JPS5916669B2 - Antigen or antibody detection method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for forming a multilayer film of immunologically complex proteins, and more specifically, a method for forming a multilayer film of immunologically complex proteins, and more specifically, for binding a specific antibody protein to an antigen protein and simultaneously controlling the activity of the antibody protein. It relates to a method of maintaining a portion of a site in an active state for subsequent immune reactions.

The present invention also provides a method for detecting immunologically active large-sized proteins and small-sized proteins that specifically react therewith, and more specifically, a method for detecting immunologically active large-sized proteins and small-sized proteins that specifically react with them. It also relates to a method for improving the contrast of. 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. Immune responses that occur within living systems such as animals and humans are important in the fight against disease. When a foreign protein (ie, an antigen) invades a biological system, antibodies specific for the antigen are produced by a process that is currently not fully understood. Such antibody molecules have complementary chemical binding sites to corresponding sites on the antigen molecule, and as a result, the antigen and antibody chemically bond, thereby producing an immunologically complex protein. . Since biological systems produce antibodies in response to the invasion of foreign proteins, detecting antibodies present within a biological system has medical diagnostic value in determining the antigens to which the system has been exposed. have Conversely, detecting the presence of certain antigens within biological systems also has medical diagnostic value. Examples of diagnosis by detection of such antigens include the detection of HCG protein molecules in urine as a pregnancy test and the detection of hepatitis-associated antigen molecules in the blood of prospective blood donors. To perform such diagnostic tests, at least one of a pair of immunologically reactive proteins must be obtained. The only sources of antigens currently known are biological systems.

More specifically, only dorsal 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 and humans that have been exposed to antigens. On the other hand, many antigens can be controllably produced in laboratory cultures. However, certain antigens, such as hepatitis-related antigens, like antibodies, can currently only be obtained from the biological systems of higher animals. While most antigens are or contain proteins as an essential component, all antibodies are proteins.

Proteins are macromolecules with high molecular weight. In other words, since proteins are polymers consisting of chains of varying numbers of amino acids, antigen and antibody proteins can each have several binding sites. Each of the five major antibody proteins (immunoglobulins GGlgM.IgA.lgE and IgD) is clearly characterized as consisting of at least two heavy peptide chains and at least two light peptide chains. The bonds between amino acid units, known as peptide bonds, are generally arranged in a Y-shape. In the case of the GG antibody protein, the active site, ie, the binding site, is located at the ends of the two arms of the Y-shape. Immune responses can be detected by a variety of techniques, including the use of appropriate substrates such as metal-coated glass slides or metal slides.

When such a substrate is exposed to a solution of antigenic protein, the antigenic protein is physically adsorbed onto the surface of the substrate in the form of a dense monomolecular layer. If the coated substrate is then exposed to serum containing antibody proteins directed against the antigenic protein, an immune reaction will occur. That is, as a result of the antibody protein selectively binding to the antigen protein through the binding site of the antibody protein molecule that is complementary to the corresponding site on the antigen protein molecule, at least one of the immunologically complexed proteins is present on the substrate surface. A partial bilayer is formed. However, a problem arises when a coated substrate having such an antigen-antibody protein complex is subsequently exposed to a solution containing the same antigenic protein or a solution suspected of containing the same antigenic protein.

That is, even after such exposure, the antigen protein usually no longer binds to the antibody protein. This is because the antigen protein molecules are in close contact with each other in the first antigen protein layer, so all active sites of the antibody protein have already bound to the antigen protein molecules. Thus, in the case of surface immunology, antibody proteins (
If it binds to the surface of the antigen protein layer, the activity of the antibody protein (
That is, there is a problem in that the ability to bind to antigen proteins, cells, or viruses is lost. Furthermore, especially when the adsorbed antigen protein layer is relatively thick, the problem often arises that it is impossible to distinguish between a monomolecular layer and a bimolecular layer of protein on the substrate.

For example, this is true when the antigen protein molecule is at least 10 times larger than the antibody protein molecule, such as a hepatitis-related antigen. Now, in accordance with the present invention, briefly, an immunologically inactive protein is added to a solution containing an immunologically active antigenic protein.

When the substrate is then immersed in such a solution, the surface of the substrate is coated with a monomolecular layer of antigen protein molecules and inert protein molecules by adsorption. The amount of inert protein molecules in that case is selected such that the antigen protein molecules in the adsorption layer are separated by an average of several hundred angstroms by inert protein molecules. When the above-mentioned coated substrate is immersed in a solution containing an antibody protein that specifically reacts with the antigen protein, the antibody protein molecules will chemically bond with the antigen protein molecules, while some of the active sites of the antibody protein molecules will be absorbed. still remains active. As a result, subsequent immersion of the above-mentioned coated substrate in a solution suspected of containing an antigenic protein results in further attachment of the antigenic protein molecule to the active site of the antibody protein molecule bound to the first antigenic protein layer. That will happen.
In this manner, the present invention can be utilized for analysis of solutions for the purpose of detecting the presence of specific antigenic proteins. Furthermore, by alternately immersing the coated substrate described above in solutions containing the same antibody protein and antigen protein, a relatively long chain of the antigen-antibody protein complex can be extended from the surface of the substrate. Such methods can be used to immunologically identify cells and viruses. This is because even if the surface of a cell or virus is irregular, the probability that some of the antibody protein molecules at the end of the chain of an antigen-antibody protein complex will find an antigen site on a specific cell or virus is relatively high. Because I can say it. According to the invention, an immunologically inactive protein is also added to a solution containing an immunologically active, relatively large-sized antigenic protein.

When the substrate is then immersed in such a solution, the surface of the substrate is coated with a monomolecular layer of antigen protein molecules and inert protein molecules by adsorption. In that case, each antigenic protein molecule is completely surrounded by inactive protein molecules. When the above-mentioned coated substrate is immersed in a second solution containing a relatively small particle size antibody protein that specifically reacts with the antigen protein, the antibody protein molecules chemically bond with the antigen protein molecules. Thereby an at least partial bilayer of protein is formed on the substrate. By the way, the amount of inactive protein molecules in the first monolayer is determined by the amount of inert protein molecules that bind to the antigen protein molecules in a larger number than if it were assumed that the antigen protein molecules were in close contact with each other. are chosen so that they are sufficiently isolated. As a result, the contrast between the monolayer and bilayer of protein deposited on the substrate soil is significantly improved.
As described above, the present invention has a monomolecular layer containing only a large-sized antigen protein (for example, a hepatitis-related antigen) and a bimolecular layer that also includes a second layer of a small-sized antibody protein (for example, a hepatitis antibody). It provides a simple method for identification and therefore can be used for the analysis of solutions aimed at determining the presence of antibody proteins. The structure and manner of carrying out the present invention will be best understood from the following description taken in conjunction with the accompanying drawings. Referring first to FIG. 1, there is shown an enlarged elevational view of a portion of a diagnostic device comprising a sheet 10 of a suitable substrate material.

