Note: Descriptions are shown in the official language in which they were submitted.
~l~g~2~
This invention relates to the detection o antigen or antibody using
¦ immunochemical reactions. Sam?les of bodily ~luid may be tested for the
1~ presence of a particular antigen or antibody, from which a diagnostic
1l inference may be made.
-- Bacl~u~
An immunochemical reaction is here defined as the specific binding
l which takes place between an antigen and antibody. Antigens and antihodies
¦ are comps)nents of the immu~ity system whereby mammals including man
protect themselves against infectious agents. An antigen is any substance
Eoreign to the organism into which it has been introduced, which is capable
! o~ eliciting the protective immune re~ponse of that organism. Most antigens
are proteinaceous materials in whole or in part, having high molecular
weights. Antibodies are also proteinaceous macromolecules, elicited in
lS response to the presence of a foreign antigen, in the organism. Antibodies
have the property of being capable of binding with antigen molecules in
highly specific combinations. The binding i5 characterized by its high degree
of speciicity and low dissociation constant.
Normally, an animal has only those antibodies which are specifically
directed against antigens to which it has been exposed in its environment.
However, an animal can be induced to form antibodies against other antigens
by artlfically introducing them, for example by injection. Medical use is
made of this phenomenon to immunize people against disease. It is also
possible to cause a laboratory animal such as a rabbit or goat to make
antibodies against specific substances. Such antibodies may be obtained
from the blood o the animal and are exploited in highly speciîic assay
techniques or the detection o the original antigenic substance. Virtually
:`
," ~k
.
an~- prot~in can in principle be defectcd by means oE an immunochemical
! reaction.
Immunochemical reactions have been exploited in a variety of ways:
for the diagnosis oE disease: ~or the identification of a specific infecting
l1 organism, as highly specific enzyme inhibitors, to determine the location
¦ of speci~ic proteins in tissues and within cells, and for the quantitative
measurement of speciEic proteins Eor which no chemical -test is available,
Since the reaction is a binding of one component ~antigen) to another (antibody~,
there is no net cllange in the number of reactive groups as in an ordinary
chemical reaction~ Analysis of an immunochemical reaction therefore
requireS techniques for differentiating between bound and unbound component3.
A variety of methods has been employed in the prior art for the
meaSurernent oE immunochemical reactions. Thege include hemagglutination,
.~ ~ Lhdn
~t~ latex particle E~i~, agar gel diffusion, complement fixation,
counterelectrophoresis, and the use of antibodies tagged with fluorescent
dyes or radioisotopes. ¦
I ~emagglutination and Eluorescent antibody techniques have been
¦ applied in the detection oE antibodies against Toxoplasma ~ondii,p~ Toxoplasma !
is a protozoan parasite of man which lives primarily inside the cells of the
host, SO that the organism is difficult to detect by microscopic means. The
~ast rnajority of Toxoplasma infections are asyr~ptomatic, However, an
asymptomatically infected mother can pass the organism to the fetus, where
Toxoplasma infection can cause a variety of birth defects: malformations,
I hydrocephalus, mental retardatlon, eye diseases often leadin~ to blindness,
and inEant mortality. A simple, inexpenSive screening test for pregnant
Jes~rc~ble
mothers would be highly ~ as a step toward the eradication oE
congen t 1l ~oxo~la~ . Although hernagglu~lnatlon and fluore9cent antibod~
~:
:
li techniques have been used in ~he diagnosis o~ Toxoplasma in~ection, the
¦I standard method has been the dye test. Serum antibody against Toxoplasma
is detected in the dye test by taking advantage of the fact that live Toxoplasmal cells are partially lysed in the presence o~ antibody. Lysed celis are
! distinguishable from unlysed cells by the addi~ion of methylene blue whichonly stains intact organisms~ In practice the test is complicated by the
additional requirement Çor an accessory factor~ thought to be complement,
¦ which must be obtained from the serum of Toxoplasma free clonors. Great
¦ attention-to detail is required in carrying out the tests successfully. More
¦ signi~icantly, the test is dangero`us to laboratory workers since it involves
the use of live Toxoplasma. A number of laboratory infections have resulted
in individuals who have perEormed the test (see Jacobs, L., "Serodiagnosis
o~ Toxoplasmosis", in ~ Infections, S. Cohen and
E. Sadun, eds., Blackwell Scientific Publications, 1976;. An improved ~;
immunochemical method for detect;ng Toxoplasma infection is therefore
des~ra.6J~
highly e~.
Although immunochemical methods generally used in the prior art
I can be highly sensitive, especially radioimmunoassay using radioisotope-
'il tagged antibody, the widespread usefulness of immunochemical assays has
¦ been limited by three factors:
(~ ) the need ~or a convenient and inexpensive method of
separating bound immunochemically reacted components from
; ` unbound components,
¦ (2) the need Eor a convenient and inexpensive way to
Z5 1 measure the amount of immunochemical reactant bound, and
3~ the need for a procedure that can be carried out
rapidly. I
,~ .
~$
2~7
Tl~e first two o~ these ,li~ficulties have been overcome by recent
il advances in the prior art, the tllird is overcome by the present invention.
The ~irst of these recent advances in the prior art is the development
I of techniques for coupling an immunochemically reactive antigen or antibody
I to an insoluble carrier material. An antibody imrnobilized on an insoluble
carrier can, when exposed to a solution con~aining antigen, bind the antigen
and render it, in turn, immobilized. The entire immunochemical reaction
¦ occurs on the carrier and the components which are bound on the carrier
can be separated from the unreacted components by conventional techniques
LO I for separating solid phase materials from a liquid phase. For example, if
¦ the carrier is in the form of beads e~- finely divided powder, separation can
be accomplished by centrifugation or decantation. Qltern~tively, the
immobilized phase may be the inner surface of the reaction vessel itself, or
it may be in the form o~ a sponge or porous matri~ so that separation may be
L5 carried out be simple decantation or by removal of the carrier, respectively.
The second advance has been the introduction of an enzyme-tagged
antibody, which is a covalent conjugate of an antikody and an enzyme. Each
retains its characteristic reactive properties: the antibody remains
immunologically reactive and the enzyme retains its catalytic activity. When
~0 such a conjugate binds to an immobilized antigen, its presence can bedetected through the activity of the coupled enzyme, after separating unbound
conjugate by appropriate means.
These two technlques have been used in combination to develop a very
sensltive type of assay termed an enzyme-linked immunosorbent assay. The
invention which is the subject of this report embodies these basic techniques.
For convenience, two types of enzyme-linked immunosorbent assay will be
dlscussed and re~erred to as EL-1 and EL-2; see the diagram beiow:
? ~
EL -1: C Ab Ag- Ab- Enz
¦ EL-2: C l~g ~b - AntiAb-Enz
C stantls for an inert~ insoluble carrier;
¦j Ab symbolizes antibody;
¦¦ Ag symbolizes antigen;
C--Ab symbolizes carrier-bound antibody;
l ~--Ag symbolizes carrier-bound antigen;
jl Ab-Enz symbolizes the conjugate of an antibody with an enzyme ;
1¦ AntiAb-Enz symbolizes an antibody against immunoglobulin,
1l conjugated with an enzyme,
¦~ EL-1 is a technique for detecting the presence of an antigen. Antibodyagainst the antigen iS immobilized on an insoluble carrier. The immobilized
antibody is then exposed to an antigen-containing fluid, The immobilized
antibody harvests the anti~en from the solution by binding it in place. The
carrier is then separated from the solution, washed free of contaminants, ~ l
¦ and exposed to a solution of the antibody conjugated to an enzyme. The
principle Oe operation is that the conjugate ls able to bind only at sites
occupied by the antigen, and that the number of sites so occupied determines
the amount of conjugate which can bind. Each site where antigen is bound
1 i8 thuS tagged with bound enzyme whose presence is manifested by its
ability to catalyze a reaction. The rate Or such reaction is proportional to
the arnount ol enzyme present~and becomes a direct measure of the amount
of antlgen bound,
In EL-2, the component to be detected is an antibody. Antigen iS
immobilized on the carrier, Binding of the antibody is then measured by
:j ~the subsequent binding of an anti-immunoglobulin-en~ym2 conjugate~ ~See
4 ¦ ~ Isdom, (:.D., ~n Clinical Chemistry, vol. ZZ, p. 1243 (ID76). )
-6-
~' ' . '' .
.
Il ~ntigen or antibocly molecllles may be immob~ ed on a solid carrier
¦¦ by a variety of methods kno~vn in the art, including covalent coupling, direct
I adsorption, physical entràpment ancl attachment to a protein-coated surface.
!I For references describing this methodology, see Silman, I.H. and
1¦ Katchalski, E. in Annual Review of 3iochemistry, Vol. 35, p. 873 ~1966),
I
¦ Melrose, G. J. H. J in Review of Pure and Applied hemistry, vol. 21, p. 83,
(1971); and Cuatrecasas, PO and Anfinsen, C.B., in Methods in Enzymology,
Vol. 22, (1971). `
The method oE attachment to a protein-coated surface is disclosed by
pa~e"t/l~ G~
~ , Lai et al. ~German OS 2, 539, 65~, US. Y;~l;l})~ In this method,
the internal and external surfaces of a microporous membrane are first
coated with a water insoluble protein such as zein, collagen, fibrinogen,
keratin, glutelin, polyisoleucine, polytryptophan, polyphenylalanine,
polSrtyrosine, or copolymers of leucine with p~a~ino phenylalanine. Such
~15 1 a coating renders the membrane capable of immobilizing a wide variety of
I biologically active proteins including enzymes, antigens, and antibodies. A
microporous structure is defined as one having more than 50% of its total
,
volume in-the form of pores ranging in size from 25 nanometers to 25
micrometers, preferably Erom 25 nanometers to 14 micrometers. A pore
. .,.,, . .
6ize range from 25 nanometers to 5 micrometers is~ employed in most
applications herein. Uncoated microporous membranes have as much as
70 tv 75% of their volume as pore space, The pores permit liquid flow
through the membrane. After being coated by zein, for exarnple, the pore
space Is reduced 5 to 10% with the result that the structure retains its
essential properties of having ~ high proportion of its volume as pore space
¦ and permitting liquid flow through the pores, The structure has a large
surtace area m contaot wlth any solution contained within the poreJ~
_7_
,
. '
Such a coated membrane, having immobilized antigen
or antibody, provides a compact, easy to manipulate carrier
for the immobilized antigen or antibody. Its integral
structure permits removal of bound from unbound components
by simple mechanical means.
A difficulty attending the use of microporous
membranes as carriers ~or immobili~ed antigen or antibody
is that these structures may adsorb proteins nonspecifically.
Uncoated membranes of cellulose acetate and nitrate mixed
esters can bind certain proteins and are also capable of
exchanging bound for soluble protein. The physico-chemical
basis for the binding is unclear~ Certain proteins appear
to bind more readily than others. As a result, assays based
on binding a specific antigen, antibody or conjugate in the
presence of a mixture of proteins can result in high back-
ground interference which may occur in an unpredictable
manner.
A related dificulty is presented when coated
membranes are used. Coated membranes, as disclosed by Lai,
et al, are capable of binding proteins general]y, but the
binding is neither as selective nor as variable as that
displayed by uncoated membxanes. Consequently, the use of
such mem~ranes to immobilize antibody or antigen in an EL-l
or EI.~2 assay may also result in high ~ackground inter-
erence due to the binding of unwanted protein species.
These difficulties have been effectively surmounted by
the present invention.
