Antibody purification at neutral pH utilizing immunospecific adsorbents

Serum IgM, IgY and IgA were suppressed by the embryonic administration of F(ab′)₂ fragments of goat anti-μ followed by bursectomy at hatch, indicating that the Fc fragment is not essential for antibody-mediated class suppression in chickens. Anti-L chain was also capable of mediating suppression but anti-IgY H chain was not, indicating that the mechanism for suppression involves specific binding to IgM-producing cells rather than a toxic effect of antibody-antigen complexes. At the doses used, both F(ab′)₂ anti-μ and intact anti-L antibody caused more marked suppression of IgA than of IgM and IgY.

Section: Methodsmentioning

confidence: 99%

Suppression of Chicken Immunoglobulin Ontogeny by F(ab′)<sub>2</sub> Fragments of Anti-μ Chain and by Anti-L Chain

Leslie

Martin

1973

“…However, DNPconjugates were quantitatively recovered with 4 N NaCNS as the eluting agent. This ion has been shown by Dandliker and DeSaussure [5] to be particularly effective in disso ciating antigen-antibody complexes. Since chaotropic ions such as thiocyanate are cap able of disrupting hydrogen, ionic and hy drophobic bonds, steps were taken to mini mize exposure of the antigen and antibody to this agent.…”

Section: Discussionmentioning

confidence: 99%

Isolation of Dinitrophenyl-Skin Protein Conjugates by Immunoadsorbent Chromatography

Lewis¹,

Heise²

1977

Immunoadsorbent columns charged with rabbit anti-dinitrophenyl (DNP) antibody were used to isolate and concentrate DNP-protein conjugates from the 105,000 g supernate of extracts prepared from guinea pig epidermis previously painted with dinitrofluorobenzene (DNFB). Conjugates isolated in this manner elicited positive delayed hypersensitivity skin tests when injected intradermally into DNFB-sensitized animals. Dinitrophenylated epidermal protein conjugates prepared in vitro were capable of specifically inhibiting the migration of peritoneal exudate cells obtained from DNFB-sensitized guinea pigs. These results demonstrate the application of immunoadsorbent chromatography and the migration inhibition assay to the further definition of the epitopes involved in contact sensitivity.

“…Although these observations suggested that such approaches may be feasible, insuf ficient supplies of potent auto-antisera prevented adequate exploration of these methods. Perhaps the most useful will prove to be the specific re moval of the auto-antigens from the crude extracts with insolubilized car diac auto-antibodies, followed by their recovery with dissociation of the immune complexes near neutral pH by chaotropic ions [29]. A similar approach has proven quite successful in the purification of insulin [30], or immunoglobulin A from whole serum [31].…”

Section: Discussionmentioning

confidence: 99%

The Cardiac Auto-Immune System

Lin

Halbert

Kiefer

1972

One of the human heart auto-antigens cross-reactive with rabbit anti-rabbit heart auto-antibody concentrates (designated auto-antigen D) was purified from phosphate-buffered saline extracts of human heart. The procedure employed sequential salting-out with ammonium sulfate, chromatography on DEAE-cellulose, filtration through Sephadex G-200 gel, and iso-electric focusing in a pH gradient from 4 to 6. The purified auto-antigen D contained no contaminants reactive with plasma-absorbed rabbit antisera to human kidney, human skeletal muscle and human heart, as well as duck anti-rabbit heart sera. One or two trace contaminants were found with goat antibodies to human serum. The iso-electric point of antigen D was 5.0, but it appeared to show a biphasic distribution and was heterogeneous in several other respects. The auto-antigen D had an estimated molecular weight of 68,000, a sedimentation coefficient of 3.5S and a diffusion coefficient of 6.68 × 10^––⁷ cm² sec ^––1.