Such substrate materials include metals, glass, mica, plastics,
It can be fused silica, quartz, etc. However, metals are preferred in view of the greatest difference in refractive index relative to proteins, particularly for use as metal slides or metal-coated glass slides. Substrate 10 is dipped into a first solution, which typically consists of saline water, containing the protein of interest (antigen or antibody from a biological point of view).
The protein molecules 11 form a single molecular layer and are adsorbed onto the substrate 10. In explaining the present invention, hereinafter, one molecular layer of protein molecules 11 adsorbed on the surface of the substrate 10 will be referred to as an antigen protein layer. Any protein is adsorbed in such a monolayer, but no further adsorption occurs. That is, the protein attaches to the substrate but not to itself. Therefore, such an antigen protein layer is only a monomolecular layer and cannot be thicker than that. The time required to completely coat the substrate with antigenic protein depends on the concentration of antigenic protein in the solution, the degree of agitation of the solution, and the temperature of the solution. For example, 1%
It takes approximately 30 minutes for the bovine serum albumin solution to completely coat the slide with a monolayer of antigenic protein. After a monolayer of antigenic protein molecules 11 has been formed over substantially the entire surface of the substrate 10, the coated substrate is removed from the solution of the antigenic protein and then contains an antibody protein that specifically reacts with the antigenic protein. immersed in a solution or a solution suspected of containing it.

Such a second solution may contain many components other than the specifically reactive antibody protein to be detected. However, proteins other than the specifically reactive antibody protein do not adhere to the antigen protein layer on the substrate. Therefore, immunological complexing between the antigen protein and the antibody protein that specifically reacts with it occurs only when the specifically reactive antibody protein is present in the second solution. will have a bilayer of protein. The time required for deposition of the second monolayer (of antibody protein molecules 12) on the coated substrate also depends on the concentration of antibody protein in the solution, the degree of agitation of the solution, and the temperature of the solution. In the case of antibody proteins in serum, the time can be as much as 1 tad longer. Such a second monolayer depends on the three factors mentioned above;
It may be only partial or it may be substantially complete. As shown in FIG. 1, when the antigen protein layer substantially completely covers the substrate 10,
That is, when the distance between adjacent antigen protein molecules is minimal, the antibody protein molecule uses substantially all of its active sites to bind to the antigen protein molecule and lose its activity.

In other words, substantially all active sites on the antibody protein molecule 12 bind to corresponding binding sites on the antigen protein molecule 11. Such a substrate coating method is described in a US patent application entitled "Method and Apparatus for the Detection and Purification of Proteins and Antibodies" filed on June 26, 1972 and entrusted to the same trustee as the present invention. It is described in the specification of No. 266278. No. 1
In the case of the figure, since one molecular layer of antigen protein molecules 11 is adsorbed over substantially the entire surface of the substrate 10, one molecular layer of antibody protein molecules 12 specific to the antigen protein binds thereto. Once the bilayer is formed, it is no longer possible for additional proteins, whether antigen or antibody, to bind.

This is because there are no more active sites left on the antibody protein molecules 12 in the second monolayer. By the way, in the present invention, at the same time that the antibody protein molecules 12 in the second monomolecular layer are bound to the antigen protein molecules 11 in the first monomolecular layer, they are further bound to additional antigen protein molecules during the subsequent immune reaction. A method has been discovered for maintaining a portion of the active site of the antibody protein molecule 12 in an active state so that it can be used. The basis of the invention relates to the step of forming a monomolecular layer of adsorbed antigen protein molecules 11 on the surface of a substrate 10, as shown in FIG. For convenience of illustration, the antibody proteins are shown as being of the IgG type, but this does not mean that the other four types of antibody proteins described above cannot be used in the present invention. ``Substrate 1 for forming an immunologically complex protein multilayer membrane
After selecting 0, such substrate is immersed in a first solution, which typically consists of saline containing the antigenic protein, as in the method described above in connection with FIG. However, in contrast to the above-mentioned method, in the method of the present invention an immunologically inactive protein is added to the first solution prior to immersing the substrate. As a result, as shown in FIG.
The adsorption layer formed on the surface of the substrate will consist of antigenic protein molecules 11 separated from each other (and surrounded along the substrate surface) by inert protein molecules 20. The amount of inert protein added to the first solution is selected so that the average distance between adjacent antigen protein molecules 11 attached to the substrate 10 is generally several hundred angstroms. The substrate 10 thus coated with a monolayer (of antigenic protein and inert protein) is removed from the first solution and then
is immersed in a second solution containing an antibody protein specific for the antigen protein in the solution.

Since the spacing between active sites of antibody protein molecules 12 (of IgG species) is 200 angstroms, most of the antibody protein molecules 12 in the second solution bound to antigen protein molecules 11 still possess active sites ( Depending on the type of antibody protein molecule, one or more active sites may remain per molecule). As a result of the antibody protein molecule 12 being maintained in an active state in this manner, it becomes possible to further bind to additional antigen protein molecules during subsequent immune reactions. Of course, it goes without saying that one antibody protein molecule cannot bind to more than one binding site on one antigen protein molecule. Therefore, as shown in FIG. 2, adjacent antigen protein molecules 1
1 are sufficiently isolated from each other by the inert protein molecule 20, the antibody protein molecule 12 is separated from the antigen protein molecule 11.
Generally, the active site 12a of each antibody protein molecule 12 will be maintained in an active state for subsequent immune reactions. In this way, the main objective of the present invention is to bind the antibody protein molecule to the surface (of a monolayer of antigenic protein) while at the same time maintaining a portion of the active site of the antibody protein molecule in an active state for subsequent immune reactions. One of the objectives has been achieved. When the antigenic protein is human serum albumin, the inert protein is, for example, ovalbumin, and the antibody protein is obtained from rabbit serum.
In the case of a 1% human serum albumin solution, the concentration of ovalbumin as an inactive protein is within the range of 1 to 10%. Referring now to FIG. 3, after being removed from the antibody protein solution (i.e., subjected to the steps shown in FIG. 2), the bilayer coated substrate 10 is placed in a first solution. The sample is immersed in a solution containing, or suspected of containing, the same antigenic protein as the case.

