A review of WHO International Standards for botulinum antitoxins

A review of WHO International Standards for botulinum antitoxins
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  A review of WHO International Standards for botulinum antitoxins R.G.A. Jones*, M.J. Corbel, D. Sesardic  Division of Bacteriology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire EN6 3QG, UK  Received 27 September 2005; accepted 23 November 2005 Abstract Clostridium botulinum  produces the most potent known toxins, with seven distinct serotypes currently defined (A e G). These toxins can causea life threatening systemic toxicity whether through natural causes such as food poisoning, infant botulism, wound botulism, or through use asbio-terror agents (e.g. inhalational botulism). It was realised early on that standard reference botulinum antitoxins were required to reduce thevariation between assays and ensure a consistent potency of therapeutic antitoxins and vaccines, and to define the serotype. This led to the In-ternational Unit being defined by the World Health Organisation (WHO) in the 1960s with the establishment of the first International Standards(IS) for serotypes A e F. Since then botulinum antitoxin ISs have been used world wide as the ‘yard stick’ to measure the neutralising potency of antitoxins. These primary WHO ISs are used to calibrate in house working reagents that are more extensively utilised. A definition of theInternational Unit for serotype G antitoxin has yet to be defined or accepted by the WHO and urgently needs addressing. However, beforeSeptember 11th 2001 there was very little interest in botulinum antitoxin IS and as a result stocks of most of the srcinal preparations arenow completely exhausted or depleted and replacements long overdue. We have reviewed the extensive history and availability of the primaryWHO ISs and interim materials. All type A and B antitoxin materials were recently assayed and their relative activities confirmed against thesrcinal IS preparations. The recent increase in demand for these materials has further exacerbated the shortage. We describe here the productionand characterization of stable freeze dried potential candidate replacements along with a new prospective first IS for type G antitoxin. Availabletoxin A reference preparations are also briefly reviewed.   2005 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.  Keywords:  Antitoxin; Standard; Assay; Botulinum 1. First WHO International Standards Material for the first recognised botulinum antitoxin stand-ards was srcinally produced in horses at the MicrobiologicalResearch Establishment, Porton Down (UK) against  Clostrid-ium botulinum  types A e E and these served as Britishreference preparations between 1954 and 1960. Since this ma-terial was considered suitable for a potential WHO Interna-tional Standard, 100 vials of each were diluted with distilledwater and 1.0 ml/ampoule filled and freeze dried in 1960 atthe National Institute for Medical Research, London, andsubsequently established during the same year as the FirstBritish standards for types A e E antitoxins [1,2].Antitoxins for types A and B were produced by immunisinghorses with gradually increasing doses of formalin inactivatedtoxoid/alum over a long period of time. Toxins used were typeA, strain 4587; and type B, strain Beans. Antitoxins for typesC e E were produced using toxin from type C, strain Cid (asubculture of 178c); type D, strain D6f (a subculture of thetype strain); and type E, strain Nanaimo, and included a finalboost with large doses of active toxin of up to 150 ml of undiluted toxic culture filtrate [1,3].Following agreement with the participants in a collaborativestudy, the International Units were defined by the National In-stitute for Medical Research (UK) as the neutralising activitycontained in 0.1360 mg of the International Standard for type * Corresponding author. Tel.:  þ 44 1707 641 000; fax:  þ 44 1707 663796.  E-mail address:  (R.G.A. Jones).1045-1056/05/$32.00    2005 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.biologicals.2005.11.009Biologicals 34 (2006) 223 e 226  A (or 500 IU/ampoule); 0.1740 mg of the International Stan-dard for type B (or 500 IU/ampoule); 0.0800 mg of the Inter-national Standard for type C (or 1000 IU/ampoule); 0.0121 mgof the International Standard for type D (or 1000 IU/ampoule);and 0.0691 mg of the International Standard for type E (or1000 IU/ampoule) [1,4].In 1964 the Statens Serum Institut (Copenhagen, Denmark)put forward a candidate botulinum type F antitoxin raised inrabbits for use as an International Standard [4]. In 1966 thismaterial was accepted as the first type F International Standardand the Unit defined by the Statens Serum Institut as that con-tained in 7.33 mg (or 4 IU/ampoule) [5].Original IU were based on each unit neutralising approx.10,000 mouse intra-peritoneal LD 50  doses for types A e D orF, or 1000 LD 50  for type E toxin [3,6]. 