We are developing a peptide ELISA for IgM and IgG detection.
We infected mice with parasite 1; for cross-reactivity analyses, we infected mice with two parasites (groups 2 and 3) and included a negative control group. Peptides were synthesized by solid phase peptide synthesis (Fmoc), with ~80% purity, from a native antigenic protein from parasite 1. Sera from different periods of infection were collected and frozen.
We coated the plates with BSA (40μg/mL) for 1 hour, and treated with glutaraldehyde 5% for 1 hour. Then, we added the peptide and incubated it overnight. Finally, we added glycine for 30min. We blocked the plate with PBS-BSA 5% for 1 hour, added the diluted samples and incubated for 1 hour under mild agitation. We added anti-biotinylated IgM or anti-IgG-HRP for 1 hour. For IgM, we added avidin-HRP for 30 minutes. For both, we added TMB for 10min, stopped with sulfuric acid and read at 450nm.
The results are not consistent inter-assays (low reproducibility). The ODs from the negative control group are higher than blank wells and similar to those from parasite 1. Sera from groups 2 and 3 resulted in higher ODs than those of the parasite 1 group. We did not expect this cross-reactivity, given the peptides appeared specific to parasite 1 in certain periods of infection and showed no homology to other parasites or microorganisms on Protein BLAST.
Does anyone have any experience with peptide ELISA that could help us?
We washed the plates after every step, as shown below:
•BSA: washed once with carbonate-bicarbonate buffer (pH 9,6)
•Glutaraldehyde: washed twice with carbonate-bicarbonate buffer (pH 9,6)
•Peptides: washed twice with carbonate-bicarbonate buffer (pH 9,6)
Glycine: washed once with PBS
•IgM
Blocking and sera: washed 3 times with PBS-Tween 20 0,05%
Anti-biotinylated IgM: washed 5 times with PBS-Tween 20 0,05%
Avidin-HRP: washed 7 times, 5 minutes each, with PBS-Tween 20 0,05%
•IgG
Blocking: washed 3 times with PBS-Tween 20 0,05%
Sera: washed 3 times with PBS-Tween 20 0,05%
Secondary antibody: washed 3 times, 5 min each, with PBS-Tween 20 0,05%
With glutaraldehyde, you'll crosslink N-termini and lysines with the substrate (BSA). Depending on your peptides' sequences, you might generate adducts which are not recognized by the antibodies you're fishing for, by masking the epitope(s) or generating crosslinking artifacts (crosslinked peptides).
Do you have the option to obtain peptides with only an N- or C-terminal cysteine and use e.g. SMCC (maleimide/NHS ester chemistry) for immobilization? (For more options, longer spacers, please have a look at https://www.iris-biotech.de/global/linkerology .
If there are internal cysteines or lysines present, it will be a bit tricky, and you need protection groups during synthesis which are only removed after coupling the peptide to the carrier protein.
If possible, couple the peptides to BSA first and then use this as a stock to coat your plates, to reduce variation between tests.
Hi Wolfgang! Thank you for your reply.This peptide, in particular, does not have lysine, but it does have an internal cysteine.The quickest way right now would be to couple the peptides with BSA.I have other peptides with the same issue, but they are already diluted in DMSO. Is it possible to couple those peptides with BSA?
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