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.
Animal ; Antibodies, Monoclonal/*immunology/isolation & purification ; Antibody Affinity ; Apolipoprotein A-I/*immunology ; Blotting, Western ; Hybridomas ; Lipoproteins, HDL/immunology ; Mice ; Mice, Inbred BALB C ; Tumor Cells, Cultured

It has been hypothesized that blood infusion of reconstituted HDL (rHDL) is a possible therapeutic strategy for the treatment of coronary artery disese. To compare short-term anti-inflammatory activity of wildtype (WT) apoA-I and point mutants, each rHDL containing WT, V156K, or R173C was infused into apo-E deficient atherosclerotic mice. Each rHDL was injected via the tail vein at a dosage of 120 mg/kg of body weight in 0.4 ml of tris-buffered saline (TBS), and blood was then collected at 24 and 48 h post-injection. Although regression activity was observed in each of the rHDL infused groups, a 30% reduction in the lipid-stained area of the aortic sinus was observed in the V156K and R173C-rHDL groups when compared to that of the WT-rHDL group, and this reduction was well correlated with an approximately 60% reduction in the accumulation of macrophages in the lesion area. Additionally, the groups that received the V156K and R173C-rHDL treatments showed smaller increases in the GOT, GPT, interleukin-6, myeloperoxidase (MPO) and lipid hydroperoxide (LPO) serum levels than those that received the WT-rHDL treatment. In addition, the strongest serum paraoxonase and ferric reducing ability was observed in the V156K and R173C-rHDL groups. In vitro nitration and chlorination of apoA-I by MPO treatment revealed that V156K-rHDL and R173C-rHDL were less susceptible to chlorination. Furthermore, rHDL treatment inhibited cellular uptake of oxidized LDL by macrophage cells and the production of proatherogenic species in culture media. In conclusion, blood infusions of the rHDLs exerted in vivo regression activity with anti-inflammatory and antioxidant activity in apo-E deficient mice and THP-1 cells, especially in those that were treated with V156K and R173C apoA-I.
Animals ; Anti-Inflammatory Agents/immunology/*therapeutic use ; Apolipoprotein A-I/blood/genetics/immunology/*therapeutic use ; Apolipoproteins E/genetics ; Aryldialkylphosphatase/blood/metabolism ; Atherosclerosis/*drug therapy ; Cell Line ; Cell Membrane Permeability ; Cholesterol/blood/metabolism ; Humans ; Lipoproteins, HDL/genetics/immunology/*therapeutic use ; Lipoproteins, LDL/metabolism ; Macrophages/cytology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Oxidation-Reduction/*drug effects ; Peroxidase/blood/metabolism ; Point Mutation