|Publication number||US3770383 A|
|Publication date||Nov 6, 1973|
|Filing date||Dec 30, 1971|
|Priority date||Apr 5, 1971|
|Also published as||US3666421|
|Publication number||US 3770383 A, US 3770383A, US-A-3770383, US3770383 A, US3770383A|
|Original Assignee||Akzona Inc|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (6), Referenced by (50), Classifications (22)|
|External Links: USPTO, USPTO Assignment, Espacenet|
United States Patent 11 1 Price  Inventor:
[ DIAGNOSTIC TEST SLIDE Richard Thompson Price, Verona, NJ.
 Assignee: Akzo na Incorporated, Asheville,
'22 Filed: Dec. 30, 1971 21 App], No.: 214,213
Related US. Application Data  Continuation-in-part of Ser. No. 131,172, April 5, 1971-, Pat. No. 3,666,421, which is a continuation-in-part of Ser. No. 818,366, April 22, 1969, abandoned.
 US. Cl. .Q 23/253 TP, 424/12  Int. Cl. G01n 33/16  Field of Search ..,23/253 TP, 230 B,
,  References Cited UNITED STATES PATENTS 3/1970 Mass 23/230 B X 12/1970 Kilburn 23/230 B X 1 Nov. 6, 1973 3,616,251 10/1971 1.111011 et al 23/253 TP X 3,672,845 6/1972 Verbeck 23/253 TP 3,697,227 10/1972 Goldstcin et a1 23/253 TP FOREIGN PATENTS OR APPLICATIONS 953,414 3/1964 01w Britain 195/116 Primary Examiner.Robert M. Reese Attorney-Hugo E; Weisberger  ABSTRACT A test slide for the performance and observation of an immunochemical or diagnostic test on its surface,
which surface is insoluble in, impermeable to, nonabsorbent to, and wettable by, water, carries on its surface at least one circumscribed test area containing a singlesolid dried stable spot deposit of an immunochemical reagent providing a predetermined amount of an antigen or an antibody, or alternatively, a second solid dried spot of a buffer material, said spot or spots upon being moistened with a liquid and/or the liquid to be tested being reconstituted to the reagent and/or buffer, to form an observable area of reaction mixture.
9 Claims, 5 Drawing Figures PATENTEDNBV 6 ms 3, 770,383
FIG. I FIG.3
DIAGNOSTIC TEST suns CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of application, Ser. No. 131,172, filed Apr. 5, 1971, now U.S. Pat. No. 3,666,421 issued May 30, 1972, which is in turn a continuation-in-part of application, Ser. No. 818,366, filed Apr. 22, 1969, now abandoned.
BACKGROUND OF THE INVENTION The test slides of the present invention are adapted for the performance of immunochemical reactions on the surface thereof. These immunochemical reactions are most commonly laboratory tests which have as their objective the determination of the presence or absence of antigens or antibodies in body fluids as an aid in the diagnosis of certain physiological or pathological conditions in humans and animals. Depending upon the particular combination of reagents and test liquid employed, the reaction may result in the formation of a precipitate in which case it is known as a precipitin reaction. Where the reaction is between substances distributed in a liquid medium, at least one of which substances is a solid which becomes agglomerated, the reaction is known as an agglutination reaction. The formation of precipitates, or the agglutination or inhibition of agglutination of specially-treated particles is manifested visually in the way the precipitates form, or the particles arrange themselves following the reaction.
These immunochemical tests, which may include, for example, tests for "blood group type, pregnancy, and similar phenomena,'are customarily performed with reagents such as, for'example, a suspension of sensitized erythrocytes, and a solution of a suitable antiserum, dispensed into a test vial from a dropper. Even though the test reagent, such as the aqueous suspension of sensitized erythrocytes, may have been vtitered very carefully, the use of droppers and vials is not only expen-. sive, but is likely to result in questionable findings becauseof the limitations in the accuracy of ordinary droppers. Such droppers are usually uncalibrated, and although the accuracy of the drop size is of great importance, such accuracy is rarely attained and the size of the drops, and their content of reagent is variable and undependable.
