JPS61180141A - Quantitative analyzing method of protein by immunodiffusion - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a technique for quantitative analysis of proteins present in a liquid mixture, and particularly to a method for quantitative analysis when the amount of sample is minute.

In recent years, with the rapid development of knowledge regarding the role that proteins play in health and disease, there has been a general need to rapidly and relatively economically quantitatively measure proteins in fluids such as serum, spinal fluid, and cell extracts. There is.

Proteins in such liquids are often identified (qualitative analysis) by an immunochemical method that utilizes precipitation of each protein caused by antibodies specific to a particular protein. These specific antibodies can be produced when foreign proteins (antigens) invade and stimulate the body. Antisera are mixtures of such known antibodies. By reacting a protein sample with such an antiserum in vitro and observing the presence or absence of precipitate as a result, it is possible to obtain powerful clues about the type of protein present in the sample.

The main purpose of the present invention is to determine the quantitative value of the concentration of one or more types of proteins present in an antigen solution in the immunodiffusion method, which has conventionally been considered to be a purely qualitative method.

The present invention is a method for measuring the protein concentration in an antigen sample by performing immunodiffusion with an antigen sample and an antibody source containing an antibody specific to a specific protein. That is, normally, proteins and antibodies first diffuse into each other in a support that does not contain a reactant, and a contact reaction occurs to form at least one sedimentation zone with a finite length, which is quantitatively measured. In order to do this, each position in a two-dimensional array distributed inside and outside the zone is optically scanned, an electrical signal corresponding to the sediment concentration at each position is generated, and the parameters of the zone that vary characteristically depending on the protein concentration are determined. is electrically induced from that signal. The results are then compared with several parameter control values determined from a control zone obtained by performing the same immunodiffusion as in the sample on a control antigen solution containing a known amount of the above-mentioned proteins. It's a method.

The above experimental results and calculations regarding the results can be performed manually, if desired, or the data can be obtained semi-automatically using optical and electrical scanning devices. The necessary data processing can also be performed fully automatically by a general-purpose computer of appropriate capacity.

In the present invention described above, various proteins in the initial mixture of antigens are first fractionated by moving in one direction in proportion to the differences in properties between the various proteins. Such selective transfer can be achieved, for example, by simple diffusion, electrophoresis, or even more complex methods such as chromatography, but the sedimentation zone formed by subsequent immunodiffusion is It remains basically unchanged even when fractionated by different methods. For example, in the case of electrophoresis, each protein moves and distributes along the direction of an electric field depending on the difference in mobility due to the difference in migration mobility between different proteins.

After such an initial fractionation, the proteins migrate in different directions and come into contact with the antiserum, generally resulting in basic separation by interdiffusion in a suitable support such as agar or agarose. form a linear distribution. The precipitation zones for each protein are generally completely separated and clearly differentiated.

If antigen and antibody can be made to diffuse into each other in such a way that they form a sedimentation zone of some finite length along a direction transverse to their initial direction of diffusion, how many distinct proteins can be separated? In some cases, each sedimentation zone can be separated without going through an initial fractionation step. There are sufficient differences in the diffusive mobilities of different proteins, or alternatively antibodies, to permit a clear distinction between such sedimentation zones. Since the zones have limited overlap, the endpoints of the zones can be clearly distinguished. The quantitative method according to the present invention is suitable for application to such types of immunodiffusion.

Similarly, if you put anti-Yu and antibodies in a hole to perform immunodiffusion and apply an electric field in a manner such as electrical immunodiffusion to increase the speed at which the antigen and antibody move toward each other, The resulting sedimentation zone maintains the same basic shape as it would have without the electric field applied. The same applies if the immunodiffusion step in immunoelectrophoresis is accelerated by an appropriate electric field. Therefore, the term immunodiffusion in this specification refers to both cases with and without an accelerating electric field.

Examples of the present invention will be described below with reference to the accompanying drawings, but these examples are not intended to limit the scope of the present invention.

Immunoelectrophoresis is well known as a qualitative method, and a variety of devices have been published to perform it, but all of them are largely the same and have minor differences. In the present invention, electrophoresis and subsequent diffusion and immunoreaction are typically performed in a monolayer gel, from a fraction of a millimeter to several millimeters thick, mounted on an optically transparent plate. It will be done. Support media commonly used today are those with a pH of about 8.6 and an ionic strength of about 0.
Agarose saturated with 1 barbital buffer is used. The sample is a hole cut into the gel layer,
It is inserted into the hole made by molding when the gel layer is placed on the carrier.

FIG. 1 shows a plate 20, on which is formed an antibody groove 24 along an axis 25 extending parallel to the direction of electrophoresis, and circular antigen holes 21 and 2 equidistantly spaced on either side of the antibody groove 24.
There are 2. Depending on the arrangement of the holes, two separate solutions or two samples of the same solution will move simultaneously towards the same antibody solution on each plate. Two additional antibody grooves may be added outside of the two antigen holes on each plate, and two additional antigen holes may be added outside of the two antigen holes to double the capacity of the plate. Similarly, additional antigen holes may be added at a sufficient distance along the direction of electrophoresis from holes 21 and 22 to prevent pattern overlap.

Shaded areas 26 and 28 in FIG. 1A represent four types of proteins a in a pair of samples of the same type after being placed in holes 21 and 22 and subjected to electrophoresis in the direction of arrow 23 for a certain period of time.

This is an approximate representation of typical distributions of b, c, and d. Normally, all proteins move in the same direction in a liquid medium, but the solvent itself has the property of carrying a net charge, resulting in gel flow and electroosmosis. Proteins therefore move in both directions relative to the hole.

After electrophoresis is completed, the antibody is placed in the groove 24, and as shown in FIG.
A sedimentation zone is formed by interdiffusion of proteins a, b, c, and d with their corresponding antibodies. B in Figure 1
, C, and D show each stage of typical zone appearance.

Precipitating arcs of unrelated antigens react with each antibody and form separately, but when they come close enough, they intersect as shown in the first diagram, and immunochemically related precipitation arcs form Fuse as a continuous reaction line. Figure 1 C and D
The sedimentation zone d' in Figure 1A actually contains two separate proteins, and even if the two proteins have the same electrophoretic mobility, they will form separate sedimentation zones. It illustrates the facts that form. Zones d and d' can also be thought of as two different proteins placed in the wells 21 and subjected to immunodiffusion without going through an initial electrophoresis step. The reason why each zone end is clearly separated is because the diffusion rate of each protein acid or antibody is different. In either case, each such zone can be analyzed separately using the methods described below.

According to the present invention, the sedimentation zone by immunoelectrophoresis can be measured directly and quantitatively. Such measurements allow the determination of the specific characteristic physical arrangement of each precipitation zone of the protein of interest on the plate, as well as measurements of light intensity by optical methods. Both methods add a temporal element, which is an important element of observational data.

However, such time measurements are meaningless if the incubation is allowed to reach equilibrium and wait for the formation of a stable zone.

Position measurement on the slide 20 can be performed, for example, with a low magnification microscope. As shown in FIG. 2, a plate 20 rests over a variable aperture 32 of a light source box 30 having a light source lamp 36 and a dark field 34 of light absorbing material such as black velvet. The microscope 40 has an objective lens 41
, a crosshair 43 for sighting is attached to the eyepiece lens 42 and the focal plane. A double slide mechanism 45 is installed on the light source box 30.
Although not shown in detail, it is equipped with a screw drive device 46 and accurate scales, which allows the position of the microscope placed on it to be accurately read with respect to the two coordinate axes. is now possible. In this figure, only one coordinate axis is shown for clarity. It is usually convenient to place the X-axis in the direction of electrophoresis, ie, parallel to the antibody groove, and to place the origin of the coordinate axes on or near axis 25, as shown in FIG. 1A.

In order to measure the density of light, the microscope is equipped with a diagonal half mirror 48 for splitting the light beam, which sends part of the light to the eyepiece and sends the other part onto the diaphragm 52. It brings together the real image. The diaphragm 52 is connected to the plate 20
Only the light from the range that matches the crosshair 43 on the
to a photosensitive transducer 50. transducer 5
0 is electrically connected to the amplifier circuit 54 and the meter 56. Instead of the meter 56, it may be possible to directly view the light using a silver screen and record it manually, or it may be possible to connect a printed circuit or an AD converter that automatically records the intensity of the light in response to a command signal. In FIG. 2, for example, instead of dark field illumination,
Various modifications are possible, such as direct illumination or overhead illumination where the reflected light from the zone can be measured directly. Examples of methods and apparatus for performing fully automated measurements are described below.

FIG. 3 illustrates certain features of the sedimentation zone defined for locating the zone according to the present invention as it appears. The horizontal line 61 at y-y is the edge of the antibody groove on the immunoelectrophoresis slide. Points E and F at coordinates (Xe, Ye) and (Xf, Yf) are the left and right end points of the extending zone 60.

In addition to zone end points E and F, it is better to measure several midpoints within each zone.

In FIG. 3, a point G at the coordinates On, Ya) is an example of such a point.

Since the zone has a certain width in the y-direction, the y-coordinate of each intermediate point, such as It is convenient to define one or more points 64, including the strongest point.

As the incubation time progresses, the absolute and relative positions of points E, F and G change as zones emerge. As a function of time, Xe and
The value of f represents a typical protein concentration. Direct protein concentration can also be determined by comparing the position value of such an unknown solution to a control value obtained with a known concentration of protein at the same time. However, more reliable and accurate results are usually obtained if one or more functions considered to be useful as parameters are derived from these initial measurements.

One of the important parameters of the settling zone used in the present invention
One is the coordinate difference (Xf - Xe), which is the maximum L of the sedimentation zone at a particular measurement time. As the data represented in FIG. 4, FIG. 5 shows changes in this parameter with respect to incubation time.

Location parameters other than zone length can be calculated as zones emerge. For example, the curvature of the zone and the variation of that curvature along the length of the zone are useful parameters that provide information about abnormal proteins (see below). Approximate curvature measurements over the entire zone, or measurements on specific portions of the zone, are relatively easily determined by comparing the x and y coordinates of three correlated points along the axis of the zone. To obtain a more accurate value for the curvature of the zone, the axis of the zone should be y-r.
It must approximately match the curve represented by (x). Here, f(x) represents an appropriate function of X. Therefore, the radius of curvature R is given by the general formula. where y' and y are the first and second derivatives of V with respect to X.

