33
Journal of Immunological Methods, 147 (1992) 33-41 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06185
The development of an enzyme-linked immunosorbent assay (ELISA) for cephalexin Edward H. Kachab a
School of Pharmaceutics,
b
a,
Wen-Yang Wu
b
and Colin B. Chapman
a
School of Pharmaceutical Chemistry, VICtOrian CoUege of Pharmacy (MOfUlSh University), Parkville 3052, Australia
(Received 23 July 1991, revised received 27 September 1991, accepted 7 October 1991)
Cephalexin was structurally modified by the attachment of a spacer at the carboxylic acid through which it was subsequently covalently attached to BSA. This method permitted the molecule to be attached without cleavage of the f3-lactam ring giving a conjugate distinct from previously described immunogenic preparations of penicillins and cephalosporins. This approach required the development of a novel spacer molecule, and its synthesis and characterisation are reported. Rabbits were used to raise antisera and the antibodies produced were characterised with respect to their reactivity with cephalexin and various analogues, other cephalosporins, and a number of penicillins. Key words: Spacer; Hapten; Competitive inhibition; ELISA; Conjugate
Introduction Amongst the most widely used antibiotics for the treatment of bacterial infections in man and animals are the penicillins and the chemically related cephalosporins. In animals, they are also used for growth promotion (Kiser et aI., 1971). Hence, food derived from these animals such as milk, eggs, and meat may contain residues of these antibiotics which in tum may cause toxico-
Correspondence to: E.H. Kachab, School of Pharmaceutics, Victorian College of Pharmacy (Monash University), 381 Royal Parade, Parkville 3052, Australia (Tel.: 387-7222). Abbreviations: BSA, bovine serum albumin; sulpho-NHS, N-hydroxysulphosuccinimide; EOC, l-ethyl-3-{3-dimethylaminopropyJ) carbodiimide; KLH, keyhole limpet haemocyanin; ABTS, 2,2' -azino-bis{3-ethylbenzthiazoline-6-sulphonic acid); 7-ADCA, 7-aminodesacetoxycephalosporanic acid; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; 00, optical density.
logical and microbiological problems in man (WHO, 1969; FAO, 1985), including allergic side effects in sensitised individuals (Zimmerman, 1959; Borrie and Barret, 1961). Consequently, maximum residue levels have been specified for food products to try and control the levels of the drugs and drug residues reaching the public (Council of Europe, 1986; WHO, 1990). Whilst ELISAs are commercially available for the penicillins (Penicillin Assays Inc. of Maiden, MA, USA), none are available for the cephalosporins, including cephalexin which is one of the most commonly used cephalosporins. Although the cephalosporin family of antibiotics are not as widely used as the penicillins, their use in animals is increasing and will continue to increase as these drugs become cheaper and more readily available. Current assays for the cephalosporins are based on microbiological methods and rely on measuring the antibacterial effects of the drug (Corry et
34
aI., 1983). Such tests usually fail to detect metabolites and degradation products lacking antibacterial activity, but these compounds can have potent allergic properties due to structural similarities with the parent drug (Rico, 1986). Hence, the availability of an immunoassay which can detect the parent drug as well as metabolites and degradation products would be valuable. Traditionally, methods of coupling p-Iactam compounds onto macromolecular protein carriers to induce immunogenicity have involved hydrolysis of the p-Iactam ring and the direct formation of a covalent bond between the p-Iactam carbonyl carbon and the E-amino group of lysines in the protein molecules at high pH (Batchelor et aI., 1966; Chase and Williams, 1971; Haan et aI., 1985; Kitteringham et aI., 1987). In the present work coupling of cephalexin was achieved through a spacer (p-nitrobenzoxycarbonylmethyl chloride (pNB-methyl chloride» attached to the carboxylic acid thereby permitting the molecule to be exposed to the immune system in a more rigid and natural conformation. Our approach required the application of organic synthesis to achieve the desired cephalexin hapten (hetacephalexin carboxymethyl ester), and it was hoped that antibodies raised against this would provide new data on the immunogenic determinants of cephalexin. Additionally, and more importantly, it was hoped that the antisera would show high specificity for cephalexin with little if any cross-reactivity against other cephalosporins or penicillins, and could be used in the development of a specific ELISA for cephalexin.
Materials and methods Materials
4-nitrobenzyl alcohol was purchased from Aldrich, USA. Sulpho-NHS and EDC hydrochloride were from Pierce, USA. KLH, BSA (9899%), ABTS diammonium salt, cephalexin, penicillin G, cefotaxime, ampicillin sodium salts, moxalactam diammonium salt, phenethicillin potassium salt, 7-ADCA, and cefaclor were all purchased from Sigma, USA. Cephazolin sodium salt was a gift from Lilly, USA. Sephadex G-25 was from Pharmacia, Sweden. Flucloxacillin, ticar-
02N-0-CHzOH
02N~CH20COCH2CI
p-nilruben1.yl alcuhul
pNIl-melhyl chi uri de
Fig. 1. Scheme for the synthesis of p-nitrobenwxycarbonylmethyl chloride (pNB-methyl cIoride).
cillin sodium salts, and HRP, were obtained from Commonwealth Serum Laboratories, Australia. Microtitre plates were from Nunc, Denmark. All other reagents were of analytical grade, and solutions were made up in de ionised water. Synthesis of pNB-methyl chloride (Fig. 1)
Freshly distilled chloroacetyl chloride (3.3 g, 29 mmoI) was added dropwise, with stirring, to a mixture of p-nitrobenzyl alcohol (3 g, 20 mmoI) and triethylamine (2.17 g, 20 mmoI) in dry dichloromethane (100 mI) at O"c. Stirring was continued for 3-4 h as the temperature was slowly taken up to room temperature. The organic solution was then washed with an ice-cold solution of hydrochloric acid (0.1 M, 3 X 30 mI), water (3 X 30 mI), a solution of sodium carbonate (saturated, 3 X 30 ml), water (3 X 30 mI), and dried over magnesium sulphate. The solvent was then evaporated under vacuum to leave behind the desired product (4.4 g, 98%), which after recrystallisation from ethyl acetate / light petroleum (b.p. 6O-70"C) gave white needles, m.p. 70-71 0c. Thin layer chromatography (TLC) (ethyl acetate/ petrol (3 : 7» gave a single spot at R F 0.29 confirming the purity of this compound. Mass spectrometry gave a molecular ion at 229.016 corresponding to the molecular formula C 9 H s N04CI which confirmed the synthesis of this compound. Hapten preparation (Fig. 2) Hetacephalexin. Ice-cold triethylamine (0.75
ml, 5.4 mmoI) was added dropwise, with stirring, to a suspension of cephalexin (1 g, 2.9 mmol) in acetone (5 ml) at O°c. After 20-30 min the solid dissolved. The reaction mixture was then left to
, t-~-~--.----..""0s 00" 1"~:~:Ae>roNH H 0
_
I
I NH z
I
J!C-N
~
2) KOH SOLlmON
~O
~N---/SJ H]C
x
f ,
I
CH]
Cephalexin
I "'('
Hetacephalexin salt
~-lN ~KCH3 potassium
0
pNB.melbyl C1IJoridelDMF
Qr{-n
0 S
Ifetacephalexin p-nitroben:r.oxyCH] carbonylmethyl ester C=O
H3C~H3 of.- N ~ I
o
NazS·9H 201
(ACETONE/WATBR)
~o
! 9:0 fHZ C=O bI
2
~k---/?X I I H]C CH] f.- N ~ o
lIapten CH 3
C=O I
o I
CHz I
COOH Fig. 2. Scheme for the synthesis of hetacephalexin carboxymethyl ester (hapten).
