GB1564333A - Method for contrast in surface immunological tests - Google Patents

Description

(54) METHOD FOR IMPROVING CONTRAST IN SURFACE IMMUNOLOGICAL TESTS (71) We, GENERAL ELECTRIC COMPANY, a corporation organized and existing under the laws of the State of New York, United States of America, of I River Road, Schenectady 12305, State of New York, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to a method for detecting immunologically reactive antigens and immunologically reactive antibodies specific to the antigen, and in particular, for improving the contrast between the single and double layers of the resulting antigenantibodies complex.

The present invention relates to the inventions described in our Patent Nos.

1,443,181, and 1,443,182, 1,479,661 and 1,486,826.

Immunological reactions are highly specific interactions in which an antigen interacts with a second biological constituent specific to the antigen and generally known as the antibody to form an immunological complex. Immunological reactions taking place within a biological system such as an animal or a human being are vital in combating disease. In a biological system, the entry of a foreign biological constituent, i.e., the antigen, causes the biological system to produce the specific antibody to the antigen in a process not fully understood at this time. The antibody molecules have available chemical combining or binding sites which complement those on the antigen molecule so that the antigen and antibody combine or bond to form the immunological complex.

Antibodies are produced by biological systems in response to invasion thereof by foreign bodies. Consequently, the detection of antibodies present in a biological system is of medical diagnostic value in determining the antigens to which the system has been exposed. It would be useful, for example, to test for the presence of the antibody to syphilis or gonorrhea in human blood, plasma or tissue. Conversely, the detection of certain antigens in a biological system also has medical diagnostic values; examples of diagnostic detection of antigens include detection of human chorionic genodotrophin (HCG) protein molecules in urine as a test for pregnancy, and detection of hepatitisassociated antigen (HAA) molecules in blood of prospective blood donors.

Most antigens are proteins or contain proteins as an essential part, whereas all antibodies are proteins. Proteins are large molecules of high molecular weight, specifically, they are polymers consisting of chains of amino acids. The antigen and antibody protein may each have several combining sites. 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 heavy (long) peptide chains of amino acids and at least two light (short) peptide chains of the acids wherein 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 combining sites are the extreme ends of the two arms of the Yshaped antibody for the Ig G antibody.

In addition to the immunological reaction which occurs between specific protein antigens and specific protein antibodies resulting in the formation of a protein antigen-protein antibody complex, other immunological complexing reactions between immunologically reactive antigens and antibodies are also contemplated by this invention. In addition, specific reactions between other biological particles, such as enzymes and substrates are also among the test methods contemplated and are embraced by the term "immunological reaction" as used herein. As used herein, the terms "antigen" and "antibody" include enzymes and substrates and similar biological particles.

For instance, the following systems include biological particles which are capable of undergoing the specific reactions described herein: Viruses Bacteria and Bacterial toxins Fungi Parasites Animal tissue and Animal body fluids.

With respect to viruses, the antigens are viral cultures or parts thereof and the antibody specific thereto can be produced by administration of the antigens to a living host. Illustratively, antigen-antibody complexes in the following virus systems are useful in the hereindisclosed procedure: Rubella virus culture (antigen) - Rubella virus antibody; polio virus culture (antigen) polio virus antibody; vesicular stomatitis virus (VSV) culture (antigen) VSV antibody.

Regarding bacteria and bacterial toxins, the antigens are the particular bacteria and bacterial toxin or parts thereof and the antibody is produced by injection of the antigens into a living host. The following are illustrative examples of antigen-antibody pairs which can be used in the present method: tetanus toxoid suspension (antigen) - tetanus antibody; diptheria toxin suspension (antigen) - diptheria antibody; Neisseria gonorrhoeal suspension (antigen) - gonorrhea antibody; Treponema pallidum suspension (antigen) syphilis antibody.

As for fungi, the antigens are antigenic extracts of fungal suspensions and the antibody is the fungal antibody produced by injection into a living host. Antigenantibody complexes of fungi systems are illustrated by the following: Aspergillus extract suspension (antigen) - aspergillus fungus antibody; Candida extract suspension (antigen) candida fungus antibody.

Antigens and antibodies in parasites systems are tested in a similar fashion to those of fungi. The system Toxoplasma gondii extract (antigen) Toxoplasma gondii antibody is a typical example.

By the term polysaccharides is meant a system wherein the antigen is a carbohydrate antigen. An example of such an antigen-antibody containing system is pneumococcus polysaccharides (antigen) pneumococcus antibody.

In addition to the typical enzyme-enzyme substrate reaction which is intended to be covered herein, enzymes themselves or parts thereof may be utilized as antigens and the antibody is the particular enzyme antibody elaborated by a living host after injection. Illustrative antigen-antibody complexes of enzyme systems are: trypsin extract -- trypsin antibody chymotrypsin extract - chymotrypsin antibody pepsin extract -- pepsin antibody ribonuclease extract - ribonuclease antibody thrombin extract -- thrombin antibody amylase extract - amylase antibody penicillinase extract - penicillinase antibody With respect to hormones, the antigenic constituent is usually found in a hormone extract and the antibody is the particular hormone antibody elaborated by the living organism after injection.An exemplary antigen-antibody complex is: insulin -- insulin antibody.

Although the ensuing discussion is directed for the most part to immunological interactions between specific protein antigens and specific protein antibodies, it is understood that it applies to the systems and the immunologically reactive antigens and antibodies hereinabove described with the proviso that when immunologically reactive antigen is a protein and the antibody is a protein specific to the protein antigen, the immunologically inert organic compound to be described is not a protein.

As is fully described in the aboveidentified Patent Specifications immunological interactions can be detected by various techniques including the use of a suitable substrate such as a metallized glass or metal slide. Exposure of the substrate to an aqueous medium containing antigen, such as a solution or suspension, will result in the antigens being physically adsorbed in a dense monomolecular layer onto the surface of the substrate.Subsequent exposure of the antigen-coated substrate to a serum or other medium containing antibodies specific to the antigen results in the immunological reaction wherein the antibodies selectively attach themselves to the antigens by means of the binding sites on the antibody molecule which complement those on the antigen molecule to thereby form at least a partial bimolecular layer of immunologically complexed antigenantibody on the substrate surface. A problem which often arises in the abovementioned methods is the inability to distinguish between the monomolecular and bimolecular layers of the complex on the substrate, especially if the adsorbed (antigen) layer is relatively thick as in the case of the HAA layer since the hepatitisassociated antigen molecule has at least ten times the molecular weight as that of the antibody to the HAA.A solution to this problem has now been discovered, and this is the subject matter of the present invention.

The present invention is a modification or improvement of the invention described in Patent Specification No. 1,486,826.

The present invention provides a method for forming multimolecular immunologically complexed films comprising the steps of treating a substrate with an immunologically reactive antigen and an immunologically inert organic compound to form a monomolecular layer of said antigen and said compound so that the antigen molecules are separated from each other by the inert organic compound molecules, and subsequently exposing the treated substrate to an aqueous medium containing an immunoSogically reactive antibody specific to the antigen to cause an immunological reaction therewith in which the antibody molecules bond with the antigen molecules to form a bimolecular layer on the substrate, and the antibody molecules retain a significantly greater number of active sites for a further immunological reaction than if the antigen molecules were closely spaced together, the immunologically inert organic compound being other than a protein when the immunologically reactive antigen is a protein and the antibody is a protein specific to the protein antigen.

The present invention also provides a method for forming multimolecular immunologically complexed films comprising the steps of treating a substrate with an immunologically reactive antigen by forming a dilute first solution of said antigen, immersing the substrate in said first solution sufficiently to coat the substrate with a partial monomolecular layer of the antigen, and removing the partially coated substrate from the first solution, and immersing the partially coated substrate in a serum solution comprising an immunologically reactive antibody and an immunologically inert organic compound whereby inert organic compound adheres to the substrate to complete a monomolecular layer of antigen molecules separated from each other by inert organic compound molecules, and the antibody molecules in the serum forms a second layer, the immunologically inert organic compound being other than a protein when the immunologically reactive antigen is a protein and the antibody is specific to the protein antigen.

The present invention also provides a method for binding reactive constituents to a surface so that they will remain active comprising the steps of coating a metallized slide with a monomolecular layer of a reactive first constituent and immunologically inert organic compound so that the first constituent molecules are separated from each other by inert organic molecules, and subsequently exposing the coated slide to an aqueous medium containing a reactive second constituent specific to the first constituent to cause a reaction therewith in which the second constituent molecules bond with the first constituent molecules to form a bimolecular layer on the slide, the inert organic compound being sufficient quantity to obtain sufficient average spacing between adjacent first constituent molecular layer so that the second constituent molecules retain active sites for a further immunological reaction, the inert organic compound being other than a protein when the reaction first constituent is a protein antigen and the reaction second constituent is a protein antibody specific to the protein antigen.

The present invention also provides a method for binding antibodies to a surface so that they remain active comprising the steps of forming an aqueous medium containing an immunologically reactive antibody, adding a sufficient quantity of an immunologically inert organic compound to said medium, and exposing a metallized slide to the aqueous medium to coat at least a portion of the slide by adsorption with a monomolecular layer of the antibody and the inert organic compound wherein the antibody molecules are sufficiently spaced from each other by the sufficient quantity of inert organic molecules so that the antibody molecules present a greater number of active sites which are retained for a further immunological reaction then if the antibody molecules were closely spaced together, such that when the immunologically inert organic compound is an inert protein, the reaction is other than an immunological reaction between a protein antigen and a protein antibody specific to the antigen.

The present invention also provides a method for determining the presence or absence of a suspected constituent in an aqueous medium comprising the steps of forming a first aqueous medium containing an immunologically reactive antigen, adding a quantity of an immunologically inert organic compound to the first aqueous medium, contacting a metal slide or a metallized glass slide with said aqueous medium to coat all or part of the slide with a monomolecular layer of the antigen and the inert organic compound, the quantity of inert compound being sufficient to provide that the antigen molecules are separated from each other by the inert organic molecules, removing the monomolecular layer coated substrate from the first aqueous medium, subsequently exposing the coated substrate to a second aqueous medium containing an immunologically reactive antibody specific to the antigen to cause an immunological reaction therewith in which the antibody molecules bond with the antigen molecules to form a bimolecular layer on the substrate, and the antibody molecules retain a significantly greater number of active sites from a further immunological reaction than if the antigen molecules were closely spaced together, removing the bimolecular layer coated substrate from the antibody containing medium, exposing the coated substrate to a third aqueous medium suspected of containing the same antigen which is in the first aqueous medium and causing an immunological reaction to occur with the antibody in which the remaining active sites on the antibody molecules bond with the antigen molecules in the third aqueous medium, and examining the coated substrate to determine whether there is a third layer thereon thereby indicating the presence of the antigen in the third aqueous medium, such that when the immunologically inert compound is an inert protein, the immunological reaction is not between a protein antigen and a protein antibody specific to the antigen.

In accordance with one aspect of this invention, one adds an immunologically inert organic compound to an aqueous medium containing an immunologically reaction antigen. A substrate is then contacted with the aqueous medium and the surface or a part of the substrate is coated, by adsorption, with a monomolecular layer of the antigen molecules and immunologically inert organic compound molecules wherein each antigen molecule is generally surrounded by inert organic molecules. Subsequent immersion of the coated substrate in a second aqueous medium containing an immunologically reactive antibody specific to the antigen results in the antibody molecules bonding with the antigen molecules to form at least a partial bimolecular layer on the substrate.

The quantity of inert organic compound molecules in the first layer is sufficient so that the spacing between antigen molecules permits more antibody molecules to be bound to the antigen molecules than if the antigen molecules were densely packed together, and thereby produces a much greater change in contrast between single and double layers coated on a substrate. It appears that the contrast is also improved because the average thickness of the first monolayer is reduced (lightened).Thus, this embodiment of the invention provides an especially useful procedure for distinguishing between a single layer of a large size antigen, such as hepatitis-associated antigen, and a double layer which includes a second layer of a relatively smaller antibody such as the hepatitis antibody, and thus can be utilized in the analysis of the second solution to determine the presence of the antibody therein.

In accordance with another aspect of this invention, an immunologically inert organic compound is added to an aqueous medium containing an immunologically reactive antigen. A substrate is then treated with the aqueous medium and the surface of the' substrate is coated by adsorption, with a monomolecular layer of the antigen molecules and inert organic compound molecules. The quantity of inert organic compound molecules is sufficient so that the adsorption layer consists of reactive antigen molecules separated from each other to average distances of several hundred Angstroms by the inert organic molecules.

The immersion of the coated substrate in an aqueous medium containing an immunologically reactive antibody specific to the antigen can result in the antibody molecules bonding with the antigen molecules, but leaving the remaining antibody sites active. As a result, subsequent immersion of the coated substrate to an aqueous medium suspected of containing the antigen results in further attachment thereof to the active sites of the antibodies that are bound to the first antigenic layer. Thus, this invention can be utilized in the analysis of an aqueous medium for detecting the presence of a particular antigen. A subsequent immersion of the coated substrate in aqueous medium alternately containing the same antibody and antigen can build up relatively long chains of antigen-antibody complexes from the surface of the substrate. The method can be used to identify cells or viruses immunologically since the probability for several of the antibody molecules on the ends of the chains of antigen-antibody complexes finding an antigenic site on particular cells or viruses is relatively high irrespective of the irregularity of the surface of the cell or virus.

The invention, both as to its organization and method of operation, together with further objects and advantages thereof, may best be understood by reference to the following description, by way of example only, taken in connection with the accompanying drawing wherein like parts in each of the several figures are identified by the same reference character.

The present invention will be further described by way of example only with reference to the accompanying drawings in which: Figure 1 is an elevation view of a substrate after it has been contacted first with an aqueous medium containing antigen by a known method and then immersed in aqueous medium containing antibody specific to the antigen; Figure 2 is an elevation view of the substrate after it has undergone two steps as in Figure 1, but with the first contact being with a mixed antibody-organic compound in accordance with the invention; Figure 3 is an elevation view of the substrate of Figure 2 after a subsequent immunological reaction wherein the coated substrate is immersed in an aqueous medium containing an antigen to the antibody in the second aqueous medium, and;; Figure 4 is an elevation view of the substrate of Figure 3 after a further immersion of the substrate in an aqueous medium containing the s me antibody and wherein the antibody molecules on the ends of the chains of the antigen-antibody complexes find antigenic sites on a virus or cell for identification thereof immunologically.

Figure 5 is a plan view of a substrate having a small region thereon of antigen and immunologically inert organic compound surrounded by said inert organic compound in accordance with the invention.

