Immunology
From Immunology 5e, Goldsby et al.
Practical Program 2016
Prof. Peter Smooker
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Coordinator:
Prof. Peter Smooker School of Applied Sciences Biotechnology and Environmental Biology RMIT-University Plenty Road, Bundoora Melbourne VIC 3083 (: 9925 7129 Fax: 9925 7110 :: peter.smooker@rmit.edu.au Practical Class Times: Friday: 12:00 – 5:00pm Practical Class Outline: Practical 1: Day 1 Preparation of Antigen – Sonication 19 August Protein Determination – Bradford method
Preparation for ELISA – coating antigen onto microtitre plate
Day 2 ELISA 26 August
SEMESTER BREAK (29/08/2016 – 02/09/2016)
Practical 2: Day 1 Invasion & Adherence Assay 09 September Day 2 Analysis of results 16 September
Practical 3: 23 September Immuno-bioinformatics
Depending on time constraints, some methods may be demonstrated to
you. Your demonstrators will advise.
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Laboratory Safety:
1. The teaching laboratories in Building 223 are PC2 laboratories and are subject to Australian Standards for Laboratories AS/NZS 2243.3:2010.
2. Long-sleeved laboratory gowns/coats are to be worn at all times in the laboratory. If you do not have a laboratory gown/coat one can be hired from the APS Preparation room after the hiring fee has been paid. These are stored in the plastic bag in a designated place in the laboratory.
3. Suitable footwear must be worn; open sandal or thongs are not acceptable. Long hair must be tied back securely as a protection from Bunsen burners and interference with work being carried out. When working with infectious material wear gloves to ensure no contamination of hands.
4. Keep your bench free of non-essential material at all times. Bags are not permitted near the laboratory benches and therefore must be stored in a designated storage area. Regard all bench tops and other surfaces as potential sources of contamination.
5. When working at the benches avoid all hand-to-mouth operations. Never smoke, eat/drink or put anything in your mouth while in the laboratory.
6. Report any accidents involving cuts, burns, broken glass or spilled cultures immediately to a demonstrator. Tissues and Sodium Hypochlorite disinfectant are provided in the event of a spill.
7. Never place contaminated pipette tips on the bench.
8. Where to discard contaminated and non-contaminated waste:
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9. Use of the Fume hood: All procedures involving dangerous chemicals will be performed in the fume hood. Students will be instructed in the session.
10. Procedures before leaving the laboratory: a. Replace all empty sterile tip boxes used. b. Empty out non-sterile tips and eppendorf tubes into their snap-lock bags
and return racks to designated trolley. c. Return all plastic racks, pipettes and bottles of reagents to the designated
trolley. d. Ensure all used contaminated material is stored/disposed of appropriately. e. Clean your bench area. f. Close lids of all Biohazard Sharps containers and place them in the
designated storage area. g. Put chairs neatly under your bench. h. Wash hands thoroughly with soap and water.
Important Safety Considerations:
• In immunology practical classes you are handling potentially dangerous bacterial cultures, please take care and adhere to good aseptic technique at all times.
• Any student who may be at increased risk of infection is urged to discuss the matter confidentially with their demonstrator.
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GUIDELINES FOR WRITTEN WORK IN ADVANCED IMMUNOLOGY AND CELL TECHNOLOGY:
For each of the three practicals within this subject there is a written component. Practical 1 (ELISA) and Practical 2 (Infection &Invasion) require a scientific report and Practical 3 (Bioinformatics) has an assignment sheet. All of the formal scientific reports must follow this set of guidelines in order to be passed. Please take note of the marking scheme for each report for detailed allocation of
marks for each section within the report. Word Count Limit: The upper limit for these reports is 750 words. Turnitin: All reports are to be submitted to Turnitin for similarities to other student’s reports and already published works. All reports must be written in Third Person/passive text (this means no personal pronouns- No; I saw/ we saw/ we did/ we showed). A correct example of passive writing is “The data shown represents….”. All sections are to be written in PAST TENSE, except when mentioning a known fact, e.g. “It was shown that some cells stained…”
HEADINGS FOR THESE REPORTS INCLUDE:
1. Introduction & Aims
The introduction should include the necessary background information required to understand the topic (for this you will need to read current literature) and an aim/purpose of the practical. Be sure to correctly reference any information provided from a reliable source. This section is usually allocated the second most marks in a marking scheme.
