JPS59160763A - Detector for protein - Google Patents

Abstract

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

Description

[Detailed description of the invention] The present invention relates to a protein detection device.

More specifically, the present invention relates to a protein detection device that performs an antigen-antibody reaction on the surface of a substrate.

The wood patent application was accepted on June 26, 1972 and was entrusted to the same trustee as the present invention.
No. 2,662,789 (1aevcr).

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 the animal in its fight against disease. A process that is not understood by foreign proteins or antigens causes biological systems to produce antibody proteins specific for that antigen. 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 in determining whether the biological system has been exposed to a foreign antigen. It has medical diagnostic value. Conversely, the detection of specific antigens within biological systems also has medical diagnostic value.

Examples include detecting HCG protein molecules in the urine as a pregnancy test, as well as detecting hepatitis-associated antigen molecules in the blood of prospective blood donors.

In order to perform such a diagnostic test, at least one of the seven pairs 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, only vertebrates are known to date to exhibit an immune response to the introduction of foreign proteins. For example, a large number of corresponding antibodies are found in the serum of an animal exposed to a large number of 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 use 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 carrying out this test, a certain amount of HCG antiserum is mixed into the urine sample. A large number of polystyrene spheres coated with HCG protein are thus introduced into the pretreated urine specimen. Such polystyrene spheres can be used when HCG protein is not present in the urine sample.
Furthermore, only in that case should agglomeration occur. That is, when HCG protein is not present in the urine sample, the polystyrene-bound HCG protein is complexed with the IgG antiserum (previously introduced into the urine sample), so that the polystyrene spheres aggregate. On the other hand, if HCG protein is present in the unspecified sample, it will interact 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 it is no longer possible for the pre-introduced pressure G antiserum to complex with the ICG protein (from the polystyrene method). Based on this idea, it should be possible to simplify the pregnancy test using the HCG protein described above. That is, one could attach the HCG antiserum to a polystyrene stop and then use it to directly test the urine specimen.

Then, if HCG protein is present in the urine sample,
However, only in that case will the polystyrene spheres aggregate.

However, such a simple method has not been used so far. The reason seems to be that the available HCG anti-suppressant is a complex mixture containing a large amount of components other than HCG antibodies. Moreover, there is the fact that extracting antibodies from HCG antiserum requires extra effort.
This is one of the reasons why inhibition tests that directly use CG antiserum have traditionally been preferred. However, according to one embodiment of the present invention, a method for efficiently separating HCq antibodies from serum is provided. Moreover, such a method also yields a diagnostic device that allows simpler direct testing to be carried out. 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, thus reducing the reliability of the test.

Therefore, although agglutination tests are typically performed with great care by skilled technicians, diagnostic errors are still inevitable during the purification and concentration of l/1° antibodies. Current methods for preparing antibodies [e.g., introduction of an antigen into an animal's biological system stimulates production of antibodies;
Serum containing antibodies in a highly diluted state is collected from the animal, and then an amount of antigen is mixed into the serum. Such antigen and antibody mixtures are complexed and precipitated from the serum. After aspirating off the residual components of the serum, the antigen-antibody precipitate is dissolved in acid 1, 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.

Incidentally, it has been shown that the antigen-antibody complex reaction also occurs when the antigen is adsorbed on the surface.

Such complex reactions on surfaces have been observed using ellipsometers. An ellipsometer is a complex optical instrument that can be used to measure film thicknesses on the order of 0.7A. However, ellipsometers are expensive and require skilled operators. Two methods have been used in traditional immunoreaction studies using ellipsometers.

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. There is another method,
The immunoreaction is carried out 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.

Now, in the present invention, any protein is adsorbed onto a substrate in the form of a monomolecular 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 It is therefore an object of the present invention to provide methods and devices useful for medical diagnostic and pharmaceutical applications by application of such discoveries.

Accordingly, the first principal object of the present invention is to provide a method and apparatus for economically detecting immune reactions occurring on surfaces.

It is also an object of the present invention to provide the above-mentioned method and apparatus for detecting such an immune response by electrical means.

Furthermore, it is also an object of the present invention to provide the above method and apparatus for detecting such an immune response by direct visual observation.

The second main object of the present invention is to provide a method and apparatus for concentrating and purifying proteins and antibodies through a controlled immune reaction that occurs on the surface.

Briefly, according to two embodiments of the invention, first a thin plate of substrate material is immersed in a solution of a first protein, so that a monolayer of the first protein is deposited on the substrate. The substrate thus coated with the first protein is then dipped into the second solution.

The second solution is known to contain a protein that specifically reacts with the first protein in the first example, and is known to contain a protein that specifically reacts with the first protein in the second example. It is suspected that it contains a protein that specifically reacts with

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 a first embodiment of the invention, 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 a second embodiment of the invention, the substrate after being immersed in a solution suspected of containing a specific reactive protein is electrically or optically examined, thereby It is determined whether the protein layer attached to the substrate is a bilayer or a monolayer. the result,
It will be determined whether the second solution contains a protein that specifically reacts with the first protein.

The patentable novel features of the invention are pointed out in the appended claims. The invention, together with its objects and advantages, will be better understood from the following description taken in conjunction with the accompanying drawings.

First, FIG. 1 is a process flow diagram illustrating various steps related to the implementation of the present invention. 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 the process diagram is that, according to the various embodiments of the present invention, any seven of the steps described at a predetermined vertical level can be selectively arranged. means that it will be carried out.

According to a first embodiment of the invention, operation begins at block 16 in FIG. First, metal, glass, mica,
A thin sheet of substrate material such as 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.

The first protein is biologically an antigen or an antibody, which is available as a highly concentrated solution, and a protein with which it specifically reacts (biologically speaking, it is an antigen or an antibody, respectively). to detect or purify antigens).

The first protein forms a single molecular layer and is adsorbed onto the substrate. Note that any protein will be adsorbed by releasing one molecular layer, but no further adsorption will occur. In other words, the protein attaches to the substrate, but not to itself. After a monomolecular layer of protein has been formed over the entire surface of the substrate, the 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 the bovine serum albumin solution in section 2, it takes about 30 minutes to completely coat a glass slide with a single molecular layer of protein.

The next step, shown at block 14 in FIG. 7, is to immerse the coated substrate in a highly concentrated solution 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 will occur between the first protein and the protein that specifically reacts with it, so that after a certain period of time the substrate will become the second protein. It will have a molecular layer. In addition, the first
It should be noted that the steps shown in blocks 16 and 14 in the figure are common to all embodiments of the invention. The time required for complete deposition of the second protein layer on the coated substrate also depends on the 11k of the specifically reactive protein in solution. For antibodies in serum, this time can extend over a day. According to one embodiment of the invention, the next step is to immerse the coated substrate in a hot weak acid solution as shown in block 15 in FIG.
At the same time, the coated substrate is observed using an ellipsometer, as indicated by block 22 in FIG. 1st
Formation of a specifically reactive protein layer on a protein-coated substrate can take a long time;
Immunological bonds between species proteins are very rapidly cleaved by weak acids. Therefore, 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 measurement of the detachment of a specifically reactive protein layer by the action of a weak acid can be achieved much more quickly than the conventional observation of the formation of a specifically reactive protein layer. Therefore, preparing a number of test slides according to block 16, exposing each of them to one of a number of serum samples as shown in block 14, and then sequentially testing such slides according to blocks 15 and 22; Thereby, it can be determined in a short time which serum sample contains 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, those immersed in a solution containing a specifically reactive protein will have a film thickness change of about 2 to 1/2 minutes 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 embodiment of the present invention is useful for protein concentration and purification, and is shown in block 13 of FIG.
．． 14. Consists of the steps shown in 15 and 26.
First, as shown in block 16I/C, the substrate is immersed in a solution of the available protein of the antigen/antibody pair. Normally this protein is an antigen, but the invention does not depend on the biological nature of the first protein. 1st
A coated substrate prepared according to block 16 in the figure is then immersed in a solution containing a protein that specifically reacts with the first protein, as shown in block 14.

Typically, the specifically reactive protein is an antibody and the solution used in block 14 is serum from an animal exposed to the first protein.

When the substrate thus coated with a bilayer of protein is immersed in a weak acid shown in block 15 in FIG. 1, the specifically reactive protein layer is separated from the first protein layer. be done. At this point, the substrate is coated with a monolayer of the first protein, while a purified solution of the specifically reactive protein is obtained in the weak acid. The next step, as shown in block 23 in FIG. 1, is to return the substrate with the first protein layer attached to the solution containing the specifically reactive protein, collect the second protein layer, It is then stripped again with a weak acid, thereby increasing the concentration of specifically reactive proteins in the weak acid. If such operations are continued, purified concentrates of specifically reactive proteins will be recovered in weak acids.

