CH617012A5 - Process for the preparation of substrates with a bimolecular coating and the use thereof. - Google Patents

Description

The present invention relates to a method for the production of substrates bimolecularly coated by means of complex-bound proteins and the use thereof for the detection of further immunologically reactive substances.

Immunological reactions are highly specific biochemical reactions in which a first protein known as an antigen combines with a second protein which is specific to the antigen and is known as an antibody and thus forms an immunologically complexed protein. Immunological reactions that take place in a biological system, such as an animal or a human, are vital for fighting diseases. In a biological system, the entry of a foreign protein, i.e. H. of the antigen, the biological system to produce the specific antibody proteins to the antigen in a process that is not yet fully understood. The antibody-protein molecules have available chemical combination or binding sites that complement those on the antigen molecule so that the antigen and the antibody can chemically combine to form the immunologically complexed protein.

Because antibodies are generated by biological systems in response to the invasion of foreign proteins into such systems, the detection of antibodies present in a biological system is of medical diagnostic value for the determination of the antigens to which the system has been exposed. Conversely, the detection of certain antigens in a biological system has medical diagnostic value. Examples of diagnostic detection of antigens include the detection of HCG protein molecules in the urine as evidence of pregnancy and the detection of hepatitis-related antigen molecules (HAA) in the blood of prospective blood donors. In order to carry out such diagnostic tests, the appropriate protein must be obtained from at least one immunologically reactive couple.

The only known source of an antibody protein is a living biological system. Furthermore, it is currently only known to vertebrates that they show immunological reactions to the introduction of a foreign protein. So z. For example, many antibodies have been found in the blood serum of animals and humans exposed to this corresponding antigen. However, many antigens can be produced in a controllable manner in laboratory cultures. However, some antigens, such as B. The antigens associated with hepatitis are currently, like antibodies, only available from higher living biological systems.

Most antigens are proteins or contain proteins as an essential component, while all antibodies are proteins. Because proteins are large, high molecular weight molecules, i. H. Polymers consisting of chains of variable numbers of amino acids can each have some binding sites on the antigen and antibody protein. The five main classes of antibodies (immunoglobulins IgG, IgM, IgA, IgE and IgD) are each obviously characterized by at least two heavy (long) peptide chains of amino acids and at least two light (short) peptide chains of the acids, with the bond between the amino acid units is known as a peptide bond. These heavy and light peptide chains are oriented in the general shape of the letter “Y” and the active or combination sites are the extreme ends of the two arms of the Y-shaped antibody for the IgG antibody.

Immunological reactions can be detected using various techniques, including the use of a suitable substrate, such as a metallized one

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Glass or a metal plate. If the substrate is exposed to a solution of the antigen, the antigen is physically adsorbed in a dense monomolecular layer on the surface of the substrate. Subsequently exposing the antigen-coated substrate to a serum which contains antibodies for the antigen leads to an immunological reaction in which the antibodies selectively attach to the antigens by means of the binding sites on the antibody molecule which complement those on the antigen molecule and thereby form at least one bimolecular partial layer of immunologically complexed protein on the substrate surface.

The problem becomes apparent when the coated substrate containing the antigen-antibody complex protein is subsequently exposed to a solution that contains or is expected to contain the same antigen. This subsequent exposure will generally not lead to a further binding of antigen to the antibody, since all active sites of the antibodies are already bound to the first antigen layer, since the active antigen molecules are at a very short distance from one another. For example, there is a problem with surface immunology that the antibodies bind to an (antigen) surface and lose their activity (i.e. their ability to further bind to an antigen, a cell or a virus).

The other problem that often arises is the inability to distinguish between the monomolecular and bimolecular layers of protein on the substrate, especially when the adsorbed (antigen) layer is relatively thick, as in the case of the HAA layer, because that the antigen molecule associated with the hepatitis is at least ten times as large as the antibody to the HAA.

The process according to the invention for the production of bimolecular layer coverings on substrates, the layer coverings containing immunologically complex bound proteins, and the use of these bimolecularly coated substrates for the detection of further immunologically reactive substances are defined in the preceding patent claims 1 and 13.

According to a particular embodiment of the present invention, an immunologically inert protein is added to a solution which contains an immunologically reactive antigen. A substrate is then immersed in the solution and the surface of the substrate is coated by adsorption with a monomolecular layer of antigen molecules and inert protein molecules. The amount of the inert protein molecules is preferably sufficient for the adsorption layer to consist of reactive antigen molecules which are separated from one another by an average of a few hundred by the inert protein molecules. Subsequent immersion of the coated substrate in a solution containing an immunologically reactive antibody protein specific to the antigen protein leads to a chemical association of the antibody molecules with the antigen molecules, but the remaining antibody sites remain active. As a result, the subsequent immersion of the coated substrate in a solution that is believed to contain the antigen results in further binding of the antigen to the active sites of the antibodies bound to the first antigen layer. The present invention can therefore be used in the analysis of a solution for detecting the presence of a specific antigen. Subsequent immersion of the coated substrate in solutions that alternatively contain the same antibody and the same antien can build up relatively long chains of antigen-antibody complexes from the surface of the substrate. The method can be used to immunologically identify cells or viruses because the probability of some of the antibody molecules finding an antigen site on certain cells or viruses at the ends of the chains of the antigen-antibody complexes is relatively high, regardless of the irregularity the surface of the cell or virus.