An example of a suitable function to fit a curve is a parabola with an axis parallel to the y-axis. Such a parabola can be expressed by any of the formulas below.

y−a6+alx+a2x2−−・・−(2a)y
-A (x-8)2+C・-・-・・-(2b) where A-a 2 , B--a I/2a 2 , C-
a.

-at2/4a2.

The values of the constants in equations (2a) and (2b) can be determined from the coordinates of any three points on the axis of the zone, or by the least squares method for any of these points, or It can be determined by other known methods. The axis of symmetry of a parabola is X −3, and the curve on the axis is from the X axis to C
is far away.

The radius of curvature R is expressed as follows using equation (1).

This radius becomes the maximum value Ro at the point of the axis of symmetry, and formula (3) becomes Ro=2A...(whole).

The above values derived from at least three of the zone axes can be used as parameters for the present invention.

Another important parameter for measuring protein concentration according to the invention is the time of first appearance of the zone TO. It is difficult to determine this time by direct observation. An example of the present invention allows the first appearance time to be determined with high reliability and reproducibility.

In FIGS. 4 and 5, the solid lines are plots of values obtained from actual experiments. These figures also include
The value obtained by extending (extrapolating) the solid line in the direction of less time is shown. This extension is indicated by a dashed line. In FIG. 4, the point 66 where the extension lines intersect represents the point where the length of the zone was O.

The extension (extrapolation) line in FIG. 5 intersects the time axis at point 67, and TO can be similarly determined. Such an extension (extrapolation) allows the time of first appearance of the sedimentation zone to be determined with practical accuracy. This method for determining TO is advantageous because it does not require constant continuous observation of the plate.

Next, quantitative measurement of the optical density (brightness) of the zone will be explained. Protein concentration cannot be practically measured by simply reading the brightness.

The reason is that the shape of the zone and the rate of zone formation change,
This is because the brightness changes as the zone becomes larger.

On the other hand, variations in these factors can be compensated to a large extent by taking a series of brightness readings at several appropriately chosen locations and processing them collectively to determine the brightness parameters. The method is to measure the brightness at equal intervals along a straight line across the sedimentation zone, usually in the y direction at a particular value of x. In such a series of readings, it is desirable to determine brightness values at several locations on both sides of the zone. These offset values are averaged to determine the background intensity, which is used to subtract the background value from the luminance value within the zone. As a result, the corrected luminance values are aggregated and
A linear integral of brightness along a line across the zone at a particular value of X can be determined. Such a linear intensity sum, lx, increases with increasing incubation time in a regular and reproducible manner, and its value varies with the concentration of reacting protein at any given time over a wide range of experimental conditions. It was found that there is a tendency to increase with

A typical example of a plot of changes in brightness observed by this cross-zone scanning is shown in FIG. The two curves were plotted with the semi-automatic apparatus described below, scanned in the y direction at the point X(+) where the sedimentation zone was closest to the antibody groove.

In FIG. 9, beaks 74 and 17 are due to zones created on either side of the antibody groove by samples of the same protein placed in the two antigen wells in a plate similar to FIG. The two small beaks at 75 and 16 are due to the respective edges of the antibody groove and provide convenient controls for measuring the distance from the groove to a particular point in the zone. The parameter ix defined above essentially corresponds to the area under the beak 74 or 77 in FIG. 9, for example.

The two beaks 74 and 77 in Figure 9 were produced by the same sample of human serum albumin. They representatively show the growth of the sedimentation zone when the incubation time is 2 hours (solid line Kerr 170) and 4 hours (dashed line Kerr 772). It can be seen that the position of the zone is very stable between these two sets of measurements;
The area under each beak has increased noticeably. beak 74
The curves 77 and 77 are shifted by an appropriate distance in the vertical direction to make the diagram easier to understand, so there is no need to worry about the discrepancy.

FIG. 10 is a plot of plates made with various concentrations of protein scanned in the y direction at the same incubation time. The graph clearly shows that as the protein arIX increases from A to C, the area of each peak increases, and the entire zone moves toward the antibody groove.

Since the parameter [X is extremely useful in determining protein concentration, such a linear intensity sum is obtained for several values of will further improve. A typical method is to find the linear intensity sum at xg and a number of points chosen at appropriate intervals on both sides of xg. By averaging or aggregating the intensity sums at several points predetermined at equal intervals, experimental errors can be reduced and the accuracy of protein concentration measurement as a whole can be improved.

Additionally, in the method described above, as the length of the settling zone increases, the number of linear sums included in the linear sum calculation etc. increases each time the plate is scanned. An example of a method for this purpose is to determine the linear intensity sum at Xg and then continue measuring until the linear intensity sum values on both sides of Xg become equal to or less than a certain threshold value.

The sum of all linear intensity sums is parameter 12 (total intensity sum), which is essentially the integral of the brightness as scanned through the sedimentation zone. This approximation can be improved as desired by incrementing the changes in x and V with each measurement to the extent that the resolution of the instrument allows. The rzQ width varies particularly strongly as a function of protein concentration over a wide range of experimental conditions. The reason is that as the concentration increases,

■This is because both dimensions increase and the average brightness of the zone also tends to increase. Because of this strong dependence on protein concentration, the parameter Iz, which is the sum of linear intensities, is particularly effective as a criterion for concentration measurements.

Once experimental values for one or more parameters have been obtained for the sample being analyzed, these values are compared to an appropriate combination of standard values for each protein. These standards were made using a series of solutions containing known amounts of the protein of interest under the same conditions as the measurements. In order to obtain such a combination of standard values, standard procedures are carried out using such standard protein solutions and subsequent measurements are taken at corresponding points on each plate as the incubation progresses. It is desirable that the standard operation be performed under all conditions as similar as possible to the measurement operation to which the standard is to be applied. In fact, it is desirable that the control value is unique and corresponds individually to each measurement operation. However, in routine measurements where the slope of the control curve is known from a previous measurement, one standard operation may be sufficient.

Standard values for desired parameters are derived by performing several such standard operations for each concentration.

The standard values of the parameters thus measured are therefore considered to be a function of both concentration and time.

When considering each linear intensity sum lx separately, a specification of the value of X is required in order to measure them all.

Using the first appearance time of the zone as a parameter has the advantage that the standard value TO does not include time as a variable. That is, in order to determine TO using the method described above, measurements must be made several times at a specific time. However, once 7'o is determined by these measurements, each measurement value becomes unnecessary. Thus, T.

The values of can be plotted as a function of protein concentration to yield one standard curve. Such a curve is illustrated in FIG. This was created by determining the values for each concentration using the extrapolation method described in connection with FIGS. 4 and 5. From the curve in FIG. 6, the concentration value can be directly read as long as the TO of the unknown protein is known.

For example, for parameters like L, lx, rz, creating a standard curve becomes more indirect.

These values are related to the time at which the measurements were taken, so the reference standards must be made to cover a range of time. It is difficult to measure data for all concentrations simultaneously. Each measurement is therefore related to the time of measurement so that the final standard value for each protein concentration is plotted as a function of time on a separate curve.

FIG. 7 is a representative example of such a group, in which standard values of parameters for three m degrees are plotted against the time axis. Extending these curves in the direction of L=○ gives the standard value of To, from which To can be plotted in Figure 6, or T for comparison with Figure 6.
(18) Experimental values can be given. In FIG. 7, an arbitrary time tl is shown.

A vertical line is drawn at t2 and t3. These combined values of L can be replotted as separate curves as a function of concentration. The resulting set of curve combinations shows the growth as a function of protein concentration for each time. This combination is shown in FIG. 8 and is more convenient than the plot versus time of FIG. 7 when comparing experimental values.

Standard values to be compared with experimental values for other parameters can be obtained in a similar manner as described for parameter L.

The total intensity sum lz is measured continuously during the formation of the zone and is found to increase linearly with time. This linear characteristic is illustrated in Figure 11, which plots Iz versus time for four solutions containing known amounts of immunoglobulin protein. Immunoglobulin tSa values are derived using an appropriately programmed general-purpose computer from automatic measurements of two-dimensional combinations of zone positions by the general method described herein. The points shown in FIG. 11 were originally plotted automatically, and the straight lines were matched to the points obtained by the computer. This figure was replotted manually to change the scale.

The linear relationship shown in FIG. 11 serves to pre-plot a standard or control against which the ΑΑΑ corresponding to the measured value of the parameter Iz can be read. For this example, the value of lz as a function of the concentration for 2,4 o'clock B(o, i day) is derived from FIG. 11, and the line 7 is shown in FIG.
It is indicated by 0. The linear dependence of lz on time indicates that the rate of change of the time differential dIz/dt or iz with respect to time is constant. Therefore, it is a practically advantageous parameter because it is time-independent. Curve 72 in FIG. 12 is a representative example of that parameter as a function of protein concentration, with each point representing the slope of one of the curves in FIG. As previously discussed with respect to the parameter TO and FIG. 6, a curve, such as curve 72 of FIG. 12, is useful as a control curve for the parameter dlZ/dt.

Another feature of the present invention is that it improves accuracy, particularly when the concentration of one or more proteins of interest in the sample to be measured is relatively low. In such cases, it is desirable to supplement the sample solution with several known amounts of such proteins by converting them into integers. Measurement operations are then performed in parallel with this solution and a standard containing a known amount of protein.

If values are needed to determine the required parameters for each solution, they can be determined for all times by the interpolation described above for time. The parameter value P obtained for the regular standard is expressed as kerf 80 in FIG.
Plot against protein concentration in the usual way as shown. Similarly, score the value of P for the original antigen sample and a portion to which a known amount of protein was added, and for t, j, and k, calculate as if the solution contained only the added protein. It is plotted as follows. Therefore, the value for the original sample is plotted at the concentration (18) position. A curve 81 is drawn through these points.

In this data processing example, the protein concentration of the original sample can be calculated in several ways, all of which should give essentially the same value. In this way, calculations can be made using several desired methods, and the results can be averaged.

First, a straight line extending horizontally from h and intersecting the curve 80 at h' indicates the density of the value indicated by ch.

This corresponds to the general comparative method mentioned earlier. Furthermore, in the same way, extension lines drawn from i, j and k to intersect the straight line 80 are i→1', j→j' and k -+k'
gives the concentration according to the length of the straight lines, and their lengths are logically all the same.

If the value of P at h is uncertain for some reason, e.g. because the observed zone is unclear, a reliable value can be obtained by averaging over all four intervals.