2. Materials & Methods
The materials and methods section will contain all of the materials used in the practical and the procedures followed. As these are provided for you in detail you do not need to re-write these, however you must mention any changes made to a protocol and reference them accordingly.
3. Results
The results section should have your data organized and presented as clearly and precisely as possible. Avoid repeating results. Data presented in the results section must be the final result (either a graph or table); any raw data (All OD readings, individual group colony counts, calculations etc.) should be placed in an appendix and referred to in text.
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In some cases you may be required to provide results from a calculation. In this case you may show written calculations for one example within the results section and provide the answers from the rest. All other calculations should be in an appendix and referred to in text. All presented data must have a legend (a statement describing what the data represents). For tables the legend is above the table (see Example 1) for figures the legend is below the figure (see Example 2). Example 1: How to include a table in your data
Table 1) Class average of colony counts of E.coli and Salmonella on LB agar E. coli
(Adherence) Salmonella (Adherence)
E. coli (Invasion)
Salmonella (Invasion)
QC Controls
10-2 10-3 10-2 10-3 10-1 10-2 10-1 10-2 10-1 10-2
132 14 85 7 0 0 22 2 0 0
Example 2: How to include a figure in your data
Figure 2) ELISA Detection of Salmonella Typhymurium 82/6915 H-antigen using anti-Salmonella H-antigen antibody and anti-Shigella antibody (negative control). 4. Discussion & Final Conclusions
The discussion section is where you “discuss” the results you saw and how it applies to current scientific research.
Any unexpected result should be discussed, i.e. if something gave a positive response and it was supposed to be negative. However don’t spend the entire discussion on the reasons why the experiment did/didn’t respond as expected. Any references mentioned in the introduction or in literature can be used as a
0
0.5
1
1.5
2
2.5
3
1 4 16 64 256 1024
O D 4 50
Titre
ELISA for Detection of Salmonella H- antigen
1/400 anti-Salmonella
1/400 Anti-Shigella
Anti-Salmonella antibody
Anti-Shigella antibody
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comparison to our experiments. Use this real knowledge to explain the relevancy of our model practical experiment. Finally there should be a sentence or two concluding statement that summarizing the findings of this report.
This section is usually allocated the most marks in a marking scheme. 5. References
The Referencing section is important part of any scientific communication. Any comment or result that is not your own must be referenced, unless it is widely accepted or known information (e.g., “Escherichia coli is a gram negative bacillus” does not need to be referenced).
Any information retrieved from a journal article or website on the Internet must be written in your own words and properly referenced. It is only acceptable to take material word-for-word from another source if you place in within a parenthesis. Direct quotations such as these are generally boring to read and should only be used if there is a special reason.
All reports must have in-text referencing as well as a reference list.
WIKIPEDIA and its constituent websites (anything with a Wiki attached or forum related references) are NOT suitable referencing material!!!
You also cannot reference spoken sources (like lecturers or demonstrators).
It is highly recommended that you use a Referencing Manager program (like Endnote/Procite/Refworks/Mendeley, etc.) to keep references properly managed. They will generate a reference list and will ensure the formatting of references automatically.
Failure to include references will result in a zero mark for the report.
6. Appendices
The appendix section includes any supplementary data or material that does not belong within the report. All raw data and calculations go in this section numbered separately.
Example:
Appendix 1: Raw calculations for the CFU/mL of E. coli and Salmonella for infection and
invasion assay.
OD600 (E.coli) = 2.65 (2.65 * 7.65) – 0.3 = 19.97 X 109 CFU/mL
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BEFORE SUBMITTING:
• Keep your report concise. DON’T WAFFLE.
• To get the highest possible marks, follow the individual marking scheme and these guidelines for each practical.
• Make sure the in text citations match the references list.
• We understand that you all come from different backgrounds, so to ensure your report follows correct spelling, language and formatting it would be beneficial if you proof-read your assignment and perhaps get someone else to proof-read it to make sure any spelling/grammatical errors are not overlooked.
