U.S. patent application number 12/767157 was filed with the patent office on 2010-10-21 for differential labelling method.
This patent application is currently assigned to KREATECH BIOTECHNOLOGY B.V.. Invention is credited to Robert Jochem Heetebrij, Eduard Gerhard Talman, Robertus Petrus Maria van Gijlswijk, Jacky Theo Maria Veuskens.
Application Number | 20100267056 12/767157 |
Document ID | / |
Family ID | 8180378 |
Filed Date | 2010-10-21 |
United States Patent
Application |
20100267056 |
Kind Code |
A1 |
Talman; Eduard Gerhard ; et
al. |
October 21, 2010 |
DIFFERENTIAL LABELLING METHOD
Abstract
The invention relates to a method for differentially labelling
one or more entities, together comprising distinct reactive sites.
The invention further relates to an entity that has been labelled
by a method according to the invention and to a diagnostic kit
comprising a labelled entity and to a diagnostic kit to employ a
method according to the invention.
Inventors: |
Talman; Eduard Gerhard;
(Leiderdorp, NL) ; van Gijlswijk; Robertus Petrus
Maria; (Alphen aan de Rijn, NL) ; Heetebrij; Robert
Jochem; (Leiden, NL) ; Veuskens; Jacky Theo
Maria; (Hasselt, BE) |
Correspondence
Address: |
HOFFMANN & BARON, LLP
6900 JERICHO TURNPIKE
SYOSSET
NY
11791
US
|
Assignee: |
KREATECH BIOTECHNOLOGY B.V.
Amsterdam
NL
|
Family ID: |
8180378 |
Appl. No.: |
12/767157 |
Filed: |
April 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10156730 |
May 28, 2002 |
7704755 |
|
|
12767157 |
|
|
|
|
Current U.S.
Class: |
435/7.9 ;
435/188; 436/525; 530/363; 530/391.3; 530/400; 536/23.1 |
Current CPC
Class: |
G01N 33/84 20130101;
G01N 33/58 20130101 |
Class at
Publication: |
435/7.9 ;
530/363; 530/391.3; 530/400; 536/23.1; 435/188; 436/525 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07K 14/765 20060101 C07K014/765; C07K 16/00 20060101
C07K016/00; C07K 14/485 20060101 C07K014/485; C07K 14/00 20060101
C07K014/00; C07H 21/04 20060101 C07H021/04; C12N 9/96 20060101
C12N009/96; G01N 33/553 20060101 G01N033/553 |
Foreign Application Data
Date |
Code |
Application Number |
May 28, 2001 |
EP |
01202007.9 |
Claims
1. A method for selectively forming a complex of a platinum
compound and a sulphur-containing reactive site of an entity or of
at least one entity of a mixture of two or more entities, the
entity or the mixture of two or more entities comprising one or
more sulphur-containing reactive sites and one or more
nitrogen-containing reactive sites, the method comprising: forming
a complex of the platinum compound and the entity or the at least
one entity of a mixture of two or more entities at a pH below the
lowest pKa of the nitrogen-containing reactive sites, whereby the
platinum compound is reacted with the entity such that
substantially only sulphur-containing reactive sites are linked to
the platinum compound; thereby selectively forming a complex of a
platinum compound and a sulphur-containing reactive site of an
entity or of at least one entity of a mixture of two or more
entities.
2. A method according to claim 1, wherein the entity or the
entities are selected from the group consisting of amino acids,
peptides, oligopeptides, polypeptides, proteins, immunoglobulins,
enzymes, synzymes, phospholipids, glycoproteins, nucleic acids,
nucleosides, nucleotides, oligonucleotides, polynucleotides,
peptide nucleic acids, peptide nucleic acid oligomers, peptide
nucleic acid polymers, amines and aminoglycosides.
3. A method according to claim 1, wherein the pH is 5 or less.
4. A method according to claim 1, wherein the platinum compound
comprises a label.
5. A method according to claim 1, wherein the platinum compound is
a trans platinum compound.
6. A method for differentially forming a complex of a first
platinum compound and a sulphur-containing reactive site of an
entity or of at least one entity of a mixture of two or more
entities, and a second platinum compound and a nitrogen-containing
reactive site of the entity or of at least one entity of the
mixture of two or more entities, the entity or the mixture of two
or more entities comprising one or more sulphur-containing reactive
sites and one or more nitrogen-containing reactive sites, the
method comprising: (a) forming a first complex of the first
platinum compound and the entity or the at least one entity of a
mixture of two or more entities at a pH below the lowest pKa of the
nitrogen-containing reactive sites, whereby the first platinum
compound is reacted with the entity such that substantially none of
the nitrogen-containing reactive sites, and substantially all of
the sulphur-containing reactive sites, are linked to the first
platinum compound; and (b) starting with the first complex, forming
a second complex of the second platinum compound and the entity or
the at least one entity of a mixture of two or more entities at a
pH at which only nitrogen-containing reactive sites are linked to
the second platinum compound; thereby differentially forming a
complex of a first platinum compound and a sulphur-containing
reactive site of an entity or of at least one entity of a mixture
of two or more entities, and a second platinum compound and a
nitrogen-containing reactive site of the entity or at least one
entity of the mixture of two or more entities.
7. A method according to claim 6, wherein the entity or the
entities are selected from the group consisting of amino acids,
peptides, oligopeptides, polypeptides, proteins, immunoglobulins,
enzymes, synzymes, phospholipids, glycoproteins, nucleic acids,
nucleosides, nucleotides, oligonucleotides, polynucleotides,
peptide nucleic acids, peptide nucleic acid oligomers, peptide
nucleic acid polymers, amines and aminoglycosides.
8. A method according to claim 6, wherein the pH in part (a) is 5
or less.
9. A method according to claim 6, wherein the pH in part (b) is 7
or above.
10. A method according to claim 6, wherein the first platinum
compound comprises a label.
11. A method according to claim 6, wherein the second platinum
compound comprises a label.
12. A method according to claim 6, wherein the first platinum
compound is a trans platinum compound.
13. A method according to claim 12, wherein the trans platinum
compound comprises a label.
14. A method according to claim 13, wherein the label is removed
from the trans platinum compound.
15. A method according to claim 13, wherein the first platinum
compound and the second platinum compound are the same trans
platinum compound comprising a label, further comprising removing
the label of the first trans platinum compound.
Description
[0001] This application asserts the priority of European patent
application 01202007.9 filed May 28, 2001 and parent U.S. patent
application Ser. No. 10/156,730 filed May 28, 2002, each of which
is incorporated by reference in its entirety.
[0002] The invention relates to a method for differentially
labelling one or more entities, together comprising distinct
reactive sites, to an entity that has been labelled by a method
according to the invention and to a diagnostic kit for employing a
method according to the invention.
[0003] An entity may be labelled with a detectable marker to
detect, visualise, quantify or monitor the entity e.g. in chemical,
biological or medical research or diagnosis. A wide variety of
labelling methods are known from the art (for a review see
Hermanson, 1996, Bioconjugate techniques, Academic Press, ISBN
0-12-342335-X).
[0004] Many factors may play a role in choosing a particular
detectable marker and a particular method of labelling. Such
factors include the nature of the entity, reaction conditions,
detection limits of the labelled entity, sensitivity during the
labelling reaction and specificity towards the entity.
[0005] Methods using platinum compounds to label bio-organic
molecules have been considered interesting for a very long time.
Various types of detectable marker moieties can be adhered to ionic
platinum. Platinum compounds may react with a variety of reactive
moieties on an entity.
