JP2006090864A - Method for calculating content of specific component in vaccine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for calculating the content of a specific component in vaccine. <P>SOLUTION: An antigen is added to a gel containing an antibody, the sedimented ring formed by the antigen-antibody reaction in the gel is captured in a computer as an image by an image capture means, the captured image is displayed on a display by the computer, and the end point of the sedimented ring displayed as the image is determined using visual aid to automatically measure the length of its diameter. Further, an X-Y table is used and automatically moved to successively capturing all of the sedimented rings as images by a camera to successively display the images on the display, and the end points of all of the sedimented rings are determined by visual aid to automatically measure the lengths of the diameters of the sedimented rings. By this method, required labor is reduced as compared with a conventional method, a work time required in measurement is shortened and the occurrence of the reading mistake caused by fatigue is reduced. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for determining the content of a specific component in a vaccine. Specifically, the present invention measures the content of a specific component in a vaccine by measuring the diameter of a settling ring obtained by a single radial immunodiffusion method (SRD or SRID) using an image analyzer. It is related to the method of seeking.

  In SRD or SRID technology, a constant concentration of antibody is placed in a support such as agarose gel, a well is opened, an antigen solution is injected into the well, and then diffused to form a sedimentation ring or sedimentation line by an antigen-antibody reaction. The diameter of the sedimentation ring is obtained, and the antigen concentration is calculated.

  As an example of applying the SRD or SRID technique, a method for determining the hemagglutinin (HA) content in an influenza vaccine can be mentioned.

  The HA content, which is a major component in influenza vaccines, can be calculated from the size of the sedimentation ring produced by the antigen-antibody reaction in the gel by adding the vaccine to an agarose gel containing antiserum against HA. Recommended by the World Health Organization (WHO).

  The method for measuring the HA content is currently performed as follows (see, for example, Non-Patent Document 1): an agarose film is primed on a glass plate and dried. The mold with the interior hollowed out is sealed to the glass plate and then agarose containing the appropriate concentration of influenza HA antiserum is injected into the mold. The gel is cured and a template is used to open a well in agarose and remove the mold.

  Next, a standard antigen of known concentration and a test antigen of unknown concentration are appropriately diluted and introduced into wells in agarose. After diffusing the antigen at a constant temperature for a certain time, a filter paper moistened with water is placed on the agarose surface. Place a weight on it and press the gel. The weight is removed, the gel is dried in warm air, and after complete drying, the filter paper is peeled off and the glass plate is colored with a staining solution. The glass plate is then decolored until a clearly demarcated colored area becomes visible and finally dried in warm air.

The diameter of the colored area surrounding the antigen well is measured at a right angle in two directions using a micrometer scale loupe, and the HA content is determined from the obtained diameter value by a parallel line quantification method.
Journal of Biological Standardization, 1977, 5, 237-247

  The HA content measurement method currently used requires that the diameter of the sedimentation ring is about 6 to 9 mm and measures to the 0.1 mm unit, and the measurement is a scale with a scale of 0.1 mm and a scale of 10 times. It is done visually using a loupe.

  In this visual measurement, when there are a large number of specimens to be measured, the operator looks into the loupe for a long time, which causes eye strain and results in poor work efficiency. In addition, there is a possibility that an error occurs in the measurement value due to operator fatigue.

  In addition, there are differences in the properties of the specimens themselves, many of which are difficult to automatically measure the diameter by the machine because the settling ring boundary is not clear enough to be recognized by the optical machine. However, the actual situation is that measurement must be performed visually. For this reason, differences in values are likely to occur due to individual differences among operators. In addition, depending on the specimen, the boundary is irregular, or the coloring of the specimen and the background is the same, and it is difficult for even a skilled operator to define the boundary.

  Therefore, there is a demand for a measurement method that makes use of human superior visual ability, is easier and less labor-intensive than conventional work, and is less likely to cause errors and measurement errors.

  In view of the above circumstances, the object of the present invention is to improve the workability and reduce the accuracy of sedimentation wheel measurement by reducing the eye strain of the operator when measuring the diameter of the sedimentation wheel caused by the antigen-antibody reaction. That is.

  The inventors of the present invention diligently studied to solve the above-described problems, and achieved the object of the present invention by taking the settling wheel as image data and examining the size of the settling ring using an image analysis technique. As a result, the present invention has been completed.

