Note: Descriptions are shown in the official language in which they were submitted.
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BIOSENSOR WITH NOVEL PASSIVATING LAYER
1. Field of the Invention
The invention relates to a biosensor having an
immobilized biocomponent for detecting a specific biomolecule
(or analyte)in an aqueous matrix (or solution) and having a
novel, highly effective passivating layer for suppressing
nonspecific binding. The invention moreover relates to a
process for producing the biosensor.
2. Prior Art
The mode of operation of biosensors is based on
combining a biocomponent capable of specifically binding an
analyte that is a biomolecule present in an aqueous matrix,
with a transducer (or signal converter) which converts a
primary chemical or physical signal created by the binding
into an optical or electrical secondary signal which can be
amplified and evaluated in a conventional manner. There have
been disclosures of reversibly operating biosensors
(biosensors in the narrower sense) and also irreversible
biosensors (probes), which are discarded after a single use.
Examples of biospecific bind pairs composed of the
biocomponent of the sensor and the biomolecule as analyte are
an antibody and an antigen, a receptor and a ligand, and also
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an enzyme and a substrate. In modern biosensors, the
biocomponent is normally immobilized on a surface of the
transducer.
The transducer may, for example, be a piezoelectric
crystal which reacts to the change in mass on its surface as a
result of the specific binding of the biomolecule with a
change in its oscillation behaviour, the change indicating the
existence of binding (qualitatively) and, if desired, its
extent (quantitatively). Instead of using piezoelectric
oscillations for the process of converting the signal, it is
also possible to use surface acoustic waves (SAW) of
piezoelectric substrates for this purpose. The depth of
penetration of the surface acoustic wave is in the range of
its wavelength, and therefore biomolecules which are
specifically bound at the surface have an effect on the
transmission of the waves. Biosensors which operate with
transducers based on piezoelectric oscillations or on surface
acoustic waves are mass sensors, since they fundamentally give
information on the change in mass (i.e., weight) at the
transducer surface. Another known biosensor type utilizes
transducers which convert the signals received into optical
phenomena and determine the analyte by making use of changes
in radiation absorption, in emission after excitation of the
probe, in reflection, refraction and diffraction, or in
polarization.
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The signals emitted by the transducer are
electronically amplified and subjected to comparative
evaluation, the result being a yes/no statement or, in the
case of quantitative analysis, a numerical value.
The sensitivity of detection of a biomolecule in a
matrix with a biosensor is diminished by the fact that the
transducer converts into electrical or optical signals not
only the signals deriving from specific binding of the
biomolecule to the biocomponent of the sensor but also signals
deriving from nonspecific binding of other components of the
matrix. This nonspecific binding may be adsorptive,
absorptive, covalent or ionic in nature and produces false
analysis results, since the signal passed on by the transducer
for evaluation is composed of the sum of the signal from
specific and from nonspecific binding. The error caused by
the nonspecific signal could be tolerated if it were constant
for a particular analytical task in the same matrix, or at
least a matrix of the same type. This is, however, not the
case. For example, the extent of nonspecific binding during
determination of antigens in blood differs from donor to
donor, and therefore even if the antigen is absent the values
obtained have a scatter the extent of which can be signified
using the standard deviation of the blank measurements from
the mean value. The greater the standard deviation, the lower
the detection sensitivity, since a test is usually only judged
to be positive if the measured value exceeds three times
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times the standard deviation.
It is therefore desirable to prevent nonspecific
binding, or at least to suppress the same, so that the
standard deviation is minimized. In a variant which has been
disclosed (see, for example, K.A. Davis, T.R. Leary:
Continuous Liquid Phase Piezoelectric Biosensor for Kinetic
Immunoassays, Analy. Chem. 61 (1989), 1227-1230), nonspecific
binding of proteins is suppressed with bovine serum albumin
(BSA) as passivating layer in the determination of
immunoglobulin G (IgG) in phosphate-buffered sodium chloride
solution (PBS). However, the effectiveness of this
passivating layer is not ideal because, on the one hand, the
macromolecules of the BSA do not seal the transducer surface
hermetically, so that there are unoccupied positions remaining
for nonspecific binding and, on the other hand, BSA in its own
right nonspecifically binds hydrophobic groups, e.g. in fatty
acid molecules.