warm up to room temperature, and stirred overnight. This solution was cooled in an ice bath and added dropwise, with stirring, to water (5 mO at 0-100C whilst carefully maintaining the pH at 2.5-3 with ice-cold sulphuric acid (2 M). The solution was then stirred at O-lOoC for 1 h and at pH 2.5-3, the acetone removed under vacuum at 24°C, and the remaining solution lyophilised. The residue was then extracted into acetone and the acetone removed under vacuum to leave behind
35
the free acid (0.85 g, 76%) as a colourless viscous oil which later gave a white foam. The purity of this compound was confirmed by TLC (methanolj ethyl acetate/0.1 M hydrochloric acid (3 : 6 : 1)), R F 0.38. Mass spectrometry gave a molecular ion peak at 387 corresponding to the molecular formula C19H21SN304 which confirmed the synthesis of this compound. The potassium salt was obtained by the dropwise addition of an ice-cold potassium hydroxide solution (0.5 M), with stirring, to a suspension of hetacephalexin (0.92 g, 2.4 mmon in water (5 mO at ODe. The pH was monitored throughout the addition until pH 8 was reached. The solution was then Iyophilised to yield the hetacephalexin potassium salt as a white solid. Hetacephalexin p-nitrobenzoxycarbonylmethyl ester. A mixture of hetacephalexin potassium salt (6.24 g, 14.7 mmoO, pNBCI 0.5 g, 6.6 mmol), potassium iodide 0.5 g, 9 mmol) in dry dimethylformamide (75 ml) was stirred under an atmosphere of nitrogen at room temperature for 24 h. The reaction mixture was diluted with ethyl acetate (200 ml) and then washed with an ice-cold solution of hydrochloric acid (0.1 M, 50 ml), a sodium chloride solution (saturated, 3 X 60 mI), and dried over magnesium sulphate. The solvent was then removed under vacuum at 24°C and the residue chromatographed on a silica gel column, 25 X 4.5 cm, with ethyl acetate. Fractions containing the product were combined together, the solvent evaporated, and the residue stirred in acetone (30 mI) overnight at room temperature. Evaporation of the solvent yielded the product (1.6 g, 46%) as a white fluffy solid. TLC (ethyl acetate) gave a single spot at RF 0.40 which confirmed the purity of this compound. Mass spectrometry gave a molecular ion peak at 580.197 corresponding to the molecular formula C28H2sN40sS which confirmed the synthesis of this compound. Hetacephalexin carboxymethyl ester (hapten). An ice-cold solution of sodium sulphide (0.2 M, 4.5 mO was added dropwise, with stirring, to a solution of hetacephalexin p-nitrobenzoxycarbonylmethyl ester (0.5 g, 0.88 mmol) in a mixture of acetone/water (4: 3) (36 mO at ODe. When the addition was complete, ice-cold ethyl acetate (20 mO was added and stirring was continued for 1
36
min to remove any unreacted starting material. The two layers were separated and ice-cold ethyl acetate (20 mI) added to the aqueous layer. The pH of the mixture was then adjusted to 3 by the dropwise addition of hydrochloric acid (0.1 M) and stirring. The ethyl acetate layer was separated and the aqueous layer reextracted with ethyl acetate (20 mI). The last two ethyl acetate extracts were combined together, washed with a sodium chloride solution (saturated, 3 x 10 mI), and dried over magnesium sulphate. Evaporation of the solvent under vacuum at 24°C gave the desired product (0.2 g, 52%) which recrystallised from acetone as a white solid, m.p. > 341°C. TLC (methano1! ethyl acetate/O.l M HCI (3: 6: 1)) gave a single spot at R F 0.39 confirming the purity of this compound. Mass spectrometry gave a molecular ion at 445.139 corresponding to the molecular formula of C21H23N306S which confirmed the synthesis of this compound. The synthesis of all the above compounds was further confirmed by 1H n.m.r. and infrared spectroscopy.
Preparation of hapten-protein conjugates To a solution of 50 mg of BSA in 0.1 M PBS pH 7.2 (4 mI) was added EDC (27 mg, 0.14 mmoI) and sulpho-NHS (30 mg, 0.14 mmoI) dissolved in 1 ml PBS. To this solution were then added 500 1'1 of hapten (50 mg, 0.11 mmoI) solution in dimethylformamide (1 mI), the pH adjusted to 7.2 with sodium hydroxide solution (1 M), and the reaction mixture stirred for 30 min at room temperature before the remainder of the solution was added. Stirring was continued for an additional 1 h and any precipitated material was removed by centrifugation. The reaction mixture was then purified by passing it through a Sephadex G-25 column and the protein conjugate eluted with de ionised water, lyophilised, and the solid stored desiccated under phosphorous pentoxide for 2 days at 4°C and then at - 200C. The coupling efficiency was estimated using differential UV absorbance against a BSA solution at 262 nm. The extinction coefficient in PBS was 6854 M - I. By this method 5 mol of hapten were found to be conjugated to 1 mol of BSA. A second conjugate was prepared with 9 mol of hapten
conjugated to 1 mol of BSA by increasing the ratio of reactants to BSA. A hapten-KLH conjugate was also prepared in a similar way to the BSA conjugates for use as an antigen to coat the ELISA plates. The number of moles of hapten bound per mole of KLH was found to be 171.
Immunisation of experimental animals Both of the hapten-BSA conjugates were used for raising antibodies. For each conjugate two New Zealand White rabbits were each initially injected intramuscularly with 1 ml of an emulsion of 2 ml Freund's complete adjuvant with 1 mlof hapten-BSA conjugate in 0.9% saline. The animals were subsequently boosted monthly with 1 ml of a 2 ml Freund's incomplete adjuvant and 1 ml hapten-BSA conjugate in 0.9% saline emulsion. Blood was obtained from each animal before each immunisation and the serum which was separated from each sample was stored at - 200C.
