Note: Descriptions are shown in the official language in which they were submitted.
2 10
GIST-BROCADES N.V.
2628 S
DETECTION OF ANTIBIOTICS
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 incubation 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
ao penicillin requires longer test times.
CA 02107856 2003-08-12
the f:onf-.eesi,,,.%.:~tions of a4ot.^'..3.;.'.., atics present in mh..~ k are
generalw y very low, t,..m~ constraint mean w~ ~7~,
~.4h4 Lu a
detection met4aod based ."`i; ~~ ,
~? e~E~ew s ~> ~",2.,~giu:d ant;e.biotict:s is not
convenient for testincz milk 'Ã~ple~ r ~4
:~ a"~,~,~
It is therefore an object of the present invention
tO PrOvide a simple test Msigned to detect low levxels of
ax c qh 'wa ?. ;~ 3 liquid " b;.o'~,~., ~ med:.i'~ suc~"'s as milk{ urine or
blood4
The time required for the fõ`4~'S'Mts should ideally ~ ~t e41. y,cSii {!y
n~naN ed about 15 m.. nõtes4 The present invention ~~~ovide, a process
Y C for the df:?te~" ="',.'3 r
~ =.. %.. r~ h ~ ~ least one unti:'sinti,, in a liquid
medium such as milk, urine and blood which - ~ comprises
(a) F%rin~in>~ togÃ:wher a fluid sample of the liquid
medium, at least one labelled antibiotic binding protein,
and at least one i[ smf 3 b:#. l's, ~ ed anti' 5iot.~' c,
(b) allowing the labe3 .~~& antibiotic binding protein
to ;3 i~ ~~ d with a the ... mm:.: bi'. w zed a: =:. iw ioticf
(c) re3::oving label3ed antibiotic b~..; .".f,% %.n"'
{=~~. protein
J
which #, is not : 3Ci unLt to immx:%'s..^ ilõ,. zesn a " ' ~..~ .'., C~'~.
,7,. ~', and
=-.C 4 4 =u
;"-^5t
(d) determining the amount of the labelled aa antibiotic binding protein
tiJ.%ui:d to the immobilized
antibiotw.o"
The process may be used to detwct a wide variety of
antib:s.otic..~'i such as 0M^lactams ;includ;Ã.ng penicillins such as
~
benm-srlpe~:icil: ~~, and ce~~ alr~>spc;ri ,sf , ~~~~acx{ cl3 ~
"3e~ r
2. ge_ntas:?Xl 3:., .'".~"<':.::lw' a compounds such c.3~'.a s'"~ .%'~
methazine, s'~'=,~...r~a ~'and
comb:. ~~ tions ther}~of% The immobilized S ,~',:~i~rib~..,w.,..Z~''4h
.int.~,.b.},.,
!i{JaY^..w w ~~r is
generally the same ak, that pr;?went in the liquid sample, but
i,e:fa' be different. "~^.a~ ."i t~., e the ...nfiea 'r.~>."'i}3 S. f...i: may
~ For . .t . 4.ed antibiotic $''sIL'~~~
be an 4 analogue < ~. the 5,,. ~ antibiotic ~ a ~
~ f'; ~. . o " ,`-~.. s:. <":# ~% %., ~;.=~ % .5. ^;f the :4o immo.~ ~ilize&
an (= ~M- ~~b?J~ and ~ ~y,{+,$.if~ t~i.~.f.w.i. '~^{ they
antibiotic v44;~ :~3?}~m.~i are different ~~S~yi
ao44L.
~.^houõd b; th, be able to bind with the binding p%. ~~`~"a.; :
ein4
The label used in the present ,. s . vention may ~e an
enz^y 4a~y . ' r-rr ~y, ~^. . ...
or a fl`..:~.2.ir.~''''~.s~.'~,:.'e ',~".. b ,~'i~:i~.;o{.~S~t=~.."f. ,,
i",'"su~'E}~ .'c as IT'~.~.. ~ ~luoresc3.~in
W~.'`i ,:hio.:;..'y'"s?i"?Cste; or w K.Iõ . ; t%;;.tra.'T?e~.. ': Y .~.
'Y's.3t~ c
s .:~ ~i i; ~.e Ee ~,.:;a~3'~`"w s <,'s. r.,'. ,,,
:iw t'; yanCf. a:,' eS , Pr;.' fe"h ably an n 4? nM. ym'"~' "" .,. as.~;'s
el.. ed antiPvS .3 o'% ic ` : 7
~: ~;. =., w Ã~ s"`s
is used in step ; N ; 4
2 c781
- 3 -
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
s 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
15 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, and 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
25 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 ooli, preferably
Bacillus stearoõthermophilus microorganism is used. Also
so 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-
sa binding protein may be purified by techniques as affinity
chromatography or gel filtration.
