TITLE: Enzyme linked immunosorbent assays (ELISAs) for mouse IL-10, IL-6, IL-1β and TNF-α
AUTHORS: Anja De Lange, Avril Walters, Nai-Jen Hsu, Muazzam Jacobs, Joseph V Raimondo

[STEPS]
?. Acquire the necessary reagents and antibodies.
?. Salts (all available from Sigma-Aldrich):34.56 g Na2HPO4192 g NaCl5.76 g of KH2PO44.8 g of KCl2.23 g NaN30.12 g MgCl2.6H2OOther reagents for solutions (all available from Sigma-Aldrich):10 ml Tween 2025 g BSA14.6 ml di-ethanolamine37 % HCL (about 10 ml)
?. Prepare stock solution of all antibodies
?. 
?. 
?. Acquire the required reagents and antibodies
?. Salts (all available from Sigma-Aldrich):34.56 g Na2HPO4192 g NaCl5.76 g of KH2PO44.8 g of KCl2.23 g NaN30.12 g MgCl2.6H2OOther reagents for solutions (also all available from Sigma-Aldrich):10 ml Tween 2025 g BSA14.6 ml di-ethanolamine 37 % HCL (a few ml)
?. Protein-binding 96 well plates:Nunc Maxisorp 96 well Immuno Plates (available from Thermo Fisher)
?. Antibodies & recombinant cytokinesWe utilise antobodies from R&D systems (a biotechne brand) - in South Africa Whitehead Scientific can supply these.IL10Capture antibody: Mouse IL‑10 Antibody (#MAB417)Detection antibody: Mouse IL-10 Biotinylated Antibody (#BAF417)Recombinant cytokine: Recombinant Mouse IL-10 (#417-ML)IL6Capture antibody: Mouse IL‑6 Antibody (#MAB406)Detection antibody: Mouse IL-6 Biotinylated Antibody (#BAF406)Recombinant cytokine: Recombinant Mouse IL-6 (#406-ML)IL1βCapture antibody: Mouse IL1β/IL-1F2 Antibody (#MAB401)Detection antibody: Mouse IL1β/IL-1F2 Biotinylated Antibody (#BAF401)Recombinant cytokine: Recombinant Mouse IL1β/IL-1F2 (#401-ML)TNFαCapture antibody: Human/Mouse TNFα Antibody (#AF-410-NA)Detection antibody: Mouse TNFα  Biotinylated Antibody (#BAF410)Recombinant cytokine: Recombinant Mouse TNFα aa 80-235 (#410-ML)
?. Prepare stock solutions of antibodies and recombinant cytokines.
?. Coating antibodies:Reconstitute 100 μg of the primary antibody in 1000 μl of autoclaved 1X PBS (100 μg/ml).Label 20 x 500 μl microcentrifuge tubes with name of the cytokine, 1st antibody, concentration and aliquot volume (eg. TNFa, 1st AB, 100 μg/ml, 50 μl).Pipette 50 μl of the reconstituted antibody solution into each microcentrifuge tube.Place in labelled freezer box and store aliquots in at -80 oC.
?. Detection (biotinylated) antibodies:First stock:Reconstitute 50 μg of the secondary/biotinylated antibody in 500 μl of 1X PBS. Label 10 x 500 μl microcentriguge tubes with name of the cytokine, 2ndantibody, concentration and aliquot volume (eg. IL6, 2nd AB, 100 μg/ml, 50 μl).Pipette 50 μl of the reconstituted antibody solution into each microcentrifuge tube.Second stock:Add 1 x 50 μl aliquot of stock 1 to 450 μl 1X PBS.Label 10 x 500 ul microcentrifuge tubes with name of the cytokine, 2nd antibody, concentration and aliquot volume (eg. IL6, 2nd AB, 10 μg/ml, 50 μl).Pipette 50 μl of the reconstituted antibody solution into each microcentrifuge tube.Place in labelled freezer box and store aliquots at -80 oC.Second stock:Add 1 x 50 μl aliquot of stock 1 to 450 μl 1X PBS.Label 10 x 500 ul microcentrifuge tubes with name of the cytokine, 2nd antibody, concentration and aliquot volume (eg. IL6, 2nd AB, 10 μg/ml, 50 μl).Pipette 50 μl of the reconstituted antibody solution into each microcentrifuge tube.Place in labelled freezer box and store aliquots at -80 oC.
