TITLE: Indirect ELISA protocol (abcam)
AUTHORS: Heather Robeson, Jing Jin, Mohammed S. Orloff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol is based on the abcam Indirect ELISA protocol.</div></div>

[STEPS]
?. [Reagent Preparation]
PARP1 Standard: A seven point standard curve using 2-fold serial dilution in Plate Coating Buffer or 1X PBS is recommended. Prepare 500 uL of high standard (1000 ng/mL) per plate assayed from stock solution.
?. [Reagent Preparation]
Sample Dilutions: Using proteins extracted from tissue via Qiagen All-Prep DNA/RNA/protein Mini kit, make dilutions with 1X concentration Plate Coating Buffer (provided in Ancillary DuoSet kit) or PBS at a final volume of 50 uL per set of replicates to be run: a.A 1:50 dilution has been the optimal dilution; to make 1:50 dilution, dilute 2.2 uL protein sample with 107.8 uL 1X Plate Coating Buffer or PBS for a total volume of 110 uL for each set of replicates, which will allow for 50 uL to be pipetted twice.b.For ease and efficiency of pipetting samples onto ELISA plate, prepare sample dilutions in a 96-well PCR plate; make sure samples are in same order as plate layout.
?. [Reagent Preparation]
Wash Buffer: If crystals have formed in the concentrate, warm to room temperature and mix gently iuntil the crystals have gone into solution.
?. [Reagent Preparation]
Reagent Diluent: Calculate amount of working concentration (1X Reagent Diluent) needed for plate blocking and antibody preparation for both antibodies. Dilute 10X concentrate Reagent Diluent to make enough 1X Reagent Diluent for all preparations plus extra to account for pipetting error.
?. Substrate:Immediately prior to step 9 of Assay Procedure, prepare Substrate by mixing Color Reagent A and Color Reagent B in 1:1 ratio. Substrate reagents used are from either the DuoSet Ancillary Reagent Kit 2 (cat #DY008) or Substrate Reagent Pack (R&D Systems cat #DY999), and constitute hydrogen peroxide and TMB solution for detection of HRP conjugated reagents. Read the optical density after adding Stop Solution (2N sulfuric acid) at 450 nm.
?. 
?. [Reagent Preparation]
Final Concentration for each standard for the seven point curve (from highest to lowest): 1000 ng/mL, 500 ng/mL, 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31.25 ng/mL and 15.625 ng/mL.
?. [Reagent Preparation]
After preparing 1000 ng/mL standard, add 250 uL of Plate Coating Buffer (or PBS) to 6 other microcentrifuge tubes for 2-fold serial dilution for the other standards; label tubes appropriately. The table below shows the 2-fold serial dilution series to create the rest of the standards.  AB1  Standard
  Concentration    2-fold Serial
  Dilution  2  500 ng/mL standard    250 uL of 1000 ng/mL standard + 250 uL Plate Coating
  Buffer  3  250 ng/mL standard    250 uL of 500 ng/mL standard + 250 uL Plate Coating
  Buffer  4  125 ng/mL standard    250 uL of 250 ng/mL standard + 250 uL Plate Coating
  Buffer  5  62.5 ng/mL standard    250 uL of 125 ng/mL standard + 250 uL Plate Coating
  Buffer  6  31.25 ng/mL standard    250 uL of 31.5 ng/mL standard + 250 uL Plate Coating
  Buffer  7  15.625 ng/mL standard    250 uL of 31.25 ng/mL standard + 250 uL Plate Coating
  Buffer
AB1  Standard
  Concentration    2-fold Serial
  Dilution  2  500 ng/mL standard    250 uL of 1000 ng/mL standard + 250 uL Plate Coating
  Buffer  3  250 ng/mL standard    250 uL of 500 ng/mL standard + 250 uL Plate Coating
  Buffer  4  125 ng/mL standard    250 uL of 250 ng/mL standard + 250 uL Plate Coating
  Buffer  5  62.5 ng/mL standard    250 uL of 125 ng/mL standard + 250 uL Plate Coating
  Buffer  6  31.25 ng/mL standard    250 uL of 31.5 ng/mL standard + 250 uL Plate Coating
  Buffer  7  15.625 ng/mL standard    250 uL of 31.25 ng/mL standard + 250 uL Plate Coating
  Buffer
?. [Reagent Preparation]
There should be 250 uL of each of the first 6 standards and 500 uL of the 7th standard when done. This is enough to run two sets of standards. Aliquot into 120 uL amounts and store one set of standards at -20oC immediately if not using. Place remaining set of standards on ice until use.
