Note: Descriptions are shown in the official language in which they were submitted.
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DETECTION OF ANTIBIOTICS
This application is a division of application
2,107,856, filed October 6, 1993.
The present process relates to the detection of
antibiotics in a liquid medium such as milk, urine and
blood.
In US-A-4,239,852 and US-A-4,239,745 a process of
detecting the presence of an antibiotic in a liquid sample
is disclosed in which the sample is incubated with cell
parts of a microorganism. Any antibiotic molecules in the
liquid sample bind to the receptor sites of the cell parts.
After this incubatiort step a tagged antibiotic is added
io which is then allowed to bind to the remaining receptor
sites. After the liquid has been separated from the cell
parts and washed, the amount of tagged antibiotic bound to
receptor sites can be determined. In the examples of these
patents, radioactively tagged antibiotics are used. Also the
commercially available tests, developed on the disclosure of
the patents, are available which utilize the competition
between radioactive labelled antibiotic and sample
antbiotic. Although this technique is very sensitive, it
requires trained personnel and expensive equipment. Another
2o disadvantage is the usual radiation hazard. Therefore there
has not been available a fast, acceptable test for
antibiotics which can be used routinely, particularly by
farm and dairy plant workers.
In the above US patents, a test based on the
competition of an enzyme tagged antibiotic and a sample
antibiotic is also mentioned. In example 2 of US-A-4,239,852
a test kit is described which is able to detect 0.05 units
of penicillin per milliliter (= 30 ppb) within a minimum of
20 minutes. The detection of smaller concentrations of
penicillin requires longer test times.
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Since the concentrations_of antibiotics present in
milk are generally very low, time constraint mean that a
detection method based on enzyme tagged antibiotics is not
convenient for testing milk samples daily.
It is therefore an object of the present invention
to provide a simple test designed to detect low levels of
antibiotics in liquid media such as milk, urine or blood.
The time required for the tests should ideally not exceed
about 15 minutes. The present invention provides a process
io for the detection of at least one antibiotic in a liquid
medium such as milk,.urine and blood which comprises
(a) bringing together a fluid sample of the liquid
medium, at least one-labelled antibiotic binding protein,
and at least one immobilized antibiotic.
(b) allowing the labelled antibiotic binding protein
to bind with the immobilized antibiotic,
(c) removing labelled antibiotic binding protein
which is not bound to immobilized antibiotic, and
(d) determining the amount of the labelled
2o antibiotic binding.protein bound to the immobilized
antibiotic.
The-process may be used to detect a wide variety of
antibiotics such as Q-lactams (including penicillins such as
benzylpenicillin, and cephalosporins), tetracyclines,
gentamycin, sulpha compounds such as sulpha methazine, and
combinations thereof. The immobilized antibiotic is
generally the same as that present in the liquid sample, but
may be different. For example the immobilized antibiotic may
be an analogue of the antibiotic in the sample. If the
immobilized antibiotic and the antibiotic are different they
should both be able to bind with the binding protein.
The label used in the present invention may be an
enzyme or a fluorescent compound such as FITC (fluorescein
isothiocyanate) or TRITC (tetramethyl rhodamine isothio-
cyanate). Preferably an enzyme-labelled antibiotic protein
is used in step (a).
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According to one embodiment of the invention an
assay can be used to detect a combination of different
antibiotics within one test. In such a test different
labelled antibiotic binding proteins in combination with the
several antibiotics immobilised are present.
Surprisingly it has been found that by labelling the
antibiotic binding protein with an enzyme, and immobilizing
the antibiotic of interest on a solid phase such as a test
tube or dipstick, a test kit is obtained which is sensitive
lo to at least 5 ppb benzylpenicillin (1 mg corresponds to
1592 i.u. benzyl penicillin-K-salt). Moreover the time
required for such a test does not exceed about 12 minutes.
The test is inexpensive and easy to perform and does not
require trained personnel. Smaller concentrations may be
detected if the incubation time is extended.
According to a preferred embodiment of the invention
step (a) comprises bringing the labelled antibiotic binding
protein into contact with the liquid sample, allowing the
antibiotic in the sample to bind to the labelled antibiotic
2o binding protein, aric3 subsequently adding immobilized
antibiotic. The binding protein and liquid sample are
generally incubated for 1 to 4 minutes, prior to the
addition of the immobilized antibiotic.
The labelled antibiotic binding protein comprises
any antibiotic binding protein for example those which may
be obtained from an antibiotic-sensitive microorganism, such
as a Bacillus stearothermophilus, Bacillus subtilis,
Streptococcus thermophilus or Escherichia coli, preferably
Bacillus stearothermophilus microorganism is used. Also
3o antibiotic binding proteins such as antibodies are embodied
in the present invention. Suitable antibodies can be
obtained by immunisation of animals, see for example E.H.
Kachab et al, The Journal of Immunological Methods, vol.
147, no. 1, January 1, 1992, page 33-41. The antibiotic-
binding protein may be purified by techniques as affinity
chromatography or gel filtration.
.` . _ . _~ . . . = -
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The labelled antibiotic binding protein has a
reactive site for binding to the antibiotics of the sample
as well as to the immobilized antibiotics. The antibiotic
binding protein is linked to the label. All methods
available that are known to generate a protein/protein
interaction could be suitably used to obtain the above
mentioned complex. For instance, linkage could be realized
by means of bifunctional reagents. Besides a covalent
interaction, binding between different proteins could also
lo be based on local charge differences on adjacent surfaces
(Van der Waals forces) and/or hydrophobic binding (G.E.
Davies and G.R. Stark (1970): Use of dimethyl suberimidate,
a cross-linking reagent, in studying the subunit structure
of oligomeric proteins. Proc. Natl. Acad. Sci. USA, 66: 651-
656; F. Wold (1972): Bifunctional reagents: Methods Enzytnol.
25: 623-651; J.R. Knowles (1972): Photogenerated labels for
biological receptor-site labelling: Acc. Chem. Res. 5: 155-
160; K. Peters and F.M. Richards (1977): Chemical cross-
linking: reagents and problems in studies of membrane
structure: Ann. Rev. Biochem. 46: 523-551; W.S. Jacoby and
M. Wilchek (eds.): Affinity labelling: Methods Enzymol. 46;
M. Das and C.F. Fox (1979) Chemical cross-linking in
biology: Ann. Rev. Biophys. Bioeng. 8: 165-193; M.R.
Bosshard (1979): Mapping of contact areas in protein-nucleic
acid and protein-protein complexes by differential chemical
modification: Methods Biochem. Anal. 25: 273-301; Bayer and
Wilchek (1978): The avidin-biotin complex as a tool in
molecular biology: Trends biochem. Sci. 3: N257-259; Bayer
et'al (1979): Meth. Enzymol. 62: 319-326. Furthermore
3o application of molecular biology could also be possible. By
this means, new proteins (fusion proteins) that are based on
the genetic information of both the antibiotic binding
protein and the enzyme label could be created.
A preferred enzyme label is horse-radish peroxidase,
3s which is known for its stability but also other enzymes can
be used. For instance peroxidase, alkaline phosphatase or
/3-galactosidase in general (all enzymes that are useful in
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an Enzyme-Linked Immunosorbent Assay, ELISA) (J.W. Goding
(1983): Monoclonal antibodies: principles and practice (ISBN
0-12-287020-4). The means of detecting the enzyme will
depend on the specific enzyme used. Typically the enzyme
label may be detected when the enzyme acts as a catalyst,
for example to catalyse a reaction giving rise to a colour
change, or when the enzyme inhibits a reaction. Generally,
when the presence of the enzyme is detected by means of a
colour change, a suitable substrate is added, upon which the
io enzyme acts. The degree of colour change is then related to
the amount of enzyme present. A suitable substrate for a
horse-radish peroxidase is for example a chromogenic colour
substrate which is easily oxidized by the formation of
oxyqen such as tetramethylbenzidine, o-phenylenediamine or
azinodiethylbenzthiazoline.