In view of the shortcomings of the vial-dropper test methods, it has been proposed in the prior art to carry out agglutination or agglutination-inhibition reactions by depositing a drop of a test reagent on the surface of a slide or cardboard, allowing the drop to be dried, and then applying test liquid thereto to observe the results. Thus, U.S. Pat. No. 2,770,572 describes a card for the determination of blood groups which consists of a solid sheet with at least one area carrying the dried residue of a test serum with specific agglutination against red blood corpuscles. In U.S. Pat. No.. 3,074,853 there is disclosed a test card having a plastic coating which is water-wettable and impermeable on which there is deposited a single dried spot of either an antigen or an antibody admixed with a finely divided substance having a contrasting color such as activated carbon against which to view the formation of the precipitate. The antibody or antigen is applied as a suspension by means of a dropper and then dried, and the serum to be tested is applied to the dried test spot by a dropper. This device has the drawback that the test spots are applied by an ordinary dropper, with attendant inaccuracy of amount deposited, and requires the presence of the colored contrast pigment.
U.S. Pat. No. 3,272,319 describes an immunological test kit utilizing a control card with a relatively nonabsorbent surface on which are deposited a dried serum or plasma, and a corresponding control card with deposits of active and inactive serum or plasma to check the antigen suspension used. A blood sample is tested against a spot known to be reactive with the antigen of the disease being checked.
There has existed a need for a simplified immunochemical test slide or card on which there would be present an accurate amount of the reagent or reagents necessary for a given test, and to which the body fluid to be tested could be applied, so as to make the test result immediately observable by the person making the test.
GENERAL DESCRIPTION OF THE INVENTION A principal object of the present invention is to provide on a single test slide or card, the necessary reagent or reagents for performing an immunochemical or diagnostic test of the type previously described, in the form of a solid, dried, stable spot deposit, accurate in unit content, and containing a predetermined amount of immunochemical test reagent to yield optimal results, which upon being moistened is reconstituted to the test reagent with the liquid to be tested, forming a spot of reaction mixture.
Another object of the invention is to provide a method for the manufacture of said slides or cards which is inexpensive and practical and which does not require elaborate equipment but which may be carried out conveniently for commercial purposes on a large scale. Still another object is to make the reagent for diagnostic testing available in convenient form on a slide or card while at the same time insuring the accuracy of the amounts of reagent present to provide dependable test results. A further object is to provide a person performing a diagnostic test with a simple card or slide on which the reagent is present so that the test can be made in situ and the card kept for record purposes. These and other objects and advantages will become apparent as the description proceeds.
The novel' test slide and method of its preparation will be illustrated with respect to a reagent for an immunochemical or diagnostic test for the detection of the antibody to gonorrhea in human blood, plasma or tissue. However, it will be understood that'this is solely for purpose of illustration of the practice of the invention, which is not to be regarded as limited thereto.
In accordance with the present invention, there is provided a test slide orcard adapted for the performance and observation of an immunochemical or diagnostic test on the surface thereof. The test slide or card is particularly adapted for demonstrating the presence or absence of an antibody to a bacterial or viral antigen or antigen extract by means of a direct agglutination between such antibody and antigen, and the invention will be described with respect to this type of test, although it is not limited thereby. I
The test slide or card of the invention comprises a substantially plane strip of a substrate material having at least one test surface thereof which is substantially insoluble in, impermeable to, non-absorbent to, and wettable by, water, and carrying on said surface at least one circumscribed test area containing a single solid, dried, stable spot deposit, accurate in unit content, of an aqueous immunochemical reagent, providing a predetermined amount of an antigen or an antibody, which upon being moistened with the liquid to be tested, is reconstituted to the test reagent. The agglutination or inhibition of agglutination may be observed visually. The antigen may be absorbed on a suitable carrier, such as f nely divided latex particles, prior to deposition on the slide, thereby affording better visual observation of the reaction.
