Verfahren und Kit zur Quantifizierung von Beta-Laktam Penizillinen
Randox Laboratories Ltd., Northern Ireland, GB
McConnell, Robert Ivan, County Antrim BT29 4QY, Northern Ireland, GB; Benchikh, El Ouard, County Antrim BT29 4QY, Northern Ireland, GB; Fitzgerald, Stephen Peter, County Antrim BT29 4QY, Northern Ireland, GB; Lamont, John Victor, County Antrim BT29 4QY, Northern Ireland, GB
derzeit kein Vertreter bestellt
AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, IE, IT, LI, LU, MC, NL, PT, SE, SK, TR
The present invention relates to a method and kit for detecting, or
determining the quantity of, β-lactam penicillins, as well as, haptens, immunogens,
conjugates and antibodies useful therein.
By "detecting" is meant qualitatively analysing for the presence or
absence of a substance.
By "determining" is meant quantitatively analysing for the amount
of a substance.
The present invention is intended to have broad applicability across
the main first generation β-lactam penicillins such as ampicillin, penicillin
G, amoxycillin, cloxacillin, dicloxacillin and oxacillin, but is not intended to
be limited to these specific β-lactam penicillins.
Antibiotics are routinely used in animal husbandry for both prophylactic
and therapeutic purposes. The β-lactam class of antibiotics are commonly used
in the meat and dairy industry, as growth enhancers. This class, also known as the
penicillins, is used to treat dairy cow mastitis, thereby increasing milk yields
and the productive life span of the cow. β-lactams may also be included in
animal feed, with the purpose of enhancing the growth of poultry and pigs. Through
disease prevention, or by inhibiting the activity of natural gut flora in such animals,
the antibiotic causes the animals to reach marketable size faster than without the
use of such enhancers.
Problems, however, may arise when residues of the β-lactams are
present in meat and dairy products. As with any antibiotic, continuous exposure
of humans to the β-lactams can result in a reduction of the efficiency of the
drugs used to treat diseases, due to the development of resistant strains of pathogenic
bacteria. The presence of the β-lactams in consumed food may also result in
allergic reactions in penicillin sensitive humans. Dairy products containing these
antibiotics may also interfere with bacterial cultures used in processing.
As a result, strict guidelines have been imposed throughout the European
Community regarding withdrawal times and the maximum recommended levels (MRLs) of
the β-lactams in milk and meat. Milk and meat samples are routinely tested
to ensure that they comply with this EC legislation. Various methods are used to
test for antibiotics such as the β-lactams. Many of these tests are based on
microbial inhibition tests, which are time consuming and may be specific for individual
β-lactams. The development of a method for the rapid detection of the β-lactams
in milk and meat would be especially valuable if the method was generic, i.e., detected
most, if not all, β-lactam antibiotics.
Many attempts have been made to raise antibodies in β-lactam
sensitised animals, with the aim of producing an immunoassay for the detection of
β-lactams in general. The first stage of such a process is to produce an immunogen
which will elicit an immune response in the animal host. This is problematic due
to the failure of the β-lactam ring to,remain intact during conjugation to
a carrier protein. Known conjugation methods based on the open lactam ring are disclosed
in, for example, US-A-4,347,312, US-A-5,128,240, in de Haan et al, 1985 and in Faghihi
Shirazi et al, 1991. This results in the production of antisera sensitive to the
open form of the β-lactam ring, which may not necessarily be sensitive to the
generic ring structure.
Alternatively, for example, the free carboxyl group of the closed
β-lactam ring may be esterified, as is disclosed in EP-A-309,299 and in Usleber
et al, 1994. Antisera raised to β-lactam antibiotics conjugated in this manner
are specific for the acyl side chains and only cross react with other β-lactam
antibiotics if they have similar side chains, as is the case with the isoxazolyl
penicillins. Further, alternatively, conjugation can occur by way of the 6-amino
group of 6-amino penicillanic acid, as is disclosed in EP-A-309,299 and de Leuw
et al, 1997. In such cases, where the β-lactam ring remains intact during conjugation,
the antibodies display high cross reactivity with the main first generation β-lactams.
