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Description  |
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The present invention relates to analytical systems using reagents
molecularly tagged with radiant energy emitters, and more particularly to
reagents useful in the detection of antigenic-type reactants and a method
of using that reagent for the stated purpose.
The technique of detecting and classifying reactants in a highly specific
reaction, such as the antigen in an antigen-antibody reaction, by tagging
one of the reactants (such as the antibodies) with a radiant energy
emitter, is known in the art. This method depends upon the ability of the
observer to distinguish between the product or complex of the antigen with
tagged antibody, and the uncomplexed tagged antibodies. For this purpose
various radiant energy detection instruments may be utilized depending on
the nature of the energy emitter.
Current methods for detecting antigens with tagged antibodies suffer from a
number of serious disadvantages, particularly the lack of sufficient
signal level associated with small quantities of antigen. Many cases of
serious disease having an antigenic etiology thus remain unnoticed,
resulting in unnecessary suffering, and in some cases, death.
In cases where the radiant energy emitter is radioactive, the reagent
containing the tagged antibody produces a potential hazard to the
manufacturers of the reagent and to the clinical laboratory people
carrying out an assay with the reagent. Additionally, transportation of
such radioactive reagents is becoming subject to increasingly onerous
restrictions. The photoemission from each radioactive atom is, or course,
very meager. Lastly, if the half-life of the radioactive emission is
short, as is often the case, the signal level of the radioactive emission
may diminish so rapidly that the shelf life of the reagent is severly
limited.
In many cases, antibody tagging is done with a coupled fluorescent dye
molecule, e.g. either a dye which is capable of fluorescent emission with
a reasonably high quantum efficiency when directly excited by radiation in
its absorption band, or a fluorochrome dye which fluoresces with a
substantially greater quantum efficiency when bound to the antibody than
when present as a free dye molecule. Typically, dyes such as fluorescein,
rhodamine, pyronine, eosin, acridine, acriflavine, safranine, methylene
blue and a host of other dyes have been used in this prior art technique
together with appropriate photometric detection devices. The signal level
of fluorescent dyes bound to an antibody-antigen complex in the prior art
was generally too low for individual particle detection to be made. Prior
art efforts to improve this sensitivity by increasing the dye loading (the
number of bound dye molecules coupled to each antibody), resulted in a
reduction of the specificity and sensitivity of the antibody-antigen
reaction, and a number of reasons can be postulated for this reduction:
(1) when enough dye molecules become coupled to the antibody, some of them
will be close enough to the antigen-specific bonding site to produce
partial steric shielding;
(2) changes in the overall hydrophilicity and net charge of the resulting
dye-antibody molecule will alter its reactivity and solubility;
(3) steric and hydrophilicity stresses on the dye-antibody molecule, as
well as possible changes in its vibrational behavior, may distort the
protein's tertiary structure and consequently its specificity;
(4) chemical interactions at the dye-antibody bond site may cause
alteration of the atomic bonding for some distance away from the link's
location, possibly involving the specific binding site.
The present invention overcomes these problems of the prior art by a novel,
advantageous system which increases the number of fluorescent dye
molecules bonded to the antibody molecule consistent with maintaining the
antibody-antigen specificity essentially unimpaired. To this end, the
present invention is embodied in a reagent comprising an antibody molecule
to which has been covalently attached a large number of fluorescent dye
molecules through a polymeric backbone having reactive, functional groups
along the length of its chain. Thus, the attachment, at a single site on
an antibody, of a polymeric chain having a multiplicity of fluorescent
moieties, increases very substantially the magnitude of fluorescent
emission (under appropriate excitation) from the combined molecule while
minimally affecting the specificity of the reactivity of the antibody with
its antigen. It is thus a principal object of the invention to increase
dye loading in specific antibody immunofluroescence without substantially
impairing specificity.
It is a further object of the invention to improve the sensitivity of
antibody immunofluorescence techniques by at least two orders of magnitude
over the prior art.
The terms "first reactant" and "second reactant" (hereinafter termed
"analyte") as used herein are intended to be construed in a broad physical
sense. The first reactant can be an antibody or any chain molecule having
two or more reactive sites (or functional groups) so sterically separated
from one another and disposed that one of the sites can be bonded to the
carrier polymer without impairing substantially the specific reactivity of
the other of the sites with the analyte body. Typically, the first
reactant can be biological, i.e. blood serum proteins, the formation of
which is biologically mediated in response to the presence of an analyte
in the form of an antigen, or nonbiological, e.g. ligands including
organic sequestering and chelating agents, and the like. Analytes are
substances which react, preferably with high specificity, with a
particular first reactant, each analyte particle or body having a
plurality of reactive sites so that it couples with a plurality of
molecules of first reactant. Analytes thus are deemed to include
biological antigens which typically are high molecular weight (e.g.
>10,000) complex organic molecules, such as enzymes, toxins, proteins,
possibly polysaccharides and lipoproteins, whole microorganisims, such as
bacteria, viruses, protozoa and the like, both live and dead, and haptenes
or substances that can react with an antibody but cannot of themselves
engender biological formation of an antibody. Such biological analytes or
antigens of course are specifically reactive with corresponding biological
antibodies by definition. Non-biological analytes which are reactive with
corresponding ligands can be as simple as a metallic ion, molecular
cluster or the like.
The carrier or backbone molecule polymer chosen has reactive sites
dispersed along the length of the chain, with a chemically different
reactive site at the end of the chain. This carrier polymer molecule
should either be rigid or tend to fold to a globular configuration in
water, so as to prevent steric impediments arising out of its unfolding or
twisting around. Its net ionic charge (when aggregated with dye molecules
attached to its side linkages) per unit volume should be essentially the
same as that of the first reactant when the latter is an antibody, at the
working pH. The reactive sites dispersed along the length of the chain of
the carrier polymer molecule should have a low affinity for the first
reactant and should not act as fluorescence quenchers. Preferably the
carrier polymer molecule has hydrophilicity similar to the first reactant.
The backbone of the polymer molecule should include covalent bonding sites
separated by a sufficient distance to avoid disruption of the useful
spectral properties of the dye moieties caused by perturbation effects of
one dye molecule interacting through space with another dye molecule.
The term "specific" as used herein is intended to describe a reaction in
which the reactants will react substantially only with each other and to
much lesser or negligible degree with other reactants, such reaction being
particularly exemplified by an antibody-antigen reaction.
Polymer backbone molecules suitable for the practice of this invention are
polyethylenimines, suitably of molecular weight in the range of
1200-60,000,, polypeptides such as polylysines, polyamides such as
nylon-6, low molecular weight [100-10,000] polymeric carboxylic acids, and
other polymeric materials containing repeating reactive functional groups
along the length of their chain.
The polymeric backbone substance is tagged with dyes by being allowed to
react with fluorescent dye molecules, each of which has a reactive group
so that it can react covalently with the repeating functional groups of
the polymeric material. Prior to reaction with the fluorescent dye
molecule, the reactive end groups of the polymer molecule are temporarily
blocked or chemically protected as by reaction with a carbonyl compound.
Suitable carbonyl compounds for this purpose are exemplified by
benzaldehyde, glutaraldehyde, etc.
Fluorescent dyes suitable for use in the present invention when
functionalized include, but are not limited to, the following:
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Acid Violet 4BL C.I. No. 42575
Acridine Brilliant Orange
C.I. No. 46005
Acridine Orange C.I. No. 46005
Acridine Yellow C.I. No. 56025
Acriflavine C.I. No. 46000
Auramine 0 C.I. No. 41000
Aurophosphine G C.I. No. 46035
Benzo Flavine C.I. No. 46035
Berberine Sulfate C.I. No. 75160
Brilliant Phosphine C.I. No. 46035
Brilliant Sulfo Flavine
C.I. No. 56205
Chrysoidine C.I. No. 11270
Coerulein S C.I. No. 45510
Coriphosphine 0 C.I. No. 46020
Coriphosphine Fuchsin
C.I. No. 42755
Euchrysine 2G C.I. No. 46040
Euchrysine 3 RX C.I. No. 46005
Flavo Phosphine R. C.I. No. 46035
Fluorescein C.I. No. 45350
Geranine G C.I. No. 14930
Methylene Blue C.I. No. 52015
Morin C.I. No. 75660
Neutral Red C.I. No. 50040
Orange G C.I. No. 16230
Phosphine 3R C.I. No. 46045
Primuline C.I. No. 49000
Pyronin GS (Pyronin extra)
C.I. No. 45005
Rhoduline Orange C.I. No. 46005
Rhoduline Violet C.I. No. 29100
Rosole Red B C.I. No. 43800
Safranin C.I. No. 50210
Scarlet R C.I. No. 26105
Sulpho Rhodamine B C.I. No. 45100
Tartrazine 0 C.I. No. 19140
Thiazine Red R C.I. No. 14780
Thiazol Yellow C.I. No. 19540
Thioflavine S. C.I. No. 49010
Thionin C.I. No. 52000
______________________________________
After the dye molecules are coupled through the reactive side groups of the
polymer, two slightly different procedures are used depending upon whether
a monoaldehyde or a polyaldehyde has been used to block the reactive end
group of the polymer. If a monoaldehyde such as benzaldehyde is used as a
protective agent, following dye coupling to the polymer the benzaldehyde
moiety is removed, as by hydrolysis, to reactivate the end site. The
latter is then functionalized in preparation for covalent attachment to
the antibody molecule.
Alternatively, if a polyaldehyde such as glutaraldehyde (propanedial) is
used (preferably in a large excess) as a protective agent for the reactive
end site of the polymer, the protective glutaraldehyde moiety need not be
removed but can serve as a linking agent for covalent attachment of the
polymer to the antibody or other first reactant.
The dye-polymer complex is then coupled to the first reactant, such as an
antibody, to provide a dye/polymer/antibody complex. The reagent is
buffered if necessary so that the dye/polymer/antibody molecules are
preferably electrically neutral, such neutrality minimizing impairment of
the specific reactivity between the first reactant and the analyte,
particularly an antibody-antigen reaction. One can change the pH of the
reagent with known buffers in the range between pH values at which, for
antibody-antigen type reactions, protein denaturing may occur, i.e.
between pH 4 and 10 approximately. Thus, any excess charge on the reagent
molecules should be in weakly ionized groups, such as carboxy or amino
groups, the pK of which is between about 4 and 10.
The dye/polymer/antibody complex or reagent will, when mixed with a
solution containing antigen specific to the antibody in the complex,
couple to the antigen. Because the analyte body with which the reagent of
the present invention is reactive, may possess several attachment sites,
each analyte body then may have coupled to it two or more of the reagent
molecules. Observation of a flow stream of minute cross-section (or some
other known technique for segregating molecules from one another)
irradiated with light in the absorption band of the dye of the complex
will detect fluorescence from each reagent molecule in the flow stream
sequentially passing the area of irradiation. By threshholding the
measurement of the amplitude of each fluorescent pulse detected, one can
readily discriminate between each point source which produced low level
signals due to simple unbound reagent molecules and the greater amplitude
signals obtained from each plurality of reagent molecules bound to a
single analyte body, thereby identifying the presence of the analyte.
Obviously, one cannot by this technique discriminate between a group of
reagent molecules bound to a single analyte body and a group of
cross-linked reagent molecules. For this reason, it is important in
preparing the reagent of the invention to guard against cross-linking as
with appropriate agents temporarily blocking reactive groups, and
preferably, before use, the reagent should be subjected to a separation
procedure, such as by silica gel chromatography, to fractionate out
substantially all cross-linked reagent molecules. In instances where,
following reaction between the reagent and an analyte, unreacted reagent
can be physically removed, detection of analyte coupled to single reagent
molecules becomes feasible.
Preparation of a typical reagent of the present invention is exemplified by
reacting polyethylenimine 200 (molecular weight 20,000) with a
stoichiometric excess of glutaraldehyde and fractionating the mixture as
with a Sephadex column to eliminate excess glutaraldehyde and polymer that
has become cross-linked by the polyaldehyde. The protected polymer is
reacted with an excess of functionalized dye such as fluoroscein
isothiocyanate and the mixture again fractionated to separate free dye
from dyed polymer.
Many dyes in functionalized form, such as fluoroscein isothiocyanate, are
commercially available. Typically, the fluoroscein is functionalized by
the known technique of first adding an extra, non-chromophoric amino group
to the fluoroscein molecule, as by nitrating the fluorescein with
NHO.sub.3 and reducing the nitrate with nascent hydrogen produced by the
addition of zinc and HCl. The isothiocyanate is then formed by adding
thiophosgene. Of course, other techniques are known to produce
functionalized dye by converting them, for example, to isothiocyanate form
or by adding other groups such as sulfonyl chloride, or a 2-bromoethyl
side chain.
Antibody, commercially obtainable, is preferably fractionated, as with
Sepharose, to separate out immunoglobin-m from gamma globulin. The latter
fraction is mixed with the dyed polymer and the reaction terminated, as
with ethanolamine or trimethylaminomethane hydrochloride, appropriately
buffered. This latter reaction is an amino-aldehyde reaction which arrests
further linking between the dyed polymer and other antibodies. The mixture
must then be fractionated as with a Sephadex column to separate the
antibody/polymer/dye molecules according to the number of polymer
molecules coupled to each antibody molecule. Fractions which are antibody
only or antibody with two or more polymers are discarded, the former
because it is useless being untagged and the latter because it has less
specificity and sensitivity than the selected fraction.
