ABSTRACT
Leishmania donovani-soluble antigens capable of antibody production in rabbits were separated from the total antigenic make-up of the parasite by adsorption on to anti-Leishmania immunoglobulins coupled to CNBr-activated Sepharose 4B (ALIG). Immune rabbit sera were produced either by intravenous inoculation of living promastigotes or by intradermal and subcutaneous inoculation of soluble antigens. Approximately four times as much soluble antigen was bound to ALIG produced by i.v. than i.d. and s.c. inoculation although the percentage recovery was less. The antigenic fractions recovered were subjected to immunoelectrophoresis, isoelectric point and molecular weight determination. The results of affinity chromatography indicate high antigen-antibody interaction affinity, the recovery of at least 28 antigens (with 0.15 M NaCl, pH 11.0) and complete loss of serological activity (determined by immunoelectrophoresis) on treatment with 8 M urea.
Subject(s)
Antigens/analysis , Leishmania/immunology , Leishmaniasis, Visceral/immunology , Animals , Antibody Formation , Antigens/immunology , Chromatography, Affinity , Immunization , Immunoelectrophoresis , Isoelectric Point , Molecular Weight , RabbitsABSTRACT
Rat serum 5'-nucleotidase, L-leucyl-beta-naphthylamidase and beta-glycerophosphatase activities are increased whilst alkaline p-nitrophenylphosphatase and alkaline phosphodiesterase activities are unchanged or decreased three days after bile duct ligation. Affinity chromatography on an immobilised antiserum raised against highly purified liver plasma membranes showed that although 5'-nucleotidase in normal serum is unrelated to the 5'-nucleotidase of liver plasma membrane, the 5'-nucleotidase of bile and much of the 5'-nucleotidase in the jaundiced serum are closely related to the plasma membrane enzyme. Since bile is rich in 5'-nucleotidase, the changes in level of this enzyme after bile duct ligation are most simply explained by leakage of bile into the blood; changes in the patterns of the other enzymes are shown to be consistent with this explanation. The jaundiced serum was examined by gel exclusion chromatography and flotation in sucrose gradients for the presence of small fragments of plasma membrane as reported in human jaundiced sera, but no such fragments could be detected three days after bile-duct ligation.
Subject(s)
Bile/enzymology , Cholestasis/enzymology , Nucleotidases/blood , Animals , Centrifugation, Density Gradient , Chromatography, Affinity , Immunoelectrophoresis , Male , RatsABSTRACT
Plasma-membrane fragments recovered in the microsomal fraction of rat liver homogenates were shown to be heterogeneous in density. It was demonstrated that 5'-nucleotidase, the most commonly used plasma-membrane marker, is concentrated in the lightest subfraction. Two of the published procedures for the isolation of plasma-membrane fragments from the microsomal fraction (Touster et al., 1970; Hinton et al., 1971) are shown to give products which are not representative of all the plasma-membrane fragments of microsomal size, and it is argued that a third procedure (House & Weidemann, 1970) is likely to give a similar product.