Although the first embodiment described above improves to a practical degree the efficiency of detecting immune reactions with an ellipsometer,
Nevertheless, it may be economically desirable to completely eliminate the need for an ellipsometer. Another embodiment of the invention is therefore provided for measuring the relative thickness of a protein layer by electrical means. In this example,
A metallic substrate may be used, or a substrate of another material may be first coated with a thin metal film, as shown in block 11 of FIG. Such metal or metal-coated substrates are then immersed in a protein solution according to the steps shown in blocks 16 and 14. The protein layer thus attached to the substrate has electrical insulation properties. The next step is as shown in block 16 in FIG.
A mercury drop or other electrode may be placed on top of the upper protein layer attached to the substrate. The upper protein layer consists of the first protein when the specifically reactive protein is not contained in the solution in question, and the upper protein layer consists of the first protein when the specifically reactive protein is contained in the solution in question. Consists of protein. 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.
dge) The electrical enclosure of the capacitor described above is measured by any suitable capacitance measuring instrument such as a). In addition,
As expected, the capacitance of the capacitor with a bilayer of protein was found to be about 2 to 5 times lower than that of the capacitor with a monolayer of protein.

Another embodiment of the present invention, which is closely related to this, 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 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 is used.
For example, a substrate made of glass is used. Then, after subjecting such gas to the steps of blocks 16 and 14,
A drop of mercury is placed on top of the top protein layer, also as shown in block 16. However, no electrical measurements are made in this embodiment. That is, the next step in this example is to visually observe the coated substrate and thereby measure the time that elapses until a visible amalgam forms between the thin metal film on the substrate and the mercury. be. This embodiment of the invention is based on the discovery that it takes approximately 7 minutes for mercury to diffuse through a monolayer of protein, whereas it takes more than 10 minutes to diffuse through a bilayer of protein. Based on.

Since mercury forms a thin metal film and visible amalgam on the substrate after diffusing through the protein layer, there is a time delay between placing a mercury drop on top of the upper protein layer and the appearance of visible amalgam. 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 appreciated by those skilled in the art, capacitance measurements in the last example described above must be made within 7 minutes 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 will also be appreciated that according to the above examples, it is possible to roughly quantitatively measure the concentration of a specifically reactive protein in the solution in question.
This can be accomplished by fabricating a number of substrates in the step of block 16 and then simultaneously immersing them in the solution of interest as shown in block 14, while sequentially removing them from the solution of interest over an extended period of time. Since the rate at which a specifically reactive protein layer is formed depends on the degree of specifically reactive proteins in the bath solution, mercury droplets are Comparing the diffusion times through the protein layers provides a rough quantitative measure of the concentration of the specifically reacting protein in the solution in question.

In a related embodiment of the invention, block 11 in FIG.
Accordingly, the substrate is coated with a thin metal film. The substrate is then dipped into the solution as shown in blocks 16 and 14. The next step in this example is to coat the top protein layer with a second metal layer, as shown in block 17 in FIG. It is best to observe it using reflected light. Note that the second metal layer is preferably installed by electroplating. Essentially, this embodiment provides a structure that acts as a diffraction grating, and the difference between a bilayer and a monolayer of a protein can be determined from the spectral components observed in the reflected light.

A device based on this embodiment is shown in FIG. First, a first protein layer 82 is placed on a 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 of interest, and if a protein that specifically reacts with the first protein layer 82 is present in the solution of interest, the second protein layer 86 is removed. is formed. 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 light a! The spectral components of the reflected light represented by 87 will change.

Another embodiment of the invention shown in FIG. 1 consists of the steps shown in blocks 12, 13, 14 and 18 in FIG. For this embodiment, 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, as shown in block 12 in FIG.
The substrate is coated with a large number of metal spheres by vapor deposition. For example, indium is slowly deposited onto a glass substrate through a tantalum port in a conventional vacuum of about s x'1o-5 mercury columns. The indium atoms deposited on the substrate aggregate into small particles because the indium atoms have strong mobility over the surface of the substrate and therefore do not significantly wet the glass substrate. 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 has a diameter on the order of 1000 amps. 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 step is to immerse the sphere-coated substrate into a solution of the first protein, as shown at block 16 in FIG.
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 of interest, as indicated by block 14 in FIG. the result,
If a specifically reactive protein exists, a bilayer of the 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. Next, as shown in block 18 in FIG. A determination is therefore made regarding the presence of a specifically reactive protein. The thickness of the protein layer corresponds to the changes in brown color observed in the coated substrate. These changes are quite pronounced. Therefore, 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 significantly influenced by a thin insulating coating placed on the sphere. It is based on the fact that

An actual photograph of a diagnostic device based on this embodiment is shown in FIG. In each photograph in FIG. 5, the test slide is viewed under transmitted light. Photo 5aV
i One table Ifi10θ shows a slide with indium spheres on it. Photo 5b is also a slide prepared in the same manner as the slide in photo 5a, but its left end is immersed in a solution of bovine serum albumin, thereby adsorbing a monolayer of bovine serum albumin 101.