As a result, an immune reaction occurs again, so the antigen protein molecule 3
0 selectively binds to the remaining active site 12g of the antibody protein molecule 12. Such methods can be utilized for the analysis of solutions for the purpose of easily detecting the presence of specific antigens. It can also be used to extend multiple chains of antigen-antibody protein complexes starting from the surface of the substrate 10, thereby forming a multilayer membrane of immunologically complex proteins. Such a multilayer film can be easily confirmed by observing the coated substrate using an optical instrument such as an ellipsometer. Alternatively, such polylayer films can be electrically tested, as detailed in the aforementioned U.S. Patent Application No. 266,278 and U.S. Patent Application No. 384,113 filed July 30, 1973. . For this purpose, the capacitance of a capacitor consisting of a metal or metallized substrate, a conductive plate formed by a mercury drop or other suitable electrode, and a dielectric formed by a protein layer is measured. Furthermore, such multilayer films can be optically inspected by visual observation, as described in the above-mentioned patent application. To this end, it is sufficient to measure the time required for visible amalgam to form between the mercury droplet placed on the protein layer and the metal thin film coated on the substrate. Finally, and most importantly, such polylayer films can also be inspected optically by reflected or transmitted light as described in the above-mentioned patent applications. Among these optical inspections using reflected or transmitted light, the first technique that has brought success (optical inspection using transmitted light) is shown below. First, a substrate 10 made of a light-transmissive material such as glass, plastic, fused silica, mica, quartz, etc. is provided. Note that a suitable substrate material is glass, and in particular, it is convenient to use a glass slide for a microscope. Such a substrate is coated with a number of metal spheres by vapor deposition of a metal (eg indium). For this purpose, for example, about 5×1 mercury column
Indium may be slowly deposited from a tantalum boat onto a glass substrate in a 0-5 mL commercial vacuum. Since indium atoms exhibit high mobility on the surface of the substrate and hardly wet the glass substrate, indium deposited on the substrate aggregates into small spheres. Any metal can be used as long as it has similar properties and thus forms a sphere when deposited onto a substrate. In fact, in addition to indium, gold, silver,
Success has also been achieved using tin and lead. This metal deposition is continued until the substrate becomes a light brown color. At this point, the metal sphere has a diameter on the order of 1000λ. The exact dimensions of the metal sphere are not critical, but its diameter should be equal to the wavelength of most of the visible spectrum. The next step involves placing a substrate 10 coated with metal spheres in a first solution containing an immunologically active first protein (e.g., an antigenic protein) and an immunologically inactive protein.
It is to soak. The first protein and the inactive protein are also deposited in a monolayer on the substrate and metal sphere. After the monolayer is formed, the coated substrate is removed from the first solution and then immersed in a second solution containing a protein that specifically reacts with the first protein. The result is a bilayer of protein attached to the substrate and metal spheres, as in FIG. 2 (apart from the absence of metal spheres). The coated substrate is removed from the second solution and then immersed in a third solution containing or suspected of containing the same first protein as in the first solution. If the first protein is present, a third monolayer (or partial monolayer) is formed on the substrate. The coated substrate thus obtained is observed under transmitted light, and a determination can be made from the appearance of the coated substrate as to the thickness of the protein layer and thus the presence or absence of the protein of interest. The thickness of the protein layer then corresponds to the change in brown tone observed in the coated substrate. Such changes are very pronounced and detection of the protein layer is therefore quite a simple task. If a metal sphere sitting alone on a substrate appears a certain shade of brown, a metal sphere coated with a monolayer of protein appears a darker shade of brown, and a metal sphere coated with a bilayer of protein appears a certain shade of brown. exhibits a darker shade of brown, and metal spheres coated with a trilayer of protein exhibit an even darker shade of brown. Such a detection method is
It has been shown that electromagnetic radiation is significantly scattered by a conductive sphere with a diameter equal to the wavelength of most of the incident energy, and that the degree of scattering is largely determined by the thin insulating coating to which the sphere is attached. Based on facts. Next, a second technique (optical inspection with reflected light) that has led to success is presented below. First, a gold base is prepared. However, for economical reasons, it is preferable to use a thin metal plated on another metal. When such a substrate is immersed in the first solution as described above, the first protein 11 and one molecular layer of the inactive protein are adsorbed. By the way, gold has an absorption band within the visible spectrum, which gives it a unique color. This fact provides the operating principle of such optical inspection techniques. As the metal substrate, it is convenient to first use a thin indium layer and then a glass slide coated with metal. Helps improve. The relative reflectance of the gold substrate as a function of wavelength gives gold its characteristic bright yellow color when no protein layer is attached. However, the presence of a monolayer of protein on the substrate gives the test slide (i.e., the substrate) a dull yellow appearance. The test slide will then have a bilayer of protein and its It has a green appearance due to its reflective properties. The presence of a further third monolayer gives an even more green appearance. Of the tests conducted to date, optical inspection using transmitted light using a substrate containing a metal sphere and optical inspection using reflected light using a gold substrate appear to be the most useful overall. Ta. Furthermore,
It was also confirmed that these two techniques have different sensitivities depending on the thickness of the protein layer in question. More specifically, the maximum sensitivity of techniques using substrates containing metal spheres is achieved for film thicknesses below about 200λ, whereas the maximum sensitivity of techniques using gold substrates is achieved for film thicknesses greater than 30λ. . Therefore, the type of substrate 10 to be used in each analysis is determined depending on the detection method used. That is, it is determined whether a metal slide or a metal-coated glass slide as a substrate should have a flat metal (e.g. gold) coating or metal (e.g. indium) spheres, also as described in the aforementioned patent application. will be done. Note that for the sake of simplicity, the base body 10 is illustrated as having a flat surface;
It should be understood that the surface to which a monolayer of is adsorbed may include the metal spheres described above. As shown in FIG. 3, the method of the present invention for forming immunologically complex protein multilayer films (trilayers) is particularly important when testing solutions for certain proteins.

