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Claims  |
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What is claimed is:
1. A method of preparing a stable immobilized hapten reagent for use in the
specific binding assay determination of a hapten or binding analog thereof
in a liquid test sample, the method comprising the steps of:
(a) reacting a hapten moiety with a polyacrylamide gel particle comprising
a plurality of external and internal chemically active functional groups
wherein the reaction is performed in a solvent in which the gel particle
is substantially nonswollen and under conditions to form a covalent bond
between the hapten moiety and said external active functional groups which
is substantially stable in aqueous solutions;
(b) washing the gel particle resulting from step(a) with a nonswelling
solvent;
(c) washing the gel particle resulting from step(b) with an aqueous
solution; and
(d) isolating the immobilized hapten reagent resulting from step(c)
comprising said gel particle and said hapten moieties bound thereto
wherein substantially all of said bound hapten moieties are covalently
linked to the external surface functional groups by a linking group which
is substantially stable in aqueous solutions.
2. The method of claim 1 wherein the nonswelling solvent is an organic
solvent.
3. The method of claim 2 wherein said organic solvent is selected from the
group consisting of dimethylformamide, dimethylsulfoxide, acetone,
chlorinated hydrocarbons, and cyclic and acyclic alkylethers.
4. The method of claim 1 wherein the hapten moiety is selected from the
group consisting of digoxin, digitoxigenin, digitoxin, digoxigenin, and
12-0-acetyldigoxigenin.
5. The method of claim 4 wherein the linking group is selected from the
group consisting of bifunctional residues of 1,6-hexamethylenediamine,
6-aminohexanol, 1,12-diamino-4,9-dioxadodecane,
1,17-diamino-3,6,9,12,15-pentaoxaheptadecane, 6-aminocaproic acid, and
bovine serum albumin.
6. The method of claim 4 wherein the linking group is a bifunctional
residue of 6-aminocaproic acid and the hapten moiety is digitoxigenin.
7. The method of claim 1 wherein the hapten moiety is a glycosylated
peptide sequence.
8. The method of claim 7 wherein the linking group is selected from the
group consisting of bifunctional residues of
1,1'-[methylenedi-4,1-phenylene]bis-maleimide, bismaleimido-hexane and
bismaleimido-hexethylene glycol.
9. The method of claim 7 wherein said glycosylated peptide sequence
corresponds with the glucosylated N-terminal peptide sequence in the
beta-subunit of human hemoglobin and the linking group is
bismaleimido-hexaethylene glycol.
10. The method of claim 1 wherein less than from about 1.times.10.sup.-12
moles hapten/g gel particle dissociates from the gel particle of the
immobilized hapten reagent upon standing in an aqueous solution for one
week.
11. The method of claim 10 wherein less than from about 1.times.10.sup.-13
moles hapten/g gel particle dissociates from the gel particle of the
immobilized hapten reagent upon standing in an aqueous solution for one
week.
12. The method of claim 1 wherein the polyacrylamide gel particle in its
nonswollen state is substantially impervious to the hapten moiety.
13. A substantially stable immobilized hapten reagent for use in the
specific binding assay determination of a hapten or binding analog thereof
in a liquid test sample, the immobilized hapten reagent comprising a
polyacrylamide gel particle which is substantially swellable in water and
a plurality of hapten moieties bound thereto, said gel particle comprising
a plurality of external and internal functional groups wherein
substantially all of said bound hapten moieties are covalently linked to
said external functional groups by a linking group which is substantially
stable in aqueous solutions.
14. The immobilized hapten reagent of claim 13 wherein the hapten moiety is
selected from the group consisting of digoxin, digitoxigenin, digitoxin,
digoxigenin, and 12-0-acetyldigoxigenin.
15. The immobilized hapten reagent of claim 14 wherein the linking group is
selected from the group consisting of bifunctional residues of
1,6-hexamethylenediamine, 6-aminohexanol, 1,12-diamino-4,9-dioxadodecane,
1,17-diamino-3,6,9,12,15-pentaoxaheptadecane,, 6-aminocaproic acid, and
bovine serum albumin.
16. The immobilized hapten reagent of claim 14 wherein the linking group is
6-aminocaproic acid and the hapten moiety is digitoxigenin.
17. The immobilized hapten reagent of claim 13 wherein the hapten moiety is
a glycosylated peptide sequence.
18. The immobilized hapten reagent of claim 17 wherein the linking group is
selected from the group consisting of bifunctional residues of
1,1'-[methylene-4,1-phenylene]bismaleimide, bismaleimido-hexane and
bismaleimido-hexaethylene glycol.
19. The immobilized hapten reagent of claim 17 wherein said glycosylated
peptide sequence corresponds with the glucosylated N-terminal peptide
sequence in the beta-subunit of human hemoglobin and the linking group is
bismaleimido-hexaethylene glycol.
20. The immobilized hapten reagent of claim 13 wherein less than from about
1.times.10.sup.12 moles hapten/g gel particle dissociates from the gel
particle of the immobilized hapten reagent upon standing in an aqueous
solution for one week.
21. The immobilized hapten reagent of claim 20 wherein less than from about
1.times.10.sup.-13 moles hapten/g gel particles dissociates from the gel
particle of the immobilized hapten reagent upon standing in an aqueous
solution for one week.
22. In an immunometric assay method for determining a hapten or analog
thereof in a liquid test sample, wherein the test sample is contacted with
a labeled antibody reagent capable of binding with said hapten or analog
thereof and with an immobilized form of the hapten capable of binding with
said antibody reagent, and wherein the amount of labeled antibody reagent
which becomes bound to said hapten or analog thereof in the sample
compared to that which becomes bound to the immobilized reagent is
determined and related to the presence of the hapten or analog thereof in
the test sample,
the improvement which comprises employing as the immobilized reagent a
substantially stable immobilized hapten reagent comprising a
polyacrylamide gel particle which is substantially swellable in water and
a plurality of hapten moieties bound thereto, said gel particle comprising
a plurality of external and internal function groups wherein substantially
all of said bound hapten moieties are covalently linked to said external
functional groups by a linking group which is substantially stable in
aqueous solutions.
23. The assay method of claim 22 wherein the hapten moiety is selected from
the group consisting of digoxin, digitoxigenin, digitoxin, digoxigenin,
and 12-0-acetyldigoxigenin.
24. The assay method of claim 22 wherein the linking group is selected from
the group consisting of bifunctional residues of 1,6-hexamethylenediamine,
6-aminohexanol, 1,12-diamino-4,9-dioxadodecane,
1,17-diamino-3,6,9,12,15-pentaoxaheptadecane, 6-aminocaproic acid, and
bovine serum albumin.
25. The assay method of claim 23 wherein the linking group is
6-aminocaproic acid and the hapten moiety is digitoxigenin.
26. The assay method of claim 22 wherein the hapten moiety is a
glycosylated peptide sequence.
27. The assay method of claim 26 wherein the linking group is selected from
the group consisting of bifunctional residues of
1,1'-[methylene-4,1-phenylene]bismaleimide, bismaleimido-hexane and
bismaleimido-hexaethylene glycol.
28. The assay method of claim 26 wherein said glycosylated peptide sequence
corresponds with the glucosylated N-terminal peptide sequence in the
beta-subunit of human hemoglobin and the linking group is
bismaleimido-hexaethylene glycol.
29. The assay method of claim 22 wherein less than from about
1.times.10.sup.-12 moles hapten/g gel particle dissociates from the gel
particle of the immobilized hapten reagent upon standing in an aqueous
solution for one week.
30. The assay method of claim 29 wherein less than from about
1.times.10.sup.-13 moles hapten/g gel particle dissociates from the gel
particle of the immobilized hapten reagent upon standing in an aqueous
solution for one week.
31. In a reagent system for determining a hapten or analog thereof in a
liquid test sample, which reagent system comprises (1) a labeled reagent
capable of binding with said hapten or analog thereof and (2) an
immobilized reagent comprising an immobilized form of the hapten capable
of binding with said labeled reagent,
the improvement which comprises employing as the immobilized reagent a
substantially stable immobilized hapten reagent comprising a
polyacrylamide gel particle which is substantially swellable in water and
a plurality of hapten moieties bound thereto, said gel particle comprising
a plurality of external and internal functional groups wherein
substantially all of said bound hapten moieties are covalently linked to
said external functional groups by a linking group which is substantially
stable in aqueous solutions.
32. The reagent system of claim 31 wherein the hapten moiety is selected
from the group consisting of digoxin, digitoxigenin, digitoxin,
digoxigenin, and 12-0-acetyldigoxigenin.
33. The reagent system of claim 32 wherein the linking group is selected
from the group consisting of bifunctional residues of
1,6-hexamethylenediamine, 6-aminohexanol, 1,12-diamino-4,9-dioxadodecane,
1,17-diamino-3,6,9,12,15-pentaoxaheptadecane, 6-aminocaproic acid, and
bovine serum albumin.
34. The reagent system of claim 32 wherein the linking group is
6-aminocaproic acid and the hapten moiety is digitoxigenin.
35. The reagent system of claim 31 wherein the hapten moiety is a
glycosylated peptide sequence.
