Rescue, expression, and analysis of a neutralizing human anti-hepatitis A virus monoclonal antibody.
A human anti-hepatitis A virus mAb was rescued from a hybridoma cell line by conventional cDNA cloning, and expressed in CHO cells. The full nucleotide sequences of the mAb H and L chains were determined, revealing a VHI/V lambda II V region combination. Comparisons with germline V genes suggest that the V regions had undergone somatic mutations characteristic of an Ag-driven immune response. A comparison of the binding to hepatitis A virus between mAb derived from the CHO cells and the original hybridoma cell line using ELISA, radioimmunoprecipitation, and solid-phase competition RIA, indicated that the CHO cell-derived mAb fully retained the specificity of the mAb produced by hybridoma cells. Analysis of viral neutralization using a radioimmunofocus inhibition assay demonstrated the retention of antibody functionality after expression in CHO cells, demonstrating the use of this technique in the rescue and high level expression of unstable efficacious human mAb.
Department of Cell Biology, Wellcome Research Laboratories, Beckenham, Kent, UK.
A P
AP
Lewis
S M
SM
Lemon
K A
KA
Barber
P
P
Murphy
N R
NR
Parry
T C
TC
Peakman
M J
MJ
Sims
J
J
Worden
J S
JS
Crowe
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D035922:Hepatitis A Antibodies; D006508:Hepatitis Antibodies / Q000235:genetics*; D006507:Hepatovirus / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D009500:Neutralization Tests
D000911:Antibodies, Monoclonal; D035922:Hepatitis A Antibodies; D006508:Hepatitis Antibodies