JPS6130215B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a protein detection device. More specifically, the present invention relates to a protein detection device that performs an antigen-antibody reaction on the gold surface of the substrate.

This patent application is related to Giaever, US Patent Application No. 266,278, filed on June 26, 1972, and assigned to the same trustee as the present invention.

By the way, an immune reaction is an extremely specific biochemical reaction. According to this method, a first protein called an antigen binds to a second protein called an antibody specific to the antigen to produce an immunologically complex protein. The immune response that occurs within a living system such as an animal is important for that animal in its fight against disease. When foreign proteins or antigens invade, biological systems produce antibody proteins specific for that antigen, by a process that is currently not fully understood. Since such antibody protein molecules have chemical bonding sites that complement corresponding sites on the antigen molecule, the antigen and antibody chemically bond to produce an immunologically complex protein.

Since antibodies are produced by biological systems in response to the invasion of foreign proteins, detection of antibodies present in a biological system is useful for medical diagnosis in that it determines what antigen the biological system has been exposed to. have a higher value. Conversely, the detection of specific antigens within biological systems also has medical diagnostic value. Examples include detecting HCG protein molecules in urine as a pregnancy test, as well as detecting hepatitis-associated antigen molecules in the blood of a prospective donor.

In order to perform such diagnostic tests, at least one of a pair of immunologically reactive proteins must be obtained in a concentrated and purified state. by the way,
The only known source of antibody proteins is biological systems. More specifically, to date, only vertebrates are known to exhibit an immune response to the introduction of foreign proteins. For example, large numbers of corresponding antibodies are found in the serum of animals exposed to multiple antigens. However, serum is an extremely complex mixture, and the required antibodies are only present in extremely low concentrations among numerous other species components. On the other hand, many antigens can be controllably produced in laboratory cultures. However, at present, certain antigens (eg, hepatitis-related antigens), like antibodies, can only be obtained from the biological systems of higher animals.

Currently, both the concentration and purification of immunologically active proteins and their diagnostic uses rely on the precipitation or aggregation properties of proteins based on immunological complex reactions. A classic example of such diagnostic use is blood group determination. In that case, a blood sample is mixed with alpha and beta serum antibodies, and the blood type is then determined by observing the presence or absence of agglutination in the blood sample.

The HCG protein pregnancy test currently being conducted is an inhibition test. When conducting such a test, a certain amount of HCG antiserum is mixed into a urine sample. In the urine sample pretreated in this way,
A number of polystyrene spheres coated with HCG protein are introduced. Such polystyrene spheres should aggregate if and only if HCG protein is not present in the urine sample. That is, when HCG protein is not present in the urine sample, the HCG protein on the polystyrene spheres is complexed with the HCG antiserum introduced into the urine sample in advance, so that the polystyrene spheres aggregate. On the other hand, if HCG protein is present in the urine sample, it will complex with the previously introduced HCG antiserum, and the complex thus generated will precipitate from the urine sample. As a result, the polystyrene spheres do not aggregate, since the previously introduced HCG antiserum can no longer be complexed with the HCG protein on the polystyrene spheres. According to this idea, it should be possible to simplify the pregnancy test using the HCG protein described above. That is, HCG antiserum could be deposited on polystyrene spheres and then used to test urine specimens directly. Then, the polystyrene spheres would aggregate if and only if HCG protein was present in the urine sample.

However, such a simple method has not been used so far. The reason seems to be that the available HCG antiserum is a complex mixture containing a large amount of components other than HCG antibodies. On top of that,
The fact that extracting antibodies from HCG antisera requires extra effort is also one of the reasons why inhibition tests using HCG antisera directly have traditionally been preferred. Therefore, in the reference examples described below in connection with the present invention, a method for efficiently separating HCG antibodies from serum is described. According to the embodiments of the present invention, a detection device is provided that enables simpler direct testing.
Typically, each antigen molecule is complexed with multiple antibody molecules. The antibody molecules may be associated with different particles, thus producing visible aggregates of coated polystyrene spheres or red blood cells, for example. However, agglutination tests have the disadvantage that the particles in question tend to agglutinate for various reasons unrelated to the immune response, thereby reducing the reliability of the test. Therefore, although agglutination tests are typically performed with great care by skilled technicians, diagnostic errors are still inevitable.

According to current methods for preparing antibody production concentrates, antibody production is stimulated by introducing antigen into the animal's biological system, and serum containing antibodies in a highly diluted state is produced. is taken from the animal and then a certain amount of antigen is mixed into the serum. Such antigen and antibody mixtures complex and precipitate from the serum. After aspiration of residual serum components, the antigen-antibody precipitate is dissolved in acid, thereby cleaving the complex bonds. This results in a solution of antigen and antibody molecules in acid. Because antigen and antibody molecules have different physical properties (eg, weight), it is possible to separate them by mechanical means, such as centrifugation.