Measurement of antibody activity The optimal concentration of antigen coating to the microtitre plates was determined by titrating the rabbit antisera against hapten-KLH conjugate which was bound to the plates at concentrations ranging from 0.03 to 10 #Lg/ml. This concentration was found to be 5 #Lglml and was used in all plate coatings for subsequent competitive inhibition assays. Rabbit antisera were diluted from 1/2000 to 1/264,000 in PBS-Tween 20 containing 0.25% (w Iv) BSA. The presence of antibodies bound to the plates was detected using HRP enzyme conjugated to affinity purified antirabbit IgG antibody diluted 1/500 and employing H 20 21ABTS in citrate buffer as the enzyme substrate. All incubation steps were for 1 h at room temperature and were followed by washing three times with PBS-Tween 20 and three times additionally with de ionised water. Plates were coated with hapten-KLH conjugate by incubating overnight in PBS at 4°C.
Competitive inhibition assays Sera which were obtained late in the immunisation protocol and which were of the highest titres were used in competitive inhibition assays to assess the specificity and the reactivity of the
37
antibodies towards cephalexin and related compounds. Antisera raised with both the 5: 1 and the 9: 1 conjugates were used in this study, and the specificity of the antibodies was evaluated by measuring their inhibition from binding onto the plate by prior incubation of antisera with increasing concentrations of various antibiotics and derivatives of cephalosporin. The results of each assay were assessed with a Titertek Multiscan microtitre plate reader using a 480 om filter. Calculations and statistical analysis
Competitive inhibition curves were plotted as percentage inhibition against the logarithm of standard antigen concentration. Percentage inhibition was calculated as [1-(00 in the presence of antigen - 00 of background)/(OD in the absence of antigen - 00 of background)] X 100%. Background 00 values were measured using KLH alone as the antigen coating on the plate and typically were less than 2% of OD values obtained using the hapten-KLH conjugate as antigen coating. The coefficient of variation was less than 5% (n = 3). Results and discussion Antisera
Titres as high as 1/20,000 were obtained with both conjugates after only one or two booster injections. There was no difference in the immunogenicity of the 5 : 1 and the 9: 1 conjugates. The titres obtained for both conjugates were very similar throughout the immunisation protocol, and the highest titre of 1/40,000 found with the 5 : 1 conjugate (serum A) was also obtained with the 9: 1 conjugate (serum B) after seven booster injections. Preimmune sera from all rabbits immunised with the above conjugates did not show any reactivity with the hapten-KLH antigen coating.
TABLE I CONCENTRATION OF VARIOUS CEPHALOSPORINS REQUIRED TO INHIBIT 50% OF ANTIBODIES FROM BINDING ONTO THE PLATE Sera A and B were raised with the 5 : 1 and the 9: 1 conjugates respectively Compound
Drug concentration required for 50% inhibition (ng/ml) Serum B
Serum A Hapten Hetacephalexin Cephalexin Cephalexin cephalloic acid Cephadroxil Hetacephadroxil 7-ADCA Cefaclor Cefaloglycine Cephazolin Cefotaxime Moxalactam
2 10 50
7 50 250
60 150 40
127 750 400
4,000 50 19,000
350 40,000
50% inhibition was 50 ng/ml which is 25 times higher than the concentration of hapten required to achieve the same inhibition. The main difference in structure between cephalexin and its synthetically derived hapten is the presence of an isopropylidene group linking the amide nitrogen and the free amine in the aminophenylacetyl amino (APA) group via a five membered ring system and the spacer attached to the carboxylic acid (Fig. 2). The isopyrolodine group was not removed prior to conjugation or immunisation as 100.---------------~~~~~~h
Competitive inhibition with cephalexin and hapten
Competitive inhibition assays carried out using serum A showed a higher reactivity towards the hapten rather than cephalexin itself. The results are given in Table I and Fig. 3. The concentration of free cephalexin in the assay required to achieve
10
100
1000
10000 100000
Antigen concentration (ng/ml)
Fig. 3. Competitive inhibition curve with serum A diluted 1/30,OOO.Hapten, &; hetacephalexin, 0; cephaiexin, .; cefaclor, x; cephalexin cephalloic acid, A; hetacephadroxil, 0; cephadroxil, .; cefaloglycine, .; 7-ADCA, HI.
38
it has been previously shown to hydrolyse readily under physiological conditions in hetacillin (Kjaer et aI., 1977), a compound structurally related to the hapten (Hardcastle et aI., 1966), yielding the original AP A group. It was therefore hoped that the same would occur with the hapten. Additionally, during the conjugation step, the isopropylidene group helped to protect the ~-Iactam ring from hydrolysis (Tsuji et aI., 1977), thus preventing conjugation from occurring through the ~ lactam carbonyl carbon giving an impure conjugate. Also, the presence of the isopropylidene group shielded the amine group on the hapten and ensured that reaction occurred only between amine groups on the BSA and the carboxylic acids on the hapten molecules. However, the expected cleavage may not have occurred and consequently antibodies with a much higher reactivity towards the hapten were obtained. Competitive inhibition with hetacephalexin Since the reactivity of the antibodies towards the spacer skeleton could also result in a higher reactivity towards the hapten rather than cephalexin, hetacephalexin (Fig. 2) was included in the competitive inhibition assay. Hetacephalexin is identical in structure to the hapten except it lacks the attached spacer. With this compound, the concentration required for 50% inhibition was only 10 nglml which compared well with the original 2 nglml found with the hapten. This indicated that our antibodies had little reactivity towards the spacer and the differences in drug concentrations required for 50% inhibition between the hapten and cephalexin was clearly mainly due to a high reactivity of the antibodies towards the isopropylidene group. Similar results were observed with serum B (Table I and Fig. 4) demonstrating a high reactivity towards the hapten with 50% inhibition occurring at drug concentrations of 7 nglml of hapten, and also with 50 and 250 nglml of hetacephalexin and cephalexin respectively.
