Love:elisa
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Eli Papa 18:09, 6 February 2008 (CST)
Test Bleed
- Anesthesize animal
- Incision on the tail vein
- collect necessary amount
Serum Prep
- Let collected blood clot for 30-60min at 37degC, RT for 15min
- (optional) Separate clot from walls with a pasteur pipette
- (optional) allow clot to contract o/n at 4degC
- remove serum from clot by spinning (eg. 6000rpm, 10000g, 10min, 4degC)
- Use or store at -20degC (can use .02% Na azide if long storage)
Buffers
- Washing buffer: PBST
- 500ml PBS
- 0.5ml tween for .1% (classic), 0.25ml tween for .05% (good for elisa,what I use)
- Dilution buffer: PBS/.05%tween/.5% fetal calf serum (cheapest serum)
- 500ml PBS
- 0.25ml tween
- 2.5ml FCS
- Blocking buffer: PBS/.5% FCS
- 500ml PBS
- 2.5ml FCS
Antibody capture assay - indirect elisa
- Use ELISA plates (absorbing bottom)
- add 50ul of antigen to each well ( at least 20ugrams/well for PVC, antigen at 10ugrams/ml is plenty with maxisorp)
- dilute using carbonate buffer to maximize absorption or PBS.
- leave plate o/n at 4degC (cover with parafilm) [ or 2hrs at RT, 30min 37degC ]
- discard solution (flick over waste)
- wash plates 2x w/ blocking buffer (! don't use detergent or other protein as it could desorb some antigen from surface)
- incubate with 100ul of blocking buffer for 1hr at 37degC [ or 2hrs at RT, o/n at 4degC ]
- wash 3x with 100ul of washing buffer
- add 50ul of serum/supernatant (do serial dilutions 1/50 to 1/10000 is a valid range)
- leave 1hr at 4degC
- wash 3x with 100ul of washing buffer
- add anti-mouse HRP secondary Ab (we use Pierce rabbit Ab with 1:1000 dilution in dilution buffer)
- wash 3x with 100ul of washing buffer
- add 50ul of TMB (or other HRP reagent)
- leave for ~1hr until it turns blue
- could read directly here or..
- block with 50ul of .5M H2SO4 and read at 50ul