Note: Descriptions are shown in the official language in which they were submitted.
~F' ~:
wo 9S/24631 PCr/US95/02547
~18~282
COVALENT IMMOBILIZED ANTIBODIES ON POLYMERIC BEADS
RELATED lN V~N l-lON
Thi~ invention is related to copending patent
application, Serial No. 07/961,157, filed October 15, 1992
and entitled POLYMERIC PARTICLES HAVING A BIODEGRADABLE
GELATIN OR AMINODEXTRAN COATING AND PROCESS FOR MAKING
SAME.
~ lCAL FIELD
This invention relates generally to polymeric
colloidal particles having a short, low molecular weight
diamine spacer or a polymeric cro~slinked aminodextran
coating that is functionalized to bind a pendent protein
such as antibody, which particles are freeze-dried and
reconstituted with an aqueous media, to the thermal
stability of such particles, to methods of making such
particles, and to the use of such particle~ in biological
assays.
R~RqRouND ART
A sensitive measure of polymer structure on colloidal
particles is light scattering, related to the size, shape,
and refractive index of the scattering object. The
modulating effect of a particle coating on light
scattering intensity can be significant if the polymeric
structures approach the size of the uncoated particles.
The Coulter VCS instrument, a~ described in International
Patent Application Publication WO90/13013, to Russell et
al., and in PCT application PCT/US91/08590, provides a
histogram of radiofrèquency (rf) versus median angle light
~cattering tLS) for lysed and quenched whole blood
~amples, wherein lymphocytes (L), monocytes (M), and
granulocytes (G) are separated into non-overlapping
: regions. Subpopulations of lymphocytes can be enumerated
by shifting targeted sub-cell population to a different
unoccupied part of the histogram. Monoclonal antibody-
W095/24631 PCT~S95102547
218~Z82
coated polystyrene beads mixed with lysed and quenched
whole blood (EDTA anticoagulated) may be used to shift the
targeted sub-cell population. The magnitude and direction
of this shift will depend on the size, ~hape, and
refractive index of the beads and their relative effect on
light scattering from the targeted cells.
Non-targeted cells will also have collisional
interactions with these beads. For example, uncoated 2
micron diameter polystyrene beads mixed with lysed and
quenched whole blood changed the distributions of
lymphocytes, monocytes, and granulocytes cells in the
radiofrequency versus median angle light scattering
histogram 80 that each was broadened slightly along the LS
axis. Thus, coated beads will have varying bro~çn;ng
effects on targeted and the non-targeted white blood cell
populations along the LS axis of the histogram, depending
on the relative stickiness of collisions between beads and
cells.
Pending patent application Serial No. 07/961,157,
discloses the use of aminodextran (T-2M), referred to as
5X-Amdex, to coat aldehyde/sulfate polystyrene latex
beads. In this antibody-spacer-bead system, the spacer is
coupled to the aldehyde groups on the polystyrene bead and
the antibody is then conjugated to the unreacted amino
groups on the surface of the 5X-Amdex-polystyrene bead.
T4 and T8 antibody are conjugated to these coated beads,
which are then used on the Coulter VCS instrument to give
CD4 and CD8 percentages in total lymphocyte populations in
whole blood. sroadening in the light scatter histogram of
unshifted and non-targeted lymphocytes, monocytes, and
granulocytes in the rf versus LS histograms was apparent
in the use of T4, T8-SX-Amdex-bead.
Broadening along the light scattering axis of
histogram causes overlap between targeted and non-targeted
cell populations which makes it difficult to accurately
enumerate cell populations. Light scattering from non-
targeted white blood cells will be increased by the
W09Sl24631 PCT~S95tO2547
2I ~8f~
greater size of cells to which only a few beads become
attached in a reversible, sticky collision or by non-
specific cellular adsorption. Similar size (about two
micron diameter) beads coated only with adsorbed antibody
do not show the extreme broadening along the light scatter
axi~ of the histogram. Thus, a compact layer of antibody
molecules adsorbed on the bead surface shows very little
non-specific interaction with non-targeted cells. Storage
of T4, T8-5X-Amdex-polystyrene beads at higher
temperatures of about 20C and 37C for two weeks instead
of at a refrigerated temperature of about 5C further
increased the broadening along the light ~cattering axis
of the histogram. Unraveling of aminodextran ch~ or
denaturation of conjugated antibody from its compact Y- or
T-shaped structure to some uncoiled protein strand
structure can further increase the preponderance of non-
specific interactions with cells that lead to an increase
in light scattering from non-targeted cell populations.
Also, this denaturation of surface antibody does promote
aggregation of the beads into large clusters which also
~how more intense light scattering.
It has been shown proteinaceous material, such as
enzymes and antibodies, show increase thermal stability
when such proteinaceous materials are immobilized by
covalent attachment to solid surfaces or polymers. For
example, urease on phospholipid-bound silica, (Kallury et
al., ~nal Chem., 64: 1062-1068 (1992)); chymotrypsin on
aldehyde/agarose gels, (Guisan et al., Biotechnol.
Bioeng., 38: 1144-1152 (1991)); glucoamylase on various
periodate oxidized polysaccharides, Lenders and Crichton,
siotechnol. Bioen~., 31: 267-277 (1988)); and K. Mosbach,
Ed., Methods in Enzymology, Academic Press, New York,
1987, Vol. 135. Urease on a phospholipid-coated silica
~urface showed about 80-90~ activity after being treated
at 100C for one hour or stored at 25C for 168 hours.
Furthermore, U.S. Patent 5,120,6423, issued June 9, 1992
to Ching et al., describes proteins that have been dried
Wo95/24631 2 1 8 4 2 8 2 PCT~S95/02547
onto solid substrates and resolubilized to provide active
protein. Various proteins are used in immunoassay kits,
containing large millimeter size plastic beads coated with
an antibody, and distributed by Abbott Laboratories, North
Chicago, Illinoi~.
In addition, sugars have been used in preservation in
order to increase the stability of biological molecules
and structures. For example, monosaccharides,
disaccharides such as trehalose and sucrose, and other
sugars have been found to be very effective in
preservation of macromolecular structures (N. Hanafusa,
Freezin~ and Dryinq of Microorqanisms, Ed., T. Nei,
University Park Press, Baltimore, 1969, pp. 117-129) as
well as in protection of cell membranes during
lyophilization and reconstitution (U.S. Patent 5,059,518
issued Oct. 22, 1991; and U.S. Patent 5,171, 661 issued
Dec. 15, 1992). Also, the naturally occurring
polysaccharide, pectin, that is present in cell walls of
all plant tissues and binds cells together, is widely used
in the preservation of jellies and similar food products.
There is evidence that some macromolecules such as
triple helical soluble collagen show reversible reduction
by about two-fold of layer thickness on glass as
temperature is lowered from 15C to 7.7C (A. Silverberg,
Polvmer Adsorption and DisPersion StabilitY, Eds., E.D.
Goddard and B. Vincent, ACS Symposium Ser. 240, Amer.
Chem. Soc., Washington, D.C., 1984, pp. 161-167).
The present invention improves prior systems by u~ing
two approaches to reduce the light scatter from the non-
specific interaction between antibody-conjugated beads and
cells and still retain a covalently-linked antibody-bead
~ystem. First, a short, low-molecular weight spacer,
diaminoalkane is used as the coating for the beads to make
the antibody-bead system structurally more closely
resemble the adsorbed antibody-bead complex. Second,
antibody-5X-aminodextran-coated beads were freeze-dried
and reconstituted with distilled water to reduce non-
~Wo95/24631 2 1 8 4 2 ~ 2 rcT~sg~/02s47
specific cell interactions with the polymeric sugarcoating of the beads. Thus use of the present invention
improves prior systems by decreasing the broadening along
the light scatter axi~, even at temperatures higher than
that used in prior system. Freeze-drying and
reconstitution of antibody-5X-aminodextran-coated beads
further improves prior systems by increasing the stability
of these beads.
DISCLOSURE OF lNV~N ~ lON
The present invention provides a method for reducing
light ~catter shift of non-targeted white blood cell
population by using colloidal particles coated with
diaminoalkane to make an antibody-bead system.
The present invention provides a method for reducing
light scatter shift of non-targeted white blood cell
population by freeze drying antibody-5X-aminodextran-
coated beads.
The present invention also provides a method of
- increasing the stability antibodies when linked to 5X-
aminodextran- and diaminoalkane-coated beads.
The present invention also provides a method of
increasing the thermal stability of antibody-5X-
aminodextran-coated beads by freeze drying said beads.
The present invention further provides a method for
making antibody-diaminoalkane-coated beads.
The present invention provides antibody-colloidal
particle systems for use on flow cytometry and VCS based
instruments.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows the reaction scheme for coating DAP on
latex beads.
Figure 2 shows the binding curve (surface versus
total antibody concentration) for CD4, CD8 antibody on
DAP-coated latex beads.
WO95/24631 2 1 8 4 2 8 2 PCIIUS9S/02547
Figure 3 shows the dependence of mean CD4/CD8 cell
percent in whole blood lymphocytes on antibody (CD4, CD8)
concentration in preparation of latex beads.
Figure 4 ~hows the binding curve for CD4, CD8
5 antibody on DAP-coated latex beads.
Figure 5 shows the binding curve for CD4, CD8
antibody on 5X-Amdex-coated latex beads.
Figure 6 shows the bead titer ~L of 1~ w/v solids
CD4, CD8-DAP-PS latex beads) dependence of mean CD4/CD8
10 cell percent in whole blood lymphocytes.
Figure 7 shows the histograms of lysed/quenched whole
blood mixed with T4-DAP-PS beads stored at 5C (a), 20C
(b), and 37C (c) for two weeks. Same with T8-DAP-PS
beads.
Figure 8 shows the histograms of lysed/quenched whole
blood mixed with three preparations of T4-DAP-PS beads,
pairwise presented for beads stored at 5C and beads stored
at 50C (a-f). Same with T8-DAP-PS beads (g-l).
Figure 9 shows the histograms of lysed/quenched whole
20 blood mixed with T4-5X-Amdex-PS beads (d-f) stored at 5C
(a), 20C (b), and 37C (c) for two weeks. Same with T8-
SX-Amdex-PS beads.
Figure 10 show~ the histograms of lysed/quenched
whole blood mixed with T4-5X-Amdex-PS beads stored at 5C
25 (a); freeze-dried and reconstituted and stored at 5C (b),
20C (c), and 37C (d) for two weeks. Same with T8-5X-
Amdex-PS beads (e-h).
~.
Figure 11 shows the histograms of lysed/quenched
whole blood mixed with T4-5X-Amdex-PS bead~ freeze-dried ,~
WO95/24631 PCT~S95/02547
2184~8~
and recon~tituted and stored at 5C (a) and 50C tb) for
two weeks, and frozen and thawed (c). Same with T8-SX-
Amdex-PS beads (d-f).
.
Figure 12 shows the pairwise histograms, before and
after freeze-drying and reconstitution, of T4-5X-Amdex-PS
beads, T8-5X-Amdex-PS bead~, and 5X-Amdex-PS beads.
MODES FOR CARRYING OUT THE l~v~llON
While the present invention i~ satisfied by
embodiments in many different forms, there i9 shown in the
drawings and will herein be de~cribed in detail a
particular embodiment of the invention, with the
understanding that the follo~ing description is to be
considered as exemplary of the principles of the invention
and is not intended to limit the scope of the invention as
determined by the appended claims and their equivalent.
The present invention provides a method for the
preparation of colloidal particles with antibody
covalently coupled to the bead surface, and for the
verification of the suitability of these bead conjugates
for use on flow cytometry and VCS based instruments ~uch
as the COULTER~ STKS 2A and VCS instruments (Coulter
Corporation, Miami, Florida) to enumerate white blood cell
population in whole blood.
As u~ed herein and in the claims, colloidal particles
refer to polyacrolein beads, polyaldehyde beads and
polystyrene beads, preferably polystyrene aldehyde/~ulfate
latex beads. The aldehyde/~ulfate polystyrene latex
particles used herein have reactive aldehyde groups which
may combine with a ~ubstance such as an aminodextran and
form covalent bonds. The size of the polymer particles
- ranges from about 0.2 to about 3.0 microns. The preferred
particle ~ize iq in the range of about 0.6 to 3.0 micron~,
most preferred 1.5 to about 3Ø ~ecause of the cell size
dependence of ~hifts in the position of lymphocytes in
forward versus side scatter and rf versus light scatter
WO95124631 PCT~S9~/02547
2184282
hi~tograms caused by surrounding cells of lymphocyte
subsets with antibody-bead conjugates, the largest
available beads of diameter about two microns are the most
effective in giving a well-resolved shifted population of
S cells in the histograms.
As used herein and in the claims the term "alkane" in
"diaminoalkane" refers to straight chain hydrocarbon of 2
to 6 carbon atoms, such as ethane, propane, butane,
pentane hexane, preferably 3 and most preferably 1,3-
diaminopropane (DAP).
All reference to the monoclonal antibodies (mAb) usedherein i5 by the identifying designations used by Coulter
Corporation, Miami, Florida for monoclonal antibodie~ made
by Coulter Corporation. The following information further
identifies the antibodies used herein. The use of these
monoclonal antibodies is by way of example only and is not
to be understood as limiting the invention. The term "CD"
refers to "Cluster Designation" adopted by the
International Workshops on Human Leukocyte Differentiation
Antigens. A.T.C.C. is the American Type Culture
Collection, Rockville, Maryland.
Antibodv CD Descri~tion or Reference
Tll CD2 Derived from hybridization of
mouse NS/l-AG4 cells with spleen
celis of BALB/cJ mice immunized
with T cell chronic lymphocytic
leukemia cells.
T4 CD4 As Tll, but immunized with
peripheral human T lymphocytes.
T8 CD8 As Tll, but immunized with human
thymocytes.
lD3 -- U.S. Patent No. 4,93l,395;
A.T.C.C. Deposit No. HB 9445.
KC-48 -- U.S. Patent No. 4,865,971;
A.T.C.C.. Deposit No. HB 9584.
W09S/24631 218 4 2 ~ 2 PCT~S9S/02547
M02 CD14 R. F. Todd et al., J. Immunol.,
126: 1435-1442 (1981) .
B4 CDl9 L . M . Nadler et al., J. Immunol.,
134 :2228 (1985) .
Bl CD20 T. Stashenko et al., J. Immunol.,
25: 1678-1685 (1980) .
NKH-l CD56 J. D. Griffin et al., J. Immunol.
130: 2947-2951 ~19B3) .
MY4 CD14 J . D . Griffin et al., J . Im~unol.
68: 932-941 (1981) .
T3 CD3 P . C . L . Beverly and R.E. Callard
(IgG1) Eur. J. Immunol, 11:329-334
~1981) .