Such proteins include hormones, in view of the general availability of themselves and antisera against them. Indeed, if the third monolayer is the hormone in question, it can be easily detected. lastly,
The method of the present invention for forming immunologically complex protein multilayer membranes is also important in the immunological identification of cells and viruses. That is, as shown in FIG. 4, if a large number of chains of antigen-antibody protein complexes are extended from the surface of the substrate 10, some of the antibody protein molecules at the end of the chain will reach the antigen site on the cell or virus 40. The probability of finding is relatively high. Moreover, even if the surface of the cell or virus is irregular, the probability is still high. As is well known, from the cell membrane that surrounds each cell,
Molecules called "transplant antigens" extend outward. cell 4
In the case of a two-layer system for the immunological identification of 0.0, it is the transplanted antigen that forms the first monolayer 11 and the cells of interest constitute the third protein layer. Alternatively, in the case of a four-layer system as shown in Figure 4, the first and third monolayers (11 and 30, respectively) consist of the transplanted antigen. It is likewise known that viruses have an envelope consisting of protein molecules, which can be cleaved off. The first molecular layer in a two-layer system and also the first in a four-layer system for immunological identification of viruses 40.
and it is such protein molecules that form the third monolayer. Note that the antibody proteins (12 and 41, respectively) forming the second and fourth monolayers are, of course, specific to the specific cell or virus to be identified.
As is clear from the above description, according to the present invention, a multilayer film of immunologically complexed proteins is formed on a substrate soil by binding antibody proteins to the surface while maintaining an active state. A novel method is provided.

For this purpose, a sufficient amount of immunologically inactive protein is added to a first solution containing immunologically active antigenic protein. When a suitable substrate is immersed in such a first solution, a monomolecular layer of antigen protein molecules and inert protein molecules is formed on the surface of the substrate by adsorption. In that case,
Antigen protein molecules are separated from each other by inactive protein molecules. After the formation of such a single molecular layer, if the coated substrate is immersed in a second solution containing an antibody protein specific to the antigen protein, the antibody protein molecules will bind to the antigen protein molecules, while the antibody protein molecules will bind to the antigen protein molecules. A portion of the active site of the molecule remains active for subsequent immune responses. Therefore,
The coated substrate can be subsequently dipped into a solution containing, or suspected of containing, the same antigenic protein as in the first solution. By repeating such operations, it is possible to extend many chains of antigen-antibody protein complexes. Referring now to FIG. 5, there is shown an enlarged elevational view of a portion of a diagnostic device also comprising a sheet 10 of a suitable substrate material.

Such substrate materials include metals, glass, mica, plastics,
It can be fused silica, quartz, etc. However, metals are preferred in view of the greatest difference in refractive index relative to proteins, particularly for use as metal slides or metal-coated glass slides. Substrate 10 is dipped into a first solution, which typically consists of saline water, containing the protein of interest (antigen or antibody from a biological point of view).
The protein molecules 11 form a single molecular layer and are adsorbed onto the substrate 10. In explaining the present invention, hereinafter, one molecular layer of protein molecules 11 adsorbed on the surface of the substrate 10 will be referred to as an antigen protein layer. Any protein is adsorbed in such a monolayer, but no further adsorption occurs. That is, the protein attaches to the substrate but not to itself. Therefore, such an antigen protein layer is only a monomolecular layer and cannot be thicker than that. The time required to completely coat the substrate with antigenic protein depends on the concentration of antigenic protein in the solution, the degree of agitation of the solution, and the temperature of the solution. For example, 1η
It takes about 10 minutes for a hepatitis-associated antigen solution at a concentration of 1/ml to completely coat a slide with a monolayer of antigenic protein.
After a monolayer of antigenic protein molecules 11 has been formed over substantially the entire surface of the substrate 10, the coated substrate is removed from the solution of the antigenic protein and then contains an antibody protein that specifically reacts with the antigenic protein. immersed in a solution or a solution suspected of containing it.

In explaining the present invention, the antigen protein molecule 11 is smaller than the antibody protein molecule that specifically reacts with the antigen protein molecule 11 adsorbed on the surface of the substrate 10 to form a second monomolecular layer. It is always assumed to have a large particle size.
The above-mentioned second solution may contain a large number of components in addition to the specifically reactive, small-sized antibody protein to be detected. However, proteins other than the specifically reactive antibody protein do not adhere to the antigen protein layer on the substrate. Therefore, immunological complexing between the antigen protein and the antibody protein that specifically reacts with it occurs only when the specifically reacting antibody protein is present in the second solution.
After a certain period of time, the substrate will have a bilayer of protein. The time required for the second monolayer (of antigen protein molecules 12) to adhere to the coated substrate substrate also depends on the concentration of antibody protein in the solution, the degree of agitation of the solution, and the temperature of the solution. In the case of antibody proteins in serum, the time can be as much as 1 tad longer. Such second monolayer may be only partial or substantially complete, depending on the three factors listed above. As shown in FIG. 5, when the antigen protein layer substantially completely covers the substrate 10, that is, when the distance between adjacent antigen protein molecules is minimal, the antigen protein layer binds to individual antigen protein molecules. The number of antibody protein molecules 12 obtained is limited to one.

This is because the antigen protein molecule surface area available for such immunological conjugation is limited. Incidentally, such a substrate coating method is described in the above-mentioned U.S. Patent Application No. 26.
No. 6278. In the case of Figure 5,
Since one molecular layer of antigen protein molecules 11 is adsorbed over substantially the entire surface of the substrate 10, one molecular layer of small-sized antibody protein molecules 12 specific to the antigen protein is adsorbed thereon. When combined, a bilayer is formed. However, the antibody protein molecules 12 in the second monolayer have a smaller particle size than the antigen protein molecules 11 in the first monolayer;
Moreover, since the second monomolecular layer is not as dense as the first monomolecular layer, it is clear that it is quite difficult to distinguish between a monomolecular layer and a bimolecular layer of protein on the substrate 10. be. A good illustration of this situation is that the antigen protein molecule 11 in the first monolayer is a hepatitis-related antigen, and the antibody protein molecule 1 in the 22nd monolayer is a hepatitis-related antigen.
An example is the case where 2 is an antibody specific to a hepatitis-related antigen. Examination of a coated substrate using an optical instrument such as an ellipsometer also distinguishes between a monolayer (of antigen protein) and a partial bilayer (of antigen-antibody-protein complex) on the substrate. That is not easy. Therefore, such diagnostic tests are of little value since it cannot be easily determined whether the second liquid fluid actually contains antibodies to hepatitis-associated antigens. However, in the present invention, a larger number of antibody protein molecules 12 are bound to the antigen protein molecules 11 in the first monomolecular 5-layer, thereby significantly increasing the contrast between the monolayer and bilayer of the protein. A way to improve has been discovered. The basis of the invention relates to the step of forming a monomolecular layer of antigenic protein molecules 11 adsorbed on three surfaces of a substrate 10, as shown in FIG. For convenience of illustration, the antibody protein is Ig
Although it is shown as a type G, other 4 types such as those mentioned above
This is not to say that species antibody proteins cannot be used in the present invention. After selecting the substrate 10 for forming a bilayer of immunologically complexed proteins, it typically consists of a saline solution containing the antigenic protein, as in the method described above in connection with FIG. The substrate is immersed in the first solution.

