Obtaining Conjugate Antibody for Sandwich ELISA - How do I obtain/create the appropriate Alkaline-phosphatase Antibody (Sep/26/2008 )
Hi all,
I am learning about ELISA as an alternative to Western Blotting. I am an Ecologist/Evolutionary Biologist by training. I am interested in finding a relatively affordable, high-throughput technique that is more accurate/quantitative than Western Blotting.
My target protein is a HSP found in Arabidopsis thaliana.
I have a very high-affinity primary antibody produced in Rabbit that I have been using for Westerns.
What I need to find out is if I can use that antibody as the Capture-antibody, and then simply do some reaction that binds the antibody to Alkaline-phosphatase and use that for the final part of the "sandwich"?
If not, how affordable/easy is it to obtain an appropriate antibody? Does it need to be developed specifically for my HSP?
Finally, does anyone have a comparison of the relative accuracy difference between Western Blotting and ELISA? That is to say, what is the smallest difference detectable in ELISA and the smallest detectable in Western Blotting? Something like, 5% difference minimum in ELISA and 30% difference in Western blotting is what I am looking for. I just need to understand the differences in resolution.
Thanks so much,
M
hi,
if ur high affinity antibody is a polyclonal antibody.
take a protion of it and perfrom HRP conjuagtion or biotinylation.
so that unmodified antibody u can use as capture and modified antibody as detector.
coming sensitivity, ELISA is much better compared western blot, except that with ELISA u can not differentiate whether your signal is coming from specific or nonspecific binding.
gud luk
if ur high affinity antibody is a polyclonal antibody.
take a protion of it and perfrom HRP conjuagtion or biotinylation.
so that unmodified antibody u can use as capture and modified antibody as detector.
coming sensitivity, ELISA is much better compared western blot, except that with ELISA u can not differentiate whether your signal is coming from specific or nonspecific binding.
gud luk
Okay. I think mine may be monoclonal, which is not a good sign, but I will try and find out for sure.
Regarding Western Blots versus ELISA:
One of the problems I have with Western blotting is not that I don't get a precise measurement of protein amount, but it is repeatability. In other words, I can put the same sample in every well of a single gel and get considerable variation in blotting intensity. On top of that, there is the issue of comparing between gels as I get even more variation among gels than within, even with the same exact sample. This means that to gain statistically significant comparison of samples I need many replicates across many gels.
That is a pain and potentially quite expensive.
If I run say 4 replicates of the same sample using ELISA, what sort of variation in measurement should I expect?
Are there literature or other sources for this information?
Thanks so much,
M
If it is a MAb you may be in trouble unless it is a repeating epitope. Keep your fingers crossed... Otherwise you may have to invest in a second antibody.
Variation when it comes to ELISA's varies from assay to assay. A rule of thumb says you should get less than 5% RSD in a sandwich ELISA, or you should rerun your samples. So, it's fair to assume your results should have less than 5% Stdev.
I wish you the best of luck.
Variation when it comes to ELISA's varies from assay to assay. A rule of thumb says you should get less than 5% RSD in a sandwich ELISA, or you should rerun your samples. So, it's fair to assume your results should have less than 5% Stdev.
I wish you the best of luck.
SatanClause: Can I ask you to clarify. What is MAb? Also, to what does 5% RSD refer? Do you mean that running the same sample in replicate will produce on average variation of only 5% for that sample?
If so, do you know where I could find some sort of published or other proof of this? For citing in a grant proposal?
Thanks,
M
Sorry if I was a bit unclear. RSD is Relative Standard Deviation - your standard deviation as percentage of the sample average (i.e. CV in percentage). Since 5% is a rule of thumb you can't really find a paper on it, but for a grant application I would use papers where ELISAs have been used in similar setups (sandwiches of HSPs). I'm pretty sure you'll find all the results will have a RSD of less than 5% (note however that when it comes to competitive assays this rule goes out the window).
Mab is simply Monoclonal Antibody. Since monoclonals bind to one specific epitope you can't make a sandwich unless the epitope repeats on the protein (which isn't something I would count on). You can't bind the detection antibody if you already have a catcher antibody occupying the epitope.
Hope that was a bit clearer. And good luck with the grant application.
hi,
i hope u made sure that ur pipettes and instruments used are working fine. this event could lead to weird results, despite you load same volume and same quantity. or find out that protein is sticky to avoid variation.
gud luk
if ur high affinity antibody is a polyclonal antibody.
take a protion of it and perfrom HRP conjuagtion or biotinylation.
so that unmodified antibody u can use as capture and modified antibody as detector.
coming sensitivity, ELISA is much better compared western blot, except that with ELISA u can not differentiate whether your signal is coming from specific or nonspecific binding.
gud luk
Okay. I think mine may be monoclonal, which is not a good sign, but I will try and find out for sure.
Regarding Western Blots versus ELISA:
One of the problems I have with Western blotting is not that I don't get a precise measurement of protein amount, but it is repeatability. In other words, I can put the same sample in every well of a single gel and get considerable variation in blotting intensity. On top of that, there is the issue of comparing between gels as I get even more variation among gels than within, even with the same exact sample. This means that to gain statistically significant comparison of samples I need many replicates across many gels.
That is a pain and potentially quite expensive.
If I run say 4 replicates of the same sample using ELISA, what sort of variation in measurement should I expect?
Are there literature or other sources for this information?
Thanks so much,
M
Unless I am mistaken the word "rabbit" was used. I am certain you have a polyclonal ab...NOT monoclonal.
Perform the conjugation with Alk Phos and HRP. Make sure you purify the product and remove unbound enzyme.
When you do your testing and titering don't overlook doing both sequential and simultaneous incubations of sample and conjuate.
good luck.