CA1048409A - Method for improving contrast in surface immunological tests with large size proteins - Google Patents

Abstract

METHOD FOR IMPROVING CONTRAST IN SURFACE
IMMUNOLOGICAL TESTS WITH LARGE SIZE PROTEINS
Abstract of the Disclosure An immunologically inert protein is added to a solution containing a large size immunologically reactive protein (antigen), and a substrate is then immersed in the solution to form, by adsorption, a monomolecular layer of the reactive protein molecules separated from each other and surrounded by the inert protein molecules.
Subsequent immersion of the coated substrate in a solution containing a small size immunologically reactive protein (antibody) specific to the first reactive protein results in a dense bimolecular layer being formed on the substrate which produces a much greater change in contrast between the single and double layers than if the large size protein molecules were closely spaced together.

Description

My invention relates to a method for detecting large size immunologically reactive proteins and small size reactive proteins specific to the first protein, and in particular, for improving the contrast between single and double layers of the protein molecules.
This application is related to my Canadian application S.N.~o7~o ~ , filed ~ 5~ /q~ , entitled "Method for Binding Antibodies To a Surface Such That They Remain Active"
Immunological reactions are highly specific biochemical reactions in which a first protein known as the antigen com-bines with a second protein specific to the antigen and known as the antibody to form an immunologically complexed protein.
Immunological reactions taking place within a biological sys-tem such as an animal or a human being are vital in combating disease. In a biological system, the entry of a foreign pro-tein, i.e., the antigen, causes the biological system to produce the specific antibody proteins to the antigen in a process not fully understood at this time. The antibody pro-tein molecules have available chemical combining or binding sites which complement those on the antigen molecule so that the antigen and antibody chemically combine or bond to form the immunologically complexed protein.
Because antibodies are produced by biological systems in response to invasion thereof by foreign proteins, the detection of antibodies present in a biological system is of medical diagnostic value in determining the antigons to which the system has been exposed. Conversely, the detection of certain antigens in a biological system also has medical diagnostic values; examples of diagnostic detection of antigens include detection of HCG

proteln molecules in urine as a test for pregnancy, and detection of hepatitis-sssociated antigen (HAA) molecules in blood of prospective blo~d donors.
Tn order to perform such dlagnostic tests, the apprapriate protein of at least an immunologically reacting pair must be obtained. The only known source of sn antibody protein is a living biological system. More particularly, only vertebrates are known at this time to exhibit immunological reactions to the introduction of a foreign protein. For example, many antibodies are found in the blqod serum of animals and human beings which have been exposed to this corresponding antigen. Many antigens, however, ~y be co~trollably prod~ced in laboratory cultures. However, some antigens, fo~ example, hepatitis-associated ~ntigen~, are at present, like antibodies,only obtainable from the higher living biological systems.
Most antigens are proteins or contain proteins as an essential part, whereas all antibodies are proteins.
Since proteins are large molecules of high molecular welght, i.e., are polymers consisting of chains of variable number~ of amino ncids, the antigen and antibody protein may each have several combining sites~ The ~ive ma~Dr classes of antibodies (immunoglobulins Ig-G, Ig M, Ig A, Ig E and Tg D) are each apparently characteriz~d by at lea6t two heavy (lcng) 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 '~"
and the active or combining sites are the extreme ends of 10484~9 RD-6735 the two arms of the Y-shaped antibody for the Ig G
antibody.
Immunological reactions can be detected by various techniques lncluding the use of a suitable substrate S ~uch as a metallized glass or metal slide. Expos~re of the substrate to a solution of antigen will result in the antigens being physically adsorbed in a dense monomolecular layer onto the surface of the sub~trate. Subsequent expo8ure of the antigen-coated substrate to a serum containing antibodies towards the antigen re~ults in the i~munological reaction wherein the antibodies selectively attach themselves to the antigens by means of the binding sites on the antibody molecule which complement tho~e on the antigen molecule to thereby form at least a partial bimolecular layer of immunologically complexed protein on the substrate surface. The problem which often arises i~ the inability to distinguish between the monomolecular and bimolecular layers of protein on the substrate, especially if the ad~orbed (antigen) layer is relatively thick as in the case of HAA layer since the hepatitis-associated antigen molecule is at least ten times as large as the antibody to the HAA.
Therefore, one of the principal objects of my invention is to provide a new method in surface immunological te~ts for improving the contrast between single and double protein layers when one of the layers consists of large ~ize proteins.
Another ob~ect of my invention i8 to provide the $mproved contrast when the layer adsorbed on the surface con8igt~ of large antigen proteins.