Referring now to Figure 1, there is shown a highly magnified elevation view of a portion of diagnostic apparatus in the form of a thin wafer 10 of a suitable substrate material which may be metal, glass, mica, plastic, fused silica, quartz or similar material with metal being preferred as having the greatest difference in refractive index to protein and preferably is in the form of a metal or metallized glass slide. A detailed discussion of substrate metallization is found in the aforementioned U.K. Patent Nos. 1,443,181, 1,443,182 and 1,479,661.

Substrate 10 when contacted with a first aqueous medium, which may be biologically an antigen or antibody dissolved in salt water, has it adsorbed onto the substrate in a monomolecular layer 11. For the purpose of this invention, the contact may be by applying a drop of solution or by partial or complete immersion in the solution, and, to simplify the following description, layer 11 adsorbed on the surface of substrate 10 will hereinafter be described as an antigen layer.

Any antigen or antibody will adsorb in such monomolecular layer, but no further adsorption will take place, that is, the antigen or antibody will attach to the substrate, but will not attach to itself. Thus, the antigen layer 11 can only be monomolecular and not of greater thickness. The time required to completely coat the substrate with the antigen is a function of the concentration of the antigen in the solution, the degree of agitation of the solution and the solution temperature. As an example, a concentration of 1 milligram per milliliter of hepatitis-associated antigen solution completely coats a slide in approximately 10 minutes with a monomolecular antigen layer.

After the monomolecular layer of antigen 11 has formed over substantially the entire surface of the substrate 10, the coated substrate is removed from the aqueous medium containing the antigen, and is next immersed in a second aqueous medium containing, or suspected of containing, the specifically reacting antibody to the antigen.

For purposes of this invention, it will be assumed that the antigen 11 adsorbed on the surface of substrate 10 is always of greater site than the antibody which is immunologically reacted therewith and forms a second layer on the substrate. The second aqueous medium may contain many constituents in addition to the specifically reacting smaller antibody whose presence it is desired to detect. However, nothing other than the specifically reacting antibody will adhere to the first antigen layer on the substrate. Thus, only if the specifically reacting antibody is present in the second aqueous medium will immunological complexing between the antigen and its specifically reacting antibody takes place and the substrate will, after a time, have a bimolecular layer thereon.The time required for the adhesion of the second (antibody) molecular layer 12 onto the coated substrate is again a function of the concentration of the specifically reacting antibody in the solution, the degree of solution agitation, and solution temperature. For antibodies in blood serum, this timing may be as long as one day, or as short as minutes, depending on the concentration. The second layer may be only a partial one, or substantially complete, depending upon the above three enumerated factors.

As illustrated in Figure 1, in the case wherein the antigen layer 11 substantially completely coats substrate 10, i.e., the spacing between adjacent antigen molecules is minimal, the number of antibody molecules 12 that can bind to any particular antigen molecule is limited due to the limited surface of the antigen molecule which is available for such immunological complexing. This substrate coating process is described in our Patent Specification No.

1,443,181.

Due to the monomolecular antigen layer 11 being adsorbed over substantially the entire surface of substrate 10 in Figure 1, the second layer 12 of the specific antibody for such antigen bonds thereto in a thin layer to form the bimolecular layer.

However, in the case when the antibodies in the second layer 12 are of smaller size than the antigens in the first layer 11 and also in case when the second layer is much less dense than the first (antigen) layer, it is clearly evident that there can be considerable difficulty in distinguishing between this single and double layer of antigen and antibody on the substrate 10. A good example of this situation is the case of first layer 11 being hepatitis-associated antigen and the second layer 12 being an antibody specific to HAA.Examination of the coated substrate with an optical instrument such as the ellipsometer does not readily allow one to distinguish between the single (antigen) and a partial or dilute double (antigen-antibody complex) layer on the substrate and thus it cannot readily be determined whether the second aqueous medium did indeed contain any of the antibodies to HAA and thus such diagnostic test is of little value. In the present invention, a method is provided in which a much greater number of antibody molecules in the second layer 12 can be bonded to the antigen molecule in the first layer 11 to thereby produce a much greater change in contrast between the single and double layers while at the same time having sites remaining active for further combining with other antigen molecules in a further immunological reaction.Also since the first layer has a reduced average thickness (close to that of albumin), contrast is improved.

The basis of the present discovery is illustrated in Figure 2 and is involved in the method of forming the initial monomolecular layer of antigen molecules 11 which are adsorbed onto the surface of substrate 10. For purposes of illustration, the antibodies depicted herein are of the Ig G class.

After selection of the substrate 10 on which the bimolecular immunologically complex film is to be formed such substrate is contacted with a first aqueous medium, e.g., a salt water solution containing the antigen as in the case of the method described hereinabove with reference to Figure 1. But in contradistinction with such previous method, an immunologically inert constituent has been added to the first aqueous medium prior to contact of the substrate therewith such that the adsorption layer formed on the surface of substrate 10 consists of the relatively large reactive antigen molecules 11 separated from each other, and surrounded along the substrate surface, by inert organic compound molecules 20 as seen in Figure 2.The inert organic compound molecules 20 are of a size at least somewhat smaller, and preferably substantially smaller than the antigen molecules 11 in order to allow the greatest surface are (and therefore more combining sites) to remain available on the antigen molecules 11 for subsequent bonding with antibody molecules.The quantity of the immunologically inert organic compound 20 added to the solution is sufficient so that, in general, the inert organic compound 20 covers 1/2 to 9/10 of the area of the complete adsorption layer, that is, the antigen corresponding covers 1/2 to 1/10 of the area, aithough this is not a limitation on the invention since the average spacing between adjacent antigen molecules is also determined by the number of active sites of the particular antigen molecule and the manner in which such molecules adhere to the substrate, i.e., the number of antigenic active sites which remain exposed.

Thus, in the case wherein the antibody molecule 12 is very much smaller than the antigen, a larger spacing of the antigen molecules is desirable, whereas if the antibody molecule is only slightly smaller than the antigen, a smaller spacing between antigen molecules may be tolerated in order to obtain the maximum benefit from the invention. In general the spacing between adjacent immunologically reactive antigen molecules adsorbed on the surface of the substrate will generally be to distances of several hundred Angstroms. As a result of the large size of the antigen relative to the inert organic molecule, a major portion of the surface of the antigen molecule will remain exposed and has its corresponding bonding sites availabe for combining with antibody molecules in a subsequent immunological reaction.

The inert organic compound can vary widely so as to chemical type, it being only required that protein not stick to it. Merely by way of illustration, one can use bovine serum albumin, egg albumin and insulin.

The concentrations employed will in general range from 0.05 to 100 mg./ml. of the aqueous medium, with the amount selected to depend on the concentration of antigen.

