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Description  |
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BACKGROUND OF THE INVENTION
The present invention relates to a method for covalent attachment of
antibodies or other molecules to a solid support using extended length
heterobifunctional reagents.
In diagnostic assays, the reaction between a specific binding member and
its complement is often employed to detect whether (and in some assays,
how much) specific binding member or complement is present in a sample. In
one type of diagnostic assay, a specific binding member (e.g. an antibody)
is detected in a sample by introducing its complement (e.g. an antigen)
into the sample and determining if any reaction occurs between the two
reagents. Alternatively, the complement itself can be detected in a sample
by introducing the specific binding member into the sample and determining
whether any reaction occurs. Because it is often difficult to detect
whether any reaction has occurred, a second specific binding member may be
added to the sample. The second specific binding member can react either
with the first specific binding member or its complement, and the second
member bears a detectable label. Of course, it is impossible to determine
beforehand how much labelled second specific binding member must be added
because it is unknown how much, if any, of the substance to be detected is
in the sample. Thus, the labelled specific binding member is added in
excess of the maximum concentration of the substance typically found in
such samples. However, the labelled specific binding member which does not
bind with the substance must be separated from the sample so that only the
bound labelled member is detected, indicating that the substance is indeed
in the sample.
A common approach to separate bound from unbound labelled member is to
employ solid phase separation. A typical example of such separation
involves linking the first specific binding member (in the case of assays
for complement to the first member) or complement (in the case of assays
for specific binding member) to a solid phase (such as microparticles)
which can be separated from the sample, for example, by filtration or
gravity sedimentation. The label associated either with the solid phase or
still in the sample is proportional to the amount of substance to be
detected in the original sample.
Other variations to this general solid phase separation scheme have been
developed, but most such schemes involve the binding of the substance to
be analyzed to a specific binding member linked to a solid phase. This
binding is crucial to assay performance. However, the linkage between the
solid phase and the specific binding member can subsequently affect
binding of the substance to be analyzed. An example will illustrate the
point. Antibodies have extremely specific structural, spacial, and polar
configurations which endow them with the ability to recognize and bind to
one type of analyte, and virtually none other. When antibodies are
employed in assays for the detection of antigens, antibodies can be linked
to solid phases. However, the proximity of the solid phase to the antibody
can block sites on the antibody where antigen binds. Alternatively, the
linkage (usually covalent) between the antibody and solid phase can alter
the structure (conformation) of the antibody so that the linkage may
deleteriously affect binding of the antibody to the analyte. The same
situation holds for conjugation of analytes, particularly proteins, to
solid phases. The analyte conformation can change upon conjugation so the
free antibody in the sample can no longer recognize it.
Covalent attachment of proteins to solid phases using heterobifunctional
reagents has been accomplished with mixed results in the past. In some
cases the proteins were directly conjugated to the solid phase. Generally,
the connecting tether has been quite short in comparison with the size of
the bound protein. This is disadvantageous in that the bound protein can
still be hindered in performing its biological function due to steric
crowding, inaccessability of binding sites, etc. This has been a problem
which has limited the bioactivity and stability of derivatized solid
phases in the past.
SUMMARY OF THE INVENTION
This invention involves conjugates of solid phases with novel linking
groups which can be used to link solid phases to substances such as
proteins, antibodies, antigens and the like. These conjugates are
hydrophilic, so they tend to be quite stable in aqueous solutions, and
they preserve the conformation of such substances when such substances are
linked to solid phases. At the same time, these linking groups are of such
lengths that the solid phases tend not to interfere with binding sites on
such substances.
This invention involves chemically derivatized solid phase materials of the
formula:
##STR1##
wherein B is an amine bearing solid phase material; X is a substituted or
unsubstituted amino acid having from three to ten carbon atoms in a
straight chain; n is from one to ten; and R is an alkyl, cycloalkyl, an
alkyl-cycloalkyl or an aromatic carbocyclic ring.