Another method not only can improve the measurement inaccuracy in It also has the advantage of being able to determine the value. In the latter case, the protein on curve 81 is obtained by connecting the obtained points i, J and c, and is further extended (extrapolated) towards the P axis to determine P at h. In this way, curve 81
Curve 80 serves as a guide in extending . For example, curve 80 in FIG. 13 has been moved generally to the left until it represents points i, j, and k. The intersection of the thus-moved curve 80 and the P axis gives a point value, from which the density ch can be determined. Furthermore, if the curve 81 is extended to the negative side of the horizontal axis -Ch, the density that matches the density value ch can be directly read.

The supplemented standard line 81 method described above has the great advantage that the same solution can be used for experimental and control purposes. This advantage can eliminate any small errors caused by extending the standard line, especially if the sample is human serum from a patient with a particular disease. The reliability of this extrapolation can be increased almost unlimitedly by making measurements at several locations rather than just the three points shown in Figure 13, and a correct knowledge of the measurement errors involved can be obtained. can be obtained.

The present invention can also automatically detect certain protein abnormalities in antigen samples. For example, normal and abnormal gamma globulins usually have small differences in their electrophoretic mobility ranges, yet react with the same antibodies, form abnormal sedimentation zones, and are generally short in length. form asymmetrically distributed sediments along the Such an anomaly causes the measured value of the intensity parameter lx to change, for example, to
can be detected by comparing different values of .

By observing asymmetry or other abnormalities in such values, it is possible to know that an abnormality has occurred. The existence of this abnormality is indicated if several independent measurements are performed at a certain value of X and the results of calculating the measurement error show a statistically significant change.

This method is considered to be a special case of the general method of comparing actually measured values of parameters that respond differently to a target abnormality. For certain parameters that exhibit such behavior, it is more effective to compare protein concentration values corresponding to the measured values of each parameter than to directly compare the parameter values themselves. For example, the abnormal distribution of brightness along the zone caused by the presence of abnormal gamma globulins, as described above, causes the zone to appear faster than in the normal case, resulting in a higher calculated concentration, whereas the total intensity Parameter 12 tends to show approximately equal concentration values for normal and abnormal proteins. A significant discrepancy between the concentration values obtained by TO and I2 thus indicates the presence of an abnormal protein.

Another parameter sensitively responsive to the presence of abnormal proteins is the axial curvature of the zone.

Due to the presence of anomalies, the curvature along the length of the zone tends to vary abnormally and asymmetrically, while the overall curvature tends to be below normal.

For example, like the above-mentioned brightness or position parameter, 2
It is further advantageous to use multivalued parameters consisting of a singular function of one or more parameters. For example, the sum of the zone length and brightness parameters becomes a new parameter that, taking proper account of measurement errors, gives more reliable results than using the individual components one by one.

Also of interest are the derivatives of two other parameters, one increasing and one decreasing with increasing protein concentration. In this way, the quotient of these parameters,
Using the differences, parameters can be obtained that have a sharper dependence on concentration than using them individually.

The first appearance time TO of the zone is an example of an inverse relationship to the protein concentration, and can be used particularly when determining such a quotient. The distance between the antibody groove and the zone at any desired value XO on the x-axis is also inversely related to the protein concentration, and such a quotient is also used as a parameter.

In general, it is better to divide a parameter that directly depends on concentration by a parameter that is inversely dependent on concentration so that the resulting multivalent parameter depends directly on concentration rather than inversely. .

For an unknown sample, reference values suitable for comparison with multivalent parameters determined by actual measurements can be derived from the reference values of the parameters of each component, as described above.

As mentioned above, measurement of position and brightness and derivation of parameters can be performed by direct observation or manual methods.
A feature of the invention is that these operations are particularly suitable for automation in part or in whole.

A particularly convenient and effective method for making the measurements required for the present invention is to optically scan the slide, using a charge-coupled scanning device, such as a television camera, or an equivalent method to scan the scanning range. This is a method of obtaining a video signal that represents the apparent brightness of a two-dimensional combination of The signal of each element is generally digitally converted and stored electrically along with the x and y coordinates corresponding to the position on the plate or the measurement time. The combination of signals for all such locations or the signals for a particular location can be retrieved for subsequent data processing. Also, any part of the image on the slide may be exposed to a CRT or equivalent method.
It can be displayed during or after scanning. Systems for scanning, storing images, reproducing and extracting specific points of an image as digital signals are known in electronics technology and can be purchased in a way that suits this requirement.

FIG. 14 illustrates an example of such a device.
A television camera 90 focuses an image of the plate 20 through a lens 91 onto the camera's photosensitive surface, a monitor CRT 92, a scanning controller 94, and a general purpose computer 100. By adjusting the lens 91, the plate 20 can create an image at any magnification. In order to center the plate at an arbitrary position, for example, it is necessary to change the position of the plate on an illuminated support such as the light source box 30 in FIG. 2, or to use conventional electronic bias control using a camera circuit. You may go.

In particular, if a scanning mechanism synchronized with the monitor CRT can be used as the scanning device, it is possible to drive the mechanism in fine steps such that the movement in both coordinate axes on the screen is continuous and equal. The image is divided into area elements, identified, for example, by x and y coordinates. For manual or automated operation, the monitor display usually has a cursor, and an electronically generated bright spot indicates the position on the screen from which the video signal is extracted from the area element for each scan. . There is a manual control switch for the operator, who can freely move the cursor visually. The device can also position the cursor directly on the desired point according to the values of the X and y coordinates by means of a digital command address. Such signals can be obtained manually or from a general purpose computer program.

As illustrated in FIG. 14, a counter 102 counts pulses from a clock 103 and continuously provides a digital signal representing the X coordinate to a branch line 104. The circuit 106 generates an analog step voltage for each minimum unit of the X coordinate on the line 1 (17) according to the signal.
8 performs scanning counting of the beam in the x-axis direction and branches Ii! 1
09 is given a digital signal representing the y coordinate for each sweep. In response to this count, the circuit 110 sends out an analog steep voltage for each minimum unit of the y-coordinate to the line 111. Step voltages on lines 1 (17 and 111 control the scanning of camera 90 and CRT 92 in the and reproduces the 11 degree change of slide 2o on the screen surface.

The selection circuit 118 typically consists of an X and Y counter, which counts up or down by counting one or more pulses when the control stick shown at 119 is manually moved. The resulting digital signal represents the x and y coordinates of the location being measured and registers 1
17. Comparison circuit 114 includes register 117
Coordinates of a specific measurement target position and lines 104 and 10 from
Constantly compare the x and y coordinates of the scanning beam from 9 onwards. When the scanning spot comes to the memorized position (address),
An operating signal is sent from comparison circuit 114 to switching circuit 120, typically once for each scan. Register 125 thus receives on its line 123 a digital signal corresponding to the brightness when scanning the identified measurement point on the slide. The motion signal from circuit 114 is also sent to cursor control circuit 126, which superimposes an intensity modulation pulse on the video signal to identify the selected point on the monitor screen, as shown at 127.

Register 125 also receives the X and Y coordinate signals from lines 115 and 116 and also receives the continuous time signal produced by time circuit 128. All of these signals are stored in register 125 and are read by computer 100 under manual control on line 129 or under computer control on line 130.
sent to.

Computer 100 individually identifies measurement points and accepts input information for spots on plate 20 as required by any program. This measurement method is
The circuit is similar to the selection circuit 118, the register 117, and the comparison circuit 114, and the selection circuit 118 operates according to an electrical signal that selectively indicates the direction of movement of the measurement point or indicates the digital address of the required position. do. Rather than creating two such devices, as shown in FIG.
8 is controlled by computer 100 via branch line 132. In order to perform such control by the computer 100, as described above, the computer 100 has built-in programs for measuring position and brightness and for guiding general parameters.

In order to scan the zones automatically, the plate should be suitably illuminated and transferred from the incubation chamber to a precisely defined scanning position. Especially when the optical scanning device is lightweight and compact, such as a charge-coupled device.
For example, it is preferable to mount the scanning device on a platform that can move in two dimensions over which immunodiffusion or immunoelectrophoresis plates are lined up. In this way,
It can also be used to place antigens or antibodies into holes or grooves using a scanning device. For this purpose, a pipette that can be controlled by digital signals is mounted slightly outside the scanning device. The video signal from the scanning device is sent to a computer that is programmed to identify the shape of each hole and to allow automatic reading. Attach the scanner pipette and the platform can be moved over the plate by computer control and stopped when the scanning axis is correctly positioned over a certain defined antigen hole. The platform is then moved a predetermined distance such that the pipette is just over the hole and a constant curve of the sample is properly injected into the hole.

When the antigen solution is injected into all the holes while washing the bed appropriately after each operation, electrophoresis begins.

Similarly, after electrophoresis is completed, or if electrophoresis is not performed, immediately after the antigen is placed in the antigen hole, the antibody is placed in the antibody groove by this B position. After a period of diffusion, the same scanning device moves over the settling zone over the plate combination to perform the scan.

The scanning capability of the scanning device is also exercised prior to injecting the sample into the hole. The computer is programmed to measure the relative positions of the antigen holes and antibody grooves, and remembers any incorrect relative positions or dimensions of holes or grooves in the plate. Those that deviate greatly from the standard are eliminated at the sample injection stage, and those that slightly deviate from the standard are automatically corrected at the final parameter derivation stage.

The initial zone scanning process obtains a video signal from an address that is continuously measured by a specific X value and a Y value moving at regular intervals over the area where the zone is expected to form. This is a stage controlled by a computer.

The video brightness value received from the register 125 is stored as data in x, y, and [for each measurement point. At the end of each such y-scan, the x-coordinate is moved a fixed distance and a similar y-scan is performed, the results recorded, and so on until the entire area of interest has been covered. Each measured brightness value is typically compared to a previously measured and stored value. It is determined that if the measured brightness is above a background region threshold, it indicates the presence of a sedimentation zone. Also, for each y-scan, the maximum brightness across the zone is determined and stored, and the axis of the zone is determined from a series of y values for equally spaced true values.

The end point of each zone is recognized by the extreme value of x such that the highest brightness peak is recognized in each direction of x. If more precise position measurement is required, a certain area near the endpoint is programmed to be scanned at even finer x and y intervals.

For each scan across the zone in the y direction, we apply the intensity sum parameter ix, described above, directly to the data recorded for the actual sedimentation zone located and recorded. obtain.

Then, the sum of these six values becomes the total intensity parameter Iz for that zone. Subtracting xll[ of the zone end yields the parameter L. Other parameters may be determined by appropriate calculations as required.

