Fucosyl monosialoganglioside: Quantitative analysis of specific potential biomarkers of lung cancer in biological matrices using immunocapture extraction/tandem mass spectrometry.

RATIONALE: Certain lung cancer patients express elevated Fucosyl Monosialoganglioside (Fuc-GM1) in circulation compared to control groups. Several sensitive methods involving characterization of Fuc-GM1 have been reported. However, a highly specific and sensitive method for quantifying multiple potential Fuc-GM1 biomarkers present in various biological matrices has not been reported to date. METHODS: Individual Fuc-GM1 analogs in a commercially obtained standard mixture were characterized using HPLC/UV/MS and high-resolution mass spectrometry (HRMS). Proprietary antibodies, mAb1 and mAb2, were used to selectively capture and pre-concentrate the soluble and drug-bound forms of Fuc-GM1 molecules present in human serum and whole blood, eliminating the background matrix components. Immunocapture extraction (ICE) followed by HPLC/MS/MS was used to quantify specific Fuc-GM1 analogs in biological matrices. RESULTS: The concentration of individual Fuc-GM1 analogs in the standard mixture was estimated to be 7-34%, using HPLC/UV/MS. Using the standard mixture spiked into the biological matrices (100 µL), the lower limit of quantification (LLOQ) of each analog was 0.2-0.4 ng/mL with a dynamic range of up to 200 ng/mL. The applicability of the ICE-HPLC/MS/MS method was demonstrated by detecting endogenous Fuc-GM1 analogs present in rat blood and in several lung cancer cell lines. CONCLUSIONS: This highly specific and sensitive HPLC/MS/MS method for quantifying individual potential Fuc-GM1 biomarkers in serum and whole blood can play a critical role in patient stratification strategies and during drug treatment. This method can be employed for monitoring both free (soluble) form and antibody drug-bound Fuc-GM1.

Dramatic elevation in urinary amino terminal titin fragment excretion quantified by immunoassay in Duchenne muscular dystrophy patients and in dystrophin deficient rodents.

Enzyme-linked and electrochemiluminescence immunoassays were developed for quantification of amino (N-) terminal fragments of the skeletal muscle protein titin (N-ter titin) and qualified for use in detection of urinary N-ter titin excretion. Urine from normal subjects contained a small but measurable level of N-ter titin (1.0 ± 0.4 ng/ml). A 365-fold increase (365.4 ± 65.0, P = 0.0001) in urinary N-ter titin excretion was seen in Duchene muscular dystrophy (DMD) patients. Urinary N-ter titin was also evaluated in dystrophin deficient rodent models. Mdx mice exhibited low urinary N-ter titin levels at 2 weeks of age followed by a robust and sustained elevation starting at 3 weeks of age, coincident with the development of systemic skeletal muscle damage in this model; fold elevation could not be determined because urinary N-ter titin was not detected in age-matched wild type mice. Levels of serum creatine kinase and serum skeletal muscle troponin I (TnI) were also low at 2 weeks, elevated at later time points and were significantly correlated with urinary N-ter titin excretion in mdx mice. Corticosteroid treatment of mdx mice resulted in improved exercise performance and lowering of both urinary N-ter titin and serum skeletal muscle TnI concentrations. Low urinary N-ter titin levels were detected in wild type rats (3.0 ± 0.6 ng/ml), while Dmdmdx rats exhibited a 556-fold increase (1652.5 ± 405.7 ng/ml, P = 0.002) (both at 5 months of age). These results suggest that urinary N-ter titin is present at low basal concentrations in normal urine and increases dramatically coincident with muscle damage produced by dystrophin deficiency. Urinary N-ter titin has potential as a facile, non-invasive and translational biomarker for DMD.

Immunoassay-based measurement of clinical biomarkers for monitoring changes in nasal cavity.

BACKGROUND: Many drugs for treatment of allergies, migraine headaches, inflammation, and other indications are administered into the nasal cavity providing access to the immune and central nervous systems. One of the concerns for using this route of administration is potential damage to the nasal epithelium and mucosal regions. We assembled a panel of clinical biomarkers that can be used to monitor changes in the nasal epithelium, mucosa, and olfactory regions in preparation for clinical trials involving drugs administered via intranasal route. These biomarkers included albumin, elastase, IL-6, IL-8, lactoferrin, myeloperoxidase and nerve growth factor. METHODS: Immunoassays were developed and used to measure changes in these biomarkers in nasal lavage samples collected twice daily from 30 assumed-healthy volunteers over a 2-day period. Various statistical methods including analysis of variance (ANOVA), paired t-test and Pearson's product-moment correlation were used to evaluate the data. RESULTS: Although the basal levels of these biomarkers were varied among subjects, the data show that the concentrations of albumin, elastase and IL-8 were significantly higher in samples collected in the morning compared to samples collected later during the day. Pre-washing nasal cavity prior to collecting nasal lavage samples did alter the measurement of elastase and albumin, but did not influence the levels of the other biomarkers. CONCLUSIONS: These data show that this panel of biomarkers can be used to monitor changes in the nasal cavity including those affected by diurnal fluctuations. These results also provide useful baseline values and sources of variability for each biomarker that could be used to help design clinical trials.