W O m D HEALTH ORGANIZATION
ORGANISATION MONDIALE DE LA SANTE
E;&WEI(iT COMRTITTEIE ON BIOLOGICAL STANDAmIZATION Geneva, 15 to E9 No~rember 2004
MXOIBS104.1998 ENGLISH OXLY
Evaluation of purified
monoeIona1 antibodies toBordeteZlapertussis firnbriae type 2 and 3
Study Report and proposal to establish WHO reference reagents
D. Xing, P. Newland and M. Corbel. Division of Bacteriology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, EN6 3QC, UK
Preparations containing S no no clonal antibodies to Bordetellapertussis
serotype 2 (coded 0411 54) and serotype 3 fimbriae (coded 04/ 156) which are intended to be used for both serotyping of clinical isolate strains and also monitoring pertussis vaccine production consistency, have been assessed using a standard strain panel in a collaborative study by eleven laboratories in nine countries for their suitability to serve as reference reagents for B, pertzdssis serotyping in two major methods, namely microplate agglutination method and slide agglutination. Rabbit polyclonal
antibodies to FIM 2 (coded 89/598) and FIM 3 (coded 89/600) were included as control and these polyclonai antibodies have been in use since 1989 as typing
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WHO!BS!04.1998 Page 2
reagents for B. pertussis serotype in the slide agglutination method. In parallel.
monoclonaI antibodies produced from a second resource of hybridoma cell lines were also compared with preparations 0 4 1 54 and 04/156. Under the recommended assa) conditions, there was close concordance between the results obtained between laboratories using either methods. Preparations 0.111 54 and 0411 56 showed good specificit]r. in both typing methods evidenced by >90% sensitivity on the
corresponding strains and 0.1 % cross-reaction. lllonoclonal antibodies obtained from the second source showed less sensitivity than preparations 041154 and 041156.
On the basis of the results of this study, it is therefore recommended that monoclonal antibody to fimbriae 2 (0411 54) be established as the WHO Reference Reagent (2004) for B. perfussis serotype 2 and monoclonal antibody to frmbriae 3 (041156) be
established as the WHO Reference Reagent (2004) for B. pertzrssis serotype 3.
All participants agreed with the interpretation of their data and the proposals to establish the materials. These preparations are currently available as liquid fiIIs and are proposed as interim reagents. Studies on the stability of freeze-dried preparations are in progress. According to current demand, an estimated 5 years supply of the antibodies is available, but arrangements exist for almost unlimited production.
Introduction
W O has recommended that whole cell Bordetetlapertmis vaccines should contain strains expressing agglutinogen 2 (equivalent to Fim 2) and agglutinogen 3 (equivalent to Fim 3) ( W O Technical Report Series, No. 800, 1990). The identity of agglutinogen I is not known but it is not appropriate for discriminating between B.
pertussis isolates as it does not vary. Serotype 2 and 3 epitopes were found to be associated with fimbriae, designated serotype 2 and serotype 3 fimbriae (Fim2 and Fim 3). Monitoring of fimbrial expression is recommended not only as a simple means for detecting changes in B. pertussis populations, but also because fimbriae could be important protective antigens.
Serotyping is one of the traditional typing methods used for typing B. pertussis strains. Serotyping with polyclonal antisera has been used routinely for many years, However, the stocks of these antisera are nearly exhausted. Furthermore, inter- laboratory comparison of serotyping results is rendered difficult due to the different sera and assays used. There is a need to standardize the assays and at the first stage to standardize the typing reagents. In May 1999, representatives from laboratories involved in epidemiological research on pertussis met at the Institut Pasteur, Paris, France. The outcome of the meeting suggested that it would be sensible to use monoclonal antibodies for typing fimbriae and that NIBSC in UK would be responsible for preparing these monoclonal antibodies for distribution.
Two sets of hybridoma cell lines for B. pertussis fimbriae type 2 and 3 were used for production of monoclonal antibodies. One set was originally developed by Dr M. Brennan and kindly donated by Dr B. Meade, FDA, USA, and another set was kindly donated by Dr A. Robinson, GAMR, UK. Ascites from these cell lines have been prepared in NIBSC, Evaluation of these 4 monoclonal antibodies and 2 polyclonal anti-agg 2 and anti-agg 3 as controls, was carried out in the first
WHOlBSl04.1998 Page 3
collaborative study initiated in early 2002. A total of 9 participants were involved using microplate agglutination and slide agglutination methods. A total of 8 B.
perfussis reference strains for Fim2 and Fim3 (in freeze-dried form) were kindly provided by Dr Hans Hallander (Sweden). These strains were selected as reference strains from clinical isolates within the EU pefistrain project for B, perfussis
characterisation of serolype. pertactin and peflussis toxin genes. However, the results of the first collaborative study were not conclusive due to raw ascites fluid being used for typing, and different dilutions being used by different laboratories. Later in the year, a small scale collaboration which involved with only 4 laboratories using
purified monoclonal antibodies was carried out. The purpose of this small study was to try to optimize basic assay conditions. It was found that both purity and concentration of Mabs are important for the test. In November 2003, a further collaborative study was carried out involving l l laboratories. The results of this latest study are reported here.
Participants
Eleven laboratories perfoming serotyping assays participated in the study.
These participants are listed in Table I. Throughout this report participants are identified only by a randomly assigned code number from l to I I.
Materials and Methods
a. Typing reagents Hybridoma cells
Hybridoma cell lines BPF2 (anti FIMS 2) and BPGlO (anti FIMS 3) had been
originally developed by Drs Brennan, Manclark and Li, GBEWFDA, USA (US Patent 5,162,223 (Nov 1992) and kindly donated by Dr B. Meade, FDA, USA in Oct 2001 via a material transfer agreement between GBEW FDA and NIBSC to allow NIBSC to use the cell lines to produce monoclonal antibodies for use in reagent preparations which will be distributed free of charge (only handling fees will apply) world-wide.