A further potential conjugation site for β-lactam penicillins is via the α
amino group of the D-α-aminoacetamido group of penicillin, as is disclosed
in, for example, Nagakura et al, 1991. The cell lines disclosed in Nagakura et al,
1991, Abp4 and Abp7, concern haptens and conjugates using a MBS (maleimidobenzoyl-N-hydroxysuccinimide)
cross-linker and the document concludes that one of the cell lines (Abp4) recognises
the thiazolidine ring whilst the other of the cell lines (Abp7) recognises the acyl
side chain. Abp4 cross reacts with penicillin G, 6-aminopenicillanic acid and certain
cephalosporins, while Abp7 is highly specific, displaying little or no cross reactivity
with the main first generation β-lactams.
DE 4 013 004 concerns itself with the use of labelled haptens in ELISA.
The present invention describes the conjugation of a novel hapten
(ampicillin derivative) at the α amino group of the D-α-aminoacetamido
group of penicillin to an antigenicity-conferring carrier material to produce an
immunogen. It also describes how antibodies generated to this immunogen are employed
in the development of a generic assay which can be used to test milk and meat and
the like for the presence of β-lactam antibiotics.
Detailed Description of Invention
In a first aspect, the invention provides a hapten comprising a 6-[D-α-aminoacetamido]
penicillin derivative crosslinked at the α-amino group with a substituted
or unsubstituted phenyldicarbaldehyde selected from the group consisting of substituted
or unsubstituted phthalaldehyde, substituted or unsubstituted isophthalaldehyde
and substituted or unsubstituted terephthalaldehyde.
A representative 6-[D-α-aminoacetamido]penicillin derivative has the structural
and the conjugation site is identified by an arrow.
Preferably, the phenyldicarbaldehyde is a substituted or unsubstituted
terephthalaldehyde, most preferably unsubstituted terephthalaldehyde. Suitable substitutions
include the addition of aldehyde, thioisocyanate and N-hydroxysuccinimide functional
groups at the para- and ortho- positions.
The haptens are prepared by reacting a substituted or unsubstituted
phenyldicarbaldehyde with a 6-[D-α-aminoacetamido]penicillin derivative in
a suitable solvent, of which dimethylformamide and dimethylsulfoxide are suitable
In a further aspect, the present invention concerns an immunogen comprising
the hapten of the present invention coupled to an antigenicity-conferring carrier
material. Preferably, the carrier material is a protein, a protein fragment, a synthetic
polypeptide or a semi-synthetic polypeptide.
In a still further aspect, the present invention concerns antibodies
raised against the immunogen of the present invention, the antibodies being capable
of binding with at least one structural epitope of an intact β-lactam ring.
Preferably, the antibodies are fixed on a backing substrate.
The invention further provides a process of preparing the antibodies,
the process comprising the steps of immunising an non-human animal, preferably a
vertebrate animal, most preferably a mammalian animal by repeated administration
of an immunogen of the present invention; and collecting the resulting serum antibodies
from the immunised animal. Preferably, the process further comprises fixing said
serum antibodies to a backing substrate, preferably a solid support, most preferably
a polystyrene solid support. Preferably the antibodies are polyclonal. Alternatively
the antibodies are monoclonal.
In a still further aspect, the present invention comprises a conjugate
comprising the hapten of the present invention covalently bonded to a detectable
labelling agent. Preferably, the labelling agent is selected from an enzyme, a luminescent
substance, a radioactive substance, or a mixture thereof. Preferably, the labelling
agent is an enzyme, preferably a peroxidase, most preferably horseradish peroxidase
(HRP). Alternatively or additionally, the luminescent substance may be a bioluminescent,
chemiluminescent or fluorescent material.
In a still further aspect, the present invention comprises a method
for detecting, or determining the quantity of, β-lactam penicillins in a sample,
the method comprising contacting the sample with the conjugate of the present invention,
or a mixture thereof, and with antibodies of the present invention, or a mixture
thereof; detecting or determining the quantity of bound conjugate; and deducing
from a calibration curve the presence or amount of β-lactam penicillins in
Preferably the antibodies are polyclonal.
In a further aspect, the invention includes a kit for detecting, or
determining the quantity of, β-lactam penicillins, the kit including the conjugate
of the present invention, or a mixture thereof, and the antibodies of the present
invention, or a mixture thereof. The kit may optionally include instructions for
the use of said conjugate(s) and said antibodies for detecting, or determining the
quantity of, β-lactam penicillins in a sample.