By the way, it is known that the antigen-antibody complex reaction also occurs when the antigen is adsorbed on the surface.
Complex reactions on such surfaces have been observed using an ellipsometer. An ellipsometer is a complex optical instrument that can measure film thicknesses on the order of 0.1 Å. However, ellipsometers are expensive and require skilled operators. In traditional studies of immune responses using ellipsometers, two methods have been used. According to one method, after the immune reaction to be studied is performed on a slide, the slide is mounted in an ellipsometer and the film thickness is measured. According to another method, the immunoreaction is performed on a slide mounted on an ellipsometer and changes in film thickness are observed. In this case, extreme care must be taken in measuring the absolute film thickness. On the other hand, measuring relative changes in film thickness is easier than measuring absolute film thickness, but takes a long time when the antibody concentration in solution is low. Therefore, for economic reasons, ellipsometric detection of immune reactions at surfaces has not been adopted for diagnostic purposes.

According to the present invention, any protein is adsorbed onto a substrate in the form of a single molecular layer, while a bimolecular layer is formed on the substrate when an antibody (or antigen) specific to such protein binds to it. It is based on the discovery that Therefore, an object of the present invention is to provide a protein detection device useful for medical diagnosis and pharmaceutical use by applying such a discovery.

It is therefore an object of the present invention to provide a device for economically detecting the immune reaction occurring on the surface of a substrate.

It is also an object of the present invention to provide the above-described apparatus for detecting such an immune reaction by direct visual observation.

To briefly describe a reference example related to the present invention, first, a thin plate made of a substrate material is immersed in a solution of a first protein, so that a monolayer of the first protein is attached to the substrate. The substrate thus coated with the first protein is then dipped into the second solution. In the first example, the second solution is known to contain a protein that specifically reacts with the first protein, and in the second example, it is known to contain a protein that specifically reacts with the first protein. It is suspected that it contains a protein that reacts with other substances. Such a specifically reactive protein, and only that protein, forms a second monolayer superimposed on the monolayer of the first protein on the substrate. Next, according to the first example, the bilayer coated substrate is immersed in a weak acid solution. As a result of the weak acid solution cleaving the immunological bond between the two protein layers, the protein that specifically reacts with the weak acid solution is recovered in a purified state. On the other hand, according to the second example, the substrate after being immersed in a solution suspected of containing a specifically reactive protein is electrically or optically inspected, thereby determining whether it is attached to the substrate or not. It is determined whether the protein layer formed is a bilayer or a monolayer. As a result, the second
It will be determined whether the sample contains a protein that specifically reacts with the first protein.

Embodiments of the invention will now be described with reference to reference examples and in conjunction with the accompanying drawings.

First, reference examples related to the present invention will be explained. FIG. 1 is a reference process flow diagram illustrating the various steps of this reference example. When reading such a process diagram from top to bottom, each vertical level represents one of the sequential work steps in time. The reason why a plurality of processes are arranged horizontally at a predetermined level in the vertical direction in a process flow diagram is that when following various reference examples, any one of the processes described at a predetermined vertical level is selectively performed. It means to do something.

According to the first reference, work begins at block 13 in FIG. First, a thin sheet of substrate material such as metal, glass, mica, plastic, fused silica, quartz, etc. is provided. Such a substrate is preferably made of a metal since it has the largest difference in refractive index for proteins, and is particularly preferably made of a glass slide coated with a metal. Next, the substrate is immersed in a solution containing the first protein of interest. Biologically speaking, such a first protein is an antigen or an antibody, which is available as a highly concentrated solution, and is also a protein that specifically reacts with it (biologically speaking, it is an antigen or an antibody, respectively). ) should be used to detect or purify. Such first
The proteins form a single molecular layer and are adsorbed onto the substrate.
Note that any protein is adsorbed in one molecular layer, but no further adsorption occurs. In other words, the protein attaches to the substrate, but not to itself. After a monolayer of protein has been formed over the entire surface of the substrate, the coated substrate is removed from the first protein solution. Note that the time required to completely coat the substrate depends on the concentration of protein in the solution and the degree of stirring of the solution. For example, when using a 1% bovine serum albumin solution, it takes approximately 30 minutes to completely coat a glass slide with a single molecular layer of protein. The next step, shown in block 14 in FIG. 1, is to immerse the coated substrate in a solution suspected of containing a protein that specifically reacts with the first protein. Not only can this solution contain many components other than the specifically reacting protein to be detected, but such a case is even more typical. However, proteins other than those that specifically react will never adhere to the first protein layer on the substrate. Therefore, if a specifically reactive protein is not present in the solution in question, the substrate after immersion will still have only a monolayer of protein. On the other hand, if a specifically reactive protein is present in the solution in question,
An immunological complex occurs between the first protein and the protein that specifically reacts with it, so that after a certain period of time the substrate will have a bilayer of protein.
It should be noted that the steps shown in blocks 13 and 14 of FIG. 1 are common to all of the reference examples. The time required for complete deposition of the second protein layer on the coated substrate also depends on the concentration of the specifically reactive protein in the solution. For antibodies in serum, this time can extend over a day. According to one reference example, the next step is to immerse the coated substrate in a weak acid solution as shown in block 15 of FIG. Observation of the coated substrate is performed using a polarimeter. Although the formation of a specifically reactive protein layer on a substrate coated with the first protein may take a long time, the immunological bond between the two proteins is cleaved very rapidly by weak acids. . Therefore, the observation performed using an ellipsometer may be a relatively simple observation of changes in film thickness, rather than a complicated measurement of absolute film thickness. Moreover, the observation of the detachment of specifically reactive proteins by the action of weak acids can be achieved much more quickly than the conventional observation of the formation of a specifically reactive protein layer. Therefore, a number of test slides are prepared according to block 13, each of which is exposed to one of a number of serum samples as shown in block 14, and then such slides are sequentially prepared according to blocks 15 and 22. It is thereby possible to determine in a short time which serum samples contain a protein that specifically reacts with the first protein. Because a weak acid does not strip the first protein from the substrate, a coated substrate immersed in a solution that does not contain a specifically reactive protein will have a similar film thickness when immersed in a weak acid and observed with an ellipsometer. Shows no change. On the other hand, a film immersed in a solution containing a specifically reactive protein changes in film thickness by about 2 to 5 times when immersed in a weak acid and observed with an ellipsometer. Each observation can be accomplished in a few minutes, resulting in an efficient and therefore diagnostically meaningful test method.

Another closely related example is useful for protein concentration and purification and consists of the steps shown in blocks 13, 14, 15 and 23 in FIG. First, as shown in block 13, a substrate is immersed in a solution of an available protein of a pair of antigens and antibodies. In the normal case, this protein is an antigen, but this does not depend on the biological nature of the first protein. The coated substrate made according to block 13 in FIG.
The first protein is immersed in a solution containing a protein that specifically reacts with the first protein as shown in FIG. In normal cases, the protein that specifically reacts is an antibody,
The solution used in block 14 is the serum of the animal exposed to the first protein. When the substrate thus coated with a bimolecular layer of protein is immersed in a weak acid as shown in block 15 in Figure 1, the specifically reactive protein layer is peeled off from the first protein layer. . At this point, the substrate is
A purified solution of the protein, which is coated with a monomolecular layer of the protein, reacts specifically in the weak acid, has been obtained. The next step is block 23 in Figure 1.
As shown in FIG. The goal is to increase the concentration of specifically reactive proteins in the protein. If such operations are continued, purified concentrates of specifically reactive proteins will be recovered in weak acids.

Although the first reference example above improves the efficiency of detecting immune reactions with an ellipsometer to a practical extent, it is still not economically viable to completely eliminate the need for an ellipsometer. It can be said that it is desirable. Therefore, there is another reference example for measuring the relative thickness of a protein layer by electrical means, in which a metal substrate is used or block 11 in FIG. As shown in Figure 1, a substrate made of another material is first coated with a thin metal film. Such a metal substrate or metal coated substrate is
It is then immersed in a protein solution according to the steps shown in blocks 13 and 14. The protein layer thus attached to the substrate has electrical insulation properties. The next step is to place a mercury drop or other electrode on top of the top protein layer attached to the substrate, as shown in block 16 in FIG. The upper protein layer consists of the first protein if the specifically reactive protein is not contained in the solution in question, while it consists of the first protein if the specifically reactive protein is contained in the solution in question. Consists of. The metal substrate or thin metal film and the mercury droplets thus constitute two electrode plates of a capacitor separated by an insulating layer consisting of a monolayer or bilayer of protein. Therefore, as shown in block 19 in FIG.
Impedance bridge (Heathkit)
The capacitance of the capacitor is measured by any suitable capacitance meter, such as an Impedance Bridge. As expected, the capacitance of the capacitor having a bimolecular layer of protein was found to be about 2 to 5 times lower than that of the capacitor having a monomolecular layer of protein.