[0006] The use of a cis-platinum compound has been described in
European patent application no. 95201197.1. Herein a method is
disclosed for linking bio-organic molecules and markers through
cis-platinum compounds, of which two co-ordination sites are
occupied by two ends of a stabilising bridge, such as an
ethylenediamine group. These known cis-platinum compounds are
suitable for linking labels to several kinds of bio-organic
molecules, such as peptides, polypeptides, proteins, and nucleic
acids. Methods using trans-platinum compounds have also been
reported (EP application 97201066.4) to be suitable to label a
variety of bio-organic molecules.
[0007] The reactivity of platinum compounds towards a variety of
reactive sites is a benefit in many applications, since it may
allow fast labelling reactions and an excellent sensitivity towards
a wide variety of entities.
[0008] It may however be desired to direct the label to a specific
reactive site of an entity, e.g. to improve the selectivity of the
labelling. Also, pre-selected sites may be labelled in complex
samples such as those samples comprising various types of
bio-organic compounds. Differential or selective labelling often
circumvents the need of sample purification and may be directed in
such a way that targeted entities do not loose their native
characteristics, e.g. 3D structure, activity, avidity, etc.
[0009] Furthermore it may be advantageous to label an entity at a
controlled number of reactive sites. This may improve accuracy of
the quantification and facilitate identification of a labelled
entity. Such an improvement would be very valuable for various
applications such as in the organochemical, biological or medical
fields.
[0010] Moreover it is often a challenge in labelling chemistry to
find a labelling method that does not affect the structure or the
activity of an entity, e.g. of an enzyme, an immunoglobulin or a
DNA-probe, to a high extent.
[0011] It is an objective of the present invention to provide a
method to differentially label one or more entities together
comprising distinct reactive sites, at a targeted reactive
site.
SUMMARY OF THE INVENTION
[0012] A method for differentially labelling one or more entities
through a platinum-linker, said entities together comprising one or
more sulphur containing reactive sites and one or more nitrogen
containing reactive sites, wherein a complex of a platinum compound
and said one or more entities, such as a marker, is formed, and
wherein said platinum compound is reacted with said one or more
entities such that substantially only sulphur containing reactive
sites or substantially only nitrogen containing reactive sites are
linked to said platinum compound.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows abduct formation between Pt and N-Ac-Methionine
at RT;
[0014] FIG. 2 shows abduct formation between Pt and Histidine at
RT;
[0015] FIG. 3 shows labelling of goat sera, non-immunized and
immunized with mouse IgG. Mouse IgG coated/DNP-ULS labelled whole
serum/anti-DNP-HRP detection;
[0016] FIG. 4 shows labelling and detection of IgG serum proteins
after ammonium sulphate precipitation: pellet;
[0017] FIG. 5 shows labelling and detection of IgG serum proteins
after ammonium sulphate precipitation: supernatants; and
[0018] FIG. 6 shows mp-11 double labelling.
DETAILED DESCRIPTION OF THE INVENTION
[0019] Surprisingly it has been found that according to the
invention one or more entities can be labelled through a
platinum-linker. In a preferred embodiment said linker is a
platinum-linker, and said entities together comprise one or more
sulphur containing reactive sites and/or one or more nitrogen
containing reactive sites, wherein a complex of a platinum compound
and a marker is formed, and wherein said platinum compound is
reacted with said one or more entities. In a preferred embodiment
of the invention substantially only sulphur containing reactive
sites or substantially only nitrogen containing reactive sites are
linked to said platinum compound.
[0020] Entity as used herein is to be interpreted as something that
comprises one or more sulphur containing reactive sites and/or one
or more nitrogen containing reactive sites. In particular an entity
relates to an inorganic or organic compound, including a
bio-organic compound. A bio-organic compound as used herein refers
to a biological carbon containing compound. Also, a bio-organic
compound refers to a compound capable of inducing or affecting an
action in a biological system, e.g. by inducing or affecting a
therapeutic or prophylactic effect, an immune response, a metabolic
process etc. "Entity" further relates to a micro-organism, a virus
or a prion, or to a material comprising one or more of said sulphur
reactive or nitrogen reactive types of reactive sites, or a product
made thereof, such as a micro-array, a microtitre plate, a test
strip or a test tube. Distinct reactive sites--which are to be
labelled differentially--may be present together in one entity or
in a combination (a mixture, a solution, a dispersion etc.) of more
entities having only one or some of the to be labelled reactive
sites, but together comprising said distinct reactive sites. Such a
combination is for example a combination of an entity with only a
nitrogen containing reactive site and an entity with only a sulphur
containing reactive site.
[0021] In principle, any type of nitrogen containing reactive site
or sulphur containing reactive site may be labelled using a method
according to the invention. Preferred reactive sites include
reactive sites comprising a primary amine, a secondary amine, a
tertiary amine, an aromatic amine, a thiol, a thioether, a sulfide,
a disulfide, a thioamide, a thion, an amide, an imide, an imine, an
iminoether, or an azide. Examples of entities that can be labelled
are entities chosen from the group of amino acids (preferably
methionine, cysteine, histidine, lysine, and tryptophan), peptides,
oligopeptides, polypeptides, proteins, immunoglobulins, enzymes,
synzymes, phospholipides, glycoproteins, nucleic acids,
nucleosides, nucleotides, oligonucleotides, polynucleotides,
peptide nucleic acids, peptide nucleic acid oligomers, peptide
nucleic acid polymers, amines, aminoglycosides, nucleopeptides, and
glycopeptides. Preferably in accordance with the invention, the
entity is chosen from the group of amino acids, peptides,
oligopeptides and polypeptides.
[0022] An entity linked to a platinum compound may be referred to
as a Pt--S adduct (when attached to a sulphur containing reactive
site), as a Pt--N adduct (when attached to a nitrogen containing
reactive site), or in general as a Pt-adduct.
[0023] A sulphur containing reactive site may hereafter be referred
to as an S-reactive site, and a nitrogen containing reactive site
may hereafter be referred to as an N-reactive site.
[0024] A platinum linker is a platinum moiety that can be used to
couple a marker to an entity. A preferred linker compound as used
in this invention is a platinum compound to which ligands are
bound.
[0025] It has been found that a method according to the invention
is highly suitable to direct the labelling of an entity towards a
specified reactive site within an entity or a group of entities
that together comprise a variety of reactive sites. Furthermore a
method according to the invention has been found to have excellent
sensitivity towards the targeted (reactive site of the) entity,
even in complex matrices. The prowess of a method according to the
invention to distinguish to which reactive site a marker is
labelled is inter alia extremely beneficial for analytical
purposes. Not only may the excellent selectivity contribute to the
accuracy and the dynamic range of quantification, but it also may
improve the homogeneity of the labelled entity. The improved
homogeneity generally has a beneficial effect upon band broadening
during separation of a sample for analysis or for purification,
e.g. by a chromatographic or electrophoretic method.
[0026] Furthermore it has been found possible to selectively label
an entity without significantly affecting the structure or activity
of the labelled entity, even if such an entity contains a fragile
or labile part. This is a highly advantageous feature of the
invention since it facilitates the detection or monitoring of a
labelled entity while the entity retains activity--preferably
substantially all of its activity--in vivo or in vitro. To the
benefit of retaining activity, it has been found possible to direct
labelling of an entity, such as an immunoglobulin, an enzyme, a
hormone, or a nucleic acid in such a way that essentially no marker
is labelled at one or more N- or S-reactive sites at a functional
part of said entity.