Hybridoma cell lines Ag 2 (anti FIMS 2) and Ag 3 (anti FIMS 3) were kindly donated by Dr A Robinson, CAMR (Centre for Applied Microbiology and Research) Porton Down, UK in Jan 2000 via a material transfer agreement between CAMR and NIBSC.
Monoclonal antibodies produced by tissue cultwe
The four hybridoma cell lines were grown in Iscove's modified Dulbecco's medium, with foetal calf serum and supplemented with insulin, transfenin and 2-
mercaptothanol. Maximum densities of the cells were grown in 75cm flasks, the supematant being harvested by centrifugation ofthe cell suspension followed by coltection of the resulting cell supematant containing the monwlonal antibody. The cell supematant from multiple flasks was then pooled and frozen before purification.
itlbnoclonal antibodies producedfrom axcitic fluid
Hybridoma cell lines were grown as described above. The hybridoma cell suspensions were concentrated approximateiy 15 times and given by intraperitoneal injection into pristane primed BalbiC mice ARer 7 days ascitic fluid was collected, centrifuged and frozen at -200C before further treatment.
WHO/BS/04.1998 Page 4
Purifcaion of monoclonal antibodes
Both the cell supernatant and ascitic fluid were purified/ concentrated by ammonium sulphate precipiration. The resulting protein was then dialysed against phosphate buffered saline (PBS). The optical density (OD) of the dialysed solutions was measured by OD at 280nm and then adjusted with PBS to give 4 mgiml IgG for the aseites and 5 mg/mf IgG for the cell supernatant. These were dispensed with Eppendorf tubes in 0.5 m1 volumes using a calibrated Cilson pipette and frozen (-2043 prior to use in the study. The precision of 511 was < CV 2.5% and but is not critical as serotyping is a qualitative test.
Two sets of monoclonal antibodies have been included in this study. One set was Mabs from mouse ascites. Another set was prepared fiom tissue culture supematant.
A11 these preparations have been purified/ concentrated by ammonium sulfate precipitation as described above. MonocIonal antibodies purified from both tissue culture ( l ) and ascitic fluid (2) were incIuded in the study.
Polycfonal antibodies
NIBSC catalogue materials 891598 (anti-agglutinogen 2) and 891600 (anti-
agglutinogen 3) (freeze dried rabbit anti-serum) was also included in this study for comparison purposes.
Participants 2, 3, 6 and 7 also used their own in-house typing reagents in the study.
b. Bacterial strains
A total of 8 B, pertussis reference strains for Fim2 and Fim3 (in freeze-dried form) used in this study was kindly provided by Dr Hans Hallander (Sweden). All strains were coded as S 1, 52, S3, SS, S6 S8 and S9 plus FDA 460. Some laboratories which did not keep the strains sent out during the first phase studylor new participants not involved in the first study, used their own reference strains.
In general, the method used to grow strains involved re-suspending the freeze-dried bacteria in approx. 0.5 m1 of PBS or broth and inoculation of the bacteria on charcoal horse-blood agar or Bordet-Gengou agar at 34-37OC for 48 -72 hours.
Apart from the reference strain panel, a total of 23 other strains and 48 clinical isolates were also typed by participants 2,4, 5,6,7, 10 and l 1 respectively. The results are presented in Table 5.
The details of the panel of antibodies and strains used in this study are presented in Table 2.
c. Typing method
The participating laboratories was asked to type 8 strains with I0 antibody preparations provided. Protocols for the slide method ( from lab I l) and for the rnicrotibe plate method (from Lab 3) have been provided to participants for
infornation. A dilution of 1 : 100 of Mabs and a bacteria1 dilution of 1 : 1 from OD 1.2 at 650 nm was found to be optimum for the microtitre plate method in the second
WHO/BS/03.1998 Page 5
small scale study. Therefore participants were requested to perform the test according to the recomended dilution factors.
Laboratories were also encouraged to perform their own in-house assay in parallel with the suggested method. It was recommended that each strain is to be typed at least twice with each antibody.
A total of 9 laboratories (participants 1.2,3,4,5,6, 7, 9 and 10) performed serotjiping using microtitre plate assay and 5 laboratories (participants 4, 5'6.8 and 11) performed serotyping using slide agglutination assay in which laboratory 4, 5 and 6 perfomed both assays. Two other new methods were also used for typing by participant 8 (Flow c~qometry method) and participant 2 (ELISA method). Details of these assay methods are presented in Appendix 5-8.
d. Collection Data:
Data were collected by NIBSC. Laboratories have been identified by a code in all analyses to maintain confidentiality.
e. Stability study:
Hitherto the monoclonal antibody preparations have not been freeze-dried. For the stability study, the frozen monoclonal antibody samples were removed from -20°C freezer for ten cycles of freezing and thawing. Control samples for each monoclonal antibody were kept frozen and take out just before assay. Since the ELISA method may give more precise results on the stability of antibody than microplate or slide agglutination test, the stability of these samples was assessed by ELISA method using Fim 2&3 (NIBSC code 851549 ) (2pgiml) as antigen to coat plate and ELISA carried out with the monoclonal antibodies. OD readings at 450nm for test samples were compared with the control sample at each time point.
Results and Discussion
Results on strain panel by microtitre plate aggluiination:
A total of 9 laboratories provided results using the microtitre plate method. The dilutions of monoclonal antibodies used for the typing were from l/lOO up to 1/6400 of the original solutions. The summary results are presented in Table 3 and details of results from each participant are presented in Appendix 1 . In general, all results obtained by different laboratories showed good agreement for the panel of strains with the exception that a high level of autoagglutination for strains S5 and S8 was found by Laboratory 7. Different dilutions (>loo) of the antibodies seems not to affect the results.