The above measurements should be carried out as a unit measurement on a set of plates containing one or more unknown samples and their associated several standard solutions, such that a complete determination of the unknown samples can be made. desirable. After each scan of a set of such plates, the computer derives the concentration of each protein for which a "sedimentation zone" is found. If, as already mentioned, a large number of standard measurements are included, then the possible measurements The concentration is determined for each calculated concentration value. It is known to perform such calculations by computer. From the concentration of each protein in the sample,
Several independent measurements can be made. Generally, several different parameters are referenced and weighted according to the expected error to calculate the average.

If the calculated value of the error with respect to the average value falls within a certain range for the sample being measured, there is no need to perform any other measurements. The computer thereby performs general printouts or recording of the final results and collects as much original data as necessary for the program. For example, a doctor requesting an analysis can request that all data be stored on a magnetic tape for future reference. In addition, a photograph of the plate subjected to immunodiffusion can be taken either through a monitor CRT or directly. Normally, if the concentration of the protein is not high, it is not good to determine the concentration of the target protein with one cycle of scanning.

a period of time, from a few minutes to an hour or more,
After additional incubation, the above scanning is repeated, and the measurement plate and its corresponding standard plate are scanned. The computer usually looks for new zones that appear where no zones were present in the previous scan, or
It is also programmed to scan areas where subsidence zones have previously been found and recorded, looking for movement or extent of zone expansion since previous recordings. Thus, the computer continues to process data with each scan.

Sometimes two precipitation zones of distinct proteins come so close that a normally formed precipitate intersects at the end. The zones created by immunoelectrophoresis are programmed to predict crossings of such zones, predict when crossings will occur, and make appropriate corrections in the process of inducing various parameters. .

If the protein being measured is of the type that crosses over, each plate is scanned early in the appearance of the zone, before cross-over has occurred, as shown in FIG. 1B. The axis and end points of the zone are then reliably localized. The computer is also programmed to check for actual intersections as part of each regular scan. For example, (X) of the measurement point of the axis of each zone.

y) Determine the coincidence of coordinates at a certain threshold indicating intersection by comparing the coordinates with those of neighboring zones.

All scans also include a check for possible intersections. For example, the computer extends all endpoints beyond their actual measurements and compares them to the extended endpoints of adjacent zones. The axis is simply extended in the direction of the slope of the axis near each end point. Alternatively, the observed zone axis may be aligned with a parabola or other curved line to properly extend it.

Intersections of zones can also be detected by calculating the rate of change of the slope of the zones' axes. Alternatively, the radius of curvature of the axis can be calculated and detected by a series of points along the zone. Any deviation from the smooth normal variation of these functions indicates that the zone contains two or more intersecting areas.

Even when the actual crossing spans half of the zone, a sufficiently accurate correction can be made by replacing the brightness values observed within the crossing area with the brightness values observed in the remaining uncrossed half. The correspondence between two points in such immunoelectrophoretic zones is determined by centering on the point XΩ closest to the antibody groove and at equal distances of X on both sides, and also in the zone where direct immunodiffusion has occurred. is determined at intervals of equal values of y on both sides of the zone axis.

Axes that lie within the intersecting area are localized by extrapolation.

More accurate correction methods are also possible if necessary. For example, the computer can be programmed to adjust the measurements at symmetrical points chosen on the non-intersecting halves of the zone to take into account the asymmetry of the zone. Once an asymmetry is detected, it may be compared, for example, with two previously observed points that intersect, and the ratio between these values determined as a correction factor.

Another example of a correction is to take into account that the actual brightness values measured in the actual intersecting areas are partly due to one zone and partly due to the other zone. This is a method of correcting by adding The computer is programmed to divide the brightness value at the intersection area into appropriate values. In order to determine a suitable division ratio, the respective values of the intersecting area and the symmetrical area may be compared, as described above, with or without the above-mentioned adjustment.

If possible, it is desirable to apply two or more separate calculations to correct the intersection area and average the results to determine the error. However, originally the crossing area was only a small part of the entire zone. Therefore, the error in approximating the correct value within the area of the intersection is acceptable and does not significantly affect the final result.

It is also possible to determine the density for each of the above-mentioned types of intersection by the parameters obtained by the part of the zone that is not affected by the intersection. That is, for a method similar to immunoelectrophoresis, measurement near the center of the zone is suitable, and for a method similar to direct immunodiffusion, measurement near the end of the zone is suitable.

Those skilled in the art will appreciate that many features of the methods previously described can be implemented with a variety of equivalent alternative techniques within the scope of the invention.

For example, parameter values that do not require continuous measurements of the incubation time can be determined after the incubation has reached equilibrium. Such measurements have the advantage that the configuration of the zones is static and stable and the time to take the measurements is not a critical factor.

Furthermore, measurements of the sedimentation zone at any stage of incubation can be carried out with specific light.

This method does not require precise time measurements, so it is usually
It is convenient after the reaction has reached equilibrium.

In summary, methods and apparatus have been described for making truly quantitative measurements of the concentration of individual proteins in physiological fluids using immunodiffusion, immunoelectrophoresis and analogue means. In actual use, the method described herein has been demonstrated to measure protein concentrations with an accuracy of 5% or less. The present invention thus provides measurement techniques for a wide range of experimental or diagnostic purposes.

[Brief explanation of drawings]