Competitive inhibition with 7-ADCA In order to determine the extent of reactivity towards the cepham nucleus (Table II) which is
l00~----------------~~~-=~
.1
1
10
100
1000
10000 100000
Antigen concentration (ng/ml)
Fig. 4. Competitive inhibition curve with serum B diluted 1/30,OOO.Hapten, .; hetacephalexin. 0; cephalexin •• ; cefac1or, x ; cephalexin cephalloic acid, 1:>.; hetacephadroxil, 0; cephadroxil, . ; cefaloglycine. e; 7-ADCA, III.
the common structural moiety to all cephalosporins, and the APA group, a competitive inhibition assay was carried out using 7-ADCA. This compound forms the original cepham nucleus and lacks the AP A group. Any strong inhibition by this compound may serve as a measure of preferential reactivity towards the cepham nucleus. Competitive inhibition assays carried out with serum A gave 50% inhibition at a concentration of 4 p.g/ml which is 80-fold less reactive than cephalexin and 2000-fold less reactive than the hapten. With serum B, 7-ADCA acted as an extremely weak inhibitor and 50% inhibition could not be measured even at concentrations as high as 64 p.g/ml which is 32,OOO-fold more than the hapten concentration required to achieve this inhibition. These results would suggest that the antibodies raised with both conjugates had the strongest reactivity towards the AP A group, with little reactivity towards the cepham nucleus. However, we must bear in mind that since 7ADCA represents only about half of the cephalexin molecule, antibodies bound to 7ADCA may have a lower avidity compared to the same antibodies bound to the intact cephalexin molecule. Competitive inhibition with other cephalosporins Cefaclor has an identical chemical structure to cephalexin except that the methyl group at C-3 is replaced by a chlorine. The standard inhibition curve observed for cefaclor was almost identical to that of cephalexin using both antisera. The 50% inhibitory concentrations were 50 ng/ml
39
TABLE II STRUcruRES OF CEPHALOSPORINS TESTED
R,C .... \I
~:t!~ ~~IH 7
6
2"
ON.,&::
o
4
H R'
COOH Cepham nucleus Compound
R Group
CH 3
7-NH2
7-ADCA Cephadroxil
R' Group
CH 3
HO-o-CHNH2
Hetacephadroxil HO--G-::r-i.° CH 3 N-C-7 NH H3C
X
CH 3
Cefaclor
o-CHNH 2
a
Cefaloglycine
o-CHNH 2
OOCCH 3
Cefotaxime H2N Cephazolin
N"f(C, )l_) ~NOCH3 S
N=N
N-N
N ....
C
I
~
I
N
\I
'CH2
-0-
Moxalactam HO (1-0 analogue of cepham nucleus)
"CHCOOH '\'\" 7-OCH3 ~
' OT.
o
Cephalexin cephalloic acid
CHZS.....
-
1\
'S..... C 'CH 3 N-N
~
1\
CH S ..... , ;N 2 ~ CH 3
110
CII_HN---/S
NH2 HOOC N .,&:: H
CH 3
COOH
and 350 ng/ml for sera A and B respectively which compared very closely with values of 50 ng/ml and 250 ng/ml obtained using the same sera with cephalexin. This initially indicated that
both antisera had no reactivity towards the methyl group and the previously observed reactivity towards the cepham nucleus was likely to have been directed towards and along the C2-S-C6-C7 skeleton. However, it should be noted that since the chlorine group is much smaller than the methyl group, it may not cause significant steric interference and reduction in the reactivity of the antibodies along the C2-C3 skeleton. In order to investigate this possibility a competitive inhibition assay was carried out using cefaloglycine which contains an acetyl group at C-3. With this slight increase in the size of the substituent at C-3 the amount of drug required to achieve 50% inhibition increased dramatically to 19,000 and 40,000 ng/ml for sera A and B respectively. Thus, in contrast to the results obtained with 7-AOCA and cefaclor, the reactivity of the antibodies may also be directed towards the cepham nucleus and especially towards the methyl group at C-3. Such antibody specificity would be in agreement with previously reported data on the immunogenicity of cephalexin (Peterson and Graham, 1974). The hydrogen at position four on the benzene ring in cephalexin is replaced in cephadroxil with the much larger hydroxyl group and this could create steric hindrance for antibodies with a reactivity towards the benzyl group thereby resulting in a weaker antigen-antibody interaction around that group. In the competitive inhibition assay, cephadroxil was a less efficient inhibitor than both cephalexin and the hapten with 50% inhibition occurring at concentrations of 150 ng/ml for serum A and 750 ng/ml for serum B. However, with hetacephadroxil, the concentrations required for 50% inhibition were 40 and 400 ng/ml for sera A and B respectively. This reduction in concentration again suggested that the antibodies exhibit a higher reactivity towards the isopropylidene group, as discussed earlier. In order to assess whether or not cross-reactivity occurred with other cephalosporins, three compounds with a much greater structural dissimilarity to cephalexin were used in the competitive inhibition assay. These compounds; cephazolin, cefotaxime, and moxalactam, exhibited detectable inhibition only at very high concentrations (Table I) and none were able to achieve 50% inhibition even at concentrations as high as 64 #£g/ml.
40
Competitive inhibition with cephalexin cephalloic acid
Cephalexin cephalloic acid is an analogue of cephalexin in which the J3-lactam ring has been hydrolysed. It was important to perform a competitive inhibition assay with this drug in order to explore the possible presence of any reactivity of the antibodies towards the J3-lactam ring. The concentration of drug required to achieve 50% inhibition was 60 and 127 nglml for sera A and B respectively. This compared very closely with the values obtained with cephalexin which showed that the antibodies had little or no reactivity towards the J3-lactam ring. Competitive inhibition with penicillins
The main differences between the cepham nucleus and the penam nucleus, the common structural moiety of all penicillins, is that the cepham nucleus contains a dihydrothiazine ring with two hydrogens at position two and the carboxylic acid being located at position four, whereas the penam nucleus contains a thiazolidine ring system with two methyl groups in place of the two hydrogens at position two and the carboxylic acid at position three in the ring. Also, the angle between the two fused rings in the penam nucleus is different from that of cepham. In our studies, it was important to include some commonly used penicillins in a competitive inhibition assay since differences in their nuclei may not be significant enough to deter high cross-reactivities from occurring and especially since cross-reactivity may also occur towards the AP A group which penicillins such as ampicillin also have. The chemical structures of the various penicillins studied are given in Table III. None of the penicillins (benzylpenicillin, ampicillin, ticarcillin, phenethicillin and flucloxacillin) gave 50% inhibition values even at concentrations as high up as 64 /Lg/ml. Hence, the results of the competitive inhibition assays suggest that the reactivity of the antibodies is directed towards and along the C7-C6S-C2-C3 skeleton of the cepham nucleus and the acyl chain, with the methyl group at C-3 and the isopropylidene group playing an important role in the reactivity, and with little reactivity occurring along the spacer skeleton or the J3-lactam ring.