[.~ l; :.=~ (I . (=
i .li 1
- 4 -
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
io 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-
is 656; F. Wold (1972): Bifunctional reagents: Methods Enzymol.
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
20 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
25 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,
35 which is known for its stability but also other enzymes can
be used. For instance peroxidase, alkaline phosphatase or
P-galactosidase in general (all enzymes that are useful in
- 5 -
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
s 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
oxygen such as tetramethylbenzidine, o-phenylenediamine or
1s 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
20 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
25 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.
30 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-
35 molecular conjugation is described by J. Carlsson et al.
(1978) in Biochem. J. 173, 723-737), in which the heterobi-
CA 02107856 2003-08-12
... ~ u,
propionate 5 ~~~~ ~ is ~sedv
Materials such as gla~~ or plastics can be used as
matrix ~aterialK NH, E~~oupa of the matrix can be used to
a obtaMn immobwliz~~tionA Covalent coupling between materials
~~ch a~ plastwcs (e, q< ~ol~,K ~ty~~~~ ) and a protein (for
example BSA) can also be used to iÃm~bilise the antibiotic
of interest, We~"/ esgK RRHv $.$urMoSS and P<& van Kil.i.~+.p'rv+LLbergf
Laboratory techniques in B.~~chemw stry and Molecular Biol~gyf
.ck Elsevier, w985<
According to a preferred embodiment, the antibiotic
is i~ ~biliz:~d onto the in~~rio.~ surface of a container such
as a test tube, preferably via a linking compound such as
BW
16 The pres ez 2 t d, n 6K'2 en5.. i?.~'i n aVro. / o YA+. +.~ o'F i'vS.es a
Xoit 3.. '.~~ r carrying out the detection, which comprises at l~~st one
enz5~~~-l~bell~~ antibiotic binding protein and at least one
immobilized antibior ir; o
ia the ~ollowi<~ examples preferred embodiments are
i,'"z described to id'.,,e. u:7trat;u the inYOzcr4.ntõ ion , 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 m%~thwdf -.a may use other tests which can Ã~~
equally used for the purpose of the present invention. These
tà aw. t~ ~ations are included in the scope of the m~ ~~~ ntion4
~~~~le I
Antibiotic residue '~~st,
.s~ In this example will ~~e described a method for
detecting bemzwrlpenwcilli:. residues as l~~ as 5 ppb i~ milk.
CA 02107856 2003-08-12
7
~ grOwn culture of an antibiotic sensitive
microo~~anism in this example ~~onw;.n"O'.~s culture arto # 108 ~~~~~~
~~~du~~s LtdY ~~~) was
ly-sed o~~s~t at 4uC with lYsozY~m-, MA,~e and triton xwloo-M,
in 0.1 ~ phosphate pH 7 ,09 The ~~~~at~ was centrifuged for
30 minutes at approximately noo x g ;&Qx After ~en~rif~~~-
atio~ the supernatant was mixed with an antibiotic affinity
#n gel nawrisz, f~r example to prepare a 7-ami~~~~phalo:~~~~anic
acid 47ACcx3.f affinity gel matrix th.~.' following method was
usedr
0,34 rz of 7ACA was mixed with 25 ml 0.1 ~~ phosphate
~s"~,
"y,~ 7<o., "} ~ rg~ ~~11 ~or. terr3f ~ to 7). To this ~ ~.~~
'y.~~:~~ f so~s,u~.~. + ~"'a was "~ ~~~i~,
tfi ml ~~ ~~ ~ affigel 101 (BioRr d) (washed with 1 1 04 ~~
~
phosphate pH 7e ~ )~ This was mixed gently for 2 hours at
:0"CY The ~~~CA-affic~~l 10 was filtered and sucked off using
v~~uu= The '~ACA-affi}';~el was then ~~she& again with OU N
phosphate pH 7n O and was ready for use5
The 1ACA-a~fi~el and the ~~pe~~ataÃ~~ of the lysed
culture was mixed for 3 hours at 20 C gently on a shaz~er,.