Photo 5C is a similar slide, the left end of which has been 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 coated portions of the slides in Photo 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 the portion of the slide that has been previously coated with bovine serum albumin. In other words,
Photo 5C illustrates the previously mentioned finding that any protein will attach to the substrate, but not to any protein layer. In preparing the slide shown in Photo 5d, a monomolecular layer 101 is attached by dipping its left end into a solution of bovine serum albumin, and a monomolecular layer 101 is deposited by dipping its right end into a solution of ovalbumin. 102 was attached and finally its lower end was dipped into a solution of rabbit antiserum against bovine serum albumin. Thus, 106 is a monolayer of rabbit antiserum against bovine serum albumin. □ Judging from the appearance of the lower right corner, the rabbit antiserum against bovine serum albumin does not adhere to the ovalbumin layer 102, so the lower right corner of the slide in photo 5d is covered with only a monolayer of ovalbumin. I know that there is.

The lower left side of photo 5d is noticeably darker, which is due to the first 7-molecule layer of pre-adsorbed bovine serum albumin and the immunologically complex rabbit antiserum against bovine serum albumin. This indicates the presence of a bilayer of protein consisting of a second monolayer. in this way,
It should be seen that only the specifically reacting proteins give a bilayer on the slide, all other protein layers are monolayers.

According to a variant embodiment of the above embodiment, providing a medical diagnostic tool, a substrate having metal spheres on its surface is partially immersed (to a first depth) in a solution of a seventh antigen. .

As a result, for the area immersed in the solution, the substrate is coated with a monolayer of the first antigen. After drying, such substrate is placed deeper into a solution of a second antigen (up to a second depth).
immersed. The second antigen does not attach to the seventh 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. The third antigen attaches only to portions of the substrate 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 according to the last described embodiment of the invention. The figure shows a part of a base 61 to which a large number of vapor-deposited metal spheres 62 are attached. Metal sphere 32tj: preferably formed by vapor depositing indium on the substrate 61 as described above, but other metals with similar atomic transfer and non-wetting properties such as gold, silver, soot, lead, etc. may be vapor deposited. It may be formed by 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 61 and metal spheres 62 is coated with a monolayer of protein molecules 64, as shown at 66 in FIG.
The device designated 66 is a diagnostic tool to be used to test solutions suspected of containing proteins that specifically react with protein molecules 34. That is, when a device designated as 66 is exposed to a protein that specifically reacts with protein molecule 64, such device will have an appearance as shown as 65.

In that case, the substrate 61 and the metal spheres 52 are coated with a bilayer of protein, which bilayer forms a first monolayer overlapping the substrate 61 and the metal spheres 62. a first protein forming a second monolayer overlapping the protein molecules 64 and the first monolayer, the substrate 61 and the metal sphere 62 and immunologically bonded to the first monolayer; Specifically reactive protein molecules 6
It consists of 6.

A modified embodiment of the above embodiment for increasing the contrast in such diagnostic coated slides is shown in FIG. In this case, as seen in FIG. 2, a slide 31 having metal spheres 52 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 61 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.
Preferably, 1 is a metallic liquid, especially mercury.
When a non-metallic liquid is used, the optical angle of incidence and observation angle to be adopted when using the diagnostic apparatus of FIG. 7 are important if total reflection is to be performed. The external appearance of the diagnostic device observed according to this modified embodiment is as follows.
Similar to the case shown in the figure, but with an increase in contrast.

