Note: Descriptions are shown in the official language in which they were submitted.
~L2~
PARTICLE-BOUND BINDING COMPONENT IMMUNOASSAY
BACKGROUND OF TIIE INV~NTION
FIELD OF Tll~ INVENTION
Tl~e field o~ this invention relates to a sol;d phase system Eor
performing an immunoassay for detection and quantitation of an
analyte suspected of being in a specimen. More particularly,
the invention relates to a method using a solid support surface
5 consisting of particles to which can be bound a binding component
or antigen.
PRIOR ART
Nurnerous methods utilizing immunoassay techniques for more
precise and reliable ways to quantitate an amount of analyte in
10 a specimen. The rapid, quantitative and cost-efficient results
des;red by the medical and diagnostic testing industry have
providecl the impetua for novel means of accomplishing these
goals. There are two types of assay systems currently used. One
is a homogeneous system whereby the assay is performed in a
15 single phase. Antibody anci analyte are allowed to react together
ancl a labelled material is introduced which binds to the antibody
or antigen after an antibody:antigen complex has formed. The
label generates a signal wllich is readable when present in tlle
reaction zone or solution. The signal is correlative with the
amount of analyte present in solution. This indirect measurement
is generally used because of the dif~iculty of direct measurement
of many analytes.
'
The second type of system is a heterogeneous assay which is
a two-phase system where there is a solid, or bound, phase and a
li.qui.d, or an unbound phase, requiring an additional step so
separate the bound from unbound material. Typically, a solid
support surface is used as the bound phase, to which is attaclled
an antibody or antigen via a chemical bond or ac1sorptio1l.
Various types of solid support surfaces have been developed to
improve the effi.ci.ency of the immunological reaction betwee
anti.body and antigen, and, to increase the efEici.ency of the
separation step. An inefficient or incomplete separation of
bound from unbound can result in unbound label remaining in the
reaction zone after the separation step, which will cause a
positive signal to be read where there is no antigen present. ~n
efficient binding and subsequent separation are two of the most
important aspects of a desirable assay.
Particles have been used in agglutination assay procedures
for some time in order to overcome the drawbacks of ineffici.ent
binding. In this type of system soluble antigens will combine
with their specific antibody to form a precipitate, in whi.ch the
antigen-antibody complexes form large aggregates which are
insoluble. The same antigens, if attached to particulate matter
such as latex particles or bacterial cells, will form
agglutinates or clumps. Ihe agglutination reaction can be
detected and quantifi.ed using visual or instrumentat:ion means
such as light-scattering or absorption techniques (Bellanti,
"Immunology II", W.B. Saunders Company, Philadelphia, 197~, p~
212). 'rhough the test is commonly used in the clinical
laboratory~ it suffers fro1n several limitations~ such as serum
inter~erences, insensitivity, and, most importantly, from the
pcrspective of the technician or scientist performing the test,
the subjective judgment of the assay endpoint. While the method
i9 COnvenieht for qualitative analys;s, it is inadequate for
quantitative analytsis, espccially of very low concentrations of
analytes. Latex particles have been used as labels for the
analytc of interest, whereby the assay relies on the use of an
agglutination reaction to decrease the number of particles of a
particular size present in the assay mixture (Boguslaski, et aL.,
"Clinical Immunochemistry," Little, Brown and Company, Boston,
1984, p.211).
Various material have been used as support support surEaccs~
including glass rods~ glass beads, silica impregnated strips,
glass fiber, filter paper, cellulose or its derivatives,
Sepharose beads, plane polymeric surfaces, magetîzable solid
phase ce~lulose/iron oxide particles, ferratin, etc.
Coated test tubes and trays have the lim;tation that only
the inner surface of the vessel is coated with the solid phase.
Material in the center of the solution will not be in intimllte
contact with the solid phase until and unless agitatcd; and even
t~-en only over a comparatively long period of time. The lack of
surface area prevents the rapid establisllment of an equilibr;um
between the bound and unbound phase.