Thus, according to the present invention, the following inventions 1 to 8 are provided.
1. The antigen was added to the gel containing the antibody (antiserum), and the sedimentation ring generated by the antigen-antibody reaction in the gel was captured and captured as an image on a computer (or personal computer, hereinafter referred to as a computer) by the image capturing means. A method in which an image is displayed on a display by a computer, the end point of the settling wheel displayed as an image is determined with the help of visual observation, and the length of the diameter is automatically measured.
2. An antigen is added to a gel containing an antibody, a measurement sample having a plurality of sedimentation rings generated by an antigen-antibody reaction in the gel is set on an XY table, and one sedimentation ring is photographed with a camera and captured as an image. After that, the XY table is automatically moved so that all the settling wheels are sequentially captured as images by the camera, and then one of the captured images is sequentially displayed on the display by the computer connected to the camera. A method of automatically determining the length of the diameter of the settling wheel by determining the end point of the settling wheel with the help of visual observation.
3. 3. The method according to 1 or 2 above, wherein the diameter of the sedimentation wheel displayed as an image is sandwiched between two parallel lines on the X-axis and Y-axis on the screen, and the length is obtained.
4). 4. The method according to any one of 1 to 3 above, wherein a straight line is drawn by clicking both ends of the diameter of the settling wheel displayed as an image, and the length is obtained.
5. The method according to any one of 1 to 4 above, wherein the length of the diameter of the settling wheel displayed as an image is measured by the number of pixels.
6). 6. The method of claim 5, further comprising determining a length per pixel or the number of pixels per mm using a reference scale.
7). 3. The method according to 1 or 2 above, wherein the antibody is an antiserum against hemagglutinin.
8). 3. The method according to 1 or 2 above, wherein the antigen is a vaccine.

  The method of the present invention reduces the labor required compared to the conventional method, can reduce the work time required for measurement, and causes less reading errors due to fatigue. In addition, since fatigue is small, there are few errors in the measured values due to fatigue. Furthermore, since it is possible to measure a plurality of persons with the same appearance by projecting them on the display, it is possible to reduce differences due to individual differences among persons who measure. That is, the operator can easily take over. According to the method of the present invention, the eye strain of the operator can be reduced, and the working environment of the operator can be improved. Still further, since the scale used in the conventional method is not used, errors due to erroneous reading of the scale can be eliminated.

  The measurement sample to be measured according to the present invention has one or more samples in which a sedimentation ring is formed by an antigen-antibody reaction. Preferably, the sample has one or more sedimentation rings formed by the reaction between the antiserum and the vaccine.

  Examples of the antibody used in the present invention include antiserum against HA. HA is a substance that causes hemagglutination, and examples thereof include those having a glycoprotein as a hemagglutinin such as influenza virus, mumps virus, and Japanese encephalitis virus.

  The antigen used in the present invention is a vaccine, preferably an influenza vaccine.

Hereinafter, the present invention will be described in detail with reference to the drawings.
FIG. 1 shows an example for carrying out the method of the invention. An antigen is added to a gel containing an antibody (antiserum), and a measurement sample 1 having a plurality of sedimentation rings generated by an antigen-antibody reaction in the gel is prepared as described in Non-Patent Document 1. The measurement sample 1 is set on the XY table 2. The illumination 3 is turned on and the measurement sample 1 is illuminated. The camera 4, for example, a CCD camera is focused on one settling wheel of the measurement sample 1, and the entire settling ring is photographed and captured as an image. The XY table 2 is moved, and all the settling wheels are sequentially captured as images by the camera 4. The camera 4 is connected to the computer 5, and all images taken by the camera 4 are taken into the storage device of the computer 5.

  In a preferred embodiment of the present invention, the plurality of settling wheels of the measurement sample 1 are prepared so that the number and pitch in the horizontal direction and the vertical direction are constant. When the numerical information of the number and pitch is input to the program of the computer 5 and the camera 4 finishes photographing one settling wheel, the computer 5 recognizes the number and the number and pitch to the XY table controller 6 connected to the computer 5. A command is sent based on the numerical information of the pitch. The XY table controller 6 is connected to the XY table 2 and automatically moves the XY table 2 in accordance with a command from the computer 5 to perform the next settling wheel photographing as before.

  Although one measurement sample 1 has a plurality of circular patterns, if the camera field of view and the focus are aligned with the reference (first) circular pattern, the subsequent positioning and photographing can be performed automatically.