FIG. 3 is an enlarged elevational view of an apparatus for diagnostic purposes and for concentrating and purifying proteins and antibodies according to another embodiment of the present invention. A device comprising a substrate 41 coated with a molecular layer is shown at 40. Next, a monolayer of protein molecules 44, which specifically react with protein molecules 42, is immunologically bound, also as described above. The above device is shown as 46 after the above procedure.On the other hand, a droplet 46 of a weak acid solution is placed on another substrate 45.The mutually facing surfaces of the substrate 41 and 45 are each coated with protein. The bilayer and the droplet of a weak acid solution (e.g., 0.IN citric acid solution) are brought into physical contact.Based on the findings of the present invention, the droplet 46 of the weak acid solution is
2 and 440. At this time, the biochemical properties of any of the proteins are not affected, and the bond between the first protein molecule 42 and the substrate 41 is not broken. Therefore, when substrates 41 and 45 are pulled apart again as shown at 47,
One molecule layer of first protein molecules 42 is attached to the substrate 41, while one molecule layer of specifically reactive protein molecules 44 is attached to the substrate 45. As a result, the substrate 41 with attached protein molecules 42 can be used to repeat the process of producing a substrate coated with a monolayer of specifically reactive proteins, or alternatively, in another embodiment of the invention described above. You can also use test slides based on . On the other hand,
The substrate 45 with the specifically reactive protein 44 attached thereto can be used as a test slide for determining the presence of the seventh protein in a solution according to another embodiment of the invention described above. This embodiment of the invention is believed to be particularly meaningful. This is because, as already pointed out, among ordinary 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, prior to the present invention, it was generally not possible to prepare test slides for determining solutions suspected of containing antigens.
However, the method and apparatus shown in FIG. 3 makes it possible, for example, to prepare a test slide consisting of a substrate 45 sewn with a monolayer of antibody molecules 44, which can be used to detect antigens. This makes it possible to determine which solutions are suspected of being present.

The structure of a diagnostic device according to another embodiment of the invention is shown in FIG. 3, and a graph representing its operating principle is shown in FIG. g. As shown in FIG. 9, the diagnostic device according to this embodiment emits a monomolecular layer 92 of a first protein from a gold substrate 91 onto which it has been 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. The entire unique color is explained by this fact, which allows this embodiment of the invention to work.

The principle of operation of this embodiment is shown in FIG. g. In the figure, seven groups of reflectance curves are drawn in which relative reflectance is plotted against wavelength. Curve 96 represents the native reflectance of a gold substrate, whereas curve 94 represents the reflectance of a gold substrate with a monolayer of protein, and curve 95 represents the reflectance of a gold substrate with a bilayer of protein. represents the reflectance of

Thus, if curve 96 is followed, a gold substrate such as 91 will exhibit a unique bright yellow color.

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 often has a bilayer, so its appearance is distinctly green, as shown by curve 95.

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

According to one diagnostic method carried out according to 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-associated antigens were mixed into human blood samples to be tested for the presence of hepatitis. The amount of antibodies was such that, if present in the blood sample, hepatitis-associated antigens 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 slide immersed in the hepatitis-negative blood sample had a bilayer of protein as confirmed by its greenish appearance. The bimolecular addition of such a protein consisted of one molecular layer of a pre-installed hepatitis-associated antigen and one molecular layer 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 thus 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 appreciated that the above test is an inhibition test.According to another diagnostic method carried out according to the invention, a direct test is also possible.

Therein, glass-indium-gold 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 patients with hepatitis 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 diagnostic method. This is because the direct test has a relatively simple procedure, and in this case, the direct test has more 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 therefore it is possible to observe all of the remarkable contrast.

The concentration of hepatitis-associated antigens in the blood of diseased humans is a function of time. In other words, the concentration of hepatitis-related antigen IW is extremely low just before the onset of hepatitis symptoms.
However, the concentration of hepatitis-related antigens in human blood long after the onset of symptoms is very low. From a medical perspective, all of these conditions are interesting. 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 much later than the onset of symptoms is a challenge in the screening of blood from prospective donors. There is value in that. As a practical matter, simple and sensitive direct tests are preferred for diagnostic purposes, given the relatively high 'a# of hepatitis-associated antigens in blood samples and their ease of implementation.

The inhibition test described above for screening purposes was experimentally evaluated to determine its value relative to known screening methods. In these tests, hepatitis-positive serum was diluted with known hepatitis-negative serum.

When 7 parts of a hepatitis-positive co-infusion serum was diluted with 32 parts of a known hepatitis-negative serum, reliable detection was achieved in 7 hours following the above procedure. In addition, hepatitis-positive co-injected serum/
Reliable detection was achieved in 1.20 hours when one part was diluted with 3000 parts of known hepatitis negative serum. It will be seen that these results are very similar to currently known immunoelectrophoresis and radioimmunoassay methods in terms of advantages and time requirements, 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. be.