• Ensure all bacterial names are italicised and written out in full once before you start using abbreviations, e.g. “Escherichia coli (E. coli) is a gram-negative rod shaped bacteria. E. coli is a commonly studied organism.”
• Ensure all Chemical names are written out in full once before you start using abbreviations, e.g. Bovine Serum Albumin (BSA)
• Any report submitted without one of abovementioned sections (with the possible exception of an appendix) will be marked down.
• Any report submitted after the assigned due date (without prior extension or confirmation from Prof. Peter Smooker) will receive a penalty as per the course guide.
• Reports submitted to Turnitin must be the same report as the one submitted for marking. Any report that has is found to be plagiarised from another student or published works will receive a mark of zero
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PRACTICAL 1
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) Background (ELISA) 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 illnesses such as typhoid fever, paratyphoid fever, and foodborne illness(1). Serotyping is the process by which the Salmonella genus is classified into further serovar subtypes is 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 (2). In this practical, we will determine the presence of a specific antigen in lysates of Salmonella and E. coli. 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 presence of H-antigen of Salmonella Typhimurium 82/6915. E. coli DH5α will be used as a negative control as it does not express the same H-antigen as Salmonella. Day 1: A. Isolation of antigen by sonication B. Determination of protein concentration using the Bradford assay C. Coating antigen onto microtitre plate Day 2: D. Indirect ELISA References: 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.
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Day 1 A. 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). The cell lysate is then centrifuged at high speed to recover the bacterial proteins, which 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, which 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 (negative) control, we use a strain that does not express the antigen (E coli DH5α). Reagents and Equipment – E coli DH5α / Salmonella Typhimurium 82/6915 cultures – 10 mM Tris-HCl, pH 7.4 – 10 ml centrifuge tubes – Benchtop centrifuge – Sonicator – Esky + ice – Pipettes and tips (non sterile) – Eppendorf tubes (non sterile)
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Procedure 1. A 10 ml overnight culture of E. coli DH5α or Salmonella Typhimurium
82/6915 is used to inoculate 150 ml 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.
This step has been done for you. Students start here: Label your tubes with your group initials 2. Re-suspend the cell pellet in 1 ml of 10 mM Tris-HCl, pH 7.4 and
centrifuge at 5,500 rpm for 2 minutes. Remove the supernatant and then repeat the re-suspension and centrifugation steps.
3. Resuspend cells in a final volume of 2 ml of 10mM Tris-HCl, pH 7.4 and place tubes on ice for sonication.
The demonstrators will combine the cultures of E. coli DH5α and Salmonella Typhimurium 82/6915, as a larger volume of culture is required to assist in
sonication. N.B Ear muffs must be worn at all times during sonication of the cells 4. Sonicate cells (as demonstrated) using the following programme:
Pulse – 30 secs.
6 times Rest – 30 secs.
Demonstrators will aliquot sonicated samples back into Eppendorf tubes and return to students. Label your tubes. 5. Transfer the cell lysate to Eppendorf tubes and centrifuge at 14,000 rpm
for 5 minutes. Transfer the supernatant to a clean Eppendorf tube.
This part will be demonstrated to you. Your demonstrators will advise.
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B. 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). Binding of the dye to different amounts of a standard protein, usually Bovine Serum Albumin (BSA) is quantitated by measuring the absorbance at 600 nm and used to generate a standard curve. This can then be used to quantify the unknown protein(s). Reagents and Equipment
– Eppendorf tubes (non sterile) – 96-well microtitre plates – Pipettes and tips
– Bio-Rad Protein Assay Dye Binding Reagent:
This reagent is commercially purchased as a concentrate o Dilute 1 part Dye Binding Reagent Concentrate in 4 parts Distilled,
Deionised (DDI) Water. Then filter through 0.45 µm filter, store at 4°C, filter required amount again before use.
