YoucantestwhetherornotyouhavegottenanimmuneresponsetothepeptideandhowstrongthatimmuneresponseisbydoingELISAsagainstpeptideconjugatedtoBSA.ByconjugatingtoBSA,youwilleliminateanysignalforantibodiesgeneratedtoKLHduringimmunization.

Protocol:

A.CouplingPeptidetoBSA

Youneed:

• 1mlof2mg/mlBSAin0.1MNaHCO3.
• 1mlof0.2glutaraldehydein0.1MNaHCO3.
• 0.5mlpeptide(1mg/mlinDMSO).Peptidecanbeaddedasasolidifsoluble.

1. Mix,addingglutaraldehydelast.PeptideandBSAturnalittleyellowevenbeforeaddingglutaraldehyde.
2. Incubate90'at37degC.
3. Add0.1volumesof0.1MNaBH4in0.1MNaHCO3.Therewillbesomebubbling.AddsameamountofNaBH4after15'.Doaquickmicrofugespiniftherearetoomanybubbles.

B.ELISAwithPeptideconjugatedtoBSA

1. Coat10ug/mlantigen(peptideconjugatedtoBSA)dilutedinTBS(50ul/well)ONat4degC.
2. Removeantigenandrinsewells2XswithTBST.
3. Block2hrwith200ulof5NFDMinTBST.
4. Removeblockingreagentandrinsewells2XswithTBST.
5. IncubateinprimaryantibodydilutedinBlockingBufferfor2hratRT.Idotriplingdilutionsbeginningat1/10(50ul/well).
6. Removeprimaryantibodyandrinsewells4XswithTBST.
7. Incubateinsecondaryantibody(1/5000Goatanti-rabbitconjugatedtoAP)dilutedinBlockingBufferfor1hratRT(50ul/well).
8. Removesecondaryantibodyandrinsewells4XswithTBST.
9. Rinsewells2Xswith50mMHCO3;0.5mMMgCl2,pH10.
10. Developin1mg/mlp-Nitrophenylphosphatein50mMHCO3;0.5mMMgCl2,pH10(50ul/well).
11. ReadA410inELISAreader.

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