[0027] Furthermore it was found that the present invention can be
used to label an entity in such a way that the configuration of the
entity largely remains unaffected after the entity has been
labelled. This embodiment of the invention is for example
particularly suitable for labelling an antibody-antigen complex or
a double stranded oligo- or polynucleotide without disturbing the
complex. This aspect of the invention may also be very useful for
visualisation of the entity and/or certain chemical or biochemical
processes in vivo or in vitro.
[0028] Examples of preferred platinum compounds are cis- or
trans-platinum compounds of the formula [Pt(II)(X1)(X2)(A)(D)] or a
cis-platinum compound of the formula [Pt(II)(X3)(A)(D)].
[0029] Herein, Pt represents platinum (Pt), A and D represent the
same or different reactive moieties, respectively involved in the
complexation of the platinum compound to a marker and the linking
of the platinum compound to the entity. The entities, X1 and X2
represent the same or different inert moieties, and X3 represents
an inert moiety that may act as a stabilising bridge, e.g. a
bidentate ligand.
[0030] A structural representation of some examples of such
platinum compounds is shown below:
##STR00001##
[0031] A platinum(II) compound, for use in a method of the
invention can be prepared via any method known in the art.
References can for example be found in Reedijk et al. (Structure
and Bonding, 67, pp. 53-89, 1987). The preparation of some
trans-platinum compounds is disclosed in EP-A 97201066.4. Further
preparation methods can be found in EP-A 96202792.6 and EP-A
95201197.1. Methods described in any of these publications are
incorporated herein by reference. In a preferred embodiment of the
invention platinum compounds are prepared according to the
spacer-tert butoxycarbonyl/NHS-label pathway.
[0032] The reactive moieties (A, D) of a platinum compound are
preferably good leaving ligands. A platinum compound, wherein A
and/or D are independently chosen from the group of Cl.sup.-,
NO.sub.3.sup.-, HCO.sub.3.sup.-, CO.sub.3.sup.2-, SO.sub.3.sup.2-,
ZSO.sub.3.sup.-, I.sup.-, Br.sup.-, F.sup.-, acetate, carboxylate,
phosphate, ethylnitrate, oxalate, citrate, a phosphonate, ZO.sup.-,
and water has been found to be particularly suitable for use in a
method according to the invention. Z is defined herein as a
hydrogen moiety or an alkyl or aryl group having from 1 to 10
carbon atoms. Of these ligands, Cl.sup.- and NO.sub.3.sup.- are
most preferred.
[0033] Any type of inert moiety may be chosen. Inert as used herein
indicates that the moiety remains attached to the platinum compound
during the labelling process and thereafter without chemically
reacting with an entity. A platinum compound comprising one or two
inert moieties chosen from the group of NH.sub.3, NH.sub.2R, NHRR',
NRR'R'' groups, wherein R, R' and R'' preferably represent an alkyl
group having from 1 to 6 carbon atoms have been found to be
particularly suitable for use in a method of the present invention.
H.sub.2NCH.sub.3 is a particularly preferred inert moiety for use
in a method according to the invention. An alkyl diamine, wherein
the alkylgroup has 2 to 6 carbon atoms is a preferred bidentate
inert moiety in a cis-platinum compound (e.g. X3 in formula 1c). In
a particularly preferred embodiment X3 represents ethylene
diamine.
[0034] Preferred platinum compounds for use in a method according
to the invention include cis[Pt(en)Cl.sub.2],
cis[Pt(en)Cl(NO.sub.3)], cis[Pt(en)(NO.sub.3).sub.2],
trans[Pt(NH.sub.3).sub.2Cl.sub.2],
trans[Pt(NH.sub.3).sub.2Cl(NO.sub.3)], and
trans[Pt(NH.sub.3).sub.2(NO.sub.3).sub.2].
[0035] The term labelling is used herein to refer to connecting a
marker with an entity, possibly via a platinum linker. A marker as
used herein may be any moiety that can be attached to the platinum
compound or the entity, and that can be used to detect, monitor or
visualise the entity. A marker may be reacted with the platinum
compound at any time. Hence, in accordance with the invention it is
possible that a platinum linker is first reacted with a marker to
obtain a linker-marker complex, which is then reacted with the
entity, or that the order is reversed. In a preferred embodiment,
the platinum linker is first reacted with the marker.
[0036] Any type of marker may be used as long as it can be attached
to the platinum compound. Such a marker may be a radioactive
marker, an enzyme , a specific binding pair component such as
avidin, streptavidin or biotin, biocytin, iminobiotin, a colloidal
dye substance, a phosphorescent marker (e.g. an Europium chelate, a
platinum porphyrine), a chemiluminescent marker (e.g. luminol), a
fluorochrome, including a cyanine, a Alexa dye (Molecular Probes),
or Bodipy-colourant (Molecular Probes), a rhodamine, dinitrophenol
(DNP), carboxyrhodamine, tert-butoxycarbonyl, a reducing substance
(eosin, erythrosin, etc.), a (coloured) latex sol, digoxigenin, a
metal (ruthenium), a metal sol or another particulate sol
(selenium, carbon and the like), dansyl lysin, a UV dye, a VIS dye,
Infra Red Dye, coumarine (e.g. amino methyl coumarine), an
antibody, protein A, protein G, etc.
[0037] Particular preferred are DNP, fluorescein, cyanine-colorants
and tetramethylrhodamine, inter alia because they can form stable
complexes with platinum linked to an entity and they may give rise
to excellent limits of detection. These markers can very suitably
be used for a technique referred to as multi-colour labelling. Thus
several colorants of this kind, optionally having similar chemical
structures while having different colours, may be used. Other
preferred markers include biotin, avidin, streptavidin and
digoxygenin.
[0038] In an embodiment of the invention the marker and/or a
reactive site of the entity may be connected to platinum through a
spacer. Preferably such a spacer comprises a chain having at least
four atoms, and preferably not more than 20 atoms, which chain
comprises an electron donating moiety on one end and a moiety for
reacting with a marker or an entity on the other end, wherein the
chain is attached to platinum through the electron donating moiety.
Of course, the spacer(s), the marker, the entity and the platinum
linker may be attached to each other in any order. For instance,
the spacer(s) may first be attached to the linker followed by
reacting the obtained compound with a marker and the entity. It is
also possible first to attach the spacer(s) to the marker before
the reaction with the linker. The electron donating moiety of the
spacer may for example be an amine group or a thiolate anion.
Preferably the chain further comprises at least one hetero-atom.
Highly preferred spacers are 1,6-diaminohexane and
1,8-diamino-3,6-dioxaoctane. In a preferred embodiment of the
invention use is made of 1,6-diaminohexane tert-butoxycarbonyl, as
an intermediate linker-spacer complex, prior to attaching to a
marker and/or entity. It goes without saying that the labelling
complex may contain more than one platinum, e.g. two platinum
atoms, such as for example described in European Patent Application
97201066.4.
[0039] One of the reaction parameters that have been found
particularly useful to choose such that an entity is differentially
labelled in a method according to the invention, is the pH value.
The pH as used herein should be interpreted as the pH value of a
composition or product according to the invention in water at
20.degree. C. In case an embodiment of the invention is employed in
an environment leading to an altered solvent autoprotolytic
constant (pK.sub.w), (e.g. presence of organic solvents, altered
temperature) a pH mentioned herein should be interpreted based upon
the pH range at 20.degree. C. in water.