A total of 18 typing tests were performed using the microplate method for the panel of strains except for strain S5 and S8 where the results from only 13 typing tests were taken into consideration due to the problem of autoagglutination. With the strain panel, using monoclonal antibodies A and B as the Vping reagents, the results
obtained from digerent laboratories showed very good concordance and aImost 100
WHOIBSIO4.I 998 Page 6
percent positive results for all serotype 2 strains using monoclonal antibodies A I and A2 and for all serotype 3 strains using monoclonal antibodies B 1 and B2. Almost no cross-reaction was found between A and B for all the strains with the exception of strain S5 (serotype 3) where only 1 out of 13 typing tests s h o ~ e d a positive response with A1 (anti-fim2). For the serotype 2&3 strain (FDA460), monoclonai antibodies A and B showed a similar percentage of positive responses to the corresponding
serotype as above, with 100% and 97% respectively.
Monoclonal antibodies C1 and C2 were approximately 10% less sensitive in typing type 2 strains than monoclonal antibodies AI and A2 (83%-94% positive responses in comparison with 100%). A rather different profile was found for type 3 strains using monoclonal antibody D1 and D2 which showed poor performance ranging from 0 to 33 percent positive response, varying according to different strains.
D1 appeared to be better than D2 (average 19% compared to 2%). An average of 4%
cross-reaction with type 3 strains was found for C1 monoclonal antibody. For the serotype 2&3 strain (FDA460), monoclonal antibodies C l and C2 showed 97% and 94% positive response respectively. However, this strain was not recognized by either D1 or D2 using the micro plate method.
Results on strain panel by slide agglutination:
A total of 5 laboratories provided results from serotyping using the traditional slide agglutination method. The concentration of monoclonal antibodies used for these typing tests were: neat, 1/10, 1/50 and 11100 dilution of the original solutions.
The summary results of the slide agglutination method are presented in Table 4 and details of results from each participant are presented in Appendix 2. Again in general, all results obtained by different laboratories showed good agreement for the panel of strains.
A total of 9 typing experiments were performed for each strain using the slide agglutination method for the panel of strain. With the strain panel, using monoclonal antibodies A and B as the typing reagents, the results obtained fiom different
laboratories showed very good agreement. This has been evidenced by 100 percent positive results to all serotype 2 strains using monoclonal antibodies A1 and A2.
When monoclonal antibodies B 1 and B2 were used for typing type 3 strains, almost 100% of positive response was obtained for strains S3, S5 and S6. However, for strain S8, there was only 89% positive response. Almost no cross-reaction was found between A and B for all the strains. For the serotype 2&3 strain (FDA460),
monoclonal antibodies A showed 98% of positive response. However, monoclonal antibodies B l and B2 only showed 67% positive response.
In agreement with the results obtained by the microplate method, monoclonal antibodies C1 and C2 showed less sensitivity in typing type 2 strains than monoclonal antibodies A1 and A2 (70%-63% positive responses in comparison with 100%).
Although monoclonal antibodies D1 and D2 were less sensitive than B1 and B2, they were found to perform better for type 3 strains S3, SS, SS, with >80% positive
response in comparison with microplate method. It is interesting to note that for strain S8, D1 and D2 only showed 28% positive response. Cross-reaction varied according to strains for all D and C monocIona1 antibodies (Table 7). For the serotype 2&3 strain (FDA460), monoelonal antibodies C l and G2 showed only 33% positive
W HO43S!O4. l 998 Page 7
response. Again this strain was not recognised by antibody D1 and only 11% were recognized by D2.
Results on participanfs own in-house strains by boot mmicruplafe avrd slide aggIuIination methods:
A summary of the results on the typing of strains which were not included in the strain panel is presented in Table 5 and detaiis of the results from each laboratop are presented in Appendix 3. A total of 8 participants used microplate method and 3 laboratories used the slide agglutination method to type their own strains.
Laboratories 2,4,5,6, 7 and 1 l using the monocIona1 antibodies performed typing of their own strains in parallel with the strain panel. Laboratories 9 and I0 did not perform typing on the strain panel because they did not participate in the first phase study in which the panel of strains had been sent out. Laboratory 9 performed typing on clinical isolates in paraiiel with 2 of their own in house reference strains of known serotype. Laboratory 10 perfomed typing on 8 of their in-house strains of known serotype.
Taking all the results obtained from both methods together, they showed a similar trend with that found for the strain panel but with slight lower sensitivity. For type 2 strains, 83%, 63% positive response were found for monoclonal antibodies A and C respectively and for type 3 strains, approximately 90% and 35% positive response when B and D monoclonal antibodies were used respectively. However, it seems slightly higher cross-reaction occurred with monoclonaI antibodies A and C against type 3 strains (Table 5). For type 2&3 strains, in general approximately 70%
or more positive responses were found except for monoclonal antibody D which did not recognize these strains.
Potyclonal antibodies:
Rabbit polyclonal antibodies were used for comparison purposes in the present study. The concentrations of the polyclonal antibodies used for typing were: 1/20 and 1/40 dilution in the microplate assays and neat, 1/10, 1/50 and 1/100 dilution of the original solutions in slide agglutination assays.
Using the microtitre plate agglutination assays, the anti-type 2 polyclonal antibody P2 performed poorly, showing negative response to all type 2 strains and 2&3 strain but approximately 34% cross-reaction to type 3 strains S5 and S6 was found. PoIyclonal antibody P3 showed 100% positive response to type 3 strains S5 and S8, but S3 and S6 only had positive responses with 50% and 90% respectively.
For the serotype 2&3 strain (FDA460), P3 only showed 50% positive response.
There was no cross-reaction observed with P3 to type 2 strains.
