US 3770380 A
An article, useful for detecting and assaying immune-adherence-reaction agglutinates, which article includes a component which (i) is a light-transparent base material coated with a dried film of diluted calf serum, (ii) has on top of said coating an antigen-containing overcoating, and (iii) is adapted to function in assembled arrangement in said article to provide a test cell of which a surface is said overcoating; and an assay method employing said article which method includes (a) contacting a liquid which contains specific antibody-coated indicator particles with said overcoating to form agglutinates at the interface of the liquid and overcoating, and (b) then detecting and determining the agglutinates formed at the interface.
Description (OCR text may contain errors)
United States Patent [1 1- Smith Nov. 6, B973 [5 ARTICLE AND METHOD FOR MULTIPLE 3,415,361 12/1968 Chambliss et al 424 12 x IMMUNE ADHERENCE ASSAY  inventor: James E. Smith, Camillus, NY. Primary Exami" JSePh Scovmnek Attorney-Edward J. Kelly et al.  Assignee: The United States of America as represented by the Secretary of the Army, Washington, DC.  ABSTRACT  Filed: Apr. 19 1971 An article, useful for detecting and assaying immuneadherence-reaction agglutinates, which article includes l l pp 135,048 a component which (i) is a light-transparent base material coated with a dried film of diluted calf serum, (ii) s Cl. n B R has on [0p Of said coating an antigen-containing 0V6!- 358/39 coating, and (iii) is adapted to function in assembled  Int. Cl. G01 n33/l6 arrangememlin Said article to Provide a test of  Field of Search 23/230 B 253 T? which a Surface is Said and an assay 23/253 R 253 Us 259 424/11 12 method employing said article which method includes 3523/36 6 4 (a) contacting a liquid which contains specific antibody-coated indicator particles with said overcoating  References Cited to form agglutinates at the interface of the liquid and overcoating, and (b) then detecting and determining UNITED STATES PATENTS the agglutinates formed at the interface. 3,502,437 3/1970 Mass 23/253 TP 3,666,421 5/1972 Price 23/253 TP 6 Claims, 12 Drawing Figures l/ V 23 I PATENIEDuov 5191s 3; 7 70,300
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JAMES E. SMITH INVENTOR 'BY 21 MW PAM ATTORNEYS PAIENTEDnuv 61975 3.770.380
SHEET 2 OF 3 Fig. 9
JAMES E. SMITH INVENTOR BY img MM ATTORNEYS Pmmmuv ems 3770.380 SHEET 38? 3 Fig. IO Fig. i I
- JAMES E. SMITH INVENTOR BY 21 1 NM ATTORNEYS ARTICLE AND METHOD FOR MULTIPLE IMMUNE ADHERENCE ASSAY The invention herein described was made in the course of or under-a contract with the Department of the Army, United States of America.
This invention relates to the biomedical laboratory art for microbial and serological assay of antigenantibody reactions and also to detection and assay of immune-adherence-reaction agglutinates. More particularly, the invention concerns an article and a method for multiple immune adherence assay. The article is of a small size and generally adapted for placement on the stage of a light microscope. It comprises components adapted for disassembly and re-assembly to provide a plurality of nonconnected cells with access to each cell possible; in its disassembled arrangement a component thereof, which is a light-transparent solid with a surface having a coating of dried calf serum thereon, is receptive to an overcoating with an antigen-containing overcoating; and in its assembled arrangement the antigencontaining overcoating functions as a cell surface. The method involves bringing a liquid which contains specific antibodycoated indicator particles into contact with the antigen-containing overcoated surface of a cell so that .immune-adherence-reaction agglutinates form at the interface of the liquid and overcoating, and then subsequentlydetermining the amount of formed agglutinates at the interface.
ta i mun hsrsns was winedipfiii by R. A. Nelson and is sometimes used synonymously with serological adhesion. In general use-the term has come to mean the specific agglutination of erythrocytes by micro-organisms which have been sensitized with homologous antibody and complement. The latter substance is not always required, however, and there has been some confusion as to the limits of this definition. Nelson and Nelson (Yale J. Biol. Med., 31, 185-200, 201-212, 1959) and D. S. Nelson (Adv. lmmunol, 3, 131-180, 1963) have attempted to distinguish immune adherence from'other adherence and hemagglutination reactions such as acid adhesion, complementdependent mixed aggregation, conglutination etc. Lamana (Bact. Rev., 21, 3045, 1957) has reviewed the history of adhesion of extraneous microscopic particles to a wide variety of particulate antigens in the presence of complement and antibody.
Microbial and serological assays based on immune adherence have proved to be sensitive. As little as 0.005 to 0.0] ug of antibody can be detected (Nelson and Mayer, J. Exptl. Med., 89,,369-393, 1949). Taveme (Brit. J. Exptl. PathoL, 38, 377-384, 1957) used immune adherence to titer S labeled T2 phage. She was able to detect 10 PFU by HA patterns and observed that after light centrifugation, the S label was associated with the pellet. Plant viruses such as cauliflower mosaic virus have been detected by D. S. Nelson (supra 1963) using either crude plant sap or purified virus as the source of antigen. The reviews mentioned above describe a wide variety of soluble antigens (ovalbumin, dextrans, lipopoly-saccharides, toxins, human serum albumin) which have been measured by immune adherence at levels as low as 0.00l0.005 ug.
In view of the foregoing illustrative art teachings, and
adherence reaction of concern herein isconsidered to v be the attaching of indicator particles to antigen sensitized by antibody and to antigen sensitized by antibody and complement. Thus in accord therewith, the words herein of agglutinate and agglutinates are the resulting product and products, respectively, of the immuneadherence reaction and are a bound-together mass and masses, respectively, composed of indicator particles, antigen, and antibody and composed of indicator particles, antigen, antibody, and complement. ln most instances, and in accord with a definition of D. S. Nelson (supra), the indicator particles are untreated primate erythrocytes or nonprimate platelets or other indicator particles that attach by a mechanism of the nature by which human erythrocytes attach to micro-organisms sensitized with antibody and complement.