[0040] In general, the formation of Pt--S adducts is pH independent
whereas formation of Pt--N adducts is pH dependent. In a preferred
embodiment one or more S-reactive sites are selectively labelled
over one or more nitrogen containing sites by making use of the
pH.
[0041] As a guideline, in a preferred embodiment, one may choose
the pH of the invention at a pH below the lowest pKa of any of an
entity's N-reactive sites that should not be labelled, allowing
differential labelling of one or more S-reactive sites. As the
skilled professional will understand, besides pKa, other factors
may play a role, including the influence of the micro-environment
in the vicinity of an entity that is to be labelled.
[0042] In a preferred embodiment the S-reactive site or sites are
selectively labelled at a neutral or acidic pH. In a more preferred
embodiment the S-reactive site or sites are differentially labelled
over N-reactive sites at a pH of 5 or less.
[0043] It has also been found possible to label histidine residues
distinctively over other N-reactive sites at a pH between about 6
and 8. A residue of a compound as used herein should be interpreted
as the compound itself or as part of a larger entity, e.g. an amino
acid residue in a protein.
[0044] An overview on the formation of Pt--S and Pt--N adducts at
various pH values is given in Table 1.
TABLE-US-00001 TABLE 1 pH dependent formation of Pt--S and Pt--N
adducts in proteins pH > 10 pH = 7 pH < 5 S donor(s) all all
all N donor(s) all Histidine only none
[0045] In theory, the formation of a Pt--S adducts is an one step
process. A reactive group leaves the platinum compound upon S
donating an electron pair to platinum. This process, the direct
conversion Pt--X into Pt--S, is believed to be pH independent. On
the other hand, N donors require replacement of a reactive group of
the platinum compound by oxygen prior to N substitution. First,
Pt--X becomes Pt--O and eventual Pt--N. This is a two step scheme
in which the first step can be controlled by changing pH. Factors
influencing pH of a solution might interfere with Pt--N adduct
formation.
[0046] The presence of ions may also be used to control the
selectivity of the platinum compound for N-reactive sites. In an
embodiment one or more leaving ligands, preferably anionic
moieties, are used in the inhibition of labelling a platinum
compound to a N-reactive site, in order to enhance differentiated
labelling of a S-reactive site. Examples of such leaving ligands
include Cl.sup.-, NO.sub.3.sup.-, HCO.sub.3.sup.-, CO.sub.3.sup.2-,
ZSO.sub.3.sup.-, SO.sub.3.sup.-, I.sup.-, Br.sup.-, F.sup.-,
acetate, carboxylate, phosphate, ethylnitrate, oxalate, citrate, a
phosphonate, ZO.sup.-, and water. Z is defined herein as a hydrogen
moiety or an alkyl or aryl group having from 1 to 10 carbon atoms.
Particularly good results have been achieved by using salts
comprising an anionic moiety, of which chloride is particularly
preferred. The counter ions are preferably alkali cations, alkali
earth cations or cations also used to direct the labelling. In a
preferred embodiment the total ionic strength of said anionic
moieties used in the inhibition of labelling to a N-reactive site
is at least 0.1 mol/l. More preferably the total ionic strength is
in the range of 0.1 to 0.5 mol/1.
[0047] The presence of metal ions, such as transition metal ions,
may also be used for selection of the reactive site to be labelled.
In particular such ions have been found suitable to prevent or slow
down labelling of an S-reactive site or to make a labelled Pt--S
adduct labile, so that effectively one or more N-reactive sites are
differentially labelled over said S-reactive site. Within a method
according to the invention it is also possible to direct the
labelling by making use of geometrical isomers of a platinum
compound--e.g. a cis-platinum compound and a trans-platinum
compound,--such that the platinum compound is specifically labelled
to either a sulphur containing reactive site or to a nitrogen
containing reactive site
[0048] The presence of a bulky inert moiety at the platinum
compound may for example be used to prevent labelling at a reactive
site of an entity, wherein said reactive site is partially shielded
from a platinum compound with a particular stereochemical structure
by the structure of the entity. This may for example be the case if
the entity has a complex 3D structure, e.g. a protein, a
conglomerate of molecules, etc.
[0049] It is also possible to differentially label an entity
according to the invention by first shielding one or more reactive
sites that should not be labelled with a shielding moiety and
thereafter react a targeted reactive site of the entity with the
platinum compound to which also a marker is attached.
[0050] Shielding as used herein is to be interpreted as
deactivation of the affinity of a reactive site for a marker, by
reaction of the reactive site with a moiety that prevents
attachment of a marker directly to said reactive site or
complexation of a marker with platinum linked with the reactive
site. Preferably the shielding moiety is present in excess over the
number of reactive sites that are to be shielded. The preferred
reaction time for the shielding process will depend upon the
application, and it will be clear to the skilled professional how
to choose the reaction conditions.
[0051] In another preferred embodiment the shielding moiety is
selectively removed from the shielded reactive site, after the
platinum compound has been reacted such that said platinum compound
is differentially linked to said entity.
[0052] In a preferred embodiment one or more S-reactive sites may
be shielded, e.g. by a trans-platinum compound under conditions as
described above, prior to selectively labelling one or more
N-reactive sites of one or more entities. Particularly good results
have been achieved with Rhodamine trans-Pt
(trans[Pt(II)(NH.sub.3).sub.2(NH.sub.2--(CH.sub.2).sub.6--NH-rhodamine)Cl-
](NO.sub.3)) as the shielding moiety. To improve shielding even
further the reaction was performed at a pH chosen between 2 and 5,
after which the pH was increased to an alkaline pH for labelling
N-reactive sites. Other preferred shielding compounds are cadmium,
mercury, or zinc complexes.
[0053] The addition of transition metal ions, such as Cu(II),
Zn(II) or a mixture thereof has been found to be particularly
suitable to selectively remove a trans-platinum compound from an
S-reactive site , whilst a labelled N-reactive site of a Pt-adduct
substantially remains stable.
[0054] The type of solvent may also be used to differentiate the
labelling. In particular the reactivity towards N-reactive sites
can vary depending upon the solvent. In particular solvents that
may act as a ligand to the platinum compound may decrease the
reactivity towards N-reactive sites, and thus such a solvent may
favour the labelling of S-reactive sites .
[0055] In addition to the parameters as mentioned above a method
according to the invention may further be fine tuned by parameters
such as temperature, preferably varied in the range between
0.degree. C. and 120.degree. C., more preferably in the range
between 20.degree. C. and 70.degree. C.; reaction time, commonly in
the range between 1 min and 48 hours, preferably in the range
between 10 min and 24 hours, more preferably in the range between
25 min and 15 hours; concentration of the reagents, molar ratio of
the reagents, overall net charge of the platinum labelling complex,
and the like. These parameters may be adjusted depending upon the
particular application in any way known in the art. The overall net
charge of the platinum labelling complex, for example, affects the
specificity of Pt--N adduct formation in histidine at neutral pH.
Neutral Pt-complexes, such as fluorescein- and cyanine Pt
complexes, form Pt--N adducts whereas positively charged platinum
labelling complexes, e.g. rhodamine- and dinitrophenol Pt
complexes, do not. Positively charged Pt labelling complexes
display differential labelling towards N adducts above the
isoelectric point of the peptide, protein, and the like. Apart from
allowing the selective labelling of N-reactive sites over
S-reactive sites or vice versa, a method according to the present
invention also makes it possible to differentiate between distinct
N-reactive sites or distinct S-reactive sites, by choosing the
correct conditions, such as described herein.
[0056] For example, one or more N-reactive sites of histidine
residues may be labelled differentially over one or more other
N-reactive sites by linking a platinum compound with a marker, and
choosing the reaction conditions such that said platinum compound
is differentially linked to a histidine residue of said entity.
Such a method can be employed in the presence of S-reactive
sites--which may be shielded during the labelling of histidine--but
also in the absence thereof.
[0057] Thus an entity, such as a peptide or a protein, can be
selectively labelled at one or more histidine residues in a mixture
of amino acids or other N-reactive site containing entities. In a
preferred embodiment differentially labelling of histidine is
accomplished by choosing a pH of about 7 and a Pt labelling
complex, with an overall neutral charge.
[0058] The selective labelling of a particular type of S-reactive
sites or a particular type of N reactive site offers a solution in
several application areas. It may for example be used to screen for
a particular type of reactive site in an entity of unknown
composition or the presence of a particular entity in a sample.
(e.g. the presence of histidine in an amino acid mixture). Thus in
a repeated differential labelling process, several entities can one
after another be labelled with a different marker, which may be
useful for screening of several components without requiring
separation of a sample, e.g. by chromatography, electrophoresis
and/or mass spectrometry.
[0059] It may also add further specificity towards the labelling in
order to avoid labelling at an undesired reactive site (e.g. at a
functional part of an entity).
[0060] Furthermore discrimination between distinct N-reactive site
or distinct S reactive sites, allows the creation of an entity with
a multitude of different markers.
[0061] With a method according to the invention one or more
labelled entities can be prepared. The invention also relates to
such entities, differentially linked with a platinum compound at a
N-reactive site or a S-reactive site. The invention further relates
to a labelled entity wherein a marker is attached to the entity via
a platinum compound linked to a specific reactive site of the
entity.
[0062] In a particular embodiment according to the invention, at
least one other reactive entity is differentially or
non-differentially labelled, after selective labelling of a first
reactive site of an entity or a mixture of entities. Such
subsequent labelling may take place with a different marker that is
reacted with a platinum compound according to the invention, but it
is also possible to use another type of labelling reaction known in
the art. For example, after differentially labelling an S-reactive
site a subsequent labelling may take place with a label that is
reactive towards amines.
[0063] In a preferred embodiment subsequent labelling also involves
differential labelling. Thus it is possible to prepare an entity to
which different markers are labelled at distinct reactive
sites.
[0064] Thus it has been found possible to label an entity or a
mixture of entities with several of different markers. Accordingly,
the invention relates to entities having two or even a plurality of
markers. Labelling with more than one marker can be very useful in
various applications. It may for example be used to screen for
particular entities in a mixture, without needing an analytical
separation, e.g. screening for the presence of methionine and
histidine in an amino acid mixture. In another embodiment it may be
used to monitor a process in which a labelled entity is involved,
e.g. a process in which an entity is split into several entities,
each having a different label or vice versa. It goes without saying
that the invention is not restricted to qualitative analyses but
also includes quantitative analyses of differential labelled
entities. In principle, a labelled entity may be subsequently
analysed using any liquid based analyte analysis system. In a
particularly suitable method according to the invention, comprising
the analysis of a labelled entity, the labelled entity is analysed
using a high throughput screening liquid based multiple analyte
analysis system, e.g. a flow cytometry-system.
[0065] The present invention further relates to a diagnostic kit
comprising an entity according to the invention. A diagnostic kit
according to the invention preferably comprises one or more
preparations selected from the group formed by entities
differentially linked to a platinum compound at one or more
nitrogen containing reactive sites and/or one or more sulphur
containing reactive sites, platinum-linker preparations, buffers,
marker preparations, transition metal ion preparations,
preparations for adjusting the ionic strength and preparations
comprising a shielding moiety.
[0066] Another embodiment of the invention relates to a diagnostic
kit, for employing a method according to the invention. Such a kit
may for example comprise reaction instructions, one or more
platinum compounds for labelling the entity, one or more markers,
one or more entities according to the invention, one or more test
samples, one or more other reagents, one or more test tubes or
strips and the like.
[0067] The invention will now further be illustrated by the
following non-limiting examples.
Example 1
[0068] Two amino acids (histidine and methionine, 0.1 mmol each)
were dissolved in 500 .mu.l deuterated sodium phosphate buffer (50
mM, pD=7.00) and incubated at room temperature with a slight excess
(0.44 mmol) of [Pt(en)(NH.sub.2--NH-Boc)Cl](NO).sub.3
(=PtN.sub.3--Cl), wherein Boc is a marker ((en)=ethylenediamine,
Boc=tert-butoxycarbonyl). The reaction process was monitored using
high-resolution NMR (Bruker DPX-300) visualising .sup.1H and
.sup.195Pt nuclei. The results are shown in FIGS. 1 and 2. The data
showed almost completion of the reaction for the S-reactive sites
(methionine, FIG. 1) within 120 mM, demonstrated by change in
signal from PtN.sub.3--Cl to Pt N.sub.3--S-adduct whereas the
reaction between the N-reactive sites and the platinum compound
proceeded slow (FIG. 2). After 24 hours only a quarter of the
histidine molecules had been labelled.
Example 2
[0069] Bovine serum albumin (BSA) was dissolved in 0.5.times.PBS
(phosphate buffered saline, pH=7.4) to a 1 mg/ml solution. To 1 ml
sample of the BSA solution, 0.5 mg Rhodamine cis-Pt
(cis[Pt(II)(en)(NH.sub.2--(CH.sub.2).sub.6--NH-rhodamine)Cl](NO.sub.3))
was added. To another 1 ml sample of BSA solution, 0.5 mg Rhodamine
trans-Pt
(trans[Pt(II)(NH.sub.3).sub.2(NH.sub.2--(CH.sub.2).sub.6--NH-rho-
damine)-Cl](NO.sub.3)) was added. Both samples were allowed to
react for 16 hrs at 37.degree. C. Thereafter unbound fluorophores
(unbound Rhodamine and unbound Rhodamine-Pt compound) were removed
by gel filtration (10 ml Sephadex G50 column, 10 cm length, 1 cm
diameter) using 1.times.PBS as an eluent. Next, the ratios of bound
fluorophore per protein (F/P ratio) were determined using the
following formula:
F / P ratio = 112.4 .times. A 521 95.0 .times. [ BSA ]
##EQU00001##
[0070] wherein A521 (absorbance at 521 nm) was determined using a
Ultrospec 4000 spectrophotometer (APB), and [BSA] (protein
concentration in .mu.g/.mu.l was determined with BCA reagent (BCA
protein assay kit nr. 23225, Pierce)
[0071] Platinum compound to protein ratios (Pt/P ratio) was
determined using the following formula:
Pt / P ratio = 68 , 000 .times. [ Pt ] 195.0 .times. [ BSA ]
##EQU00002##
wherein [Pt] (platinum concentration in .mu.g/1 was determined by
atomic absorption spectroscopy. Briefly, the extend of
platinum-protein binding was determined by a Perkin Elmer Atomic
Absorption Spectrometer 3100 set to a slitband of 0.70 nm to
monitor the Pt line at 265.9 nm. The linear range for
quantification was 100-1500 ng/mL. Deuterium background correction
was used throughout analysis and the sample volume was between
0.020-0.060 mL. Furnace parameters were: drying 120.degree. C./90
sec., ashing 1300.degree. C./60 sec., flushing 20.degree. C./15
sec. and atomization at 2650.degree. C./5 sec. Argon gas was used
to purge the furnace.
[0072] The results were as follows:
TABLE-US-00002 Platinum compound F/P ratio Pt/P ratio Cis 4.1 4.0
Trans 0.9 3.6
[0073] BSA is rich in methionine and cystein residues (S-reactive
sites), at the above conditions reaction to N-reactive sites is
slow. The Pt/P ratio shows that both the cis and the trans-Platinum
compound successfully react with the protein. The F/P ratio shows
however that under the conditions of this experiment only the
marker (rhodamine) is released from the trans-platinum compound,
while the cis-platinum compound remains bound to the protein . This
illustrates that a trans-platinum compound may be used to shield a
reactive site from attachment of a marker to the trans-platinum
bound reactive site.
Example 3
[0074] Bovine serum albumin (BSA, Sigma; A-9647), Avidin-D (Vector;
A-2000) and Goat IgG anti-mouse IgG (total IgG fraction; Dept.
Nephrology, Leiden University Medical Centre) were used to be
labelled with biotin-Pt
(cis[Pt(II)(en)(NH.sub.2--(CH.sub.2).sub.2--CO--(CH.sub.2).sub.2--CO--(CH-
.sub.2).sub.2--NH-biotin)CL](NO.sub.3)) (KREATECH, ULK001), DNP-Pt
(cis[Pt(II)(er)(NH.sub.2--(CH.sub.2).sub.6--NH-DNP)CL](NO.sub.3))
(KREATECH, ULK003), Rhodamine-Pt
(cis[Pt(II)(en)(NH.sub.2--(CH.sub.2).sub.6--NH-rhodamine)CL](NO.sub.3))
(KREATECH, ULK101) and dGreen-Pt
(cis[Pt(II)(en)(NH.sub.2--(CH.sub.2).sub.6--NH-dGreen)CL](NO.sub.3))
(KREATECH, ULK301).
[0075] For each labelling of BSA and IgG, 250 .mu.g protein in 250
.mu.l PBS was mixed with 250 .mu.l water containing 125 .mu.g
labelling reagent (protein to label ratio=1:0.5). When needed the
volume was adjusted to 0.5 ml with distilled water. The reaction
was allowed to proceed for 16 hrs at 37.degree. C. Unbound
labelling reagents were removed by gel filtration (SephadexG25,
PD10; APB) with TBS/0.05% Tween 20 as eluent. DNP-Pt labelling of
avidin-D was chosen to optimise labelling of proteins with none or
non-accessible cysteine and methionine amino acids. Avidin-D was
labelled at different protein:label ratios and at fixed ratios in
75 mM and 500 mM Na-phosphate-, TrisHCl- or Na-carbonate buffers
with pH varying from 7 to 10. Protein concentrations during
labelling remained 0.5 mg/ml, whereas label-Pt reagent
concentrations varied between 0.25 to 0.75 mg/ml.
[0076] Fluorochrome to protein ratios (F/P ratio) as well as DNP to
protein ratios (D/P ratio) were calculated by measuring the
absorption at the fluorochrome absorption maximum (DNP: 363 nm,
dGreen: 507 nm and rhodamine: 521 nm). A correction factor is
introduced which adjusts the measurement for cis-platinum
contributions at a particular wavelength and protein concentrations
are determined using BCA reagent (Pierce; 23225). Calculating
protein concentrations at 280 nm is disrupted by A280 nm
contributions of the Pt reagent and can not be used. F/P-ratio
formulas were then extracted using UV/VIS spectroscopy and Platinum
flameless atomic absorbance spectroscopy (Pt--FAAS). Pt--FAAS was
used to determine the number of protein-bound platinum compounds,
which provided an accurate measurement of bound fluorochromes or
DNP-molecules. The formulas used to calculate F/P and D/P-ratios
are listed in Table 2.
TABLE-US-00003 TABLE 2 Formulas used to calculate fluorochrome to
protein and DNP to protein ratios BSA-DNP 3.78 .times. A363 IgG-DNP
11.67 .times. A363 Av-DNP 5.5 .times. A363 [BSA] [IgG] [Avidin]
BSA-Rhod 1.29 .times. A521 IgG-Rhod 3.63 .times. A521 Av-Rhod 1.95
.times. A521 [BSA] [IgG] [Avidin] BSA-dGreen 1.66 .times. A507
IgG-dGreen 3.85 .times. A507 Av-dGreen 2.37 .times. A507 [BSA]
[IgG] [Avidin]
[0077] Table 3 shows that BSA and IgG contain more platinum bound
fluorochromes compared to avidin-D. In case of Rhodamine-Pt: BSA
contains 1 fluorochrome/16.6 kD, IgG has 1 fluorochrome/19.5 kD and
avidin 1 fluorochrome/82.5 kD. Furthermore, DNP-Pt and Rhodamine-Pt
have comparable reactivity and both are more reactive than
dGreen-Pt.
TABLE-US-00004 TABLE 3 F/P- and D/P-ratios obtained from labelling
experiments protein:label F/P ratio ratio labeling or protein label
(.mu.g:.mu.g) buffer D/P ratio BSA Rhodamine 1:0.5 0.25 .times. PBS
pH 7.4 4.0 0.5 .times. PBS 4.1 .sup. 1 .times. PBS 3.6 DNP 1:0.5
0.5 .times. PBS 6.1 dGreen 1:0.5 0.5 .times. PBS 2.4 Goat IgG DNP
1:0.5 0.5 .times. PBS 8.4 Rhodamine 1:0.5 0.5 .times. PBS 7.7
dGreen 1:0.5 0.5 .times. PBS 3.9 Avidin-D DNP 1:0.5 0.5 .times. PBS
1.6 Rhodamine 1:0.5 0.5 .times. PBS 0.8 dGreen 1:0.5 0.5 .times.
PBS 0.3 Avidin-D DNP 1:0.5 TrisHCl 500 mM: pH 7 0.2 75 mM; pH 7 1.6
75 mM; pH 8 1.4 75 mM; pH 9 1.6 Avidin-D DNP 1:0.5 Na carbonate 500
mM: pH 8 0.8 500 mM; pH 9 1.2 500 mM; pH 10 1.9 75 mM; pH 8 1.6 75
mM; pH 9 1.5 75 mM; pH 10 2.0 1:1.0 75 mM; pH 10 2.4 1:1.25 75 mM;
pH 10 2.9
[0078] Experiments performed to increase D/P-ratios for avidin
labelling are also listed in Table 3. It is shown that increase in
pH of the labelling solution from pH 7 to pH 10 hardly increases
the D/P-ratio at low salt conditions. A significant increase is
found when the same experiment is performed at high salt
conditions, however, a maximum D/P-ratio of 2 was found that could
not be raised by varying salt or pH conditions. Increase of the
label-Pt concentration during labelling was found to increase
D/P-ratios further.
Example 4
[0079] Normal goat serum and serum of a goat immunised with mouse
IgG, were labelled with DNP-Pt
(cis[Pt(II)(en)(NH.sub.2--(CH.sub.2).sub.6--NH-DNP)CL](NO.sub.3))
at a total protein to DNP-Pt ratio of 2:1 (w/w) for 16 hrs at
37.degree. C. Mouse IgG was immobilised on a micro titre plate in a
dilution series of coating concentrations of 0, 0.1, 0.3, 1, 3, 10,
30, 100, 300 and 1000 ng/ml per well. After this coating step the
plates were rinsed with PBS-0.05% Tween 20 for three successive
times and finally post-coated with 125 .mu.l PBS/2% casein/3% BSA
for 30 minutes at 37.degree. C.