Using the slide assays, the anti-type 2 polyclonal antibody P2 was found to perform better (>60% positive response) in comparison with microplate agglutination assays (0%) for type 2 strains, but it showed less sensitivity to serotype 2&3 strain (FDA460) with only 33% positive response. Approximately 1 10/0 cross-reaction to type 3 strain S5 and 56 was found for P2. PoIyclonaI antibody P3 showed
approximate1y 90% positive response to type 3 strains S3, S5 and S6 with less
WHOfBSIO4.1998 Page 8
sensitivity to S8 (72%). For the serotype 2&3 strain (FDA460), P3 only showed 55%
positive response. There was no cross-reaction observed with P3 to type 2 strains.
These polyclonal reagents were prepared for the slide agglutination assay in the 1980s and since then have been widely used for serotyping. They have been
recommended to be used neat in the slide method after re-constitution of the freeze- dried material in 0.5mI of water. There was no evidence of loss of activity on storage since the undiluted material still worked we11 in slide agglutination tests in the present study. However, it is noted that far greater dilutions than the originally recommended concentration were used by most panicipants and this may at Ieast partially explain the poor performance found in this study.
Comparison of microplate and slide agglutination met!zods
Figure l gives a summary of combined results for the strain panel using both microplate and slide methods. Since the results obtained from this study using monoclonal antibodies A and B were the most promising, comparison of microplate and slide methods are made using monoclonal antibodies AI and B1 only as
examples. The results are presented in Figure 2. Overall, these two methods showed comparable results. Both methods showed 100% positive response when A1 and B1 were used for typing the corresponded strains, except that the slide method showed slightly less sensitivity to type 3 strain SS. For type 2&3 strain (FDA 4601, although the B I was less effective than AI preparation in the plate method, again the slide method was less sensitive than the plate method, in particular when B 1 used as the typing reagent. In general, both methods showed good agreement in the results and the sensitivity was >90% up to 100% for the strain panel with the exception of the B l preparation in the slide method for the type 2&3 strain.
Other typing methods
In parallel with the microplate and slide methods described above, two
laboratories also reported their results obtained from their own in-house methods for the strain panellor in addition with in house strains. Participant 2 used indirect whole cell ELISA method and participant 8 used flow cytometry method. These results and antibody dilutions used in the typing are presented in Table 6 and the details of the method are given in Appendix 7 and 8. The results obtained from both the ELISA and flow cytometry method using monocIonal antibodies A and B preparations appeared to be in good agreement with the results obtained by microplate and slide methods. Preparations C and D did not perform well either in the flow cytometry method or in microplate and slide methods, although the C preparation gave a better result in the indirect ELISA method.
Laboratoly performance
In general, results obtained from all laboratories were in good agreement. There was no particular problem trend obsemd on laboratory performance.
WHOIBSI04.1998 Page 9
Monoclorzal antibody performance from combined results obtained by all typing methods
Both monoclonal antibody preparations A and B performed very we11 in relation to positive response (>95%) except for strain S8 (89% positive response) and cross-reaction (51%). Slightly less sensitiviv was found for preparations C l and G2 (>'75% of positive response). However, discordant results with different strains were obtained for preparations D1 and D2 using both methods. Figure 3 presents overall results for all strains of known serotype tested and by all methods. Details of data are presented in Table 7 .
In terms of overall sensitivity. the monoclonal antibody preparations A and I3 showed the best performance of all the preparations. It is evidenced that these two reagents react with the corresponding fimbrial components effectively. It has also been reported that the preparation C and D detect fimbrial components. The lower sensitivity found for preparation D in the present study was not in agreement with the results obtained from an early study in which this preparation showed superior to preparation B in the slide agglutination method when used for typing clinical isolates.
However, it is not clear that the monoclonal antibodies actually detect identical epitopes on these strains. Thus there may be incomplete overlap of specificity. This could explain the discrepancies noted in this and in the previous study. Another factor influencing agglutinating activity could be the isotypelsubclass of the antibody. The C and D reagents were reported to be of IgM isotype (although anti-Agg 3 reacted with anti-IgG in our tests) whereas the A and B antibodies were IgG.
Monoclonal antibodies produced by both the mouse ascites and tissue culture methods performed well in the present study regardless of typing methods. This gives a good indication that firture production of these typing reagents could be done by tissue culture method.
For determining the presence of Fim 2 and Fim 3, the monoclonal antibody reagents A and B seem to offer the best sensitivity and we suggest that these should be adopted as the references as confirmed by the multi-laboratory collaborative study.
Stabiliq study
Currently, monoclonal antibodies A and B at 4mglml IgG are kept as frozen (- 2042) in 0.5 m1 per vial. Stability was assessed by removing these reagents from -20°C freezer for ten cycles of freezing and thawing. Comparison of results obtained from ELISA on samples over 10 cycles of freezing and thawing with a sample kept at -20°C did not show decrease in optical density (Figure 4) which indicates that these antibodies are stable under current storage conditions.
CONCLUSIONS AND RECOMMENDATIONS
Inter-laboratory comparison of seroQping resuits is rendered diMicult due to the different sera and assays used. The need for inter-laboratory comparability of results from seroQping B. perfussis is recognized. AIthough it is clear that reference
WHO/GS/04.1998 Page 10
preparations alone will not achieve this goal, characterised reference preparations nevertheless provide an initial point of comparison.
Results from this study confirmed that monoclonal antibodies produced by BPF2 and BPC 10 hybridoma cells were effective for typing B. pertzdssis using different typing methods. Monoclonal antibodies produced by Ag2 and Ag3
hybridoma cells were less effective than that produced by BPF2 and BPC 10. On the basis of the results of this study, it is therefore recommended that monoclonal
antibody to fimbriae 2 (0411 54) be established as the WHO Reference Reagent (2004) for B. pertussis serotype 2 and monocfonal antibody to fimbriae 3 (041156) be
established as the WHO Reference Reagent (2004) for B. per*f.tussis serotype 3. All participants agreed with the interpretation of their data and the proposals to establish the materials. These are proposed as interim preparations pending investigation of the stability of freeze dried materials.
