 |
Description  |
|
|
This invention relates to media for use in direct and indirect bioassay
processes and to the method of making and using such media.
Competitive radioimmunoassay has been employed as an analytical technique
for several years and the general procedure has been extended into
non-immune systems as described by Solomon A. Berson and Rosalyn S. Yalow
in IMMUNOBIOLOGY, R. A. Good and D. W. Fisher, Editors, Chapter 30,
"Radio-immunoassay-Current Status", pages 287-293, (Sinauer Associates,
Inc., Stamford, Conn.). As conventionally carried out, competitive
radioassay procedures involve mixing a sample of solution containing a
test substance, e.g., a hormone, with specific known amounts of antibody
followed by the corresponding radioactively labelled hormone to which the
antibody was made, after which the mixture is incubated for an extended
period of time, usually a day or more, to permit the labelled hormone to
compete with the unlabelled hormone in approximate proportion to their
relative concentrations for binding the antibody. Thereafter the residual
labelled free hormone is separated from the mixture, e.g., by adsorption
on talc or charcoal, by paper chromatoelectrophoresis or double antibody
precipitation, and the radioactivity in the complex of bound
hormone-antibody is measured with an isotope counter. By comparing the
count with that obtained using a solution containing a known quantity of
hormone, the amount of hormone in the test sample is determined. Another
method for performing the competitive radioimmunoassay includes mixing of
specific amounts of labelled antigen with the test sample containing
unknown amounts of unlabelled antigen, then bringing both simultaneously
into reaction with the antibody and finally following the previously
described separation and counting procedure. In still another competitive
assay procedure, the test antigen can be brought into contact with a
complex consisting of the labelled antigen and specific antibody. The
amount of radioactivity released is proportional to the concentration of
the antigen in the test solution.
Non-competitive or so-called sandwich technique radioimmunoassays are also
used to determine the concentration of an antigen in test samples. In this
technique the antigen in the sample is first reacted with immobilized
antibody to form an antigen-antibody complex. Radioactive antibody, either
antigen specific or antibody specific, is caused to react with the
complex, whereupon the labelled antibody occupies a number of sites
proportional to the amount of antigen in the test solution and can be
measured after removing the unreacted labelled antibody by washing.
The competitive and non-competitive assays may involve the use of
indicators other than radioactivity. U.S. Pat. No. 3,641,235 describes the
use in filter paper of indicator dyes attached to immobilized antigens (or
antibodies) which release in proportion to the amount of antibody (or
antigen if the antibody is immobilized). The released dye can then be
measured by fluorimetry, spectrophotometry, refractometry or the like and
related to concentration of the antigen (or antibody) being determined.
U.S. Pat. No. 3,654,090 describes the determination of a component of the
antigen-antibody reaction using one component (antigen or antibody) in an
insolubilized form and the other one covalently linked to an enzyme. The
enzyme such as glucose oxidase, peroxidase, etc. is released upon reaction
and can be determined by a reaction either with a colored substrate, or it
may itself yield a colored end product, as described for example in Weiss
U.S. Pat. No. 3,641,235 in which there is described the application to a
strip of filter paper a mixture of immunological reagent particles and
fluorescein, after which the fluorescein is released and transported by
capillary movement of an antiserum sample. A variety of such reactions are
known. In addition, direct measurements of the captured or released
materials, or of materials which after release have been recaptured, can
be made where applicable by refractometry, interferometry, fluoresence,
bioassay or spectrophotometry, turbidimetry, bioluminescence, etc.
The assays described are not limited to immune systems. Enzymes and their
substrates can be determined as well. In these determinations the enzyme
or substrate is insolubilized depending upon which substance is to be
determined. If the enzyme is to be determined, the substrate is
insolubilized. If the substrate is to be determined, the enzyme is
insolubilized. The test solution is brought into contact with the
immobilized enzyme (or substrate) whereupon reaction occurs forming an
immobilized complex. Radioactively labelled substrate (or enzyme) is
introduced and reacts at unoccupied sites. Determination of the
radioactivity in the complex gives a measure of the amount of enzyme (or
substrate) in the test sample. In a modification of this procedure, a
specific amount of the radioactively labelled enzyme (or substrate) can be
added directly to the test sample and a competitive one step reaction
effected after which determination of the radioactivity present in the
immobilized complex provides a measure of the enzyme in the test solution.
