description): The investigators hypothesize that fetal cells have unique markers, and propose to construct a large antibody library to search for these markers for use in prenatal diagnosis. The initial effort will focus on cell surface markers of fetal erythroblasts. The proposed study has two specific aims:
Aim 1 : Construct a recombinant antibody (rAb) phage display library with a repertoire of sufficient size and diversity to any antigen epitopes of fetal erythroblasts. A phage antibody expression library will be constructed from the spleens of mice immunized with human fetal hepatic erythroblasts. The antibody repertoire of about 10 to the 10th power single-chain functional V-gene fragments (scFv) will be cloned in a phagemid vector, enabling both phage displayed and soluble rAb to be produced in E. coli. Theoretically, this library will produce rAbs that recognize any antigen epitopes of fetal erythroblasts.
Aim 2 : Select rAbs specific for cell surface markers that are exclusively expressed in fetal erythroblasts but not in the adult counterpart. The rAb phage expression library will be subject to positive selection for fetal erythroblasts by panning, followed by negative selection to deplete rAbs cross-reacting with adult erythroblasts. The enriched pool of rAb phages will be expanded in individual clones for determining the specificity and sensitivity to detect and isolate fetal erythroblasts in the maternal blood.