– BSA standard: Standard Albumin solution: 1 mg/ml – 0.15 M NaCl
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Procedure 1. Label tubes as follows:
Blank S1 S2 S3 S4 S5 S6 S7 S8
2. Set up the standards as follows:
µL Blank S1 S2 S3 S4 S5 S6 S7 S8
BSA (1 mg/ml) 0 0 3 6 9 12 15 18 21
0.15 M NaCl 100 100 97 94 91 88 85 82 79
Final protein amount (µg) 0 0 3 —- —- —- —- —- —-
Fill in the blanks as how much protein is in each tube (µg)
3. For test samples, aliquot 10 µL into an Eppendorf tube, then add 90 µL of 0.15 M NaCl (This makes a 1/10 dilution).
4. Add 900 µL Dye Binding Reagent to each tube, mix thoroughly, stand for 2
minutes at room temperature.
5. Aliquot 2 x 200 µL to 96-well microtitre plates as shown below and read on ELISA reader at 595 nm. Pipette standards and test samples as follows:
Make sure that there are no air bubbles present as this can alter the reading.
1 2 3 4 5 6 7 8 9 10 11 12 A B B 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 F G H
B – Blank S1 to S8 – Standards T1 & T2 – Samples
KEEP YOUR SAMPLE as it will be used in the next step!
Sample
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Calculations to determine your protein sample: Students will be provided an excel spreadsheet containing Raw data (OD595 readings) from the Bradford Assay. From these results students will create a standard curve showing the absorbance versus protein amount (µg) for the standards and this can be used to determine the protein content of your cell lysate. Creating the Standard Curve In Excel:
Use these instructions in conjunction with Bradford calculations tutorial given in class.
1. The first thing to be done is to average all of your blank wells, e.g., Average Blank =Average(A1:A4).
2. Then you must average all of your standards in duplicates E.g., S1 (average) =Average(C1:D1), S2 (average) =Average(C2:D2) and so on.
3. Finally you must do the same for all of your samples. 4. Once you have done this, you must then normalise against background. In order to do
this you take all OD readings and minus the Average Blank. 5. Once all of the readings are normalised, you can create the standard curve using the BSA
standard protein concentration (calculated page 13, step 2) as the X-axis, and the Standard OD readings (minus blank) as the Y-axis. To do this fill in the standard concentrations in a column next to standard OD readings and highlight both columns, then go to Chart and select Scatter plot (Marked Scatter).
6. 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.
7. From the equation you can determine your total protein concentration. Calculating your protein concentration: Once you have your standard curve you can calculate the protein concentration (µg/µl) of your original sample (obtained in part A of this practical). You will need to take into account the dilution factor and the volume of your diluted sample (from step 3 of this procedure).
This is an example of the calculations required for determining your protein
concentration. Use these to assist your own calculations
but do not use the values included.
If your equation is
y=0.0246x and your Average OD (minus blank) is 0.13338, substitute y for
your OD value: y=0.0246x
0.13338=0.0246x
1. Then you solve for x (your protein concentration) y=0.0246x 0.13338=0.0246x
x=0.13338/0.0246 x= 5.422µg (in 10 µL)
= 5.422µg/10
= 0.5422 µg/µL this is your final concentration!!!
(This is the volume we added to the assay, so to determine µg/µL (final)
concentration you must divide by 10).
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B. COATING ANTIGEN ONTO MICROTITRE PLATE Reagents and Equipment – Antigen (from last week’s practical) – Coating buffer (0.016 M Na2CO3, 0.034 M NaHCO3, pH 9.6) – 96-well flat bottom ELISA plate – Yellow tips ProcedureS Note that some wells are treated differently to others- carefully follow the protocol below (particularly note the underlined points). 1. Use Bradford assay results to estimate total protein content in the
samples, and dilute protein samples to 0.005 µg/µL in Coating buffer (which has been provided).