If desired, a second solid dried spot deposit of a buffer reagent may be included within the circumscribed area in close proximity to the reagent spot deposit, and wetted at the same time as the reagent spot deposit itself. Alternatively, the buffer may be incorporated into the antigen or antibody suspension and the latter together with the buffer deposited as a single spot.
In both situations, an observable area of reaction mixture is formed upon wetting with the liquid to be tested.
DESCRIPTION OF THE PREFERRED EMBODIMENTS The nature and preparation of the test slides or cards of the invention will be better understood by reference to the accompanying drawings, which illustrate a presently preferred embodiment, but are not to be considered as limiting the invention thereto. In the drawings:
FIG. 1 shows a plan view of the upper surface of a test slide having thereon a single solid, dried test spot of an immunochemical reagent incorporating a latex carrier and a buffer;
FIG. 2 is a cross-sectional view of the slide of FIG. 1, taken along the line l 10;
FIG. 3 is a plan view of an alternative embodiment having a solid dried test spot of an immunochemical reagent in close proximity to a solid dried spot of a buffer;
FIG. 4 is a plan view of the test slide of FIG. 1 enclosed in an aluminum foil protective container; and
FIG. 5 is a cross-sectional view of the slide and container of FIG. 4, taken along the line 11 e 11.
Referring now to the drawings, FIG. 1 shows a test slide suited for the performance of a test for gonorrhea. The slide comprises a substantially rectangular sheet or strip of substrate material 11 having the impermeability toward water and the wettability referred to previously. The substrate material may, for example, be glass, glazed porcelain, or a synthetic material, e.g. a plastic material such as a phenol-formaldehyde resin the surface of which has been treated so as to make it wettable, for example, by sand blasting or rubbing with an abrasive, or a transparent plastic such as nitrocellulose or methyl methacrylate.
In the embodiment shown in FIG. 1, the substrate is a thin sheet of cardboard having one or both surfaces coated with a coating of a water-impermeable and water-wettable material such as plasticized nitrocellulose, having a thickness which will maintain the flexilibity of the cardboard, e.g. about 0.02 inch. In all cases, the surface of the substrate which carries the test reagent area should be water-insoluble.
The test card 10 bears on its coated surface a circle 12 of contrasting color, which serves to demarcate the location of the area containing the test reagent. In the embodiment shown in FIG. 1, there is a single test reagent 13. This is a solid dried spot of an antigen suspension, for example a dried aqueous suspension of carrier particles such as polystyrene latex, bentonite particles, cholesterol, quartz crystals, erythrocytes and stroma (cell walls) thereof, sensitized with Neisseria antigen or antigen extract. The test reagent spot advantageously has an average diameter between about 5 and about 15 mm., and is wetted by the liquid to be tested when this is applied to the slide.
In the alternative embodiment shown in FIG. 3, the spot of test reagent has in close proximity a spot of additional reagent such as a buffer material 14, so that both spots may be wetted by the liquid to be tested when this is applied to the slide or, alternatively, each spot may be wetted separately with different'liquids, such as the liquid to be tested and water, and then admixed.
Where the substrate is glass, the test results may be readily studied under suitable magnification.
FIG. 4 illustrates an arrangement whereby the test slide of FIGS. 1 or 3 is enclosed in a protective package 30 fabricated from two sheets 31 and 32 made of metal foil such as aluminium foil or of metal foil laminate such as aluminum foil-plastic laminate material, the package being heat sealed or bonded with adhesive around the peripheral margin 34. The package is formed by coating the edge portions of aluminum foil with a solution of a suitable adhesive, such as a vinyl resin, e.g. polyvinyl chloride, a butadiene-acrylonitrile polymer, polyethylene, or other plastic material conventionally used for bonding metal foil, followed by heat sealing. I
Where aluminum or other metal foil-plastic laminate material is employed, the package is formed by superimposing the plastic face of the sheets comprising a readily sealable adhesive film such as polyethylene, and heat sealing to provide a hermetic seal for the package. In the filling and bonding or heat sealing operations, which are preferably performed under sterile conditions, the individual slide should be deposited on the bottom metal foil sheet in such position that it is sufficiently away from the sealing portions so as not to be affected by the heat of the sealing operation. It will be understood that a series of slides may be deposited on a lower metal foil web and an upper metal foil web bonded or sealed to the lower web at suitable intervals so as to provide a continuous series of packages which may be separated by a suitable tear line provided between adjacent envelopes.
The preparation of the test reagent which when dried forms the test deposit or spot on the slide or card is described in the accompanying example.
In performing a diagnostic test, such as, for example, a test for gonorrhea, a measured amount of the body fluid to be tested, such as blood or blood components, is placed on or in proximity of a dried reagent spot. Additionally, the dried antigen reagent spot can be reconstituted with a liquid, such as water. The spot or spots and the test liquid are then mixed by stirring with a toothpick, glass rod, or plastic stirring device, as to unite the reagent or reagent-buffer to a single area of reaction mixture in which the immunochemical reaction takes place.
The presence or absence of gonorrhea in the blood is detected by the agglutination or inhibition of agglutination of the sensitized carrier particles, the reaction being manifested within a few minutes. Similar aggluti- "nation or inhibition of agglutination, or precipitation,
is to be observed in the case of other types of immunochemical reagents and test liquids.
In the preparation of the test slides or cards, the reagents are constituted as solutions or suspensions in a volatile liquid medium, advantageously an aqueous medium. Such solutions or suspensions may also contain potentiating or resuspending aids. It has been found, in accordance with the invention, that a number of adjuvants contribute to the ease of resuspension of the dried reagents and serve to produce a firm bonding of the dried'reagent to the test slide surface. These reagents include, for example, bovine serum albumin in concentration up to an including'5% (wt/vol), l percent being optimal; lactalbumin hydrolysate in concentrations up to and including 5 percent, 1 percent being optimal; and gum arabic in concentration of about 0.5
' It'has also been found, in accordance with the invention, that the inclusion of a saccharide, such as sucrose or mannitol in the reagent provides, upon drying, a
hard, glaze-like finish to' the dried test spot which protects it against abrasion and mechanical damage, thereby aiding in packing and storage.
As mentioned previously, it is an important aspect of the invention that quantitatively accurate amounts of EXAMPLE 1 DIRECT LATEX AGGLUTINATI ON '(GONORRHEA) Antibody to Neisseria gonorrhoeae may be detected in human blood or tissue fluids by means of complement fixation test or indirect fluorescent antibody test. However, these current methods are thought to lack both sensitivity and specificity. What will be described is a rapid and sensitive method for the determination of the presence or absence of such antibody in serum or whole blood.
Polystyrene latex particles, having a particle size range of about 0.1 to about 1.5 microns (average 0.5) serving as a carrier, are sensitized with a known weight of Neisseria antigen or antigen extract and suspended in a borate buffer. The latex is sensitized with the Neisseria antigen by first treating the latex with bovine serum albumin (approximately 45 micrograms per ml.), centrifuging and incubating the latex in a solution of Neisseria antigen. Excess antigen is removed by centrifuging and washing, and then the latex is suspended in the buffer; 0.03 ml. drops are measured onto slides and dried.
Simultaneously with the dropping of the latex suspension onto the slide, a drop of glycine buffer may be dropped in close proximity. The composition of this buffer is as follows:
Glycine 0.945 gm. Sodium Chloride 1.223 gm. Sucrose 6.0 gm.
To the dried antigen spot on the slide there is added .a measured amount of water or buffer for the hydration of the reagent. A drop of blood or serum is placed beside the wetted reagent when the one drop reagent test.
is employed, or in the instance where the buffer has been dried as a separate spot, the blood or serum in proper amount is placed upon the buffer. This is mixed and the two liquefied reagents admixed. Agglutination will occur within two minutes if the sample tested contains antibody to the antigenic component'utilized; in the case where no antibody is present, a homogeneous suspension will be noted. For the purpose of better visualization, a dye such as RhodamineB or Orange G may be added to either of the reagents before drying.
EXAMPLE 2 Direct Latex Agglutination (Rheumatoid Factor) Rheumatoid factors can be detected in human serum by means of a well known test using human gamma globulin affixed to a latex particle. In this test when the thus sensitized latex particle is brought into contact with human serum, blood or synovial fluid containing rheumatoid factors, an agglutination of the sensitized latex particle results. Polystyrene latex particles, 1.5 percent solids are suspended in a pH 8.6 glycine buffer of the following composition. They are sensitized with human immunoglobulin (lgG) (see Singer and Plotz, Amer. J. Med. 21:888, 1956) and are made one percent with respect to bovine serum albumin. 0.03 ml drops are measured onto slides and dried.
Glycine (Aminoacetic acid) 9.45 gm. Sodium Chloride l2.23 gm. Sucrose 60.00 gm.
Dissolve in 800 ml distilled water and adjust pll-l to.
8.6 with IN motif/add distilled water to 1" liter total volume.
Simultaneously to the dispensing of the drop of latex suspension onto the slide, a drop of glycine buffer of the same composition as given is dried onto the slide in a position immediately adjacent to but separate from the drop of latex suspension.
To' test for rheumatoid factor in human serum the serum is diluted 1:20 with 0.9 percent saline or with glycine buffer. (previously described) and one drop (0.03 ml). is measured onto the dried buffer spot. One drop of water (0.03 ml) is added to the latex spot to reconstitute th is reagent. The diluted, buttered serum and latex are mixed and spread over the circumscribed area delineated on the slide. Rheumatoid Factor is demonstrated by an agglutination of the particles within one minute.
What is claimed is:
l. A test slide adapted for the performance and observation of an immunochemical or diagnostic test for gonorrhoea on the surface thereof, comprising a substantially plane strip of a substrate material having at least one test surface which is insoluble in, permeable to, non-absorbent to, and wettable by, water, and carrying on said surface at least one demarcated circumscribed test area including a single deposited solid dried aqueous test reagent, consisting essentially of particles of a carrier sensitized with a known weight of Neisseria gonorrhoea antigen or antigen extract providing a pre determined amount of said test reagent, which upon being moistened with blood components or tissue fluids to be tested, is reconstituted to said test reagent, forming an area of reaction mixture.
2. The test slide of claim 1, in which said deposited test reagent has an average diameter between about and about millimeters.
3. The test slide of claim 1 in which said substrate material is glass.
4. The test slide of claim 1, in which a deposited solid dried spot of an additional reagent is positioned in close proximity to said reagent deposit, within said circumscribed area.
5. The test slide of claim 1 in which said substrate I material is a substantially rectangular sheet of cardboard carrying on one surface thereof a coating of a water-impermeable and water-wettable synthetic material.
6. The test slide of claim 1 in which said circumscribed test area including said test reagent is demarcated by a circle of contrasting color.
7. The test slide of claim 1 in which said carrier particles are latex particles.
8. The test slide of claim l in which said test reagent includes an adjuvant to bind the deposit to the surface and to promote resuspension upon moistening.
9. The test slide of claim 1 which is enclosed within a protective container comprising a base sheet of metal foil and a co-extensive cover sheet of metal foil bonded to said base sheet around the periphery of said sheets to provide a hermetic enclosure for said test slide.
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|U.S. Classification||422/425, 436/826, 436/534, 436/531, 436/509, 436/543, 436/527, 436/825, 435/7.36, 436/805, 436/811|
|International Classification||G01N33/543, G01N33/552|
|Cooperative Classification||Y10S436/811, G01N33/552, G01N33/54313, Y10S436/805, Y10S436/825, Y10S436/826, Y10S436/806|
|European Classification||G01N33/552, G01N33/543D|