Effect of the addition of complement in 21 fresh and 227 stocked serum samples.

<p>Distribution of serum samples is shown for neutralizing antibody titers assayed without inactivation and for those assayed after inactivation with the addition of complement, using 21fresh serum samples (left panel). 227 stocked serum samples were assayed in a similar manner (right panel).</p

Genome construction of the recombinant mumps Hoshino strain expressing GFP and expression of GFP.

<p>Vero cells were infected with GFP Hoshino mumps strain at m.o.i. = 0.02 and subjected to experiments for GFP expression with fluoro EIA and microscopic examination on day 1, 3 and 5 of infection in comparison with mock-infection. Infectivity was assayed in culture supernatants on day 1, 3, and 5 of infection.</p

Relationship between the appearance of CPE and GFP expression.

<p>Serial two-fold dilutions from 1∶4 to 1∶256 were mixed with an equal volume of challenge virus. In the left panel, the schematic results of two neutralization methods are shown. CPE was observed in one of the two wells at 1∶32, and the conventional neutralizing antibody titer was 1∶16 by 100% inhibition of CPE. The mean FU value of the two cell control wells was 202 and that of the 1∶32 dilution was 450, showing 1∶16 of neutralizing antibody titer. Using 1,452 serum samples, the consistency of neutralizing antibody titers was compared based on different cut-off values for GFP expression: 1.5-fold, 2.0-fold, and 2.5- fold of FU values of the cell control wells.</p

Effects of freeze-thawing and inactivation at 56°C for 30 min on neutralizing antibody titers.

<p>Upper panel shows the neutralizing antibody titers of eight fresh sera (A–H), without inactivation and after three or five rounds of freeze-thawing. Lower panel shows the results of neutralizing antibody titers after inactivation.</p

Neutralizing antibody titers of non-inactivated and inactivated sera with the addition of complement.

<p>Neutralizing antibody titers were examined in five sera (A–E) before and after inactivation. Complement was added at 1∶200, 1∶400, 1∶800, and 1∶1600 to the neutralizing mixture when inactivated sera were used. Each experiment was done in triplicate and mean titers were shown.</p