Polystyrene aldehyde/sulfate latex beads have
15 chemically reacti~e aldehyde functional groups on their
surface, which are an intrinsic part of the polymer
ch~;n¢ that make up the latex particle. These surface
aldehydes are first chemically coupled to a short, low-
molecular weight spacer such as 1, 3 -diaminopropane or to a
polymeric polyamine such as 5X-aminodextran which, in
turn, are activated with a heterobifunctional crosslinker
to covalently link antibody molecules to the bead surface.
The resulting bead-antibody conjugates contain tightly
bound antibody which i8 not released into the medium when
suspended in bovine serum albumin ~BSA) buffer solutions
or in whole blood with anti-coagulants and red blood cell
lysing and fixing agents.
The chemistry of the initial step of coupling
diaminoalkane to the surface of polystyrene/sulfate latex
beads i8 outlined in the chart in Figure 1. The reaction
of the aldehyde moiety with amine groups to form covalent
Schiff's base linkages proceeded best at an alkaline pH of
about 10 or above. Thus, the beads which were suspended
in distilled water were mixed with DAP to give a pH above
11. Schiff's base carbon-nitrogen bonds are stable if
they originate from aromatic aldehydes. The surface
Woss/24631 218 ~282 PCT~Sg5/02547
aldehyde on the polystyrene beads might be aromatic if it
were attached to the styrene ring in the polymer. Since
the method that was used to prepare the beads at
Interfacial Dynamic~ Corporation (IDC), Portland Oregon,
is not divulged, the Schiff~s bases must be reduced with
alkaline sodium borohydride solution to yield a stable
carbon-nitrogen bond. The latter reductant also plays an
additional role in the reduction of the unreacted surface
aldehyde groups to surface alcohol groups that cannot
further react non-specifically with amine groups in
antibodies-or antigens expressed on cell 6urfaces.
In principle, the direct reaction of amine groups
within the antibody with the surface aldehyde groups of
the beads is also possible. In practice, however, this
direct reaction gave very low surface antibody coverages
and very poor test results on the Coulter VCS instrument.
The negative results from this direct coupling of antibody
to bead probably stem from several adverse factors.
Steric hindrance can prevent the close approach of the
amino groups on or near the surface of the antibody
molecule to the surface aldehyde groups of the beads.
Since antibody molecules can be denatured at the high pH
of 10 needed for optimum amine-aldehyde coupling and by
reaction with borohydride solutions, optimum stabilization
of the Schiff's base formed by direct coupling could not
be achieved. Therefore, the direct antibody coupling
reaction was not further pursued.
The well-established heterobifunctional cross-linker,
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-
carboxylate (sulfo-SMCC), was used to activate DAP-coupled
beads for conjugation to iminothiolane activated antibody.
Because of the longer arms projecting from the surface of
sulfo-SMCC activated DAP-beads, this conjugation proces~
works well to yield high surface antibody coverages on the
beads and good test results on the VCS. The conjugation
depend~ on the reaction between maleimide groups of the
activated beads and sulfhydryl groups (intrinsic or from
W095/24631 2 ~ 8 4 2 8 2 PCT~S95/02547
iminothiolane activation of amine groups) of the antibody.
Methods for the preparation of aminodextrans with
various degrees of amine substitution and of polystyrene
aldehyde/sulfate latex beads with a covalent, first layer
of aminodextran to which antibodies were conjugated are
described in pending patent application, Serial No.
07/961,157, filed October 15, 1992, which is a
Continuation-in-Part of U.S. Patent 5,169,759, issued
December 8, 1992.
Methods of the Invention
Primary reagent~ were optimized in the following
sequence: 1. DAP concentration; 2. sulfo-SMCC
concentration; 3. bead ~ w/v solids; 4. antibody
concentration during conjugation. Initial runs of T4-
beads were done with 1~ w/v solids beads andconcentrations of sulfo-SMCC and iminothiolane-activated
antibody taken from established runs with previous beads,
i.e., 6.75 ~L of 10 mg/mL sulfo-SMCC per mL of 1~ w/v
solids beads and 0.625 mg I.T.-antibody per mL of 1~ w/v
2~ solids beads.
1. DAP Concentration
DAP concentration was varied over a wide range. The
stoichiometric amount of aldehyde on the bead surface was
not analytically determined by the manufacturer; but, an
upper limit on the amount of surface aldehyde was provided
by knowing the amount of aldehyde that was used in the
manufacture of the beads. This was 1 aldehyde per 50 A2 of
~urface area. For beads with a specific ~urface of 2.6464
mZ/g, this gives 0.088 ~mol aldehyde per mL of 1~ w/v
solids beads. This requires 6.5 ~g of diaminopropane per
mL of 1~ w/v solids beads by stoichiometry. In order to
avoid aggregation of the beads, a much larger amount of
DAP had to be used. In a series of runs with 2.0, 1.0,
0.5, 0.25, and O.125~ DAP with 1~ w/v ~olids beads, the
first trial with 2~ DAP showed the least number and
W095/2463l 218 4282 PCT~S95/02~47
smallest aggregates of beads after reduction with sodium
borohydride and washing. Further trials with 2.0 to 16.0
mg/mL DAP were continued to conjugate T4 antibody and
obtain VCS data. A DAP concentration of 4.0 mg/mL was
sufficient to obtain reproducible ~CD4 cells when 1~ w/v
solids beads were used. All higher DAP concentrations
were also acceptable but the 4.0 mg/mL DAP concentration
was selected for further work.
2. Sulfo-SMCC Concentration
o Next, several trials with 1/2X, lX, 2X and 3X the
initial 6.75 ~L of 10 mg/mL of sulfo-sMcc per mL of 1~ w/v
solids beads were made to activate the DAP-coated
polystyrene aldehyde/sulfate beads. The process was
carried through to conjugation of T4 antibody to the beads
activated at different levels and testing of these beads
in the VCS assay. The data show that 1/2X to 2X
activation is acceptable but 3X activation is not
acceptable for 1~ w/v solids beads. Thus, the lowest
activation level, 3.375 ~L of 10 mg/Ml sulfo-SMCC per Ml
of 1~ w/v solid~ beads was used in further work.
3. Bead Concentration
Further trials were made with varying bead
concentrations: 1~, 2.5~, and 4.2~ w/v solids beads.
Since the beads were supplied by the manufacturer at 4.2
w/v solids, this was used as the highest concentration of
beads. In working with polystyrene aldehyde/sulfate beads
of different mean diameters all at 1~ w/~ solids, it was
found that beads of diameter 0.6 ~m and smaller showed a
much greater tendency to aggregate. Therefore, the
particle number densities and mean free paths for these
particle suspensions at 1~ w/v solids were calculated and
listed below:
Mean Diameter, Particles/cc Mean Free Path,
~m ~m
1. 0.294 7.19 x 1011 1.12
2. 0.604 8.29 x 101 2.29
Wo95/24G3l 218 ~ 2 8 2 PCT~S95/02547
3. 1.01 1.78 x 101 3.83
4. 1.59 4.55 x 109 6.03
5. 2.15 1.84 x 109 8.16
Compared at a fixed percentage of solidR, the much
larger numbers of smaller particles and, thus, the smaller
mean free path will produce many more collisions per unit
time that can result in aggregation. Thus, 2 ~m diameter
beads at 4.2~ w/v solids will have a mean free path of
about 2 ~m, 80 that aggregation might become a serious
problem. The previously selected amounts of DAP and
sulfo-SMCC for 1~ w/v solids beads were multiplied by 2.5
and 4.2 for the respective trials, since surface area or
number of aldehyde groups on the spherical bead surface is
directly related to the mass of beads expressed for a
constant volume through the specific surface in units of
m2/g. The VCS data for T4-conjugated beads at various bead
concentrations showed that 2.5~ and 4.2~ w/v solids beads
can be used throughout the preparative process a well as
1~ w/v solids beads. The highest bead concentration,
4.2~, was used in further work to obtain the greateRt
yields of antibody-conjugated beads in any one trial.