In past practice of the immune adherence reaction, the agglutinates which formed were frequently detected and assayed by permitting them to clump together and settle out of a liquid or as by turbidimeter measurements thereof while in a dispersed or suspended state. U.S. Pat. No. 2,770,572, Eldon, discloses a blood grouping card or means having thereon small discrete surface areas carrying the dried residue of dextran solution and a serum containing a specific agglutinin, and teaches blood grouping testing through contacting the small discrete surface areas with saline solution and blood and subsequently estimating intensities of the reaction mixture. In U.S. Pat. No. 3,074,853, there are teachings of agglutination formation by successively contacting anti-body-containing and antigencontaining liquids with a dried coating containing carrier particles (e.g., charcoal) of observations thereof for the appearance of agglomeration in the liquid, and of these evaluations with different dilutions of the liquids to obtain an idea of the titer of the liquid being tested. U.S. Pat. No. 3,463,614, Leslie, discloses light transmission measurements being used for detection and assay of agglomeration within a capillary tube of a pulsed fluid mixture of red blood cells and a reagent which may or may not cause agglutination. U.S. Pat. No. 2,666,355, Trumit, includes teachings of a bovine serum albumin coating on top of a light-reflective coating, of contacting'the bovine serum albumin coating with antigen-containing liquid, and of studying progress of the resulting antibody-antigen reaction by means of light reflected from the reflective coating through the reacting field of the bovine serum albumin coating and the antigen-containing liquid in contact therewith.
From the foregoing discussed art, it appears that a major restriction or sensitivity of an immune adherence assay is the manner and means of determining when specific agglutination of the indicator particles occurs and of determining and measuring the amount of agglutinates formed.
Accordingly, it is an object of the invention to provide a useful article and method which will enhance sensitivity and reliability over prior art practices for practices for multiple immune adherence assay. Another object of the invention is to provide an article and method for detecting and assaying the immune adherence reaction to determine the presence of as few as l X 10 antigen particle concentrations. Still another object is to provide a method and apparatus wherein agglutinates forming in an immune adherence reaction are formed and adhere to an interface of an indicatorcontaining liquid and an antigen-containing coating overlying a dried serum coating adhered to a lighttransparent solid. An additional object is to provide a rapid and reliable light-microscopic assay for qualitative and quantitative assay of agglutinates formed in an immune adherence reaction. Still additional objects are to provide a method and apparatus wherein agglutinates formed by an immune adherence reaction will be formed in a two-dimensional display or arrangement on the coated surface of a light-transparent material to permit light-microscopic detection and counting of the formed agglutinates. A further object is to provide a plate-like member for an article useful for detecting and assaying immune-adherence reaction agglutinates, which plate-like member on a flat surface thereof has a light-transparent normal blood serum coating with an antigen-containing overcoating thereover.
Advantageously the method and article of the invention have a capability and sensitivity of detecting and measuring amounts of antigen as low as 1 X '6 g. and lower to provide a sensitivity of over 1,000 times greater than known heretofore published methods.
There exists a built-in amplifying system in the method and article of the invention in that more than one antibody-coated indicator particle attaches to the dried serum film as a result of repeated contacts with the antigen on it. This action apparently provides at least a part of the observed significantly increased sensitivity. Statistical data substantiate the feasibility of the methodology and the practicability of the article of the invention in providing this advantage. The article and method of the invention permit not only qualitative analyses so as to identify a particular antigen or antibody, but also enable assay of the concentration of the antigen or antibody. Additionally the invention is useful for detecting and assaying a wide variety of antigens including not only animal and human viruses, but also free viruses as well as virus in lyophilized preparations of tissue cultures, mouse brain homogenates, and serum proteins.
All of the foregoing objects and advantages as well as some additional ones will be readily apparent from the description of the invention which follows and from the drawings thereof, of which:
FIG. 1 is a perspective view of a broken-away fragment portion of a component of an embodiment of an article of the invention, which-component is a means forming surfaces of a plurality of recessed cells with each cell having a top-opening access thereto;
FIG. 2 is a perspective view of a broken-away fragment portion of another component of the article, which component is a means serving as a cover for closing the top-opening access of the recessed cells illustrated in the component of FIG. 1;
FIG. 3 is a cross-sectional view on line 33 of FIG. 1 illustrating a fragment portion of the FIG. 1 component forming the surfaces of one of its recessedcells before placement of a dried serum on the illustrated cells floor;
FIG. 4 is a cross-sectional view on line 3-3 of FIG. 1 illustrating a fragment portion of the FIG. 1 component having a light-transparent dried serum coating on the illustrated cells floor;
FIG. 5 is a cross-sectional view on line 33 of FIG. 1 illustrating a fragment portion of the FIG. 1 component having the illustrated cells floor coated with a light-transparent dried serum coating which is overcoated with a light-transparent antigen-containing overcoating;
FIG. 6 is a cross-sectional view on line 33 of FIG. 1 illustrating a fragment portion of the FIG. 1 component in association with a fragment portion of the FIG. 2 component and having the illustrated cells floor bearing a light-transparent dried serum coating overcoated with a light-transparent antigencontaining overcoating and having the illustrated cell filled with a liquid containing homologous antibody-coated indicator particles;
FIG. 7 is an exploded perspective view of another embodiment of an article of the invention;
FIG. 8 is a side plan view in assembled arrangement of the article illustrated in FIG. 7;
FIG. 9 is an end plan view in assembled arrangement of the article illustrated in FIG. 7;
FIG. 10 is a side plan view of a cover plate bearing on a Hat surface thereof a coating containing antibody;
FIG. 11 is a side plan view of a cover plate bearing on a flat surface thereof a coating containing antibody or antibody and complement and having an antigencontaining overcoating on top thereof; and
FIG. 12 is an exploded perspective view of another embodiment of an article of the invention.
With reference to the drawings, wherein for purposes of clarity and brevity of illustration, the drawing figures are not necessarily drawn to true scale and in accurate proportion:
FIG. 1 illustrates a broken-away portion of a component of an embodiment of an article of the invention. The illustrated component is a plate-like member 20, serving as a means providing a plurality of unconnected recessed open-on-top cells 21 or reaction chambers for carrying forth therein assays for immune-adherencereaction agglutinates. The plate-like member 20 is of a light-transparent material, such as polystyrene or polymethacrylate injection molded in its illustrated configuration, and desirably coated at least on its cells inner surfaces with a very thin coating improving water wettability. U.S. Pat. No. 3,097,070 teaches an illustrative useful adherent coating of between 0.1 and 1.5 microns in thickness of a material selected from the group consisting of silicon oxide, tantalum, aluminum, magnesium fluoride, and aluminum oxide for improving the water-wettability of inner surfaces of containers employed as laboratory ware. The individual recessed cells 21 illustrated in plate-like member 20 are dis posed in rows and columns each provided with a separate letter identification to facilitate ready identification of various individual cells 21. Member 20 may be provided, as desired, with a larger or smaller number of recessed cells 21 and other manner of identification of individual cells than illustrated, and also may be of any convenient size with it to be noted that a 3% inch wide by 5 inches long plate-like member 20 is capable of providing as high as about or more individual cells 21. Plate-like member 20 is employed in the article of the invention in association with a means serving as a cover closing the top-opening accesses to its individual cells 21. In FIG. 2 there is illustrated a unitary cover means, which is a several mil thick sheet 22 of a lighttransparent material. In the article, sheet 22 is in association with member 20 as by placing on top thereof so as to cover cells 21, and held in association by a conventional clamp (not illustrated) or like holding means.