  For example, when the sample is composed of 16 4 × 4 settling wheels arranged at an equal pitch as shown in FIG. 8, the computer 5 finishes shooting the settling ring as the first reference. A command to move the XY table 2 leftward by one pitch is output to the XY table controller 6. Upon receiving the command, the XY table controller 6 converts the signal into a drive signal for a drive device such as a motor in the XY table 2, and outputs the signal to the drive device in the XY table 2. -Y table 2 moves to the left by one pitch. Thereafter, the same process is repeated twice, and after the fourth settling wheel has been shot, the computer 5 gives an instruction to move the XY table 2 backward by one pitch and rightward by three pitches. -Output to Y table controller 6. Receiving the command, the XY table controller 6 activates the driving device in the XY table 2 and moves the XY table 2 so that the fifth settling wheel is directly under the camera. Thereafter, the same processing is repeated to photograph all 16 settling wheels, and then the computer 5 moves the XY table 2 so that the reference settling wheel is directly below the camera, and the photographing is completed.

When the image capture is completed, the photographed image is displayed on the display, and the end point of the settling ring is visually determined to measure the outer diameter of the image (settling ring). Before measuring the outer diameter of the image, a reference scale whose length and height (for example, 8 mm × 8 mm) are known is taken in as follows.
(A) Shoot the reference scale under the same conditions as the sample shooting conditions.
Various kinds of reference scales may be used, and those that are easy to use may be used.
(B) The length (horizontal length) and height (vertical length) of the reference scale are measured.
(C) Since the number of pixels of each dimension is displayed, the numerical value is set as a reference scale value. That is, the coefficient of length per pixel is obtained. Thereby, the length and height per pixel on the display can be obtained without requiring particularly complicated setting and calibration. The reason why both the length and the height are measured is that image processing boards often have different vertical and horizontal resolutions.

The outer diameter of the image can be measured at a right angle in two directions and can be measured by the following two methods.
Four-cursor method: Two parallel lines on each of the X-axis and Y-axis on the screen are moved so as to be in contact with the outer periphery of the sample, and measured. The position of the parallel lines can be adjusted with the mouse and the arrow keys.
Click method: Clicking the start point and end point to be measured or both the left and right and upper and lower ends with the arrow keys of the mouse draws a straight line between the two points, and measures the length.

  The outer diameter of the image on the screen may be a raw image (or a color image) or a binarized image (or a monochrome image). A binarized image can be created by determining an appropriate threshold value (or reference value) and making an image brighter than the threshold value white or the like and an image darker than the threshold value black or the like. As mentioned above, the boundary line of the settling wheel is very vague and it is often difficult to set an automatic threshold value by a computer, but such a binarized image has a particularly clear boundary line. This is effective for the measurement of the standard scale (calibration round seal).

  Next, the measured length and height of the settling wheel are converted into actual lengths and heights. That is, by calculating the number of pixels between the left and right and upper and lower ends, and multiplying the obtained number of pixels by the length and height per pixel obtained using the reference scale described above ( Alternatively, the actual length and height of the settling wheel is calculated by dividing the number of pixels obtained by the number of pixels per unit length (eg, 1 mm). The measured data and / or the calculated actual length and height are sequentially stored in a storage device in the computer 5.

  The HA content value is obtained from the obtained diameter value by a parallel line quantitative method.

  The obtained results can be automatically stored in a computer and can be stored in a database, so that the results can be easily managed.

  The measurement sample, apparatus, and specific operation method actually used will be described below, but the present invention is not limited thereto.

1. Measurement sample Measurement sample 1 was prepared as described in Non-Patent Document 1, using influenza HA antiserum as an antibody and influenza vaccine as an antigen. As shown in FIG. 2, the measurement sample 1 has a circular agarose gel with a diameter of about 90 mm pressed and completely dried on a glass plate (180 × 145 mm). FIG. 3 is an enlarged view of a circular agarose gel, in which four vertical and horizontal four well circles and concentric settling wheels are formed at equal pitches. FIG. 4 is an enlarged view showing one settling wheel. The horizontal outer diameter (d1) and the vertical outer diameter (d2) of the settling wheel will be measured.

2. The equipment used is as follows:
1) Personal computer and image processing board (a) Personal computer system that can start Windows XP (desktop personal computer and 17-inch liquid crystal display)
O / S is Windows XP
(B) Image processing (FDM-PCI IV) + standard measurement software • Maximum resolution = 640 × 480 pixels • Maximum frame rate = 30 frames / second • Supported video source = NTSC signal • Supported bus = PCI bus (board size: PCI short standard)
・ Current consumption = 5V-1A, 12V-0.15A
2) Monochrome CCD camera and zoom lens 3) Camera illumination unit ・ Illumination method = White LED transmission illumination

