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Potency Testing of Swine Erysipelas Vaccinesby Serology - Results of a Pre-validation StudyUte Rosskopf-Streicher, Sigrid Johannes, Manfred Wilhelm, Heike Gyra, and Klaus CusslerPaul-Ehrlich-Institut, D-Langen

SummaryThe European Pharmacopoeia (Ph. Eur.) monograph on SwineErysipelas Vaccine (inactivated) (Ph. Eur., 1997) requires thepotency of each batch to be demonstrated in a mouse protec-tion test. In this parallel-line bioassay, mice are challengedwith a virulent strain of Erysipelothrix (E.) rhusiopathiae afterimmunisation with different doses of either the standardpreparation or of the test vaccine. More than one hundredanimals are necessary for the routine testing of a single batch.In previous studies (Beckmann und Cuj3ler,1994; Rosskopf-Streicher et al., 1998), we have shown that an indirect enzyme-linked immunosorbent assay (ELISA) may be used to quantifythe humoral response of mice. This method can replace thechallenge model for the purposes of potency testing in thefollowing manner: Ten mice are immunised subcutaneouslywith 1/10 of the vaccine dose requiredfor pig vaccination.After three weeks, the mice are bled under anaesthesia. Serumsamples are pooled and the antibody content is compared tothat of a reference serum.In view of animal welfare the advantages of the alternativemodel are obvious: A highly reduced number of animals andthe replacement of challenge exposure. This includes thediscontinuation of the control group with a mortality rate of100%.A pre-validation study was initiated to evaluate the perfor-mance of the serological method. Eight laboratories used thetest kits to evaluate pooled serum samples from vaccinatedmice. Both the reagents and the test protocol were shown to besatisfactory. The intra- and inter-laboratory reproducibilitythat was achieved indicates that the method is a strongcandidate for validation.

Zusammenfassung: Priifung der Wirksamkeit von Rotlaufimpf-stoffen durch Serologie - Ergebnisse einer PriivalidierungDie Anforderungen an die Qualitat der Rotlaufimpfstoffe sindim Deutschen Arzneibuch; 10.Ausgabe (DAB 10),festgelegt.Die Wirksamkeit muj3derzeit in einem Tierversuch; der eineBelastungsinfektion an Miiusen einschliej3t,nachgewiesenwerden. Fur die Priifung einer Impfstoffcharge sind mindestens106 Tiere notig. Das Ergebnis (Sterberate) dient zur Berech-nung der Schutzkraft in Internationalen Einheiten (I.E.).In einem Entwurf zur Neufassung der Monographie von

Rotlaufimpstoffen wird folgende Methode for die Chargenprii-fung vorgestellt: leweils einer Gruppe von 10Miiusen werden1/10 der Zieltierdosis der Testvakzine bzw. des Referenrimpf-stoffes verabreicht. Eine ungeimpfte Kontrollgruppe wirdmitgefiihrt. Drei Wochen nach der Immunisierung werden dieTiere einer Belastungsinfektion mit einer virulenten Kultur vonErysipelothrix rhusiopathiae unterzogen. Die ungeimpftenTiere miissen innerhalb von 2 bis 5 Tagen an dieser 1nfektionsterben. Aus der Sterberate der geimpften Tiere laj3tsich dieWertigkeit des 1mpfstoffes errechnen.Die Infektionsversuche sind mit schweren Belastungenfiir dieTiere verbunden. In der Regel verenden mehr als 50% derMause. Der Tierversuch erfordert den Umgang mit infektiosemMaterial und ist mit hohen Kosten verbunden. Zudem wird dieStabilitdt des Internationalen Standardpriiparats in jiingsterZeit angezweifelt. Eine Ersatzmethode ist daher dringenderforderlich. Da die Immunitiit gegeniiber der Rotlaufinfektioniiberwiegend humoral bedingt ist, konnen serologischeNachweissysteme herangezogen werden.In einem vorlduferprojeki wurde ein ELISA entwickelt (Gyra etal., 1994; Beckmann et al., 1994) und auf seine Einsetzbarkeitfor die Chargenprufung getestet. Es ist das Ziel dieses An-schluj3projektes, den ELISA zu optimieren und in einemRingversuch tu validieren, um eine Akzeptanz als Ersatzmetho-de rum Belastungsversuch zu erreichen.In Anlehnung an den Entwurf der iiberarbeiteten Monographieschlagen wir folgende Methode zur Chargenpriifung vor: 10Mdusen wird 1110der Schweinedosis des zu testenden Impf-stoffes s.c. appliziert. Anstelle der Infektion wird das Blut derTiere 3Wochen spiiter unter Narkose gewonnen. Anschliej3endwerden die Seren gepoolt und im ELISA auf ihren Antikorper-gehalt gepriift. Dabei wird die Probe mit einem Referenzserummit bekanntem Antikorpergehalt verglichen.Das Referenzserum im Test wurde von uns durch Immunisie-rung von Miiusen mit 5 internationalen Einheiten des Rotlauf-standards (Rf2) gewonnen. Die Auswertung erfolgte durch einSoftware Programm, welches die relative Wirksamkeit desTestimpfstoffes zur Referenz bewertet.Ziel diesel' Priivalidierungsstudie war es, festzustellen, ob dieELISA Ergebnisse in verschiedenen Laboratorien reproduzier-bar sind. Anwendbarkeit der Referenzmaterialien und desTestprotokolls sollten ebenfalls beurteilt werden.

Keywords: 3R, refinement, reduction, Erysipelas vaccines, batch potency test, mouse model, ELISA, pre-validation study

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1 Introduction

Swine erysipelas is a bacterial disease ofworld-wide importance. Immunisation isvery effective in preventing this infection.Vaccines have already been used for 50years and are safe, efficient and easy toproduce.The requirements for the quality con-

trol of erysipelas vaccines are prescribedby the Ph. Eur.(1997). The potency has tobe demonstrated using an animal challen-ge model. At least three dilutions of theInternational Standard and of the test vac-cine are used to immunise separate groupsof mice and an additional group of ten un-vaccinated mice is also required. Threeweeks after vaccination, all mice are chal-lenged with a virulent culture of E. rhu-siopathiae. The unvaccinated control ani-mals have to die within five days of infec-tion in order for the test to be valid. Thepotency of the test product is evaluatedby comparing the survival rates (protec-tion dose) of mice vaccinated with theStandard vaccine with those of mice gi-ven the test vaccine. The value obtainedis expressed in International Units (IU).At least 106 animals are required to test

one batch of vaccine. This potency testuses a large number of animals and it isalso an extremely severe procedure. The-refore, the highest priority should be gi-ven to the development and validation ofalternatives (Hendriksen et aI., 1998).The mechanism bywhich erysipelas vac-

cines induce a protective immune responseis clearly related to humoral immunity (Ro-the, 1982a). Thus serological methodswhichmeasureprotective antibodiesagainstE. rhusiopathiae are promising candidatesto replace the challengeprocedure.Recentlywe developed an ELISA, which is suitablefor this purpose (Rosskopf et al., 1998).Theestablishment of this in vitro method is ba-sed on data gathered in a pre-validation stu-dy involving eight laboratories of varioustypes, e. g. Control Laboratories, manufac-turers and academic institutions.Each of theparticipating laboratorieswas providedwiththe test kit and protocol.

2 Animals, material and methods

time of immunisation. Animals werehoused under conventional conditionsin Makrolon cages (size 3) and were fedcommercial pellets; water was availa-ble ad libitum.

2.2 VaccinesNine erysipelas vaccines were used, inclu-ding both monovalent and combined pro-ducts (see table 1). All vaccines contai-ned serovar 2, while two products alsocontained serovar 1. Combined vaccinesincluded a porcine parvovirus component.Three vaccines (VI, V2 and V3) were di-luted with complete vaccine base by themanufacturer and only differed in theirantigen content. Two vaccines (V6 andV7) were diluted with saline to obtainproducts of lower quality.