Tn accordance with the present invention, there is
provided a method for de-te~ting the presence OL one component
of an immunochemical reaction between a first component
that is selected from the group consisting of antibodies
,~
~b~
and antigens, and a second component that binds immuno-
chemically with the first component, comprising:
immobilizing the first component on the internal and
external surfaces of a microporous membrane that has upper
and lower external membrane sur~aoes ancl that is at least
50% microporous, the membrane having an interconnecting
network of pores extending throughout il:s thickness between
the upper and lower external membrane surfaces, providing
internal membrane surfaces, the membrane having pore si~es
in the range from 25 nanometers to 25 mi.crometers, and the
network of pores permitting fluid flow through the membrane,
thereby providing internal and external surface areas that
are exposed to any fluid flow into and through the membxane
rom the upper surface to the lower surface thereof, the
aggregate internal surface area being many times largér than
the external surface area, app].ying a solution of the second
component to the upper surface of the membrane onto which
the first component is immobilized and then passing it
through the membrane, whereby the second component is brought
into reactive contact with the immobilized first component
on the external and internal surfaces of the membrane, to
permit immunochemical binding of the second component to
the immobllized.first component, then flowing a rinse solution
through the membrane from the upper surface to discharge from
the lower surface and to flush away residual solution of the
second component, applying a solution of an enzyme labelled
immunochemical reactant for the second component to the
upper surface of the membrane and then passing it through
the me~rane, whereby the labelled reactant is brought into
reactive contact with the bound second component, ~lowing
a rlnse solution through the membrane from its upper surfaGe
~a~
:
to discharge from i.ts lower surface, to flush away any
residual solution of the enzyme-labelled immunochemical
reactant, and then detecting the presence on the surfaces of
the membrane of the second component by observation of a
colour change on the surfaces of the membrane produced by
a reaction of the enzyme with a chromagen.
~ he present invention also includes a device for use
in a diagnostic test for detecting the presence
in a test solution of an immunochemically reactive substance
selected from the group consisting of an antibody ancL an
antigen tha~ are immunochemically reactive with each other,
comprising: a microporous membrane having a pair of opposed
external surfaces, one of which is an upper surace and the .
other of which is a lower surface, the membrane being
formed with an intercommunicatiny network of pores extending
throughout its thickness, with pore sizes in the range from
25 nanometers to ~5 micrometers, the membrane being at least
~ 50% porous and having a large internal surface area, the
:~ pores providing passageways ~rom its upper surface to its
20 lower surface and permitting fluid flow through the membrane,
the membrane having applied to its surfaces an immunochemically
neutral protein dried in situ thereon, the membrane having
immobilized thereto a biolo~ically active substance selected
from the g.roup, a support structure that is formed with an
opening, the membrane having its upper surface sealed to the
suppor~ structure about the opening to aispose the mem~rane
~ across the opening, whereby a test solution applled in the
opening to the upper surface of the membrane can flow
through the membrane for contact with its internal surfaces
3Q ana the immunochemical reactant immobilized thereon, for
discharge ~rom the lower surface of the membrane.
In addition, the present invention provides a test
9a ~
kit for use with a solutisn to be tested compris1.ng the
device and reagent materials comprising, as a first reagent,
a solution of a conjugate of an antibody and an enzyme, and
as a second reagent material, a solution of a substrate
for the enzyme moiety of the conjugate, and a rinse buffer
solution.
In one of its specific embodiments, the present
invention provides a method for detecting in a bodily fluid
one componenk of a two component immunochemical reaction
between a first component that is selected from the group
consisting o~ antigens and anti~odies, second component
from the group that binds immunochemically with the ~irst
component, the method comprising: providing as a test device
a plastic support structure having at least two generally
~up-shaped wells each of which is opened at its lower end
and has a substantially circular transverse section, each of
the wells having sealed thereto about its bottom opening a
microporous membrane, each microporous membrane being
disposed to have upper and lower external membrane surfaces
and being at least 50~ microporous, each membrane having
an intercommunicating netwo.rk of pores extending throughout
its thickness ~etween its upper and lower membrane surfaces
to provide internal surface areas, ha~ing pore sizes in the
range from 25 nanometers to 25 micrometers, and permitting
fluid flow therethrough, thereby providing internal and
external surface areas that are exposed to any fluid flow
into and through the porous membrane from the upper surface
to the lower surface thereof, the aggregate internal surface
area being many times larger than the external su.rface
area, each membrane having immo~ilized thereto one component
selected from the group, applying a sample of bod~ly fluid
that may contain the complementary one of the components to
.
.i~ ~ 9b ~
the upper surface of the membrane of a first one of the
wells and then passing it through the membrane from the
upper surface thereof to the lower surface thereof through
the pores of the membrane for discharge from the lower
surface of the membrane, the complementary component if
present in the bodily fluid binding specifically to the
immobilized component; applying to the upper surface of
the membrane in a second cup a negative control fluid
known to be free of the complementary component and then
passing the negative con~rol fluid through the membrane from
its upper surface to its lower surface or discharge from the
lower surface of the membrane; passing washing fluid uni-
directionally through each of the membranes to remove
unbound materials; applying a solution of an enzyme conjugate
for the complementary component to the upper surface of each
of the membranes, the conjugate solution binding specifically
to any of the complementary component that is hound on the
membrane, and then flowing the enzyme conjugake solution
through each of the membranes ~rom the upper surface of
each respectively through the membrane for contact with its
internal surfaces and for discharge from its respective
lower surface; flowing washing fluid unidirectionall~
through each of the membranes to remove unbound conjugate
solution; applying to the upper surface of each of the
membrane means for detecting bound enzyme by observation of
a colour change on the surfaces o~ the membrane produced by
; a reaction of the enz~me with a chromagen, and comparing
~ the two membranes to determine the substantial absence of
enzyme from the mem~rane trea~ed with the negative control
. solution and the presence or absence of enzyme on the
; membrane treated with the bodily fluld sample; the method
- 9c-
f~ .