~1~)484Q9 A further object of my invention is to provide improved contrast between a monomolecular and bimolecular layer by means of a third molecular layer of protein coated on a substrate when the adsorbed-first layer consists of large size antigens and the third layer is an antibody to the antibody in the second layer.
Briefly, and in accordance with the objects of my invention, I add an immunologically inert protein to a solution containing a relatively large size immunologically reactive antigen protein. A substrate is then immersed in the solution and the surface of the substrate is coated, by adsorption, with a monomolecular layer of the antigen protein molecules and inert protein molecules wherein each antigen molecule is generally surrounded by inert protein molecul~s. Subsequent immersion of the coated substrate in a second solution containing a relatively small size immunologically reactive antibody protein specific to the antigen protein results in the antibody molecules chemically bonding with the antigen molecules to form at least a partial bimolecular layer on the substrate. The quantity of inert protein 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 of the two proteins coated on a substrate.
Thus, my invention provides a simple procedure for distinguishing between a single layer of a large size antigen, such as hepatitis-associated antigen, and a i04~4Q9 double layer which include~ a second layer of a smal1 size 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. ~~ .
The features of my invention which I desire to protect ~erein are pointed out with particularity in the appended claim~. The invention itself, however, 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 taken ln connection with the accompanying drawing wherein like ; parts in each of the severai figures are identified by the same reference character, and wherein:
FIGURE 1 is an elevation view of a substrate after - 15 it has been immersed first in a~solution of a large size antigen by a known method and then in a solution of a smaller size antibody specific to the antigen;
FIGURE 2 is an elevation view of the substrate after it has undergone two immersion steps as in FIGURE l,-but with the first immersion being in accordance with my - 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 a solution containing an antibody to the antibody in the second solution; and FIGURE 4 is a plan view of a substrate having a small region thereon of active-inert proteins surrounded by ' inert protein in accordance with my invention.
Referring now to.FIGURE 1, there is shown a highly magnified elevation view of a portion o~ diagnostic ~04~409 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. Substrate 10, when immersed in a first solution generally of salt water containing a first protein of interest, which may be biologically an antigen or antibody, is adsorbed onto the substrate in a monomolecular layer 11. For the purpose of my invention, the layer 11 adsorbed on the surface of substrate 10 will herein-after be described as an antigen layer. Any protein will adsorb in such monomolecular layer, but no further adsorption will take place, that is, the protein will attach to the substrate, but will not attach to itself. Thus, the antigen 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 agitation of the solution and the solution temperature. As an example, a concentration of 1 milligram per cubic centimeter of hepatitis-associated antigen solution completely coats a slide in approximately lO
minutes with a monomolecular antigen protein layer.
After the monomolecular layer of antigen protein 11 has formed over substantially the entire surface of the substrate 10, the coated substrate is removed from the solution of the antigen protein, and is next immersed in a second solution containing, or suspected of containing, the specifically reacting antibody ~04~4~9 protein to the antigen protein. For purposes of my invention, it will be assumed that the protein 11 adsorbed on the surface of substrate 10 is always of greater size than the protein which is immunologically reactive there-with and forms a second layer on the substrate. The second solution may contain many constituents in addition to the specifically reacting smaller antibody protein whose presence it is desired to detect. However, no protein other than the specifically reacting antibody protein will adhere to the first antigen protein layer~on the substrate. Thus, only if the specifically reacting antibody protein is present in the second solution will immunological complexing between the antigen and its specifically reacting antibody take place and the substrate will, after a time, have a bimolecular protein 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 protein 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. 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 described in my Canadian application, S.N. 172,639, entitled "Method and Apparatus for Detection and Purification of Proteins and Antibodies", filed May 29, 1973, and assigned to the assignee of the present invention.
Due to the monomolecular antigen layer 11 being adsorbed over substantially the entire surface of substrate 10 in FIGUR~ 1, the second layer 12 of the smaller size specific antibody for such antigen bonds thereto in a thin layer to form the bimolecular layer.
However, due to the proteins in the second layer 12 being of smaller size than the proteins in the first layer 11 and also due to the second layer being much less dense than the first tantigen) layer, it is clearly evident that there can be considerable difficulty in distinguishing between this single and double layer of protein 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 solution did indeed contain any of the antibodies to HAA and thus such diagnostic test is of little value. In the present invention, I have discovered a method by which a much ~reater 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 10484~ .
`:

double layers. The basis of my 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, but there are no reasons why antibodies of the other four major classes enumerated above cannot be utilized in my invention.
After selection of the substrate 10 on which the - 10 bimolecular immunologically complex film is to bfifo ~ edgenera y o s~1t water such substrate is immersed-in a-first solutio~ 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 immuno-logically inert protein has been added to the first solution prior to imme~sion of the substrate therein such that the adsorption layer formed on the surface of substrate 10 consists of the relatively large reactive antigen protein molecules 11 separated from each other, and surrounded along the substrate surface, by inert protein molecules 20 as seen in FIG~RE 2. The inert protein molecules 20 are of a size at least 80mewhat smaller, and preferably substantially smaller than the antigen molecules 11 in order to allow the greatest surface area (and therefore more combining 8ites) to remain available on the antigen molecules 11 for subsequent bonding with antibody molecules. The quantity of the immunologically inert protein 20 added to the ~olution is sufficient so that, in general, the inert protein 20 covers 1/2 to 9/10 of the area of the complete adsorption layer, that is, the antigen corresponding covers _g_ .

16~484Q9 l/2 to 1/lO of the area, although this is not a limitation on my invention since the average spacing between adjacent antigen molecules is also determined by the number of active sites on 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 my invention. As a result of the large size of the antigen protein relative to the inert protein molecule, a major portion of the surface of the anti-gen molecule will remain exposed and has its corresponding bonding sites available for combining with antibody molecules in a subsequent immunological reaction.
The (antigen-inert protein) monomolecular coated sub-strate 10 is then removed from the first solution and is then immersed in a second solution 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 protein 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.
Thus, as seen in FIGURE 2, a greater number of antibody molecules 12 will, in general, combine with each antigen 3~ 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 ~0484~9 contrsst between the single and double layers is obtaine~ in the FIGURE 2 embodiment of my invention (due tO the greater number of antibody molecules that are in the second layer).
For sn exsmple of my invention, the first solution has a con-centration of hepatitis-associated antigen (HAA) and bovine serwm 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 8econd solution is a dilute solution of the HAA antibody. The method described hereinabove can thus be utilized in the analy$is of a solution 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 sub-sequently be immersed-in a third solution or a serum contain-ing antibodies 30 to the antibodies 12 in the second layer in order to build up a third layer as illustrated in FIGUKE 3.
Since each antibody molecule 12 has several antigenic deter-minants, 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 antibody 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 monomolecular and greater contrast between the/bimolecular antigen-first anti-body layers. In this latter case, the inert protein mole-cules 20 between the large antigen molecules 11 in the first layer also aid in preventing sticking to the substrate of the non-specific protein matter in the serum which contains the antibodies to the antibody of such antigen. Thus, there is a two-fold gain in that I obtain more specifically bonded antibodies for each antigen molecule and there are less ~0484Q9 RD-6735 non-specifically bonded protein on the substrate, all result-ing from the use of the inert protein for spacing the large antigsn molecules from each other and for covering the sur-face of the substrate surrounding each antigen molecule with such inert protein. The procedure used in forming the third layer in FIGURE 3 is, therefore, useful in the analysis of the second solution suspected of containing antibodies 12 since the subsequent immersion of the coated substrate in the third solution will provide considerably enhanced contrast between the monomolecular layer and bimolecular (actually now a trimolecular layer if a bimolecular layer exists due to the second solution in fact containing the antibody 12).
The presence of the second (antibody 12) protein layer in the FIGURE 2 embodiment, which is also indicated by means of the third (antibody 30) protein layer in the FIGURE 3 embodiment can be readily verified by viewing the coated substrate with an optical instrument such as an ellipsometer.
Alternatively, and as described in greatex detail in my afore-mentioned Canadian application S.N. 172,639, as well as my Canadian application S.N. 204,262, filed July 8, 1974, the protein layers can also be examined electrically by measuring the electric capacitance of a capacitor having conducting plates formed by the metal or metal coated sub-strate and a mercury drop or other suitable electrode, and the capacitor dielectric being the protein layers. Also, as described in my canadian applications, the protein layers can be examined optically by unaided visual observation by deter-mining the length of time before a visible amalgam is formed between a drop of mercury and metal film coated on the sub-strate with the protein layers therebetween. Finally, and most importantly, the protein layers can be examined optically RD-~735 by reflected light or transmitted light as explained in my Canadian 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, quartz, or the like, 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 sub-strate in an ordinary vacuum of about 5 x 10 5 mm of mercury.
Because the indium atoms have high mobility on the surface of the substrate and do not wet the glass substrate sig-nificantly, the indium evaporated onto the substrate agglom-erates into small particles. Any metal having similar characteristics so that it will form globules on the sub-strate when evaporated thereon may be used. In addition to indium, gold, silver, tin, and lead have been successfully used. The evaporation of metal is continued until the sub-strate appears light brown in color. At this point, the metal globules have diameters on the order of 1000 ~. The precise size of the globules is not cirtical but they must have diameters equal to a large fraction of a wavelength of visible light. The next step is to immerse the globule-covered substrate 10 in a first solution of a first immuno-logically reactive protein such as the antigen 11 and the inert protein 20. The first reactive protein and inert protein again adhere in a monomolecular layer over the sub-strate and the metal globules thereon. When a monomolecular layer has formed, the coated substrate is then removed from the first solution and immersed in a second solution con-taining (or suspected of containing) the specifically reacting protein 12 to the first protein and results(in the presence of such protein 12 in the second solution) in the substrate and metal globules having a bimolecular protein 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 solut-ion and immersed in a third solution which contains a reactive protein to the reactive protein