JPS6255105B2 - - Google Patents

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 fluids 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 a living 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.

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. A detection method (Japanese Unexamined Patent Publication No. 50-107123) is known, but the parameters for quantitative analysis obtained by this method are values corresponding to the precipitate concentration in the gel at one measurement point. It is inaccurate because it is greatly affected by the diffusion rate in the gel, and
The drawback is 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 yet 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.

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 in addition to the above object.

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 heretofore 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, 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, we optically scan each location in a two-dimensional array distributed inside and outside the zone, generate an electrical signal corresponding to the sediment concentration at each location, and detect the specific changes in the protein concentration. A parameter relating to the first appearance time of the represented 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 above experimental results and calculations regarding the results are as follows:
You can also do it manually if necessary,
Data can also be determined semi-automatically using optical and electrical scanning devices. Further, the necessary data processing can be performed fully automatically by a general-purpose computer with an appropriate capacity.

The present inventors have determined that the first appearance time of an elongated sedimentation zone of finite length formed by mutual diffusion of protein and antibody, that is, the time from the start of diffusion until the appearance of the sedimentation zone, is the quantification of protein. The present invention has been based on the discovery that there is an effective parameter for measuring a plurality of electrical signals measured at respective locations in a two-dimensional array distributed inside and outside an elongated sedimentation zone. It can be derived from various parameters derived from the signal, such as the distance between both ends of the sedimentation zone, the total value of light intensity, etc. These preliminary parameters, including sedimentation zone shape factors, are correlated to protein concentration and are also a function of time. Therefore, when the time of first appearance of the sedimentation zone cannot be directly measured, the time of first appearance can be determined by extending (extrapolating) the values of these preliminary parameters to the zero point. In the present invention, an elongated sedimentation zone with a finite length is formed by mutual immunodiffusion, and this has an important meaning in performing quantitative protein analysis. That is, since this elongated sedimentation zone normally appears as a symmetrical shape, various corrections can be made by utilizing the symmetry when deriving the preliminary parameters.

In the present invention described above, when the initial antigen mixture contains various proteins, firstly, in proportion to the difference in characteristics between the various proteins, the direction of mutual diffusion with the antibody source is perpendicular to the direction of diffusion between the various proteins. A portion is fractionated by moving it in the direction of Such selective transfer can be effected, for example, by simple diffusion, electrophoresis, or even by more complex methods such as chromatography, but is formed by subsequent immunodiffusion. The sedimentation zone remains essentially the same no matter which method is used for fractionation.
For example, in the case of electrophoresis, the difference in migration mobility between different proteins causes each protein to move and distribute along the direction of the electric field in accordance with the difference in mobility. After such an initial fractionation, the proteins migrate in different directions and come into contact with the antiserum, generally by interdiffusion in a suitable support medium such as agar or agarose. form a basic linear distribution. Sedimentation zones for each protein are generally completely separated;
clearly discriminated.

There are inherently sufficient differences in the diffusive mobilities of different proteins, or alternatively antibodies, to allow for the identification and discrimination of such sedimentation zones. Since the zones have only a 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 an antigen and an antibody in a hole to perform immunodiffusion and apply an electric field in a way such as electrical immunodiffusion to increase the rate at which the antigen and antibody move toward each other, the result is The formed 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 involving and not involving 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 followed by diffusion and immunoreaction are
It is usually carried out in a single layer of gel, from 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 a barbital buffer with a pH of about 8.6 and an ionic strength of about 0.1. The sample is then placed into a hole cut into the gel layer or molded into the gel layer when it 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 22 equally spaced on either side of the antibody groove 24. There is. 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. At the same time, 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 correspond to holes 21 and 22.
The representative distribution of four types of proteins a, b, c, and d in a pair of samples of the same type after electrophoresis is performed for a certain period of time in the direction of arrow 23 is approximately shown. It is something. Normally, all proteins move in the same direction in a liquid medium, but the solvent itself carries 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,
A sedimentation zone is formed by interdiffusion of proteins a, b, c, and d of FIG. 1A and their corresponding antibodies. B, C, and D in FIG. 1 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 Figure 1D, forming immunochemically related precipitation arcs. merge as a continuous reaction line. The sedimentation zone d' in Figures 1C and D indicates that region d in Figure 1A actually contains two separate proteins, even though the two proteins have the same electrophoretic mobility. , illustrating the fact that separate sedimentation zones are formed. Zones d and d' are formed by placing two different proteins in the hole 21.
It can also be considered that immunodiffusion is applied without going through the initial electrophoresis step. The reason why each zone end is distinct is that the diffusion rate of each protein 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 immunoelectrophoretic sedimentation zone 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. You can also do it. Both methods add a time element, which is an important element of observational data.

