Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library.
We have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and kappa light-chain variable and constant region domains, were inserted into modified bacteriophage lambda expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2% in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. We estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.
Mullinax
R L
RL
Stratagene, La Jolla, CA 92037.
Gross
E A
EA
Amberg
J R
JR
Hay
B N
BN
Hogrefe
H H
HH
Kubitz
M M
MM
Greener
A
A
Alting-Mees
M
M
Ardourel
D
D
Short
J M
JM
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D005821:Genetic Techniques; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D013745:Tetanus Toxoid / Q000276:immunology*
D007128:Immunoglobulin Fragments; D012333:RNA, Messenger; D013745:Tetanus Toxoid