
Determinationofthe5-hmCrabbitpolyclonalantibodytiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionoftherabbitpolyclonalantibodydirectedagainst5-hmCinantigencoatedwells.TheantigenusedwasBSAcoupledtothe5-hmCbase.Byplottingtheabsorbanceagainsttheantibodydilution,thetiteroftheantibodywasestimatedtobe1:3,500.
AnhydroxymethylatedDNAIP(hMeDIP)wasperformedusingtherabbitpolyclonalantibodydirectedagainst5-hydroxymethylcytosine
TheIgGisotypeantibodiesfromrabbit(Cat.No.25204)wasusedasnegativecontrol.TheDNAwaspreparedwiththeGenDNAmoduleofthehMeDIPkitandsonicatedtohaveDNAfragmentsof300-500bp.1μgofhumanHeLacellsDNAwerespikedwithnon-methylated,methylated,andhydroxymethylatedfragments.TheimmunoprecipitatedmaterialhasbeenanalysedbyqPCRusingtheprimerpairspecificforthe3differentcontrolsequences.Theobtainedresultsshowthattherabbitpolyclonalfor5-hmCishighlyspecificforthisbasemodification(noIPwithnon-methylatedormethylatedCbasescontainingfragments).
Dotblotanalysisofthe5-hmCrabbitpolyclonalantibodywiththeC,mCandhmCPCRcontrols
100to4ng(equivalentof5to0.2pmolofC-bases)ofthehmC,mCandCPCRcontrolswerespottedonamembrane(AmershamHybond-N+).Themembranewasincubatedwiththerabbit5-hydroxymethylcytosinepolyclonalantibody(cat.no.25204,dilution1:200).Themembraneswereexposedfor30seconds.
Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.
ELISA(1:500)
DB(1:200)
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一般来说多克隆的阳性率高一些,但出现假阳性的比例也高一些。
制备抗体都需要进行纯化处理
抗体的特异性鉴定 抗体的特异性是指与相应抗原或近似抗原物质的识别能力。抗体的特异性高,它的识别能力就强。衡量特异性通常以交叉反应率来表示。交叉反应率可用竞争抑制试验测定。以不同浓度抗原和近似抗原分别做竞争抑制曲线,计算各自的结合率,求出各自在 IC50时的浓度,并按下列公式计算交叉反应率。 如果所用抗原浓度IC50浓度为pg/管,而一些近似抗原物质的IC50浓度几乎是无穷大时,表示 这一抗血清与其他抗原物质的交叉反应率近似为 0,即该血清的特异性较好。
抗体的亲和力 是指抗体和抗原结合的牢固程度。亲和力的高低是由抗原分子的大小、抗体分子的结合位点与抗原决定簇之间立体构型的合适度决定的。有助于维持抗原抗体复合物稳定的分子间力有氢键、疏水键、侧链相反电荷基因的库仑力、范德华力和空间斥力。亲和力常以亲和常数K表示,K的单位是L/mol,通常K的范围在 108 ~1010 /mol,也有多达 1014 /mol。抗体亲和力的测定对抗体的筛选,确定抗体的用途,验证抗体的均一性等均有重要意义。向左转|向右转
又由于自然存在的抗原大都存在多个抗原表位,会刺激机体产生多种针对同一抗原的不同抗原表位相应的不同抗体.