FIGS. 3 through 6 are cross-sectional views taken on line 3-3 of FIG. 1 and illustrate with exaggerated detail an individual cell 21 or reaction chamber and its environs. The illustrated recessed cell 21 is open on top and comprises a cylindrical side wall 23 and a fiat floor 24. In FIG. 4 the cells flat floor 24 is shown with a light-transparent dried serum coating 25 thereon, and in FIG. 5 the cells flat floor 24 is shown having thereon a light-transparent dried serum coating 25 which has been overcoated with a light-transparent antigencontaining overcoating 26. In FIG. 6, sheet 22 is shown in association with plate-like member to provide a roof 27 for cell 21. In FIG. 6 the flat floor 24 has thereon a light-transparent dried serum coating which has been overcoated with a light-transparent antigen-containing overcoating 26. The cell 21 in FIG. 6 also is illustrated as filled with a liquid 28 containing homologous-antibody-coated particles with some immune-adherence-reaction agglutinates 29 shown on an enlarged scale as formed at the interface of the liquid 28 and the antigen-containing overcoating 26 and as adhering to the antigen-containing overcoating 26.
FIGS. 7, 8, and 9 illustrate another embodiment of an article, generally designated 30, of the invention. Article 30 comprises: an elongated flat bottom plate-31; a notched elongated flat plate 32 provided along both side edges with a plurality of notches 33; a plurality of cover plates 34 with one each cover plate 34 provided for each notch 33; a second notched elongated flat top plate 35, also provided with a corresponding plurality of notches 36 along both side edges, and with plates and 32 being substantially alike in configuration; and three bolts 37 and three wing nuts 38-for bolts 37. In assembled arrangement each bolt 37 passes through a hole 39 in plate 32 and through a hole 40 in plate 35 with a wing nut 38 assembled thereon and tightened to hold plate 32 against bottom plate 31 and cover plate 34 between plates 32 and 35 with each cover plate 34 covering and overlapping a notch 33 and underlying a notch 36. In assembled arrangement, the combination of plates 31 and 32 and cover plates' 34 provide a plurality of cells 41 or test chambers corresponding to the number of notches 33 provided in plate 32 with access possible to each cell 41 from its'open side at the side of plate. In an illustrative-useful example of the article illustrated in FIGS. 7, 8, and 9, the plates 31, 32, and 35 are approximately '7 cm'wide by 20.3 cm long with plate 31 being 3 mm. thick, plate 32 beingl mm.thick, and plate 35 being 2 mm. thick, and are of a polyacrylate resinous solid material, such as Lucite. The notches 33 and 36 in plates 32 and 35, respectively, approximate a 16 mm, width at the edge of the plates and extend substantially at right angles a distance of approximately 18 mm. inwardly from the side edges of these plates. The cover plates 34 approximate 25 X 25 mm. and are 1 mm. thick glass. 1
In FIGS. 10 and 11 are illustrated a cover plate 34 with the cover plate 34 illustrated in FIG. 10 shown with a light-transparent dried serum coating 45 on a flat surface thereof. In FIG. 11, the illustrated cover plate 34 is shown with a flat surface covered with a lighttransparent dried serum coating 45 having a lighttransparent antigen-containing overcoating 46 thereover.
With reference to FIG. 12, it illustrates another embodiment of an article, generally designated 50, of the invention. Article 50 comprises: an elongated base plate means 51 provided with a plurality of open-on-aside and open-on-top recessed cells 52 or reaction chambers located along the uppermost of both side edges of base plate means 51; an elongated cover plate 53; and two elongated flexible channel-like clamping means 54 and 55 for holding the base plate means 51 and elongated cover plate 53 clamped together when in assembled arrangement. Each of clamping means 54 and 55 are provided with a plurality of channels 56 or holes therethrough so located that in assembled arrangement at least one channel 56 lies in correspondence with the open side of each cell 52 making access thereto possible. In an illustrative useful example of the article 50 of the invention in assembled arrangement, its overall size approximated 20 cm. long by 7 cm. wide by about 6 cm. thick, each cell 52 approximated 18 X 16 X 1 mm., base plate means 51 is of a molded polyacrylate resinous solid material, cover plate 53 is of 2 mm. thick glass, and channel-like clamping means 54 and 55 of an extruded material, such as polystyrene, metal, or the like, having channels 56 punched or drilled therethrough.
A prerequisite for the article of the invention is that the articles component, which provides a flat portion thereof having thereon a light-transparent dried serum coating, be of a light-transparent solid material. This light-transparent requirement exists in that agglutinates to be assayed upon their subsequent formation at the interface of a liquid containing homologous-antibodycoated indicator particles and a light-transparent antigen-containing overcoating on the light-transparent dried serum coating are detected and assayed by a light-detection means with light passed through such articles component. In the instance of the embodiment of the article illustrated in FIGS. 1 through 6, having recessed cells 21 in member 20 with the requisite coatings on flat floor 24, member 20 is of a lighttransparent solid material. Alternatively and in notillustrated modification, if such requisite coatings are placed on a flat surface of sheet 22 so that sheet 22 in association with member 20 provides a roof 27 for cell 21 with these coatings then serving as the cells ceiling, then in such an instance sheet 22 necessarily is of a light-transparent solid material. In the article s embodiments illustrated in the other FIGS. 7-12, cover plates 34 of article 30 and cover "late 53 of article 50 are of a light-transparent solid material. However, inmodifications of these embodiments of the article in which bottom plate 31 provides the floor of cells 41 and base plate 51 provide the floor of cells 52 and these floors have thereon the requisite coatings, then in such in stances plates 31 and 51, respectively, are of a lighttransparent solid material. Glass is most useful and a preferred light-transparent solid material for the articles component which is of a light-transparent solid material. However, light-transparent solid materials, such as polyacrylate, polystyrene, polycarbonate, and polyethyleneterephthalate resins, are useful and may be employed for the articles components. The remainder of the components comprising the article may be of the same material making up the article's component which is of a light-transparent solid material. However, they do not necessarily have to be of a light-transparent material and may be of a material which is only lighttranslucent, or even opaque, so long as the material or materials employed are such that the surfaces thereof which will serve to provide the cells or reaction chambers are biologically indifferent to the immune adherence reaction and materials participating therein and do not deleteriously affect or interfere with the immune adherence reaction to be performed in the cells. Conveniently these components are molded, machined, or otherwise fabricated out of synthetic polymeric resinous materials commonly known as plastics. Conveniently those components of the article which serve to hold the article in an assembled configuration, such as bolts 37 and wing nuts 38, are of metal, for example, stainless steel. Those components can be of almost any construction material having the requisite strength, flexibility, and other physical properties for their particular purpose.
