Note: Descriptions are shown in the official language in which they were submitted.
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PROCESS FOR RINSING FABRICS
TECHNICAL FIELD
The present invention generally relates to a process for
rinsing fabrics. More in particular, it relates to a process
for rinsing fabrics whereby a benefit agent is delivered to
a fabric during the rinse cycle of a washing process.
BACKGROUND AND PRIOR ART
A conventional fabric cleaning process as it is carried out
in an automated washing machine, comprises a main wash cycle
(which may comprise a number of sub-cycles) in which soiled
fabrics are contacted with a liquid aqueous cleaning
composition or wash liquor. Such a composition comprises
surfactants builders and optionally other ingredients such
as enzymes, bleaching agents, etc. Subsequently, the now
cleaned fabrics are subjected to a rinse cycle in which the
cleaning composition is removed by pumping it off and
spinning the wash load followed by soaking in fresh water.
In the rinse cycle so-called softening agents may be added
which improve the feel of the washed fabrics. Such softening
agents conventionally comprise cationic softening compounds
of the quaternary ammonium type. Other benefit agents
include perfumes.
The compositions which are added during the rinse cycle have
a relatively short contact time with the fabrics and most of
the compounds are immediately removed again in the last
centrifuge step.
It is therefore an object of the present invention to
provide an alternative or improved process for rinsing
fabrics whereby a benefit agent is delivered to a fabric
during the rinse cycle of a washing process.
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Surprisingly, we have now found that these and other objects
of the invention may be achieved by the rinsing process of
the invention, whereby a benefit agent is delivered to a
fabric during the rinse cycle of a washing process, said
benefit agent being deposited onto the fabric by means of a
reagent having a high binding affinity for the fabric.
WO-A-98/00500 (Unilever) discloses a composition comprising
a benefit agent attached to a peptide or protein deposition
aid which has a high affinity for fabric. The composition is
claimed to effectively deposit the benefit agent onto the
fabric during the wash cycle.
According to DE-A-196 21 224 (Henkel), the transfer of
textile dyes from one garment to another during a washing or
rinsing process may be inhibited by adding antibodies
against the textile dye to the wash or rinse liquid.
WO-A-98/07820 (P&G) discloses amongst others rinse treatment
compositions containing antibodies directed at cellulase and
standard softener actives (such as DEQA).
DEFINITION OF THE INVENTION
According to a first aspect of the invention, there is
provided a process for rinsing fabrics whereby a benefit
agent is delivered to a fabric during the rinse cycle of a
washing process, said benefit agent being deposited onto the
fabric by means of a reagent having a high binding affinity
for the fabric.
According to a second aspect, there is provided a rinse
composition for use in the process of the invention,
comprising a reagent having a high binding affinity for the
fabric and a benefit agent.
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DETAILED DESCRIPTION OF THE INVENTION
1.1 The benefit agent
In its first aspect, the invention relates to a process for
rinsing fabrics whereby a benefit agent is delivered to a
fabric during the rinse cycle of a washing process. The
benefit agent can be selected from softening agents,
finishing agents/ protective agents, fragrances (perfumes),
bleaching agents.
Examples of softening agents are clays, cationic surfactants
or silicon compounds. Examples of finishing agents/
protective agents are polymeric lubricants, soil repelling
agents, soil release agents, photo-protective agents
(sunscreens), anti-static agents, dye-fixing agents, anti-
bacterial agents and anti-fungal agents. The fragrances or
perfumes may be encapsulated, e.g. in latex microcapsules.
Suitable examples of bleaches are photobleaches. Examples of
photobleaches are given in EP-A-379 312 (British Petroleum),
which discloses a water-insoluble photobleach derived from
anionically substituted porphine, and in EP-A-035 470 (Ciba
Geigy), which discloses a textile treatment composition
comprising a photobleaching component.
1.2 The reagent having a high binding affinity.
In the rinsing process according to the invention, the
benefit agent is deposited onto the fabric by means of a
reagent having a high binding affinity for the fabric. A
specific example of such a reagent is for instance an
antibody.
Generally speaking, the degree of binding of a molecule A to
another molecule B can be generally expressed by the
chemical equilibrium constant Kd resulting from the
following reaction:
(AJ + /BJ r~ (A =_ BJ
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The chemical equilibrium constant Kd is then given by:
- (AJx(BJ
KJ_
(A ---- BJ
Whether the binding of a molecule to the fabric is specific
or not can be judged from the difference between the binding
(Kd value) of the molecule to one type of fabric, versus the
binding to another type of fabric material. For applications
in laundry, said material will be a fabric such as cotton or
polyester. However, it will usually be more convenient to
measure Kd values and differences in Kd values on other
materials such as a polystyrene microtitre plate or a
specialised surface in an analytical biosensor. The
difference between the two binding constants should be
minimally 10, preferably more than 100, and more preferably,
more that 1000. Typically, the reagent should bind to the
fabric, with a Kd lower than 10-9 M, preferably lower than
10-6 M and could be 10-1° M or even less . Higher binding
affinities (Kd of less than 10-5 M) and/or a larger
difference between the one type of fabric and another type
of fabric (or background) binding would increase the
deposition of the benefit agent. Also, the weight efficiency
of the reagent in the total rinse composition would be
increased and smaller amounts of the reagent would be
required.
Several classes of reagent or molecules can be envisaged
which deliver the capability of specific binding to fabrics,
to which one would like to deliver the benefit agent. In the
following we will give a number of examples of such
molecules having such capabilities, without pretending to be
exhaustive.
1.2.1. Antibodies.
Antibodies are specific binding proteins. Their function in
nature is to protect against disease by recognising (and
binding) foreign bodies, such as viruses or Bacteria, but
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not self-cells. Furthermore, methods are well-known in the
art to generate antibodies that are specific for almost any
protein, organic molecule, or cell surface, that is likely
to be encountered. This binding specificity has been
5 exploited in the Biotechnology industry, principally for
medical diagnostics. For example, many home-based pregnancy
test kits comprise an antibody that specifically binds to
the pregnancy marker hormone, human chorionic gonadotropin
(hCG), but not to other hormones present in urine.
More recently, the use of antibodies in laundry products has
been described (Henkel, Procter and Gamble, Unilever). In
particular, Unilever has described the use of stain-specific
antibodies to target bleaching enzymes exclusively to stains
but not to dyes - thus achieving efficient stain removal
without damaging surrounding fabric.
Antibodies are well known examples of molecules which are
capable of binding specifically to compounds against which
they were raised. Antibodies can be derived from several
sources. From mice, monoclonal antibodies can be obtained
which possess very high binding affinities. From such
antibodies, Fab, Fv or scFv fragments, can be prepared which
have retained their binding properties. Such antibodies or
fragments can be produced through recombinant DNA technology
by microbial fermentation. Well known production hosts for
antibodies and their fragments are yeast, moulds or
bacteria.
A class of antibodies of particular interest is formed by
the Heavy Chain antibodies as found in Camelidae, like the
camel or the llama. The binding domains of these antibodies
consist of a single polypeptide fragment, namely the
variable region of the heavy chain polypeptide (HC-V). In
contrast, in the classic antibodies (murine, human, etc.),
the binding domain consists of two polypeptide chains (the
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variable regions of the heavy chain (Vh~ and the light chain
(V1)). Procedures to obtain heavy chain immunoglobulins from
Camelidae, or (functionalized) fragments thereof, have been
described in WO-A-94/04678 (Casterman and Hamers) and WO-A
94/25591 (Unilever and Free University of Brussels).
Alternatively, binding domains can be obtained from the Vh
fragments of classical antibodies by a procedure termed
"camelization". Hereby the classical Vh fragment is
transformed, by substitution of a number of amino acids,
into a HC-V-like fragment, whereby its binding properties
are retained. This procedure has been described by Riechmann
et al. in a number of publications (J. Mol. Biol. (1996)
259, 957-969; Protein. Eng. (1996) 9, 531-537,
Bio/Technology (1995) 13, 475-479). Also HC-V fragments can
be produced through recombinant DNA technology in a number
of microbial hosts (bacterial, yeast, mould), as described
in WO-A-94/29457 (Unilever).
Methods for producing fusion proteins that comprise an
enzyme and an antibody or that comprise an enzyme and an
antibody fragment are already known in the art. One approach
is described by Neuberger and Rabbits (EP-A-194 276). A
method for producing a fusion protein comprising an enzyme
and an antibody fragment that was derived from an antibody
originating in Camelidae is described in WO-A-94/25591. A
method for producing bispecific antibody fragments is
described by Holliger et al. (1993) PNAS 90, 6444-6448.
A particularly attractive feature of antibody binding
behaviour is their reported ability to bind to a "family" of
structurally related molecules. For example, in Gani et al.
(J. Steroid Biochem. Molec. Biol. 48, 277-282) an antibody
is described that was raised against progesterone but also
binds to the structurally-related steroids, pregnanedione,
pregnanolone and 6-hydroxy-progesterone. Therefore, using
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the same approach, antibodies could be isolated that bind to
a whole "family" of stain chromophores (such as the
polyphenols, porphyrins, or caretenoids as described below).
A broad action antibody such as this could be used to treat
several different stains when -coupled to a bleaching enzyme.
1.2.2. Peptides.
Peptides usually have lower binding affinities to the
substances of interest than antibodies. Nevertheless, the
binding properties of carefully selected or designed
peptides can be sufficient to deliver the desired
selectivity in an oxidation process. A peptide which is
capable of binding selectively to a fabric to which one
would like to deliver a benefit agent, can for instance be
obtained from a protein which is known to bind to that
specific fabric. An example of such a peptide would be a
binding region extracted from an antibody raised against
that fabric. A suitable peptide could be analogous to the
active center of a protein analogous to a non-catalytic
binding domain of a protein, e.g. a receptor.
Alternatively, peptides that bind to such substance can be
obtained by the use of peptide combinatorial libraries. Such
a library may contain up to 101° peptides, from which the
peptide with the desired binding properties can be isolated.
(R. A. Houqhten, Trends in Genetics, Vol 9, no &, 235-239).
Several embodiments have been described for this procedure
(J. Scott et al., Science (1990) 249, 386-390; Fodor et al.,
Science (1991) 251, 767-773; K. Lam et al., Nature (1991)
354, 82-84; R.A. Houghten et al., Nature (1991) 354, 84-86).
Suitable peptides can be produced by organic synthesis,
using for example the Merrifield procedure (Merrifield
(1963) J.Am.Chem.Soc. 85, 2149-2154). Alternatively, the
peptides can be produced by recombinant DNA technology in
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microbial hosts (yeast, moulds, bacteria)(K.N. Faber et al.
(1996) Appl. Microbiol. Biotechnol. 45, 72-79).
1.2.3. Pepidomimics.
In order to improve the stability and/or binding properties
of a peptide, the molecule can be modified by the
incorporation of non-natural amino acids and/or non-natural
chemical linkages between the amino acids. Such molecules
are called peptidomimics (H. U. Saragovi et al. (1991)
Bio/Technology 10, 773-778; S. Chen et al. (1992)
Proc.Natl.Acad. Sci. USA 89, 5872-5876). The production of
such compounds is restricted to chemical synthesis.
1.2.4. Other organic molecules.
It can be readily envisaged that other molecular structures,
which need not be related to proteins, peptides or
derivatives thereof, can be found which bind selectively to
fabrics to which one would like to deliver a benefit agent.
For example, certain polymeric RNA molecules which have been
shown to bind small synthetic dye molecules (A. Ellington et
al. (1990) Nature 346, 818-822). Such binding compounds can
be obtained by the combinatorial approach, as described for
peptides (L. B. McGown et al. (1995), Analytical Chemistry,
663A-668A).
This approach can also be applied for purely organic
compounds which are not polymeric. Combinatorial procedures
for synthesis and selection for the desired binding
properties have been described for such compounds (Weber et
al. (1995) Angew.Chem.Int.Ed.Engl. 34, 2280-2282; G. Lowe
(1995), Chemical Society Reviews 24, 309-317; L.A. Thompson
et al. (1996) Chem. Rev. 96, 550-600). Once suitable binding
compounds have been identified, they can be produced on a
larger scale by means of organic synthesis.
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The reagent has a high binding affinity for the fabric. The
reagent is a protein or a peptide, it may be that one part
of its polypeptide chain is responsible for the binding
affinity to the fabric, and part of the reagent comprises an
enzyme part capable of providing a benefit. In the first
situation, the bleaching enzyme may be a fusion protein
comprising two domains, which may be coupled by means of a
linker. Alternatively, the reagent having the high binding
affinity may be covalently coupled to a benefit agent by
means of a bivalent coupling agent such as glutardialdehyde.