36. The reagent system of claim 35 wherein the linking group is selected
from the group consisting of bifunctional residues of
1,1'-[methylene-4,1-phenylene]bismaleimide, bismaleimido-hexane and
bismaleimido-hexaethylene glycol.
37. The reagent system of claim 35 wherein said glycosylated peptide
sequence corresponds with the glucosylatjed N-terminal peptide sequence in
the beta-subunit of human hemoglobin and the linking group is
bismaleimido-hexaethylene glycol.
38. The reagent system reagent of claim 31 wherein less than from about
1.times.10.sup.-12 moles hapten/g of particle dissociates from the
particle of the immobilized hapten reagent upon standing in an aqueous
solution for one week.
39. The reagent system reagent of claim 38 wherein less than from about
1.times.10.sup.-13 moles hapten/g gel particle dissociates from the
particle of the immobilized hapten reagent upon standing in an aqueous
solution for one week. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
The present invention relates to specific binding assays and reagents for
use therein for the determination of an analyte in a liquid test medium.
In particular, the present invention relates to heterogeneous immunoassays
for determining haptens employing immobilized hapten reagents for the
separation of bound and free forms of a labeled reagent.
Various heterogeneous specific binding assays have been developed which
generally involve specific binding interactions between the analyte to be
detected, a specific binding partner for the analyte, and a labeled
reagent, which can be the same or different as the binding partner for the
analyte. When performing such assays, the labeled reagent becomes bound to
its corresponding binding partner to generate a bound species, any of the
labeled reagent not so bound being the free species, wherein the extent of
binding is a function of the amount of analyte present. Where the
detectable response of the labeled reagent is essentially
indistinguishable in the bound species and the free species, it is
necessary to physically separate such bound and free species from each
other in order to effectively determine the amount of analyte present.
Accordingly, once the bound and free species of the labeled reagent have
been separated from each other, the amount of label present in either
fraction thereof is determined by measuring the activity of the particular
label of the labeled reagent and correlating such activity with the amount
of analyte present.
The physical separation of the bound species from the free species can be
accomplished in many ways. In the heterogeneous specific binding assay
known as the immunometric assay, the labeled reagent comprises a labeled
form of an anti-analyte antibody. According to such format, separation of
the free labeled reagent from the bound labeled reagent is accomplished by
addition of an immobilized form of the analyte under determination or an
analog thereof which will bind with the antibody of the free labeled
reagent. For example, U.S. Pat. No. 4,200,436 discloses an immunoassay for
the detection of antigen employing an immobilized form of the antigen to
be measured to separate the bound and free forms of a labeled monovalent
antibody to the antigen. The immobilized form of the antigen is prepared
by chemically binding or physically adsorbing the antigen to solid
supports or carrier materials, such as polysaccharides or plastics,
according to methods known in the art.
Similarly, U.S. Pat. No. 4,551,426 discloses a heterogeneous immunoassay
for the hapten digoxin employing an immobilized form of ouabain (a digoxin
analog) to separate the bound and free forms of an anti-digoxin labeled
antibody. The immobilized form of ouabain is prepared by coupling ouabain,
either directly or through a spacer arm such as a protein, polyamino acid,
or synthetic linker, to a support material, such as beaded agarose, beaded
dextran, polyacrylamide, or glass, according to methods known in the art.
Such support materials, however, together with the processes employed to
couple the desired analyte or analog thereof (ligand) to such support
materials, result in relatively unstable reagents, reagents exhibiting
substantial release or leakage of the ligand into the surrounding liquid
when used in an immunoassay as heretofore described. Such instability is
believed to be the result of an instability in the linkage between the
ligand and the support as well as nonspecific binding of the ligand to the
support material. Such instability of the linkage and nonspecific binding
of ligand results in a substantial amount of the ligand being slowly
released or leaching into the surrounding medium. The leaching of the
ligand into the test medium primarily occurs as a result of the ligand
being nonspecifically bound to internal and external portions of the
support material. Although inconvenient, ligand nonspecifically bound to
the external surface can be removed by washing the support material with
an aqueous wash solution prior to use in an assay procedure. However,
ligand nonspecifically bound to internal portions cannot be effectively
removed and such internalized ligand leaches out from the interior of the
support material and into the liquid test medium where it can be
substantially indistinguishable from analyte from a test sample and free
to bind to the labeled reagent, resulting in an inaccurate measurement of
the amount of analyte actually present in the test sample.
The synthesis and use of support materials, particularly crosslinked
polymer supports, having chemical structures which are physiochemically
compatible with the backbone structure of a peptide have also been
described for use in solid-phase peptide synthesis. In particular,
techniques for coupling peptides to a polymer (Stahl, et al., J. Amer.
