Three monoclonal antibodies to the VHS virus glycoprotein: comparison of reactivity in relation to differences in immunoglobulin variable domain gene sequences.
Three monoclonal antibodies (MAbs) to the VHSV G protein were compared in different immunoassays and the variable domain cDNA sequences from the respective immunoglobulin (Ig) genes were determined. One MAb (IP1H3) was non-neutralising and recognised different virus isolates equally well in ELISA. The other two were neutralising and recognised the same or closely related epitopes. One of these two MAbs (3F1H10) was more restricted in its ability to neutralise heterologous VHSV isolates than the other (3F1A2). A semi-quantitative relationship between binding of the two neutralising MAbs in ELISA 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 MAbs. The Ig variable heavy (VH) and light (V kappa) domain gene sequences of the three hybridomas were compared. The inferred amino acid sequence of the two neutralising antibody VH domains differed by three amino acid residues (97% identity) and only one residue difference was evident in the V kappa domains. In contrast, IP1H3 shared only 38 and 39% identity with the 3F1A2 and 3F1H10 VH domains respectively and 49 and 50% identity with the 3F1A2 and 3F1H10 V kappa domains respectively. The neutralising antibodies were produced by hybridomas originating from the same fusion and the high nucleotide sequence homology of the variable Ig gene regions indicated that the plasma cell partners of the hybridomas originated from the same virgin B lymphocyte. The few differences observed in the VH and V kappa amino acid sequences were probably due to somatic mutations arising during affinity maturation and might explain the observed reactivity differences between the two MAbs.
Danish Veterinary Laboratory, Aarhus, Denmark.
N
N
Lorenzen
P M
PM
Cupit
C J
CJ
Secombes
C
C
Cunningham
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics; D000956:Antigens, Viral / Q000276:immunology*; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000276:immunology*; D008969:Molecular Sequence Data; D009500:Neutralization Tests / Q000662:veterinary; D014759:Viral Envelope Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; C096089:G protein, viral hemorrhagic septicemia virus; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins