CA1047918A - Method and apparatus for detection and purification of proteins and antibodies - Google Patents

Abstract

Abstract of the Disclosure Method and apparatus for the detection and purification of proteins and antibodies based upon the effect that any arbitrary protein will adsorb onto a substrate in a monomolecular layer only, but that a specific antibody (or antigen) for such arbitrary protein will bond thereto to from a bi-molecular protein layer on the substrate is disclosed. In practicing this invention, a first layer of protein is adsorbed onto a substrate and the coated substrate is then exposed to a solution suspected of containing the specific antibody (or antigen) of interest. The substrate is then examined to determine whether a monomolecular or bi-molecular layer of protein is adhering thereon.
Optical, electrical, And chemical means for examining the coated substrate are disclosed. Purification is provided by severing the immunological bond with weak acid.

Description

~4791~ RD-5719 IMPROVED METHOD AND APPARATUS FOR DETECTION
AND PURIFICATION OF PROTEINS AND ANTIBODIES

, This invention relates to method and apparatus for detection and purification of proteins and antibodies.
More particularly, this invention relates to detection and purification of proteins and antibodies where the antigen-antibody reaction takes place at the surface of a substrate.
This application is related to the Canadian application of Giaever, Serial No. 172,639 filed May 29, ~ ~
1973~ and assigned aæ herein. ~ `
Immunological reactions are highly specific bio- `
chemical reactions in which a first protein called the antigen combines with a second protein specific to the antigen called its antibody to form an immunologically complexed protein. Immunological reactions taking place within a biological system such as an animal are vital to ~ `
the animal in combating disease. In a biological system, the entry o~ a foreign protein, i.e., the antigen, causes the biological system to produce the specific antibody /i ::;
proteins to the antigen in a process not fully ~mderstood at this time. The antibody protein molecules have available chemical bondin~ sites which complement those on the antigen molecule and so the antigen and antibody chemically combine to form an immunologically complexed protein. `
Because antibodies are produced by biological systems in response to invasion thereo~ by foreign proteins, .
` 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. Conversel~, the ~

.:

i~47~. RD-5719 detection of certain antigens in a biologic system also has medical diagnostic value; examples of diagnostic detection of ant~ens include detection of HCG protein molecules in urine as a test for pregnancy, and detection of hepatitis associated antigen molecules in blood of prospective blood donors.
In order to perform ~uch diagnostic tests, the appropriate protein of at least an immunologically react-ing pair must be obtained in concentrated purified form.
The only known source of antibody proteins is a living biological system. More particularly, only vertabrates are known at this time to exhibit immunological reactions to the introduction of a ~oreign proteln. For example, many antibodies are found in the blood serum of an~lals which have been exposed to the corresponding antigens.
Blood serum, however, is a very complex mixture in which the antibodies required areipresent in very low - concentrations amid a large plurality of other constituents.
Many antigens, on the other hand, may be controllably produced in laboratory cultures. However, some antigens, ~or examle, hepatitis associated antigen, are at present, like antibodies, only obtainable from the higher living biological systems.
As presently practiced, both the collection and purification and the diagnostic utilization of immuno- -logically actiue proteins rely upon the precipitating or agglutinating characteristic of the proteins resulting from the immunological complexing reaction. The classic -~
example of these diagnostic uses is the blood typing procedure in which blood samples are mixed with alpha and

2-.~, . . "
...... . ., - ~

l~il8 RD-5719 beta type serum antiboclles and blood type is detenmined by observing any agglutination occurring in the blood samples.
The HCG protein pregnancy test as currently `practiced is an inhibition ~est. The ~est is performed by mixing a quantity of HCG anti-serum into a urîne specimen. -`
A plurality of polystyrene spheres which have been coated ~.
with HCG protein are then introduced into the previously :~
prepared urine specimen~ The polystyrene spheres will agglutinate if, but only if, HCG protein is ab~ent from the urine specimen. If HCG protein is a~bsent from a uri.ne speclmen, the HCG protein on the polystyrene~spheres complexes .
with the HCG anti-serum previously introduced in the urine ~:.
specimen and the spheres agglutinate. If, on the other hand, HCG protein is present in the urine specimen, it complexes with the previously introduced HCG anti-serum forming a ; ;
: .
complex which precipitates out of the specimen so that the previously introduced anti-serum is no longer available to `
complex with the HCG protein on the spheres to cause agglut n- .. ..;;
ation thereoE. In accordance with the teachings of this - 20 disclosure, the present HCG protein pregnancy test could be simpliEied by adharing HCG anti-serum onto the polystyrene spheres and directly testing a urine specimenO In this case, the polystyrene's spheres would agglutinate if, but only if, HCG protein is present in the specimen.
It appears that the reason this simpler procedure has not been employed is that the avail.able HC& anti-sera are complex mixtures containing a large proportion of ~
constituents other than HCG antibodi.es. The additional ; effort requi.red in the prior art to extract the antibodies rom the HCG.anti-sera make the inhibition test, utilizing , ;.

,, , , . ~ ~ . .. ..... . . . . . . .

