Note: Descriptions are shown in the official language in which they were submitted.
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BEHRINGWERKE AKTIENGESELLSCHAFT 8b/B 035 - Ma 555
Dr. Ha/8n
Porous solid phases having bioaffin;ty,
a process for preparing porous solid phases having
bioaffinity, and their use
The invention relates to a porous solid phase having
bioaffinity and to a process for preparing a porous solid
phase to which one or more binding partners of a binding
; system having bioaffini$y are bound. The invention fur-
thermore relates to the use of porous solid phases prep-
ared in such a fashion for binding to the counter partner
corresponding to the binding partner.
A large number of porous solid phases are known, to which
the binding partners of binding systems hav;ng bioa~finity
are bound the binding partners being enzymes, lec~ins,
antigens, antibodies or partners of other systems having
bioaffinity.
Porous solid phases which have binding partners having
bioaffinity and in which the binding partners are bound
adsorptively or covalently to tne material o-f the porous
solid phases are described, for example in EPA 0,063,810
and US Patent 4,366,241.
In addition, porous solid phases exist which contain
binding partners having a bioaffinity and in which latex
particles to ~hich the binding partners are coupled are
incorporated in film coatings (EPA 97,952 and German
Offenlegungsschrift 3,329,728).
Surprisingly, it has been found that it is possible to
incorporate particles to which one or more different bind-
ing partners having bioaffinity are coupled into preformed
materials having an isotropically porous, i.e. foamed, or
anisotropically porous, i.e. fibrous, structure in a
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fashion such that the particles are fi%ed in these materials.
The invention relates to a porous sol;d phase which con-
tains a binding partner having a bioaffinity and which
can be obtained by incorporating a dispersion or suspen-
sion of particles to which one or more binding partners
having bioaffin;ty are bound~ into a porous material,
comprising fibers, and subsequently removing the disper-
sion or suspension agent.
The invention also relates to a process for preparing
porous solid phases which contain a binding partner having
; a bioaffinity, wherein a dispersion or suspension of par-
ticles to which one or more binding partners having a bio-
affinity are bound ;s incorporated into a porous material,
and the dispersion or suspension agent is subsequentLy
removed~
; The invention furthermore relates to the use of such a
porous solid phase which conta;ns a binding partner having
a bioaffinity, ;n apparatuses for separating off and det-
ecting the corresponding counterpartners having a bioaffin-
ity and for detecting the binding partner in dissolved form.
; 25 The porous material can be designed in various shapes.
Examples are spheres, cylinders and flat structures, such
as fleeces, membranes and papers.
The porous structures can be produced by foaming or by
precipitating using a precipitation bath of solutions or
dispersions of synthetic, semisynthetic or natural poly-
mers or by evaporating the solvent from solutions of mem-
brane~forming polymers~ and by press-molding fibers which
are prepared from the polymers mentioned below.
For the preparation of porous solid phases for diagnostic
agents, fleeces, papers or sponges made from celluLose or
cellulose derivatives, and also membranes made from
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cellulose derivatives or polyamides are preferred.
The particles may be latex particles wh;ch are composed
entirely of a hard polymer or in the core of a hard poly-
S mer. Examples of such polymers are polymers which can beprepared by suspension or emulsion polymerization, such
as polystyrenes or polyacrylatesn
The lattices xhich have a core/shell structure with a
hydrophilic shell are preferred.
The binding partners hav;ng b;oaff;n;ty are bound adsorp-
tively or preferably covalently to the surface of the
lattices, it being possible ~or the binding partners to
be receptors, lectins, protein A of Staphylococcus aureus,
protein G from Streptococcus, antigens, haptens, anti-
bod;es, enzymes or inhibitors thereof.
The particLes may also be composed of stabilized cells or
cell fragments.
Such cells or fragments preferably carry components which
are capable of specific binding, such as, for example, re-
ceptors wl1ich bind the antibodies via the Fc portion, of
these, for example, protein A of Staph. aureus or protein G
of Streptococcus of group C or G, lectins, antigens, haptens,
antibodies and antibodies, enzymes or inhibitors thereof
wh;ch are bound via protein A. Entire cells or membrane
fragments of Staph. aureus, COWAN I stra;n, to wh;ch anti-
bodies are bound as described by SuWO Kessler (J. Immunol.,1975, 115, 1617-1624~, are particularly preferred.
The process according to the invention can be carried out
by apply;ng dispersions and suspensions of the particles
onto the porous materials described above or incorporat-
ing them by dipping and subsequently removing the liquid
incorporated during this by evaporation. The pore and
particle sizes, and the number of particles and the volume
lS,~
of the porous matrix are matched so that the l;quid flow
in the matrix ~hich is necessary for the respective test
structure is not hindered.
In the case of membranes, pore sizes from 0.1-12 ~m and par-
ticle sizes from ~.û1~1 ~m are preferred. The particle dis-
persions or suspensions applied have a concentration of 1-
100 g/l. The suspension can be appl;ed to the support in
point or line form by dropping-on, pipett;ng~on or with the
aid of pumps and cannulars. Drying can be effected by allow-
ing to lie in air, or by blowing-over air at room temperature
or elevated temperature ;n open or closed systems at atmos-
pheric pressure or in vacuo. The drying condit;ons, ;n par-
ticuLar the thermal load permitted, are determined by the
stab;lity of the active component coupled to the part;cle.
The solid phases prepared accord;ng to the ;nvention can
be used as biologically active solid phases, such as, for
example, enzyme, antibody, antigen or hapten solid phases.
These are preferably employed in diagnostic test elements
having zones which are alongside one another, above one
another or mixed alongside and above one another.
In the following Examples, the preparation of latex and
cellular solid phases and their use in diagnostic test
elements having function zones lying alongside one another
are shown. The solid phases here are both enzyme and
antibody solid phases.
The Examples should in no way be regarded as limiting,
but serve merely to further illustrate the subject-matter
of the invention.
Example 1
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Porous solid phase containing glucose oxidase
3 ml of a dispersion containing 50 g/l of latex having
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acetal groups and prepared as described in Example 2b of
EPA 0,080,614 were incubated for one hour at 20C with
aqueous solutions of 300 ~l of 1 normal HCl and 300 ~l of
200 g/l (~ een 20. A pH of 6.5 was then produced by
adding 250 ~l of 1 normal NaOH and a saturated solution of
Na2HPO4. 1.5 ml of a solution of 1 g/l of glucose
oxidase having an activity of 250 U/mg (~oehringer Mann-
heim, degree of purity 1r Order No. 105 139) in phosphate-
buffered physiological sodium chloride solution~ pH 7.Z
(PBS) and 1.5 ml of a solution of 5 g/l of sod;um cyano-
borohydride in PBS were subsequently added, and the m;x-
ture was kept at 4C for 15 hours. 1.2 ml of û.5 mol/l
ethanolamine, adjusted to pH 8.5 using HCl, and 0.3 ml of
a solution of 25 g/L of sodium borohydride ~ere then
added, and the mixture was kept at 4C for a further hour.
After centrifugation, the sediment was resuspended in PBS
~ith 2 g/l (R)Tween 20, a voLume of 15 ml of the disper-
sion of latex/glucose oxidase conjugate being obtained.
Filter paper strips from Macherey und Nagel, Duren, Federal
Republic of Germany, Prod. No. 215, were soaked with 40~l/
cm of the dispersion of latex/glucose oxidase conjugate,
and subsequently dried at 20-50C. In the same fashion, mem-
branes of cellulose coesters from Millipore, USA, Prod. No.
SCWP, were soaked with 10 ~l/cm2 of dispersion and dried.
Example 2
Antibodies against porous soLid phase containing human
myoglobin
2.1 A conjugate of latex and rabbit IgG with antibodies
against human myoglobin was prepared by proceeding
in accordance with Example 1, with the change that
1.5 ml of a solution of 2.0 mg/mL of rabbit Ig~ with
antibodies against human myoglobin was used in place
of the solution of glucose oxidase. Porous solid
phases were prepared as described in Example 1.
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2~2 A conjugate of cells of Staphylococcus aureus (COWA~
I strain) and antibodies against human myoglobin was
prepared as follows:
16 mL of a solution of 2 g/l of rabbit IgG containing
antibodies against human myoglobin were added to
50 ml of 100 g/l suspension of Staphylococcus aureus
cells in a 0.05 mol/l aqueous ssd;um phosphate buffer
solution of pH 7.4 con$a;ning 9 g/l of sod;um chlor-
ide. The suspension was stirred for one hour. The
cells~ charged with antibodies, were subsequently
separated from the supernatant by centrifuging and
decant;ng. The cells were resuspended ;n 45 ml of
the abovementioned buffer and, for tovalent binding
of the ant;body to the ceLls, were treated with con-
stant stirr;ng w;th 50 ml of a solut;on of 2 g/l of
glutaraldehyde ;n the abovement;oned buffer and, after
stirring for one hour, with 50 ml of a solution of
5û g/l of sodium sulfite in the abo~ementioned buffer.