The gel was washed six times with Vw ~~ phosphate + 1 ~ NaCl
pH 7x 0v For each wash was used 500 ml,
20 m~ el~tionwbuffer (0.05 M phosphate + 0.5 N NaCl
to + OM triton ~ -100 + Ã3 s A M hy droxylamine pH s 0; was added
to the moist gel cake and mixed for 20 minutes at ~~~~
gently on a shaker. The mixture was then centrifuged at VCs
~'s minutes at approximately 300 x gr The supernatant was
dialysed in 32 mm tubing >.12-14 kD cut-off3 y
Of The first dialysis wa..~`z against 0.05 M phosphate +
0.5 N ~aC~ pH 7.0 everniqht at 46Cx the second up to the
fifth dialysis was against 0,1 M carbQ;.zate pH 9<4 with a
change of buffer every 4-6 hours.
The lysate was cenwriwugate& 20 ~inut~s at approximately
n 1000 x g at W~ and concentrated in an ~ICOW '` ~~~ceritrawor
(ultra filtration) (model 0 82~,~07 WR Grace and W)
according to the ma:~~~~acturer4s standard operation procedure
- 8 -
(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
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
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
(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.
Co~jiaciation qf a 0-lactam to a prot ein
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.
I.U :iT .,i
_ 9 -
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 1N 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 p-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:
CA 02107856 2003-08-12
Mono basic ~odium ~~~~~~hate 11ry'~ ~~am/l
Dibasic sodium phb=~~~ ~~e Ux ~ ~~~~~ ~
~enzalkani.;m ch A.wride 3.57 gram/l
Glycerol 500 MI/I
~~~n 2 1V5 ml/I
pH adjusted to 6a5,.
This :~~lution ;~~s nade 50 x woncentrated for
stability i. {. year) and cr~~~enient transportation of the
f:~ ~~ ~ ~ ~ ~t kit. For the test a 50 x dilution in di~tilled
s water (or tap water) is ~~~edv Also other low salt and
r
surfactant containing solutions may be po~siblev
For the s'=:rb:atrat-~.'~. of HRPO it is possible to us's.,.'a a
chromogenic colour substrate which is easily oxidized by the
formation of oxygena in this example is used a ~~~~~cial
product TM-blu~~ frQm TSI (US-A-5r 01:~ ,646) which has a good
(for ~
stability ~x ~=~~. ' eavat ~. year at V ~~ and oper~:t~,onr The
2, :M~~our deve;~~~~ent can he measured by optical density at 650
nanometer wavelength or after acidification at 450 nano-
~,....._, . .............
The use of a stop solution such as 1.54 :~aF for
strong acids) is highly stable and gives a ~ett~r
q-uantiw~ti5~a approach in case a difference in colour
development is being measured at a ti~~~baseN
~~~õ}~."õ''T,~~.~.t=,"7'`~,,,.,~~t~1~~~ F ;~~''~~~~..G~~ s~i;.~'1~~.. ..
V2 mi of m:;l.~ sample and O< 2 ml of a diluted abp
~-~nxyme conjugate was added to an empty reaction ampoule.
3`S Af'~ ~ r incubation for 2 min' t: w es at 64'C the contents of th~.-y.
tube was transterred to the coated tube. After the second
incubation of I minutes at 64'; the contents of the coated
W L 0
- 11 -
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
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 %
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.
Table II
/3-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
is 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 P-lactam-assay. We will therefore
give in this example the same outline as the Q-lactam assay
of Example 1 with description of differences.
Obtainina of antibiotic binding protein
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 sulfosuccinicmidylsuberate-BSA
and 2 mg of unreacted BSA is mixed with the raw antiserum in
e5 the ratio of 10 to 1. This removes all detectable reactivity
with BSA.
~ .~. ~ ~ ., ... ~
13 -
Conjugation of antibiotic binding protein with an enzyme
label
Absorbed anti-gentamycin antiserum is reacted with
horseradish peroxidase labelled staphylococcus aureus
s 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.
Conjugation of qentamycin 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 J3-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
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.
- .+ 2 ,= ~ f- ~>
.~ 0 ~ ~~ ~~ ,.~
- 14 -
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-carbonvl-3-methylcet3h-3-
is em-7-yl)aminocarbonvlethyldithioethylcarbonyllbovine serum
albumin (1)
Step A:
Preparation of 78- (2-Pyridyldithiopropionamido)-3-methyl-3-
cebhem-4-carboxylic acid (2)
~
~1 / =
S,. S
NH g
0 (:~N
0
(?) 0 OH
7Q-Amino-3-methyl-3-cephem-4-carboxylic acid (34.28
33 mg; 0.16 mmol) was suspended in water (7 ml) and, with
- 15 -
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 MgSO4, 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"1.
