JP6202893B2 - Method for evaluating the expression level of target protein in cells - Google Patents

Description

  The present invention relates to a method for evaluating a target protein expression level of a cell by immunostaining using an enzyme-labeled antibody.

  Cytodiagnosis and tissue diagnosis for diagnosing a patient from morphological information of cells and tissues play an important role in pathological diagnosis. For example, cells are stained using hematoxylin / eosin staining for tissue diagnosis, and general staining methods such as Papanicolaou staining and Giemsa staining for cytodiagnosis. And the presence or absence of abnormalities in the shape of the nucleus.

  As a method for examining the expression of a specific protein in a cell, an immunostaining method in which immunostaining is performed using an antibody that specifically binds to a target protein is common. In the immunostaining method, the presence or absence and expression intensity of the target protein are identified in each cell based on the presence or absence of staining and the staining intensity. Immunostaining includes an enzyme antibody staining method using an enzyme-labeled antibody and a fluorescent antibody staining method using a fluorescently labeled antibody. However, since an expensive fluorescence microscope is not required, enzyme antibody staining is used in medical settings. Law is preferred.

  Combining both the morphological information and the expression information of a specific protein serving as a pathological marker enables highly accurate pathological diagnosis. Both dye staining for morphological observation and immunostaining for examining the expression of the target protein to be analyzed are methods for staining cells in a specimen. Coloring occurs when the same specimen is stained twice. Therefore, it is very difficult to evaluate the expression level of the target protein for each cell in the specimen. In general, therefore, separate pathological specimens are used for dye staining and immunostaining. When both stainings are performed on the same specimen, the specimen is divided into two by a cell transfer method or the like (for example, see Non-Patent Document 1), one is dyed, and the other is immunostained. , Color mixing is avoided.

  In addition, there is known an apparatus that measures a change in calcium ion concentration in real time while taking a fluorescence image of a cell, and associates the position of the image with a time-series signal. (For example, refer to Patent Document 1). If such an apparatus is applied, cell morphology information and molecular information can be obtained together by performing immunostaining by a fluorescent antibody staining method.

JP 2006-3154 A

Edited by Cytologists Association, “Cytology Preparation Manual”, 2008, pages 19-20.

The morphological information and the expression information of the target protein are preferably acquired for the same cell. However, when both dye staining and immunostaining are performed on the same specimen, dye staining becomes the background, and accurate image analysis of color intensity by immunostaining cannot be performed, and target protein expression information There is a problem of low reliability.
On the other hand, there is a method of dividing one specimen into two or more by a cell transfer method or the like, but this method is cumbersome and time-consuming, and it may be impossible to transfer depending on the type and state of the specimen. is there.

  In the immunostaining using an enzyme-labeled antibody, the present invention is similar to cells that have been double-stained with a dye stain. It aims at providing the method which can evaluate a target protein expression level accurately.

  As a result of diligent research to solve the above-mentioned problems, the present inventor uses the enzyme-labeled antibody method to change the color development intensity (coloration intensity) over time, so that the color derived from staining other than the enzymatic reaction can be changed. From the cell image as a background, it was found that accurate image analysis of color intensity by enzyme reaction was possible, and the present invention was completed.