The current invention also involves protein-solid phase conjugates of the
formula:
##STR2##
wherein Q is a SH or a thiol bearing peptide polypeptide or protein: and
wherein B, X. n, and R are as defined previously.
DETAILED DESCRIPTION OF THE INVENTION
As indicated above, the present invention involves conjugates of "amine
bearing solid phases." Such solid phases include those polymers, glasses,
and natural products which bear amine (either primary, secondary, or
tertiary) groups which can be reacted with a maleimide moiety to form a
stable covalent bond. A wide variety of solid phases are possible,
consistent with this definition: commercially available polystyrene
aminated particles, amino silica gels, partially hydrolyzed nylon,
partially reduced polyacrylamides, partially reduced cyanoacrylates and
copolymers containing such polymers. Solid phases containing nitrile
groups which can be reduced to yield amine groups to produce
"amine-bearing solid phases" consistent with this invention.
While the Examples which follow generally deal with microparticle solid
phases, other solid phase configurations are possible: beads, sheets,
spheres, filters, and the like. However, one solid phase configuration of
particular interest is fibers. Many of the polymers mentioned previously
are available in fiberous form. These fibers can be chopped
(discontinuous) or continuous, but the latter are preferred. Continuous
fibers can be derivitized with the linking groups of this invention, and
proteins such as antibodies can be conjugated to the linking groups. The
protein-bearing fiber can then be sewn or woven into a solid support such
as cloth, a mat, or a woven or non-woven filter media. The fiber can be
sewn or woven into distinctive patterns. For example, the fibers can be
arranged in a plus (+) sign with the vertical bar having positive controls
and the horizontal bar having negative controls, as disclosed in copending
U.S. application Ser. No. 831,013 which is incorporated herein by
reference. Thus, when an assay is performed using the sewn fibers, a plus
sign will appear if the sample passed through the media has analyte in it,
and a minus (-) (horizontal bar only) will appear if no analyte is in the
sample.
Another approach is to attach the linking groups of this invention to the
fiber, sew the fiber into an inert backing material (i.e. a material which
lacks maleimide groups), and react the protein to the exposed maleimide
moieties on the linking groups.
The term "alkyl-cycloalkyl" as used herein includes alkyl groups linked to
cycloalkyl ring structures where the alkyl group links the cycloalkyl to
the maleimide or the carbonyl groups in the chemical structures shown
above. The term "alkyl" includes straight or branched alkyl groups,
preferably lower alkyl groups having from one to six carbon atoms.
The substituent "Q" is a --SH or thiol-bearing peptide, polypeptide or
protein. For convenient reaction with the linking groups of this
invention, the peptide, polypeptide or protein to be conjugated to the
linking groups bear reactive thiol (mercapto) or sulfhydryl (--SH) groups.
It is recognized that mercapto groups contain sulfhydryl groups, but this
invention contemplates that peptides, polypeptides or proteins lacking
sulfhydryl groups may be artificially derivatized with sulfhydryl groups
which are not mercapto groups, mercapto groups being organic compounds
bearing sulfhydryl groups. The sulfhydryl group on the peptide,
polypeptide or protein reacts with maleimide moiety on the linking groups
of this invention to form a covalent bond between the linking groups and
the peptide, polypeptide or protein.
The Examples which follow illustrate this invention. Examples 1-6 describe
the synthesis of various linker groups of this invention. Examples 7-18
describe the uses of the linker groups to conjugate solid phases to
proteins. These examples are not intended to limit the invention.
Example 1
##STR3##
Trans-4-(aminomethyl)-cyclohexanecarboxylic acid was purchased from Aldrich
Chemical Co. and converted to N-(4-carboxycyclohexylmethyl) maleimide
N-Hydroxysuccinimide active ester by the method of Yoshitake et al. (J.
