An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ionexchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40°C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (Km) and the maximal rate of hydrolysis (Vmax) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate L-histidyl-i-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.

A lymphoblastoid cell line (B1) was isolated in culture following a brief exposure to 5-azacytidine from peripheral-blood mononuclear cells of a boar previously injected with cells (Shimozuma) producing porcine retrovirus (Tsukuba-1) and suffering a severe non-neoplastic syndrome at autopsy. B1 cell line and 5 of its sublines were propagated for more than 100 generations, retaining doubling times comprised between 16.8 and 27.5 hr and growing readily in agarose or agar (plating efficiency: 5 to 50%). Karyotype analyses showed that 4 sublines were nearly diploid, except for cells of L14, which displayed a monosomy affecting chromosome 18 pair. Two sublines (L35 and L45) were considered as being of T-cell lineage, since MSA, antigen was observed on the surface of approximately 30% of cells. Three sublines (L23, L14 and L52) were considered of B-cell lineage, since membrane immunoglobulins were observed on the cell surface. In addition, sublines L23 and L52 were actively secreting immunoglobulin of mu isotype. Retrovirus particles were evidenced in gradient-purified preparation of 200-fold-concentrated cell culture supernatants of the B1 cell line, L14, L35 and L52 sublines, using both a reverse transcriptase activity assay and electron microscopic observation. These cell lines can be used to select for porcine retrovirus variants with transforming potential for lymphocytes of B and T lineages.

Affinity antibodies purified against native ovalbumin were found more reactive (higher avidity) against heat-denatured ovalbumin than against the native molecule by three different immunochemical methods. Quantitative immunoprecipitation in soluble phase revealed that more antigen-antibody complexes were insoluble with native ovalbumin than with heat-denatured ovalbumin; 25% of antibodies were still present in supernatant at equivalence as measured by ELISA. At the same conditions with heat-denatured ovalbumin, very small amounts of antibodies were precipitated while almost no activity was found in the supernatant. Classical ELISA or competitive ELISA test allowed detection down to 100 ng/mL of native ovalbumin and 10 ng/mL of denatured ovalbumin.

A B S T R A C T The specificity of rabbit anti-native ovalbumin antibodies ugainst native ovalbumin ( N O A ) or heat-denatured ovalbumin

A method to identify and quantify additives in canned food was developed. Ninety to ninety five percent of proteins from canned mushrooms were solubilized by treatment with O.lN NaOH for 3 days. The extracted, solubilized proteins were then detected by direct ELISA test. An ELISA test which allowed quantification of ovalbumin in canned mushrooms was developed. Immunoblotting also showed the same sensitivity and accuracy with ELISA.