Design and analysis of PCR primers for the amplification and cloning of human immunoglobulin Fab fragments.
We have developed PCR primers for the amplification and cloning of the genes encoding human antibody fragments. Two sets of primers were designed to amplify the coding sequence of the complete variable region and the first constant domain of immunoglobulin heavy chains. One set of primers was designed to amplify the coding sequence of complete kappa light chains. These three sets of primers were used in PCR amplifications with cDNA prepared from EBV transformed B cells as the template. The amplified fragments were cloned and their nucleotide sequences were determined. Analysis of the DNA sequences of such PCR generated antibody fragments suggested that our primer sets were able to amplify the major subsets of the heavy and light chain variable region genes. Comparison between the estimated frequency of the different subsets of heavy chain variable region genes and the distribution of our subclones further indicated that one set of our primers was able to proportionally amplify the subsets of the heavy chain family.
Department of Immunology, Cetus Corporation, Emeryville, CA 94608.
M
M
de Boer
S Y
SY
Chang
G
G
Eichinger
H C
HC
Wong
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics*; D005784:Gene Amplification; D015723:Gene Library; D005803:Genes, Immunoglobulin; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments