Immunology Prac report 2: Bradford Assay & ELISA
Immunology Prac report 2: Bradford Assay & ELISA
Total 50 marks (worth 10%)
Introduction & Aim 10 marks
Provide an overview of the project & give background on key aspects such as Bradford assay & ELISAGive real life examples of when techniques are usedRead current literature & reference correctly in-textProvide the aim in the last sentence (be clear & concise)
What was the final goal? (note: performing techniques isnt the aim)
Materials & methods 2 marks
You must refer to the prac manual with a proper IN TEXT citation (one sentence)
DO NOT list all the materials & methods or any alterations
Results 15 marks
Logical & clear presentation, showing only final results (not raw data)
Include table & figure legends & make sure all gel images & graphs are correctly annotated
All results must ALSO be explained in words!
Show Bradford results (both Salmonella & E. coli)
Include the graph with equation & R2 value (dont tabulate antibody readings)
State final sample concentrations
Calculate the volume of protein needed for ELISA plate coating (in ul)
Show ELISA results (both Salmonella & E. coli)
Tabulate control well results (pass/fail) in table format
Include graphs to determine titre, with all units clearly labelled
State the titres you have determined, in wordsDiscussion 15 marks
Actually DISCUSS your results
What did you see? What does it mean? In terms the key principles of the pracWas it the expected result? Why/Why not?
If not, what would you expect to see?
What might have caused unexpected results? (dont waffle about technical/human error)
You MUST discuss any data you included in your resultsRefer back to background & examples provided in the introduction & discuss further
Last paragraph should be the conclusion summarise the findings. Was the aim achieved?
References 4 marks
Must have AT LEAST 3 different references, follow one formatting style no WikipediaPrac manual must be one of your references (for the methods only)
Formatting 4 marks
Logical layout, with correct scientific & English expression and spelling
IMMUNOLOGY PRACTICAL 2
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
Background (ELISA)
In practical 1 we learned how bacteria can adhere to, and sometimes invade, mammalian cells. This practical follows on from that by using molecular techniques to determine the presence of virulent bacteria, for example in an infection. Salmonella enterica subsp. enterica serovar Typhimurium is a Gram-negative, rod-shaped, flagellated, facultative anaerobic bacterium. It is a member of the genus Salmonella. Many of the pathogenic serovars of the S. enterica species are in this subspecies.
Salmonella are found worldwide in cold and warm-blooded animals (including humans), and in the environment. They are commonly the cause of illnesses such as typhoid fever, paratyphoid fever, and foodborne (gastrointestinal) illness ADDIN EN.CITE <EndNote><Cite><Author>HERIKSTAD</Author><Year>2002</Year><RecNum>468</RecNum><DisplayText>(1)</DisplayText><record><rec-number>468</rec-number><foreign-keys><key app="EN" db-id="ewp0t5dz72xsene9et65wtrtx9ea9x9wxr2v">468</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>HERIKSTAD,H.</author><author>MOTARJEMI,Y.</author><author>TAUXE,R.</author><author>nbsp</author><author>V.</author></authors></contributors><titles><title>Salmonella surveillance: a global survey of public health serotyping</title><secondary-title>Epidemiology & Infection</secondary-title></titles><periodical><full-title>Epidemiology & Infection</full-title></periodical><pages>1-8</pages><volume>129</volume><number>01</number><dates><year>2002</year></dates><isbn>1469-4409</isbn><urls><related-urls><url>http://dx.doi.org/10.1017/S0950268802006842</url></related-urls></urls><electronic-resource-num>doi:10.1017/S0950268802006842</electronic-resource-num><access-date>2002</access-date></record></Cite></EndNote>(1).
Serotyping is the process by which the Salmonella genus is classified into further serovar subtypes. This can be performed due to immunogenic surface marker variation in the O-polysaccharide (O-Antigen) and the flagellin protein (H-antigen). Fritz Kauffmann and P. Bruce White initially proposed serotyping in 1934 as a classification scheme for Salmonella ADDIN EN.CITE <EndNote><Cite><Author>McQuiston</Author><Year>2011</Year><RecNum>407</RecNum><DisplayText>(2)</DisplayText><record><rec-number>407</rec-number><foreign-keys><key app="EN" db-id="ewp0t5dz72xsene9et65wtrtx9ea9x9wxr2v">407</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>McQuiston, John R.</author><author>Waters, R. Jordan</author><author>Dinsmore, Blake A.</author><author>Mikoleit, Matthew L.</author><author>Fields, Patricia I.</author></authors></contributors><titles><title>Molecular Determination of H Antigens of Salmonella by Use of a Microsphere-Based Liquid Array</title><secondary-title>Journal of Clinical Microbiology</secondary-title></titles><periodical><full-title>Journal of Clinical Microbiology</full-title></periodical><pages>565-573</pages><volume>49</volume><number>2</number><dates><year>2011</year><pub-dates><date>February 1, 2011</date></pub-dates></dates><urls><related-urls><url>http://jcm.asm.org/content/49/2/565.abstract</url></related-urls></urls><electronic-resource-num>10.1128/jcm.01323-10</electronic-resource-num></record></Cite></EndNote>(2).
In this practical, we will determine the presence of a specific antigen, flagella protein (H-antigen), in lysates of Salmonella and E. coli. Lysates of cell cultures will be made and then probed with an antibody specific to the antigen.
We will be using the Bradford Assay for the determination of protein concentration, and an Enzyme-Linked-Immunosorbent Assay (ELISA) in order to determine the titre of H-antigen of Salmonella Typhimurium 82/6915. E. coli DH5 will be used as a control as it does not express the same H-antigen as Salmonella. Note that there may be some cross-reactivity however.
As a true negative control Shigella lysate is used. These non-motile bacteria do not have an H-antigen.
Step 1 (in the lab this is day 1):
1. Isolation of antigen by sonication of cells
2. Determination of protein concentration of the lysate using the Bradford assay
3. Coating antigen onto microtitre plates
Step 2 (in the lab this is day 2):
4. Indirect ELISA
References:
ADDIN EN.REFLIST 1.HERIKSTAD, H., Y. MOTARJEMI, R. TAUXE, nbsp, and V. 2002. Salmonella surveillance: a global survey of public health serotyping. Epidemiology & Infection 129:1-8.2.McQuiston, J. R., R. J. Waters, B. A. Dinsmore, M. L. Mikoleit, and P. I. Fields. 2011. Molecular Determination of H Antigens of Salmonella by Use of a Microsphere-Based Liquid Array. Journal of Clinical Microbiology 49:565-573.
1. ISOLATION OF ANTIGEN BY SONICATION
Background
Sonication can be defined as the disruption of cells by high frequency sound waves. This technique is commonly used to isolate bacterial proteins and involves harvesting and washing of the bacterial cells, followed by sonication on ice (see method below and the sonication video).
The cell lysate is then centrifuged at high speed to remove the cell debis, while the released proteins are found in the supernatant. These proteins can then be used as soluble antigens in the Enzyme Linked Immunosorbent Assay (ELISA).
In this practical we are using a strain of Salmonella Typhimurium that expresses flagella protein (H-antigen). When a lysate is made, it will contain this antigen, in addition to all the other bacterial proteins. As a control, we use a strain that does not express the same H-antigen (E. coli DH5), and as a true negative a species that does not express an H-antigen at all (Shigella).
PROCEDURE
A 10 ml overnight culture of E. coli DH5 or Salmonella Typhimurium 82/6915 is used to inoculate 150 ml Luria Broth (LB) and is grown to an OD of 0.3- 0.6. The bacteria from ten millilitre samples of these are collected by centrifugation at 5,500 rpm, and the pellets stored frozen.
The cell pellet is resuspended in a final volume of 1.5 mL and sonicated for 3 minutes to break down the cell walls (watch the posted video on how sonication is performed).
The sonicated sample is centrifuged and you take 1mL of the clear supernatant from the top of the tube, being careful to avoid the cell debris pellet below. This clear supernatant contains mostly proteins and nucleic acids from the bacterial culture.
Determine the protein concentration of each sample (E. coli and Salmonella) by performing a Bradford assay.
