Note: Descriptions are shown in the official language in which they were submitted.
Z~;03
B~CKGROUND OF THE INVENTION
This invention relates to a method for determin-
ing the presence of selected immunochemically responsive
substances such as antibodies or antigens in body fluids.
These selected substances will be referred to hereinafter
as analytes.
The determination of such analytes provides
much useful medical information. For example, pregnancy
testing, syphillis testing and blood factor testing are
all now done by conventional immunochemical methods.
Most of these methods involve a visual inspection for
the formation of precipitated or agglutinated antigen-
antibody complexes. These methods sometimes require
several replications at different reagent ratios to
insure accuracy and also require an individual visual
inspection for each test result. Use has also been
made of radioactive and fluorescent tags to avoid the
subjective visual determination. However, these methods
sometimes have problems either in the use of expensive
or transient tags, or in sensitivity or in safety and
handling of radioactive materials. The method of this
invention uses immune reagents tagged with particles
such as magnetic materials which are safe to handle
and which can be detected readily with highly sensitive
and readily available electronic equipment. This method
is readily adaptable to automated or semi-automated
operation.
~2~:50:~
The use of magnetic particles in serological
testing is known, but prior to this invention the magnetic
particles have been used only to remove or sequester various
components from a liquid sample. For example, U.S.
4,018,886 and U.S. 3,970,518, teach the use of magnetically
active particles to collect selected proteins, followed by
cleaving the proteins from the magnetic particles and a
visual examination for precipitated proteins. Hersh et al.,
U.S. 3,333,997, teaches the use of magnetic particles to
concentrate radioactive tags on a test substance.
None of the references teach the use of magnetic
detection to obtain the desired result.
SUMMARY OF THE INVENTION
The presence or absence in a body fluid of an
analyte, such as a specific antigen or antibody, can be
determined by depositing a sample of the body fluid on a
surface coated with a receptor reagent specifically ~eactive
with the analyte. If the analyte is present, it will form
a complex with the receptor reagent. The surface is then
contacted with an immune reagent comprising an immune
component which is specifically reactive with either the
receptor reagent or with a complex of the receptor reagent
and the analyte. The immune reagent also comprises particles
which have characteristics capable of affecting electrical
reactance. That is, they are minute fragments which alter
the permittivity, conductivity or magnetic permeability of
the surface. These particles will be referred to in this
case as reactance tags. ~etal or metal oxide particles are
preferred, and magnetically active particles are more
preferred. The immune reagent is generally applied to the
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surface as a liquid suspension. Unreacted immune reagent is
then removed from the surface and the surface is examined
for changes in dielectric constant, conductivity or magnetic
permeabiLity. In a direct test, the immune reagent is
reactive with the receptor reagent-analyte complex and if the
reactance tags are found to be attached to the surface, the
presence of the analyte in the body fluid is indicated.
Similarly, in an indirect test, the immune reagent is reac-
tive with the receptor reagent but not with the receptor
reagent analyte complex. If no reactance tags are detected
on the surface, the presence of the analyte in the body
fluid is indicated. In a competitive test th~ body 1uid
and immune reagent are deposited on the surface simultan-
eously. The amount of reactance tags on the surface is a
measure of the amount of analyte in the body fluid. This
system may be used manually, but it is readily adaptable to
automated operation.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a drawing of a test card useful in
this invention. Figure 2 is a schematic representation of
the reactions involved in this invention.
DETAILED DESCRIPTION OF THE INVENTION
Conventional means can be used to detect the
presence of the reactance tags on the test sur~ace. For
example, if the reactance tags affect the dielectric
properties of the surface, a capacitance measurement will
reveal whether the particles are present. In another
example, if the particles ar~ magneti~, magnetic pick up
heads such as those found in standard tape recording equip-
ment may be used. Capacitive, conductive and magnetic
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permeability measurements that do not require direct contactwith the test surface are preferred, but measurements using
direct contact between the surface and the detector are not
excluded. In order for the presence of the particles to be
detected, it is preferable that the test surface be inert
with respect to the property to be measured. However, it is
possible to arrange the receptor reagent on the surface in
bands or to induce a periodic magnetization on the surface
as that produced by a magnetic tape recording system and
then 'o move the surface at a particular rate past a detec-
tor which will monitor frequency. The detected frequency
will be a function o~ the arrangement of the bands of
receptor reagent or induced periodic magnetization and the
rate of mo~ement of the surface. Thus, the test surface
itself need not be entirely free from electrical reactance
to be considered inert for the purposes of this invention.