Biochem., 101:395-399 (1979)). This material (100 mg) is then dissolved in
dry dimethylformamide (DMF) (1.0 ml), 6-aminocaproic acid (39.2 mg; 1.0
eq) is added, and the resulting mixture is stirred overnight at room
temperature under nitrogen atmosphere. The following morning
dicyclohexylcarbodiimide (DCCI) (67.8 mg; 1.1 eq) is added, and the
reaction mixture is stirred for an additional six hours. Precipitated
dicyclohexylurea (DCU) is removed by filtration, and the resulting DMF
solution is evaporated under reduced pressure to give a tacky solid, which
is purified by flash chromatography upon silica gel (5%
methanol/chloroform) to give compound 1 (71 mg) as a white solid in 53%
overall yield. (R=cyclohexylmethyl; n=1; X=6-aminocaproyl).
Example 2
##STR4##
Compound 1 (100 mg; synthesis described in Example 1) is dissolved in dry
DMF (1.0 ml), 6-aminocaproic acid (29.3 mg; 1.0 eq) is then added and the
resulting mixture is stirred overnight at room temperature under nitrogen
atmosphere. The following morning DCCI (50.7 mg; 1.1 eq) is added, and the
reaction mixture is stirred for an additional six hours. Solid precipitate
(DCU) is removed by filtration, and the resulting DMF solution is
evaporated under reduced pressure to give a tacky solid, which is purified
by flash chromatography upon silica gel (10% methanol/chloroform to give
compound 2 (60 mg) as a white solid in 48% overall yield.
(R=cyclohexylmethyl; n=2; X=6-aminocaproyl).
Example 3
##STR5##
Compound 2 (100 mg; Gynlhesis described in Example 2) is dissolved in dry
DMF (2.0 ml), 6-aminocaproic acid (23.4 mg; 1.0 eq) is then added, and the
resulting mixture is stirred overnight at room temperature under nitrogen
atmosphere. The following morning, DCCI (40.5 mg; 1.1 eq) is added, and
the reaction mixture is stirred for an additional six hours. Solid
precipitate (DCU) is removed by filtration, and the resulting DMF solution
is evaporated under reduced pressure to give a tacky solid, which is
purified by flash chromatography upon silica gel (10% methanol/chloroform)
to give compound 3 (60.0 mg) as a white solid in 50% overall yield.
(R=cyclohexylmethyl; n=3; X=6-aminocaproyl).
Example 4
##STR6##
Compound 3 (100 mg; synthesis described in Example 3) is dissolved in dry
DMF (10.0 ml), 6-aminocaproic acid (19.5 mg; 1.0 eq) is then added, and
the resulting mixture is stirred overnight at room temperature under
nitrogen atmosphere. The following morning DCCI (33.7 mg; 1.1 eq) is
added, and the reaction mixture is stirred for an additional six hours.
Solid precipitate (DCU) is removed by filtration, and the resulting DMF
solution is evaporated under reduced pressure to give a tacky solid, which
is purified by flash chromatography upon silica gel (10%
methanol/chloroform) to give compound 4 (53 mg) as a white solid in 45%
overall yield. (R=cyclohexylmethyl; n=4; X=6-aminocaproyl).
Example 5
##STR7##
CBZ-triglycine (4.0 g; Bachem Chem. Co.) is dissolved in 50.0 ml dry DMF.
N-hydroxysuccinimide (1.42 g; 1.0 eq), and DCCI (2.55 g; 1.0 eq) are added
and the resulting mixture is stirred overnight at room temperature under
nitrogen atmosphere. The following morning, precipitated DCU is removed by
filtration, and the resulting DMF solution is evaporated under reduced
pressure to give a yellow oil. Recrystallization from ethyl
acetate/chloroform gives the intermediate compound 7 (3.0 g) in 57% yield.
##STR8##
Glycine t-butyl ester hydroxhloride (0.54 g; Sigma Chem. Co.) is suspended
in dry DMF (25.0 ml). Compound 7 (1.35 g; 1.0 eq) from Part (a) is then
added, along with triethylamine (1.62 g; 5.0 eq). The resulting solution
is allowed to stir overnight at room temperature under nitrogen
atmosphere. The following morning, solvent is removed under reduced
pressure to give a crude product. Recrystallization from ethyl
acetate/chloroform gives intermediate compound 8 (0.95 g) in 68% yield.
##STR9##
Compound 8 (0.95 g) from Part b is dissolved in dry methanol (300 ml).
Glacial acetic acid (0.45 ml) is then added and the solution is purged
with nitrogen for 15 minutes. Palladium on carbon (1.5 g; palladium
content 10%) is then carefully added, with stirring. A stream of hydrogen
gas is bubbled through the stirring solution for three hours at room
temperature. The solution is carefully purged with nitrogen for 15
minutes, then filtered. The filtrate solution is concentrated under
reduced pressure to give intermediate compound 9 (700 mg) as the acetate
salt.
##STR10##
Compound 9 (700 mg: acetate salt) from Part c is dissolved in dry DMF (25
ml). N-(4-carboxycyclohexylmethyl)maleimide (697 mg) from Example 1 is
then added, and the mixture is allowed to stir overnight at room
temperature under nitrogen atmosphere. The following morning DMF is
evaporated under reduced pressure to afford a crude product.
Recrystallization from ethyl acetate/hexane affords intermediate compound
10 in 22% yield.
##STR11##
Compound 10 (225 mg) from Part d is suspended in chloroform (1.5 ml). Dry
trifluoroacetic acid (1.5 ml) is then added, and the mixture is stirred at
room temperature under a nitrogen atmosphere for a period of three hours.
Solvent is evaporated under reduced atmosphere to give a crude product.
Trituration with ethyl acetate gives intermediate compound 11 (127 mg) in
61% yield.
##STR12##
Compound 11 (100 mg) from Part e is dissolved in dry DMF (7.0 ml) along
with N-hydroxysuccinimide (37.1 mg; 1.5 eq) and DCCI (221.5 mg; 5.0 eq).
The reaction mixture is stirred overnight at room temperature under a
nitrogen atmosphere. The following morning, precipitated DCU is removed by
filtration, and DMF is evaporated under reduced pressure to give a crude
solid. Trituration with chloroform gives compound 12 (86 mg) in 60% yield.
(R=cyclohexylmethyl; n=4; X=glycyl).
Compound 12 can be used for conjugating proteins (e.g. antibody and
enzymes) to solid phases using the procedures outlined in the following
Examples.
Example 6
##STR13##
A round bottom flask equipped with a magnetic stirrer is charged with
m-maleimidobenzoyl-N-hydroxysuccinimide ester (0.314 g; 0.001 mole)
obtained from Pierce Corporation dissolved in DMF (5.0 mL). 6-Aminocaproic
acid (0.131 g; 1 equiv.) is added, and the resulting solution is stirred
overnight at room temperature under nitrogen. After 18 hours,
olicyclohexylcarbodiimide (DCCI; 0.206 g; 1.1 equiv.) is added followed by
N-hydroxysuccinimide (0.115 g, 1 equiv.). The reaction solution is stirred
for additional eight hours at room temperature under nitrogen.
Precipitated dicyclohexylurea (DCU) is removed by filtration, and the
resulting DMF solution is evaporated under reduced pressure. The resulting
solid is purified by silica gel chromatography (5% methanol in chloroform)
to give compound 13 in 50% yield. This compound is treated with
aminocaproic acid in a manner identical to the method described in
examples 2, 3 and 4 of this application to produce compounds where n is up
to ten, and R=phenyl).