2.3 Procedure of immunisationEach vaccine was administered to 50 mice(see 2.l.) in order to enable sufficientamounts of serum to be collected for thestudy. Mice received one subcutaneousvaccination of 1110 of the pig dose. Threeweeks later, the animals were bled underanaesthesia. Blood samples were incuba-ted at room temperature for one hour andcentrifuged for 2 minutes at 6000 g (Sar-stedt micro tubes No 41.150.005) to sepa-rate out the blood clots. The sera from eachgroup were pooled, aliquotted, coded andstored at -200 C.

2.4 Reference serumMouse reference serum was prepared byinjecting each of 500 mice (see 2.1.) sub-cutaneously with 5 IU of the InternationalStandard in a volume of 0.2 mI. The pro-cedures for bleeding and serum preparati-

on were as described in 2.3.

2.5 Negative serumThe negative control serum was preparedusing unvaccinated mice (see 2.1.). Theprocedures for bleeding and serum prepa-ration were as described in 2.3.

2.6 Preparation of coating antigenThe E. rhusiopathiae mouse challengestrain, Frankfurt XI, serovar N was usedto prepare the coating antigen (Moos,1983). It was extracted with EDTA andalkaline treatment according to the methodof Groschup et al. (1991). Briefly, wet bac-teria were suspended in buffer (Tris HCl)containing EDTA (ImM) and incubatedfor half an hour. After centrifugation, thecell pellet was resuspended in O.OIMNaOH and incubated with constant stir-ring for eighteen hours. Following neutra-lisation with 2M HCl, the cells were re-moved by centrifugation. The supernatantwas concentrated by filtration (Bottle topfilter, 45 11m, Nalgene) and aliquotted intovials. Each vial contained 530 ug of pro-tein. Antigenic components were separa-ted by SDS-polyacrylamide gel electro-phoresis according to the method of La-emmli (1970). Proteins were visualisedwith Coomassie blue. The major protecti-ve proteins of E. rhusiopathiae have mo-lecular weights of 64 to 66 kDa and 40 to35 kDa (Groschup et aI., 1991; Lachmannand Deicher, 1986).

2.7 Study designEight laboratories participated in the pre-validation study.The participants aremen-tioned at the end of the report (Annex).The order of listing does not correspond

Table 1: Specifications of the inactivated erysipelas vaccines used in the pre-validation study

Vaccine (V) Mono (M) Adsorbate (A)Comment

Serum (S*) Combined (C) Lysate (L)

Vi (81) M A

V2 (82) M A Erysipelas antigen reduced (dilution of Vi)

V3 (83) M AErysipelas antigen reduced again

(dilution of V2)

V4 (84) C L Parvocomponent

V5 (85) C A Parvocomponent

V5 (85) M L diluted with saline

V7 (87) M A diluted with saline

V8 (88) M A

V9 (89) M A

2.1 Laboratory animalsFemale NMRI mice were purchasedfrom Charles River, Kisslegg, Germa-ny. The mice weighed 18-20g at the S* = serum pool S optained by immunisation with the corresponding vaccine

124 ALTEX 16, 3199

~~ ROSSKOPF-STREICHER ET AL.--~~--------------------------------~v~

80,070.060,050.0

II40.0>- 30.0o 0 •.c 20,0CI) 8 8.•. 10.0 00 V • 'iJ Va. ~ QCI) /.::: 1,0/ /

ECI)

'V • Lab A0::

~0 Lab C

0,5T Lab D

• Lab Ef--

'V• • Lab G0 Lab H

0,0S 1 S2 S3 S4 S 5 S6 S7 S8 S9

5era

Figure 1: Distribution of the relative potency for each serum. Shown are the mean valu-es of 3 measurements for each laboratory.

to the codes denoted throughout this re-port by capital letters (Laboratory A-H).Serum samples were coded (SI-S9) andwere tested blind by the participants. Eachlaboratory was provided with the ELISAkit (sera, coating antigen, conjugate, sub-strate, buffer and microtitre plates) and theStandard Operating Procedure (SOP). TheELISA was performed as described byRosskopf-Streicher et al. (1998). The pla-tes were coated with antigen and then onereference and 3 test sera were applied toeach plate. A negative serum control andconjugate control were also included oneach plate.The ELISA was carried out with nine

test sera (see table 1) and repeated threetimes (to assess intra-laboratory variati-on). Test and reference sera were dilutedin twofold steps (11 dilutions). In additi-on, each serum was diluted 1:1000 andplated into 22 wells (to evaluate intra-as-say precision).