A full review of chemistries appropriate for coupling two
biomolecules is provided in "Bioconjugate techniques" by
Greg T. Hermanson, Academic Press Inc (1986). Alternatively,
if the reagent having the high binding affinity is a peptide
or a protein, it may also be coupled to the enzyme by
constructing a fusion protein. In such a construct there
would typically be a peptide linker between the binding
reagent and the enzyme. An example of a fusion of an enzyme
and a binding reagent is described in Ducancel et al.
Biotechnology 11, 601-605.
A further embodiment would be for the reagent with a high
binding affinity to be a bispecific reagent. Such a reagent
could fulfil the requirement of accumulating the benefit
agent on the fabric either by supplying said reagent
together with the benefit agent as a pre-formed non-covalent
complex or by supplying the two separately and allowing them
to self-assemble either in the rinse liquor or on the
fabric.
In a preferred embodiment, the rinse process of the
invention is carried out using micro-particles sensitised
with antibody, and configured such that the micro-particles
are loaded with the benefit agent and the antibody has a
high affinity or specificity for a substance (or "marker
molecule") typically found on some regions of fabrics but
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not on others. Examples of such marker molecules include
bleach-damaged dyes and microbes known to be associated with
malodour. The antibody targets the benefit agent to its
intended site of action and binds it there. For example,
5 Microbe-specific antibodies may target fragrance-containing
particles to the regions of malodour. Thus, a more efficient
use of expensive ingredients is achieved. Alternatively,
antibodies specific for bleach-damaged dyes can target dyed
particles to faded regions, thus replenishing the colour
10 lost in the main wash cycle.
Previously, such micro-particles have been sensitised with
antibody and used to generate a coloured end-point in
medical diagnostic devices, when they are applied manually
onto a test strip. According to the present invention,
analogous particles are being specifically bound to some
cotton swatches but not others, depending on which marker
molecules are present on the swatches. The binding of
particles is being driven not by manual application but by
agitating a bulk liquid phase (e. g. a rinse liquor)
containing said particles and swatches. The agitation
increases the number of collisions between fabric and
particles and thus increases specific binding: particles
sensitised with specific antibody result in productive
collisions and binding is permanent; particles sensitised
with non-specific antibody result in non-productive
collisions and do not bind permanently. Such an agitation
could be readily achieved during the rinse cycle in an
automatic washing machine.
Another advantage of the present invention is that it is
possible to target some benefit molecules to particular
regions of fabric during the rinse. For example, dyes can be
targeted to colour-bleached regions to replenish dye lost in
the main wash or fragrance can be targeted to regions where
it is most needed (i.e. those regions where microbes
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associated with malodour are present - such as the
"underarm" regions). However, methods for targeting small
molecules (such as a dye or a fragrance) to particular
regions of fabric have not previously been described. The
inventors have approached this problem by loading small
molecules (such as a dye) onto a micro-particle and then
sensitising the particles with an antibody. The advantage of
this is that a single antibody binding event can deposit
many dye molecules onto the target-region of fabric. Whereas
antibody-sensitised particles have been described previously
(as component parts of medical diagnostic devices) they are
used in a fundamentally different way: in the medical
device, the particles are manually applied to the target
surface (typically nitro-cellulose paper) and then eluted
with a solution. If specific antibody is present, the
particles remain stuck on. Otherwise they do not. In
contrast, antibody-sensitised particles have not previously
been used to target a small chemical compound (such as a dye
or a fragrance) from a bulk liquid phase to a particular
target site on a surface. Furthermore, if an analogous
interaction between the particles and the target surface
(i.e. if the swatches are manually placed in the bulk liquid
and left static) little or no binding is observed. However,
the inventors have been able to achieve surprisingly
specific binding to cotton swatches by agitating a bulk
liquid phase (of rinse liquor or tap water) containing said
particles and swatches.
1.3 The fabrics
For laundry detergent applications, several classes of
natural or man-made fabrics can be envisaged, in particular
cotton. Such macromolecular compounds have the advantage
that they can have a more immunogenic nature, i.e. that it
is easier to raise antibodies against them. Furthermore,
they are more accessible at the surface of the fabric than
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for instance coloured substances in stains, which generally
have a low molecular weight.
An important embodiment of the invention is to use a binding
reagent (as described above) that binds to several different
types of fabrics. This would have the advantage of enabling
a single benefit agent to be deposited to several different
types of fabric.