Position measurement on the plate 20 can be performed using a low magnification microscope, for example. As shown in FIG. 2, a light source box 30 with a light source lamp 36 and a light absorbing dark field 34 such as black velvet is provided.
A plate 20 is placed over the variable opening 32 of. The microscope 40 has an objective lens 41, an eyepiece lens 42, and a crosshair 43 for aiming at the focal plane. A double slide mechanism 45 is installed on top of 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. It is becoming possible to do this. 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 uses the other part to form a real image on the diaphragm 52. are tied together. Diaphragm 52 transmits only light from the area on plate 20 that corresponds to crosshair 43 to photosensitive transducer 50 . Transducer 50 is electrically connected to amplifier circuit 54 and 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 a printed circuit or an AD converter may be connected to automatically record the intensity of light according to a command signal. In FIG. 2, various modifications are possible, such as direct illumination instead of dark field illumination, or illumination from above so that the reflected light from the zone can be directly measured. Examples of methods and apparatus for performing fully automatic measurements are described below.

FIG. 3 illustrates certain features of the sedimentation zone that are defined for locating the zone in accordance with the present invention when the zone appears. y=
The horizontal line 61 in yAb is the edge of the antibody groove on the immunoelectrophoresis slide. Coordinates Xe, Ye and Xf, Yf
Points E and F at are the left and right end points of the spreading 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 coordinates Xg and Yg is an example of such a point.

Since the zone has a certain width in the y direction, G
The y-coordinate of each intermediate point is the leading edge 63 closest to the antibody groove, the trailing edge 65 of the zone, or one or more points within the zone, including the part of the zone with the highest light intensity. It is convenient to specify 64.

As the incubation time progresses, the absolute and relative positions of points E, F and G change as zones emerge. Time of first appearance of sedimentation zone T ₀
is usually difficult to determine by direct observation, and this parameter T ₀ can be derived based on various preliminary parameters expressed as a function of time. According to the preliminary parameters described below, the parameters can be determined with high reliability and reproducibility.
T ₀ can be found. As a function of time,
The values of Xe and Xf illustrated in Figure 4 are correlated with protein concentration, and the point 66 where the extended lines intersect in Figure
represents the point at which the zone first appears.

One of the important preliminary parameters for deriving the parameter related to the first appearance time of the sedimentation zone used in the present invention is the coordinate difference (Xf - Xe), which is the maximum L of the sedimentation zone at a specific measurement time. be. As the data represented in FIG. 4, FIG. 5 shows the variation of this parameter with respect to incubation time.

In FIGS. 4 and 5, the solid lines plot values obtained from actual experiments. These figures also show values obtained by extending (extrapolating) the solid line in the direction of less time. This extension is shown as a dashed line. Point 6 where the extension lines intersect in Figure 4
6 represents the point where the length of the zone was 0. The extension (extrapolation) line in FIG. 5 intersects the time axis at point 67, and T ₀ can be similarly determined. Such an extension (extrapolation) allows the time of first appearance of the sedimentation zone to be determined with practical precision. T ₀
This method for determining is advantageous because it does not require constant continuous observation of the plate.

Another preliminary parameter for deriving the first appearance time T ₀ is the sum of light intensities within the zone. Protein concentration cannot be practically measured by simply reading light intensity. This is because the shape of the zone and the rate of zone formation change, and as the zone becomes larger, the light intensity changes.