~O ~7 ~L~ RD-5719 sera directly, preferable in the prior art. However, in accordance with one embodiment of this invention, a pro-cedure is provided whereby HCG antibodies are efficiently separated from sera and, which procedure, furthermore, produces diagnostic apparatus whereby the simpler, direct test is performable. Each antigen molecule will typically complex with a plurality of antibody molecules which may be assoclated with different particles thereby forming visible clumps of, for example, coated polystyrene spheres or red blood cells. A shortcoming of agglutination tests is that the particles involved may tend to agglomerate for any of a variety of reasons having nothing to do with i.mmunological agglutination thereby decreasing the reliability of the test.
Typically, agglutination tests are performed with great care ~`~
by skilled technicians but never~heless occasional diagnostic errors occur.
The present procedure for obtaining purified concentrations of antibodies comprises the steps of stimu-lating the production of antibodies in an animal by introducing the antigen into the animal's system, obtaining blood serum from the animal which contains the antibodies in a highly dilute form, and mixing a quantity of the speciic antigen into the serum. The mixture of antigen and antibody complexes~and precipitates out of the serum solution. The remaining constituents of the serum are drawn off and the antibody-antigen precipitate is dissolved in an acid which severs the complexing bonds. At this point one has a solution of antigen and antibody molecules in acid. Since the antibody and antigen molecules have differing physical characteristics, for example, weight, they may be separated from each other by mechanical means, for example, by centrifugingO

~79~8 ~-5719 It is known that ~he antibody-antigen complexing reaction will take place when an antigen is adsorbed at a surface. The complexing reaction at a surface has been observed by means of an ellipsometer. An ellipsometer is a complex optical instrument by means o which it îs possible to measure the thicknesses of films on the order ;
of 0.1 A, Ellipsometers are expensive and require skilled operators. In studies of immunological reactions uslng ellipsometers performed to date, ~wo methods have been used.
In one method, the reaction to be studied is allowed to take place and then the slide on which the reaction has taken place is mounted in an ellipsometer and an actual measurement of film thickness i8 made. In the other `
method, the slide is mounted in the ellipsometer while the immunological reaction is taking place and the change of ` !
film thickness is observed with the ellipsometer. The` ; `
measurement of absolute thickness requires extreme care. ~ `On the o~her hand, when the concentration o antibodies in the solution is low, the measurement of relative change `~
~n o thlckness, while easier than a measurement of ab801ute thickness, will ~ake a long time. Accordingly, for economic reasons, the detection of immunological reactions at a surface using an ellipsometer has not been adopted or diagnostic purposes. ;;
This invention includes the discovery that any arbitrary protein will adsorb onto a substrate in a mono- -molecular layer only, but that a specific antibody (or antigen) or such arbitrary protein will bond thereto to form a bi-molecular protein layer on the substrate. The objects of this invention therefore comprise the application ~O 4~ 9 ~ RD-5719 of this discovery to the provision of me~hod and apparatus for exploiting thls discovery in medical diagnostic and pharmacological applications.
Accordingly, it is a first principal object of this invention to provide method and apparatus for economically detecting immunological reactions occurring at a surface.
It is an ob~ect of this invention to provide such method and apparatus wherein such immunological reactlon~
are detectable by electrical mean~.
It i8 another object o this invention to provide such method and apparatus for detecting such immunological reactions by direct visual observation.
A second principal object of this invention is lS to provide method and apparatus for concentrating and an fi~J~S
purifying proteins and sw~UuilL-} by means ~ controlled immunological reactions occurring at a surace.
Briefly, ~nd in accordance with two embodiments o this invention, a wafer of substrate material i~ ~irst immersed in a solution of a first protein so tha~ a mono- ~`
molecular layer of such first protein adheres to the substrate. The substrate coated with the first protein `
is then immersed in a second solution, which in a first embodiment is known to contain the specifically reacting `~
protein to the first protein, and in a second embodim~nt is suspected of containing the specifically reacting protein to the first protein. The specifically reacting protein, and only the specifically reacting protein, forms a second monomolecular layer overlying the monomoIecular layer of th~ first protein on the substrate. Then, in ~; ~

~ ~7 9 ~ RD-5719 accordance with a first embodiment o this inventlon, the bi-molecular coated substra~e is immersed in a weak acid solution which severs the immunological bond be~we~n the two protein layers and provides or the collection of the specifically reacting protein in a purified form in a weak acid solution; in a second embodiment of this invention, the coated substrate, after having been immersed in the solutlon suspected of ~ontaining specific-~ ;
ally reactlng protein, is examined electrically or optically to determine whether a bi-molecular layer or ~ `
monomolecular layer of protein is adhering thereto, thereby ` `
determining whether or not the second solution conta~ned the specifically reacting protein to the first protein.
The novel features of this invention sought to be patented are set forth with particularity in the appended claims. The invention, together with further objects and advantages thereof,`may be unders~ood Erom a reading of the following speciication and appended claims in view of the accompanyLng drawings in which:
FIG. 1 is a flow chart illustrating the proce3s steps o the various embodiments of this invention.
FIG. 2 is an elevation view of diagnostic apparatus in accordance with one embodiment o this inven~ion illustrating the method o making and using same.
FIG. 3 is an elevation view of apparatus useul for diagnostic purposes and for the purification and concentration of antibodies in accordance with another embodiment of this invention, ~O 47 9 ~ ~D-5719 FIG. 4 is a mechanlcal schematic dlagram of apparatus in accordance wi~h an embodiment of this inven~ion for concentrating and purifying proteîns and antibodies.
FIG. 5 is a photograph illustrating the operation ;;
of diagnostic apparatus in accordance with this invention.
FIG. 6 is an elevation view of diagnostic apparatus in accordance with another embodlment of this invention.
FIG. 7 is an elevation view of diagnostic appara~us in accordance with this invention i~lustrating a modification in the method of using the apparatus of ~IG. 2.
FIG. 8 is a graphical representation illustrating the principle of operation of diagnostic apparatus in ;;
accordance with the embodiment of this invention illustrated in FIG. 9.
FIG. 9 is an elevation view of another embodiment ;
of diagnostic apparatus in accordance with this invention.
FI~. 1 is a flow chart illustrating the process steps involved in practlcing this invention. In reading the ~low chart from top to bottoml each vertical level represent~ one time sequential step of the process. The appearance of several steps arrayed horizontally at a given vertical level in the flow chart indicates the alternative performance of one of the indicated steps at the indicated sequential position in accordance with the various embodiments of this invention.
In accordance with a first embodiment of this invention, the process begins at block 13 of FIG. 1 in which a wafer of substrate material which may be metal, glass, mica, plastic, fused sili~a, quartz, or similar `' ~

