Problem	Possible cause(s)	Solution
Weak signal or no plateau reached in virus titration	i) low NA enzyme activity ii) virus stock not stored under optimal conditions	i) confirm that the diluent has pH that is optimal for NA activity; if pH is optimal,  prepare a new virus stock or concentrate virus ii) Regrow and aliquot stock; snap-freeze vials on dry ice before storing at -80 °C
Weak or no color in positive cell control wells	i)   Virus dilution incorrectly assigned ii) Vial-to-vial variability in frozen virus aliquots iii)  PNA- HRPO denatured or diluted too much iv) OPD incorrectly prepared	i)  Repeat virus titration ii) Titrate several vials from the same batch to ensure there is no variability. If significant variability, prepare fresh aliquots iii) Use optimum virus dilution to retitrate PNA- HRPO iv) Repeat with fresh preparation of OPD
Weak or no inhibition by positive control sera	i)   Too much virus used in assay ii)  Serum deteriorated	i)   Repeat virus titration ii)  Obtain new antisera      Check storage conditions
Inhibition by negative control sera	i)  Inadequate  heat-treatment of serum ii) Too little virus used in assay	i)  Repeat heat-inactivation of serum ii) Repeat virus titration
High background	i) Possible contamination of plates ii) PNA- HRPO concentration too high/too low	i) Repeat using freshly coated plates ii) Titrate PNA-HRPO to identify correct dilution to use
Virus titration shows apparent inhibition of NA activity at low dilutions	i) Allantoic fluid may contain substrate for NA	ii) Use virus that has been pelleted through a sucrose cushion