Fast, low sample volume cholesterol and triglyceride determination
A cholesterol and reagent technology, applied in the field of fast, low sample volume cholesterol and triglyceride determination, can solve the problem of increased cost of lipoprotein cholesterol sub-fraction analysis
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Embodiment 1
[0203] Embodiment 1 chemical method and reagent
[0204] Cholesterol Determination Chemistry:
[0205] The assays disclosed herein can be used to form colored products from reactions with cholesterol and cholesterol derivatives (eg, cholesteryl esters) found in blood samples. As shown in Figure 1, cholesteryl esters can react with cholesterol esterase to generate free cholesterol. Free cholesterol can react with cholesterol oxidase and oxygen to form hydrogen peroxide. The hydrogen peroxide formed in this reaction can be used to quantify the amount of cholesterol in the sample. Hydrogen peroxide can react with peroxidases to form colored products, for example in the presence of or through the use of colorants, and such reactions can be detected and quantified. For example, hydrogen peroxide in horseradish peroxidase and colorants such as aminoantipyrine compounds (eg 4-aminoantipyrine) and N-ethyl-N-(2-hydroxy-3-sulfopropyl )-3,5-dimethoxyaniline to form colored produc...
Embodiment 2
[0224] Example 2 - Cholesterol Determination
[0225] 1) Combine 30 μL of sample (neat plasma or serum diluted 1:30 with water or phosphate-buffered saline (PBS)) with 20 μL of reagent AC in the wells of a 384-well microplate reader (MTP) at 37°C.
[0226] 2) Shake the plate at 1800 rpm for 2 seconds.
[0227] 3) Incubate the plate at 37°C for 5 minutes.
[0228] 4) Add 20 μL of reagent BC. The plate was shaken at 1800 revolutions per minute (rpm) for 2 seconds.
[0229] 5) Incubate the plate at 37°C for 12 minutes in a spectrophotometer capable of reading absorbance at given time intervals.
[0230] 6) Record the absorbance (A) at 560 nm and 700 nm every 30 seconds (measured with a M5 spectrophotometer (Molecular Devices, Sunnyvale, CA)). Note that other intervals, including longer intervals, are also suitable.
[0231] sample:
[0232] figure 2 Lipid content of lipoproteins (results including triglycerides) measured in clinical serum samples used for the studies ...
Embodiment 4
[0273] Example 4 Combined Measurement of Blood Cholesterol and Triglycerides
[0274] Cholesterol (C) and triglycerides (TG) can be measured on aliquots of the same blood sample or on different blood samples taken from the same patient in order to compare with what would be achieved by measuring C alone or measuring D alone Provide more complete clinical information. Additionally, TG measurements can be used to provide a measure of VLDL in a blood sample. Therefore, the measurements of LDL-C, HDL-C, TC, VLDL-C and TG can be obtained by the methods of Examples 1, 2 and 3, and VLDL-C can be estimated by the following formula:
[0275] VLDL-C=TG / 5.
[0276] TC can be measured directly according to the methods and assays disclosed herein; similarly, HDL-C and LDL-C can be measured directly according to the methods and assays disclosed herein. Since TC is a measure of the total cholesterol in the sample, TC is the sum of the cholesterol in the lipoprotein fraction of the sampl...
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