A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pM sensitivity each consuming only 1 l of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibodybased probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.

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Heat resistance of Bacillus cereus spores (ATCC 7004, 4342, and 9818) heated in different types of milk (skim, whole, and concentrated skim milk), skim milk containing stabilizing additives (sodium citrate, monopotassium phosphate, or disodium phosphate, 0.1%), and cream was investigated. Thermal resistance experiments were performed at temperatures within the range of 92 to 115 degrees C under continuous monitoring of pH. For strain 4342 no significant differences (P < 0.05) in D values were detected in any case. For strains 7004 and 9818 higher D values of about 20% were obtained in whole and concentrated skim milk than those calculated in skim milk. From all stabilizing additives tested, only sodium citrate and sodium phosphate increased the heat resistance for strain 9818. However, when the menstruum pH was measured at the treatment temperature, different pH values were found between the heating media. The differences in heat resistance observed could be due to a pH effect rather than to the difference in the substrates in which spores were heated. In contrast, when cream (fat content 20%) was used, lower D values were obtained, especially for strains 7004 and 9818. z values were not significantly modified by the milk composition, with an average z value of 7.95+/-0.20 degrees C for strain 7004, 7.88+/-0.10 degrees C for strain 4342, and 9.13+/-0.16 degrees C for strain 9818.

The effects of post‐treatment environmental factors on the heat resistance of Bacillus stearothermophilus spores (ATCC 12980, 7953, 15951 and 15952) were investigated. Nutrient Agar (NA), Antibiotic Assay Medium (AAM), Dextrose Tryptone Agar (DTA) and Tryptic Soy Agar (TSA) with Ca2+ added to a final concentration of 100 p.p.m. were used as recovery media. No significant differences were seen between D‐values obtained except in the case of strain 12980 when comparing TSA with the other media and for strain 7953 comparing AAM and DTA. The optimum incubation temperature was slightly lower for heated than for unheated spores of each strain, although, in general, 50 °C was adequate. Higher D‐values were obtained at 50–55 °C. The effects of the pH of the medium in the range 5.0–7.0 and the addition of starch and phosphate on heat resistance have also been investigated. Maximum colony counts of heated spores were obtained at pH 7.0 and decreased as pH fell. D‐values were significantly lower at pH ≤ 5.5. Increasing the concentration of phosphate in the recovery medium from 0 to 0.2% resulted in a progressive decrease in spore recovery and D‐values. The addition of starch improved recoverability. The z‐values obtained for the four strains studied under the different recovery conditions were similar with a mean value of 7.58 °C ± 0.28.