Determination of sulphamethazine in animal tissues by enzyme immunoassay

Download Determination of sulphamethazine in animal tissues by enzyme immunoassay

Post on 15-Jun-2016




1 download


<ul><li><p>Analytzca Chtmtca Acta, 275 (1993) 323-328 Elsener Science Publishers B V , Amsterdam </p><p>323 </p><p>Determination of sulphamethazine in animal tissues by enzyme immunoassay </p><p>C Renson, G Degand and G Maghum-Roglster Laboratoue dhtyse des Den&amp;es Ahnentaues dOngme Ammale, Faculti de M&amp;iecme Vthnaue, Unwers~te de L&amp;e, </p><p>B&amp; 842 Sart-Tdman, B-4ooo LGge (Be&amp;m) </p><p>Ph Delahaut </p><p>Centre dEconomte Rurale, B-6900 Ma&amp;e (Belguun) (Received 30th June 1992, revised manuscnpt received 29th September 1992) </p><p>Abaa-act </p><p>An enzyme mmmnoassay for sulphametbazme was developed usmg an antiserum rzused m rabtuts by unmumaa- tlon agamst a sulphamethazme dlazo derwatrve coupled to bovme serum albumm Horseradish perox~dase coupled to sulphametbazme by a carbodurmde method was selected as a tracer The dose of sulphametbazme that caused 50% bmclmg mhiilfion was 3 8 ng per well wth a 2-h mcubation tune at 37oC and 12 ng per well wrth a 16-h mcubatlon tune at 4C The assay was evaluated by usmg control and sulphamethazme-fortdied mu&amp;e and bver samples These samples were treated by the matnx sohd-phase dqerslon method for the subsequent lsolaaon of the drug The method allowed a dete.&amp;on lmut well below the tolerance tit (0 1 mg kg-) generally appbed for sulphamethazme </p><p>+r&amp; Enzymatic methods, Immunoassay; Ammal &amp;sues, Sulphamethazme, Trues </p><p>Sulphonanudes have a broad spectrum of an- tlbactenal atiwty and are frequently mcorpo- rated 111 feeds as a practical method for the prevention or treatment of a variety of so-called confinement diseases 111 ammals, mamly pigs However, there are concerns for human health because of the possible presence of residues of the drug 111 meat [l] The US Food and Drug Admmlstratlon (FDA) requires a 19day mth- drawal period of feed contammg sulphametha- zme before slaughter and has estabhshed Its tol- erance hmlt m tissues for human consumption at 0 1 mg kg- [2-51 Although dtierent sulphon- </p><p>Correspondence to G Maghum-Roglster, Laboratolre dAnalyse des De&amp;es Abmentares dOngme Ammale, Fa- cult6 de Midecme WtQmlure, Umversk de L&amp;ge, Bit B42 Sart-Tdman, B-4000 Loge (Be&amp;urn) </p><p>armde-antlblotx combmatlons have been ap- proved for use m swme feed, sulphamethazme has been xdenttied as the major problem m ca 95% of all sulphonanxde tissue vlolatlons [6] This paper reports a competltlve enzyme munu- noassay (EIA) for routme screening of sulpha- methazme m tissues </p><p>EXPERIMENTAL </p><p>Reagents and eqtupment Sulphamethazme was obtamed from Janssen </p><p>Chmuca (Geel/Belgmm), sulphaguamdme, sul- phachloropyrldazme, sulphaqumoxahne, sulpha- merazme and sulphadnnethoxme from Sigma (St LOUIS, MO), sulphamlamtde, sulphadlazme and sulphathlazole from Aldrich Wlwaukee, WI), </p><p>ooO3-2670/93/$06 00 Q 1993 - Elsevler Science Pubhshers B V All nghts reserved </p></li><li><p>324 C Rmson et aL /Anal Chzm Acta 275 (1993) 323-328 </p><p>bovme serum albumm (BSA) from Serva (Heldel- berg, Germany), horseradish peroxldase (HRP) from Boehrmger (Mannhenn, Germany), 3,3, 5,5-tetramethylbenzldme (TMB) mlcrowell per- oxldase system from firkegaard &amp; Perry (Galthersburg, MD), Tween 20, cytochrome c and thlmerosal (merthlolate) from Merck (Darmstadt, Germany), aprotmme (trasylol) from Bayer (Brussels), Freunds complete adjuvant from DIFCO (Brussels) and bulk C,, (40 pm, 18% load, end-capped) octadecylsdyl-denvatlzed silica from AnalytIchem International (Harbor City, CA) All other chemicals were of analytlcal- reagent grade or better and were used as re- ceived Delomzed water was purified mth a Mllh- Q system (Waters) </p><p>Buffers were prepared as follows (A) carbon- ate-hydrogencarbonate buffer solution (coatmg buffer) 50 mM pH 