1. Use sera from immunized animals, control sera from nonimmunized animals, and supernatants from empty wells to control and, if needed, adjust screening protocols. All experimental conditions have to be pretested and optimized in preliminary experiments.
2. Start screening 10-12 d after the fusion when wells containing hybridoma clones are 25-50% confluent. The entire screening procedure should yield final results no later than 48 h to avoid overgrowth and death of the tested clones.
3. Transfer 150 p,L of the hybridoma supernatant from the top of positive wells into empty prelabeled sterile 96-well plates. Replace the wells with 150 p,L of complete HAT medium.
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