U.S. patent application number 09/783758 was filed with the patent office on 2001-11-22 for bleaching enzymes and detergent compositions comprising them.
This patent application is currently assigned to Unilever Home & Personal Care USA, Division of Conopco, Inc.. Invention is credited to Berry, Mark John, Convents, Daniel, Davis, Paul James, Gidley, Michael John, Van Der Logt, Cornelis Paul.
Application Number | 20010044397 09/783758 |
Document ID | / |
Family ID | 8235207 |
Filed Date | 2001-11-22 |
United States Patent
Application |
20010044397 |
Kind Code |
A1 |
Berry, Mark John ; et
al. |
November 22, 2001 |
Bleaching enzymes and detergent compositions comprising them
Abstract
There is provided a bleaching enzyme capable of generating a
bleaching chemical and having a high binding affinity for
non-colored compounds present in stains on fabrics, said
non-colored compounds having a molecular weight of at least 100,
preferably of at least 1,000 and more preferably of at least 5,000.
Furthermore, there is provided an enzymatic bleaching composition
comprising said bleaching enzyme and a surfactant and a process for
bleaching stains present of fabrics.
Inventors: |
Berry, Mark John;
(Sharnbrook, GB) ; Convents, Daniel; (Vlaardingen,
NL) ; Davis, Paul James; (Sharnbrook, GB) ;
Gidley, Michael John; (Sharnbrook, GB) ; Van Der
Logt, Cornelis Paul; (Sharnbrook, GB) |
Correspondence
Address: |
UNILEVER
PATENT DEPARTMENT
45 RIVER ROAD
EDGEWATER
NJ
07020
US
|
Assignee: |
Unilever Home & Personal Care
USA, Division of Conopco, Inc.
|
Family ID: |
8235207 |
Appl. No.: |
09/783758 |
Filed: |
February 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09783758 |
Feb 15, 2001 |
|
|
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09456805 |
Dec 8, 1999 |
|
|
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Current U.S.
Class: |
510/302 ;
510/305; 510/306; 510/309; 510/372; 510/374; 510/380; 510/392;
510/530 |
Current CPC
Class: |
C07K 16/468 20130101;
C12N 9/0004 20130101; A61K 47/6815 20170801; C07K 2317/31 20130101;
C11D 3/3845 20130101; C11D 3/38654 20130101; C12N 9/0061 20130101;
C07K 2319/00 20130101; A61K 47/61 20170801 |
Class at
Publication: |
510/302 ;
510/305; 510/306; 510/309; 510/372; 510/374; 510/380; 510/392;
510/530 |
International
Class: |
C11D 003/386; C11D
003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 11, 1998 |
EP |
98310204.7 |
Claims
1. Bleaching enzyme capable of generating a bleaching chemical and
having a high binding affinity for non-coloured compounds present
in stains on fabrics, characterised in that said non-coloured
compounds have a molecular weight of at least 100, preferably of at
least 1,000 and more preferably of at least 5,000.
2. Enzyme according to claim 1, wherein the enzyme comprises an
enzyme part capable of generating a bleaching chemical which is
coupled to a reagent having the high binding affinity for
non-coloured compounds.
3. Enzyme according to claim 1, wherein the enzyme part is an
oxidase and the bleaching chemical is hydrogen peroxide.
4. Enzyme according to claim 1, wherein the enzyme part is an
oxidase selected from the group consisting of glucose oxidase,
galactose oxidase and alcohol oxidase.
5. Enzyme according to claims 1-2, wherein the enzyme part is a
haloperoxidase and the bleaching chemical is hypohalite.
6. Enzyme according to claim 1, wherein the enzyme part is a
chloroperoxidase and the bleaching chemical is hypochlorite.
7. Enzyme according to claim 1, wherein the wherein the enzyme part
is a Vanadium chloroperoxidase and the bleaching chemical is
hypochlorite.
8. Enzyme according to claim 17, wherein the Vanadium
chloroperoxidase is Curvularia inaequalis chloroperoxidase.
9. Enzyme according to claim 1, where the enzyme part is a laccase
or a peroxidase and the bleaching molecule is derived from an
enhancer molecule that has reacted with the enzyme.
10. Enzyme according to claim 1, wherein the enzyme part is coupled
to a reagent having a high binding affinity for pectin or
beta-lactoglobulin when the pectin or beta-lactoglobulin is
adsorbed onto a surface.
11. Enzyme according to claim 1, wherein the enzyme part is coupled
to a reagent having a high binding affinity for pectin or
beta-lactoglobulin when the pectin or beta-lactoglobulin is
adsorbed onto a surface selected from cotton, polyester, or
polyester/cotton fabric.
12. Enzyme according to claim 1, capable of binding to tomato
stains present on fabrics.
13. Enzyme according to claim 1, wherein the reagent having a high
binding affinity is a protein or a peptide.
14. Enzyme according to claim 1, wherein the reagent having a high
binding affinity is an antibody, an antibody fragment, or a
derivative thereof.
15. Enzyme according to claim 1, which is a fusion protein
comprising a bleaching enzyme and all or part of a heavy chain
immunoglobulin that was raised in Camelidae and has a specificity
for non-coloured compounds present in stains on fabrics, said
non-coloured compounds having a molecular weight of at least 5,000,
preferably at least 10,000.
16. Enzyme according to claim 1, wherein the reagent having a high
binding affinity has a chemical equilibrium constant K.sub.d for
the substance of less than 10.sup.-4 M, preferably less than
10.sup.-6 M.
17. Enzyme according to claim 1, wherein the chemical equilibrium
constant K.sub.d is less than 10.sup.-7M.
18. A multi-specific antibody or antibody or an analogous structure
arranged so that at least one specificity is directed to
non-coloured compounds present in stains on fabrics, said
non-coloured compounds having a molecular weight of at least 5,000,
preferably at least 10,000, and the others are directed to one or
more bleaching enzymes.
19. An antibody or antibody fragment or an analogous structure
according to claim 18, having one specificity directed to
non-coloured compounds present in stains on fabrics and one to a
bleaching enzyme.
20. Bleaching enzyme comprising an antibody or antibody fragment
according to claim 18.
21. Enzymatic bleaching composition comprising a bleaching enzyme
according to claim 1 and one or more surfactants.