2.PROTEIN DETERMINATION: BRADFORD METHOD
Background
The Bradford method utilises the ability of a dye, for example Bio-Rad Protein Assay Dye Reagent, to bind to proteins (specifically arginine, histidine and the aromatic amino acids). To determine the concentration of your unknown samples, you must generate a standard curve from samples of known protein concentration. Binding of the dye to different amounts of a standard protein, usually Bovine Serum Albumin (BSA) is quantified by measuring the absorbance of each standard at 600 nm, this absorbance is used to generate a standard curve. This can then be used determine the concentration of protein in your unknown samples.
Procedure
739140316865Sample
00Sample
Set up test tubes for the blanks and standards as follows: The numbers are microlitres added.
L S1 S2 S3 S4 S5 S6 S7 S8
BSA (1 mg/ml) 0 3 6 9 12 15 18 21
0.15 M NaCl 100 97 94 91 88 85 82 79
Final protein
amount (g) 0 3 ---- ---- ---- ---- ---- ----
Fill in the blanks as how much protein is in each tube (g)
For your test samples (E. coli and Salmonella) use 10L in a total volume of 100 L with NaCl.
Add 900L dye to each sample and aliquot 200 L of each sample in duplicate into a 96-well plate as shown below. Wells that are blank have nothing added to them.
Measure the absorbance at 595 nm and determine protein concentration in both the E. coli (T1) and Salmonella (T2) samples.
1 2 3 4 5 6 7 8 9 10 11 12
A B B B S1 S2 S3 S4 S5 S6 S7 S8 C S1 S2 S3 S4 S5 S6 S7 S8 D T1 T2 E T1 T2 B Blank (duplicates). Contains only the dye.
S1 to S8 Standards (duplicates)
T1 & T2 Samples (duplicates).
Calculating protein concentrations in Excel:
You will be given the Bradford Assay raw data in an excel spreadsheet. From these results, create a standard curve showing the absorbance versus protein amount (g) for the standards and this can be used to determine the protein concentration of samples.
Creating the Standard Curve In Excel:
Use these instructions in conjunction with Bradford calculations tutorial.
Start by averaging both of your blank wells, e.g. Average Blank =Average(A1:A2).
Then, average all of your standards in duplicate, e.g. S1 =Average(B1:C1),
S2 =Average(B2:C2) and so on.
Finally you must also average your samples. e.g. E. coli = Average(D1:E1)
Once you have done this, you must then normalise against background absorbance. In order to do this you subtract the Average Blank from all of your standards and sample averages.
Once all the readings are normalised, you can create the standard curve using the BSA standard protein concentration (calculated page 3, step 2) as the X-axis, and the Standard OD readings (minus blank) as the Y-axis. To do this place the standard protein concentrations in a column next to standard OD readings and highlight both columns, then go to Chart and select Scatter plot.
Once you have a graph on the page (ensuring Protein Concentration is X-axis and OD readings is Y-axis) Right click on one of the points and select Add trend line. Once this opens up select Linear and ensure the intercept = 0, and make the equation and R2 value visible on the graph.
From the equation you can determine your total protein concentration as follows.
Calculating your protein concentration:
Once you have your standard curve you can calculate the protein concentration (g/l) of your original sample. You will need to take into account the dilution factor (10) and the volume of your diluted sample (from step 3 of this procedure).
See the Bradford calcs for Protein Concentration for a full description of this.
3. COATING ANTIGEN ONTO ELISA PLATE
ProcedureSUse Bradford assay results to determine protein concentration in the samples as per page 12 instructions.
Once you have done this, determine the volume of each sample needed to dilute each sample to a final concentration of 0.005 g/L in a final volume of 3 mL.68580021590C1= your sample protein concentration (g/L)
V1= what we are trying to find out (L)
C2=0.005 g/L (final concentration)
V2= 3 mL= 3000L (total volume required to coat a 96-well ELISA plate)
00C1= your sample protein concentration (g/L)
V1= what we are trying to find out (L)
C2=0.005 g/L (final concentration)
V2= 3 mL= 3000L (total volume required to coat a 96-well ELISA plate)
Use C1V1=C2V2 calculation to determine volume of protein needed:
Example:
If my protein concentration is 0.5422 g/L then the volume I need is:
C1V1=C2V2
V1= C2 x V2 = 0.005 g/L X 3000 L = 27.665 L
C1 0.5422 g/L
Now try it for yourself:
If your protein concentration is g/L then the volume needed is:
C1V1=C2V2
V1= C2 x V2 = 0.005 g/L X 3000 L = L
C1 g/L
Coat the wells with 100 L of diluted samples.