i~79~ RD-5719 material, with metal being preferred, as having the greatest difference in refractive index to protein, and preferably is in the form of a metallized glass slide, is immersed in a solution containîng a first protein of interest which may be biologically an antigen or antibody, ~ `
whic~ is available in a relatively ~oncentrated solution and which is to be used to detect or purify its correspond-ing specifically reacting protein, which will be blologically respectlvely an an~ibody or an antigen. The first protein adsorbs onto the substrate in a monomolecular`
layer. Any protein will adsorb in such monomolecular layer but no further adsorption will take place. That i8, the protein wlll attach to the substrate, but wlll not attach to itself. After a monomolecular layer of protein ha~ Eormed over the entire surface of the substrate, the coated substrate is removed from the solution of the ~irst protein. The t-ime required to completely coat the substrate is a function of the concentration of the protein in the solution and the degree o agitation of the solution. As an example, a 1 percen~ bovine serum albumin ~olution completely coats a slide in approximatel~ 30 minutes with a monomolecular protein layer. The next s~ep, illustrated in block 14 of FI~. 1 is to immerse the protein coated substrate in a solution suspected of containing the specifically reacting protein to the first protein. This solution may, and typically does9 contain many constituents in addition to the specifically reacting protein whose presence it is desired to detect. However, no protein other than a specifically reacting protein will adhere to the first protein layer on the substrate. Accordingly, _9_ ,' ~L~ ~7 9 ~3 RD-5719 if the specifically reacting protein is not present, the substrate following immersion in the suspected solution will still contain only a monomolecular protein layer thereon. If, on ~he other hand, the specifically reacting ~;
protein is present in the solution, immunological complexing between the first protein and its specifically reacting protein will take place and the substrate will, after a time, have a bi-molecular protein layer thereon.
It is to be noted that the steps illustrated in blocks 13 and 14 of FIG. 1 are common to all of the embodiments of this invention. The time required for the adhesion of a complete second molecular layer onto the coated substrate ; `~
~ ., is again a function of the concentration of specifically reacting protein in the solution. For antibodies in lS blood serum, this time may be as long as one day. In one embodiment of this invention, the next step is to immerse the coated substrate in a weak acid solution as illustrated in block 15 of FIG. 1 while simultaneously observing the coated subætrate in an ellipsometer as illustrated in block 22 of FIG. 1. While the formation of the specifically reactin~ protein layer on the first protein coated substrate may take an extended period of time, the immuno-logical bond between the two proteins i9 severed very quickly by the weak acid. Accordingly, the observation made with the eIlipsometer can be the relatively simple ~;-observation of change of film thickness rather than the more complicated measurement of absolute thickness. At the same time, observing the stripping away of the specifically reacting protein layer by action of the weak ~;
acid may be performed mNch more rapidly than the prior art -10~

~79~8 RD-5719 ~ .
method of observing the building of the specifically reacting protein layer. Accordingly, a large plurality of test slides may be prepared in accordance with block 13 and each exposed to one of a large plurality of serum samples as indicated in block 14, and then in a short time each test slide may be examined serially in accordanca with blocks 15 and 22 to determine which of the serum samples contained the specifically reacting protein to the first protein. Since the acid will not strip the first protein from the substrate, those coated substra~es which were immersed in ~olutions which did not contain the specifically reacting protein will exhibit no change o f film thickness when immersed in acid and observed in an ellipsometer. On the other hand, those which were immersed in a solutian which did contain the specifically reacting protein will exhibit approximately a factor o~
2-5 change in thickness when immersed in acid and obs~rved in an ellipsometer. Each observation can be made in a ~ew minutes thereby providing an efficient, and thereore diagnostically significant, test procedure.
~ second and closely related embodiment of this invention provides for the concentration and purification of proteins and comprises the steps illustrated in FIG. 1 at blocks 13, 14, 15, and 23. The substrate is first immersed in a solution of the available protein of the antigen-antibody pair as illustrated in block 130 In the usual case, this will be the antigen, but this invention is not dependent upon the biological identity of the first protein. The coated substrate produced in accordance with block 13 of FIG. 1 is then immersed in a solution -11- . :

, ., ~ , . . : , - :

7~i8 RD-5719 containing the specifically reacting pro~ein to the first protein as shown in block 14. In the usual case, the specificall~ reacting protein will be an antibody and the ~`
solution employed in block 14 will be the blood serum of an animal which has been exposed to the first protein.
The substrate, now coated with a bi-molecular protein layer, is next immersed in a weak acid as shown in block 15 of FIG. 1 which strips the speci1cally reacting protei~
layer from the first protein layer. At this point the substrate is coated with a monomolecular layer o the first protein and a purified solution of specifically r~act-ing protein in the weak acid has been provided. The next step, as shown in block 23 of FIG. 1, is to~return the substrate with its first protein layer adhering thereon to lS the solution containing the specifically reacting protein to pick up a second layer thereof which is again stripp~d by the acid thereby increasing the concentration o spec~ically reacting protein in the acid. This`process ~`
is continued and provides for the collection of a concentration af pure specifically reacting protein in the weak a~id bath.
While the first described embodiment improves the ` ~
efficiency of ellipsometric immunological detection to ; ~ -the point of practical feasibility~, it is nevertheless ~ `
economically desirable to eliminate the need or the `~
ellipsometer entirely. Accordingly, another embodiment af this invention provides for determination o relative protein film thickne~s by electrical means. In this embodiment, either a metallic substrate is chosen, or a `
~0 substrate of another material is first coated with a metal -1?~

... - ....................... . :

