A neutralization-inhibition enzyme immunoassay for anti-idiotypic antibodies that block monoclonal antibodies neutralizing Semliki Forest virus

Abstract

In this paper we compare a solid-phase enzyme immunoassay (EIA) with a neutralization-inhibition enzyme immunoassay (NI-EIA) for the determination of anti-idiotypic antibodies against Semliki Forest virus (SFV)-neutralizing monoclonal antibodies (MAs) UM 5.1 (IgG2a) and UM 1.4 (IgG2a). Against these MAs strong immune sera were induced in female BALB/c mice by two subcutaneous injections, 3 weeks apart, with keyhole limpet hemocyanin coupled MA mixed with the saponin Quil A. Rabbit immune sera were prepared by intracutaneous injections of purified MA mixed with either FCA (first immunization) or IFA (second and third immunization). In the NI-EIA serum is preincubated with neutralizing MA, in wells of 96-well plates, before SFV is added. Binding of anti-idiotypic antibodies to MA results in a diminished capacity of that MA to neutralize SFV. After 1 h incubation with SFV L cells are added and residual infectious virus is allowed to multiply for 6 h at 37°C. Then the monolayers are fixed with glutaraldehyde and subsequently SFV is quantified with a horseradish peroxidase-labelled SFV-specific MA. Low absorbance values indicate that the neutralizing capacity of MA is intact and that blocking antibodies were not present in serum. In contrast high absorbance values indicate that blocking (anti-idiotypic) antibodies had abrogated the neutralizing capacity of MA. With the strongly neutralizing MA UM 5.1 as idiotypic antigen the NI-EIA proved to be at least as sensitive as the solid-phase EIA. Furthermore both normal mouse serum-absorbed rabbit immune sera and mouse immune sera were not cross-reactive in both solid-phase EIA and NI-EIA.