Optionally, and if desired, the articles components may be provided with suitable letters, numbers, or appropriate markings (not illustrated) to facilitate their assembly, disassembly, and reassembly, as required, and to assure providing of a proper assembled arrangement thereof for employment for assay purposes. Additionally where a cover plate, such as cover plate 33 of article 30, serves in the assembled arrangement as a cover plate for more than one cell, optionally such a cover plate may be provided with peripheral boundary markings or the like (not illustrated) outlining discrete surface area portions serving as a wall of the various cells. Such peripheral boundary markings for a cover plate facilitate providing of requisite coatings on the specific discrete surface area portions where they are required. Also the articles components optionally may be provided with suitable markings or other identification, such as illustrated in the component shown in FIG. 1, to permit a ready correlation and convenient identification of each cell and thus avoid confusion as to specific assays performed in the various cells of the article.
Generally the article is so fabricated that each cell is of a small size so that an amount of indicatorcontaining liquid between a fraction of one drop and up to several drops will suffice easily to fill each cell. For most purposes, including quantitative assays, a cell having a flat floor or ceiling surface providing a surface area for the serum coating between 0.3 to 0.8 square inch is more than adequate to permit accurate analysis. Desirably each cell is relatively shallow with a 1 mm. depth being quite useful. Depth of the cell is of considerable importance,'since the total number of indicator particles which impinge on the antigen per unit of time is influenced by the distance which they must settle. For optimum performance the cell depth is between 1 and 2% mm. This permits maximum multiple contacts between antigen andantibody in a short period of time. It also allows easy charging of the cells through a surface wetting and/or capillary phenomena upon placing a small amount of the indicator liquid at an access into the cell.
For commercial sales and merchandising of the article of the invention, the articles components can be in a disassembled or a fully or partially assembled arrangement. In' all arrangements and kits for providing the article, there is included a component of the article, which component is of a light-transparent solid material and serves or is to serve as a means forming the floor or the ceiling of a cell or reaction chamber with the surface of this component, serving as the floor or ceiling as the case may be, being a flat surface having thereon a light-transparent dried serum coating. In these kits or packages, the component including the light-transparent dried serum coating generally is wrapped and/or otherwise protected by suitable packaging or the like to preserve its integrity and aseptic condition and to prevent contamination of its lighttransparent dried serum coating. For usage of the article, the user or analyst overcoats the light-transparent dried serum coating with the appropriate antigencontaining overcoating for the specific analysis to be performed, and then proceeds with the desired assay with or without disassembly and/or reassembly in full or part of the article, as the case may be, depending on the manner in which the article or its components are provided. In other instances of merchandising of the article and for specific assay purposes therewith, the requisite light-transparent component thereof, having a flat surface having thereon a light-transparent dried serum coating, includes on top of the serum coating a light-transparent overcoating of a specific antigencontaining overcoating. In such instances, the user or analyst then can proceed directly with the desired specific assay without any need to overcoat the serum coating with an antigen-containing overcoating before analysis.
In practice of the invention, it is prerequisite that a flat surface area of the cell serving as the floor or the ceiling of the cell have thereon a film-like lighttransparent dried serum coating. This dried film of serum serves as an undercoating and substrate for subsequent placement thereon of an antigen-containing overcoating and also prevents nonspecific adherence of antibody-coated indicator particles. The following is an illustrative example of the preparation of one such calf serum coated cover plate useful as a cover plate component for the article shown in FIGS. 7-11.
New 25 X 25 mm. square by 1 mm. thick glass cover slips or cover plates are placed in racks, cleaned by sonification in a 7X tissue culture detergent for 30 minutes, and then rinsed in flowing tap water and followed by a distilled water rinse. 7X tissue culture detergent (Linbro Chemical Corporation) is understood to be a commercially available liquid cleaner for laboratory glassware which cleaner is substantially nonalkaline and nonacidic, having a pH of about 7.8, and contains about 5 percent of water and an anionic surfaceactive detergent in a solvent therefor. The wet cover slips then are heated in a boiling solution of 0.05 M tris buffer or pH 7.5 in a l N sodium chloride solution, rinsed with a fresh amount of the tris buffer/saline solution, and then drained and dried between layers of lens paper. A useful tris buffer is trishydroxy methylaminemethane hydrochloride. Alternatively, in lieu of the preceeding preparation of cover slips or plates for subsequent coating, one may utilize any of numerous well-known art methods for providing scrupulously clean and sterile appropriately buffered surfaces on the cover slips or plates.
The cover slips are then coated with a film-like lighttransparent coating of normal mammalian serum. This may be accomplished by dipping the clean and sterile cover slips or plates in a 50 percent calf serum, aqueous solution, placing them on an absorbent pad of cheese cloth in a petri dish, and then drying in a forced-air draft infrared oven for about 3 hours at C. Cover slips, coated with calf serum in the manner just described, can be stored at room temperature'(about 20-25C.) and about a 40 percent relative humidity for lengthy periods of time (i.e., longer than several months) and be of utility in the invention. Alternatively, instead of dip coating, one may apply the serum coating by numerous conventional means, such as spraying, knife-blade application, and the like means which provide a uniformly distributed coating of a relatively uniform filmlike thickness, generally in the order of about 0.1 to 1.0 microns or less thick. While calf serum is preferred and highly useful in the article and method of the invention asthe first coating on the cover slips or plates, it is contemplated other serums containing therein serum alpha-2-globulin and/or the complex of serum albumin and alpha-2-globulin also may be utilized as the first coating. Such materials as gelatin, plant lectins, a variety of purified enzymes and serum proteins, as well as serum lipoproteins, serum albumin, gamma globulin, beta globulins, and the like are ineffective substitutes for calf serum in that they are of little to no utility and/or lack storage stability for extended times.