2. Second WHO International Standards In 1976 a report was noted by the WHO Expert Committeeon Biological Standardization (ECBS) indicating that  C. botu-linum  type B toxin was heterogeneous and requested from theStatens Serum Institut, together with the Chiba Serum Institute(Japan), to investigate this matter in relation to the InternationalStandard for  C. botulinum  type B antitoxin [7]. However, sub-sequently in 1977 it was noted that stocks of the 1st Interna-tional Standard held by the Statens Serum Institut weredepleted and that a candidate replacement did not give widelydifferent potencies when titrated against toxins prepared fromproteolytic and non-proteolytic strains [8]. Further materialwas obtained for processing into the proposed replacementstandard and a collaborative study initiated with the ChibaSerum Institute [8 e 11]. Despite collaborative studies showinga significantly higher potency using B toxin produced from thesame strain (Okra) as used for raising the new antitoxin, theWHO ECBS authorized a proposed replacement as the 2nd In-ternational Standard for  C. botulinum  type B [12]. Since dis-crepancies in results from the collaborating laboratories werereported, a further collaborative study was planned. In themean time the candidate preparation was distributed, as stocksof the original standard held by the Statens Serum Institutwere exhausted [13,14]. Following this study the material cod-ed BUSB 5004 was recognised as the 2nd International Stan-dard for type B antitoxin and on the basis of the resultsassigned an activity of 31 IU/ampoule [15] (Table 1). 3. Availability In 1989 the WHO ECBS noted that stocks of the Interna-tional Standard for  C. botulinum  type C antitoxin were solow that distribution had ceased [16]. Toxins of types A, B and E are those for which therapeutic antitoxins are most fre-quently required for humans, and the continued supply of these was therefore considered important. However, the Com-mittee was of the opinion that as C, D and F are mainly of in-terest for typing purposes, that they should be retained as theydefined the relevant International Units, but that the Interna-tional Standard for type C antitoxin need not be replaced[16]. The recent generation or development of new bio-defence polyclonal and oligoclonal (a combination of severalmonoclonals per serotype) antitoxins and vaccines againstall seven serotypes has, however, changed this perspectiveand highlighted the urgent need for replacement antitoxinsto calibrate potency assays [17,18].This is in contrast to the use of these standards in serotyp-ing, which has probably decreased in recent years due in partto the use of genetic testing and the increased availability of alternative commercial polyclonal antibodies (e.g. StatensSerum Institut).In 1996 the custodian for the remaining stocks of Interna-tional standards was changed from the Statens Serum Institut(Denmark) to the National Institute for Biological Standardsand Control (UK).Unfortunately, over the years many of the other officialWHO antitoxin standards have also become depleted or totallyexhausted, with serotype A, C, E and F antitoxins no longeravailable for distribution. Furthermore, there is still no definedInternational Unit for a botulinum type G antitoxin. Stocks of serotype D are also very low and are currently being rationedbut will soon no longer be available for distribution.Two reserve type A and B batches of antitoxin were alsoproduced in 1960 but utilised equine antiserum donated byBurroughs, Wellcome (Beckenham, UK). Unlike the 1st Inter-national Standards these materials were first diluted withnormal horse serum (Table 2). Since stocks of the InternationalStandard for type A have been exhausted, the reserve batch 59/ 21 has been issued as a replacement (Table 2). Progress has,however, been made in recent years to provide candidate Table 1Official WHO International Standards for botulinum antitoxinsSerotype Botulinum antitoxin standard Type Code/availabilityType A 1st International/1st BritishStandard (500 IU/ampoule)Equineserum60/18 (BUSA),not availableType B 1st International/1st BritishStandard (500 IU/ampoule)Equineserum60/21, notavailableType B 2nd International Standard(31 IU/ampoule)EquinepurifiedF(ab # ) 2 BUSB 1390,availableType C 1st International/1st BritishStandard (1000 U/ampoule)Equineserum60/22 (BUSC),not availableType D 1st International/1st BritishStandard (1000 IU/ampoule)Equineserum61/01 (BUSD)360, availableType E 1st International/1st BritishStandard (1000 IU/ampoule)Equineserum60/23 (BTUSE),not availableType F 1st International Standard(4 IU/ampoule)RabbitserumBUSF, notavailableTable 2Reserve batch botulinum antitoxin materials made in 1960Serotype Botulinum antitoxinstandardType Code/availabilityType A NIBSC reagent(2000 IU/ampoule)Equineserum59/21 Approx. 