Physical separation of the bound phase from the unbound
phase is required in heterogeneous assays. Most of ten the bound
phase is retained for measurement. Separation is generally
accomplished by one oE several methods including chromatography,
125~ 30
filtration, electrophoresis, adsorption, precipitation, or centrifugation. It is preferable to use a method
which is adaptable for use in automated equipment or in a system that can be used by a trained
technician.
Thus, Micheal E. Jolley, Ph.D., discloses in Pandex Laboratories Research Report No. 1, July
5 1983, a particle concentration tluorescence immunoassay wherein 0.6-0.8um polystyrene particles are
bound to the antigen of interest. A first antibody directed against the antigen and a labelled second
antibody directed against the first antibody are contacted with the bound antigen. The label is limited
to a fluorescent molecule and is read after separation by front-surface fluorimetry.
Such procedure contains deficiencies in that it is limited to fluorescent signal ancl detection
10 means and it is also limited to front-surface reading; which does not appear to be pragmatic for rear
surface Yiewing . Moreover, the particles disclosed are only composed of polystyrene and 0.~0.8um
in size, rather than a broad range of useful particle compositions and si~es.
U.S. Patent No. 4,201,763, issued to Monthony, et al., discloses water insoluble hydrophilic
polymeric particles in the performance of a sandwich or competitive immunoassay. The method is
15 limited to the use of a fluorescent label and therefore lacks the ~rersatjljty of uses in situations where
nonfluorescent labels are preferable.
U.S. Patent No. 4,415,700, issued to Batz, et al., describes hydrophilic latex particles consisting
of a homo- or co-polymer of monomers containing at least one epoxy group and at least one
~ri
2~30
1)olymeri~able carbon-carbon double bo~d in the molecule. The
mc~11od using the particles is a competitive assay whercin
labelled first antibody bound analyte and unlabelled Pirst
alltibody bound analyte compete for binding sites on a particle
5 bound second (anti-first antibody) antibody. Again, ~he
invcntion is limited to one type of label, in this instance an
cnzyme, and is also limited to a particular particle composition.
With the emergence of the physician's office and self-
tcsting markets, there is a need for a separation technique that
10 can ~e used by relatively unskilled users. A soli~ phase that
woul~ be compatible with an easy-to-use separation system, that
provid~d accurate and repeatable results would be advantageous.
Such a method should have a solid phase that permits a rapid
equilibrium to be established between the antibody and antigen so
15 as to promote rapid binding of the two; it should also be
convenient and inexpensive; and, it should be usable in settings
in a~dition to a hospital or laboratory, i.e., the physician's
offic~ or in the home.
SUMMARY OF THE INVENTION
The present invention relates to a method for per-
forming a heterogeneous immunoassay for the detection and
quantitation of an analyte suspected of being in a specimen
which overcomes the problems heretofore encountered~
Thus, according to one aspect of the invention there
is provided an immunoassay method for the detection or
quantitation of an analyte suspected of being in a spec;men
~z~
solution comprising: (a) passing said specimen, insoluble
particles, a first binding component capable of being bound
to said particles, and second binding component labelled
with a signal generating material to a filter means having
a suficient pore size such that said particles are
trapped within said filter yet permitting rapid passage of
fluid therethrough in such a manner that an immunological
reaction occurs if analyte is present in said specimen,
resulting in the formation of an immunocomplex of
l~ insolubili~ed first binding component:analyte:second
labelled binding component on or within said filter means;
(b) separating bound from unbound material; and
(c) determining the presence and/or amount of signal
produced which is correlative with the amount of analyte
present in the solution.
In more detail, the present invention, at least in its
preferred form, is directed to a method comprising a
relation between finely divided particulate solid material
capable of forming a stable suspension in a liquid
,~
32~8~
mcdium and an inert porous filter matrix means of sufficient pore
size to retain the particles on or within the filter matix, yet
permitting the flow of liquid the~ethrough. Material thus
retained remains available to interact in subsequent assay
5 procedures.
A solid phase is provided to which has been bound a binding
component. In the performance oE the assay, which can be
simultaneous or sequential, an analyte in solution is mixe-l with
an amoullt of particle-bound binding COmpOnent and aLlowed to
10 react. A second binding component labelled with a signal-
generating material, is added and likewise allowed to react,
forming an immunocomplex of particle-bound binding
componcnt:antigen:labelled second binding component. Tile complex
is then waslled to remove unbound labelled binding component and
15 the reaction area read to measure the amount of signal present,
which is correlative with the presence or quantitiy of analyte
present in the specimen.
