DK151240B - PROCEDURE FOR THE PREPARATION OF MULTIMOLECULAR LAYERS OF IMMUNOLOGICALLY REACTIVE PROTEINS ON A SUITABLE SUBSTRATE - Google Patents

Description

151240

The invention relates to a process for producing multi-molecular layers of immunologically reactive proteins in which (a) a suitable substrate is coated with a monomolecular protein layer consisting of an immunologically reactive first protein, and (b) the coated substrate is then treated with a solution of an immunologically reactive second protein specific to the first protein to produce an immunological reaction by which the molecules of the second protein bind to the molecules of the first protein to form a bimolecular layer.

Immunological reactions are highly specific biochemical reactions whereby a first protein, known as the antigen, unites with a second for the antigen-specific protein known as antibody to form an immunological protein complex. Immunological responses that occur in a biological organism, such as an animal or human, are indispensable for disease control. In a biological organism, the intrusion of a foreign protein, that is, the antigen, causes the biological organism to produce specific antibody proteins against the antigen in a process that is not yet fully understood. The antibody protein molecules have available chemical combination or binding centers that are complementary to the antigen molecule so that the antigen and antibody are chemically combined or linked together to form the immunological protein complex.

Because antibodies are formed in biological organisms in response to the entry of foreign proteins into them, the detection of antibodies present in a biological organism is of diagnostic value in determining the antigens to which the organism has been exposed. Conversely, the detection of certain antigens in a biological organism also has diagnostic value; Examples of diagnostic detection of antigens include detection of HCG protein molecules in urine as a pregnancy test, and detection of HAA molecules, which are antigens associated with hepatitis, in the blood of any blood donors. To conduct such diagnostic tests, the protein, from at least one immunological pair, must be provided.

35 The only known source of antibody protein is * a living biological organism. Specifically, only vertebrates are currently known to exhibit immunological responses upon introduction of a foreign protein. Eg. Many antibodies have been found in blood serum in animals and humans that have been exposed to the corresponding antibodies. However, many antigens can be produced under control in laboratory cultures. However, certain antigens, e.g. Antigens associated with hepatitis, like antibodies, are currently only obtained from the higher living biological organisms.

Most antigens are proteins or contain proteins as a major component, whereas all antibodies are proteins. Since proteins are large molecules of high molecular weight, that is, polymers consisting of chains of amino acids of different numbers, the antigen and antibody protein can each have several combination centers.

The five major classes of antibodies (immunoglobulins Ig G, Ig M, Ig A, Ig E and Ig D) are each apparently characterized by at least two long peptide chains of amino acids and at least two short peptide chains of the acids in which the binding between the amino acid units is known as and peptide bond. These long and short peptide chains are oriented 15 in the form of the letter "Y", and the active centers or combination centers are the outer ends of the two arms of the Y-shaped antibody in the 1 g G antibody.

Immunological reactions can be detected by various methods, including the use of a suitable substrate such as a metal-coated glass or metal plate. Exposing the substrate to an antigen solution will cause the antigens to be physically adsorbed in a dense monomolecular layer on the surface of the substrate. Subsequent exposure of the antigen-coated substrate to a serum containing antibodies to the antigen results in the immunological reaction whereby these antibodies selectively bind to the antigens at the antibody molecule binding centers complementary to those on the antigen molecule, thereby forming in it at least a partial bimolecular layer of immunological protein complexes on the substrate surface.

The problem arises from subsequent exposure of the coated substrate containing the antigen-antibody complex protein to a solution containing or suspected of containing the same antigen. The subsequent exposure will generally not provide additional binding of antigen to the antibody, since all the active centers 35 of the antibody proteins are already bound to the first antigen layer due to the fact that the active antigen molecules are very close to each other. Thus, in surface immunology, there is a problem in that antibodies that bind to a (antigenic) surface lose their activity (i.e., the ability to form additional bonds with an antigen, a cell, or a virus).

3 151240

Another problem that often arises is the inability to distinguish between the monomolecular and bimolecular protein layers on the carrier, especially if the adsorbed (antigenic) layer is relatively thick, as with HAA layers, since it has anti-hepatic inflammation. gene molecule is at least 10 times the antibody to HAA.

It is an object of the invention to improve said prior art so that more than bimolecular layers of immunologically reactive proteins can be prepared.

This is achieved by the method having the preliminary characteristics set forth in the present invention in that the substrate is further coated with an immunologically inert protein which is adsorbable to the substrate and in such a way that the molecules of the first protein are separated from each other. of the inert proteins.

Other embodiments of the method according to the invention are characterized by the subclaims 2-16.