?. [Reagent Preparation]
Do not make more than enough standard for two sets unless using them immediately; degradation of store protein standard is highly likely, and it is best to make new standards every one or two runs.
?. [Reagent Preparation]
Sample Dilutions: Using proteins extracted from tissue via Qiagen All-Prep DNA/RNA/protein Mini kit, make dilutions with 1X concentration Plate Coating Buffer (provided in Ancillary DuoSet kit) or PBS at a final volume of 50 uL per set of replicates to be run:
?. [Reagent Preparation]
A 1:50 dilution has been the optimal dilution; to make 1:50 dilution, dilute 2.2 uL protein sample with 107.8 uL 1X Plate Coating Buffer or PBS for a total volume of 110 uL for each set of replicates, which will allow for 50 uL to be pipetted twice.
?. [Reagent Preparation]
For ease and efficiency of pipetting samples onto ELISA plate, prepare sample dilutions in a 96-well PCR plate; make sure samples are in same order as plate layout.
?. [Reagent Preparation]
Wash Buffer: If crystals have formed in the concentrate, warm to room temperature and mix gently iuntil the crystals have gone into solution.
?. [Reagent Preparation]
Reagent Diluent: Calculate amount of working concentration (1X Reagent Diluent) needed for plate blocking and antibody preparation for both antibodies. Dilute 10X concentrate Reagent Diluent to make enough 1X Reagent Diluent for all preparations plus extra to account for pipetting error.
?. [Reagent Preparation]
Substrate:Immediately prior to step 9 of Assay Procedure, prepare Substrate by mixing Color Reagent A and Color Reagent B in 1:1 ratio. Substrate reagents used are from either the DuoSet Ancillary Reagent Kit 2 (cat #DY008) or Substrate Reagent Pack (R&D Systems cat #DY999), and constitute hydrogen peroxide and TMB solution for detection of HRP conjugated reagents. Read the optical density after adding Stop Solution (2N sulfuric acid) at 450 nm.
?. [Assay Procedure]
Add 50 uL of sample or standards diluted in Plate Coating Buffer, or an appropriate diluent (PBS), per well. Cover with an adhesive strip and incubate overnight at 4oC.
?. [Assay Procedure]
In case strips may fall out from frame during washes, number each strip respectively according to plate layout.
?. [Assay Procedure]
The coating incubation time may require some optimization.
?. [Assay Procedure]
Remove the coating solution and wash the plate three times by filling the wells with 200 μl of ELISA Wash Buffer, or PBS.
?. [Assay Procedure]
The solutions or washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel.
?. [Assay Procedure]
Complete removal of liquid at each step is essential for good performance.
?. [Assay Procedure]
Block plates by adding 200 uL of Reagent Diluent to each well. Incubate at room temperature for a minimum of 2 hours.
?. [Assay Procedure]
Wash the plate twice with ELISA Wash Buffer, or PBS.
?. [Assay Procedure]
Dilute the Primary Antibody to the working concentration in Reagent Diluent. Immediately coat the microplate with 100 uL per well of the diluted Primary Antibody. Seal the plate and incubate a minimum of 2 hours at room temperature.
?. [Assay Procedure]
Although 2 hours is usually enough to obtain a strong signal, if a weak signal is obtained, stronger staining will often observed when incubated overnight at 4°C.
?. [Assay Procedure]
Wash the plate four times with ELISA Wash Buffer, or PBS.
?. [Assay Procedure]
Dilute Secondary Antibody to the working concentration in Reagent Diluent, then coat each well of the microplate with 100 uL of the antibody solution. Cover with a new adhesive strip and incubate 1 – 2 hours at room temperature.
?. [Assay Procedure]
Wash the plate four times with ELISA Wash Buffer, or PBS.
?. [Assay Procedure]
Add 50 uL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light (cover with aluminum foil if needed).
?. [Assay Procedure]
Immediately prior to step 9 of Assay Procedure, prepare Substrate by mixing Color Reagent A and Color Reagent B in 1:1 ratio. Reagents are from the DuoSet Ancillary Reagent kit, or R&D Systems Substrate Reagent Pack.
?. [Assay Procedure]
Add 50 uL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
?. [Assay Procedure]
Read plate within 30 minutes of adding Stop Solution.
?. [Assay Procedure]
Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings to 450 nm. This subtraction will correct for optical imperfections in the plate. Reading made directly at 450 nm without correction may be higher and less accurate.
?. [Assay Procedure]
Microplates are read using the SpectraMax spectrophotometer.
?. [Assay Procedure]
Standard curves and adjusted concentrations are calculated using the SoftMax Pro 6.5 software for the SpectraMax spectrophotometer.