Enzyme Linked Immuno Sorbent Assay(ELISA) - Types of ELISA Assays - Science Assignment Help

Assignment Task

Introduction 
The enzyme linked immunosorbent assay (ELISA) exploits antibody to antigen binding specificity for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones. There are four types of ELISA assay (see Fig. 1): competitive, sandwich, direct and indirect. In this practical exercise, you will be setting up a competitive assay between an unlabelled substance and a labelled- enzyme. In a competitive assay, the labelled probe competes with unlabelled substance for binding to the immobilised ligand. The enzymatic reaction is inversely correlated with the amount of unlabelled antigen present. 
Fig. 1 Types of ELISA assays 
An ELISA assay is typically carried out in a 96-well plate where a ligand (e.g. an antigen) is covalently linked to an activated polystyrene well surface. Antibodies labelled with enzyme are then introduced which bind specifically to the antigen. This binding can be quantified by addition of substrate for the labelled enzyme following a wash to remove unbound substance. The strength of the enzymatic reaction is proportional to the amount of bound labelled substance present. In this practical exercise, you will be working with horseradish peroxidase (HRPO) conjugated biotin. HRPO reacts with the substrate TMB to produce a yellow colour detected at 450 nm (or ABTS to produce a blue-green colour detected at 405 nm). Biotin forms a covalent amide link with HRPO via its carboxyl group to form an enzyme-labelled biotin, i.e. HRPO-biotin conjugate. Note: This experiment is technically not an ELISA, as it does not involve an antibody-antigen interaction. Instead, the binding specificity between biotin and avidin will be exploited to competitively measure free biotin relative to a HRPO-biotin conjugate. 
• In week 1, you will first find the limit of detection of avidin using this labelled enzyme. Secondly you will set up a competitive ELISA assay between biotin and HRPO-biotin to establish a standard curve for the detection of biotin in this assay.
Methods 
Part A: determine the detection limit of Avidin

1. Add 10 μL of the original Avidin solution (1 mg/mL) to 490 μL of PBS in a microfuge tube to obtain a stock concentration of 20 μg/mL.

2. Place 50 μL of PBS in wells 1-11 of rows A to D – leave the well 12 in all the rows empty

3. Add 100 μL of stock Avidin solution (20 μg/mL) to well 12 of row A and B and 100 μL of PBS to well 12 of row C and D (these rows will be references for no Avidin, BSA blocked and unblocked respectively)

4. Prepare a dilution series by transferring 50 μL from well 12 to well 11 of rows A to D, then 50 μL from well 11 to well 10 and so on (pipette up and down to mix before removing 50 μL).

5. Discard 50 μL of solution from well 1 of rows A to D.

6. Incubate the plate at room temperature for 20 min to coat the wells with Avidin – please remember to cover the plates!

7. Plate Blocking: Add 50 μL of BSA solution (10 mg/mL) to all wells of rows A to C (but NOT D)without removing the Avidin solution and incubate the plate for 15 min

8. Wash the plate by flipping the contents out in the sink and then using the “Elisa Wash Buffer” – repeat this step at least 3 times

9. Adding HRPO-biotin. Place 50 μL of diluted HRPO-biotin provided to each well of rows A to D 10. Incubate the plates for 15 min 11. Wash the plate with “Elisa Wash Buffer” at least 3 times 12. Add 100 μL of TMB substrate to each well –blue colour indicates bound HRPO activity

13. After 5 min (or up to 15 min if colour is slow to develop) add 100 M μL of H2SO4

14. Measure the absorbance at 450 nm using the Microplate reader

15. Save the results in an excel file and keep a copy for your reports. 
Competition Assay for Biotin Part B: Plate coating and plate blocking 
1. Choose a dilution of Avidin from Part A that is in the upper limit of a (hopefully) linear section, giving an absorbance that is approximately 80% of the maximum measured. Make 2 ml of diluted avidin solution at this concentration. For example if column 10 gave 80% of max in part A, this is 1⁄4 of the column 12 concentration of 20 μg/mL (i.e. 5 μg/mL). In this example, dilute 10 μL of original Avidin solution (1 mg/mL) to in 1.99 mL of PBS (1/200) in a microfuge tube to obtain a diluted stock of 5 μg/mL. You may want it more dilute than this i.e. less than 10 μL Avidin, eg 5.0 or 2.5 μL.

2. Place 50 μL of diluted Avidin solution in all wells of rows A to C.

3. Place 50 μL of PBS in all wells of row D (row D is taken as a negative control (Blank) in the absence of avidin).

4. Incubate the plate at room temperature for 20 min to coat the wells with Avidin – please remember to cover the plates!