DESCRIPTION OF THE INVENTION
This invention is directed to a binding component attached
to a particulate material used as a sol;d phase in a
heterogeneous immunassay procedure.
In the course of the invention described herein the
following terms will be described and have the following applied
meanings:
binding componenC - any molecule, compound, or combination
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thereof capable of recognizing and binding to a distinct spatial
or polar organization of a molecule (commonly referred to as an
antigenic determinant or an idiotype);
solid phase - material that has been rendered insoluble by
5 binding to the parti.culate material, as distinguished from the
liquid phase which contains soluble material;
label - a substance that generates or can be made to
~nelaLe a signal capable of being de~ected or measured by visual
or i.nBtrumentation means; and
analyte (of interest) - a compound, substance or organism
suspected of being present in a patient sample solution; the
presence or absence of which is of interest to the user, and
whicll contains at least one unique spati.al or polar or~anization
capable of being recognized and bound by a binding component.
Soli.d phase immunoassays generally use some type of solid
support surface to which can be bound, directly or indirectly,
components which are thereby rendered insoluble with respect to
the liquid medium in wh;ch the components were di.ssolved. The
principle involved is that by removing the material from the
20 liquid phase, and subsequently washing the solid phase, a more
complete separation can occur, which increases the overall
sensitivity of the assay. To this end various types and
structures of materals have been chosen for different purposes.
Systems use sheets of filter paper, glass, cellulose, or like
25 types of materials, to which the binding component is attached.
In somc cases the i.nner or outer surface of a test tube or
microtiter tray well is used so as to provide simultaneously a
reacti.on vessel integral with the solid phase. Additionally,
beads of various compositions have been used in an effort to
increase the surface area of the solid support surface material.
rlle present invention uses a nnvel relation between finely
divided particulate solid material capable of forming a stable
5 suspension in a liquid medium and an inert porous Eilter matri~
means of sufficient pore size to retain the particles on or
within the filter matrix, yet permitting the flow oE liqtlid
therethrough. Material thus retained re~nains available to
interact in subsequent assay procedures such as but not limited
10 to washing to deparate bound form unbound material, reagent
addition, optical mea.surement, and the lilce.
The ~articles can be made of any so]ld material that meets
two requirements. First, it must be suspendable in solution; by
this is meant that the particles are insoluble in solution but
15 are small enough to be suspended in the solution. This increases
favorable reaction kinetics through Brownian motion, thereby
establishing equilibrium faster than a system with less available
surface for binding. Second, the material must be filterable:
particulate material is capable of being retained on or within a
20 semi-permeable substance, preferably inert with respect to the
chemical reactions. Filter materiaL is composed oE a substance
such as paper, glass fiber, scintered glass, cotton, synthetic
polymer, plastic, cellulose, cellulose acetate,
polytetrafluoroethylene, polyethylene, polypropylene or
~5 polyvinylidine fluoride, and the like. The particles can be made
of a wide variety of materials including, but not limited to,
glass, cellulose, synthetic polymers or plastics, proteins (e.u.,
. ~L2g~8~ .
gll1teraldehyde or albumin), bacterial cells (e.g., protein A
staphylococcus)~ and the l;ke. Latex is a preferred material
becausc of its availability, co~st-ef~ec~;veness and ease of use.
The size of the particles is limited, again, only by the
requirelllents of suspendability and filterabilityO Tlle particles
should be large enough to be trapped by a given fiLter material,
yet small enough to be suspendable in a liquid medium. They arc
preferably spheroidal in shape, but tlle structural and spatial
configuration i6 not critical; for instance, the particles could
be slivers, ellipsoids, cubes, and the likeO A suitable particle
size ranges from a diameter of 0.2um to 50.0um, preferably
between 0.4um to l.Oum.