That is, the method for evaluating the expression level of the target protein in the cell according to the present invention is the following [1] to [ 4 ].
[1] (a) performing an antigen-antibody reaction using an enzyme-labeled antibody or enzyme-labeled antibody complex that specifically binds to a target protein on cells in a cell sample;
(B) after the step (a), adding a chromogenic substrate to the cells in the cell sample and causing a color to develop by an enzymatic reaction;
(C) obtaining a transmitted light image of cells in the cell sample at at least two or more time points after the addition of the chromogenic substrate, including the time point immediately after the addition of the chromogenic substrate in the step (b);
(D) based on two or more transmitted light images acquired in the step (c), examining the change over time of the transmittance in each cell;
(E) a step of evaluating the expression level of the target protein in each cell based on the change over time of the transmittance determined in the step (d);
Have a, in the step (d), the time course of the transmittance, the amount of change in transmittance and obtaining as an enzyme reaction time required to reach a predefined constant amount, the target cell A method for evaluating the expression level of a protein.
[2] (a) performing an antigen-antibody reaction using an enzyme-labeled antibody or enzyme-labeled antibody complex that specifically binds to a target protein on cells in a cell sample;
(B) after the step (a), adding a chromogenic substrate to the cells in the cell sample and causing a color to develop by an enzymatic reaction;
(C) obtaining a transmitted light image of cells in the cell sample at at least two or more time points after the addition of the chromogenic substrate, including the time point immediately after the addition of the chromogenic substrate in the step (b);
(D) based on two or more transmitted light images acquired in the step (c), examining the change over time of the transmittance in each cell;
(E) a step of evaluating the expression level of the target protein in each cell based on the change over time of the transmittance determined in the step (d);
Have
A method for evaluating the expression level of a target protein in a cell, wherein, in the step (d), a change in permeability over time is determined as a rate of change in enzyme reaction time per permeability.
[ 3 ] The method for evaluating the expression level of a target protein in a cell according to [1] or [2] , wherein the cells in the cell sample are stained with a dye before the step (b).
[ 4 ] The method for evaluating the expression level of a target protein in a cell according to [1] or [2], wherein the transmittance is expressed as the number of gradations of an image.

  According to the method for evaluating the expression level of a target protein in a cell according to the present invention, the expression level of the target protein can be accurately determined even for cells having a coloration other than the enzyme reaction of the enzyme-labeled antibody using the enzyme antibody staining method as a background. Can be evaluated well. For this reason, for example, cells with different expression levels of the target protein can be identified even for cell samples that have been subjected to double staining with dye staining for morphology observation.

In Example 1, the transmitted light image acquired about each cell sample is shown. In Example 1, the figure which plotted the number of gradations for every enzyme reaction time about each cell is shown.

The method for evaluating the expression level of a target protein of a cell according to the present invention (hereinafter sometimes referred to as “expression level evaluation method according to the present invention”) is based on the presence or absence of staining by the enzyme antibody staining method and the staining intensity. A method for evaluating the expression level of a target protein in each cell, comprising the following steps (a) to (e).
(A) performing an antigen-antibody reaction using an enzyme-labeled antibody or enzyme-labeled antibody complex that specifically binds to a target protein on cells in a cell sample;
(B) after the step (a), adding a chromogenic substrate to the cells in the cell sample and causing a color to develop by an enzymatic reaction;
(C) obtaining a transmitted light image of cells in the cell sample at at least two or more time points after the addition of the chromogenic substrate, including the time point immediately after the addition of the chromogenic substrate in the step (b);
(D) based on two or more transmitted light images acquired in the step (c), examining the change over time of the transmittance in each cell;
(E) A step of evaluating the expression level of the target protein in each cell based on the change over time of the transmittance determined in the step (d).

  Examples of cell samples used in the expression level evaluation method according to the present invention include biological samples (samples collected from living organisms), cells separated from biological samples, cultures of cells separated from biological samples, And cultures of cultured cells. Biological samples include tissue fragments collected from living organisms, blood, lymph, bone marrow, ascites, exudates, amniotic fluid, sputum, saliva, semen, bile, pancreatic juice, urine and other body fluids, feces, intestinal lavage fluid, and lung lavage fluid , Bronchial lavage fluid, or bladder lavage fluid. Examples of cells separated from biological samples include mononuclear cell components (buffy coat) separated and collected from blood, cell components separated and collected from lymph, etc. Examples include separated solutions, cell components separated and recovered after the tissue pieces are separated. In addition, the collection | recovery method from the biological body of a tissue piece is not specifically limited, A biopsy specimen, a surgical sample, etc. are mentioned. The cell sample provided in the present invention preferably contains two or more types of cells with different expression levels of the target protein, more preferably a biological sample, a cell separated from the biological sample, etc. Are more preferred.

  As the cell sample provided in the present invention, a cell sample that has been dyed at least before step (b) (before the enzyme reaction), preferably before performing the antigen-antibody reaction in step (a) is particularly preferable. preferable. The dye staining is preferably general staining in pathology for tissue observation or cell morphology observation. Specifically, hematoxylin / eosin staining (HE staining), Papanicolaou staining, Giemsa staining and the like are preferably used.

  The greater the amount of target protein present, the stronger the staining intensity by immunostaining. For this reason, in the cell which performed only immunostaining, the expression intensity | strength of the target protein in each cell can be compared by comparing the staining intensity | strength in immunostaining. In the transmitted light image of the immunostained cell, the staining intensity is reflected in the transmittance, and the transmittance is smaller as the staining intensity is higher.

  However, when cells stained with a dye are further stained with an enzyme antibody staining method, the color of the colored region due to the enzyme reaction and the colored region due to the dye staining overlap with each other. From the light image, it is not possible to distinguish between coloration due to enzyme reaction and coloration due to dye staining. In other words, in cells that have been double-stained with dye staining, the staining intensity in the transmitted light image after the enzyme reaction does not accurately reflect the expression level of the target protein. The expression level cannot be evaluated. On the other hand, in the expression level evaluation method according to the present invention, not the transmittance (staining intensity) in the transmitted light image but the change over time of the transmittance as an index, so for the cells that are dye-stained, The expression level of the target protein can be evaluated with high accuracy.

  First, as a step (a), a target protein is labeled with an enzyme using an antigen-antibody reaction to cells in a cell sample. The enzyme that labels the target protein may be any enzyme that can be used in the enzyme antibody staining method. As the enzyme, peroxidase or phosphatase is widely used and preferable.

  Enzyme labeling of the target protein may be performed using an enzyme-labeled antibody (enzyme-labeled primary antibody) obtained by directly enzyme-labeling an antibody that specifically binds to the target protein (primary antibody). Alternatively, an enzyme-labeled secondary antibody obtained by enzyme-labeling an antibody that specifically binds to the antibody (secondary antibody) may be used. In the latter case, the target protein is labeled with a complex of a primary antibody and an enzyme-labeled secondary antibody. Furthermore, even if a target protein is enzyme-labeled with these complexes using a primary antibody, a biotin-labeled secondary antibody (biotin-labeled secondary antibody), and enzyme-labeled streptavidin (or enzyme-labeled avidin). Good. In the present invention, the “enzyme-labeled antibody complex” includes a complex of a primary antibody and an enzyme-labeled secondary antibody, and a streptavidin (or enzyme-labeled) that is enzyme-labeled with a primary antibody and a biotin-labeled secondary antibody. Any of the complexes with avidin) are included.

  The antibody used as the primary antibody or secondary antibody may be a monoclonal antibody or a polyclonal antibody. Such antibodies may be commercially available antibodies, or antibodies prepared by immunizing laboratory animals such as mice, rats, rabbits and the like with conventional methods.

  Enzyme labeling of the target protein can be performed by conventional methods. For example, first, cells in a cell sample are fixed to a cell observation container that can be observed with a microscope using transmitted light (bright field), and then the enzyme-labeled primary antibody or primary antibody and enzyme are immobilized on the fixed cells. The target protein possessed by the cell is enzyme-labeled by bringing a labeled secondary antibody or the like into contact at a time or sequentially as necessary. When contacting a plurality of antibodies or the like sequentially, it is also preferable to perform a washing treatment with PBS (phosphate buffered saline) or the like before contacting the next antibody.

