The use of red cells with fused Semliki Forest virus envelope proteins in antibody determinations by hemolysis in gel

Chicken red blood cells with fused Semliki Forest virus (SFV) proteins on the cell membrane were used in the hemolysis-in-gel (HIG) plates. Optimally the plate contained a 1.5 mm thick gel of 1% agarose with 1% red cells and 1 unit/ml gel of complement. 400 ng of SFV was fused to the red cells in 1 ml of the gel (about 20 virions fused to one red cell). Five μl of inactivated (56°C, 30 min) serum samples were pipetted into wells in the gel. After 20 h of incubation at 37°C the diameters of the hemolytic zones were directly proportional to the logarithm of the serum dilution. This made it possible to calculate the antibody titers for the samples using an experimental formula T = 10φk (T is the titer, φ the diameter in mm and k a variable coefficient, which had to be determined for each batch of the plates using a standard serum). Using regression and residual analyses, the formula was shown to fit the experimental results. The fusion-based HIG could be read after as early as 2 h of incubation. The specificity of the test was studied using antisera against Western equine encephalomyelitis, Eastern equine encephalomyelitis, Chikungunya and Uruma viruses, which all gave negative results in the SFV HIG test. Antisera against SFV E1 and E2 proteins were positive, but anti-E3 serum was negative when measured in the SFV HIG test.