Rosa Pizzano

Coupling Immunomagnetic Separation on Magnetic Beads with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Detection of Staphylococcal Enterotoxin B

ABSTRACT The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly “on beads” by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, “off beads” after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.

Fast isoelectric focusing and antipeptide antibodies for detecting bovine casein in adulterated water buffalo milk and derived mozzarella cheese.

Plasmin hydrolysis of water buffalo casein (CN) can liberate a peptide comigrating with bovine gamma(2)-CN. Occurrence of this peptide may lead to false-positive detection of cows milk for a genuine water buffalo cheese when it is analyzed by applying a fast version of the European official method for detecting bovine casein in water buffalo cheese. After isoelectric focusing of CN plasminolysates, performed according to the official method, immunoblot analysis with antipeptide antibodies was assayed to distinguish between gamma(2)-CN and the interfering bovine gamma(2)-CN-like peptide. Small, synthetic peptides containing partial sequences of bovine gamma(2)-CN were used as immunogens for antipeptide antibodies raised in rabbits. The antibody preparation directed toward the synthetic peptide containing the first five amino acid residues of gamma(2)-CN cross-reacted with native and in vitro generated gamma(2)-CN from bovine and water buffalo CN, but it did not recognize the bovine gamma(2)-CN-like band in the electrophoretic profile of pure water buffalo CN.

Detection of pH 4.6 Insoluble β-Lactoglobulin in Heat-Treated Milk and Mozzarella Cheese

Different protein aggregates including beta-lactoglobulin (beta lg) were detected in the pH 4.6 insoluble fraction recovered from actual heat-treated milk samples by gel electrophoresis and immunoblotting. A competitive enzyme-linked immunosorbent assay (ELISA) using anti-beta lg polyclonal antibodies was developed to analyze the beta lg partition in the protein fractions obtained upon acidification of both milk and Mozzarella cheese at pH 4.6. According to ELISA determinations, nearly 90% of the pH 4.6 soluble beta lg included in raw milk was found in the pH 4.6 insoluble fraction of ultrahigh temperature (UHT)-treated milk. As concerns Mozzarella cheese analysis, ELISA results indicated that about 36% of the total beta lg milk content was transferred from pasteurized milk to Mozzarella cheese, whereas less than 0.5% was transferred from raw milk. The pH 4.6 insoluble beta lg proved to be a suitable indicator of the intensity of the heat treatment applied to milk. The ELISA-based detection of this parameter was suggested for quality control of both drinking milk and raw milk cheese.

Isoelectric Focusing and ELISA for Detecting Adulteration of Donkey Milk with Cow Milk

Donkey milk has been recently revalued intensely due to its nutritional properties. Moreover, donkey milk has been proposed as an effective alternative food for some infants with cow milk allergy. Two fast analytical methods were proposed to detect the fraudulent practice of blending cow milk to donkey milk. Detection of cow αs1-casein bands along the profiles of experimental donkey-cow milk mixtures analyzed by isoelectric focusing was adequate to estimate cow milk used as adulterant of donkey milk starting from 5% (v/v). An ELISA-based method using the antipeptide antibodies raised against the 1-28 sequence stretch of cow β-casein was also developed for an accurate definition of composition of donkey-cow milk mixtures. The presence of cow milk at levels as low as 0.5% (v/v) was detected in donkey-cow milk mixtures prepared at laboratory scale and assayed by ELISA.

Occurrence of major whey proteins in the pH 4.6 insoluble protein fraction from UHT-treated milk.

A clear picture of the protein rearrangement in milk following UHT-treatment was drawn by a comparative analysis of the pH 4.6 soluble protein fraction (SPF) and the pH 4.6 insoluble protein fraction (IPF) recovered from raw and UHT-treated milk samples. The two protein fractions were analyzed by mono- or bidimensional gel electrophoresis under reducing and nonreducing conditions, and protein bands were identified by specific immunostaining. Results showed that bovine serum albumin, β-lactoglobulin, and, to a lesser extent, α-lactalbumin coprecipitated with caseins in UHT-treated milk samples at pH 4.6. These proteins were almost exclusively involved in high molecular weight aggregates held together by disulfide bonds. Partition of α-lactalbumin and bovine serum albumin in the protein fractions obtained upon acidification of milk at pH 4.6 was evaluated by competitive immunoassays. The ELISA-based results suggested the possibility of using pH 4.6 insoluble α-lactalbumin and bovine serum albumin, in addition to pH 4.6 insoluble β-lactoglobulin, as indicators of the intensity of the heat treatment applied to milk.