AGKNO WLEDGMENTS
Grateful acknowledgements are due to Drs Brennan, Manclark, Li and Meade CBEWFDA, USA and Dr A Robinson, CAMR for kindly donating the hybridoma cell lines. Special thanks given to Dr H. Hallander, SIIDC, Sweden and Dr N. Guiso, Institut Pasteur, France for provision of the strain panel and helpful advice. We also thank all of the participants for their contributions without which this study would not have been possible.
REFERENCES
Brennan et al. United States Patent No 5 162,223. 1992
A.R. Gorringe, L.A.E. Ashworth, L. I. Irons and A. Robinson, Effect of monoclonal antibodies on the adherence of Bordetella pertussis to Vero cells. FEMS Microbiology letters. 1985,26:5-9.
F.R. Mooi, H. Hallander, C.H. Wirsing von Konig, B. Hoet, N. Guiso,
Epidemiological typing of Bordetellapertussis isolates: recommendations for a standard methodology. Eur. J. Clin. Microbiol. Infect. Dis. 2000, 19: 174- 1 8 1.
N. Guiso, CH. Wirsing von Konig, H. Hallander, Fimbrial typing of Bordetella pertussis isolates: Agglutination with polyclonal and monoclonal antisera. J. Clin.
Microbiol. 2001, 39: 1684-1685.
D.K.L. Xing, S. Ramakrishnan, P. Newland, M.J. Corbel, Fimbrial typing of Bordetella pertussis isolates: Agglutination with polyctonal and monocfonal antibodies. J. Clin. Microbiol. 2001, 39: 4220.
WHO!BS/04.1998 Page I I
Table 1 Participants
Dr Hans Hallander and Mr Abdolreza Advani Swedish Institut for Infectious Disease Control Doktorsringen 25
SE-I71 82 SOLNA Sweden
Prof. Dr. Fritz C.H.Wirsing von Kiinig Institut fuer Hygiene und Labormedizin Klinikum Krefeld
Lutherplatz 40 D-47805 Krefeld Gemany
Dr. N. Guiso
Molecular Prevention and Therapy of Human Diseases Institut Pasteur
25, Rue du Dr. Roux F-75724 Paris Cedex 15
France
Dr Dorothy Xing and Miss Penny Newland Division of Bacteriology
National Institute for Biological Standards and Control Blanche Lane
South Mimms Hertfordshire EN6 3QC, UK Dr Raymond Tsang,
GNS Infection and Vaccine Preventable Bacterial Diseases Division, National Microbiology Laboratory,
Population and Public Health Branch, Health Canada, 10 15 Arlington Street,
Winnipeg, Manitoba, Canada R3E 3R2.
Dr Tim C Harrison and Mr John Duncan Health Protection Agency
Respiratory and Systemic Infection Laboratory Specialist and Reference M i c r o b i o l o ~ Division 6 1 Colindale Avenue
London
NW9 5WT, U K
\HO/OS/O4.1998 Page 12
Table l
cont.Dr Ditte Dragsted and Mrs Lene Berthelsen
Department of Bacteriology. Mycology and Parasitology Statens Serum Institut,
Artillerivej 5, 2300 Copenhagen S Denmark
Dr Qiushui He and Miss Mia Antila Pertussis Reference Laboratory National Public Health Institute Kiinamyllynkatu 13
FM-20520 Turku Finland
Dr Frans A.G. Reubsaet and Mrs Dieneke Hoeve
Special Reference Department for Identification of Bacteria (BBD), National Institute of Public Health and the Environment (RIVM) P.O. Box 1
3720 BA Bilthoven
Antonie van Leeuwenhoeklaan 9 372 1 BA Bilthoven
The Netherlands Dr Andrew J Lawrence,
Microbiology and Infectious Diseases Department.
Women's and Children's Hospital 72 King William Road
North Adelaide, South Australia 5006
Dr Jayasena Rajiv and Mr John Suendemann QC Development
CSL Ltd
45 Poplar Rd, Parkville Victoria 3052
Australia
WI-10!BSi04.1998 Page 13
Table 2a Antibody Sample Information
1
Antibody Code/
Description l1
A1I
Monoclonal antibody to FIM 2 (NIBSC code 04154) 1 l/
from hybridoma cell line BPF2 (F2), Mouse ascites purifiedI
by ammonium sulphate precipitation.1
i I SuppIied diluted to Jmgiml in PBS, frozen without sodium
1
1
azide IA2
I
Monoclonal antibody to FIM 2. from hybridoma cell line j BPF2 (F2), Tissue culture supematant purified and Iconcentrated by ammonium sulphate precipitation
.
Supplied diluted at Smgiml, frozen without l
; sodium azide
I
IB1 ' Monoclonal antibody to FIM 3 (NIBSC code 041'1 56)
I
From hybridoma cell line BPC I0 (F316), t II
B21
from hybridoma celi line BPCIO (F3/6), tissue culture1
mouse ascites, prepared as above. !
Monoclonal antibody to FIMS 3,
C l
supematant, prepared as above. l
Monoclonal antibody to FIM 2, from hybridoma cell line Ag
1
C2
/
Freeze dried preparation.l
2, mouse ascites, prepared as above.
Monoclonal antibody to FIMS 2, from hybridoma cell line Ag
PoIyclonal2
Table 2b B. pertussis strains
Freeze dried preparation.
Rabbit polyclonal antibody to FIM 3 (NIBSC code 89/600),
Strain ] Serotj7pe
!
-
FDA 460
1
2, 3 IW HO/BS/O4. I998 Page I4
Table
3Microtitre plate ag~futination: - Summary
l i l l I 1
*auioagglairtaiion with these strains excluded from this table
Data present of positive results to number of tests p r f o r m d at all antibody dilutions using the bacterial strain panel Results scored as folfows :
+ = 1 ;
-
= 0; +/- 4 . 5 ; t r a c d . 1WHO/BS!O.I. 1998 Page 15
Table 4 Slide agglutination: Surnrnaw
FDA 2,3
Data present positive results to number of tests performed at all antibody dilutions using the bacterial strain panel.