  In addition, when the target protein is present in the cells, it is also preferable to perform permeabilization on the cells previously contained in the cell sample before contacting the primary antibody. By the permeabilization treatment, a hole is made in the cell membrane or the nuclear membrane, and the antibody or the like easily penetrates into the cell or nucleus. Examples of the permeabilization treatment include conventional methods such as treatment of immersing cells in a surfactant solution. As the surfactant, Triton X-100, Tween 20, NP-40, saponin, digitonin, or the like can be used.

  The cell observation container is not particularly limited as long as it is generally used for cell staining, but is preferably a slide glass or a multiwell plate. By using an imaging cytometer or the like using a slide glass or a multi-well plate, a large number of specimens can be processed quickly and easily.

  The method for fixing cells to the cell observation container is not particularly limited, and can be performed by a conventional method. For example, the cells adhered to the cell observation container may be naturally dried, or may be treated with an immobilization treatment solution such as formaldehyde, paraformaldehyde, glutaraldehyde, methanol, ethanol, acetone, or the like. When the immobilization treatment solution is used for fixation, the immobilization treatment solution is washed with PBS or the like after the immobilization treatment to remove the immobilization treatment solution. In addition, before the reaction with the primary antibody, it is also preferable to block the cell observation container on which the cells are fixed using skim milk-containing PBS or the like.

  Next, as a step (b), a chromogenic substrate is added to the cells labeled with the enzyme, and color is developed by an enzymatic reaction. The chromogenic substrate may be any compound that, as a product, produces a chromogenic substance that changes the transmittance at a specific wavelength in the visible region when chemically modified by an enzymatic reaction. The color-developing material can be visually recognized from a microscopic observation image as a color that deposits on a chemically, physically, and electrostatically generated site and changes the transmittance of the deposited site. For example, diaminobenzidine (DAB), aminoethylcarbazole (AEC), BCIP / NBT, Fast Red, New Fuchsin, etc. are used as the color developing substrate for peroxidase.

  In the expression level evaluation method according to the present invention, as step (c), a transmitted light image is acquired at two or more time points for the same cell during the enzyme reaction in step (b). One of the transmitted light images to be acquired is acquired immediately after the addition of the chromogenic substrate. Instead of the transmitted light image immediately after the addition of the chromogenic substrate, a transmitted light image before the addition of the chromogenic substrate may be obtained. The acquisition time point of the transmitted light image can be appropriately adjusted in consideration of the type of enzyme, the temperature of the enzyme reaction, the type and concentration of the chromogenic substrate, and the like. For example, since the difference in reaction due to the amount of enzyme tends to appear earlier than the end point of the enzyme reaction, at least one of the plurality of transmitted light images acquired in step (c) is at the chromogenic substrate addition point ( It is preferable to obtain 0.5 to 3 minutes after the enzyme reaction start time).

  Specifically, an optical microscope equipped with an imaging device capable of acquiring a transmitted light image is installed with a cell observation container on which cells labeled with the target protein are enzyme-labeled. Transmitted light images are acquired at two or more points including. As the optical microscope, an optical microscope capable of observation at a magnification of about 20 to 400 times is preferable. As the imaging apparatus, an imaging apparatus that is normally used for imaging cells such as a CCD camera is used. The optical microscope preferably further includes an information processing apparatus for performing a predetermined image analysis process on the acquired transmitted light image.

  Based on the two or more transmitted light images acquired in the step (c), the temporal change in transmittance in each cell is examined (step (d)), and the target of each cell is determined based on the obtained temporal change in transmittance. The expression level of the protein is evaluated (step (e)). The change in transmittance with time is, in other words, a change in color with the passage of time. As will be shown below, the change in transmittance with time associated with color development is proportional to the expression level of the target protein (abundance in cells). For this reason, the expression level of the target protein is larger as the cell has a larger change in permeability over time, and the expression level of the target protein is lower as the cell has a smaller change in permeability over time.

  In enzyme-labeled immunostaining, the substrate reacts with the enzyme-labeled antibody to cause color development. This reaction is represented by the following formula (1). In the formula (1), E means an enzyme, S means a substrate, ES means an enzyme substrate complex, and P means a colored substrate.