2.8 Data calculationThe data were expressed as Relative Po-tencies (RP) which compare the poten-cy of the test preparation with that of areference preparation (calculations per-formed using the Relative Potency Cal-culation Software, USDA) (Wilbur,1993). The reference serum has the ar-bitrary value of 1. This value representsthe pass mark for the test vaccines. Serawith a value ~ 1 have the same or a hig-her potency than the reference serum

ALTEX 16, 3/99

and pass the requirements. Sera with po-tencies lower than I fail in the test.

2.9 Statistical methodsSources of variation in the relative po-tency results were assessed using a fi-xed effects linear model which tookinto account the serum tested, the la-boratory which carried out the test andthe day on which the test was conduc-ted.The variability between laboratories

(reproducibility) and the day-to-day va-riation within a laboratory (repeatabi-lity) were calculated by comparingpairs of relative potencies. Lin's con-cordance correlation coefficient Pc (Lin,1992) is an appropriate index to use inorder to quantify the degree to whichpairs deviate from total agreement, i.e.the 45° line through the origin (Pc = I).

3 Results

3.1 Data returned and data excludedResults were received from all laborato-ries. The results from two laboratories (Band F) were not evaluated either becausedata were submitted too late, or becausethe values for the blanks and negative se-rum controls were not within the speci-fied range (negative serum: optical densi-ty 0,060 and conjugate control: opticaldensity 0,025). The only data that couldbe used from laboratory F was that rela-ting to intra-assay precision .

3.2 Inter-laboratory reproducibilityFigure 1 shows the results of measure-ments performed on the various sera(mean value of day 1-3) by each laborato-ry. As described under 2.9. a vaccine willpass the test, when the serum RP is ~ 1. Ifthe RP is below 1, a vaccine will fail thetest and will not meet the potency requi-rements. All laboratories assessed sera S1,S2, S4, S5, S7, S8 and S9 as indicating a"pass" and S3 and S6 as indicating a "fail"in the test.Table 2 illustrates the potency ranking

of the vaccines according to the serologi-cal results. Number 1 indicates the pro-duct that induced the highest immune re-sponse and No 9 the product that inducedthe lowest response. There is good agree-ment between the results from different la-boratories.

3.3 In vivo-in vitro comparisonThe results for sera SI-S3 in the serologi-cal test system showed a gradation that re-lated to the ranking of the respective vac-cines (VI- V3) in the mouse-challenge-test(as assessed by the manufacturer). Vacci-ne 3 was quantified at 28 IU (2.8 IU per

Table 2: Ranking of the sera with a score of 1 to 9. 1 corresponds to the product inducing thehighest immune response and 9 to that inducing the lowest response. Five sera were ranked in thesame position by each laboratory. Four sera showed differences in only one of the assigned ran-kings.

Lab. S1 S2 53 54 55 S6 S7 S8 S9

A 4 7 8 3 5 9 6 2 1

C 4 7 8 2 5 9 6 3 1

D 4 6 8 2 5 9 7 3 1

E 4 6 8 2 5 9 7 3 1

G 4 6 8 3 5 9 7 2 1

H 4 7 8 3 5 9 6 2 1

125

ROSSKOPF-STREICHER ET AL. ~r:"\----------------------------~~--~c,

14

13 • Lab. A012 0 Lab. C

11 'V ~ Lab. D

• 'V Lab. E>- 10

(27 LU.) • Lab. G0r::: 9 0 0 Lab. H<I>.•..

80c.. •<I> 7>:;:: 6C1I 'Va; 50:: 0

4 I (14LU.)

30

•2

0~ (2,8LU.)