Immobilization of the antibiotic of interest may be
carried out in a manner known per se, for example by
covalent or non-covalent adsorption (P. Tijsen, Practice and
Theory of Enzyme Immunoassays, Elsevier, 1985) to a solid
matrix (e.g. plate, tube, dipstick or beads (Fe, latex,
etc.)). An antibiotic having a lactam-ring can be covalently
conjugated to a carrier, optionally via a spacer. All
methods available to construct chemical bonds could be
suitably used, unless they are detrimental to the
antibiotic.
It will be obvious that many coupling techniques can
be applied, for instance those known from peptide chemistry
and that many bifunctional compounds are suitable as a
spacer.
Suitable procedures for instance are the methods
described by H.R. Yocum et al. (J. Biol. Chem. (1980), 255,
3977-3986), in which spacers of the general form X(CH2),-COOH
- are used.
A very efficient and reliable method for inter-
molecular conjugation is described by J. Carlsson et al.
(1978) in Biochem. J. 173, 723-737), in which the heterobi-
_ti
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functional reagent N-succinimidyl-3-(2-pyridylthio)-propionate (SPDP) is used.
Materials such as glass or plastics can be used as matrix material.
NH2 groups of the matrix can be used to obtain immobilization. Covalent
coupling
between materials such as plastics (e.g. polystyrene) and a protein (for
example
BSA) can also be used to immobilize the antibiotic of interest, see e.g.
R.H. Burdon and P.H. van Knippenberg, Laboratory Techniques in Biochemistry
and Molecular Biology, Elsevier, 1985.
According to a preferred embodiment, the antibiotic is immobilized
onto the interior surface of a container such as a test tube, preferably via a
linking
1 o compound such as BSA.
Hence, in another aspect, the invention relates to an antibiotic-
Bovine Serum Albumin conjugate selected from the group consisting of 7 amino-
cephalosporanic acid-spacer-Bovine Serum Albumin and N-pentadecakis [N-(4-
carbonyl-3-methylceph-3-em-7-yl)amino-carbonylethyldithioethylcarbonyl]Bovine
Serum Albumin.
The present invention also provides a kit for carrying out the
detection, which comprises at least one enzyme-labelled antibiotic binding
protein
and at least one immobilized antibiotic.
In the following examples preferred embodiments are described to
illustrate the invention. However, it is to be understood that the invention
is not
limited to the specific embodiments and that a person skilled in the art who
is
familiar with the methods may use other tests which can be equally used for
the
purpose of the present invention. These alterations are included in the scope
of
the invention.
Example 1
Antibiotic residue test.
In this example will be described a method for detecting
benzylpenicillin residues as low as 5 ppb in milk.
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Extraction of antibiotic bindincr orotein
A grown culture of an antibiotic sensitive
microorganism in this example Bacillus stearothermophilus
s(continuous culture art. # 108 Porton Products Ltd, UK) was
lysed overnight at 4 C with lysozyme, Mose and tritooa X-100TM
in 0.1 M phosphate pH 7.Ø The lysate was centrifuged for
30 minutes at approximately 1600 x g(4'C). After centrifug-
ation the supernatant was mixed with an antibiotic affinity
tio gel matrix, for example to prepare a 7-aminocephalosporanic
acid (7ACA) affinity gel matrix the following method was
used.
0.34 g of 7ACA was mixed with 25 ml 0.1 M phosphate
pH 7.0 (pH corrected to 7). To this solution was added 100
1s ml beads affigel 10' (BioRad) (washed with 1 1 0.1 M
phosphate pH 7.0). This was mixed gently for 2 hours at
200C. The 7ACA-affigel 10 was filtered and sucked off using
vacuum. The 7ACA-affigel was then washed again with 0.1 M
phosphate pH 7.0 and was ready for use.
20 The 7ACA-affigel and the supernatant of the lysed
culture was mixed for 3 hours at 20'C gently on a shaker.
The gel was washed six times with 0.1 M phosphate + 1 M NaCl
pH 7Ø For each wash was used 500 ml.
20 ml elution-buffer (0.05 M phosphate + 0.5 M NaCl
25 + 0.1% triton X-100 + 0.8 M hydroxylamine pH 7.0) was added
to the moist gel cake and mixed for 20 minutes at 20'C
gently on a shaker. The mixture was then centrifuged at 4'C,
6 minutes at approximately 300 x g. The supernatant was
dialysed in 32 mm tubing (12-14 kD cut-off).
