Love:elisa

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)
 * 2) * dilute using carbonate buffer to maximize absorption or PBS.
 * 3) leave plate o/n at 4degC (cover with parafilm) [ or 2hrs at RT, 30min 37degC ]
 * 4) discard solution (flick over waste)
 * 5) wash plates 2x w/ blocking buffer (! don't use detergent or other protein as it could desorb some antigen from surface)
 * 6) incubate with 100ul of blocking buffer for 1hr at 37degC [ or 2hrs at RT, o/n at 4degC ]
 * 7) wash 3x with 100ul of washing buffer
 * 8) add 50ul of serum/supernatant (do serial dilutions 1/50 to 1/10000 is a valid range)
 * 9) leave 1hr at 4degC
 * 10) wash 3x with 100ul of washing buffer
 * 11) add anti-mouse HRP secondary Ab (we use Pierce rabbit Ab with 1:1000 dilution in dilution buffer)
 * 12) wash 3x with 100ul of washing buffer
 * 13) add 50ul of TMB (or other HRP reagent)
 * 14) leave for ~1hr until it turns blue
 * 15) * could read directly here or..
 * 16) block with 50ul of .5M H2SO4 and read at 50ul