TheFluoroSpotassayisaninventivedevelopmentoftheELISpotassayandusesfluorescentdetectioninsteadofcolorimetric.Thisallowsforsimultaneousdetectionofseveralanalytes.ThemonkeyIgG/IgAB-cellFluoroSpotkitenablesdetectionofIgG-andIgA-secretingcellsinthesamewell.
Thekitcontainsmatchedquantitiesofcaptureantibodiesandfluorophore-conjugateddetectionantibodies,lowfluorescentPVDFplatesandafluorescenceenhancer.ThepolyclonalactivatorsR848andrecombinanthumanIL-2areincludedforpre-stimulationofmemoryBcells.
TheantibodiesinthiskitreactwithhumanIgGandIgAandcross-reactwithIgGandIgAfromnon-humanprimates.
Foranalysis,werecommendtheuseofanautomatedFluoroSpotreaderequippedwithfiltersforFITC(excitation490nm/emission510nm)andCy3(excitation550nm/emission570nm).Filtersshouldhavehighspecificitytoavoidbleed-throughartifacts.
AnalyteInformation
IgA
IgA(ImmunoglobulinA)representsonly10-15%ofthetotalIgmoleculesinserumbutisthepredominatingIgisotypeinsaliva,tears,breastmilkandinthegastrointestinalandurogenitaltract.Inserum,theIgAmoleculeiscommonlyfoundasamonomerofeitherIgA1orIgA2subclassbutcanalsoexistasdimersorlargerpolymericforms.TheIgAfoundinsecretions(secretoryIgAorsIgA)iscommonlyadimerwithtwoIgAmoleculesjoinedbytheJchain.SecretoryIgAisthemostimportantIgisotypeinmucoussurfaces,theareaofentryformostpathogens.
IgG
IgG(ImmunoglobulinG)isthemostabundantIgisotypeinserumandmakesupapproximately80%ofallserumImmunoglobulins.Inhumans,therearefoursubclassesofIgGwithhighestserumconcentrationsofIgG1followedbyIgG2,IgG3andIgG4.InmicetheIgGsubclassesaredefinedIgG1,IgG2a/c,IgG2bandIgG3.TheIgGmoleculeconsistsoftwoheavyandtwolightschains(κorλ)resultinginamoleculewithtwoarmsforantigenbinding.HighlevelsofIgGantibodiesareinducedfollowingtheinitialIgMresponseinatypicalimmuneresponsetoantigens.
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| Otherformats: | ClicktoviewallNHPIgA,IgGproducts |
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尤其是获得SCI文献引用的产品，
绝对ok的
用Mabteck公司的ALP标记抗体吧，配合Millipore公司的Multiscreen-IP96孔板，所有的试剂都可以减半，照样可以出非常好的结果，4*96孔的试剂盒我一直做8*96孔，但BCIP/NBT显色液没试过减半，因为它很便宜。记得当年第一次做实验就出了很好的结果，经过不断摸索，做出的蓝紫色斑点确实非常漂亮，几乎没有非特异斑点，嗯，ELISPOT是我最拿手的实验之一。
关于PBMC:
拿到血液标本后，首先我要做的是尽快分离PBMC（一般2h内），国产的分离液里天津灏洋的比较好，然后用冻存液冻存PBMC。PBMC的冻存过程比一般的细胞株要更为严格，最好用冻存器。ELISPOT试验的第一天，在包被抗体的同时，将细胞复苏，完全培养液培养过夜，第二天收集细胞并计数即可。