It is also possible to use as a solicl particle organ;sms
which can bind to the antibody directed against them. In such an
embodiment the organism would be of a size or diameter that would
be suspendable and filterable. An example is Toxoplasma gondii,
a parasite of micron dimensions which binds to the antibody
specific for it. A labelled anti-antibody directed against the
Toxoplasma ~ antibody is used to attach a signal gener~tor
to the colDplex.
While the specific particle size is relevant~ the primary
focus is on tlle relationship between the particle size, filter
thickness and filter pore size. The filter pore size is defined
by the construction of the filter material. In the case of glass
fiber filters the size density of the fibers def;nes the amount
and size of tlle ;nterstices therein. The filter is designed to
contain pores or interstices large enough to entrap particles
within or on the f;lCer snd prevent their passing therethrou&h.
~2~
!
Moreover, the depth or thickness of the filter should be
sufficient to permit an effective amount of particles to become
entrapped. Were the filter too thin, there sould be inadequate
space for enough particles to be entrapped to perform a
5 quantitative assay.
It is preferable for the particles to be caught within the
filter, but some of the particles will remain blocked on its
surface, wllether because of ciumping, aggregation or random
non~lniformly sized particles. One advantage of interstice or
10 pore entrapment is the favorable reaction kinetics that result.
When the particles are immobi]ized within the porous filter and
fluid must be contacted with the surface of the particles, such
as in a wash step, there is greater surface area availaable for
contact if the particles are suspended, as it were, in the
15 filter, with their surfaces virtually completely exposed to the
fluid in contact therewith. Where the particles are trapped
solely Qn the surEace of a thin membrane incapable of re~aining
particles in a three dimensional network of pores or interstices,
there is less surface area available because the particles are in
20 contact with each other, resulting in a decreased surface area;
therefore~ a less efficient wash or fluid contact occurs,
decreasing the efficience of the assay procedure.
Another advantage of internal, as opposed to external,
entrapment is the convenience of physical transport of devices
25 embodying the assay materials. Where particle bound binding
component is pre-spotted in the filter~ lyophilized, or otherwise
immobilized, the fllter effect;vely binds the particles witl
~L292~
;
little possibility of the particles dissociating from the filter
during rough handling. Where particles are pre-spotted on top of
the surface of a thin membrane, there is a greater likelihood of
the particle layer coming ofE the membrane if dropped, jostled or
otherwise disturbed. ~dditionally, if the particles are
deposited as a layer on a membrane~ and dried thereon, when
reconstituted tlle layer mi~ht float off the membrane and sl-ift
its position, potentially reducing the amount of material within
a narrowly defined reaction zone. This deficiency is not present
in the instant invention because pre-spotted particles are
securely immobili~ed within the filter and not subject to the
same mistreatment. It is inevitable, however, that a certain
fraction of the particles will be blocked on the surface of thc
filter. The method o theis invention comtemplates the
enablement of the procedure regardless of the amount of surface
entrapment that occurs.
The filter itself has the unique characteristic of being
able to wick fluid away from the point of application by
capillary action while the particles are entrapped in the filter
matrix. l~lis permits a large volulme o~ fluid to be transported
from an area in contact with the particles and other bound
components and to an area away therefrom. Since the efficiency
of an assay technique is, in part, dependent upon the
completeness of separation between bound and unbound material,
t~le greater the volume of wash fluid that is in contact Witll the
components, the more effective the wash. The completeness of the
wash reduces the amount of background noise present wllere unbound
signal generating material remains in the zone of measurement.
11
92~c~
The ilter contemplated by the present invention can provide an
improved means for accomplishing th;s desired separation.
The particles have bound to ~hem a binding component~ which
is rendered insoluble. The binding component i6 chosen to be
5 able to recogllize and I>ind to an analyte in solu~ion. Typically,
where the analyte is an antigen (any substance capable of
eliciting an immune response, e.g., group A strep or human
chorionic gonadotropin) the bincling component is an antibody that
will bind to an antigenic determinant on the antigen. Where the
10 analyte is an antibody, such as when testing for rubella or
hepatitis~ the binding component is an antigen. It is also
po~sible for the analyte to be un antibody and the binding
component to be an (anti)antibody directed to the analyte
antibody. Moreover, it is useful to employ monoclonal antibodies
15 for the particle-bound binding component as well as for the
labelled binding component because of the high degree of
selectivity and sensitivity associated with such antibodies.