[E] ₀ is the total enzyme concentration ([E] ₀ = [E] + [S] ₀ ), [S] is the substrate concentration, [E] is the free enzyme concentration, and k is the reaction rate constant. The time change of the concentration ([E]) is expressed by the following formula (2).

At this time, assuming that the substrate is largely excessive and the change of S is poor, [S] is a constant value, and therefore, the following equations (3) and (4) are obtained.

The time change of the transmittance is proportional to the time change of the colored substrate concentration [P]. Here, since the time change d [P] / dt = k ₃ [ES] of the substrate concentration [P], the following equation (5) is obtained.

Assuming that the color reaction has started and the enzyme reaction has reached a steady state, the change in time of [ES] is small, so it can be assumed that the following equation (6) is satisfied. Therefore, the speed (v ₀ ) of the color reaction is expressed by the following formula (7). That is, in the enzyme-labeled immunostaining, the expression level of the target protein is proportional to [E] _0, and thus the speed of the color reaction, that is, the amount of change in the transmittance during the enzyme reaction time is proportional to the expression level of the target protein.

  On the other hand, the substrate concentration [P] is represented by the following formula (8) under the condition that the substrate is excessive and does not change and reaches a steady state.

Therefore, the enzyme reaction time t when [P] reaches a certain value P ′ is the following formula (9), and [E] ₀ proportional to the expression level of the target protein is inversely proportional to the enzyme reaction time. To do. Conversely, [P] _{t = t ′} at a constant reaction time t ′ is the following formula (10), which is proportional to the expression level of the target protein.

  Since the coloration by the enzyme reaction is an independent chemical reaction, the proportional relationship is maintained regardless of the presence or absence of background staining. For this reason, even under conditions having a background, the expression level of the target protein can be compared by comparing the amount of change in permeability during the enzyme reaction time.

  Specifically, the target protein expression level of each cell can be evaluated by comparing the time change rate of the transmittance. The rate of change in transmittance over time is, for example, for each cell, obtained from the two or more transmitted light images acquired in step (c), with the transmittance as the vertical axis and the enzyme reaction time as the horizontal axis. When the transmittance is plotted, the slope of the approximate line for each plot is obtained.

  It can be evaluated that a cell having a higher rate of change in permeability with time has a higher target protein expression level and a cell with a lower rate of change in permeability with time has a lower target protein expression level. If the expression level of the target protein varies depending on the cell type, it can be evaluated that cells with the same rate of change in permeability over time are likely to be the same type of cells, and the rate of change in permeability over time is greatly different. It can be evaluated that there is a high possibility that the cells to be treated are different types of cells.

  The target protein expression level can also be evaluated by comparing the amount of change in the transmittance per predetermined time. For example, when two transmitted light images acquired at different points in time are compared, the difference in the transmittance of the same cell in the two transmitted light images can be said to be the amount of change in the transmittance per fixed time. It can be evaluated that a cell with a higher amount of change in permeability per predetermined time has a higher target protein expression level, and a cell with a lower amount of change in permeability per predetermined time has a lower target protein expression level.

  In addition, as shown in the above formula (9), the target protein expression level can also be evaluated by comparing the enzyme reaction time required for the change amount of the transmittance to reach a predetermined amount. It can be evaluated that the shorter the enzyme reaction time required for the transmittance change amount to reach a predetermined amount, the larger the target protein expression amount, and the longer the enzyme reaction time, the smaller the target protein expression amount.

  Similarly, the target protein expression level of each cell can be evaluated by comparing the rate of change in enzyme reaction time per permeability. It can be evaluated that the target protein expression amount is larger as the enzyme reaction time change rate per transmittance is smaller, and the target protein expression amount is smaller as the enzyme reaction time change rate per transmittance is larger. The rate of change in enzyme reaction time per transmittance was determined, for example, for each cell from the two or more transmitted light images obtained in step (c), with the enzyme reaction time on the vertical axis and the transmittance on the horizontal axis. When the transmittance of each cell is plotted, it is obtained as the slope of the approximate straight line for each plot.

