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| [ 文章来源：http://iacf.bsd.uchicago.edu | 文章作者：
| 发布时间：2006-12-15|
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There are many different types of ELISAs, which can detect the presence of protein in serum or supernatent. One of the most common types of ELISA is the so-called "sandwich ELISA." It is termed this because the antibody that you are detecting gets sandwiched between an antigen and a chromogenically-conjugated antibody. Below you will find a basic protocol for this assay.
- To coat the plate with the appropriate antigen, fill the microwells of a Nunc Maxi-Sorp Immuno Plate with 100uL of the diluted antigen.
- Incubate at 4C overnight
- Wash the unbound antigen off the plate by flicking the contents of the plate into the sink, fill the wells with DI water, flick again, repeat 2X with PBS-Triton.
- Block non-specific binding by adding 200uL of 1%BSA/PBS
- Incubate for 30-60minutes at Room Temperature (RT)
- Wash plate as above
- Add 100uL of (diluted) samples to appropriate wells. Be sure to include positive and negative controls, and, if necessary, a standard curve.
- Incubate for 1hour at RT
- Repeat washing step
- Prepare appropriate dilution of the second step antibody conjugated either with Alkaline Phosphatase or Horseradish Peroxidase. (antibodies should be titrated for optimal dilution)
- Add 100uL of second step antibody to wells and incubate for 1hr
- Repeat washing step
- Prepare substrate solution*
- Add 100uL of substrate to well and incubate at RT for 60min
- add stopping solution if appropriate
- Read plates on an ELISA plate reader*
*Common Substrates and the appropriate plate reader setting
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Cellular ELISA Protocol