22. Enzymatic bleaching composition according to claim 21, wherein
the enzyme produces hydrogen peroxide and comprising an activator
which generates peracetic acid.
23. Enzymatic bleaching composition according to claim 21, wherein
the enzyme produces hydrogen peroxide and comprising and a
transition metal catalyst.
24. Process for bleaching stains present on fabrics, wherein
stained fabrics are contacted with a solution comprising a
bleaching enzyme according to claim 1.
25. Process for bleaching stains present on fabrics, wherein
stained fabrics are contacted with an enzymatic bleaching
composition according to claim 21.
Description
TECHNICAL FIELD
[0001] The present invention generally relates to bleaching
enzymes. More in particular, it relates to bleaching enzymes
capable of generating a bleaching chemical and having a high
binding affinity for non-coloured compounds present in stains on
fabrics. The invention also relates to a detergent composition
comprising said enzymes and to a process for bleaching stains
present on fabrics.
BACKGROUND AND PRIOR ART
[0002] Detergent compositions comprising bleaching enzymes have
been described in the prior art. For instance, GB-A-2 101 167
(Unilever) discloses an enzymatic bleach composition in the form of
a hydrogen peroxide-generating system comprising a C.sub.1-C.sub.4
alkanol oxidase and a C.sub.1-C.sub.4 alkanol. Such enzymatic
bleach compositions may be used in detergent compositions for
fabric washing, in which they may provide a low-temperature
enzymatic bleach system. In the wash liquor, the alkanol oxidase
enzyme catalyses the reaction between dissolved molecular oxygen
and the alkanol to form an aldehyde and hydrogen peroxide. In order
to obtain a significant bleach effect at low wash temperatures,
e.g. at 15-55.degree. C., the hydrogen peroxide must be activated
by means of a bleach activator. Today, the most commonly used
bleach activator is tetra-acetyl ethylene diamine (TAED), which
yields peracetic acid upon reacting with the hydrogen peroxide, the
peracetic acid being the actual bleaching agent.
[0003] Although this and several other enzymatic bleach systems
have been proposed, there is still a need for alternative or
improved enzymatic bleach systems. In particular, the enzymatic
bleach system should be capable of bleaching stains which are
otherwise difficult to remove, the so-called "problem stains" such
as tomato, tea, blackberry juice, or red wine. Such stains would
require a significant amount of bleaching for their removal, which
might negatively affect the colours of the garment.
[0004] In conventional laundry bleach systems, fabrics are
uniformly exposed to the same concentration of bleach, whether
"problem stains" are present or not. Moreover, repeated washing
with conventional bleach systems, which may contain relatively high
concentrations of bleach, may cause damage to garments such as the
fading of dyes.
[0005] It is therefore an object of the present invention to
provide alternative or improved enzymatic bleach systems which, in
particular, should be capable of bleaching stains which are
otherwise difficult to remove, and should preferably be more
selective in its bleaching action. It is a further object of the
present invention to provide an alternative or improved enzymatic
process for bleaching stains on fabrics.
[0006] We have now surprisingly found that it is possible to
control the bleaching reaction in an enzymatic bleach process by
using the bleaching enzyme according to the invention, which is
capable of generating a bleaching chemical and has a high binding
affinity for non-coloured compounds present in stains on fabrics,
said non-coloured compounds having a molecular weight of at least
100, preferably at least 1,000. Even more preferably, the
non-coloured compounds have a molecular weight of at least 5,000
and especially preferred are compounds having a molecular weight of
at least 10,000. Preferably, the enzyme comprises an enzyme part
capable of generating a bleaching chemical, coupled to a reagent
having a high binding affinity for the non-coloured compounds
present in stains on fabrics. The new bleaching enzyme is
particularly attractive for treating "problem stains" which occur
only occasionally, such as fruits and vegetables. These stains are
not present on most garments and when they are present they are
likely to be present in different positions than habitual stains
such as those found on collars and cuffs. According to the
invention, it is possible to optimise the in-use concentration of
the new bleaching enzyme so that threshold concentrations of bleach
are only reached if stain is present and the new bleaching enzyme
binds to and accumulates on said stain. When this happens, the high
local concentration of enzyme generates a high local concentration
of bleach near to the stain and thereby exerts a selective
bleaching action where it is required. Therefore, the unstained
part of the garment (typically the majority) is not exposed to high
levels of bleach and thereby this fabric is protected from
bleach-associated damage. Moreover, the next time the same garment
has a stain such as fruit or vegetable stains, it is likely to be
in a different position on the garment. Therefore, a different
position on the garment will be exposed to high levels of bleach.
Therefore, problems associated with several washes in conventional
bleaching systems, such as dye-fade, will be reduced or eliminated
altogether. This is in stark contrast to conventional bleaching
systems where all garments are uniformly exposed to high
concentrations of bleach, in every wash, regardless of whether
problem stains are present or not.
DEFINITION OF THE INVENTION
[0007] According to a first aspect of the invention, there is
provided a bleaching enzyme capable of generating a bleaching
chemical and having a high binding affinity for non-coloured
compounds present in stains on fabrics, said non-coloured compounds
having a molecular weight of at least 100, preferably at least
1,000. Preferably, the enzyme comprises an enzyme part capable of
generating a bleaching chemical, coupled to a reagent having a high
binding affinity for the non-pigmented compounds present in stains
on fabrics.
[0008] According to a second aspect, there is provided an enzymatic
bleaching composition comprising one or more surfactants and the
bleaching enzyme according to the invention.
[0009] According to a third aspect, there is provided a process for
bleaching stains present of fabrics, wherein stained fabrics are
contacted with an a solution comprising the bleaching enzyme of the
invention.
DESCRIPTION OF THE INVENTION
[0010] 1. The Bleaching Enzyme
[0011] In its first aspect, the invention relates to a bleaching
enzyme which is capable of generating a bleaching chemical and has
a high binding affinity for stains present on fabrics. Preferably,
the enzyme comprises an enzyme part capable of generating a
bleaching chemical which is coupled to a reagent having a high
binding affinity for non-coloured compounds present in stains on
fabrics. Said non-coloured compounds have a molecular weight of at
least 100, preferably of at least 1,000, more preferably of at
least 5,000. Especially preferred are non-coloured compounds having
a molecular weight of at least 10,000.
[0012] 1.1 The Enzyme Part, Capable of Generating a Bleaching
Chemical.
[0013] The bleaching chemical may be enzymatically generated
hydrogen peroxide. The enzyme for generating the bleaching chemical
or enzymatic hydrogen peroxide-generating system may in principle
be chosen from the various enzymatic hydrogen peroxide-generating
systems which have been disclosed in the art. For example, one may
use an amine oxidase and an amine, an amino acid oxidase and an
amino acid, cholesterol oxidase and cholesterol, uric acid oxidase
and uric acid or a xanthine oxidase with xanthine. Alternatively, a
combination of a C.sub.1-C.sub.4 alkanol oxidase and a
C.sub.1-C.sub.4 alkanol is used, and especially preferred is the
combination of methanol oxidase and ethanol. The methanol oxidase
is preferably isolated from a catalase-negative Hansenula
polymorpha strain. (see for example EP-A-244 920 (Unilever)). The
preferred oxidases are glucose oxidase, galactose oxidase and
alcohol oxidase.
[0014] A hydrogen peroxide generating enzyme could be used in
combination with activators which generate peracetic acid. Such
activators are well-known in the art. Examples include
tetraacetylethylenediamine (TAED) and sodium
nonanoyloxybenzenesulphonate (SNOBS). These and other related
compounds are described in fuller detail by Grime and Clauss
in-Chemistry & Industry (Oct. 15, 1990) 647-653. Alternatively,
a transition metal catalyst could be used in combination with a
hydrogen peroxide generating enzyme to increase the bleaching
power. Examples of manganese catalysts are described by Hage et al.
(1994) Nature 369, 637-639.
[0015] Alternatively, the bleaching chemical is hypohalite and the
enzyme part is then a haloperoxidase. Preferred haloperoxidases are
chioroperoxidases and the corresponding bleaching chemical is
hypochlorite. Especially preferred chloroperoxidases are Vanadium
chloroperoxidases, for example from Curvularia inaequalis.
[0016] Alternatively, peroxidases or laccases may be used. In this
case the bleaching molecule is derived from an enhancer molecule
that has reacted with the enzyme. Examples of laccase/enhancer
systems are given in WO-A-95/01426. Examples of peroxidase/enhancer
systems are given in WO-A-97/11217.
[0017] 1.2 The Part Having the High Binding Affinity.
[0018] The new bleaching enzyme has a high binding affinity for
non-coloured compounds present in stains on fabrics, said
non-coloured compounds having a molecular weight of at least 100,
preferably at least 1,000, more preferably of at least 5,000
Daltons. It will be understood that the non-coloured compounds may
also have higher molecular weights of at least 10,000, 100,000 or
even 1,000,000 Daltons or more. It may be that one part of the
polypeptide chain of the bleaching enzyme is responsible for the
binding affinity, but it is also possible that the enzyme comprises
an enzyme part capable of generating a bleaching chemical which is
coupled to a reagent having the high binding affinity for the
non-coloured compounds present in stains on fabrics. In the first
situation, the bleaching enzyme may be a fusion protein comprising
two domains which may be coupled by means of a linker. In the
second situation, the reagent having the high binding affinity may
be covalently coupled to the enzyme part for generating the
bleaching chemical, by means of a bi-valent 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. Bio/technology 11, 601-605.
[0019] A further embodiment would be for the reagent with a high
binding affinity to be a bispecific reagent, comprising one
specificity for non-coloured compounds present in stains on
fabrics, said non-coloured compounds having a molecular weight of
at least 1,000, preferably of at least 5,000 and more preferably of
at least 10,000. Such a reagent could fulfil the requirement of
accumulating enzyme on the stain either by supplying said reagent
together with enzyme as a pre-formed non-covalent complex or by
supplying the two separately and allowing them to self-assemble
either in the wash liquor or on the stain.
[0020] The novel bleaching enzyme according to the invention is
based on the presence of a part having a high binding affinity for
non-coloured compounds present in stains on fabrics, said
non-coloured compounds having a molecular weight of at least 100,
preferably of at least 1,000 and more preferably of at least
5,000.
[0021] The degree of binding of a molecule A to another molecule B
can be generally expressed by the chemical equilibrium constant
K.sub.d resulting from the following reaction:
[A]+[B][A.ident.B]
[0022] The chemical equilibrium constant K.sub.d is then given by:
1 K d = [ A ] .times. [ B ] [ A B ]
[0023] Whether the binding of a molecule to a non-coloured compound
present in stains on fabrics is specific or not can be judged from
the difference between the binding (K.sub.d value) of the molecule
to stained (i.e. a material treated so that stain components are
bound on), versus the binding to unstained (i.e. untreated)
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 a values and differences in K.sub.d
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 compound should bind the stain, or the stained
material, with a K.sub.d lower than 10.sup.-4 M, preferably lower
than 10.sup.-6M and could be 10.sup.-10M or even less. Higher
binding affinities (K.sub.d of less than 10.sup.-5M) and/or a
larger difference between the non-coloured substance and background
binding would increase the selectivity of the bleaching process.
Also, the weight efficiency of the molecule in the total detergent
composition would be increased and smaller amounts of the molecule
would be required.
[0024] Several classes of molecules can be envisaged which deliver
the capability of specific binding to non-coloured compounds
present in stains one would like to bleach. In the following we
will give a number of examples of such molecules having such
capabilities, without pretending to be exhaustive.
[0025] 1.2.1. Antibodies.
[0026] 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.
[0027] 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 consist of two
polypeptide chains (the variable regions of the heavy chain
(V.sub.h, and the light chain (V.sub.l)). 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).
[0028] Alternatively, binding domains can be obtained from the
V.sub.h fragments of classical antibodies by a procedure termed
"camelization". Hereby the classical V.sub.h 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).
[0029] 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.
[0030] 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 the same approach,
antibodies could be isolated that bind to a whole "family" of stain
chromophores (such as the polyphenals, 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.
[0031] 1.2.2. Peptides.
[0032] 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 a oxidation
process. A peptide which is capable of binding selectively to a
substance which one would like to oxidise, can for instance be
obtained from a protein which is known to bind to that specific
substance. An example of such a peptide would be a binding region
extracted from an antibody raised against that substance. Other
examples are proline-rich peptides that are known to bind to the
polyphenols in wine.
[0033] Alternatively, peptides which bind to such substance can be
obtained by the use of peptide combinatorial libraries. Such a
library may contain up to 10.sup.10 peptides, from which the
peptide with the desired binding properties can be isolated. (R. A.
Houghten, 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).
[0034] 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 microbial hosts (yeast,
moulds, bacteria)(K. N. Faber et al. (1996) Appl. Microbiol.
Biotechnol. 45, 72-79).
[0035] 1.2.3. Pepidomimics.
[0036] 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.
[0037] 1.2.4. Other Organic Molecules.
[0038] 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 substances one
would like to oxidise with the desired binding properties. 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).
[0039] 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.
[0040] 1.3 The Non-coloured Compounds Present in Stains on
Fabrics
[0041] For laundry detergent applications, several classes of
coloured substances one would like to bleach can be envisaged, in
particular coloured substances which may occur as stains on fabrics
can be a target. It was found to be advantageous to target the
bleaching enzymes not directly to such coloured stains themselves,
but rather to macro-molecular compounds which themselves are not
coloured but which are associated with the stains. 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 stains than coloured substances, which generally have a low
molecular weight. Finally, it is important to emphasise that
although many stains are heterogeneous, certain non-coloured
compounds are commonly present in a variety of stains.
[0042] It the context of the present invention, a non-coloured
compound is defined as a compound which, in purified form in
solution and after correcting for effects such as the scattering of
light, has an optical density (or adsorption) for all wavelengths
in the visible spectrum (i.e. from 325 nm to 900 nm) and for a
light path of 1 cm at a concentration of 1 mg/ml in solution of
less than 0.2 and preferably less than 0.05.
[0043] An important embodiment of the invention is to use a binding
molecule (as described above) that binds to several different, but
structurally-related, non-coloured molecules in a class of "stain
substances". This would have the advantage of enabling a single
enzyme species to bind (and bleach) several different stains. Some
examples of classes of non-coloured compounds associated with
stains are given below:
[0044] 1.3.1. Pectins.
[0045] Pectins are a heterogeneous group of polysaccharides which
are rich in D-galacturonic acid. They are one of the most important
components in the cell wall matrix of plant cells. For a review see
A. Jauneau et al. (1998) Int. J. Plant Sci. 159 (1) 1-13.
[0046] 1.3.2. beta-lactoglobulin
[0047] Beta-lactoglobulin (BLG) is the major whey protein in the
milk of various species including cows, sheep, goats, horses, and
pigs. For a review see J. Godovac-Zimmermann and G. Braunitzer
(1987) Milchwissenschaft 42 (5) 294-297.
[0048] 2. The Detergent Composition.
[0049] The bleaching enzymes of the invention can be used in a
laundry detergent composition which is specifically suited for
stain bleaching purposes, and this constitutes a second aspect of
the invention. To that extent, the composition comprises one or
more surfactants and optionally other conventional detergent
ingredients. The invention in its second aspect provides an
enzymatic detergent composition which comprises from 0.1-50% by
weight, based on the total detergent composition, of one or more
surfactants. This surfactant system may in turn comprise 0-95% by
weight of one or more anionic surfactants and 5-100% 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).
[0050] 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 Hauser Veriag, 1981.
[0051] 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 C.sub.6-C.sub.22
alkyl phenol-ethylene oxide condensates, generally 5 to 25 EO, i.e.
5 to 25 units of ethylene oxide per molecule, and the condensation
products of aliphatic C.sub.8-C.sub.18 primary or secondary linear
or branched alcohols with ethylene oxide, generally 5 to 40 EO.
[0052] 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.sub.8-C.sub.18 alcohols, produced for example from tallow or
coconut oil, sodium and potassium alkyl C.sub.9-C.sub.20 benzene
sulphonates, particularly sodium linear secondary alkyl
C.sub.10-C.sub.15 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 C.sub.11-C.sub.15 alkyl benzene sulphonates and sodium
C.sub.12-C.sub.18 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.
[0053] 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 C.sub.16-C.sub.18
primary alcohol sulphate together with a C.sub.12-C.sub.15 primary
alcohol 3-7 EO ethoxylate.
[0054] The nonionic detergent is preferably present in amounts
greater than 10%, e.g. 25-90% by weight of the surfactant system.
Anionic surfactants can be present for example in amounts in the
range from about 5% to about 40% by weight of the surfactant
system.
[0055] The 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 enzymatic bleaching detergent composition
according to the invention will generally be used as a dilution in
water of about 0.05 to 2%.
[0056] The bleaching enzyme used in the present invention can
usefully be added to the detergent composition in any suitable
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 enzyme to a liquid detergent product is in
the form of a slurry containing 0.5 to 50% by weight of the enzyme
in a ethoxylated alcohol nonionic surfactant, such as described in
EP-A-450 702 (Unilever).
[0057] The enzymatic bleaching compositions of the invention
comprise about 0.001 to 10 milligrams of active bleaching enzyme
per liter. A detergent composition will comprise about 0.001% to 1%
of active enzyme (w/w).
[0058] The enzyme activity can be expressed in units. For example,
in the case of glucose oxidase, one unit will oxidise 1 sole of
.beta.-D-glucose to D-gluconolactone and H.sub.2O.sub.2 per minute
at pH 6.5 at 30.degree. C. The enzyme activity which is added to
the enzymatic bleaching composition will be about 2.0 to 4,000
units per liter (of wash liquor).
[0059] The invention will now be further illustrated in the
following, non-limiting Examples. In the examples, two types of
analytical experiment are described. The first type of experiment
(as described in examples 1,2 and 5) involves using a surface that
is specially designed to investigate antibody binding: the
microtitre plate. These experiments investigate whether the
non-coloured components of stains, such as pectin in fruit and
vegetables or beta-lactoglobulin in milky beverages, can be
specifically targeted by antibodies in the presence of other
components of the stain. In these experiments, the staining
material was sometimes diluted many-fold so as to optimise antibody
binding specificity. This is standard practise when working with
microtitre plate assays as the technique is so sensitive and works
best when using small amounts of sample. Furthermore, as the
surface of the microtitre plate has been specially designed to
adsorb biological material and to have very low on-specific binding
effects, these experiments are the best check on antibody
specificity.