TABLE III STRUCTURES OF PENICILLINS TESTED H R...... C N)jCJ
Journal of Immunological Methods, 147 (1992) 33-41 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06185
The development of an enzyme-linked immunosorbent assay (ELISA) for cephalexin Edward H. Kachab a
School of Pharmaceutics,
b
a,
Wen-Yang Wu
b
and Colin B. Chapman
a
School of Pharmaceutical Chemistry, VICtOrian CoUege of Pharmacy (MOfUlSh University), Parkville 3052, Australia
(Received 23 July 1991, revised received 27 September 1991, accepted 7 October 1991)
Cephalexin was structurally modified by the attachment of a spacer at the carboxylic acid through which it was subsequently covalently attached to BSA. This method permitted the molecule to be attached without cleavage of the f3-lactam ring giving a conjugate distinct from previously described immunogenic preparations of penicillins and cephalosporins. This approach required the development of a novel spacer molecule, and its synthesis and characterisation are reported. Rabbits were used to raise antisera and the antibodies produced were characterised with respect to their reactivity with cephalexin and various analogues, other cephalosporins, and a number of penicillins. Key words: Spacer; Hapten; Competitive inhibition; ELISA; Conjugate
Introduction Amongst the most widely used antibiotics for the treatment of bacterial infections in man and animals are the penicillins and the chemically related cephalosporins. In animals, they are also used for growth promotion (Kiser et aI., 1971). Hence, food derived from these animals such as milk, eggs, and meat may contain residues of these antibiotics which in tum may cause toxico-
Correspondence to: E.H. Kachab, School of Pharmaceutics, Victorian College of Pharmacy (Monash University), 381 Royal Parade, Parkville 3052, Australia (Tel.: 387-7222). Abbreviations: BSA, bovine serum albumin; sulpho-NHS, N-hydroxysulphosuccinimide; EOC, l-ethyl-3-{3-dimethylaminopropyJ) carbodiimide; KLH, keyhole limpet haemocyanin; ABTS, 2,2' -azino-bis{3-ethylbenzthiazoline-6-sulphonic acid); 7-ADCA, 7-aminodesacetoxycephalosporanic acid; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; 00, optical density.
logical and microbiological problems in man (WHO, 1969; FAO, 1985), including allergic side effects in sensitised individuals (Zimmerman, 1959; Borrie and Barret, 1961). Consequently, maximum residue levels have been specified for food products to try and control the levels of the drugs and drug residues reaching the public (Council of Europe, 1986; WHO, 1990). Whilst ELISAs are commercially available for the penicillins (Penicillin Assays Inc. of Maiden, MA, USA), none are available for the cephalosporins, including cephalexin which is one of the most commonly used cephalosporins. Although the cephalosporin family of antibiotics are not as widely used as the penicillins, their use in animals is increasing and will continue to increase as these drugs become cheaper and more readily available. Current assays for the cephalosporins are based on microbiological methods and rely on measuring the antibacterial effects of the drug (Corry et
34
aI., 1983). Such tests usually fail to detect metabolites and degradation products lacking antibacterial activity, but these compounds can have potent allergic properties due to structural similarities with the parent drug (Rico, 1986). Hence, the availability of an immunoassay which can detect the parent drug as well as metabolites and degradation products would be valuable. Traditionally, methods of coupling p-Iactam compounds onto macromolecular protein carriers to induce immunogenicity have involved hydrolysis of the p-Iactam ring and the direct formation of a covalent bond between the p-Iactam carbonyl carbon and the E-amino group of lysines in the protein molecules at high pH (Batchelor et aI., 1966; Chase and Williams, 1971; Haan et aI., 1985; Kitteringham et aI., 1987). In the present work coupling of cephalexin was achieved through a spacer (p-nitrobenzoxycarbonylmethyl chloride (pNB-methyl chloride» attached to the carboxylic acid thereby permitting the molecule to be exposed to the immune system in a more rigid and natural conformation. Our approach required the application of organic synthesis to achieve the desired cephalexin hapten (hetacephalexin carboxymethyl ester), and it was hoped that antibodies raised against this would provide new data on the immunogenic determinants of cephalexin. Additionally, and more importantly, it was hoped that the antisera would show high specificity for cephalexin with little if any cross-reactivity against other cephalosporins or penicillins, and could be used in the development of a specific ELISA for cephalexin.
Materials and methods Materials
4-nitrobenzyl alcohol was purchased from Aldrich, USA. Sulpho-NHS and EDC hydrochloride were from Pierce, USA. KLH, BSA (9899%), ABTS diammonium salt, cephalexin, penicillin G, cefotaxime, ampicillin sodium salts, moxalactam diammonium salt, phenethicillin potassium salt, 7-ADCA, and cefaclor were all purchased from Sigma, USA. Cephazolin sodium salt was a gift from Lilly, USA. Sephadex G-25 was from Pharmacia, Sweden. Flucloxacillin, ticar-
02N-0-CHzOH
02N~CH20COCH2CI
p-nilruben1.yl alcuhul
pNIl-melhyl chi uri de
Fig. 1. Scheme for the synthesis of p-nitrobenwxycarbonylmethyl chloride (pNB-methyl cIoride).
cillin sodium salts, and HRP, were obtained from Commonwealth Serum Laboratories, Australia. Microtitre plates were from Nunc, Denmark. All other reagents were of analytical grade, and solutions were made up in de ionised water. Synthesis of pNB-methyl chloride (Fig. 1)
Freshly distilled chloroacetyl chloride (3.3 g, 29 mmoI) was added dropwise, with stirring, to a mixture of p-nitrobenzyl alcohol (3 g, 20 mmoI) and triethylamine (2.17 g, 20 mmoI) in dry dichloromethane (100 mI) at O"c. Stirring was continued for 3-4 h as the temperature was slowly taken up to room temperature. The organic solution was then washed with an ice-cold solution of hydrochloric acid (0.1 M, 3 X 30 mI), water (3 X 30 mI), a solution of sodium carbonate (saturated, 3 X 30 ml), water (3 X 30 mI), and dried over magnesium sulphate. The solvent was then evaporated under vacuum to leave behind the desired product (4.4 g, 98%), which after recrystallisation from ethyl acetate / light petroleum (b.p. 6O-70"C) gave white needles, m.p. 70-71 0c. Thin layer chromatography (TLC) (ethyl acetate/ petrol (3 : 7» gave a single spot at R F 0.29 confirming the purity of this compound. Mass spectrometry gave a molecular ion at 229.016 corresponding to the molecular formula C 9 H s N04CI which confirmed the synthesis of this compound. Hapten preparation (Fig. 2) Hetacephalexin. Ice-cold triethylamine (0.75
ml, 5.4 mmoI) was added dropwise, with stirring, to a suspension of cephalexin (1 g, 2.9 mmol) in acetone (5 ml) at O°c. After 20-30 min the solid dissolved. The reaction mixture was then left to
, t-~-~--.----..""0s 00" 1"~:~:Ae>roNH H 0
_
I
I NH z
I
J!C-N
~
2) KOH SOLlmON
~O
~N---/SJ H]C
x
f ,
I
CH]
Cephalexin
I "'('
Hetacephalexin salt
~-lN ~KCH3 potassium
0
pNB.melbyl C1IJoridelDMF
Qr{-n
0 S
Ifetacephalexin p-nitroben:r.oxyCH] carbonylmethyl ester C=O
H3C~H3 of.- N ~ I
o
NazS·9H 201
(ACETONE/WATBR)
~o
! 9:0 fHZ C=O bI
2
~k---/?X I I H]C CH] f.- N ~ o
lIapten CH 3
C=O I
o I
CHz I
COOH Fig. 2. Scheme for the synthesis of hetacephalexin carboxymethyl ester (hapten).