Furthermore, according to a particular embodiment of the present invention, an immunologically inert protein is added to a solution which contains a relatively large immunologically reactive antigen protein. A substrate is then immersed in the solution and the surface of the substrate is coated by adsorption with a monomolecular layer of antigen protein molecules and inert protein molecules, in which each antigen molecule is generally surrounded by inert protein molecules. Subsequent immersion of the coated substrate in a second solution, which contains relatively small immunologically reactive antibody proteins specific to the antigen protein, leads to a chemical binding of the antibody molecules to the antigen molecules with formation of at least one bimolecular partial layer on the substrate. The amount of the inert protein molecules in the first layer is sufficient to allow the distance between the antigen molecules to allow more antibody molecules to bind to the antigen molecules than when the antigen molecules are tightly packed together, and there is a much greater change in the contrast between them a single and a double layer on a substrate coated with two proteins. The invention thus provides a simple procedure for distinguishing between a single layer of a large antigen, such as the antigen associated with hepatitis, and a double layer, which includes a second layer of small antibodies, such as the hepatitis antibody, and can thus be used in the Analysis of the second solution can be used to determine the presence of the antibody.

The invention is explained in more detail below with reference to the drawing. In detail show:

1 is a side view of a substrate after it has been immersed in a solution of an antigen by a known method and then in a solution of the antibody specific for the antigen;

FIG. 2 shows a side view of a substrate produced according to the invention after it has been subjected to two immersion stages as in FIG. 1, but the first immersion being carried out in accordance with the invention;

3 shows a side view of the substrate of FIG. 2 produced according to the invention after a subsequent immunological reaction, in which the coated substrate was immersed in a solution containing the same antigen; N-

FIG. 4 shows a side view of the substrate of FIG. 3 produced according to the invention after a further immersion of the substrate in a solution which contained the same antibody and in which the antibody molecules at the ends of the chains of the antigen-antibody complexes antigen sites on a virus or Find cells to identify them immunologically;

5 is a side view of a substrate after first being immersed in a large antigen solution by a known method and then in a solution of a smaller antibody specific for the antigen;

FIG. 6 shows a side view of a substrate produced according to the invention after it has been subjected to two immersion stages as in FIG. 5, but the first immersion being carried out in accordance with the present invention;

FIG. 7 shows a side view of the substrate of FIG. 6 produced according to the invention after a subsequent one

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immunological reaction in which the coated substrate was immersed in a solution containing an antibody to the antibody in the second solution, and

8 shows a plan view of a substrate produced according to the invention, which has a narrow range of active inert proteins, which is surrounded by inert protein.

1 is a greatly enlarged side view of part of the diagnostic apparatus in the form of a thin plate 10 made of a suitable substrate material, which can be metal, glass, mica, plastic, molten silicon dioxide or similar material, with metal being preferred, because it has the biggest difference in refractive index compared to protein. The substrate preferably has the form of a metal or metallized glass plate. A monomolecular layer 11 adheres to the substrate 10 when it is immersed in a solution of 15, generally salt water, containing a first protein of interest, which may be biologically an antigen or an antibody. For the purpose of the present invention The layer 11 adsorbed on the surface of the substrate 10 is described below as an antigen layer. Each protein is adsorbed in such a monomolecular layer, but no further adsorption takes place, i. H. the protein adheres to the substrate, but not to itself. The antigen protein layer 11 can therefore only be monomolecular and not of greater thickness. The time required to completely coat the substrate with the antigen protein is a function of the concentration of the protein in the solution, the degree of stirring of the solution and the temperature of the solution. So covered z. B. a 1% bovine serum albumin solution completes a slide in about 30 minutes with a monomolecular antigen protein layer.

After the monomolecular layer of the antigen protein 11 is formed on substantially the entire surface of the substrate 10, the coated substrate is removed from the solution of the antigen protein and next immersed in a solution which contains the antibody protein which reacts specifically to the antigen protein or which is believed to contain this.

This second solution can be in addition to the specific 40

reacting antibody protein, the presence of which is to be detected, contain many components. However, no protein other than the specifically reacting antibody protein will adhere to the first layer of antigen protein on the substrate. Therefore, only when the specifically reacting antibody protein is present in the second solution will immunological complexation take place between the antigen and its specifically reacting antibody, and after a certain time the substrate will have a bimolecular protein layer. 50 The time required for the adhesion of the second (antibody) molecular layer 12 to the coated substrate is again a function of the concentration of the specifically reacting protein in the solution, the degree of solution movement and the solution temperature. For antibodies in 55 blood serum, this time can be up to one day. The second layer may be only a partial layer or an essentially complete layer depending on the three factors mentioned above.

As shown in Fig. 1, in the case where 60 the antigen layer 11 substantially completely covers the substrate 10, i. H. if the distance between adjacent antigen molecules is very small, the antibody molecules essentially all of their combination sites to bind to the antigen molecules and thus lose their activity, i.e., their activity. H. essentially all active sites on the antibody molecules 12 combine with corresponding active sites on the antigen molecules 11. This

Substrate coating process is described in CH-PS 579 777.