Indirectly, enzyme inhibitors can be measured by these processes by
determining the degree to which the enzyme-substrate complexing is
inhibited and comparing with proper controls.
All of these bioassay procedures involve in common the reaction of the test
substance with an appropriate reagent, e.g., hormone-antibody,
antigen-antibody, enzyme-substrate, or their converse reactions, together
with direct or indirect quantitative measurement of the amount of reagent
so reacted by measurement of a characteristic of the reagent (or of
another substance, an indicator, which has reacted with the reagent), such
as color, radioactivity, or other physical characteristics.
The present invention provides a solid medium, preferably a gel medium, for
use in such assay procedures in which there is disposed in contact with
the medium and fixed against diffusive movement therethrough a fixed
quantity of desired reagent capable of reaction with the test substance,
the reagent being confined to a localized, restricted or defined zone or
aea of the medium. The reagent is immobilized or fixed against diffusive
movement through or across the medium preferably either by bonding it
chemically to the substance of the medium or by bonding it chemically or
physically, e.g. by adsorption to a particulate solid material insoluble
in the gel medium, the particles being dispersed or embedded in the zone
or area of medium and being sufficiently large, i.e., having at least one
dimension greater then 0.01 so that they are incapable of substantial
movement through the medium at ordinary temperatures, i.e., from 0.degree.
to 60.degree.C., the particles thus serving as anchors to fix the reagent
in place in the desired zone or area of the medium.
The medium can be used in bioassay procedures by applying to it a sample of
the test substance and a known quantity of an indicator substance also
capable of reacting with the reagent. In the preferred method, the
indicator is applied to the medium at a zone or area more remote from that
of the reagent than is the test substance so that the test substance will
come into contact with and react with the reagent before the indicator
reaches and reacts with it. Diffusion of the test substance and of the
indicator through or across the medium so as to react sequentially with
the fixed reagent can be accelerated by any of the usual means such as
gravity, centrifugal force, osmotic pressure, or electrical field. After
the test substance in the sample has completely reacted with the fixed
reagent and the indicator substance has reacted sequentially with the
excess or remainder of the fixed reagent, the residual unreacted indicator
is removed from the zone or area by washing and/or continuance of the
diffusion process. The amount of the indicator which has reacted with the
reagent and which consequently remains in the desired zone or area is then
measured by determining a suitable characteristic such as radioactivity.
From this value it is possible to determine the proportion of the fixed
reagent which first reacted with the test substance, hence the quantity of
the test substance present in the sample.
The medium of the present makes possible a great increase in the speed of
the assay procedure, shortening the total time required to as little as 2
hours or even less, and in addition eliminates the precipitation step and
separation of the precipitate which is involved in many of the
conventional assay procedures. The medium may be any of the solid media
commonly employed for molecular diffusion procedures such as any of the
usual materials which form gels when exposed to water or aqueous systems
and which are commonly employed as media for chromatography or
electrophoresis. Such materials include cellulosic materials such as
micro-crystalline regenerated cellulose, hydroxypropyl cellulose,
carboxymethyl cellulose, and diethylaminoethyl cellulose, as well as
non-cellulosic materials such as polymers and copolymers of acrylamide,
hydroxyacrylate or hydroxymethacrylate polymers or copolymers, gelatin,
and various polysaccharides such as starch, dextran, agar, agarose and
carrageenan, cross-linked forms of any of the foregoing as well as other
water sorptive gel-forming solid materials as well as mixtures of any two
or more of the foregoing. The medium preferably is in the form of a sheet,
film or layer of the material of the order of 0.1 to 2 millimeters in
thickness when hydrated and supported on a relatively stiff backing sheet
or film such as glass or polyester or cellulose acetate film of the type
used for photographic film, which support is inert to the test substances
and reagents employed.
The reagent which is fixed or anchored in the gel medium may be any of
those employed in conventional assay procedures. In general, these
reagents are proteinaceous or peptidal, steroidal, terpenoidal,
polynucleosidic carbohydrate, or polysaccharide materials, the precise
identity in each case depending, as is well known, on the identity of the
test substance. For example, in an immune system, for test substances
containing antigens such as hormones, the reagent employed is the
appropriate antibody; among the hormones to which the procedure has been
applied are insulin, ACTH, parathyroid hormone, glucagon, proinsulin,
vasopressin, oxytocin, gastrin, calcitonin, testosterone, estradiol,
aldosterone, estrone, and dihydrotestosterone. Antibodies are also used as
reagents in immune systems for other antigens such as intrinsic factor,
esterases and other enzymes, digitoxin, Australian antigen, alkaline
phosphatase, tumor-associated antigens such as alpha fetoglobulin,
carcinoembryonic antigen, and other tumor-associated antigens, rheumatoid
factor, folic acid, neurophysin and morphine. In non-immune systems in
which the test substance is a hormone such as thyroxine, cortisol,
corticosterone, cortisone, 11-desoxycortisol, or progesterone, there may
be used as the reagents any of the specific binding proteins in plasma, as
is well known, while for test substances such as vitamin B.sub.12, folic
acid, cyclic AMP, GMP, or messenger RNA, there may be used as the reagent,
respectively, intrinsic factor, FA reductase, phosphodiesterases, and
complementary DNA. Similarly, for test substances which are enzymes, the
corresponding substrate may be used as the reagent. It is also possible in
all cases to reverse the procedure, e.g., when the test substance contains
an antibody or binding protein from plasma, the corresponding antigen or
hormone can be used as the reagent.
The reagent may be chemically bonded to the material of the medium itself,
using appropriate coupling agents which do not interfere with its
reactivity toward the test substance, or it may be chemically bonded,
either directly or by means of appropriate coupling agents, to a solid
material different from and insoluble in the material of the medium, which
solid material is embedded or dispersed in the medium and is in the form
of sufficiently large particles or pieces (at least the dimension greater
than 0.01 micron) so as to be immobile and incapable of movement by
diffusion through the medium even under the influence of the accelerating
forces employed during the test procedure. It may also be adsorbed onto
the surfaces of or absorbed into certain solid materials and held there by
physical or chemico-physical forces which are not primary valence bonds.
In addition, in the case of some reagents, it is possible by reacting them
with appropriate chemical cross-linking agents to convert them into
insoluble solid materials which, while retaining their desired reactivity
with the test substance, are in the form of sufficiently large molecules
or particles to be immobilized within or on the surface of the medium and
not subject to transport by diffusion.
The size and shape of the zone or area of the medium within which the
reagent is confined or fixed is a matter of choice provided that it is
less than all of the medium. It may and preferably does extend throughout
the thickness of the medium when the latter is in the form of a sheet or
film, and its location, size and shape as well as the number and spacing
when more than one is present can be varied depending upon whether or not
external forces are to be applied to accelerate diffusion during the assay
procedure, and to facilitate proper positioning of the sample of test
substance and of other substances for rapid and effective conduct of the
assay.
The coupling agents which can be used to bond the reagent chemically to the
material of the medium or to another solid material are well known and
function through well known reactive groups. In the case of materials
containing hydroxyl groups such as cellulosic materials and
polysaccharides, cyanogen halides such as cyanogen bromide can be used as
the binding agent by reacting it first with the hydroxyl-containing
material, then bringing proteinaceous or peptidal reagent into contact
with the thus activated material. In the case of acrylamide polymers,
glutaraldehyde may be similarly used as a binding agent by first reacting
it with the polymer. It is also possible to react the hydroxyl-containing
materials, after activation with cyanogen bromide, with alkylene diamines,
then couple them to proteinaceous reagents by using a water-soluble
carbodiimide, or an 0-bromoacetyl-N-hydroxysuccinimide as coupling agent.
The latter coupling agent can also be used to couple with reagents which
contain peptide, amino, phenolic hydroxyl, or imidazole groups. Other
coupling agents which can be used to replace the foregoing for coupling to
a variety of reagents include succinic anhydride, diazotizing agents, and
mercapto compounds, as taught by Cuatrecasas, Nature, 228, 1327-8, and
Biochemistry, 11 (12), 2291-9. Many other coupling agents and processes
have been described in the literature for chemically bonding materials
containing hydroxyl groups to enzymes, antibodies, antigens, steroidal
hormones, proteins, and peptides.
The solid materials onto the surfaces of which the reagents can be adsorbed
for embedding in the gel material can be in the form of films, sponges,
sieves, gauzes, fibrous webs or the like but are preferably in the form of
small particles ranging in size from 0.01 microns to 100 mils or more in
diameter in order to provide a large surface to volume ratio. Among useful
materials are Fuller's earth, talc, porous glass beads, hydroxy apatite,
zirconyl phosphate, charcoal, polyethylene, polypropylene, or polystyrene
particles, ion-exchange resins such as polysulfonate or polycarboxylate
resins, diethylaminoethyl cellulose, or the like.
Reagents which can be insolubilized by reaction with cross-linking agents
comprise antibodies, enzymes, and proteins, including protein antigens,
all of which can be reacted with such cross-linking agents as
glutaraldehyde or ethyl chloroformate, or glyoxal.
The bound reagents may be placed in or confined to a localized zone of the
medium by various means. For example, the binding agent may be reacted
with a solid material in the form of a sheet, web, sponge, sieve, gauze or
in the form of a mass of granules or particles after which the solution of
reagent is brought into contact therewith (e.g., by electrophoresis or
diffusion) and allowed to react so as to become bonded thereto. A measured
quantity of this complex can then be fixed in or adhered to any desired
portion or zone of a conventional sheet or layer of gel medium used for
chromatography or electrophoresis. A plug or insert of the complex of the
appropriate size and shape can be inserted in a well or opening cut in a
conventional sheet or layer of medium and can be bonded or adhered in
place, or the well can be filled with a mass of the complex in granular
form which is adhered in place by any suitable adhesive, including
gel-forming materials of the same type useful for forming the medium
itself. The gel-forming material is normally tacky when swollen with water
and is readily adherent to itself under these conditions. A layer of
granules or particles of the complex may also be bonded to the surface of
a conventional sheet or layer of gel-forming material, being confined to
the desired location by means of a fence or jig or other masking device
until it has become adequately adhered.
Instead of bonding a plug of complex into a well in a conventional sheet of
medium, the plug may be adhered by conventional procedures to a suitable
backing or support sheet, e.g., glass or polyester such as polyethylene
terephthate, and then a sheet or layer of conventional gel-forming
material can be formed or deposited on the backing around the plug. It is
also possible to bring the binding agent into contact with only a
restricted localized zone of a conventional sheet or film of paper or
cellulosic material or of hydrated or hydratable gel material, the extent
of contact (and hence of reaction) being confined to the desired zone by
means of a mask or jig, and thereafter to bring the desired reagent (or a
solution thereof) into contact with the same zone to cause the reagent to
become bonded in that zone only.
In the case of media made of gel-forming materials which can be dried for
storage and then rehydrated immediately prior to use, as for example
agarose made in accordance with U.S. Pat. No. 3,527,712, the medium of the
present invention containing reagent bonded to a localized zone thereof
can also be dried, for example by lyophilization, for storage and can be
rehydrated shortly before use for radioassay molecular diffusion
procedures. In a preferred embodiment, the medium of the present invention
consists essentially of an inert flexible backing or support film to one
face of which is bonded a layer or sheet of dried rehydratable agarose
having a localized zone of restricted area much smaller than the total
area of the layer or sheet within which zone there is bound to the
agarose, either at the exposed surface of the layer or throughout the body
of the layer or both, the desired reagent.