9 6 Na,CO, (159 g), NaHCO, (2 99 g) and dlstllled water (to 1 0, (B) phos- phate-buffered salme (PBS)-gelatme solution (0 01 M PBS-O 2% gelatme) (EIA buffer) NaCl (8 6 g), Na,HPO, 2H,O (16 8), NaH,PO, H,O (0 14 g), thunerosal (0 1 g), gelatme (2 g) and distilled water (to 10, (C) washmg solution NaCl (8 76 g), Tween 20 (0 5 ml), dlstdled water (to 1 0, (D) stoppmg solution 6 M H$O, </p><p>All stock and workmg standard solutions were stored at 4C </p><p>ELISA plates (Nunc-Immuno Plate Maxlsorp F96) were obtamed from Nunc (Roslulde, Den- mark) Absorbance of plate wells was measured with a MultIscan MCC/340 nucroplate reader from ICN Flow Laboratorles </p><p>Zmmwwgen preparatton Sulphamethazme was dlazotlzed and coupled </p><p>to bovme serum albumm [7,8] Prepratwn of the drazo denvatrve Sulpha- </p><p>methazme (57 mg, 0 27 mm00 was dissolved m 4 ml of 0 5 M H,SO, and the solution was left overnight wth stlrrmg at 4C A solution of sodmm mtnte (19 mg, 0 27 mm00 m dlstllled water (1 ml) was added drowse to this nuxture wth stvrmg m the dark at 4C The formation of dlazotlzed sulphamethazme was detected by the appearance of a deep yellow colour after reaction wth N,ZV-dnnethylamlme 191 The formatton of </p><p>the dlazo derlvatlve was also momtored by thm- layer chromatography on silica gel 60 Alu Rol sheets (Merck) after nugratlon m the solvent mnc- ture ethyl acetate-ammoma solution-methanol (30 + 1 + 15, v/v/v) After spraymg of the sheet vvlth a 0 1% solution of 2,7-dlchlorofluoresceme m ethanol (Merck), it appeared that the R, value of sulphamethazme m this system was 0 79, whereas its dlazo derlvatwe did not migrate The reaction was contmued until the disappearance of the sulphamethazme spot The reaction was stopped by addltlon of a solution of ammonium sulphamate (7 mg) m water (140 ~1) </p><p>Couplrng to bovrne serum ahunm The solution of the dlazo derwatlve was added m 50-~1 por- tions to 4 ml of carbonate buffer (pH 10) contam- mg 100 mg of BSA The reactlon mixture was left for 18 h at 4C Unreacted material was sepa- rated from the conjugate by chromatography on Sephadex G-50 Hrlth 0 05 M ammomum carbon- ate solution (pH 8) The conjugate was then lyophlllzed </p><p>Anthdy pregaratwn The mununogen was used to prepare an anti- </p><p>serum Fwe rabbits (New Zealand breed) were munumzed by the protocol of Valtukatls et al [lo] with 1 mg of duuotlzed sulphamethazme- BSA conjugate m complete Freunds adJuvant The ammals recewed five mJectlons vvlth an mter- val between inJectIons of 1 month After the thud mJectlon, blood samples were taken from the ear vem at lo-day intervals to follow the appearance of the antibodies </p><p>The crude antiserum obtamed was dlmded mto ahquots and stored at - 20C after dllutlon m 0 1 M PBS-glycerol nuxture (serum-PBS-glycerol, 1 + 9 + 10) correspondmg to a fmal dilution of 1+ 19 </p><p>Enzyme conjugate preparatwn The sulphamethazme horseradlsh peroxldase </p><p>coqugate was prepared by a m&amp;led carbodl- mude method [11,12] Sulphamethazme (10 mg) was dissolved m a nuxture of pyrldme (400 ~1) and dlstdled water (400 ~1) and the solution was added to horseradish peroxldase (20 mg) drs- solved m distilled water (2 ml) A lo-mg amount </p></li><li><p>C Rensm et at! /Anal Chm Acta 275 (1993) 323-328 325 </p><p>of l-ethyl3-(3-drmethylammopropyl)carbodr- lrmde hydrochlorrde was added and the solution was strrred at 4C for 18 h Excess of sulphameth- azme was ehmmated by chromatography on Sephadex G-25 column m PBS buffer, the col- umn was first washed wrth PBS buffer, the solu- tron was then added to the column and the en- zyme conjugate preparatron was eluted wrth 3 5 ml of PBS buffer </p><p>Tins conjugate was then diluted (1 + 20, v/v&gt; m a buffer contammg 80 ~01% of a solution of 75 mlofPBS+2gofBSA+12Smlofglycerol+20 mg of thunerosal, 20 ~01% of aprotmme and 0 1 wt % of cytochrome c </p><p>Pretreatment of the samples The muscle and hver samples were treated </p><p>accordmg to the matrrx