Since the monomolecular antigen layer 11 is adsorbed over substantially the entire surface of the substrate 10 in FIG. 1, the second layer 12 of the specific antibody for this antigen combines with it to form the bimolecular layer and a further combination with additional protein, be it antigen or antibody, is not possible since there are no further active sites on the antibody molecules in layer 12. In the present invention, a method has now been found by means of which the antibody molecules in the second layer 12 can be connected to the antigen molecules in the first layer 11 and at the same time have remaining active sites for a further connection with other antigen molecules in a further immunological reaction. The basis of the present invention is shown in FIG. 2. For illustration, the antibodies depicted therein are of the IgG class, but there are no reasons why antibodies from the other four main classes mentioned above cannot be used in the present invention.

After selecting the substrate 10 on which the multimolecular immunologically complexed film is to be formed, this substrate is generally immersed in a salt water solution containing the antigen, as in the case of the method described in connection with FIG. 1. In contrast to this known method, however, an immunologically inert protein was added to the first solution before immersing the substrate in this solution, so that it was on the surface of the substrate

10 formed adsorption layer consists of reactive antigen-protein molecules 11, which are surrounded by each other and along the substrate surface by inert protein molecules 20, as shown in Fig. 2. The amount of the immunologically inert protein added to the solution is sufficient that the average distance between adjacent antigen molecules 11 attached to the substrate 10 is generally several hundreds.

The monomolecularly coated substrate 10 (with antigen and inert protein) is then removed from the first solution and immersed in a second solution which contains specific antibodies for the antigen of the first solution. Since the distance between the active sites of an antibody molecule 12 (of the IgG class) is approximately 200 Â, a large proportion of the antibody molecules 12 with the second solution, which bind to the antigen molecules 11, will keep active binding sites (there can be more than one remaining binding sites per molecule exist for a further combination with additional antigen molecules in a subsequent immunological reaction, depending on the specific class of the antibody molecule. Of course, any antibody molecule cannot bind to more than one active site on any antigen molecule. As shown in FIG. 2 , The antibody molecules 12 are due to the sufficient distance between the adjacent antigen molecules 11 by the intermediate immunologically inert protein molecules 20 to the antigen molecules

11 bound, and in general each antibody molecule 12 has an active binding site 12a for a subsequent immunological reaction. Is the active antigen z. B. human serum albumin, then is an example of the immunologically inert protein egg albumin and the antibodies are obtained from rabbit serum. In the case of a 1% albumin solution from human serum, the concentration of the inert egg albumin protein is therein

1 to 10 %•

After substrate 10 is removed from the antibody solution (i.e., as shown in FIG. 2)