3. Summary of the Invention
It has now been found that organic hydrogels give
passivating layers for biosensors which have excellent
effectiveness with respect to nonspecific binding. The
invention therefore provides a biosensor with an immobilized
biocomponent for specific binding of a biomolecule (analyte)
which is present in an aqueous matrix. A significant feature
of this biosensor is a layer of an organic hydrogel as
passivating layer with respect to nonspecific binding.
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The invention further provides a process for
producing the biosensor by the use of an organic hydrogel for
a passivating layer.
The invention also provides a method for determining
a biomolecule or analyte in an aqueous matrix by the use of
the novel biosensor.
The novel biosensors are distinguished by a
considerably smaller standard deviation of blank measurements
from the mean value, and therefore by greater sensitivity than
known biosensors with or without a passivating layer. This
may be due to a hydrated envelope of the hydrogels, which
deters the approach of, and nonspecific bind of, molecules
which differ from the analyte. The passivating layer adheres
particularly firmly, and therefore the biosensors are
particularly suitable as reversible sensors if the hydrogel is
applied by well known graft polymerization techniques. A
further advantage is that the cross-sensitivity is reduced (or
the specificity for the sought-for analyte is increased).
4. Description of Preferred Embodiment of the Invention
2o The passivating layer of an organic hydrogel is the
significant feature of the biosensor of the invention. It is
preferably a mass sensor and the biocomponent which it
contains is generally an antibody, an enzyme or a receptor,
desirably sonically or covalently bound to the transducer,
directly or via a polymeric intermediate layer. The
transducer may be composed of conventional materials known for
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this purpose, e.g. silicon nitride (Si3N4), silica or
lanthanum niobate (LaNb03). The novel biosensor likewise has
no particular features with regard to the processing and
evaluation of the secondary signal created in the transducer.
In a preferred embodiment of the novel biosensor,
the passivating layer of the organic hydrogel has been applied
to the surface of the transducer directly or, preferably
indirectly via an intermediate layer, especially desirably by
grafting hydrophilic monomers, and contains on its free
surface, covalently or sonically bound or integrated into the
hydrogel, the biocomponent for specific binding with the
biomolecule in the matrix. Biosensors having such a
passivating layer grafted onto an activated polymeric
intermediate layer and containing the biocomponents sonically
or covalently bound on their free surface are distinguished by
particularly low sensitivity.
4.1. Passivatinq Layer/Hydrophillic Vinyl Monomers
The organic hydrogel of the passivating layer may be
produced by polymerization, preferably by graft
polymerization, of hydrophilic vinyl monomers, optionally
together with proportions of hydrophobic vinyl monomers. The
hydrophilic polymer becomes a hydrogel on contacting an
aqueous medium, a condition which is always fulfilled in the
use as specified. Preparation of hydrogels or of polymers
which give a hydrogel may be conducted as described for
example in German Patent Application Nos. 197 00 079.7 (O. Z.
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5146), 197 15 449.2 (O. Z. 5176) and 197 27 556.7 (O. Z. 5210).
The hydrogel of the passivating layer is, of course,
itself hydrophillic. Preferred passivating layers are made of
hydrogels which exhibit a contact angle of less than 40°, more
preferably less than 30°, when the contact angle of an air
bubble is determined by the method of R.J. Good et al.,
Techniques of Measuring Contact Angles in Surface and Colloid
Science (ed. R.J. Good) Vol. 11, Plenum Press, New York, N.Y.
This is done by allowing an air bubble to form below the
specimen immersed in water, and this bubble coats the surface
to a greater or lesser extent depending on its hydrophilicity.