Results scored as follows:-
+
I-
0M-
0.5trace 0.1
Neat 1/10 1/50 1/100
1 2 3 3
1/1 1.112 U3 313
1/1 212 313 313
1/1 I R 3/3
1 /3
1/1 in O/I on 011 on
3/3 113
213 1 /3 213 113
011 012
01 1 012 0/3
0/3
1 13 013
WHOlBSIO4.1998 Page 16
Table 5. Summaw of microplate and slide results using participants own strains of known serotvpe
Data present positive scored /total number of tests performed Results scored as follows :-
+
I0
+/- 0.5
Trace 0.1
WHOIBSi04.1998 Page 17
Table
6Results of serotvping
usingother methods
a) Indirect whole cefi ELISAb) Flow Cytometry
WHOIsS/04.1998 Page 18
Table 7 Surnrnaw
-combined results of ail methods used with the strain panettVHO!BSl04. I 998 Page 19
WHO/BS~O4.1998 Page 20
Figure 1. Combined results for strain panel by microplate and slide methods
-- -P
Serotype 2 strains
A I A2 B1 B2 C l C2 D1 D2 Antibodies
- -
Serotype 3 strains
A I A2 B1 82 Cl C2 D1 02 antibodies
WHO/BS/04.1998 Page 2 1
Figure 2. Comparison of microplate and slide methods
S1
S
2 S9 FDAStrains
Type 3 strains
Plate I =AI, PlateZ=B I ; Slide I =A I , Slide 2=B l
a
Plate A I ;I
Slide A I
'
II
o
Plate B11 Slide B1
WHOIBSIO4. I998 Page 22
Figure
3.Overall results for all strains tested by all methods
Monoclonal antibodies
Serotype 3 strains
100
p
803
60 - --g
40 q Negatim resp. I'6 m Positim resp.
20 -- --
n
A I A2 B1 B2 C1 C2 D1 D2 Monoclonal antibodies
Serotype 2&3 strains
100 80
3
60 -8
40 ~ N e g a t i w r e s p .'6 m Positim resp.
20 - -
n "
A I A2 B1 B2 C1 C2 D1 D2 Monoclonal antibodies
WHOiBSiO4.1998 Page 23
Figure
4. Stabilivassessment
ofmonoclonal antibody preparations
-p--
Stability of anti FlMS Mab F2
"
'
4
". ."
, a-. U, m W m X i i0
1 2 3 4 5 6 7 8
Dilution 112 from 1:50
+
F2 ascites freeze thaw X 10 F2 super' -206---- F2 s u M r e e z e thaw XI 0 --?K- Dilumt -- blank - -- - -
-- -- - - -- - -- Stability of anti FlMS Mab F316
". ."
W 11 2 3 4 5 6 7 8
Dilution 112 from 1:50
- - - -
1
-+-
F316 ascftes -20c+
F316 ascltes freeze thaw X 10 I F316 supet -2OC-+ F316 supet freeze thaw X 10
+
Diluent blankWHOIBSI04.1998 Page 24
Appendix 1.
Microtitre plate results with concentrations of bacteria1 suspension and dilutions of antibodies used against the bacterial strain ~ a n e l
WHO!BS!04.1998 Page 25
Appendix 1 cont. Microtitre late results with concentrations of bacterial suspension and dilutions of antibodies used against the bacteria1 strain panel
WWOIBSIO4.1998 Page 26
Appendix 1 cont. Microtitre plate results with concentrations of bacterial suspension and dilutions of antibodies used a ~ a i n s t the bacterial strain r>anel
WHO!BS/04.1998 Page 27
Appendix 2
Slide Agglutination with all dilutions of antibodies used apainst the bacterial strain pane!
WHOIBSIO4.1998 Page 28
Apaendix 2 cont.
-
Slide Apglutination including all dilutions of antibodies used against the bacterial strain panelWHO/BS!04.1998 Page 29
Appendix 3
Agglutination results using the panel of antibodv with participant's own strains Microtitre plate
WHOIBSI04.1998 Page 30
Appendix 4
Participants in - house or other &ping reagents
Slide Agglutination
WWOiBSJ0.l.. 1998 Page 3 1
Appendix 5:
Protocol for microtiter plate method for serotlrping of B. ttert~ssis
Materials
96 we11 plate (V-bottom, Nunc products. Denmark) Spectrophotometer (Phamacia Biotech, Novaspec 11)
Plate sealers 8.3s13.3cm @uric products, Fasson S695, Denmark) Guvettes (Plastibrand, CMBH, Germany)
Tubes (sarstedt yellow top tubes) sterile loops
Reagents
Moraoclonal antibodies F2g2C8 anti-Fim2 (provided by Nicole Guise) Stock sol'ution: a 15mgiml solution in gIycerolDBS 1 X 50% at -20°C
For one month: a 4mglml solution in PBS IX at 4'G. For agglutination techniques dilute the stock solution 11600, in PBS IX on the day of the experiment according to the provider.
Monoclonal antibociies CIOC2D5 anti-Fim3 (provided by Nicole Guise) Stock solution: a 24mg/ml solution in glycerol/PBS 1X 50% at -20°C
For one month: a 4mg/ml solution in PBS IX at 4OC. For agglutination techniques dilute the stock solution 116400, in PBS 1X on the day of the experiment according to the provider.
Reference strain: FDA460 expressing both fim 2 and 3
Protocol
*
Bacterial strainsAll determinations are performed twice on the same plate.
50yl of bacterial strain of strain FDA460 with an Or)e5(jnm = 1.0* is placed in wells A1 to A6.
5 0 ~ 1 of bacterial strain of strain FDA460 with an ODGSonm = O S * is placed in wells A7 to A12.