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stages), the bimolecularly coated substrate can be used further by next immersing it in a solution which contains the same antigen as the first solution or in which such an antigen is suspected and these antigen molecules 30 are selectively transferred to the remaining active sites 12a Antibody molecules 12 bound in a further immunological reaction. The result is shown in FIG. 3. This use can thus be used to easily detect the presence of a specific antigen when analyzing a solution. On the other hand, this use can also be used to construct different chains of alternating antigen-antibody molecules, in which each chain originates from the surface of the substrate 10, and to form a multimolecular immunologically complexed film of different chains of alternating antigen-antibody molecules on it . The multimolecularly complexed film is easily detected by viewing the coated substrate with an optical instrument such as an egg lipometer. On the other hand, the multimolecularly complexed film can also be examined electrically by measuring the capacitance of a capacitor with conductive plates formed by the metal or metal-coated substrate and a drop of mercury or other suitable electrode, the protein layers being the capacitor dielectric, as in CH-PS 579 777 is described in detail. Furthermore, this CH patent describes the method for optical examination by simple visual observation by determining the time before a visible amalgam is formed between a drop of mercury and the metal film coating the substrate through the intermediate protein layers. Finally, and this is of the greatest importance, the multimolecularly complexed film can also be examined optically in reflected or continuous light, as is also described in the aforementioned Swiss Patent No. 579,777. In this latter optical inspection by reflected or continuous light, the following is a first continuous light technique that has been successfully used: The substrate 10, which must be a translucent substrate, such as glass, plastic, molten silicon dioxide, mica, quartz or the like and which is preferably glass, with slides being a conveniently available embodiment, is first coated with a plurality of metal beads by vapor deposition of a metal, e.g. B. indium, covered on the substrate. The indium is e.g. B. slowly evaporated from a tantalum crucible on the glass substrate in an ordinary vacuum of about 5 X 10 ~ s mm Hg. Since the indium atoms have a high mobility on the surface of the substrate and do not noticeably wet the glass substrate, the indium evaporated onto the substrate agglomerates into small particles. Any metal with similar properties so that it also forms spheres when it evaporates on the substrate can be used. In addition to indium, gold, silver, tin and lead have been used successfully. Evaporation of the metal continues until the substrate appears light brown in color. At this point in time, the metal spheres have diameters in the order of 1000 Â. The exact size of the beads is not critical, but they must have a diameter equal to a large proportion of the wavelength of visible light. The next step is to immerse the bead-coated substrate 10 in a first solution of a first immunologically reactive protein, such as antigen 11 and inert protein 20. The first reactive protein and the inert protein again adhere to the substrate in a monomolecular layer the metal balls on it. When a monomolecular layer has formed, the coated substrate is removed from the first solution and immersed in a second solution containing the protein 12 that specifically reacts with the first protein, and the result is the adherence of a bimolecular protein layer similar to that in FIG. 2 shown on the substrate and the metal beads. Then the coated substrate is taken from the second solution and immersed in a third solution which contains or presumably contains the reactive protein of the first solution. If this protein is present, a third layer or partial layer is formed on the substrate. The coated substrate is then viewed in transmitted light and the appearance of the coated substrate determines the thickness of the protein layer adhered to it, and accordingly whether the expected protein was present in the solution or not. The detection of the protein layers corresponds to the variations in the brown tone that is observed in the coated substrate. These variations are quite clear and the detection of the protein layers is therefore a simple procedure. The beads alone on the substrate appear as a first brown tone, the beads covered with a monomolecular protein layer appear as a darker brown tone and the beads covered with a bimolecular protein layer appear as an even darker brown tone and finally the beads covered with a trimolecular protein layer are even browner. This detection method is based on the fact that electromagnetic radiation is largely dispersed by conductive spheres with diameters equal to a large proportion of the wavelength of the incident energy and that in the case of scattering by such spheres, the scattering is strongly by a thin dielectric layer on the spheres being affected. A second technique for optical inspection with reflected light that has been used successfully is as follows: a gold substrate, which for economic reasons preferably consists of a thin gold layer on another metal, has adsorbed thereon a monomolecular layer of the first reactive protein 11 and of the inert protein 20 after immersion in the first solution described above. Gold has an absorption band within the visible spectrum and this fact determines the characteristic gold color and allows the execution of this special optical examination technique. The gold substrate can conveniently be a glass slide that is covered with a thin layer of indium and has a gold layer over it, the indium layer both improving the adhesion between the glass and the gold as well as the optical properties of the slide. The relative reflectivity of the gold substrate as a function of wavelength results in the substrate having the characteristic light yellow color of the gold metal, if not any protein layer adhered to it. In the presence of a monomolecular layer on the substrate, the slide (substrate) has a dark yellow appearance. After the slide has been exposed to a solution which contains the protein 12 which reacts immunologically against protein 11 and the slide has a bimolecular protein layer, the reflection properties give a greenish appearance. A third layer of protein gives it an even greener look. In tests that have been carried out so far, optical investigations of the coated substrate by reflected or transmitted light, using a substrate comprising a metal bead or a gold substrate, appear to be the most useful methods in general. It was further found that these two techniques have different sensitivities as a function of the thickness of the protein films of interest. In individual cases, the

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Greatest sensitivity in technology with a substrate that has metal spheres, with film thicknesses below about 200 Â. The greatest sensitivity of the gold substrate lies in film thicknesses that are greater than 30 Â. The detection method used in individual cases thus determines the type of substrate 10 used in each analysis, i. H. whether the substrate is a metal or a metallized glass slide that has a flat metal coating (made of gold as an example) or metal beads (made of indium as an example) on the surface, as also explained above. For the sake of simplicity, substrate 10 is illustrated as having a flat surface, although the surface on which the first antigen layer is adsorbed could also contain the aforementioned metal spheres.

The use for forming multimolecular layers of immunologically complexed proteins, as shown in Fig. 3, is particularly important when examining a solution for certain proteins, e.g. B. a hormone, since hormones and antiserum for hormones are generally available. Therefore, if the third layer is the hormone of interest, it is easily detectable.

Finally, the formation of multimolecular, immunologically complex layer coverings is also important in the immunological identification of cells or viruses. As illustrated in FIG. 4, the use according to the invention makes it possible to build up a multiplicity of chains of antigen-antibody complexes from the surfaces of the substrate 10, so that the probability for the different antibody molecules at the ends of the different chains is relatively high to find an antigenic site on a cell or virus 40. And this probability remains high regardless of the irregularity of the surface of the cell or the virus. As is well known, the cell membrane, which includes each cell, has outwardly extending molecules called "transplant" antigens. These antigens are the ones used to form the first layer 11 in a two-layer system for the immunological identification of cells, the cells of interest being the third layer. In the case of a four-layer system of antigen-antibody chains, as depicted in Figure 4, the first and third layers (11 and 30) would consist of the transplantation antigens. It is also known that a virus has a protein layer of protein molecules that can be split and separated and such molecules are used to form the first, first and third layers in two and four layer systems of antigen-antibody chains, respectively used to immunologically identify a virus 40. Antibodies 12 in the second layer and 41 in the fourth layer would, of course, be specific to the particular cell or virus being examined.

