Quantitative elution studies in experimental immune complex and nephrotoxic nephritis.
Optimal conditions have been determined for elution of antiglomerular basement-membrane antibodies and antigen-antibody complexes from nephritic kidneys, obtained from rabbits with nephrotoxic nephritis and various forms of BSA-induced immune complex disease. Elution from kidney homogenates was performed with acid and alkaline buffers, solutions of chaotropes, urea, sodium and magnesium chloride and other agents. Functional activity of antibodies exposed to elution procedures was tested by immunofluorescence, or by a modified Farr's technique. Antibodies of progressively greater binding capacity were eluted by a stepwise acid gradient (pH 3-2--2-8) using low or high ionic strength glycine--HCl buffer. Antigen-antibody complexes were best eluted using 3 M KBr (pH 9) using several extractive steps. A stepwise alkaline gradient (pH 10-5--11-1 using 0-1 M glycine-NaOH buffer) or 8 M urea were found to be satisfactory alternative methods of eluting immune complexes. Elution procedures were best carried out in the cold in all circumstances. Other eluting agents were found to be less successful or less practical.
Quantitative elution studies in experimental immune complex and nephrotoxic nephritis. Optimal conditions have been determined for elution of antiglomerular basement-membrane antibodies and antigen-antibody complexes from nephritic kidneys, obtained from rabbits with nephrotoxic nephritis and various forms of BSA-induced immune complex disease. Elution from kidney homogenates was performed with acid and alkaline buffers, solutions of chaotropes, urea, sodium and magnesium chloride and other agents. Functional activity of antibodies exposed to elution procedures was tested by immunofluorescence, or by a modified Farr's technique. Antibodies of progressively greater binding capacity were eluted by a stepwise acid gradient (pH 3-2--2-8) using low or high ionic strength glycine--HCl buffer. Antigen-antibody complexes were best eluted using 3 M KBr (pH 9) using several extractive steps. A stepwise alkaline gradient (pH 10-5--11-1 using 0-1 M glycine-NaOH buffer) or 8 M urea were found to be satisfactory alternative methods of eluting immune complexes. Elution procedures were best carried out in the cold in all circumstances. Other eluting agents were found to be less successful or less practical.