I need to measure IL-6 and TNF-α in human plasma samples. I need high sensitivity kits however ELISA kits are too expensive. Does anyone know if there is a way to increase sensitivity in the plates that are not pre-coated?
Sensitivity of the plate will be increased if you pre-coat them (the plates with Capture Antibody) for more than 24 hours (best would be 48 hours) and during the work to increase the number of washings. Self made kits would it mean that you can prepare the Assay Diluent, Coating Buffer, and the Wash Buffer by yourself, but not any antibodies or enyzmes! So you could order Standard kits (antibodies, enyzme and standards) which cost less then other kits.
Use a fluorescent HRP substrate. It can work only if the kit's antibodies are good enough to support it. False positive signal can become the limiting factor. You also need a good fluorescence plate reader of course. I've seen QuantaRed make a 20x difference, and I believe that might have been with that very IL6 kit.
We have tried to use the "ready set go" kits with little success for the sensitivies that most human research studies require. We now tend to use DuoSets where you make much of it yourself (some aspects described above).
https://www.rndsystems.com/products/human-tnf-alpha-duoset-elisa_dy210
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