Love:elisa

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Eli Papa 18:09, 6 February 2008 (CST)

Test Bleed

  1. Anesthesize animal
  2. Incision on the tail vein
  3. collect necessary amount

Serum Prep

  1. Let collected blood clot for 30-60min at 37degC, RT for 15min
  2. (optional) Separate clot from walls with a pasteur pipette
  3. (optional) allow clot to contract o/n at 4degC
  4. remove serum from clot by spinning (eg. 6000rpm, 10000g, 10min, 4degC)
  5. 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)
  1. 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.
  2. leave plate o/n at 4degC (cover with parafilm) [ or 2hrs at RT, 30min 37degC ]
  3. discard solution (flick over waste)
  4. wash plates 2x w/ blocking buffer (! don't use detergent or other protein as it could desorb some antigen from surface)
  5. incubate with 100ul of blocking buffer for 1hr at 37degC [ or 2hrs at RT, o/n at 4degC ]
  6. wash 3x with 100ul of washing buffer
  7. add 50ul of serum/supernatant (do serial dilutions 1/50 to 1/10000 is a valid range)
  8. leave 1hr at 4degC
  9. wash 3x with 100ul of washing buffer
  10. add anti-mouse HRP secondary Ab (we use Pierce rabbit Ab with 1:1000 dilution in dilution buffer)
  11. wash 3x with 100ul of washing buffer
  12. add 50ul of TMB (or other HRP reagent)
  13. leave for ~1hr until it turns blue
    • could read directly here or..
  14. block with 50ul of .5M H2SO4 and read at 50ul