Example 7
Preparation of Monoclonal anti-CA-125 IgG-Derivatized Microparticles using
a 30 Atom Linkage
(a) Pretreatment of Amine Microparticles
Amine microparticles (Seradyn; 0.164 micron; 2.5% solids; 1 ml) are mixed
with 0.5 g Biorex ion exchange resin (Biorex MSZ501D; 20-50 mesh; catalog
#142-7425). The mixture is rotated end-over-end for one hour at room
temperature, then vacuum filtered through a course sintered glass funnel
with washing. The filtrate is collected and centrifuged at 15,000 rpm for
30 minutes. Supernatant is discarded, and the microparticle pellet is
resuspended in distilled water with vortexing, and adjusted to 2.5% solids
by addition of distilled water.
(b) Derivatization of the Particles
Resuspended, pre treated particles from part a at 2.5% solids are mixed
with an equal volume of compound 3 (Example 3) solution (2 mg/ml in DMF)
and allowed to react at room temperature for one hour with end-over-end
rotation. The reaction mixture is then diluted ten-fold with
phosphate-buffered saline "PBS" (pH 7.2), and centrifuged at 15,000 rpm
for 30 minutes. The resulting supernatant is discarded, and the
microparticle pellet is resuspended with phosphate-buffered saline. The
centrifugation-resuspension sequence is repeated twice, the solution is
again centrifuged, supernatant is discarded, and the pellet is resuspended
to a concentration of 2.5% solids with Tris buffer (0.05 M tris; 0.1 M
NaCl; pH 8.0).
(c) Preparation of the Antibody
A solution of monoclonal anti-CA-125 IgG (7.4 mg/ml; in phosphate-buffered
saline) is incubated with DTT (dithiothreitol; 25 mM in final reaction
mixture) for twenty minutes at room temperature with stirring on a rotary
agitator. The solution of partially reduced antibody is then desalted by
chromatography upon a pre-equilibrated Sephadex G-25 (coarse) column with
pH 7.0 phosphate buffer (0.1 M phosphate; 0.1 M NaCl, 5 mM EDTA) as
eluent. Fractions are collected, protein-containing fractions are pooled,
and the protein concentration of the pooled solution is estimated by
measuring absorbance at 280 nm.
(d) Reaction of Maleimide-Derivatized Microparticles with Partially Reduced
IgG
The partially reduced antibody from part c (1 ml; 1 mg/ml) is combined with
the maleimide-derivatized microparticles from part (b) (1 ml; 2.5%
solids). The mixture is rotated end over-end at room temperature
overnight. The following morning, the reaction mixture is diluted ten-fold
with wash buffer (0.01 M phosphate; pH 7.2; 1% Tween), then centrifuged at
15,000 rpm for 30 minutes. The resulting supernatant is discarded, the
microparticle pellet is washed twice (vortexing in 1 ml wash buffer;
diluting tenfold with wash buffer, then centrifuging at 15,000 rpm for 30
minutes followed by discarding supernatant). The washed pellet is
resuspended to a final concentration of 0.125% solids in storage buffer
(0.01 M Tris; pH 8.1; 0.1 M NaCl; 0.1% sodium azide; 13.6% sucrose). The
final microparticle suspension is first passed through a 23, then a 25
gauge needle. The resulting microparticle conjugate has the anti-CA-125
IgG antibody conjugated to the microparticle with a 30 atom linker arm
from Example 3. Microparticles in storage buffer are stored until future
use in an immunoassay for the detection of CA- 125 antigen.
Example 8
Preparation of Monoclonal anti-CA-125 IgG-Derivatized Microparticles using
a 23 Atom Linkage
The procedure of Example 7 is repeated using the 23 atom linker group
(Compound 2) from Example 2 instead of the 30 atom group of Example 7.
Example 9
Preparation of Monoclonal anti-CA-125 IgG-Derivatized Microparticles using
a 16 Atom Linkage
The procedure of Example 7 is repeated using the 16 atom linker group
(Compound 1) from Example 1 instead of the 30 atom group of Example 7.
Example 10
Preparation of Polyclonal anti-CA-125 IgG-Derivatized Microparticles using
a 30 Atom Linkage
The procedure of Example 7 is repeated using a polyclonal anti-CA-125 IgG
antibody instead of the monoclonal antibody of Example 7. Polyclonal
anti-CA-125 antibody was obtained by immunizing sheep subcutaneously and
intramuscularly with 50,000 units of CA-125 antigen in Freund's adjuvant.