In a similar manner, as just described, one can place a light-transparent dried serum coating on the flat surface of the cover plate 53 utilized in the articles embodiment shown in FIG. 12 and, if desired, place this serum coating over the entire surface or only selected discrete portions of the surface of cover plate 53. For the articles embodiment illustrated in FIGS. l-6, floor 24 of a cell 21 can be provided with a light-transparent dried serum coating by placing thereon a very small amount of diluted aqueous calf serum and then drying the same, or alternatively the serum coating placed on sheet 22 alike the described placement of the serum coating on cover plate 53.
In general, practice of the invention also involves providing an antigen-containing overcoating on top of the light-transparent dried serum coating. This overcoating is readily provided through placing a liquid containing 'a specific virus or other specific antigen on top of the dried serum coating and drying the same thereon. Upon providing of the light-transparent antigen-containing overcoating on the light-transparent dried serum coating for the articles embodiment illustratedin FIGS; 1-6, it is ready for assays to be performed therewith. Following the providing of the lighttransparent antigen-containing overcoating on the light-transparent dried serum coating on the cover slips and plates of the article s embodiments shown in FIGS. 7-l2, these slips and plates are placed in assembled arrangement to provide the article having a multiplicity of cells or reaction chambers.
The article of the invention is placed in a suitable position so that the antigen-containing overcoating provides the floor of the cells and, each cell then is filled with antibody-coated sheep erythrocytes (red blood cells) or a like indicator-containing liquid. In the instance of the article of FIGS. 1-6, filling is through the top opening accesses to the cells before a covering, such as sheet 22, is placed thereon to cover the cells. In the instances of the articles of FIG. 7-12, filling is through the open side of the cells. Filling is by a convenient means, such as by employing a Pasteur pipette or a hypodermic syringe. The filled cells are permitted to remain in the position, where the antigen-containing overcoating serves as the cells floor, so that the red bloodcells or like indicator particles settle on to the antigen-containing overcoating, usually within about 10 to minutes. The article is then inverted or placed in a position so that the antigen-containing overcoating serves as the ceiling of the cells; and permitted to remain in that position so that unattached red blood cells or like indicator particles through gravity move away from the overcoating and settle out in the liquid contained in the cells, usually within 12 to l5 minutes. Then by microscopic light-detection means or the like means there are determined the agglutinates formed of those attached red blood cells or other indicator particles, remaining on the interface of the liquid and the overcoating, which have become attached to antigens in the overcoating sensitized by the antibody or antibody and complement of the undercoating. It has been found that the numbers of red blood cells and other indicator particles attaching to the antigen particles are greater than the number of antigen particles present in the overcoating. This built-in amplification factor in the assay is particularly advantageous and unique to this method of display. The number of agglutinates thus determined is proportional to the number of antigens contained in the overcoating.
The following are illustrative preparations of several indicator-containing liquids useful for practice of the method of the invention.
Illustrative preparations of useful liquids containing erythrocytes:
Procedure A The Bing et al. (Proc. Soc. Exp. Biol. Med., 124, pages l,l66l,170 (1967), procedure may be used. For example, twenty-five percent aqueous glutaraldehyde (Fischer Scientific practical grade) is diluted to a 1 percent concentration. The diluent (PBS) contains 0.15 M Na PO,, pH 8.2; nine volumes of 0.l5 M NaCl; and 5 volumes of distilled water. Packed sheep red blood cells (RBC) are diluted to l-2 percent with the cold 1 percent gluteraldehyde solution and incubated for 30 minutes at 4C. with stirring. The fixed red blood cells are then washed five times with 0.15 M NaCl and five times with distilled water. All red blood cell suspensions are then resuspended to a concentration of 2 X 10 RBC/ml in the 0.15 M phosphatebuffered saline (PBS), pH 7.2.
Procedure B The Stavitsky (J. ImmunoL, 72, page 360 (1954), procedure also may be used. For example, glutaraldehyde fixed red blood cells are tanned with an equal volume of buffered 120,000 tannic acid (J. & Baker Co., reagent grade) for 10 minutes at 37C. The tanned cells then are washed and resuspended to original volume in phosphate-buffered saline, pH 6.4. Procedure C Tanned red blood cells also may be coated with protein. For example, tanned red blood cells are coated with gamma globulin by mixing one volume of phosphate-buffered saline, pH 6.4, and 2 volumes of the tanned red blood cell suspension. The mixture is in.
cubated at room temperature (about 25C.) for 15 minutes and then washed with phosphate-buffered saline, pH 7.2, plus 1:100 normal rabbit serum. All sera are heated at 56C. for 30 minutes prior to use and tested for nonspecific agglutins.
As should be readily apparent, the invention permits that the various individual cells in the one article may be used all for the same assay, some cells for one assay with other cells used for different assays, and even control or standard assays to be performed in some cells of the article Thus, different indicator-containing liquids may be used in the various different cells, as desired and depending on the particular assay to be carried forth in that cell. Likewise, one particular virus or antigen can be assayed in one and/or several cells, a different dilution thereof or another specific antigen in still other cells, etc. as well as a control or standard assay in other cells, such as by employing an overcoating devoid of antigen or containing a specified amount of a known antigen, and all sorts of assay combinations of the preceeding, as appropriate and desired. In the instance of employing a multiplicity of cover slips or plates, different antigen-containing overcoatings, which differ in the amount and/or particular kind of antigen, may be placed on those particular dried-serumcoated cover slips or plates utilized with certain cells; and in the instance of a cover plate functioning to cover several or all the cells, such different antigencontaining overcoatings may be placed only on the specific discrete surface areas of the cover plate functioning to cover one only and/or several specified particular cells, as desired.
The invention will be further understood from the specific illustrative examples thereof which follow.
