Medical Microbiology

70 2 BasicPrinciplesofImmunology

ProductionofMonoclonalAntibodies

Fig.2. 10 Monoclonalantibodiesareproducedwiththehelpofcelllinesobtained

fromthefusionofaBlymphocytetoanimmortalmyelomacell.Inthefirstin-

stance,miceareimmunizedagainstanantigen.Theythenreceiveasecond,in-

travenous,doseofantigentwotofourdaysbeforecellfusion.Thenspleencellsare

removedandfusedtothemyelomacelllineusingpolyethyleneglycol(PEG).Those

spleencellsthatfailtofusetoamyelomacelldiewithinonedayofculture.Next,

thefusedcellsaresubjectedtoselectionusingHATmedium(hypoxanthine,ami-

nopterin,thymidine).Aminopterinblocksspecificmetabolicprocesses,butwith

thehelpoftheintermediarymetabolites(hypoxanthineandthymidine)spleen

cellsareabletocompletetheseprocessesusingauxiliarypathways.Themyeloma

cells,ontheotherhand,haveametabolicdefectwhichpreventsthemfromutiliz-

ingsuchalternativepathwaysandresultinginthedeathofthosecellsculturedin

HATmedium.However,onceaspleencellhasfusedwithamyelomacell,thefused

spleen-myelomaproduct(hybridoma)isHAT-resistant.Inthiswayonlythesuc-

cessfullyfusedcellswillbeabletosurviveseveraldaysofcultureonHATmedium.

Afterthistime,thecellcultureisdilutedsuchthatthereis,ideally,onlyonehy-

bridomawithineachwell.Individualwellsarethentestedforthepresenceofthe

desiredantibody.Iftheresultispositive,thehybridomacellsaresubclonedseveral

timestoensureclonality;withthespecificityoftheproducedantibodybeing

checkedfollowingeachroundtosubcloning.Productionofpurelyhumanmono-

clonalantibodiesiscarriedoutusingmicewhoseIggeneshavebeencompletely

replacedbyhumanIggenes.

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Kayser, Medical Microbiology © 2005 Thieme