However, unlike the above method, in the method of the present invention, an immunologically inactive protein is added to the first liquid solution prior to immersing the substrate. As a result, as shown in Figure 6,
The adsorption layer formed on the surface of the substrate 10 will consist of relatively large antigen protein molecules 11 separated from each other (and surrounded along the substrate surface) by inert protein molecules 20. In order to make a larger surface area (and thus more binding sites) of the antigen protein 11 available for binding to antibody protein molecules during subsequent immune reactions, the inert protein molecules 20 are It has a particle size that is at least somewhat (preferably substantially) smaller than 11. The amount of inactive protein added to the first fermentation solution is generally such that the amount of inactive protein is 1 of the total area of the adsorption layer.
/2 to 9/10 of the total area, and the antigen protein is selected so that it occupies 1/2 to 1/10 of the total area. However, this is not a limitation on the invention. This is because the factors that determine the average spacing between adjacent antigen protein molecules include the number of binding sites on each antigen protein molecule and the manner in which such molecules attach to the base, in other words, the binding sites that are kept exposed. This is because the number of can also be considered. In short, in order to take full advantage of the benefits of the present invention, it is necessary that the antibody protein molecule 12 be the antigen protein molecule 1
If the distance between the antigen protein molecules is much smaller than 1, it is desirable to increase the distance between the antigen protein molecules.
2 is only slightly smaller than the antigen protein molecule 11, there is no problem even if the distance between the antigen protein molecules is small. As a result of the antigenic protein molecule 11 having a smaller particle size than the inert protein molecule 20, most of the surface of the antigenic protein molecule is maintained in an exposed state, and the corresponding binding site is thus protected during subsequent immune reactions. In other words, it can be used for binding with antibody proteins. The substrate 10 thus coated with a monolayer (of antigenic protein and inert protein) is removed from the first liquid and then contains antibody proteins specific for the antigenic protein in the first liquid. Immersed in a second solution.

Unlike in Fig. 5, in the case of Fig. 6, the antigen protein molecule is surrounded by inert protein molecules of small particle size.
A greater surface area (and therefore a greater number of binding sites) is exposed on the antigen protein molecule. Therefore, the antigen protein molecules in FIG. 5 can more easily bind immunologically to a larger number of antibody proteins than those in FIG. In this way, in the case of FIG. 6, generally speaking, a larger number of antibody protein molecules 12 are present in each antigen protein molecule 11 than in the case of FIG.
Combine with.

If we compare Figures 5 and 6 here, we can see that between the monolayer and the bilayer of the protein (in view of the large number of antibody protein molecules present in the second monolayer) It is clear that the contrast in the case of FIG. 6 is greater. To give an example, in the first liquid, hepatitis-related antigen occupies 1/4 of the total area of one monolayer, and bovine serum albumin occupies the remaining 3.
Contains hepatitis-related antigens and bovine serum albumin at a concentration that accounts for /4? The second solution is a dilute hepatitis antibody solution. In this way, the above method
It can be used for analysis of solutions for the purpose of easily detecting the presence of antibody proteins against relatively large antigen proteins. The substrate of FIG. 6 thus coated with a bilayer is also coated with a second antibody directed against the antibody protein in the second monolayer to form a third monolayer as shown in FIG. A subsequent immersion in a third solution or serum containing protein can be performed.

Since each antibody protein molecule 12 in the second monolayer has several antigen determinants, it will generally bind to several molecules 30 of the second antibody protein, thereby forming a relatively dense layer. A monolayer of 3 can be formed. The presence of such a dense third monolayer not only indicates the presence of the second monolayer, but also the presence of a monolayer (of the antigen protein) and a second monolayer (of the antigen protein and the first antibody protein). This results in a significant improvement in the contrast between molecular layers. In this case, the presence of inactive protein molecules 20 between large-sized antigen protein molecules 11 in the first monolayer also means that the second
It also serves to prevent non-specific proteins in serum containing antibody proteins from adhering to the substrate. in this way,
As a result of isolating large-sized antigen protein molecules using inert proteins and coating the substrate surface around each antigen protein molecule, antibody protein molecules that specifically bind to each antigen protein molecule are isolated. There will be a dual benefit in that the number of non-specifically bound protein molecules on the substrate will be reduced at the same time as the number increases. All in all, in view of the fact that immersing the coated substrate in the third solution significantly improves the contrast between the monolayer and the bilayer (and ultimately the trilayer), the It can be said that the step of forming a monomolecular layer in step 3 is useful when analyzing a second liquid solution suspected of containing the first antibody protein. 6th
The presence of a second monolayer (of antibody protein molecules 12) in the case of Figure 7 and also of the third (of antibody protein molecules 12) in the case of Figure 7
The presence of a monolayer (of antibody protein molecules 30) can be easily confirmed by observing the coated substrate using an optical instrument such as an ellipsometer. Alternatively, such protein layers can be electrically interrogated, as detailed in the aforementioned US Patent Application Nos. 2,662,78 and 3,841, and as explained above. For this purpose, the capacitance of a capacitor consisting of a conductive plate formed by a metal substrate or metal coating and a mercury drop or other suitable electrode and a dielectric formed by a protein layer is measured. . Furthermore, such protein layers can be optically inspected by visual observation, as described in the aforementioned patent applications and explained above with respect to protein multilayers. To this end, it is sufficient to measure the time until a visible amalgam is formed between the mercury droplet placed on the protein layer and the metal thin film coated on the substrate. Finally, and most importantly, as described in the aforementioned patent application and also explained above with respect to multilayer films of proteins, such protein layers can be optically illuminated by reflected or transmitted light. It can also be inspected. However, in this case, after the coated substrate is removed from the second hemolytic solution, a protein that specifically reacts with the protein in the second hemolymph solution (for example, a second antibody protein for the first antibody protein) is added to the coated substrate. By immersion in the third solution,
A third monolayer (or partial monolayer) is formed over the second monolayer, if present. Such coated substrates are observed using transmitted light. From the appearance of the coated substrate, a determination can be made as to the thickness of the protein layer attached thereto, and thus the presence or absence of the second monomolecular layer. To further improve the contrast between monolayers and bilayers of protein, a drop of a solution containing the antigenic protein and the inert protein can be placed on the substrate 10. When the substrate coated with droplets is immersed in a liquid solution containing only inactive proteins, as shown in FIG.
A monolayer consisting of a small antigen-inactive protein region completely surrounded by inactive protein 20 is formed. Such methods help solve the problem of non-specific adsorption when antibody proteins are present in serum. That is, the inert protein coated on the remaining surface of the substrate prevents non-specific proteins in serum from adhering to the substrate, thereby providing improved contrast. As a result of the development of simple methods such as those described above, it is now possible to detect hepatitis with a test that has the same sensitivity as standard radioimmunoassay tests.

