It is a prac reportAs its worth 5% its not many wordsaround 300-400 words
RMIT Classification: Trusted 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(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 (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 DH5α will be used as a negative control as it does not express the same H-antigen as Salmonella. 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 plate Step 2 (in the lab this is day 2): 4. 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. 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). 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 (negative) control, we use a strain that does not express the H-antigen (E coli DH5α). PROCEDURE 1. A 10 ml overnight culture of E. coli DH5α 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. 2. 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). 3. 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. 4. 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 1. Set up test tubes for the blanks and standards as follows: The numbers are microlitres added.Sample 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 (g) 0 0 3 ---- ---- ---- ---- ---- ---- 2. Fill in the blanks as how much protein is in each tube (µg) 3. For your test samples (E. coli and Salmonella) use 10L in a total volume of 100 L with NaCl. 4. Add 900L dye to each sample and aliquot 200 L of each sample in duplicate into a 96-well plate as shown below. 5. 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 – BlankS1 to S8 – StandardsT1 & T2 - Samples 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. 1. Start by averaging both of your blank wells, e.g. Average Blank =Average(A1:A2). 2. Then, average all of your standards in duplicate, e.g. S1 =Average(B1:C1), S2 =Average(B2:C2) and so on. 3. Finally you must also average your samples. e.g. E. coli = Average(D1:E1) 4. 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. 5. Once all of 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. 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 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). 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(This is the volume we added to the assay, so to determine g/L (final) concentration you must divide by 10). x= 5.422g (in 10 L) = 5.422g/10 = 0.5422 g/L this is your final protein concentration!!! 3. COATING ANTIGEN ONTO ELISA PLATE ProcedureS 1. Use Bradford assay results to determine protein concentration in the samples as per page 12 instructions. 2. Once you have done this, determine the volume of each sample needed to dilute each sample to a final concentration of 0.005 µg/µL in a final volume of 3 mL. C1= your sample protein 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 ELISA plate) Use C1V1=C2V2 calculation to determine volume of protein needed: Example: If my protein concentration is 0.5422 µg/µL then the volume I need 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 protein 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 3. Coat the wells with 100 µL of diluted samples. 4. 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 only 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 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,