In the above hepatitis diagnostic method, it is preferable to use a substrate coated with gold rather than a substrate coated with metal spheres. This is because a hepatitis-associated antigen molecule has a diameter of approximately zlo A. This film thickness is within a range suitable for obtaining highly sensitive indications using a substrate coated with gold, but it is within a range suitable for obtaining highly sensitive indications using a substrate coated with gold group spheres. It's too thick.

FIG. 4 is a mechanical schematic diagram of a protein or antibody concentration M device according to another embodiment of the present invention.

At the outset, it will be helpful in understanding this embodiment to state that in terms of its operating principle, the embodiment shown in FIG. 4 is a modified embodiment of the embodiment shown in FIG. 3 described above. . 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 56 containing the protein to be concentrated in mW. After the immunological complex between the two specifically reactive protein members has taken place in the container 52, the belt 51, which now has a bilayer of proteins, is placed in a container 58 containing a weak acid solution 59. Moving.

As a result, the immunological bond between the two specifically reactive proteins is cleaved by the weak acid solution 59, and the second protein layer is therefore recovered in the weak acid solution 59. Therefore, the belt 51 leaving the container 58 will again have only one monolayer of the original protein.

Next, when the belt 51 is 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 connected to capstan 60 which cooperates with pinch rollers 61 and 62, respectively.
and 66 through liquid 56 and weak acid solution 59. Although the capstans 60 and 66 may be driven by any suitable means, it is convenient to use a shaped electric motor connected to the capstans 60 and 66 via a reduction gear. 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 install a large number of upper idle rollers 54 and lower idle rollers 55 to allow the vent 51 to pass through the liquid 56 multiple times. In this way, the portion of belt 51 that is in contact with liquid 56 at a given time is lengthened, and therefore the time that a given section of belt 51 is exposed to liquid 56 is lengthened. On the other hand, it is sufficient for the VC to pass the belt 51 through the weak acid solution 59 only once to exfoliate the second protein layer. Follow? So, weak acid solution 59
To guide the belt 51 therein, l pairs of upper idle rollers 56 and only one 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. In order to ensure renewal of the liquid or weak acid solution in contact with the containers 52 and 58 Vcfi and the belt 51, stirring means 6 penetrate liquid-tight through the outer wall and stir the liquid 53 and the weak acid solution 59.
5 and 66, respectively. Container 52 is also equipped with a drain line 67 controlled by valve 68 for draining liquid 56 when the concentration of the desired protein decreases to such an extent that efficient recovery is no longer possible. Note that after draining, fresh liquid 56 can be injected into the container 56 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 the protein in the weak acid solution is reached. After discharging, fresh weak acid solution is also poured into the container 5 from the upper open end.
Just inject into 8. As pointed out in connection with FIG. 3, belt 51 can be coated with any desired protein (whether antigen or antibody), so long as an immune reaction involving the desired protein is present. The illustrated method and apparatus are useful for preparing purified concentrates of any desired protein. Furthermore, with slight modifications to this embodiment, the apparatus of FIG. 4 can also be used to selectively remove certain undesirable proteins from a mixture such as serum. To this end, it is sufficient to operate the apparatus of Figure 1 for an extended period of time, with the liquid 56 not being replaced, but the weak acid bath g, 59 being replaced periodically as necessary. In this case, the product obtained from the outlet tube 67 is serum depleted of the desired A protein, the extent of which being removed depends largely on the operating time of the apparatus.

Although the invention has been described above in connection with specific embodiments,
It will be apparent to those skilled in the art that other modifications and variations are possible in accordance with the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than in the embodiments described.

Next, the embodiments of the present invention are listed below.

A: Depositing metal on a thin plate made of a light-transmitting substrate material, and immersing the thin plate in a bath solution of a protein that specifically reacts with a specific protein to form a monomolecular layer of the protein that specifically reacts with the specific protein. Coating the thin plate, immersing the thin plate in a solution in which the presence of the specific protein is suspected, and then inspecting the thin plate to determine whether the protein attached to the thin plate is a bilayer or a monolayer. A method for testing a solution suspected of containing a specific protein.

2. The method of claim 1, wherein said step of depositing metal comprises coating said sheet with a thin metal film.

3. Placing a mercury drop on the top surface of the protein on the thin plate,
3. The method of claim 2, wherein the testing step consists of then measuring the capacitance between the mercury drop and the metal thin film.

France The metal is one that forms a visible amalgam with mercury, a mercury drop is placed on the top surface of the protein layer on the thin plate, and then the time elapses from the placement of the mercury drop to the appearance of a visible amalgam. 3. The method of claim 2, wherein said testing step consists of measuring time.