  Similarly to the rate of change in transmittance over time, the amount of change in transmittance per predetermined time, the enzyme reaction time required for the amount of change in transmittance to reach a predetermined amount, or the enzyme reaction per transmittance Even when the rate of time change is used as an index, cells with similar values can be evaluated as highly likely to be the same type of cells, and cells with greatly different values can be different types of cells. It can be evaluated that the property is high.

  In each transmitted light image, the transmittance of each cell is calculated based on the gradation data (number of gradations) of the color component of the target pixel constituting the cell image of each cell. Specifically, the average value of the gradation values of all the pixels constituting the cell image of the cell is the gradation value of each cell and serves as a transmittance index.

  By performing the expression level evaluation method according to the present invention on the dye-stained cells, a dye-stained image that is an important information source of the cell morphology is obtained for the same cells in which the expression level of the target protein is evaluated. Can do. For this reason, for example, in a pathological specimen, by using a disease marker as a target protein, it is possible to examine the presence or absence of morphological change of cells peculiar to the disease, the presence or absence of expression of the disease marker, and the amount of expression. Thereby, the morbidity, presence / absence, severity, etc. of the disease can be more accurately evaluated than the conventional method in which tissue staining and immunostaining are performed on different cell specimens.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
For the prostate cancer cell line (PC-3 line), the expression level evaluation method according to the present invention was performed on a Papanicolaou-stained cell specimen using Ki-67 as a growth marker as a target protein.
First, the PC-3 strain adhered to the glass bottom of the cell culture vessel and cultured was treated with a 4% by volume paraformaldehyde solution for 30 minutes and fixed. After washing with distilled water, the cells on the slide glass were stained with hematoxylin (Merck, product number: 10924490500). The stained cells were washed with distilled water and then immersed in a 0.5% by volume Triton X-100 solution for 5 minutes. Then, after washing twice with PBS, the cells on the glass bottom were immersed in Dako Real peroxidase Blocking solution (manufactured by Dako, product number: S202386) for 10 minutes for blocking treatment. The blocked glass bottom was washed twice with PBS and then immersed in PBS containing 5% by volume of goat serum for 30 minutes. Next, the cells on the glass bottom were reacted with a primary antibody against Ki-67 (Dako, product number: M724029) at room temperature for 30 minutes. After the reaction, the plate was washed twice with PBS, and then an enzyme-labeled secondary antibody (manufactured by Dako, product number: K1390) was reacted at room temperature for 30 minutes, and after the reaction, washed twice with PBS (sample).

  A treatment similar to the above except that the cell sample treated as above was used as a positive control, except that hematoxylin staining was not performed, hematoxylin staining was not performed, and no primary antibody against Ki-67 was added. The cell sample subjected to was used as a negative control.

  Thereafter, the cell culture container of each cell specimen is placed on the stage of an optical microscope (Olympus, product number: IX70) equipped with an imaging device (Olympus, product number: DP72), and the visual field to be imaged is determined. A DAB chromogenic substrate solution (manufactured by Dako, product number: K1390) was added to the cells on the glass bottom, 0 minutes after the addition (immediately after the addition), 0.5 minutes, 1 minute, and 3 A transmitted light image after a minute was acquired. For any cell of interest in the obtained image, the color component displayed in gray scale was digitized with 256 gradations using image processing software cellSens (Olympus). As the transmittance of each cell, the average gradation value of all pixels constituting each cell was calculated.

  The transmitted light image acquired about each cell specimen is shown in FIG. As a result of microscopic observation, no positive cells (cells immunostained with anti-Ki-67 antibody) were found in the negative control (upper row in FIG. 1). On the other hand, the positive control was positive in almost all cells (middle of FIG. 1). In addition, in the sample, the color due to Papanicolaou staining was confirmed immediately after DAB addition (after 0 minutes), and as the reaction time passed, the appearance of further coloration from above the background Papanicolaou staining was observed (FIG. 1). Bottom).