~04~ RD-5719 film as indicated in block 11 of FIG. 1. The metal substrate or metal-coated substrate is ~hen immersed in protein solutions in accordance with the steps of blocks 13 and 14 as discussed above. The protein layers adhering to the substra~e are electrically insulating. The next step is to place a mercury drop or other electrode upon the upper protein layer adhering to the substrate as shown in block 16 of FIG. 1, The upper protein layer is either the first protein, if the suspected solution contained no specifically reacting protein, or the specifically reacting protein if the solution did contain it. Accordingly, the ~ ~ .
metal film or metal substrate and the mercury drop comprise the two conducting plates of an electric capacitor separated by an insulating layer comprising either a mono~
molecular protein layer or a bi-molecular protein layer.
The electric capacitance of this capacitor is then measured as indicated at block 19 of FIG. 1 by means of any suitable instrument Eor measuring capacitance as is known in the art, for example, Heathkit Impedance Bridge Model IB-28.
~0 The electrical capacitance o~ such capacitors having a bi-molecular protein layer dielectric was, as expected, found to vary by approxlmately a factor of 2-5 from that of such capacitors having a monomolecular prote~n layer.
Another,closely related, embodiment of ~his invention further simplifies the detection of the specific-ally reacting protein by enabling its presence to be ascertained by unaided visual observation. This embodiment employs a substrate of a metal which forms an amalgam with mercury or ànother substrate material such as glass whlch is coated in the block 11 step with a thin film of e m~r~k -13-~0479`18 RD-5719 metal which forms an amalgam with mercury, preferably gold.
The substrate is then subjected to the block 13 and block 14 steps as before and a mercury drop is again placed on the upper protein layer as indicated in block 16. In this embodiment, however, no electrical measurement is made.
In this embodiment, the next step is to visually observe the coated substrate and determine the length of time which elapses before a visible amalgam is formed between the mercury and the metal ~ilm coated on the substrate.
This embodiment of the invention depends upon the inventive discovery that mercury diffuses through a mono-molecular protein layer in approximately 1 minute, but requires 10 minutes or longer to diffuse through a bi-molecular protein layer, Since the mercury must diffuse through the protein layer or layers to form a visible amalgam with the metal film coating on the substrate, the `
time between the placing of the mercury drop upon the upper protein layer and the appearance of the visible amalgam indicates whether a monomolecular or bi-molecular pro~ein layer Ls present on the substrate and consequently whether or not the suspected solution actually contained specifical~y reacting protein to the ~irst pro~ein. At this point, those skilled in the art will reallze that :
the capacitance measurement of the Last previously discussed ~`~
embodiment must be made within l minute of the placing of the mercury drop upon the upper protein layer lest the mercury electrode diffuse through the insulating~protein layer and short out the capàcitor. Alternatively, shorting may be prevented by coating the metal with a metal oxide insulating layer and adhering the protein onto the metal . . - . ~ . - - . .

10479~3 RD-5719 oxide. It will also be recognized ~hat a rough quanti-tative measure of the concentration of specifically reacting protein in the suspec~ed solution may be made in accordance with this embodiment by preparing a plurality of substrates in the block 13 step and immersing them simultaneously in the suspected solution as indicated in block 14, but removing them sequentially from the solution over an extended period of time. Slnce the rate at which the specifically reacting protein layer forms is a function of `~
the concentration of speciically reacting protein in the solution, a comparison of diffusion times for the mercury drop through the protein layers on a series of substrates having been exposed to the suspected solution for varying periods of time will provide a rough quantitative indica-tion of the concentration of specifically reacting protein in the suspected solution.
In anot~er related embodiment, a substrate is coated with a metal film as depicted in block 11 of FIG. 1 and as discussed in the last embodiment. The substrate is then immersed in solutions as shown in block~ 13 and 14 and discussed above. The next step in accordance with this embodiment is to coat the upper protein layer with a second ~`
metal layer as illustrated in block 17 of FIG. l and to view the structure thus produced by reflected light as illustrated in block 21 of FIG. 1. The second metal ls prPferably applied by electroplating. Essèntially, this embodiment produces a structure~which functions as a diffrac-tion grating and the difference between a monomoleculàr and bi-molecular protein insulator layer is determinable from ~
the spectral components observable in the refLected light, `

~479~8 RD-5719 FIG. 6 illustrates apparatus in accordance with this embodiment. A metallic surface 81 of a substrate which may be either a metallic substrate or a non-metallic substrate coated with a metal ilm, has a first protein layer 82 applied thereto as indicated in block 13 of FIG. 1. The substrate coated with protein layer 82 is then immersed in the solution to be tested and a second layer 83 of specifically reacting protein to the protein oE layer 82 forms thereon if it is present in the solution.
A second layer of metallization is then applied to the exposed surace o the protein film. A very small quantity o metal is applied so that the second metallization - .
tends to be in the form of a plurality of discontinuous metallized areas as, for example, 84 and 85. A beam o light represented by rays 86 is then directed onto the ~ ;
slide thus prepared. The light is reflected at metal boundaries. The distance between the reElecting surfaces of metal substrate surface 81 and metallization partieles, `~
84, ~or example, is a function of the thickness o the protein film between members 81 and 84. Accordingly, ~he observed reflected light, illustrated by rays 87~
; varies in spectral composition as the protein layer varies between a monomolecular layer and a bi-molecular layer.
Another embodiment of ~his invention illustrated in FIG. 1 comprises the steps illustrated at blocks 12, 13, 14, and 18 o FIG. 1. In this embodiment, the substrate 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 be~ng a conveniently available source, is firs~ coated with a ~3 RD_5719 plurality of metal globules by evaporating a metal, for example, indium, onto the substrate as indicated in block 12 of FIG. 1. For example, the indium is evaporated slowly from a tantalum boat onto the glass substrate 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 g]ass 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, and lead have been success- ``
fully used. The evaporation of metal is continued untLl the substrate appears light brown in color. ~t this point, O
the metal globules have diameters on the order o~ 1000 A. ~ ~
, The precise size of the globules is not critical but they `;
must have diameters equal to a large fraction of~ a wave-length of visible light. The next step is to immerse the globule-covered substrate in a solution of a first protein `~
as illustrated in block 13 of FIG. 1. The first protein again adheres in a monomolecular layer over the substrate and the metal globules thereon. When a monomolecuLar layer protein has formed, the coated substrate may then be ~ ;
used to test suspected solutions for the presence of a ~5 specifically reacting protein to the first protein by immersing the coated substrate in the suspected solution as indicated in block 14 o FIG. 1. If~the suspected specifically reacting protein was present, the substrate and metal globules have a bi-molecular protein layer adhering thereto; if the specifically reacting protein was ~ RD-5719 not present, only a monomolecular protein l~yer overlies the substrate and metal globules. The coated sub~trate is then viewed by either reflected or transmitted light as indicated in block 18 of FIG. 1 and a determinatlon is S made from the appearance o the coated substrate as to the thickness of the protein layer adhering thereto and accordingly as to the presence or absence of the suspected speciically reacting protein. The detection of protein layers corresponds to variations in the shade of brown which is observed in the coated substrate. These variations are quite pronounced and the de~ection 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 brownJ and the particles covered with a bi-molecular protein layer appear as a still darker shade o~ brown. 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 ~ncident energy and that in the case of scattering from such spheres, the scattering is strongly influenced by a thin dielectric coating applied to the spheres.
FIG. 5 is an actual photograph of diagnostic apparatus in accordance with this embodiment. In each view o FIG. 5 the test slide is viewed by transmitted light.
View 5a is a glass slide having indium globuLes over one ~
suriace 100 thereof. View 5b shows a slide prepared as ~ ~ ;
the slide of View 5a whose left edge has~`been immersed in a solution of bovine serum albumin, thereby adsorbing a , ,~
;. ;.. .. . . . .