Another reference example closely related to this is one that further simplifies the detection of specifically reactive proteins by making it possible to confirm the presence of specifically reactive proteins by visual observation. In this reference example, a mercury- and amalgam-forming metal substrate is used, or another material (preferably gold) coated with a thin film of a mercury- and amalgam-forming metal (preferably gold) according to the steps in block 11. For example, a substrate made of glass is used. Then, after subjecting the substrate to the steps of blocks 13 and 14, a drop of mercury is placed on top of the upper protein layer, also as shown in block 16. However, no electrical measurements are performed in this reference example. That is, the next step in this reference example is to visually observe the coated substrate and thereby measure the time that elapses until a visible amalgam is formed between the thin metal film on the substrate and the mercury. It is. This reference example is based on the discovery that mercury takes about 1 minute to diffuse through a single molecular layer of protein, but more than 10 minutes to diffuse through a bilayer of protein. There is. Since mercury forms a thin metal film and a visible amalgam on the substrate after diffusing through the protein layer, the time from placing a mercury droplet on the top protein layer to the appearance of a visible amalgam is represents whether a bilayer or a monolayer of protein is present on the substrate, and thus represents whether the solution in question actually contains a protein that reacts specifically with the first protein. As will now be understood by those skilled in the art, capacitance measurements in the last reference example above must be made within one minute of placing the mercury drop on top of the top protein layer. Otherwise, mercury would diffuse through the insulating protein layer and short circuit the capacitor. Otherwise, such shorting can be prevented by coating the metal with an insulating layer of metal oxide and then depositing the protein on the metal oxide. It should be noted that if the above-mentioned reference example is followed, it will also be recognized that it is possible to roughly quantitatively measure the concentration of a specifically reactive protein in the solution in question. To do this, after producing a large number of substrates in the process of block 13,
4, they may be immersed simultaneously in the solution in question, while being removed sequentially from the solution in question over time. Because the rate at which a specifically reactive protein layer forms depends on the concentration of specifically reactive protein in the solution, mercury droplets are Comparing the diffusion times through the layers provides a rough quantitative measure of the concentration of the specifically reactive protein in the solution in question.

In a related embodiment, the substrate is coated with a thin metal film according to block 11 in FIG. 1, as in the last embodiment described above.
The substrate is then dipped into the solution as shown in blocks 13 and 14. The next step in this reference example is to coat the upper protein layer with a second metal layer as shown in block 17 in FIG.
The structure thus obtained is then observed by reflected light as shown in block 21 in FIG. Note that the second metal layer is preferably installed by electroplating. Essentially, this reference example provides a structure that acts as a diffraction grating, and the difference between a bimolecular layer and a monomolecular layer of a protein can be determined from the spectral components observed in the reflected light.

A device based on this reference example is shown in FIG. First, a first protein layer 82 is deposited on the metal surface 81 of a metal substrate or a non-metallic substrate coated with a metal thin film according to block 13 in FIG. The substrate coated with the protein layer 82 is then immersed in the solution in question, resulting in a first protein layer 82.
A second protein layer 83 is formed if a protein that specifically reacts with 2 is present in the solution in question. A second metal layer is disposed on the exposed surface of such protein layer. If a very small amount of metal is used then the second metal layer will consist of a large number of discrete metallized regions (eg 84 and 85). A beam of light, represented by ray 86, is applied onto the substrate thus obtained. Such light is reflected at the metal interface. The distance between the reflective surface of the metal surface 81 and the metallized region (for example) 84 is then a function of the thickness of the protein layer therebetween. Therefore, depending on whether the protein layer is a bilayer or a monolayer, the spectral components of the reflected light represented by the light beam 87 will change.

Another reference example shown in FIG. 1 consists of the steps shown in blocks 12, 13, 14 and 18 in FIG. In this reference example,
An optically transparent substrate material such as glass, plastic, fused silica, mica, quartz, etc. must be used. Glass is particularly preferred, and a readily available substrate is a microscope slide glass. First, a substrate is coated with a number of metal spheres by depositing a metal (eg, indium) as shown in block 12 in FIG. For example, indium is slowly deposited onto a glass substrate from a tantalum boat in a commercial vacuum of about 5 x 10 ^-5 mm of mercury. Indium atoms have large mobility characteristics on the surface of the substrate,
Therefore, since the glass substrate is not significantly wetted, the indium deposited on the substrate aggregates into small particles. Any metal that has similar properties such as forming spheres when deposited onto a substrate can be used. In fact, besides indium, gold, silver, tin, and lead were also useful. This metal deposition is continued until the substrate becomes light brown in color. At this point, the metal sphere is
It has a diameter of about 1000 Å. The exact dimensions of the sphere are not important, but its diameter must be equal to the wavelength of the main part of visible light. The next process is
As shown in block 13 of FIG. 1, the sphere-coated substrate is immersed in a solution of a first protein. The first protein is also deposited in a monolayer on the substrate and metal sphere. Once a monolayer of protein is formed, such a coated substrate can be used to test a solution suspected of containing a protein that specifically reacts with the first protein. That is, the coated substrate is immersed in the solution in question, as shown in block 14 of FIG. As a result, if a specifically reactive protein is present, a bilayer of protein will adhere to the substrate and metal sphere. On the other hand, in the absence of a specifically reactive protein, only a monomolecular layer of protein is attached to the substrate and metal sphere. Then the first
As shown in block 18 of the figure, the coated substrate is observed under reflected or transmitted light. As a result, from the appearance of the coated substrate, a determination can be made regarding the thickness of the protein layer adhering thereto, and therefore regarding the presence of specifically reactive proteins. The thickness of the protein layer corresponds to the change in brown tone observed in the coated substrate. These changes are so pronounced that detection of the protein layer is a very simple task. Particles coated with one monolayer of protein exhibit a darker tone of brown, and particles coated with a bilayer of protein exhibit a darker tone of brown, compared to the brown tone of the particles alone on the substrate. Shows brown color. Such detection methods require that electromagnetic radiation is significantly scattered by a conductive sphere with a diameter equal to the wavelength of the main portion of the incident energy, and that such scattering is prevented by a thin insulating coating placed on the sphere. This is based on the fact that

