JPH0762666B2 - Method for detecting abnormal migration image of protein fraction - Google Patents

Description

TECHNICAL FIELD The present invention relates to an improved method for detecting an abnormal migration image of a protein fraction obtained by electrophoresis from a sample such as plasma.

(B) Conventional technology The protein fractionation of a sample by electrophoresis, particularly the plasma (serum) protein fractionation, is widely performed as one item of the screening test. Plasma proteins are composed of a large number of proteins of over 80 kinds. They have important physiological significance in plasma coagulation, immune system, metabolic control, etc., are involved in maintaining plasma oncotic pressure, and are involved in substance exchange in peripheral tissues. Abnormalities in the composition of plasma proteins are caused by abnormalities such as in vivo synthesis and degradation. This abnormality appears as an abnormal migration image of the plasma protein fraction and contains a lot of pathological condition information.

By the way, in recent years, the above-mentioned detection of abnormal electrophoretic images of plasma protein fractions is being automated by using a computer.
This automated detection method is described below.

FIG. 6 (c) shows a densitogram of the sample plasma protein content image. This densitogram may be obtained by subjecting a sample plasma to fractionation by electrophoresis on a support such as a cellulose acetate membrane and measuring the optical density of the fractionated image with a densitometer (Japanese Patent Publication No. 61-16019). See the bulletin).

The densitogram is divided into 5 fractions each containing one peak. The fraction a ₀ containing the largest peak at the left end is due to albumin in plasma. The α ₁ -globulin fraction a ₁ and the α ₂ -globulin fraction containing peaks in order on the right of this albumin fraction a ₀
a ₂ , β-globulin fraction a ₃ , and γ-globulin fraction a ₄ appear.

This densitogram was compared with a control densitogram (see FIG. 6 (a)) obtained from the protein fraction of the serum managed for control (hereinafter referred to as the control serum), so normalization was performed by computer. To be done. This is because the migration length of the sample plasma differs depending on the migration conditions, and as it is, the number of data making up the sample densitogram and the control densitogram will differ, making it impossible to compare the two. The data of the densitogram is corrected and interpolated, and the number of the data is adjusted to the control densitogram so that the data of the both have a one-to-one correspondence.

For this normalization, first, the albumin peak position was determined from the sample densitogram and the control densitogram.
x _sm and x _cj are extracted [see FIGS. 6 (a) and 6 (c)].

Next, the sample densitogram is (x _cj −x _ci ) /
(X _sn −x _sm ) times to make the sample densitogram and the control densitogram have the same length [see FIG. 6 (b)]. The thus-normalized sample densitogram is compared with a control densitogram showing a healthy state, and an abnormal electrophoretic image of the sample plasma protein fraction is detected.

(C) Problems to be Solved by the Invention In the above-mentioned conventional method for detecting an abnormal migration image of a protein fraction,
In some cases, the β-globulin peak position of the sample densitogram cannot be accurately extracted, and the normalization of the sample densitogram may be inaccurate. In that case, there is a disadvantage that an abnormal migration image cannot be detected.

When the β-globulin peak position of the sample densitogram cannot be accurately extracted, as shown in FIG. 6 (d),
The case where β-γ linking L occurs or the case where peaks of β lipoprotein and various M proteins appear appears.
Furthermore, depending on the type of cellulose acetate membrane,
In some cases, the sample is applied to the β-globulin fraction position, but in this case, the non-migrated substance in the sample remains at that position and is detected as noise. Even in the case of a cellulose acetate film in which a sample is applied to the α ₁ -globulin fraction position, noise may appear in the β-globulin fraction. Thus, when peak noise is contained in the densitogram, it is eliminated by computer processing, and the β-globulin peak position is accurately determined.
It is difficult to extract x _sn .

In addition, even if the normalization of the sample densitogram is performed correctly, it is not necessary for the computer processing to compare the normalized sample densitogram with the control densitogram and detect the abnormal migration image. It was complicated and required a long time.

The present invention has been made in view of the above inconveniences, and a method for detecting an abnormal migration image of a protein fraction, which ensures normalization of a sample densitogram and accurately and prepares a detection process of an abnormal migration image, particularly a detection process by a computer. Is intended to provide.