Figure 1 is a diagram of a plate for immunoelectrophoresis, where A in Figure 1 shows the distribution of typical various proteins after electrophoresis, and B, C, and D in Figure 1 show the subsequent diffusion and immunity. Each stage of the precipitation reaction is shown. FIG. 2 is a longitudinal sectional view of the device for measuring slides. FIG. 3 is a diagram illustrating the characteristics of a settling zone with respect to the present invention. FIG. 4 is a graph showing a typical relationship between zone end points and time. FIG. 5 is a graph showing a typical relationship between zone length and time. FIG. 6 is a graph showing a typical relationship between the first appearance time of a zone and protein concentration. FIGS. 7 and 8 are graphs showing typical relationships between time and protein concentration versus zone length. FIG. 9 is a graph showing an example of scanning the density of the sedimentation zone at two different incubation times. FIG. 10 is a graph obtained by scanning the shading of the sedimentation zone formed by each protein concentration. FIG. 11 is a graph showing an example of the relationship between parameter Iz and time. FIG. 12 is a graph showing an example of the relationship between the parameter rz and the protein concentration parameter dlZ/dt. FIG. 13 is a graph showing an example of inducing a protein concentration value by adding a known protein to an unknown protein. FIG. 14 is a block diagram of an embodiment of an electronic scanning device according to the present invention. 20...Plate 21.22...Antigen hole 24.
...Antibody groove 90.91.92...Optical means 94.
...Patent applicant for control means Frederick J.A. Alagem (1 other person) Patent attorney Satoru Yoshimura State - Amendment Procedures August 16, 1985 Michibe Uga, Commissioner of the Patent Office1, Indication of the case Patent Application No. 156185 of 19852, Title of the invention Method for quantitative analysis of proteins by immunodiffusion 3. Relationship with the case of the person making the amendment Patent applicant address 911(15) for California, United States of America
845° Las Palmas Road, Pasadena Name Frederick J. Alagem Address 911 (15) for California, United States of America 845° Las Palmas Road, Pasadena Name Badomasini Kei Aggar 4, Agent 2-chome Shimochiai, Shinjuku-ku, Tokyo 14 No. 1 No. 5, Date of amendment order Amended statement filed at the same time as request for examination of application 1, Title of invention Method for quantitative analysis of proteins by immunodiffusion 2, Claims (1) Antigen sample containing protein , a method for quantitatively measuring the protein concentration in an antigen sample by immunodiffusion with an antibody source containing an antibody specific to the protein in the antigen sample, the protein and antibody being initially mixed with these reactive substances. forming at least one elongated sedimentation zone of finite length by reacting and diffusing through areas of the support medium where neither is present; and scanning said sedimentation zone by optical means so that a portion of the sedimentation zone is generating a plurality of electrical signals in response to the sediment concentration at each position of a two-dimensional array distributed within the sedimentation zone and partially distributed outside the sedimentation zone; electronically deriving a parameter value related to the distance between the ends of the elongated sedimentation zone representing a change from a small number of the electrical signals; and obtaining an index for quantitatively measuring the protein concentration in the antigen sample. comprising comparing the parameter value with a reference parameter value derived from a control sedimentation zone generated by equivalent immunodiffusion of a plurality of control antigen solutions, each containing a known concentration of the protein. A method for quantitative analysis of proteins. (2) the step of scanning a sedimentation zone includes a plurality of scan paths intersecting the sedimentation zone and a plurality of scan paths beyond each end of the sedimentation zone, the scans being spaced apart from each other; intersecting said sedimentation zone along parallel scanning paths, further deriving the value of said parameter selecting a scanning path adjacent to a respective sedimentation zone edge to transmit said plurality of electrical signals. The method for quantitative protein analysis according to claim 1, which comprises comparing the selected scanning paths and measuring the mutual distances of the selected scanning paths. 3) The method for quantitative protein analysis according to claim 1, which comprises generating an image representing the sedimentation zone on a screen surface of a cathode ray tube in response to the plurality of electrical signals. (4) the step of deriving parametric signals comprises deriving a first signal that increases with increasing protein concentration, deriving a second signal that decreases with increasing protein concentration, and The method for quantitative protein analysis according to claim 1, which comprises deriving a third signal representing a differential combination of the two signals as a combined parameter signal. (5) The combined parameter signal is
The quantitative protein analysis method according to claim (4), wherein the method represents the ratio between the signal of the second signal and the second signal. (Q) the scanning of the sedimentation zone, wherein the array includes at least one location on each side within the sedimentation zone and in an area overlapping another sedimentation zone, and wherein the scanning step of the sedimentation zone generates a plurality of electrical signals; generates an electrical signal at a second location on the other side of the first zone and in a non-overlapping area that overlaps the other sedimentation zone; A method for quantitative protein analysis according to claim 1, which comprises using the second position signal for deriving the position signal. The concentration of the protein is increased by previously adding different known amounts of the protein, and the scanning step, the step of deriving the parameter value, and the step of comparing the control parameter value and the determined parameter value are performed on the sample. The protein quantitative analysis method according to claim (1), which comprises performing the quantitative analysis on a sedimentation zone formed in each portion. (1) Each portion of the antigen sample is used for immunodiffusion of each sample portion. The resulting sedimentation zone of each sample portion is compared to the added protein concentration in the scanning step, parameter value derivation step. Claim 1, further comprising the step of effectively plotting said obtained parameter values for each sample portion and determining the value of said protein concentration in the original sample by extrapolation of said obtained curve. 1
Quantitative protein analysis method described in section ). (91) Claim 1 (1) comprising the step of staining said sedimentation zone with a dye prior to the step of scanning said sedimentation zone.
Quantitative protein analysis method described in section ). 0ω electronically transmitting, from the plurality of electrical signals, a corresponding value of a second parameter of a zone that is characteristically different from the first parameter depending on the presence of a certain abnormality of the protein; the value of the first parameter and the second parameter
2. The method for quantitative protein analysis according to claim 1, comprising the step of detecting the presence of the abnormality by comparing the value of the parameter. (11) A method for quantitatively measuring the protein concentration in an antigen sample by immunodiffusing an antigen sample containing a protein and an antibody source containing an antibody specific to the protein in the antigen sample, the method comprising: Prior to immunodiffusion, the antigen sample is subjected to selective protein migration in a direction perpendicular to the direction of mutual diffusion with the antibody source, and the protein and antibody are initially reacting and diffusing through areas of absent support medium to form at least one elongated settling zone of finite length;
The sedimentation zone is scanned by optical means and a plurality of electrical charges are generated in response to the sedimentation concentration at each position of a two-dimensional array, with some portions distributed within the sedimentation zone and portions distributed outside the sedimentation zone. electronically deriving from a plurality of said electrical signals a parameter value relating to the distance between the ends of said elongated sedimentation zone representative of a characteristic change in said protein concentration; Reference parameters derived from control sedimentation zones generated by equivalent immunodiffusion of multiple control antigen solutions, each containing a known concentration of the protein, in order to obtain an index for quantitatively measuring the concentration of the protein in the sample. A method for quantitative protein analysis, comprising comparing the value with the above parameter value. [+2 wherein said step of scanning a settling zone comprises a plurality of scanning paths intersecting said settling zone and a plurality of scanning paths beyond each end of said settling zone, said scanning comprising mutually spaced parallel scanning paths; the step of deriving the value of the parameter selects a scan path adjacent to a respective sedimentation zone edge and compares the plurality of electrical signals; 12. The protein quantitative analysis method according to claim 11, further comprising: measuring the mutual distance of the selected scanning paths. (Theft) The method for quantitative protein analysis according to claim 111, which comprises generating an image representing the sedimentation zone on a screen surface of a cathode ray tube in response to the plurality of electrical signals. said step of deriving a parametric signal comprises deriving a first signal that increases with increasing protein concentration, deriving a second signal that decreases with increasing protein concentration, and said first and second signals. The quantitative protein analysis method according to claim (1), comprising deriving a third signal representing a differential combination of as a combined parameter signal. The protein quantitative analysis method according to claim (b), wherein C represents the ratio of the first signal to the second signal. a first zone including at least one location on each side and in a region overlapping with another sedimentation zone, wherein the scanning step of the sedimentation zone for generating a plurality of electrical signals overlaps with the other sedimentation zone; generating an electrical signal at a second location on the other side of and in a non-overlapping region, and using the second location signal to roughly derive the electrical signal at the first location. A method for quantitative protein analysis according to claim 11, comprising: (b) each portion of the antigen sample contains a different known amount of the protein prior to the immunodiffusion of each sample portion; The scanning step, the step of deriving the parameter value, and the step of comparing the control parameter value with the determined parameter value are performed on the sedimentation zone formed in each sample portion. A method for quantitative analysis of proteins according to claim 11, comprising: (2) each portion of the antigen sample being treated with a different known amount of protein prior to immunodiffusion of the multi-sample portion. The obtained sedimentation zone of each sample portion, enriched by the addition of the protein, is determined in the scanning step, the parameter value derivation step, and the obtained parameter for each sample portion with respect to the added protein concentration. Claim 1, further comprising the steps of effectively plotting the values and determining the value of said protein concentration in the original sample by extrapolation of said curve obtained.
11) The protein quantitative analysis method described in item 11). 09 The method of claim 1 further comprising the step of staining the sedimentation zone with a dye prior to the step of scanning the sedimentation zone.
11) The protein quantitative analysis method described in item 11). (21) Electronically transmitting, from the plurality of electrical signals, a corresponding value of a second parameter of a zone that is characteristically different from the first parameter depending on the presence of a certain abnormality of the protein. and the step of detecting the presence of the abnormality by comparing the value of the first parameter and the value of the second parameter, the method for quantitative protein analysis according to claim (11). . 3. Detailed Description of the Invention The present invention relates to a technique for quantitative analysis of proteins present in a liquid mixture, and particularly to a method for quantitative analysis when the amount of a sample is minute. In recent years, with the rapid development of knowledge regarding the role that proteins play in health and disease, there has been a need to quickly and relatively economically quantitatively measure proteins in fluids such as serum, spinal fluid, and cell extracts. generally increasing. Proteins in such liquids are often identified (qualitative analysis) by an immunochemical method that utilizes precipitation of each protein caused by antibodies specific to a particular protein. Such specific antibodies are produced when a foreign protein (antigen) invades and stimulates the body. Antiserum is a mixture of such known antibodies. By reacting a protein sample with such an antiserum in vitro and observing the presence or absence of precipitate as a result, it is possible to obtain powerful clues about the type of protein present in the sample. A method for quantitative protein analysis using such an immunochemical method involves bringing one reagent, antibody or antigen, into contact with the surface of a gel containing the other reagent, and detecting scattered light from the formed precipitate. Detection method (Japanese Patent Application Laid-Open No. 50-1 (1983)
17123> is known, but the parameter for quantitative analysis obtained by this method is the precipitate in the gel at one measurement point! Since the value corresponds to 2 concentrations,
It is inaccurate because it is greatly affected by the diffusion rate in the gel, and it has the disadvantage that it can only be applied to single-component products. Therefore, such immunochemical methods are generally considered to be qualitative methods in protein analysis, and no specific method effective for quantitative analysis has been found. An object of the present invention is to provide a method for quantitative protein analysis using an immunochemical method that is highly reliable and reproducible. In addition to the above object, another object of the present invention is to provide a method for quantitatively analyzing each protein in a sample containing a wide variety of proteins. The present invention is a method for measuring the protein concentration in an antigen sample by performing immunodiffusion with an antigen sample and an antibody source containing an antibody specific to a specific protein. That is, proteins and antibodies first diffuse into each other in a support medium that does not contain these reactants, and a contact reaction occurs to form at least one elongated sedimentation zone with a finite length, which can be quantitatively measured. To do this, each location in a two-dimensional array distributed across the interior and exterior of the zone is optically scanned, generating an electrical signal corresponding to the sediment concentration at each location, and elongated strips representing specific changes in the protein concentration. A parameter relating to the distance between the zone ends along the sedimentation zone is electrically derived from the signal. Then, the results are compared with parameter control values obtained from a control zone obtained by performing the same immunodiffusion as in the case of the sample on a control antigen solution containing a known amount of the above protein. . The experimental results and calculations related to the results described above can be performed manually, if desired, or data can be obtained semi-automatically using optical and electrical scanning devices. The necessary data processing can also be performed fully automatically by a general-purpose computer of appropriate capacity. The present inventors have discovered that the distance between the ends of an elongated sedimentation zone of finite length formed by mutually diffusing proteins and antibodies has an effective parameter for quantitative measurement of proteins, In accordance with the present invention, this parameter is derived from a plurality of electrical signals measured at respective locations in a two-dimensional array distributed within and outside the elongated sedimentation zone. Since the elongated settling zone typically appears as symmetrical, the end position of the settling zone can be corrected or supplemented by calculations based on its axis of symmetry or the curvature of the settling zone. In the present invention described above, when the initial antigen mixture contains various proteins, first, the direction of mutual diffusion with the antibody source is perpendicular to the direction in which the various proteins diffuse in proportion to the difference in characteristics between the various proteins. A portion is fractionated by moving in the direction. Such selective transfer can be achieved, for example, by simple diffusion, electrophoresis, or even more complex methods such as chromatography, but the sedimentation zone formed by subsequent immunodiffusion is It remains