![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Journal of Clinical Microbiology, May 2005, p. 2449-2451, Vol. 43, No. 5
0095-1137/05/$08.00+0 doi:10.1128/JCM.43.5.2449-2451.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vaccine Preventable Bacterial Diseases Division, National Microbiology Laboratory, Population and Public Health Branch, Health Canada, Winnipeg, Manitoba, Canada,1 Swedish Institute for Infectious Disease Control, Solna, Sweden,2 Division of Bacteriology, National Institute for Biological Standards and Controls, Blanche Lane, Potters Bar, United Kingdom3
Received 26 November 2004/ Returned for modification 4 January 2005/ Accepted 6 January 2005
 |
ABSTRACT |
|---|
 Top Abstract TextReferences |
|---|
|
TEXT |
|---|
|
TopAbstractTextReferences |
|---|
Based on reactions with specific antisera, B. pertussis can be divided into three types: serotype 2, serotype 3, and serotype 2,3. The major serotyping antigens of B. pertussis have been determined to be associated with their fimbriae. Traditionally serotyping is done by the bacterial agglutination test using the slide agglutination method with bacteria mixed with specific serotyping antisera on glass slides. Hybridoma monoclonal antibodies to the serotype 2 and serotype 3 fimbria antigens have also been produced and characterized (7). Attempts to standardize the serotyping method by means of microagglutination have also been made and published (9). Nevertheless, the bacterial agglutination method is still subjective and depends on the ability of the bacteria to form a smooth suspension. Therefore, we have explored the possibility of using an objective method of indirect whole-cell ELISA for the serotyping of B. pertussis isolates. This assay development was evaluated independently at two laboratories with strains of B. pertussis that had been serotyped by the microagglutination method in one laboratory and then tested blindly in a second laboratory by the indirect whole-cell ELISA using different batches of the same serotyping monoclonal antibodies. In this communication, we report our findings and compare the two methods for the determination of serotypes of B. pertussis isolates.
B. pertussis isolates used in this study were mostly from the culture collection of the Swedish Institute for Infectious Disease Control (SIIDC) and were selected to represent isolates from different periods as well as expressing different serotyping antigens of Fim2, Fim3, and Fim2,3. All isolates were retyped before they were sent blindly to the National Microbiology Laboratory (NML) for testing by ELISA. A few Canadian patient isolates were typed by ELISA at NML and sent blindly to SIIDC for testing by the bacterial microagglutination assay.
Monoclonal antibodies that recognize serotype 2 and 3 fimbria antigens were made from hybridoma cell lines BPF2 (anti-Fim2) and BPC10 (anti-Fim3), which were originally developed by Brennan, Manclark, and Li (November 1992; U.S. patent 5,162,223) (7). Antibodies from the hybridoma cell lines were produced at the National Institute of Biological Standards and Control and made available to NML and SIIDC.
Serotyping of isolates by the bacterial microagglutination method was done as essentially described by Mooi et al. (9). Traditional slide agglutination was carried out according to the method described by Preston (10). Indirect whole-cell ELISA was done according to a procedure described for the serotyping of meningococci (1). Briefly, a smooth suspension of a loopful of bacteria grown for 48 h on a Bordet-Gengou agar plate was prepared in pH 7.4 sterile phosphate-buffered saline and heat inactivated at 56°C for 1 hour. The inactivated bacterial suspension was cooled and stored at 4°C until ready for testing. Such inactivated cell suspensions for the ELISA were found to be stable at 4°C for months and can be reused as antigens (e.g., as controls) in multiple assays. Antigen coating was done by adding 100 µl per well of the inactivated B. pertussis bacterial antigen, diluted in phosphate-buffered saline to give an optical density of about 0.1 at 620 nm, to a Nunc Maxisorp 96-well flat-bottomed Immuno microtiter plate (Nalge Nunc International, Rochester, NY). Detection of binding of the serotyping monoclonal antibodies to the bacterial cells was done by addition of a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G F(ab')2 fragment-specific antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.). Each assay was done in the presence of both a Fim2-positive control strain and a Fim3-positive control strain. Details of the different serological methods are available from the authors.
Optimal dilutions of the anti-Fim2 and anti-Fim3 monoclonal antibodies for use in the indirect whole-cell ELISA were determined by titration of each antibody against Fim2-positive and Fim3-positive reference strains. A dilution of 1:1,000 was chosen for both antibodies in subsequent ELISAs for the serotyping of B. pertussis isolates.
Out of the 54 Swedish isolates tested blindly by the indirect whole-cell ELISA method, 53 (98%) gave concordant results with the bacterial microagglutination method (Table 1). Of these 53 Swedish isolates that gave concordant results with both methods, 23 were serotype 2, 26 were serotype 3, and 4 were serotype 2,3. The only isolate that gave a discordant result was an isolate that was found to be serotype 3 by bacterial microagglutination but that was identified as serotype 2,3 by indirect whole-cell ELISA. This strain was retyped by both laboratories, and the results did not change. Potential reasons for this discrepant result may include the following. The microagglutination test was done on bacteria grown on charcoal agar plates for 72 h while the indirect ELISA was done on antigens prepared from cells grown for 48 h on Bordet-Gengou agar plates. Cells grown under different conditions may have different expression of their fimbria antigens (e.g., cells grown on charcoal agar may express only Fim3 antigen, or the Fim2 antigens may be expressed at low quantities or different qualities or at a subsurface location and therefore not be accessible to antibodies).
|
Our data presented here show that the indirect whole-cell ELISA method for serotyping of B. pertussis is at least as good as the bacterial microagglutination assay (9). Several advantages of the indirect whole-cell ELISA method may make it a potentially attractive alternative method for serotyping pertussis strains. These advantages may include the method's objectivity and reproducibility and use of very small amounts of antibodies as well as its suitability for screening large numbers of strains. Therefore, based on this preliminary result, we propose to further evaluate and validate this method for the routine serotyping of B. pertussis in an interlaboratory trial involving several laboratories. Such studies may also involve the exchange of antigen preparations by different participating laboratories to compare antigenic presentation in cells prepared under different laboratory conditions, including the type of medium used to grow the bacteria, as well as to allow different laboratories to test the same batch of cells or antigens.
|
ACKNOWLEDGMENTS |
|---|
R. Tsang also thanks Nicole Guiso of Institut Pasteur, France, for the gift of the reference strains 2558 and Hav.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractTextReferences |
|---|
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Antimicrob. Agents Chemother. | Clin. Microbiol. Rev. |
|---|---|
| Clin. Vaccine Immunol. | ALL ASM JOURNALS |
|---|