00 The first dialysis was against 0.05 M phosphate +
0.5 M NaCl pH 7.0 overnight at 4'C, the second up to the
fifth dialysis was against 0.1 M carbonate pH 9.4 with a
change of buffer every 4-6 hours.
The lysate was centrifugated 20 minutes at approximately
35 1000 x g at 4 C and concentrated in an AMICONTM concentrator
(ultra filtration) (model f 8200, WR Grace and Co.)
according to the manufacturer's standard operation procedure
. ` .. `
` . .. '` . .
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(SOP). The purified antibiotic binding protein is now ready
for conjugation.
Conjugation of antibiotic binding protein (abp) with an
enzyme label
In this example horse-radish peroxidase (HRPO) is
used. 1 mg HRPO (suitable for labelling) in 1 ml of
lo distilled water (d.i.-water) and 0.2 ml 0.1 M Na-periodate
was mixed 20 minutes at 20 C and dialysed overnight at 4 C
against 0.001 M Na-acetate pH 4.4. This dialysate was
adjusted to pH 9.0-9.6 by adding 25 l of 0.1 M carbonate.
Directly hereafter 1 mg abp (Pierce protein assay) was added
1s into the dialysate. The mixture was gently shaken for 2
hours at room temperature. Thereafter 150 l of 4 mg Na-
borohydride/ml d.i.-water was added to the mixture, this was
incubated for 2 hours at 4 C.
The solution was dialysed against PBS (0.01 M
20 phosphate + 0.9% (m/v) NaCl pH 7.0) with four buffer changes
every 4 hours.
After the dialysis was completed, the dialysate was
diluted in 10% goat sera (inactivated) + 0.03% 4-aminoanti-
pyrine. This is named antibiotic binding protein-enzyme
25 (HRPO) conjugate. The highest dilution which gave a
fast colour development with the colour-substrate is used in
the test-format. Preserving the diluted conjugate with
thiomersal gives a highly stable test-kit reagent which can
be stored for at least 6 months at 4 C.
ConZugation of a Q-lactam to a protein
In this example the basis structure of the
cephalosporins (7 amino-cephalosporanic acid (7ACA)) is used
for conjugation to Bovine Serum Albumine (BSA). A spacer
between the 7ACA and the BSA is used to obtain the best
affinity and specificity,for P-lactams.
=~. ~~
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40 mg of 7ACA was added to 4 ml of 50mM Hepes
(pH 7.5) solution. After dissolving the pH was adjusted to
pH 7.0 with iN NaOH. Hereafter 20 mg BSA and 40 mg
Bis(sulfosuccinicmidyl)suberate (spacer) and an extra 2 ml
of 50mM Hepes solution was added. The mixture was gently
shaken for 45 minutes at 20 C.
After mixing the solution was dialysed (tubing cut-
.off 12-14 kD) for 48 hours against PBS with three buffer
changes. This dialysate is used for tube coating after
1o dilution.
Coating of p-lactam-spacer-protein conjugate to a solid-
phase
Coating of this conjugate to a solid-phase makes it
possible to use a convenient separation between bound and
unbound abp-conjugate. In this example the following method
was used:
0.125 ml dialysate (7ACA-spacer-BSA conjugate) was
added to 500 ml carbonate pH 9.6. 0.5 ml of this solution
was added to the polystyrene star tubes (NUNC MAXISORB ).
Tubes were covered and incubated overnight at 4 C. After
incubation the /3-lactam spacer protein conjugate dilution
was removed from the tubes and 2 ml 0.05 M phosphate + 0.5%
BSA + 2% sucrose + 0.1 M glycine pH 7.2 was added to each
tube. After 1 hour at 20 C the tubes were emptied and dried
for 48 hours at 22-27 C with less than 30% humidity. Dried
tubes are stable for at least 1 year at 4 C.
Wash-solution
A convenient separation method is washing the solid-
phase (tubes) with a solution as described below.