A schematic diagram of a detection device based on this reference example is shown in FIG. In each diagram in FIG. 5, the test slide is shown as viewed under transmitted light. Figure 5a shows a slide with indium spheres on one surface 100. FIG. 5b is also a slide prepared in the same manner as the slide of 5a, but its left end is immersed in a solution of bovine serum albumin, thereby adsorbing a monolayer of bovine serum albumin 101. Figure 5
c is a similar slide, the left end of which is immersed in a solution of bovine serum albumin, thereby depositing a monolayer of bovine serum albumin 101. Further, its lower end is immersed in an ovalbumin solution, thereby adsorbing a monomolecular layer 102 of ovalbumin. By the way, it should be noted that the appearance of the covered parts of the slides in Figure 5c are all similar. This indicates that only one monolayer of protein is present on the slide. Therefore, it can be seen that although bovine serum albumin and ovalbumin are adsorbed onto the slide, ovalbumin does not adhere to areas of the slide that have been previously coated with bovine serum albumin. In other words, Figure 5c illustrates the aforementioned finding that any protein will attach to the substrate, but not any protein layer. Figure 5d
In preparing the slide, the monomolecular layer 101 is attached by dipping its left end into a solution of bovine serum albumin, and the monolayer 101 is deposited by dipping its right end into a solution of ovalbumin. A molecular layer 102 was deposited and finally its lower end was dipped into a solution of rabbit antiserum against bovine serum albumin. 103 is therefore a monolayer of rabbit antiserum against bovine serum albumin.
From the appearance in the lower right corner, the rabbit antiserum against bovine serum albumin is in the ovalbumin layer 1.
It can be seen that the lower right corner of the slide in Figure 5d is coated with only a monolayer of ovalbumin. The lower left hand corner of Figure 5d is noticeably darker due to the first monolayer of preadsorbed bovine serum albumin and the second monolayer of rabbit antiserum against bovine serum albumin immunologically complexed therewith. This indicates the existence of a bilayer of protein consisting of a monolayer of . It should thus be seen that only the specifically reactive proteins provide a bilayer on the slide, and all other protein layers are monolayers.
Note that FIG. 5-1 is an actual photograph of the reference example detection device schematically shown in FIG. 5, and photographs 5a-1,
5b-1, 5c-1 and 5d-1 show actual photographs corresponding to schematic diagrams 5a, 5b, 5c and 5d, respectively.

According to a variation of the above reference example, which provides a medical diagnostic tool, a substrate having metal spheres on its surface is partially immersed (to a first depth) in a solution of a first antigen. As a result, for the area immersed in the solution, the substrate is coated with a monolayer of the first antigen. After drying, the substrate is immersed more deeply (to a second depth) into a solution of a second antigen. The second antigen does not attach to the first antigen, but does attach to the uncoated portion of the substrate immersed in the solution. After drying again, such a substrate is immersed even deeper (to a third depth) into a solution of a third antigen. Third
The antigen attaches only to the portion of the substrate that is not coated with the first or second antigen. Such operations are repeated for any number of antigens. As a result, a substrate coated with a monomolecular layer of various antigens forming a large number of stripes is obtained. The coated slide thus becomes a diagnostic tool. In performing a medical diagnosis, such a slide may be immersed in a sample of blood, for example, for a certain period of time (typically several hours). Next, the slide is removed from the specimen and washed with water, followed by observation using reflected light or transmitted light. Such slides display dark and light stripes representing the antibodies present in the specimen, allowing medical diagnosis to be made based on this information.

FIG. 2 is an enlarged elevational view of a portion of a diagnostic device based on the last reference example described above. In the figure, a part of a base body 31 is shown, to which a sphere 32 of vapor-deposited metal is attached. Although the metal spheres 32 are preferably formed by depositing indium on the substrate 31 as described above, other metals with similar atomic transfer and non-wetting properties such as gold, silver, etc.
It may also be formed by vapor depositing tin, lead, or the like. Many metals should exhibit such properties as the temperature of the substrate changes. Upon immersion in a solution of the first protein, the device consisting of substrate 31 and metal spheres 32 is coated with a monolayer of protein molecules 34, as shown at 33 in FIG. The device designated as 33 has a protein molecule 34
It is a diagnostic tool that should be used to test solutions suspected of containing proteins that specifically react with the protein. That is, if a device shown as 33 is exposed to a protein that specifically reacts with protein molecule 34, such device will have the appearance shown as 35. In that case, the substrate 31 and the metal spheres 32 are coated with a bilayer of protein, which bilayer forms a first monolayer overlapping the substrate 31 and the metal spheres 32. protein molecules 34 and a first protein forming a second monolayer overlapping the first monolayer, the substrate 31 and the metal sphere 32 and immunologically bonded to the first monolayer. and a protein molecule 36 that specifically reacts with the protein.