This example employs the article illustrated in FIGS. 1-6. The flat floor 24 of each cell 21, if not already provided with a light-transparent dried serum coating 25, is provided with serum coating 25 as follows. A 0.05 mil. of calf serum, diluted 1:10 in 0.15 M phosphatebuffered saline (PBS), is pipetted into each cell 21, and with the fiat floors 24 level dried thereon for 2 hours at 90C. If desired not to be used immediately, member 20 providing a plurality of cells 21 having their fiat floors 24 coated with light-transparent dried serum coating 25, can be stored in a vacuum desiccator, or otherwise packaged, wrapped, or stored, to maintain aseptic condition, and kept for extended time of weeks and even months and remain useful. Then about a 0.025 to 0.2 ml sample of a virus to be assayed, after being diluted suitably with a commercial tryptose phosphate broth (Difco), is placed in a cell 21 and dried on top of serum coating 25 to provide the antigencontaining overcoating 26 thereon. This commercial tryptose phosphate broth (TPB) is described on pages l-l0l of the Difco Manual, 9th Edition, 1967, of Difco Laboratories, Detroit, Mich. Drying can be at about 37C. overnight, or alternatively at 80C. for 1.5 hours. Similarly other cells 21 are provided with their respective antigen-containing overcoating 26 on top of their serum coating 25, with the particular virus or antigen employed in a cells specific overcoating being that determined or selected by the particular assay to be performed in the cell 21 in which it is placed. Member 20, now provided with cells 21 having their flat floors 24 coated with dried serum coating 25 overcoated with antigen-containing overcoating 26, also can be stored or packaged and utilized for assays later, orr used immediately, as desired. Assays then are performed by pipetting, or otherwise introducing into cells 21, a liquid 28 of 0.05 ml. of sheep red blood cells (SRBC) which had been fixed in glutaraldehyde, tanned, coated with antiviral gamma globulum and diluted to 0.625 percent packed volume with tryptose phosphate broth (Difco) containing 2 percent normal rabbit serum. At this time a prefabricated sheet 22 is placed and held in association with member so as to serve as a cover closing the top-opening accesses of cells 21. Alternatively, in lieu of utilizing a prefabricated sheet 22, an individual cover sheet is fabricated in situ for each cell 21 so as to serve as cover closing the cells top-opening access.
This in-situ cover formation is accomplished by placing one drop of a 4 percent collodion solution in amyl acetate in each cell 21. Following the closing of the cells top-opening accesses, the introduced liquid 28 is permitted to stand for 12 minutes in contact with overcoating 26 so that the SRBC are able to settle out on nunrnnofinn "K In "on incfanrn nf' nfilivinn an in c;h|
fabricated cover being formed from the collodion solution for closing the cell's top-opening accesses, the amyl acetate evaporates and a collodion film deposits on the surface of the liquid 28 in the cell 21 to provide the cover sheet during the time in which the SRBC are permitted to settle out on overcoating 26. The article of member 20 in association with sheet 22 or its collodion-film covers as the case may be, then is inverted so that floors 24 of cells 21 now are the ceilings of cells 21. After a short time in the inverted position, generally 3 to l5 minutes, SRBC settled out on overcoating 26 and failing to attach thereto will fall away from overcoating 26 sufficiently to be out of the focal plane of the interface of liquid 28 and overcoating 26. At this time a photographic record is made of the interface, generally at lOOX total magnification by photographing through the floors 24 of member 20. After the unattached SRBC have settled away from the focal plane of the interface, the number of attached SRBC remains relatively constant so long as the member 21 is not heated by a microscope lamp, nearby burner or the like. A fluorescent or a 7 V. incandescent lamp is a preferred illumination source for making a photographic record or for a preliminary scanning. A preliminary scanning of the interface of various cells usually makes it unnecessary to photograph interfacesof all cells utilized in a particular dilution series of assays.
Advantageously this example permits an accomplishing of one hundred or more assays in the same time that 12 assays are accomplished through assembling and employing the article 30 as described in the next example. Additionally the article and its use, as described in this example, is readily adapted for use in conjunction with automatic scanning-counting systems for counting particles and thus also readily adaptable for automated assay procedures and apparatus. With the article being furnished with the cells floors coated with lighttransparent dried serum coating, or with the cells floors coated with light-transparent serum coated overcoated with the requisite antigen-containing overcoating, or with prepackaged materials for providing the overcoating and/or serum coating, the article and assay procedure are so noncomplicated and straight forward that it is suitable for use in an average clinical laboratory or physician's office for such purposes as testing for measles antibody and venereal disease screening.
Other advantages of the article employed in this example include: an ability to provide the article with a very large number of cells 21 for assay purposes and thus enable assay of a large number of dilution, treatments, and the like; and an ability to provide the article fabricated of relatively low-cost materials so that it is a disposable item withno need to clean and then recoat for additional assays to economize on assay costs.
The article employed in this example in common with all articles of the invention include such unique features and provide uniqueness in assay methodology through: cell depth being such that the employed depth for the liquid containing the antibody-coated indicator particles approximates about 2% mm. to provide settling times closely coinciding with release of dried antigen from the overcoating; nonspecific adherence to the overcoating being avoided through employing a dried serum film under the overcoating; multiple contacts occurring between antibody-coated erythrocytes and antigens to provide a built-in amplification of the immune adherence reaction; and an antigen-containing overcoating provided by application of the antigen in a tryptose phosphate broth and which after drying and upon gen at the critical moment when the erythrocytes settle out on the overcoating. g
In another example, the article 30 illustrated in FIGS.