in 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 viewed by transmitted light, and a determination is made from the appearance of the coated substrate as to the thickness of the protein layer adhering thereto and accordingly as to the presence or absence of the second protein 12. The detection of protein layers corresponds to variations in the shade of brown which is observed in the coated substrate. These vari-ations are quite pronounced and the detection of protein layers is therefore a simple straightforward procedure. The particles alone on the substrate appear as a first shade of brown, the particles coated with a monomolecular protein layer appear as a darker shade of brown, the particles covered with a bimolecular protein layer appear as a still darker shade of brown, and the particles covered with a trimolecular protein layer appear darkçr 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 energy and that in the case of scattering from such spheres, the scattering is strongly influenced by a thin dielectric coating ~048409 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 adsorbed thereon a monomolecular layer of the first reactive protein 11 and inert protein 20 after immer-sion in the hereinabove identified first solution. Gold has an absorption band within the visible spectrum, and this fact accounts for the characteristic color of gold and pro-~ides for the operation of this particular optical examin-ation technique. The gold substrate may conveniently be a : glass slide coated with a thin indium layer and overcoated the - with the gold layer wherein/indium layer improves both the adhesion between the glass and~ the gold as well as the optical characteristics of the slide. The relative reflect-ivLty 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 protein layer a & ering thereon. In the presence of a monomoiecular layer on the substrate, the appearance of the test slide (substrate) has a dull yellow appearance. After the test slide has been exposed to a solution cobtaining the immuno-logical reacting protein 12 to the protein 11 which is in the first layer along with the inert protein 20, the test slide, has a bimolecular protein layer thereon and a reflectivity characteristic which provides a greenish appearance. A
third protein layer provides an even more greenish appearance.
In tests which have been performed to date, it appears 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 the~e two techniques have different sensitivities as functions o~ the thicknesses of protein film~ 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 ~ubstrate technique has the greatest sensitivity for films exceeding 30 A in thickne3s. The partlcular detection method employed thus determines the type of substrate 10 utilized in each analysis, that is, whether the substrate iB a metal or metallized glass ~lide, with a flst metal (gold, as one example) coating or metal (indlum, as one example) globules on the surface, as again explained in my copending applications. For purposes of slmplification, I have illustrated the sub~trate 10 herein as having a flat surface, although it i~ to be understood that the surface to which the antigen layer adsorbs could also contain the aforementioned metal globules.
The cor.tra~t between single and double protein layers can be enhanced to an even greater extent by depositing the antigen and inert protein on substrate 10 as a single drop of the solution, and subeequently immersing the drop-coated sub~trate into a solution containing only the inert protein 20 so as to form a monomolecular layer of the small antigen-inert protein area completely surrounded by the inert protein as depicted in FIGURE 4. This feature results in overcoming the problem of nonspecific adsorption in the case where the antibodies are in a serum since the inert protein along the remaining surface of the substrate prevents the nonspec~fic proteins in the serum from adhering to the substrate and thereby improve the contrast.
The simple procedures described hereinabove have now lO9U3 ~ 9 RD-6735 resulted in the ability to detect hepatitis by a test whic~.
is as sensitive as the standard radioimmuneassy tes~.
Finally, the procedure described hereinabove is important in identifying viruses and enzymes immunologically. All anti-~ens of the virus type are larger than their specific anti-bodies, whereas the antigens of the enzyme type may be larger or smaller, depending upon the particular type of enzyme.
A procedure for identifying a particular virus or large size enzyme ~ known as the inhibition test, is accom-plished in the following manner: The particular virus or large size enzyme is put into a first solution (generally salt water) with the inert protein and the substrate is then immersed therein, or alternatively, a drop of the solution is placed on the substrate and then the substrate is immersed in a solution of only the inert protein. After a monomolecular layer of the virus or enzyme (i.e., antigen) and inert protein has been adsorbed on the substrate, the coated substrate is removed from the first solution.
Samples of human serum to be tested for the presence of the ~0 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 procedu~es 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 cont~ined the particular virus or enzyme.
However, if a bimolecular layer is detected, then this 10484~9 RD-6735 indicates that Lhe serum did not originally contain such virus or large size enzyme antigen since the antibodLes did not immunologically combine with their specific antigen tvirus or enzyme) in the serum sample, and therefore were free to combine with the antigen of the first layer adsorbed on the substrate.
From the foregoing description, it can be appreciated that my invention makes available a new method for improv-ing the contrast in surface immunological tests between 10 - single and double layers of protein by the addition of an immunologically inert protein of sufficient quantity to a first solution containing an immunologically reactive ; antigen protein. ~-~ ~ ~
Having described my invention with reference to the par~icular embodiments and examples, it is believed obvious that modification and variation of my invention is possible - in the light of the above teachings. Thus, the inert protein could be in a solution separate from the antigen, and the substrate would be immersed therein as a preliminar~
step, or as an intermediate step between immersions in a dilute solution of the antigen and the antibody solution.
As another approach, in the case where the antibodies are in a serum, a dilute solution of the antigen would be utilized in forming an incomplete first layer on the substrate, and the inert protein would be omitted in such solution since the nonspecific protein in the serum would function as the inert protein in adhering to the substrate 8nd thereby act as the spacing agents between the ant~gen molecules. Finally, although ~he reactive protein adsorbed on the substrate surface has been described 1~)48409 hereinabove as being an antigen, it should be evident that such first lay~r reactive protein could be an antibody, and the second layer protein would then be the specific antigen, the latter arrangement being useful in cases S 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 my invention as described which is within the full intended scope of the invention as defined by the following claims.