Based on the present description, it is clear that the present new method is important for binding antibodies to a surface so that they remain active and can form multimolecular immunologically complexed films on a substrate. The immersion of a suitable substrate in the first solution forms a monomolecular layer of reactive antigen protein molecules, which are separated from one another by the inert molecules, by adsorption on the surface of the substrate. The formation of such a special monomolecular layer then allows the subsequent immersion of the coated substrate in a second solution which contains the antibody specific to the first antigen, so that the antibody molecules are bound to the antigen molecules and at the same time have other active sites free to be used in subsequent immunological Reactions to be used. The coated substrate can also subsequently be immersed in a solution containing or suspected of having the same antigen as the first solution, and this procedure can be repeated to cause different chains of alternating antigen-5 antibody molecules to build up.

5 shows a greatly enlarged side view of a part of the diagnostic apparatus in the form of a thin plate 10 made of a suitable substrate material, which can be metal, glass, mica, plastic, molten silicon dioxide, quartz or similar material, with metal being preferred is because it has the greatest difference in refractive index from the protein, and preferably the substrate is in the form of a metal or metallized glass plate. If the substrate 10 is immersed in a first solution, generally of salt water, which contains the first protein of interest, which can be biologically an antigen or an antibody, this is adsorbed on the substrate in a monomolecular layer 11. For the purposes of the present explanation, the layer 11 adsorbed on the surface of the substrate 10 is described below as an antigen layer. Each protein will adsorb in such a monomolecular layer, but no further adsorption takes place, i.e. H. the protein adheres to the substrate, but not to itself. The antigen-protein layer 11 can therefore only be monomolecular and have no greater thickness. The time required to completely coat the substrate with the antigen protein is a function of the concentration of the protein in the solution, the degree of stirring of the solution and the solution temperature. So coated z. For example, a concentration of 1 mg / cm3 of the antigen associated with the hepatitis completely slides in about 10 minutes with a monomolecular layer of antigen protein.

After the monomolecular layer of the antigen-35 protein 11 has formed on substantially the entire surface of the substrate 10, the coated substrate is removed from the solution of the antigen protein and next immersed in a second solution, which is the antibody protein which reacts specifically with the antigen protein ent-40 holds or presumably contains. For the purposes of the present invention, it is assumed that the protein 11 adsorbed on the surface of the substrate 10 is always larger than the protein immunologically reactive therewith, which forms a second layer on the substrate. The second solution 45 can contain many components in addition to the specifically reacting smaller antibody protein whose presence is to be detected. However, no protein other than the specifically reacting antibody protein will adhere to the first layer of antigen protein on the sub-50 strat. Immunological complexation will therefore only take place between the antigen and its specifically reacting antibody, and after a certain time the substrate will only carry a bimolecular protein layer if the specifically reacting antibody-5S protein is present in the second solution. The time required for the adhesion of the second (antibody) molecular layer 12 to the coated substrate is in turn a function of the concentration of the specifically reacting protein in the solution, the degree of solution movement and the solution temperature. For antibodies in the blood serum, this time can be up to one day. The second layer can be either only a partial layer or an essentially complete one, depending on the three factors mentioned above.

6S In the case as shown in Fig. 5, in which the antigen layer 11 substantially completely covers the substrate 10, i. H. if the distance between adjacent antigen molecules is very small, the number of

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Antibody molecules 10 that can be bound to any particular antigen molecule are limited due to the limited surface area of the antigen molecule available for such immunological reactions. This substrate coating process is described in the above-mentioned CH-PS 5 No. 579 777.

Since the monomolecular antigen layer 11 is adsorbed over essentially the entire surface of the substrate 10 in FIG. 5, the second layer 12 composed of the smaller specific antibody for this antigen combines with it in a thin layer to form a bimolecular layer. However, since the proteins of the second layer 12 are smaller in size than the proteins of the first layer 11 and accordingly the second layer is less dense than the first (antigen) layer, it is clear that there are considerable difficulties in distinguishing between can give this single and the double protein layer on the substrate 10. A good example of this situation is the case where the first layer 11 consists of the antigen associated with the hepatitis and the second layer 29 12 consists of the specific antibody to the HHA. Examination of the coated substrate with an optical instrument such as the egg lipometer does not allow one to easily distinguish between the single (antigen) and partial or dilute double (antigen-antibody complex) layer 25 on the substrate, and so on cannot be easily determined whether the second solution actually contained any of the antibodies to HAA and so such a diagnostic procedure is of little value. In the present invention, a method has been found by means of which a much larger number of antibody molecules in the second layer 12 can be bound to the antigen molecules of the first layer 11, so that a much larger change in the contrast between the single and the double layer is caused. The substrate produced according to the invention is also shown in FIG. 6. 35

After selecting the substrate 10 on which the bimolecular immunological complex film is to be formed, this substrate is immersed in a first solution which generally consists of salt water which contains the antigen, as is the case in the case of the method in connection with FIG 5 has been described. In contrast to this previous method, however, an immunologically inert protein was added to the first solution before immersing the substrate in the solution, so that the 45 adsorption layer formed on the upper layer of the substrate 10 consists of relatively large reactive antigen protein molecules 11, which are separated from one another along the substrate surface and are surrounded by inert protein molecules 20, as shown in FIG. 6. The inert protein molecules 20 have a size that is at least somewhat smaller and preferably considerably smaller than that of the antigen molecules 11 in order to make the largest possible surface area and thus more binding sites of the antigen molecules 11 accessible for the subsequent connection with the antibody molecules. The amount of immunologically inert 35 protein 20 added to the solution is sufficient to generally cover with the inert protein 20 1/2 to 9/100 of the area of the complete adsorbent layer, i. H. the antigen accordingly covers Va to V10 of the area, although this is not a limitation of the present invention, since the average distance between adjacent antigen molecules is also determined by the number of active sites on the respective antigen molecule and the way in which such molecules are attached to the Adhere substrate is determined, d. H. the number of active antigen sites, 65 that are exposed. In the case where the antibody molecule 12 is much smaller than the antigen, a larger distance between the antigen molecules is desirable, whereas with a only slightly smaller antibody molecule compared to the antigen, a smaller distance between the antigen molecules can be tolerated in order to maximize the benefit of to obtain the present invention. As a result of the large antigen protein relative to the inert protein molecule, a major portion of the surface of the antigen molecule will remain free and its corresponding binding sites are accessible for combination with antibody molecules in a subsequent immunological reaction.

The monomolecularly coated substrate 10 (with antigen and inert protein) is then removed from the first solution and immersed in a second solution which contains the antibodies specific for the antigen of the first solution. As a result of the larger surface area and the more numerous antigen sites exposed on the antigens, since the antigens are surrounded by the smaller inert proteins in Fig. 6, compared to Fig. 5, the antigen molecules in Fig. 6 can be immunologically more easily with a larger number of antibodies than combine in Fig. 5.

Therefore, as shown in Fig. 6, a larger number of antibody molecules 12 will generally combine with each antigen molecule than in the case of Fig. 5, and it is obvious from the comparison of Figs. 5 and 6 that because of the larger number of antibody molecules which is present in the second layer, a much larger change in contrast between the single and double layers is obtained in the embodiment of the invention of FIG. 6. So z. B. the first solution is such a concentration of the hepatitis-related antigen (HAA) and bovine serum albumin (BSA) which is the inert protein that the HAA V4 of the surface in the monomolecular layer and the inert BSA protein are the remaining 3A of the Surface covered and the second solution is a dilute solution of the HAA antibody. The method described above can therefore be used in analysis to easily detect the presence of a specific antibody to a particular, relatively large antigen.

The double-layered substrate of FIG. 6 can then be used to be immersed in a third solution or serum containing antibodies 30 to the antibodies 12 of the second layer to build up a third layer, as shown in FIG. 7 is shown. Since each antibody molecule 12 has different antigen-determinants, it can generally combine with different molecules 30, which are antibodies to the antibodies 12, and thereby form a relatively thick third layer from such second antibody molecules 30. The presence of the dense third layer 30 shows the presence of the second layer 12 and thus creates an even greater contrast between the monomolecular and the bimolecular layer of antigen and the first antibody. In the latter case, the inert protein molecules 20 between the large antigen molecules 11 in the first layer also help to prevent non-specific protein material in the serum, which contains the antibodies to the antibody of such an antigen, from adhering to the substrate. Thus, a twofold gain is achieved by obtaining more specifically bound antibodies for each antigen molecule and less nonspecifically bound protein on the substrate, and all of this results from using the inert protein to keep the large antigen molecules apart and to cover the surface of the substrate surrounding each antigen molecule with such inert protein. The procedure used to form the third layer in Fig. 7 is therefore useful in analyzing the second solution, which is believed to contain antibody 12, since the subsequent immersion of the coated substrate in the

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third solution creates a significantly enhanced contrast between the monomolecular layer and the bimolecular layer, which is now actually a trimolecular layer when a bimolecular layer was present due to the fact that the second solution actually contained the antibody 12.

The presence of the second (antibody 12) protein layer in the embodiment of FIG. 6, which is also indicated by the third (antibody 30) protein layer in FIG. 7, can easily be demonstrated by viewing the coated substrate with an optical instrument such as a Egg ellipsometer. The protein layers, on the other hand, can also be examined electrically, as described in detail in the above-mentioned CH-PS 579 777, by measuring the electrical capacitance of a capacitor which has conductive plates made up of the metal or metal-coated substrate and a mercury drop or another suitable electrode and in which the capacitor dielectric is formed by the protein layer. Furthermore, the protein layers, as described above with respect to the multimolecular complexed film, can also be examined optically by observing them visually and thereby determining the length of time before a visible amalgam is formed between a drop of mercury and the metal film on the substrate, whereby the protein layers are in between. Finally, and this type of examination is the most significant, the protein layers can also be examined optically with reflected or continuous light, as described above with respect to the multimolecular complexed film. In the case of the protein layers, the coated substrate is then removed from the second solution and immersed in a third solution containing a reactive protein with respect to the reactive protein in the second solution (such as an antibody to the antibody) to a third layer (or a Sub-layer) to form on the substrate when the second layer is present. The coated substrate is then observed with continuous light and a determination is made of the thickness of the protein layer adhering to the substrate from the appearance of the coated substrate and, accordingly, a determination of the presence or absence of the second protein 12.