All subsequent boosts were done every two weeks using 50,000 units of
CA-125 antigen in Freund's incomplete adjuvant.
Example 11
Preparation of Monoclonal anti-PAP IgG-Derivatized Microparticles using a
30 Atom Linkage
(a) Preparation of Reduced anti-PAP antibody
Dithiothreitol (DTT; 1.93 mg) is placed in a reaction vial. Mouse
monoclonal anti-PAP antibody (0.5 ml; 6.98 mg/ml) is then added, and the
mixture is uncubated at room temperature for 20 minutes at room
temperature. The reaction mixture is then applied to a pre-equilibrated
Sephadex G-25 column (coarse 1.times.20 cm) and eluted with pH 7.2
phosphate buffer (0.2 M phosphate; 0.1 M NaCl; 5.0 mM EDTA). Fractions are
collected and absorbance at 280 nm is measured. Protein-containing
fractions are combined, the pool is diluted ten-fold with chromatography
buffer, and protein concentration of the resulting diluted pool of
activated antibody is estimated by measuring absorbance at 280 nm.
(b) Reaction of Derivatized Microparticles with Reduced Anti-PAP Antibody
Reduced antibody from part a (0.5 mg (e.g. 336 ul at 1.49 mg/ml)) is placed
in a reaction vial. The derivatized microparticle solution (0.5 ml) from
Example 7 part b is then added, and the mixture is incubated overnight at
room temperature on a rotary agitator. The following morning, the reaction
mixture is centrifuged (20 minutes at 12,000 rpm), supernatant is removed,
and absorbance at 280 nm is measured. The antibody-derivatized
microparticles are then resuspended in PBS-Tween buffer (0.1 g Tween in
100 ml PBS; 1.0 ml). The mixture is again centrifuged (20 minutes at
12,000 rpm), supernatant is discarded, and 5.0 ml storage buffer (0.01 M
Tris; 0.1 M NaCl; 0.1% sodium azide; 13.6% sucrose; pH 8.1) is then added.
The microparticles are passed first through a 23 gauge needle, then a 25
gauge needle. The resulting microparticle suspension is then transferred
to a 10 ml plastic screw-cap vial for storage until use in an enzyme
immunoassay for the detection of PAP antigen.
Example 12
Covalent Attachment of B-12 Intrinsic Factor to Aminated Polystyrene
Microparticles
A 10% solution of 100 ul of aminated polystyrene microparticles (0.164
micron, purchased from Seradyn) is placed in a reaction vial. A solution
of Compound 2 in DMF (9 ul; 1.21 mM) is added with a solution of B-12
intrinsic factor (210 ul; 0.1537 mg/ml) in phosphate-buffered saline. The
reaction mixture is rotated end-over-end overnight at room temperature.
The following morninq, the reaction mixture is centrifuged (30 minutes;
13,000 rpm). Supernatant is discarded, the remaining solid is resuspended
in distilled water, and the centrifuging-resuspension step is repeated.
The product produced is a B-12 intrinsic factor/microparticle conjugate
linked with a 23 atom linker of Example 2. The particles are then
suspended in 750 ul of buffer (0.01 M Tris; 0.1 M NaCl; 0.1% sodium azide;
13.6% sucrose; pH 8.1) and used in an assay for the detection of B-12.
Example 13
Covalent Attachment of Recombinant Hepatitis B Core Antigen to Aminated
Polystyrene Microparticles Using Compound 3
(a) Derivitization of Microparticles with Compound 3
Amino microparticles (Polysciences; 0.5 micron) are placed in a reaction
vial. A solution of Compound 3 (Example 3) in DMF is then added, and the
mixture is treated as described in Example 7 part b.
