U.S. patent number 3,770,383 [Application Number 05/214,213] was granted by the patent office on 1973-11-06 for diagnostic test slide.
This patent grant is currently assigned to Akzona Incorporated. Invention is credited to Richard Thompson Price.
United States Patent |
3,770,383 |
Price |
November 6, 1973 |
DIAGNOSTIC TEST SLIDE
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, non-absorbent to, and wettable by,
water, carries on its surface at least one circumscribed test area
containing a single solid dried stable spot deposit of an
immuno-chemical 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.
Inventors: |
Price; Richard Thompson
(Verona, NJ) |
Assignee: |
Akzona Incorporated (Asheville,
NC)
|
Family
ID: |
26829211 |
Appl.
No.: |
05/214,213 |
Filed: |
December 30, 1971 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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131172 |
Apr 5, 1971 |
3666421 |
May 30, 1972 |
|
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818366 |
Apr 22, 1969 |
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Current U.S.
Class: |
422/425; 436/509;
436/531; 436/543; 436/811; 436/826; 435/7.36; 436/527; 436/534;
436/805; 436/825 |
Current CPC
Class: |
G01N
33/54313 (20130101); G01N 33/552 (20130101); Y10S
436/805 (20130101); Y10S 436/825 (20130101); Y10S
436/826 (20130101); Y10S 436/811 (20130101); Y10S
436/806 (20130101) |
Current International
Class: |
G01N
33/552 (20060101); G01N 33/551 (20060101); G01N
33/543 (20060101); G01n 033/16 () |
Field of
Search: |
;23/253TP,23B,23R
;424/12,13 ;195/99 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Primary Examiner: Reese; Robert M.
Parent Case Text
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.
Claims
What is claimed is:
1. 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 predetermined 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 5 and about 15
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 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 1 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.
Description
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 titered very carefully, the use of
droppers and vials is not only expensive, but is likely to result
in questionable findings because of 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 non-absorbent 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 or card 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.
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 finely 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 10 -- 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 -- 11.
Referring now to the drawings, FIG. 1 shows a test slide suited for
the performance of a test for gonorrhea. The slide 10 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 15 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.
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 agglutination 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), 1 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 percent.
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 the reagents be applied to
the slide, in order to provide optimal sensitivity to the test, in
advance. This obviates the inaccuracy of the conventional test
methods which employ medicine dropper dispensing of test
reagents.
The following examples, which are to be regarded as illustrative,
and not as limiting, show the preparation of various test reagents,
as described above.
EXAMPLE 1
DIRECT LATEX AGGLUTINATION (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 Rhodamine B 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 (IgG)
(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 12.23 gm.
Sucrose 60.00 gm.
Dissolve in 800 ml distilled water and adjust pH to 8.6 with IN
NaOH. 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 this reagent. The diluted, buffered 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.
* * * * *