Serum 1 Serum 2 Serum 3

Figure 2: Three vaccines, which differed only in their antigen content, showed a grada-tion of results in the challenge test (values in brackets, 1/10 of the pig dose) as well asin the serological test. The vaccine that failed the animal test had an RP value of < 1 andtherefore also failed in the serological test.

mouse dose). All laboratories reached thesame conclusion in assigning an RP valuebelow 1 for Serum 3.Thus, the vaccine was demonstrated to

have failed the potency requirements inboth systems. The increasing antibodycontent of Serum 2 and Serum 1 wasmeasurable. These data correlated with theincreasing antigen content of vaccine 2and vaccine 1. Vaccines 1 and 2, in thesame way as sera 1 and 2, were shown tohave potencies> 50 IU expressed in RPvalues as a multiple of 1.This gradation in results provides the

first demonstration of the correlation bet-ween the in vivo and the serological me-

A c GLaboratory

Figure 3: For the Intra-assay precision wecompared the variation coefficients oftwelve sera. Each serum had been diluted1:1000 and tested in the ELISA 22fold byall laboratories. The mean value of theoptical density, standard deviation and thecoefficient of variation (CV) had been cal-culated for each serum separatly, The co-lumns represent the portion of sera (outof 12) with a CV lower than 15% in per cent.

126

thod (see fig. 2) and demonstrates the im-portance using the complete vaccine basefor dilution.Vaccines diluted with saline (V6 and

V7) showed a high deviation from the IUgained in the animal test and those resultsobtained by serology. Antibodies inducedby V6 (diluted from 260 IU per pig doseto 50 IU per pig dose) were extremely lowand a calculation of the RP value was notpossible. In contrast, V7 (diluted from 140IU to a pig dose of 14 IU) showed RP va-lues in the range of 3 to 4.

3.4 Intra-assay precisionIn order to evaluate the precision withinlaboratories, each of twelve sera was di-luted 1:1000 and repeatedly tested 22foldon one plate. Afterwards the mean value(MV) of the optical density, standard de-viation (SD) and the coefficient of varia-tion (CV) had been calculated for eachserum (Formula: CV = SD x 100/MV).So we gained twelve Cvs from the labs.

The columns in figure 3 show the portion(in per cent) of sera (out of 12) with a CVequal or lower than 15% per laboratory.

3.5 Statistical analysisAn analysisof variancereveals that the sour-ce of variation in the relative potencies isdue to the highly significant (p < 0.001)factors of test serum and laboratory. Thefactor relating to the day of testing, whichis nested within the factor for the laborato-ry in the linear model, does not show anysignificantinfluence (p = 0.058) ata= 5%.The serum effect has to be significant,because the value of the ELISA lies in itsability to detect different antibody levelsin sera. Multiple comparisons between allpairs oflaboratories, using simulation-ba-sed simultaneous 95% confidence inter-vals, show a tendency to significant diffe-rences between those pairs which havelaboratory A or laboratory H in common(see also table 3).Multiple pairwise comparisons using

Lin's concordance correlation coefficientdemonstrate the reproducibility to be in therange acceptable to very good (i\ 00() .78,table 3) and the repeatability to be in therange good to very good 0\ 00() .85, table4).The soleexception is themoderate agree-ment between days 2 and 3 in laboratory A,which was apparently due to results obtai-ned for sera 8 and 9 G\ = 0.62 , table 4).

4 DiscussionVaccines prepared from E. rhusiopathiaeserovar 2 have been successfully used toprevent swine erysipelas for more than 50years. The vaccines induce protection inmice and swine challenged with differentserovars of E. rhusiopathiae, including themost common isolates of serovar 1 and 2(Takahashi et al., 1984).The potency requirements for erysipe-

las vaccines are part of the Ph. Eur. (1997).The monograph requires a minimum an-

H

Table 3: Inter-laboratory (lab-to-lab) variation showing Lin's Pc for pairs of meanrelative potencies from three consecutive days in each laboratory.

Laboratory A C 0 E G

C 0.86

0 0.78 0.98

E 0.81 0.99 0.99

G 0.83 0.98 0.99 0.98

H 0.96 0.91 0.83 0.87 0,85

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tigen content of 50 IU as determined in amouse protection test. By definition, oneIU of the International Standard protect50% of the animals within a populationof mice. More than 100 mice are neededin order to test the potency of a single batchof vaccine. At least half of the animalssuccumb from erysipelas during the test.Therefore, there is an urgent need for analternative test system that avoids the chal-lenge procedure and uses fewer animals.Recently, a serological test system ba-

sed on an antigen extract, which includesthe protective antigen fraction of E. rhu-siopathiae, has been developed (Rosskopfet al., 1998). However, the relevance ofsuch new test systems as indicators of ef-ficacy in pigs has to be demonstrated.Unfortunately, the relevance for the tar-get species of the established laboratoryanimal challenge test has never been de-monstrated. The IU value has been defi-ned only in the mouse system. To bridgethis gap, a challenge test has been carriedout in swine using different doses of thestandard vaccine. An injection of 50 IUprotected pigs against virulent challenge(challenge dose: 106 cfu/O,l ml of serovar1 and 107 cfu/O,l ml of serovar 2) (unpu-blished results).A potency test should be able to con-

firm that the potency of a test vaccinebatch is at least equivalent to the potencyof a batch with demonstrated efficacy inpigs. Immunisation of mice with an anti-gen dose of 5 IU (1/10 of a pig protectiondose) seems suitable for this purpose. Aserum pool obtained from mice vaccina-ted with 5 IU is therefore a representativereference for calculation of the relativepotency of a test product.An alternative method must be fully de-

veloped and properly validated before itcan be considered for regulatory use.However, apart from general guidelines ontoxicological studies, little has been pu-blished on the validation of alternativemethods in vaccine quality control. Re-cently, a workshop on validation studiesfor alternative methods for the potencytesting of vaccines was held (Hendriksenet al., 1998). The criteria indicating thereadiness of a test for inter-laboratory va-lidation were defined:

Test development in the laborato-ry of originPre-validation (informal inter-laboratory study)

ALTEX 16, 3/99

Table 4: Intra-laboratory (day-to-day) variation showing Lin's Pc for pairs of relativepotencies in each laboratory

Laboratory A C 0 E G H

day1 - day2.-

0.85 0.87 0.97 0.97 0.97 0.99

day2 - day3 0.62 0.88 0.90 0.98 0.99 0.97l-

day1 - day3 0.89 0.99 0.94 0.98 0.99 0.96

Validation (formal inter-laborato-ry study)Assessment of study andproposalsProgression towards regulatoryacceptancePre-validation includes three main pha-

ses: protocol refinement, protocol trans-fer, and protocol performance (Curren etal., 1995). The informal inter-laboratorystudy carried out at the pre-validation sta-ge involves assessing the inter-laboratorytransferability of the method, undertakingany optirnisation and standardisation of theprotocol which may be considered neces-

sary, and identifying any unexpected pro-blems with the test procedures. The ob-jective of the pre-validation stage is toensure that the method tested fulfils thecriteria for inclusion in a formal validati-on study.The requirements for pre-validation and

the results of the erysipelas serological me-thod are shown in table 5.The aim of the pre-validation study was

to prove the reproducibility in different la-boratories of a serological method for thebatch potency testing of erysipelas vacci-nes. To perform this study it was necessa-ry to use a reference serum prepared by a

Table 5: Overview of criteria operating before and during the pre-validation study. Thedefinitions refer to the guideline of VICH and the report and recommendations of ECVAMworkshop 31 [Hendriksen et aI., 1998 and VICH, 1997].

Criteria Prevalidation Comment

1. Protocol +Important for work according to GLP orGMP

1.1. Protocol transfer and All laboratories were able to perform theperformance + test according to the protocol

1.2. Protocol refinement +The study lead to an improvement of theprotocol

Expresses the closeness of agreement

2. Precisionbetween a series of measurements of thesame sample under prescribed condi-tions. Criteria: 2.1.-2.3.

Expresses the precision under the same2.1. Repeatability + conditions over a short interval of time

(intra-assay precision).

Expresses variations within laboratories:2.2. Intermediate precision + different days, different analysts, different

equipment. etc.

Expresses the precision between2.3. Reproducibility + laboratories. for the standardisation of a

method

The ability to obtain test results which are3. Linearity + directly proportional to the concentration

of analyte in the sample

The interval between the upper and lowerconcentration of analyte in a sample has

4. Range +a suitable level of precision, accuracy andlinearity. The range between 0,5 and 5 isimportant for the assessment of the RPusing our method.

5. Preparation and Coating antigenproviding of reference + Reference serummaterial Test sera

127

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vaccine of known potency (5 IV). In fu-ture (European Pharmacopoeia Com-mission, 1999) companies should pre-pare in-house reference vaccines.However, due to our experience the useof an in-house reference serum wouldalso be possible and reduce the numberof animals necessary for the test per-formance.

The assay performed well when used ac-cording to the SOP. Minor changes to theprotocol were suggested by the study par-ticipants. The reproducibility of results forthe test sera, both within and between thelaboratories, was demonstrated. There wasgood agreement between all participantsregarding the potency ranking of the dif-ferent sera (table 2).

Three vaccines with varying antigencontent were evaluated by the manufac-turer in a mouse challenge test (fig. 2. va-lues in brackets). All laboratories wereable to identify the batch of low qualityand the gradation of the antigen contentwas reproducible. The other approach,which was to produce batches of inade-quate potency by diluting the product withsaline, failed. The two vaccines (V6 andV7) did not reveal the theoretically expec-ted results. This demonstrates the im-portance of diluting a vaccine with thecomplete vaccine base (see 3.3.) in orderto obtain results, which correlate with tho-se from the in vivo model. In view of therevision of the Pharm. Eur. monograph(1999) this fact should be respected forthe formulation of product specific refe-rence material. The serological method issuitable for discrimination between vac-cines inducing different levels of antibo-dies.

In conclusion, the ELISA for the detec-tion of protective antibodies against E.rhusiopathiae fulfilled the requirementsfor a pre-validation study. Therefore, thetest is suitable for in-process controls andfor showing the consistency of the pro-duction process (cases where an interna-tionally validated system is not necessa-ry). Such a pre-screen for non-regulatorychecks already offers the possibility tosave many animals. However, a formal va-lidation study will also be initiated so asto obtain international acceptance and theintroduction into the Pharmacopoeia re-quirements of a serological method for thepotency testing of swine erysipelas vacci-nes.

128

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AcknowledgementsThe study was supported by the GermanMinistry of Education, Science, Researchand Technology. We are very grateful tothe participants for their interest and par-ticipation in this study. Special thanks aredue to Keith Redhead (Hoechst RousselVet., UK) for providing vaccine samplesand test results. We also thank MarliesHalder, Lukas Bruckner and Krys Bottrilfor constructive discussions and the helpin preparing the manuscript.

Annex: List of participantsDr. E. Borrmann / P. Methner

ZEBETBundesinstitut fur gesundheitlichenVerbraucherschutz u. VeterinarmedizinD-Jena

Dr. L. Bruckner / C. SennhauserInstitut fur Viruskrankheitenund ImmunprophylaxeCH -Mittelhausern

Dr. H. Gyra / S. Johannes /U. RoBkopf-Streicher / D. HausleithnerPaul- Ehrlich- InstitutBundesamt fur Sera und ImpfstoffeD-Langen

Dr. M. HalderECVAMJoint Research CentreI-Ispra

Dipl. Chem. JakelImpfstoffwerk Dessau-Tornau GmbHD-RoBlau

DVM U. Polster / Sylvia GroBklausBundesforschungsanstalt furViruskrankheiten der TiereD-Insel Riems

Dr. B. von Wangenheim / S. AnknerLandesuntersuchungsamt fur dasGesundheitswesen SudbayernD-OberschleiBheim

Dr. R. WeiJ3Institut fur Hygiene undInfektionskrankheiten der TiereD-GieJ3en

Correspondence toDipl.-Biol. Ute Rosskopf-StreicherPaul- Ehrlich- InstitutPaul-Ehrlich-Str.51-59D-63225 LangenTel.: +49-6103-77-7415/ Fax: 1254E-mail: rosut@pei.d

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