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I would like to find an antibody for coating on ELISA plate. The binding target is adenovirus so I think ab24155 may fit to my requirement but I still need to confirm it. Is there antibody only without other protein? Can it be used for coating plate? Do you have protocal for coating on ELISA plate (is it the same as your ELISA protocal but exchange antigen to antibody)?
Asked on Oct 14 2005
Thank you for your enquiry. To answer your question. Yes, this antibody can be used by ELISA. It has been purified using Protein G and therefore should contain just Goat IgG. I would recommend performing the sandwich ELISA protocol as follows, coating the plate as described. SANDWICH ELISA PROTOCOL Buffers and Reagents: For accurate quantitative results, always compare signal of unknown samples against those of a standard curve. Standards (duplicates or triplicates) and blank must be run with each plate to ensure accuracy. Incubation with Primary and Secondary antibody 1. Add 100 µl of diluted primary antibody to each well (10 µg/mL in PBS). 2. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature. 3. Wash the plate four times with PBS. 3. Add 100µl of appropriately diluted samples to relevant wells. Ensure that appropriately diluted standards are included. Samples or standards should preferably be run in triplicate. Incubate 90 minutes at 37oC. (Dilute samples and standards in PBS). Wash x4 in wash buffer. 4. Add 100µl of conjugated secondary antibody, diluted at the optimal concentration (according to the manufacturer) in blocking buffer immediately before use. 5. Cover the plate with an adhesive plastic and incubate for 1-2 h at room temperature. 6. Wash the plate four times with PBS. Detection Although many different types of enzymes have been used for detection, horse radish peroxidase (HRP) and alkaline phosphatase (ALP) are the two widely used enzymes employed in ELISA assay. It is important to consider the fact that some biological materials have high levels of endogenous enzyme activity (such as high ALP in alveolar cells, high peroxidase in red blood cells) and this may result in non-specific signal. If necessary, perform an additional blocking treatment with Levamisol (for ALP) or with 0.3 % solution of H2O2 in methanol (for peroxidase). Solutions ? Blocking solutions: commonly used blocking agents are BSA, non-fat dry milk, casein, gelatin. PBS containing 1 % Bovine Serum Albumin (BSA) ? Wash solution: usually PBS or Tris-buffered saline (pH 7.4) with detergent such as 0.05 % (v/v) Tween20 (TBST) ? Antibody solution: primary and secondary antibody should be diluted in 1x blocking solution to prevent non-specific binding. I hope this information helps. Please do not hesitate to contact me should you require further assistance.
Answered on Oct 17 2005