(D) Means for Solving Problems As a means for solving the above-mentioned inconvenience, the method for detecting an abnormal electrophoretic image of a protein fraction of the present invention comprises subjecting a sample sample and a control sample to electrophoresis on a membranous support. , A protein densitogram of each protein is obtained, and a sample densitogram and a control densitogram are obtained from these protein images by densitometry, and both densitograms are compared to detect an abnormal migration image of the protein fraction. From each of the control densitogram and the control densitogram, a position at which the albumin peak position and the γ-globulin peak tail are equal to a predetermined threshold value is extracted, the sample densitogram is normalized based on these positions, and the normalized sample densitogram The difference between the control densitogram and the control densitogram is taken, and the abnormal electrophoresis image of the protein fraction is detected based on this difference. That.

(E) Action In the method for detecting an abnormal electrophoretic pattern of a protein fraction of the present invention, a position at which the tail of the γ-globulin peak is equal to a predetermined threshold value is extracted. The γ-globulin peak value itself varies greatly depending on the sample, but this position can be reliably extracted without being affected by the presence of the β-riboprotein peak or the sample application position on the membranous support, and the sample residue Accurate normalization of the togram.

In addition, the difference waveform obtained by taking the difference between the control densitogram and the normalized sample densitogram shows that the excess or deficiency of the specific protein in the sample is due to the upward or downward peak in the fraction of the difference waveform. Since it appears clearly, the process of detecting an abnormal migration image can be performed accurately, simply and quickly.

(F) Embodiment An embodiment of the present invention is shown in FIGS. 1 (a) to 1 (c), 2 (a) to 2 (c), 3 (a), 3 Based on FIG. (B), FIG. 4 and FIG.
This will be described below.

This example is an application of the present invention to a protein fraction of human plasma. First, densitometry of plasma protein fraction will be described.

FIG. 4 shows a plan view of the cellulose acetate membrane (membrane support) 1. A specimen sample is applied on the cellulose acetate film 1 and fractionated by the electrophoresis method. 2, ..., 2 show the sample plasma protein image thus obtained.

FIG. 5 shows an example of the processing device. In this device, light from the light source 3 is applied to the cellulose acetate film 1 through the lens 4, the filter 5 and the slit 6. Then, the light passing through the cellulose acetate film 1 is detected by the light receiving element 7.

The cellulose acetate film 1 is fed in the right direction R in FIG. 5, for example. This direction R is also shown in FIG. Each minute image 2 is scanned in the moving direction of the cellulose acetate film 1, that is, in the direction perpendicular to the direction R, and photometry is performed. The output signal of the light receiving element 7 is amplified by the preamplifier 8 and converted into a logarithmic value by the logarithmic converter 9.
The output signal of the logarithmic converter 9 is converted into a digital signal by an analog / digital (A / D) converter 10 and taken into a microcomputer (CPU) 11 at regular intervals. In this way, a densitogram of the sample plasma protein fraction is obtained. Two examples of this sample densitogram are shown in FIG. 1 (b) and FIG. 1 (c). The densitograms shown in FIG. 1 (b) and FIG. 1 (c) are displayed in analog form by connecting individual sample concentration data.

FIG. 1 (a) shows a densitogram of control serum (control densitogram) obtained as described above. This control serum is commercially available for use as a standard serum for clinical tests and the like. In the sample densitogram of FIG. 1 (b), when the migration length of the sample plasma protein fraction is shorter than the migration length of the protein fraction of the control serum, the sample densitogram of FIG. 1 (c) is the sample plasma protein The case where the migration length of the fraction is longer than that of the protein fraction of the control serum is shown.

Next, from the control densitogram and the sample densitogram, albumin peak positions x _ci , x _sm and points x _cj , x at which the predetermined threshold value Δy of the right skirt b of the γ-globulin peak is equal to x _cj , x.
_sn is extracted by the CPU 11 [see FIG. 1 (a), FIG. 1 (b) and FIG. 1 (c)].