'H NMR (360 MHz; CDC13; d-value in ppm; TMS): 2.06 (3H, CH );
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).
t8 enB:
Introduction of 2-pyridyl disulphide group into bovine serum
albumin by N-succinimide 3-(2-pyridylthio)propionate and
subsequent thiolation by specific reduction.
Na
S B
B S S CO-CH2-CH2-SH
A A
~ 0 n (4) (5) "
B
(3) S
A
CA 02107856 2003-08-12
This +ria~ ~erH'isrmed s;.~,.=a ;wau:s: h 3~3 .~ ~ ~~ ~ by J" Marl~so$#x Hx
Drevi~ and R. Axd:s^a [Bv of". he:? o ~,.~" ,. 11978)
8)
A solution of N <, ~ccinimi~yl 3-(2-pyridyldithio~
~ropi~nate 5Ioo mgf 3to gm>;iJ in absolute ethanol (4 ml) was
~ added dropwise into a stirred ~olutiom-. of bovine serum
albumin (BSA) (1) (265 mg> 00 Amok. ) in &I K sed~ UM,
~"s~~~phate buffer{t 0. 1 M NaCl, pH 7.53 (4 ml) at room
, ".emp~ratur'".'~.. (20C)e After stirring for .:~5 ~>,~~~ ~~,
4~ r about 24"Cr
the rer ctz on mi~=eture was ~ ~parat~~ by gel fM lt~ ation on
seep'nadex Gw250 (3a q ) (elutdol:. i:eliam4 0,1 id ~culitga rouselAlattr
b}hffsur,{`Vw M MCI, pH 7.51). Fractions containing the
;.~~otein-2-pzrridyl disulphide derivative (A) were ~ollected9
Y4'ew.gi i", fi 25o 66-.i0 gn
The w'V-upfM: tr'~;;~m measu~~~ent:~ of V09828 g (1) (from
ia the above combined fractions) after reduction Y{ait:~
dithio~~~ reito: ; a~ 1";a 340 nm? sh~ ~~ ed that 24 ~ ol of 2
-
~.~ ~~ idyl ~ ~ sulp>.~. ide struct>~ resI mM l of bn wY i ~ serum albumin
have been wncoh porateCh' ,
The.~eafterz the combined fractions [~ontaining 4
2o p:~ol of bovine serum alb}.~min-2-pyridyld:~~ulphi~e derivative
(A)] were f ~'', ee e `=h.' riads again dissolved in water (10 ml) and
then, with help of gel filtration on ~ephadex G-25r the
buffer of the concentrated y~~~~einm bound-/-pyridvYl
disulphide ~ ~ rivf%~ tiYf'M> Ay was ~ ~ ~~~ed to pH 5,81 with 0.1 M
<-5 citric aM id-sodium c iM.f ate bt;".~ fer/V! M NaÃ." lr pH 5.81. The
fractions containing bovine seru:~ albumin-o-p.~ridyl
disulph.~~~ derivative ; Af were combined and treated with
dwWhioth~eitol (6wa 7 mg; 4{wx mmol) at room temperature
(234C) and further sw x. r.~ed for 1.5 hours at the same
3 temperature. Then, the ps_>,"'.te3"sn a,.~f"2E:ha d tf3 `.:"as. ~f ' r
Fw~"...u,~ ~; der"k. r
`~v~a'~",s~,~fe
(1) was separated by gel filtration on ~~~~~~~~ex-25 through
elation with ~.~ ,1 M Q...tN ic acid-sodium citrate :~~~~~~r/O4,~. M.
Na~.~ls pH 5o 31~ These fractions were combined asd freeze
dried.
:35 The 6uf~~~ pH of the $.3 ee~e dried prodmcw containing
bovine serum a'.bum"a.n""s~',i-ouX'sd th:#.ol groups derivative (1)
(4 gmM l) was t3'a:a 3 sf~.} rmed to 7,53 by diw;x :;z a.i,{ ving in 's. y 1
M
~,,, .. .
17 -
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 7Q-(2-tayridyldithiopropionamido)-3-methyl-3-
cephem-4-carboxylic acid (2) with bovine serum albumin-bound
lo thiol aroup derivative (5) to form N-pentadecakisrN-(4-
carboxyl-3-methylceph-3-em-7-yl)aminocarbonvl-
ethvldithioethylcarbonyl]bovine serum albumin (1)
g 0
A S *~'~~ NH S
0
/'EN n
0
(1) : n=15 0 OH
A solution of 70-(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 Xõax 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
- 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 ~.ax 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)aminocarbonylethyldithioethylcarbonyl]-
bovine serum albumin (1) were combined and freeze dried.
lo 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 CH3 signal at
1.92 ppm.