Wash solution for separation of conjugate bound to
antibiotics coated on solid-phase (tubes) from conjugate
bound to 'sample'-antibiotics is prepared as follows:
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Mono.basic sodium phosphate 11.7 gram/1
Dibasic sodium phosphate 21.6 gram/1
Benzalkonium chloride 3.57 gram/1
Glycerol 500 ml/1
Tween 20TM 12.5 ml/1
pH adjusted to 6.5.
This solution was made 50 x concentrated for
stability (1 year) and convenient transportation of the
final test kit. For the test a 50 x dilution in distilled
,o water (or tap water) is used. Also other low salt and
surfactant containing solutions may be possible.
Substrate
~s For the substrate of HRPO it is possible to use a
chromogenic colour substrate which is easily oxidized by the
formation of oxygen. In this example is used a commercial
product TM-blue from TSI (US-A-5,013,646) which has a good
stability (for at least 1 year at 4'C) and operation. The
20 colour-development can be measured by optical density at 650
nanometer wavelength or after acidification at 450 nano-
meter.
Stop solution
The use of a stop solution such as 1.5% NaF (or
strong acids) is highly stable and gives a better
quantitative approach in case a difference in colour
development is being measured at a time-base.
Test-performance (sequential assay)
0.2 ml of milk sample and 0,2 ml of a diluted abp-
enzyme conjugate was added to an empty reaction ampoule.
After incubation for 2 minutes at 64'C the contents of the
tube was transferred to the coated tube. After the second
incubation of 2 minutes at 64'C the contents of the coated
- . . =>
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tube was dumped into the sink and the tubes were washed
three times with 50 x diluted wash solution by filling the
tubes, dumping the contents into the sink and removing
residuals by tapping on absorbent paper. After the wash, 0.5
ml of TM-blue colour-substrate was added into the tube.
This incubation for 4 minutes at room temperature was
stopped by adding 0.5 ml of stop solution (1.5% NaF). The
colour development at 650 nm was compared with an antibiotic
standard tested along with the sample.
A preserved, freeze-dried antibiotic standard is
included in the test kit for convenience (stable for at
least 1 year).
With this method milk samples with antibiotic
residues as little as 5 ppb benzyl penicillin can easily be
i5 detected with a total incubation time of 8 minutes. Results
of the test are shown in Table I.
Table I
sample ppb Pen. G Reader units*
present in sample expressed in t
1 0 100
2 1.25 96
3 2 69
4 3 65
5 4 51
6 5 47
7 10 32
* Reader units: measured at 650 nanometer wavelength
This test according to this example is also sensitive for
other p-lactams see Table II for examples.
.. -.ti . = ;` . ';~ . =~ -
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Table II
p-lactam antibiotic sensitivity ppb
Amoxicillin 5
Ampicillin 10
Cephapirin 5
Ceftiofur 5
A competitive assay can be performed by combining the first
and second incubation step together. This test format will
be somewhat faster by elimination of the incubation time of
the second step. However the sensitivity as the sequential
assay will be smaller.
Example 2
Antibiotic residue test
In this example a method will be described for
detecting gentamycin residues as low as 30 ppb in milk.
The gentamycin assay is in general developed with
the same basics as the Q-lactam-assay. We will therefore
give in this example the same outline as the J3-lactam assay
of Example.1 with description of differences.
Obtaining of antibiotic binding arotein
A commercial anti-gentamycin-rabbit antibody is
obtained from.Biodesign International, Kennebunkport, Maine,
USA. The anti-gentamycin-rabbit antiserum is absorbed to
remove the bovine serum albumin (BSA) carrier antibodies. A
solution containing 10 mg/mi suifosuccinicmidylsuberate-BSA
and 2 mg of unreacted BSA is mixed with the raw antiserum in
the ratio of 10 to 1. This removes all detectable reactivity
with BSA.
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Conjugation of antibiotic binding protein with an enzyme
label
Absorbed anti-gentamycin antiserum is reacted with
horseradish peroxidase labelled Staphylococcus aureus
protein A to form an antibiotic binding protein enzyme
conjugate. An optimal ratio of antibody to protein A is
determined by forming the assay with checkerboard titrations
of the two components diluted in phosphate buffered saline
containing 1% BSA.