Preferably, the sample is a solution, such as a biological fluid,
including milk; or a cellular tissue cutting, such as meat.
In the method and kit of the present invention, the respective crosslinkers
(of the immunogen and the conjugate), crosslinked at the α-amino position,
may be the same or different.
In a further aspect, the present invention involves use of the conjugates
according to the present invention, or a mixture thereof, with the antibodies according
to the present invention, or a mixture thereof, to test samples such as milk and
meat for detecting, or determining the quantity of, β-lactam antibiotics.
The present invention relates to novel haptens which are employed
in the preparation of novel immunogens by conjugation to conventional antigenicity-conferring
carrier materials. The resulting immunogen is then administered to animals, preferably
vertebrate hosts, most preferably mammalian hosts, to elicit production of avid
polyclonal antisera which are then used to develop a generic immunoassay for the
β-lactam penicillins, employing a conjugate (hapten-labelling agent), or a
mixture thereof, as the detection reagent.
The chemical structure of ampicillin and the other β-lactam penicillins
are summarised in the following table, having regard to the structural formula set
Chemical structures of the β-lactam penicillinsPenicillinRPenicillin GPhCH2COPenicillin VPhOCH2COAmpicillinD-PhCH(NH2)COAmoxycillinD-(p-Hydroxy)PhCH(NH2)COOxacillin5-methyl-3-phenyl-4-isoxazolyl-carbonylCloxacillin5-methyl-3-(O-chlorophenyl)-4-isoxazolyl-carbonylDicloxacillin5-methyl-3-(O,O'-dichlorophenyl)-4-isoxazolyl-carbonyl6-Aminopenicillanic AcidH
The focus of the present invention is the preparation of antibodies
specific for the entire group of β-lactam penicillins. In order to achieve
this broad specificity, ampicillin is derivatized through the amino group employing
a bifunctional cross linker such as a substituted or unsubstituted phenyldicarbaldehyde,
preferably substituted or unsubstituted terephthalaldehyde (unsubstituted terephthalaldehyde
is shown in Figure 1 of the accompanying drawings). The β-lactam ring of ampicillin
is conserved during derivatization to ensure that the epitopes common to the penicillin
group are retained.
Although the hapten of the present invention (ampicillin derivative)
provides defined structural epitopes, it is not in itself immunogenic and therefore
must be conjugated to a suitable antigenicity-conferring carrier material, so that
the thus-formed immunogen will elicit an immunogenic response when injected into
a host animal. Suitable antigenicity-conferring carrier materials include proteins
and proteins fragments such as albumins, serum proteins e.g. globulins, ocular lens
proteins and lipoproteins. Illustrative protein carriers include bovine serum albumin,
egg ovalbumin, bovine gamma globulin, thyroxine binding globulin, keyhole limpet
haemocyanin etc. Alternatively, synthetic poly(amino acids) having a sufficient
number of available amine groups such as lysine may be employed, as may other synthetic
or natural polymeric materials bearing reactive functional groups. In particular,
carbohydrates, yeasts or polysaccharides may be conjugated to the hapten to produce
an immunogen of the present invention.
The hapten (ampicillin derivative) is also conjugated to a labelling
agent such as an enzyme (for example, horseradish peroxidase), a fluorescent substance
or a radioactive substance to produce a detection reagent for use in the immunoassay.
The fluorescent substance may be, for example, a monovalent residue of fluorescein
or a derivative thereof.
Preparation of the hapten and its conjugation either to the carrier
material or to the labelling agent (e.g. enzyme or other label) is performed according
to Reaction Scheme 1 set out in Figure 1. Thus, for example, ampicillin is reacted
with terephthalaldehyde in dimethylformamide at room temperature for 18 hours to
produce a Schiff base intermediate. The intermediate is reacted with either the
carrier material (for example, bovine serum albumin) or with the labelling agent
(e.g., enzyme or label) in acetate buffer at pH4-5 and this is followed by reduction
of the Schiff base with, for example, sodium cyanoborohydride to yield either the
immunogen of the present invention or the conjugate of the present invention, respectively.
In order to confirm that adequate conjugation of hapten to carrier
material has been achieved, prior to immunisation, each immunogen is evaluated using
matrix-assisted UV laser desorption/ionization mass spectrometry (MALDI MS). In
the case of the preferred carrier material, bovine serum albumin, a minimum of 6
molecules of hapten per carrier molecule is preferred.
In order to generate polyclonal antisera, the immunogen is mixed with
Freund's Adjuvant and the mixture is injected into a host animal, such as a rabbit,
sheep, mouse, guinea pig or horse. Further injections (boosts) are made and serum
is sampled for evaluation of antibody titer. When the optimal titer has been reached,
the host animal is then bled to yield a suitable volume of specific antiserum. The
degree of antibody purification required depends on the intended application. For
many purposes, there is no requirement at all for purification, however, in other
cases, such as where the antibody is to be immobilised on a solid support, purification
steps can be taken to remove undesired material and reduce or eliminate nonspecific
The antibodies generated to ampicillin are useful as reagents in biochemical
assays for the determination of the presence of β-lactam penicillins in biological
fluids such as milk and in food products such as meat.
In the drawings:-
Figure 1 concerns Reaction Scheme 1, which is a general reaction scheme for
the preparation of a hapten according to the present invention and its subsequent
conjugation to either a carrier material or a labelling agent, to form an immunogen
according to the present invention or a conjugate according to the present invention;
Figure 2 schematically illustrates a competitive ELISA titration assay on a
Figure 3 is a calibration curve for a competitive ELISA; and
Figure 4 is a calibration curve generated employing each of the beta lactam
penicillins as a standard in an ELISA.
EXAMPLE 1Preparation of hapten
183mg (1.364mmole) of terephthalaldehyde was added under nitrogen
to a solution of 500mg (1.24mmole) of ampicillin trihydrate in 10ml dimethylformamide
at 20°C. The mixture was protected from light and stirred for 24 hours at room temperature.
To confirm the reaction was complete, thin layer chromatography (TLC) (80% chloroform,
20% methanol v/v) was performed which showed no remaining starting materials and
the formation of a new spot less polar than ampicillin. The hapten solution was
stored under nitrogen at -20°C (stable for 1 year).
EXAMPLE 2Preparation of immunogen (hapten-bovine serum albumin)
The hapten solution prepared in Example 1 was added dropwise to a
solution of 200mg of bovine serum albumin (BSA) in 10ml of 0.1M sodium acetate buffer,
pH 4.1. The mixture was protected from light and stirred at room temperature for
4 hours. Reduction of the Schiff base was accomplished by addition of 30mg of sodium
cyanoborohydride. The mixture was stirred for 90 mins and 5mg sodium borohydride
was added. After stirring for a further 10 mins, the mixture was dialysed against
phosphate buffered saline, pH 7.2, at 4°C for 24 hours (3 changes). The extent of
conjugation of hapten to BSA was evaluated by MALDI MS, which revealed a conjugation
ratio of 6.3 hapten molecules to one molecule of BSA.
EXAMPLE 3Preparation of conjugate (hapten-HRP)
The conjugation of the hapten of Example 1 to HRP was performed in
a similar manner to that described for the preparation of the immunogen. 40µl of
the hapten solution prepared in Example 1 was added to 20mg HRP (horseradish peroxidase)
in 2 ml of 0.1M sodium acetate buffer at pH4-5. The mixture was protected from light
and stirred for 4 hours at room temperature. Sodium cyanoborohydride (0.7mg) was
added and the mixture was stirred for 90 mins. The conjugate was purified using
two PD-10 columns (Pharmacia Biotech) and dialysed overnight, protected from light,
against double deionised water at 2-8°C.
EXAMPLE 4Preparation of antibodies raised against the immunogen of Example 2
An aqueous solution of the immunogen of Example 2 was formulated with
Freund's Complete Adjuvant (FCA) to form an emulsion consisting of 2mg/ml immunogen
in 50%(v/v) FCA. Three sheep were immunised with this emulsion, 0.25ml being subcutaneously
injected at each of 4 sites in the flank of each animal. Subsequent immunisations
(boosts) contained 1mg/ml immunogen emulsified in 50%(v/v) Freund's Incomplete Adjuvant
(FIA) and were administered in the same manner at monthly intervals for 1 year.