?. Recombinants:First stock: Prepare a 10 μg/ml solution by adding the appropriate volume of dilution buffer to the antibody. Divide into 100 μl aliquots.Second stock: 500 ng/ml, 20 x 100 μl aliquots (add 1900 μl dilution buffer to stock 1 aliquot)Third stock: 100 ng/ml, 5 x 100 μl aliquots (add 400 μl dilution buffer to stock 2 aliquot)Last stock: 10 ng/ml, 10 x 100 μl aliquots (add 400 μl culture media/dilution buffer to stock 3 aliquot)
?. Coat the 96-well plate with coating antibody.
?. For IL10, IL6 and IL1β:Prepare 5100 μl of approx 3 μg/ml coating antibody by adding 3 x 50 μl aliquots of the coating antibody stock solution (100 μg/ml) to 4900 μl of 1X PBS.For TNFα:Prepare 5100 μl of approx 1 μg/ml coating antibody by adding 1 x 50 μl aliquots of the coating antibody stock solution (100 μg/ml) to 5050 μl of 1X PBS.For all:Add 50 μl of the newly prepared coating antibody solution to each well of a Nunc Maxisorp 96 well Immuno Plates. Cover the plate with parafilm or foil and leave overnight at 4 oC (in the fridge).
?. Wash 4X with 1X washing buffer.
?. 1. Prepare 1L of 1X washing buffer by adding 50 ml of 20X washing buffer to 950 ml of diH2O.2. Fill a plastic squeeze wash bottle with 1X washing buffer.3. Remove the foil from the plate and shake the coating antibody out of the plate by inverting the plate and shaking/flicking it over a wash basin.4. Fill each well of the plate with washing buffer using the squeeze wash bottle.5. Remove the wash buffer by inverting the plate and shaking/flicking it over a wash basin.6. Repeat this washing process another 3 times.7. After the final wash, try to remove as much of the washing buffer from the wells as possible by shaking/flicking the plate, and then bang the plate (upside down) on a clean towel/thickly layered tissue paper until you have managed to remove practically all fluid from the wells.
?. "Block" the 96-well plate with blocking buffer.
?. Add 200 μl of blocking buffer to each well. Cover with parafilm/foil and leave overnight at 4 oC (in the fridge)/or incubate at 37 oC for 2hr. (Plates may be blocked and left at 4oC for up to 1 week).
?. Wash 4X with 1X washing buffer.
?. 1. Fill a plastic squeeze wash bottle with 1X washing buffer.2. Remove the foil from the plate and shake the blocking buffer out of the plate by inverting the plate and shaking/flicking it over a wash basin.3. Fill each well of the plate with washing buffer using the squeeze wash bottle.4. Remove the wash buffer by inverting the plate and shaking/flicking it over a wash basin.5. Repeat this washing process another 3 times.6. After the final wash, try to remove as much of the washing buffer from the wells as possible by shaking/flicking the plate, and then bang the plate (upside down) on a clean towel/thickly layered tissue paper until you have managed to remove practically all fluid from the wells.
?. Create the recombinant cytokine standard curves.
?. 1. Prepare 250 μl of 4 ng/ml recombinant cytokine by adding 150 μl dilution buffer/culture media to 100 μl of 10 ng/ml recombinant cytokine stock.2. Pipette 50 μl of the 4 ng/ml recombinant cytokine into well A1, A2, B1 and B2.3. Add 50 μl of dilution buffer/culture media to wells A2 and B2.4. Add 50ul of dilution buffer to wells A3-A12 and B3-B12.5. Using a pipette set to 50 μl, mix well A2 by drawing up and then expelling 50 μl of the solution in the well.6. Draw up 50 μl of the solution in well A2 and add it to well A3. Again, mic the solution using the pipette.7. Now add 50 μl of the solution in well A3 to well A4.8. Repeat this diluting/mixing process through to well A11. Dispose of the last 50ul that you draw up of well A11. This will create a standard curve, through serial dilution, of 4 ng/ml to 3.9 pg/ml, with a dilution buffer/culture medium blank in well A12.9. Repeat this whole process in row B, to produce the standard curve in duplicate.