The size of the γ-globulin peak varies greatly depending on the protein composition of the sample plasma. Therefore, the threshold Δy
Is set according to the size of the γ-globulin peak of the sample densitogram so that the data position x _sn can be extracted reliably. The threshold value Δy is set by an input device (not shown) connected to the CPU 11, such as a keyboard.

Subsequently, normalization processing of the sample densitogram is performed. To do so, first multiply the sample densitogram by x and align it with the length of the control densitogram. The value of this x is shown by the following formula.

x is larger than 1 in the case of the short sample densitogram shown in FIG. 1 (b) (x> 1), and smaller than 1 in the case of the long sample densitogram shown in FIG. 1 (b) (x <
1)

Here, when the sample densitogram is multiplied by x, the data becomes insufficient or excessive, and individual data of the sample densitogram do not correspond one-to-one with many data of the control densitogram.

First, the correction / interpolation processing when the sample densitogram data is insufficient will be described below.

The case where the data is insufficient is the case where the migration length of the sample densitogram is shorter than that of the control densitogram, as shown in FIG. 1 (b).

For example, let x _cj = 1000, x _ci = 100, x _sn = 750, x _sm = 150. In this case, the value of x becomes 1.5, and the sample densitogram is stretched. As a result, x
The data interval of the doubled densitogram is widened and does not correspond to the individual data of the control densitogram, and the number thereof is insufficient [see FIG. 3 (a)].

Therefore, data position before normalization analyte densitograms x _s = 150, 152, 154, when it is ..., i.e. x doubling analyte electronic data chromatogram position x 's ₌ 150,153,156, with ... when there is data position x _s = 150, 152, 154, the data position of the normalized sample densitograms data at ...... x "s ₌ 150,153,156, will put in order ....

Data position before normalization analyte densitograms x _s = 151,153,155, when a ......, i.e. x doubling sample densitograms data position x 's ₌ 151.5,154.5,157.5, when it is ... is , The data at the data positions x _s = 151, 153, 155, ... Are put in order at the data positions x ″ _s = 152, 155, 158, ... In the normalized sample densitogram.

The data position of the normalized sample densitogram x ″ _s = 151, 154, 157, ... Has the data position of the sample densitogram before normalization x _s = 15
Arithmetic mean of 0, 151, x _s = 152, arithmetic mean of 153, x _s =
Enter the arithmetic average value of 154 and 155 in order [Fig. 3 (a)]
Refer to the dashed arrow in the middle.

On the other hand, when the data of the sample densitogram becomes excessive, the migration length of the sample densitogram becomes longer than that of the control densitogram as shown in FIG. 1 (c).

For example, _assume that x _cj = 1000, x _ci = 100, x _sn = 1580, and x _sm = 80. In this case, the value of x becomes 0.6, and the sample densitogram is compressed. As a result, the data position x ′ _s interval of the sample densitogram multiplied by x is shortened, the number of data is increased, and the data does not correspond to the individual data of the control densitogram [see FIG. 3 (b)]. Processing is performed.

First, the data position x _s = 80,85 of the sample densitogram before normalization, ie, the data position x ′ _s = 80,83,86, ... Put it in the data position x ″ _s = 80, 83, ... of the chemical specimen densitogram as it is.

In the data position x ″ _s = 81 of the normalized sample densitogram, the data position x _s = 81, 82 of the sample sample densitogram before normalization, that is, in the data position x ′ _s of the x-fold sample densitogram.
= The arithmetic average value of the data at 81.6 and 81.2.

In the case of x ″ _s = 82, the arithmetic mean value of the data in x _s = 83, 84 is obtained.
The data at x ″ _s = 84, 85, 87, 88, ... Are sought (see the broken line arrow in FIG. 3 (b)).

FIG. 2 (a) shows the normalized sample densitogram as described above, and FIG. 2 (b) shows the control densitogram. Further, FIG. 2 (c) is a difference waveform obtained by subtracting the data of the corresponding control densitogram from the data constituting the normalized sample densitogram.

