==================================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