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How much saliva do the human salivary glands produce per day? |
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A slightly alkaline secretion, comprised of water (mostly), mucus, proteins, salts, and enzymes |
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NO test is specific for saliva Idntification of amylase indicates saliva |
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Enzyme that cleaves polysaccharidse that are composed of D-glucose units at the alpha1-4 linkages. AKA cleaves starch. Digests carbohydrates in the oral cavity |
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Contains two glucose polymers: amylose and amylopectin |
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Long, unbranced glucose connected at the alpha1-4 |
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Branched alpha1-6 connections and linear alpha1-4 |
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beta-amylase in plant and bacterial sources alpha-amylase in human, human saliva (HSA), and human pancreatic (HPA) |
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Human alpha-amylases: Two isoforms |
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Differ in amino acid sequence Human alpha-amylsase (HSA) Human pancreatic amylase (HPA) |
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Human Alpha-Amylase (HSA) |
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From saliva Inactivated by stomach acids AMY1 locus |
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Human Pancreatic Alpha-Amylase (HPA) |
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From pancreas Inactivated in the intestines with some activiy remaining in the feces. AMY2 locus |
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Semen, tears, milk, persperation, vaginal secretions, serum, and urine |
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Visual examination with ALS or microscope Starch-iodine assays Colorimetric Assays |
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Visual Examination: ALS or Microscope |
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Can see mucous cells to test if it is saliva ALS: Fluorescence of saliva is less intense than that of semen Microscopic: stain for buccal epithelial cells |
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Iodine used to test for starch NOT specific because it is technically for STARCH Starch appears blue, amylopectin appears reddish-purple in the presence of iodine Albumin and other proteins produce a false positive |
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Dye-labeled amylase substrates are employed Phadebas Test Press Test |
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Clinical diagnosis & forensic use Procion Red Amylopectin (PRA) or phadebas reagent: -starch is linked to the dye forming insoluble complex -dye becomes sluble, creating a colored product measured by a spectrophotometer -done via paper or tube method -may not detect low levels |
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-filters are sprayed with Phadebas solution and allowed to air dry -paper soaked with dried Phadebas solution is pressed over the area to be tested -initial time, single minutes for the first 10 minutes, and 5 minutes for up to 40 minutes total = intervals to note observations Appears light blue on surface of paper |
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Immunochromatographic Assays - RSID-Saliva ELISA - monoclonal HSA antibody RNA-based Assays -based on certain genes specifically expressed in certain types of cells -based on mRNA & htRNA |
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Confirmatory Assays: Immunochromatographic Assays |
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RSID-Saliva Sensititivy: 1 microL saliva necessary specificity: no cross reaction with animal saliva, earwax, etc. most body fluids are okay. utilizes 2 anti-salivary alhpa-amylase monocolonal antibodies in a lateral flow immunochromatographic strip test formation to detect the presence of salivary amylase rather than the activitiy of the enzyme No high dose Hook effect |
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Confirmatory Assays: ELISA |
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monoclonal HSA (human salivary alpha-amylase) antibody Only cross-reactivity for chimpanzee, mouse, rat, and eel Did not work on old stains Specific and can be used for quantification |
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Confirmatory Assays: RNA-based Assays |
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mRNA that is expressed only in the oral cavity Uses reverse transcriptase - PCR (RT-PCR) to detect levels of mRNA HTN3 encodes histain 3 - a protein involved in non-immune response host defense STATH encodes statherin - inhibits precipitation of calcium salts in the salivary glands & oral cavity Highly specific and can be automated, BUT RNA is unstable due to degradation |
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