Monoclonal antibody production and characterization for the measurement of plasma high density lipoprotein

We prepared two monoclonal antibodies-A-I30 and A-I4 to HDL apo A-I-with the ultimate goals of expressing and overproducing the valuable immunodiagnostic single chain Fv by phage display libraries in E, coli. Monoclonal antibodies were produced by immunizing mice with apolipoprotein A-I, and purified from ascitic fluid by affinity chromatography on a Protein A Sepharose CL-4B column. The specificity of A-I30 and A-I4 was confirmed by ELISA and Western blotting. The dissociation constants(Kd) of antigen-antibody complex obtained by enzyme linked immunosorbent assay(ELISA) method were 2.25 x 10(-8) M for A-I30 and 2.15 x 10(-8) M for A-I4. The experimental values of Kd are shown to be close to those obtained by immunoprecipitation of the radiolabeled antigen. From the above results, it was shown that A-I30 and A-I4 could be provided as excellent reagents for the determination of plasma HDL concentrations in clinical specimens using ELISA.

Distribution and characters of peptide YY receptor in rat jejunum epithelial cell / 西安交通大学学报(医学版)

Objective To detect normal rat jejunum epithelial cells to learn about the distribution of peptide YY(PYY)receptor and measured two important parameters of PYY receptor so as to explore the target that PYY binds to and display its physiological function.Methods Receptor radioligand assay was conducted by using 125I-PYY to detect normal rat jejunum epithelial cells.We also measured receptor dissociation constant(Kd),the affinity or the ability that the receptor could bind ligand;maximum binding capacity(Bmax),the concentration of binding site;and Hill coefficient.Results There were PYY receptors in normal rat jejunum epithelial cells.Dissociation constant(Kd)of PYY receptor was(386.69?129.95)pmol/L.Maximum binding capacity(Bmax)was(303.21?116.85)fmol/mg protein.Hill coefficient was 1.PYY receptor had high affinity.Conclusion There are distributions of PYY receptors in normal rat jejunum epithelial cells.Kd means that PYY receptor has high affinity.Bmax means that the number of receptors is limited and decides the characters of saturability.We think that PYY directly inhibits secretion by binding to PYY receptor in jejunum epithelial cells when PYY inhibiting jejunum secretion.