ELISA+Protocol

Original protocols Kurt has - [[file:ELISA original protocols.pdf]]
= = =__**ELISA Protocol**__=

- 0.2M Na2CO3 - 0.2M NaHCO3 - 12 Channel pipette - 96 Well ELISA plate (may be a good idea to use a flat bottom plate) - Automated ELISA Plate Washer - Automated ELISA Plate reader - 1X PBS - 3% BSA - PBST solution (1X PBS + 0.05% Tween-20) - Anti-species HRP (0.5mg/ml in 50% glycerol) - 37C incubator - Nunc flat-bottom Maxisorp plate or another plate suitable for ELISA
 * Equipment/Materials**
 * Detection Reagent
 * either TMB substrate
 * or ABTS
 * last seen in 4 C fridge containing stock antibodies
 * Stopping reagent
 * either 0.5M HCl for TMB
 * or 1% SDS for ABTS
 * last seen on Bart's lab bench

>
 * Procedure**
 * Make the coating buffer by adding:
 * 3mL of 0.2M Na2CO3 (Disodium carbonate) to
 * 7mL of 0.2M NaHCO3 (sodium bicarbonate)
 * At pH 9.5
 * Example preparation
 * 0.2*mol/(1*L)*105.99*g/(1*mol)*0.3*L = 6.36 g
 * 0.2*mol(1*L)*84.0*g/(1*mol)*0.7*L = 11.76 g
 * pH of one solution without adjustment was 9.54 (4-7-12)
 * This is enough for one plate.
 * Cover with foil, as solution is light sensitive
 * Add antigen to the coating solution to a concentration of 1µg of antigen/mL buffer (often may want to do 10 ug/mL or another antigen concentration)
 * Add 100µL of coating solution with antigen to each well of the plate (make sure that you use a suitable plate such as a Nunc flat-bottom Maxisorp plate).
 * Incubate overnight at 4C or 1 hr at 37 C
 * Note that the plate can also be coated at 4C over the weekend. Bart even mentioned that a paper showed that the best results were obtained after 3-7 days of coating.
 * Wash plate 4x with PBST solution.
 * First empty solution in plate by shaking plate in sink. Then you can use the automated plate washer. If you transfer the tube attached to the Biotek automatic plate washer, make sure that you run a prime first (Run->Prime->then use options to cycle through to "prime 200"). Put plate in the plate holder. Hit “run” – “wash” – make sure program “05” is selected. Hit “enter”, then hit “start.”
 * Block for 1 hour with 200µL PBST + 3% BSA or 5% nonfat milk in TBST at 37C.
 * Example 5% nonfat milk preparation: 1.25 g dried milk powder in 25 mL TBST.
 * Wash plate 4x with PBST solution
 * Can also use automated plate washer. Put plate in the plate holder. Hit “run” – “wash” – make sure program “05” is selected. Hit “enter”, then hit “start."
 * Add 100 uL PBST to each well. Dilute serum 1:200 in PBST
 * Example Plate Layout [[file:example elisa plate layout.xlsx]]
 * Then make serial two-fold dilutions in PBST on the plate (probably all the way across the plate except for the last column which could be saved for a secondary only if this isn't already somewhere else on the plate) (probably want duplicates of each sample). The last well should only have 100 uL of solution.
 * probably want to have some duplicate coated wells with secondary only with no primary (a serial dilution does not need to be performed). You may also want some coated wells with naive sera with secondary and primary
 * Add 100µL to the appropriate well, and incubate for 1 hour at 37C.
 * Wash 4x with PBST (same as before).
 * Dilute 2º antibody (anti-species-HRP) 1:2000 (as recommended by Shen) or 1:1000 (or 2.5 nM as recommended by Bart) in PBST. Add 100µL to each well of plate and incubate for 1 hours at 37C.
 * Example 1:1000 dilution: 10 uL secondary stock antibody in 10 mL PBST
 * Example 2: 0.5mg/mL*1g/(1000mg)*1mol/(150000g)*1000mL/(1L)=3.33E-6 M
 * Need 400 uL of 2.5E-9 M so 3.33E-6M*y/(400uL)=2.5E-9M -> y=0.3 uL in 400 uL PBST
 * Wash plate 4x with PBST, then wash 4x with PBS (the second wash can often be skipped).
 * Add detection reagent
 * Note that in some situations you may want to read the absorbance, allow the plate to keep developing, and read the absorbance again.
 * If TMB is used: Add 100µL of TMB to each well and incubate 10 minutes at room temperature. (Note: amount of TMB should be 1µL TMB per 1µL that was in the well)
 * If ABTS is used: Add 100 uL ABTS to each well solution and incubate at 37 C 30 min
 * Stop detection reagent reaction
 * If TMB is used: add 0.5M HCl (use the same amount that you used for TMB (100 uL)) (make sure there are no bubbles)
 * If ABTS is used: add 50 uL 1% SDS (make sure there are no bubbles)
 * Read absorbance at 405nm.
 * Load protocols -> ELISA -> HRP and TMB. Click the read button. You may need to tell the software to select COM port before reading.
 * Plate-> Settings can be used to set the wavelength
 * Save data (the data can be exported to an excel file if you want)
 * If Biotek automatic plate washer was used, perform Day Rinse (Main menu->Run->Prime->then cycle through options to select P_Day_rinse). The waste container (probably on the floor) may need to be changed)

Subtract the average secondary signal from the sample signals. To calculate the titer, average the normal serum readings (exclude negative values) and multiply by 2 (total must be greater than or equal to 0.05). The antibody titer is defined as the highest dilution that generates an absorbance greater than this value.