Claims (10)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for improving contrast in surface immuno-logical tests with relatively large size antigen proteins comprising the steps of selecting a suitable substrate, coating the substrate with a monomolecular layer of an immunologically reactive antigen protein and inert protein of smaller size than the antigen protein so that the antigen molecules are separated from each other and surrounded along the substrate surface by the smaller inert protein molecules, and subsequently exposing the coated substrate to a solution of an immunologically reactive antibody protein specific to the antigen protein and of smaller size relative thereto to cause an immunological reaction there-with in which a relatively large number of the antibody protein molecules bond with the antigen protein molecules to form a bimolecular layer on the substrate, the relatively large number of antibody molecules bonding with the antigen molecules resulting from the larger number of active sites being exposed on the antigen molecules due to their being surrounded along the substrate surface by the smaller inert protein molecules, the relatively dense second layer of smaller size antibody molecules providing significantly greater contrast between the single and double layers of protein when the substrate is examined than if the antigen molecules were closely spaced together.

2. The method set forth in claim 1 wherein the step of coating the substrate with a monomolecular layer of an antigen protein and inert protein consists of the steps of forming a first solution of the immunologically reactive antigen protein, adding the smaller immunologically inert protein of sufficient quantity to the first solution, immersing the substrate in the first solution to coat the substrate with the monomolecular layer of the antigen protein and the inert protein, the inert protein being of sufficient quantity so that the antigen protein molecules are sufficiently separated from each other and surrounded along the substrate surface by the inert protein molecules, and removing the monomolecular layer coated substrate from the first solution.