Particular antigens detected by the process of this invention are typified
by viruses such as Hepatitis B antigen and Echo 12 virus, Hoof and Mouth
disease antigen and Swine vesicular disease virus antigen. It will be
recognized, however, that the present process is not limited to the
detection of those viruses specifically named but is generally applicable
to all antigens for which the appropriate antibody is available.
The invention will appear more fully from the examples which follow. These
examples are given by way of illustration only and are not to be construed
as limited either in spirit or in scope as many modifications both in
materials and in methods will be apparent to those skilled in the art.
EXAMPLE I
The formation of a polymer/dye complex is achieved as follows:
To a solution of 2 mg. of polyethylenimine 200 (molecular weight 20,000) in
1 ml. of 0.1 M sodium cacodylate at pH 7.0, 0.1 ml. of 25% aqueous
glutaraldehyde is added with vigorous stirring. The resulting reaction
mixture is stirred for about 5 minutes and excess glutaraldehyde then
removed by passage through a Sephadex G-25 (0.9.times.15 cm.) (silica gel)
column. The column is eluted with 0.1 M, pH 7.2 aqueous sodium cacodylate
buffer and to the eluate is added 50 mg. of fluorescein isothiocyanate
dissolved in 1.5 ml. of aqueous 0.5 M. pH 9.5 sodium carbonate buffer. The
mixture is stirred continuously during the addition and stirring continued
for about 16 hours, during which time the mixture is excluded from light.
The excess dye is removed by passage through a Sephadex G-25 (silica gel)
column (0.9.times.30.0 cm.) and subsequent elution of the column with 0.1
M, pH 7.0 aqueous sodium cacodylate. 2 ml. fractions are collected.
The resulting polymer/dye complex is analyzed by the Folin-Ciocaulteau
protein assay. That assay gives a linear curve with polyethylenimine and
thus is suitable for estimation of the amount of polymer present. The
Extinction Coefficient of fluorescein isothiocyanate at 495 nm. is
73.times.10.sup.3 and drops to 75% of this value on binding. By measuring
both polymer and dye present in a given sample of the complex, the degree
of dye binding is estimated. This degree of binding depends upon the dye
concentration in the initial reaction mixture. Limited fractionation is
achieved by gel filtration. The complex prepared by the process of this
Example contains approximately 70 dye molecules per molecule of
polyethylenimine.
EXAMPLE II
Thee procedure of Example I is followed however altering the molar ratio of
dye to polyethylenimine progressively resulting in polymer/dye complexes
containing approximately 65 and 80 molecules, respectively, of dye per
molecule of polyethylenimine 200.
EXAMPLE III
The procedure of Example I is followed, employing however 0.1 ml of a
solution of 25% benzaldehyde is dioxane in place of the glutaraldehyde, to
produce a polymer/dye complex similar to that of Example I except that the
reactive end sites of each polyethylenimine molecule is coupled to a
benzaldehyde moiety.
EXAMPLE IV
When the procedure of Example I is repeated substituting polyethylenimine
600 (molecular weight 60,000) for polyethylenimine 200 the polymer/dye
complex obtained contains approximately 130 dye molecules per molecule of
polyethylenimine.
EXAMPLE V
The procedure of Example I is repeated, using under similar conditions,
polylysine (mol. wt. 8,000-20,000) in place of polyethylenimine, the
isothiocyanate of lissamine Rhodamine-B in place of fluoroscein
isothiocyanate, thereby providing a polylysine/rhodamine complex in which
each backbone molecule of the complex has a plurality of dye molecules
bound thereto. Lissamine Rhodamine B has the structure described in page
379 of Dyeing and Chemical Technology of Textile Fibres, Trotman, 45th
Ed., C. Griffin & Co., London.
EXAMPLE VI
The substitution of sulfonyl chloride of Lissamine Rhodamine-B in the
procedure of Example V also results in the corresponding rhodamine/polymer
complex.