[0080] Next serum was diluted in maleic acid buffer (Roche
Diagnostics) to a solution with a protein concentration of 0.5
ng/.mu.l. Next 100 .mu.l of labelled serum was added to the
immobilised mouse IgG and was allowed to react for 60 min at
37.degree. C. The micro titre plate was washed with
1.times.PBS-0.05% Tween 20 followed by an 1 hour incubation at
37.degree. C. with an HRP labelled anti-DNP antibody (#NEN 7-1-99)
diluted in maleic buffer. Unbound anti DNP-HRP was removed by 3
washes with 1.times.PBS-0.05% Tween 20, 1 min. each. Next, 100
.mu.l TMB substrate, diluted in a citrate-phosphate buffer pH 5.3,
was added to the wells and allowed to react in the dark for 30 min
at room temperature (20-22.degree. C.). To stop the reaction 100
.mu.l of 1N H.sub.2SO.sub.4 was added. Absorption at 450 nm was
determined as a measure for the anti Mouse IgG--labelled according
to the invention--bound to Mouse IgG. The results are shown in FIG.
3. In contrast to the non-immunised goat serum the experiment with
the immunised goat serum showed a signal of bound anti DNP,
indicating that anti-mouse IgG has specifically bound to mouse
IgG.
[0081] This experiment was repeated with biotin as the marker
instead of DNP and anti-biotin instead of anti-DNP. Similar results
were observed.
Example 5
[0082] Micro titre plates (MB, 762070, Griener) were coated with
either Rabbit anti-humane IgG (DAKO, A0424), Rabbit anti-humane IgA
(DAKO, A0092), Rabbit anti-humane IgM (DAKO, A0426), Rabbit
anti-humane IgD (DAKO, A0093), or Rabbit anti-humane IgE (DAKO,
A0094). Each antibody was dissolved in 1.times.PBS at a
concentration of 10 .mu.g/ml. The micro titre plates were coated
with 100 .mu.l overnight at room temperature. Next, the plates were
rinsed with rinsing buffer (0.15 M NaCl, 4.9 mM
Na.sub.2HPO.sub.4.2H.sub.2O, 1.2 mM KH.sub.2PO.sub.4, 0.05% Tween
80, 0.005% thimerasol) and post coated with 150 .mu.l 1.times.PBS,
2% casein, 3% BSA (30 min at 37.degree. C.). Untreated whole human
serum, at various dilution rates ranging from 1:250 up to
1:9.10.sup.5 (in serum dilution buffer: 0.1 M Tris pH 7, 0.15 M
NaCl, 1% BSA, 2% casein, 0.05% Tween 80, 0.025% thimerasol), was
added (100 .mu.l) to the anti-humane IgG and anti-humane IgA coated
plates and incubated for one hour at 37.degree. C. The wells were
rinsed thoroughly and the detection limit established by using
anti-humane IgG-HRP (DAKO, P-214/stock solution: 1:20 dilution in
Stabilzyme Select (Surmodics), finally 1:100 diluted in serum
dilution buffer) and anti-humane IgA-HRP (DAKO, P-216/1:35 dilution
in Stabilzyme Select (Surmodics), finally 1:100 diluted in serum
dilution buffer) conjugates and TMB substrate according to standard
procedures.
[0083] The same untreated whole humane serum sample was labelled by
adding DNP-Pt
(cis[Pt(II)(en)(NH.sub.2--(CH.sub.2).sub.6--NH-DNP)CL](NO.sub.3))
in a total protein to DNP-Pt ratio of 4:1 (w/w) and allowing the
mixture to react overnight at room temperature. Next, the sample
was diluted, added to the plates (100 .mu.l/well), and incubated as
above. Detection limit was determined by using anti-DNP-HRP
conjugate (#NEN 7-1-99, 1:1000 dilution in serum dilution buffer;
100 .mu.l/well; 1 hour at 37.degree. C.) and TMB substrate (30 min.
at room temperature).
[0084] The results were as follows:
TABLE-US-00005 Entity Classical sandwich ELISA DNP-Pt format IgG
1:3.10.sup.5 1:2.10.sup.5 IgA 1:8.10.sup.4 1:4.10.sup.4 IgM n.a.
1:2.10.sup.4 IgD n.a. 1:2.10.sup.3 IgE n.a. 1:2.10.sup.3
[0085] All subclasses were shown to maintain their antigen binding
capacity.
Example 6
[0086] The effect of ammonium sulphate was evaluated. First
proteins were precipitated with either 50, 100, 200 or 400 .mu.l of
a saturated (NH.sub.4).sub.2SO.sub.4 solution (30 min on ice-30 min
room temperature-centrifugation). The supernatant was separated
from the precipitate. The precipitates were dissolved to a 0.5
mg/ml concentration in 0.5.times.PBS (without dialysis). The
protein concentration was determined with BCA reagens (Pierce, see
above). Next, the re-dissolved precipitate was labelled with DNP-Pt
(cis[Pt(II)(en)(NH.sub.2--(CH.sub.2).sub.6--NH-DNP)CL](NO.sub.3))
at a 4:1 ratio (w/w) for 4 hrs at 50.degree. C. The results are
shown in FIG. 4.
[0087] Also, the supernatants, transferred to new tubes, were
labelled with DNP-Pt. To 0.5 mg protein (in the supernatant) 0.125
of DNP-Pt
(cis[Pt(II)(en)(NH.sub.2--(CH.sub.2).sub.6--NH-DNP)CL](NO.sub.3))
was added. The mixture was allowed to react for 4 hrs at 50.degree.
C. The results are shown in FIG. 5.
[0088] The results demonstrate that a method according to the
invention can be used to label either a entity that has been
precipitated in ammonium sulphate or an entity that is dissolved in
an ammonium sulphate solution without need for remove any excess
ammonium sulphate. The latter is not possible with standard
labelling moieties, e.g. HNS-esters.
Example 7
[0089] In this example differential labelling is demonstrated by
making use of fluorescence resonance energy transfer (FRET). The
bio-organic molecule of choice is microperoxidase. Microperoxidase
mp-11 (Sigma M6765) consists of 11 amino acids with two N reactive
sites (lysine and histidine) and two S-reactive sites (cysteine).
The full length sequence of mp-11 is: valine-glutamine-lysine
(N)-cysteine (S)-alanine-glutamine-cysteine (S)-histidine
(N)-threonine-valine-glutamine. Mp-11 was dissolved in
0.5.times.PBS (pH 7.2) at a concentration of 1 mg/ml. A aliquot of
this solution (0.25 mg) was labelled with Flu-ULS at a 1:0.25 ratio
in 0.5.times.PBS (final volume 499.5 .mu.l) at 50.degree. C. for 4
hours. The fluorescein labelled mp-11 solution was purified over a
PD-10 column (APB, nr. 17-0851-01). Prior to the purification of
the solution the column was rinsed three times with 5 ml
0.5.times.PBS. The fluorescein labelled mp-11 solution was analysed
on a Ultrospec 4000 spectrophotometer (APB) Subsequent, fluorescein
labelled mp-11 was labelled with rhodamine-ULS (ratio 1:0.25).
Labelling was allowed to take place overnight at 4.degree. C. Next,
the solution was purified and analysed as described above.