A modification of the above-mentioned reference example that increases the contrast in such a coated slide for diagnosis is the seventh example.
As shown in the figure. In this case, as seen in FIG. 2, a slide 31 having metal spheres 32 on its surface and coated with protein molecules 34 is immersed in a light-reflecting liquid 71 with the coated side facing downward.
The slide 31 is then observed from its top surface using reflected light. In order to completely reflect the light incident on the diagnostic device shown in FIG. 7, the light reflective liquid 71 is preferably a metallic liquid, particularly mercury. When a non-metallic liquid is used, the optical incident angle and observation angle to be adopted when using the diagnostic apparatus of FIG. 7 are important if total reflection is to be performed. The appearance of the diagnostic device viewed according to this modification is similar to that shown in FIG. 5, but an increase in contrast can be seen.

FIG. 3 is an enlarged elevational view of an apparatus for diagnostic purposes and for concentrating and purifying proteins and antibodies based on another reference example. In the figure, protein molecules 4 are shown as described above.
A device consisting of a substrate 41 coated with a monolayer of P.2 is shown as 40. The device is then shown at 43 after immunological binding of a monolayer of protein molecules 44 that specifically react with protein molecules 42, also as described above. On the other hand,
On another substrate 45 a droplet 46 of a weak acid solution is placed. The mutually facing surfaces of substrates 41 and 45, each having a bilayer of protein and a droplet of a weak acid solution (eg, 0.1N citric acid solution), are brought into physical contact.
Accordingly, droplet 46 of weak acid solution cleaves the immunological bond between protein molecules 42 and 44.
At this time, the biochemical properties of any protein are not affected, and the bond between the first protein molecule 42 and the substrate 41 is not broken.
Therefore, if substrates 41 and 45 are pulled apart again, as shown at 47, substrate 41 will have the first
A monolayer of protein molecules 42 is attached to the substrate 45, while a protein molecule 4 that reacts specifically is attached to the substrate 45.
A monolayer of 4 is attached. As a result, the substrate 41 with attached protein molecules 42 can be used to repeat the process of creating a substrate coated with a monomolecular layer of a specifically reactive protein, or in a test based on another reference example described above. It can also be used as a slide. On the other hand, the substrate 45 to which the specifically reactive protein 44 is attached can be used as a test slide for determining a solution in which the presence of the first protein is suspected according to the other reference example described above. This reference example is considered to be particularly meaningful. This is because, as already pointed out, among the common proteins of biological interest, antigens are quite easily available in purified form, and therefore, by adsorbing them onto a substrate, the presence of antibodies can be suspected. This is because while test slides can be prepared to determine which solutions are used, antibodies are not readily available. Therefore,
Conventionally, it has generally been impossible to prepare test slides for testing solutions in which the presence of antigens is suspected. However, the method and apparatus shown in FIG. 3 allows for example the preparation of test slides consisting of a substrate 45 coated with a monolayer of antibody molecules 44, which can be used to detect the presence of antigen. Therefore, it is possible to determine which solutions are suspected.

Next, examples of the present invention will be described. FIG. 9 shows the structure of a detection device according to an embodiment of the invention. Further, FIG. 8 shows a graph representing the principle of operation. As shown in FIG.
It consists of a gold base 91 on which 2 is adsorbed.
However, for economical reasons, it is preferable that the substrate 91 is a thin layer of gold plated on another metal. Gold has absorption bands within the visible spectrum. This fact explains the unique color of gold and allows embodiments of the invention to work.

The principle of operation of an embodiment of the invention is illustrated in FIG. In the figure, a group of reflectance curves are depicted in which relative reflectance is plotted against wavelength. Curve 93 represents the original reflectance of a gold substrate, whereas curve 94 represents the reflectance of a metal substrate with a monolayer of protein, and curve 95 represents the reflectance of a metal substrate with a bilayer of protein. Represents reflectance. In this way, if the curve 93 is followed, a gold substrate such as 91 will have a bright yellow color characteristic of gold.
When a monomolecular layer 92 of the first protein is deposited on the substrate 91, following the curve 94, the test slide will have a dull yellow appearance. If the test slide is then exposed to a solution suspected of containing a protein that specifically reacts with the first protein, the test slide will be exposed to the protein if a specifically reacting protein is present in the solution. It will have a bilayer and therefore its appearance will be distinctly green as shown by curve 95.

Based on tests conducted to date, the reference examples using a substrate with metal spheres attached and the examples using a metal substrate appear to be generally the most useful. Furthermore, it was also confirmed that these two cases exhibit different sensitivities depending on the film thickness of the protein in question. More specifically, the maximum sensitivity of the reference example using a substrate with metal spheres attached is achieved for film thicknesses of about 200 Å or less. However, the maximum sensitivity of the embodiments using gold substrates is achieved for film thicknesses greater than 30 Å.

