MFE-23 is a single-chain Fv (scFv) antibody ti•agment that was selected from a phage-display library for high affinity binding to carcinoembryonic antigen (CEA), and has important clinical uses in the detection, monitming and targetting of colon cancer.CEA is a seven-domain cell adhesion molecule which is expressed at high levels on colon carcinoma cells.150: 256 residues) consists of anN-terminal VH domain, a IS-residue linker, a VL domain and a 12-residue tag.MFE-23 was expressed in E. coli and purified on CEA coupled to Sepharose.Cr-ystals were produced by the hanging-drop method.MFE-23 at 2 mg/ml was mixed 1:1 with precipitant (100 mM Tris-HCI at pH 6.5 containing 45% saturated ammonium sulfate), and a 10 ~ll drop of this mixture was equilibrated against 0.5 ml of precipitant at l8°C.Well-fom1ed crystals fom1ed within several days, and diffracted to 0.2 nm resolution.Diffraction data were collected using an R-A.'CIS-ITC mounted on an RU200 rotating anode X -ray source.The crystal belongs to the trigonal space group P3221 and has unit cell dimensions of a= b = 6.170 nm and c = J 2.794 nm.A single crystal was used for structure detennination.Data to a resolution of 0.28 nm were processed using DENZO and the CCP4 program package.The structure was solved by molecular replacement using the Fv coordinates from a mmine IgA Fab fragment (Brookhaven code 2FBJ).The antibody complementarity determining regions, the linker and the tag were omitted for the calculation of initial 2Fo-Fc electron density maps.Refinement of the MFE-23 structure is cunently in progress, utilizing the rigid-body refinement and the positional refinement algorithms ofX-PLOR ar1d manual rebuilding of the model in 0. PS04.15.12 ARE "HOT SPOTS" [SUPER- FLEXIBLE REGIONS]
Hybridomas secreting a human monoclonal IgGi antibody against a variant of the rh^G antigen were produced from B lymphocytes of an Rh-negative donor with serum antibodies to D and C antigens. The antibody reacts by indirect hemagglutination with nearly all C- or D-positive cells, confirming the strong association in the expression of G with D or C antigens. The lack of antibody reactivity to C-negative D^u cells suggests a particular epitope on the D complex associated with the G antigen.
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