Multiplex Assay for Quantification of Acute Phase Proteins and Immunoglobulin A in Dried Blood Spots.

Inflammation is the first line defense mechanism against infection, tissue damage, or cancer development. However, inappropriate inflammatory response may also trigger diseases. The quantification of inflammatory proteins is essential to distinguish between harmful and beneficial immune response. Currently used immunoanalytical assays may suffer specificity issues due to antigen-antibody interaction and possible cross-reactivity of antibody with other protein species. In addition, immunoanalytical assays typically require invasive blood sampling and additional logistics; they are relatively costly and highly challenging to multiplex. We present a multiplex assay based on selected reaction monitoring (SRM) for quantification of seven acute-phase proteins (i.e., SAA1, SAA2-isoform1, SAA4, CRP, A1AT-isoform1, A1AG1, A1AG2) and the adaptive immunity effector IGHA1 in dried blood spots. This type of sample is readily available from all human subjects including newborns. The study utilizes proteotypic isotopically labeled peptides with trypsin-cleavable tag and presents optimized and reproducible workflow and several important practical remarks regarding quantitative SRM assays development. The panel of inflammatory proteins was quantified with sequence specificity capable to differentiate protein isoforms with intra- and interday precision (<16.4% coefficient of variation (CV) and <14.3% CV, respectively). Quantitative results were correlated with immuno-nephelometric assay (typically greater than 0.9 Pearson's R).

Dynamics of allergy development during the first 5 years of life.

Allergic diseases have increased in developed countries during the past decades. A cohort of Slovak children was followed from birth to track allergic symptoms dynamics in early childhood. Information on allergic symptoms (atopic dermatitis = AD, rhino conjunctivitis = RC, wheezing = Wh, urticaria = Ur) and food allergies among children was based on clinical evaluation of children by allergists at three developmental stages (infant, toddler, preschool). Out of 320 cases of allergies, 64 infants, 145 toddlers, and 195 preschool children suffered from AD, RC, Wh, Ur, or their combinations (i.e., significant increase with age, p < 0.001). AD first appeared in infants, Wh and/or RC rose mainly in toddlers, and Ur among preschool children. AD in infants or toddlers disappeared in the subsequent developmental stage in approximately one third of all cases. Single AD persistence without remission or extension was not common and accounted only for 6.9% of AD infants' allergic manifestations. In addition to single-symptom allergic diseases, this study also identified several combinations of atopic symptoms.Conclusions: The proportion of multi-symptom allergies increased while single-symptom forms decreased. The observed temporal trends of allergic symptoms correspond to the atopic march. What is Known: • The observed temporal trends of allergic symptoms correspond to the atopic march. What is New: • Allergic diseases in children were first manifested as single forms, with atopic dermatitis (AD) commonly functioning as the "entry point" to allergies. • The overall proportion of single-symptom allergic disorders decreased over time while the proportion of multi-symptom allergies increased.

Early-life exposure to household chemicals and wheezing in children.

The prevalence of the asthmatic symptoms among children increases globally over the time. Reduced exposure to pathogens in early childhood and increased exposure to anthropogenic irritants result in increased risk of wheezing in children, and all of this may be related to the usage of household chemicals. Objective of this analysis thus was to study the potential effects of overall exposure to home chemicals in the early life on the phenotypes of wheezing from birth until five years of age. 3411 mother-infant pairs from the Czech part of the European Longitudinal Study of Pregnancy and Childhood (ELSPAC-CZ) participated in the study. The exposure was estimated by the composite household chemical score from 18 chemical-based products. Social, medical and environmental factors were taken into account as covariates in multivariable multinomial logistic regression using phenotypes of wheezing as a study outcome. We were able to determine the association between several wheezing childhood phenotypes and the frequent usage of household chemicals in the fully adjusted model. Statistically significant odds ratios (OR) for increasing exposures per 1 SD of exposure score were obtained for the intermediate onset transient (OR 1.27, 95% CI 1.10-1.47), intermediated onset persistent (OR 1.23, 95% CI 1.03-1.46), and early onset persistent phenotypes (OR 1.36, 95% CI 1.04-1.77) in comparison to never wheezing children. Moreover, the persistent phenotypes were significantly associated with school age asthma. Our study has shown the negative role of the increased household chemicals usage on the respiratory outcomes in children up to five years of age. Overall evaluation of the household chemical exposure may be useful tool for any large epidemiological studies.