Suitable hydrophilic vinyl monomers contain at least
one olefinic double bond, and also at least one hydrophilic
group. The olefinic double bonds may be present in functional
groups of various types, for example in alkenyl groups, such
as vinyl or allyl radicals, or in radicals derived from
unsaturated carboxylic acids or from their derivatives, for
example acrylic acid or methacrylic acid, or from the amides
of these carboxylic acids or malefic acid. There is also great
scope for variation with regard to the hydrophilic groups.
Examples which may be mentioned of suitable hydrophilic groups
are: hydroxyl groups, ether groups, acyloxy groups, carboxyl
groups, carboxylic ester groups, carboxamide groups,
carboalkoxy groups and nitrile groups; 1,2-epoxy groups;
sulfuric esters, sulfonic acid, sulfinic acid groups,
phosphoric acid groups, phosphonic acid groups and phosphinic
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acid groups, including the salts and esters corresponding to
these; primary, secondary and tertiary amino groups; acylamino
groups, which may be in open chains or incorporated into a
ring; polyalkylene oxide groups, such as polyethylene oxide
groups and polypropylene oxide groups, with or without a
terminal hydroxyl group; polyester groups, polyesteramide
groups and polyetheresteramide groups; the pyrrolidone ring
and similar heterocycles, and also radicals of olefinically
functionalized sugars. The hydrophilicity of a monomer is, of
course, dependent of the balance between hydrophilic and
hydrophobic fractions in its molecule. Monomers suitable for
the invention are soluble in water at 20°C to an extent of at
least 1% by weight, preferably at least 10% by weight and in
particular at least 40% by weight, based in each case on the
entire solution.
The hydrophilic vinyl monomers used for the
invention preferably contain one olefinic double bond and one
hydrophilic group. However, they may also have more than one
olefinic double bond and/or hydrophilic group. Open-chain
polyalkylene oxides having two terminal vinyl, allyl, acryloxy
or methacryloxy groups, for example, are therefore very
suitable.
Examples of suitable hydrophilic vinyl monomers
which may be mentioned are: acrylic acid and its derivatives,
e.g. acrylamide, N,N-diemethylacrylamide, acrylonitrile,
methyl acrylate, 2-hydroxyethyl acrylate, 2-hydroxypropyl
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acrylate and 2-methoxyethyl acrylate; 2-ethyoxyethyl acrylate,
4-hydroxybutyl acrylate and 1,4-butanediol diacrylate, and
also methacrylic acid and its corresponding derivatives; vinyl
derivatives of carboxylic acids, such as vinyl acetate, N-
vinylactetamide and N-vinylpyrrolidone; vinylsulfonic acids
and their alkali metal salts, such as sodium vinylsulfonate;
alkenylarylsulfonic acids and their alkali metal salts, such
as styrenesulfonic acid and sodium styrene sulfonate, vinyl
ethers, such as vinyl methyl ether, vinyl ethyl ether, vinyl
glycidyl ether, diethylene glycol divinyl ether and vinyl n-
butyl ether; vinyl ketones, such as vinyl methyl ketone, vinyl
ethyl ketone and vinyl n-propyl ketone; vinylamines, such as
N-vinylpyrrolidine; polyalkyleneoxy compounds with terminal
allyl, vinyl, acrylyl or methacylyl groups, such as
ethoxytetraethoxyethyl acrylate or methacrylate, n-
propoxydodecaethyleneoxyethyl vinyl ether, polyethylene glycol
mono- or diacrylates with molecular weights of 600 or 1200,
poly(ethylene/propylene) glycol mono- or dimethacrylates with
molecular weights of 400 and 800, and also allyloxyoctapropyl-
eneoxyethanol; sugar derivatives, such as vinyl-substituted
arbinose or acryloylated hyroxypropylcellulose; and
functionalized polyalkylene glycols, such as triethylene
glycol diacrylate or tetraethylene glycol diallyl ether.
In cases where trifunctional or higher-functional
vinyl monomers are used concomitantly, the result is a
crosslinked copolymer and this, in particular in the graft
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polymerization described subsequently, achieves a particularly
good seal of the surface of the transducer and therefore a
greater accuracy of measurement of the biosensor. Examples of
suitable trifunctional or higher-functional vinyl monomers are
polyacrylates, such as trimethylolpropane triacylate,
trimethylolpropane trimethacrylate and pentaerythritol
tetraacrylate; polyvinyl ethers, such as the trivinyl ether of
glycerol-12E0 and the tetraallyl ether of pentaerythritol.
The crosslinking vinyl monomers may be used in amounts of
preferably from 0.01 to 1 mol%, based on all of the monomers.
4.2. Hydrophobic Vinyl Monomers as Comonomers
Besides the hydrophilic monomers, concomitant use
may be made, in preparing the hydrogels or the polymers which
are hydrogels on contact with an aqueous medium, of certain
amounts, e.g. up to 60 mol%, based on all of the monomers, of
hydrophobic vinyl monomers, e.g. a-olefins, such as propene,
1-butene and 1-octene; vinyl chloride; and vinyl aromatics,
such as styrene, a-methylstyrene and vinyl toluene.
Instead of the crosslinking hydrophilic vinyl
monomers mentioned which may optionally be used concomitantly,
it is also possible to make concomitant use of trifunctional
or higher-functional hydrophobic vinyl monomers in the amounts
mentioned, the effect achieved being essentially the same as
that described.
The molar proportion of the hydrophobic vinyl
monomers should be judged in such a way that the hydrogel
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shows a contact angle of less than 40°, preferably less than
30°C, in the test of R.J. Good et al. described above.
4.3. Preparation of the Organic Hydro~els in Bulk
The monomers, in each case individually or else as a
mixture appropriate for the respective application, may be
polymerized in a conventional manner, preferably by free-
radical-initiated solution or emulsion polymerization. The
monomers are generally used as solutions of concentrations of
from 1 to 40% by weight, preferably from 5 to 20% by weight.
It is expedient for the solvent to be water. The resultant
polymer is then immediately a hydrogel. If an organic solvent
is used for the operation, the polymer becomes a hydrogel at
the latest during use of the biosensor in contact with the
aqueous matrix. Use may be made of conventional
polymerization initiators, e.g. organic peroxy compounds or
hydroperoxides, azo compounds, peracids, persulfates or
percarboneates, in the conventional amounts, the
polymerization being initiated thermally or by irradiation.
The solutions or emulsions are immediately suitable for
coating the transducers, preferably by spincoating, by which
means passivating layers with thicknesses of from 0.1 to 100
~.m can be created from the polymer.
Alternatively, the passivating layer may be created
by grafting the hydrogel polymer depicted above onto the
surface of the transducer. In this case, it is preferred to
apply an intermediate layer of a (co)polymer before grafting;
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the intermediate layer is, or may be, activated so as to
permit grafting. Grafting is preferred, since it gives a
firmly adhering, particularly thin but nevertheless coherent
coating on the surface of the transducer.
4.4. Polymeric Intermediate Layer and its Activation
Suitable polymers for the intermediate layer are
commodity polymers with or without radiation-sensitive groups,
for example polyurethanes, polyamides, polyesters, polyethers,
polyether-block-amides, polyester-block-amides, polysiloxanes,
polystyrene, polyvinyl chloride, polycarbonates, polyolefins,
polysulfones, polyisoprene, polychlorprene, polytetra-
fluoroethylene, polyacrylates, polymethacrylates, and also the
corresponding copolymers and blends. The intermediate layer
may be applied preferably by spincoating from organic
solutions and generally likewise has a thickness of from 0.01
to 100 Vim.
The surface of the intermediate layer may be
activated by any of a large number of methods in preparation
for grafting, and mention is made of the most important of
these.
(1) Commodity polymers without W-radiation-sensitive
groups may advantageously be activated by W radiation, e.g.
in the wavelength range from 100 to 400 nm, preferably from
125 to 310 nm. Particularly good results are achieved with
continuous and largely monochromatic radiation, as produced,
for example, by Excimer W sources (Heraeus, Kleinostheim,
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Germany), for example using F2, Xe2, ArF, XeCl, KrCl or KrF as
the medium for the lamp. However, other radiation sources,
such as mercury vapor lamps with a broad radiation spectrum
and a proportion of visible radiation are suitable as long as
they emit considerable proportions of radiation in the
wavelength ranges mentioned. It has been found that the
presence of small amounts of oxygen is advantageous. The
preferred partial pressures of oxygen are from 2x10-5 to 2x10-
2 bar. The operation may, for example, be carried out at a
reduced pressure of from l0-4 to 10-1 bar, or using an inert
gas, such as helium, nitrogen or argon, with an oxygen content
of from 0.02 to 20 parts per thousand. The ideal irradiation
time depends on the polymeric substrate, on the composition of
the surrounding gaseous medium, on the wavelength of the
radiation, and also on the power of the radiation source, and
may readily be determined by exploratory trials. The
substrates are generally irradiated for from 0.1 seconds to 20
minutes, in particular from 1 second to 10 minutes. These
very short irradiation times give only very little heating of
the polymeric substrate and, even with radiation whose
wavelength is at the hard end of the wider range mentioned,
there is no occurrence of undesirable side reactions which
could cause damage to the exposed surfaces.
(2) According to the invention, it is also possible to
achieve activation by using a high-frequency plasma or
microwave plasma (Hexagon, Technics Plasma, 85551 Kirchheim,
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Germany) in air or in a nitrogen or argon atmosphere. The
exposure times are generally from 30 seconds to 30 minutes,
preferably from 2 to 10 minutes. The energy input, for
laboratory apparatus, is from 100 to 500 W, preferably from
200 to 300 W.
(3) It is also possible to use corona apparatus (SOFTAL,
Hamburg, Germany) for activation. The exposure times in this
case are generally from 1 second to 10 minutes, preferably
from 1 to 60 seconds.
(4) Activation by electron beams or gamma rays (e. g.
from a cobalt 60 source) permits short exposure times,
generally from 0.1 to 60 seconds.
(5) Flame treatment of surfaces likewise leads to their
activation. Suitable apparatus, in particular that which has
a barrier flame front, may readily be constructed or, for
example, purchased from ARCOTEC, 71297 Monsheim, Germany.
Such apparatus may be operated using hydrocarbons or hydrogen
as the gas for combustion. In all cases, overheating which
damages the intermediate layer has to be avoided, and this is
readily achieved by intimate contact between a cooled metal
surface and the surface opposite to that being flame-treated.
The exposure times generally amount to from 0.1 second to 1
minute, preferably from 0.5 to 2 seconds, the flames in all
cases being nonluminous and the distance of the surface of the
intermediate layer to the outer flame front being from 0.2 to
5 cm, preferably from o.5 to 2 cm.
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(6) The surfaces of the intermediate layers may moreover
be activated by treatment with strong acids or bases. Among
the strong acids which are suitable, mention may be made of
sulfuric acid, nitric acid and hydrochloric acid. For
example, polyamides may be treated for from 5 seconds to 1
minute with concentrated sulfuric acid at room temperature.
Particularly suitable strong bases are alkali metal hydroxides
in water or in an organic solvent. For example, the substrate
may be exposed to dilute sodium hydroxide solution for from 1
l0 to 60 minutes at from 20 to 80°C. Alternatively, polyamides,
for example, may be activated by exposing the substrate
surface to 2% strength KOH in tetrahydrofuran for from 1
minute to 30 minutes.
(7) Finally, monomers with W-radiation-sensitive groups
may be incorporated at an early stage, during preparation of
the polymers for the intermediate layer. Examples of suitable
monomers of this type are furyl and cinnamoyl derivatives,
which may, for example, be used in amounts of from 3 to 15
mol%. Monomers of this type which have good suitability are
20 cinnamoylethyl acrylate and methacrylate.
In many cases, e.g. in the case of highly
hydrophobic polymers, it may be advisable to activate the
surfaces of the intermediate layer by a combination of two or
more of the activation methods mentioned. The preferred
activation method is W-radiation as in number (1).