5. Plate Blocking: Add 50 μL of BSA solution (10 mg/mL) to all wells of rows A to D without removing the Avidin solution and incubate the plate for 15 min

6. Wash the plate by flipping the contents out in the sink and then using the “Elisa Wash Buffer” – repeat this step at least 3 times 

Part C: Preparing the biotin dilution series 1. Prepare a Biotin dilution series in row H of the same plate. 2. Place 50 μL of PBS in wells 1 to 11 of row H and 95 μL of PBS in well 12 of row H. 3. Add 5 μL of Biotin (1 mM) to well 12 of row H. Total volume in row 12 is now 100 μL and biotin is 
diluted to 0.05 mM. 4. Prepare a biotin dilution series by transferring 50 μL of diluted biotin solution from well 12 (pipette up and down to mix before removing 50 μL) to well 11 of row H, then 50 μL from well 11 (again pipette up and down) to well 10 and so on to well 2. – Remove 50 μl from well 2 and discard. DO NOT add biotin to well 1 
Part D: Setting up the plate for competition assay 1. Add 50 μL of 1/5 diluted HRPO-biotin provided to each well of row H and mix properly. Start at well 
1 and can continue with same tip as biotin becomes more concentrated to 12

2. Transfer 20 μL of diluted biotin / HRPO-biotin solution from well 1 of row H to well 1 of rows A, B, C and D, then from well 2 of row H to well 2 of rows A, B, C and D, and so on. This can be done with 6 tips on a multi-well pipette.

3. Incubate the plate at room temperature for 20 min – please remember to cover the plates.

5. Wash the plate by flipping the contents out in the sink and then using the “Elisa Wash Buffer” – repeat this step 4 times

6. Add 100 μL of TMB substrate to each well – blue colour shows bound HRPO activity.

7. After 5 min (or up to 15 min if colour is slow to develop) add 100 M μL of H2SO4

8. Measure the absorbance at 450 nm using the Microplate reader 9. Save the results in an excel file and keep a copy on USB drive or email for your reports. 
Part 2 (Week 2): Competitive ELISA (enzyme-linked “immuno-sorbent” assay) between unknown samples of Biotinylated protein and HRPO-Biotin conjugate. 
Introduction 
As for Part 1, for this practical exercise, you will be working with horseradish peroxidase (HRPO) conjugated biotin. HRPO reacts with the substrate TMB to produce a yellow colour detected at 450 nm. Note: This experiment is technically not an ELISA, as it does not involve an antibody-antigen interaction. Instead, the specific binding between biotin and avidin will be exploited to measure the concentration of biotinylated protein in 2 samples of unknown concentration by competition with a HRPO-biotin conjugate. 

Introduction (8 marks in total) 
1. Abstract, Introduction and Aims must not be more than 2 pages of text 2. Must include 1-2 diagrams 3. In-text references must be provided at appropriate places and full reference entry must be given at 
the end of the report in the bibliography 4. Provide general information about immunochemistry / ELISA / competitive ELISA with reference to practical applications and on HRPO, biotin/ avidin and advantages of using the avidin-biotin system. 
Aims (2 marks in total) brief – just a couple of sentences. 
Results and Calculations (12 marks in total) 
Results must be presented as narrative text describing why you are doing something, what you did and what you found. The text refers to figures in appropriate positions and in order. Do not just paste in figures without explanation. Figures must include legend that explains enough to understand figure. 
Part 1A 
1. Calculate the concentration of avidin in each well – remember that you have serially diluted the stock avidin solution; each well will have a different dilution factor!

2. Calculate the amount of avidin i.e. A(avidin) in each well. 
Formula you can use: 
A(avidin) = C(avidin) X Volume 
MW Where C(avidin) is the concentration of avidin in μg/mL, volume is the total volume in the well in mL and MW is the molecular weight of avidin – you will have to find the molecular weight of avidin from literature! 3. Subtract the averaged no-avidin control baseline data from all other data. Average all triplicates, remembering to exclude obvious outliers or anomalous data (explain which and why excluded). Draw a graph with amount of avidin (pmol) on the X-axis, which is best plotted on a Log scale, and absorbance on Y-axis. Please include standard deviation or standard error in the mean error bars. 
Find out the limit of detection of avidin (above baseline) from the graph. You may observe a linear section above baseline and perhaps a plateau region. What could explain a plateau towards an apparent maximum absorbance? Can any linear section be fit with a linear trendline? You may need to replot just the linear section of data to get a good fit. Note down the equation of the trendline and R2 value. What do these tell you? 

Part 1B 4. Calculate the amount of unlabeled biotin added to each well) - remember that you have serially diluted 
the stock biotin solution; each well will have a different dilution factor! 5. Calculate the concentration of Biotin (μM) in each well, taking into account the dilution from the stock. 6. Again, average and subtract baseline absorbance data. It is then worth normalizing your absorbance measures to the maximum absorbance (with no biotin added) by dividing all other data by averaged no-biotin data. Now 1.0 is maximum or can multiply by 100 if you wish to express as a %. Average your normalized replicate data and determine the standard deviation (SD) or preferably standard error in the mean (SEM) for inclusion as error bars. 7. Pasting transposed data into columns helps with graphing. Draw a graph with biotin concentration (pmol) on the X-axis (again log scale will help here) and normalized absorbance on the Y-axis. This type of competition assay is best fit with an inhibitory Hill plot, also know as a 4-parameter logistic (4PL) equation, which unfortunately is not available as a trendline in Excel. Remember this is a competition assay so the highest biotin concentration gives the lowest absorbance. 
 

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