solid-phase drspersron method [13] The matenal CC,,, octadecylsrlyl-de- rrvatrzed srlrca) was washed usmg a vacuum box successrvely with 30 ml each of hexane, drchloromethane and methanol to remove con- tammants, then 0 5 g of tissue was added to 2 g of the prewashed C,, matenal m a glass mortar The sample was gently blended wrth a glass pestle to produce a semi-dry, homogeneous mater&amp; Tlns was poured mto a syrmge barrel column (10 ml) contammg two filter-paper drscs Whatman No 1) Two other filter-paper discs were placed on the top of the materud and a syrmge plunger was used to compress the sample to a volume of 4 ml A RIO-~1 drsposable pipette up was attached to the end of the column to increase the resr- dence tnne of the elutmg solvent m the column The column was washed wtth 8 ml of hexane by gravrty flow (excess of hexane was removed by applymg a posrtrve pressure) and sulphametha- zme was eluted with 8 ml of drchloromethane The eluted fraction was evaporated to dryness under nitrogen and the residue was drssolved in 250 ~1 of PBS-gelatme (buffer B) </p><p>Assay procedure The sulphamethazme antrserum drluted 1+ </p><p>15 000 111 coatmg buffer A (100 ~1) was added to the umer 60 wells of a polystyrene nucrotrtratron plate (the well to well varratrons are greatly re- duced by onnttmg results from the peruneter </p><p>wells) The plate was covered wrth a plastrc fihn (plate sealer from Tech Gen, Zelhk, Belgmm) and stored at 4C for 16 h. The coatmg solutron was asprrated, then 50 ~1 of standard solutions of sulphamethazme (range 0 2-100 ngl or trssue ex- tracts from control or sprked samples were drs- pensed, m tnphcate, mto mdrvrdual wells on the nucrotrtratron plate already coated wrth the spe- crfic anubodres Subsequently, 100 ~1 of the sul- phamethazme-enzyme conjugate solution drluted 1 + 10000 111 PBS-gelatme (buffer B) was added The plate was covered with a plate sealer and, after shakmg, mcubated overmght at 4C or for 2 h at 37C The wells were washed three times wrth washing solutron C to remove unbound ma- terial TMB solution (150 ~1) was then added to each well and the plate was mcubated at 37C m the dark After 30 mm the reacuon was stopped by addmg 50 ~1 of 6 M H,SO., to each well, m the same order and at the same rate as the substrate solutron was added so as to keep the reaction ume constant for all of the samples The plate was gently shaken before absorbances were measured at 450 MI wrth the nucrotrtre plate reader </p><p>The cross-reactrvrtres (CR) of the antrserum were determmed by preparmg dose-response curves for a range of sulphonanndes and were calculated vvlth the equatron </p><p>CR= Mm1 sulphametllaPne requucd to reduce colour production by 50% </p><p>nmol other drug rrxtured to reduce colour pmductmn by 50% </p><p>Catiratwn The percentage bmdmg was calcu- lated from the absorbance obtamed m the ab- sence (I?,) and the presence U?) of sulphametha- zme 111 standards and samples as follows bmdmg (o/o) = (B - NS/B, - NS) X 100, where NS rep- resents the non-specrfrc bmdmg, I e., the ab- sorbance measured m wells uncoated wrth the antrserum Cahbratron graphs were prepared by plotting log(sulphamethazme concentratron) against percentage bmdmg or agamst logltiper- centage bmdmg) (logrt-log plot) </p><p>Calculatwn Results were calculated accordmg to the method of Rodbard and Frazrer [14] by mterpolatron from a cahbratron graph where the bound actrvrty, expressed as the logrt of the ratio (m percent) between absorbance mcrease per 30 </p></li><li><p>326 C Renson et aL /AnaL Chm Acta 275 (1993) 323-328 </p><p>rmn, at each concentration of sulphamethazme (B) tided by the bound acttvrty m the absence of unlabelled sulphamethazme (B,), was plotted agamst IogGulphamethazme concentration) The Rodbard and Frazier procedure of calculation was adapted (Logrvet, Wodecq, Belgmm) to en- zyme mmnmoassay usmg EXCEL (Microsoft) on a Macintosh computer </p><p>a </p><p>40- </p><p>20- </p><p>RJZSULTS </p><p>Typtcal