# Must be 1500 word essay, on either topic 1 or 2. Harvard referencing with in text and reference list

i need to complete this assignment which is related to assay design course PHRM 4120: ELISA Calculations – Project Name: Date:
View complete question i need to complete this assignment which is related to assay design course PHRM 4120: ELISA Calculations – Project Name: Date: Example #1: Free T3 ELISA Principle of the assay (modified from the information provided in the kit insert): In this assay, the samples (containing fT3), assay buffer and fT3 enzyme conjugate are simultaneously added to the wells coated with an anti-fT3 monoclonal antibody. FT3 in the samples competes with a fT3 enzyme conjugate for the binding sites. Unbound fT3 and fT3 enzyme conjugate are washed off using washing buffer. Upon the addition of the substrate, a blue color is developed, which is changed to yellow upon the addition of an acid solution. The intensity of the yellow color is inversely proportional to the concentration of FT3 in the samples. A standard curve is prepared relating color intensity to the concentration of the FT3. Plate setting and concentrations of standards: Reading of the plate at 450 nm: Answer the following questions about Example #1: Identify the following: Antigen: Antibody: Which one has been conjugated to the enzyme? Draw a schematic representation of this assay and identify the ELISA format. Explain your answer. Is this a competition or inhibition ELISA? Explain your answer (identifies the control and test substances, if applicable). In the product insert, it is indicated that the substrate contains TMB. What is TMB? What is the complete composition of the substrate and why? Based on the fact that the color in the wells before stopping is blue and after stopping is yellow, which is read at 450 nm, what is the most likely enzyme linked to fT3 in the conjugate?
Calculating the results: First, complete the columns labeled OD1 and OD2 in the table below using the data from page #1. Then, calculate the mean OD for each sample. Write the mean value in the appropriate column. Construct the standard curve using the calculated values from the table. Plot the absorbance for the fT3 standards (mean) in the vertical axis (y) versus fT3 standard concentrations (T3 Levels pg/mL) in the horizontal axis (x) on a graph paper (you can use excel for this). Draw the best curve through the points. Attach the graph with your report. Read the concentration for each unknown sample from the curve. Record their value in the table below. Explain how the calculations of the unknown samples were performed under conclusions. Also under conclusions, provide a 3-5 sentences reflection of what you learned from this example. T3 levels Sample Identity OD 1 OD 2 Mean pg/ml Std1= 0.00 Std2= 1.20 Std3= 2.50 Std4= 5.00 Std5= 8.50 Std6= 18.00 Sample1= Sample2= Conclusions for Example #1: ___________________________________________________________________________________
Example #2: Quantikine Human PCSK9 Immunoassay kit Principle of the assay (modified from the information in the kit insert): A monoclonal antibody specific for PCSK9 has been pre-coated onto a microplate. Following blocking for a period of time, standards and samples are pipette into wells and any PCSK9 present in the samples binds to the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for PCSK9 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and blue color develops in proportion to the amount of PCSK9 bound in the initial step. The color development is stopped changing the color to yellow and the intensity of the color is measured using a plate reader. Plate setting and concentrations of standards: Reading of the plate at 450 and 540 nm: Answer the following questions about Example #2: Draw a schematic representation of this assay. Explain your answer including the identification of the antigen and antibodies. Are the enzyme/substrate combination used in this assay the same as for Example #1? Explain your answer. Provide an explanation to why it would be necessary to correct the data using the reading at 540 nm? Is this a competition or inhibition ELISA? Explain your answer. Calculate the results: First, complete the columns labeled OD1 and OD2 for each wavelength in the table below using the data from page #5. Then, calculate the mean OD for each wavelength for each sample. Write the mean value in the appropriate columns. Subtract the mean OD at 540 nm from the mean OD at 450 nm (450 540; plate imperfection correction) for each sample and write the values in the appropriate column. Subtract standard 8 (blank or standard with concentration 0) from all the other samples (background subtraction) and write the values in the appropriate column. Construct the standard curve using the calculated double corrected values (*) from the table. Plot the absorbance for the PCSK9 standards in the vertical axis (y) versus PCSK9 standard concentrations (PCSK9 ng/mL) in the horizontal axis (x) on a graph paper (you can use excel for this). Draw the line through the points. Attach the graph with your report. Read the concentration for each unknown sample from the line. Record their value in the table below. Explain how the calculations of the unknown samples were performed under conclusions. Also under conclusions, provide a 3-5 sentences reflection of what you learned from this example. Sample Identity OD 1 (450 nm) OD 2 (450 nm) Mean OD 1 (540 nm) OD 2 (540 nm) Mean Correction (Subtract 540 from 450) Correction (Subtract Std 8 from all)* PCSK9 ng/ml Std1= 40 Std2= 20 Std3= 10 Std4= 5 Std5= 2.5 Std6= 1.25 Std7= 0.625 Std8= 0.000 0 Sample1= Sample2= Sample3= Sample4=
Conclusions for Example #2: ___________________________________________________________________________________________
Questions about the assays in Examples #1 and #2: Compare and contrast the two ELISA procedures. Include at least 8 points of comparison. Why is never washed after the addition of the substrate in any ELISA assay? Of all the steps performed in ELISA, which is the most important and why? _____________________________________________________________________________
Example #3: ELISA to detect PCSK9/LDL receptor complexes Schematic representation of this assay: Plate setting and concentrations of standards: Answer the following questions about this assay: Compare and contrast this ELISA with the ELISA in example #2.

Reading of the plate at 450 and 540 nm: Answer the following questions about Example #3: Identify the following in this assay: Antigen: Antibodies: What type of ELISA is this assay? Explain your answer. Compare and contrast Examples #2 and #3. Use at least 8 points of comparison. Calculate the results: First, complete the columns labeled OD1 and OD2 for each wavelength in the table below using the data from page #12. Then, calculate the mean OD for each wavelength for each sample. Write the mean value in the appropriate columns. Subtract the mean OD at 540 nm from the mean OD at 450 nm (450 540; plate imperfection correction) for each sample and write the values in the appropriate column. Subtract standard 7 (blank or standard with concentration 0) from all the other samples (background subtraction) and write the values in the appropriate column. Construct the standard curve using the calculated double corrected values (*) from the table. Plot the absorbance for the complex standards in the vertical axis (y) versus complex standard concentrations (complex ng/mL) in the horizontal axis (x) on a graph paper (you can use excel for this). Draw the line through the points. Attach the graph with your report. Read the concentration for each unknown sample from the line. Record their value in the table below. Explain how the calculations of the unknown samples were performed under conclusions. Also under conclusions, provide a 3-5 sentences reflection of what you learned from this example. Sample Identity OD1 (450 nm) OD2 (450 nm) Mean OD1 (540 nm) OD2 (540 nm) Mean Correction (Subtract 540 from 450) Correction (Subtract Std 7 from all)* PCSK9/LDL receptor complexes (ng/ml) Std 1 = 20 Std 2 = 10 Std 3 = 5 Std 4 = 2.5 Std 5 = 1.25 Std 6 = 0.625 Std 7 = 0.000 0 Sample 1 = Sample 2 = Sample 3 = Sample 4 = Sample 5 = Conclusions about example #3: ___________________________________________________________________________________

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