The invention will be better understood from the following description taken in conjunction with the drawings, in which:

FIG. Figure 1 is a stand of substrate after, in known manner 20, immersed first in a solution of antigen and then in a solution of the antibody specific for the antigen,

FIG. 2 is a stand of a substrate after having been subjected to two immersion steps, as in FIG. 1, but wherein the first immersion is according to the invention,

FIG. 3 is a view of the substrate of FIG. 2, following a subsequent immunological reaction whereby the coated substrate is immersed in a solution containing the same antigen; FIG. 4 is a view of the substrate of FIG. 3, after further immersing the substrate in a solution containing the same antibody, and wherein the antibody molecules at the ends of the chains of the antigen-antibody complexes find antigen centers on a virus or cell for immunological identification in cation thereof,

FIG. 5 is a stand of a substrate after it has been known to be immersed first in a solution of a large size antigen and then in a solution of a smaller antibody specific to the antigen;

FIG. 6 is a stand of a substrate after being subjected to two immersion steps as in FIG. 5, but where first immersion is according to the invention,

FIG. .7 is a view of the substrate of FIG. 6, after a subsequent immunological reaction in which the coated substrate is immersed in a solution containing an antibody to the antibody in the second solution; and FIG. 8 is a plan view of a substrate on which there is a small region of active-inert proteins surrounded by inert protein.

10 In FIG. 1 is a highly enlarged stand of part of a diagnostic apparatus in the form of a thin plate 10 of suitable substrate material, which may be metal, glass, mica, plastic, molten silica, quartz or a similar material where metal is preferred, since it has the greatest difference in refractive index in relation to protein, and is preferably in the form of a metal plate or sheet of metallized glass. When the substrate 10 is immersed in a solution, usually saline containing a first protein of interest, which may biologically be an antigen or antibody, a monomolecular layer 11 on the substrate is adsorbed. As far as the invention is concerned, the layer 11 adsorbed on the surface of the substrate 10 will hereinafter be referred to as antigenic layer, but the antigenic layer may well be a layer of antibody. Any protein will adsorb in such a monomolecular layer, but no further adsorption will take place, that is, the protein will bind to the substrate but not to itself. Thus, the antigenic protein layer 11 can 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. For example, a 1% solution of bovine serum albumin covers a plate completely in about 30 minutes. with a monomolecular antigenic protein layer.

After the monomolecular layer of antigen protein 11 has been formed over substantially the entire surface of substrate 10, the coated substrate is removed from the solution of the antigen protein and then immersed in a solution containing or suspected to contain the specifically reacting antibody protein to antigen protein.

This second solution may contain many components in addition to the specifically reacting antibody protein, the presence of which it is desired to detect. However, no protein other than the specifically reacting antibody protein will adhere to the first antigenic protein layer on the substrate. Thus, only if the specifically reacting antibody protein is present in the second solution will immunological complexation between the antigen and its specifically reacting antibody be present, and after a while, a bimolecular protein layer will be present. The time required for the adhesion of the second (antibody) molecule layer 12 to the coated substrate is again a function of the concentration of the specifically reacting protein in the solution, the degree of stirring in the solution and the temperature of the solution. For 10 antibodies in blood serum, this period can be as long as one day.

The second layer may be only a partial layer or a substantially complete layer, depending on the three factors mentioned above.

As shown in FIG. 1, the antibody molecules utilize in the case where the antigen layer 11 covers the substrate 10 substantially completely, that is, the distance between adjacent antigen molecules is minimal, essentially all of their combination centers to bind the antigen molecules so that their activity is lost. that is, substantially all of the active centers of the antibody molecules 12 associate with correspondingly active centers of the antigen molecules 20 11. This substrate coating process is described in the specification of GB Patents Nos. 1,443,181 and Nos. 1,443,182.

Due to the monomolecular antigenic layer 11 adsorbed on substantially the entire surface of the substrate 10 of FIG. 1, the second layer 12 of the specific antibody to this antigen 25 binds thereto to form the bimolecular layers, and no further connection with additional protein, neither antigen nor antibody, is possible as there are no remaining active centers on the antibody molecules. in layer 12. In the method of the present invention, the antibody molecules of the second layer 12 are bound to the antigenic molecules of the first layer 11, but there are still centers active for further binding formation with other antigen molecules in yet another immunological reaction. The basis of the method of the present invention is shown in FIG. 2 and relates to the method of forming the first monolayer molecular layer of antigen molecules 11 which are adsorbed on the surface of substrate 10. For purposes of illustration, the antibodies depicted herein are the IgG class, but there is no reason why antibodies of the the other four main classes listed above cannot be used in connection with the invention.