4) Automatic feeder (a) XY table (KS262-50)
・ Outer dimensions = 236.5 x 236.5 x 70 mm
(Stage surface size = 60 x 60 mm)
・ Movement = 50mm × 50mm ・ Resolution = 4μm / pulse ・ Maximum speed = 20mm / sec
・ Withstand load = 8.8kgf
(B) Controller driver-External dimensions = 200w x 200D x 88h
・ Power supply = AC100-240V ± 10%
-Control method = RS-232C
(C) Handy terminal: Manually move the XY table (used to adjust the reference settling wheel to the focus of the camera).
(D) Sample fixing table Sample table area = 180 to 200 w × 145 to 200 h
-Work set method = One-touch type-Weight = approx. 15kg

5) Camera set base and base Z-axis movement range = approx. 50 mm 6) Measurement software Software for external diameter measuring device 7) Reference scale

3. The main operation screen of the application software is as follows:
1) Main menu: Displays basic functions.
2) Setting screen: Sets important data managed by the system, such as basic data and reference scale values. Plate switching (1 or 2), data recording location setting screen 3) Shooting screen: Samples are automatically shot.
4) Measurement screen: Measures the outer diameter of the captured image.
5) Measurement data management screen: Performs file management of measured data.
6) Maintenance screen: Manages information necessary for the system.

4). The basic operation is as follows:
1) Turn on the power of the device and the personal computer, start WindowsXP, and then start the program of the outer diameter measuring device.
2) Transition to the setting screen and set the basic data.
(B) Basic data setting (confirmation)
・ Moving pitch (10.0 to 15.0 mm)
・ Plate switching (1 → 2 or 2 → 1)
(In this example, two plates containing the same specimen were used, and the average value of each plate was adopted as the measurement value.)
・ Data recording locations (16 locations for Plate 1 and Plate 2)
・ Measurement method for external dimensions (four cursor method, click method)
(B) Input of data relating to measurement sample Input specific information of the sample necessary for data management (see FIG. 5).

3) Set the measurement sample 1 (SRD plate) at a predetermined position (on the illumination table (backlight)).
(A) While viewing the raw image displayed on the monitor, use the handy terminal to align the first sample so that it is at the center of the monitor screen.
(B) Adjust the focus / aperture of the camera, shooting range, and lighting conditions.
It can be confirmed with both raw images and binarized images.
4) Reference scale capture (photographing)
(A) A reference scale jig is prepared (for example, a calibration circle seal (diameter 8 mm) is pasted on the SRD plate), and an image is captured.
(B) The scale value is read from the binarized image and registered in the reference data.
That is, it is confirmed how many pixels the diameter of the calibration circular seal is on the screen. Dividing the obtained number of pixels by 8 mm gives the number of pixels per mm (or dividing the 8 mm by the number of pixels gives the length (mm) per pixel).
Description of reference scale capture (a) The reference scale is photographed in the same state as the sample photographing conditions. Prepare multiple reference scales and use one that is easy to use according to the application.
(B) The length (horizontal length) and height (vertical length) of the reference scale are measured.
(C) The number of pixels of each dimension is displayed, and the numerical value is registered as a reference scale value. That is, a coefficient of length per pixel is obtained.
Since image processing boards often have different vertical and horizontal resolutions, the same operation is performed both vertically and horizontally. That is, both vertical and horizontal reference scales are required.
(D) If a reference scale value is not input, an error message is output during dimension measurement.
(C) After taking (photographing) the reference scale, set a reference value for the measurement value.

5) Capture of sample image When the start of image capture is designated, the XY stage 2 starts operation, and images are taken in order from the first sample. Then, all the 16 samples are photographed and finished. When all the photographing is finished, the XY stage 2 automatically returns to the first sample position.

6) Measurement of outer diameter (a) When image capture is completed and measurement is specified, the screen transitions to a screen for measuring outer dimensions.
(B) The outer diameter can be measured by the following two methods.
(A) Four cursor method The operation of the four cursor method is performed as shown in FIG. Move the cursor up / down, left / right to touch the outer circumference of the sample by dragging with the mouse, adjust the position with the cursor fine adjustment arrow keys, and measure.
(B) Click Method The click method is performed as shown in FIG. Move the mouse arrow key to the outer circumference of the image to be measured, and left-click to confirm.
(C) Measurement is performed while visually checking an image automatically taken by the inspection apparatus.
The 16 measurement results (X-axis direction data and Y-axis direction data) are displayed in a list on the operation screen.
(D) If you want to re-measure, recall the captured image again.