(b) Reaction of Recombinant Core Antigen With Derivatized Microparticles
Recombinant hepatitis B core antigen is placed in a vial. Derivatized
microparticles (from part a) are then added, and the mixture is treated as
described in Example 7 part d to yield microparticles conjugated to the
antigen with a 30 atom linker which can be used in assays for detection of
hepatitis B.
Example 14
Preparation of E. coli .beta.-Galactosidase-Derivatized Microparticles With
A 30 Atom Linkage
E. coli .beta.-galactosidase (1 ml; 1 mg/ml) in pH
e buffered saline (0.M phosphate; 0.1M 7.0 phosphate buffered saline (0.1 M
phosphate; 0.1 M NaCl) is added to maleimide-derivatized microparticles
from Example 7, part b. The reaction mixture is rotated end-over-end
overnight at room temperature. The following morning the reaction mixture
is centrifuged at 15,000 rpm for 30 minutes. The resulting supernatant is
discarded, and the microparticle pellet is resuspended to 2.5% solids with
pH 7.0 phosphate buffered saline (0.M phosphate; 0.M NaCl). The
centrifugation, decanting, resuspension sequence is repeated twice. The
microparticle pellet is finally resuspended in storage buffer (0.1 M tris;
pH 7.0; 0.1 M NaCl; 0.1% sodium azide; 13.6% sucrose). The resulting
microparticle suspension is passed through first a 23, then a 25 gauge
needle. The product is E. coli .beta.-galactosidase conjugated to amino
microparticles with a 30 atom linkage. Enzyme-derivatized microparticles
in storage buffer are then stored until future use.
Example 15
Preparation of Calf Intestinal Alkaline Phosphatase-Derivatized
Microparticles Using a 30 Atom Linkage
(a) Thiolation of the Enzyme
Calf intestinal alkaline phosphatase (0.5 ml; 10 mg/ml) in pH 8.0 Tris
buffer (0.05 M Tris; 10 mM MgCl.sub.2 ; 0.1 mM ZnCl.sub.2) is placed in a
reaction vial. Iminothiolane hydrochloride is then added to a
concentration of 4.0 mM. The mixture is stirred for 30 minutes at room
temperature, then desalted on a Sephadex G-25 (coarse) column with
phosphate-buffered saline (0.1 M phosphate; 0.1 M NaCl; 10 mM MgCl.sub.2,
0.1 M ZnCl.sub.2 ; pH 7.0) as eluent. Fractions are collected,
protein-containing fractions are pooled, and protein concentration of the
pooled solution of thiolated enzyme is estimated by measuring absorbance
at 280 nm.
(b) Reaction of Thiolated Enzyme with Maleimide Derivatized Microparticles
Thiolated enzyme from part a (1 ml; 1 mg/ml) is combined with the
maleimide-derivatized microparticles from Example 7, part b (1 ml; 2.5%
solids). The reaction mixture is rotated end-over-end overnight at room
temperature, then treated as described in Example 7, part d to provide a
suspension of microparticles covalently derivatized with calf intestinal
alkaline phosphatase.
Example 16
Preparation of Monoclonal Anti-CA-125 IgG-Derivatized Microparticles
(a) Preparation of Thiolated Microparticles
Resuspended, pretreated amine microparticles from Example 7, part a (1 ml;
2.5% solids) are mixed with iminothiolane HCl to achieve a final
iminothiolane concentration of 50 mM. The reaction mixture is stirred at
room temperature for one hour, then treated as described in Example 7,
part b.
(b) Derivatization of the Antibody
A solution of monoclonal anti-CA-125 IgG (7.4 mg/ml) in phosphate buffered
saline is incubated with 30 molar equivalents of a DMF solution of
Compound 3 (5.0 mM). The reaction mixture is stirred for 30 minutes at
room temperature, then desalted on a Sephadex G-25 (coarse) column with pH
7.0 phosphate buffer (0.1 M phosphate; 0.1 M NaCl) as eluent. Fractions
are collected, protein-containing fractions are pooled, and protein
concentration of the pooled solution is estimated by measuring absorbance
at 280 nm.