~O 47 9 ~ RD-5719 monomolecular layer 101 of bovine serum albumin thereon.
View 5c shows a similar slide in which the left edge has been immersed in bovine serum albumin thereby adhering `~
a monomolecular 101 of bovine serum albumin thereon, and whose lower edge has been immersed in a solution of ov-albumin thereby adsorbing a monomolecular layer 102 of ovalbumin thereon. It is important to note that the appearance of the coated portions of the slide of View 5b are similar, indicating that only a monomolecular protein layer is present on the slide. This demonstrates that bovine serum albumin and ovalbumin adsorb onto the slide and that the ovalbumin does not adhere to the portion o the slide previously coated with bovine serum albumin.
In other words, View 5c illustrates the discovery discussed above that any arbitrary protein will adhere to the substrate but a protein will not adhere to an arbitrary protein film. The slide of View 5d was prepared by immersing the left edge thereo~ in bovine serum albumin in solution to adhere monomolecular layer 101 thereon; the right edge was then immersed in an ovalbumin solution to adhere monomolecular layer 102 thereon; and finally the lower edge was immersed in a solution o rabbit anti-~erum to bovine serum albumin. Accordingly, 103 is a mono-molecular layer of rabbit anti-serum to bovine serum -albumin. The appearance of t~e lower right-hand cornqr indicates that the rabbit anti-serum to bovine serum albumin did not adhere to ovalbumin layer 102 and accord-ingly the lower right-hand corner of the slide of View Sd is coated with only a monomolecular layer of ovalbumin.
The lower left-hand corner of View 5d is pronouncedly 19- , ~79~ - R~-5719 darkened indicating the presence thereon of a bi-molecular protein layer comprising a first monomolecular layer of bovine serum albumin, previously adsorbed ~hereon, ha~ing a second monomolecular layer of rabbit anti-serum to bovine serum albumin immulogically complexed with the underlying bovine serum albumin. It will accordingly be seen that only the specifically reacting prote.in pair formed a bi-molecular layer on the slide, all other coatings ~ :
thereon being monomolecular.
In a modification of this embodiment which provides for a medical diagnostic system, the substrate having metal spheres thereon is partially immersed, to a first depth, in a solution of a first antigen. The substrate is coated in a monomolecular layer by the irst~antigen over the area which is immersed in the solution. The substrate is then dried and is more deeply immersed, ~o a second depth, in a solution of a second antigen. The second antigen does not adhere to t~e first antigen but does adhere to the uncoated portion of the substrate which is immersed in the solution. The substrate is then again dried and immersed in a solution o a third antigen to a `~
third and greater depth. The third antigen a&eres to that portion and only the portion of the substrate which ~ ~`
is not coated with the first or second antigen. The process is repeated for any number of antigens of intere~t.
The resulting product is a substrate coated with a plurality of bands of monomolecular layers of different antigens.
This coated slide is the diagnostic toolO The medical diagnostic procedure is then to ~mnerse the slide in a specimen of, for example, blood for a period of typically ; '', 1~479~8 RD-5719 several hours in duratlon. The slide is then removed from the specimen~and after washing in water ls viewed by reflected or transmitted light. The slide then exhibits a pattern of lighter and darker bands which are indicative of the antibodies present in the specimen from which information a medical diagnosis is made.
FIG. 2 is a highly magnified elevation view of a portion of diagnostic apparatus in accordance with the last discus~ed embodiment of this invention. FIG. 2 shows a portion ofsubstrate material 31 having a plurality of globules of evaporated metal 32 attached thereto.
Particles 32 are preferably formed by the evaporatio~ of indium onto substrate 31 as discussed above but may al80 be formed by evaporation of gold, silver, tin, lead, or other metal having similar non-wetting and atomic mobility characteristics. Any of a large number of metals will exhiblt such characteristics as the temperature o~ the substrate is varied. AEter immersion in a solution of a Elrst protein, the slide segment comprislng substrflte 31 2~ and me~al globules 32 is coated wlth a monomolecular layer o molecules 3~ o~ the ~lrst proteln as indicated in ` FIG. 2 generally at 33. The apparatus indicated at 33 is ~;
the diagnostic instrument which is used to test suspected solutions for the presence of specifically reacting protein ;~`
to protein molecules 34. If the apparatus indicated generally at 33 is exposed to specifically reacting protein to the protein of molecules 34, the apparatus wiLl acquire the appearance indicated generally at 35 in which substrate 31 and metal globules 32 are coated with a bi-molecular ~;
proteln layer comprlsing the molecules 34 of the first -21~
' ~ :' ~7 9 ~
RD_5719 protein forming a first monomolecular layer overlying substrate 31 and globules 32 and a second monomolecular layer of protein consisLing of the molecules 36 of the specifically reacting protein to the first protein, immunologically bonded to the molecules of the first -protein and overlying the first protein layer, the metal globules,and the substrate.
FIG. 7 illustrates a modification of this embodi-ment of the invention whic~ provides for increased contrast in the coated diagnostic slîde. In FIG. 7, the slide 31 having metal globules 32 applied thereto and coated with protein molecules 34, as in FIG. 2, is immersed, the : .
coated side downwardly, in a quantity 71 of light re~lect~
ing liquid. Slide 31 is then viewed from its upper surface ~ ~;
by reflected light. In order to provide for ~otal reflection of light incident on the diagnostic apparat~
of FIG. 7 ? reflecting liquid 71 is prefera`bly a metallic `
liquid, and ls more particularly, preferably mercury. If a nonmetallic liquid is employed, the optical incidence ~;
and viewing angles employed in using the FIG. 7 embodiment ~;
become critical if total re1ection is to be observed. The appearance of a test slide viewed in accordance with these ;
teachings is similar to that shown in FIG. 5, but with -enhanced contras~
FIG. 3 is a highly magnified sectional elevation view of apparatus useful for diagnostic purposes and for the purification and concentration of proteins and anti-bodies in accordance with another embodiment of this invention. Indicated generally at 40 is a substrate 41 coated with a monomolecular layer of protein lecules 42 which has been prepared as discussed abo~e. Indicated -~2 lO ~ 9 1~ RD-5719 generally at 43 is substrate 41 and protein layer 42 to which has been immunologically bonded a second mono-molecular layer o~ protein molecules 44 of the speci~ically reacting protein to molecules 42 in accordance with the procedures discussed above. A second substrate 45 has thereon a drop 46 of a weak acid solution. The mutually facing surfaces of substrates 41 and 45 having thereon respectively a bi-molecular protein layer and a weak acid drop of, for example, citric acid in a 0.1 normal solution, are then physically brought into contact with each other.
In accordance with an inventive discovery of this invention, the weak acid drop 46 severs the immunological bonds between molecules 42 and molecules 44 without affecting the bio-chemical characteristics of either protein and without severing the adhesion bond between the first~protein molecules 42 and substrate 41. When substrates 41 and 45 ~
are again separated as indicated generally at 47, substrate .
41 has adhering thereon a monomolecular layer o~ Eirst protein molecules 42 and substrate 45 has adhering thereon a monomolecular layer of speci.fically reacting protein molecule~ 44. Substrate 41 with molecules 4~ thereon may then be used to repea~ the process producing another substrate coated with a monomolecular layer of specifi.cal~y reacting protein or may be used as a test slide in accord-2S ance with the other previously discussed embodiment Oe this invention. Substrate 45 with specifically reacting protein 44 adhering thereto may be used as a test sllde to :
test suspected solutions for the presence of molecules of its corresponding ~irst protein in accQrdance with the other previously discussed embodiments of this invention.
This embodiment of this invention is considered to be : ' ~04791~ RD-5719 particularly significant because~ as has already been noted, in the case of the usual proteins of biological interes~, antigens are fairly readily available in purified form and so may be adsorbed onto a substrate to form a test slide for the detection of the presence of antibodies in suspected solutions but antibodies are not so available.
Therefore, previously to this invention it has generally not been possible to produce test slides for testing -suspected solutions for the presence of antigen3. The method and apparatus illustrated in FIG. 3, however, provides for the production of test slides comprising substrate 45 coated with a monomolecular layer of, or example, antibody molecules 44 which may be used to test suspected solutions for the presence of antigen.
Another embodiment of diagnostic apparatus in ~-~
accordance with this invention is illustrated structurally in FIG. 9, and its principle of operation is illustrated graphically in FIG. 8. As illustrated in FIG. 9, diagnostic apparatus in accordance with this invention comprises a gold substrate 91, which, for reasons of economy, is preferably a thin gold layer plated onto another metal, has adsorbed thereon a monomolecular layer 92 of first protein. Gold has an absorption band within the ~;
visible spectrum. This fact accounts for the characteristic color of gold and provides for the operation of this embodiment of this invention. ~ ~
FIG. 8 ill~strates the operation of this embodiment ;~ ~-and constitutes a set o reflectivity curves in which relative reflectivity is plotted as a function of wavelength.
Curve 93 represents the reflectivity of gold metal. Curve 94 ~ ~ , ~ 47 9 ~ RD-5719 represents the reflectivlty of gold metal having a mono-molecular protein layer ~hereon. Curve 95 represents the reflectivity of gold metal having a bimolecular protein layer thereon. Accordingly, a gold substrate such as 91 has, in accordance with curve 93, the characteristic bright yellow color of gold metal. When the test protein layer 92 is applied to substrate 91, the appearance o the test slide, in accordance with curve 94, has a dull yellow appearance. After the test slide has been exposed to a solution suspected o~ containing immunological reacting protein to the protein comprising layer 92, the test slide, if such specifically reacting protein was present in the solution,has a bi-molecular protein layer thereon and a reflectivity characteristic indicated by curve 95 which provides a distinctly green appearance. ~ ~
In tests which have been performed to date, it appears that the embodiments of this invention which employ a substrate including metal globules and that employ-lng a gold substrate are the most generally useful.
Furthermore, it has been determined that these two embodi-ments have differing sensitivities as ~unctions of the thlcknesses of protein films o~ interest. Specifically, the greatest sensitivity of the embodiment having a substrate including metal globules occurs with films having thicknesses below approximately 200 A. The gold substrate embodiment has the greatest sensitivity for films exceeding 30 A in thickness.
In one diagnostic procedure which has been per~ormed in accordance with this invention, glass slides were coated with a thin indium layer and overcoated with a gold layer, ~Lo ~7 ~ ~8 RD-5719 The use of indium undercoating was required to improve the adhesion between the glass and the gold. It was found further that the diffusion of the indium into the over-lying gold improved the optical characteristics of the test slides. The slides thus prepared were coated with a monomolecular layer of hepatitis associated antigen.
Samples of human blood to be tested for the presence ~o hepatitis were prepared by mixing therein a quantity o antibodies to hepatitis associated antigen sufficient to be immunologically removed from the mixture if hepatitis ~;;
associated antigen be present in the sample. The test slides were then immersed in the previously prepared blood ` ~
samples. When removed, the slides which had been immer~ed ',! ' in hepatitis negative samples had a bi-molecular protein layer thereon co~lprising the hepatitis associa~ted antiger previously applied thereto and a second layer of anti-bodies to hepatitis associated antigen immunologically i~
bonded thereto as evidenced by a greellish band on the slide. ;~ ;
Slides having been immersed in hepatitis positive samples, ;`
on the ot~er hand, retained their original dull yellow appearance indlcating that only the monomolecular hepatiti~
associated antigen layer was present thereon the anti- ~-~
bodies which had been introduced into the samp~le~having ~;
complexed with the antigen therein and precipitated out of the sample to be unavailable to react with the antigen -on the test slide.
It will be recognized that the test described above is an inhibition test. In another diagnostic procedure which has been performed, a direct test in accordance with this invention was employed. In the direct -26~
. , .
'~