Use C1V1=C2V2 calculation to determine dilution of antigen:
Example:
If my lysate concentration is 0.5422 µg/µL then the volume I need to add is:
C1V1=C2V2 V1= C2 x V2 = 0.005 µg/µL X 3000 µL = 27.665 µL
C1 0.5422 µg/µL
Now try it for yourself: If your lysate concentration is µg/µL then the volume needed is: C1V1=C2V2 V1= C2 x V2 = 0.005 µg/µL X 3000 µL = µL
C1 µg/µL
2. Coat the wells with 100 µL of diluted samples (equivalent to 0.5µg of antigen per well) to all wells.
3. Incubate the plate at 40C overnight.
C1= your sample lysate concentration (µg/µL) V1= what we are trying to find out (µL) C2=0.005 µg/µL (final concentration) V2= 3 mL= 3000µL (total volume required to coat a 96-well plate)
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Day 2
B. INDIRECT ELISA Background The indirect ELISA is used for the screening of antisera for specific antibodies and utilises semi-purified or purified antigen. Q: What does a direct ELISA identify? Antibodies are detected by coating the wells of microtitre plates with antigen, incubating the coated plates with test solutions containing specific antibodies, and washing away unbound antibodies. A solution containing a secondary antibody (against the test antibodies) conjugated to an enzyme such as horseradish peroxidase is then added to the plate. After incubation, unbound conjugate is washed away and substrate solution is added. After incubation, the amount of substrate hydrolysed is assessed by measuring the absorbance at 450 nm, using an ELISA plate reader. The measured amount is proportional to the amount of specific antibody in the test solution. Figure 3. Indirect ELISA to detect specific antibodies. Ag = antigen, Ab =
antibody, E = enzyme.
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Reagents and Equipment – 96-well flat bottom ELISA plate coated with antigen (prepared previously) – Yellow tips – Wash bottles – 1x PBS – PBS/Tween (1x PBS with 0.05% Tween 20) – Distilled water – Blotto (5% skim milk in PBS with 0.05% Tween 20) – Diluent (1% skim milk in PBS with 0.05% Tween 20) – Anti-Salmonella H-antigen polyclonal antibody – Anti-Shigella polyclonal antibody – Goat anti rabbit IgG – HRP conjugate – TMB substrate – 2 M H2SO4 – ELISA plate reader
Note that some wells are treated differently to others- carefully follow the protocol below (particularly note the underlined points).
1-3 done by staff)
Table 1) Template for ELISA assay.
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Procedure 1. Wash plate with 1X PBS/Tween once, and add 200 µL of Blotto solution
(to all wells except A3, add 100 µL PBS to A3) incubate for 1 hour at 370C with gentle shaking.
2. Discard Blotto solution, and wash wells with 3 times with PBS/Tween.
3. Add 100 µL of diluent into well A1& A3, C2-12 and E2-12.
4. Add 200 µL of Salmonella antiserum into well C1, and 200 µL of Shigella antiserum into well E1. In well A2 & A3 add 100 µL of Salmonella antiserum.
5. Make a serial dilution from C1 to C12 as following:
6. Repeat this process for Row E exactly as you have done for C.
7. Incubate the plate at 37oC for 1 hour with gentle shaking.
8. Discard primary antibody and wash wells 3 times with PBS/Tween.
9. Add 100 µL of 1/5000 diluted Goat anti-rabbit IgG-HRP conjugate to all wells, except A2. Add 100 µL of diluent in well A2.
10. Incubate the plate at 37oC for 1 hour with gentle shaking.
11. Discard antibody solution and wash 3 times with PBS/Tween, and once with distilled water.
Perform Step 12 & 13 to be completed in the Fume hood:
12. Add 100 µL 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).
13. Stop the reaction by addition of 100 µL of 2M H2SO4 per well (TMB substrate will turn yellow).
14. The ELISA plates will be read at 450nm.
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PRACTICAL 2
ADHERENCE and INVASION ASSAYS
Background Most pathogenic bacteria causing infection require virulence determinants that enhance their ability to adhere to sites of infection and invade through the membrane. Humans possess several physical and chemical barriers to infectious agents, which are the same in all individuals. The similarity of these barriers has enabled microbial evolution that use mechanisms based on a similar theme. Adherence is the first and most important microbial mechanism for initiating the infectious disease process. Attachment or adhesion requires the involvement of a receptor on the host and a molecule on the surface of the microbe called an adhesin. Generally host receptors are carbohydrates while adhesins are usually proteins. For example, strains of E. coli possess one type of pilus, referred to as type 1 pili that bind to receptors containing the sugar mannose. Pili are adhesins found in many Gram-negative species. Gram-positive organisms, such as Streptococcus pyogenes, adhere to epithelial cells of the skin and nasopharynx. Epithelial cells are covered with the plasma glycoprotein fibronectin. Fibronectin acts as a receptor for the lipoteichoic acid adhesin of S. pyogenes. Some microbes do not remain on the epithelial surface but instead penetrate to subepithelial layers. The ability to penetrate below the epithelium is referred to as invasiveness. Salmonella Typhimurium is capable of invading the human intestinal epithelium. Salmonella, and some enteropathogenic E. coli strains, use a terminally differentiated epithelial cell, the M cell of the Peyer’s patch in the terminal ileum and in other gut-associated lymphoid tissue, as a portal of entry into the host. The M cells found in Peyer’s patches are thought to internalise luminal contents for delivery to underlying antigen-presenting cells. M cells possess fewer lysosomes and a sparse mucus layer that makes them a prime target for invasive bacterial pathogens. In this experiment, the invasiveness of Salmonella Typhimurium 82/6915 will be determined. E. coli DH5α is used as a control (this strain of E. coli is not invasive). References:
1. Boyd, Robert F., Basic medical microbiology. 5th ed. 1995. Little Brown & Company (Inc.)
2. Neidhardt, Frederick C., Escherichia coli and Salmonella. 2nd ed. 1996.
ASM Press.
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Figure 4. Electron Micrograph of E. coli adhering to the intestinal epithelium (From H. W. Moon, B. Naggy, and R. E. Isaacson, J. Infect. Dis. 136[Suppl.]:124, 1977)
Cancer Cells in Culture Both normal cells and cancer cells can be cultured in vitro in the laboratory. However, they behave quite differently. Normal cells pass through a limited number of cell divisions (50 is about the limit) before they decline in vigor and die. This is probably caused by their inability to synthesize telomerase. Cancer cells may be immortal; that is, proliferate indefinitely in culture. For example INT407 cells are cultured in laboratories around the world. They are all descended from cells removed from the human embryonic intestinal epithelium. Cancer cells in culture produce telomerase.
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Day 1 Materials
– DMEM (Dulbecco’s Modified Eagle Medium) – Salmonella Typhimurium 82/6915 culture – E. coli DH5α culture – 24-well plate (12 wells seeded with INT407 cells) – PBS (phosphate buffered saline) – Gentamycin (200 µg/mL) – Triton X-100 (0.1%) – LB agar plates – Sterile spreaders – Sterile Eppendorf tubes – Plastic dropper – Incubator (37°C, 5% CO2)
Procedure Note: Salmonella Typhimurium 82/6915 is a non-attenuated wild type strain and hence, is virulent and infectious. Wear gloves at all times during the experiment. 1. Each pair is provided with Salmonella Typhimurium 82/6915 and
Escherichia coli DH5α. Determine the cell count for both bacterial cultures. You are given the optical density reading of the cell growth at 600 nm. Using the following formula, dilute your bacteria to 5 x 107 CFU in a total volume of 1 mL in 1 x DMEM.
(OD600 x 7.65) – 0.3 =? X 109 CFU/mL
2. You are supplied with a 24 well plate, which has 12 wells containing a
monolayer of INT407 cells. The cells have been seeded out at 105 cells per well. Wash the monolayer in each well gently with PBS 3 times (500 µl of PBS each time).
Growing INT407 cells are adherent – that means, they stick to the bottom of the well and multiply. Hence, you can discard growth medium/PBS by carefully tipping the medium off.
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3. Label the plate with columns 1-3 and rows a-d (Figure 5).
Blank = 1 x DMEM Column 1 – Control (blank – DO NOT ADD ANY BACTERIA) Column 2 – Adhesion and invasion (Stripes) Column 3 – Invasion only (Spots) 4. Add 200 µL of 1 x DMEM to all wells in column 1. 5. Add 200 µL of 5 x 107/mL bacteria (in DMEM) according to the diagram
shown above. This will equate to 1 x 107 bacteria per well.
6. Incubate at 37 °C in CO2 incubator for 1.5 hour.
During this incubation both E. coli and Salmonella will adhere to the INT407 cells. Only Salmonella will invade the INT407 cells, as E. coli DH5α is incapable of invading host cells. During this incubation period, label the tubes you will need for subsequent steps as outlined on page 23.