basically unchanged even when fractionated by different methods. For example, in the case of electrophoresis, each protein moves and distributes along the direction of an electric field depending on the difference in mobility due to the difference in migration mobility between different proteins. After such an initial fractionation, the proteins migrate in different directions and come into contact with the antiserum, generally resulting in basic separation by interdiffusion in a suitable support medium such as agar or agarose. form a linear distribution. The precipitation zones for each protein are generally completely separated and clearly differentiated. There are sufficient differences in the diffusive mobilities of different proteins, or alternatively antibodies, to permit a clear distinction between such sedimentation zones. Since the zones overlap only to a certain extent, the endpoints of the zones can be clearly distinguished. Furthermore, even if adjacent sedimentation zones overlap, the position of the overlapping end can be calculated by utilizing the symmetry of the sedimentation zones and based on the axis of symmetry or the curvature of the sedimentation zones. When using scattered light or light intensity at one measurement point as a quantitative parameter as in the prior art, the density of the protein and the degree of diffusion over time have a significant influence on the measurement results, and it is necessary to correct this. However, since the quantitative method according to the present invention uses the shape factor of the sedimentation zone as a parameter, it is possible to perform such selective movement as a fractionation process before interdiffusion. It is. Even if you put the antigen and antibody into a hole to perform immunodiffusion and apply an electric field in a way like electroimmunodiffusion to increase the speed at which the antigen and antibody move towards each other, the resultant The sedimentation zone maintains the same basic shape as when no electric field is applied. The same applies if the immunodiffusion step in immunoelectrophoresis is accelerated by an appropriate electric field. Therefore, the term immunodiffusion in this specification refers to both cases with and without an accelerating electric field. Examples of the present invention will be described below with reference to the accompanying drawings, but these examples are not intended to limit the scope of the present invention. Immunoelectrophoresis is well known as a qualitative method, and a variety of devices have been published to perform it, but they all have little differences. In the present invention, electrophoresis and subsequent diffusion and immunoreaction are performed in a monolayer gel, typically a fraction of a millimeter to several millimeters thick, mounted on an optically transparent plate. The support medium used is agarose, commonly used today, saturated with barbital Il solution with an E)H of about 8.6 and an ionic strength of about 0.1.The sample is then cut into gel layers. A plate 20 is shown in Figure 1, on which a plate 20 is shown extending parallel to the direction of electrophoresis. An antibody groove 24 along the axis 25 and circular antigen holes 21 and 2 arranged at equal intervals on both sides thereof.
There are 2. Depending on the arrangement of the holes, two separate solutions or two samples of the same solution will move simultaneously towards the same antibody solution on each plate. Two additional antibody grooves may be added outside of the two antigen holes on each plate, and two additional antigen holes may be added outside of the two antigen holes to double the capacity of the plate. Similarly, additional antigen holes may be added at a sufficient distance along the direction of electrophoresis from holes 21 and 22 to prevent pattern overlap. Shaded areas 26 and 28 in FIG. 1A represent four types of proteins a in a pair of samples of the same type after being placed in holes 21 and 22 and subjected to electrophoresis in the direction of arrow 23 for a certain period of time. This is an approximate representation of typical distributions of b, c, and d. Normally, all proteins move in the same direction in a liquid medium, but the solvent itself has the property of carrying a net charge, resulting in gel flow and electroosmosis. Proteins therefore move in both directions relative to the hole. When the electrophoresis is finished, the antibody is placed in the groove 24, and as shown in FIG.
A sedimentation zone is formed by interdiffusion of proteins a, b, c, and d with their corresponding antibodies. B in Figure 1
, C, and D show each stage of typical zone appearance. Precipitating arcs of unrelated antigens react with each antibody and form separately, but when they come close enough, they intersect like a first cause, and immunochemically related precipitating arcs Fuse as a continuous reaction line. Figure 1 C and D
The sedimentation zone d' in Figure 1A actually contains two separate proteins, and even if the two proteins have the same electrophoretic mobility, they will form separate sedimentation zones. It illustrates the facts that form. Zones d and d- can also be thought of as two different proteins placed in the wells 21 and subjected to immunodiffusion without going through an initial electrophoresis step. The reason why each zone end is clearly separated is because the diffusion rate of each protein acid or antibody is different. In either case, each such zone can be analyzed separately using the methods described below. According to the present invention, the sedimentation zone by immunoelectrophoresis can be measured directly and quantitatively. Such measurements allow the determination of the specific, characteristic physical arrangement of each precipitation zone of the protein of interest on the plate, as well as measurements of light intensity by optical methods. Both methods add a temporal element, which is an important element of observational data. However, such time measurements are meaningless if the incubation is carried out until equilibrium is reached and a stable zone is formed. Position measurement on the plate 20 can be performed using a low magnification microscope, for example. As shown in FIG. 2, a plate 20 is placed over a variable opening 32 of a light source box 3o having a light source lamp 36 and a light absorbing dark field 34 such as black velvet. Objective lens 41 for microscope 4°
, a crosshair 43 for sighting is attached to the eyepiece lens 42 and the focal plane. On top of the light source box 3o is a double slide mechanism 45.
Although not shown in detail, it is equipped with a screw drive device 46 and accurate scales, which allows the position of the microscope placed on it to be accurately read with respect to the two coordinate axes. is now possible. In this figure, only one coordinate axis is shown for clarity. It is usually convenient to set the X-axis in the direction of electrophoresis, ie, parallel to the antibody groove, and to set the origin of the coordinate axes on or near axis 25, as shown in FIG. 1A. In order to measure the density of light, the microscope is equipped with a diagonal half mirror 48 for splitting the light beam, sending part of the light to the eyepiece and using the other part to form a real image on the diaphragm 52. are tied together. The diaphragm 52 is connected to the plate 20
Only the light from the range that matches the crosshair 43 on the
Send to photosensitive transducer 5°. transducer 5
o is electrically connected to the amplifier circuit 54 and the meter 56. Instead of the meter 56, it may be possible to directly observe the light with the naked eye and record it manually, or it may be possible to connect a printed circuit or a converter that automatically records the intensity of the light according to a command signal. In FIG. 2, for example, instead of dark field illumination,
Various modifications are possible, such as direct illumination or overhead illumination where the reflected light from the zone can be measured directly. An example of a method and ti[ for performing a fully automated measurement is described below. FIG. 3 illustrates certain features of the sedimentation zone defined for locating the zone according to the present invention as it emerges. The horizontal line 61 in y-y is the edge of the antibody groove on the immunoelectrophoresis slide. Points E and F at coordinates (Xe, Ye) and (Xf, Yf) are the left and right end points of the extending zone 60. In addition to the zone end points E and F, it is better to measure several intermediate points within each zone. In FIG. 3, a point G at Y(1) with coordinates <xa is an example of such a point. Since the zone has a certain width in the y-direction, the y-coordinate of each intermediate point, such as It is convenient to define one or more points 64, including the strongest point. As the incubation time progresses, the absolute and relative positions of points E, F and G change as zones emerge. As a function of time, Xe and
The value of f is correlated with the protein concentration, and the difference in this coordinate (Xf
-Xe ), ie the length L of the sedimentation zone at a particular measurement time, can be directly determined by comparing the length L of the sedimentation zone with a control value obtained with a known concentration of protein at the same time. However, even more reliable and accurate results can be obtained by deriving one or more functions considered to be effective as parameters from such measurement results and correcting the above parameters. good. As the data shown in FIG. 4, FIG. 5 shows changes in this parameter with respect to incubation time. In FIGS. 4 and 5, the solid lines are plots of values obtained from actual experiments. These figures also include
The value obtained by extending (extrapolating) the solid line in the direction of less time is shown. This extension is indicated by a dashed line. In FIG. 4, the point 66 where the extension lines intersect represents the point where the length of the zone was O. The extension (extrapolation) sl in Figure 5 intersects the time axis at point 67,
TO can be found in the same way. By such extension (extrapolation), the time of first appearance of the sedimentation zone can be determined practically accurately, and this time of first appearance can also be used as a temporal reference for determining the length of the sedimentation zone. Once experimental values for determining parameters are obtained for the sample to be analyzed, these values are compared with appropriate combinations of standard values for each protein. These standards were made using a series of solutions containing known amounts of the protein of interest under the same conditions as the measurements. To obtain such a combination of standard values, standard procedures are carried out using such standard protein solutions and subsequent measurements are taken at corresponding points on each plate as the incubation progresses. It is desirable that the standard operation be performed under all conditions as similar as possible to the measurement operation to which the standard is to be applied. In fact, it is desirable that the reference value is unique and corresponds individually to each measurement operation. However, in routine measurements, if the slope of the control curve is known from the previous measurement, 1
Sometimes standard operations are sufficient. Standard values for desired parameters are derived by performing several such standard operations for each concentration. The standard values of the parameters thus measured are therefore considered to be a function of both concentration and time. Creating a standard curve is more indirect. These values are related to the time at which the measurements were taken, so the reference standards must be made to cover a range of time. It is difficult to measure data for all concentrations simultaneously. Each measurement value is therefore related to the time of measurement so that the final standard value for each protein concentration is plotted on a separate curve as a function of time. FIG. 6 is a representative example of such a group, in which standard values of parameters for three concentrations are plotted against the time axis. In FIG. 6, vertical lines are drawn at arbitrary times t, t2, and t3. These multiple values of L can be replotted as separate curves as a function of concentration. The resulting set of curve combinations shows L as a function of protein concentration for each time. This combination is shown in FIG. 7 and is more convenient than the plot versus time of FIG. 6 when comparing experimental values. Another feature of the present invention is that it improves accuracy, particularly when the concentration of one or more proteins of interest in the sample to be measured is relatively low. In such cases, it is desirable to supplement the sample solution with several known amounts of such proteins in whole number ratios. Measurement operations are then performed in parallel with this solution and a standard containing a known amount of protein. If values are needed to determine the required parameters for each solution, they can be determined for all times by the interpolation described above for time. The parameter values P obtained for the authentic standards are plotted against protein concentration in the conventional manner as shown in curve 80 of FIG. Similarly, score each value of P for the original antigen sample and a portion to which a known amount of protein was added, and for i, j, and k, calculate as if the solution contained only the added protein. It is plotted as follows. Therefore, the value for the original sample is plotted at the concentration (18) position. A curve 81 is drawn through these points. In this data processing example, the protein concentration of the original sample can be calculated in several ways, all of which should give essentially the same value. In this way, calculations can be made using several desired methods, and the results can be averaged. First, a straight line extending horizontally from h and intersecting the curve 80 at h' indicates the density of the value indicated by ch. This corresponds to the general comparative method mentioned earlier. Furthermore, in the same way, extension lines drawn from +, j, and k to intersect the straight line 80 are i −4i ′, j 4j ′, and k
It gives the concentration according to the length of the straight line -k', and all of those lengths are logically the same. If the value of P at h is uncertain for some reason, e.g. because the observed zone is unclear, a reliable value can be obtained by averaging over all four intervals. Another method not only can improve the measurement inaccuracy in It also has the advantage of being able to determine the value. In the latter case, the protein above curve 81 is obtained by connecting the obtained points i, j and k and is further extended (extrapolated) towards the P axis to determine P at h. In this way, curve 81
Curve 80 serves as a guide in extending . For example, the curve 80 in FIG. It has been moved as a whole to the left until it now represents j and k. The intersection of the thus-moved curve 80 and the P axis gives a point value, from which the density ch can be determined. Furthermore, if the curve 81 is extended to the negative side of the horizontal axis -ch, the concentration value c
The concentration matching h can be read directly. The supplemented standard line 81 method described above has the great advantage that the same solution can be used for experimental and control purposes. This advantage can eliminate any small errors caused by extending the standard line, especially if the sample is human serum from a patient with a particular disease. The reliability of this extrapolation can be increased almost indefinitely by increasing the number of samples supplemented with various amounts of protein and by making measurements at several locations rather than just the three shown in Figure 8. It is possible to obtain a correct understanding of the measurement errors involved. The present invention can also automatically detect certain protein abnormalities in antigen samples. For example, normal and abnormal gamma globulins usually have small differences in their immunodiffusion and electrophoretic mobility ranges, yet react with the same antibodies, forming abnormal precipitation zones and generally forming sediments that are asymmetrically distributed along the length of the . The occurrence of such abnormalities can be detected by observing the asymmetry of the elongated sedimentation zone or abnormalities in other values. The existence of this abnormality is indicated if several independent measurements are performed at a certain value of X and the results of calculating the measurement error show a statistically significant change. Another parameter that responds sensitively to the presence of abnormal proteins is the axial curvature of the zone. Due to the presence of anomalies, the curvature along the length of the zone tends to vary abnormally and asymmetrically, while the overall curvature tends to be below normal. It is further advantageous to use multivalued parameters which consist of a unique function of two or more parameters. For example, the sum of the zone length L and the light intensity parameter becomes a new parameter that gives more reliable results than using the individual components one by one, if measurement errors are taken into account appropriately. . Also of interest are the derivatives of the two parameters, one increasing and one decreasing with increasing protein concentration. In this way, the quotient of these parameters,
Using the differences, it is possible to obtain parameters that have a sharper dependence on concentration than using them individually. The first appearance time TO of a zone is an example of an inverse relationship to the protein concentration, and can be used particularly when determining such a quotient. The distance between the antibody groove and the zone at any desired value XΩ on the X-axis is also inversely related to protein concentration, and such a quotient is also used as a parameter. In general, it is better to divide a parameter that directly depends on concentration by a parameter that is inversely dependent on concentration so that the resulting multivalent parameter depends directly on concentration rather than inversely. . For an unknown sample, reference values suitable for comparison with multivalued parameters determined by actual measurements can be derived from the reference values of the parameters of each component, as described above. As mentioned above, the measurement of position and light intensity and the derivation of parameters can be performed by direct observation or manual methods, but as a feature of the present invention, these operations can especially be automated in part or in whole. It means that it is suitable. A particularly convenient and effective method for making the measurements required for the present invention is to optically scan the slide, using a charge-coupled scanning device, such as a television camera, or an equivalent method to scan the scanning range. This is a method of obtaining a video signal that represents the apparent light intensity of a two-dimensional combination of The signal of each element is generally digitally converted and stored electrically along with the X and Y coordinates corresponding to the position on the plate or the measurement time. Such a combination of signals for all locations or signals for a specific location can be retrieved during subsequent data processing 1. Also, any portion of the image on the slide can be displayed on a CRT or equivalent, either during or after scanning. Systems for scanning, storing images, reproducing and extracting specific points of an image as digital signals are known in electronics technology and can be purchased in a way that suits this requirement. FIG. 9 illustrates an example of such a device, in which a television camera 90 is connected to a photosensitive surface of the camera by a lens 91.
An image of the plate 20 is formed on a monitor CRT 92, a scanning control device 94, and a general-purpose computer 100. By adjusting the lens 91, the plate 20 can create an image at any magnification. In order to center the plate at an arbitrary position, for example, it is necessary to change the position of the plate on a support for illumination material such as the light source box 30 in FIG. 2, or to use conventional electronic bias control using a camera circuit. You may go. In particular, if a scanning mechanism synchronized with the monitor CRT can be used as the scanning device, it is possible to drive the mechanism in fine steps such that the movement in both coordinate axes on the screen is continuous and equal. The image is divided into area elements, identified, for example, by X and y coordinates. For manual or semi-automatic operation, the monitor display usually includes a cursor with an electronically generated bright spot indicating the location on the screen from which the video signal is being extracted from the area element on each scan. There is a manual control switch for the operator, who can freely move the cursor visually. The device can also position the cursor directly on the desired point depending on the x and y coordinate values via digital command addresses. Such signals can be obtained manually or from a general purpose computer program. As illustrated in FIG.
Count the pulses from 03 and continuously apply X to the branch line 104.
Gives a digital signal representing the coordinates. The circuit 106 generates an analog step voltage for each minimum unit of the X coordinate on line 1 (17) according to the signal.Divider circuit 108
performs scanning counting of the beam in the x-axis direction and provides a digital signal representing the y-coordinate to the branch line 109 for each sweep. In response to this count, the circuit 110 sends out an analog step voltage for each minimum unit of coordinates to the line 111. The step voltages on lines 101 and 111 are
Controls the scanning of the RT92 in the X and Y directions to maintain synchronization between the two. The video signal from camera 90 is on line 11
2, the light is sent to the monitor CRT 92, and changes in the light intensity of the plate 20 are reproduced on the screen surface. The selection circuit 118 typically consists of an X and Y counter, which counts up or down by counting one or more pulses when the control stick shown at 119 is manually moved. The resulting digital signal represents the x and y coordinates of the location being measured and registers 1
17. Comparison circuit 114 includes register 117
Coordinates of a specific measurement target position and lines 104 and 10 from
Constantly compare the x and y coordinates of the scanning beam from 9 onwards. When the scanning spot comes to the memorized position (address),
Normally, an operation signal is sent from the comparison circuit 1j4 to the switching circuit 120 once for each multi-scan. Register 125 thus receives on its line 123 a digital signal corresponding to the light intensity when scanning the specified measurement point on the slide. The motion signal from circuit 114 is also sent to cursor control circuit 126, which applies light intensity modulated pulses to the video signal to identify the selected point on the monitor screen, as shown at 127. Register 125 also receives the X and Y coordinate signals from lines 115 and 116 and also receives the continuous time signal produced by time circuit 128. All of these signals are stored in register 125 and are read by computer 100 either by manual control on line 129 or by computer control on line 130.
sent to. Computer 100 individually identifies measurement points and accepts input information for spots on plate 20 as required by any program. This measurement method is
The circuit is similar to the selection circuit 118, the register 117, and the comparison circuit 114, and the selection circuit 118 operates according to an electrical signal that selectively indicates the direction of movement of the measurement point or indicates the digital address of the required position. do. Rather than creating two such devices, as shown in FIG.
is controlled by computer 100 via branch line 132. In order to perform such control by the computer 100, as described above, the computer 100 has built-in programs for measuring position and light intensity and for guiding general parameters. In order to scan the zones automatically, the plate should be suitably illuminated and transferred from the incubation chamber to a precisely defined scanning position. Especially when the optical scanning device is lightweight and compact, such as a charge-coupled device.
For example, it is preferable to mount the scanning device on a platform that can move in two dimensions over which immunodiffusion or immunoelectrophoresis plates are lined up. In this way,
It can also be used to place antigens or antibodies into holes or grooves using a scanning device. For this purpose, a pipette that can be controlled by digital signals is installed slightly outside the scanning rig. The video signal from the scanning device is sent to a computer that is programmed to identify the shape of each hole and to allow automatic reading. The pipette-mounted platform of the scanner can be moved over the plate by computer control and stopped when the scanning axis is correctly positioned over a defined antigen well. The platform is then moved a predetermined distance such that the pipette is just over the hole and a volume of sample is correctly injected into the hole. When the antigen solution is injected into all the holes while washing the pipette appropriately after each operation, electrophoresis begins. Similarly, after electrophoresis is completed or, if electrophoresis is not performed, immediately after the antigen is placed in the antigen hole, this device places the antibody in the antibody groove. After a period of diffusion, the same scanning device moves over the settling zone over the plate combination to perform the scan. The scanning capability of the scanning device is also exercised prior to injecting the sample into the hole. The computer is programmed to measure the relative positions of the antigen holes and antibody grooves, and remembers any incorrect relative positions or dimensions of holes or grooves in the plate. Those that deviate greatly from the standard are eliminated at the sample injection stage, and those that slightly deviate from the standard are automatically corrected at the final parameter derivation stage. The initial zone scanning process obtains a video signal from an address that is continuously measured by a specific X value and a Y value moving at regular intervals over the area where the zone is expected to form. This is a stage controlled by a computer. The video light intensity values received from register 125 are stored as x, y, and t data for each measurement point. At the end of each such y-scan, the x-coordinate is moved a fixed distance, and a similar y-scan is performed, the results recorded, and so on until the entire area of interest is covered. Each measured light intensity value is typically compared to a previously measured and stored value. It is determined that if the measured light intensity is above a background region threshold, it indicates the presence of a sedimentation zone. Also, for each y-scan, the maximum light intensity across the zone is determined and stored, and the axis of the zone is determined from a series of y values for equally spaced x values. The end point of each zone is recognized by the extreme value of X such that the highest point (peak) of light intensity is recognized in each direction of X. If more precise position measurement is required, a certain area near the endpoint is programmed to be scanned at even finer x and y intervals. A direct mathematical process is applied to the data recorded for the actual sedimentation zone located and recorded, and the X value of the zone end is subtracted, resulting in the parameter L. Other parameters may be determined by appropriate calculations as required. The above measurements should be carried out as a unit measurement on a set of plates containing one or more unknown samples and their associated several standard solutions, such that a complete determination of the unknown samples can be made. desirable. Upon each scan of a set of such plates, the computer derives the concentration of each protein where a sedimentation zone is found. If, as already mentioned, a number of standard measurements are involved, the possible measured concentrations are determined for each calculated concentration value. It is known to perform such calculations by computer. It is possible to perform several independent measurements based on the concentration of each protein in the sample. In general, several different parameters are used as reference, weighted according to the expected error, and the average determined. If the calculated error with respect to the average value falls within a certain range for the sample being measured, there is no need to perform any further measurements, and the computer will then print out the final result. Records and collects as much original data as necessary for the program.For example, the doctor requesting the analysis can put all the data on a magnetic tape and request it for future reference. A photograph of the plate after diffusion can be taken either through a monitor CRT or directly. Usually, the protein concentration is high (if not, one scan cycle is sufficient to determine the concentration of the protein of interest). is not good. For a certain amount of time, from a few minutes to an hour or more,
After incubation is added, the above scanning is repeated, and the measurement plate and the corresponding standard plate are scanned. Computers typically look for new zones that appear where no zones were present in previous scans, or they scan areas where previously subsidence zones have been found and recorded, and determine the extent of zone movement and expansion from previous records. programmed to explore. Thus, the computer continues to process data with each scan. Sometimes two precipitation zones of distinct proteins come so close that a normally formed precipitate intersects at the end. The zones created by immunoelectrophoresis are programmed to predict crossings of such zones, predict when crossings will occur, and make appropriate corrections in the process of inducing various parameters. . If the protein being measured is of the type that crosses over, each plate is scanned early in the appearance of the zone, before cross-over occurs, as shown in FIG. 1B. The axis and end points of the zone are then reliably localized. The computer is also programmed to check for actual intersections as part of each regular scan. For example, by comparing the ( Possibility checks are also included; for example, the computer extends all endpoints beyond their actual value and compares them with the extended endpoints of adjacent zones. Such axis extensions are A simple method is to extend the zone linearly in the direction of the slope of the axis.Alternatively, the observed zone axis may be properly extended to match a parabola or other curve.Intersection of Zones can also be found by calculating the rate of change of the slope of the axis of the zone. Alternatively, it can be found by calculating the radius of curvature of the axis by a series of points along the zone. Any value that deviates significantly from normal variation indicates that the zone contains two or more intersecting areas. A sufficiently accurate correction can be made by deriving the position of the zone edge observed in the other half that does not intersect, assuming that it is symmetrical. Correspondence between two points is determined at equal value intervals of X on both sides of the point XQ closest to the antibody groove, and for zones where direct immunodiffusion is performed, on both sides of the zone axis. are determined at intervals of equal values of ■.Axes that lie within the intersecting area are localized by extrapolation.More precise methods of correction are possible if desired, e.g. is programmed to take into account the asymmetry of the zone by adjusting the measurements at symmetrical points chosen on the non-intersecting halves of the zone.If an asymmetry is detected, e.g. The correction factor can also be determined by comparing the two points observed at the two points and determining the ratio between these values.Another example of correction is the light intensity actually measured in the area where the intersection actually intersects. A method that takes into account that the values are partly due to one zone and partly due to the other zone.The computer adjusts the light intensity values at the intersection area appropriately. To determine the appropriate division ratio, as mentioned above, it is sufficient to compare the respective values of the intersecting area and the symmetrical area, without making the above adjustments. You don't have to do it either. If possible, it is desirable to apply two or more separate calculations to correct the intersection area and average the results to determine the error. However, originally the crossing area was only a small part of the entire zone. Therefore, the error in approximating the correct value within the area of the intersection is acceptable and does not significantly affect the final result. Those skilled in the art will appreciate that many features of the methods previously described can be implemented with a variety of equivalent alternative techniques within the scope of the invention. For example, parameter values that do not require continuous measurements of the incubation time can be determined after the incubation has reached equilibrium. Such measurements have the advantage that the configuration of the zones is static and stable and the time to take the measurements is not a critical factor. Furthermore, measurements of the sedimentation zone at any stage of incubation can be carried out with specific light. This method does not require precise time measurements, so it is usually
It is convenient after the reaction has reached equilibrium. In summary, methods and apparatus have been described for making truly quantitative measurements of the concentration of individual proteins in physiological fluids using immunodiffusion, immunoelectrophoresis, and analog means. When actually using the method described here,
It has been demonstrated that the protein concentration can be measured with an accuracy of 5% or less. The present invention thus provides measurement techniques for a wide range of experimental or diagnostic purposes. 4. Brief explanation of the drawings Fig. 1 is a diagram of an immunoelectrophoresis plate, where A in Fig. 1 shows the distribution of typical various proteins after electrophoresis, B, C in Fig. 1, D shows each step of subsequent diffusion and immunoprecipitation reaction. FIG. 2 is a longitudinal sectional view of the device for measuring slides. FIG. 3 is a diagram illustrating the characteristics of a settling zone with respect to the present invention. FIG. 4 is a graph showing a typical relationship between zone end points and time. FIG. 5 is a graph showing a typical relationship between zone length and time. Figures 6 and 7 are graphs showing typical relationships between time and protein concentration versus zone length. FIG. 8 is a graph showing an example of inducing a protein concentration value by adding a known protein to an unknown protein. FIG. 9 is a block diagram of an embodiment of an electronic scanning device according to the present invention. 20... Plate 21.22... Antigen hole 24...
・Antibody groove 90.91.92...Optical means 94...
・Control means patent applicant Frederick J.A. Alagem (1 other person) Representative Patent attorney Satoru Furumura Figure 1 Figure 2 Figure 3 Figure 6 To Figure 7