The medium of the present invention is used for bioassay by first hydrating
it, in the case of media in the form of dry hydratable gel-forming
materials, and then applying a sample of the test substance, which may be
but need not be in the form of a solution or dispersion in water or in an
aqueous medium, the sample being applied either directly to the zone
containing the fixed reagent or to a portion of the medium spaced from the
margin of the zone, after which it is permitted or caused to diffuse into
the zone by conventional procedures to enter into the desired reaction.
The amount or proportion of the fixed reagent which has reacted is then
measured by any of the direct or indirect procedures described, using
appropriate indicators as desired. When an indicator material is used, it
is brought into contact with the zone containing the fixed reagent, after
the test substance has reacted with the reagent, in order to react
irreversibly with residual reagent, and any excess of the indicator is
removed by diffusion out of the zone into the other portion of the medium,
or indeed out of the medium completely, using any of the conventional
means for accelerating diffusion if desired. The amount of indicator
remaining in the zone is then determined by measurement of its
characteristic property--e.g., radioactivity, biological or chemical
activity, color, luminescence, fluorescence, or light refractivity. The
results can be compared with those obtained by following the same
procedures but using control samples containing known quantities of the
test substance.
The following specific examples are intended to illustrate more fully the
nature of the invention without acting as a limitation upon its scope.
EXAMPLE 1
Beads of aqueous agarose gel such as those used for affinity chromatography
are activated by reacting with cyanogen bromide, then reacted or coupled
with antibody associated with hepatitis (HB Ab) to bond the antibody
reagent to the agarose. The beads are then suspended in an aqueous
solution (0.5%) of agarose at 37.degree.C. which is then poured into a
petri dish and allowed to gel, forming a layer about 1 mm. thick
containing the antibody reagent fixed within it.
A conventional counterelectrophoresis strip (MCI Biomedical Type 35-II)
consisting of a layer of dried rehydratable agarose prepared as described
in U.S. Pat. No. 3,527,712 bonded to a flexible polycarbonate support film
having a row of spaced wells 4 mm. in diameter arranged along each margin,
opposite each other, is immersed in water for 45 minutes to rehydrate it,
then in 0.05 molar sodium barbital at pH 8.2 for 15 minutes to prepare it
for electrophoresis.
Discs 4 mm. in diameter are cut from the layer of gel containing the fixed
antibody reagent by means of a cork borer, and each disc is placed in a
well on the anodal side of the strip and adhered in place with a warm
aqueous solution of agarose to provide a capture zone.
The medium thus prepared is used for measuring hepatitis antigen in test
substances, i.e., the sera of patients, by placing 10 microliters of each
sample of serum in a cathodal well of the medium opposite one of the plugs
containing fixed antibody. A standard sample containing a known quantity
of hepatitis associated antigen (HB Ag) is placed in another of the
cathodal wells, and in still another is placed a negative control. The
strip thus prepared is placed in a standard electrophoresis apparatus
using a buffer solution the same as that with which the strip was
equilibrated and subjected to electrophoresis for 60 minutes at 45 ma and
30 V. D.C. At this point there is introduced into each of the cathodal
wells 10 microliters of a standard indicator solution of radio-labelled
antigen (HB Ag containing bound I.sup.125) and electrophoresis is
continued for another 60 minutes.
The medium or strip is then removed from the electrophoresis unit, washed
for 2 hours with stirring in aqueous 0.85% sodium chloride, rinsed in
distilled water, and dried in air at 60.degree.C. Each of the capture
zones containing the fixed antibody is then removed by means of a punch
and counted in an isotope counter. The results obtained from the test
substances can be compared with the results from the known control and the
negative control.
If desired, a series of known samples of different amounts of HB Ag can be
subjected to the procedure to provide a standardization or calibration
curve to facilitate precise movement of unknown samples.
EXAMPLE 2
Beads of aqueous agarose gel are activated with cyanogen bromide as in
Example 1, then reacted or coupled with purified gamma globulin (Ig G)
reagent. The beads are then fixed in a well of a conventional
electrophoresis strip as described in the preceding Example to provide a
medium having a plurality of capture zones, each containing fixed Ig G
reagent, along the cathodal side of the strip.