1760,availableType B NIBSC reagent(2400 IU/ampoule)Equineserum60/001 Approx. 1600,available224  R.G.A. Jones et al. / Biologicals 34 (2006) 223 e  226   replacements where required (C e F), together with the first ref-erence material for serotype G utilising equine antiserum kindlydonated by USAMRIID in 1998, and filled and freeze dried atNIBSC (Table 3). These materials have yet to be fully cali-brated in terms of the srcinal WHO International Standards(where relevant) and an accepted unit for serotype G has stillto be defined. 4. Calibration Users of BUSB have reported in the past that this prepara-tion differs in its relative behaviour to reserve batch 60/01which at the time was assumed to have the same activity asthe 1st IS (500 IU/ampoule). This difference was also subse-quently confirmed at NIBSC in November 2000. These find-ings were noted in a WHO Annex which suggested that itshould be investigated further and if necessary steps weretaken to obtain a replacement.Subsequent in house studies performed in 2005 (Fig. 1) us-ing the local flaccid paralysis assay [19] to verify the activityof interim reference reagents against the srcinal ISs revealedno significant difference between the activity of the 1st and2nd ISs using B toxin from the Okra strain. Following thisconfirmation of BUSB (2nd IS) activity, it will now continueto be issued by NIBSC. However, the interim (reserve) refer-ence materials 59/21 and 60/01 were found to contain approx.2000 and 2400 IU/ampoule, respectively, relative to the appro-priate primary International Standards (Fig. 1).Candidate replacement standards for types E and F werealso calibrated in house against the srcinal ISs and assignedactivities of 197 and 125 IU/ampoule, respectively (Fig. 2).All currently available International Standards and candi-date replacement preparations are available from NIBSCwith further details and ordering information at Botulinum antitoxins for general invitro/diagnostic use are also available from a number of suppliers including the Statens Serum Institut ( sw379.asp). 5. Human botulinum antitoxin A human botulinum antiserum (code no. 00/500) preparedfrom the pooled serum of individuals vaccinated witha pentavalent (A e E) vaccine has also been filled and freezedried for distribution for potential use in human serologywork. Each ampoule contains  w 0.5 IU against type A and Btoxins. 6. Toxin reference preparations Following the emergence of therapeutic botulinum toxinsand the perceived need for International Standards or commonreference toxins, it was proposed by the WHO that NIBSCshould seek candidate materials and arrange a collaborativestudy [20]. Three candidate botulinum type A toxin referencematerials were obtained and a collaborative study was per-formed in 10 laboratories in five countries [21]. The study Table 3Candidate reference replacements for botulinum antitoxinsSerotype US srcinal lot#approx. potencyFreeze driedpotencyestimateCodenumberNumber of ampoules(approx.)Type A 027-0001 @673 IU/ml ND None NDType B 067-0006 @2257 IU/ml ND None NDType C 4000-012 @7617 IU/ml Unknown 01/508 2840Type D 3500-007 Activity unknown Unknown 01/510 2810Type E 072-0003 @166 IU/ml 197 IU/ampoule 02/318 2570Type F 022-0013 @257 IU/ml 125 IU/ampoule 01/506 2700Type G 176-0006 Activity unknown Unknown 01/512 2840ND ¼ not done. 1 10 1000123 Antitoxin mIU/ml        S     c     o     r     e Fig. 1. Mouse, local flaccid paralysis assay (48 h) of botulinum type A and Breference antitoxins. Type A antitoxins (solid line) tested against a fixed toxindose (from Hall strain). 1st IS 60/18 ( - ), and 59/21 ( : , assuming 2000 IU/ ampoule). Type B antitoxins (dotted line) tested against a fixed toxin dose(from Okra strain). 1st IS 60/21 ( ; ), 2nd IS BUSB ( A ), and 60/01 ( C ,assuming 2400 IU/ampoule).   SEM ( n ¼ 8). 0.1 1 10 1000123 Antitoxin mIU/ml        S     c     o     r     e Fig. 2. Mouse, local flaccid paralysis assay (24 h) of botulinum type E and Freference antitoxins. Type E antitoxins (purple) tested against a fixed toxindose (from Alaska strain). 1st IS BTUSE ( - ), and 02/318 ( : , assuming197 IU/ampoule). Type F antitoxins (red) tested against a fixed toxin dose(from Langland strain). 1st IS BUSF ( ; ), and 01/506 ( A , assuming 125IU/ampoule).   SEM ( n ¼ 8).225  R.G.A. Jones et al. / Biologicals 34 (2006) 223 e  226   showed that expression of results relative to a standard reducedbetween-laboratory variation, and one candidate material wasproposed as a potential International Reference Reagent. How-ever, not all the study participants agreed with this choice be-cause of concerns about the instability of the candidatestandard if saline was used as the diluent in the assay, andalso due to concerns over the long term stability of the toxin[22]. The Committee suggested that the authors considerwhether there was a need for product-specific reference mate-rials [22]. No International Standards for botulinum toxin A orother serotypes currently exist. Nevertheless, some manufac-turers have since moved to using their own in house referencematerial to express potencies as relative values as outlined ina recent European Pharmacopoeia monograph [23]. 