The (antigen-inert constituent) monomolecular coated substrate 10 is then immersed in a second solution suspected of containing the antibodies specific to the antigen in the first solution. As a result of the greater surface area (and more antigenic sites) remaining exposed on the antigens due to the antigens being surrounded by the smaller inert constituent in Figure 2, as compared to Figure 1, the antigen molecules in Figure 2 can more readily immunologically combine with a greater number of antibody molecules than in Figure 1.Since the spacing between the active sites of an antibody molecule 12 (of the Ig G Class) is approximately 200 Angstrom, a large fraction of the antibody molecules 12 in the second medium which combine with the antigen molecules 11 will have their remaining combining sites (and there may be more than one remaining active site per molecule, depending upon the particular class of antibody molecule) remain active for further combination with additional antigen molecules in a subsequent immunological reaction. Any one antibody molecule cannot in general combine with more than one active site on any one antigen molecule.Thus, as seen in Figure 2, due to the sufficient spacing between adjacent antigen molecules 11 by means of the immunologically inert organic compound molecules 20, the antibody molecules 12 are bound to the antigen molecules 11 and in general each antibody molecule 12 has a combining site 12a remaining active for a subsequent immunological reaction. One of the principal objects of this invention has thus been achieved, antibodies have been bound to a monomolecular antigen layer surface such that combining sites of the antibodies remain active for further immunological reactions. In the case of the active antigen being human serum albumen, the immunologically inert protein is egg albumen, as one example, and the antibodies are obtained from a rabbit serum.In the case of a 1% human serum albumen solution, the concentration of the inert egg albumen protein therein is in the range of ll00X.

As seen in Figure 2, a greater number of antibody molecules 12 will, in general, combine with each antigen molecule than in the Figure 1 case, and it is obvious from a comparison of Figures 1 and 2 that a much greater change in contrast between the single and double layers is obtained in the Figure 2 embodiment of this invention (due to the greater number of antibody molecules that are in the second layer and the more uniform thickness, better matching the background). For an example of this invention, the first solution has a concentration of hepatitis-associated antigen (HAA) and bovine serum albumin (BSA) inert protein sufficient to have the HAA cover 1/4 of the surface area in the monomolecular layer and the BSA inert protein cover the remaining 3/4 area and the second solution is a dilute solution of the HAA antibody.The method described hereinabove can thus be utilized in the analysis of an aqueous medium for readily detecting the presence of an antibody specific to a particular relatively large size antigen.

The double layer coated substrate of Figure 2 can subsequently be immersed in a third aqueous medium such as a serum containing antigen 30 to the antibodies 12 in the second layer in order to build up a third layer as illustrated in Figure 3. Since each antibody molecule 12 has several antigenic determinants, it can, in general, combine with several molecules 30 that are antibodies to the antibody 12 to thereby form a relatively dense third layer of such second antigen molecules 30. The presence of the dense third layer 30 is indicative of the presence of the second layer 12 and thus provides an even greater contrast between the monolayer or the layer containing immunologically complexed antigen-first antibody.In this . patter case, the inert organic compound molecules 20 between the large antigen molecules 11 in the first layer also aid in preventing non-specific sticking to the substrate of the protein matter in the serum. Thus, there is a twofold gain in that one obtains specifically bonded constituent on the substrate, all resulting from the use of the inert organic compound for spacing the large antigen molecules from each other and for covering the surface of the substrate surrounding each antigen molecule with such inert organic compound.The procedure used in forming the third layer in Figure 3, is, therefore, useful in the analysis of the second aqueous medium suspected of containing antibodies 12 since the subsequent medium will provide considerably enhanced contrast between the monomolecular layer and bimolecular (actually now a trimolecular layer if a bimolecu lar layer exists due to the second aqueous' medium in fact containing the antibody 12).

Referring to Figure 3, after the substrate 10 has been removed from the antibody containing aqueous medium (i.e., after the steps shown in Figure 2), the bimolecular coated substrate 10 is next immersed in an aqueous medium containing, or suspected of containing, the same antigen as in the first aqueous medium, and such antigen molecules 30 will selectively bind to the remaining active sites 12a of the antibody molecules 12 in a further immunological reaction. This procedure can thus be utilized in the analysis of a solution for readily detecting the presence of a particular antigen therein.Alternatively, this procedure can be used to cause a buildup of several chains of alternating antigen-antibody molecules wherein each chain has its origin at the surface of substrate 10 to thereby form a multimolecular immunologically complexed film of the several chains of alternating antigen-antibody molecules thereon.

The presence of the second (antibody 12) layer in the Figure 2 embodiment, which is also indicated by means of the third (antibody 30) layer in the Figure 3 embodiment can be readily verified by viewing the coated substrate with an optical instrument such as an ellipsometer.

Alternatively, the layers can also be examined electrically by measuring the electric capacitance of a capacitor having conducting plates formed by the metal or metal coated substrate and a mercury drop or other suitable electrode, and the capacitor dielectric being the layers. Also, as described in the aforementioned copending applications, the layers can be examined optically by unaided visual observation by determining the length of time before a visible amalgam is formed between a drop of mercury and metal film coated on the substrate with the layers therebetween. Finally, and most importantly, the layers can be examined optically by reflected light or transmitted light as explained in the aforementioned copending applications.In this latter optical examination by reflected or transmitted light, the following is a first (transmitted light) technique which has successfully been used. The substrate 10 which must be a light transmissive substrate such as glass, plastic, fused silica, mica or quartz, and is preferably glass, with microscope slides being a conveniently available source, is first coated with a plurality of metal globules by evaporating a metal, for example, indium, onto the substrate. For example, the indium is evaporated slowly from a tantalum boat onto the glass substrate in an ordinary vacuum of about 5x105 mm of mercury. Because the indium atoms have high mobility on the surface of the substrate and do not wet the glass substrate significantly, the indium evaporated onto the substrate agglomerates into small particles.Any metal having similar characteristics so that it will form globules on the substrate when evaporated thereon may be used. In addition to indium, gold, silver, tin, bismuth, and lead have been successfully used. The evaporation of metal is continued until the substrate appears light brown in color. At this point, the metal globules have diameters on the order of 1000 A. The precise size of the globules is not critical but they must have diameters equal to a large fraction of the wavelengths of visible light. The next step is to immerse the globule-covered substrate 10 in a first medium containing a first immunologically reactive antigen 11 and the inert organic compound 20. The first reactive antigen and inert organic compound again adhere in a monomolecular layer over the substrate and the metal globules thereon.When a monomolecular layer has formed, the coated substrate is then removed from the first medium, preferably a solution and immersed in second medium, preferably a solution containing (or suspected of containing) the specifically reacting antibody 12 to the first antigen and results (in the presence of such antibody 12 in the second solution) in the substrate and metal globules having a bimolecular layer adhering thereto similar to that shown in Figure 2 (i.e., without the metal globules).

The coated substrate is then removed from the second aqueous medium and immersed in a third aqueous medium, preferably a solution which contains a reactive antigen to the reaction antibody in the second aqueous medium to form a third layer (or partial layer) on the substrate if the second layer is present. The coated substrate is then viewed by transmitted light, and a determination is made from the appearance of the coated substrate as to the thickness of the layer adhering thereto and accordingly as to the presence or absence of the second antibody 12. The detection of layers corresponds to variations in the shade of brown which is observed in the coated substrate. These variations are therefore a simple straightforward procedure.The particles alone on the substrate appear as a first shade of brown, the particles coated with a monomolecular layer appear as a darker shade of brown, the particles covered with a bimolecular layer appear as a still darker shade of brown, and the particles covered with a trimolecular layer appear darker still. This detection method is based on the fact that electromagnetic radiation is scattered to a large degree by conducting spheres having diameters equal to a large fraction of a wavelength of the incident light and that in the case of scattering from such spheres, the scattering is strongly influenced by a thin dielectric coating applied to the spheres.