The contrast between single and double protein layers can be increased to an even greater extent by applying the antigen and inert protein to the substrate 10 as a single drop of solution and then immersing the drip-coated substrate in a solution containing only the inert protein 20 to form a monomolecular layer in which the small anti-gene inert protein region is completely surrounded by the inert protein, as shown in FIG. 8. This embodiment solves the problem of non-specific adsorption in the event that antibodies are present in a serum, since the inert protein on the surface of the substrate prevents the non-specific proteins in the serum from sticking to the substrate and thus improves the contrast .

Finally, the procedure described above is important in the immunological identification of viruses and enzymes. All antigens of the virus type are larger than their specific antibodies, while the antigens of the enzyme type can be larger or smaller, depending on the specific type of enzyme. A procedure for identifying a specific virus or large enzyme known as an inhibition test is carried out in the following way:

A special virus or large enzyme is introduced into a first solution (generally salt water) together with the inert protein and the substrate is immersed in this solution or a drop of the solution is placed on the substrate and the substrate is then placed in a solution of only immersed in the inert protein. After a monomolecular layer of the virus or enzyme (i.e., the antigen) and the inert protein was adsorbed on the substrate, the coated substrate was removed from the first solution. Samples of human serum to be tested for the presence of the particular virus or enzyme are prepared by mixing an amount of known antibodies to the specific virus or enzyme in an amount sufficient to immunologically remove it from the mixture when that Virus or enzyme is present in the serum sample. The monomolecularly coated substrate is then immersed in or exposed to the previously prepared serum sample and, after removal thereof, examined according to any of the procedures described above. If the examination of the substrate indicates the presence of only a monomolecular layer on it, then one knows that the human serum examined originally contained the specific virus or enzyme. However, if a bimolecular layer is detected, this shows that the serum originally did not contain such a virus or large enzyme antigen, since the antibodies did not immunologically bind with their specific antigen (virus or enzyme) in the serum sample and they were therefore able to combine with the antigen of the first layer adsorbed on the substrate.

It is clear from the above description that the substrate obtained according to the invention contributes to improving the contrast in immunological surface tests between a single and a double layer of protein.

In addition to the specific embodiments and examples that have been described, various modifications and variations are possible within the scope of the present invention. For example, For example, the inert protein could be used in a solution separate from the antigen, and the substrate would be immersed in this separate solution as a precursor to the antigen and antibody solutions.

The invention can also be used to detect enzymes by using a dilute solution of the enzyme to obtain the antigens in the first layer and adding bovine serum albumin as the inert protein to the first solution and a solution of an antibody to the enzyme (from one Rabbit, which had previously been injected with the enzyme) in the second solution and then immersed the bimolecularly coated substrate in a third solution which is believed to contain the enzyme and then examining the substrate for the presence of a third layer that would be formed from the special enzyme. However, although the reactive protein adsorbed on the substrate surface has been described as an antigen, it is clear that the first layer reactive protein could also be an antibody and the second layer protein would then be the antigen specific for that antibody and the third layer of protein would then be the antibody again. Such an arrangement of a first antibody layer and a second antigen layer, which is specific to the antibody, would be useful in cases in which an antibody molecule is larger than its specific antigen molecule, which e.g. B. is the case with regard to the antibody to insulin. i

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Claims (15)