The binding component can be directly attached to the
particle material via absorption or covalent chemical bonding,
20 the methods for which are well Icnown in the art. Direct adhering
of binding component can be achieved by reacting carbodiimide
with the particle and the binding component. Alternatively,
serum Protein A is used in a well known procedure. Indirect
insol~blization of binding component can be achieved by attaching
25 to the particle material a member of a binding pair, such as
biotin, and separately attaching to the binding component the
other member o~ the binding pair, such as avidin. ~hen particle-
12
125~L8~)
bound biotin is mixed with the binding component-bound avidin the
avidin and biotin bond together, forming a particle-avidin-
biotin-binding component linkage. Such a method wou]d be
advantageous when it is desirable to insolublize the binding
5 component:analyte complex after it has formed, rather than
before. Another technique for indirectly attaching the binding
component to the particle material is hy binding an antibody to
the particle material, separately binding to the binding
COolpOllent tl different, (anti~antibody directed against the
10 particle-bound antibody and subsequently mixing the products of
each reaction together to form a particle-
antibody:(anti)antibody-binding component structure.
~ here latex particles and glass ~iber filter paper is used,
conditioning of the filter is necessary prior to, or
15 contemporaneously with, the addition of insolublized material to
the filter means. The purpose is twofold; first, to enhance the
immobilization of latex particles within the interstices;
second, to prevent the nonspecific sticking of unbound antibody
or other extraneous materials to the filter. The conditioning
20 material can be different for both, such as but not limited to
gelatin, horse serum, albumin, dry milk, and the like. The
material can be added to tlle filter prior to the addition of
commponents to the filter. ~lternat;vely, the conditioning
mtlterial may be added simultaneously with the other reactants,
25 or, it can even be mixed with the specimen fluid as a diluent or
the particle suspension.
The particles coated with binding component are then
contacted in a reaction vessel or container with a biolo&ical
lZ~2~
. . .
fluid suspected of containing an analyte sucll as bacterial,
viral, fungal or parasitic antigens and immunoglobulins~
antibodies~ hormones~ serum protei.ns~ en~ymes9 drugs~ and the
li.ke. Biological Eluids from which samples can be obtained
5 include urine, feces, blood, serum, mucus, sputum, semen, and the
li.ke. Analyte will fonn an immunological comp:Lex with the
insolublized bi.nding component.
Simultaneously~ or sl~bsequently, depending on tlle assay
procedure, a second binding component labelled with a signal-
10 generating material is added to the reaction vessel. Thislabelled binding component commonly is an antibody, directed
against the analyte. The anti.body has been conjugated with a
label prior to its addition to the reacti.on vessel. Several
types oE labels are available for use in the present invention,
15 depending upon the type of assay bei.ng conducted, including
enzyme, luminescent, bioluminescent, cheml-luminescent and
radioi.sotopic materials. Ihe label must be capable of emitting
or assi.st in emitting a signal detectable by visual or
instrumentation means. Tlle labelled binding component will bind
20 to the complex thereby providing an indirect means for
identifying the presence and quantity of analyte present in the
rcaction vessel.
The newly formed insoluble immunocomplex of i.nsolublized
binding component:analyte:labelled binding component is then
~5 washed to remove unbound material whicll could interfere with the
accurate representati.on of the label present; namely, label which
has nonspeci~ically bound to material in the reaction vessel
14
8~
other than the insolublized immunocomplex~ such as proteins~ the
reaction vessel itself or thc particles. The separation is
conducted by a filtration procedure involving passing through or
into a filter material the reaction mixture, wllich is retained
5 because of its size in the filter interstices. The
immunochemical reactions can occur within the filter or cxternal
thercto. Unbound materials wash through or out of the filter and
away from the reaction zone which is to be read. Separation is
accorIl;plished by passing fluid through the filter by gravity,
10 capillary action, or by using positive or negative pressure, such
as a vacuum or pump. Tlle completeness of the separation is
critical to the sensitivity of the assay; the more unbound
material which could emit a detectable s;gnal that is removed the
less background noise there will be to interfere or obscure a
15 weak signal being emitte~t from a very low concentration of bound
label.
