A new method for the detection of neutralizing antibodies against mumps virus

Abstract

Neutralization test is the most reliable method of evaluating immunity against viral diseases but there is no standard procedure for mumps virus, with tests differing in the infectivity of the challenge virus, 50% plaque reduction or complete inhibition of cytopathic effects (CPE), and usage of complement. A reliable, easy, and simple neutralization test for mumps virus was developed in this study. A recombinant mumps virus expressing GFP was generated as a challenge virus. Complement was added to the neutralizing mixture at 1∶200 when stocked serum samples were used. Neutralizing antibody titers were expressed as the reciprocal of the highest dilution that did not exceed two-fold of FU values (GFP expression) of the cell control wells. A total of 1,452 serum samples were assayed by inhibition of GFP expression in comparison with those examined by conventional 100% inhibition of CPE. 1,367 (94.1%) showed similar neutralizing antibody titers when examined by both methods. The GFP expression inhibition assay, using a recombinant mumps virus expressing GFP, is a simple and time- saving method.

Figure 1. Genome construction of the recombinant mumps Hoshino strain expressing GFP and expression of GFP.

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.

Figure 2. Relationship between the appearance of CPE and GFP expression.

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.

Figure 3. Effects of freeze-thawing and inactivation at 56°C for 30 min on neutralizing antibody titers.

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.

Figure 4. Neutralizing antibody titers of non-inactivated and inactivated sera with the addition of complement.

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.

Figure 5. Effect of the addition of complement in 21 fresh and 227 stocked serum samples.

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).