Chem. Soc., Vol. 101(18) p. 5383(1979)]and the crosslinking of various
polymers [Varadarajan, et al., Biopolymers, Vol. 22, p. 839 (1983)]using
reverse-phase suspension polymerization in aqueous organic solvent
mixtures have been employed to obtain favorable swelling properties of
such support materials in order to provide increased external and internal
reaction sites.
Accordingly, it is an object of the present invention to provide an
immobilized hapten reagent which is stable in aqueous solution and which
does not slowly release or leach hapten into a surrounding aqueous
solution.
Further, it is an object of the present invention to provide a process of
covalently binding a hapten moiety substantially only to the surface of a
carrier material with substantially no internalization of nonspecifically
bound hapten which would otherwise be slowly released or leached into the
surrounding medium.
Another object of the present invention is to provide a stable, immobilized
hapten reagent for use in an immunoassay for the effective immobilization
and separation of the free species of a labeled reagent from its bound
species.
It is still a further object of the present invention to provide a highly
sensitive liquid immunoassay having a low, analytically insignificant
initial background signal for the accurate determination of a hapten or
binding analog thereof in a liquid test sample.
SUMMARY OF THE INVENTION
The present invention provides an immobilized hapten reagent which is
substantially stable in an aqueous environment for use in a specific
binding assay, particularly an immunoassay, for the determination of a
hapten or binding analog thereof from a liquid test sample. The
immobilized hapten reagent comprises a carrier material composed of a
polyacrylamide gel particle and a plurality of hapten moieties bound
thereto. The gel particle comprises a plurality of external and internal
functional groups on the internal and external surfaces of the gel
particle, respectively, wherein substantially all of the bound hapten
moieties are covalently linked to the external surface functional groups
by a linking group which is substantially stable in aqueous solutions,
particularly immunoassay test mediums, and an analytically insignificant
amount of the hapten moiety remains nonspecifically bound to the carrier
material. Accordingly, substantially all of the the hapten moiety remains
covalently bound to the carrier material during the performance of an
immunoassay and only an insignificant amount, if any, of the hapten moiety
dissociates or leaches from the carrier material into the test medium.
According to the present invention, there is also provided a method of
preparing the stable immobilized hapten reagent which minimizes the
nonspecific binding of the hapten moiety to the gel particle, particularly
the nonspecific binding of the hapten moiety to internal surface areas of
the gel particle. The method comprises the steps of (a) reacting the
hapten moiety with a polyacrylamide gel particle comprising a plurality of
external and internal chemically active functional groups in a solvent in
which the gel particle is substantially nonswollen and under conditions to
form a covalent bond between the hapten moiety and the external functional
groups which is substantially stable in aqueous solutions, (b) washing the
gel particle resulting from step (a) with a nonswelling solvent, (c)
washing the gel particle resulting from step (b) with an aqueous buffer
solution, and (d) isolating the immobilized hapten reagent resulting from
step (c) comprising the gel particle and the hapten moieties bound thereto
wherein substantially all of the bound hapten moieties are covalently
linked to the external surface functional groups.
The gel particle is substantially nonswellable when reacted with the hapten
moiety in the presence of the nonswelling solvent and, accordingly, is
substantially impervious to the hapten moiety and the covalently binding
thereof is limited substantially only to the external surface functional
groups of the gel particle. Any of the hapten moiety which becomes
nonspecifically bound to the external surface of the gel particle in step
(a) is removed with the nonswelling and aqueous wash solutions of steps
(b) and (c), respectively.
The immobilized hapten reagent is particularly useful in a heterogeneous
specific binding assay involving binding between a hapten or binding
analog thereof and a labeled reagent comprising a labeled binding partner
for the hapten or binding analog thereof, wherein it is necessary to
physically separate any of the labeled reagent which becomes bound to the
hapten or binding analog thereof from the labeled reagent not so bound.
Any of the labeled reagent which does not bind to the hapten or binding
analog thereof from the test medium is separated from the bound labeled
reagent by binding to the hapten of the immobilized hapten reagent wherein
the extent of binding is a function of the amount of hapten or binding
analog present in a liquid test sample.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph which illustrates the dose response to digoxin generated
in an immunoassay employing digitoxigenin as the hapten moiety in an
immobilized hapten reagent of the present invention.
FIG. 2 is a graph which illustrates the reactivity of an immobolized
glycopeptide reagent of the present invention in an immunoassay for the
determination of the amount of glycosylated hemoglobin HbAlc in a whole
blood sample.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The immobilized hapten reagent of the present invention can be employed in
conventional heterogeneous specific binding assay methods, particularly
heterogeneous enzyme immunoassays, involving binding between a hapten or
binding analog thereof and a labeled reagent comprising a labeled form of
a specific binding partner for the hapten or binding analog thereof.
Furthermore, the hapten component of the immobilized hapten reagent can be
varied for use in such specific binding assay for the detection of any
hapten or binding analog thereof for which a specific binding partner
exists in biological systems or can be synthesized. Where the specific
binding partner for the hapten or binding analog thereof is an
anti-hapten, such as an antibody to the hapten or fragment thereof, such
specific binding assay method is referred to as an immunometric method.
According to such heterogeneous specific binding assay methods,
particularly the immunometric method, the hapten or binding analog thereof
being detected is generally combined with the labeled reagent to form a
reaction mixture wherein the labeled reagent binds to the hapten being
detected. The extent of such binding is then determined and related to the
hapten to be determined. The ratio of the amount of labeled reagent bound
to the hapten being detected (i.e., the bound species) to the amount of
labeled reagent not so bound (i.e., the free species) is a function of the
amount of hapten present. Since the signals generated by the label of the
labeled reagent of both the bound and free species thereof are
indistinguishable, it is necessary to physically separate the free species
from the bound species to permit the independent determination of the
amount of label present in the one or the other, which determination can
then be correlated to the amount of hapten present.
The immobilized hapten reagent of the present invention is particularly
useful for the separation of the free species of a labeled anti-hapten
antibody from the bound species of such anti-hapten antibody in a specific
binding assay wherein the free species binds to and is immobilized by the
immobilized hapten reagent to achieve the necessary separation step. In
particular, the immobilized hapten reagent of the present invention
comprises an appropriately functionalized polyacrylamide gel particle
carrier material and a plurality of hapten moieties bound thereto. The gel
particle comprises a plurality of external and internal functional groups
and substantially all of the bound hapten moieties are covalently linked
substantially only to the external surface groups of the gel particle by a
linking group. The linking group provides a covalent bond which is
substantially stable in aqueous solutions, particularly in aqueous
solutions comprising the liquid test medium of an immunoassay, wherein the
hapten moiety remains covalently bound to the carrier material during the
performance of an immunoassay to effectively separate the free species
from the bound species. It is to be appreciated that such stability in
aqueous environments prevents the dissociation of the hapten moiety from
the carrier material and, accordingly, leaching of the hapten moiety into
the test medium environment to thereby result in decreased assay
sensitivity and accuracy, as will be described in greater detail
hereinafter.
According to a preferred embodiment of the present invention, the
immobilized hapten reagent is particularly useful in an immunoassay for
the detection of digoxin from a liquid test sampel. The labeled reagent
comprises an enzyme-labeled monovalent antibody fragment derived from a
monoclonal antibody against digoxin, preferably the Fab' fragment of a
monoclonal IgG antibody to digoxin, generally obtained according to
methods known in the art and labeled with an enzyme, preferably
.beta.-D-galactosidase. Preferably, the labeled reagent is
electrophoretically purified on an electrophoretic polyacrylamide gel to
result in a substantially pure monoconjugate preparation thereof
comprising a single monovalent antibody fragment component covalently
linked to a single enzyme component according to the method described in
the copending U.S. patent application entitled, "Substantially Pure
Enzyme-Antibody Monoconjugate Preparation" (U.S. Ser. No. 939,640), filed
on even date herewith.
The immunoassay for digoxin is performed by reacting the labeled reagent,
preferably the monoconjugate preparation thereof, with a test sample
containing digoxin, and then adding the immobilized hapten reagent of the
present invention comprising digoxin or an analog thereof, such as
digitoxigenin, covalently linked to the external surface amine groups of
an amine functionalized polyacrylamide carrier material by a linking
group, as will be described in greater detail hereinafter. Where the
hapten moiety of such immobilized hapten reagent comprises digoxin or an
analog of digoxin, such as digitoxigenin, the digoxin from the liquid test
sample binds to the antibody fragment of the labeled reagent to form the
bound species thereof, and any of the free species of the labeled reagent
which does not bind to digoxin from the test sample binds to the
immobilized hapten reagent for the immobilization and subsequent
separation thereof from the bound species, wherein the bound species
remains in solution and the free species settles out from solution. The
amount of digoxin in the test sample is then determined by measuring the
enzyme activity of the bound species of the labeled reagent.
