U.S. patent number 4,166,105 [Application Number 05/535,095] was granted by the patent office on 1979-08-28 for dye tagged reagent.
This patent grant is currently assigned to Block Engineering, Inc.. Invention is credited to Tomas Hirschfeld.
United States Patent |
4,166,105 |
Hirschfeld |
August 28, 1979 |
Dye tagged reagent
Abstract
Reagents for the detection of specific reactants, such as
antigens in biological fluids, comprising a polymer coupled to an
antibody, the polymer having a plurality of dye molecules coupled
thereto. The reagent is prepared by covalently linking an end
functional group of polymers such as polyethylene amine to an
antibody, typically through an aldehyde linkage, the polymer
molecule having had a plurality of dye molecules, coupled to the
polymer through side reactive sites.
Inventors: |
Hirschfeld; Tomas (Framingham,
MA) |
Assignee: |
Block Engineering, Inc.
(Cambridge, MA)
|
Appl.
No.: |
05/535,095 |
Filed: |
December 20, 1974 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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383892 |
Jul 30, 1973 |
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Current U.S.
Class: |
436/536; 435/5;
436/800; 436/820; 525/54.1; 530/389.4; 530/815 |
Current International
Class: |
G01N
31/22 (20060101); G01N 31/00 (20060101); A61K
029/00 (); G01N 031/00 (); G01N 031/22 (); G01N
033/16 () |
Field of
Search: |
;424/3,7,8,12
;260/6,112R ;250/461B,459 ;23/23B |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Unger Waron CA vol. 79, 1973, No. 103454m. .
Nature, vol. 249, No. 5452, pp. 81-83, May 3, 1974..
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Primary Examiner: Moyer; Donald B.
Attorney, Agent or Firm: Schiller & Pandiscio
Parent Case Text
This application is a continuation-in-part of copending application
Ser. No. 383,892, filed July 30, 1973 now abandoned.
Claims
What is claimed is:
1. A reagent reactive with an analyte body, said reagent
comprising;
a solution containing a plurality of individual molecules of a
first reactant, each of said molecules being a chain molecule
having a plurality of reactive sites, substantially only one of
said sites being specifically reactable with said analyte body in a
reaction wherein several of said molecules can become coupled to
said analyte body;
like plurality of polyfunctional polymeric backbone molecules
substantially each of which is covalently bonded to only a
corresponding one of said molecules of first reactant at another of
said sites sterically separated from said one site so as not to
impair substantially the specificity of said reaction, each said
backbone molecule having coupled thereto radicals or fluorescent
dye in a plurality limited so as not to impair substantially the
specificity of said reaction.
2. The reagent of claim 1 wherein said first reactant is a protein
molecule.
3. The reagent of claim 1 wherein said analyte body is an antigen
and said first reactant is an antibody.
4. The reagent of claim 3 wherein said antibody is that which is
specific to Hepatitis B antigen.
5. The reagent of claim 1 wherein said analyte body is a polyvalent
metal and said first reactant is a ligand.
6. The reagent of claim 5 wherein said ligand is dibenzyl
gloxime.
7. The reagent of claim 1 wherein said fluorescent dye is
fluorescein.
8. The reagent of claim 1 wherein said dye is Lissamine
Rhodamine-B.
9. The reagent of claim 1 wherein said polymeric backbone is a
polyethylenimine of molecular weight in the range of
1200-60,000.
10. The reagent of claim 1 wherein said polymeric backbone is a
polyethylenimine of molecular weight of substantially 1200.
11. The reagent of claim 1 wherein said polymeric backbone is a
polyethylenimine of molecular weight of about 20,000.
12. The reagent of claim 1 wherein said polymeric backbone is a
polyethylenimine of molecular weight in the range of 1200 to 60,000
and said fluorescent dye is fluorescein.
13. The reagent of claim 1 wherein the ratio in said reagent
molecules of said first reactant to said backbone molecules is
substantially 1:1.
14. Method of manufacturing a reagent reactive within an analyte
body, said method comprising the steps of
reacting a polyfunctional polymer molecule with a carbonyl compound
to block one or more reactive sites at the ends of the polymeric
backbone and thereby provide a protected polymer;
reacting functionalized molecules of dye with side reactive sites
on said polymeric backbone of said protected polymer molecule to
thereby provide a dye-tagged polymer molecule; and
covalently coupling said dye-tagged polymer molecule through one of
said end reactive sites to a reactant molecule specifically
reactable with said analyte body.
15. Method as defined in claim 14 wherein said carbonyl compound is
a monoaldehyde and said step of covalently coupling comprises first
removing said monoaldehyde from said one end site by mold
hydrolysis so as to reactivate said end site and then reacting the
reactivated end site with said reactant molecule.
16. Method as defined in claim 15 wherein said reactant molecule is
polyethylenimine.
17. Method as defined in claim 15 wherein said reactant molecule is
a polyvalent metal ligand.
18. Method as defined in claim 15 wherein said carbonyl compound is
benzaldehyde, said dye is fluorescein isothiocyanate and said
polymer is polyethylenimine.
19. Method of detecting analyte bodies in solution, comprising the
step of producing a mixture by mixing with said bodies a reagent
comprising molecules of reactant each of which has a plurality of
reaction sites, substantially one of said sites being specifically
reactable with a corresponding one of said analyte bodies, each of
said molecules having covalently coupled thereto a single
polyfunctional polymeric backbone molecule having in turn coupled
thereto a plurality of molecules of fluorescent dye in a number
limited so as not to impair substantially the specificity of the
reaction between said analyte bodies and said reactant.
20. Method as defined in claim 19 wherein said analyte bodies are
antigens and said reactant is antibody.
21. Method as defined in claim 20 wherein said antigens and said
antibody are mixed with one another under conditions in which the
dye/polymer/antibody in the reagent is substantially electrically
neutral.
22. Method as defined in claim 19 including the step of
illuminating the mixture of analyte bodies and reactant with
radiation in an absorption band of said dye.
23. Method as defined in claim 22 including the additional steps of
observing the amplitude of fluorescent emission from each point
source thereof when said dye molecules are illuminated and
selecting only the emissions having amplitudes above a selected
threshold.
24. Method as defined in claim 19 wherein said analyte bodies are
polyvalent metal and said reactant is a ligand.
Description
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:
______________________________________ 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.
* * * * *