A second technique for optical examination by reflected light which has successfully been used is as follows: A gold substrate, which, for reasons of economy, is preferably a thin gold layer plated onto another metal, has absorbed thereon a monomolecular layer of the first reactive antigen 11 and inert organic compound 20 after immersion in the hereinabove identified first aqueous medium. Gold has an adsorption band within the visible spectrum, and this fact accounts for the characteristic color of gold and provides for the operation of this particular optical examination technique. The gold substrate may conveniently be a glass slide coated with a thin indium layer and overcoated with the gold layer wherein the indium layer improves both the adhesion between the glass and the gold as well as the optical characteristics of the slide.The relative reflectivity of the gold substrate as a function of wavelength results in the substrate having the characteristic bright yellow color of gold metal in the absence of any antigen layer adhering thereon. In the presence of a monomolecular layer on the substrate, the appearance of the test slide (substrate) has a dull yellow appearance.

After the test slide has been exposed to an aqueous medium containing the immunologically reacting antibody 12 to the antigen 11 which is in the first layer along with the inert organic compound 20, the test slide, has a bimolecular layer thereon and a reflectivity characteristic which provides a greenish appearance. A third layer provides an even more greenish appearance. In tests which have been performed to date, it apepars that the optical examinations of the coated substrate by reflected or transmitted light and which employ a substrate including metal globules or a gold substrate are the most generally useful. Furthermore, it has been determined that these two techniques have different sensitivities as functions of the thicknesses of films of interest.Specifically, the greatest sensitivity of the technique having a substrate including metal globules occurs with films having thicknesses below approximately 200 A. The gold substrate technique has the greatest sensitivity for films exceeding 30 A in thickness. The particular detection method employed thus determirtes the type of substrate 10 utilized in each analysis, that is, whether the substrate is a metal or metallized glass slide, with a flat metal (gold, as one example) coating or metal (indium, as one example) globules on the surface, as again explained in the above-described copending applications.For purposes of simplification, the substrate 10 is illustrated herein as having a flat surface, although it is to be understood that the surface to which the antigen layer adsorbs could also contain the aforementioned metal globules.

The herein disclosed method of forming multimolecular (3 layer) immunologically complexed films, as depicted in Figure 3, is especially important in testing an aqueous medium for a certain biological constituent for example a hormone, since hormones and antiserum to hormones are generally available. Thus, when the third layer is the hormone of interest, it is easily detectable.

The forming of multimolecular immunologically complexed films in accordance with this invention is also important in identifying~~ cells or viruses immunologically. Thus, as illustrated in Figure 4, by building up a plurality of chains of antigen-antibody complexes from the surface of substrate 10, the probability for several of the antibody molecules on the end of the various chains to find an antigenic site on a cell or virus 40 is relatively high. And this probability remains high irrespective of the irregularity of the surface of the cell or virus. As is well known, the cell membrane which encloses each cell has molecules extending outward therefrom which are called the "transplantation" antigens.These antigens are the ones utilized in form the first layer 11 in a twolayer system for identifying cells immunologically wherein the cell of interest would constitute 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, respectively) would consist of the transplantation antigens. In like manner, it is well known that a virus has a protein coat of protein molecules which can be split up and separated, and such molecules respectively form the first, and first and third layers in two and four-layer systems of the antigen-antibody chains utilized for identifying a virus 40 immunologically. The antibodies 12 in the second layer and 41 in the fourth layer would, of course, be specific to the particular cell or virus being investigated.

It should be mentioned that the configuration of the substrate used herein is not critical to the invention. Preferably, it is slide shaped, e.g., in the form of metallized glass slides because of the ready availability of such slides. However it can be in the form of a belt as described in our Patent Specification No. 1,433,182. The only limitation imposed on the substrate is its ability to allow the formation of a monomolecular layer thereon. The dimensions of form will be dictated by the manner in which the test is carried out including subsequent analysis and its objective. If it is for collection of a particular immunologically active biological constituent a belt is preferred for reasons discussed in Patent Specification No.

1,433,182.

The term "immunologically inert organic compound" embodies any organic substrate, e.g., proteinaceous or nonproteinaceous material which is nonreactive to the interacting constituents and has no effect on the complexing molecules.

It must, however, have the ability to adhere to the substrate, and to separate and surround the antigen molecules.

The contrast between single and double adsorption layers can be enhanced to an even greater extent by depositing the antigen and inert organic compound on substrate 10 as a single drop of the aqueous medium, and subsequently immersing the drop-coated substrate into an aqueous medium containing only the inert organic compound 20 so as to form a monomolecular layer of the small antigeninert organic compound area completely surrounded by the inert organic compound as depicted in Figure 5. This feature results in overcoming the problem of non-specific adsorption in the case where the antibodies are in a serum since the inert organic compound along the remaining surface of the substrate prevents non-specific sticking by constituents in the serum to the substrate and thereby improve the contrast.

The following examples, which are to be regarded as illustrative and not limiting, show how surface immunological tests are carried out according to this invention.

EXAMPLE 1 A diagnostic test for hepatitis antibody is carried out as follows: A glass slide is metallized with a layer of indium metal globules. A solution of 1 mg./ml. of hepatitis-associated antigen in 0.85% silane is prepared, and to this is added bovine serum albumin (BSA) to provide a concentration of O.1Jng./ml. of BSA. A drop of the solution of antigen and inert protein is placed in the center of the slide. The slide is incubated in a moist chamber (plastic box filled with wet sponges) at 230C until the antigen-protein adheres to the metallized surface (10--30 minutes). The slide is washed with distilled water and blown dry with a jet of air. The inert protein covers about 3/4 of the circular area of the dry adsorption layer, and the antigen about 1/4, leaving a major portion of the latter molecules exposed.The slide can be stored for later use or used immediately. In use, the test slide is exposed to human blood serum which is suspected of containing antibody to hepatitis-associated antigen. This is a total volume of 3 ml. The slide and antibody solution are incubated at 200C in a plastic box attached to a shaker, for 12 hours. The slide is dried and observed. A sharply contrasting visible spot indicates a positive reaction. In cases where the blood serum does not contain antibodies to hepatitisassociated antigen, no bimolecular layer will form, and no spot will be seen.

EXAMPLE 2 The procedure of Example 1 is repeated except that the sample to be tested is 3 ml.

of human blood serum suspected of containing hepatitis-associated antigen, which has been mixed with anti-HAA rabbit serum (diluted approximately to 1:100,000 into the human serum). In this case, a sharply contrasting visible spot indicates the absence of HAA in the human blood serum.

EXAMPLE 3 A diagnostic test for gonorrhoea is carried out as follows: A glass slide is metallized with a layer of titanium and the surface layer is then oxidized to produce a cover layer of an oxide of titanium.

Neisseria gonorrhoeae extract is dissolved in salt water to produce a concentration of 1 mg./ml., then to this is added bovine serum albumin to provide a concentration of 3 mg./ml. A drop of the solution of antigen and inert protein is placed in the center of the slide. The slide is incubated in a moist chamber (plastic box filled with wet sponges) at room temperature (230C) until the antigen adheres to the oxide surface (10--30 minutes). The slide is washed with distilled water and blown dry with a jet of air. The slide can be stored at this point for later use. To use, the test slide is immersed in blood serum which is suspected of containing antibody to the Neisseria antigen. This is in total volume 3 ml. The slide and antibody solution are incubated at 20"C in a plastic box attached to a shaker, for 12 hours. If a sharply contrasting visible spot is observed in the center of the slide, this indicates a positive reaction. In cases where the blood serum does not contain gonorrhoeae antibodies, no bimolecular layer will form, and no spot will be seen.

EXAMPLES 11 Diagnostic tests according to this invention are carried out by the procedure of Example 1 substituting the following substrates, antigens, immunologically inert organic compounds, and antibody preparations: Inert Organic Example Substrate Antigen Compound Antibody Source 4 indium metallized hepatitis- egg albumin human blood serum glass associated 5 indium metallized hepatitis- insulin human blood serum glass associated 6 gold metallized hepatitis- bovine serum human blood serum glass associated albumin 7 tantalum hepatitis- bovine serum human blood serum metallized glass associated albumin 8 gold metallized rubella virus bovine serum human blood serum glass extract albumin 9 gold metallized polio virus bovine serum human blood serum glass extract albumin 10 gold metallized vesicular bovine serum humand blood serum glass stomatitis virus albumin 11 gold metallized Treponema bovine serum human blood serum glass pallidum albumin suspension EXAMPLE 12 A diagnostic test for hepatitis antigen is carried out as follows: A glass slide is metallised with a layer of indium metal globules. A solution of 1 mg./ml. of hepatitis-associated antigen in 0.85% saline is prepared, and to this is added bovine serum albumin to provider concentration of 0.1 mg/ml. (BSA). A drop of the solution of antigen and inert protein is placed in the center of the slide. The slide is incubated in a moist chamber (plastic box filled with wet sponges) at 230C until the antigen adheres to the metallized surface (10--30 minutes).

The slide is washed with distilled water and blown dry with a jet of air. The inert protein covers about 3/4 of the circular area of the dry adsorption layer, and the antigen about 1/4, leaving a major portion of the latter molecules exposed. The side is then exposed to a preparation of anti rabbit HAA serum which contains antibody to hepatitisassociated antigen. This produces a bimolecular layer in which a large fraction of the outwardly exposed antibody molecules have remaining combining sites.

The slide and antibody solution are incubated at 20"C for 10 minutes. After incubation, the slide is again washed with distilled water. The test slide is exposed to a solution suspected of containing the hepatitis-associated antigen as described above. The formation of a sharply contrasting visible spot on the slide indicates a positive reaction. In cases where the suspected solution does not contain antibodies to hepatitis-associated antigen, no trimolecular layer will form.

EXAMPLE 13 Diagnostic tests according to this invention are carried out by the procedure of Example 12 substituting the following substrates, antigens, immunologically inert organic- compounds, and antibody preparations: Inert Organic Example Substrate Antigen Compound Antibody Source 14 indium metallized hepatitis- egg albumin rabbit blood serum glass associated 15 indium metallized hepatitis- insulin rabbit blood serum glass associated 16 gold metallized hepatitis- bovine serum rabbit blood serum glass associated albumin 17 tantalum hepatitis- bovine serum rabbit blood serum metallized glass associated albumin 18 indium metallized insulin bovine serum rabbit blood serum glass albumin In addition to the above specific examples, quantitative tests for antibodies may be carried out in accordance with this invention using slide shaped substrates metallized with gold or tantalum upon which are deposited aqueous solutions of bovine serum albumin in admixture with the following antigenic preparations: tetanus toxoid (tetanus) diphtheria toxin (diphtheria) asperigillus extract (fungus) candida extract (fungus) toxoplasma gondii (parasite) nucleoprotein and DNA thyroglobulin (Hashimoto's disease) collagen fractions human chorionic gonadotrophin (pregnancy) insulin keyhole lymphet hemocyanin (antibody response) pencillinase extract (large enzyme) It is also evident that the procedure described herein-above can also be used to identify viruses and enzymes immunologically.All antigens of the virus type are larger than their specific antibodies, whereas the antigens of the enzyme type may be larger or smaller, depending upon the particular type of enzyme. As an obvious variation, one procedure for identifying a particular virus or large size enzyme, known as the inhibition test, is accomplished in the following manner: The particular virus or larger size enzyme is put into a first aqueous medium (generally salt water) with the inert organic compound and the substrate is then immersed therein, or alternatively, a drop of the aqueous medium containing the enzyme is placed on the substrate for a short time, rinsed, and then the substrate is immersed in an aqueous medium containing only the inert organic compound.After a monomolecular layer of the virus or enzyme (i.e., antigen) and inert organic compound has been adsorbed on the substrate, the coated substrate is removed from the first aqueous medium. Samples of human serum to be tested for the presence of the particular virus or enzyme are prepared by mixing therein a quantity of known antibodies to the particular virus or enzyme sufficient 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 exposed to, the previously prepared serum sample and upon removal therefrom is examined in accordance with any of the procedures described hereinabove. If inspection of the substrate indicates the presence of only a monomolecular layer thereon, then it is known that the human serum under test originally contained the particular virus or enzyme.However, if a bimolecular layer is detected then this indicates that the serum did not originally contain such virus or large size enzyme antigen.

From the foregoing description, it can be appreciated that this invention makes available a new method for improving the contrast in surface immunological tests between single and double layers of immunologically reactive biological antigen antibodies by the addition of an immunologically inert organic compound of sufficient quantity to a first aqueous medium containing an immunologically reactive antigen.

Having described the invention with reference to the particular embodiments and examples, it is believed obvious that modification and variation of this invention is possible in the light of the above teachings. Thus, the inert organic compound could be in an aqueous medium separate from the antigen, and the substrate would be immersed therein as a preliminary step, or as an intermediate step between immersions in a dilute aqueous medium containing the antigen and the antibody.As another approach, in the case where the antibodies are in a serum, a dilute aqueous medium containing antigen would be utilised in forming an incomplete first layer on the substrate, and the inert organic compounds would be omitted in such solution since the non-specific compounds in the serum would function as the inert organic compounds in adhering to the substrate and thereby act as the spacing agents between the antigen molecules.

Finally, although the reactive constituent adsorbed on the substrate surface has been described hereinabove as being an antigen, it should be evident that such first layer reactive constituent could be an antibody, and the second layer constituent would then be the specific antigen, the latter arrangement being useful in cases wherein an antibody molecule is larger than its specific antigen molecule, an example being the antibody to insulin. It is, therefore, to be understood that changes may be made in a particular embodiment of this invention as described which is within the full intended scope of the invention as defined by the following claims.

WHAT WE CLAIM IS: 1,. A method for forming multimolecular immunologically complexed films comprising the steps of treating a substrate with an immunologically reactive antigen and an immunologically inert organic compound to form a monomolecular layer of said antigen and said compound so that the antigen molecules are separated from each other by the inert organic compound molecules, and subsequently exposing the treated substrate to an aqueous medium containing an immunologically reactive antibody specific to the antigen to cause an immunological reaction therewith in which the antibody molecules bond with the antigen molecules to form a bimolecular layer on the substrate, and the antibody molecules retain a significantly greater

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (35)

1. **WARNING** start of CLMS field may overlap end of DESC **.

   candida extract (fungus) toxoplasma gondii (parasite) nucleoprotein and DNA thyroglobulin (Hashimoto's disease) collagen fractions human chorionic gonadotrophin (pregnancy) insulin keyhole lymphet hemocyanin (antibody response) pencillinase extract (large enzyme) It is also evident that the procedure described herein-above can also be used to identify viruses and enzymes immunologically. All antigens of the virus type are larger than their specific antibodies, whereas the antigens of the enzyme type may be larger or smaller, depending upon the particular type of enzyme.As an obvious variation, one procedure for identifying a particular virus or large size enzyme, known as the inhibition test, is accomplished in the following manner: The particular virus or larger size enzyme is put into a first aqueous medium (generally salt water) with the inert organic compound and the substrate is then immersed therein, or alternatively, a drop of the aqueous medium containing the enzyme is placed on the substrate for a short time, rinsed, and then the substrate is immersed in an aqueous medium containing only the inert organic compound. After a monomolecular layer of the virus or enzyme (i.e., antigen) and inert organic compound has been adsorbed on the substrate, the coated substrate is removed from the first aqueous medium.Samples of human serum to be tested for the presence of the particular virus or enzyme are prepared by mixing therein a quantity of known antibodies to the particular virus or enzyme sufficient 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 exposed to, the previously prepared serum sample and upon removal therefrom is examined in accordance with any of the procedures described hereinabove. If inspection of the substrate indicates the presence of only a monomolecular layer thereon, then it is known that the human serum under test originally contained the particular virus or enzyme. However, if a bimolecular layer is detected then this indicates that the serum did not originally contain such virus or large size enzyme antigen.