?. Add samples.
?. 50 μl of experimental samples should be added into available wells.If you have never before run samples from a specific experimental set-up before, it is wise to dilute samples threefold over three wells (ie. in one well you will have the undiluted sample, in an adjacent well you will have a sample which you have diluted threefold, and in a well next to that you will have a sample that you have diluted ninefold).Cover plate with parafilm/foil and incubate overnight at 4 oC or for 2hr at 37 oC.
?. Wash 4 X with 1X washing buffer.
?. 1. Fill a plastic squeeze wash bottle with 1X washing buffer.2. Remove the foil from the plate and shake the cytokines/experimental samples out of the plate by inverting the plate and shaking/flicking it over a wash basin.3. Fill each well of the plate with washing buffer using the squeeze wash bottle.4. Remove the wash buffer by inverting the plate and shaking/flicking it over a wash basin.5. Repeat this washing process another 3 times.6. After the final wash, try to remove as much of the washing buffer from the wells as possible by shaking/flicking the plate, and then bang the plate (upside down) on a clean towel/thickly layered tissue paper until you have managed to remove practically all fluid from the wells.
?. Add the detection (biotinylated) antibody.
?. For IL10, IL6, IL1β and TNFα:Prepare 5100 μl of approx 0.3 μg/ml coating antibody by adding 3 x 50 μl aliquots of the coating antibody stock solution (10 μg/ml) to 4900 μl of dilution buffer.Add 50 μl of the newly prepared detection antibody solution to each well. Cover the plate with parafilm or foil and and incubate for 1hr at 37oC.
?. Wash 4X with 1X washing buffer.
?. 1. Fill a plastic squeeze wash bottle with 1X washing buffer.2. Remove the foil from the plate and shake the detection antibody out of the plate by inverting the plate and shaking/flicking it over a wash basin.3. Fill each well of the plate with washing buffer using the squeeze wash bottle.4. Remove the wash buffer by inverting the plate and shaking/flicking it over a wash basin.5. Repeat this washing process another 3 times.6. After the final wash, try to remove as much of the washing buffer from the wells as possible by shaking/flicking the plate, and then bang the plate (upside down) on a clean towel/thickly layered tissue paper until you have managed to remove practically all fluid from the wells.
?. Add the streptavadin alkaline phosphatase.
?. Prepare 6000 μl of 1:1000 streptavadin alkaline phosphatase by adding 6 μl of 1 mg/ml streptavadin alkaline phosphatase to 5994 μl dilution buffer.Add 50 μl of the 1:1000 streptavadin alkaline phophatase solution to each well. Incubate at 1 hr at 37 oC.
?. Wash 4X with 1X washing buffer.
?. 1. Fill a plastic squeeze wash bottle with 1X washing buffer.2. Remove the foil from the plate and shake the streptavadin alkaline phophatase solution out of the plate by inverting the plate and shaking/flicking it over a wash basin.3. Fill each well of the plate with washing buffer using the squeeze wash bottle.4. Remove the wash buffer by inverting the plate and shaking/flicking it over a wash basin.5. Repeat this washing process another 3 times.6. After the final wash, try to remove as much of the washing buffer from the wells as possible by shaking/flicking the plate, and then bang the plate (upside down) on a clean towel/thickly layered tissue paper until you have managed to remove practically all fluid from the wells.
?. Add phosphatase substrate.
?. Prepare 6000 μl of 1 mg/ml phosphatase substrate by adding 0.006 g of phosphatase substrate to 6 ml substrate buffer.Add 50 μl of the 1 mg/ml phophatase substrate solution to each well. Cover plate and allow colour to develop for 30-45 min.
?. Streptavadin alkaline phosphatase and phosphatase substrateBD Streptavidin-alkaline phosphatase (SAv-AKP) was obtained from Ascendis Medical (#BD/554065)Phosphatase substrate was obtained from Sigma-Aldrich (#P4744-1G)
?. Read absorbance on a 96-well plate spectrophotometer.
?. Absorbance should be measured 405 nm with reference wavelength of 492 nm.
?. Plot a 4-parameter or 5-parameter standard curve and estimate cytokine concentration in your experimental samples.
?. First, subtract the absorbance of the blanks from all samples (the software on most modern 96 well plate spectrophotometers is able to do this for you).Next, plot a 4 parameter or 5 parameter curve using your duplicate recombinant cytokine standards (the software on most modern 96 well plate spectrophotometers is also able to do this for you).Once you have the standard curve you can utilise it's formula to estimate the cytokine concentrations of your unknown samples (the software on most modern 96 well spectrophotometers is also able to do this for you).
?. Prepare the solutions you will require (these volumes will be sufficient for about 25 x 96 well plates).
?. 1.2 L Phosphate buffered saline (PBS) pH 7.4 (20X)Add the following reagents in about 500 mL diH2O IN ORDER:34.56 g of Na2HPO4192 g of NaCl5.76 g of KH2PO44.8 g of KClMake up to 1.2 L with diH2O and store at room temperature.
?. 1.5 L Phosphate buffered saline (pH 7.4) (1X)Dilute 75 ml of 20X PBS with 1425 ml diH2O
?. 1 L Washing Buffer (20x)Add the following to 990 ml 20X PBS:10ml Tween 202 g NaN3**If horse radish peroxidase is utilised instead of alkaline phosphatase, NaN3 must be left out of solutions.
?. 
?. 150 ml Substrate BufferAdd the following to 100 ml diH2O0,03 g NaN314.6 ml di-ethanolamine 0.12 g MgCl2.6H2OAdjust pH to 9.8 with 37 % HCLMake up to 150 ml with diH2O and store at 4 °C
?. 500 ml Dilution BufferAdd the following to 300 ml 1X PBS:5 g BSA0.1 g NaN3Make up to 500 ml with 1X PBS and store at 4 °C
?. 500 ml Blocking BufferAdd the following to 300 ml 1X PBS:20g BSA0,1g NaN3Make up to 500 ml with 1X PBS and store at 4 °C
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?. Streptavadin alkaline phosphatase and phosphatase substrate:BD Streptavidin-alkaline phosphatase (SAv-AKP) was obtained from Ascendis Medical (#BD/554065).Phosphatase substrate was obtained from Sigma-Aldrich (#P4744-1G).
?. Protein-binding 96 well plates:Nunc Maxisorp 96 well Immuno Plates (available from Thermo Fisher).
?. Antibodies & recombinant cytokines:We utilise antibodies from R&D systems (a biotechne brand) - in South Africa, Whitehead Scientific supplies these antibodies.IL-10Capture antibody: Mouse IL‑10 Antibody (#MAB417).Detection antibody: Mouse IL-10 Biotinylated Antibody (#BAF417).Recombinant cytokine: Recombinant Mouse IL-10 (#417-ML).IL-6Capture antibody: Mouse IL‑6 Antibody (#MAB406).Detection antibody: Mouse IL-6 Biotinylated Antibody (#BAF406).Recombinant cytokine: Recombinant Mouse IL-6 (#406-ML).IL-1βCapture antibody: Mouse IL-1β/IL-1F2 Antibody (#MAB401).Detection antibody: Mouse IL-1β/IL-1F2 Biotinylated Antibody (#BAF401).Recombinant cytokine: Recombinant Mouse IL-1β/IL-1F2 (#401-ML).TNF-αCapture antibody: Human/Mouse TNF-α Antibody (#AF-410-NA).Detection antibody: Mouse TNF-α Biotinylated Antibody (#BAF410).Recombinant cytokine: Recombinant Mouse TNF-α aa 80-235 (#410-ML).
