Hello, my question is the following:
I happen to have a murine serum against polyclonal OVA antigen and I have OVA conjugated 488 alexa fluor and I happen to generate an immune complex and then inoculate this in culture dendritic cells. So I would like to know what volume of serum I should take out of the stock to form the immune complex plus the OVA conjugated 488 alexa fluor and see if it is internalized or not in condition of viral infection. I have a 24 well plate with DCs, each well has about 1,000,000 dendritic cells.
PS: According to the literature the ideal concentration to form an immune complex of ova plus anti-ova IgG is 1 ug/ml OVA plus 25 ug/ml anti-OVA IgG.
If you intend to use the antiserum directly, you need to titrate it, in order to find out how much specific IgG you have.
If you have enough material, say at least 2 or better 5ml of antiserum, I'd recommend isolating the specific antibody by antigen affinity chromatography on immobilized OVA (e.g., on NHS activated sepharose). Then you can directly use the protein concentration for your calculations and have no possibly interfering contaminants.
As a last resort, you could isolate the already formed immunocomplexes by gel filtration (size exclusion chromatography) on Sephacryl S300 or a similar resin (e.g., Superdex Increase).
To determine the volume of serum to form the immune complex with OVA conjugated 488 Alexa Fluor, you need to consider the desired concentration of the immune complex and the concentration of the OVA conjugate in the serum stock. Here's a general procedure to help you calculate the volume:
It's important to note that this calculation assumes that the serum stock is not significantly affecting the final volume or concentration of the immune complex. If the serum volume is substantial compared to the final volume, you may need to adjust the calculation accordingly.
Additionally, ensure that you account for appropriate controls in your experimental setup, including control samples without the immune complex, controls with non-specific serum, and controls without viral infection, as these will help you evaluate the effects of the immune complex and viral infection accurately.
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