7. Summary Only two WHO International Standards for botulinumantitoxin: Type B, 2nd IS (BUSB) and a limited amount of type D, 1st IS (61/01 or BUSD), are still available fordistribution.Interim and candidate reference reagents are available fortypes A e F. Type A, B, E and F interim candidate reference re-agents have been calibrated ‘in house’ using the mouse flaccidparalysis assay. These interim reagents should, however, alsobe calibrated internationally and assigned WHO InternationalStandard status, if found to be acceptable.A definition of the International Unit for serotype G anti-toxin has yet to be defined or accepted by the WHO andurgently needs addressing. In anticipation, a candidate typeG standard has been prepared for distribution and assessment. Acknowledgements We would like to thank staff from BSS for their help inperforming the bio-assays and Peter Rigsby for statisticallyanalyzing the data. References [1] Bowmer EJ. Preparation and assay of the International standards for Clostridium botulinum  types A, B, C, D and E antitoxins. Bull WorldHealth Organ 1963;29:701 e 9.[2] WHO Expert Committee on Biological Standardization Fifteenth report.World Health Organization Technical Report Series 1963;259:25.[3] Bowmer EJ. Antitoxins of   Clostridium botulinum  types A, B, C, D and E.Preparation and assay of proposed International Standards. Faculty of Medicine University of Liverpool. MD thesis; 1962. p. 1 e 108.[4] WHO Expert Committee on Biological Standardization Sixteenth report.World Health Organization Technical Report Series 1964;274:20 e 1.[5] WHO Expert Committee on Biological Standardization Eighteenthreport. World Health Organization Technical Report Series 1966;329:17.[6] Siegel LS. Evaluation of neutralizing antibodies to type A, B, E, and Fbotulinum toxins in sera from human recipients of botulinum pentavalent(ABCDE) toxoid. J Clin Microbiol 1989;27(8):1906 e 8.[7] WHO Expert Committee on Biological Standardization Twenty-seventhreport. World Health Organization Technical Report Series 1976;594:16.[8] WHO Expert Committee on Biological Standardization Twenty-eighthreport. World Health Organization Technical Report Series 1977;610:19.[9] WHO Expert Committee on Biological Standardization Twenty-ninthreport. World Health Organization Technical Report Series 1978;626:13.[10] WHO Expert Committee on Biological Standardization Thirty-firstreport. World Health Organization Technical Report Series 1981;658:14.[11] WHO Expert Committee on Biological Standardization Thirty-secondreport. World Health Organization Technical Report Series 1982;673:18 e 9.[12] WHO Expert Committee on Biological Standardization Thirty-thirdreport. World Health Organization Technical Report Series 1983;687:19.[13] WHO Expert Committee on Biological Standardization Thirty-fourthreport. World Health Organization Technical Report Series 1984;700:14.[14] WHO Expert Committee on Biological Standardization Thirty-fifthreport. World Health Organization Technical Report Series 1985;725:15.[15] WHO Expert Committee on Biological Standardization Thirty-sixthreport. World Health Organization Technical Report Series 1987;745:13 e 4.[16] WHO Expert Committee on Biological Standardization Sixteenth report.World Health Organization Technical Report Series 1989;786:18.[17] Nowakowski A, Wang C, Powers DB, Amersdorfer P, Smith TJ,Montgomery VA, et al. Potent neutralization of botulinum neurotoxinby recombinant oligoclonal antibody. Proc Natl Acad Sci U S A 2002;99:11346 e 50.[18] Smith LA, Jensen MJ, Montgomery VA, Brown DR, Ahmed SA,Smith TJ. Roads from vaccines to therapies. Mov Disord 2004;19(Suppl.8):S48 e 52.[19] Sesardic D, Mclellan K, Ekong TA, Das RG. Refinement and validationof an alternative bioassay for potency testing of therapeutic botulinumtype A toxin. Pharmacol Toxicol 1996;78(5):283 e 8.[20] WHO Expert Committee on Biological Standardization Forty-fourthreport. World Health Organization Technical Report Series 1994;848:21.[21] Sesardic D, Leung T, Gaines DR. Role for standards in assays of botuli-num toxins: international collaborative study of three preparations of botulinum type A toxin. Biologicals 2003;31(4):265 e 76.[22] WHO Expert Committee on Biological Standardization Forty-ninthreport. World Health Organization Technical Report Series 2000;897:25 e 6.[23] Botulinum toxin type A for injection 01-2005:2113 European Pharmaco-poeia 5.03. EDQM; 2005. p. 1117 e 9.226  R.G.A. Jones et al. / Biologicals 34 (2006) 223 e  226  All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.
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