The wash solution can be a buffer, such as phosphate or
TRIS, or any other solution appropriate and compatible with the
components involved. Where an enzyme label is used the substrate
20 may be added as part of the wash solution or separately added
prior to reading.
Thc signal is read after any necessary developing, (Iuenching
or other modifîcation of the signal. Reading can be done
visually or through an instrument, such as a colorimeter to
25 measure color absorbance where an enzyme label is used; a
photometer to measure visible light where a luminescent~
bioluminescent or chemi-luminescent label is used; or a
scintillation or gamma counter to measure radiation where a
~Z9;~
raclioisotope is the label. The amount of signal produced is
correlative with the concentration oE analyte present in the
biological sample ~lu;d; the measurement is useful in diagnosis
or monitoring drug levels or disease states.
The preferred embodiment of this invention is a sandwich
cnzyme immulloassay where the binding component is an antibody and
attaclled to latex particles; tlle analyte is an antigen; the label
is an en~yme; and the substances are added sequentially to a
filter material, in which the separation occures. After washing
10 the insolubli~ed complex a substrate is added to produce a visual
indication if antigen is present. Tlle reaction area can al60 be
read by an instrument to provide a sens;tive quantitation of tlle
antigen. An advantage of sequen~ial addition is the greater
degree of control over the binding reactions that is obtained.
For example, where a very low concentration of antigen is
suspected of being present, a longer incubation period is
requ;red because of the slower reaction kinetics involved;
equilibrium will take longer to achieve. The result of tlle
flexibility and control gained is the increased sensitivity for a
quantitative measurement o~ analyte.
Another embodiment of this invention is tile simultaneous
addit;on of insolublized binding cornponent, analyte and labelled
binding component. An advantage of this embodiment is the
elimination of the separate addition steps, thereby saving time
for the user. This method is particularly useful where only a
qualitative "yes-no" result is desired.
A further embodiment oE this invention is a competitive
16
8~
assay whereby sample analyte and labelled analyte compete for
binding sites on the insolublized binding component.
Still another embodiment of this invention is the pre-
spoLting of particle-boulld binding CompOnent in the filter
5 material or separation medium prior to the addition of analyte
and otllcr substances. This embodiment has the advantage of
al]owing for storage of a test device wllereby the filter material
has been pretreated with a particular particle-bound binding
component enabling a user to perform an assay for a desircd
10 analyte and elimate one step of manipulation, thereby reducing
the time and error associated an assay. Furthermore, this
embodiment eliminates the step of separately adding the particle
bouncl antibody, thus simplifying the procedure.
An alternative embodiment of tl-is invention utilizes a
15 plurality of monoclonal antibodies insolublized on or within tlle
filter matrix and are designed to recognize and bind different
and distinct analytes simultaneously. A plurality of labels of
different wavelengths are used to identity the particular analyte
and a photorneter or other appropriate instrument, used to measure
20 the intensity of the of the signal at the different corresponding
wavelengtl~s. In this manner it is possible, in a single test
device or reaction area, using a single procedure, to obtain
accruate and sensitive determinations of multiple antigens in a
patient sarnple. A significant example of this is an assay for
25 Gonorrhea and Chlamydia, a test frequently needed in hospitals
and physicians' offices. Sucl- a test, using monoclonal
ant;bodies to the Gonorrhea and Chlamydia viruses bound to
insolublized particles and a single reaction zone or container,
could determine whether either, both or neither of the two
diseases was present, employing only a single procedure.
~ !oreover~ using the high selectivity of monoclonal
antibodies, this invention could be used to detect the serotype
oE a part;cular bacteria, virus, paratsite, or other organism
wl~ere e treatment would be different depending on the particular
patlogen present.
Where enzyme labelling is desired a plurality of substrates
is use each producing a signal readable by a photometer witll
monochromat~c light. By scanning the spectrum, or using specific
wavelengths, the instrument could distinguish the diferent
wavelength signals~ which the human eye would normally interpret
as one average color.
