ELISAIndirect ELISA Capture ELISA Troubleshooting Guide (26 Kb PDF) References Indirect ELISAReagents and Equipment ProcedureReagents and Equipment1.Phosphate buffered saline (PBS) tablet: 10 mM phosphate buffer, pH 7.4, 150 mM NaCl (Product No. P4417) and 0.1% sodium azide (Product No. S2002). 2.Carbonate-Bicarbonate buffer capsule, pH 9.6 (Product No. C3041). 3.Washing buffer (PBS-T): 10 mM phosphate buffer pH 7.4, 150 mM NaCl, 0.05% Tween 20 (Product No. P3563). 4.Monoclonal primary antibody. 5.Antibody controls: species- and isotype-matched, non-specific immunoglobulin (e.g., mouse myeloma proteins as a control for a mouse monoclonal primary antibody) 6.Alkaline phosphatase-conjugated secondary antibody or peroxidase-conjugated secondary antibody 7.Substrate for alkaline phosphatase conjugated secondary antibody (such as SIGMAFAST pNPP tablets, Product No. N1891) or substrate for peroxidase conjugated secondary antibody (such as SIGMAFAST OPD tablets, Product No. P9187). 8.Stopping reagent for alkaline phosphatase: 3 M NaOH (optional) or stopping reagent for peroxidase: 3 M HCl or 3 M H2SO4 (optional). 9.Microtiter plates 10. Microtiter plate reader equipped with a 405 nm or 450/492 nm filter (for pNPP or OPD, respectively). Procedure Antigen Coating Primary Antibody Reaction Application of Secondary Antibody Substrate Preparation Development Antigen Coating1.Prepare an antigen solution at the appropriate concentration in carbonate-bicarbonate buffer or PBS. 2.Pipette 0.2 ml of the above solution to each well of the microtiter plate. 3.Incubate at 37 C for 30 min., or incubate (covered) overnight at 4 C. 4.Remove the coating solution. Wash three times with PBS-T.Note: If problems with non-specific binding occur, an additional blocking step (30 min. 5% BSA-PBS) may be required. For further information see: Vogt, R.F., et al., J. Immunol. Meth., 101, 43 (1987). Primary Antibody Reaction1.Dilute the monoclonal primary antibody in PBS-T. The optimal dilution should be determined using a titration assay. 2.Add 0.2 ml of the diluted monoclonal antibody to each well. The negative control should be species- and isotype-matched, non-specific immunoglobulin diluted in PBS-T. 3.Incubate at room temperature for 2 hours. 4.Wash as in step 4 of Antigen Coating. Application of Secondary Antibody1.Dilute the enzyme-conjugated secondary antibody in PBS-T. Add 0.2 ml of this solution to each well. The optimal dilution should be determined using a titration assay. 2.Incubate at room temperature for 2 hours. 3.Wash as in step 4 of Antigen Coating. Substrate Preparation1.During the last incubation and immediately before use, prepare the enzyme substrate or bring the premade liquid substrate to room temperature. Development1.Add 0.2 ml of the freshly prepared substrate to each well. 2.Color should develop in positive wells after 30 minutes (yellow or orange, for pNPP or OPD, respectively). 3.Absorbance may be read directly in a microplate reader (at 405 nm or 450 nm, for pNPP or OPD, respectively) or the reaction may be stopped with 50 l per well of the appropriate stopping reagent and absorbance read later (at 405 nm or 492 nm, for pNPP or OPD, respectively). Capture ELISAReagents and Equipment Procedure Capture ELISA (also known as “sandwich“ ELISA) is a sensitive assay to quantitate picogram to microgram quantities of substances (such as hormones, cell signaling chemicals, infectious disease antigens and cytokines.). This type of ELISA design may be needed rather than a direct or indirect ELISA for several reasons. The substance to be analyzed may be too dilute to bind to the polystyrene microtiter plate (such as a protein in a cell culture supernatant) or does not bind well to plastics (such as a small organic molecule). It may be mixed with too many other substances to bind well to the plate, two Sigma kits that describe or utilize capture ELISA are the TNF-alpha kit and the Mouse Isotyping Reagents kit (Product No. ISO2). Reagents and Equipment1.Phosphate buffered saline (PBS): 10 mM phosphate buffer, pH 7.4, 150 mM NaCl tablet (Product No. P4417) and 0.1% sodium azide (Product No. S2002) 2.Carbonate-Bicarbonate buffer capsule, pH 9.6 (Product No. C3041) 3.Washing buffer (PBS-T): 10 mM phosphate buffer pH 7.4, 150 mM NaCl, 0.05% Tween 20 (Product No. P3563) 4.Capture or coating antibody 5.Control antigen to be used for standard curve 6.Labeled detection antibody 7.Substrate. Typically the most sensitive substrate is used (such as TMB or OPD if using peroxidase as the enzyme label). 8.Stop solution (optional) 9.Microtiter plates 10. Microtiter plate reader ProcedureCoating of capture antibody Application of control and samples Application of detection antibodyNote: This is provided as a general protocol. Optimal dilutions for the capture antibody, samples, controls, and detecting antibodies as well as incubation times will need to be determined empirically and may require extensive titration. Ideally, one would use