I have been doing ELISA to detect Ab in our serum samples and every time i got color in my negative control (uncoated wells and wells that only have secondary Ab) as well as the absorbance for all standards are same.
I diluted my primary Ab in 1% BSA (1ul Ab+999ul 0.05% PBST)
secondary Ab was also diluted 1:2000 in PBST
i only suck solution from the wells and wash 5 times with 0.05% PBST.
any good advice can help proceed this experiments.
If you are trying to detect antibody in your serum, why are you coating with a primary Ab? Shouldn't you coat with the antigen that your serum antibodies will recognize?
Is this a commerical kit?
I'll need your entire protocol in detail to know what is going on. Sounds like a blocking or washing issue, or the ELISA is not conducted correctly.
i am coating plate with Ag overnight at 4C then i suck the solution wash three times with 0,05 PBST then add blocking buffer (1%BSA in PBST) for 1h, wash then add my primary Ab that diluted in 1%BSA for 1h then wash. next add secondary Ab diluted 1;2000 in PBST then wash. add substrate for 30 min then add stop solution and read immediately.
washing has been done by adding270 ul of 0.05% PBST to wells then suck the solution
Try blocking with 3% BSA in 1xPBST for 2 hours.
I would also increase your wash buffer volume to 400uL/well
Do you tap the plates after your wash to make sure that the plates are properly dried?
what is the target of your primary? Usually, for indirect ELISAs like this, the "primary" is the serum, and then the "secondary" is the anti-IgG ligated to HRP.
Amended on 1 June 2019
I must sadly declare that ELISA is not a reliable and not a quantitative method at all. IgG recognizes many proteins giving false negative and false positive result.
Therefore, I must recommend the new identification method of PDMD method (please see files; HepG2 Fucoidan and JMBT Alopecia). It is noteworthy that PDMD method can only be performed by using the reliable Shimadzu PPSQ microsequencer at this time.
PDMD method requires fresh serum sample (freased sample without thaw is possible to be used) and fresh biological specimens (tissue samples freased at -80 C without thaw are also possible to be used).
Thioctic acid/lipoic acid is also determined by using fresh serum and suitably stored samples (please see file; Lipoic acid Avidin).
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