3. The method set forth in claim 1 wherein the step of coating the substrate with a monomolecular layer of an antigen protein and inert protein consists of the steps of forming a first solution of the smaller immunologically inert protein, immersing the substrate in the first solution sufficiently to coat the substrate with a partial mono-molecular layer of the inert protein, removing the partially coated substrate from the first solution, forming a second solution of the immunologically reactive antigen protein, immersing the partially coated substrate in the-second solution to coat the remaining surface of the sub-strate with a partial monomolecular layer of the antigen protein so that the monomolecular layer consists of the antigen molecules separated from each other and surrounded along the substrate surface by the inert protein molecules, and removing the monomolecular layer coated substrate from the second solution.

4. The method set forth in claim 1 wherein the step of coating the substrate with a monomolecular layer of an antigen protein and inert protein consists of the steps of forming a dilute first solution of the immunologically reactive antigen protein, immersing the substrate in the first solution sufficiently to coat the substrate with a partial mono-molecular layer of the antigen protein, removing the partially coated substrate from the first solution, forming a second solution of the smaller immunologically inert protein, immersing the partially coated substrate in the second solution to coat the remaining surface of the substrate with a partial monomolecular layer of the inert protein so that the monomolecular layer consists of the antigen molecules separated from each other and surrounded along the substrate surface by the inert protein molecules, and removing the monomolecular layer coated substrate from the second solution.

5. The method set forth in claim 1 wherein the step of coating the substrate with a monomolecular layer of an antigen protein and inert protein consists of the steps of forming a dilute first solution of the immunologically reactive antigen protein, immersing the substrate in the first solution sufficiently to coat the substrate with a partial mono-molecular layer of the antigen protein, removing the partially coated substrate from the first solution and immersing the partially coated substrate in the solution of the immunologically reactive antibody protein wherein the antibodies are in a serum, the proteins in the serum other than the antibodies functioning as the inert protein and adhering to the substrate to complete the monomolecular layer of antigen molecules separated from each other and surrounded along the substrate surface by smaller inert protein molecules, and the antibody protein molecules in the serum forming the second layer.

6. The method set forth in claim 1 wherein the step of coating the substrate with a monomolecular layer of an antigen protein and inert protein consists of the steps of forming a first solution of the immunologically reactive antigen protein, adding the smaller immunologically inert protein of sufficient quantity to the first solution, depositing at least a single drop of the first solution on the substrate to have the antigen-inert protein form a small area monomolecular layer thereon, forming a second solution of the immunologically inert protein, subsequently immersing the drop-coated substrate into the second solution so as to form a complete monomolecular layer over the entire surface of the substrate which includes the small antigen-inert protein area surrounded by the inert protein from the second solution, and removing the monomolecular layer coated substrate from the second solution.

7. The method set forth in claim 2 and further comprising the steps of removing the bimolecular layer coated substrate from the antibody solution, subsequently exposing the coated substrate to a third solution containing an antibody to the antibody in the second layer to cause an immunological reaction therewith in which the antibody molecules in the second layer combine with a relatively large number of the antibody molecules in the third solution to provide an even more significantly improved contrast between the first and second layers of protein upon examination of the coated substrate than if the antigen molecules were closely spaced together.

8. The method set forth in claim 1 wherein the step of subsequently exposing the coated substrate to a solution of an antibody protein consists of exposing the coated substrate to a solution suspected of containing the immunologically reactive antibody protein specific to the antigen protein, and further comprising the steps of removing the coated substrate from the solution suspected of containing the antibody, and examining the coated substrate to determine whether there is a second layer thereon thereby indicating the presence of the antibody in the suspect solution.

9. The method set forth in claim 1 wherein the step of subsequently exposing the coated substrate to a solution of an antibody protein consists of exposing the coated substrate to a second solution suspected of containing the immunologically reactive antibody protein specific to the antigen protein, and further comprising the steps of removing the coated substrate from the second solution suspected of containing the antibody, subsequently exposing the coated substrate to a third solution containing an antibody to the antibody which may be in the second solution to cause an immunological reaction therewith in which any antibody molecules in the second layer combine with a relatively large number of the antibody molecules in the third solution, removing the coated substrate from the third solution, and examining the coated substrate to determine whethe r there is a third layer thereon thereby indicating the presence of the antibody in the suspect second solution, the third layer providing an even more significantly improved contrast between the first and second layers of protein than if the antigen molecules were closely spaced together.

10. The method set forth in claim 2 wherein the step of adding a sufficient quantity of the inert protein to the first solution consists of adding a quantity sufficient so that the inert protein molecules occuyp 50 to 90 percent of an area on the surface of the substrate and the antigen molecules correspondingly occupy 50 to 10 percent of the area.