ZO
After stirring for a further hour, the cells were separ-
ated from the supernatant by centr;fug;ng and decanting.
The cells ~ere washed by resuspend;ng them ;n 200 ml of
0.05 mol/l aqueous sod;um phosphate buffer of pH 7.4 and
Z5 separat;ng them from the wash;ng buffer by centrifug;ng
and decant;ng.
The sol;d phases were prepared by resuspend;ng 20 g/l
of the cells thus treated ;n PaS and applying them
3û onto filter paper as in Example 1 and drying.
Exam
Test element for determination of myoglobin, containing
a porous solid phase in accordance with Example 2.1
3.1 Myoglobin peroxidase conjugate
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Electrophoretically uniform human myoglobin and peroxidase
from Boehringer Mannheim, Order No. 413 470, was used.
N-gamma-maleimidobutyryloxysuccinimide (GM~S) ~as
obtained from Behring Diagnostics and reacted with
human myoglobin as described by Tanimori et al.~ 1983,
in J. Imm. Meth. 62, 123-131. 2-Iminothiolane hydro-
chloricle (Sigma, Cat. No. I 6256) was reacted with
peroxidase as described by King et al., 1978, in Bio-
chemistry 17, 1499-1506~ A conjugate was prepared,
as de~r~d by Tanimori et al., from the product of the
reaction of GM~S with human myoglobin and the imino-
thiolane peroxidase. The crude conjugate was purified
by gel chromatography on Ultrogel ACA 44 (LKB). The
fraction in which about 1-2 peroxidase molecules were
coupled with each molecule of human myoglobir, was
used for the test. The conjugate was diluted to 100
pyrogallol units/ml with Enzygnost (R) IgE incubation
medium from Behringwerke, Order No. OSD.
3.2 Preparation of the components of a test element
3.2.1 Viscose fleece containing tetramethylben~i~ine and
glucose
Viscose fleece having a basis weight of 140-1~0 9/m2
from Kalle, ~iesbaden, Federal Republic of Germany,
was soaked with an aqueous solution containing 375
mg/l of tetramethylbenzidene dihydrochloride and
50 g/l of D-glucose, in a fashion such that the
fleece was not able to take up any further solution.
The fleece was dried at 20-50C and cut into pieces
of size 10x5 mm.
3~2.2 Paper containing myoglobin peroxidase conjugate and
glucose oxidase
A number 1 filter paper fro~ Macherey und Nagel cut
into 30x5 mm pieces was soaked in 2 separa~e areas
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once w;th 5 ~L of a solution of myoglobin perox;dase
conjugate in accordance with Example 3.1 and once with
5 ~l of a solution of 200 ~/ml of glucose oxidase, the
points of application being selected so that the wetted
areas of the paper were 5-6 mm apart. The filter paper
was dr;ed at 20-50C.
~ 3.2.3 Paper containing antibodies against myoglobin
:~'
Porous solid phase in accordance with Example 2.1 was
cut into 10x5 mm pieces.
:`
3.2.4 Non-impregnated paper
Schleicher und Schuell (Prod. No. 2668/8) paper was
cut into 20x~ wm rectangular pieces.
4.3 Preparation of the test element
Using a double-sided adhesive tape, in each case one
of the rectangular pieces was stuck successively via
its narrow sides in adsorptive contact to a polyester
f;lm in the following sequence:
a) Viskose fleece, in accordance with Example 3.2.1,
b) Paper, in accordance with Example 3.2.2, positioned
such that the area containing the glucose oxidase
was adjacent to the porous solid phase,
c) Porous solid phase, in accordance with Example
3.2.3, and
d) Paper in accordance with Example 3.2.4
4.4 Functional testing of the test element
In each case 100 ~l of a solution con~aining 10~ 100,
1,000 and 10,000 mg/ml of human myoglobin in Enzygnost
(R) IgE dilution buffer were applied to the viskose
fleece of separate test elements, and the color inten-
sities produced on the porous solid phase after 12 to
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13 minutes ~ere measured using a Quelle (R)Sanoquell
reflection photometer calibrated for the content of glu-
cose ;n blood, and the ~ollowing results were obtained:
Myoglobin Measured signal
cal;brated for
~g/ml ~mg of glucose per dl
of blood
115
10100 110
1,000 70
10,000 0
It was possible to prepare a test element which can func-
tion in the same fashion with latex-bound glucose oxidase
in accordance with Example 1 had been fixed as the porous
solid phase, as described in Example 3.2.2, and also when
the antibodies had been bound to cells of Staphylococcus
aureus and fixed in the porous solid phase according to
Example 2.2.
