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抗牛IFN-γ单克隆抗体的制备及ELISA检测方法的初步建立.docx

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抗牛IFN-γ单克隆抗体的制备及ELISA检测方法的初步建立 Introduction Interferon-γ(IFN-γ)isapro-inflammatorycytokineinvolvedintheimmuneresponseagainstviral,bacterial,andparasiticinfections.Itisalsoinvolvedintheregulationofinflammationandcellulargrowthanddifferentiation.Assuch,IFN-γplaysacriticalroleinbothinnateandadaptiveimmunity.TheabilitytodetectandquantitateIFN-γiscriticalforunderstandingimmuneresponsesanddevelopingtherapeuticsforinfectiousandautoimmunediseases.Currently,thereareseveralmethodsavailableformeasuringIFN-γ,includingenzyme-linkedimmunosorbentassay(ELISA).Inthisstudy,wedescribethedevelopmentofamonoclonalantibodyagainstbovineIFN-γandanELISAmethodforthedetectionofIFN-γinbovineserumsamples. MaterialsandMethods PreparationofmonoclonalantibodyagainstbovineIFN-γ PolyclonalantibodiesagainstbovineIFN-γweregeneratedbyimmunizingrabbitswithrecombinantbovineIFN-γ.TheIgGfractionofthepolyclonalantibodieswaspurifiedandusedastheimmunogenformicetogeneratehybridomas.HybridomasthatsecretedantibodiesagainstbovineIFN-γwereselectedusingELISAandconfirmedbyWesternblotanalysis. ELISAstandardization ELISAplateswerecoatedwithrecombinantbovineIFN-γandincubatedovernight.TheplateswereblockedwithBSAandincubatedwiththemonoclonalantibodyagainstbovineIFN-γ,followedbyasecondaryantibodyconjugatedtohorseradishperoxidase(HRP).TheplatesweredevelopedusingasubstrateforHRP,andtheopticaldensity(OD)wasmeasuredat450nm.ThestandardcurvewasgeneratedusingserialdilutionsofrecombinantbovineIFN-γ,andunknownsampleswerecomparedtothestandardcurvetodeterminetheconcentrationofIFN-γpresentinthesample. ValidationofELISAmethod ThespecificityofthemonoclonalantibodywasdeterminedbytestingitsabilitytodetectbovineIFN-γinbovineserumsamples.TheserumsampleswerespikedwithrecombinantbovineIFN-γatdifferentconcentrations,andtheELISAwasperformedasdescribedabove.Therecoveryratewascalculatedbycomparingthemeasuredconcentrationtotheexpectedconcentration. Results MonoclonalantibodyagainstbovineIFN-γ WegeneratedamonoclonalantibodyagainstbovineIFN-γusinghy