7-l l is employed. A thin even film of a laboratory stop-' cock grease (Lubrisealf Arthur H. Thomas Co.) is spread by means of a soft roller or like applicator means onto the top and bottom sides of notched elongated flat plate 32. Lubriseal is understood to be a tacky, smooth textured, odorless, stable, petroleumbase grease that is free from vegetable and mineral oil and is practically insoluble in water. Cover plates 34, bearing a coating of a thin light-transparent film of dried calf serum applied as described earlier, then are pressed into the stopcock-grease film on the top of plate 32, with each calf serum-coated plate positioned so as to cover the top of a notch 33 and to serve eventually as the top member of a cell 31 upon the article being placed in assembled arrangement. Plate 32 having calf-serum coated cover plates 34 stuck thereto by the stopcock-grease film then is inverted with the cover plates resting on a level surface. About 0.4 ml. of a liquid'containing the specific virus or other antigen to be assayed is applied in each notch 33 to each dried-calfserum coating of cover plates 34 and air dried thereon. Drying can be accelerated by flowing dry air at about 37C. over the applied liquid. The amount of this applied liquid can be reduced to 0.06 ml., if necessary or desired, and several aliquots thereof can be applied and air dried sequentially. Alternatively the virus or other antigen, if desired may be printed or otherwise applied as with pads, stamps, swabs, and the like devices to provide the overcoating. The cover slips should be maintained relatively level during drying, such as by a suitable rack or like means, to ensure a uniform distribution of the virus or other antigen particles in the dried overcoating containing the same. The plate 32, having antigen-overcoated cover plates adhered thereto now is inverted and assembled in aligned conjunction with bottom plate 31 and top plate 35 into the articles assembled arrangement with bolts 37 passing through holes 39 and by means of wing nuts 38 tight ened thereon holding-the article 30 in its assembled arrangement. In the assembled arrangement of article 30 a plurality of cells 31 are provided, each of which is a substantially water-tight chamber as the result of the grease seals between the assembled bottom plate 31 and plate 32, and the cover plates 34 except for an ac cess into each cell 31 from the side of the article. The article in its assembled arrangement now is placed in a rack or other suitable support means, so that the overcoated cover plates serve as the floors of the individual cells 31. Each cell then is filled, as by means of a Pasteur pipette of hypodermic syringe with an indicatorcontaining liquid, and in this example with one of the aforedescribed liquids containing antibody-coated sheep erythrocytes in a concentration of l X l0 red blood cells/ml. In this specific example precisely 15 minutes is allowed for the red blood cells to settle onto the virus-calf serum film coatings. Longer settling times are useful, but are unnecessary, with it desirable that all assays for comparison purposes utilize a preselected finite settling time. The article then is inverted in a smooth motion and stored upright in a rack for 12 minutes to allow unattached red blood cells to settle away from the coverslip. Here too longer times are useful, but unnecessary, with it also desirable to utilize the same preselected finite time for all assays. The undersurface of the coverslip is examined with the high dry objective of a light microscope, per.43X field, and the number of formed agglutinates from the adhered red blood cells are determined and recorded. Those red blood cells which adhere represent occurrence of antigen-antibody reactions at the interface of the overcoated cover plate and the liquid containing the settled excess red blood cells.
The light microscopic determination of the agglutinates formed at the interface of the overcoating on the cover plate and the liquid containing a mat of settledout surplus red blood cells may be by any conventional light-microscopic means known to the art. A convenient procedure is to place the apparatus at this step of the assay on the stage of a light-microscope providing a suitable magnification, generally a magnification of at least times and up to several hundredfold or more, and then adjust the microscope to focus on the field of the interface and the agglutinates formed thereon. Conveniently in this determination one also employs a standardized counting geometry and procedure, ruled guides, preselected sampling and counting positions, discrete areas of counting and the like, all of which are well known, to arrive at an accurate determination of the amount of agglutinates resulting in the particular immune-adherence assay carried forth in a particular cell.
Where terminology of light microscope, lightmicroscopic means, light-detection means" have been employed, it is intended to include and encompass by that terminology other useful techniques and determination means which may utilize alight beam only in part for the detection and determination of the formed agglutinates on the interface. For example, one may employ what is called a flying spot scanner or like means, wherein. a light beam is used to scan a field or surface to detect particles thereon having lightreflection, -transmittance, and/or -absorption characteristics differing from the surface itself or the material underlying that surface and wherein thelight signals from such scanning then are transmitted, transposed, and possibly amplified to provide visual and/or electrical signal read outs thereof.
By the procedure just described in the foregoing specific examples, immune-adherence assays are performed employing overcoatings containing as few as l X 10 virus particles or other antigen in the applied overcoating composition of the viruses and other antigens tabulated in the following Table I. In each of these assays the viruses were detectable in these low concentrations, and accurate determinations were possible of the quantity of each being assayed by microscopic evaluation of the agglutinates formed at the interface of the overcoating and the indicator-containing liquid.
TABLEI Virus or Antigen Strain West Nile Egypt lOl Western equine encephalitis California Yellow fever 17D Sindbis All-339 Influenza A/PR8/33 Influenza A/Japan 305/57 Influenza B/Lee/40 Influenza B/Great Lakes/S4 Newcastle disease California Hemmagglutinating virus Japan Sendai Poliovirus Type 1 Sabin Poliovirus Type 2 Sabin Poliovirus Type 3 Sabin Herpes simplex SOK Herpes simplex HF 378 Herpes simplex HP 490 Herpes simplex L2 Vesicular stomatitis Indiana Coliphage Tl Coliphage T2 Coliphage T3 Coliphage T4 positions in Row III are counted employing a 43X objective. Occasionally the five positions in another row, for example Row II, were counted. Employment of a ruled grid in the eyepiece of the microscope aids and facilitates making the counts and especially counts in excess of 150 agglutinates per field.
Tables 11, III, and IV which follow present results of counts typically obtained and illustrative of a multitude of evaluations made.
TABLE Il.-ASSAYS OF INFLUENZA A/PRS TPB+0.1 HAU PR8K TPB* Cell Number- Cell Number- Sampling position 1 2 3 5 6 1 2 3 5 6 a 172 145 168 132 157 101 16 5 8 31 24 9 196 I44 166 141 128 136 l2 24 33 ll 8 200 I89 169 113 153 157 7 ll 31 l9 l3 4 190 173 140 153 120 124 4 6 l2 l2 8 214 161 125 152 I 157 96 5 l2 15 21 14 ll Code:
TPB= tryptosephosphate broth. H A U hemagluttinating unit.
To illustrate the sensitivity and reliability of the assay TABLE llL-SRBC SENSITIZED WITH method of the invention, a quantitative evaluation was ANTI'TZ GAMMA GLOBUUN made with statistical data amassed to substantiate feasia. Cells contain l x 10 T2 PFU bility of the methodology and practicality of the article 35 Cell Numberof the invention. In this evaluation, the employed antlgen in the overcoating was phage T2 and its concentra- Sampli Position 1 2 3 4 5 tion was 1 X 1 0 plaque-forming units/cell; the under- I 47 100 98 105 78 coating was dried calf serum; and the SRBC-mdrcator- I76 87 90 86 103 containing liquid was in 1 percent tryptose phosphate 3. g; broth (Difco) in phosphate-buffered saline, pH 7.2. 126 3 79 97 35 The counting procedure to determine the amount of A '42 9 formed agglutinates was standardized with a standa- Yerage 98 98 87 rized counting geometry preselected to make available 2?: 1048, r'f f is mean number.