7) Save and confirm measurement data (a) When measurement is completed, save the data.
(B) Save the measurement data in a form.
Save in the format of the grade record form.
Information specific to the sample, such as the test date, test vaccine strain, and standard antigen lot, is input in advance on the setting screen.
File names are saved in Excel Book format.
(C) When correcting data once saved, call the stored file name and correct it.
It is corrected in the format of the grade record form.

5. Measurement Sample and Measurement Conditions 1) Measurement Sample As shown in FIG. 2, the measurement sample used was a sample plate comprising four rows in both vertical and horizontal directions and a total of 16 sedimentation rings.

2) Measurement location and measurement order All the external dimensions of the circular pattern that thinly protruded outside the 16 circles regularly arranged were measured. The order of measurement (photographing) is shown in FIG.

3) Measurement conditions (measurement resolution is 0.1 mm)
The maximum outer diameter part (lateral dimension d1) of the X axis is measured.
The maximum outer shape (vertical dimension d2) of the Y axis is measured.

4) Storage of measurement data The measured data is recorded (saved) in a predetermined format.
The recording (storing) format of measurement data is as shown in FIG.
Note) Record the measured data in a preset recording location.
The recording location can be set on the recording location setting screen.

6). Automatic Image Capture Image capture (photographing) is performed while moving the XY table 2 under preset conditions (see FIG. 8).
1) X-Y table movement condition setting X-axis pitch and Y-axis pitch are set based on the first sample. In the preferred embodiment, the number of samples is 4 × 4.
(A) X-axis feed pitch (10.0 to 15.0 mm)
The movement range of the X axis must not exceed 50 mm.
(B) Y-axis feed pitch (10.0 to 15.0 mm)
The movement range of the Y axis should not exceed 50 mm.

  When the same day measurement was performed by the conventional method and the method according to the present invention, no significant difference was found in the HA content in 14 samples out of 15 samples from 3 strains. In addition, when the same plate was measured at different days, no significant difference was found in 26 samples out of 27 samples from 3 strains. For samples with significant difference, the value measured by the conventional method was larger. However, there was no significant difference between the conventional method and the method according to the present invention in almost all samples as a whole. By using the method according to the invention, it is possible to reduce the working time by about 40%.

  The method of the present invention can measure the size of a sedimentation ring generally generated by an antigen-antibody reaction in a shorter time and more efficiently than conventional visual measurement. In particular, the method of the present invention is suitable for measuring the size of a sedimentation ring produced by the reaction of influenza HA antiserum and influenza vaccine in the SRD or SRID method.

1 shows an example of an apparatus for carrying out the method of the present invention. Measurement sample 1 is shown. The circular agarose gel shown in FIG. 2 is enlarged and shown. The one sedimentation ring shown in FIG. 3 is expanded and shown. An example of sample specific information is shown. An example of a four-cursor operation is shown. An example of the click method operation is shown. Indicates the measurement (photographing) order. Indicates the recording (storing) format of measurement data.

Explanation of symbols

1 Measurement Sample 2 XY Table 3 Illumination 4 Camera 5 Computer

Claims (8)

  The antigen was added to the gel containing the antibody, the sedimentation ring generated by the antigen-antibody reaction in the gel was captured as an image by the image capturing means, and the captured image was displayed on the display by the computer and displayed as an image. A method of automatically determining the length of the diameter of the settling wheel by determining the end point of the settling wheel with the help of visual observation.   An antigen is added to a gel containing an antibody, a measurement sample having a plurality of sedimentation rings generated by an antigen-antibody reaction in the gel is set on an XY table, and one sedimentation ring is photographed with a camera and captured as an image. After that, the XY table is automatically moved so that all the settling wheels are sequentially captured as images by the camera, and then one of the captured images is sequentially displayed on the display by the computer connected to the camera. A method of automatically determining the length of the diameter of the settling wheel by determining the end point of the settling wheel with the help of visual observation.   The method according to claim 1 or 2, wherein the diameter of the settling wheel displayed as an image is sandwiched between two parallel lines on the X-axis and the Y-axis on the screen, and the length is obtained. The method according to any one of claims 1 to 3, wherein a straight line is drawn by clicking both ends of the diameter of the settling wheel displayed as an image, and the length thereof is obtained.   The method as described in any one of Claims 1-4 which measures the length of the diameter of the sedimentation wheel displayed as an image with the number of pixels.   6. The method of claim 5, further comprising determining a length per pixel or a number of pixels per mm using a reference scale.   The method according to claim 1 or 2, wherein the antibody is an antiserum against hemagglutinin.   The method according to claim 1 or 2, wherein the antigen is a vaccine.

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