6 151240

After selecting the substrate 10 on which the multimolecular film of immunological complexes is to be formed, this substrate is immersed in a first solution, usually saline containing the antigen, as in the one described above in connection with FIG. 1 described procedure 5. However, in contrast to this prior method, an immunologically inert protein has been added to the first solution prior to immersion of the substrate such that the adsorption layer formed on the surface of substrate 10 consists of the reactive anti-gene protein molecules 11 separated (and over the substrate surface surrounded by inert protein molecules 20 as seen 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 adhering to the substrate 10 will usually be several hundred Angstroms.

The substrate 10 coated with antigen inert protein in monomolecular layer is then removed from the first solution and then immersed in a second solution containing the antibodies specific to the antigen in the first solution. Since the distance between the active centers of an antibody molecule 12 (of the IgG class) is about 200 angstroms, a larger fraction of the antibody molecules 12 in the second solution that associate with the antigen molecules 11 will still have their remaining combination centers (and there may be more than one remaining active center per molecule, depending on the particular class of antibody molecules) active for further combination with additional antigen molecules in a subsequent immunological reaction. Of course, any antibody molecule cannot be combined with more than one active center on any antigen molecule.

As seen in FIG. 2, thus, because of the sufficient distance between adjacent antigen molecules 11 produced by the immunologically inert protein molecules 20, the antibody molecules 12 are bound to the antigen molecules 11, and generally each antibody molecule 12 has a combination center 12a still active for a subsequent immunological reaction. . Thus, the object of the invention has been fulfilled, with antibodies bound to a surface (of a monomolecular antigenic layer) so that combination centers of the antibody proteins remain active for further immunological reactions. In the case where the active antigen is human serum albumin, the immunologically inert protein is e.g. egg albumin f and the antibody molecules are obtained from a rabbit serum. At a 1% resolution of 151240! 7 human serum albumin, the concentration of the inert egg albumin protein in the serum ranges from 1-10%.

After the substrate 10 has been removed from the antibody solution, see FIG. 3, (i.e., following the steps shown in Figure 2), the bimolecularly coated substrate 10 is then immersed in a solution containing or suspected to contain the same antigen as the first solution, and such suspected antigen molecules 30 will selectively bind to the remaining active centers 12a in the antibody molecules 12 in yet another immunological reaction. Thus, the method can be used for analysis of a solution and easy detection of the presence of a particular antigen in it. Alternatively, the method can be used to produce several chains of alternately anti-gene antibody molecules, each chain originating at the surface of the substrate 10, thereby forming, by immunologically complex formation, a multimolecular film of the numerous chains. of alternating antigen-antibody molecules thereon. The film of multimolecular-complexes is readily recognized by considering the coated substrate with an optical instrument such as an ellipometer. Alternatively and as described in more detail in the disclosure of the aforementioned GB patents 20 Nos. 1,443,181 and 1,443,182 and in the description of GB patent no.

1,479,661, the film of multimolecular complexes can also be electrically examined by measuring the electrical capacity of a capacitor with conductor plates formed from the metal substrate or metal-coated substrate and a mercury drop or other suitable electrode where the capacitor dielectric is the protein layers. As described in these patents, the film of multimolecular complexes can also be optically examined visually without visual aids by determining the length of time before a visible amalgam is formed between a drop of mercury and metal film coating on the substrate with the protein layers 30. Finally, and most importantly, the film of multimolecular complexes can be examined optically with reflected or transmitted light, as explained in the above-mentioned patents. In the latter optical study by means of reflected or transmitted light, the following is a first (transmitted light) method which has been used successfully:

The substrate 10, which is to be a light transmitting substrate such as glass, plastic, molten silica, mica, quartz or the like, and preferably glass, where microscope glass is an easily accessible source, is first coated with a plurality of small metal balls. pidampe a metal, e.g. indium p in the substrate. Eg. this indium is slowly evaporated from a tantalum peel on the glass substrate in a regular vacuum of about 5 x 10 5 mm mercury. Due to the high mobility of the indium atoms on the substrate surface 5 and not wetting the glass substrate substantially, it agglomerates the small amount of evaporated indium in the substrate. Any metal having similar properties which will cause it to form small spheres on the substrate as it is vaporized can be used. In addition to indium, gold, silver, tin and lead have been used successfully.