4. Antibody Concentration
To optimize the amount of activated antibody, T4 or
T8, used for conjugation to activated beads, a wide range
of total 2-iminothiolane-antibody concentrations between
0.1 and 2.0 mg/mL were used. For each concentration
trial, data were accumulated indirectly for surface
antibody concentrations after the conjugation period (by
filtering beads and reading absorbance at 280 nm for
antibody concentration in supernatant; knowing total
antibody concentration, the surface antibody concentration
was calculated from the difference between total and
supernatant antibody concentrations) and for ~CD4 and ~CD8
in whole blood by using the purified beads in the VCS
assay. The results for 2 ~m raw beads showed that surface
coverages of T4 and T8 antibody on beads reached a plateau
W095/24631 2 18 4 2 8 ~ PCT~S95102547
l4
at about 0.8 and 1.3 mg/mL total antibody concentration
and 0.45 and 0.75 mg/mL surface antibody concentration,
respectively; while ~CD4 and ~CD8 in whole blood by the
VCS assay levelled off to about 0.3 and 0.8 mg/mL total
antibody concentration, re~pectively. A graphical display
of the latter results i9 shown in Figure~ 2 and 3.
Similar data on a different lot of 2 ~m raw beads gave
surface coverage of T4 and T8 antibody with no well-
defined plateau, especially the curve for T8 antibody
which had a steadily climbing surface antibody load after
the inflexion point. The results are shown graphically in
Figure 4. The m~X; r~lm surface coverage of T8 antibody on
beads in both trials is about a factor of 5/3X greater
than the ~ lm T4 antibody coverage.
The above indirect method of assaying for surface
antibody coverage does not discriminate between
covalently-bound antibody and loosely-bound or adsorbed
antibody, which may later be washed away by the bead
purification steps with BSA buffer. For T4 antibody on
beads at pH = 7.2-7.3 the maximum coverage~ in the 3-4
mg/m2 range represent about monolayer surface coverage when
compared with literature data for IgG on solid polymer
surfaces. Eosinisothiolcyanate-bovine serum albumin
(EITC-BSA) on polymethyl methacrylate (PMMA) was shown-by
Tilton, Ch~nn;ng, and Gast [R.D. Tilton, Surface Diffusion
and HydroPhobic Interactions in Protein AdsorPtion, Ph.D.
dissertation, Stanford University, 1991] to have a surface
loading of about 1.5 mg/m2 at its plateau when
concentrations were determined by a surface radioactivity
technique. Similar data, about 1.5 mg/m2 saturation
coverage, were obtained with radioiodinated BSA on sulfate
polystyrene latex beads (Donald E. Brooks, Emerqinq
Concepts in Medical Diaqnostic Applications of Latexes,
Orlando, Florida, December 4-5, 1989). Bagchi and
Birnbaum (Bagchi and Birnbaum, J. Colloid Interface Sci.,
83: 460-478 (1981)) have reported relatively low surface
coverages, about 1.5 to 3.0 mg/m2, of rabbit IgG on
Wo95124631 PCT~S95/02547
21842~2
negatively charged latex beads at pH = 4.0 and pH = 10.0;
however, at the isoelectric point, pH = 7.8, of the IgG
the saturation coverage was about 7.5 mg/m2. The large
variation in surface coverage with pH was attributed to
changes in structure and/or orientation of the IgG, which
in turn would give values of surface area per molecule
between 3.1 x 103 and 12.4 x 103 A2.
Therefore, the high surface coverages of about 5.5 to
7.0 mg/m2 of T8 antibody on beads at pH = 7.2-7.3 could
represent either conformation/orientation changes in T8
antibody to accommodate more molecules per unit surface
area or multi-layer adsorption of T8 antibody on top of
the first covalently-bound T8 lay.er on the beads. sCA
.direct surface antibody assay results (vide infra) for
samples taken after washing conjugated and blocked T8-
beads with lxPBS showed that a large amount of loosely-
adsorbed T8 was removed by the washings. An explanation
for the second adsorbed layer of T8 antibody on beads but
no similar layer of T4 antibody on beads lies in the
isoelectric points of the two antibodies (pI range of T4
antibody: 5.4-5.8; pI range of T8 antibody: 6.7-7.3
from electrophoresis data) and the pH of the conjugation
mixture in lxPBS, 7.3-7.3. Since the pI range of T8 and
the pH of the medium during conjugation overlap
considerably and antibody i8 least soluble or most
attracted to itself at its isoelectric point, the excess
T8 antlbody tends to precipitate onto the solid bead
~urface on top of the first covalently-bound layer. T4
antibody on beads does not behave like T8 on beads since
there is no overlap between its pI range and the pH of the
medium during conjugation. Further, high coverages of
both T4 and T8 on the same polystyrene aldehyde/sulfate
beads, coated with polymeric 5X-aminodextran instead of
the low-molecular weight, 1,3-diaminopropane, were
observed in the 7.0-8.0 mg/m2 range. The surface coverage
results, shown in Figure 5, indicate that polyamine-coated
polystyrene latex particles are capable of increasing the
W095/24631 2 1 8 4 2 8 2 PCT~Sss/02S47
available surface for conjugation of antibody up to two-
fold. Also, the aminodextran coating alters the surface
charge of the particles-from slightly negative due to
terminal sulfate groups on polystyrene ch~ to positive
due to protonated amino groups at pH 7.~-7.3 of the lxPBS
medium. This positively charged particle surface is then
more attractive to T4 antibody which has a slight negative
charge at near neutral pH values of the medium. The T4
and T8 antibody concentrations during conjugation were
therefore chosen to be 0.7 and 0.8 mg/mL for optimum
surface coverage and VCS assay results.
Three trials of both T4 and T8 beads were done under
the above optimized conditions. The volume of about 4
w/v solids beads was kept at 75 mL for each of the six
runs, three with T4 antibody and three with T8 antibody.
All trials were prepared with total T4 and T8
concentrations of about 6.2 and 7.1 mg/m2 (0.7 and 0.8
mg/mL). The first trials gave apparent surface T4 and T8
coverages of 3.7 and 4.2 mg/m2; the second, 5.0 and 6.3
mg/m2; and the third, 5.3 and 6.2 mg/m2, respectively.
Bic; nchO~; n; c acid (BCA) direct T4 and T8 antibody assay
results for the second trial, in which samples were washed
with lxPBS at pH - 7.2-7.3 ;~e~;ately after conjugation,
gave T4 and T8 surface coverages of 1.9 and 1.1 mg/m2,
respectively. These coverages are more in line with
values expected for covalently-bound antibody on the latex
beads.