  FIG. 2 shows a plot of the number of gradations for each enzyme reaction time for each cell. The negative control cell (Cell 1) and one of the 4 cells of the sample (Cell 5) showed almost no change in the number of gradations. On the other hand, the positive number of cells (Cells 2 to 4) and the remaining 3 cells (Cells 6 to 8) of the sample showed an increase in the number of gradations with the passage of time.

  From FIG. 2, for each cell, the amount of change in the number of gradations in the enzyme reaction time of 0 to 1 minute was represented by an approximate line, and the slopes were compared. Table 1 shows the slope and intercept of the approximate expression obtained from the change in the number of gradations in 0 to 1 minute. As a result, the inclination of about 10 to 40 seen in the positive control was also observed in the samples (Cells 6 to 8) having a background by Papanicolaou staining. Further, in the positive control, there was a difference in slope between the low expression cells (Cell 2, slope 13), the medium expression cells (Cell 3, slope 20) and the high expression cells (Cell 4, slope 46) of Ki-67. Similarly to the above, in the sample, there is a difference in slope between the low expression cell (Cell 6, slope 9), the medium expression cell (Cell 7, slope 25) and the high expression cell (Cell 8, slope 34). It was. From this result, it is clear that the relative expression level of the target protein can be evaluated for the dye-stained cells by the expression level evaluation method according to the present invention.

  Since the expression level evaluation method according to the present invention can accurately evaluate the target protein expression level by the enzyme antibody staining method even for cells whose background is a color other than the color developed by the enzyme reaction of the enzyme-labeled antibody, It is suitably used in pathological fields such as cytodiagnosis and tissue diagnosis for performing both staining and immunostaining.

Claims (4)

(A) performing an antigen-antibody reaction using an enzyme-labeled antibody or enzyme-labeled antibody complex that specifically binds to a target protein on cells in a cell sample;
(B) after the step (a), adding a chromogenic substrate to the cells in the cell sample and causing a color to develop by an enzymatic reaction;
(C) obtaining a transmitted light image of cells in the cell sample at at least two or more time points after the addition of the chromogenic substrate, including the time point immediately after the addition of the chromogenic substrate in the step (b);
(D) based on two or more transmitted light images acquired in the step (c), examining the change over time of the transmittance in each cell;
(E) a step of evaluating the expression level of the target protein in each cell based on the change over time of the transmittance determined in the step (d);
I have a,
In the step (d), the expression amount of the target protein of the cell, characterized in that the change in permeability over time is determined as an enzyme reaction time required for the change in permeability to reach a predetermined amount. Evaluation method.   (A) performing an antigen-antibody reaction using an enzyme-labeled antibody or enzyme-labeled antibody complex that specifically binds to a target protein on cells in a cell sample;
(B) after the step (a), adding a chromogenic substrate to the cells in the cell sample and causing a color to develop by an enzymatic reaction;
(C) obtaining a transmitted light image of cells in the cell sample at at least two or more time points after the addition of the chromogenic substrate, including the time point immediately after the addition of the chromogenic substrate in the step (b);
(D) based on two or more transmitted light images acquired in the step (c), examining the change over time of the transmittance in each cell;
(E) a step of evaluating the expression level of the target protein in each cell based on the change over time of the transmittance determined in the step (d);
Have
  A method for evaluating the expression level of a target protein in a cell, wherein, in the step (d), a change in permeability over time is determined as a rate of change in enzyme reaction time per permeability. The method for evaluating the expression level of a target protein in a cell according to claim 1 or 2 , wherein cells in the cell sample are stained with a dye before the step (b). The method for evaluating the expression level of a target protein in a cell according to claim 1 or 2, wherein the transmittance is expressed as the number of gradations of an image.