My Ssec Capstone Project D ep artm en t o f P h arm acy A ssignm ent Topic

D ep artm en t o f P h arm acy A ssignm ent Topic

D ep artm en t o f P h arm acy A ssignm ent Topic:Production ofM onoclonalA ntibody U sing H ybridom a Technology C ourse C ode:PH R M 407 C ourse Title:Pharm aceuticalB iotechnology Section:01 Subm itted to: N ajneen A hm ed SeniorLecturer D epartm entofpharm acy Subm itted by: M aksuda A kter ID :2016-1-70-010 Subm ission D ate:06.11.2018

Introduction: A specializedclusterofcellscalledB-lymphocytesgenerateproteinmoleculescalledantibodies orimmunoglobulin's.Theinvasiveforeignsubstancespeciallyantigensattackthedefense system ofourbody.Antibodiesarepartofdefensesystem ofourbodywhichprotectfrom antigens.Eachsubstancecontainauniquesubstancedeterminant(epitops).Themainreasonfor theantigenspecificityisthecomplimentarydeterminativeregions(CDRs)ofantibodies.W hen anantigencomesincontactwithbody'sdefensesystem,variousantibodiesaremadefrom B- lymphocyte.Theseform ofantigensaretermedaspolyclonalantibodywhichmightreactwith specificantigen.Therearemanydifficultiesforusingpolyclonalantibodiesfortherapeuticand diagnosticuse. M onoclonalantibodiesaresinglekindofantibodythatisspecificforaparticularepitope.Ithad beenadream ofscientisttoproducemonoclonalantibodyforvariousantigens.W ithinafew years,animalswereimmunizedagainstaspecificantigenandB-lymphocyteshadbeencollected andculturedinvitroforproducingM ABs.ThisapproachbecamefailedsinceculturingnomalB- lymphocytesweretroublesome. In1975,GeorgeKohlerandCesarM ilsteinachievedlargescalemanufacturingofM Abs.They fusedantibodyproducingB-lymphocyteswithmyelomacellstoproduceahybridomacellsin vitro. ImmortalizedB-lymphocyteswillmultiplyindefinitelyandproduceM ABs.Theexpansionand multiplyingpropertiesofmyelomacellsecreteantibodyofB lymphocyte.Theproductionof monoclonalantibodybyhybridomacellisreferredtoashybridomatechnology.Toprovide desiredantibodyinlargeamounthybridomacellsareused.1

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

M ethod:Figure:stepsinvolvedinproductionofmonoclonalantibodybyhybridomatechnology Adaptedfrom:Cdn.biologydiscussion.com.(2018).onlineAvailableat: http://cdn.biologydiscussion.com/wp-content/uploads/2015/09/clip_image004_thumb17.jpg Accessed4Nov.2018.

Following areseveralstepsinvolvingtheproductionofmonoclonalantibodies: ? Immunization ? Fusionofcell ? Selectionofhybridomacell ? Screening ? Cloningandpropagation ? Characterizationandstorage.1 Im m unization Thefirststepinhybridomatechnologyistoimmunizeananimalwithaspecificantigen.The injectiongivenatmultiplesitesformanytimes.ThistriggersthestimulationofB-lymphocytes whicharereapondingtotheantigen.A finaldoseofantigeninintravenouslyadmininstered beforekillingtheanimal.Theproductionofmaximum immune-stimulatedcellsforsynthesisof antibodieshavegrownthroughthisapproach.Intheserum oftheanimaltheconcentrationof specifiedantibodiesisassayedatfrequenttimeintervalthroughoutthecourseofimmunization. Theanimalissacrificedwhentheserum concentrationofandibodiesisoptimum.Bymechanical orenzymaticmethod,spleenoftheanimalisasepticallyremovedanddisruptedtoreleasethe cells.Bydensitygradientcentrifugation,thelymphocytesofthespleenareseparatedfrom the remainderofthecells.1 Fusion ofcell ThelymphocyteswerewashedandmixedwithHGPRT (Hypoxanthine-GuaninePhosphoribosyl Transferase)enzyme.Themixtureofcellswereexposedtopolyethyleneglycol(PEG).The exposureshouldbeforashortperiodbecauseofitstoxicproperty.ThenPEG shouldbe removedbywashing.Afterthatthecellsarekeptinafreshmedium.Thesecellsaremadefrom myelomacellsandB-lymphocytes.Thusthecellsarecalledhybridomas(fusedcells).1

Selection ofH ybridom as Onlythehybridomacellswillgrow intheHAT(Hypoxanthine-aminopterin-thymidine)medium. Theremainderofthecellswilldisppearslowly,after7-10daysofculture.Itisveryimportantto selectahybridcellproducingasingleantibody.Ifthehybridomasareisolatedandgrown separately,itispossibletoselectonlyonehybridcell.Afterdilutionofthesuspensionof hybridomacell,theindividualaliquotescontainonecelleachonanaverage.Therequired antibodyproducedifthesecellsaregrowninaregularculturemedium. Screening Fortherequiredspecificitythehybridomacellshouldbescreenedforantibodysecretion.Each culturemedium ofhybridomacellsaretestedfortherequiredspecificityperiodically.ELISA (Enzyme-linked-immunosorbent-serologicassay)isthemostcommonassaymethod.Tothe bottom of96-wellplatesantigensareabsorbed.So,thespecificantigenwillbebindtothe anibodyandtheunboundantibodyandrestofthemedium maybewashedoff.Byscreening,the hybridomacellproducingrequiredantibodycanbeidentified.Thustheantibodysecretedfrom thehybridcellistermedasmonoclonalantibody. Cloning and propagation Afterscreening,thehybridcellproducingdesiredantibodyisseparatedandcloned.Thereare twotechniquenormallyusedforcloning. ? Limitingdilutionmethod: Inthistechnique,serialdilutionofsuspensionofhybridomacellsaredone.Insmallculturewells, thealiquotsofeverydilutionareplaced.Eachaliquotsinawellcontainsonlyonehybridcell. Forthisreasondilutionsarecreated.Thisconfirmsthattheantibodymadeismonoclonal. ? Softagarmethod: Inthistechnique,insoftagarhybridomacellsarecultured.Severalcellscanbegrowninasemi solidmedium atthesametimetoform colonies.1

Characterization and storage Fortherequiredspecificity,theantibodyissubjectedtobiochemicalandbiophysical characterization.TofindtheM Abforthesubclassorclassofimmunoglobulin,specificityforan epitope,rangeofbindingsiteitcontaincharacterizationisveryimportant. Thestabilityofmonoclonalantibodyisalsoimportant.Towithstandfreezingandthawing,the abilityofthecellshouldbecharacterized.Atmanystepsofcloningandculture,therequired cellsarefrozeninliquidnitrogen. Conclusion:Forthepreparationofmonoclonalantibodyinvitrohybridomatechnologyisan appropriatemethod.Fordiagnosing,preventingandtreatingdiseasesmonoclonalantibodyhave openedremarkableapproaches.2 References: 1.BiologyDiscussion.(2018). M onoclonalAntibodies:Production,AdvantagesandLimitations. onlineAvailableat:http://www.biologydiscussion.com/antibodies/monoclonal-antibodies- production-advantages-and-limitations/10068Accessed4Nov.2018. 2.Global-research-online.net.(2018).onlineAvailableat:http://www.global-research- online.net/volume1issue2/Article%20017.pdfAccessed4Nov.2018.

x

Hi!
I'm Ava

Would you like to get a custom essay? How about receiving a customized one?

Check it out