==================================BSR15================================== 15. The purification of monoclonal antibodies using (specifically) high pressure/performance liquid chromatography, also known as HPLC. 1 UI - 87110899 AU - Havell EA TI - Purification and further characterization of an anti-murine interferon-gamma monoclonal neutralizing antibody. AB - The R4-6A2 rat-mouse hybridoma, which secretes a rat IgG1 monoclonal antibody (MAb) capable of neutralizing murine interferon-gamma (MuIFN-gamma), was grown as ascites in athymic nude (nu/nu) mice. The neutralizing titers of ascitic fluids were 400 times higher than those of supernatants from R4-6A2 cultures. Moreover, the specific activities (MAb) neutralizing units/mg protein) of the ascitic fluids were much greater than those of R4-6A2 culture supernatants. Ascitic fluid MAb was purified by anion-exchange chromatography, with excellent recovery of applied neutralizing activity. The ability of the R4-6A2 anti-MuIFN-gamma MAb to neutralize several different activities of MuIFN-gamma was studied. The MAb neutralized the ability of MuIFN-gamma to inhibit the growth of cells (antiproliferative activity). The ability of MuIFN-gamma to render cells of a heterologous species (rat) resistant to viral replication was also neutralized by this MAb. The MAb-neutralizing titers for MuIFN-gamma antiviral activities, on both homologous (murine L929) and heterologous (rat fibroblast) cells, were inversely proportional to the antiviral activity titers on each cell type. MH - Animal ; Antibodies, Monoclonal/IMMUNOLOGY/*ISOLATION & PURIFICATION ; Ascitic Fluid/ANALYSIS ; Chromatography, DEAE-Cellulose ; Chromatography, High Pressure Liquid ; Hybridomas/METABOLISM ; Interferon Type II/ *IMMUNOLOGY ; Mice ; Neutralization Tests ; Rats ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. ; Virus Replication/DRUG EFFECTS SO - J Interferon Res 1986 Oct;6(5):489-97 2 UI - 86316785 AU - Kfir R ; Johannsen E ; Botes DP TI - Monoclonal antibody specific for cyanoginosin-LA: preparation and characterization. AB - The toxin cyanoginosin-LA (MW 909), isolated from Microcystis aeruginosa, was successfully conjugated with polylysine and muramyl dipeptide to form a high molecular weight complex consisting of a hapten, a carrier and a built-in adjuvant. This complex was used for the immunization of mice. Monoclonal antibodies specific for cyanoginosin-LA were produced using the hybridoma technique. The ten most efficient producers of these antibodies were further characterized and the monoclonal antibody produced by them was found to be identical on the basis of additivity test, isoelectric focussing and sub-class immunoglobulin typing results. The anti-cyanoginosin-LA monoclonal antibody focussed at a pH of approximately 6.55 and was found to belong to the mouse immunoglobulin subclass IgM. Large scale production of the antibody (in vivo) was followed by purification with ammonium sulphate precipitation and high performance liquid chromatography. The anticyanoginosin-LA monoclonal antibody, when reacted with six variants of cyanoginosin (other than cyanoginosin-LA), bound all with equal efficiency. MH - Acetylmuramyl-Alanyl-Isoglutamine ; Animal ; Antibodies, Monoclonal/ *BIOSYNTHESIS/ISOLATION & PURIFICATION ; Chromatography, High Pressure Liquid ; Enzyme-Linked Immunosorbent Assay ; IgM/ANALYSIS ; Isoelectric Focusing ; Mice ; Mice, Inbred BALB C ; Peptides, Cyclic/*IMMUNOLOGY ; Polylysine SO - Toxicon 1986;24(6):543-52 3 UI - 86304303 AU - Chou DK ; Ilyas AA ; Evans JE ; Costello C ; Quarles RH ; Jungalwala FB TI - Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNK-1 antibody and some IgM paraproteins in neuropathy. AB - Novel sulfated glucuronic acid-containing glycolipids have been identified in the nervous system. These glycolipids are highly antigenic and share antigenic determinants with several nervous system glycoproteins, such