Enzyme Linked Immunosorbent Assay (ELISA).ppt

Enzyme Linked Immunosorbent Assay (ELISA) ELISA Enzyme Linked Immunosorbent Assay (ELISA) Term Was Coined By Engvall and Pearlmann in 1971 Different Types Sandwich Indirect Competitive Similar To RIA, Except No Radiolabel Can Be Used To Detect Both Antibody and Antigen Very Sensitive, pg/mL Relies on Monoclonal Abs Sandwich ELISA 2 Antibodies Required Must Recognize Different Epitopes 1st Antibody Is Referred To As Capture Ab 2nd Antibody Detection Ab 2nd Antibody Is Biotinylated Enzymes Commonly Used: HRP (Horse Radish Peroxidase) And AKP (Alkaline Phosphatase) Substrate is TMB (Chromogen) ELISA Plate 96 well plate Made of plastic on which protein can be adsorbed (bind) easily Usually done overnight @ 4?C Special buffer used that will not denature Ab and maximize binding Blocking step ensures no empty spaces are left Blocking reagent is often 10% FBS Standard Curve Serial dilutions of the cytokine being measured Exact concentration is needed A plot of concentration (pg/mL or ng/mL) is plotted against OD (optical density) Sensitivity Of Elisa Typically the lowest cytokine concentration that can be detected above negative control 2-3 S.D Above Mean Background Signal Depending On Antibody Pair Used Sensitivity Varies Ex. 10 pg/mL General Protocol Dilute capture Ab @ 1-4 ?g/mL In Binding Solution Ex. Stock Solution Of Capture Ab: 0.5 mg/mL And Capture Ab Recommended Conc. 2 ?g/mL First Question To Ask Yourself ? How much volume would I use? Count 16 wells for S.C+ 3 wells for Negative Controls Your Samples (usually in triplicates) Add them up and multiply by 100 ?L (typical volume used per well) Let’s Say 4 mL Needed You will need 16 ?L of capture Ab Add capture Antibody, Seal plate (minimize evaporation) Incubate overnight at 4?C Binding Solution Pharmingen Recommended Reagent 0.1 M Na HPO4, adjust to pH 9.0 or to pH 6.0 with 0.1 M NaH2PO4 pH Is Very Important, If Wrong No Binding Some Antibodies Require pH 6.0 Ex. Antibodies for mIL-10, mMCP-1, mTNF, rGM