Incubate the plate at 40C overnight.
INCLUDE THESE CALCULATIONS IN YOUR PRAC REPORT!
4. INDIRECT ELISA
The indirect ELISA is used to detect specific proteins or antibodies in a sample. Proteins are detected by coating the wells of microtitre plates with lysate, and then incubating the coated plates with serially diluted primary antibody (antiserum) which binds to the target protein. Next, a secondary antibody that is conjugated to an enzyme (such as horseradish peroxidase) is then added to the plate. The secondary antibody will bind specifically only to the primary antibody.
After incubation, unbound secondary antibody is washed off and a substrate solution is added that reacts with the conjugated enzyme. After incubation, the amount of substrate hydrolysed is assessed by measuring the absorbance at 450 nm, using an ELISA plate reader. The titre of the protein (or antigen) can be determined as the highest dilution to give a positive reading above the negative control.
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Figure 3. Indirect ELISA to detect specific antigen. Ag = antigen, Ab = antibody, E = enzyme.
Table 1) Plate layout for ELISA assay.
1 2 3 4 5 6 7 8 9 10 11 12
A (Controls) -ve control (no 1 ab) -ve control (no 2 ab) +ve control (no blocking) B Blank row C
(E. coli or Salmonella) 1:100 1:200 1:400 1:800 1:1600 1:3200 1:6400 1:12800 1:?1:?1:?1:?D Blank Row E
(Shigella) 1:100 1:200 1:400 1:800 1:1600 1:3200 1:6400 1:12800 1:?1:?1:?1:?F Blank Row Procedure. Note that the Shigella antigen was prepared for you.
Wash plate with 1X PBS/Tween once, and add 200 L of Blotto solution (to all wells except A3, add 100 L PBS to A3) incubate for 1 hour at 370C with gentle shaking. This saturates all sites that the lysate hasnt bound to minimise subsequent non-specific binding of antibody.
Discard Blotto solution, and wash wells with 3 times with PBS/Tween.
Add 100 L of diluent into well A1-A3, C2-12 and E2-12.
Add 200 L of Salmonella 1:100 diluted antiserum into well C1 and E1. In well A2 & A3 add 100 L of Salmonella antiserum. Do not add to well A1, as this is the No primary antibody control.
Make a twofold serial dilution from C1 to C12 by transferring 100 L from C1 to C2, from C2 to C3 and so on across each well of the plate. Discard 100 from the last well after mixing well, so that all plate wells have 100 L volume. Repeat this process for Row E.
Incubate the plate at 37oC for 1 hour with gentle shaking.
Discard primary antibody and wash wells 3 times with PBS/Tween.
Add 100 L of 1/5000 diluted in Diluent Goat anti-rabbit IgG-HRP conjugate to all wells, except A2. Add 100 L of diluent in well A2.
Incubate the plate at 37oC for 1 hour with gentle shaking.
Discard antibody solution and wash 3 times with PBS/Tween, and once with distilled water.
Perform Steps 11, 12 & 13 to be completed in the Fume hood:
Add 100 L of TMB (tetramethylbenzidine) substrate to all wells (Blue colour will develop). Incubate the plates in dark for up to 30min (your demonstrator will define the length of incubation required).
Stop the reaction by addition of 100 L of 2M H2SO4 per well (TMB substrate will turn yellow).
The ELISA plates will be read at 450nm.
You will be given the ELISA data to calculate the titre of the H antigen in the E. coli and Salmonella samples, using Shigella as an internal negative control. Instructions on determining titre will be uploaded to canvas.
Note that the Bradford assay and ELISA results are given in separate Excel files.