[0060] In the second type of analytical experiment (as described in
examples 4 and 6), swatches of cotton are stained with staining
material such as tomato ketchup or milky tea under conditions that
are likely to be encountered with "real" stains i.e. undiluted
staining material. These experiments investigate whether the
non-coloured components are accessible to antibody when bound to
the porous structure of the cotton and also whether the
non-coloured components remain bound to the cotton after soaking
the cotton in surfactant. In contrast to coloured components, it is
not possible to tell whether the non-coloured molecules are bound
to the cotton simply by visual inspection. Therefore, a specific
binding probe (such as an antibody) is needed to determine whether
they are present. Unlike the microtitre plates, the cotton has not
been specially designed for immunoassay and so it can be expected
that background signal will be higher. However, the analysis of the
exposure of stain marker molecules on cotton is nevertheless of
critical importance to applications in laundry products.
[0061] Taking the results of both types of experiment together we
have found (in our view surprisingly) that some non-coloured
components of common stains are readily adsorbed onto surfaces and
can be specifically targeted by antibodies even after extensive
washing and soaking in surfactant. Moreover, the two experimental
approaches described here could be used to define other
non-coloured marker molecules in stains that could be used for the
invention by virtue of being a) accessible for binding by antibody
b) able to remain attached to surfaces in the presence of
surfactant.
[0062] Finally, having established that a particular marker
molecule is accessible and remains attached to cotton in the
presence of surfactant, it is then possible go evaluate the effect
of treating a stain with an antibody/oxidase conjugate that has a
specific binding affinity for said marker molecule. Such an
experiment is described in example 8.
Example 1
[0063] Binding of a Pectin-Specific Antibody to a Microtitre Plate
Sensitised with Tomato Products.
[0064] A rat monoclonal antibody that is specific for pectin and
known as "JIM5" (K. A. VandenBosch et al. EMBO Journal vol. 8 no. 2
pp.335-342, 1989; J. P. Knox et al. Planta (1990) 181: 512-521) was
used. However, other antibodies that bind pectin could also be
used. Methods of how to raise such antibodies by inoculation are
known. See for instance K. A. VandenBosch et al. as above and F.
Liners et al. Plant Physiol. (1989) 91, 1419-1424. The antibody
preparation used in this example was a culture supernatant that was
a gift from the John Innes Centre, Norwich, U.K.
[0065] Tomato ketchup (H.J. Heinz Co Ltd. Hayes, U.K.) and sieved
tomatoes (Valfrutta) were diluted approximately 1,000-fold in
phosphate buffered saline, PBS [0.01M
Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4, 0.15M NaCl (pH 7)]. The
diluted tomato samples were applied to the wells of microtitre
plates (high capacity, flat-bottomed ELISA plates; Greiner
Labortechnik) and incubated at 37.degree. C. for 48 hours with an
air-tight plastic seal. As a negative control, some wells were
treated with PBS only.
[0066] The sensitised plates were washed with PBST [PBS+0.15% tween
20 (Sigma)] using an automated microtitre plate washer. Then a
range of dilutions of the JIMS preparation (dilutions were made in
PBST) were applied to the wells and incubated for 1 hour at room
temperature. Control wells were treated with either a negative
culture supernatant (not containing JIM 5) or PBST only.
[0067] Plates were washed as above and then a Sheep anti-rat
IgG/alkaline phosphatase conjugate (Serotec Product No AAR02A) was
applied to the wells. The conjugate was diluted 1 in 1,000 in PBST
and incubated in the wells for 1 hour at room temperature.
[0068] The plates were washed as above and then substrate buffer
was applied. [1 mg/ml para-nitrophenyl phosphate (pNPP) in 1M
diethylamine (pH 9.8); 1 mM MgCl.sub.2]. Signal was read at 405 nm
in an automated plate reader after 5 minutes.
[0069] The results are given below in Table 1 as optical densities
or "signal" recorded at 405 nm--the higher the signal, the more
antibody has bound to the surface.
1TABLE 1 Antibody reagent applied Sensitisation JIM5/l0 JIM5/l00
JIM5/1,000 None NC/l0 Sieved 1.2 0.89 0.16 0.01 0.01 tomatoes
Tomato 0.98 0.71 0.15 0.00 0.00 ketchup None 0.01 0.01 0.01 0.01
0.01 JIM5/10 = JIM5 culture diluted 1 in 10 in PBST JIM5/100 = JIM5
culture diluted 1 in 100 in PBST JIM5/1,1000 = JIM5 culture diluted
1,1000 in PBST NC/10 = Negative culture diluted 1 in 10 in PBST
[0070] The results show that JIM- can specifically bind to both
tomato products when adsorbed onto the microtitre plate. Therefore,
it is concluded that pectin remains bound to the surface in an
accessible form even after washing the surface with a
surfactant-containing liquid (PBST).
EXAMPLE 2
[0071] Binding of a Pectin-Specific Antibody to a Microtitre Plate
Sensitised With Orange Juice
[0072] Pure, unsweetened orange juice (Safeway) was diluted
approximately 1,000-fold in phosphate buffered saline, PBS [0.01M
Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4, 0.15M NaCl (pH 7)]. The
diluted orange sample was applied to the wells of microtitre plates
(Greiner, high capacity) and incubated at 37.degree. C. overnight
with an air-tight plastic seal. As a negative control, some wells
were treated with PBS only.
[0073] The sensitised plates were washed with DBST [PBS+0.15% tween
20 (Sigma)] using an automated microtitre plate washer. Then a
range of dilutions of the JIMS preparation (dilutions were made in
PBST) were applied to the wells and incubated for 1.5 hours at room
temperature. Control wells were treated with either a negative
culture supernatant (not containing JIM 5), a non-specific rat
monoclonal antibody (Serotec product PRP04) made up to 4 .mu.g/ml
in PBST, or PBST only.
[0074] Plates were washed as above and then a Sheep anti-rat
IgG/alkaline phosphatase conjugate (Serotec Product No AAR02A) was
applied to the wells. The conjugate was diluted 1 in 1,000 in PBST
and incubated in the wells for 1 hour at room temperature. The
plates were washed as above and then substrate buffer was applied.
(1 mg/ml pNPP in 1M diethylamine (pH 9.8); 1 mM MgCl.sub.2). Signal
was read at 405 nm in an automated plate reader after 40
minutes.
[0075] The results are given in Tables 2 and 3 below as optical
densities or "signal" recorded at 405 nm--the higher the signal,
the more antibody has bound to the surface.
2TABLE 2 Antibody reagent applied Sensitisation JIM5/10 JIM5/40
JIM5/160 JIM5/640 Orange juice 1.37 0.63 0.17 0.05 None 0.06 0.06
0.07 0.07 JIM5/10 = JIM5 culture diluted 1 in 10 in PBST JIM5/40 =
JIM5 culture diluted 1 in 40 in PBST JIM5/160 = JIM5 culture
diluted 1 in 160 in PBST JIM5/640 = JIM5 culture diluted 1 in 640
in PBST
[0076]
3TABLE 3 Negative control applied Sensitisation None NC/10 NAb
Orange juice 0.02 0.02 0.03 None 0.07 0.07 0.08 None = PBST only
NC/10 = Negative culture diluted 1 in 10 in PBST NAb = Non-specific
rat monoclonal antibody at 4 .mu.g/ml in PBST.
EXAMPLE 3
[0077] Purification of JIM5 From Culture Supernatant.
[0078] 500 ml of clarified culture was concentrated to
approximately 12 ml using a stirred cell (Amicon) fitted with an
ultrafiltration membrane (Amicon PM30). The concentrate was applied
to a Protein G "Hi-Trap" column (Pharmacia). The column was then
washed with PBS to remove non-specifically bound material. JIMS
antibody was then specifically eluted by washing the column with
0.1 M glycine buffer (pH 2.5). The desorbed fraction was
immediately neutralised with 1/20 volume 3M tris (pH 8.8). The
neutralised fraction was dialysed into PBS. Recovered antibody was
determined by measuring the absorbance at 280 nm, assuming en
extinction coefficient of 1.4 for a concentration of 1 mg/ml
protein.
EXAMPLE 4
[0079] Binding of Purified Pectin-Specific Antibody to Cotton
Swatches Stained With Tomato.
[0080] Eighteen 1 cm.times.1 cm cotton swatches were cut from white
de-sized cotton fabric and labelled with a "B" pencil so that they
could be identified. Six swatches were stained by submerging them
in tomato ketchup (Heinz) and incubated at 37.degree. C. overnight
in a sealed Petri dish. Six were stained by submerging in sieved
tomatoes (Valfrutta) and incubated at 37.degree. C. overnight in a
sealed tube. Six were not stained.
[0081] The stained swatches were pre-washed so that the stains
became typical of those described as "residual" i.e. faint but
stubborn. Swatches were pre-washed in batches of six, according to
stain type, so that cross-contamination of stain material between
different batches was minimised. They were rinsed with distilled
water to remove surplus tomato and then washed vigorously in
3.times.100 ml of wash buffer (PBS+0.2% Co-Co 6.5EO). They were
recovered from each wash with a tea strainer. After the third wash,
they were blot-dried on paper towel.
[0082] Swatches were then placed in plastic tubes containing 2 ml
of antibody at 5 .mu.g/ml in wash buffer. Antibody was either
purified JIM5 (as described in example 3) or a non-specific rat
monoclonal antibody of the same sub-class (Serotec Product number
PRP04). Each type of swatch was kept separate according to stain
type to minimise cross-contamination. There were therefore a total
of six tubes, each tube contained three swatches, every swatch in a
single tube had undergone the same treatment, as summarised in
Table 4 below.
4TABLE 4 Tube Antibody Stain 1 JIM5 Ketchup 2 JIM5 Sieved tomatoes
3 JIM5 None 4 Non-specific Ketchup 5 Non-specific Sieved tomatoes 6
Non-specific None
[0083] Tubes were incubated for 2 hours at room temperature. Then
they were washed with 3.times.100 ml of wash buffer, blotted dry
and then placed in conjugate. The conjugate was a sheep
anti-ratIgG/alkaline phosphatase conjugate (Serotec product number
AAR02A) diluted 1 in 1,000 in wash buffer. The tubes were incubated
for a further 2 hours at room temperature. Again, stained and
un-stained swatches were kept apart to minimise cross-contamination
of stain material.
[0084] Swatches were washed and dried as before and then
individually placed in 1 ml of substrate buffer [1 mg/ml pNPP in 1M
diethylamine (pH 9.8); 1 mM MgCl.sub.2)]. The substrate was
dispensed into the wells of a 24-well cell culture plate (Costar,
Cambridge USA) and the swatches were incubated for 30 minutes at
room temperature, before removing 200 .mu.l for reading in a
microtitre plate reader.
[0085] Results are given in Table 5 below as optical densities or
"signal" recorded at 405 nm--the higher the signal, the more 4
antibody has bound to the cotton. Results are given for replicate
swatches and then mean figures have been calculated.
5 TABLE 5 Antibody Stain Signal (A405) Mean signal JIM5 Ketchup
1.185, 0.942, 1.214 1.1 JIM5 Sieved 1.048, 1.052, 0.947 1.0
tomatoes JIM5 None 0.494, 0.581, 0.539 0.54 non-specific Ketchup
0.427, 0.394, 0.348 0.39 non-specific Sieved 0.425, 0.429, 0.354
0.40 tomatoes non-specific None 0.527, 0.463, 0.442 0.47
[0086] The results show that the pectin-specific antibody binds to
tomato-stained swatches significantly better than to un-stained
swatches. Therefore, it can be concluded that pectin has bound to
the cotton during the staining procedure and that a significant
amount remains bound even after several washes in
surfactant-containing buffer. Furthermore, the pectin must be
accessible to antibody even when it is bound to or within the
porous structure of the cotton.
EXAMPLE 5
[0087] Binding of Lactoglobulin-specific Antibody to a Microtitre
Plate Sensitised With Milky Tea
[0088] A rabbit polyclonal reagent was used. This was prepared by
inoculating a rabbit with beta-lactoglobulin B, or "BLG" (Sigma
Product number L8005), recovering immune sera, and purifying
lactoglobulin-specific antibodies by antigen affinity
chromatography. Methods describing how to do this are published by
S. C. Williams et al. [J. Immunological Methods 213 (1998)
1-17].
[0089] Tea was made by pouring boiling water onto a tea-bag
(Typhoo) and then adding milk (Co-op half-fat milk). The tea was
allowed to cool and then applied to the wells of a microtitre plate
(Greiner, high capacity) an then incubated at 37.degree. C. for 48
hours with an air-tight seal.
[0090] The sensitised plates were washed with PBST (PBS+0.15% tween
20) using an automated microtitre plate washer. Then a range of
dilutions of the lactoglobulin-specific antibody (dilutions were
made in PBST) were applied to the wells and incubated for 2 hours
at room temperature. Control wells were treated with a non-specific
rabbit antibody i.e. a antibody that had been raised against a
different antigen (Dako rabbit anti-mouse, product number Z259,
Dako A/S, Glostrup, Denmark). Other control wells were treated with
PBST only.
[0091] Plates were washed as above and then a goat anti-rabbit
IgG/alkaline phosphatase conjugate (Zymed Laboratories Inc, San
Fransisco, Product No 62-6122) was applied to the wells. The
conjugate was diluted 1 in 1,000 in PBST and incubated in the wells
for 1 hour at room temperature. The plates were washed as above and
then substrate buffer was applied (1 mg/ml pNPP in 1M diethylamine
(pH 9.8); 1MM MgCl.sub.2). Signal was read at 405 nm in an
automated plate reader after 30 minutes. The results are given in
Table 6 below as optical densities or "signal" recorded at 405
nm--the higher the signal, the more antibody has bound to the
surface.
6TABLE 6 Antibody reagent applied anti-BLG anti-BLG Anti-BLG anti-
NAb Sensiti- at at At BLG at at sation 4 .mu.g/ml l .mu.g/ml 250
ng/ml 62 ng/ml none 4 .mu.g/ml Milky 2.12 l.09 0.28 0.07 0.01 0.06
Tea
[0092] The results show that the anti-lactoglobulin antibody can
bind lactoglobulin (both specifically and in a dose-dependant
manner) when adsorbed onto a surface in the presence of tea.
EXAMPLE 6
[0093] Binding of Lactoglobulin-specific Antibody to Cotton
Swatches Stained With Milky Tea
[0094] Eighteen 1 cm.times.1 cm cotton swatches were cut from white
de-sized cotton fabric and labelled with a "B" pencil so that they
could be identified. Nine swatches were stained by submerging them
in milky tea (made with Typhoo tea bag and Co-op half-fat milk) and
incubating at 37.degree. C. overnight in a sealed tube. Nine were
not stained.
[0095] The stained swatches were pre-washed so that the stains
became typical of those described as "residual" i.e. faint but
stubborn. They were washed vigorously in 3.times.100 ml of wash
buffer (PBS+0.2% Co-Co 6.5EO). They were recovered from each wash
with a tea strainer. After the third wash, they were blot-dried on
paper towel.
[0096] Swatches were then placed in plastic tubes containing 2 ml
of antibody at 5 .mu.g/ml in wash buffer or a negative control tube
containing wash buffer only. Antibody was either rabbit
anti-lactoglobulin ("anti-BLG" as described in example 5) or a
non-specific rabbit antibody (Dako rabbit anti-mouse, product
number Z259). Each type of swatch was kept separate according to
the type of treatment it had received so as to minimise
cross-contamination of reagents. There were therefore a total of
six tubes, each tube contained three swatches, every swatch in a
single tube had undergone the same treatment, as summarised in
Table 7 below.
7TABLE 7 Tube Stain Antibody 1 Milky tea BLG-specific 2 Milky tea
Non-specific 3 Milky tea None 4 None BLG-specific 5 None
Non-specific 6 None None
[0097] Tubes were incubated for 1.5 hours at room temperature. Then
they were washed with 3.times.100 ml of wash buffer, blotted dry
and then placed in conjugate. The conjugate was a goat
anti-rabbitIgG/alkaline phosphatase conjugate (Zymed product number
62-6122) diluted 1 in 1,000 in wash buffer. The tubes were
incubated for a further 1.5 hours at room temperature. Again,
stained and unstained swatches were kept apart to minimise
cross-contamination of stain material.
[0098] Swatches were washed and dried as before and then
individually placed in 1 ml of substrate buffer (1 mg/ml pNPP in 1M
diethylamine (pH 9.8); 1 M MgCl.sub.2). The substrate was dispensed
into the wells of a 24-well cell culture plate A (Costar) and the
swatches were incubated for 20 minutes at room temperature, before
removing 200 .mu.l for reading in a microtitre plate reader.
[0099] Results are given as optical densities or "signal" recorded
at 405 nm--the higher the signal, the more antibody has bound to
the cotton. The results are given below in Table 8 for replicate
swatches and then mean figures have been calculated.
8TABLE 8 Stain Antibody Signal (A405) Mean signal Milky tea
BLG-specific 0.702, 0.560, 0.586 0.61 Milky tea Non-specific 0.380,
0.288, 0.322 0.33 Milky tea None 0.195, 0.165 0.18 None
BLG-specific 0.412, 0.298, 0.245 0.32 None Non-specific 0.375,
0.412, 0.295 0.36 None None 0.377, 0.257, 0.329 0.32
[0100] The results show that the lactoglobulin-specific antibody
binds to cotton swatches that have been stained with milky tea
significantly better than to un-stained swatches. Therefore, it can
be concluded that lactoglobulin has bound to the cotton during the
staining procedure and that a significant amount remains bound even
after several washes in surfactant-containing buffer. Furthermore,
the lactoglobulin must be accessible to antibody even when it is
bound to or within the porous structure of the cotton.
EXAMPLE 7
[0101] Conjugation of Pectin-specific Antibody to Glucose Oxidase
Enzyme.
[0102] Antibody was chemically coupled to enzyme using a protocol
that was based loosely on the methods described in Carlsson et-al.
(1978) Biochem. J. 173, 723-737 and in "Bioconjugate Techniques" by
Greg T Hermanson, Academic Press (1996), page 70-71. There are also
several other methods for coupling two active proteins that are
well known in The Art. The details of the precise protocols used in
this example are given below.
[0103] Derivatisation of Antibody with "SAMSA"
[0104] Purified JIM5 antibody (as described in example 3) was
concentrated to 6.4 mg/ml and buffer-exchanged into 0.1 M
NaH.sub.2PO.sub.4, pH 6.5 by using a "Centricon 30" ultrafiltration
tube (Millipore). 40.mu.l of this antibody preparation was
dispensed into a glass reactivial. A solution of "SAMSA"
[S-Acetylmercaptosuccinic anhydride (Sigma product number A-1251)]
was made up. The SAMSA solution was 10 mg/ml in DMF [Dimethyl
formamide]. 2 .mu.l of the SAMSA solution was added to the antibody
and the mixture stirred vigorously for 30 minutes at room
temperature 21.degree. C..+-.1. At the end of 30 minutes the
following solutions were added at 5 minute intervals.