2. The Rinse Composition.
The rinse compositions of the invention can be used in a
detergent composition which is specifically suited for
rinsing purposes, and this constitutes a second aspect of
the invention. When formulating a rinse product, it is
important to ensure that the other ingredients of the
product are compatible with antibody activity. WO-A-98/07820
(P&G) discloses inter alia rinse treatment compositions
containing antibodies directed at cellulase and standard
softener actives such as DEQA. The rinse product according
to the present invention preferably contains no softener or
low levels of softener active (e. g. HEQ). If HEQ is present,
the rinse product contains Sodium tripolyphosphate (STP) to
stabilise antibody activity.
However, the present inventors achieved much superior
binding and specificity in rinse liquors (or tap water) by
omitting typical softener compositions. They also achieved
improved binding in the presence of softener compositions by
adding salts, especially multivalent salts such as STP. It
is also very surprising that the inventors have found
antibodies to be active in rinse liquors (or tap water).
Previously published descriptions of specific antibody
binding are typically in physiological strength buffer
(0.15M NaCl) often supplemented with 0.150 surfactant. In
many ways this mimics the environment in which the
antibodies bind in nature, namely in serum which is
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approximately 0.15M NaCl, pH 7, and where serum albumin may
be thought to act in an analogous way to a surfactant in
that it reduces the opportunity for non-specific binding
reactions.
To that extent, the rinse composition comprises one or more
benefit agents and optionally other conventional detergent
ingredients. The invention in its second aspect provides a
rinse composition which comprises from 0.1 - 50 o by weight,
based on the total composition, of one or more surfactants.
This surfactant system may in turn comprise 0 - 95 o by
weight of one or more anionic surfactants and 5 - 100 o by
weight of one or more nonionic surfactants. The surfactant
system may additionally contain amphoteric or zwitterionic
detergent compounds, but this in not normally desired owing
to their relatively high cost. It was found to be
advantageous to also include cationic surfactants into the
composition. Examples of suitable cationic surfactants are
given in WO-A-97/03160 and WO-A-98/17767 (Procter&Gamble).
In general, the nonionic and anionic surfactants of the
surfactant system may be chosen from the surfactants
described "Surface Active Agents" Vol. 1, by Schwartz &
Perry, Interscience 1949, Vol. 2 by Schwartz, Perry & Berch,
Interscience 1958, in the current edition of "McCutcheon's
Emulsifiers and Detergents" published by Manufacturing
Confectioners Company or in "Tenside-Taschenbuch", H.
Stache, 2nd Edn., Carl Hawser Verlag, 1981.
Suitable nonionic detergent compounds which may be used
include, in particular, the reaction products of compounds
having a hydrophobic group and a reactive hydrogen atom, for
example, aliphatic alcohols, acids, amides or alkyl phenols
with alkylene oxides, especially ethylene oxide either alone
or with propylene oxide. Specific nonionic detergent
compounds are Cg-C22 alkyl phenol-ethylene oxide condensates,
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generally 5 to 25 E0, i.e. 5 to 25 units of ethylene oxide
per molecule, and the condensation products of aliphatic C8-
C18 primary or secondary linear or branched alcohols with
ethylene oxide, generally 5 to 40 E0.
Suitable anionic detergent compounds which may be used are
usually water-soluble alkali metal salts of organic
sulphates and sulphonates having alkyl radicals containing
from about 8 to about 22 carbon atoms, the term alkyl being
used to include the alkyl portion of higher acyl radicals.
Examples of suitable synthetic anionic detergent compounds
are sodium and potassium alkyl sulphates, especially those
obtained by sulphating higher C$-C1$ alcohols, produced for
example from tallow or coconut oil, sodium and potassium
alkyl C9-CZO benzene sulphonates, particularly sodium linear
secondary alkyl Clo-C15 benzene sulphonates; and sodium alkyl
glyceryl ether sulphates, especially those ethers of the
higher alcohols derived from tallow or coconut oil and
synthetic alcohols derived from petroleum. The preferred
anionic detergent compounds are sodium C11-Cis alkyl benzene
sulphonates and sodium C12-C18 alkyl sulphates. Also
applicable are surfactants such as those described in EP-A-
328 177 (Unilever), which show resistance to salting-out,
the alkyl polyglycoside surfactants described in EP-A-070
074, and alkyl monoglycosides.
