how much Ag must to coat in every well for ELISA and I want to now which kind of controls can i have for each ElISA experiment?
Which Elisa? It depends..
the controls should be coating alone, coating + sup, coating+ secondary ab
If you are not working with a kit, you must do a preliminary assay with different antigen concentration. Look at the literature for a start
the solid phase in an ELISA plate has a capacity to bind about 1-2 µg/well. Therefor a concentration of 1 - 10 µg/mL is sufficient. Higher concentrations causes double layer which can be removed during the following washing steps. The optimum can be found by checkerboard titration (normally at 2 µg/mL).
Put your antigen (or antibody) in a 0.1 mol/L carbonate buffer, pH 9.6, make sure that there is no detergent present (this will block the binding sites [hydrophobic polystyrene rings on the plastic surface and hydrophobic amino acids in your proteins] effectively. Incubate overnight at 4 °C, remove next morning the liquid by beating the plates upside down on a fleece. Wrap the coated plates in aluminium foil and store them frozen until usage.
Before using, wash the plates with the washing buffer 0.01 mol/L Phospahse + 0,1% Tween 20.
Yes this method of coating antibody is best...leave for overnight incubation at 4 degree.you will get good results
I agree with Stephan too. Generally 1 - 10 ug/ml is good enough and a checkerboard method works best in defining the optimal concentration. One way to understand whether the concentration decided by you is optimal or not is to do an antigen binding experiment. Here you coat two plates overnight at 4oC and then next day, yoeu check your standard curve response first on any one of the plates. After incubation, do not discard the well contents of standard curve. Transfer them into Plate 2. Continue both plates as per designed ELISA and take OD measurements. Consider the values from Plate 1 as Master values and those from Plate 2 as Test values. Quantify the percentage binding by calculating the difference in ODs between Plates 1 & 2. If this binding is > 90%, then you know your coating concentration is good.
For coating Ag you must perform checker board titration and you have to keep controls like air blank, Blank, Conjugate control, Substrate control, Positive control and negative control.
I would also recommend running your samples and/or the controls in duplicate if room permits. We almost always run the controls and standard in duplicate here where I work, but we only run the samples in duplicate if there is room on the plate. That way you have a better chance of insuring yourself of false controls or random bad wells.
To know the quantity of Ag to coat in ELISA well you have to perform checker board titration. First let me know whether you want to do direct ELISA or indirect ELISA? Accordingly I can give protocol. Regarding controls - for any ELISA you have to keep Air blank, Blank, Conjugate control, Substrate control, Postive control, and Negative control.
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