According to another preferred embodiment of the present invention, the
immobilized hapten reagent comprises a glycosylated peptide sequence, such
as corresponding to the glycolsylated N-terminal peptide sequence in the
beta-subunit of human hemoglobin (glycopeptide), covalently linked to the
external surface sulfhydryl groups of a polyacrylamide-sulfhydryl
derivatized carrier material, such as described in the copending U.S.
patent application entitled "Crosslinked Polyacrylamide Sulfhydryl Gel and
Sulfhydryl-Functionalized Derivatives Thereof" (U.S. Ser. No. 929,904),
filed on even date herewith, which is useful in an immunoassay for the
determination of HbAlc. The labeled reagent comprises a monovalent
antibody fragment derived from a monoclonal antibody specific for the
glucosylated N-terminal peptide sequence in the beta-subunit of human
hemoglobin (see European Patent Application No. 185,870) labeled with
.beta.-D-galactosidase and purified to a monoconjugate preparation thereof
as heretofore described, and the amount of HbAlc in the liquid test sample
is determined by measuring the enzyme activity of the bound species of the
labeled reagent.
The measurement of the enzyme activity in such immunoassays is accomplished
by removing an aliquot of supernatant and depositing the aliquot into a
reagent pad incorporated with a chromogenic substrate for the enzyme
label, such as resorufin-.beta.-D-galactopyranoside,
o-nitrophenol-.beta.-D-galactopyranoside, or, more preferably, a
chromogenic acridinone enzyme substrate for .beta.-D-galactosidase
comprising a 7-hydroxy-9H-acridin-2-one chromogen derivatized with a
.beta.-D-galactose residue such as described in the copending U.S. patent
application entitled, "Chromogenic Acridinone Enzyme Substrates" (U.S.
Ser. No. 939,855), filed on even date herewith. The detectable signal
which is generated by the interaction between the enzyme and the
chromogenic substrate is then measured with, for example, a reflectance
photometer, and correlated to the amount of hapten present in the liquid
test sample.
IMMOBILIZED HAPTEN REAGENT
It is to be appreciated that in order to provide a highly sensitive
immunoassay to accurately determined the amount of hapten present in a
liquid test sample, nonspecific binding of the hapten moiety to the
carrier material should be minimized. Any of such nonspecifically bound
hapten moiety otherwise results in the dissociaton of the hapten moiety
from the carrier material and the slow release or leaching thereof into
the liquid immunoassay test medium as a free hapten species which competes
with the hapten from the test sample for binding to the labeled reagent.
Accordingly, the label of the labeled reagent bound to such dissociated
hapten moiety provides a background signal which will interefere with the
detection of the signal provided by the label of the labeled reagent bound
to the hapten from the test sample to result in an inaccurate
determination of the hapten being detected from the liquid test sample.
Accordingly, an important feature of the present invention is to provide an
immobilized hapten reagent which is substantially stable in aqueous
solutions and which comprises a hapten moiety covalently linked
substantially only to the external surface of an appropriately
functionalized polyacrylamide gel particle carrier material, with only an
analytically insignificant amount, if any, of the hapten moiety
nonspecifically bound thereto.
According to the present invention, control of nonspecifically bound hapten
to within acceptable limits is achieved by reacting the hapten moiety with
a carrier material comprising an appropriately functionalized or
derivatized polyacrylamide resin in a nonswelling solvent in the presence
of a linking group which forms a substantially stable, covalent body
therebetween to provide an immobilized hapten reagent which is
substantially stable in aqueous solutions. The nonswelling solvent limits
the extent of covalent binding substantially only to the external surface
functional groups of the carrier material, as will be described in greater
detail hereinafter.
(a) Carrier Material
According to the preferred embodiment of the present invention, the carrier
material of the immobilized hapten reagent is an aminoethyl-derivatized
polyacrylamide resin, which is generally swellable in aqueous solutions
and which can be prepared according to methods known in the art. According
to such methods, a polyacrylamide resin is first prepared by the
copolymerization of acrylamide and N,N'-methylenebisacrylamide [S. Hjerten
and R. Mosbach, Anal. Chem., Vol. 3, p. 109(1962)], to form, under
suitable conditions, crosslinked polyacrylamide chains, followed by
treatment with anhydrous ethylene-diamine [J.K. Inman and H.M. Dintzis,
Biochemistry, Vol. 8, p. 4074(1969)]to obtain an aminoethyl-derivatized
polyacrylamide gel comprising a plurality of amine functional groups for
use as the carrier material of the immobilized hapten reagent of the
present invention. In addition to the derivatization of polyacrylamide
with amine functional groups by direct activation of the polyacrylamide
resin as heretofore described, acrylamide and methylenebisacrylamide can
be copolymerized with, for example, acrylic acid esters of
N-hydroxysuccinimide or N-hydroxyphtalimide which can then be readily
reacted with haptens containing primary amino functions, such as an
aminohexyl group, which displace the active ester in the resin to provide
the immobilized hapten reagent of the present invention [see, J.K. Inman,
Methods in Enzymology, Vol. 34B, pp. 30-58(1974); G.L. Stahl, et al., J.