9L~ 4q ~ ~ RD-~719 test, glass-indium-gold slides were prepared as described above and then coated with a monomolecular layer of a~ti-bodiesto hepatitis associated antigen~ Some of the slides were then immersed into pooled serum taken from hepatitis patients and thereore known to contain hepatitis associated antigen. Others of the slides were immersed in serum samples known to be free of hepatitis associated antigen.
As expected those slides which had been immersed in the pooled serum had bimolecular protein layers thereon, while those immersed in the other blood had only mono-molecular layers thereon.
Theoretically, the clirect test described ;
immediately above is the preferred diagnostic procedure .
both because of the relative s-lmplicity thereof and also because, in this case, the direct test has a higher `~
sensitivity than the inhibition test. The higher sensitiv-ity of the direct test results from the fact that hepatitis associated antigen molecules are very much larger than mol~cules oE antibody to hepatitis associated antigen.
~here~ore, the direct test lnvolves a much larg~r percentage change in ~ilm thickness, and accordingly greater contrast is observable.
The concentration o~ hepatitis associated antigen in the blood of a person having been exposed to the disease ~5 is a function of time. The concentration of hepatitis associated antigen is quite high during the clinical and ' immediately preclinical phases of hepatitis. In the ~ar post-clinical phases, h~wever, the concentration of hepatitis associated antigen in the person's blood is very low. Both conditlons are medically of interest.
-~7-~479~ RD-S719 I)ctection of the antigen cluring the clinical and immediately ~c~Iinical ~h~Iscs is or v.lIuc ror thc pur~oses of diagnosis, L)ctcction or the alItigcn in thc far post-cIinical phase is of value for scrccn;ng thc hlood o-f prospective blood donors. As a practical matter the simplcr, more sensitive dircct test is preferable for diagnostic purposes since it may be readily performed because of the relatively high concentration of hepatitis associated antigen in the blood samplc.
The inhibition test dcscribed for screening purposes has bccn evaluated experimcntally to determIne its value rclativc to other known scrccning tcsts. In these ~ests,~ ;
hepatitis positive poolcd serum was diluted with known lIel;ltitis negative serum. IJsing the inhibition procedure ~cscril~c(l ~Ihove, a dilution o~ onc part he~atitis positive `
poolc~ serum to 32 parts known hepatitis negative serum `~
resultcd in rellable detection over a one-hour period. IJsing a dilution of one part hepatitis positive pooled serum to ;
3000 parts known negative serum, reliable detection was ohtained over a twenty-hour period. These results will be ..
sccn to hc vcry similar in sensitivity and time consumed, rcs~cctivc]y, to tho ~rescntly known counterelectrophoresis proccdure aIld radioimmIlnoassay procedurc. A screening procedure in accordance with this invention therefore is seen to provide results comparable to the best obtainable with pri~or art procedures, with the additional advantage of obtaining the results through a significantly less~expensive procedure.
The use of a gold coated substrate in preference to a metal globule coated substrate in the aforedescrlbed ~0479~8 RD-5719 hepatitis diagnostic procedure is preferred because hepatitis assoclated an~igen molecules are approximately 210 A in diameter. Such film th~cknesses are well within the capability of gold coa~ed substrates to provlde sensitive indication, but are too thick for high sensitivity of indication using the metal globule coated substrate.
FIG, 4 is a mechanical schematic diagram of apparatus in accordance with another embodiment of thi~
invention for concentrating and purifying proteins and antibodies. It will aid in under~tanding the embodiment of FIG. 4 to realize at the outset that in operation the ;
FIG. 4 embodiment is a modification o~ the FIG. 3 embodi-ment just discussed. In FIG. 4, a continuous flexlble belt of s~bstrate material 51 which i8 coated with a mono-molecular l~yer of protein which specifically reacts with the protein to be concentrated and pur~fied is first introduced in~o a con~ainer 52 which contains a quantity o~ liquid 53 which includes the protein to be puriied.
In~mological complexing between the two specifically ~0 reacting proteins takes place in container S2 and then sub~trate belt 51, n~w containing a bi-molecular prote~n layer, proceeds to container 58 containing a wea~ acid solution 59 which severs the immunological bond between the two specifically reacting proteins thereby coll~cting the molecules of the second protein layer in solution 59.
Upon exiting con~a~ner S8, there~ore, substrate 51 a~ain has thereon only the original monomolecular protein layer.
Substrate 51 is ~hen returned to container 52 to again pick up a monomolecular layer of the protein to be collected which is again stripped off ln container 58.