617 012 2nd PATENT CLAIMS 1. A method for forming bimolecular layer coverings on substrates, the layer coverings containing immunologically complex bound proteins, characterized by the following process steps: (a) coating a suitable substrate with a monomolecular protein layer which consists of an immunologically reactive first protein and an inert protein, so that the molecules of the first protein are separated from one another by the inert protein molecules and (b) contacting the coated substrate with a solution of an immunologically reactive second protein, specific to the first protein, to effect an immunological reaction therewith in which the second protein molecules combine with the first protein molecules to form a bimolecular layer on the substrate and after the formation of the bimolecular layer, the second protein molecules retain a larger number of active sites for a further immunological reaction than if the monomolecular protein layer did not contain any inert protein. 2. The method according to claim 1, characterized in that the substrate is a metallized slide. 3. The method according to claim 1, characterized in that the immunologically reactive first protein is an antigen and that the immunologically reactive second protein is an antibody specific to the antigen, preferably an antibody of the immunoglobulin Ig G class. 4. The method according to claim 3, characterized in that the antigen is an enzyme, that the inert protein is bovine serum albumin and that the antibody comes from rabbits to whom the enzyme had previously been injected. 5. The method according to claim 1, characterized in that the substrate is flat and that step (a) comprises: (1) preparing a first solution of an inert protein, (2) dipping the substrate sufficiently in the first solution to cover the substrate with a monomolecular sublayer of the inert protein, (3) removing the partially coated substrate from the first solution, (4) preparing a second solution containing immunologically reactive, first protein, (5) immersing the partially coated substrate in the second solution to cover the remaining surface of the substrate with a monomolecular sublayer of the immunologically reactive first protein, so that the monomolecular layer consists of the molecules of the immunologically reactive first protein that are separated from each other by the inert ones Protein molecules are separated, and (6) removing the substrate covered with the monomolecular layer from the second solution. 6. The method according to claim 1, characterized in that step (a) comprises: (1) preparing a diluted first solution of the immunologically reactive first protein, (2) immersing the substrate in the first solution, sufficient to cover the substrate with a monomolecular sublayer of the immunologically reactive first protein, (3) removing the partially covered substrate from the first solution, (4) preparing a second solution containing inert protein, (5) immersing the partially coated substrate in the second solution to cover the remaining surface of the substrate with a monomolecular partial layer of inert protein, so that the monomolecular layer consists of the molecules of the immunologically reactive first protein, which are separated from each other by the inert protein molecules are and (6) removing the substrate covered with the monomolecular layer from the second solution. 7. The method according to claim 3, characterized in that method step (a) comprises: (1) preparing a first solution of an antigen, (2) adding the inert protein in sufficient amount to the first solution, (3) immersing the substrate in the first solution in order to coat the substrate with the monomolecular protein layer, the inert protein being present in a sufficient amount such that the antigen molecules in the monomolecular layer are separated from one another by the inert protein molecules, (4) removing the substrate covered with the monomolecular layer from the first solution and (5) coating the substrate with the antigen-corresponding antibodies, the antigen originating from the outer parts of specific cells or viruses. 8. The method according to claim 1, characterized by the following stages: Coating a metallized slide with a monomolecular protein layer consisting of an immunologically reactive first protein and an inert protein of smaller size than the reactive first protein, so that the molecules of the immunologically reactive first protein are separated from one another by the smaller molecules of the inert protein and surrounded by them, and then treating the coated slide with a solution of an immunologically reactive second protein that is specific to and smaller in size than the first protein to cause an immunological reaction therewith in which a relatively large number of immunologically reactive second protein molecules coexist connects the immunologically reactive first protein molecules to form a bimolecular layer on the slide, and wherein the inert protein is present in a sufficient amount so that its molecules occupy 50 to 90% of the surface of the slide corners, wherein the relatively dense second layer of reactive molecules of the immunologically reactive second protein of smaller size gives a significantly improved contrast when the slide is examined than if the monomolecular layer did not contain any inert protein. 9. The method according to claim 1, wherein the substrate is a metallized slide, characterized in that a solution of an immunologically reactive first protein is prepared, a sufficient amount of an inert protein of a smaller size than that of the immunologically reactive first protein to the solution is added, the slide is brought into contact with the solution in order to cover at least a part of the slide by adsorption with a monomolecular layer of the immunologically reactive first protein and the smaller inert protein, so as to pass through the molecules of the immunologically reactive first protein separate the smaller inert protein molecules from each other and to surround the reactive protein along the surface of the slide with the smaller molecules of the inert protein, so that when the slide is examined after the further immunological reaction between the first and the second immunological reactive protein, an improved contrast between mono- and bimolecular layers of proteins is obtained. 10. The method according to claim 1, characterized by: coating the substrate with a monomolecular protein layer composed of an antigen and an inert protein 5 10th 15 20th 25th 30th 35 40 45 50 55 60 65 3rd 617 012 of smaller size than the antigen, so that the antigen molecules are separated from one another and surrounded by the smaller, inert protein molecules over the substrate surface, and subsequent treatment of the coated substrate with a solution of an antibody specific for the antigen, which is of a smaller size than in order to bring about an immunological reaction in which the antibody molecules bind to the antigen molecules to form a bimolecular layer on the substrate, a relatively large number of the antibody molecules binding to the antigen molecules resulting from the large number of active sites which are exposed on the antigen molecules since they are surrounded by the smaller inert protein molecules along the substrate surface, and the relatively dense second layer of the smaller antibody molecules produces an improved contrast between the single and double layers of the protein than if only Anti gene molecules would be arranged close together. 11. Substrates covered with bimolecular layers, obtained by the process according to claim 1. 20 12. Use of the substrates covered with bimolecular layers according to claim 11 for the detection of further immunologically reactive substances. 13. Use according to claim 12 of the substrates produced according to claim 7, characterized in that they are brought into contact with a third solution, which is suspected to contain the same antigen as the first solution, for an immunological reaction with the antibody, in which the remaining active sites on the antibody molecules 30 chemically combine with the antigen molecules that may be present in the third solution. 14. Use according to claim 13, characterized in that the substrate is then examined to determine whether a third layer has formed on the substrate, and thus whether the antigen is present in the third solution or not. 15. Use according to claim 14 of the substrates obtained according to claim 7, characterized in that they are brought into contact with a third solution which is believed to contain the specific cell or virus from which the antigen was obtained, so that the antibody molecules find antigenic sites on the cell or virus to cause binding by then examining the substrates to determine whether there is a third layer on them and thereby the presence of the particular cell or virus in the third solution that the coated substrate may be removed from the third solution, then brought into contact with a fourth solution containing the same antibody as the second solution to cause an immunological reaction in the active sites of the Antigen molecules from the fourth solution combine with antigen molecules from the third solution bind that the substrate is removed from the fourth solution, the same with the chains of the antigen-antibody complexes that are built up from the surface of the substrate, possibly with a fifth solution that is believed to be specific Contains cells or viruses in contact, so that the antibody molecules in the 60 chains of the antigen-antibody complexes find and combine antigen sites on the cells or viruses, and that one finally removes the substrate from the fifth solution and examines the same to determine whether the active sites remaining on the antibody molecules have linked to antigen sites on the cells or the virus in the fifth solution.