Preferred surfactant systems are mixtures of anionic with
nonionic detergent active materials, in particular the
groups and examples of anionic and nonionic surfactants
pointed out in EP-A-346 995 (Unilever). Especially preferred
is surfactant system which is a mixture of an alkali metal
salt of a C16-C18 primary alcohol sulphate together with a
Ci2-Cis primary alcohol 3-7 EO ethoxylate .
The nonionic detergent is preferably present in amounts
greater than 100, e.g. 25-90o by weight of the surfactant
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system. Anionic surfactants can be present for example in
amounts in the range from about 5o to about 40% by weight of
the surfactant system.
5 The rinsing detergent composition may take any suitable
physical form, such as a powder, a tablet, an aqueous or non
aqueous liquid, a paste or a gel. The complex of benefit
agent and reagent having a high affinity according to the
invention will generally be used as a dilution in water of
10 about 0 . 05 to 2 0 .
The rinse composition in accordance with the invention
comprising the complex of the reagent having a high affinity
for the fabric and the benefit aid can have any suitable
15 form, i.e. the form of a granular composition, a liquid or a
slurry of the enzyme, or with carrier material (e.g. as in
EP-A-258 068 and the Savinase (TM) and Lipolase (TM)
products of Novo Nordisk). A good way of adding the complex
to a liquid rinse product is in the form of a slurry
containing from 0.005 to 50 o by weight of the complex in an
ethoxylated alcohol nonionic surfactant.
The rinse compositions of the invention comprise about 0.001
to 10 mg, preferably from 0.01 to 10 mg of antibody per
liter of the rinse liquor in use. A concentrated rinse
composition before use will comprise about 1 to 1000 mg/l,
preferably from 10 mg to 100 mg per liter of the rinse
product.
In the figures is:
Figure 1 a Graph showing the effects of wash and rinse
liquors from Persil non-biological powder main wash on
antibody binding activity (a specific anti-hCG monoclonal
was used) compared to standard curves in PBST and tap water
bench marks). Graph shows binding signal (optical density at
405nm) versus antibody concentration.
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Figure 2: Graph showing the effects of wash and rinse
liquors from Zeus liquid main wash on antibody binding
activity (a specific anti-hCG monoclonal was used) compared
to standard curves in PBST and tap water bench marks. Graph
shows binding signal (optical density at 405nm) versus
antibody concentration.
The invention will now be further illustrated in the
following, non-limiting Examples.
Example 1
Antibody binding in Tap water to antigen.
Method: Antibody activity was determined using an immuno-
assay or 'ELISA'. The assay comprised a specially designed
nylon peg sensitised with antigen. A number of wash and
rinse liquors were used to determine the effects on antibody
activity. These liquors were obtained from wash cycles using
standard Persil non-biological powder and Zeus (concentrated
zeolite built) non-biological HDL formulation.
All reagent solutions were exposed to the peg surface in
microtitre plates. The nylon pegs were first washed with
ethanol, prior to activation of the peg surface with
gluteraldehyde. The gluteraldehyde was then washed away with
distilled water before sensitising the surface of the pegs
with hCG, and blocking with bovine serum albumin. The
blocked pegs were 'crunched' onto plastic bars, twelve pegs
per bar, and sucrose coated to preserve them before drying
and sealing in pouches for storage. Firstly, the pegs had to
be rehydrated by immersing in phosphate buffer saline (PBS)
[0.15M sodium chloride, 0.0075M di-sodium hydrogen ortho-
phosphate and 0.0025M sodium di-hydrogen orthophosphate,
pH7.2] or distilled water only if using tap water or
wash/rinse liquor as antibody diluent, for 15 minutes. Mouse
anti-hCG was assayed as a dilution curve, with
concentrations starting at l~g/ml, in phosphate buffered
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saline + 0.015° v/v tween 20 (PBST) pH7.2. Mouse anti-E3G
was assayed as a negative control to allow assessment of
non-specific binding and was also run in a dilution curve,
with equal concentrations to the mouse anti-hCG. The pegs
were exposed to the various concentrations of anti-hCG and
anti-E3G for 60 minutes, at room temperature, before washing
in PBST. This was followed by exposure to Goat anti-mouse
alkaline phosphatase conjugate, diluted in PBST, for 30
minutes at room temperature. After a final wash in PBST, the
pegs were analysed for the amount of alkaline phosphatase
bound by incubating in a colour generating substrate system
(p-nitro-phenyl phosphate) where the more antibody bound the
more colour is generated. The resulting colour was read in
an ELISA plate reader at 405nm. Figures 1 and 2 demonstrate
the ability of the antibodies to bind to its antigen in tap
waver and rinse liquors.