~ .
being capable of completion in less than one hour.
A test kit for effecting the latter process also
is provided comprising a plastic support structure that is
formed with at least two of the generally cup-shaped wells
thereinl each having a bottom opening/ lo the pe:iphery of
each of which one of the microporous membranes is sealed,
each sealed membrane extending across ilts respective opening
and having a majority of its volume as pore volume, each
membrane having immobilized on its in~ernal and exte:rnal
surfaces the immunochemical component a given one component
from the group, one of the membranes serving as a test
membrane for bodily fluid and the other of the membranes
providing a negative control; each membrane beinq disposed
to have an upper surface and a lower surface and an
intercommunicating network of pores extending throughout
its thickness, providing internal membrane surfaces, and ~ .
pro~iding passageways interconnecting the upper membrane -.
external surface and the lower membrane external surface,
the pores having sizes in the range from 25 nanometers to
25 micrometers; a negative control reagent liquid that is
free of the other immunochemical reactant component
selected from the group; a solution of an enzyme con~ugate
.~ that can immunochemically bind to the other component of the
group, and means for permitting the detection of the
: . presence of the enæyme in the conjugate on a membrane by
observation o~ a colour change on the surfaces o~ the
mem~rane produced by a reaction of the enzyme with a ~ ;
chromagen.
The present invention, therefore, is a diagnostic
3Q assay method and relates to the materials necessary to
practice the method as well as to the method itself. The
- 9d
: .
method is applicable to the detection of an antigen or an
antibody in a fluid sample. In one application of the
invention, for example, it is used to detect the presence
or absence of an antibody in a sample oi` serum. In another
application, an antigen is detected in a sample of a bodily
fluid.
The materials used for the practice of the invention
include an antigen or antibody immobilized on the surface
of an integral structure having a large surface available
for contact with the sample and treated to minimize non-
specific protein binding, means for mounting the structure
so that sample, ~7ash solutions and reagent may be permitted
to flow through the structure, a first reagent comprising a
conjugate of an anti~ody with an enzyme and a second reagent
comprising a substrate for the enzyme.
In the preferred embodiment, one component of the
immunochemical reaction, either antigen or antibody, is
immobilized on the zein-coated inkernal and external surfaces
of a microporous mem~rane, as previously defined, whereby
these surfaces are rendered immunochemically reactive.
The membrane is mounted so that the fluid to be tested, for
example, serum, may be applied to one side of the membrane,
allowed to flow through the pores of the men~rane and
collected from the other side. The desired immunochemical
~; reaction between a component in the serum and the immobilized
component takes place during the period of time in which the
serum is in contact ~ith the reactive surfaces during
passage through the membrane~
Once the desired immunochemical component is harvested
from the test fluid, its presence on the mem~rane surface may
be detected by the subsequent passage through the membrane of
- 9e ;-
~,, )~ . ' .
an antibody-enzyme conjugateO The antibody moiety of the
conjugate is specific for the component to be detected,
while the enzyme is chosen to be one whose activity is
readily detected by methods well known in the art.
Peroxidase (enzyme no. 1.11.1.71 is preferred. The enzyme
numbering scheme used herein is that set forth in Florkin
et al, "Comprehensive ~iochemistry" Vol. 13, 3rd Ed.,
Elsevier Pub. Co. New York (1973~o Immunochemical
binding of the conjugate to the harvested component occurs
during passage of conjugate solution throug~ the membrane.
The conjugate will be immunochemically bound only at sites
where the component to be detected is bound.
_
2t~
A reagent solution conl:aining substrate for the bound en~yme is ne~t
passed through the memhrane. Qualitatively, the prcsence o~ immuno-
,¦ chemically bound peroxidase is detected by the use o~ a chrl~magenic substrat~
Il and the subsequent development of color. In the absence of nonspeciic
¦ binding any color which develops is due ~o the immunochemical binding of
the conjugate, which in turn depends on the presence o the component to be
~ detected and demonstrates the existence of the component to be detected in
1I the test fluid.
¦ Quantitatively, the amount of color which is developed may be
j measured, for example, by spectrophotometry. The amount of color
developed is a measure o~ the amount of chromagenic substrate converted to
product, which in turn is a measure of the amount of enzyrne bound. In the
! absence of nonspecific binding of the conjugate, the amount of conjugate
hound is a measure of the amount of the component -to be tested harvested
on the membrane, and hence a measure of the amount oE the component
present in the test fluid.
In practice, a certain amount of nonspecific binding occurs, It is
therefore necessary to employ a control sample, in which it is kr-own that
the component to be detected is absent. ~n important feature of the invention
is the adoption of techniques which minimize the amount of nonspecific
binding and maximize the differences observed between positive samples and
controls. In this regard, the use of a zein coal;ing on the microporous
membranes, the use of a two stage immob~lization procedure, and the use
¦, of highly purified peroxidase in forrning the conjugate have been effectivelyl combirled in the preferred embodimen~ to reduce nonspecific binding. The
procedures employed are critical to the practice of the invention and wlll
be discussed in detail below~
I -10- ~
It has be~n discovered that immunochemical assays
may be carried out according to the invention using extremely
short incubation times. Complete tests can be run from start
to finish in 20-30 minutes, as compared to several hours for
prior methods. In addition, the invention provides advan-
tages of simplicity and ease of operation, adaptability
for routine use, and lack of requirement for a highly trained
technician or expensive equipm~nt. Because of these
advantages, the invention renders immunochemical assays
more readily available for use in a wide range of clinical,
industrial, and environmental tests. Examples of the
invention's applicability include the routine detection of
hepatitis B antigen in donated blood, the diagnosis of
, and the testing o Eoodstuffs or microbial
contamination or toxins.