The medium thus prepared is used for detection and analysis of antibodies
to gamma globulin by placing 10 microliters of each of the test samples of
patient serum in the opposing wells on the anodal side of the strip and
subjecting the strip to electrophoresis as in the preceding example,
followed by adding 10 microliters of solution of radioactive anti-IgG
antibodies as an indicator, further electrophoresis, washing, drying and
counting as before. A sample or series of samples of solutions containing
known quantities of antibodies are used for calibration.
EXAMPLE 3
A medium having capture zones is prepared as described in the preceding two
examples except that anti alpha-1-fetoglobulin antiserum is used as the
reagent. The medium or strip thus prepared can be used for determination
of the tumor-associated antigen known as alpha-1-fetoglobulin, employing
radioactive alpha-1-fetoglobulin as the indicator. The results are
compared with a calibration curve prepared using samples containing
various known quantities of the antigen.
Example 4
A medium having capture zones is prepared as described in Example 1 except
that the agarose beads, after activation by reacting with cyanogen
bromide, are further reacted with NH.sub.2 (CH.sub.2).sub.10 NH.sub.2,
then coupled with the HB Ab using
1-ethyl-3-(3'dimethylaminopropyl)-carbodiimide. The medium can be used in
the same manner as described in Example 1.
EXAMPLE 5
A medium is prepared as described in Example 1 except that there is used
instead of the beads of aqueous agarose gel beads of cross-linked dextran
aqueous gel. The medium is used as described in Example 1.
Similarly, there can be substituted for the beads of agarose in Example 4
porous glass beads or cross-linked dextran beads activated with
p-isothiocyanatophenoxy hydroxy propyl chloride. Also, the antibody and
antigen of Example 1 can be interchanged, i.e., the antigen can be fixed
in the medium and used as the reagent for a test substance containing the
antibody. The beads of agarose of Example 1 can also be replaced by
particles of cellulose and the cyanogen bromide replaced by
chloro-s-triazine, cyanuric chloride, or 1-(m-nitrobenzyloxy) methyl
pyridinium chloride. The agarose beads can be replaced by particles of
nylon (a superpolyamide) or by particles of aqueous polyacrylamide gel or
of diethylaminoethyl cellulose and the cyanogen bromide replaced by
glutaraldehyde. The cyanogen bromide of Example 1 can also be omitted and
there can be used, in place of the agarose beads, beads of
E-aminocaproyl-D-tryptophan methyl ester of agarose, or beads of
N-hydroxysuccinimiide ester of succinylated aminoalkyl agarose, or of
polypropylene or polystyrene, the latter two polymers adsorbing the
antibody on their surfaces. The cyanogen bromide and agarose beads of
Example 1 can be replaced by zirconyl phosphate or by tanned red blood
cells.
In Example 4, the carbodiimide can be replaced by
0-bromoacetyl-N-hydroxysuccinimide.
There may also be used in place of the antibody reagent of Example 1 an
antibody containing an indicator dye as described in U.S. Pat. No.
3,641,235; in this case the radioactive indicator need not be used, the
color liberated by the antigen of the test substance being used as the
indicator. Similarly, a conjugate of antibody and enzyme as described in
U.S. Pat. No. 3,654,090 can be used as the reagent in place of the
antibody of Example 1; the radioactive indicator can be dispensed within
this case also, the enzyme released from the conjugate by the test
substance containing the antigen serving as the indicator.
Glucose oxidase can be substituted for the antibody in Example 1 and used
as a fixed reagent to detect and measure glucose in a test sample, a leuco
dye solution being used as the indicator for the hydrogen peroxide
released from the reagent by the glucose. To determine the enzyme pyruvic
kinase (PK) in a test substances, phosphoenolpyruvate substrate can be
substituted for the antibody in Example 1, and a solution of adenosine
diphosphate used along with the test substance. The pyruvate released can
be determined using as an indicator an enzyme such as lactic dehydrogenase
and standard color reagents.
* * * * *
|
|
 |
|
 |
Description |
|