Unknown samples are allocated to the wells of rows B to H, and wells I to 6 are used for the bacterial strain sample with an OL)fj50nm = 1.0*, and wells 7 to 12 are used for the bacterial strain sample with an OD650,, = 0.5*.
*
F2D2G8 antibodies (anti.-Fim2)501.11 anti-Fim 2 at the right concentration are distributed in wells of columns l, 2, 7 &
8 of rows A to H depending on the number of samples.
WHOIBSI04.1998 Page 32
*
C1 062335 antibodies (anti-Fim3)50pI anti-Fim 3 at the right concentration are distributed in wells of columns 3,4, 9 &
l 0 of rotvs A to H depending on the number of samples.
*
PBS 1X (negative control)50pI of PBS 1X are distributed in wells 5,6, I I & f 2 of rows A to H depending on number of samples.
Cover the plate with a plate seater and incubate for 12-36 hours at 37'G.
Results
Positive resuIts are observed after formation of a diffuse layer of antigen-antibody compfex in the bo8om of the wefi. Start to read the control!
Repeat the entire assay if the control failed.
*
Negative results are observed when bacteria sediment as a bulton at the bottom of the well without forming any antibody-antigen complex.The plate should be read by two independent readers in order to validate a correct result.
If the result is uncertain document as not typeable or repeat the test
We also found that agglutinations are easier to read after incubation at 35-36°C
positive
I
*
:- It is important that the inoculum should be locally adjusted and standardized. We have found in our lab that the assay performs equally weIl or better i.e. the result is much easier to deternine, using one optical density of 0.8 (650nm) for a11 samples and controls.WHO/BS!04.1998 Page 33
Appendix 6 :
Prototol of slide agglutination for serotfping B.perfus;sis strains (NIBSCI Materials
Charcoal agar pIates Sterile glass Bijou bottles Microscope slides:-
Sterile saline Sterile loops (pIastic) (BDH Superfrost) Sterile PBS Lahoratov timer (countdown) Gilson PI0 with tips
Reference B. perfussis strains :-GL 353 serotype I 360E serotype 1,2
134 serotype 1,3 W28 seroepe 1,2.3
Polyclonal (Rabbit) reference antisera (NIBSG reagents):- B. pertussis anti
-
Agglutinogen 1 891596B. perfussis anti
-
Agglutinogen 2 891598 B. pertussis anti-
Agglutinogen 3 891600Monoclonal antibodies (mouse) from cell lines F2, F316, Ag2 and Ag3 :-
Anti FIMS 2 (F2 and Ag2) and Anti FIMS 3(F316 and Ag3) monoclonal antibodies
-
partially purified ascites at 4 mglml IgG or partially purified cell culture supernatant at 5mg/ml, both stored at -20OC
Method
1. Grow standard strains at 37OC for 48 hours on charcoal agar plates Inoculate the plate heavily to get maximum growth per plate.
2. Dispense 0.3ml of saline into a Bijou bottle. Carefully scrape growth off one plate with sterile loop.
Make a paste of this growth on side of the glass Bijou bottle using a loop and suspend this paste in the saline to form a smooth dense suspension without clumps.
3. Preparation of polyclonal antisera.
-
for 891596, 891598 and 8901600 Open ampoule and dissolve the freeze dried contents in 0.5ml PBSAliquot this solution into 1 0 0 ~ 1 aliquots and freeze at -20°C until needed
.
Monoclonal Antibodies:-
Agglutination using Mabs is concentration dependant and may require titration against the strains under test. A suitable range would be undiluted to 11100, diluted in PBS.
4. Slide agglutination :- (Use prewashed clean microscope slides e.g. BDH Supefirost)
4.1 Label slide (on frosted end with pencil or with pemanent marker)
4.2 Shake bacterial suspension by gentle agitation of the Bijou bottle and using a Cilson P10 pipette, with a IOpl tip, dispense 10p1 of the suspension onto a slide twice, to give two discrete drops approximately 30mm apart
UrHO!BS/04. 1998 Page 34
4.3 Add lop1 of the antibody to one drop. mix with the pipette tip. Try not to not form bubbles.
4.4 Immediately, using a fresh tip, add 1 Op1 of saline to the second drop and mix, this is the control.
4.5 Set timer to countdown fbr 5 minutes, start the timer at commencement of mixing.
4.6 Leave slide Rat and cover with a half plastic Petri dish
-
this reduces evaporation, important as a small volume is used for this test.4.7 At 2
-
3 minutes from start gently rock the slide for added mixing.4.8 At 5 minutes, stop timer, and observe the result with a black background, Iight and magnification, if required.
Results
If positive
-
agglutination is observed as clumping and the control drop remains as a smooth suspension. Any apparent agglutination at the edges of the drop resulting from drying is disregarded.If agglutination is observed in the control the test must be repeated
-
auto agglutination can occur.Result from reference strains:
-
polyclonal anti -Agg I will agglutinate all standard strains, anti-
Agg 2 will agglutinate strain 360E and anti-
Agg 3 will agglutinate strain 134. All polyclonal antibodies will agglutinate W28.Monoclonal antibodies will agglutinate the appropriate serotype and W28.