Blood sampling took place 7 to 14 days after each boost. Each sample was processed
to produce antiserum which was further purified by caprylic acid and ammonium sulfate
precipitation to yield an immunoglobulin G (IgG) fraction. The IgG fraction was
evaluated by competitive ELISA microtiter plate assay, as described below.
EXAMPLE 5Development of a competitive ELISA
A checkerboard titration was performed to determine optimum capture
antibody and conjugate (ampicillin-HRP) concentrations. Serial dilutions of the
IgG fraction of each antiserum to be tested (prepared in accordance with Example
4) were prepared in 10mM Tris, pH 8.5. The wells of an enhanced binding 96 well
polystyrene microtiter plate were coated with these dilutions (as shown in Figure
2) by incubation at 37°C for 2 hours (125µl/well). The plate was washed 4 times
with Tris buffered saline (pH7.4) containing Tween (Trade Mark) 20 (TBST) and tapped
dry. 50µl of a 10ng/ml solution of ampicillin (mid assay range) in TBST was added
to the appropriate wells (Figure 2). 50µl of TBST was added to the remaining (control)
wells. Serial dilutions of conjugate (ampicillin-HRP) were prepared in Tris buffer
at pH7.2 containing EDTA, D-mannitol, sucrose, thimerosal and BSA and 75µl of each
dilution was added to the wells, as shown in Figure 2. The plate was incubated at
37°C for 2 hours. The excess unbound conjugate was removed by washing 6 times over
a 10 minute period with TBST. 125µl of tetramethylbenzidine (TMB) substrate solution
was added to each well of the plate, which was then incubated for 15 to 20 minutes,
in the dark, at room temperature. The reaction was terminated by addition of 125µl
0.2M H2SO4 to each well. The absorbance was then measured
at 450nm using a microtiter plate reader. A 1/1000 dilution of capture antibody
in combination with a 1/15000 dilution of conjugate produced an acceptable top absorbance
of 2.15 and a significant drop in absorbance between the 0 and 10ng/ml antigen concentrations
A microtiter plate was then coated with the IgG fraction of anti-ampicillin
antiserum at the optimum coating dilution of 1/1000 in Tris, pH8.5, as outlined
above. Standard solutions of ampicillin (sodium salt) were prepared in TBST and
applied at the following concentrations: 0, 1, 5, 10, 50, 100, 200, 500ng/ml. The
data generated resulted in the sensitive calibration curve illustrated in Figure
3 where B is the absorbance measured at 450nm for xng/ml ampicillin and B0
is the absorbance measured at 450nm for 0ng/ml ampicillin.
EXAMPLE 6Cross reactivity of the ampicillin immunoassay with each of the β-lactam
Standard solutions of benzylpenicillin (penicillin G-PenG), amoxycillin
(Amox), cloxacillin (Clox), dicloxacillin (Diclox) and oxacillin (Oxa) were prepared
in TBST at 0, 1, 5, 10, 50, 100, 200 and 500ng/ml. Calibration curves were generated
employing each of the β-lactam penicillin standards in the ampicillin immunoassay
(Figure 4) and these were used to determine the cross-reactivity of the immunoassay
with each penicillin. The results of this study are presented in Table 2, cross
reactivity being calculated according to the following formula:
%CR = IC50amp/IC50pen X 100
where %CR is the percentage cross-reactivity, IC50amp
is the concentration of ampicillin which causes 50% displacement of signal and IC50pen
is the concentration of β-lactam penicillin, for which %CR is being evaluated,
which causes 50% displacement of signal.
The ampicillin immunoassay exhibited a high level of cross-reactivity
with each of the β-lactam penicillins (Table 2). By high level of cross-reactivity
is meant a cross-reactivity of greater than 35%, relative to 100% for ampicillin.
The present immunoassay is most specific for ampicillin (100% CR), amoxycillin (87%
CR) and benzylpenicillin (72% CR). Since the maximum recommended levels (MRL) for
amoxycillin, ampicillin and benzylpenicillin in milk are each 4µg/kg and for oxacillin,
cloxacillin and dicloxacillin are each 30µg/kg, the IC50 values determined for each
of the β-lactam penicillins suggest that the ELISA described is suitable for
use as a generic immunoassay for β-lactam antibiotics in compliance with EC
Cross reactivity of the ampicillin immunoassay with the β-lactam penicillins.β-LactamAssay 1Assay 2Assay 3Mean ResultsMRLs µg/kgIC50 ng/ml%CRIC50 ng/ml%CRIC50 ng/ml%CRIC50 ng/ml%CRAmp1.71002.51002.51002.21004Amox1.8943.0833.0832.6874PenG2.7633.2783.4743.1724Oxa4.042.57.0366.6385.93930Cloxa3.4505.8435.84354530Dicloxa3.8456.0424.9514.94630
Example 7Qualitative analysis of β-lactam antibiotics in milk employing
the generic immunoassay.
A range of milk samples were tested for the presence of β-lactam
antibiotics using standard antimicrobial methods. These samples with assigned values
were then tested using the present ELISA. Microtitre plates were coated and reagents
were prepared as described in Example 5. The immunoassay procedure was adapted as
follows. A 1% solution of milk buffer at pH7.4 was prepared by dissolving skimmed
milk powder in distilled water and 25µl of this buffer was added to the wells of
the plate. Following the addition of the milk buffer, ampicillin standards were
loaded (25µl per well), followed by milk samples (25µl per well). Both standards
and samples were run in duplicate. Conjugate (ampicillin-HRP) was then added (75µl
per well), and the microtitre plate incubated for 2 hours at 37°C for the competition
reaction to take place. After the competition reaction, the plate was washed and
developed using the same procedure as described in Example 5. The results of the
analysis are shown in Table 3, which shows the calculated concentrations for a range
of milk samples tested by the generic β-lactam ELISA described. The β-lactam
ELISA described was used to test milk samples known to be negative for the presence
of β-lactams (samples 1-6), as well as confirmed β-lactam positive samples
(samples 7 & 8). The ELISA results confirm that samples 1-6 are negative and
that samples 7 and 8 are positive for β-lactam antibiotic content (see Table
The results of the ELISA, shown in Table 3, demonstrate that the immunoassay
can be used successfully to screen milk samples for the presence of β-lactams.
Negative samples tested by ELISA were confirmed as negative, and known positive
samples were confirmed as positive.
Faghihi Shirazi M., Hung TV., Womersley DM. 1991. Polyclonal antibodies
reactive to some Beta-Lactam antibiotics. Australian Journal of Dairy Technology,
De Haan P., de Jonge A.J.R, Verbrugge T. and Boorsma D.M., 1985. Three
epitope specific monoclonal antibodies against the hapten penicillin. Int. Arch.
Allergy Appl Immun. 76: 42-46.
De Leuw P., Kapa G. and Petz M., 1997. Production and Characterisation
of Multianalyte Antibodies against penicillins in egg yolk. J. of AOAC International
Nagakura N., Souma S., Shimizu T., Yanagihara Y., 1991. Anti-ampicillin
monoclonal antibodies and their cross-reactivities to various β-lactams. J.
of Antimicrobial Chemotherapy 28: 357-368.
Usleber, E., Lorber, M. Straka M., Terplan G., Martlbauer E., 1994.
Enzyme Immunoassay for the Detection of Isoxazolyl Penicillin Antibiotics in Milk.
Analyst, 119, 2765-2768.
Hapten, umfassend ein 6-[D-αAminoacetamido]-Penicillinderivat, das an
der α-Aminogruppe mit einem substituiertem oder unsubstituiertem Phenyldicarbaldehyd,
der aus der Gruppe ausgewählt ist, bestehend aus substituiertem oder unsubstituiertem
Phthalaldehyd, substituiertem oder unsubstituiertem Isophthalaldehyd und substituiertem
oder unsubstituiertem Terephthalaldehyd, vernetzt ist.
Hapten nach Anspruch 1, worin der Phenyldicarbaldehyd einen substituierten oder
unsubstituierten Terephthalaldehyd, bevorzugt einen unsubstituierten Terephthalaldehyd
Verfahren zur Herstellung eines Haptens nach Anspruch 1 oder 2, worin ein substituierter
oder unsubstituierter Phenyldicarbaldehyd mit einem 6-[D-α-Aminoacetamido]-Penicillinderivat
in einem geeigneten Lösungsmittel zur Reaktion gebracht wird.