warm up to room temperature, and stirred overnight. This solution was cooled in an ice bath and added dropwise, with stirring, to water (5 mO at 0-100C whilst carefully maintaining the pH at 2.5-3 with ice-cold sulphuric acid (2 M). The solution was then stirred at O-lOoC for 1 h and at pH 2.5-3, the acetone removed under vacuum at 24°C, and the remaining solution lyophilised. The residue was then extracted into acetone and the acetone removed under vacuum to leave behind
35
the free acid (0.85 g, 76%) as a colourless viscous oil which later gave a white foam. The purity of this compound was confirmed by TLC (methanolj ethyl acetate/0.1 M hydrochloric acid (3 : 6 : 1)), R F 0.38. Mass spectrometry gave a molecular ion peak at 387 corresponding to the molecular formula C19H21SN304 which confirmed the synthesis of this compound. The potassium salt was obtained by the dropwise addition of an ice-cold potassium hydroxide solution (0.5 M), with stirring, to a suspension of hetacephalexin (0.92 g, 2.4 mmon in water (5 mO at ODe. The pH was monitored throughout the addition until pH 8 was reached. The solution was then Iyophilised to yield the hetacephalexin potassium salt as a white solid. Hetacephalexin p-nitrobenzoxycarbonylmethyl ester. A mixture of hetacephalexin potassium salt (6.24 g, 14.7 mmoO, pNBCI 0.5 g, 6.6 mmol), potassium iodide 0.5 g, 9 mmol) in dry dimethylformamide (75 ml) was stirred under an atmosphere of nitrogen at room temperature for 24 h. The reaction mixture was diluted with ethyl acetate (200 ml) and then washed with an ice-cold solution of hydrochloric acid (0.1 M, 50 ml), a sodium chloride solution (saturated, 3 X 60 mI), and dried over magnesium sulphate. The solvent was then removed under vacuum at 24°C and the residue chromatographed on a silica gel column, 25 X 4.5 cm, with ethyl acetate. Fractions containing the product were combined together, the solvent evaporated, and the residue stirred in acetone (30 mI) overnight at room temperature. Evaporation of the solvent yielded the product (1.6 g, 46%) as a white fluffy solid. TLC (ethyl acetate) gave a single spot at RF 0.40 which confirmed the purity of this compound. Mass spectrometry gave a molecular ion peak at 580.197 corresponding to the molecular formula C28H2sN40sS which confirmed the synthesis of this compound. Hetacephalexin carboxymethyl ester (hapten). An ice-cold solution of sodium sulphide (0.2 M, 4.5 mO was added dropwise, with stirring, to a solution of hetacephalexin p-nitrobenzoxycarbonylmethyl ester (0.5 g, 0.88 mmol) in a mixture of acetone/water (4: 3) (36 mO at ODe. When the addition was complete, ice-cold ethyl acetate (20 mO was added and stirring was continued for 1
36
min to remove any unreacted starting material. The two layers were separated and ice-cold ethyl acetate (20 mI) added to the aqueous layer. The pH of the mixture was then adjusted to 3 by the dropwise addition of hydrochloric acid (0.1 M) and stirring. The ethyl acetate layer was separated and the aqueous layer reextracted with ethyl acetate (20 mI). The last two ethyl acetate extracts were combined together, washed with a sodium chloride solution (saturated, 3 x 10 mI), and dried over magnesium sulphate. Evaporation of the solvent under vacuum at 24°C gave the desired product (0.2 g, 52%) which recrystallised from acetone as a white solid, m.p. > 341°C. TLC (methano1! ethyl acetate/O.l M HCI (3: 6: 1)) gave a single spot at R F 0.39 confirming the purity of this compound. Mass spectrometry gave a molecular ion at 445.139 corresponding to the molecular formula of C21H23N306S which confirmed the synthesis of this compound. The synthesis of all the above compounds was further confirmed by 1H n.m.r. and infrared spectroscopy.
Preparation of hapten-protein conjugates To a solution of 50 mg of BSA in 0.1 M PBS pH 7.2 (4 mI) was added EDC (27 mg, 0.14 mmoI) and sulpho-NHS (30 mg, 0.14 mmoI) dissolved in 1 ml PBS. To this solution were then added 500 1'1 of hapten (50 mg, 0.11 mmoI) solution in dimethylformamide (1 mI), the pH adjusted to 7.2 with sodium hydroxide solution (1 M), and the reaction mixture stirred for 30 min at room temperature before the remainder of the solution was added. Stirring was continued for an additional 1 h and any precipitated material was removed by centrifugation. The reaction mixture was then purified by passing it through a Sephadex G-25 column and the protein conjugate eluted with de ionised water, lyophilised, and the solid stored desiccated under phosphorous pentoxide for 2 days at 4°C and then at - 200C. The coupling efficiency was estimated using differential UV absorbance against a BSA solution at 262 nm. The extinction coefficient in PBS was 6854 M - I. By this method 5 mol of hapten were found to be conjugated to 1 mol of BSA. A second conjugate was prepared with 9 mol of hapten
conjugated to 1 mol of BSA by increasing the ratio of reactants to BSA. A hapten-KLH conjugate was also prepared in a similar way to the BSA conjugates for use as an antigen to coat the ELISA plates. The number of moles of hapten bound per mole of KLH was found to be 171.
Immunisation of experimental animals Both of the hapten-BSA conjugates were used for raising antibodies. For each conjugate two New Zealand White rabbits were each initially injected intramuscularly with 1 ml of an emulsion of 2 ml Freund's complete adjuvant with 1 mlof hapten-BSA conjugate in 0.9% saline. The animals were subsequently boosted monthly with 1 ml of a 2 ml Freund's incomplete adjuvant and 1 ml hapten-BSA conjugate in 0.9% saline emulsion. Blood was obtained from each animal before each immunisation and the serum which was separated from each sample was stored at - 200C.
Measurement of antibody activity The optimal concentration of antigen coating to the microtitre plates was determined by titrating the rabbit antisera against hapten-KLH conjugate which was bound to the plates at concentrations ranging from 0.03 to 10 #Lg/ml. This concentration was found to be 5 #Lglml and was used in all plate coatings for subsequent competitive inhibition assays. Rabbit antisera were diluted from 1/2000 to 1/264,000 in PBS-Tween 20 containing 0.25% (w Iv) BSA. The presence of antibodies bound to the plates was detected using HRP enzyme conjugated to affinity purified antirabbit IgG antibody diluted 1/500 and employing H 20 21ABTS in citrate buffer as the enzyme substrate. All incubation steps were for 1 h at room temperature and were followed by washing three times with PBS-Tween 20 and three times additionally with de ionised water. Plates were coated with hapten-KLH conjugate by incubating overnight in PBS at 4°C.
Competitive inhibition assays Sera which were obtained late in the immunisation protocol and which were of the highest titres were used in competitive inhibition assays to assess the specificity and the reactivity of the
37
antibodies towards cephalexin and related compounds. Antisera raised with both the 5: 1 and the 9: 1 conjugates were used in this study, and the specificity of the antibodies was evaluated by measuring their inhibition from binding onto the plate by prior incubation of antisera with increasing concentrations of various antibiotics and derivatives of cephalosporin. The results of each assay were assessed with a Titertek Multiscan microtitre plate reader using a 480 om filter. Calculations and statistical analysis
Competitive inhibition curves were plotted as percentage inhibition against the logarithm of standard antigen concentration. Percentage inhibition was calculated as [1-(00 in the presence of antigen - 00 of background)/(OD in the absence of antigen - 00 of background)] X 100%. Background 00 values were measured using KLH alone as the antigen coating on the plate and typically were less than 2% of OD values obtained using the hapten-KLH conjugate as antigen coating. The coefficient of variation was less than 5% (n = 3). Results and discussion Antisera
Titres as high as 1/20,000 were obtained with both conjugates after only one or two booster injections. There was no difference in the immunogenicity of the 5 : 1 and the 9: 1 conjugates. The titres obtained for both conjugates were very similar throughout the immunisation protocol, and the highest titre of 1/40,000 found with the 5 : 1 conjugate (serum A) was also obtained with the 9: 1 conjugate (serum B) after seven booster injections. Preimmune sera from all rabbits immunised with the above conjugates did not show any reactivity with the hapten-KLH antigen coating.
TABLE I CONCENTRATION OF VARIOUS CEPHALOSPORINS REQUIRED TO INHIBIT 50% OF ANTIBODIES FROM BINDING ONTO THE PLATE Sera A and B were raised with the 5 : 1 and the 9: 1 conjugates respectively Compound
Drug concentration required for 50% inhibition (ng/ml) Serum B
Serum A Hapten Hetacephalexin Cephalexin Cephalexin cephalloic acid Cephadroxil Hetacephadroxil 7-ADCA Cefaclor Cefaloglycine Cephazolin Cefotaxime Moxalactam
2 10 50
7 50 250
60 150 40
127 750 400
4,000 50 19,000
350 40,000
50% inhibition was 50 ng/ml which is 25 times higher than the concentration of hapten required to achieve the same inhibition. The main difference in structure between cephalexin and its synthetically derived hapten is the presence of an isopropylidene group linking the amide nitrogen and the free amine in the aminophenylacetyl amino (APA) group via a five membered ring system and the spacer attached to the carboxylic acid (Fig. 2). The isopyrolodine group was not removed prior to conjugation or immunisation as 100.---------------~~~~~~h
Competitive inhibition with cephalexin and hapten
Competitive inhibition assays carried out using serum A showed a higher reactivity towards the hapten rather than cephalexin itself. The results are given in Table I and Fig. 3. The concentration of free cephalexin in the assay required to achieve
10
100
1000
10000 100000
Antigen concentration (ng/ml)
Fig. 3. Competitive inhibition curve with serum A diluted 1/30,OOO.Hapten, &; hetacephalexin, 0; cephaiexin, .; cefaclor, x; cephalexin cephalloic acid, A; hetacephadroxil, 0; cephadroxil, .; cefaloglycine, .; 7-ADCA, HI.
38
it has been previously shown to hydrolyse readily under physiological conditions in hetacillin (Kjaer et aI., 1977), a compound structurally related to the hapten (Hardcastle et aI., 1966), yielding the original AP A group. It was therefore hoped that the same would occur with the hapten. Additionally, during the conjugation step, the isopropylidene group helped to protect the ~-Iactam ring from hydrolysis (Tsuji et aI., 1977), thus preventing conjugation from occurring through the ~ lactam carbonyl carbon giving an impure conjugate. Also, the presence of the isopropylidene group shielded the amine group on the hapten and ensured that reaction occurred only between amine groups on the BSA and the carboxylic acids on the hapten molecules. However, the expected cleavage may not have occurred and consequently antibodies with a much higher reactivity towards the hapten were obtained. Competitive inhibition with hetacephalexin Since the reactivity of the antibodies towards the spacer skeleton could also result in a higher reactivity towards the hapten rather than cephalexin, hetacephalexin (Fig. 2) was included in the competitive inhibition assay. Hetacephalexin is identical in structure to the hapten except it lacks the attached spacer. With this compound, the concentration required for 50% inhibition was only 10 nglml which compared well with the original 2 nglml found with the hapten. This indicated that our antibodies had little reactivity towards the spacer and the differences in drug concentrations required for 50% inhibition between the hapten and cephalexin was clearly mainly due to a high reactivity of the antibodies towards the isopropylidene group. Similar results were observed with serum B (Table I and Fig. 4) demonstrating a high reactivity towards the hapten with 50% inhibition occurring at drug concentrations of 7 nglml of hapten, and also with 50 and 250 nglml of hetacephalexin and cephalexin respectively.