Proper function of the invention is in part dependent
upon the choice of a suitable surface upon which antigen or
antibody is immobilized. The surface structure should permit
1uid flow into and through the structure and should present
a large surface area relative to the volume of fluid con-
tained within the structure. Such requirements are satisfied,
to varying degrees, by such diverse structures as hollow
fibre bundles, poxous refractory filters, microporous membrane
filters, and packed columns. The choice of support in each
case will depend upon the type of assay and the use to which
i~ is put. For a wide variety of assays, where speed,
convenience and economy must be considered, the use of a
microporous mem~rane is preferred.
The method of immobilizing antigen or antibody to the
surface of such a structure may in principle be any method
suitable for the particular surface employed and material to
.~
.!
.
be attached. The method of Lai, supra, is suitable for
immobilization of biologically active materials to a wide
variety of surfaces. In addition, the use of a coating has
the unexpected advantage of providing a means for controlling
nonspecific protein binding.
In the preferred emhodiment, ze;n~coated microporous
membranes are employed. The term microporous membrane is
here defined as having pores that fall within the size range
from 25 nanomsters to about 25 micrometers and preFerably,
from 25 nanometers to 14 micrometers. The coating may be
applied without su~stantial loss of flow-through capability
and with only a slight diminution of pore volume. Micro-
porous membrane filters present the ~urther advantage that
their external as well as their internal surfaces become
coated and thus able to immobilize antibody. Particulate
antigens, such as whole cells or cell fragments, can be bound
- 12 ~
llC4~r~7
e~,-en tho-lgh they may be too large to enter the pores of the membrane,
Preparations of antibody immobilized to zein-coated microporous membrane
filters remain immunochemically reactiYe for long periods oE time uncler
Il proper conditions. Samples may be stored under refrigeration or lyophilized
¦ asld stored in a controlled humidity environment.
i Antigens and antibodies used in the invention may be prepared by
standard techniques well known in the art. Antibodies may be prepared
I from the serum o~ animals such as rabbits, horses, or goats which have
il beer immunized against the appropriate antigen. Antigens are purified
¦¦ from the source organism lay known techniques used in the Separation and
purification of biological materials.
Since the structure having the irnmobilized component is -to be
e~osed to a bodily fluid comprising a mixture of proteinaceouS rnaterials,
any affinity between such materials and the structure's surface can result
i in nonspeciic binding. Such binding could seriously interfere with the assay,
For e~ample, the total amount of protein which could bind to the uncoated
¦¦ surfaces of a microporous membrane could exceed the amount bound by a
specific immunochemical reaction. Similarly~ where a coated microporous
membrane iS employed, the membrane may retain the ability to bind
additional protein nonspecificallyO
It has been di8covered as a part of this invention that no~specific
binding may be minimized by interposing a second stage immobilization step,
in which an immunochemically neutral protein is immobilized to the ~ilter.
Immobilization therefore occurs in two stages according to the preferred
1 embodiment of the invention: a ~irst stage in which the desired immuno-
chemical componen~ is immobilized, and a second stage, following the
completi n oE the first, ~n which an immunochemically neutral protein such
-13-
: l .
: I
l as Eetal calf serum or boville gamma globulin is next immobilized. The
te~n immunochernically neutral is defined in terms of the specific
components of the assay. Any protein which does not combine immuno-
Il chemically with a component of the assay or with one of the reagents is
1¦ considered immunochemically neutral, even though such protein might be
I¦ immunochemically reactive in another system. 'rhe combination o~ coating
¦¦ a microporous membrane by the method of Lai, immobilizing an
¦ immunochemical reactant in a first immobilization step, followed by
I immobilizing an immunochemically neutral protein in a second immobilization
LO step results is substantial reduction of nonspecific binding when themembrane
bearing its immobilized components is exposed to a mixture of proteinaceous
m ate rials .
The conjvgate o~ antibody with enzyme is made using techniques
known in the prior art. (For references, see ~vrameas, S. and Uriel, J.,
L5 in Comptes Rendus ~Iebdomadaires des Seances de l'Acadernie des Sciences,vol. 262, p. 2543, (1966); Nakane, P.K. and Pierce, G.B., in ~ournal of
Mistochemistry and Cytochemistry" vol. 14, p. 929, (1966); Nakane, P.K.,
il in Methods in Enzymology, vol. 37, p. 133, (1975). ) In an EL-1 type of
¦ assay, the antibody moiety of the conjugate should have the same
~0 ¦ immunological specificity as the immobilized antibody. In EL-2 where the¦ substance to be detected is an antibody, the immunochemically reactive
¦ moiety of the conjugate must be an antibody capable of binding immuno-
chemically with the antibody to be tested. Such antibo~ies may be obtained
by immunizing an animal with the antibody or immunoglobulin fraction of
~5 serum from the animal in which the antibody to be tested originated. For
I e}~ample, where the antibody to be tested is a human antibody, a goat antibody¦ against human antibody is obtained from the serum of a goat immunized
1~ -14-
against human immunoglo~ulin (antibody). The enzyme
moiety may be any enzyme capa~le of catalyæing a reaction
which can be detected by any method known to those skilled
in the art, and which retains its activity after conjugation
with antibody. Horseradish peroxidase (enzyme no. 1.11.1.7~
is preferred because of its convenience and suitability to a
wide range of applications. It is well known that the enzyme
catalyzes the oxidation of a variety of organic compounds
in the presence of hydrogen peroxide. Many such organic
substrates are chromagenic, i.e., undergo a colour change
upon oxidation.
It has been found in the present invention that the
purity of the enzyme preparation used in the ~ormation of
conjugate has an effect on the degree of nonspecific binding.
