The document handed out today, mentioned the use of enzymes to assay for the det
ID: 67203 • Letter: T
Question
The document handed out today, mentioned the use of enzymes to assay for the determination of [TAG] in serum. Your job is to find out the biochemistry of this assay. There are several; you just need to choose and describe one of them? What (besides serum containing and unknown [TAG]) Chemicals/biochemicals are required? What instrument is required for quantitation? At what setting? List what diseases or conditions might be indicated by elevated [TAG] levels. The handout also mentioned the use of 'biochemiluminesence' in EUSAs where the second Ab is tagged with the enzyme luminase. Find out what reaction is catalysed by luminase. What would be required for quantitation in such ELISAs? What, on the molecular level, is common to the process that take place in biochemiluminesence and fluorescence? What is different?Explanation / Answer
Ans.)
1.What instrument is used for Quantification?
A.Quantus Fluorometer.
Assay:
a) The triglyerides are hydrolysed bymixture of lipase and esterase.
b) The glycerol released is determined bykinetic fixed time analysis with use of glycerol kinase,pyruvate kinase and lactate dehydrogenase.
c) There is no need for serum for serum Blanks and reagent Blanks
d.) the method has been adapted to a centrifugal analyser and yeilds satisfactory results with regard to precision,accuracy and insensitivity.
Diseases or conditions with elevated TAG
Ans.) Hypertriglyceridemia (High Triglycerides),Fatty liver,Type 2 Diabetes, overindulgent eating.
2. The reaction catalysed by Luminiase is Firefly reaction in which ATP is directly involved in light emission.The lucerifin Lucerifase is enzyme substrate reaction ,in which the lucerifin ,the substrate is oxidized by molecular oxygen,the reaction being catalysed by Luciferse enzyme with consequent emission of light.
3. In Fluorescence,the energy from external source of light is absorbed and re emitted.The emitted light with its modified colour is only visible while the stimulated light is turned on.
In Bioluminescence,the excitation energy is supplied by a chemical reaction rather than source of light.But in Fluorescence,the energy from the source of light is absorbed and reemitted as another photon.
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