~4791~ RD-5719 Belt 51 is driven through solutions 53 and 59 by capstans 60 and 63 operating cooperatively with respectively pinch rollers 61 and 62. Capstans 60 and 63 may be driven by any suitable means which may conveniently be small electric motors coupled ,o capstans 60 and 63 by speed reducing ~ ;
gear ~rains. Because the immunological reaction is a ~uch slower reaction than the acid bond-severing reaction, it is `
desirable for efficiency that coated substrate belt 51 be ;
in contact with solution 53 containing the protein to be ~ ~`
collected for a much greater time than it is in contact ~
with weak acid solution 59. Accordingly, it is desirable ;
that container 52 be substantially larger than container 58 and that a plurality of upper and lower idler rollers 54 ;
and 55, respectively, be provided for causing multiple passage of belt 51 through solution 53. This provides for a greater len~th of belt 51 being in contact with solution 53 at any given time and therefore for any given segment oP bel~ 51 being e~posed to solution 53 for a greater perlod oE time. On the other hand, a single passage of bel~ 51 through acid solution 59 is quite sufficient to ~rip the second protein layer there~rom. Accordingly, a pair of upper idler rollers 56 and a single idler roller S7 ~ -~
are provided for guiding belt 51 through acid solution 59.
A plurality of idler rollers 64 are provided as required to mechanically support belt 51 during its passage between containers 52 and 58. Containers 52 and 58 are preferably provided with agitating means 65 and 66, respectively, which fluid-sealingly penetrate the walls thereof for agitating solutions 53 and 59 to insure the refreshment o~
3n the solutions in contact wîth belt 51. Container 52 is~
-30- ~ ~;