Org. Chem., Vol. 44, p. 3424(1979); G.L. Stahl, et al., J. Amer. Chem.
Soc., Vol. 101, p. 5383(1979)].
The aminoethyl-derivatized polyacrylamide gel is particularly preferred
because of its high capacity for the binding or immobilization of hapten
thereto, as well as its low nonspecific adsorption properties. Further,
the gel is also commercially available (Bio-Rad Laboratories, Richmond,
CA, USA) having various aminoethyl capacities, generally from between
about 1.0 and 2.0 meq. per dry gram of the resin. The gel also has a
preferred pH range from about pH 2.0 to pH 10.0, and is susceptible to the
hydrolysis of amide side groups at higher or lower pH values.
According to another embodiment of the present invention, the carrier
material of the immobilized hapten reagent is a novel polyacrylamide
sulfhydrylk gel having, as the functional groups, a plurality of
sulfhydryl groups, as described in greater detail in the copending U.S.
patent application entitled "Crosslinked Polyacrylamide Sulfhydryl Gel and
Sulfhydryl-Functionalized Derivatives Thereof" (U.S. Ser. No. 939,904),
filed on even date herewith. The polyacrylamide sulfhydryl gel is prepared
from the free radical polymerization of acrylamide, bisacrylamide, and
N,N'-bisacrylylcystamine and is particularly useful for the preparation of
an immobilized glycopeptide reagent for use in the immunoassay
determination of the glycosylated form of hemoglobin known as HbAlc. It is
to be appreciated that the polyacrylamide sulfhydryl gel is similarly
swellable in aqueous solutions. The swelling characteristics can be
controlled by the ratio of monomers employed, the degree of crosslinking
and the particular crosslinking group, and the active functional groups.
Generally, the physical structure of the polyacrylamide gel particles
comprises crosslinked polyacrylamide chains which define an external
surface area and an internal surface area wherein the accessibility of the
hapten moiety and other reagents to the internal surface area is limited
when the gel particle is nonswollen as heretofore described. It is to be
understood that the swelling characteristic of the gel particle is the
result of the structural network of the crosslinked polyacrylamide chains
which result in a generally porous natrue of the gel particle.
Accordingly, when the gel particle is nonswollen, the polyacrylamide
chains are held tightly together by the crosslinking groups to result in
an effective pore size which is smaller or impervious to the hapten moiety
or other reagents to thereby prevent the permeation and internalization
thereof into the internal surface area of the gel particle. The particles
further comprise a plurality of their respective chemically active
functional groups at the external and internal surface areas thereof which
are essential to the formation of a stable, covalent bond between the
hapten moiety and the particle by a linking group, as will be described in
greater detail hereinafter. It is to be appreciated that where a covalent
bond is not formed between the hapten moiety and a functional group of the
particle, such hapten moiety is likely to become nonspecifically bound to
the particle by nonspecific binding interactions, such as through ionic
and hydrophobic binding interactions and the like. Such nonspecific
binding of a hapten moiety to a particle results in a high degree of
dissociation of such nonspecifically bound hapten moiety from the carrier
material and subsequent leaching or slow release thereof when subjected to
immunoassay test conditions or other aqueous environments.
Although a substantially stable, covalent bond can be formed between the
hapten moiety and the functional groups as heretofore described in either
an aqueous environment or an organic environment, the use of a nonswelling
solvent is preferred according to the present invention in order to
achieve the least amount of nonspecific binding of the hapten moiety to
the gel particle, and, more particularly, substantially only to the
external surface thereof according to the present invention. In
particular, the substantially high hydrophilicity of the particle results
in the undesirable swelling of the gel and increase in the permeation or
internalization of the hapten moiety and other reagents into the gel
particle when in an aqueous solution, whereas there is essentially no
swelling or attendant increase in such permeation or internalization when
in an organic solution or solvent containing little or no water.
Accordingly, the use of a nonswelling solvent according to the present
invention prevents the substantial swelling of the gel particle, which
limits the covalent binding of the hapten moiety substantially only to the
functional groups on the external surface of the gel particle by
minimizing the permeation or internalization of the hapten moiety and
other reagents into the particle. Such internalization would otherwise
result in the undesirable formation of covalent bonds between the hapten
moiety and functional groups which are present on the internal surface
area of the gel particle as heretofore described. Such internalization of
the hapten moiety and other reagents also results in the greater
probability of the nonspecific adsorption of such internalized hapten
moiety which would be difficult to remove with a wash solution and which
could later leach out from the gel particle during the performance of an
immunoassay and thereby result in decreased assay sensitivity and
accuracy.