According to one embodiment of the invention, a glass slide was first coated with a thin layer of indium and then with a thin layer of gold. An indium bottom coating was used to improve adhesion between the glass and the gold. Furthermore, it has been found that diffusion of indium into a thin layer of gold improves the optical properties of the test slide. The slides thus prepared were coated with a monolayer of hepatitis-associated antigen. On the other hand,
Antibodies against hepatitis-related antigens were mixed into human blood samples to be tested for the presence of hepatitis. The amount of antibody was such that any hepatitis-associated antigens present in the blood sample would be immunologically removed from the mixture. A test slide was immersed in a blood sample that had been pretreated in this way. Upon removal, the slides immersed in the hepatitis-negative blood samples had a bilayer of protein as confirmed by their greenish appearance. The protein bilayer consisted of one monolayer of a pre-installed hepatitis-associated antigen and one monolayer of an antibody against the hepatitis-associated antigen immunologically bound thereto. On the other hand, slides immersed in hepatitis-positive blood samples retained their original dull yellow appearance and were therefore found to have only a monolayer of hepatitis-associated antigens. In this case, the antibody introduced into the blood sample is complexed with the internal hepatitis-related antigen and precipitated from the blood sample, so it is impossible for it to react with the hepatitis-related antigen on the test slide.

By the way, it will be recognized that the above test is an inhibition test. According to another detection method carried out according to the invention, a direct test is also possible. Accordingly, glass-indium-metal slides were first prepared as described above and then coated with a monolayer of antibodies against hepatitis-associated antigens. Some of the slides were taken from hepatitis patients and were therefore immersed in mixed serum known to contain hepatitis-related antigens. The remaining slides were also immersed in a serum sample known not to contain hepatitis-associated antigens. As expected, the slides immersed in the mixed serum had a bilayer of protein, whereas the slides immersed in another serum sample had only a monolayer of protein.

From a theoretical point of view, the above-mentioned direct test is the preferred detection method. This is because the direct test has a relatively simple procedure, and in this case, the direct test has a higher sensitivity than the inhibition test. The higher sensitivity of direct tests is due to the fact that molecules of hepatitis-associated antigens are much larger than molecules of antibodies to hepatitis-associated antigens. Therefore, in a direct test, the rate of change in film thickness is large and it is therefore possible to observe a significant contrast.

The concentration of hepatitis-associated antigens in the blood of diseased humans is a function of time. That is, the concentration of hepatitis-related antigens is extremely high just before the onset of hepatitis symptoms. However, the concentration of hepatitis-related antigens in human blood after the onset of symptoms is extremely low. From a medical perspective, both of these conditions are of interest. While the detection of hepatitis-associated antigens during and just before the onset of symptoms is valuable for diagnostic purposes, the detection of hepatitis-associated antigens at a time later than the onset of symptoms is important for screening the blood of prospective donors. It is valuable in that respect. As a practical matter, simple and sensitive direct tests are preferred for diagnostic purposes, given the relatively high concentrations of hepatitis-associated antigens in blood samples and their ease of implementation.

The inhibition tests described above for screening purposes were experimentally evaluated to determine their value relative to known screening methods. In these tests, hepatitis-positive serum was diluted with known hepatitis-negative serum. Hepatitis positive co-infusion serum 1
When one part was diluted with 32 parts of a known hepatitis negative serum, reliable detection was achieved in one hour following the procedure described above. Furthermore, when 1 part of hepatitis-positive co-injected serum was diluted with 3000 parts of known hepatitis-negative serum, reliable detection was achieved in 20 hours. It will be seen that these results are very similar in terms of sensitivity and turnaround time to currently known immunoelectrophoresis and radioimmunoassay methods, respectively. Thus, the selection method according to the invention not only gives results comparable to the best results achievable by conventional methods, but also has the advantage that the outlay required to obtain such results is significantly lower. It brings.

In the above method for diagnosing hepatitis, it is preferable to use the gold-coated substrate of the present invention rather than the metal sphere-coated substrate of the reference example. This is because the molecules of hepatitis-associated antigens have a diameter of about 210 Å. Such a film thickness is within a suitable range for obtaining highly sensitive readings using a substrate coated with gold, but is within a suitable range for obtaining highly sensitive readings using a substrate coated with metal spheres. is too thick.

