One-step purification of nisin A by immunoaffinity chromatography.

The lantibiotic nisin A was purified to homogeneity by a single-step immunoaffinity chromatography method. An immunoadsorption matrix was developed by direct binding of anti-nisin A monoclonal antibodies to N-hydroxysuccinimide-activated Sepharose. The purification procedure was rapid and reproducible and rendered much higher final yields of nisin than any other described method.

Generation of polyclonal antibodies against a chemically synthesized N-terminal fragment of the bacteriocin pediocin PA-1.

Six mice were immunized intraperitoneally (i.p.) with a chemically synthesized 9-mer fragment (PH1) designed from the N-terminal part of the bacteriocin pediocin PA-1 and conjugated to keyhole limpet haemocyanin (KLH). After three doses of the immunogen had been administered, serum-specific antibodies were detected by a competitive direct ELISA. Myeloma cells were injected i.p. into mice in order to obtain ascites polyclonal antibodies. Although four mice developed ascites, only mouse 2 had detectable specific antibodies in the ascites fluid. The serum and ascites antibodies were specific for PH1 but they did not recognize the whole pediocin PA-1 molecule. This is the first attempt to generate antibodies against bacteriocins with a chemically synthesized oligopeptide as immunogen. This approach still remains attractive for detection, quantification, mode of action studies and purification of bacteriocins, especially those for which the purification process is difficult or inefficient at present.

Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination.

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.

Partial immunological characterization of heat-stable proteinases from Pseudomonas spp. of dairy origin.

A homogeneous extracellular heat-stable proteinase from Pseudomonas fluorescens AH-70 was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relationship of the proteinases from 26 psychrotrophic Pseudomonas spp. isolated from raw milk. The inhibition of the proteinases by the antiserum, the gel precipitin reactions and the use of a double-antibody sandwich ELISA, revealed that proteinase AH-70 is immunologically related to proteinases from 8/26 other Pseudomonas strains. These results also indicate that the immunological techniques for the detection of proteolytic enzymes in raw milk require antibody preparations of broader specificity.

Sandwich ELISA for detection of horse meat in raw meat mixtures using antisera to muscle soluble proteins.

A double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) has been successfully developed for the detection of defined amounts of horse meat (1-50%) in unheated meat mixtures. The assay uses horse-specific antibodies obtained by immunoadsorption of the crude horse antisera onto immobilised sarcoplasmic extracts from chicken, beef and pig to remove cross-reacting antibodies. The purified antibodies bound to a solid support sequester horse muscle soluble proteins from meat mixtures. Further immunorecognition was made with the same antibodies conjugated to the enzyme horseradish peroxidase. Subsequent enzymic conversion of substrate gave clear optical density differences when assaying mixtures of minced beef and pig containing variable amounts of horse meat.

Effect of growth temperature on lipid composition of Streptococcus faecium.

The effect of growth temperature on the lipid and fatty acid composition of Streptococcus faecium has been studied. No differences in the qualitative composition of S. faecium lipids were observed. In all isolated fractions (neutral lipids, glycolipids, and phospholipids plus other polar lipids), the major fatty acids were palmitic (C-16:0), palmitoleic (C-16:1), octadecenoic (C-18:1), and cyclopropane (C-19:0). Changes in the fatty acid composition of the different fractions were observed which depended on growth temperature; the most significant one was the decrease of octadecenoic acid and the increase of palmitic acid in glycolipids and polar lipids as the temperature increased. The level of cyclopropane C-19:0 was approximately eightfold lower at 8 degrees C than at the other temperatures tested (20, 30, and 45 degrees C).