Finally, methods such as those described above are also important for immunological identification of viruses and enzymes.

All virus-type antigens are larger than the antibodies specific for them, whereas enzyme-type antigens can be larger or smaller depending on the type. The method for identifying specific viruses or large-sized enzymes, known as inhibition tests, is performed as follows.
A first solution, typically consisting of saline containing the specific virus or large size enzyme and inactive protein, is prepared and the substrate is immersed therein. Alternatively, a drop of such a solution is placed on the substrate and the substrate is then immersed in a solution containing only inert protein. After the virus or enzyme (ie, antigen) and one molecular layer of inert protein have been adsorbed onto the substrate, the substrate is removed from the first liquid solution. On the other hand, a sample of human serum to be tested for the presence of a particular virus or enzyme is pretreated with a mixture of known antibodies against the particular virus or enzyme. In this case, an amount of antibody is used that is sufficient to immunologically eliminate the presence of a particular virus or enzyme in the serum sample. The monolayer-coated substrate is then immersed in the pretreated serum sample and, after removal from the serum sample, examined according to any of the techniques described above.
If examination of the substrate reveals the presence of only one monolayer, the human serum to be tested is known to contain the particular virus or enzyme. However, the detection of a bilayer indicates that the human serum to be tested does not contain the particular virus or enzyme. This is because antibodies cannot immunologically bind to antigens (i.e., specific viruses or enzymes) in serum samples, and therefore cannot bind to antigens in the first monolayer adsorbed onto the substrate. Because it will be. As is clear from the above description, by adding a sufficient amount of immunologically inactive protein to the first solution containing the immunologically active antigenic protein, one of the proteins in the surface immunoassay can be detected. A novel method for improving the contrast between molecular and bilayer layers is also provided in accordance with the present invention. Although the invention has been described with respect to specific embodiments, it will be obvious that other modifications and variations are possible in light of the above description. For example, it is also possible to prepare a solution containing only an inactive protein separately from the antigen protein and immerse the substrate therein.
Such immersion is performed as a preliminary step or as an intermediate step between immersion in a dilute solution of antigen protein and a solution of antibody protein. Alternatively, if the antibody protein is present in serum, a dilute solution of the antigen protein can be used to form an incomplete monolayer on the substrate. At this time, inactive proteins are omitted from the liquid. This is because (non-specific) proteins in serum attach to the substrate as inactive proteins and serve to isolate antigenic protein molecules from each other. The invention can also be used to detect enzymes. To do this, the substrate is immersed in a first solution containing the enzyme as the antigenic protein and bovine serum albumin as the inactive protein, and the antibody protein against the enzyme (obtained from a rabbit previously injected with the enzyme) is added. The substrate is immersed in a second solution containing the enzyme, the substrate is immersed in a third solution suspected of containing the enzyme, and then the substrate is immersed in a third solution containing the enzyme.
The substrate may be tested for the presence of a monolayer of . Furthermore, although the immunologically active protein adsorbed onto the substrate surface has been described as an antigen protein, it goes without saying that such a protein may also be an antibody protein. In that case,
The protein forming the second monolayer is an antigen protein specific to the antibody protein, and the protein forming the third monolayer is again an antibody protein. Finally, it is also clear that a method is possible in which the proteins forming the first monolayer are antibody proteins and the proteins forming the second monolayer are specific antigenic proteins. This method is useful when the antibody protein molecule is larger than the specific antigen protein molecule, such as insulin antibodies. Next, the embodiments of the present invention are listed below.

1. The step of coating the substrate with a monomolecular layer of an immunologically active antigenic protein and an immunologically inactive protein prepares a first hemolyte containing the antigenic protein, and By adding a sufficient amount of the inert protein to the solution and immersing the substrate in the first solution, the protein molecules are sufficiently isolated from each other by the inert protein molecules. coating said substrate with a monolayer of said antigenic protein and said inert protein, and then removing said substrate thus coated with said monolayer from said first solution. Method.

2. The step of coating the substrate with a monolayer of an immunologically active antigenic protein and an immunologically inactive protein includes preparing a first solution containing the inert protein and coating the substrate with a monolayer of an immunologically active antigenic protein and an immunologically inactive protein. immersing said substrate in said tenth solution sufficient to coat it with a partial monolayer of inert protein; removing said partially coated substrate from said first solution; A second milk solution containing an antigen protein is prepared, and the substrate is immersed in the second milk solution, so that molecules of the antigen protein are isolated from each other by molecules of the inactive protein. It consists of coating the remaining surface of said substrate with a partial monolayer of said antigenic protein and then removing said substrate thus coated with a monolayer from said second filtrate.

Method according to the claims 3. The step of coating the substrate with a monomolecular layer of an immunologically active antigenic protein and an immunologically inactive protein includes preparing a dilute first solution containing the antigenic protein and coating the substrate with a monolayer of an immunologically active antigenic protein and an immunologically inactive protein. immersing the substrate in the first solution sufficient to coat it with a partial monolayer of the antigenic protein; removing the partially coated substrate from the first solution; By preparing a second liquid containing an inactive protein and immersing the substrate in the second liquid, the ZZ molecules of the antecedent antigen protein are isolated by the molecules of the inactive protein. The remaining surface of the substrate is coated with a partial monolayer of the inert protein, and the substrate thus coated with a monolayer is then coated with the second monolayer. A method as claimed in the preceding claims, comprising removing it from a liquid.