Coniuqation of gentamycin to a protein
In this example gentamycin is used for conjugation
to Bovine Serum Albumin (BSA). A spacer between gentamycin
and the BSA is used to obtain the best affinity and
specificity. The spacer is the same as used in Example 1.
Coating of gentamycin-spacer-protein conjugate to a solid
phase
As described in the p-lactam test, the gentamycin-
spacer-protein conjugate is coated to a solid phase.
Wash-solution, substrate, stop-solution
Wash-solution, substrate and stop-solution of
gentamycin test is the same as used for the Q-lactam test.
Test-performance (sequential assay)
The same test method as described in Example 1 for
the Q-lactam test can be used for the gentamycin test.
However, this test uses all incubations at a temperature of
so 20 C.
With this test method milk samples with antibiotic
residues as little as 30 ppb (can be lowered to 2.5 ppb
referred to Table III) can easily be detected with a total
incubation time of 8 minutes.
Results of the test are shown in Table III.
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Table III
Sample ppb gentamycine reader units*
present in sample expressed in %
1 0 100
2 2.5 55
3 5 39
4 10 31
5 20 23
6 30 20
7 36 18
*reader units: measured at 650 nanometer wavelength
Example 3
Preparation of N-pentadecakisfN-(4-carbonyl-3-methylceph-3-
is em-7-yl)aminocarbonylethyldithioethylcarbonyllbovine serum
albumin (1)
Sten A:
Preparation of 70-(2-Pyridyldithiopropionamido)-3-methyl-3-
cephem-4-carboxylic acid (2)
S.s
NHS
o
or"
0 OH
7p -Amino-3-methyl-3-cephem-4-carboxylic acid (34.28
mg; 0.16 mmol) was suspended in water (7 ml) and, with
. . , -.` .. . =, .. - , .-. . .. .,
= . _ . .
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stirring, pH was brought to 7.1 with 0.1 M phosphate buffer,
pH 8Ø Then, a solution of N-succinimidyl 3-(2-pyridyl-
dithio)propionate (100 mg; 0.32 mmol) in absolute ethanol
(10 ml) was added dropwise while keeping the temperature at
about 3 C. The reaction mixture was further stirred for
about 72 hours at 3"C, diluted with water (20 ml) and
extracted with ether to.remove the front moving components.
Thereafter, the water-layer was brought to pH 2.5 with 0.1N
hydrochloric acid and extracted with ethyl acetate. The
io combined ethyl acetate extracts were washed with water,
dried over anhydrous MgSO41 solvent removed under reduced
pressure and the product (2) dried under vacuum to a
constant weight. Yield = 28.4 mg.
IR Spectrum (KBr): 3294, 1778, 1712, 1686 cm-'.
'H NMR (360 MHz;. CDC13; S-value in ppm; TMS): 2.06 (3H, CH3);
2.68, 2.99, (2xm, 2x2H, (CH2)2]; 3.24, 3.53, (ABq, J=18.7 Hz,
C2H2); 5.02 (d, 1H, J=4.5 Hz, C6H); 5.70 (dd, 1H, J=4.5 Hz
and J=7.9 Hz, C7H); 7.21, 7.77, 7.87, 8.48 (m, 1H; m, 1H; d,
1H; dd, 1H; pyr.); 8.20 (d, 1H, J=7.9 Hz, NH).
Step B:
Introduction of 2-pyridyl disulphide group into bovine serum
albumin by N-succinimide 3-(2-pyridylthio)propionate and
subsequent thiolation by specific reduction.
N
B B
tCOCH2CH2SH
\ p, A
10 n (4) ~S) r
B
(3) S
A
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This was performed as described by J. Carlsson, H.
Drevin and R. Axen (Biochem. J. (1978) 173, 723J.
A solution of N-succinimidyl 3-(2-pyridyldithio)-
propionate (100 mg; 320 mol) in absolute ethanol (4 ml) was
s added dropwise into a stirred solution of bovine serum
albumin (BSA) (3) (265 mg; 4.0 mol) in 0.1 M sodium
phosphate buffer/0.1 M NaCl, pH 7.53 (4.ml) at room
temperature (24 C). After stirring for 35 min at about 24'C,
the reaction mixture was separated by gel filtration on
io Sephadex G-25TM (38 g ) (elution med.iian: 0.1 M sodium phosphate
buffer/0.1 M NaCl, pH 7.53).. Fractions containing the
protein-2-pyridyl disulphide derivative (4) were collected,
weight = 25.6635 g.