   From the foregoing description, it can be appreciated that this invention makes available a new method for improving the contrast in surface immunological tests between single and double layers of immunologically reactive biological antigen antibodies by the addition of an immunologically inert organic compound of sufficient quantity to a first aqueous medium containing an immunologically reactive antigen.

   Having described the invention with reference to the particular embodiments and examples, it is believed obvious that modification and variation of this invention is possible in the light of the above teachings. Thus, the inert organic compound could be in an aqueous medium separate from the antigen, and the substrate would be immersed therein as a preliminary step, or as an intermediate step between immersions in a dilute aqueous medium containing the antigen and the antibody.As another approach, in the case where the antibodies are in a serum, a dilute aqueous medium containing antigen would be utilised in forming an incomplete first layer on the substrate, and the inert organic compounds would be omitted in such solution since the non-specific compounds in the serum would function as the inert organic compounds in adhering to the substrate and thereby act as the spacing agents between the antigen molecules.

   Finally, although the reactive constituent adsorbed on the substrate surface has been described hereinabove as being an antigen, it should be evident that such first layer reactive constituent could be an antibody, and the second layer constituent would then be the specific antigen, the latter arrangement being useful in cases wherein an antibody molecule is larger than its specific antigen molecule, an example being the antibody to insulin. It is, therefore, to be understood that changes may be made in a particular embodiment of this invention as described which is within the full intended scope of the invention as defined by the following claims.

   WHAT WE CLAIM IS: 1,. A method for forming multimolecular immunologically complexed films comprising the steps of treating a substrate with an immunologically reactive antigen and an immunologically inert organic compound to form a monomolecular layer of said antigen and said compound so that the antigen molecules are separated from each other by the inert organic compound molecules, and subsequently exposing the treated substrate to an aqueous medium containing an immunologically reactive antibody specific to the antigen to cause an immunological reaction therewith in which the antibody molecules bond with the antigen molecules to form a bimolecular layer on the substrate, and the antibody molecules retain a significantly greater