4. The step of coating the substrate with a monolayer of an immunologically active antigenic protein and an immunologically inactive protein includes preparing a dilute first solution containing the antigenic protein and coating the substrate with a monolayer of an immunologically active antigenic protein and an immunologically inactive protein. immersing the substrate in the first solution sufficient to coat it with a partial monolayer of the antigenic protein, and removing the partially coated substrate from the first solution; Then, in the case where the substrate is immersed in a solution of an antibody protein that specifically reacts with the antigen protein, as a result of the presence of the antibody protein in the serum, other than the antibody protein in the serum The protein acts as the inert protein and attaches to the substrate, so that the substrate is free from the antigenic protein and the inactive protein such that the molecules of the antigenic protein are separated by the molecules of the inactive protein. 3. The method of claim 1, wherein molecules of the antibody protein in the serum form a second monolayer.

5. The substrate coated with the bilayer is removed from the antibody protein scarlet liquor and subsequently placed in a third solution suspected of containing the same antigenic protein as in the first solution. to cause an immune reaction with the antibody protein, thereby chemically binding the active sites remaining on the molecules of the antibody protein to the molecules of the antigenic protein in the third aqueous fluid; 2. The method according to item 1, further comprising the steps of determining the presence or absence of the antigen protein in the third liquid by examining whether or not a third protein layer is present on the substrate. Method.

5. In the case where the step of preparing the first liquid solution containing the antigen protein consists of separating the antigen protein present on the outermost part of a specific cell or virus and then preparing a liquid solution thereof, The bilayer-coated substrate is removed from the solution of the antibody protein, and the substrate is subsequently immersed in a third liquid solution suspected of containing the specific cell or virus. binds to the antigenic site on the specific cell or virus in the third solution, and then tests for the presence of a third protein layer on the substrate to detect the antigen in the third solution. The steps of determining the presence or absence of particular cells or viruses are additionally included.

The method according to item 1 above. 7. The bilayer-coated substrate is removed from the antibody protein solution and subsequently coated with the first bilayer. Exposure of the substrate to a third solution containing the same antigenic protein as in the solution causes an immune reaction with the antibody protein, thereby freeing the active sites remaining on the molecules of the antibody protein from the third solution. The steps of step 3 of binding with the antigen protein molecules in the liquid fluid are additionally included.

The method according to item 1 above. 8. The substrate is removed from the third liquid solution and subsequently exposed to a fourth yeast solution containing the same antibody protein as that in the second solution to cause an immune reaction. molecules of the antibody protein in the fourth solution are combined with molecules of the antigen protein derived from the third liquid solution while maintaining the active site of the substrate in an active state, thereby removing the antigen from the surface of the substrate. Steps of chain elongation of the single antibody protein complex are additionally included;
The method according to item 7 above.

9. The substrate with the chain of antigen-antibody protein complexes extending from its surface is removed from the fourth solution, and subsequently cells or cells specific for the antibody protein in the chain of antigen-antibody protein complexes are removed from the fourth solution. The substrate is exposed to a fifth liquid solution suspected of containing a virus, the substrate is removed from the fifth solution, and the active sites remaining on the molecules of the antibody protein are removed from the cell or virus. The above-mentioned fifth
9. The method according to item 8, further comprising the steps of determining the presence or absence of the cells or virus in the solution.

10. Said first? The step of adding a sufficient amount of the inert protein to the solution is an amount selected such that the average spacing between adjacent molecules of the antigenic protein adsorbed on the surface of the substrate is generally several hundred angstroms. 2. The method of claim 1, comprising adding said inert protein.

11. The antibody protein is of the immunoglobulin IgG species.

The method according to item 1 above. 12. The antigen protein in the first fluid is an enzyme, the inactive protein is bovine serum albumin, and the antibody protein in the second fluid is obtained from a rabbit that has been previously injected with the enzyme. be.

12. The method according to item 11 above. 13. A metal-coated slide is selected and the slide is coated with an immunologically active first protein and an immunologically inert protein such that molecules of the first protein are separated by molecules of an inert protein. coating with a monomolecular layer of protein and then exposing said slide to a liquid solution of a second protein that specifically reacts with said first protein to cause an immunoreaction with said first protein; the second protein molecules combine with the first protein molecules to form a bilayer on the slide, while the amount of inactive protein in the monolayer increases. an active site characterized in that the average spacing between adjacent molecules of the first protein is sufficiently large so that the molecules of the second protein possess active sites for subsequent immune reactions; A method of binding immunologically active proteins to a surface while maintaining its condition.

14. said step of coating said slide with a monolayer of an immunologically active first protein and an immunologically inactive protein, preparing a first fluid containing said first protein; A sufficient amount of the inactive protein is added to the first fermentation solution, and the slide is immersed in the first fermentation solution, so that one molecule of the first protein and the inactive protein is added to the slide. layer and then the slide thus coated with a monolayer is coated with said first layer. 14. The method of claim 13, comprising removing from the liquid.

15. The first protein is an antigen protein, and the second protein is an antigen protein.
The protein is an antibody protein specific to the antibody protein.

The method according to item 13 above. 16. The first protein is an antibody protein, and the second protein is an antigen protein specific to the antibody protein.

The method according to item 13 above. 17. Select a metal-coated slide, prepare a liquid solution containing immunologically active antibody proteins, add a sufficient amount of immunologically inactive protein to the solution, and then place the slide in the liquid solution. at least a portion of the slide is coated with a monomolecular layer of the antibody protein and the inert protein such that the antibody protein molecules are sufficiently isolated by the inert protein molecules upon exposure to As a result of these steps, the antibody protein molecules possess a larger number of active sites for subsequent immune reactions than would be the case if the antibody protein molecules were sealed together. and
A method of binding antibody proteins to surfaces while maintaining their active state.

