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Serotyping of B. pertussis
Serotyping is the detection of the expression of the fimbriae 2 or 3 at the surface of the bacteria using monoclonal antibodies.
1 Material
96 well plate (V-bottom)
Spectrophotometer
Plate sealers 8.3 x 13.3 cm.
2 Reagents
Monoclonal antibodies F2?2G8 anti-Fim 2 (reagents can be purchased from the National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Pottersbar, Hertfordshire EN6 30G, United Kingdom; contact point Dr Dororthy Xing).
Stock solution: a 15 mg/ml solution in glycerol/PBS 1 X 50% at -20°C.
For one month: a 4mg/ml solution in PBS 1X at 4°C. For agglutination techniques dilute the stock solution in PBS 1X on the day of the experiment according to the provider.
Monoclonal antibodies C10C2D5 anti-Fim 3 (reagents can be purchased from the National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Pottersbar, Hertfordshire EN6 30G, United Kingdom; contact point Dr Dororthy Xing).
Stock solution: a 24 mg/ml solution in glycerol/PBS 1X 50% at -20°C.
For one month: a 4mg/ml solution in PBS 1X at 4°C. For agglutination techniques dilute the stock solution PBS 1X on the day of the experiment.
Reference strain: Bp 460 (reagents can be purchased via WHO). This reference strain is expressing Fim 2 and Fim 3 antigens.
3 Protocol
Bacterial strains
- All determinations are performed twice on the same plate.
- 50 µl of bacterial suspension of strain Bp 460 with an OD650 nm = 1 is placed in wells A1 to A6.
- 50 µl of bacterial suspension of strain Bp 460 with an OD650 nm = 0.5 is placed in wells A7 to A12.
- Unknown samples are allocated to the wells of lines B to H: wells 1 to 6 are used for the bacterial strain sample with an OD 650 = 1, and wells 7 to 12 are used for the bacterial strain sample with an OD650 = 0.5.
F2?2G8 antibodies (anti-Fim 2)
50 µl anti-Fim 2 at the right concentration are distributed in wells 1, 2, 7 and 8 of lines A to H depending on the number of samples.
C10C2D5 antibodies (anti-Fim 3)
50 µl anti-Fim 3 at the right concentration are distributed in wells 3, 4, 9 and 10 of lines A to H depending on the number of samples.
PBS 1X (negative control)
- 50 µl of PBS 1X are distributed in wells 5, 6, 11 and 12 of lines A to H.
- Cover the plate with a plate case and leave overnight at 37°C.
4 Results
Positive results are observed after formation of an antigen-antibody complex in the bottom of the well.
Negative results are observed when bacteria sediment out at the bottom of the well without forming any antigen-antibody complexes.
The plate should be read by two independent readers in order to validate a correct result.
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