   number of active sites for a further immunological reaction than if the antigen molecules were closely spaced together, the immunologically inert organic compound being other than a protein when the immunologically reactive antigen is a protein and the antibody is a protein specific to the protein antigen.
2. 2. A method as claimed in claim 1 wherein the step of treating the substrate with an antigen and inert organic compound comprises the steps of forming a first aqueous medium containing the immunologically reactive antigen, adding the immunologically inert organic compound of sufficient quantity to the first aqueous medium, contacting the substrate with the first aqueous medium to coat all or part of the substrate with the monomolecular layer of the antigen and inert organic compound, said inert compound being of sufficient quantity so that the antigen molecules are sufficiently separated from each other by the inert organic molecules, and removing the monomolecular layer coated substrate from the first aqueous medium.
3. 3. A method as claimed in claim 1 wherein the step of treating the substrate with an antigen and inert organic compound comprises the steps of forming a first aqueous medium containing the immunologically inert organic compound, immersing the substrate in the first aqueous medium sufficiently to coat the substrate with a partial monomolecular layer of the inert organic compound, removing the partially coated substrate from the first aqueous medium, forming a second aqueous medium containing the immunologically reactive antigen, immersing the partially coated substrate in the second aqueous medium to coat the remaining surface of the substrate with a partial monomolecular layer of the antigen so that the monomolecular layer consists of the antigen molecules separated from each other by the inert organic molecules, and removing the monomolecular layer coated substrate from the second aqueous medium.
4. 4. A method as claimed in claim 1 wherein the step of treating the substrate with an antigen and inert organic compound comprises the steps of forming a dilute first aqueous medium containing the immunologically reactive antigen, immersing the substrate in the first aqueous medium sufficiently to coat the substrate with a partial monomolecular layer of the antigen, removing the partially coated substrate from the first aqueous medium, forming a second aqueous medium containing the immunologically inert organic compound, immersing the partially coated substrate in the second aqueous medium to coat the remaining surface of the substrate with a partial monomolecular layer of the inert organic compound so that the monomolecular layer consists of the antigen molecules separated from each other by the inert organic molecules, and removing the monomolecular layer coated substrate from the second aqueous medium.
5. 5. A method as claimed in claim 1 wherein the step of treating the substrate with an antigen and inert organic compound comprises the steps of forming a first aqueous medium containing the immunologically reactive antigen, adding the immunologically inert organic compound of sufficient quantity to the first aqueous medium, depositing at least a single drop of the first aqueous medium on the substrate to have the antigen-inert organic compound form a small area monomolecular layer thereon, forming a second aqueous medium containing immunologically inert organic compound, subsequently immersing the drop-coated substrate into the second aqueous medium so as to form a complete monomolecular layer over the entire surface of the substrate which includes the small antigen-inert organic compound area surrounded by the inert organic compound from the second aqueous medium, and removing the monomolecular layer coated substrate from the second aqueous medium.
6. 6. A method for forming multimolecular immunologically complexed films comprising the steps of treating a substrate with an immunologically reactive antigen by forming a dilute first solution of said antigen, immersing the substrate in said first solution sufficiently to to coat the substrate with a partial monomolecular layer of the antigen, and removing the partially coated substrate from the first solution, and immersing the partially coated substrate in a serum solution comprising an immunologically reactive antibody and an immunologically inert organic compound whereby inert organic compound adheres to the substrate to complete a monomolecular layer of antigen molecules separated from each other by inert organic compound molecules, and the antibody molecules in the serum forms a second layer, the immunologically inert organic compound being other than a protein when the immunologically reactive antigen is a protein and the antibody is specific to the protein antigen.
7. 7. A method as claimed in claim 2 and further comprising the steps of removing the bimolecular layer coated substrate from the antibody containing medium, subsequently exposing the coated substrate to a third aqueous medium suspected of containing the same antigen which is in the first aqueous medium to cause an immunological reaction with the antibody in which the remaining active sites on the antibody molecules bond with the antigen molecules in the third aqueous medium, and examining the coated substrate to determine whether there is a third layer thereon thereby indicating the presence of the antigen in the third aqueous medium.
8. 8. A method as claimed in claim 2 wherein the step of forming a first aqueous medium containing the immunologically reactive antigen consists of separating the antigens which are in the outermost portions of a particular cell or virus, and forming an aqueous medium thereof prior to adding said inert organic compound thereto, and further comprising the steps of removing the bimolecular layer coated substrate from the antibody containing medium, subsequently immersing the coated substrate in a third aqueous medium suspected of containing the particular cell or virus so that the antibody molecules find antigenic sites on the cell or virus to cause bonding therewith and examining the coated substrate to determine whether there is a third layer thereon thereby indicating the presence of the particular cell or virus in the third aqueous medium.
9. 9. A method as claimed in claim 2 and further comprising the steps of removing the bimolecular layer coated substrate from the antibody medium, subsequently exposing the coated substrate to a third aqueous medium containing the same antigen which is in the first aqueous medium to cause an immunological reaction with the antibody in which the remaining active sites on the antibody molecules bond with the antigen molecules in the third aqueous medium.
10. 10. A method as claimed in claim 9 and further comprising the steps of removing the coated substrate from the third aqueous medium, and subsequently exposing the coated substrate to a fourth aqueous medium containing the same antibody which is in the second aqueous medium to cause an immunological reaction in which first active sites of the antibody molecules in the fourth aqueous medium bond with antigen molecules from the third aqueous medium while having other combining sites remain active to thereby build up chains of antigen antibody complexes from the surface of the substrate.
11. 11. A method as claimed in claim 10 and further comprising the steps of removing the coated substrate from the fourth aqueous medium, subsequently exposing the substrate with the chains of antigenantibody complexes built up from the substrate surface to a fifth aqueous medium suspected of containing cells or virus specific to the antibody in the chains of antigen-antibody complexes, removing the coated substrate from the fifth aqueous medium, and examining the substrate to determine whether active sites remaining on the antibody molecules have combined with antigen sites on the cells or virus in the fifth aqueous medium.
12. 12. A method as claimed in claim 2 or any one of claims 7 to 11 wherein the step of adding a sufficient quantity of the immunologically inert organic compound to the first aqueous medium comprises adding a quantity sufficient so that the average spacing between adjacent immunologically reactive antigen molecules absorbed on the surface of the substrate will generally be to distances of several hundred Angstroms.
13. 13. A method as claimed in any one of the preceding claims wherein said substrate is slide shaped.
14. 14. A method as claimed in any one of claims 1 to 13 wherein the antibody is of the immunoglobulin Ig G class.
15. 15. A method for binding reactive constituents to a surface so that they will remain active comprising the steps of coating a metallized slide with a monomolecular layer of a reactive first constituent and immunologically inert organic compound so that the first constituent molecules are separated from each other by inert organic molecules, and subsequently exposing the coated slide to an aqueous medium containing a reactive second constituent specific to the first constituent to cause a reaction therewith in which the second constituent molecules bond with the first constituent molecules to form a bimolecular layer on the slide, the inert organic compound being of sufficient quantity to obtain sufficient average spacing between adjacent first constituent molecules in the monomolecular layer so that the second constituent molecules retain active sites for a further immunological reaction, the inert organic compound being other than a protein when the reactive first constituent is a protein antigen and the reactive second constituent is a protein antibody specific to the protein antigen.
16. 16. A method as claimed in claim 15 wherein the step of coating the slide with a monomolecular layer of a first constituent and inert organic compound comprises forming a first aqueous medium of the reactive first constituent, adding the inert organic compound of sufficient quantity to the first aqueous medium, contacting the slide with the first aqueous medium to coat all or part of the slide with the monomolecular layer of the first constituent and the inert organic compound and removing the monomolecular layer coated slide from the first aqueous medium.
17. 17. A method for binding antibodies to a surface so that they remain active comprising the steps of forming an aqueous medium containing an immunologically reactive antibody, adding a sufficient quantity of an immunologically inert organic compound to said medium, and exposing a metallized slide to the aqueous medium to coat at least a portion of the slide by adsorption with a monomolecular layer of the antibody and the inert organic compound wherein the antibody molecules are sufficiently spaced from each other by the sufficient quantity of inert organic molecules so that the antibody molecules present a greater number of active sites which are retained for a further immunological reaction than if the antibody molecules were closely spaced together, such that when the immunologically inert organic compound is an inert protein, the reaction is other than an immunological reaction between a protein antigen and a protein antibody specific to the antigen.
18. 18. A method for determining the presence or absence of a suspected constituent in an aqueous medium comprising the steps of forming a first aqueous medium containing an immunologically reactive antigen, adding a quantity of an immunologically inert organic compound to the first aqueous medium, contacting a metal slide or a metallized glass slide with said aqueous medium to coat all or part of the slide with a monomolecular layer of the antigen and the inert organic compound, the quantity of inert compound being sufficient to provide that the antigen molecules are separated from each other by the inert organic molecules, removing the monomolecular layer coated substrate from the first aqueous medium, subsequently exposing the coated substrate to a second aqueous medium containing an immunologically reactive antibody specific to the antigen to cause an immunological reaction therewith in which the antibody molecules bond with the antigen molecules to form a bimolecular layer on the substrate, and the antibody molecules retain a significantly greater number of active sites for a further immunological reaction than if the antigen molecules were closely spaced together, removing the bimolecular layer coated substrate from the antibody containing medium, exposing the coated substrate to a third aqueous medium suspected of containing the same antigen which is in the first aqueous medium and causing an immunological reaction to occur with the antibody in which the remaining active sites on the antibody molecules bond with the antigen molecules in the third aqueous medium, and examining the coated substrate to determine whether there is a third layer thereon thereby indicating the presence of the antigen in the third aqueous medium, such that when the immunologically inert compound is an inert protein, the immunological reaction is not between a protein antigen and a protein antibody specific to the antigen.
19. 19. A method as claimed in claim 18 wherein said examining step comprises visually the coated substrate by transmitted light.
20. 20. A method as claimed in claim 19 wherein said metal is indium and the visual determination is made by distinguishing among four shades of brown.
21. 21. A method as claimed in any one of claims 15 to 20 wherein the reactive first constituent is an antigen, and the reactive second constituent is an antibody specific to the antigen.
22. 22. A method as claimed in any one of claims 15 to 21 wherein the reactive first constituent is an antibody, and the reactive second constituent is an antigen to which the antibody is specific.
23. 23. A method as claimed in any one of claims 15 to 22 wherein the metallized slide also includes a metal oxide layer intermediate the metal of the slide and said monomolecular surface.
24. 24. A method as claimed in claim 23 wherein said metal is titanium and said oxide is an oxide of titanium.
25. 25. A method as claimed in any one of claims 15 to 22 wherein said metallized slide is a metal slide or a metallized glass slide.
26. 26. A method as claimed in claim 25 wherein the metal comprising the slide is selected from indium, gold, silver, tin and lead.
27. 27. A method as claimed in claim 25 wherein said metallized slide comprises indium on a glass slide.
28. 28. A method as claimed in claim 25 wherein said metallized slide comprises gold on a glass slide.
29. 29. A method as claimed in claim 25 wherein said metallized slide comprises a glass slide coated with a thin indium layer and overcoated with a gold layer.
30. 30. A method as claimed in any one of the preceding claims wherein said antigen is derived from a bacteria, a virus, a fungus, mammalian tissue or mammalian body fluids.
31. 31. A method as claimed in any one of the preceding claims wherein said inert organic compound is selected from bovine serum albumin, egg albumen or insulin.
32. 32. A method for forming multimolecular immunologically complexed films substantially as hereinbefore described in any one of the examples.
33. 33. A method as claimed in any one of claims 1, 6, 15 or 17 substantially as hereinbefore described and illustrated.
34. 34. A surface having a film bound to it by the method as claimed in any one of claims 1 to 17 or 33.
35. 35. A method as claimed in claim 18 substantially as hereinbefore described.