All VCS testing was done with the following
protocol: 20 ~L of 1~ w/v solids T4 or T8 beads and 100
~L of whole blood; two minute immediate mix. The standard
statistical protocol for obtaining ~CD4/CD8 data is two
donors; eleven runs per donor with lX titer (20 ~L of 1
w/v solids beads); and eleven runs per donor with 1/4X
titer (5 ~L of 1~ w/v solids beads). All three trials
gave CVs under 5~ for mean ~CD4/CD8. A typical graphical
representation of ~CD4/CD8 from VCS assays versus bead
titers with whole blood is shown in Figure 6 for trial
' Wo95124631 2 1 8 4 2 8 2 PCT~S95/02S47
one. The curves all reach a plateau at about lO ~L titer
of l~ w/v solids beads per lOO ~L of whole blood aspirated
on the VCS instrument, 80 that the standard titer of 20 ~L
of l~ w/v solids beads is more than enough for evaluating
~CD4/CD8 in all potential donor~.
THERMAL STABILITY OF T4,T8- CONJUGATED BEADS
The effect of ~torage of antibody-conjugated beads at
higher than normal temperature (about 5C) in a
refrigerator was tested in closed vials at room
temperature (about 20C) and in an incubator at 37C. The
adequacy wa6 tested in terms of number and strength of
multiple covalent binding site~ from antibody to the bead
surface to prevent the antibody from changing its active
conformation and orientation with respect to the bead
surface when the beads are brought to elevated
temperatures for extended periods of time.
In the VCS assay of beads with whole blood, the
functional test of antibody-conjugated beads stored at
elevated temperature~ i9 performed for comparison with
beads stored at 4-10C. Their ability to shift relevant
subpopulations of white blood cell6 in the rf versus LS
histogram according to the specificity of the antibody i9
evaluated. Any denaturation of the surface-bound antibody
may be reflected in non-specific interactions with non-
targeted cell populations. This typically broadens thelymphocyte and granulocyte-monocyte distributions along
the light scatter (LS) axis of the histogram and may yield
a false higher percentage of targeted cells because of
mixing of shifted and unshifted cell populations. Also,
the normal titer of beads might not be sufficient to shift
all targeted cells 80 that a fal6e lower percentage will
be measured. The supernatants of all tested bead
suspensions were also analyzed for free antibody by an EIA
assay to verify whether the antibody was strongly bound to
the bead surface.
Wogs/2463l 2 1 8 4 2 8 2 PCT~S9~/02547
Testing wa~ done on all three trials of optimized T4
and T8 beads, stored for two weeks in capped vials at 5C
in a refrigerator, at about 20C ambient room temperature,
and at 37C in an incubator. VCS test results are
presented in Table I and repre~entative rf versus LS
histograms are displayed in Figure 7. T4,T8-conjugated
beads stored at elevated temperatures showed very little
degradation as seen in the CVs ranging between 1.0 and
3.0~ for the mean percent CD4 or CD8 for three
temperatures. Individual coefficient of variations (CVs)
for measurements made in quintuplet were high, 6.9 and
7.7~, in two runs with T8-conjugated beads stored at 37C
for two weeks; however, deviations from the mean in both
cases were still less than two SDs. These T8-conjugated
beads also showed the largest broadening of non-targeted
lymphocyte and granulocyte-monocyte populations in the
histograms shown in Figure 7. The degree of broadening can
be associated with some degradation in the way in which
surface antibody pre~ents itself to cells with which it
interacts, i.e., the extent of non-specific interactions
with non-targeted cells has increased. The extent of
deterioration, however, was not great enough for the
population of non-targeted cells to interfere with the
population of targeted cells in the VCS assay.
~'VO 95124631 PCI/US95/02547
218428~
19
Table I
VCS Assay Results for ~CD4/CD8 in Whole Blood
(Donor 32442 for lots -47 and -81;Donor 53254 for lot -24)
for Accelerated Stability Run on T4, T8-Conjugated
Polystyrene Beads
~eadTrial 1 2 3 4 S Mean SD CV,%
Lot Temp. % CD4(-1) or CD8(-2)
6C 43.24 44.27 43.88 43.69 41.66 43.2S 1.00 2.30
-47-1 20C 44.22 41.79 4S.81 44.40 42.17 43.68 1.67 3.80
37C 43.73 42.78 43.39 43.89 40.12 42.78 I.SS 3.60
Mean 43.24
SD 0.4S
CV, % 1.00
6C 42.82 43.33 43.64 44.19 43.32 43.46 O.S0 1.20
- 81-1 20C 46.63 43.44 43.S0 41.20 43.74 43.70 1.93 4.40
37C 43.0S 43.0S 44.40 42.96 41.03 42.90 1.21 2.80
Mean 43.3S
SD 0.41
CV, % 1.00
6C 21.23 20.02 21.26 21.31 20.S1 20.87 O.S7 2.80
-47-2 20C 18.47 19.22 20.44 21.09 20.04 19.85 1.03 S.20
37C 18.95 19.S3 20.17 18.80 22.19 19.93 1.38 6.90
Mean 20.22
SD 0.68
cv, æ 3.00
6C 20.S2 22.57 21.07 21.70 22.S7 21.69 0.91 4.20
-81-2 20C 22.66 21.79 21.S2 22.02 22.97 22.19 0.61 2.70
37C 24.70 20.87 21.97 24.98 22.64 23.03 1.77 7.70
Mean 22.30
SD 0.68
CV, % 3.00
6C S0.4S S0.21 Sl.87 S2.11 S0.2S S0.98 0.93 1.83
- 24-1 20C 49.73 Sl.13 S0.00 S0.63 48.66 S0.03 0.94 1.88
37C 49.91 S0.20 49.81 48.14 S0.72 49.76 0.97 I.9S
Mean S0.26
SD 0.64
CV, % 1.28
6C 19.92 21.21 20.18 20.70 19.91 20.38 O.S6 2.7S
-24-2 20C 21.22 20.37 20.2S 19.92 20.S1 20.4S O.S0 2.46
37C 21.33 19.96 19.70 21.2S 20.11 20.47 0.76 3.73
Mean 20.43
SD O.OS
CV, % 0.23
~09St24631 2 18 4282 PCT~S95/02547
Duplicate samples were also stored for two weeks at
about 47-50C in an oven. Representative VCS histograms
for the heated samples and the control samples stored at
about 5C are shown in Figure 8. The T4 beads were still
functional giving CVs of 3.3, 1.6, and 2.5 for mean
percentages of CD4 of 53.09, 52.37, and 52.89,
respectively, in the three trials, compared to control
bead values of 52.73 (1.2~ CV), 52.52 (2.7~ CV), and 52.48
(2.2~ CV), even though some broadening of the non-targeted
cell populations was observed with the use of heated
beads. T8 beads stored at 47-50C showed more extensive
broadening of non-targeted cell distributions. They also
failed completely to shift the targeted cells in one trial
in which the control beads gave a mean ~CD8 of 15.43 (2.4
CV), and gave CVs of 4.4 and 7.6 for mean ~CD8 of 16.20
and 15.07, respectively, in the other two trials, for
which the control beads gave values of 15.62 (5.3~ CV) and
15.20 (7.7~ CV).
EIA analyses for antibody in the supernatant of
samples stored in closed vials at 20C and 37C ~howed
about the same low amounts of free antibody as in analyses
of refrigerated samples. Results for T4 samples gave the
following data -- trial one: 1.13, 1.40, and 2.65; trial
two: 0.75, 1.02, and 1.40; trial three: 1.43, 1.83, and
3.41 ~g/mL at 5, 20, and 37C storage, respectively. T8
samples gave the following results -- trial one: 0.58,
0.67, and 1.28; trial two: 0.44, 0.45, and 0.45; trial
three: 1.24, 1.60, 2.64 ~g/mL at 5, 20, and 37C storage,
respectively. Three samples stored at 37C for two weeks
showed larger than normal amounts, 2.65, 3.41, and 2.64
~g/mL, of free antibody. The largest amount represents
about 1.7~ of the surface-bound T4 antibody for freshly
prepared beads.
Testing of T4,T8--5X-Amdex--polystyrene beads stored
at 5C, about 20C, and 37C for two weeks in closed ~ial~
showed considerable degradation of bead performance at the
higher temperatures, especially for T4 beads, as seen in
W095/2463l 2 1 8 4 2 8 2 PCT~S~S/02547
the histograms in Figure 9. The percentage CD4/CD8
obtained with these beads stored at elevated temperatures
could not be reliably measured. EIA analyses of the
supernatants, however, showed little shedding of any
antibody at elevated temperatures: for T4 beads- 0.43,
0.80, and 1.37 ~g/mL; for T8 beads- 0.27, 0.57, and 0.55
~g/mL for 5, 20, and 37C storage, respectively.
CRYOGENIC AND DRY STORAGE ST~3ILITY OF T4, T8-CONJUGATED
BEADS
In one run, samples of T4, T8--DAP- -bead trial~ one,
two, and three, suspended in 1~ BSA, 0.1~ sodium azide in
lxPBS, were freeze-dried and stored in capped vials in the
dry state at about -20C for four days. In a second run,
duplicate samples as well as adsorbed T4 and T8 beads were
freeze-dried and stored in capped vials in the dry state
at about -20C for one week. The lyophilized samples were
then brought to room temperature and reconstituted with
distilled water. These samples were all tested on the VCS
for functionality and results are summarized in Tables II
and III. In a third run, duplicate samples, first washed
free of salt and protein with distilled water, were
freeze-dried and stored in capped vials at -20C for one
week before reconstitution with BSA buffer solution. Test
results for these beads compared to untreated samples are
compiled in Table IV. VCS test results for run one
freeze-dried beads showed excellent results for CD4 and
CD8 percentages in the lymphocyte population, with CVs
less than 5~ except in one case in run two for T8 bead~ of
trial three. Some small degree of expansion of the non-
targeted lymphocyte population in the rf versus LShistogram did occur along the LS axis for bead trials two
and three, while trial one beads showed hardly any change
from results for non-freeze-dried bead suspensions. These
results confirm the cryogenic and dry storage stability of
covalently immobilized T4 and T8 antibodies on polystyrene
beads. It is to be noted that the medium in which the
2184282
-"095t24631 PCT~S95/02547
beads are freeze-dried is crucial for successful
reconstitution o~ viable antibody-conjugated beads. When
antibody-conjugated beads were first washed with distilled
water as in the protocol for run three; then, VCS te~ting
of the resultant beads showed much more deterioration in
the functioning of T4 and T8 conjugated beads. Non-
targeted lymphocyte and granulocyte-monocyte populations
showed extreme broadening along the light scatter (LS)
axis, similar to results obtained with T4 and T8
conjugated to aminodextran-coated polystyrene latex beads.
T8 beads gave CVs greater than 5~ for CD8 percentages.
Table II
VCS Test Results for
Freeze-Dried and Reconstituted Samples (FD)
Donor
42440 % CD4 % CD8
lot- 47-l, FD - 81-1, FD - 47-2, Fl~ - 81-2, FD
trbl
Sl.16 S0.28 17.2S 17.66
249.98 S1.04 18.32 19.24
349.31 S0.02 17.97 19.67
449.72 48.08 18.13 19.70
SS0.73 Sl.99 17.77 18.11
MeanS0.18 S0.28 17.89 18.88
SD0.7S 1.42 0.42 0.94
CV, 9~ I.S0 2.80 2.40 4.90
lot- 24-1 - 24~1, FD - 24-2 - 24-2, FD
trial
48.93 49.S2 18.94 17.78
248.42 Sl.13 17.S3 18.66
349.61 SO.IS ` 17.20 17.S7
449.27 48.89 18.71 17.S2
SS0.16 S0.83 17.64 16.96
Mean49.28 S0.10 18.00 17.70
SD0.66 0.92 0.77 0.63
CV, %1.30 1,80 4.30 3.S0
' WO 95/24631 2 1 8 4 2 8 2 PCT/llS9~/02547
Tsble 111
VCS Test Results for Frce~e-Drled, Dry Stored (I Week),
Jnd r-~ 3 S-mple5 (PD)
Donor
46986 % CD4
lot- 47-1 - 47-l,FD - 81-1 - 81-1, FD - 24-1 - 24-1, PD 417-1179-5 -1179-5, PD
trl-l
S0.8S 49.08 49.31 50.47 49.93 50.77 48.41 52.83
247.29 48.98 48.56 49.98,50.03 50.62 49.52 52.00
348.50 51.32 47.48 S0.96 47.82 47.98 48.33 S0.18
447.S2 Sl.60 49.70 48.08 49.12 50.20 48.63 51.60
548.6S 51.21 49.22 50.72 48.58 49.S6 48.61 47.38
Me-n48.S6 S0.44 48.8S 50.04 49.10 49.83 48.70 50.80
SD1.41 1.29 0.87 I.IS 0.93 1.13 0.47 2.14
CV, %2.90 2.S6 1.78 2.31 1.89 2.27 0.97 4.21
X CD8
lol- 47-2 - 47-2, FD - 81-2 - 81-2, PD 24-2 - 24-2, FD 677-2342-62-2342-62,FD
lrlsl
22.97 21.86 24.31 23.50 23.58 23.48 23.86 23.4S
222.36 23.94 24.04 23.3C 23.50 23.22 22.53 23.70
322.22 22.99 25.75 25.75 23.83 20.68 24.10 23.15
21.07 23.50 24.95 24.34 22.34 21.26 22.78 23.87
523.26 21.25 24.75 25.06 21.92 22.48 22.14 22.00
Mesn22.38 22.71 24.76 24.40 23.03 22.22 23.08 23.13
SD0.85 1.12 0.66 1.02 0.85 1.22 0.85 0.69
CV, %3.78 4.95 2.66 4.18 3.68 5.49 3.70 3.00
WO 95/24631 2 1 8 ~ 2 8 2 PCIIUS95102547
Table IV
VCS Test Results for Distilled-Wa~er-Washed }~eads;
Freeze-Dried and ~q s~ ' (FD)
Donor
47274 % CD4 ~O CD8
lot- 47-1- 47-1, FD - 47-2 - 47~, FD
trbl
37.98 37.07 28.00 22.42
2 38.32 38.85 27.24 26.62
3 37.81 36.96 28.86 27.31
4 36.29 3S.76 26.38 27.S2
S 3S.94 37.40 27.S3 2S.46
Mean 37.27 37.21 2~.60 2S.87
SD1.07 1.11 0.92 2.09
CV, % 2.90 3.00 3.30 8.10
lot- 81-1- 81-1, FD - 81-2 - 81-2, FD
trial
37.87 38.02 29.80 27.00
2 36.67 36.70 30.04 27.14
3 37.40 40.27 30.26 2S.92
4 37.78 36.S4 29.54 27.98
S 39.63 37.71 28.93 30.35
Mean 37.87 37.8S 29.71 27.68
SD1.10 l .S0 O.S3 1.66
CV, % 2.90 3.90 1.80 6.00
Similar freeze-drying and dry storage of T4,T8--5X-
aminodextran--polystyrene beads followed by reconstitution
with distilled water showed a vast improvement over non-
freeze-dried samples in performance in the VCS assay.
