Three monoclonal antibodies to the VHS virus glycoprotein: comparison of reactivity in relation to differences in immunoglobulin variable domain genesequences

Three monoclonal antibodies (MAbs) to the VHSV G protein were compared in d ifferent immunoassays and the variable domain cDNA sequences from the respe ctive immunoglobulin (Ig) genes were determined. One MAb (IP1H3) was non-ne utralising and recognised different virus isolates equally well in ELISA. T he other two were neutralising and recognised the same or closely related e pitopes. One of these two MAbs (3F1H10) was more restricted in its ability to neutralise heterologous VHSV isolates than the other (3F1A2). A semi-qua ntitative relationship between binding of the two neutralising MAbs in ELIS A and their neutralising activity was evident. Binding kinetic analyses by plasmon resonance identified differences in the dissociation rate constant (kd) as a possible explanation for the different reactivity levels of the M Abs. The Ig variable heavy (VH) and light (V kappa) domain gene sequences o f the three hybridomas were compared. The inferred amino acid sequence of t he two neutralising antibody VH domains differed by three amino acid residu es (97% identity) and only one residue difference was evident in the Vk. do mains. In contrast, IP1H3 shared only 38 and 39% identity with the 3F1A2 an d 3F1H10 VH domains respectively and 49 and 50% identity with the 3F1A2 and 3F1H10 VK domains respectively. The neutralising antibodies were produced by hybridomas originating from the same fusion and the high nucleotide sequ ence homology of the variable Ig gene regions indicated that the plasma cel l partners of the hybridomas originated from the same virgin B lymphocyte. The few differences observed in the VH and V kappa amino acid sequences wer e probably due to somatic mutations arising during affinity maturation and might explain the observed reactivity differences between the two MAbs. (C) 2000 Academic Press.