as neural cell adhesion molecules, myelin-associated glycoprotein, and ependymins. The structure of the major antigenic glycolipid from human peripheral nerve was determined by chemical and enzymatic degradation, incorporation studies, sugar analysis after permethylation, pertrimethylsilylation, and gas liquid chromatography-mass spectrometry techniques as well as fast atom bombardment-mass spectrometry of the native antigen. The following structure was established for the major antigenic glycolipid. sulfate-3-GlcA beta(1---3)Gal beta(1----4)GlcNAc beta(1----3)Gal beta(1----4)Glc beta(1----1)-ceramide. The major fatty acids in the ceramide were 18:0, 18:1, 24:0, and 24:1, with C18-sphingenine as the long chain base. MH - Antibodies ; Antibodies, Monoclonal/*ANALYSIS ; Carbohydrate Conformation ; Carbohydrate Sequence ; Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Gangliosides/ISOLATION & PURIFICATION ; Glucuronates/*ANALYSIS ; Glycolipids/*ISOLATION & PURIFICATION ; Hippocampus/ANALYSIS ; Human ; IgM/*ANALYSIS ; Mass Fragmentography ; Paraproteins/*ANALYSIS ; Peripheral Nerve Diseases/*METABOLISM ; Peripheral Nerves/*ANALYSIS ; Sulfates/*ANALYSIS ; Support, U.S. Gov't, P.H.S. SO - J Biol Chem 1986 Sep 5;261(25):11717-25 4 UI - 86278569 AU - Pavlu B ; Johansson U ; Nyhl:en C ; Wichman A TI - Rapid purification of monoclonal antibodies by high-performance liquid chromatography. AB - Three different high-performance liquid chromatographic (HPLC) techniques, i.e., ion-exchange, hydrophobic interaction and hydroxyapatite chromatography, have been used to purify monoclonal antibodies from ascites fluid. The monoclonal antibodies were raised against coagulation factor VIII. Precipitation of the antibodies by ammonium sulphate prior to HPLC made it possible to purify the antibody in one chromatographic step. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis this highly pure preparation revealed only one extra polypeptide besides the light- and heavy-chain immunoglobulin polypeptides. Attempts were also made to purify the antibody without prior ammonium sulphate precipitation. A combination of ion-exchange and hydrophobic interaction chromatography resulted in an antibody preparation comparable in purity to the one obtained from ammonium sulphate-precipitated immunoglobulin. The rapid HPLC techniques were found to be very useful for purification of monoclonal antibodies on a preparative scale, where sample loadings of up to 25 mg of ascites protein were fully resolved in satisfactory yields. MH - Ammonium Sulfate ; Animal ; Antibodies, Monoclonal/*ISOLATION & PURIFICATION ; Ascitic Fluid/IMMUNOLOGY ; Chromatography, High Pressure Liquid ; Chromatography, Ion Exchange ; IgG/ISOLATION & PURIFICATION ; Mice ; Spectrophotometry, Ultraviolet SO - J Chromatogr 1986 May 30;359:449-60 5 UI - 86216636 AU - Laurent G ; Pris J ; Farcet JP ; Carayon P ; Blythman H ; Casellas P ; Poncelet P ; Jansen FK TI - Effects of therapy with T101 ricin A-chain immunotoxin in two leukemia patients. AB - Two leukemia patients, refractory to chemotherapy, were treated with T101-ricin A-chain immunotoxin (T101 IT). Patient 1 (T-ALL) received a single 13.5 mg dose of T101 IT IV (12-hour infusion). Patient 2 (B-CLL) was treated with a daily 25 mg dose of T101 IT IV (two-hour infusion) over three consecutive days. Patient 2 also received 300 mg of chloroquine IM on days two and three as enhancer. In vivo binding of T101 IT was demonstrated by FACS analysis using either an antimouse Ig-FITC or anti-A-chain-FITC antibodies. Following IT therapy, the expression of T65 antigen on target cells dropped to 50% and 20% of pretreatment levels, respectively. In patient 1, circulating blast cells remained unsaturated during therapy while in patient 2, cells were fully saturated for four to six hours following each infusion. Pharmacokinetic studies showed a rapid clearance of T101 IT after IV