10 Metal evaporation is continued until the substrate appears in a light brown color. At this point, the small metal spheres have diameters of the order of 1000 Å. The exact size of the spheres is not critical, but they must have diameters equal to a large fraction of a wavelength of visible light. The next step consists of immersing the spherical substrate 10 in a first solution of a first immunologically reactive protein, such as the antigen 11, and the inert protein 20. The first reactive protein and the inert protein again adhere to a monomolecular layer over the substrate and the metal beads. on this. Once a monomolecular layer is formed, the coated substrate is then removed from the first solution and immersed in a second solution containing the specific protein-responsive protein 12, resulting in the substrate and the metal beads obtaining a bimolecular protein layer that adheres thereto, similar to that of FIG. 2 (which is, however, without the metal-25 balls). The coated substrate is then removed from the second solution and immersed in a third solution containing or suspected to contain the reactive protein in the first solution; if the protein is present, a third layer (or partial layer) is formed on the substrate. The coated substrate is then considered by transmitted light, and a determination of the thickness of the protein layer adhering thereto, and consequently of the presence or absence of the putative protein, is made from the appearance of the coated substrate. The detection of protein layers corresponds to variations in the brown tint observed in the coated substrate. These variations are quite pronounced and the detection of the protein layers is therefore a simple process. The particles alone on the substrate appear as a first shade of brown, particles coated with a monomolecular protein layer appear as a darker shade of brown, particles covered with a bimolecular protein layer, appear as an even darker shade of brown, and particles covered with a tri-molecular protein layer seems even darker. This pivoting method is based on the fact that electromagnetic radiation is widely dissipated by conductive spheres of diameters equal to 5 by a large fraction of a wavelength of incident radiation, and that dissipation when scattering from such spheres is affected strongly by a thin dielectric coating applied to the spheres.

Another method of optical examination using reflected light that has been used successfully is the following: On a gold substrate, which for economic reasons is preferably a thin gold layer as a coating on a second metal, a monomolecular layer of the first one is adsorbed. reactive protein 11 and the inert protein 20 after immersion in the first solution described above. Gold has an adsorption band in the visible spectrum, and this fact depends on the characteristic color of the gold and the performance of the particular optical examination method in question. Suitably, the gold substrate may be a glass plate coated with a thin indium layer coated with the gold layer, the indium layer improving both the adhesion between the glass and the gold as well as the optical properties of the plate. The relative reflectivity of the gold substrate as a function of wavelength results in the substrate having the light yellow color characteristic of metallic gold in the absence of adherent protein layers on the gold. In the presence of a monomolecular layer on the substrate, the appearance of the sample plate (substrate) is dark. After the sample plate has been exposed to a solution containing the immunologically responsive protein 12 to the protein 11, wherein the protein 11 is in the first layer together with the inert protein 20, there is found on the sample plate a bimolecular protein layer and a reflectivity property which gives a greenish look. A third layer of protein gives an even greener look. In the experiments conducted so far, it appears that optical examinations of a coated substrate by means of reflected or transmitted light and using a substrate containing metal balls or a gold substrate are the most widely applicable. Further, it has been established that the above two methods have different sensitivities as a function of the thicknesses of the protein films of interest. In the method using a substrate containing metal spheres, the greatest sensitivity is found in films with thicknesses below approx.

10 Å. The Gu Id substrate method has the greatest sensitivity for films with a thickness over 30 Å. Thus, the particular detection method used determines the substrate type 10 used in the individual assay, that is, whether the substrate should be a metal plate 5 or a metallized glass plate with a flat metal (e.g., gold) coating or metal (e.g., indium) spheres on the surface, as again explained in the descriptions of the above patents. For simplicity reasons, the substrate 10 is shown with a flat surface, but it should be understood that the surface to which the first 10 antigenic layer adsorbs could also contain the aforementioned small metal spheres.

The process for forming multimolecular (3 layers) films of immunological complexes, as shown in FIG. 3, is particularly important when examining a solution for a particular protein, e.g.

15, as hormones and hormone antiserum are generally available. Thus, when the third layer is the hormone of interest, it is easily detectable.

Finally, formation of multimolecular films of immunological complexes according to the invention is also of importance in immunological determination of cells or viruses. As shown in FIG. 4, when constructing a plurality of chains of antigen-antibody complexes from the surface of the substrate 10, there is a relatively high probability that several of the antibody molecules at the end of the various chains find an antigenic center on a cell or virus 40, and this probability remains high regardless of the irregularity of the surface of the cell or virus. As is well known, the cell membrane enclosing each cell has molecules extending outward from the membrane and called untransplantation "antigens. These antigens are those used in forming the first layer 11 in a 30 layer layer. a system for immunological identification of cells in which the cell of interest will constitute the third layer, with a four-layer system of antigen-antibody chains, as shown in Figure 4, the first and third layers (11 and 30, respectively) similarly, it is well known that a virus has a protein sheath of 35 protein molecules that can be cleaved and separated, and such molecules form the first and the first and third layers, respectively, in two and four-layer systems of the antigenic cells, respectively. antibody chains used to immunologically identify a virus 40. The antibodies 12 in the second layer and 41 in the fourth layer will, of course, be specific to the particular cell or virus being examined for.

In FIG. Figure 5 shows a greatly enlarged standard view of a portion of a diagnostic agent in the form of a thin plate 10 of a suitable substrate material which may be metal, glass, mica, plastic, molten silica, quartz or a similar material wherein metal is preferred, having the greatest difference in refractive index relative to protein, and wherein the plate is preferably in the form of a metal plate or metallized glass plate. When the substrate 10 is immersed in a first solution, usually saline containing a first protein of interest, which may be biologically an antigen or antibody, it is adsorbed on the substrate in a monomolecular layer 11. In connection with the invention, the layer 11 containing adsorbed on the surface of substrate 10, hereinafter to be written as an antigenic layer.

Any protein will adsorb in such a monomolecular layer, but no further adsorption will take place, that is, the protein will bind to the substrate but not to itself. Thus, the antigenic protein layer can 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. Eg. covers a solution of the antigen associated with hepatitis at a concentration of 1 milligram per gram. cm a plate completely within approx. 10 min. with a monomolecular antigenic protein layer.

After the monomolecular layer of antigen protein 11 is formed over substantially the entire surface of the substrate 10, the coated substrate is removed from the solution of the antigen protein and then immersed in another solution containing or suspected to contain the antibody protein that reacts specifically. with the antigen protein. In connection with the invention, it is believed that the protein 11 adsorbed on the surface of the substrate 10 always has a larger size than the protein which is immunologically reactive with it and forms the second layer on the substrate. The second solution may contain many constituents in addition to the specifically reacting minor antibody protein, the presence of which it is desired to detect. However, no protein other than the specifically reacting antibody protein will adhere to the first antigenic protein layer on the substrate. Thus, only immunologically complex formation will occur between the antigen and the specifically reacting antibody if the specific reactive antibody protein is present in the second solution, and on the substrate there will, after a time, be a bimolecular protein layer. The time required for 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 stirring of the solution and the temperature of the solution. For blood serum antibodies, this period may be up to one day. The second layer may be a partial layer only or a substantially complete layer according to the three factors listed above.

In the case where the antigen layer 11 substantially covers the substrate 10 completely, that is, where the distance between adjacent antigen molecules is minimal, the number of antibody molecules 12 which can bind to a particular antigen molecule, as shown in FIG. 5, limited due to the limited surface available in the antigen molecule for the immunological complex formation. This substrate coating process is disclosed in the specification of the aforementioned GB Patents Nos. 1,443,181 and Nos. 1,443,182.

Because the monomolecular antigen layer 11 is adsorbed over substantially the entire surface of the substrate 10 of FIG.

20 5, the second layer 12 of the specific antibody of smaller size is bound to the antigen in a thin layer to form the bimolecular layer. However, because the proteins in the second layer 12 are smaller than the proteins in the first layer 11, and also because the second layer is much less dense than the first (antigenic) layer, it is quite time-consuming, however. clearly, there can be considerable difficulty in distinguishing the single and double protein layers on the substrate 10. A good example of this is the case where the first layer 11 is the hepatitis antigen (HAA) and the second layer 12 is an antibody specific for HAA. Viewing the coated substrate with an optical instrument such as an ellipse meter does not make it easy to distinguish between a single (antigenic) layer and a partial or diluted double (antigen-antibody complex) layer on the substrate, so it is not easy to determine , whether the second solution really contains antibodies to HAA, and such diagnostic sample is therefore of little value. In the method of the present invention, a much larger number of antibody molecules in the second layer 12 can be bound to an antigen molecule in the first layer 11, thereby forming a much greater contrast difference between the single and the double layer. The basis of the method of the present invention is shown in FIG. 6 and relates to the method of forming the first monomolecular layer of antigen molecules 11 adsorbed on the surface of substrate 10.

By way of illustration, the antibodies depicted in the figure are the IgG 5 class, but there is no reason why antibodies of the other four major classes listed above cannot be used according to the invention.

