7. (a) Explain how signal amplification is achieved in enzyme-linked immunosorbe

Question

7. (a) Explain how signal amplification is achieved in enzyme-linked immunosorbent assays. (b) What is the advantage of a time-resolved emission measurement with Eu3+ versus measurements of fluorescence from organic fluorophores?
10:51 PM oo AT&T; 71% K Homework Hw 3.pdf 5. Yan and colleagues developed a method for the analysis of iron based its formation of a fluorescent metal gand complex with the ligand (Anal Chim Acta 1992, 12 24) In the presence of the surfactant cetyltrimethyl ammonium bromide, the analysis iscamied out sing an excitation wavelength of 316 mm with cmission monitored at 528 nm. Standardization with extemal tandards gives the following equation for lincar regression of the data in the calibration curve. A05113gsample of dry dog food is ashed to remove organic materials and the neidue dissolved in mall amount ofHCI and diluted to volume in a 50-ml. volametric mask. Analysis of the resulting solution gives a fluorescent emission intensity of 5.72. Determine the mg Fe in the sample of dog food. 6. The concentration of acetylsalicylic acid. Cdleo in aspirin tablets is determined by it to the salicylate ion. Cnilsoh .and determining its concentration spectronaonometrical ly. A stock standard solution prepared by weighing 0.0774 g of salicylic acid.Citilo into a I-L volumetric flask and diluting to volume. A set of calibration standards is prepared by pipeting 0.200, 4.00, 6.00. 800, and 10.00 mLofthe sock solution into separate 100 ml. volumetric flasks that contain 2.00 mL of 4 M NaOH and diluting to volume. uorescence is measured at an emission wavelength of400 nm using an excitation wavelength of 310nm with results shown in the following table, Boo DEI DOTO Several aspirin tablets are ground to a fine powder in a mortar and pestle, A0.1013-g portian of the powder is placed in a 1-1. volumetric flask and luted to volume with distilled water. A ponion of this solution is fltered binders and a 1000-mL aliquot transferred 00-mL volumetric flask that coetains 200 ml of NaOH. After diluting to volume the fluorescence of the resulting solution is &69. What is the %whw acetylsalicylic acid in the aspirin tablets? 7. (a Explain how signal amplification is achieved in enzyme linked immunosorbent assays. (bo What is the advantage of time-resolved emission measurement with Eu" versus measurements of fluorescence from organic fluorophores? 8. The following procedure comes from the paper "TNTDetection Using Multiplexed Liquid Amay Displacement immunoassays .Analytical Chemistry. 2006.78. 2279. The immunoassay measures ves such as TNT in organic solvent extracts of soil The assay employs now cytometer, which counts mall particles flowing through namow tube. The cytometer in this experiment inradiates the particles with a green laser and measured nuorescence from cach panicle asit flows past the 48 Courses Calendar To Do Notifications Messages

Explanation / Answer

7. (a) The amplification of signal of ELISA can be carried out in the following number of ways such as -

(i) Incubating the samples at 4 degree Celsius for overnight; (ii) Incubating the secondary antibody for overnight; (iii) Increasing coating concentrations; (iv) Affinity of solid phase antibody, so use of higher affinity antibody is mostly recommended such as Streptavidin-Horse radish peroxidase; (v) Addition of Polyethylene glycol (1-3%) to the incubation buffer; (vi) Freeze thawing of the samples; (vii) Use of multiple layers of antibodies

7. (b) First, let's see the disadvantages of measurements of fluorescence from organic fluorophores, which are as follows -

(i) Raman and Rayleigh scattering; (ii) Quenching of fluorescence; (iii) Difficult of separation of excitation and emission wavelength; (iv) Non specific binding with the reagents; (v) Background fluorescence from cuvettes, sample and the optics; (vi) Short lived emitted fluorescence.

Thus, the advantages of a time-resolved emission measurement with Eu3+ are -

(i) Quenching of fluorescence is reduced; (ii) The emitted fluorescence is long lived; (iii) Disapperance of serum autofluorescence; (iv) Due to large stokes shift, excitation and emision wavelength are easily separable; (v) Narrow emission band width, thus allowing the use of narrow bandpass emission filters; (vi) Fluorescent Eu+3 complexes have a characteristic of absorbing radiation at the wavelength characteristic of the ligand and emission of radiation characteristic of Eu+3 due to energy transfer from ligand to metal ion.

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