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- Put 1ml of a 5% suspension of sheep red blood cells (RBCs) into each of two sterile tubes.
Mark one 'Test' and the other 'Control'.
- Add 0.5ml of purified rubella haemagglutinin (titre 1:128) to the 'Test' cells only.
- Mix gently and leave for 5min at room temperature.
- Half fill both tubes with complement fixation buffer (CFB) (a phenobarbitone based buffer containing Mg2+ and Ca2+ - Why?).
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- Centrifuge at 2002 rpm for 3-5 min.
- Carefully decant off the supernatant. The RBC's in the 'Test' bottle are now sensitized with rubella antigen.
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- Gently resuspend each of the RBC pellets in 0.5ml CFB.
- Add 0.5ml Guinea-pig complement ('GPC'), mix and warm in 43°C waterbath for a few seconds.
- Add 15ml 1% agarose in CFB (warmed to 43°C), immediately mix and pour into a sterile petri dish. Swirl to cover the entire dish.
- Allow plates to set, label 'Test' and 'Control' plates.
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- Punch out 5 equally spaced wells in each plate.
- Label the wells,'A', 'B', 'C', '+' and '-'.
- Put 30µl of the appropriate serum into each well.
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