7. Wash the monolayer gently with PBS three times (500 µl of PBS each time).
8. To columns 1 and 3 add 200 µL of 200 µg/mL gentamycin. To column 2 add 400 µL/well of 0.1% Triton X-100.
A
B
C
D
3 1 2
Blank SALMONELLA E. COLI
FIGURE 5
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Note: Gentamycin kills the bacteria that have adhered but not those that have invaded. Triton X-100 lyses INT407 cells releasing any bacterial cells that have invaded the INT407 cells.
9. Incubate at 37°C in CO2 incubator for 15 minutes.
10. Remove 20 µL from wells A2 – D2 (mix the well with a pipette first).
Immediately return the plate to the incubator.
11. Make serial dilutions (in 1X PBS) of the samples from the 4 wells of
column 2 and plate out 100 µL of 10-2 and 10-3 dilutions onto LB agar. Label these plates adhesion (E. coli or Salmonella).
12. Incubate column 1 and 3 for a further 45 minutes at 37°C in CO2 incubator.
13. Wash the monolayer three times with PBS.
14. Add 400 µL of 0.1% Triton X-100 to columns 1 and 3 and incubate at
37°C in CO2 incubator for 15 minutes. 15. Make serial dilutions (in 1X PBS) of the 4 wells of column 3 and only A1
from column 1 plate out 100 µL of 10-1 and 10-2 dilutions onto LB agar. Label plates from column 3 invasion (E. coli or Salmonella) and that from A1 control
16. Incubate LB agar plates at 37°C overnight.
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Table 2. Labelling instructions for Eppendorf tubes for dilutions in steps 11-15.
Step Well Sample Dilutions req. for plating
Tube labels No.
Tubes req.
11 Adhesion and Invasion
A2 E. coli 10-2, 10-3 A2 E -2 and A2 E -3 3
B2 E. coli “ B2 E -2 and B2 E -3 3
C2 S. Typhimurium “ C2 S -2 and C2 S -3 3
D2 S. Typhimurium “ D2 S -2 and D2 S -3 3
TOTAL (step 9) 12 tubes
15 Invasion only
A3 E. coli 10-1, 10-2 A3 E -1 and A3 E -2 2
B3 E. coli “ B3 E -1 and B3 E -2 2
C3 S. Typhimurium “ C3 S -1 and C3 S -2 2
D3 S. Typhimurium “ D3 S -1 and D3 S -2 2
A1 Blank 10-1, 10-2 A1 B -1 and A1 B -2 2
TOTAL (step 11) 10 tubes
Total for assay 22 tubes
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Day 2 Count colonies on plates and record all results as a group. Use following table to record group results. You will have duplicate plates for the same sample at the same dilution. Record your average colony count in the table.
Group
ADHERENCE INVASION QC
BLANK E. coli Salmonella E. coli Salmonella
10-2 10-3 10-2 10-3 10-1 10-2 10-1 10-2 10-1 10-2
1
2
3
4
5
6
7
8
Check for any colonies on control plates and record all results.
Results are expressed as a percentage of the total number of colonies adhered or invaded divided by the inoculum.
Total %
Adhered Bacteria Total %
Invaded Bacteria Further, calculate % of bacterial cells invaded compared to those that adhered
using the total number of cells adhered.