On the other hand, changes in these factors are largely compensated for by taking a series of light intensity readings at several appropriately chosen locations and processing them holistically to determine the light intensity parameters. be able to. The method is to measure the light intensity at equal intervals along a straight line across the sedimentation zone, usually in the y direction at a specific value of x. In such a series of readings, it is desirable to determine light intensity values at several locations on either side of the zone. These offset values are averaged to determine the background intensity, which is used to subtract the background value from the light intensity values within the zone. As a result, the modified light intensity values are aggregated and
The linear integral of the light intensity along the line across the zone can be determined at the value of .
Such a linear light intensity sum, Ix, increases with increasing incubation time in a regular and reproducible manner, and its value varies with the concentration of reacting proteins 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 light intensity observed by this cross-zone scanning is shown in FIG. The two curves were floated with the semi-automatic apparatus described below and scanned in the y direction at the point Xg where the sedimentation zone was closest to the antibody groove. In FIG. 9, peaks 47 and 77 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 peaks at 75 and 76 are due to the respective edges of the antibody groove and provide convenient controls for measuring the distance D from that groove to a particular point in the zone. . The parameter Ix defined above essentially corresponds to the area under the peak 74 or 77 in FIG. 9, for example.

The two peaks 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 curve 70) and 4 hours (dashed curve 72). It can be seen that between these two sets of measurements, the zone positions are very stable, but the area under each peak increases significantly. peak 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 their discrepancy.

Figure 10 plots plates made with various concentrations of protein scanned in the y direction at the same incubation time. As the protein concentration increases from A to C according to the graph,
It can be clearly seen that the area of each peak increases and the entire zone moves towards the antibody groove.

Since the parameter Ix is extremely useful for determining protein concentration, the method of determining the linear light intensity sum for several values of x, summing or averaging them, and determining the result using a large number of light intensity parameters yields the following results: can be further improved, and a typical method thereof is to obtain the sum of linear light intensities at Xg and several points selected at appropriate intervals on both sides thereof.
By averaging or totaling the sum of linear light intensities 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 light intensity sums included in the light intensity sum calculation etc. increases each time the plate is scanned. An example of a method for this purpose is to determine the sum of linear light intensities at Xg, and then continue measurement until the value of the sum of linear light intensities on both sides of Xg becomes equal to or less than a certain threshold value. The sum of all linear light intensity sums is the parameter Iz (total light intensity sum), which is essentially the integral of the light intensity as it scans the settling zone. This approximation can be improved as desired by scaling the changes in x and y with each measurement to the extent that the resolution of the instrument allows. The width of Lz varies particularly strongly as a function of protein concentration over a wide range of experimental conditions. The reason is that as the concentration increases, x, y
This is because both dimensions increase and the average light intensity of the zone also tends to increase. Because of this strong dependence on protein concentration, the parameter Iz, which is the sum of linear light intensities, is particularly effective as a criterion for concentration measurement.

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. To obtain such a combination of standard values, a standard procedure is carried out using such standard protein solutions, and measurements are taken successively 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 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 such standard operations several times for each concentration. The standard values of the parameters thus measured are:
Therefore, it is considered to be a function of both concentration and time. When considering each linear light intensity sum Ix separately, details of the value of x are required in order to measure all of them.

Using the first appearance time of the zone as a parameter has the advantage that the standard value T ₀ does not include time as a variable. That is, by the method described so far
To determine T ₀ , measurements must be taken several times at a specific time. However, once T ₀ is determined by these measurements,
Each measurement value becomes unnecessary. Thus, T ₀
The value of 1 is plotted as a function of protein concentration and
One standard curve can be obtained. Such a curve is illustrated in FIG. This was created by determining values for each concentration using the extrapolation method described in connection with FIGS. 4 and 5. According to the curve in Figure 6, T ₀ of the unknown protein
Once you know this, you can directly read the concentration value.

For example, for preliminary parameters such as L, Ix, Iz, building the 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 value is therefore related to the time of measurement so that the final standard value of each protein concentration is plotted as a function of time on a separate curve.

FIGS. 7 and 11 are representative examples of such groups, and the standard values of each parameter for three concentrations are plotted against the time axis.
Extending these curves in the direction of L=0 and Lz=0, respectively, gives the standard value of T ₀ , from which T ₀ in FIG. 6 can be plotted, or
Experimental values of T ₀ can be given for comparison with the figure. In Figures 7 and 11, arbitrary time
Vertical lines are drawn at t ₁ , t ₂ and t ₃ . Each of these L, Iz values can be replotted as a separate curve as a function of concentration. The resulting set of curves is, for each time,
L and Iz are shown as a function of protein concentration.
This combination is shown in FIGS. 8 and 12, respectively.