.v l w ere .s is standard deviation of mean. 25 sampling positions on each cover slip or plate. Each *p u -f rming unit. sampling position or field covered 0.96 mm. and was located 1 mm. from another field. The 25 were located five each-to-each of five parallel rows, Rows I, II, III, IV, and V, and arranged to provide five parallel columns a, b, c, d, and e, with five positions falling in each column. The first column a was located 5 mm. in from the left hand edge of the cell wall and the last row V b. (ells Contain T1B* was located 7.5 mm. in from the open edge of the cell. Cell Number Ruled guides were affixed to the microscope stage to Sampling permit rapid positioning onto a particular row for scan- P0Sltlon l 2 3 4 5 ning with the objective. To count a series of fields a to 23 47 34 53 33 e m a particular row, the operator positions the article 22 51 48 55 58 36 of the invention against the appropriate row guide and a: 2. Z2 1, moves the artlcle until the left hand edge of the cell is 2| 4! 62 61 92 53 centered in his objective. The operator then moves the 60 Avcp article 5 mm. to the left and counts field a, for field b age 3| 40 54 61 70 he moves it 1 mm. further to the of a and counts field b, for field 0 he moves it 1 mm. further to the left and so on to field e. Customarily only five specific sampling TPB tryptose-phosphate broth.
TABLE IV.CALCUI.ATED RANGE OF SAMPLE MEANS FOR VARIOUS CONCENTRATIONS OF VIRUS I test range of sample means* T2 11 n mber Standard (confidence limits) antigen RBC observed Degrees of deviation (PFU/cell) Per 43 X field freedom of the mean 95 percent 99 percent 4.5 X 10 313 29 41.2 229-397 200-426 l.3 10' 300 29 31.4 236-364 214-386 8.7 X 10 231 29 95.3 35-426 169-293 4.3 X 10 166 19 13.4 138-194 128-204 1.3 1O 116 24 31.5 52-180 28-204 8.7 X 10" 97 29 15.7 65-129 54-140 4.3 X 10 67 24 14.3 38-96 27-107 Values include the 30 RBC which constitute the background counts.
n- 1. i.e.. the number of fields counted- 1. Code:
PFU plaque-forming unit. RBC= red blood cells.
From the foregoing data, it will be observed iri orfi to obtain an absolute quantitative assay that appropriate correction must be made of the mean average agglutinate counts by deduction therefrom of a mean average background count. Depending on the nature and quality of the particular indicator-containing liquid antigen-containing overcoating, calf serum under-coating and the like, there usually results a small amount of some nonspecific adherence of coated erythrocytes and/or indicator particles at the interface. Generally the amount of observed nonspecific adherence is relatively small even though average variations between background counts can be large. Thus, variations between background counts as great as 50 percent of the mean average background count per cell and even of 50 percent of the mean count for a particular sampling position of a cell may be encountered. However, of great significance and a most important statistic is that generally background counts average only about 21 red blood cells or less per sampling position or field even when examined under 430 magnification. Several factors of possible effect on background counts were stud ied by storing an indicator-containing liquid which included antibody-coated red blood cells therein at 4C. for 0, 8, and 24 hours before performing assays therewith. No significant difference was noted in the resulting background counts whether the employed indicator-containing liquid was freshly prepared, i.e., zero storage, or was stored for either 8 or 24 hours before assay therewith.
Although the invention disclosed herein has been illustrated and taught with specificity and in detail, it will be obvious to one skilled in the art from the teachings herein that other embodiments of the invention are possible and fall within the true scope of the invention with it intended that the invention be limited only to that extent as set forth in the claims below.
1. In an article useful for detecting and assaying immune-adherence-reaction agglutinates, the improvement of: a component thereof of a light-transparent solid material having (i) a dried light-transparent filmcoating of nonnal blood serum adheringto a flat surface of the component, and (ii) an overcoating on top of said coating with said overcoating being a dried light-transparent antigen-containing film-coating and (iii) with said component adapted in assembled arrangement of said article to provide a reaction chamber in which immune adherence reactions can be performed with said overcoating providing the floor surface of said reaction chamber.
2. In an article, providing at least one cell comprised of floor, roof, and side-wall surfaces, for detecting and assaying by light-detection means of immuneadherence-reaction agglutinates subsequently formed in said cell, the improvement in combination therewith a portion of said article being of a light-transparent solid material having a flat surface having thereon a light-transparent dried cell serum film-coating overcoated with a light-transparent antigencontaining film-overcoating, said portion adapted in association with said article to function as the floor or roof of said cell with said light-transparent antigen-containing film-overcoating adapted to serve as a surface of said cell and with said lighttransparent solid material adapted for access thereto by said light-detection means;
whereby upon a liquid, containing homologousantibody-coated indicator particles, being placed in said cell in contact with said light-transparent antigen-containing overcoating, said agglutinates form at the interface of said liquidand overcoating and are detectable by said light-detection means.
3. A method, useful for detecting and assaying immune-adherence-reaction agglutinates of indicatorparticles/antigen/antibody and indicatorparticles/antigen/antibody-complement formed within a reaction chamber comprising the steps of:
a. contacting -a liquid, containing homologousantibody-coated indicator particles, with a dried antigen-containing overcoating on a dried serum under-coating which is adhered to a lighttransparent solid'suppoit serving as the floor of said chamber whereby said agglutinates form at the interface of said liquid and overcoating; I
b. subsequently positioning said liquid and said sup port in relationship .to each other so that said support faces substantially upward to serve as the roof of said chamber, whereby indicator particles not forming a portion of said agglutinates move away from said interface and settle out in said liquid; and
c. determining the amount of said agglutinates formed at said interface.
4. The method of claim 3 employing: a depth between 1 and 2% mm. of said liquid for contacting said dried antigen-containing overcoating; the contacting for between 3 and 15 minutes; and the antigencontaining overcoating which is a dried tryptosephosphate broth having the antigen therein.
5. The method of c1aim'4 in which the determining employs a light microscope to view said interface through said light-transparent solid support to detererythrocytes for the indicator particles and the dried mine numerically the agglutinates formed on a specific serum undercoating which is a film of heat-fixed calf area of said overcoating. serum.
6. The method of claim 4 employing mammalian