BD Biosciences 1-2900 Arg ent ia Road M iss iss aug a, ON • L5N 7X9 • CANADA Local Tel: (905) 542-8028 • Toll-Free Tel: (888) 259-0187 Local Fax: (905) 542-9391 • Toll-Free Fax: (888) 229-9918 Cytokine E L IS A Introduction Due to the amplifying potential of enzyme labels, immunoassays which utilize enzyme-c onj ugat ed ant ibo dies have bec ome i ncreas ing ly po pul ar beca us e of t heir high specificity and s ens itivit y. 1 In 1971, Engvall and Perlm ann 2 coined the term “ enzyme-linked imm unosorbent ass ay ” w hich is perhaps bett er know n by t he acro nym, “ E L IS A” , to d es c ri be an enzyme-bas ed im munoassay met ho d w hi c h is useful for measuring antigen concentrations. Cytokine sandwich ELISA are sensitive enzyme immunoassays that can specifically detec t and quant it ate t he c oncentrat ion o f s olubl e c ytoki ne and c hemokine proteins. The basic c yt ok ine s andw ich ELISA method makes use of highly-purified anti-c yto kine antibodies (c apt ure antibodies ) wh ich are noncovalentl y ads orb ed (“ c oated” – primarily as a result of hydrophobic interactions ) ont o pl as tic microw ell plat es. Af t er plat e w ashin gs , t he immobilized ant ibodies serve t o spec if ica lly capture s olu ble cytok ine pro t eins present in s amples w hich w ere applied to t he plat e. Af t er was hing away unbound material, the c aptur ed c ytoki ne prot eins are det ec t ed by biot in-c onjug ated anti -c ytoki ne antib odies (det ec t ion antib odies ) f ollow ed by an enzyme-labeled avidin or s t reptavidin s t age. Follow ing t he additi on of a c hromo genic substrate-cont aining s olution, t he lev el of c olour ed product generated by t he bound, enzy me- linked det ec t ion reagents can be conveniently measured spectrophotometrically using an ELISA- plat e rea der at an appro pri ate opt ica l dens it y ( OD) . Data s t orage and reanalys is are grea t ly s impl ified when t he plat e reader is connected t o a comput er. By inc luding serial dilutions of a standard c ytoki ne protein solution of known concentration, the sandwich ELISA supports the development of standard curves. S t andard curves (aka“ c alibr ati on curves ” ) are generally plo t t ed as t he s t andard c ytoki ne prot ein concentration (typic ally ng o r p g of c ytoki ne/ ml) vers us t he c orr espon din g mean OD v alue of r eplicat es. T he c once ntrat ion s of t he put ati ve c yto kine- c ont ainin g s amples can be int erpol ated from the standard curve. This process is made eas ier by u s in g an E L IS A compu t er s of t ware program. 3 Generally, it is useful to perform a dilution series of the unknown samples to be assured that t he OD w ill f all wit hin t he linear port ion of t he s t andard curve. Depending on the nature of the ELISA reagents used, investigators may choose to apply diff erent c urve fit analys is t o their data, includin g eit her linear-log, log-log, or f our-parameter trans f ormat ions . 1,4,5