?. Prepare the solutions you will require (these volumes will be sufficient for about 25 x 96 well plates).
?. 1.2 L Phosphate buffered saline (PBS) pH 7.4 (20X)Add the following reagents in 500 mL diH2O IN ORDER:34.56 g of Na2HPO4192 g of NaCl5.76 g of KH2PO44.8 g of KClMake up to 1.2 L with diH2O and store at room temperature.
?. 1.5 L Phosphate buffered saline (pH 7.4) (1X)Dilute 75 ml of 20X PBS with 1425 ml diH2O.
?. 1 L Washing Buffer (20x)Add the following to 990 ml 20X PBS:10ml Tween 202 g NaN3**If horse radish peroxidase is utilised instead of alkaline phosphatase, NaN3 must be omitted fromsolutions.
?. 500 ml Blocking BufferAdd the following to 300 ml 1X PBS:20g BSA0,1g NaN3Make up to 500 ml with 1X PBS and store at 4 °C.
?. 500 ml Dilution BufferAdd the following to 300 ml 1X PBS:5 g BSA0.1 g NaN3Make up to 500 ml with 1X PBS and store at 4 °C.
?. 150 ml Substrate BufferAdd the following to 100 ml diH2O:0,03 g NaN314.6 ml di-ethanolamine0.12 g MgCl2.6H2OAdjust pH to 9.8 with 37 % HCL.Make up to 150 ml with diH2O and store at 4 °C.
?. Prepare stock solutions of antibodies and recombinant cytokines.
?. Coating antibodies:1. Reconstitute 100 μg of the primary antibody in 1000 μl autoclaved 1X PBS (100 μg/ml).2. Label 20 x 500 μl microcentrifuge tubes with name of the cytokine, 1st antibody, concentration andaliquot volume (eg. TNF-α, 1st AB, 100 μg/ml, 50 μl).3. Pipette 50 μl of the reconstituted antibody solution into each microcentrifuge tube.4. Place in labelled freezer box and store aliquots at -80 °C.
?. Detection (biotinylated) antibodies:First stock:1. Reconstitute 50 μg of the secondary/biotinylated antibody in 500 μl of 1X PBS. Label 10 x 500 μlmicrocentrifuge tubes with name of the cytokine, 2nd antibody, concentration and aliquot volume(eg. IL-6, 2nd AB, 100 μg/ml, 50 μl).2. Pipette 50 μl of the reconstituted antibody solution into each microcentrifuge tube.Second stock:1. Add 1 x 50 μl aliquot of stock 1 to 450 μl 1X PBS.2. Label 10 x 500 μl microcentrifuge tubes with name of the cytokine, 2nd antibody, concentration andaliquot volume (eg. IL-6, 2nd AB, 10 μg/ml, 50 μl).3. Pipette 50 μl of the reconstituted antibody solution into each microcentrifuge tube.4. Place in labelled freezer box and store aliquots at -80 °C.
?. Recombinants:1. First stock: Prepare a 10 μg/ml solution by adding the appropriate volume of dilution buffer tothe antibody (the volume of dilution buffer required will vary depending on the amount of cytokine purchased). Divide into 100 μl aliquots.2. Second stock: 500 ng/ml, 20 x 100 μl aliquots (add 1900 μl dilution buffer to stock 1 aliquot).3. Third stock: 100 ng/ml, 5 x 100 μl aliquots (add 400 μl dilution buffer to stock 2 aliquot).4. Final stock: 10 ng/ml, 10 x 100 μl aliquots (add 400 μl culture media or dilution buffer to stock 3aliquot).
?. Coat the 96-well plate with coating antibody.
?. For IL-10, IL-6 and IL-1β:Prepare 5100 μl of approx 3 μg/ml coating antibody by adding 3 x 50 μl aliquots of the coatingantibody stock solution (100 μg/ml) to 4950 μl of 1X PBS.For TNFα:Prepare 5100 μl of approx 1 μg/ml coating antibody by adding 1 x 50 μl aliquots of the coatingantibody stock solution (100 μg/ml) to 5050 μl of 1X PBS.For all:Add 50 μl of the newly prepared coating antibody solution to each well of a Nunc Maxisorp 96 wellImmuno Plates. Cover the plate with parafilm or foil and leave overnight at 4 °C.