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, the value of P for the original antigen sample and a portion to which a known amount of protein was added is calculated for each point h, i, j, and k as if the solution contained only the added protein. It is plotted as follows. Therefore, the value h for the original sample is plotted at the zero concentration 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→i', j→j' and k
→Gives concentration according to the length of the straight line k', and logically all of these lengths are 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 calculate values. In the latter case, the protein above curve 81 is obtained by connecting the obtained points i, j and k, and further extended (extrapolated) towards the P axis.
and determine P at h. In this way, curve 8
Curve 80 serves as a guide in extending 1. For example, the curve 80 in FIG. 13 has points i,
It has been moved as a whole to the left until it now represents j and k. The intersection of the curve 80 thus moved and the P axis gives the value of point h, from which the density Ch can be determined. Furthermore, by extending the curve 81 to the negative side of the horizontal axis -Ch, it is possible to directly read the density that matches the density value Ch.

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. By increasing the number of samples supplemented with various amounts of protein, and by making measurements at several locations rather than just the three points shown in Figure 13, the reliability of this extrapolation can be reduced to a minimum. It can be improved without limit and a correct knowledge of the measurement errors involved can be obtained.

The present invention can also automatically detect certain protein abnormalities in antigen samples. For example, between normal and abnormal gamma globulin, there is usually
have small differences in the range of immunodiffusion and electrophoretic mobility, yet react with the same antibodies, forming unusual precipitation zones and generally forming precipitates that are asymmetrically distributed along the length of the zone. . Such an abnormality can be detected by comparing the measured value of the linear light intensity sum Ix with different values of X, for example. 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. The abnormal distribution of light intensity along the zone, as described above, due to the presence of abnormal gamma globulins, for example, causes the zone to appear faster than in the normal case, resulting in higher calculated concentrations;
On the other hand, the total light intensity sum Iz tends to show approximately equal concentration values for normal and abnormal proteins. A significant discrepancy between the concentration values obtained by the zone's first appearance time T ₀ and Iz thus indicates the presence of an abnormal protein.

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, such as for example the light intensity or position parameters mentioned above.

The derivative of the two parameters is also advantageous as a parameter, one increasing and one decreasing with increasing protein concentration. Thus, by using the quotient or difference of these parameters, it is possible to obtain parameters that have a sharper dependence on concentration than by using them individually.

The first appearance time T ₀ of the zone is an example of having an inverse relationship to the protein concentration, and can be used especially when calculating such a quotient. The distance between the antibody groove and the zone at any desired value Xg on the x-axis also has an inverse relationship 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 is not inversely dependent on concentration but linearly dependent on it. .

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 guidance of parameters can be performed by direct observation or manual methods, but as a feature of the present invention, these operations can be particularly 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. 2
The method is to obtain a video signal that represents the apparent light intensity of a combination of dimensions. 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 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. 14 illustrates an example of such a device, in which a television camera 90 focuses an image of the plate 20 through a lens 91 on 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, the plate can be moved on an illuminated support such as the light source box 30 in FIG. 2, or conventional electronic bias control using a camera circuit can be used. You may do so.

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 such fine steps 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 has a cursor with an electronically generated bright spot indicating the location on the screen from which the video signal is extracted from the area element on each scan. There is a manual control switch for the operator, which allows him to move the cursor freely and 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. 14, the counter 102 counts the pulses from the clock 103 and
A digital signal continuously representing the x coordinate is given to 04. The circuit 106 generates an analog step voltage on the line 107 for each minimum unit of the x coordinate in response to the signal. A dividing circuit 108 scans and counts the beam in the x-axis direction and provides a branch line 109 with a digital signal representing the y-coordinate for each sweep. In response to this count, the circuit 110 sends an analog step voltage to the line 111 for each minimum unit of the y-coordinate. Step voltages on lines 107 and 111 control the scanning of camera 90 and CRT 92 in the x and y directions and keep them synchronized. camera 90
The video signal from the monitor CRT 92 is sent via line 112 to the monitor CRT 92, which reproduces the changes in the light intensity of the slide 20 on the screen.