administration. Antimouse and anti-A-chain antibodies could not be detected. There were no treatment-related adverse effects. In patient 1 a rapid but transient decrease of target cells was observed, possibly related to the administration of the antibody part of T101 IT. In contrast, patient 2 showed a 40% reduction of the lymphocyte count, which remained stable over a period of 2 weeks. Such a clinical benefit following IT therapy in patient 2 could be ascribed to the absence of circulating free antigen and the complete saturation of target cells. MH - Adult ; Antibodies, Monoclonal/ANALYSIS ; Antibody Formation ; Antigens, Surface/ANALYSIS ; Chloroquine/BLOOD ; Chromatography, High Pressure Liquid ; Cytotoxicity, Immunologic ; Female ; Human ; Kinetics ; Leukemia, Lymphoblastic/*DRUG THERAPY ; Leukemia, Lymphocytic/*DRUG THERAPY ; Leukocyte Count ; Male ; Middle Age ; Ricin/*THERAPEUTIC USE SO - Blood 1986 Jun;67(6):1680-7 6 UI - 86196394 AU - Josi:c D ; Sch:utt W ; Van Renswoude J ; Reutter W TI - High-performance liquid chromatographic methods for antibodies, glycosidases and membrane proteins. AB - The broad range of applications of high-performance liquid chromatography (HPLC) in biochemistry and cell biology is demonstrated by the purification of antibodies, separation of glycosidases and isolation of a liver membrane protein with a molecular weight of 65 000-67 000 daltons. The advantage of HPLC over classical chromatographic methods is shown by the purification of the glycosidases from Streptococcus pneumoniae. These enzymes can be purified to a degree similar to what can be achieved by "classical: ion exchange, combined with affinity chromatography, but the time needed for the HPLC experiment is much shorter and the yield at least three to five times higher. Particular attention is directed to sample preparation before HPLC separation. For the best results, a combination of HPLC with other biochemical and immunochemical methods is necessary, as is also demonstrated. MH - Antibodies/*ANALYSIS ; Antibodies, Monoclonal/ANALYSIS ; Chromatography, High Pressure Liquid ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Glucosidases/*ANALYSIS ; Human ; Membrane Proteins/ *ANALYSIS ; Molecular Weight ; Support, Non-U.S. Gov't SO - J Chromatogr 1986 Feb 26;353:13-8 7 UI - 86061741 AU - Chou CH ; Cox AA ; Fritz RB ; Wood JG ; Kibler RF TI - Monoclonal antibodies to human myelin basic protein. AB - SJL/J and (SJL X PL) F1 hybrid mice were immunized with intact human myelin basic protein (MBP) or the three major peptic fragments of MBP, residues 1-38, 39-89, and 90-170. Immune spleen cells were fused with mouse myeloma P3 X 63Ag8 (NS1) cells in the presence of polyethylene glycol. Hybridoma supernatant culture fluids were screened for antibody to MBP by a solid-phase radioimmunoassay (RIA). The specificity of the monoclonal antibody (mAb) was characterized by RIA using the three major MBP peptic fragments and subfragments as well as MBP and MBP fragments of different species with known amino acid sequence differences. Six MBP mAbs were generated, one of them IgM isotype and the remainder IgG isotypes. One mAb each reacted against regions of residues 22-38, 39-69, 70-89, 90-116, and two reacted against residues 118-157. Immunoblots also showed that the five IgG mAbs were reactive against MBP and the peptic fragment of MBP containing the epitope. Immunohistochemical studies showed the IgG mAbs specifically stained myelinated fiber tracts in human brain tissue. MH - Amino Acid Sequence ; Animal ; Antibodies, Monoclonal/*IMMUNOLOGY/ ISOLATION & PURIFICATION ; Antigenic Determinants/IMMUNOLOGY ; Cattle ; Chromatography, High Pressure Liquid ; Cross Reactions ; Encephalitogenic Basic Proteins/GENETICS/*IMMUNOLOGY ; Guinea Pigs ; Human ; Mice/ IMMUNOLOGY ; Radioimmunoassay ; Rats ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. SO - J Neurochem 1986 Jan;46(1):47-53