Development and evaluation of a bead-based Multiplexed Fluorescent ImmunoAssay (MFIA) for detection of antibodies to Salmonella enterica serogroup B and C1 in pigs.

BACKGROUND: Since 1995, a surveillance program for Salmonella has been applied in the Danish pig industry in order to reduce cases of human salmonellosis. The objective of this study was to develop a bead-based Multiplexed Fluorometric ImmunoAssay (MFIA) as an improved serological surveillance method compared to the Salmonella mix ELISA, which has been the national reference immunoassay in the Danish Salmonella surveillance program for about 20 years. RESULTS: An MFIA for detection of antibodies to Salmonella serogroup B and C1 was developed and optimized with regard to coupling of beads with Salmonella lipopolysaccharide antigens and establishing suitable assay conditions. The Salmonella MFIA was validated by testing sera from experimentally infected pigs as well as field sera from non-infected and infected pig herds, and by comparing to results from the Salmonella mix ELISA, which was run in parallel. Sensitivity and specificity was evaluated using receiver operating curve analysis showing an area under curve for the serogroup B and C1 MFIA of 0.984 and 0.998, respectively. The Salmonella MFIA was shown to detect more antibody-positive samples in seropositive herds compared to the Salmonella mix ELISA, and Bayesian statistics confirmed that the MFIA had a considerably higher sensitivity (94.5%) compared to the mix ELISA (75.1%). The assay specificity was slightly lower for the Salmonella MFIA (96.8%) compared to Salmonella mix ELISA (99.5%). Coupled beads were stable for at least 1 year at 4˚C, and MFIA reproducibility and repeatability of the Salmonella MFIA were acceptable. Results from proficiency tests also indicated that the Salmonella MFIA was more sensitive than the Salmonella mix ELISA and that they had similar specificity. CONCLUSIONS: A bead-based MFIA for simultaneous detection of porcine serum antibodies to Salmonella enterica serogroup B and C1 was developed and implemented in the Danish porcine serological Salmonella surveillance program in 2018. The Salmonella MFIA can distinguish, as opposed to the Salmonella mix ELISA, between antibodies to serogroup B and C1 and the MFIA shows considerably better sensitivity.

Lifestyle and demographic associations with 47 inflammatory and vascular stress biomarkers in 9876 blood donors.

BACKGROUND: The emerging use of biomarkers in research and tailored care introduces a need for information about the association between biomarkers and basic demographics and lifestyle factors revealing expectable concentrations in healthy individuals while considering general demographic differences. METHODS: A selection of 47 biomarkers, including markers of inflammation and vascular stress, were measured in plasma samples from 9876 Danish Blood Donor Study participants. Using regression models, we examined the association between biomarkers and sex, age, Body Mass Index (BMI), and smoking. RESULTS: Here we show that concentrations of inflammation and vascular stress biomarkers generally increase with higher age, BMI, and smoking. Sex-specific effects are observed for multiple biomarkers. CONCLUSION: This study provides comprehensive information on concentrations of 47 plasma biomarkers in healthy individuals. The study emphasizes that knowledge about biomarker concentrations in healthy individuals is critical for improved understanding of disease pathology and for tailored care and decision support tools.

Blood-based biomarkers are circulating molecules that can help to indicate health or disease. Biomarker levels may vary depending on demographic and lifestyle factors such as age, sex, smoking status, and body mass index. Here, we examine the effects of these demographic and lifestyle factors on levels of biomarkers related to activation of the immune system and cardiovascular stress. Measurements of 47 different proteins were performed on blood samples from nearly 10,000 healthy Danish blood donors. Measurement data were linked with questionnaire data to assess effects of lifestyle. We found that immune activation and vascular stress generally increased with age, BMI, and smoking. As these measurements are from healthy blood donors they can serve as a reference for expectable effects and inflammation levels in healthy individuals. Knowledge about the healthy state is important for understanding disease progression and optimizing care.

Simultaneous detection of antibodies to five Actinobacillus pleuropneumoniae serovars using bead-based multiplex analysis.

We developed and made a preliminary validation of a bead-based multiplexed immunoassay for simultaneous detection of porcine serum antibodies to Actinobacillus pleuropneumoniae serovars 1, 2, 6, 7, and 12. Magnetic fluorescent beads were coupled with A. pleuropneumoniae antigens and tested with a panel of serum samples from experimentally infected pigs and with serum samples from uninfected and naturally infected pigs. The multiplex assay was compared to in-house ELISAs and complement fixation (CF) tests, which have been used for decades as tools for herd classification in the Danish Specific Pathogen Free system. Assay specificities and sensitivities as well as the corresponding cutoff values were determined using receiver operating characteristic (ROC) curve analysis, and the A. pleuropneumoniae multiplex assay showed good correlation with the in-house ELISAs and CF tests with areas under ROC curves ≥ 0.988. Benefits of multiplexed assays compared to ELISAs and CF tests include reduced serum sample volumes needed for analysis, less labor, and shorter assay time.