In this difference waveform, a maximum (upward) peak and a minimum (downward) peak corresponding to the abnormal protein composition in the sample plasma appear. For example, in the γ-globulin fraction a ₄ , the maximum peak P′a
Appears, which is due to the maximum peak Pa of the normalized sample densitogram shown in FIG. 2 (a). In this case, it is shown that the specific component in the plasma protein corresponding to the maximum peak Pa is higher than usual.

On the other hand, the albumin fraction a ₀ of the differential waveform contains a minimum peak P′b. This indicates that the specific component in the plasma protein corresponding to the minimum peak P′b is less than usual. Although FIG. 2 (c) shows the case where the differential waveform is relatively close to the baseline, various maximum and minimum peaks appear in each fraction depending on the pathological condition.

Therefore, the peak width and peak value are detected for each fraction, and these values are compared with a predetermined threshold value to detect an abnormal migration image. The threshold value may be different for each fraction, or may be changed according to the condition of the sample. In this way, the abnormal migration image appears as a peak on the differential waveform, so the detection process can be performed accurately and quickly. In addition, the abnormal migration image thus obtained is accurate and provides valuable information for pathological condition analysis.

(G) Effect of the Invention The method for detecting an abnormal electrophoretic pattern of a protein fraction of the present invention detects the position at which the albumin peak position and the γ-globulin peak tail are equal to a predetermined threshold value from the sample densitogram and the control densitogram, respectively. Extraction, normalization of the sample densitogram based on these positions, taking the difference between this normalized sample densitogram and the control densitogram, and detecting the abnormal migration image of the protein fraction based on this difference Is. Therefore, there is an advantage that the normalization of the sample densitogram is ensured, and the abnormal migration image detection process can be performed accurately, simply and quickly.

[Brief description of drawings]

FIG. 1 (a) is a diagram showing a control densitogram in one embodiment of the present invention, FIG. 1 (b) is a diagram showing an example of a sample densitogram in the same embodiment, and FIG. 1 (c).
Is a diagram showing another example of the sample densitogram in the same example, FIG. 2 (a) is a diagram showing a normalized sample densitogram in the same example, and FIG. 2 (b) is the same example. The figure again showing the control densitogram in the example, FIG. 2 (c) is a figure showing the differential waveform in the same embodiment, and FIGS. 3 (a) and 3 (b) are the normalization in the same embodiment. FIG. 4 is an explanatory diagram, FIG. 4 is a diagram showing a protein content image in the same example, and FIG. 5 is a diagram illustrating a processing apparatus of the same example.
FIG. 6 (a), FIG. 6 (b), FIG. 6 (c) and FIG. 6 (d) are diagrams for explaining a conventional abnormal migration image detection method and its problems. 1: Cellulose acetate membrane, 2 …… 2: Plasma protein image.

Claims (5)

[Claims] 1. A sample sample and a control sample are electrophoresed on a membranous support to obtain respective protein content images, and a sample densitogram and a control densitogram are obtained from these protein content images by densitometry. In the method for detecting an abnormal electrophoretic pattern of a protein fraction by detecting an abnormal electrophoretic pattern of a protein fraction by comparing a togram and a control densitogram, the albumin peak position and the γ-globulin peak are detected from the sample densitogram and the control densitogram, respectively. Extract the position that becomes equal to the predetermined threshold at the hem, normalize the sample densitogram based on these positions, take the difference between this normalized sample densitogram and the control densitogram, and based on this difference A method for detecting an abnormal migration image of a protein fraction, which comprises detecting an abnormal migration image. 2. The method for detecting an abnormal electrophoretic image of a protein fraction according to claim 1, wherein the predetermined threshold value is changed according to the protein composition of the specimen sample. 3. The detection of the abnormal migration image is performed by detecting a peak from each fraction in the difference and comparing the width and value of the peak with a predetermined value. The method for detecting an abnormal migration image of a protein fraction according to the item 2. 4. The method for detecting an abnormal migration image of a protein fraction according to claim 3, wherein the predetermined value is set for each fraction. 5. The method for detecting an abnormal migration image of a protein fraction according to claim 3 or 4, wherein the predetermined value is variable.