[0090] The results are presented in FIG. 6. The data show that
mp-11 is labelled with fluorescein (A470 FAM 50) and rhodamine
(A510 FAM 50 Rho 4). An elevated rhodamine specific emission was
obvious when the double labelled mp-11 was illuminated at 470 nm
(this is the excitation wavelength of fluorescein) (A470 FAM 50 Rho
4). After excitation fluorescein transfers sufficient energy to the
nearby rhodamine leading to fluorescence of rhodamine at 570 nm
without direct excitation of rhodamine at 510 nm, this is FRET.
Example 8
[0091] Bovine serum albumin (BSA) was labelled with cis or trans
rhodamine-Pt at pH 4 or 7. BSA was dissolved in 1.times.PBS
(phosphate buffered saline, pH=7.4) at an amount of 3%. Small
aliquots of this solution (3.3 .mu.l) were labelled according the
following scheme: (a) plus 25 .mu.l rhodamine cis-Pt
(cis[Pt(II)(en)(NH.sub.2--(CH.sub.2).sub.6--NH-rhodamine)CL](NO.sub.3))
of a 1 mg/ml stock solution in 0.075 M NaAC/citrate buffer pH 4
(final volume 1 ml); (b) plus 12.5 .mu.l Rhodamine trans-Pt
(trans[Pt(II)(NH.sub.3).sub.2(NH.sub.2--(CH.sub.2).sub.6--NH-rhodamine)CL-
](NO.sub.3)) of a 2 mg/ml stock solution in 0.075 M NaAC/citrate
buffer pH 4 (final volume 1 ml); (c) same as (a) but in
0.5.times.PBS pH 7; (d) same as (b) but in 0.5.times.PBS pH 7. In
all cases the protein to label ratio is 1:0.25. Labelling took
place at 50.degree. C. for 4 hours. Thereafter the labelled BSAs
were column purified. Visual evaluation of the samples clearly
showed no coloured solution in (b) and (d) whereas (a) and (c) were
coloured (c stronger then a).
Example 9
[0092] The effect of soft transition metals was evaluated in order
to control further the differential labelling conditions. A ten
fold excess of N-acetyl methionine (final conc. 2 mM) or N-acetyl
histidine (final conc. 2 mM) was added to a solution containing
either DNP-Pt (final conc. 0.2 mM) or Rho-Pt (final conc. 0.2 mM)
in 10 mM sodiumphosphate pH 8 and 20 mM NaCl. To each solution
either 0, 1, 2, or 5 equivalents of CdCl.sub.2 or K.sub.2PdCl.sub.4
was added to study the influence of the presence of soft transition
metals on reaction rates of marker-Pt compounds with S-reactive
sites and N-reactive sites containing amino acids, respectively.
The reactions were performed at 37.degree. C. It should be noted
that the pH dropped upon addition of the soft transition metal.
This was observed for all soft transition metals used in this
experiment. The drop in pH was the most pronounced for palladium
and the least for cadmium. The disappearance of label-Pt was chosen
as a measure and the samples lacking an additional soft transition
metal (e.g. Pd, Cd, . . . ) served as controls. The relative
changes measured in samples containing such additional soft
transition metal(s) is a measure for the effect on the presence and
concentration of such compound(s) on the labelling characteristics
of N or S-reactive site containing amino acids. The results are
presented in Table 4. The results show that labelling of methionine
is very fast. This finding is in agreement with data presented
above. Addition of considerable amounts of cadmium diminishes the
reaction rate only slightly. However addition of palladium
significantly inhibits the reaction in a concentration dependent
manner Labelling of histidine is quite slow, and decreases when
cadmium is added. A 5 fold excess of cadmium with respect to
histidine, prevented labelling to occur in the first place.
Palladium seems to speed up the reaction with histidine when
present at low concentration, at higher concentrations the reaction
is slowed down. However these changes in reaction rate might not
solely be due to the presence of a soft transition metal, or
mixtures thereof, but also in part be due to changes in pH.
Palladium has also an effect on labelling of both S-reactive site
(e.g. methionine) and N-reactive site (e.g. histidine) containing
amino acids, but more so on methionine. This offers an excellent
opportunity to selectively diminish sulfur labelling.
Example 10
[0093] Bovine serum albumin (BSA, Sigma; A-9647) was dissolved in
either 20 mM phosphate buffer pH 8 or 20 mM sodium acetate buffer
pH 4 at a concentration of 5 mg/ml. To aliquots of these solutions
was added Flu-Pt, Rho-Pt, Flu-NHS (Molecular Probes, C-6164,
dissolved in DMSO at a concentration of 10 mg/ml) or Rho-NHS
(Molecular Probes, C-6123, dissolved in DMSO at a concentration of
5 mg/ml) at a ten fold excess. The labelling reaction was allowed
to take place over night at 37.degree. C. All samples were purified
by column purification (PD10) and analysed spectrophotometrically
according to standard procedures. The results showed hardly any
labelling at low pH for the Flu-NHS label whereas the Flu-Pt label
displayed a significant higher F/P ratio. Note that the baseline
Flu-NHS value is mainly attributable to non specific binding of the
label (negative charge) to the protein (positive charge). Both
labels yielded comparable F/P ratios at neutral pH matching the
Flu-Pt value at low pH. Similar results were obtained with
rhodamine with the exception that the Rho-Pt value was lower
compared to the Rho-NHS value at low pH. In this case the data
corresponding to the low pH experiment are actual baseline values
representing no or very little labelling. This finding might at
least in part be explained as a result of the overall net charge of
the labelling compound in view of the charge of the protein.
[0094] This example demonstrates the successful use of different
labelling technologies and potential electrostatic interactions
contributing to the scope of the present invention.
Example 11
[0095] Epidermal Growth Factor (EGF, Sigma; E9644) was dissolved in
50 mM phosphate buffer pH 8 at a concentration of 1 mg/ml. Ten fold
excess of Flu-NHS (Molecular Probes, C6164; dissolved in DMSO at a
concentration of 10 mg/ml) or Flu-Pt (KREATECH, ULK004) was added
to aliquots of the EGF solution. The labelling reaction was allowed
to take place overnight at 30.degree. C. and 37.degree. C. for the
Flu-NHS and Flu-Pt markers, respectively. Next, the samples were
purified by column purification (PD10) and analysed
spectrophotometrically according to standard procedures. The
results showed a F/P ratio of 0.07 and 0.28 for the Flu-NHS and
Flu-Pt markers, respectively. EGF does not contain lysine and
therefor is not a preferred target for NHS labelling. The terminal
amino group serves as the only potential labelling site for a NHS
complex. A significant higher F/P ratio was achieved for the Flu-Pt
complex under similar conditions.
TABLE-US-00006 TABLE 4 t1/2 values from labelling reactions
containing soft transition metals DNP-Pt Rho-Pt Methionine No soft
transition metal 15 min 15 min CdCl.sub.2 1 equivalent 60 min (pH
7) 60 min (pH 7) 2 60 min (pH 6) 60 min (pH 6) 5 60 min (pH 4.5) 60
min (pH 4.5) K.sub.2PdCl.sub.4 1 equivalent .apprxeq.100 hours (pH
7) 2 .infin. (pH 5) 5 .infin. (pH 3) Histidine No soft transition
metal 3 hours 3 hours CdCl.sub.2 1 equivalent 15 hours (pH 7) 15
hours (pH 7) 2 50 hours (pH 6) 25 hours (pH 6) 5 .infin. (pH 4.5)
.infin. (pH 4.5) K.sub.2PdCl.sub.4 1 equivalent 2 hour (pH 7) 2
.infin. (pH 6) 5 .infin. (pH 3)
* * * * *