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4.5. Preparation of the Passivating Layer by Graft
(co)polymerization of monomers
The passivating layer may be created by graft
(co)polymerization, by grating the monomers onto the activated
intermediate layer. If the intermediate layers have been
activated by one of the methods described under (1) to (6), it
is expedient to expose the activated surfaces to the action of
oxygen, e.g. in the form of air, for from 1 to 20 minutes,
preferably from 1 to 5 minutes.
l0 The surfaces of the intermediate layers which have
been activated (also, if desired, as in (7)) are then coated
by known methods, such as dipping, spraying or brushing, with
solutions of the vinyl monomers) to be used according to the
invention. Solvents which have proven successful are water-
ethanol mixtures, but other solvents may also be used as long
as they have sufficient ability to dissolve the monomers) and
give good wetting of the surfaces. Depending on the
solubility of the monomers and on the desired thickness of the
finished coating, the concentrations of the monomers in the
2o solution may be from 1 to 40% by weight. Solutions with
monomer contents of from 5 to 20% by weight, for example of
about 10% by weight, have proven successful in practice and
generally in a single operation give cohesive coatings with
thicknesses which may be more than 0.1 ~m and which cover the
surface of the intermediate layers.
After the solvent has evaporated, or during its
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evaporation, the polymerization or copolymerization of the
monomers) applied to the activated surface is expediently
induced by means of radiation in the short-wavelength segment
of the visible ranged or in the long-wavelength UV range of
electromagnetic radiation. An example of radiation which has
good suitability is that with wavelengths of from 250 to 500
nm, preferably from 290 to 320 nm. Radiation in the
wavelength range mentioned is relatively soft and is selective
in polymerization, and does not attack the polymeric
intermediate layer. As in the activation of the surfaces of
the intermediate layer, it is again advantageous here to
operate with a radiation source which emits continuous and
largely monochromatic radiation. A particularly suitable
source is again the Excimer UV source with continuous
radiation, e.g. with XeCl or XeF as the medium for the
radiation. The required intensity of radiation and the
exposure time depend on the particular hydrophilic monomers
and may readily be determined by exploratory experiments. In
principle, mercury vapor lamps may also be used here, as long
as they emit considerable proportions of radiation in the
wavelength ranges mentioned. The exposure times in all cases
are generally from 10 seconds to 30 minutes, preferably from 2
to 15 minutes.
Alternatively, the passivating layer may also be
created by irradiation of the transducer which has been
provided with an activated polymeric intermediate layer and
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dipped into the monomer solution.
Depending on the concentration of the monomer
solution, on the coating conditions (application method, dip-
irradiation), on the intensity of the radiation and on the
exposure time, grafting gives a firmly adhering barrier layer
of the hydrogel, generally with a thickness of from 10 nm to
500 ~,m.
4.6. Incorporation of the Biocomponents
Since the biocomponents are generally sensitive
l0 molecules, it is preferred to incorporate them on the surface
of the barrier layer of the organic hydrogel after this layer
has been applied. The binding which links the biocomponent
with the barrier layer is preferably of covalent or ionic
type. The details depend on the nature of the biocomponent,
where the biocomponent contains, for example, free primary or
secondary amino groups, and this is frequently the case, it
may be attached ionically with an ammonium structure or
covalently via a carboxamide bridge, an example of the latter
being the known method using N-hydroxysuccinimide and a
20 carboxyl group in the hydrogel. The coat of the biocomponent
achieved in this way on the surface of the barrier layer has
good density, increases measurement sensitivity, and at the
same time is stable and resistant to abrasion.
4.7. Use of the Biosensor
The novel biosensor is suitable, as are
corresponding biosensors of the prior art, for determining a
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biomolecule (or analyte) in an aqueous matrix, the biomolecule
(or analyte) being specifically bound by the biocomponent of
the sensor. It is therefore possible by this means, for
example, to detect HIV antibodies in human blood, in order to
identify infection of donated blood.
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