Browsing by keyword "Alpha-1 antitrypsin"
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Design, Cloning, and In Vitro Screening of Artificial miRNAs to Silence Alpha-1 AntitrypsinThis protocol describes the design, cloning, and in vitro screening of artificial microRNAs (miRNAs) to silence alpha-1 antitrypsin (AAT). This method would be of interest to silence AAT in a variety of in vitro or in vivo models, and prevalidated sequences against human AAT are provided. This simple 5-day protocol may more generally be used to design artificial miRNAs against any transcript.
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Pathophysiology of Alpha-1 Antitrypsin Lung DiseaseAlpha-1 antitrypsin deficiency (AATD) is an inherited disorder characterized by low serum levels of alpha-1 antitrypsin (AAT). Loss of AAT disrupts the protease-antiprotease balance in the lungs, allowing proteases, specifically neutrophil elastase, to act uninhibited and destroy lung matrix and alveolar structures. Destruction of these lung structures classically leads to an increased risk of developing emphysema and chronic obstructive pulmonary disease (COPD), especially in individuals with a smoking history. It is estimated that 3.4 million people worldwide have AATD. However, AATD is considered to be significantly underdiagnosed and underrecognized by clinicians. Contributing factors to the diagnostic delay of approximately 5.6 years are: inadequate awareness by healthcare providers, failure to implement recommendations from the American Thoracic Society/European Respiratory Society, and the belief that AATD testing is not warranted. Diagnosis can be attained using qualitative or quantitative laboratory testing. The only FDA approved treatment for AATD is augmentation therapy, although classically symptoms have been treated similarly to those of COPD. Future goals of AATD treatment are to use gene therapy using vector systems to produce therapeutic levels of AAT in the lungs without causing a systemic inflammatory response.
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Quantification of Murine AAT by Direct ELISAThis methods chapter elaborates on how a direct enzyme-linked immunosorbent assay (ELISA) is used to specifically detect and quantify murine alpha-1 antitrypsin (AAT). As a direct ELISA, it lacks some sensitivity as compared to the "sandwich" ELISA method; however, it does reliably differentiate between samples with varying amounts of the mouse AAT protein. This protocol relies on the principle of adsorption to coat each well with sera proteins, whereas detection occurs specifically using a two-step antibody combination. This procedure effectively identifies and quantifies murine AAT from a wide variety of samples including mouse serum, cell culture medium, and cell or tissue lysate.
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Quantification of Total Human Alpha-1 Antitrypsin by Sandwich ELISAIn this chapter we describe an enzyme-linked immunosorbent assay (ELISA) to quantitatively measure human alpha-1 antitrypsin (AAT) protein levels in serum, other body fluids or liquid media. This assay can be used to measure the expression of the human AAT (hAAT) gene in a variety of gene transfer or gene downregulation experiments.A hAAT-specific capture antibody and a HRP-conjugated anti-AAT detection antibody are used in this assay. The conjugated anti-AAT used in this protocol, instead of the typical sandwich which employs an unconjugated antibody followed by a specifically conjugated IgG, makes the assay simpler and decreases variability. This provides a useful tool to evaluate the AAT levels in clinical and research samples and can allow fairly rapid testing of a large number of samples.
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Quantification of Z-AAT by a Z-Specific "Sandwich" ELISAThis protocol describes an enzyme-linked immunosorbent assay (ELISA) to specifically detect Z-alpha-1 antitrypsin (AAT), the most common protein variant associated with alpha-1 antitrypsin deficiency. This "sandwich" ELISA relies on an anti-Z-AAT specific capture antibody and a HRP-conjugated anti-AAT detection antibody. This method would be of interest to identify and quantify Z-AAT in a variety of samples such as cell culture medium, cell or tissue lysate, animal or patient serum. Because this method is specific and sensitive, it would be particularly valuable for detection of Z-AAT in the presence of background M-AAT, for instance when quantifying silencing of Z-AAT in patients undergoing M-AAT augmentation therapy.