Answer To: PHRM 4120: ELISA Calculations - Project Name: Date: Example #1: Free T3 ELISA Principle of the assay...
Deepika answered on Oct 23 2021
PHRM 4120: ELISA Calculations - Project
Name: Meera
Date: 21/10/2020
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:
1. Identify the following:
Antigen: Samples containing fT3
Antibody: anti-fT3 monoclonal antibody
Which one has been conjugated to the enzyme? – Antigen (fT3)
2. Draw a schematic representation of this assay and identify the ELISA format. Explain your answer.
This is competitive ELISA as enzyme conjugated and non-enzyme conjugated fT3 compete for the enzyme binding site on anti-fT3 monoclonal Ab (primary antibody). If the sample has lower amount of antigen (fT3), the signal (formation of yellow product) is stronger due to more concentration of enzyme conjugated fT3 in the solution. If sample has higher amount of antigen, signal is weak (lighter yellow color intensity) due to less concentration of enzyme conjugated fT3 in the solution.
3. Is this a competition or inhibition ELISA? Explain your answer (identifies the control and test substances, if applicable).
This is a competitive ELISA as fT3 antigen in the sample competes with the enzyme conjugated fT3 for its binding on the anti-fT3 monoclonal Ab for giving the final product. Control sample (Enzyme control) has fT3 enzyme instead of fT3 enzyme conjugate, therefore there is no change in the color of reaction mix. Another kind of control, known as substrate control, has anti-fT3 monoclonal Ab added in the reaction mix instead of fT3 enzyme conjugate so that there is no change in the color of the reaction mix. Enzyme and substrate controls are set so as to set a value to the wavelength measure, while taking final reading in the ELISA reader. Color intensity (signal) for the samples is checked and this value is subtracted from all the other sample readings before calculating the final result.
4. 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?
3,3′,5,5′-Tetramethylbenzidine or TMB is a chromogenic substrate which is used as a visualising reagent in enzyme-linked immunosorbent assays (ELISA). It is a white solid which forms a pale blue-green liquid in solution with ethyl acetate. This is because, in bottle TMB is present in reduced form, hence is colorless. However, in presence of HRP (horse reddish peroxidase) and H2O2, TMB gets oxidized and turns blue in color. When H2SO4 (acid) is added, it color further changes to yellow. This color change reaction is done because yellow color absorbs better at 450 nm, wavelength at which this reaction is read in an ELISA reader.
5. 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?
The enzyme linked to fT3 conjugate is HRP (Horse radish peroxidase).
6. Calculating the results:
a. 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.
b. 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.
c. 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.
d. 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 pg/ml
3.419
3.503
3.461
0
Std2=
1.2 pg/ml
3.263
3.406
3.3345
1.2
Std3=
2.5 pg/ml
3.042
3.158
3.1
2.5
Std4=
5 pg/ml
2.415
2.412
2.4135
5
Std5=
8.5 pg/ml
1.326
1.34
1.333
8.5
Std6=
18 pg/ml
0.828
0.883
0.8555
18
Sample1=
unknown
0.155
0.144
0.1495
3.302828
Sample2=
unknown
2.034
1.987
2.0105
3.014373
Conclusions for Example #1:
Standard table was drawn based on the experimental values as given in the table. fT3 concentration in sample 1 and sample 2 as calculated from the regression equation obtained from the graph are 3.3 pg/ml and 3.01 pg/ml, respectively.
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:
1. Draw a schematic representation of this assay. Explain your answer including the identification of the antigen and antibodies.
Anti-PCSK Ab is immobilized...