EXAMPLE VII
To form a reagent of the present invention (e.g. a dye/polymer antibody
complex) Anti-Echo virus antiserum (2.5 Mg.) is dissolved in 0.1 M, pH 7.0
aqueous sodium cacodylate and 1.1 mg. of the polymer/dye complex of
Example I is added with stirring. The resultant mixture is stirred for 10
minutes and 1 mg. tris/chloride is added to inhibit cross-linking between
antibody molecules. Stirring is continued for 35 more minutes and the
mixture is then applied to a Sephadex G-200 column (0.9.times.60 cm.)
(silica gel) equilibrated with 0.1 M, pH 8.5 tris/chloride buffer. The
column is eluted with the same buffer and 2 ml. fractions of the reagent
are collected.
The optical density of the reagent and of the fractions is determined at
280 nm. and 495 nm. By difference spectral analysis the amount of antibody
in each fraction is determined and the amount of dye bound per antibody
molecule is estimated. Knowing the number of dye molecules per polymer
molecule, the average number of polymer molecules per antibody molecule is
calculated. By this assay procedure it was determined that 1.2-1.3 polymer
molecules are bound to each antibody molecule.
The immunological activity of the dye/polymer/antibody complex is measured
by hemagglutination. By this method it was found that the
dye/polymer/antibody complex retained 70% of the activity of the
uncombined antibody.
The fluorescence of the dye/polymer/antibody complex is obtained using an
Aminco Bowan fluorimeter. Fluorescence is measured in relation to standard
solutions of fluoroscein isothiocyanate of concentration 0.001-1.0 ml.
Excitation is measured at 495 nm. and emission at 526 nm. Complexes are
diluted to give the same optical density at 495 nm. as do the known
dilutions of fluoroscein isothiocyanate. The quantum efficiency was
determined as 4% for the complex containing polyethylenimine with 80 dye
molecules, using for comparison free fluoroscein isothiocyanate as 100%.
The reagent thus prepared is run through a Sephadex column and all
fractions discarded except that containing reagent in which a polymer
molecule is coupled to only one antibody. When that fraction is mixed with
a solution containing Anti-Echo virus as an analyte an antibody-antigen
reaction occurs resulting in each viral particle coupling to two or more
antibody complexes.
EXAMPLE VIII
To form another reagent of the present invention, the benzaldehyde moiety
of the complex of Example III is removed by mild hydrolysis in 1 ml. of 1%
HCl in a cooled solution for about three hours, and excess acid removed by
dialyzing the solution. Thereafter, water is removed from the solution by
evaporation at reduced pressures, and the resulting polymer/dye complex is
dissolved in hot acetone. To the acetone solution, 10 equivalents of
thiophosgene is added with additional acetone and the mixture refluxed for
four hours. The acetone is then evaporated to produce a functionalized
polymer/dye complex.
The functionalized polymer/dye complex is dissolved in water, buffered at
pH 9.5 with 0.5 m Na.sub.2 CO.sub.3 and about 2.5 milligrams of Anti-Echo
virus antiserum is added. The mixture is stirred continuously for about 16
hours during which time light is excluded from the mixture. Excess dye is
then removed by passage through a Sephadex column followed with elution of
the column with 0.1 M, pH 7.0 aqueous sodium cacodylate. 2 ml. fractions
are collected containing polymer/dye antibody complex in which the
polymer-antibody ratio is 1:1.
EXAMPLE IX
Yet another reagent of the present invention useful for detecting the
presence of a polyvalent metal, (here specifically nickel) is formed as
follows of a ligand for that metal:
To a solution containing 10 mg. of the polymer/dye complex prepared
according to Example I is added 0.1 mg. of a ligand, here benzyl p-amino
benzyl diisonitrosoethane (i.e. benzyl p-amino glyoxime) and the mixture
stirred for one minute. The reaction is terminated then by adding 100 mg.
of ethanolamine which has been carbonate buffered to pH 9. The mixture is
dialyzed to remove excess ethanolamine and benzyl glyoxime. The resulting
reagent when painted onto a dried smear of nickel-containing fluid on a
glass slide will coupld approximately four molecules of the
polymer/dye/ligand to each nickel atom. The slide is then lightly washed
with water to remove excess reagent. On microscopic examination of the
slide illuminated with light is the absorption bond of fluoroscein, the
presence of nickel atoms will be indicated by the amplitude of
fluorescence from each point source which indicates a group of bound
complexes.
Certain changes may be made in the above method and produce without
departing from the scope of the invention herein involved as will be
obvious to one skilled in the art, and it is therefore intended that all
matter contained in the above description shall be interpreted in an
illustrative and not in a limiting sense.
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