Isolation of IgG antibody Fv-DNA from various mouse and rat hybridoma cell lines using the polymerase chain reaction with a simple set of primers.
To facilitate the isolation of IgG antibody Fv-DNA sequences from hybridoma cell lines, we have established a polymerase chain reaction (PCR) procedure requiring only a small number of primers. The sense primers homologous to DNA coding for the first framework sequences were designed to hybridize to all the known antibody sequences under conditions that permit a high number of mismatches. The antisense primers were homologous to DNA coding for the beginning of the constant regions of the gamma and kappa chains. Restriction sites introduced by the primers enable the DNA to be cloned into bacterial expression vectors. Only three sense VH primers and two sense VL primers paired with one backward primer for the heavy and light chains, respectively, were necessary for the amplification of Fv-DNA from a total of 17 rodent cell lines that we have so far worked with. These consisted of 12 mouse cell lines and five rat cell lines. This procedure will therefore probably be sufficient to isolate the Fv-DNA from most mouse cell lines and possibly also from most rat cell lines.
Dübel
S
S
Recombinant Antibody Research Group, Heidelberg, Germany.
Breitling
F
F
Fuchs
P
P
Zewe
M
M
Gotter
S
S
Welschof
M
M
Moldenhauer
G
G
Little
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification*; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D006825:Hybridomas / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats
D017931:DNA Primers; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D004247:DNA