solution or a sodium azide aqueous solution has been attempted (Medical Technology Separate Volume). "All about dyeing methods", published by Ito Denshaku, July 1988).

According to the method as described above, in general, in order to inactivate the endogenous peroxidase, it has to be treated for a considerable time. However, even though the endogenous peroxidase may certainly be inactivated after such a long time, the immunostained antigen is also damaged, and even if an antigen-antibody reaction is carried out later, the immune reaction will be lost. Does not proceed well, and there is a problem that it may not be dyed. Antigens susceptible to such damage include cytomegalovirus (CMV) and herpes zoster virus (V
ZV), herpes simplex virus (HSV) and the like. On the other hand, if the inactivation treatment time of the endogenous peroxidase is shortened and the treatment conditions are made mild in order to avoid such damage to the antigen, the inactivation is not sufficiently achieved.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have, however, earnestly studied a method in which endogenous peroxidase is sufficiently inactivated and the antigen to be immunostained is not damaged.

[0007]

Means for Solving the Problems The above-mentioned problems of the present invention include an endogenous substance derived from a biological sample to be stained by bringing the biological sample to be stained into contact with a solution in which hydrogen peroxide and sodium azide coexist. This is achieved by an immunostaining method which eliminates the influence of endogenous peroxidase, which comprises inactivating peroxidase and then immunostaining a biological sample with a peroxidase-labeled antibody.

The effect of the present invention is particularly well exhibited when the biological sample is a human leukocyte-containing substance suspected of being infected with a virus, especially CMV, VZV or HSV.

As described above, by coexisting hydrogen peroxide and sodium azide, the processing time can be shortened, and the endogenous peroxidase can be inactivated before the damage of the antigen occurs. .

The concentration of hydrogen peroxide and sodium azide in the coexistence and the treatment time by the coexistence can be easily determined by a simple experiment, so that it is not particularly limited, but the experiment is exemplified by CMV. The method and preferable conditions are as follows.

Experimental method 1) After leukocytes are separated and collected from blood, leukocytes are fixed on a slide glass using a cell centrifuge and fixed with acetone. It is then immersed in methanol solutions containing endogenous peroxidase inactivating agents at different concentrations for different times. In this way, test bodies having different treatment concentrations × treatment time are obtained. 2) After washing them with water, a peroxidase-labeled antibody solution is placed on the fixed cells and reacted with an antigen / antibody. 3) After washing the fixed cells, an enzyme substrate solution is added to develop the color. Then, after washing, the counter-staining solution is placed on the fixed cells and counter-stained. 4) After washing the fixed cells, the fixed cells are mounted with a cell mounting solution and sealed, and the number of staining positive cells per 50,000 white blood cells is counted using an optical microscope.

The above operation is performed on a sample from a healthy person and a sample from an infected person.

When the treatment concentration × the treatment time is too long, the antigen has been damaged, so that neither healthy nor infected samples are stained. On the other hand, if the treatment concentration x the treatment time is too short, the endogenous peroxidase is not inactivated,
Both healthy people and infected people will be stained. In between, there are suitable conditions in which healthy people are not stained and only infected people are stained.

The following is a representative example of immunostaining of white blood cells infected with human CMV.

Commercially available sodium azide and hydrogen peroxide can be used. In order to inactivate the endogenous peroxidase using these, a biological sample may be simply immersed in a methanol solution containing both of these agents and contacted for a predetermined time.

The concentration of hydrogen peroxide in the methanol solution is 0.001 to 0.8 wt%, preferably 0.006 to
0.4 wt%, the concentration of sodium azide is 0.0
1 to 2.7 wt%, preferably 0.003 to 0.9 wt
%.

These proportions are 0.001 to 2700, preferably 0.005 to 150, particularly preferably 0.005 to 2700 in terms of the weight ratio of sodium azide to hydrogen peroxide [sodium azide / hydrogen peroxide (wt / wt)]. Is 0.1 to 5.

The contact time with such a solution depends on the concentration and proportion of each component, but is usually 10 seconds to 3 seconds.
It is 0 minutes, preferably 30 seconds to 20 minutes.

The contact temperature is usually room temperature, but depending on the solvent, it may be as low as -30 ° C, and conveniently, it may be carried out within the range of 4 to 45 ° C.

As a preferable example of the contacting conditions, hydrogen peroxide is 0.006 to 0.4 wt%, sodium azide is 0.003 to 0.9 wt%, and sodium azide / hydrogen peroxide is 0.005 to 0.005 wt%. It takes 10 seconds to 20 minutes for a 150 (wt / wt) solution.

A lower alcohol such as methanol is preferably used as the solution, but various solvents such as acetone type, ether type and hydrocarbon type may be appropriately selected.

The biological sample to be stained is not particularly limited. In the case of normal tissues, tissue slices can be used as they are, but in the case of a white blood cell-containing substance that exerts a particularly remarkable effect when the present invention is applied, white blood cells are separated and collected from blood,
A white blood cell is fixed to a slide glass using a cell centrifuge and fixed with acetone, whereby a biological sample can be easily prepared. Also, alveolar lavage fluid cells and the like can be targeted.

The cell membrane of the leukocytes fixed with acetone on the slide glass becomes extremely rough, and when the slide glass is immersed in the above-mentioned methanol solution, for example, the solution immediately enters the cells and exists in the cytoplasm. Inactivates endogenous peroxidase.

In the present invention, the endogenous peroxidase present in the cytoplasm is inactivated, but the antigenic protein such as CMV present in the nucleus is not damaged.

The sample after the inactivation treatment of the above-mentioned endogenous peroxidase is immediately washed with, for example, water to remove sodium azide and hydrogen peroxide from the sample.

The biological sample thus obtained, that is, the leukocyte-containing material immobilized on a slide glass in the above case, is immunoreacted with a peroxidase-labeled antibody or an antibody fragment having an antigen recognition property by a method known per se. In the present invention, the antibody means all of these and all of the fragments. At this time, of course, an indirect immunostaining method may be used.
Since the cell membrane of the above leukocytes is extremely rough, the labeled antibody can invade the cells and react with the antigen in the nucleus. Conventionally known horseradish peroxidase may be used as the peroxidase. As the antibody, a monoclonal antibody that recognizes the target antigen can be used.

The biological sample, which has undergone the immunoreaction as described above, is then washed, and a solution containing a substrate for peroxidase is added to develop color. Then, after washing, the counter-staining solution is placed on the biological sample and counter-colored.

The stained biological sample is washed, mounted with a mounting liquid such as glycerol or polyvinyl alcohol, and mounted, and the stained state is observed using an optical microscope.

[0029]

INDUSTRIAL APPLICABILITY According to the present invention, when a biological sample containing endogenous peroxidase, such as white blood cells, is immunostained with a peroxidase-labeled antibody, the endogenous properties can be efficiently maintained while retaining the antigenic characteristics of the antigen to be stained. Peroxidase can be inactivated, and as a result, specific detection of virus-related proteins can be performed accurately.

[0030]

The present invention will be described in detail below with reference to examples. Unless otherwise stated, the operating procedures in the examples were in accordance with the following standard operating procedures.

Standard Operating Procedure 1) After leukocytes are separated and collected from blood, leukocytes are fixed on a slide glass using a cell centrifuge and fixed with acetone. Then, this is immersed in a methanol solution containing an endogenous peroxidase inactivating agent described in each Example at room temperature for a predetermined time. 2) After washing with water, put the enzyme-labeled antibody solution on the fixed cells,
React. Horseradish as a labeling enzyme
Peroxidase was used, and as the antibody, a human type monoclonal antibody that recognizes the CMV immediate-early antigen (a monoclonal antibody produced by a hybridoma of Micro Engineering Lab. No. 4030 (FERM BP-4030)) was used. 3) After the immune reaction is completed, the fixed cells are washed and then a substrate solution is added to develop the color. Then, after washing, the counter-staining solution is placed on the fixed cells and counter-stained. As the substrate solution, diaminobenzidine or aminoethylcarbazole was used. 4) After washing the fixed cells, the fixed cells are mounted with a cell mounting solution and sealed, and the number of staining positive cells per 50,000 white blood cells is counted using an optical microscope.

Glycerol or polyvinyl alcohol was used as the cell mounting liquid.

The symbols in the results of observation with an optical microscope have the following meanings.

[0034]

[Table 1]

In the present invention, basically, it is preferable that the cytoplasm such as eosinophil is not stained and the cell nucleus of the CMV-infected cell is stained. Therefore, the condition that both ● and □ are satisfied is preferable. .

[Example 1] Examination of conditions for inactivating endogenous peroxidase-nazide azide
Examination of Thorium Concentration Using whole blood of a healthy person who was not infected with CMV, the inactivating condition of endogenous peroxidase carried by eosinophils in leukocytes was examined according to a standard operating procedure.

Only sodium azide was used as the inactivating agent, and the conditions and results are shown in Table 2.

[0038]

[Table 2]

The above result is 0 when sodium azide is used alone.
It is shown that the inactivation of the endogenous peroxidase is insufficient at ˜0.9 wt% and 5 minutes.

[Example 2] Examination of inactivation of endogenous peroxidase and antigen damage
-Examination of hydrogen peroxide concentration Using whole blood of a healthy person not infected with CMV (sample (A)) and HEL cells (human fetal lung-derived cell line) infected with CMV (sample (B)) The inactivation conditions were examined according to standard operating procedures. When HEL cells are used, a cell suspension of HEL cells is used for the cell centrifuge (cytospin) in 1) of the standard operating procedure. HE
L cells do not carry endogenous peroxidase.

Table 3 shows the inactivating conditions and the results.

[0042]

[Table 3]

From the above results, when hydrogen peroxide alone is used, as understood from the results of the sample (A), 0.9 wt%, in order to inactivate the endogenous peroxidase possessed by eosinophils, It is understood that 100 seconds is not yet sufficient, and higher concentration or longer time is required. The result of the sample (B) is 0.1 wt% or less, and the CMV antigen protein is stained under the condition of 100 seconds, that is, the antigenicity is retained, but the antigenicity is maintained at 0.3 wt% or more for 100 seconds. It can be seen that it disappears and is not stained. This indicates that it is difficult to inactivate the endogenous peroxidase and retain the antigenicity under the above inactivating condition using only hydrogen peroxide.

[Example 3] Inactivation in a mixed system of sodium azide and hydrogen peroxide
Examination of Conditions The same samples (A) and (B) as those used in Example 2 were used, and a mixed system of sodium azide and hydrogen peroxide was used as an endogenous peroxidase inactivating agent. The inactivation conditions were examined in the same manner as in 2.

The conditions and results are shown in Table 4.

[0046]

[Table 4]

No. Hydrogen peroxide 0.
Although the antigenicity is retained at 1 wt% for 100 seconds, the inactivation of the endogenous peroxidase is insufficient (corresponding to No. 6 of Example 2). The retention of retention allows the inactivation of endogenous peroxidase to be achieved. However, as the concentration increases, the antigenicity disappears, so in this case, it may be necessary to shorten the time.

[Example 4] A case where blood of a patient having white blood cells infected with CMV was not used.
Examination of activation conditions-Examination of sodium azide concentration Sample (A) (blood of healthy person not infected with CMV)
And blood of a patient with CMV-infected white blood cells (sample (C))
And using a mixed system deactivator in the same manner as in Example 3, the inactivating conditions were examined according to the standard procedure.

The conditions and results are shown in Table 5.

[0050]

[Table 5]

As described above, the condition that ● and □ are satisfied at the same time is a preferable condition.
wt%, sodium azide 0.0125 to 0.2 wt
%, Immersion time of 100 seconds are preferable conditions.

[Example 5] Using a blood of a patient having white blood cells infected with CMV
Examination of activation conditions- Examination of hydrogen peroxide concentration Sample (A) (blood of a healthy person not infected with CMV)
And blood of a patient with CMV-infected white blood cells (sample (C))
And in the same manner as in Example 3 using a mixed-system inactivating agent, the inactivating conditions were examined according to the standard procedure.

The conditions and the results are shown in Table 6.

[0054]

[Table 6]

In Table 6, the conditions for satisfying both □ and ● are as follows:
Under the condition of sodium azide 0.05 wt%, hydrogen peroxide is 0.003 to 0.2 wt% and the immersion time is 100 seconds, and this condition is a preferable condition.

[Example 6] HEL cells infected with CMV and blood of a healthy person were used.
Examination of inactivation conditions-Examination of inactivation treatment time Sample (A) (blood of healthy person not infected with CMV)
Then, using the sample (B) (CMV-infected HEL cells), the inactivating agent of the mixed system was used in the same manner as in Example 3, and the inactivating condition was examined according to the standard procedure.

The conditions and results are shown in Table 6.

[0058]

[Table 7]

From the above results, when the immersion time is 30 seconds,
Hydrogen peroxide concentration is preferably 0.1 to 0.4 wt% and sodium azide concentration is preferably 0.3 to 0.9 wt%. Also, when the immersion time is 600 seconds, the hydrogen peroxide concentration is
0.00625 to 0.025 wt% and sodium azide concentration are preferably 0.003125 to 0.0125 wt%.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Shigeki Fujinaga 2-1-1 1-1 Uchisaiwaicho, Chiyoda-ku, Tokyo Inside Teijin Limited

Claims (3)

[Claims] 1. A peroxidase-labeled antibody is used after contacting a biological sample to be stained with a solution in which hydrogen peroxide and sodium azide coexist to inactivate endogenous peroxidase derived from the sample. A method for immunostaining a biological sample, which comprises immunostaining the biological sample. 2. The immunostaining method according to claim 1, wherein the biological sample contains human leukocyte cells suspected of being infected with a virus. 3. The hydrogen peroxide in the solution is 0.8-0.
001 wt% and sodium azide 2.7 to 0.
The immunostaining method according to claim 1, wherein the immunostaining method is 001 wt%.