Fresh and reconstituted sample histograms are compared in
Figure 10. The freeze-dried and reconstituted, T4,T8-5X-
Amdex-bead ~amples now perform as well as T4,T8--DAP-
polystyrene or adsorbed T4,T8--polystyrene beads (CVs
below 5~) and show histograms in which the L, G, and M
distributions are as narrow along the LS axis. It is
inferred that loose, hydrophilic ch~;n~ of the polymeric
sugar on the surface of the bead followed the water during
its re~oval by freeze-drying to co~en~e onto the
particles. Then, the outer part of the aminodextran sugar
layer collapsed or ~hrunk onto the bead without loss of
conjugated antibody activity. Since aminodextran was
2184Z82
'V095/24631 PCT/US95/02547
covalently coupled to the aidehyde bead surface and
crosslinked with glutaraldehyde, as well as blocked with
fresh aminodextran, a very low-density, polymer coating
was fixed to the polystyrene beads. Thus, it i9
appropriate that the aminodextran coating and antibody
conjugated to the outer surface of the coating might
rearrange their structures upon freeze-drying. The
antibody presentation on the composite bead surface
apparently became the lor~n~r~t surface feature 80 that
non-specific interactions with non-targeted cells were
enormously reduced.
Further, a sample of the reconstituted beads that
were stored at about 4C, was taken and stored in a closed
vial in an incubator at 37C for two weeks. These samples
were also compared in the VCS assay and showed in Figure
10 a small amount of broadening of non-targeted cell
populations for reconstituted beads kept at 37C, but did
not reverse to the broad distributions given by the
original beads. Another set of freeze-dried and
reconstituted T4,T8-5X-aminodextran bead samples, kept at
about 47-50C for two weeks, also passed specifications in
the VCS assay as shown in histograms in Figure 11, showing
that these beads were much more stable to thermal
treatment than T4,T8-DAP-PS beads by not reversing to
their original state. The mean percentage CD4 was 50.96
(1.8~ C~) with control bead~ and 51.84 (3.1~ CV) with
heated beads, and the mean percentage CD8 was 15.82 (8.3~
CV, < 2 SD) with control beads and 15.66 (7.3~ CV, c 2 SD)
with heated beads, all measured in quintuplet for a single
blood donor. Thus, when freeze-dried and reconstituted,
the polysaccharide derivative, aminodextran, coupled to
polystyrene beads is much more effective in preserving the
macromolecular structure of antibody that iB conjugated to
it than it i8 when not freeze-dried and reconstituted.
Size measurements of the T4-5X-Amdex-PS beads before
and after freeze-drying and reconstitution were made by
photon correlation spectroscopy or quasi-elastic light
'~095124631 2 1 8 4 2 8 2 PCT~S95/02~47
26
scattering on the COULTER N4MD sub-micron particle
analyzer, using a 90 scattering angle. Beads suspended at
1~ w/v solids in l~BSA, 0.1~ sodium azide in lxPBS buffer
were diluted 300-fold with two times filtered (0.2 ~m
cellulose nitrate filter) lxPss solution and measured in a
1 cm path quartz fluorescence cell with 5 sides polished.
Reconstituted bead~ gave a weighted mean diameter of 3.04
~m with an intra-assay standard deviation of 0.34 ~m while
original beads gave similar data but with an additional
large contribution (~15~) of dust, i. e., particles
greater than lo ~m in size. The raw polystyrene
aldehyde/sulfate beads gave a mean diameter of 2.07 ~m
with an intra-assay standard deviation of 0.36 ~m and an
inter-assay S.D. of 0.10 ~m for three runs of eleven
measurements each. The thickness of the T4 or T8 antibody
and 5X-aminodextran coating is therefore 0.48 ~m for
spherical particles after freeze-drying and
reconstitution. This compares with a calculated thickness
of 0.22 ~m for 5X-aminodextran on 0.30 ~m diameter ferrite
particles, as estimated from the carbon analysis for
distilled water washed and dried particles. A few large
aggregates of coated beads are responsible for large light
scattering contribution, and these large bead aggregates
are dissociated into individual, primary beads in the
freeze-drying and reconstitution process.
The same T4,T8-5X-aminodextran-PS beads were also
measured before and after freeze-drying and reconstitution
on the COULTER Profile II flow cytometer to obtain forward
light scatter versus side light ~catter histograms as
shown in Figure 12. A single predominant circular and
tight distribution was observed for both coated and non-
coated raw beads; however, both fresh and freeze-dried and
reconstituted samples showed a minor weak distribution at
higher forward and side scatter intensity showing the
presence of small aggregates of two or three particles.
No difference was observed in the histograms to indicate
-"095/24631 2 1 8 ~2 8 2 PCT~S95/02547
27
the presence of very large aggregates in the original
samples .
T4,T8--5X-Amdex--polystyrene beads that were frozen
at -20C and then allowed to thaw out in a refrigerator at
about 5C for about two hours were also tested on the VCS
instrument. These samples showed a small amount of
improvement in the histograms (Figure 11) by a narrowing
of non-targeted L, G, and M distributions along the LS
axis.
All publications cited in this specification are
indicative of the level of skill of those in the art to
which this application pertains. Each publication is
individually incorporated herein by reference in the
location where it is cited.
The following examples serve to illustrate the
present invention. The concentration of reagents,
temperatures and values of other variable parameters are
only to exemplify application of the present invention and
are not to be considered as limitations thereof.
EXAMPLE I
COUPLING OF 1,3-DIAMINOPROPANE
TO POLYSTYRENE LATEX BEADS
To 150 mL of 4.2~ w/v solids polystyrene
aldehyde/sulfate beads (Interfacial Dynamics Corp., about
2 ~m diameter) in a 250 mL-polypropylene centrifuge tube
were added 16.8 mg of liquid DAP (0.888 g/mL specific
gravity) per mL of bead suspension or 2.838 mL DAP. The
resulting suspension was mixed well by vortexing,
sonicated for 30 sec, and roller mixed for 24 to 72 hours.
Reduction of Schiff's base groups and unreacted
aldehyde on bead surface.
Solid sodium borohydride, 47.46 mg per mL of 4.2~ w/v
solids beads that were roller mixed with DAP, or 7.119 g
were added directly to the bead reaction mixture. No
excessive effervescence took place in the presence of
. -vo gs/2463l 2 1~ ~ 2 ~ ~ PCT~S95102547
excess DAP at a pH of about 11.5. The reaction mixture
was roller mixed for three hours with occasional brief 30
sec sonication and then the beads were separated by
centrifugation at about 2000 g for 5 min. The supernatant
was discarded and the residue of beads was resuspended in
150 mL distilled water. Redispersion of the beads was
accomplished by vortexing and brief sonication. The
washing procedure by centrifugation was repeated four
times and the final bead suspension was adjusted to 150 mL
total volume with lxPBS solution.
EXAMPLE II
ACTIVATION OF DAP COUPLED BEADS WITH SUBFO-SMCC
About 14.175 ~L of freshly prepared 10 mg/mL sulfo-
SMCC in lxPBS was used per milliliter of 4.2~ w/v Rolids
DAP-coated polystyrene bead suspension. In a typical
preparation, 1.063 mL of the sulfo-SMCC solution was added
to 75 mL of 4.2~ w/v solids beads. The mixture was then
roller mixed in a 250 mL plastic centrifuge tube for about
one hour, separated by centrifugation, and washed a
plurality of times with lxPBS solution.
The functionalized, DAP-coated particles resulting
from the above series of steps have pendent maleimidyl
groups and are suitable for conjugation to a variety of
biological molecules. If the substance which i9 desired
to be conjugated to the particles has a sufficiency of
active sulfhydryl groups, activation of that substance is
not necessary, and the following Example III may be
skipped.
EXAMPLE III
ANTIBODY ACTIVATION WITH 2-IMINOTHIOLANE
HYDROCHLORIDE
A 35.99 mg/Ml concentrate of T4 monoclonal antibody
in lxPBS containing 0.1~ sodium azide was prepared. For
-vo9s124631 2 1 8 ~ 2 ~ 2 PCT~S9StO2547
29
100 mg T4 (or T8) antibody and 15 mg/mL antibody
concentration during coupling, the total reaction volume
should be 6.667 mL. Using a 15:1::IT:T4 activation ratio,
9.375 ~mol (1.29 mg) IT (0.65 mL of 2 mg/mL IT) in lxPBS
i8 required. Therefore, 3.238 mL of lxPBS solution was
added to 2.779 mL of T4 concentrate, to which resulting
solution an additional 0.65 mL of 2 mg/mL IT solution was
added. The net re~ulting solution was roller mixed in a
tube reactor for 1 hour. The contents of the reaction
tube were then applied to the top of a 200 mL G-50
Sephadex column, equilibrated and washed with 500 mL
lxPBS. The derivatized antibody was eluted using lxPBS
and a plurality of 5 mL fractions were collected with the
aid of a W monitor. Fractions in the middle of the band
absorbing at 280 nm were pooled and the A280 value was
used to determine the T4/IT antibody concentration.
Typically, the T4/IT or T8/IT concentration was about 5
mg/mL.
EXAMPLE IV
CONJUGATION OF T4/IT OR T8/IT WITH
SULFO-SMCC DERIVATIZED PARTICLES
With a total volume 100 mL, the concentration of
particles was 3.15~ w/v solids and the T4/IT concentration
was 0.7 mg/mL or the T8/IT concentration was 0.8 mg/mL.
In one run, when the purified T4/IT solution concentration
was 5. 013 mg/mL, then 13.964 mL of T4/IT antibody solution
in lxPBS was added to 75 mL of 4. 2~ w/v solids sulfo-SMCC
activated particles which had been preconcentrated by the
removal of 5 mL of supernatant. In another run, the
purified T8/IT antibody solution concentration was 5.703
mg/mL, 80 that 14.028 mL of T8/IT antibody solution in
lxPBS was added to 75 mL of 4. 2~ w/v solids sulfo-SMCC
activated particles which had been preconcentrated by the
removal of 5 mL of supernatant. The antibody/IT solution
was added to the particles in 2 mL increments with
W095/24631 2 1 8 4 2 8 ~ PCT~S95/02547
vortexing and ultrasonication between additions. The
resultant mixture was then roller mixed in a 250 mL-tube
for about two hours.
EX~MPLE v
BLOCKING UNREACTED MALEIMIDYL AND SU~FHYDRYL GROUPS
Unreacted maleimidyl groups on the sulfo-SMCC
activated particles were blocked with L-cysteine after
antibody conjugation. Typically, 11.88 mL of 5 mg/m~ L-
cysteine in lxPBS were added to the conjugation mixture of
the previous step and the resulting suspension was roller
mixed for 15 min. Unreacted sulfhydryl groups were
blocked by the addition of 13.31 mL of 20 mg/mL
iodoacetamide in lxPBS followed by the addition of 2.48 mL
of lM, pH 9.8 sodium borate buffer solution. The resulting
suspension was roller mixed for 30 min, the blocked
conjugation mixture was separated by centrifugation and
the particles washed two times with a solution of 1~
bovine serum albumin (Pentex Fraction V, protease free)
and 0.1~ sodium azide in lxPBS (BSA buffer solution).
After washing, the particles were resuspended in BSA
buffer Rolution to a total volume of 75 mL ( 4.2~ w/v
solids), roller mixed for 1 hour, stored at about 4C for a
time in the range of 8-16 hours, separated by
centrifugation and washed three times with BSA buffer
solution.
EXAMPLE VI
COUPLING OF AMINODEXTRAN COATING TO BEADS
5X-aminodextran was prepared, purified, and freeze-
dried according to methods described in a copending patent
application, Serial No. 07/961,157, incorporated herein by
reference. The same aldehyde/sulfate polystyrene latex
j particles of about 2 ~m diameter from Interfacial Dynamics
W095t24631 2 1 8 ~ 2 8 2 PCT~S95/02S47
31
Corporation (Portland, Oregon) were used here as for the
preparation of DAP-coated beads.
59.524 mL of polystyrene bead concentrate, 4.2~ w/v
solids, was added to a 250 mL centrifuge tube. 1.250 g of
solid 5X-aminodextran were di~solved in loo mL of
distilled water and transferred into the tube containing
the polystyrene particles. Then, 90.476 mL of distilled
water was further added to the same tube to make a 1~ w/v
solids suspension of beads and a 5 mg/mL solution of 5X-
aminodextran. Further, the suspension was made 1 mM in
potassium hydroxide by the addition of 50 ~L of 5M KOH
solution to promote the reaction of aldehyde groups on the
bead ~urface with amino groups of the aminodextran at a pH
of about 10Ø The mixture in the tube was then roller
mixed for a period of 8 to 16 hours.
The aminodextran coated beads prepared in the above
manner were separated by centrifugation at about 2000 g
for 10 min, the supernatant was discarded, and the residue
was resuspended in a 2~ w/v solution of 5X-aminodextran to
make a total volume of 250 mL. The bead suspension was
vortexed to redisperse the particles, made 1 mM in KOH by
addition of 50 ~L of 5M KOH solution, and then roller
mixed for 1.5 hours. 2.925 mL of 25~ glutaraldehyde
solution (0.778 mmol or 1 mole per mole amino groups in
5X-aminodextran) were added to the bead suspension and
roller mixed for one hour. The beads were then separated
from free glutaraldehyde and aminodextran by
centrifugation, the supernatant liquid discarded. To
block unreacted aldehyde groups, the beads were
resuspended in 250 mL of 1~ w/v 5X-aminodextran and roller
mixed for a time in the range of 8-16 hours. The beads
were then separated by centrifugation, the supernatant was
discarded, the residue was resuspended in 250 mL of a 10
mG/mL sodium borohydride in l mM KOH solution, and the
bead mixture was roller mixed for another one hour.
Subsequently, the beads were washed four times using
centrifugation and lxPBS solution. The wash supernatant
~vogs/24631 ~ 282 PCT~S95/02547
liquids were discarded and the washed beads were suspended
in sufficient lxPBS to yield 250 mL of 1~ w/v solids 5X-
- aminodextran coated poly~tyrene latex beads.
The beads and antibody were activated and conjugated
by procedures similar to tho~e described above for T4 or
Ta antibody and DAP-coated polystyrene beads except the
bead concentration was 1~ w/v solids, the sulfo-SMCC
concentration was 4.2 times lower, and the quantity of
T4/IT and T8/IT that was used during conjugation wa~ 8
mg/m2 x 2.6464 m2/g x 1 g/100 mL or 0.212 mg/mL.
The invention has been described with reference to
specific examples, materials and data. As one skilled in
the art will appreciate, alternate means for using or
preparing the various aspects of the invention may be
available. Such alternate means are to be construed as
included within the intent and spirit of the present
invention as defined by the following claims.
.
