Int. Archs Allergy appl. Immun. 54: 475-478 (1977)
Studies on Acid Eluates from Kidneys of Sheep with Glomerulo nephritis Mediated by Antibody to Glomerular Basement Membrane1 S. R. Batsford and J. Hardwicke Abteilung für Innere Medizin IV (Schwerpunkt Nephrologie), Albert Ludwigs-Universität, Frei burg i. Br., and Department of Experimental Pathology, Medical School, University of Birmingham, Birmingham
Sheep that are immunised with heterolo gous glomerular basement membrane (GBM) develop a progressive, generally fa tal glomerulonephritis [9]. This disease is caused by an autoimmune process in which the sheep produces antibodies cross-reacting with its own GBM [4, 10, 11]. While much useful data has been accumulated from studies employing acid eluates of diseased human kidneys [2, 3, 5, 7, 8] few detailed analyses of eluate content have been made. To improve this situation data is presented here on the composition of acid eluates from the kidneys of six sheep immunised with heterologous basement membrane. The sheep received intramuscular injec tions of 10 mg of rabbit [1, 2] or human GBM [3-6] incorporated into Freund’s complete adjuvant. This procedure was re 1 This work was carried out with the aid of an MRC advance training grant.
peated monthly and when the serum crea tinine level rose sharply (which occurred in all cases) the animal was bled out, the kid neys being removed and frozen at -20 °C. Antibody was eluted from sheep kidneys at acid pH as follows: the cortex was cut into small pieces which were homogenised in ice-cold PBS. The homogenate was washed in 1-litre volumes of PBS and the optical density of the supernatant was mea sured at 280 nm, generally this reading de creased rapidly to a low level which then persisted undiminished (for up to 30 washes in one case). To one part of the washed homogenate were added 9 parts of citrate buffer, pH 2.6, 0.1 M and the mixture (final pH 3.0) was stirred at room temperature for 2 h. The supernatant was decanted, centri fuged, neutralised with 1 N sodium hydrox ide (slowly with stirring), dialysed against PBS and concentrated to between 1 and 2 ml. Significant quantities of protein could
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 1:04:00 AM
Abstract. Kidneys from six sheep having glomerulonephritis mediated by antibody to glomerular basement membrane (GBM) were extracted at acid pH. Each preparation was characterised using immunological techniques and the eluates contained between 3.6 and 13% anti-GBM antibody of IgG class. It appears that this low antibody content is due to the presence of contaminants, mainly serum proteins.
476
Batsford/Hardwicke
Tabic I Sheep Immunised with GBM No. from
Fraction Protein IgG extracted eluted eluted mg mg
IgG in eluate %
Eluted IgG Anti-GBM Anti-GBM Anti-GBM binding to antibody antibody antibody GBM, % eluted, mg in eluate, % /
Studies on Acid Eluates from Kidneys of Sheep with Glomerulo nephritis Mediated by Antibody to Glomerular Basement Membrane1 S. R. Batsford and J. Hardwicke Abteilung für Innere Medizin IV (Schwerpunkt Nephrologie), Albert Ludwigs-Universität, Frei burg i. Br., and Department of Experimental Pathology, Medical School, University of Birmingham, Birmingham
Sheep that are immunised with heterolo gous glomerular basement membrane (GBM) develop a progressive, generally fa tal glomerulonephritis [9]. This disease is caused by an autoimmune process in which the sheep produces antibodies cross-reacting with its own GBM [4, 10, 11]. While much useful data has been accumulated from studies employing acid eluates of diseased human kidneys [2, 3, 5, 7, 8] few detailed analyses of eluate content have been made. To improve this situation data is presented here on the composition of acid eluates from the kidneys of six sheep immunised with heterologous basement membrane. The sheep received intramuscular injec tions of 10 mg of rabbit [1, 2] or human GBM [3-6] incorporated into Freund’s complete adjuvant. This procedure was re 1 This work was carried out with the aid of an MRC advance training grant.
peated monthly and when the serum crea tinine level rose sharply (which occurred in all cases) the animal was bled out, the kid neys being removed and frozen at -20 °C. Antibody was eluted from sheep kidneys at acid pH as follows: the cortex was cut into small pieces which were homogenised in ice-cold PBS. The homogenate was washed in 1-litre volumes of PBS and the optical density of the supernatant was mea sured at 280 nm, generally this reading de creased rapidly to a low level which then persisted undiminished (for up to 30 washes in one case). To one part of the washed homogenate were added 9 parts of citrate buffer, pH 2.6, 0.1 M and the mixture (final pH 3.0) was stirred at room temperature for 2 h. The supernatant was decanted, centri fuged, neutralised with 1 N sodium hydrox ide (slowly with stirring), dialysed against PBS and concentrated to between 1 and 2 ml. Significant quantities of protein could
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 1:04:00 AM
Abstract. Kidneys from six sheep having glomerulonephritis mediated by antibody to glomerular basement membrane (GBM) were extracted at acid pH. Each preparation was characterised using immunological techniques and the eluates contained between 3.6 and 13% anti-GBM antibody of IgG class. It appears that this low antibody content is due to the presence of contaminants, mainly serum proteins.
476
Batsford/Hardwicke
Tabic I Sheep Immunised with GBM No. from
Fraction Protein IgG extracted eluted eluted mg mg
IgG in eluate %
Eluted IgG Anti-GBM Anti-GBM Anti-GBM binding to antibody antibody antibody GBM, % eluted, mg in eluate, % /