The Examples which follow futher define and illustrate
various embodiments of Applicant s invention. They are by way of
illustration only and not by way of limitation. Other methods
are contemplated as being within the scope and spirit of tllis
invention.
EXAMPLES
EXAMPLE 1 - PROCEDURE FOR SE~UENrIAL ASSAY
A test to determine the presence of human chorionic gonadotropin
(hCG) as an indication of pregnancy is performed on a urine
specimen.
First, 5 drops of 0.5% gelatin in Tris buEEered saline
are added to a glass iber filter paper (Micro Filtration
Systems) to condition the filter. Then 4 ml of 2.5% latex
particles (average diameter oE 1 um~ Polysciences) are added to 1
~92~
mg/ml solution of a monoclonal antibody directed against hCG and mixed for three hours, then
centrifuged. One drop of the urine specimen is added to 1 drop of the antibody-coated latex particlcs
and the mixture permitted to incubate for one minute. One drop of a second monoclonal antibody
directed against a different and distinct epitope on the hCG molecule and labelled with alkaline
5 phosphatase is then added and the mixture is added to the filter and the filtrate washed with 1 ml Tris
bufferred saline solution. One drop of indoxyl phosphate substrate is added to the washed mixture
to develop color. Appearance of a blue color is indicative of a positive result.
EXAMPLE 2 -PROCEDURE FOR SIMULTANEOUS SANDWICH ASSAY
The procedure according to EXAMPLE 1 is used; but, the antibody coated latex particles,
10 urine specimen and labelled second antibody are mixed simultaneously and then incubated. Again,
appearance of a blue color indicates a positive result.
EXAMPLE 3 -PROCEDURE FOR SIMWLTANEOUS COMPETITIVE ASSAY
A test to determine the presence andtor quantitation of digoxin.
First, 5 drops of ~.5% gelatin in Tris bufferred saline solution are added to a glass fiber fillter
15 paper (Micro Filtration Systems~ to condition the filter. Then 4 ml of 2.5 % latex particles (1 um
average diameter, Polysciences) are added to 1 mg/ml solution of a monoclonal antibody directed
against digoxin and rnixed for 3 hours, then centri~uged. One to 10 drops
19
.~
8~
of a serum specimen suspected of containing digoxin and 1 drop of
alkal;.ne phosphatase labelled digoxin are combined with an excess
amount of latex coated monoclonal anti.body and incubated for
hollr. Thle mi.xture is added to the filter and washed witl- 1 ml
5 of Tri.s buffered saline to remove unbound digoxi.n. Then 1 drop
of indoxyl phosphate substrate i.s added to the washed mixture.
Tlle appearance of a blue color is indicative of the presence of
digoxin.
EXA~5PLE 4 - PROCEDURE FOR SE~UENTIAL CO~IPETITIVE ASS~Y
A test to determine the presence of thyroid stimulating hormone
(TSII).
First, 5 drops of 0.5% gelatin in Tris buffered saline are
added to a glass fiber filter paper (~5icro Filtration Systems) to
condi.tion the filLer. Then, ~I ml oE 2.5% latex particles
(average diameter of 1 um, Polysciences) are added to 1 mg/ml
solution of a monoclonal antibody directed against TS~I and mixed
for 3 hours~ then centrifuged.
One to 10 drops of alkaline phosphatase conjugated TSi! are
ad(led to 1 drop of the latex bound monoclonal antibody and
incubate(l. The mixture is added to the conditioned Eilter and
washed with 1 ml of Tris buffered saline to remove unbound
material. One drop of blood serum specimen suspected of
containing TSI~ i9 added to the Eilter and allowed to react. Tlle
mixture is again washed with Tris buffered saline to remove
unbound material. Then, 1 drop of indoxyl phosphate is added.
The appearance of a blue color is indicative of the presence of
TS~I. A quantitative measurement of TSH can be made by reacling
the reaction zone with a colorimeter or similar instrument.
Wllile the invention has been described in connection with a
preferred embodiment~ it is not intended ~o limit tlle scope of
thc invention to the particular form set forth, buC, on the
contrary, it is intended to cover such alternatives,
modifications, and equivalents as may be included within the
spirit and scope of tlle invention as deEined by the appended
claims.