Competitive inhibition with 7-ADCA In order to determine the extent of reactivity towards the cepham nucleus (Table II) which is
l00~----------------~~~-=~
.1
1
10
100
1000
10000 100000
Antigen concentration (ng/ml)
Fig. 4. Competitive inhibition curve with serum B diluted 1/30,OOO.Hapten, .; hetacephalexin. 0; cephalexin •• ; cefac1or, x ; cephalexin cephalloic acid, 1:>.; hetacephadroxil, 0; cephadroxil, . ; cefaloglycine. e; 7-ADCA, III.
the common structural moiety to all cephalosporins, and the APA group, a competitive inhibition assay was carried out using 7-ADCA. This compound forms the original cepham nucleus and lacks the AP A group. Any strong inhibition by this compound may serve as a measure of preferential reactivity towards the cepham nucleus. Competitive inhibition assays carried out with serum A gave 50% inhibition at a concentration of 4 p.g/ml which is 80-fold less reactive than cephalexin and 2000-fold less reactive than the hapten. With serum B, 7-ADCA acted as an extremely weak inhibitor and 50% inhibition could not be measured even at concentrations as high as 64 p.g/ml which is 32,OOO-fold more than the hapten concentration required to achieve this inhibition. These results would suggest that the antibodies raised with both conjugates had the strongest reactivity towards the AP A group, with little reactivity towards the cepham nucleus. However, we must bear in mind that since 7ADCA represents only about half of the cephalexin molecule, antibodies bound to 7ADCA may have a lower avidity compared to the same antibodies bound to the intact cephalexin molecule. Competitive inhibition with other cephalosporins Cefaclor has an identical chemical structure to cephalexin except that the methyl group at C-3 is replaced by a chlorine. The standard inhibition curve observed for cefaclor was almost identical to that of cephalexin using both antisera. The 50% inhibitory concentrations were 50 ng/ml
39
TABLE II STRUcruRES OF CEPHALOSPORINS TESTED
R,C .... \I
~:t!~ ~~IH 7
6
2"
ON.,&::
o
4
H R'
COOH Cepham nucleus Compound
R Group
CH 3
7-NH2
7-ADCA Cephadroxil
R' Group
CH 3
HO-o-CHNH2
Hetacephadroxil HO--G-::r-i.° CH 3 N-C-7 NH H3C
X
CH 3
Cefaclor
o-CHNH 2
a
Cefaloglycine
o-CHNH 2
OOCCH 3
Cefotaxime H2N Cephazolin
N"f(C, )l_) ~NOCH3 S
N=N
N-N
N ....
C
I
~
I
N
\I
'CH2
-0-
Moxalactam HO (1-0 analogue of cepham nucleus)
"CHCOOH '\'\" 7-OCH3 ~
' OT.
o
Cephalexin cephalloic acid
CHZS.....
-
1\
'S..... C 'CH 3 N-N
~
1\
CH S ..... , ;N 2 ~ CH 3
110
CII_HN---/S
NH2 HOOC N .,&:: H
CH 3
COOH
and 350 ng/ml for sera A and B respectively which compared very closely with values of 50 ng/ml and 250 ng/ml obtained using the same sera with cephalexin. This initially indicated that
both antisera had no reactivity towards the methyl group and the previously observed reactivity towards the cepham nucleus was likely to have been directed towards and along the C2-S-C6-C7 skeleton. However, it should be noted that since the chlorine group is much smaller than the methyl group, it may not cause significant steric interference and reduction in the reactivity of the antibodies along the C2-C3 skeleton. In order to investigate this possibility a competitive inhibition assay was carried out using cefaloglycine which contains an acetyl group at C-3. With this slight increase in the size of the substituent at C-3 the amount of drug required to achieve 50% inhibition increased dramatically to 19,000 and 40,000 ng/ml for sera A and B respectively. Thus, in contrast to the results obtained with 7-AOCA and cefaclor, the reactivity of the antibodies may also be directed towards the cepham nucleus and especially towards the methyl group at C-3. Such antibody specificity would be in agreement with previously reported data on the immunogenicity of cephalexin (Peterson and Graham, 1974). The hydrogen at position four on the benzene ring in cephalexin is replaced in cephadroxil with the much larger hydroxyl group and this could create steric hindrance for antibodies with a reactivity towards the benzyl group thereby resulting in a weaker antigen-antibody interaction around that group. In the competitive inhibition assay, cephadroxil was a less efficient inhibitor than both cephalexin and the hapten with 50% inhibition occurring at concentrations of 150 ng/ml for serum A and 750 ng/ml for serum B. However, with hetacephadroxil, the concentrations required for 50% inhibition were 40 and 400 ng/ml for sera A and B respectively. This reduction in concentration again suggested that the antibodies exhibit a higher reactivity towards the isopropylidene group, as discussed earlier. In order to assess whether or not cross-reactivity occurred with other cephalosporins, three compounds with a much greater structural dissimilarity to cephalexin were used in the competitive inhibition assay. These compounds; cephazolin, cefotaxime, and moxalactam, exhibited detectable inhibition only at very high concentrations (Table I) and none were able to achieve 50% inhibition even at concentrations as high as 64 #£g/ml.
40
Competitive inhibition with cephalexin cephalloic acid
Cephalexin cephalloic acid is an analogue of cephalexin in which the J3-lactam ring has been hydrolysed. It was important to perform a competitive inhibition assay with this drug in order to explore the possible presence of any reactivity of the antibodies towards the J3-lactam ring. The concentration of drug required to achieve 50% inhibition was 60 and 127 nglml for sera A and B respectively. This compared very closely with the values obtained with cephalexin which showed that the antibodies had little or no reactivity towards the J3-lactam ring. Competitive inhibition with penicillins
The main differences between the cepham nucleus and the penam nucleus, the common structural moiety of all penicillins, is that the cepham nucleus contains a dihydrothiazine ring with two hydrogens at position two and the carboxylic acid being located at position four, whereas the penam nucleus contains a thiazolidine ring system with two methyl groups in place of the two hydrogens at position two and the carboxylic acid at position three in the ring. Also, the angle between the two fused rings in the penam nucleus is different from that of cepham. In our studies, it was important to include some commonly used penicillins in a competitive inhibition assay since differences in their nuclei may not be significant enough to deter high cross-reactivities from occurring and especially since cross-reactivity may also occur towards the AP A group which penicillins such as ampicillin also have. The chemical structures of the various penicillins studied are given in Table III. None of the penicillins (benzylpenicillin, ampicillin, ticarcillin, phenethicillin and flucloxacillin) gave 50% inhibition values even at concentrations as high up as 64 /Lg/ml. Hence, the results of the competitive inhibition assays suggest that the reactivity of the antibodies is directed towards and along the C7-C6S-C2-C3 skeleton of the cepham nucleus and the acyl chain, with the methyl group at C-3 and the isopropylidene group playing an important role in the reactivity, and with little reactivity occurring along the spacer skeleton or the J3-lactam ring.
TABLE III STRUCTURES OF PENICILLINS TESTED H R...... C N)jCJ
