| Kitsize | 12x8 |
| Method | ELISA |
| Incubationtime | 1x1h,1x30min,1x30min |
| Standardrange | 0.5-18µg/L,AssayRange:0.029-180µg/L |
| Specimen/Volumes | 5µLserum,plasma/withoutextraction |
| Substrate/isotope | TMB450nm |
| RegulatoryStatus: | Forresearchuseonly |
- issuitedforIGF-Ideterminationinserumandplasmaofmiceandrats
- highsensitive:0.029ng/mlanalyticalsensitivity;requiredsamplevolumeisverysmall
- isfast:incubationtimeatotalof2hours
- SingleStandardswith0.5,2.5,6,12,18ng/mlrecombinantIGF-IareprovidedintheKit
- 2ControlSeraareprovidedforqualitycontrol
- useshighaffinityantibodiesagainstm/rIGF-I
- Microtiterplatesareseparatelybreakapart
IntendedUse
MeasurementofIGF-Iinmouse/ratserumandplasma.
INTRODUCTIONBesidedifferentcellculturemodelsandstudieswithhumanpatients,miceandratsaresuitablemodelorganismsforbasicresearchandpre-clinicalstudies.Thus,wedevelopedthistestsystemasatoolforIGF-Imeasurementsinmiceandratforusageinresearchandpreclinicalstudies.EvenifthecomparABIlityofmiceandhumansislimitedweoffersomebackgroundinformationonthehumanIGF-Isysteminthefollowingsection:
Insulin-likegrowthfactors(IGF)IandIIplayapivotalroleinregulatingtheproliferationanddifferentiationofmanycelltypes.IGF-IisidenticalwithSomatomedinC(Sm-C)andhasamolecularweightof7649daltons.Itsmajorregulatorsaregrowthhormone(GH)andnutrition(6).Incontrasttomanyotherpeptidehormones,IGFsareavidlyboundtospecificbindingproteins(IGFBP).ThesevenIGFBPswhichareknownatpresenteitherbindIGF-IandIGF-IIwithsimilaraffinitiesorshowapreferenceforIGF-II.
AmajorproblemofIGF-ImeasurementresultsfromtheinterferenceofIGFBPsintheassay.DirectdeterminationsinuntreatedserumsamplesgivefalsevaluesbecauseoftheextremelyslowdissociationoftheIGF-I/IGFBP-3complexesduringtheassayincubation.DependingontheratioIGF-ItoIGFBPinthesampleinterferencecomesup.
Therefore,varioustechniqueswereappliedtophysicallyseparateIGF-Ifromitsbindingproteinsbeforemeasurement,including(a)sizeexclusionchromatographyunderacidicconditions,(b)solid-phaseextractionand(c)acid-ethanolextraction.Thesetechniques,however,areeitherinconvenientortime-consumingorgiveincompleteandnotreproducIBLerecoveries.Themostwidelyusedmethodistheacid-ethanolextractionwitharecoveryofonly70-80%ofIGFBP-boundIGF-Iasaresultofco-precipitation.Theabsoluteresultsofsuchanextractionarethereforefalselow.TheextractionremovestheIGFBPsonlyinsufficientlyandleadstoreductioninsensitivityoftheassayduetopredilutionofthesamplesbytheextractionprocedure.FurThermore,theremainingIGFBPmaystillinterfereintheassay.Inaddition,theacid-ethanolextractionisineffectiveinspecimensotherthanserumorplasma(e.g.cellculturemedia),inwhichdeterminationofIGF-IisalreadydifficultenoughduetothefactthatIGFBPsarefrequentlypresentatlargeexcess.
Toavoidthesedifficulties,anuncomplicatedassaywasdeveloped,inwhichspecialsamplepreparationisnotrequiredbeforemeasurement.