18. A suitable substrate is selected and the substrate is coated with the immunologically active antigenic protein and more by molecules of the inert protein such that the molecules of the antigenic protein are separated from each other and surrounded along the surface of the substrate. coating with a monolayer of an immunologically inert protein of small particle size, and then exposing the substrate to a solution of an antibody protein that specifically reacts with the antigenic protein and has a smaller particle size. As a result of the steps involved in causing an immune reaction with the antigenic protein, there are no relative molecules on the antigenic protein molecules because they are surrounded along the surface of the substrate by molecules of the inert protein of small particle size. A relatively large number of binding sites are exposed, and therefore a relatively large number of molecules of the antibody protein bind to molecules of the antigen protein to form a bilayer on the substrate, thus allowing the small particle size of the antigen to bind to molecules of the antigen protein. Because the second monomolecular layer consisting of protein molecules is relatively dense, the monomolecular layer of protein during the examination of the substrate is smaller than that in the case where the molecules of the antigen protein are assumed to be in close contact with each other. and a method for improving contrast during a surface immunoassay using a relatively large antigen protein, characterized in that the contrast between bilayers is significantly improved.

19. A metal-coated slide is selected and the slide is coated with an immunologically active first protein such that molecules of the first protein are separated from each other and surrounded along the surface of the substrate by molecules of an inert protein. and coated with a monolayer of immunologically inactive protein of smaller particle size,
Next, the steps of exposing the slide to a liquid solution that specifically reacts with the first protein and having a smaller particle size than the second protein to cause an immunoreaction with the first protein; As a result, molecules of the inert protein occupy 50-90% of the surface area of the slide and correspondingly molecules of the first protein occupy 50-10% of the surface area.
Since the amount of the inactive protein is selected such that it occupies a large number of binding sites, there are more binding sites on the first protein molecule than if it were assumed that the first protein molecules were in close contact with each other. The exposed and therefore relatively large number of molecules of said second protein combine with molecules of said first protein to form a bilayer on said slide, so that said second protein of small particle size
Because the second monolayer consisting of protein molecules is relatively dense, the protein concentration during examination of the slide is greater than if it were assumed that the first protein molecules were in close contact with each other. A method for improving contrast during surface immunoassays using immunologically active proteins of relatively large particle size, characterized by significantly improved contrast between monolayers and bilayers. .

20. Select metal-coated slides containing immunologically active proteins? Molecules of the active protein are prepared by preparing a solution, adding to the solution a sufficient amount of an immunologically inert protein having a particle size smaller than the active protein, and then exposing the slide to the solution. coating at least a portion of the slide with a monolayer of the active protein and the inactive protein such that the molecules of the inactive protein are sufficiently isolated from each other and surrounded along the surface of the slide; As a result of these steps, a greater number of binding sites are exposed on the active protein molecules than would be possible assuming that the active protein molecules were in close contact with each other, thus making it easier for the slide to undergo subsequent immune reactions. Contrast during surface rabies testing with relatively large particle size immunologically active proteins, characterized by improved contrast between monolayers and bilayers of proteins during testing. How to improve.

Brief Explanation of Page 1 Figure 1 shows that the antigen protein is first immersed in a solution containing an antigen protein by a known method, and then the antigen protein is immersed in a solution containing the antigen protein. an elevational view of the substrate after being immersed in a solution of a specific antibody protein;
FIG. 2 is an elevational view of a substrate subjected to two dipping steps in the same manner as the substrate of FIG. 1, but the first dipping step was according to the invention; FIG. Elevated view of the substrate of FIG. 2 after further immune reaction by immersion in a solution containing the protein, FIG. 4 containing the same antibody protein? Elevated view of the substrate of FIG. 3 when the antibody protein molecule at the end of the chain of the antigen-antibody protein complex finds the antigenic site on the virus or cell to be immunologically identified after being re-immersed in the solution. , FIG. 5 shows the results after first being immersed in a filtrate of a large-sized antigen protein by a known method and then immersed in a filtrate of a small-sized antibody protein specific for the antigen. 6 is an elevational view of a substrate which has been subjected to two dipping steps in the same manner as the substrate of FIG. 5, but where the first dipping step is in accordance with the invention; FIG. 7 is an elevational view of the substrate of FIG. 6 after undergoing further immunoreaction by immersion in a solution containing an antibody protein against the antibody protein in the second solution, and FIG. 1 is a plan view of a substrate having a small antigen-inactive protein region surrounded by inactive protein according to the present invention; FIG.

In the figure, 10 is a substrate, 11 is an antigen protein, 12 is an antibody protein against the antigen protein 11, 20 is an inactive protein, 30
represents the same antigen protein as antigen protein 11, 40 represents a cell or virus, and 41 represents the same antibody protein as antibody protein 12.

Claims (1)

[Scope of Claims] 1. Select a suitable substrate, and prepare the substrate in such a way that molecules of the immunologically reactive first protein are separated by molecules of the immunologically inactive protein. coated in a monolayer with a mixture of said first protein and an immunologically inert protein, and then a solution of an immunologically reactive second protein that specifically reacts with said first protein. exposing the coated substrate to a substance to cause an immunoreaction with the first protein, so that molecules of the second protein bind to molecules of the first protein and bind to the substrate. On the other hand, compared to the case where the monolayer of the protein does not contain immunologically inactive proteins, the molecules of the second protein are more susceptible to subsequent immune reactions after the formation of the bilayer. characterized by possessing a significantly large number of active sites for
A method for detecting antigens or antibodies by forming a multilayer membrane of immunologically complex proteins. 2 Selecting a suitable substrate and infusing the substrate with immunologically active proteins such that molecules of the antigenic protein are separated from each other and surrounded along the surface of the substrate by molecules of the immunologically inactive protein. A mixture of an antigen protein and an immunologically inactive protein with a smaller particle size is coated in a single molecular layer, and then a solution of an antibody protein that specifically reacts with the antigen protein and has a smaller particle size. exposure of said substrate to cause an immune reaction with said antigenic protein, resulting in encirclement along the surface of said substrate by molecules of said immunologically inactive protein of small particle size. Therefore, a relatively large number of binding sites are exposed on the molecules of the antigen protein, and therefore a relatively large number of molecules of the antibody protein bind to the molecules of the antigen protein, forming two molecules on the substrate. layer, so that the second monolayer consisting of molecules of the antibody protein of small particle size is relatively dense, so that the monolayer containing the antigenic protein is an immunologically inactive protein. A surface using an antigenic protein having a relatively large particle size, characterized in that the contrast between a single molecular layer and a bimolecular layer of the protein during examination of the substrate is significantly improved compared to a case where the substrate does not contain A method according to claim 1, useful for improving contrast during immunological tests.