ABSTRACT
BACKGROUND: CC chemokine TARC (thymus and activation-regulated chemokine), a potent chemoattractant for Th2 lymphocytes, is thought to play important roles in inflammatory diseases. We developed a new sensitive enzyme-linked immunoassay (ELISA) for human TARC (hTARC) to accurately measure and evaluate its concentrations in blood. METHODS: An ELISA was developed using two established monoclonal antibodies against hTARC. Using this assay, we observed changes of hTARC concentrations in serum and plasma obtained from individual subjects. Improvements to the assay were made to allow use for the clinical evaluation of samples from atopic dermatitis (AD). RESULTS: The lower detection limit of the ELISA was 1.4 pg/ml for a 25 microl sample volume. Other assay characteristics were enough to satisfactorily measure hTARC in biological fluids. This ELISA revealed that changes in serum and plasma concentrations were related to sample handling before separation from blood. With appropriate sample preparation, significant increases of hTARC were observed in patients with AD in comparison with normal subjects. CONCLUSIONS: Appropriate sample preparation is important for clinical studies on hTARC. Accurate measurement using our ELISA method offers a suitable clinical index for evaluating the severity of allergic diseases of Th2-dominant disorders, such as AD.
Subject(s)
Antibodies, Monoclonal/immunology , Chemokines, CC/blood , Chemokines, CC/immunology , Enzyme-Linked Immunosorbent Assay/methods , Calibration , Chemokine CCL17 , Dermatitis, Atopic/blood , Edetic Acid , Female , Humans , Male , Sensitivity and Specificity , Time FactorsABSTRACT
BACKGROUND: Brain natriuretic peptide (BNP) is a vasoreactive peptide hormone, which is synthesized and secreted mainly from the heart ventricles. METHODS: Molecular forms of immunoreactive human brain natriuretic peptide (BNP) were examined. Chemically synthesized human BNP was added to whole blood samples from a healthy volunteer. The immunoreactive peptide was recovered by immunoaffinity chromatography followed by reversed-phase HPLC (RP-HPLC). Molecular form of immunoreactive BNP in plasma from heart failure patients was also examined. RESULTS: Sequential analysis and amino acid analysis of the peptide revealed that two amino acid residues were deleted from the amino terminus of BNP. When roughly classified according to molecular weight (MW), two forms of BNP (high-MW BNP and low-MW BNP) were observed. The estimated MW of high-MW BNP (36 kDa) was three times that of pro-BNP (12 kDa). CONCLUSIONS: Analysis of low-MW BNP by RP-HPLC revealed that a small amount of BNP 1-32 or des-SerPro-BNP (BNP 3-32) was contained in plasma from heart failure patients.