?. Wash 4 times with 1X washing buffer.
?. 1. Prepare 1L of 1X washing buffer by adding 50 ml of 20X washing buffer to 950 ml of diH2O.2. Fill a plastic squeeze wash bottle with 1X washing buffer.3. Remove the foil from the plate and shake the coating antibody out of the plate by inverting the plateand shaking/flicking it over a wash basin.4. Fill each well of the plate with washing buffer using the squeeze wash bottle.5. Remove the wash buffer by inverting the plate and shaking/flicking it over a wash basin.6. Repeat this washing process another 3 times.7. After the final wash, try to remove as much of the washing buffer from the wells as possible byshaking/flicking the plate, and then hit the plate (upside down) on a clean towel/thickly layeredtissue paper until you have managed to remove practically all fluid from the wells.
?. Add blocking buffer to all wells to prevent non-specific antibody binding.
?. Add 200 μl of blocking buffer to each well.Cover with parafilm/foil and leave overnight at 4 °C (in the fridge)/or incubate at 37 °C for 2 hr. (Platesmay be blocked and left at 4 °C for up to 1 week).
?. Wash 4 times with 1X washing buffer.
?. 1. Prepare 1L of 1X washing buffer by adding 50 ml of 20X washing buffer to 950 ml of diH2O.2. Fill a plastic squeeze wash bottle with 1X washing buffer.3. Remove the foil from the plate and shake the coating antibody out of the plate by inverting the plateand shaking/flicking it over a wash basin.4. Fill each well of the plate with washing buffer using the squeeze wash bottle.5. Remove the wash buffer by inverting the plate and shaking/flicking it over a wash basin.6. Repeat this washing process another 3 times.7. After the final wash, try to remove as much of the washing buffer from the wells as possible byshaking/flicking the plate, and then hit the plate (upside down) on a clean towel/thickly layeredtissue paper until you have managed to remove practically all fluid from the wells.
?. Prepare serial dilutions of recombinant cytokines to produce "known concentration" standard curves.
?. 1. Prepare 250 μl of 4 ng/ml recombinant cytokine by adding 150 μl dilution buffer/culture media to100 μl of 10 ng/ml recombinant cytokine stock.2. Pipette 50 μl of the 4 ng/ml recombinant cytokine into well A1, A2, B1 and B2.3. Add 50 μl of dilution buffer/culture media to wells A2 and B2.4. Add 50  μl of dilution buffer to wells A3-A12 and B3-B12.5. Perform two-fold serial dilution from A2 to A12. Using a pipette set to 50 μl, mix well A2 by drawing up and then expelling 50 μl of the solution in the well about 3 to 5 times.6. Draw up 50 μl of the solution in well A2 and add it to well A3. Again, mix the solution as described above, using apipette set to 50 μl.7. Now add 50 μl of the solution in well A3 to well A4.8. Repeat this diluting/mixing process through to well A11. Dispose of the last 50 μl that you draw up ofwell A11. This will create a standard curve, through serial dilution, of 4 ng/ml to 3.9 pg/ml, with adilution buffer/culture medium blank in well A12.9. Repeat this whole process in row B, to produce the standard curve in duplicate.
?. Add samples.
?. 50 μl of experimental samples should be added into wells reserved for samples.If you have not run samples from a specific experimental set-up before, it is wise to dilutesamples threefold over three wells (ie. in one well you will have the undiluted sample, in an adjacent wellyou will have a sample which you have diluted threefold, and in a well next to that you will have asample that you have diluted ninefold).Cover plate with parafilm/foil and incubate overnight at 4 °C or for 2hr at 37 °C.
?. Wash 4 times with 1X washing buffer.