Immunogen, umfassend ein Hapten nach Anspruch 1 oder 2, das an ein Antigenität
verleihendes Trägermaterial gekoppelt ist.
Immunogen nach Anspruch 4, worin das Trägermaterial ein Protein, ein Proteinfragment,
ein synthetisches Polypeptid oder ein semisynthetisches Polypeptid darstellt.
Antikörper, gebildet gegen ein Immunogen nach Anspruch 4 oder 5, worin die Antikörper
zur Bindung mit mindestens einem strukturellen Epitop von einem intakten β-Lactamring
Antikörper nach Anspruch 6, worin die Antikörper auf einem Trägersubstrat fixiert
Verfahren zur Herstellung der Antikörper nach Anspruch 6 oder 7, wobei das Verfahren
die Schritte des Immunisierens eines nicht humanen Tieres durch wiederholte Verabreichung
eines Immunogens nach Anspruch 4 oder 5 und des Sammelns der sich ergebenden Serumantikörper
aus dem immunisierten Tier umfasst.
Verfahren nach Anspruch 8, worin das Verfahren weiter das Fixieren der genannten
Serumantikörper an einem Trägersubstrat umfasst.
Konjugat, umfassend ein Hapten nach Anspruch 1 oder 2, das kovalent an ein nachweisbares
Markierungsmittel gebunden ist.
Konjugat nach Anspruch 10, worin das Markierungsmittel aus einem Enzym ausgewählt
ist, das bevorzugt eine Peroxidase, am bevorzugtesten Meerrettich-Peroxidase; eine
lumineszierende Substanz, die bevorzugt aus einer biolumineszierenden, chemilumineszierenden
oder fluoreszierenden Substanz ausgewählt ist; eine radioaktive Substanz; oder ein
Gemisch davon darstellt.
Verfahren zum Nachweis oder zur Bestimmung der Menge an β-Lactam-Penicillinen
in einer Probe, wobei das Verfahren Folgendes umfasst: Kontaktieren der Probe mit
einem Konjugat nach Anspruch 10 oder 11 oder einem Gemisch davon, und mit Antikörpern
nach Anspruch 6 oder 7 oder einem Gemisch davon; Nachweis oder Bestimmung der Menge
an gebundenem Konjugat; und Ableitung von einer Kalibrationskurve das Vorliegen
oder die Menge an β-Lactam-Penicillinen in der Probe.
Kit zum Nachweis oder zur Bestimmung der Menge an β-Lactam-Penicillinen,
wobei das Kit ein Konjugat nach Anspruch 10 oder 11 oder ein Gemisch davon und Antikörper
nach Anspruch 6 oder 7 oder ein Gemisch davon einschließt.
A hapten comprising a 6-[D-α-aminoacetamido] penicillin derivative crosslinked
at the α-amino group with a substituted or unsubstituted phenyldicarbaldehyde
selected from the group consisting of substituted or unsubstituted phthalaldehyde,
substituted or unsubstituted isophthalaldehyde and substituted or unsubstituted
A hapten according to claim 1, wherein the phenyldicarbaldehyde is a substituted
or unsubstituted terephthalaldehyde, preferably an unsubstituted terephthalaldehyde.
A process for preparing a hapten according to Claim 1 or 2, wherein a substituted
or unsubstituted phenyldicarbaldehyde is reacted with a 6-[D-α-aminoacetamido]penicillin
derivative in a suitable solvent.
An immunogen comprising a hapten according to Claim 1 or 2 coupled to an antigenicity-conferring
An immunogen according to Claim 4, wherein the carrier material is a protein,
a protein fragment, a synthetic polypeptide or a semi-synthetic polypeptide.
Antibodies raised against an immunogen according to Claim 4 or 5, wherein the
antibodies are capable of binding with at least one structural epitope of an intact
Antibodies according to Claim 6, wherein the antibodies are fixed on a backing
A process of preparing the antibodies according to Claim 6 or 7, the process
comprising the steps of immunising an non-human animal by repeated administration
of an immunogen according to Claim 4 or 5, and collecting the resulting serum antibodies
from the immunised animal.
A process according to Claim 8, wherein the process further comprises fixing
said serum antibodies to a backing substrate.