As a reference example, FIG. 4 shows a mechanical schematic diagram of an apparatus for concentrating and purifying proteins and antibodies. At the outset, it should be stated that in terms of the operating principle, the reference example shown in Figure 4 is a modification of the reference example shown in Figure 3 mentioned above.
This reference example will help you understand. Shown in the figure is a continuous flexible belt 51 of substrate material coated with a monolayer of protein which specifically reacts with the protein to be concentrated and purified. First of all, such a belt is introduced into a container 52 containing a liquid 53 containing the protein to be concentrated and purified.
After the immunological complex between the two specifically reactive proteins has taken place in the container 52, the belt 51, which now has a bilayer of proteins, moves into the container 58 containing the weak acid solution 59. . As a result, the immunological bond between two specifically reactive proteins was
9 and the second protein layer is thus recovered in a weak acid solution 59. Therefore, the belt 51 leaving the container 58 will again have only one monolayer of the original protein. When the belt 51 is then returned to the container 52, a single molecular layer of the protein to be concentrated and purified is again collected and then collected again into the container 58. Belt 51 is driven through liquid 53 and weak acid solution 59 by capstans 61 and 63 cooperating with pinch rollers 61 and 62, respectively. capstan 60 and 6
3 may be driven by any suitable means, but it is convenient to use a small electric motor connected to the capstans 60 and 63 via a speed reduction device. Since the immune reaction is a much slower reaction than the bond cleavage reaction caused by weak acids, the time that the belt 51 is in contact with the liquid 53 containing the protein to be concentrated and purified is much longer than the time that the belt 51 is in contact with the weak acid solution 59. From the point of view of efficiency, it is desirable to make the length longer than that.
Therefore, it is desirable to make the container 52 substantially larger than the container 58 and to provide multiple upper idle rollers 54 and lower idle rollers 55 to allow the belt 51 to pass through the liquid 53 multiple times. In this way, the portion of belt 51 that is in contact with liquid 53 at a given time is lengthened, and therefore the time that a given section of belt 51 is exposed to liquid 53 is lengthened. On the other hand, in order to peel off the second protein layer, it is sufficient to pass the belt 51 through the weak acid solution 59 only once. Therefore, weak acid solution 59
In order to guide the belt 51 therein, a pair of upper idle rollers 56 and a single lower idle roller 57 are provided. Further, in order to mechanically support the belt 51 when moving between the containers 52 and 58, a large number of idle rollers 64 are installed as necessary. Containers 52 and 58 are provided with stirring means 65 for stirring liquid 53 and weak acid solution 59 through the outer wall in a liquid-tight manner to ensure renewal of the liquid or weak acid solution in contact with belt 51.
and 66, respectively.
Container 52 is also equipped with a drain tube 67 controlled by a valve 68 for draining liquid 53 when the concentration of the desired protein decreases to such an extent that efficient recovery is no longer possible. . Note that after the discharge, fresh liquid 53 may be injected into the container 52 from the upper open end. Container 58 is also equipped with a drain tube 69 controlled by valve 70 to drain the desired solution when the desired concentration of purified protein in the weak acid solution is reached. After discharging, fresh weak acid solution may be injected into the container 58 from the upper open end. As noted in connection with FIG. 3, belt 51 can be coated with any desired protein (whether antigen or antibody);
The method and apparatus of Figure 4 is useful for preparing purified concentrates of any desired protein, so long as an immune response involving the desired protein is present. Furthermore, by slightly modifying this reference example, the apparatus of FIG. 4 can be used to selectively remove specific undesirable proteins from a mixture such as serum. To this end, it is only necessary to operate the apparatus of FIG. 4 for an extended period of time, while not replacing the liquid 53, while periodically replacing the weak acid solution 59 as necessary. In this case, the product obtained from the outlet tube 67 is serum free of undesirable proteins, the extent of which being removed depends entirely on the operating time of the apparatus.

Although embodiments of the present invention have been described above along with reference examples, it will be obvious to those skilled in the art that other modifications and changes can be made in accordance with the above teachings.

[Brief explanation of the drawing]

Figure 1 is a reference process flow diagram illustrating the work steps in various reference examples, Figure 2 is an elevation view showing how to fabricate and use a detection device based on one reference example, and Figure 3 is a diagram for another reference example. Fig. 4 is a mechanical schematic diagram of an apparatus for concentrating and purifying proteins and antibodies based on another reference example, and Fig. 5 is based on another reference example. A schematic diagram of the detection device; FIG. 5-1 is an actual photograph corresponding to the schematic diagram in FIG. 5; FIG. 6 is an elevation view of the detection device based on another reference example; FIG. 7 is a diagram of the device in FIG. FIG. 8 is an elevation view showing a modified reference example of the usage method; FIG. 8 is a graph showing the operating principle of the detection device shown in FIG. 9 showing an embodiment of the present invention;
FIG. 9 is an elevational view of a detection device according to an embodiment of the present invention. In the figure, 31 is a substrate, 32 is a metal sphere, 34 is a first protein molecule, 36 is a protein molecule that specifically reacts with the first protein, 41 is a substrate, 42 is a first protein molecule, 44 is a protein molecule that specifically reacts with the first protein, 45 is another substrate, 46
71 represents a weak acid solution, 71 represents a light-reflecting liquid, 91 represents a gold substrate, and 92 represents a monolayer of the first protein.

Claims (1)

[Claims] 1. A gold surface, a substrate having a layer of another metal between the gold surface and the substrate to improve the adhesion between the two, and a specific antigen-antibody reaction with the protein to be detected attached to the surface. A device for detecting the presence of a specific protein in a liquid, consisting of both elements of a monolayer of a protein.