Left: The method according to claim 2, wherein the inspection step consists of coating the upper surface of the protein layer on the thin plate with a second metal layer and then observing the thin plate with reflected light.

5. The method of claim 5, wherein said step of coating with said second metal layer comprises electroplating said second metal layer onto said sheet.

Z. The method of claim 1, wherein the step of depositing a metal having high atomic migration properties and low surface wetting properties comprises depositing the metal onto the thin plate.

g. The method of claim 7, wherein said step of inspecting comprises visually inspecting said sheet, and the determination is made by identifying differences in brown tones.

Z. The method of claim 7, wherein the metal is indium, and the indium is deposited onto the sheet from a tantalum port in a vacuum of about j x 10-5 meters of mercury.

10. The method of item 1, wherein said step of inspecting comprises visually inspecting said sheet, and the determination is made by identifying three shades of brown.

//, immersing a thin plate made of a base material in a solution of a protein that specifically reacts with a specific protein, coating the thin plate with a monolayer of the protein that specifically reacts with the specific protein; immersing the thin plate in the solution suspected of being present;
A method for testing a solution in which the presence of a specific protein is suspected, comprising the steps of immersing the thin plate in a weak acid solution while observing the thin plate using an ellipsometer.

1. The method according to item 1, wherein the thin plate has a surface made of a material having a refractive index substantially different from that of the protein.

/3. A belt made of substrate material is immersed in a solution of a protein that specifically reacts with the immunologically active protein to coat the belt with a monolayer of the specifically reactive protein. immersing a first portion of the belt in a solution containing an immunologically active protein to complex the immunologically active protein with the specifically reactive protein; moving the seventh portion of the belt from the solution containing active protein into a weak acid solution while immersing the first portion in the weak acid solution;
moving and immersing a second portion of the belt into the solution containing the immunologically active protein, and then from the weak acid solution to the solution containing the immunologically active protein; A method for preparing a purified concentrate of immunologically active proteins, comprising the steps of returning said first belt of said belts into said first belt.

/lA A thin plate made of a substrate material having a metal surface is immersed in a solution of a protein that specifically reacts with a specific protein, the thin plate is immersed in a solution in which the presence of the specific protein is suspected, and then the Testing a solution in which the presence of a specific protein is suspected, consisting of the steps of determining whether the protein layer attached to the thin plate is a bilayer or a monolayer by electrically testing the thin plate. how to.

/Left The metal surface in contact with the first surface of the protein layer constitutes a first electrode, while a second electrode is installed on the second surface of the protein layer, and then the seventh and second electrodes are placed on the second surface of the protein layer. The method according to item 1≠, wherein the testing step consists of measuring the capacitance between the electrodes.

/B. The method according to item 1j, wherein the second electrode comprises a mercury drop.

/'7 Immerse a thin plate made of a substrate material with a metal surface in a solution of a protein that specifically reacts with a specific protein, immerse the thin plate in a solution in which the presence of the specific protein is suspected, and then A method for preparing a solution in which the presence of a specific protein is suspected, comprising the steps of chemically testing the thin plate to determine whether the protein layer attached to the thin plate is a bilayer or a monolayer. How to test.

/B The metal surface is a metal that forms a visible amalgam with mercury, a mercury droplet is placed on the top surface of the protein layer, and then the time elapses from the placement of the water@drop to the appearance of visible amalgam. 78. The method according to item 77, wherein the testing step consists of measuring .

/T The testing step consists of immersing the thin plate in a weak acid solution to cleave the immunological bonds in the protein layer while observing the thin plate with an ellipsometer.
78. The method according to item 77.

, 20. A substrate is immersed in a solution of a protein that specifically reacts with an immunologically active protein, and a monolayer of the specifically reacting protein is removed from the substrate. The immunologically active protein is complexed with the specifically reactive protein by immersing the substrate in a solution containing the active protein, and then the substrate is immersed in a weak acid solution to complex the immunologically active protein with the specifically reactive protein. A method for purifying an immunologically active protein, comprising the steps of cleaving the immunological bond between the immunologically active protein and the specifically reactive protein.

2/, the weak acid solution is placed on the surface of the second substrate, and the step of immersing in the weak acid solution connects the surface of the substrate having the protein layer and the second substrate having the weak acid solution. 2. The method of claim 2, comprising contacting the surface of the

22. A base member, a number of metal spheres attached to one side of the base member, and a monolayer of immunologically active protein covering at least a portion of the base member and the metal spheres. A device for medical diagnosis.