In a preferred system for detecting the presence of magnetic
tag, the test surface is adjacent to a reference surface
coated with magnetic particles. Following the immune reac-
tions, both the test surface and the reference surface canbe magnetized in~a periodic fashion. The detector circuit
can be made to detect only the portion of the signal from
the test sur~ace which is in place with the signal from the
reference surface. This arrangement decreases the effects
of system noise and provides increased sensitivity.
The test surface must also be capable of bonding
with the receptor reagent and non-interfering with the test
result. The surface can be flat or it can have depressions
or dimples in which each test is conducted. While many
materials are suitable test surfaces, plastic cards or
.ZZ~Q3
tapes are preferred. The test surface on which receptor
reagent is bonded may be within enclosed plastic packs or
pouches. These packs can also contain the immune reagents
in a separate area designed for addition to the test surface
following sample addition.
The presence of a change in electrical reactance
such as magnetic activity can be determined, even when a
very small amount of the reactance tags is present. There-
fore, each test will usually require only a droplet of body
fluid and the test area can be very small. Consequently,
a large number of discrete test areas and if desired,
re~erence areas, can be arrayed on a single card or tape.
The discrete areas may be either a series of tests for
different select proteins or a series of tests for the same
protein where the concentration of immune reagent on the test
surface is varied over a range to give an indication of not
only the presence of the select protein but also its approx-
imate concentration in the body fluid.
The receptor reagent can be attached to the test
~0 surface by surface adsorption, gel entrapment, covalent
bonding or other similar methods. Of these, covalent bond-
ing is preferred. Any system of receptor attacnment capa~le
of orienting the reange molecules on the tes~ surface so
that they will have maximum activity is also preferred.
Receptor reagents include not only antibodies, but also
tissue or cellular receptor proteins, serum transport pro-
teins and lectins. Of these, antibodies probably haYe the
widest applicability. Other classes of compounds, such as
chelating agents, may also ser~e as useful receptor reagents
if they react with the substance to be determined in the
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body fluid with sufficient specificity to avoid false results
caused by competing reactions.
The materials useful as reactance tags which
combine with immune compounds to form the immune reagents
are those which will alter the electrical reactance of the
test surface. That is, these materials, if distributed as
a finely divided powder on the test surface, alter the
dielectric, conductive or the magnetic properties of the
surface. The advantage of this invention resides in using
particles which are detectable in very small quantities by
very sensitive but well developed and readily available
electrical components. Further, the use of such materials
avoids the problems of transient activity and handling
hazards which one encounters in the use of radioactive tags.
The preferred materials are metals and metal oxides. The
more preferred metals and metal oxides are those which
exhibit ferromagnetism. Such materials can be reacted with
the antibodies and applied to the test surace while in a
demagneti~ed s-tate. Once they have been applied, the entire
surface can be exposed to a magnetic field to magnetize the
particles for detection purposes. This magnetic activity
is then readily detectable by various well-known means such
as a standard magnetic tape system or Hall effect detector.
Magnetic materials include, but are not limited to, metals
and alloys such as iron powder, nickel, cobalt, CrO2,
FERROFLUID* (a ferromagnetic liquid produced by Ferrofluids
Corp.), CoO, NiO, Mn203, magnetoplumbites, magnetic iron
oxides and ALNICO*, an alloy of aluminum, nickel cobalt
and copper. Other us~ful materials are organic charge
complexes with the high electrical conductivity such as
* denotes trade mark
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N-methylphenazinum tetracyanoquinodimethane, ~Ce(NO3)6Mg
(H2O)~]3-6H2O crystal, Bi2Se3 crystals and tetrahiofulvalene
complexes with tetrocyano-p-quinodimethane or K2Pt(CN4)BrO3-
H2O and amorphous materials with magnetic properties such
as the chalcogenides, e.g., the europium chalcogenides and
chalcogenide glass particles.
Preferred magnetic materials are the magnetic
oxides of iron,cobalt, nickel, chromium and maganese and
oxide coated particles of iron or nic~el.
In order to provide sufficient surface area for
bonding the immune compounds, it is pre~erred that the
reactance tags have a large surface area, at least 100 m2/gm.
Particles in the range of 0.01 to 50~m in diameter are
preferred.