% Invasion compared to
adhesion
= Number of colonies adhered X dilution factor X 100 Original inoculum
=
= Number of colonies invaded X dilution factor X 100 Number of colonies adhered =
= Number of colonies invaded X dilution factor X 100 Original inoculum =
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PRACTICAL 3
Immunology Prac 3. Bioinformatics
For your write-up of this practical, give an introduction on computational methods of epitope prediction (approx half a page), noting both T and B cell, and the differences. Answer the questions posed below in bold, with reasoning. 1. Use the following sequence: >gi|45384056|ref|NP_990483.1| ovalbumin [Gallus gallus] MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRT QINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASR LYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGII RNVLQPSSVDSQTAMVLVNAIVFKGLWEKTFKDEDTQAMPFRVTEQESKPV QMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINF EKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLS GISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFL FCIKHIATNAVLFFGRCVSP Using http://www.syfpeithi.de/home.htm , determine which is the dominant CD8 epitope in mice. Use H2-kb, and search for octomers. Try human HLA-B*08, again octomers. Is the same peptide dominant? Is it predicted that HLA-B*08 can present this peptide? This peptide is widely used in antigen presentation assays in immunology. Does BIMAS http://www-bimas.cit.nih.gov/molbio/hla_bind/ also predict this as the dominant peptide? Using the Hopp-Woods tool at http://www.vivo.colostate.edu/molkit/hydropathy/index.html find the most hydrophilic region of the protein. Where is it? Why might it be important to know this? 2. If you were going to design a peptide vaccine to (A) induce CTL’s, or (B) induce antibodies from the following sequence, which regions would you choose? Assume you will test in H2-Db mice. LPKSFDARVEWPHCPSISEIRDQSSCGSCWAFGAVEAMSDRICIKSKGKHK PFLSAENLVSCCSSCGMGCNGGFPHSAWLYWKNQGIVTGDLYNTTNGCQP YEFPPCEHHVIGPLPSCDGDVETPSCKTNCQPGYNIPYEKD 3. Using PAProc, http://www.paproc.de/ predict the proteolytic products from ovalbumin after processing by the human wild-type 1 proteosome.
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Is the dominant H2-kb epitope in mice available for loading onto human MHC? Around what proportion of the protein would be available for presentation? 4. B cell epitope prediction. These are much more complex, as often epitopes represented by antibody are not linear, and therefore are derived from different regions of the polypeptide chain. If detailed knowledge of the protein structure is known, then prediction may be easier. Generally, prediction revolves around the prediction of surface regions. Go to http://tools.immuneepitope.org/tools/bcell/iedb_input Input the ovalbumin sequence. Use each of the algorithms to see the output. What does each predict for the ovalbumin sequence, and why is this important? Are any of the peptides predicted by more than one method?
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APPENDIX: Material Safety Data Sheet for Tetramethyl Benzidine (TMB)
EMERGENCY OVERVIEW NON-CARCINOGENIC ANALOG OF BENZEDINE, MILD OXIDIZING AGENT
PRODUCT IDENTIFICATION Form: liquid Colour: Colourless to light yellow Boiling point: approx. 100 °C Solubility in water: miscible: Stability: The product is stable for a minimum of 1 year at 2°-25°C. Protect from direct UV light. Avoid elevated temperatures.
Incompatibility: Strong oxidizing agents and metals.
HEALTH HAZARD INFORMATION Primary routes of Exposure: Routes of exposure: may be absorbed by ingestion. Inhalation: Inhalation of vapours is unlikely at normal temperature. Skin: skin contact may cause irritation. Eyes: Splashes may cause irritation. Ingestion: May cause irritation. Accidental exposure/spillage information: Personal precautions: Use personal protection. Ventilate area of leak or spill. Methods for cleaning up/collecting: absorb spillage with an inert material and place spillage in a suitable container for disposal.
PRECAUTIONS FOR USE Personal protective equipment: respiratory protection: none with normal use. Skin protection: use gloves of rubber or plastic. Eye protection: wear tight fitting safety goggles when risk of splashing.
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First Aid: Inhalation: Remove to fresh air. Keep at rest. If needed: get medical attention. Skin contact: Remove contaminated clothing and wash with soap and water. Eye contact: Immediately flush with water or physiological salt water, holding eye lips open, remember to remove contact lenses, if any. If needed: get medical attention.
Ingestion: Rinse mouth and drink plenty of water. Keep under surveillance. If needed get medical attention.
Information: show this Safety Data Sheet to doctor or emergency ward. SAFE HANDLING INFORMATION Flammability: Not flammable. Personal precautions: Use personal protection. Ventilate area of leak or spill. Methods for cleaning up/collecting: absorb spillage with an inert material and place spillage in a suitable container for disposal.
Safe storage: in a well closed container. Storage is recommended at 2 to 25°C. Disposal: Do not empty large amounts into water or drains.
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