Appendix 7:
Flow Cvtometrv method (IFJiIL)
Typing of Reference Strains
1. Antibodies: AI, B1, C l , D1 Dilution of l .ab: 1 : 200
2. Antibody: goat anti mouse Ig (FITC), DAKO Dilution of 2. Ab: 1 :50
Centrifugation; 1500 U/min 20°C; 10 min, brakes off Buger: PBS
Strains: S1, S2, S3, SS, S6, S8, S9, FDA 460 from MicroBank Procedure:
1 . Make suspension of strain at McFarland 1,5 -1 ,8 in PBS 2. 5 tube per strain
a. l x neg. control b f x A l
c 1 x B l d 1 x G 1 e l x D 1
WHO/BS/04.1998 Page 35
3. 100 1.11 suspension per tube
4. 20 1.11 l .ab (1 :200) (for neg.control)
5. incubate 2 X 10 min at room temperature, shake in behveen 6. add 2ml PBS
7. centrifuge
8. discard supernatant
9. suspend pellet in l 00pl PBS 10. add 20pl 2.ab
l l . incubate 2 x l0rnin at room temperature in the dark, shake in between 12. add 2rnI PBS
13. centrifuge
14. discard supernatant
15. resuspend pellet in 5001.11 PBS 16. measure in cporneter
Results:
2500 events in cpometer were reproducibly achieved after 8 -9 sec
WHO!BSI04.1998 Page 36
Appendix 8:
Serotvpin~ of Bordetella itlertussis bv indirect whole cell ELISA using rnonoclonal antibodies to Fim2 and Fim3 antigens.
Antigen preparation
1) From a 48 hours culture plate of a freshly grown B, perfussis isolate on Bordet Gengou agar plate. a fu11 Ioopful of the bacteria is resuspended in 3 to 5 rnl of pH 7.4 sterile phosphate buffered saline (PBS) to give a unifom suspension (vortexing in a tube may be required). The bacterial suspension is heat inactivated at 56'C for one hour, and the inactivated bacterial suspension is cooled and stored at 4°C until ready to be tested.
2) For coating ELISA plates, the inactivated bacterial suspension is diluted in sterile PBS to give an optical density of0.l (range 0.095 to 0.12) at 620 nm.
ELISA Reagents and Method
Reagents
a) Nunc Maxisorp 96-well flat bottom Immuno microtiter plates (Nunc catalog number 439454)
b) Bovine serum albumin, BSA (Sigma catalog number A-3803) c) Sodium choloride (Sigma catalog number S-7653)
d) Polyoxyethylene sorbitan monolaurate, Tween 20 (Sigma catalog number P- 7949)
e) Anti-Fim2 and anti-Fim3 monoclonal antibodies (NIBSC, UK)
f) Horseradish perxodiase conjugated goat anti-mouse IgG F(ab')z fragment specific antibody (Jackson ImmunoResearch Laboratories, Inc. catalog number 1 15-036-072)
g) ABTS substrate (Roche Diagnostics GmbH, catalog number 1 204 521) h) Buffer for ABTS (Roche Diagnostics GmbH, catalog number 1 204 530)
Equipment
a) ELISA washer: Denley WeIlWash 4, MK2
b) ELISA reader: Automated Microplate reader E1x808 (Bio-Tek Instruments, Inc.)
c) Microtiter plate shaker (Lab-Line Instruments, Inc.) d) Plate sealer (Costar catalog number 3095)
Method
a) Antigen coating is done by adding LOO p1 per well of the inactivated B.
perturnis bacterial antigen suspension diiuted to give an optical density of about 0. I at 620 nm and the bacteria is allowed to dry onto the wells of
WHO!BS/04.1998 Page 37
microtiter plate at 37°C (usually takes overnight incubation in a 37°C incubator).
b) Plates with coated bacteria are washed three times with 0.9% saline with 0.05% Tween 20 (saline-Tween).
c) Excess binding sites on the wells are blocked by addition of300 p1 per wet1 of 2% BSA-PBS for incubation at 37°C for 90 minutes.
d) Plates are again washed three times with saline-Tween.
e) Suitably diluted serotyping monoclonal antibodies are added to the bacteria in a volume of LOO p1 per we11 for incubation at 37°C for 90 minutes.
f) Plates are then tvashed four times with saline-Tween.
g) S u i ~ b l y diluted horseradish peroxidase conjugated goat anti-mouse antisera is added in volumes of 100 p1 per well for incubation at 37°C for 90 minutes.
h) Plates are washed four more times and ABTS added for incubation at room temperature on a shaker for 30 to 60 minutes.
Notes
a) Blocking solution: 2% (wiv) of BSA in PBS, must be filtered through hvo layers of Whatman Number 1 filter papers after the powder has dissolved into the PBS and should be stored at -20°C at a11 times except during use to prevent microbial contamination and growth.
b) During incubation, with the exception of the blocking step, plates should be covered with plate sealers.
c) Both primary antibody (serotyping monoclonal antibody) and secondary antibody (goat anti-mouse enzyme conjugate) are diluted in 2% BSA-PBS.
d) With the brand of goat anti-mouse conjugate, we have been using a dilution of 1 5000 for our tests.
e) Substrate solution for horseradish peroxidase enzyme: we use reagents from Roche, 1 tablet of ABTS in 5 m1 of buffer; a more economic way is to use ABTS powder, hydrogen peroxide solution, and citrate buffer (recipe available upon request).
f ) We always include strains known to be Fim2 +ve and Fim3 +ve as control on all plates. Also each serotyping monoclonal antibody, anti-Fim:! as well as anti-Fim3 is always tested against both Fim2 +ve as well as Fim3 i-ve control strains on each run.
g) Since the test results are most of the time very clear cut, there is usually no problem in differentiating behveen positive and negative findings. As a general guide, the ELISA OD for the general backgound should be less than 0.2. Positive reactions (based on OD from known +ve control strains) should be at least several fold higher than negative reactions (based on OD from known -ve control strains). Also positive strains should give ELISA OD of more than 0.3. Strains giving intermediate ELISA readings with the seroQping monoclonal antibodies may suggest mixed population of cells with some expressing while other not expressing the Firn antigens.
h) To make the antigen suspension for plate coating, I have allowed for some variations in the cell concentrations. One may assume if the range of OD is smaller, e.g. from 0.1 to 0.105, the positive ELISA ODs obtained with positive strains may be more uniform (or less fluctuations in the ELISA ODs).
i) Depending on the concentrations of the primary antibody (serotyping rnonoclonal antibodies), plates may be read sool~er than 60 ininutes or even sooner than 30 ~ninures of incubation with the substrate.