, 23. Apparatus according to the second clause, wherein there are multiple types of said monolayers, each covering a corresponding portion of said substrate and said metal sphere.

244. The device according to item 22, wherein the base member is made of a light-transmitting thin plate, and the metal spheres are indium particles having a long axis within a range of 70 to 20θ0λ.

, 23. A device for detecting the presence of a protein of interest in a liquid, consisting of both a substrate having a surface containing a metal and a monolayer of a protein that specifically reacts with the protein of interest attached to said substrate.

2B. The device of item 2J, wherein the surface comprises a number of metal spheres attached to the substrate.

27. The device according to item 26, wherein the metal sphere is made of a metal that has high atomic migration properties on the substrate and low surface wetting properties.

2g, the pregnancy diagnosis according to item 2j, wherein the protein in question is HCG protein, the liquid is urine, and the specifically reactive protein is H (a protein that specifically reacts with HCG protein); equipment.

[Brief explanation of the drawing]

Fig. 1 is a process flow diagram illustrating the work steps in each embodiment of the present invention, Fig. 2 is an elevational view showing a method of manufacturing and using a diagnostic device based on an embodiment of the present invention, and Fig. 3 11 is an elevation view of an apparatus for concentration and purification of antibodies and for diagnostic purposes according to another embodiment of the present invention, and Figure 1 is a mechanical view of an apparatus for concentration and purification of proteins and antibodies according to yet another embodiment of the present invention 5 is a photograph showing the operation of the diagnostic device based on the present invention, FIG. 4 is an elevation view of a diagnostic device based on yet another embodiment of the present invention, and FIG. 7 is a photograph showing the operation of the diagnostic device based on the present invention. FIG. 1 is an elevational view showing a modified embodiment of the method of using the device, FIG. 1 is a graph showing the operating principle of the diagnostic device of FIG. It is a front view. In the figure, 61 is the substrate, 62 is a metal sphere, 64 is a molecule of the first protein, 66#'i is a molecule of a protein that specifically reacts with the first protein, 41 is the substrate, and 42 is the first protein molecule. molecule, 44￥i A molecule of a protein that specifically reacts with the first protein, 45 is another substrate, 46 is a weak acid solution, 71 is a light-reflecting liquid, 91 is a gold substrate, and 92 is the first Represents a single molecular layer of protein. Patent applicant General Electric Hisashi ■ agent (763
0) Birth: Tokuji Numa Showa (1999) Commissioner of the Patent Office Kazuo Wakasugi 2. Name of the invention: Protein detection device 3. Name of the person making the correction: General Electric Company Representative Name: Andon J. Willy Telephone (588) 5
200-5207 February 8, 1980 6, Detailed Description of the Invention and Brief Description of the Drawings in the Specification Subject to Amendment, Figure 5 of the Drawings, Reference Photograph, and Explanation of the Reference Photograph. 7. Contents of amendment (I) The following corrections will be made in the detailed description of the invention column of the specification. (1) On page 27, line 10, change ``actual photo'' to ``1 schematic drawing.'' (4) On page 27, lines 14-15, change "Photo 5b is also a photo" to "Figure 5b is also a figure." 5) On page 27, line 19, change "photo" to "figure." (6) On page 28, lines 4-5, change "photo" to "figure."(7') On page 28, line 14, change "photo" to "figure." (8) Line 28, line 15, change “photo” to “figure.” (9) Line 5, page 29, change “photo” to “figure.” (10) Line 8, line 29, "Photograph" is changed to "Diagram". (II) Page 54, page 16 in the brief explanation of drawings in the specification
In line 17, ``Photograph showing operation'' has been changed to ``Schematic diagram.'' (I[l) Figure 5 of the drawings has been amended as shown in the attached sheet. (IV) Submit reference photos 58 to 5d and a description of the reference photos as shown in the attached sheet. 8. List of attached documents (I) Figure 5 5a to 5e 1 copy (II) Reference photos 5a to 5d 1 copy (DI) Reference photo description 1 copy F/1)j (5a) (5C) 101 100 (5b) (5d)

Claims (1)

[Claims] A substrate having a gold surface and a layer of another metal between the entire surface and the substrate to improve adhesion between the two, and a substrate that causes a specific antigen-antibody reaction with the protein to be detected attached to the surface. Consisting of both elements of a monolayer of protein,
A device that detects the presence of specific proteins in liquids.