?. 1. Prepare 1L of 1X washing buffer by adding 50 ml of 20X washing buffer to 950 ml of diH2O.2. Fill a plastic squeeze wash bottle with 1X washing buffer.3. Remove the foil from the plate and shake the coating antibody out of the plate by inverting the plateand shaking/flicking it over a wash basin.4. Fill each well of the plate with washing buffer using the squeeze wash bottle.5. Remove the wash buffer by inverting the plate and shaking/flicking it over a wash basin.6. Repeat this washing process another 3 times.7. After the final wash, try to remove as much of the washing buffer from the wells as possible byshaking/flicking the plate, and then hit the plate (upside down) on a clean towel/thickly layeredtissue paper until you have managed to remove practically all fluid from the wells.
?. Add the detection (biotinylated) antibody.
?. For IL-10, IL-6, IL-1β and TNF-α:Prepare 5100 μl of approx 0.3 μg/ml coating antibody by adding 3 x 50 μl aliquots of the coatingantibody stock solution (10 μg/ml) to 4950 μl of dilution buffer.Add 50 μl of the newly prepared detection antibody solution to each well. Cover the plate with parafilmor foil and and incubate for 1 hr at 37 °C.
?. Wash 4 times with 1X washing buffer.
?. 1. Prepare 1L of 1X washing buffer by adding 50 ml of 20X washing buffer to 950 ml of diH2O.2. Fill a plastic squeeze wash bottle with 1X washing buffer.3. Remove the foil from the plate and shake the coating antibody out of the plate by inverting the plateand shaking/flicking it over a wash basin.4. Fill each well of the plate with washing buffer using the squeeze wash bottle.5. Remove the wash buffer by inverting the plate and shaking/flicking it over a wash basin.6. Repeat this washing process another 3 times.7. After the final wash, try to remove as much of the washing buffer from the wells as possible byshaking/flicking the plate, and then hit the plate (upside down) on a clean towel/thickly layeredtissue paper until you have managed to remove practically all fluid from the wells.
?. Add the streptavadin alkaline phosphatase.
?. Prepare 6000 μl of 1:1000 streptavadin alkaline phosphatase by adding 6 μl of 1 mg/ml streptavadinalkaline phosphatase to 5994 μl dilution buffer.Add 50 μl of the 1:1000 streptavadin alkaline phophatase solution to each well. Incubate at 1 hr at 37 °C.
?. Wash 4 times with 1X washing buffer.
?. 1. Prepare 1L of 1X washing buffer by adding 50 ml of 20X washing buffer to 950 ml of diH2O.2. Fill a plastic squeeze wash bottle with 1X washing buffer.3. Remove the foil from the plate and shake the coating antibody out of the plate by inverting the plateand shaking/flicking it over a wash basin.4. Fill each well of the plate with washing buffer using the squeeze wash bottle.5. Remove the wash buffer by inverting the plate and shaking/flicking it over a wash basin.6. Repeat this washing process another 3 times.7. After the final wash, try to remove as much of the washing buffer from the wells as possible byshaking/flicking the plate, and then hit the plate (upside down) on a clean towel/thickly layeredtissue paper until you have managed to remove practically all fluid from the wells.
?. Add phosphatase substrate.
?. Prepare 6000 μl of 1 mg/ml phosphatase substrate by adding 0.006 g of phosphatase substrate to 6ml substrate buffer.Add 50 μl of the 1 mg/ml phophatase substrate solution to each well. Cover plate and allow colour todevelop for 30-45 min.
?. Read absorbance on a 96-well plate spectrophotometer.
?. Absorbance should be measured 405 nm with reference wavelength of 492 nm.
?. Plot a 4-parameter or 5-parameter standard curve and estimate cytokine concentration in your experimental samples.
?. First, subtract the absorbance of the blanks from all samples (the software on most modern 96 wellplate spectrophotometers is able to do this for you).Next, plot a 4 parameter or 5 parameter curve using your duplicate recombinant cytokine standards(the software on most modern 96 well plate spectrophotometers is also able to do this for you).Once you have the standard curve you can utilise it' formula to estimate the cytokine concentrationsof your unknown samples (the software on most modern 96 well spectrophotometers is also able to dothis for you).