Claims (20)

[Claims] (1) A method for quantitatively measuring the protein concentration in an antigen sample by immunodiffusing an antigen sample containing a protein and an antibody source containing an antibody specific to the protein in the antigen sample, the method comprising: by optical means, allowing the protein and antibody to react and diffuse into each other through a region of the support medium initially free of either of these reactants to form at least one elongated sedimentation zone of finite length; A plurality of electrical signals are generated in response to the sediment concentration at each position of a two-dimensional array that scans the sedimentation zone and has a portion distributed within the sedimentation zone and a portion distributed outside the sedimentation zone. electronically deriving from a plurality of the electrical signals a parameter value relating to the distance between the ends of the elongated sedimentation zone representing a characteristic change in the protein concentration; To obtain an index for quantitatively measuring protein concentration, reference parameter values derived from control sedimentation zones generated by equivalent immunodiffusion of multiple control antigen solutions, each containing a known concentration of the above protein, and above.
1. A method for quantitative protein analysis, comprising comparing with a parameter value. (2) the step of scanning a sedimentation zone includes a plurality of scan paths intersecting the sedimentation zone and a plurality of scan paths beyond each end of the sedimentation zone, the scans being spaced apart from each other; intersecting said sedimentation zone along parallel scanning paths, further deriving values of said parameters;
Claim 1, further comprising selecting scan paths adjacent to respective sedimentation zone ends, comparing the plurality of electrical signals, and determining the mutual distances of the selected scan paths. Quantitative protein analysis method described in . (3) The method for quantitative protein analysis according to claim (1), which comprises generating an image representing the sedimentation zone on a screen surface of a cathode ray tube in response to the plurality of electrical signals. (4) the step of deriving parametric signals comprises deriving a first signal that increases with increasing protein concentration, deriving a second signal that decreases with increasing protein concentration, and The method for quantitative protein analysis according to claim (1), comprising deriving a third signal representing a differential combination of the two signals as a combined parameter signal. (5) The method for quantitative protein analysis according to claim (4), wherein the combined parameter signal represents a ratio between the first signal and the second signal. (6) the scanning of the sedimentation zone, wherein the array includes at least one location on each side within the sedimentation zone and in an area overlapping another sedimentation zone, and generates a plurality of electrical signals; a step generating an electrical signal at a second location on the other side of the first zone and in a non-overlapping area that overlaps the other settling zone; and further generating the electrical signal at the first location. 2. The method for quantitative protein analysis according to claim 1, which comprises using the second position signal to derive . (7) each part of said antigen sample is enriched by adding different known amounts of said protein prior to said immunodiffusion of each sample part, and said scanning step, derivation of parameter values; Quantification of protein according to claim (1), wherein the step of comparing the determined parameter value with the step and control parameter value is performed for a sedimentation zone formed for each sample portion. Analysis method. (8) Each portion of the antigen sample is enriched by adding a different known amount of the protein prior to immunodiffusion of each sample portion, and the resulting sedimentation zone of each sample portion is a scanning step, a step of deriving a parameter value, and a step of effectively plotting said obtained parameter value for each sample portion against added protein concentration and said obtained parameter value in the original sample. Claim No.
1) Quantitative protein analysis method described in section 1). (9) The first aspect of the present invention includes the step of staining the sedimentation zone with a dye prior to the step of scanning the sedimentation zone.
Quantitative protein analysis method described in section ). (10) Electronically transmitting, from the plurality of electrical signals, a corresponding value of a second parameter of a zone that is characteristically different from the first parameter depending on the presence of a certain abnormality of the protein. and a step of detecting the presence of the abnormality by comparing the value of the first parameter and the value of the second parameter, the method for quantitative protein analysis according to claim (1). . (11) A method for quantitatively measuring the protein concentration in the antigen sample by immunodiffusion of an antigen sample containing a protein and an antibody source containing an antibody specific to the protein in the antigen sample, comprising: Prior to immunodiffusion, the antigen sample is subjected to selective protein migration in a direction perpendicular to the direction of mutual diffusion with the antibody source, and the protein and antibody are initially reacting and diffusing through regions of absent support medium to form at least one elongated settling zone of finite length; and scanning said settling zone by optical means so that a portion of said settling zone settles. generating a plurality of electrical signals in response to the sedimentation concentration at each position of a two-dimensional array distributed within the zone and partially distributed outside the sedimentation zone; and generating a specific change in the protein concentration. electronically deriving a parameter value related to the distance between the ends of the elongated sedimentation zone along the elongated sedimentation zone from the plurality of electrical signals; and obtaining an index for quantitatively measuring the protein concentration in the antigen sample. comparing the parameter value with a reference parameter value derived from a control sedimentation zone generated by equivalent immunodiffusion of a plurality of control antigen solutions each containing a known concentration of the protein. Quantitative protein analysis method. (12) the step of scanning a sedimentation zone includes a plurality of scan paths intersecting the sedimentation zone and a plurality of scan paths beyond each end of the sedimentation zone, the scans being spaced apart from each other; intersecting the sedimentation zone along parallel scanning paths, further deriving the value of the parameter, selecting a scanning path adjacent a respective sedimentation zone edge to transmit the plurality of electrical signals. 12. The method for quantitative protein analysis according to claim 11, which comprises comparing and measuring mutual distances of the selected scanning paths. (13) The method for quantitative protein analysis according to claim (11), which comprises generating an image representing the sedimentation zone on a screen surface of a cathode ray tube in response to the plurality of electrical signals. (14) the step of deriving a parametric signal derives a first signal that increases with increasing protein concentration;
Claims comprising: deriving a second signal that decreases with increasing protein concentration; and deriving a third signal representing a differential combination of the first and second signals as a combined parametric signal. The method for quantitative protein analysis according to item (11). (15) The method for quantitative protein analysis according to claim (14), wherein the combined parameter signal represents a ratio between the first signal and the second signal. (16) the arrangement is located on one side within the sedimentation zone;
said scanning step of said settling zone generating a plurality of electrical signals includes at least one location in each of said settling zones in said other settling zone and said first zone overlapping said other settling zone; A patent comprising generating an electrical signal at a second location in side and non-overlapping regions and further using said second location signal to derive said electrical signal at said first location. A method for quantitative protein analysis according to claim (11). (17) each portion of the antigen sample is enriched by adding a different known amount of the protein prior to the immunodiffusion of each sample portion, and the scanning step
According to claim 11, the step of deriving the parameter value and the step of comparing the determined parameter value with the reference parameter value are performed for a sedimentation zone formed for each sample portion. Quantitative protein analysis method. (18) Each portion of the antigen sample is enriched by adding a different known amount of the protein prior to immunodiffusion of each sample portion, and the resulting sedimentation zone of each sample portion is a scanning step, a step of deriving a parameter value, and a step of effectively plotting said obtained parameter value for each sample portion against added protein concentration and said obtained value of said protein concentration in the original sample. 12. The quantitative protein analysis method according to claim 11, wherein the method is performed in the step of determining by extrapolation of a calculated curve. (19) The scope of claim 1 further comprising the step of staining the sedimentation zone with a dye prior to the step of scanning the sedimentation zone.
11) The protein quantitative analysis method described in item 11). (20) Electronically transmitting, from the plurality of electrical signals, a corresponding value of a second parameter of a zone that is characteristically different from the first parameter depending on the presence of a certain abnormality of the protein. and the step of detecting the presence of the abnormality by comparing the value of the first parameter and the value of the second parameter, the method for quantitative protein analysis according to claim (11). .