A conjugate comprising a hapten according to Claim 1 or 2 covalently bonded
to a labelling agent which is detectable.
A conjugate according to Claim 10, wherein the labelling agent is selected from
an enzyme, which is preferably a peroxidase, most preferably horseradish peroxidase;
a luminescent substance which is preferably selected from a bioluminescent, chemiluminescent
or fluorescent substance; a radioactive substance; or a mixture thereof.
A method for detecting, or determining the quantity of, β-lactam penicillins
in a sample, the method comprising contacting the sample with a conjugate according
to Claim 10 or 11, or a mixture thereof, and with antibodies according to Claim
6 or 7, or a mixture thereof; detecting, or determining the quantity of, bound conjugate;
and deducing from a calibration curve the presence, or amount of, β-lactam
penicillins in the sample.
A kit for detecting, or determining the quantity of, β-lactam penicillins,
the kit including a conjugate according to Claim 10 or 11, or a mixture thereof,
and antibodies according to Claim 6 or 7, or a mixture thereof.
Haptène comprenant un dérivé de la 6-[D-α-aminoacétamido]pénicilline réticulé
au groupe α-amino avec un phényldicarbaldéhyde substitué ou non substitué
choisi parmi le groupe constitué par un phthalaldéhyde substitué ou non substitué,
un isophthalaldéhyde substitué ou non substitué et un téréphthalaldéhyde substitué
ou non substitué.
Haptène selon la revendication 1, dans lequel le phényldicarbaldéhyde est un
téréphthalaldéhyde substitué ou non substitué, de préférence un téréphthalaldéhyde
Procédé de préparation d'un haptène selon la revendication 1 ou 2, dans lequel
un phényldicarbaldéhyde substitué ou non substitué est mis à réagir avec un dérivé
de la 6-[D-α-aminoacétamido]pénicilline dans un solvant approprié.
Immunogène comprenant un haptène selon la revendication 1 ou 2 couplé à un véhicule
Immunogène selon la revendication 4, dans lequel le véhicule est une protéine,
un fragment de protéine, un polypeptide synthétique ou un polypeptide semi-synthétique.
Anticorps dirigés contre un immunogène selon la revendication 4 ou 5, dans lequel
les anticorps sont capables de se lier avec au moins un épitope structurel d'un
noyau β-lactame intact.
Anticorps selon la revendication 6, dans lesquels les anticorps sont fixés sur
un substrat support.
Procédé de préparation des anticorps selon la revendication 6 ou 7, le procédé
comprenant les étapes consistant à immuniser un animal non humain par administration
répétée d'un immunogène selon la revendication 4 ou 5, et à recueillir les anticorps
du sérum résultant à partir de l'animal immunisé.
Procédé selon la revendication 8, dans lequel le procédé comprend en outre la
fixation desdits anticorps du sérum à un substrat support.
Conjugué comprenant un haptène selon la revendication 1 ou 2 lié de façon covalente
à un agent de marquage qui est détectable.
Conjugué selon la revendication 10, dans lequel l'agent de marquage est choisi
parmi une enzyme, laquelle est de préférence une peroxydase, de manière préférée
entre toutes une peroxydase de raifort ; une substance luminescente qui est de préférence
choisie parmi une substance bioluminescente, chimioluminescente ou fluorescente
; une substance radioactive ; ou une mélange de ces substances.
Procédé pour détecter les pénicillines à noyau β-lactame, ou pour en déterminer
la quantité, dans un échantillon, le procédé comprenant la mise en contact de l'échantillon
avec un conjugué selon la revendication 10 ou 11, ou avec un mélange de ces conjugués,
et avec les anticorps selon la revendication 6 ou 7, ou avec un mélange de ces anticorps
; la détection du conjugué lié ou la détermination de sa quantité ; et la déduction
à partir d'une courbe d'étalonnage de la présence, ou de la quantité, de pénicillines
à noyau β-lactame dans l'échantillon.
Kit pour détecter les pénicillines à noyau β-lactame, ou pour en déterminer
la quantité, le kit comprenant un conjugué selon la revendication 10 ou 11, ou un
mélange de ces conjugués, et les anticorps selon la revendication 6 ou 7, ou un
mélange de ces anticorps.