[0105] (i) 20 .mu.l of EDTA to stabilise the derivatised
antibody.
[0106] (ii) 100 .mu.l of 0.1M Tris pH 7.0 to adjust the pH.
[0107] (iii) 100 .mu.l of 1M NH.sub.2OH pH 7.0 to deprotect the
SAMSA and expose thiol groups.
[0108] At 45 minutes, the mixture was made up to 2.5 ml (with 0.1 M
NaH.sub.2PO.sub.4, pH 6.5) and desalted on a PD10 column
(Pharamacia) previously equilibrated in 0.1 M NaH.sub.2PO.sub.4+5
mM EDTA pH 6.5. The derivatised antibody was recovered by eluting
the column with 3.2 ml of buffer (in 0.1 M NaH.sub.2PO.sub.4+5 mM
EDTA pH 6.5).
[0109] Derivatisation of Glucose Oxidase with "SPDP"
[0110] Glucose oxidase (or "Gox") type XS [Genencor OxyGo HPL 5000
(commercial grade)] was made up to 12.8 mg/ml in 0.1 M
NaH.sub.2PO.sub.4 (pH 7.5). 62.5 ul of this enzyme preparation (0.8
mg) was placed in a reactivial with stirring, 0.5 ml of 0.1 M
NaH.sub.2PO.sub.4 pH 7.5 was added. A solution of "SPDP"
[3-(2-Pyridyldithio)propionic acid N-Hydroxy succinimide ester
(Sigma product number P-3415)] was made up. The SPDP solution was
13.15 mg/ml in DMSO [Dimethyl sulfoxlde]. 30.4 .mu.l of the SPDP
solution was added to the reactivial and the mixture was stirred
for 30 minutes at room temperature. Then the mixture was made up to
2.5 ml (with 0.1 M NaH.sub.2PO.sub.4 pH 7.5) and desalted on a PD10
column (Pharamacia) previously equilibrated in 0.1 M
NaH.sub.2PO.sub.4 pH 6.5. The derivatised enzyme was recovered by
eluting the column with 3.2 ml of buffer (0.1 M NaH.sub.2PO.sub.4
pH 6.5).
[0111] Reaction of Derivatised Antibody With Derivatised Glucose
Oxidase
[0112] The antibody and Gox preparations were pipetted into
separate centricon 30 tubes (Millipore) and centrifuged at 4000 RPM
until each preparation had been concentrated to a volume of about
400 .mu.l (from a starting volume of 3.2 ml). 135.mu.l of the Gox
preparation was mixed with all of the antibody preparation (400
.mu.l) in a glass vial. The vial was placed at 4.degree. C.
overnight to allow conjugation to proceed.
EXAMPLE 8
[0113] Bleaching of Tomato Stain With Pectin-Specific Antibody/Gox
Conjugate
[0114] De-sized white cotton cloth was stained by submerging in
sieved tomatoes (Valfrutta) and boiling for 1 hour. The cloth was
rinsed with cold water and dried at 37.degree. C. overnight. Dried
cloth was cut into 2.times.2 cm square swatches and pre-rinsed with
"wash buffer II" [PBS+0.0375% Coco 6.5EO, 0.0375% Las (pH 8.0)] to
leave a residual stain.
[0115] 6 identical swatches were added to each of 4 glass vials.
Each vial was then treated with 1 ml of a different solution: vial
1 was treated with wash buffer II only; vial 2 was treated with
glucose oxidase "Gox" type XS [Genencor OxyGo HPL 5000 (commercial
grade)], diluted in wash buffer II; vial 3 was treated the
pectin-specific antibody/Gox conjugate (as prepared in Example 7),
diluted in wash buffer II; and vial 4 was treated with a
non-specific antibody/Gox conjugate (comprising an antibody that
does not bind pectin), again made up in wash buffer II. The enzyme
preparation and the conjugates had been diluted so that they all
contained an enzyme activity equivalent to approximately 2.8 .mu.g
of unconjugated Gox. The vials were incubated at room temperature
for two minutes. Then 8 ml of wash buffer II was added to each vial
followed by 90 .mu.l of 1M glucose. The vials were inverted to mix
the contents and placed at 37.degree. C. for 35 minutes (Vials 2-4
now contained an enzyme activity approximately equivalent to 300
ng/ml of unconjugated Gox).
[0116] The swatches were removed and rinsed in distilled water.
Each set of swatches were dried by placing in a 37.degree. C.
incubator for 3 hours. Dry cloths were analysed
spectrophotometrically (using a "Color Eye 7000" spectrophotometer,
Macbeth). Stain removal was expressed as .DELTA.R.sub.440 and
.DELTA.E, read against stained, untreated controls. The results are
shown below.
9TABLE 9 Removal of tomato stain with pectin-specific antibody/GOx
conjugate Average Average Vial R440 .DELTA.R440 .DELTA.R440
.DELTA.E .DELTA.E l. Buffer only 63.9 8.2 8.6 6.1 6.2 63.4 7.8 5.7
63.9 8.3 6.2 64.8 9.2 6.2 65.1 9.4 6.7 64.5 8.8 6.4 2. Gox in 62.1
6.4 7.7 4.9 5.6 buffer 62.2 6.5 5.6 62.1 6.4 4.8 64.6 8.9 6.2 66.1
10.4 7.2 63.1 7.6 5.6 3. Pectin- 69.1 13.5 11.8 8.7 8.0 specific
67.8 12.2 8.2 antibody/Gox 66.5 10.9 7.4 conjugate 65.5 9.8 7.4 (in
buffer) 66.8 11.2 7.8 68.8 13.2 8.6 4. Non-specific 61.3 5.7 7.8
4.9 5.7 antibody/GOx 65.5 9.8 6.7 conjugate 62.2 6.6 4.7 (in
buffer) 64.0 8.4 6.1 64.1 8.5 6.1 62.3 7.7 5.5
[0117] The results show that the pectin-specific conjugate removes
more stain than the non-specific conjugate or unconjugated
enzyme.
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