The greater the purity of the enzyme preparation, the less
the nonspecific binding. In part, the reduction is made
possible because the total amount of conjugate protein
required is reduced as the specific activity of the enzyme
is increased. The opportunity ~or nonspecific ~inding is
therefore reduced as well. In the preferred embodiment, the
use of a highly purified peroxidase preparation has been found
to significantly reduce the amount of colour reaction observed
in control samples as compared with known positives.
The first reaction step in the assay procedure
involves bringing together the immobilized component of the
irnmunochemical reaction and the test fluid, which may contain
the component to ~e detected, such that reactive contact
occurs. It is at this step that the choice of a suitable
carriex for the immobiliæed component, having a large surface
area in relation to the volume contained within the structure
becomes important~ As the test fluid flows through the
- 15 -
`~.`1
. _ . .
structure, the pxobability that a solute molecule will
contact one of the surfaces during liquid flow through the
structure is very high. The probabil.ity of an immunochem-
ically reactive contact is further increased as the probabil-
ity of such collisions per unit of surface area is maximized.
It is believed that the probability of such collisions
depends, in a general way, upon the parameters of pore size
and flow rate, where solute
- 15a -
' ,"
concentra~ion and reaction temperature are held constant.
Thus, the more constricted the passageway through which the
solute molecules pass, relative to their mean free path,
the greater the likelihood of collision with the surface.
The lower the flow rate~ the longer the residence time of
the solute within the structure itself, and the greater
the probabillty of collision. In accordance with the fore~
going considerations, structures having large ratios of
surface exposed to the volume of fluid flowing therethrough
are employed in the present invention in an attempt to
increase the probability of reactive contact with the
surface and to reduce the time required to carry out the
reactions involved.
The invention is descrihed further, by way of
illustration, with reference to the accompanying drawings,
wherein: '
Figure 1 is a perspective view of a device for use in
the diagnostic assay of multiple samples, constructed in
accordance with one embodiment of the invention;
Figure 2 is a fragmentary section, taken on the line
2-2 of Figure 1, looking in the direction of the arrows;
Figure 3 is a fragmentary top plan view thereof; and
Figure 4 is a fragmentary top plan view of a device
made in accordance with a different em~odiment of the
inventionO
Referring now in detail to the drawings by numerals
of referencel the de~ice 10 is formed from a molded plastic,
preferably transparent and tou~h, base on a tray 12. The
tray 12 has a bottom panel 13 and upstanding walls 14 that
are formed with an internal lip 16. A holder 18 is de~achably
seated on ~he lip 16. This holder is also formed from tough,
,
- 16 -
. ~ ,
.
i2~
transparent plastic and it in turn is formed with a plurality
of generally cylindrical, cup-like recessed parts 20. The
bottom opening of each cup 20 is covered with a membrane 22
that is heat sealed to the lower rim of the cup. In the
embodiment of the invention shown in Figure 4, the holder
18' is formed with a much larger number of cups 20', thus
permitting a single device to be used for a larger number
of assays.
In use, a sample is poured into a cup. The sample
flows through the membrane into the tray. As will be
described in detail hereafter, the membrane is washed
and treated with reagents r as needed, to detect the
presence o~ absence of a particular material in the sample.
In the preferred embodiment, microporous membrane~
are employed as carrier for the immobilized component. The
membrane is mounted in a convenient holder in order to permit
fluid flow of sample, wash solutions
:
~ I - 16a -
~.~
and reagel~ts tl~rough the pore~ o~ the membr~le. Such mernbranes are
a~-ailable in a variety of pore sizes. The optimum pore size will clepend on
tlle requirements of the specific assay, includins the flolv characteristics of
the sample to be tested and the amount of fluid to be processed. The
incubation time is governed by the flo~,v rate through the filter and ma~- be
controlled by a variety oE means known in the art. ~ simple and effective
technique is to apply fluid dropwise to the upper sur~ace of the membrane so
that the rate of flow oE fluid is governed only by the h~ dro3tatic pressure
exerted by the drop as it rests on top of the membrane. IE the droplet is
small enough, i. e., approaching the retention volume of the membrane, the
- i' fluid will tend to ~low into the pores of the membrane arld be retained there
by capillary attraction until displaced by additional fluid, Reaction time
under these circumstances is controlled by t'ne residence time of the sarnple
! within the membrane pores,
When the structure is mounted so that reactants and reagents can flour 1 -
through the structure ~rom one side to the other, the entire seg~:lence of
steps is easily and rapidly performed, Flrst, test fluid is per~itted to ~low
, through the structure in order to harvest an~- of the component to be tested ~ -
I present therein. Second, a solution of conjugate is permitted to flow through
I at a controlled rate so that conjugate may immunochemically bind to any
¦ component to be tested harvested in the first step. Thirdg substrate solution
! is perrnitted to flow through the structure at a controLled rat~, in order to
' permit immunochemically bound enzyme to convert the substrate to a ~
measureable product,. Washing steps m~y be interposed between the threa
reaction steps if desired. The incubation temperature ma~ be controlled
within the range 15C to 45C to suit ;ndividual needs~ Room te~nperature
is preferred Eor the sake of convenience, in a qualitati~-e assay. In the
practice of the preferred embodiment, using To:{oplasma ant;gen imrrobilized
_ . , .
17
Oll a microporolls n~embrar:e, tlle presence o~ antibody to Toxopla ,ma in
ser~ull is readil~ detected using a series of 5 minute incul)ations for each
step ~vitll a ~vaslling step interposed between each, so that t'ne entire sequence
of reactions is completed in 20-30 minutes, as described in detail in Ex~mple
1.
There is a variety of chromagenic subsl:rates available for the
Jes~a l /~
qV detection o peroxidase activity, For qualitatiYe tests, it is ~e~Pe~te to
employ either a chromagen which precipitates on the mem~rane when acted
upon by peroxidase. or one in which the peroxidase reaction product binds
preferentially to the membrane,. An example of the former is 3-amino-
ethylcarbazole. An exarnple of the latter is 4-amino antipyrine. Quantitative
measurements may be carried out using a soluble chromagen product and
. .