These materials can be bonded to immune compounds
by known techniques. The reactance tags can be encapsulated
with an organic polymer to which the immune compounds can
be bonded, or the surface can be silanized by conventional
techniques. Organic compounds can then be attached to the
silane linkage. U.S. 3,954,666 teaches polymer encapsulation
o~ core materials and U.S. 3,983,299 teaches the use of
silane linkages to bond organic compounds to inorganic
particles. Techniques for immobilization of en~ymes on
magnetic supports which would be equally applicable to
antibodies are described in methods in Enzymology, XLIV
pp. 324-326. The manner of attachment of the reactive
organic compounds to the reactan~e tags does not form the
basis of this invention. Thus, other bonding methods may
also be used so long as they do not interfere with the
complexing ability of the immune compounds. The immune
~l~2Z,~C~3
compounds are chosen to be speciically reactive with either
the receptor reagents or with the complex of the receptor
reagent with the body fluid substance to be determined.
Since the method of this invention utilizes
electronic rather than visual inspection it is readily
adaptable to automated operation. As used herein, the term
"automated" is n,ot meant to exclude the possibility that
some of the operations may be performed manually. Referring
to Figure 1, a preferred test card 1 is shown having several
areas 2 on which receptor reagents are bonded. Separate
areas having different receptor reagents can be provided or
the same receptor reagent can be bonded to each area in
different concentrations. The card has a printed test
identification 3, as well as a machine readable code 4,
which contains the necessary information for automated
operation. Optionally, the card can have a space 5, for
entering a pati~nt identification code.
Figure 2 shows schematically a test area 2 of a
test card of Fig. 1. Receptor reagents lQ are bonded to
the test area. Patient body fluid containing the antibodies
11, the presence of which is to be determined is added to
each test area. The antibody 11, if present, forms a
complex 12 with the receptor reagent. The test area is
then exposed to immune reagent 13 comprised of an immune
compound 14 bonded to a reactance tag 15~ This i~mune
reagent forms complex 16 ~ith the receptor reagent-patient
antibody comple~, if present, on the test area.
A preferred system for the practice o~ this
invention would include a station for entering the ne&essary
test cards for the desired tests. Cards ma~ be entered
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individually or in group as required. The cards are moved
automatically to a sample addition station where a droplet
of a patient's body fluid is applied to each of the test
areas on the card. An automated device may include
temperature control and a station for equilibration o~ the
~ody fluid on the test areas for predetermined times. The
card is then moved to a station where the immune reagent is
applied to each of the test areas. Again, there may be
temperature control and equilibration systems incorporated.
Next, excess immune reagent is removed, and the card is
then examined for the presence of reactance tags remaining
in the test by measuring changes in electrical reactance o
the card test areas.
It should be noted that in the particular embodi-
ment described the test card is moved through each station.
Other embodiments where each operation is conducted on a
card maintained at a single location in an automated or
semi-automated device are equally operable.
The method of this in~ention can be applied to
the detection of a wide variety of materials including but
not limited to antigens and antibodies of viral, bacterial,
cellular and human origin in addition to hormones, drug
me~abolites and specific proteins. For example, the
method is applicable to the detection of human auto-
antibodies, e.g, an antithyroglobulin; antigenic proteins
such as complement factors, protein hormones, immunoglobu-
lins and free light and heavy chains, e.g , th~roxine-
binding globulin, and various species of microorganism
antigens and antibodies such as hepatitis A and B virus.
Application o~ this approach is primarily dependent on the
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availability of a suitable material to serve as a receptor
reagent specifically reactive with the item to be determined.
EX~MPLE 1
In the determination of a human antibody, IgG, a
serum sample can be prediluted with a protein buffer solution
containing 0.12~ bovine serum albumin and phosphate buffered
saline at pH 7.7 + 0.2. Sodium azide, 0.1~, an antimicro-
bial agent can be added in the diluent as a preservative.
The diluted patient sample can be quantitatively
transferred to a test reaction card. The solid phase
support consisting of anti-human IgG covalently bonded to
derivatized polyamide sheets is prepared as follows:
Nylon-6 film tlO g) can be suspended in 3N HCl
(300 ml~ and stirred at 30C for four hours, removed from
the solution and washed exhaustively with water, ethanol
and ether. The carboxyl content of a Nylon-6 film hydro-
lyzed for four hours is about 30-90 ~MoLes/gm.
The coupling of the antibody to the depolymerized
Nylon-6 support can be carried out by carbodiimide activa-
tion. The overall reaction involves the condensation~etween the carboxyl group on the support and the amine
groups of the antibody. The reaction is carried out in
two steps.
One gram of the acid-treated Nylon-6 film is
added to a solution of 50 mg of 1-cyclohexyl-3-(2-morpho-
linoethyl)-carbodiimide metho-p-toluenesulfate in 10 ml of
water. The reaction mixture is adjusted to pH 4-5 with
6N HClr and the reaction allowed to continue at 30C for
two-four hours. The film is then removed and washed several
times with water to remove the excess carbodiimide.
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The acti~ated film is then introduced into a
solution containing commercially available anti-numan IgG
in concentrations of 1 to 5Q mg protein in 10 ml of water.
After adjusting the solution to pH between 4-5, the reaction
is continued for two to four hours at room temperature or
overnight at 4C. The reaction solution is then decanted
and the film washed with water or buffer.
The test surface can then be equilibrated with
the diluted sample for sufficient time to permit antibody
binding to o~cur. The first equilibration may typically
be about 30 minutes at 32C. The reaction zones can then
be washed with the same protein buffer solution and shaken
to wash unabsorbed antigen from the surface.
The test reagent`zones can then be immersed in an
excess of goat anti-human IgG tagged with magnetic particles.
For equilibration on the card, the immune particles are
appropriately diluted in phosphate saline buffer at pH 7.5
+ O.5 containing 0.12% bovine serum albumin. After equili-
bration, the test reaction suxfaces can be washed witn the
BSA phosphate buffer at pH 7.7 + 0.2.
The reaction surfaces are magnetized in a magnetic
field and the test card processed through a detector capa-
ble of determining the presence of magnetic particles.
E ~LE 2
-
This Example demonstrates the determination of
antibodies directed against specific antigens.
A serum sample was prediluted with a protein
buffer solution containing 0.1~ bovine serum albumin and
Tris-(hydroxymethyl)-aminomethane (Tris) buffered at pH6.5
+ O.4. Sodium azide, 0.1~, an antimicrobial agent, was
added in the diluent as a preservative.
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The diluted sample was quantitatively transferred
to a test reaction surface. The solid phase support consist-
ing of human serum albumin covalently bonded to derivatized
to either polyamide or polymethacrylic acid polymers was
prepared as follows:
The overall coupling reaction between albumin
and films can involved cross linking bet~een the amino or
carboxyl groups of the film and protein. Cross linking
can be achieved through the use of a polyfunctional aziri-
dine reagent which is highly reactive with materials con-
taining active hydrogens.
The films for attachment of the human serum
albumin were first suspended in a 1~ solution of X~A poly-
functional aziridine ~Cordova Chemical~ Sacramento,
California). Following stirring at 27C for thirty minutes,
the films were removed from solution and wahsed with dis-
tilled water. The washed films were then stirred in a
solution containing human serum albumin (lmg~ml) ror twelve
hours at 4C. The immobilized albumin film reatent can then
be washed repetitively with Tris buffer (pH6.5 + 0.4) at
4C. The washings are continued until no further albumin
is released from the film. Representative levels of
albumin attachment to the films are set out below:
Film (~g/cm?)
Surface Groups
Surlyn~ 3.3
Mylar~ ionomer resin
polyester (Flame Treated) 7.3
No Surface Groups
Polycarbonate
Nylon (HCl Treated) 16.0
Activated Surface Groups
Ethylene Maleic Anhydride <1.0
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The test surface for anti-HSA was equilibrated
with the diluted sample for sufficient time to permit anti-
HSA binding. The reaction surface was then washed with the
same Tris buffer solution (pH7.4 + 0.4) used in Example 1
to remove unattached sample debris from the surface.
The test reasent card was then immersed with
excess of goat anti-human serum albumin tagged with magnetic
particles. For equilibration, the immune anti-HSA particles
were appropriately diluted in a Tris buffer a pH6.5 + 0.5
containing 0.12~ goat serum albumin. After equilibration
the test reaction surfaces were washed free of unattached
particles with the goat serum albumin Tris buffer.
The magnetic particles remaining on the reaction
surface can be magnetized in response to a magnetic signal
of known frequency. The test card can then be processed
through a detector, capable of detecting the presence of
the magnetic particles. The detection of the reactance
properties of the test surface can be adided by the instru-
ment techniques of phase lock application, phase sensitive
~0 rectification and other standard electronic amplification
systems known to those skilled in the art.
As used herein, the phrase "consisting essentially
of" is not use~ to exclude tne additional operation steps
or elements which do not prevent the advantage of this
invention from being realized.
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