The selection circuit 118 typically consists of an X and Y counter that counts up or down by counting one or more pulses when the control stick 119 is manually moved. The resulting digital signal represents the x and y coordinates of the location being measured and is stored in register 117. Comparison circuit 114 compares the coordinates of a specific measurement target position from register 117 and lines 104 and 10.
Constantly compare the x and y coordinates of the scanning beam from 9 onwards. When the scan spot comes to a stored location (address), an activation signal is sent from comparator circuit 114 to switching circuit 120, typically once for each scan. Thus, the register 125 receives a digital signal corresponding to the light intensity when scanning the specified measurement point on the slide on the line 123. The actuation signal from circuit 114 is also sent to a cursor control circuit 126 which superimposes light intensity modulation pulses on the video signal to identify selected points on the monitor screen, as shown at 127.

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

The computer 100 individually identifies the measurement points;
It also accepts input information for spots on the plate 20 as required by any program. In terms of circuit, such a measurement method uses the selection circuit 11.
8. Similar to the register 117 and the comparison circuit 114, the selection circuit 118 operates according to electrical signals that selectively indicate the direction of movement of the measurement point or indicate the digital address of the required position. Rather than constructing two such devices, as shown in FIG. Controlled by computer 100. In order to perform such control by the computer 100, as described above, the computer 100 has built-in programs for measuring the position and light intensity and for guiding general parameters.

In order to scan the zone automatically, the plate should be suitably illuminated and moved from the incubation chamber to a precisely defined scanning position. Particularly when the optical scanning device is lightweight and compact, such as a charge-coupled device, it is recommended to mount the scanning device on a platform that can move in two dimensions, such as on a side-by-side array of immunodiffusion or immunoelectrophoresis plates. is desirable. 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 controlled by digital signals is mounted at a distance from 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. The pipette-mounted platform of the scanning device can be moved over the plate by computer control and stopped when the scanning axis is properly 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 antigen holes and antibody grooves, and remembers 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 scanning process of the first zone consists of video signals from addresses that are successively measured by measuring x values and y values moving at regular intervals over the area where the zone is expected to form. It is a step controlled by a computer to obtain the
The video light intensity values received from register 125 are stored as x, y, and t data for each measurement point. After each such y-scan, x
The coordinates are moved a fixed distance, a similar y scan is performed, the results are recorded, and this is repeated 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 a series of y-scans for equally spaced x values are determined and stored.
The axis of the zone is determined from the value of . The end point of each zone is recognized by the extreme value of x at which the highest point (peak) of light intensity is observed in each direction of x. If more precise position measurement is required, in a certain area near the end point,
It is programmed to scan with even finer x and y intervals.

We perform direct mathematical processing on the data recorded for the actual sedimentation zone located and recorded in this way, and for each scan across the zone in the y direction, the parameter is the linear light intensity sum as described above. Get Ix. Then, the sum of these values becomes the total light intensity sum parameter Iz for that zone. Parameter L is obtained by subtracting it from the x value of the zone end. 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 such a set of plates, the computer derives the concentration of each protein for which a sedimentation zone was 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 computers. Several independent measurements can be made from the concentration of each protein in the sample. 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. Also, photographs of the plates undergoing 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. After an additional period of incubation, ranging from a few minutes to an hour or more, the above scanning is repeated, and the measurement plate and its corresponding standard plate are scanned. Computers typically search for new zones that appear where no zones were present in previous scans, or scan areas where subsidence zones have been previously found and recorded, and determine the extent of zone movement or 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 the normally formed precipitate intersects at the end. 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. I do.

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 programmed to check for actual intersections as part of each regular scan as well. For example, by comparing the x,y coordinates of the measured points of the axes of each zone with those of adjacent zones, a match of coordinates at a certain threshold indicating an intersection is determined. All scans also include a check for possible intersections. For example, the computer extends all endpoints beyond the actual value,
Compare with extended endpoints of neighboring zones. A simple method for extending the axis in this manner is to linearly extend the axis in the direction of the axis' gradient 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 intersection spans half of the zone, a sufficiently accurate correction is made by replacing the light intensity values observed within the intersection area with the light intensity values observed in the other half that is not intersected. be able to. The correspondence between two points in the zone by such immunoelectrophoresis is determined by centering on the point Xg closest to the antibody groove, and at intervals of equal values of x on both sides, and by performing direct immunodiffusion. For zones, it 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 may 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 light intensity values actually measured in the intersecting areas are partly due to one zone and partly due to the other zone. This is a method of correcting it by putting it in. The computer is programmed to divide the light intensity value at the intersection area into appropriate values.
In order to determine a suitable division ratio, the respective values of the intersection 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 for each of the above-mentioned types of intersections to use preliminary parameters obtained by parts of the zone that are 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, measurements of the sedimentation zone at any stage of incubation can be made with a specific light. Since this method does not require precise time measurements, it is usually convenient after the reaction has reached equilibrium.

In summary, in order to obtain true quantitative measurements of the concentration of individual proteins in physiological fluids, immunodiffusion techniques,
Methods and apparatus for carrying out measurements using immunoelectrophoresis and analog means have been described. In practice, 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 the parameter Iz and time. FIG. 12 is a graph showing an example of the relationship between the parameter Iz and the protein concentration parameter dIz/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, 2
4...Antibody groove, 90, 91, 92...Optical means, 94...Control means.

Claims (1)