~47~18 RD-5719 provided with drain pipe 67 controlled by valve 68 for draining solution 53 therefrom when the concentration of the desired protein therein has been decreased to the point at which efficient collection thereof is no longer possible. A fresh sample of solution 53 may then be added to container 53 through the open upper end thereof.
Container 58 is provided with drain pipe 69 controlled by valve 70 for draining off the desired solution of purified protein in weak acid when the desired concentra-tion has been reached. A fresh charge of the acid may similarly be added to container 58 through the open upper end thereof. Since as disclosed above with reference to ; `
FIG. 3, substrate belt 51 may be coated with any desired protein, be it antigen or antibody, the method and lS apparatus o FIG. 4 is useful for collecting a purified ~ ;
concentration of any arbitrary protein desired so long as an immunological reaction involving the desired protein exists. Additionally, by slightly modifying its operation, the apparatus of E'IG. 4 may be used to selectlvely remove a speci-Eic undesired protein from a mixture such as a serum. To accomplish this, the apparatus of FIG, 4 i~
simply run for an extended period of time without refresh-ment of solution 53 and with periodic refreshment as may be required of solution 59. In this case, the product obtained at drain pipe 67 is serum 53 from which the undesired protein has been eliminated to any required degree depend-ing only on the time of operation of the system.
Whi~e this invention has been described with .
reference to particular embodiments and examples 9 other modifications and variations will occur to those skilled , ., ~0479~8 RD-5719 : ;

in the art, in view of the above teachings. Accordingly, it should be understood that within the scope of the :
appended claims, the invention may be practiced otherwise than is specifically described.

'~, ~'.".;,'~

Claims (13)

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

1. A diagnostic method for determining the presence or absence of a specific antibody or antigen in a biological sample comprising the steps of:
depositing a metal onto a substrate material;
contacting said substrate material with an aqueous medium containing either a corresponding specifically reacting antigen to said specific antibody or a corresponding specifically reacting antibody to said specific antigen, -to coat all or part of said substrate material with a mono-molecular layer of said specifically reacting antigen or antibody;
immersing said coated substrate material in said sample; and examining said coated substrate material to deter-mine whether said substrate has a bimolecular or mono-molecular layer adhering thereto.

2. The method of Claim 1 wherein said step of depositing a metal comprises evaporating a metal having high atomic mobility and poor surface wetting characteristics as metal globules onto a light transmissive substrate material.

3. The method of claim 1 to determine the absence or presence of a specific antigen wherein the aqueous medium contains a corresponding specifically reacting antibody to said specific antigen.

4. The method of claim 1 wherein said step of depositing a metal comprises coating said substrate material with a metal film.

5. The method of claim 4 wherein the substrate material is slide like and also includes a metal oxide layer intermediate the metal of the slide and said monomolecular layer.

6. The method of claim 5 wherein said metal is titanium and said metal oxide is an oxide of titanium.

7. The method of claim 1 wherein said step of depositing a metal comprises evaporating a metal selected from the group consisting of indium, gold, silver, tin and lead, as metal globules onto a light transmissive substrate.

8. The method of claim 7 wherein said light trans-missive substrate is selecced from the group consisting of glass, plastic, fused silica, mica and quartz.

9. The method of claim 7 wherein said metal is indium and said examining step comprises visually examining said substrate and said determination is made by distin-guishing between different shades of brown.

10. The method of claim 1 wherein said step of depositing a metal comprises coating said substrate with a thin layer of gold.

11. The method of claim 10 wherein said examining step comprises visually examing said substrate and said determination is made by reflectivity measurement.

12. The method of claim 10 wherein another metal is placed between said gold layer and said substrate.

13. The method of claim 12 wherein said other metal is indium.