Example 2
Binding antibody loaded particles to fabric senstised with
antigen.
Method: Two centimetre squares of cotton fabric were treated
with cellulase/hCG conjugate (to sensitise fabric with
antigen) and were then exposed to specific and non-specific
antibody sensitised latex particles, diluted to O.lo solids
in PBS + 0.2o non-ionic surfactant Co-Co 6.5E0 and tap
water. Exposure was static, for 60 minutes, at room
temperature. The exposed cotton squares were then washed
thoroughly with large volumes of tap water and vigorous
shaking.
Results: The results presented in the table below indicate
the ability to target particles to an antigen on fabric via
a specific antibody binding event in tap water. The greater
the DE the greater the particle deposition.
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Table 1. Binding of targeted particles to cotton in tap
water (non-shaking/static exposure)
Cotton Particle Cotton/ DE OReflectan
Surface Sensitisation Particle values ce (OR)
Treatment interaction at 650nm
Cellulase/hCG Specific Targeted 9.0 20.8
antibody
(anti-hCG)
No conjugate Specific Untargeted 1.2 2.9
antibody
(anti-hCG)
Cellulase/hCG Non-specific Untargeted 1.9 4.7
antibody
(anti-E3G)
No conjugate Non-specific Untargeted 1.5 0.8
antibody
(anti-E3G)
The examples illustrated above relate to monoclonal antibody
molecules. The following examples demonstrate similar
effects using antibody fragments.
Example 3
Functional bihead antibody molecule in tap water.
This example describes the confirmation that engineered
antibody molecules (with the potential of large-scale
exploitation) can function effectively in a non-buffered tap
water environment.
Method: Bi-head molecule (denoted 1349) will bind red wine
stains (anti-polyphenolic) and the enzyme Glucose oxidase
(Gox). Bi-Head 1349 was diluted at 10~.g/per ml in tap water
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19
or Phosphate buffered saline + tween 20, PBST [0.01M
Na2HP09/NaH2P09, 0. 15M NaCl and 0. 15 o Tween 20] each
containing 5~g/ml Gox. Additionally a control of Gox only at
5~g/ml was diluted in tap water or PBST to be used as a
control. The prepared solutions were dispensed in lml
volumes to two stained swatches and two unstained swatches.
Tap water and PBST only were added to two unstained swatches
as a further control. Samples were left at 21°C(~ 1) for 30
minutes without shaking. The samples were placed in bottle
containing either tap water or PBST (as appropriate) and
agitated on a rototorque (Cole Parmer instrument Co.
Chicargo Model 7637-10) for 2 minutes. This rinsing process
was repeated twice more. The swatches were then placed in a
clean 24 well tissue culture plate and a substrate was added
comprising O.1M Na Phosphate buffer pH 6.5, lOmM Glucose,
l~g/ml Horse Radish Peroxidase [Sigma] and 10~.g/ml
Tetramethyl Benzidine [Sigma]. This would produce a coloured
endpoint that was proportional to the amount of Gox present
on the cloth. To stop the reaction 300.1 of 1M HCL was added
to each well, samples of 1001 were then transferred to
microtitre plate wells (Sterilin) and the absorbance at
450nm was measured with a plate reader (Dynatech MR5000).
The results are:
1349/Gox Gox
Tap water/Wine 2.065 1.26
PBST/Wine 1.511 0.002
Tap water /Unstained 0.258 0.043
PBST/ Unstained 0.002 0.001
The data show that the binding signal in tap water on red
wine stain by 1349+Gox is approximately twice that of Gox
when it is untargeted. For a comparison the signal in PBST
for untargeted Gox is less than 0.01 whereas that for
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1349+Gox (targeted) is 150 times greater. There is an
increase in the non-specific binding of Gox to stained cloth
in tap water. However, on unstained cloth the background
signal for 1349+Gox is small and the signal to noise ratio
5 is high at 8:1, this is acceptable and demonstrates
significant specific binding by the bi-head molecule to the
red wine stain. The controls for tap water and PBST only
resulted in no signal.