Accordingly, the immobilization of the hapten moiety to the gel particle is
performed in a nonswelling organic solvent such as dimethylsulfoxide,
dimethylformamide, acetone, chlorinated hydrocarbons, cyclic and acyclic
alkylethers, and the like, preferably containing little or no water, which
results in essentially no swelling of the gel particle and, accordingly,
essentially no attendant increase in the permeation or internalization of
the hapten moiety or other reagents into the particle. Since the
negligible increase in particle size will result in a gel particle which
is substantially impervious to and will effectively exclude the permeation
of a hapten moiety and other reagents into the gel particle, any
nonspecific binding of the hapten moiety is limited substantially only to
the external surface of the gel particle. Any of the hapten moiety which
becomes nonspecifically bound to the external surface of the gel particle
is effectively removed with a nonswelling wash solution followed by an
appropriately buffered rinse or wash solution, such as with an acidic salt
solution. Upon the removal of such external, nonspecifically bound hapten
moiety, the resulting immobilized hapten reagent comprises substantially
all of the hapten moiety covalently bound to the external surface of the
gel particle. The reagent thus is substantially stable in aqueous
solutions as a result of the insignificant amount of the hapten moiety
which is nonspecifically bound to the carrier material. In particular, it
is to be appreciated that according to the present invention, less than
from about 1.times.10.sup.-12 moles of the hapten moiety/g. of the resin,
more usually less than from about 1.times.10.sup.-13 moles of the hapten
moiety/g. of the resin, preferably less than about 1.times.10.sup.-14
moles of the hapten moiety/g. of the resin, will dissociate from the gel
particle upon standing in an aqueous liquid, such as a buffer solution,
e.g., the phosphate-chloride assay buffer described in Example 5, to
potentially result in an insignificant amount of leakage thereof during
the performance of an immunoassay.
(b) Hapten Moiety
The hapten moiety of the immobilized hapten reagent can be the hapten under
determination, or an analog thereof which is capable of binding to the
specific binding partner thereof of the labeled reagent, and which hapten
or binding analog thereof can be covalently linked to the external surface
functional groups of a carrier material particle according to the present
invention.
In particular, the hapten moiety of the immobilized hapten reagent of the
present invention can be selected for the determination of any hapten for
which a binding partner exists in a biological system or can be
synthesized, and includes, but is not intended to be limited to, digoxin,
digitoxigenin, digitoxin, digoxigenin, 12-0-acetyldigoxigenin, and
glycosylated peptide sequences such as the glucosylated N-terminal peptide
sequence in the beta-subunit of human hemoglobin, as well as other general
classes of drugs, metabolites, hormones, vitamins, toxins and the like
organic compounds. Haptenic hormones include thyroxine and
triiodothyronine. Vitamins include vitamins A, B, e.g., B.sub.12, C, D, E
and K, folic acid and thiamine. Drugs include antibiotics such as
aminoglycosides, e.g., gentamicin, tobramycin, amikacin, sisomicin,
kanamycin, and netilmicin, penicillin, tetracycline, terramycine,
chloromycetin, and actinomycetin; nucelosides and nucleotides such as
adenosine diphosphate (ADP) adenosine triphosphate (ATP), flavin
mononucleotide (FMN), nicotinamide adenine dinucleotide (NAD) and its
phosphate derivative (NADP), thymidine, guanosine and adenosine;
prostaglandins; steroids such as the estrogens, e.g., estriol and
estradiol, sterogens, androgens, and adrenocortical steroids; and others
such as phenobarbital, phenytoin, primidone, ethosuximide, carbamazepine,
valproate, theophylline, caffeine, propanolol, procainamide, quinidine,
amitryptiline, cortisol, desipramine, disopyramide, doxepin, doxorubicin,
nortryptiline, methotrexate, imipramine, lidocaine, procainamide,
N-acetylprocainamide, amphetamines, catecholamines, and antihistamines.
Toxins include acetyl T-2 toxin, alfatoxine, cholera toxin, citrinin,
cytochalasins, staphylococcal enterotoxin B, HT-2 toxin, and the like.
(c) Linking Group
The linking group of the immobilized hapten reagent of the present
invention employing the aminoethyl-derivatized polyacrylamide gel particle
can be selected from a number of linking groups known in | | |