(c) Reaction of Thiolated Microparticles with Maleimide-Derivatized
Antibodies
Thiolated microparticles from part a (1 ml; 2.5% solid) are mixed with
maleimide-derivatized antibodies from part (b) (1 ml; 1 mg/ml). The
reaction mixture is rotated end-over-end overnight at room temperature.
The following morning, the antibody-derivatized microparticles are treated
as described in Example 7, part (d) to produce a microparticle/IgG
conjugate which can be used in an assay for the detection of CA-125
antigen.
Example 17
Preparation of Monoclonal anti-CA-125 IgG-Derivatized Nylon Fibers
(a) Pretreatment of Nylon Fibers
Nylon monofilement fishing line (Berkley, 6 inches, 2 lb. test) is
incubated for 30 minutes at room temperature with 3N HCl (10 ml) with
shaking. The fiber is then washed twice with 20 ml distilled water. The
washed, partially hydrolyzed fiber is stored in distilled water until
further use.
(b) Maleimide Derivitization of Partially Hydrolyzed Nylon Fiber
A two inch section of partially hydrolyzed nylon fiber from part (a) is cut
into 1/8 inch pieces. The 1/8 inch nylon lengths are placed in a reaction
vial along with a DMF solution of Compound 3 (1.0 ml; 5.0 mM). The
reaction mixture is shaken vigorously for two hours, then filtered through
a course sintered glass funnel. The maleimide-derivatized nylon fibers are
washed several times with distilled water, then stored until further use
in distilled water.
(c) Reaction of Maleimide-Derivatized Nylon Fibers with Partially Reduced
anti-CA-125 IgG
Partially reduced monoclonal anti-CA-125 IgG from Example 7, part (c) (1
ml; 1 mg/ml) is added to maleimide-derivatized nylon fibers from part b.
The reaction mixture is incubated overnight at room temperature with
end-over-end rotation. The following morning, the reaction mixture is
filtered through a coarse sintered glass funnel and the
antibody-derivatized fibers are washed several times with wash buffer (0.1
M phosphate; 0.1 M NaCl; pH 7.0). The resulting fibers contain covalently
attached monoclonal anti-CA-125 IgG via a 30 atom spacer group. The
resulting fibers are stored in fiber storage buffer (0.1 M, phosphate; 0.1
M NaCl; 1% BSA; 0.1% sodium azide) for use in an assay for the detection
of CA-125 antigen).
Example 18
Preparation of Monoclonal anti-CA-125 IgG-Derivatized Wool Fibers
(a) Partial Reduction of Wool Thread
Wool thread is cut into 1/2 inch pieces. A piece of thread is then immersed
in 1 ml 5 mM DTT solution and shaken vigorously for one hour. The reaction
mixture is then filtered through a coarse sintered glass funnel, and
washed five times with buffer (pH 7.0; 0.1 M phosphate; 0.1 M NaCl; 5 mM
EDTA).
(b) Reaction of Maleimide-Derivatized Antibody With Partially Reduced Wool
Thread
Partially reduced wool thread from part a is placed in a reaction vial.
Maleimide-derivatized monoclonal anti-CA-125 IgG from Example 7, part d (1
ml; 1 mg/ml) is then added, and the mixture is rotated end-over-end
overnight at room temperature. The following morning, the reaction mixture
is filtered through a coarse sintered glass funnel and washed five times
with wash buffer (0.1 M phosphate; 0.1 M NaCl; pH 7.0). The resulting
fiber contains monoclonal anti-CA-125 IgG covalently attached via a 30
atom spacer group. The resulting fibers are stored in fiber storage buffer
(0.1 M phosphate; 0.1 M NaCl; 1% bovine serum albumin; 0.1% sodium azide)
until used in an assay for the detection of CA-125 antigen.
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