calibration graphs obtamed (a) after a short mcubatron time (2 h at 37C) and (b) after a long mcubation time (overnight at 4C) are pre- sented m Fig 1 The day-to-day variability of the loglt-log plot was determmed from ten cahbra- tion graphs for both types of mcubation For 2-h competitions at 37C, the mean slope f standard deviation (S D ) and relative standard deviation (RSD) was -(062fO02) (3%) and the mrd- point of the curve at 50% bmdmg mhibmon (ED501 was (3 8 f 0 25) ng per well (6 6%) For competrttons overmght at 4C, these values were respectively -(O 69 f 0 011 (2%) for the slope and (12 f 0 05) ng per well (4%) for the mid-pomt (n = 10) </p><p>0 - --..---I - . .-.-I . . --..-. * - - *I 1 10 100 Ma0 </p><p>ng/rrll </p><p>B/no (*I </p><p>b </p><p>40- </p><p>20- </p><p>Nme sulphonanndes were assessed for cross- reactivity with the anti-sulphamethazme anti- serum The cross-reactlvmes (CR) of the antibody are given m Table 1 It is clear that other com- monly used sulphonamrdes, except sulphamera- zme, had no srgmficant effects Compared with sulphamethazme, sulphamerazme, with the unique dtfference of one methyl group at the pmrdmyl C-4 position, showed 13% cross-reac- t1v1ty </p><p>o+ . . . . ...I . . . . . ... . . . 91 1 10 100 </p><p>ng/vell </p><p>Fig 1 Cahbratlon graphs for the enzyme unmunoassay of sulphamethazme (a) 2-h mcubatlon at 3PC, (b) 16-h mcuba- tlon at 4C </p><p>Evaluations of the accuracy and reproducibil- rty of the assay m muscle and hver samples are given m Table 2 The accuracy of the method was checked by recovery experunents performed on </p><p>TABLE 1 </p><p>Percentage cross-reactlon of the antwrum antl-dlazotied sulphamethazme coupled to bovme serum albumm </p><p>Substance Cross-reaction (%) B Substance Cross-reactlon (%) a </p><p>Sulphamethazme loo Sulphamerazme 13 2 Sulphadlmethoxme 01 Sulphaguamdme &lt; 0 01 Sulphaqumoxahne </p></li><li><p>C Renson et aL /AnaL Chun Acta 275 (1993) 323-328 327 </p><p>TABLE 2 </p><p>Accuracy and reproduclblhty of the assay m muscle and hver samples a </p><p>Muscle samples </p><p>ACCUWY </p><p>Added (Pg kg-) </p><p>Measured &amp;gkg-Y </p><p>Recovery (%6) </p><p>100 &amp;J13*34 81 250 2225*93 89 </p><p>Reprodunblhty </p><p>Added (tig kg-) R S D (%I </p><p>100 250 </p><p>Intra-assay (n = 8) Inter-assay (n = 8) </p><p>74 113 82 12 2 </p><p>Lver samples </p><p>AccUl?lCy </p><p>Added (CLg kg-1 </p><p>100 250 </p><p>Measured Recovery (CLg kg- ) (%) </p><p>918+42 92. 2353*112 94 </p><p>Added (fig kg-l) R SD (%I </p><p>100 250 </p><p>Intra-assay (n = 8) Inter-assay (n = 8) </p><p>81 102 78 124 </p><p> Sulpbamethazme concentrations were determmed by en- zyme unmunoassay after addltlon of known amounts of sul- phamethazme to a muscle or bver sample from an untreated pig Values are means of e&amp;t determmatlons (blank values subtracted) f S D </p><p>spked tissue samples Known amounts of sul- phamethazme were added to tissue samples at two concentrations and assayed m tnphcate </p><p>The recovenes were near to 100% and the difference between the true value and the mean of the determmatlons was well below the limits (-20% to + 10% for real concentrations &gt; 10 pg kg-) recommended by the EEC Commlsslon Declslon (87/410) WI The reproductblhtles also fitted the recommendations of the EEC (for real concentrations &gt; 10 pg kg-, R S D &lt; 0 15%) </p><p>When used to analyse muscle samples, the hmlt of detection (mean determmed concentra- tron for 20 blank samples + 3 tnnes the S D &gt; 1161 of thrs assay was established to be 4 8 pg kg- </p><p>and the hnut of determmatlon (mean of 20 blank samples+6tnnestheSD)was54pgkg- In liver tissue, the hnut of detection and hmlt of determmatlon were calculated to be 5 1 and 5 7 c1g kg-, respectively </p><p>DISCUSSION </p><p>The enzyme nnmunoassay developed for the determmatlon of sulphamethazme residues m pig tissues extracts 1s based on a competrtion be- tween sulphamethazme present m the sampl...</p></li></ul>


View more >