After selecting the substrate 10 on which the bimolecular film of immunological complexes is to be formed, this substrate is immersed in a first solution, usually saline containing the antigen, as in the above in connection with FIG. 5. However, in contrast to this prior method, an immunologically inert protein has been added to the first solution prior to immersing the substrate second, such that the adsorption layer 15 formed on the surface of substrate 10 consists of the relatively large reactive antigenic protein molecules 11 separated. apart and surrounded over the substrate surface 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 significantly smaller than the antigen molecules 11, so that the largest surface area (and therefore several combination centers) remains available on the antigen molecules 11 for later binding with antibody molecules. The amount of the immunologically inert protein 20 added to the solution is sufficient for the inert protein 20 to normally cover 1/2 to 9/10 25 of the area of the entire adsorption layer, i.e., the antigen similarly covers 1/2 to 1/10 of the area, though this is not always a necessary condition, since a sufficient average distance between adjacent antigen molecules is also determined by the number of active centers on the particular antigen molecule and the way 30 such molecules bind to the substrate, i.e. the number of antigenic active centers that remain free. In the case where the antibody molecule is much smaller than the antigen, a greater distance between the antigen molecules is desirable, whereas a smaller distance between the antigen molecules can be tolerated if the antibody molecule is only slightly smaller than the antigen to obtain the best effect of the practice of the invention. Due to the large size of the antigen protein relative to the inert protein molecule, a larger area of the surface of the antigen molecule will remain free and have its corresponding binding centers available for combination with antibody molecules in a subsequent immunological reaction.

The (with antigenic inert protein) monomolecularly coated substrate 10 is then removed from the first solution and then immersed in a second solution containing the antibodies specific for the antigen in the first solution. As a result, a larger surface area (and several antigen centers) remain free on the antigens when the antigens are surrounded by the smaller, inert protein of FIG. 6, the antigen molecules of FIG. 6 in comparison with those in FIG. 5 more easily form immunological compounds with a greater number of antibody molecules.

As seen in FIG. 6, a greater number of antibody molecules 12 will thus generally associate with each antigen molecule than in the one shown in FIG. 5, and it is from a comparison of FIG. 5 and 6, it is clear that a much larger contrast difference is obtained between 15 single and double layers in the case of FIG. 6 because of the greater number of antibody molecules contained in the second layer). As an example of the practice of the invention, a first solution has a sufficient concentration of the hepatitis B antigen (HAA) and inert protein in the form of bovine serum albumin (BSA) that HAA covers 1/4 of the surface area of a monomolecular layer. and the inert BSA protein covers the remaining 3/4 of the area and another solution is a diluted solution of antibody to HAA. Thus, the method described above can be used in analyzing a solution for easy detection of the presence of an antibody specific to a particular antigen of relatively large size.

The substrate of FIG. 6, which is coated with a bilayer, can then be immersed in a third solution or serum containing antibodies 30 to the antibodies 12 contained in the second layer to form a third layer, as shown in FIG. 7. Since each antibody molecule 12 has several antigenic determinants, it can generally form compounds with several molecules 30, which are antibodies to antibody protein 12, thereby forming a relatively dense third layer of such secondary antibody molecules 30. The presence of the dense third layer 30 is an indication of the presence of the second layer 12 and provides an even greater difference between the monomolecular layer and the bimolecular antigen-primary antibody layer. In this latter case, the inert protein molecules 20 between the major antigen molecules 11 in the first layer also help prevent the non-specific protein material in the serum containing antibodies to the antibody protein for this antigen from adhering to the substrate.

Thus, there is the double benefit that more specifically bound antibodies are obtained for each antigen molecule and that there is less 5 non-specifically bound protein on the substrate, which is due to the use of the inert protein to separate the large antigen molecules from one another and to cover of the surface of the substrate surrounding each antigen molecule with this inert protein.

The method used to form the third layer of FIG. 7, is therefore useful in analyzing a second solution suspected of containing antibody 12, since subsequent immersion of the coated substrate in the third solution will provide significantly improved contrast between the monomolecular layer and the bimolecular layer (in fact now a trimolecular layer, 15 if a bimolecular layer exists because the second solution actually contains the antibody 12).

The presence of the second (antibody) protein layer in the embodiment of FIG. 6, which is also indicated by the third (antibody 30) protein layer in the embodiment shown in FIG. 7, can easily be verified by considering the coated substrate with an optical instrument such as an ellipsometer. As described above and in more detail in the descriptions of the aforementioned GB patents Nos. 1,443,181, Nos. 1,443,182, and Nos. 1,479,661, another possibility is that the protein layers can be electrically examined by milking the electrical capacity of a capacitor with conductive plates formed of metal substrate or metal coated substrate and a mercury drop or other suitable electrode, the capacitor dielectric being the protein layers. Also, as described in the descriptions of the above GB patents and above in connection with the multimolecular film, the protein layers can be examined optically by visual observation without aids by determining the length of time before a visible amalgam is formed between a drop of mercury and a metal film coating of the substrate with the protein layer in between. Finally, and most importantly, the protein layers can be examined optically with reflected or transmitted light as explained in the aforementioned patents and above in connection with the multimolecular film.

However, in the case of protein layers, the coated substrate is then removed from the second solution and immersed in a third solution containing a protein that is reactive to the second solution (such as an antibody to the antibody). , to form a third layer (or partial layer) on the substrate if the second layer is present. The coated substrate is then observed in transmitted light, and from the appearance of the coated substrate, a determination is made of the thickness of the protein layer adhering to the substrate, and thus a determination of whether the second protein 12 is present or not.

The difference between single and double protein layers can be further enhanced by depositing the antigen and inert protein on substrate 10 as a single solution droplet and then immersing the substrate coated with the droplet in a solution containing only the inert protein 20 such that a monomolecular layer of the small antigenic inert protein region is formed completely surrounded by the inert protein, as shown in FIG. 8. This aspect causes the problem of non-specific adsorption to be overcome in the case where the antibody proteins are present in a serum as the inert protein on the residual surface of the substrate prevents nonspecific proteins in the serum from adhering to the substrate, and thereby improving the contrast.

The simple methods described above have made it possible to detect liver inflammation in a sample as sensitive as the radioimmuno-standard test.

Finally, the method described above is important in the immunological identification of viruses and enzymes. All virus type antigens are larger than their specific antibodies, whereas the enzyme type antigens may be larger or smaller depending on the particular enzyme type. A method for identifying a particular virus or large enzyme known as the inhibition assay is carried out as follows:

The particular virus or major enzyme is placed in a first solution (usually saline) with the inert protein, and the substrate is then immersed in this solution, or alternatively, a drop of the solution is applied to the substrate and the substrate is then immersed in a solution of only the inert protein. After a monomolecular layer of the virus or enzyme (i.e., the antigen) and the inert protein has been adsorbed on the surface, the coated substrate is removed from the first solution.

Human serum samples to be tested for the presence of the particular virus or enzyme are prepared by mixing therein an amount of known antibodies for the specific virus or enzyme which is adapted to be immunologically removed from the mixture. , if the virus or enzyme is present in the serum sample. The monomolecular coated substrate is then immersed in or subjected to the pre-made serum sample and examined after removal from it by one of the methods described above. If the examination of the substrate shows the presence of only one monomolecular layer, it is known that the human serum examined initially contained the particular virus or enzyme. However, if a bimolecular layer is detected, this indicates that the serum did not initially contain this viral or major enzyme antigen, as the antibody proteins did not form immunological compounds with their specific antigen (virus or enzyme) in the serum sample and were therefore free to form compounds. with the antigen in the first adsorbed layer on the substrate.

Although the reactive protein adsorbed on the substrate surface is described above as an antigen, it should be understood that the reactive protein in the first layer may be an antibody and the protein in the second layer will then be the antigen specific for this antibody. while the third layer protein is again the antibody protein. Thus, the reactive protein adsorbed on the substrate surface as a first layer of reactive protein and described above as an antigen may be an antibody, and the second protein layer will then be the specific antigen thereof, which arrangement is particularly useful in cases where the antibody molecule is larger than its specific antigen; An example of this is the antibody to insulin.

Claims (16)

151240 A process for producing multimolecular layers of immunologically reactive proteins, wherein (a) a suitable substrate is coated with a monomolecular protein layer consisting of an immunologically reactive first protein, and (b) the coated substrate is then treated with a solution of an immunologically reactive second protein specific to the first protein to produce an immunological reaction wherein the second protein's molecules are bound to the first protein's molecules to form a bimolecular layer, characterized in that the substrate is further coated with a immunologically inert protein adsorbable to the substrate and in such a way that the molecules of the first protein are 15 spaced apart by the inert proteins. Process according to claim 1, characterized in that the substrate is a metallized plate. Method according to claim 1 or 2, characterized in that the immunologically reactive first protein is an antigen and the immunologically reactive second protein is an antibody specific for the antigen. Method according to any one of the preceding claims, characterized in that the antibody is an antibody of the immunoglobulin Ig G class. The method of claim 4, characterized in that the antigen is an enzyme, the inert protein is bovine serum albumin, and the antibody is obtained from a rabbit previously injected with the enzyme. A method according to any one of the preceding claims, characterized in that the coating of the substrate with a monomolecular layer of the first protein and inert protein comprises: i) forming a first solution of a protein antigen, ii) adding a sufficient amount of the inert protein for the first solution; iii) immersing the substrate in the first solution for coating the substrate with a monomolecular protein layer, the amount of the inert protein sufficient for the protein antigen molecules to separate from the monomolecular layer iv) removing the substrate coated with the monomolecular layer from the first solution. Method according to any one of claims 1 to 5, characterized in that the substrate is flat and the coating of the substrate with a monomolecular layer of the first protein and inert protein comprises: i) forming a first solution of inert protein, ii) sufficient immersion of the substrate in the first solution to coat the substrate with a partial monomolecular layer of the inert protein, iii) removal of the partially coated substrate from the first solution, iv) formation of a second solution of immunologically reactive first protein; 15 v) immersion of the partially coated substrate in the second solution to coat the residual surface of the substrate with a partial monomolecular layer of the immunologically reactive first protein such that the monomolecular layer comprises the immunologically reactive first protein molecules separated from each other by the inert proteins, and vi) removing the monomolecular layer coated sub strat from the second solution. Process according to any one of claims 25 to 5, characterized in that the coating of the substrate with a monomolecular layer of the first protein and inert protein comprises: i) forming a diluted first solution of the immunologically reactive first protein, ii) sufficient immersion of the substrate in the first solution to coat the substrate with a partial monomolecular layer of the first protein, iii) removal of the partially coated substrate from the first solution, iv) formation of a second solution of the v) immersing the partially coated substrate in the second solution to coat the residual surface of the substrate with a partial monomolecular layer of the inert protein such that the monomolecular layer consists of the molecules of the immunologically reactive first protein separated and (vi) removing the monomolecular layer-coated substrate from the a. than the solution. A method according to any one of claims 1-5, characterized in that a protein antigen solution is formed that the substrate is sufficiently immersed in the antigen solution to coat the substrate with a partial monomolecular layer of the antigen that the partially coated substrate is removed from the the antigen solution and the partially coated substrate is immersed in a solution of the corresponding antibody where the solution is a serum and the proteins present in the serum in addition to the antibodies act as the inert protein and adhere to the substrate to complete the monomolecular protein layer containing antigen molecules that are separated from each other by inert protein molecules, and the antibody molecules from the serum form the second layer. Process according to claim 6, characterized in that the inert protein is added in a sufficient quantity that the average distance between adjacent protein antigen molecules in the monomolecular layer on the substrate will be several hundred Angstroms. A method according to any one of claims 6-10, characterized in that it further comprises: (c) removing the bimolecular layer-coated substrate 25 from the antibody solution, (d) subsequently treating the coated substrate with a third one. a solution suspected of containing the same antigen present in the first solution to produce an immunological reaction with the antibody, where the remaining active centers of the antibody molecules are chemically bound to the antigen molecules if present in the third one. and (e) examining the coated substrate to determine if there is a third layer thereon which indicates, if appropriate, the presence of the antigen in the third solution. A method according to any one of claims 6-10, characterized in that the first solution of the immunologically reactive first protein is prepared in such a way that the antigens present in the outermost parts of a particular cell or cell in particular, virus is separated, and a solution thereof is formed, and: (c1) the substrate coated with the bimolecular layer is removed from the antibody solution, (d ') the coated substrate is subsequently immersed in a third suspected solution. to contain the particular cell or virus, such that the antibody molecules are bound to the antigenic centers of the cell or virus, and (e1) the coated substrate is examined to determine if there is a third layer thereon indicating the presence of the particular cell 10. or the special virus in the third solution. The method of claim 12, further comprising: (f) removing the coated substrate from the third solution, and (g) subsequently treating the coated substrate with a fourth solution containing the same antibody, which is in said second solution, to produce an immunological reaction in which active centers on the antibody molecules in the fourth solution are bound by antigen molecules from the third solution. A method according to claim 13, characterized in that it further comprises: (h) removing the coated substrate from the fourth solution; (j) subsequent treatment of the substrate having antigen-antibody chains constructed from the substrate surface. , with a fifth solution suspected of containing cells or virus, so that the antibody molecules in the chains of antigen-antibody complexes find antigenic centers on the cell or virus to produce binding thereon, (k) removing the coated substrate from the fifth solution, and (l) examination of the substrate to determine whether residual active centers on the antibody molecules have been combined with antigenic centers on the cells or virus particles of the fifth solution. The method of claim 1 or 2, characterized in that the immunologically reactive first protein is an antibody and the immunologically reactive second protein is an antigen to which the antibody is specific. The method of claim 1 used to improve the contrast of a surface immunological sample of relatively large size protein antigens, characterized in that the substrate is coated with a monomolecular protein layer consisting of a protein antigen and an inert protein of smaller size than the protein antigen, thus that the antigen molecules are separated from each other and surrounded along the substrate surface by less inert protein molecules, and that the coated substrate is then treated with a solution of an antibody specific to the protein antigen and of a size smaller than that to produce an immunological reaction therewith, the antibody molecules are bound to the protein antigen molecules to form a bimolecular layer on the substrate.