[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. allowing the protein and antibody to react and diffuse into each other through a region of the support medium initially free of any of these reactants to form at least one elongated sedimentation 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 first appearance time of said sedimentation zone representing a characteristic change in said protein concentration;
In order to obtain an index for quantitatively measuring the concentration of the protein in the antigen sample, a control sedimentation zone generated by equivalent immunodiffusion of multiple control antigen solutions, each containing a known concentration of the protein, was derived. 1. A method for quantitative protein analysis, comprising comparing a reference parameter value with the above parameter value. 2 the step of scanning the sedimentation zone is repeated at a plurality of specified times according to the appearance of the sedimentation zone;
the step of deriving the value of the parameter from the plurality of electrical signals for each scan includes a second parameter that varies characteristically as a function of time and takes a known value at the time of first appearance of the sedimentation zone; Quantification of the protein according to claim 1, which comprises deriving the value of the parameter and further extrapolating the time for the value of the second parameter to restore to the known value to derive the first appearance time. Analysis method. 3. The protein quantitative analysis method according to claim 2, wherein the second parameter is a distance between a plurality of ends of the sedimentation zone. 4. The protein quantitative analysis method according to claim 2, wherein the second parameter is the sum of light intensities within the zone. 5. The protein quantitative analysis method 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. 6. said step of scanning a sedimentation zone is repeated at a plurality of times of occurrence of said sedimentation zone, said plurality of electrical signals providing measurements of light intensity at a number of said positions, each said position corresponding to each measurement; and the x-coordinate value and y-coordinate value at that point in time.The quantitative protein analysis method according to claim 1. 7. 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 A method for quantitative protein analysis according to claim 1, comprising deriving a third signal representing a differential combination of signals as a combined parameter signal. 8 the combined parameter signal is
8. The quantitative protein analysis method according to claim 7, wherein the method represents the ratio between the signal of the second signal and the second signal. 9, 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. , generating an electrical signal at a second location on the other side of the first zone and in a non-overlapping region that overlaps the other sedimentation zone, and further generating the electrical signal at the first location. 2. The protein quantitative analysis method according to claim 1, which comprises using the second position signal to derive the protein. 10 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, parameter value derivation step and 2. The method for quantitative protein analysis according to claim 1, wherein the step of comparing the control parameter value and the determined parameter value is performed for a sedimentation zone formed in each sample portion. 11 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 step of scanning, a step of deriving parameter values, and a step of effectively plotting the obtained parameter values for each sample portion against the added protein concentration, as well as plotting the obtained parameter values for each sample portion against the added protein concentration. 2. The protein quantitative analysis method according to claim 1, wherein the method is performed in the step of determining by extrapolation of a curve obtained by the method. 12. The method for quantitative protein analysis according to claim 1, further comprising the step of staining the sedimentation zone with a dye prior to the step of scanning the sedimentation zone. 13 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; 2. The method for quantitative protein analysis according to claim 1, comprising the step of comparing the value of the first parameter and the value of the second parameter to detect the presence of the abnormality. 14. Claim 13, wherein said second parameter comprises a sum of light intensities within a zone, and said first and second parameter values are compared by respective protein concentrations derived from these values. Quantitative analysis method for the described protein. 15 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 this, 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 free of any of these reactants. reacting and diffusing through a region of the support medium to form at least one elongated settling zone of finite length; scanning said settling zone by optical means so that a portion of said settling zone is within said settling zone; generating a plurality of electrical signals in response to a precipitated concentration at each position of a two-dimensional array, with a portion distributed within the sedimentation zone, and a portion of which is distributed outside the sedimentation zone; In order to electronically derive parameter values related to the first appearance time of the sedimentation zone from the plurality of electrical signals, and to obtain an index for quantitatively measuring the concentration of the protein in the antigen sample, each of the proteins has a known concentration. A method for quantitative protein analysis, 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 containing. 16. The step of scanning the sedimentation zone is repeated at a plurality of specified times according to the appearance of the sedimentation zone, and the step of deriving the value of the parameter from the plurality of electrical signals for each of the scanning, deriving the value of a second parameter that changes characteristically as a function of time and takes a known value at the time of first appearance of said sedimentation zone, and further the time at which the value of said second parameter restores to said known value; 16. The method for quantitative protein analysis according to claim 15, which comprises extrapolating the time to derive the first appearance time. 17. The protein quantitative analysis method according to claim 16, wherein the second parameter is a distance between a plurality of ends of the sedimentation zone. 18. The protein quantitative analysis method according to claim 16, wherein the second parameter is the sum of light intensities within the zone. 19. The protein quantitative analysis method according to claim 15, comprising generating an image representing the sedimentation zone on a screen surface of a cathode ray tube in response to the plurality of electrical signals. 20 said step of scanning a sedimentation zone is repeated at a plurality of times of occurrence of said sedimentation zone, said plurality of electrical signals providing measurements of light intensity at a number of said locations, each said location corresponding to each measurement; and the x-coordinate value and y-coordinate value at that point in time.The quantitative protein analysis method according to claim 1. 21 said step of deriving a parametric signal derives a first signal that increases with increasing protein concentration; a second signal that decreases with increasing protein concentration;
deriving a signal of the first and second
16. The method for quantitative protein analysis according to claim 15, comprising deriving a third signal representing a differential combination of signals as a combined parameter signal. 22. The method for quantitative protein analysis according to claim 21, wherein the combined parameter signal represents a ratio between the first signal and the second signal. 23, 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. , generating an electrical signal at a second location on the other side of the first zone and in a non-overlapping region that overlaps the other sedimentation zone, and further generating the electrical signal at the first location. 16. The protein quantitative analysis method according to claim 15, which comprises using the second position signal to derive . 24. Each portion of said antigen sample is enriched by adding different known amounts of said protein prior to said immunodiffusion of each sample portion, and said scanning step, parameter value derivation step and 16. The protein quantitative analysis method according to claim 15, wherein the step of comparing the control parameter value and the determined parameter value is performed for a sedimentation zone formed for each sample portion. 25 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 step of scanning, a step of deriving parameter values, and a step of effectively plotting the obtained parameter values for each sample portion against the added protein concentration, as well as plotting the obtained parameter values for each sample portion against the added protein concentration. 16. The quantitative protein analysis method according to claim 15, wherein the method is performed in the step of determining by extrapolation of a curve obtained by applying a curve. 26. The method for quantitative protein analysis according to claim 15, further comprising the step of staining the sedimentation zone with a dye prior to the step of scanning the sedimentation zone. 27 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; 16. The protein quantitative analysis method according to claim 15, comprising the step of comparing the value of the first parameter and the value of the second parameter to detect the presence of the abnormality. 28. Claim 27, wherein said second parameter consists of a sum of light intensities within a zone, and said first and second parameter values are compared by respective protein concentrations derived from these values. Quantitative analysis method for the described protein.