The UV-spectrum measurements of 0.09828 g(4) (from
is the above combined fractions) after reduction with
dithiothreitol (at I,,,x 343 nm) showed that 24 mol of 2-
pyridyl disulphide structures/mol of bovine serum albumin
have been incorporated.
Thereafter, the combined fractions (containing 4
20 mol of bovine serum albumin-2-pyridyldisulphide derivative
(4)) were freeze-dried, again dissolved in water (10 ml) and
then, with help of gel filtration on Sephadex G-25; the
buffer of the concentrated protein-bound-2-pyridyl
disulphide derivative (4) was changed to pH 5.81 with 0.1 M
25 citric acid-sodium citrate buffer/0.1 M NaCl, pH 5.81. The
fractions containing bovine serum albumin-2-pyridyl.
disulphi.de derivative (4) were combined and treated with
dithiothreitol (61.7 mg; 400 mol) at room temperature
(23'C) and further stirred for 1.5 hours at the same
30 temperature. Then, the protein-bound thiol groups derivative
(5) was separated by gel filtration on Sephadex-25 through
elution with 0.1 M citric acid-sodium citrate buffer/0.1 M
NaCl, pH 5.81. These fractions were combined and freeze
dried.
35 The buffer pH of the freeze dried product containing
bovine serum albumin-bound thiol groups derivative (5)
(4 mol) was transformed to 7.53 by dissolving in 0.1 M
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sodium phosphate buffer/0.1 M NaCl, pH 7.53 (5 ml) and then
followed by gel filtration over Sephadex-25 using the same
buffer as the eluent. The fractions containing the bovine
serum albumin derivative (5) were collected, weight =
22.8398 g.
Step C-
Reaction of 7l3-(2-pvridyldithiopropionamido)-3-methyl-3-
cephem-4-carboxylic acid (2) with bovine serum albumin-bound
lo thiol ctroup derivative (5) to form N-pentadecakis LN-(4-
carboxyl-3-methylceph-3-em-7-yl)aminocarbonyl-
ethyldithioethylcarbonyl]bovine serum albumin (1)
B 0
A S ~S NH S
O
(1): n=15 0 OH
A solution of 7p-(2-pyridyldithiopropionamido)-3-
methyl-3-cephem-4-carboxylic acid (2) (34.28 mg) from Step A
in 0.1 M sodium phosphate buffer/0.1 M NaCl, pH 7.53 was
so added to a stirred solution of bovine serum albumin-bound
thiol groups derivative (5) from Step B(9.1433 g; 1.6 mol)
at 20 C. The reaction mixture was further stirred for 2
hours at 20 C and left for two days at 0 C. The UV-spectrum
measurements of the reaction mixture (at X,aX 343 nm) showed
that 15 mol of N-(4-carboxyl-3-methylceph-3-em-7-
yl)aminocarbonylethyldithioethylcarbonyl units (n=0)/mol of
bovine serum albumin have been introduced. Thereafter, the
CA 02655875 2008-12-22
- 18 -
reaction mixture was purified by gel filtration on Sephadex
G-25 (elution medium: 0.1 M sodium phosphate buffer/0.1 M
NaCl, pH 7.53). The fractions having Imax 265 nm were
collected and freeze dried.
The freeze dried product was unsalted with help of
gel filtration on Sephadex G-25 using water as the eluent.
The fractions containing N-pentadecakis(N-(4-carboxy-3-
methylceph-3-em-7-yl)aminocarbonylethyldithioethylcarbonylJ-
bovine serum albumin (1) were combined and freeze dried.
io Yield = 93.6 mg. The product (1) has been identified through
'H NMR spectrum (600 MHz; D20; 6-value) showing Q-lactam
protons at 5.60 and 4.98,ppm respectively and a C~ signal at
1.92 ppm-
.+.`
. . .
. .- _,
