Isolation and amplification of human IgE Fd encoding mRNA from human peripheral blood lymphocytes.
In order to establish the feasibility of applying recombinatorial library technologies to investigate human in vivo IgE responses, and as a pre-requisite of recombinatorial library construction, we have attempted to determine workable peripheral blood sample volumes required for isolation of mRNA for polymerase chain reaction (PCR) amplification of human IgE Fd encoding sequences. Cells secreting chimeric human IgE monoclonal antibody specific for the hapten NIP were used to establish the conditions for specific amplification of C epsilon 1 domain and Fd encoding sequences, as determined by Southern hybridisation. Amplification of C epsilon 1 domain sequences could be achieved using as few as ten cultured cells as the source of RNA. Specific IgE+ B cell enrichment using immuno-magnetic particles prior to RNA extraction was, however, required to obtain amplification of IgE C epsilon 1 and Fd fragments from lymphocytes prepared from 40 ml human peripheral blood. IgG1+ B cell enrichment from similar samples was not required for detectable amplification of human C gamma 1 cDNA sequences. However, this procedure improved amplification efficiency. Optimisation of methods to separate specific B cell populations, or specific RNA/cDNA sequences, will facilitate in vitro generation of human IgE Fab fragments from peripheral blood.
Walker
M R
MR
Molecular Biology Research Group, University Department of Clinical Biochemistry, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, UK.
Bevan
L J
LJ
Daniels
J
J
Rottier
M M
MM
Rapley
R
R
Roberts
A M
AM
D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA / Q000032:analysis; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood*; D007128:Immunoglobulin Fragments / Q000032:analysis*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D014066:Palatine Tonsil / Q000276:immunology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D007128:Immunoglobulin Fragments; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; C100277:immunoglobulins, Fd; D007073:Immunoglobulin E; D004247:DNA