' measuring the development of color by suitable methods such`a~ spectro-
photometry.
',~ The speed and operaSing convenience of the present invention ~nake l
it eminently suited ~or routine analysis. All the compon~nts can be provided
in a stable ~orm, dried or lyophillzed, so that a techrlician has only to
dissolve the preweighed mixture of bu~er salts or reagents in apredetermined
amount o~ water prior to use. A test kit embodying these advantages is
l~ contemplated. For example, a test kit for the detection of Toxoplasma
l~ antibodies in serum would comprise -the following: microporous membranes
~¦ having Toxoplasma antigen ~nmobilized thereon mounted in a suil:able holder
for adding reaction components to the upper surface OI the membrane and for
,
collecting the materials from the under surface which have flowed t'nrough,
2S lyophilized antibody-enzyme conjugate, dried buffer salts preweighed, anddry substrate preweighed. Where the enzyme moiety of the conjugate is
peroxidase, a substrate mixture containing 4-amino antipyrine, 4-hydro~cy-
~enzy~e n~ 3-/4)
benzoate, lyophilized glucose oxidase~, glucose and buffer saLts is
advantageous. The action of glucose oxidase on glucose generates the
'7
~ drogen peroYiùe substr~te used in the pero:;ida~e reaction, t'n~reb~-
elilrlinatincS the need to provide h~drogen pe~oxic~e, lvhich is di~ficult to
stabilize îor long periods o~ storage. When oxidized 'oy pero~;ide in the
presence of peroxidase~ 4-amino antipyrine forms a colored substance which
preferentially adsorbs to the microporous rnembrane~ In this manner, all
of the components o~ the test can be provided in stable, dry, water soluble
form.
The described invention presents significant advances over ~he prior
~ art in terms of speed, operating simplicityr convenience and expense.
Specific e~amples of procedures embodying aspects of the invention will ne~
be presented, in order to further demonstrate the in~-ention.
, .
~ ! E:~ample 1 - ~Antibody
I I Screening Assay
~' A. A microporous membrane filter having 3 micron nominal pore
ii size ~Type SS> manufactured ~y Millipore Filter Corp., Bedford, ~Iass. )
was coated with zein according to the Eollowlng p ocedure. Zein ~79g) was
dissolved in a solvent mixture containing 180 ml ethanol, 34~ ml n-butanol,
,, 80 ~nl ~ater and 30 ml cellosolve, by mixing in a ball mill until completely ~ -
¦I dissolved, Filters were submerged in the zein solution, allowed to soak
!I for 16 hQurs9 removed and air dried.
¦ B. Toxoplaama antigen was immobilized on coated membrane filters
¦ by immersing the coat. d filters overnigm at 4C in 1. 5 ml of an antigen
I preparation containing 6mg - 12mg total protein, The filters were then air
dried at room temperature, washed with MilliRO (Reglstered Trade ~ark
2~ ; of Millipore Corporation, Bedford, Mass. ) water and again aiI dried at room temperature~
The ~a antigen preparation was a lysate o~ fresh Toxoplasrna
cells obtained from peritoneal exudates of infected mLce by techniques known
in the art.
.~, ., 19- ,
'' ',, :
. I ' . . . .. . . . . . ..
C~. Tlle second-s~age immobilization ~as carried O-lt a~ter the
immol~iliæation witll To-.;oplasm~ ;lntigen tvas c:ompleted ancl the ~ilters ~vere
dry. The filters were placed in a beaker containin~ a solution oE bovine
~ r~
gamma globulin ~raction II at a concentration of 20 mg per ml in ~IilliPIO
water and allo~ved to soak îor appro~;imatelS 16 hours. The filters were
T~iI
then air dried at room temperature, washed once with ~IilliP~O water and
air dried again at room temperature.
D. Serum to be tested was obtained ~rorn patient's blood samples
, which were allo~ved to clo$ to rernove red blood cells and clotting proteins.
The serum sample ~as diluted with an equal volume oE tris-saline buPfer,
5 (Tris-saline huFEer contained 0. l ~I 2-amino-2-hydro~cymethyl-1, 3-
propanediol and 0.15 ~ sodium chloride, pH 8, 0),
E. A coated membrane filter having immobilized Toxoplasma antigen
was mounted in a suitable holder to permi$ added fluid to enter one side of
li .
l the membrane, flow through the membrane pores and exit from the other ~l
I~ side of the membrane. Serum (l00 ,ul, diluted l:l in tris-saline buEfer) was
3 passed into the membrane's pores and allowed to remain there ~or 5 minutes
at room temperature. Serum was then washed out oE the filter by addition of
Il 1 ml tris-saline buf~er.
¦~ F. The ~ilter was then treated by the addition of 100~ of antibody-
¦, enzyme conjugate. The conjugate was composed of horseradish peroxidase
5 (Worthington, HPOFF) coupled to goat anti-human IgG ant~body (Meloy
Laboratories~ Inc., ~pringEield, Va., lot No. G5l621, 34. 2 mg/ml). Coupling
was carried out by the metaperiodate activation method, as descrihed by
Nakane, P,K, and Ka~vaoi9 ~., in ~. Histochem. Cytochem., vol. 22,
p. I084 ~1974). The conjugate solution added to the filter hacl 6.6 ,uglml
total conJugate protein and was diluted 500-fold from a stock solution~ using
l0% ~etal calf sçrum as diluent. The conjugate tvas incubated with the îilter
for 5 minutes, lthen washed l,vith 1 ml tris-saline bu~er.
,
-20-
G. Q~lalitative staining was done using 3-an ino-eth~Llcarbazole as
the cllromagen. Tlle chromagen (10 mg) ~ as clissol;-ed in 6 ml dimeth~
sulfo~ide, rollowed by the ~urther addition of aO ml 0. 02 ~ sodillm ace~ate
pH 5. O. Just before use, 0~ 5 ml o~ 3% (v/~-) h5~drogen peroxide was acided.
The solution was then allowed to react S minutes wi~n the components
immobilized on the membrane filter while flowing through the filter, The
presence of an~ibc dy to To~oplasma in the test serum was indicated b~- a red
color developed on the test filter. A filter treated ~Yith a sample of normal
serum remained white or faintly colored.
Example 2
. __
~; ~n this experiment, the e~fect of varying the incubation times for the
,i ~
', serum incubation step (as described in Example I/E) and for the conju~ate
i incubation step (Example I/F) was tested. The experimental procedure o~
~! Example I was followed, except that the second-stage immobilization ~step
1, I/C) was omitted. Test sarnples of serum were known to be negative ~lacking
Toxoplasma antibody) or positive (containing Toxoplasma antibody) by .he
!, fluorescentantibodytest, (Kelen, ~E., Ayllon-Leindl, L. andLabzoffsky,
¦I M.A., Canad. 3. Microbiol., vol 8, pp 545-554 tl962)).
¦ A. The time o~ serum incubation was varied, while conjugate
2~ I incubation time and aubstrate incubation time wère held constant. Results
are shown in Table 1, where a minus sign (-) indicates lack of color
development, and plus signs (+) indicate shades oE color on a scale îorm
i ligb~ pink (+) to doric pink (++~
" .':
,~
-21 -
. , .
"'' .. , . - . '' ~ ,'
Table
rr~lbe number j Serum I Ser~ Result
~ ~ontents ~ incubation I
l ! time
I (nli~lUteS) !
1 negative 1
2 negative 15
3 positive ~ ++~
4 positive 5 +++
l O 5 positive 10 1 +~+
6 positive 10 +++
7 positive 1~ ++-~
8 positive 15 _ ++~
The 5 minute samples developed a5 much color as the 15 minute samples,
indicating that a 5 minute incubation was adequate.
f ' e Xper;~
B. In -this _ 7 serum incubation tirne and substra~e
1, incubation time were held constant at S minutes, and conjugate incubation
,'~ time was varied ~rom 5 minutes to 15 minutes. The results are shown in
~! .
~, Table 2.
i' rrable 2
" Tube number Serum I Result ,
,~ contentsincubation
(minutes)
. .
2 5 ' 1 negative 15 +
, . 2 negative 15 -t
l l 3 positive 5 ++~
! 4 positive 5
positive 10
6 positive 10 ++-~
7 po sitive l S ~+ ~
l 8 positive_ _ 15 _ +~+
I
.
It was concluded that 5 minutes reaction times were adequate ïor both
.
the se~um incubation and the conjugate incu~ation.
Exampl_ 3
This representative experiment was designed to test the effectiveness
o~ the second-stage immobili~.~,ation, described in Example I/C, in reducing
the hackground ~olor oï negative control samples. The serum sarnples
employed were knowrl negative ~T ~coplasma antibody absent) or positive
(having ~ma antibody ) samples as previously determined by the
- 2 2 -
fluorescent antibody test, or the hemagglutination assay.
(Jacobs, L. and Lunde, M.N., J. Parasitol, vol. 43, pp 303-
314 (1957)). The tests were carried out as described in
Example 1, except that for one-step immobilization samples,
step I/C was omitted, and for two-step immobilization samples,
100~ (W/v) fetal calf serum was substltuted for bovine gamma
globulin in step I/C. The results are shown in Table 3. The
symbols employed have the same meaning as in Example 2.
Table 3
¦ Tube Sample Method of Result
Inumber Immobilization
. . . _ .
1 negative l-step +
2 negati~e l-step + .-
3 negative 2-step
4 negative 2-skep
negative 2-step _
6 negative 2-step _
7 positive 2-step ~
8 positive 2-step +++
~t was concluded that 2-step immobilization significantly
decreased background colour in negative samples, and improved
the colour differential between negative and positive samples.
The present invention relates to the detection of
antigens and antibodies in bodily fluids for diagnostic pur-
poses, exploiting immunochemical reactions. A combination of
factoxs and principles has been brought to bear in reducing
the time required to carry out the sequence o~ steps and in
improving the operating simplicity and economy of the entire
procedure.
In the preferred embodiment the use of zein or collagen
coated microporous membxanes pxovides signific~nt advantages
in the practice of
, ~ ' ' .
. . .
: ~ - 23 ~
4~Z','
the invention. ~irst, immobilization of specific antigens or antibodies is
readily effected. Second, the binding properties of coated micropo~ous
membranes make them sufficiently manageable that nonspeci~ic binding can
Il be suppressed to an acceptable level.
1¦ The method is applicable to any test fluid capable of flowing through
~¦ the type of structure described, or any fluid capable of being rendered
¦ Elowable therethrough, by dilution, prefiltration, and the like. The method
Il is suita~le for use with either EL-l or EL-2 assays, i. e., eiLher an antigen!j or an antlbody may be detected in the test fluid. Adaptation of the inventionfor the assay of a particular antigen or antibody is primarily a matter of
choosing the appropriate component to be immobilized on the structure, and
choosin~ the proper immunochemical component to be conjugated to an
enzyme.
While the invention has been described in connection with specific
embodiments thereof, It will be understood that it is capable of further
¦ modifications and this application is intended to cover any variations, uses,
¦ or adaptations of the invention followIng in general, the principles of the
¦ invention and including such departures from the present disclosure as come
~ within known or customary practice within the art to which the invention
ZO I pertains and as may be applIed to the essential features hereinbefore set
¦ forth, and as follows in the scope of the appended claims.
I ,~ ~ ~
! ~:
