Decrease in serum antibody titers - (Apr/01/2008 )
Hi,
We have been using an ELISA protocol for several years with reproducible results. However, recent results for our antibody titers have decreased by 10 fold (log scale). Not sure what is going on since the protocol and antibodies to IgG and SA-HRP have remained the same. Basically, coat our plates with antigen diluted in PBS O/N at 4C. Next morning, we wash the plates with pbs and block in PBS-3%BSA. While this is happening, we dilute our serum 1:40 serially (8 dilutions total)in pBS-3%BSA. We wash the plates in PBST (0.05%) and plate O/N at 4C. The next day, we wash with PBST, then add anti-mouse IgG (diluted 1:2000) for 1hr at RT. Then, we wash again and add SA-HRP (1:3000) for 30 min at RT. We wash again, add TMB Start solution and let develop for 2 min before adding Stop and reading at 450nm. Generally our numbers read 0.065 - 2.0, with dilution 4 of 8 at 1.0 and dilution 5 of 8 at 0.6. Now our numbers read 0.065 - 2.0, with dilution 4 of 8 reading 0.6 and dilution 5 of 8 reading 0.2. Therefore, when we interpolate the amount of antibody in the serum, there is a 10 fold decrease since the curve of the titration has shifted to the left. Any suggestions?
Thanks!
Hi Ichor,
there are a number of possibilities, which you would probably have to check out one by one. Starting from the plastic plates - did you change your supplier, have they been exposed to any weird conditions, etc.
Otherwise my best bet would be that your serum concentration is lower than it used to be. You didn't say what kind of serum you're using, so it's difficult to say. I can say that our hybridoma cell production varies quite a bit from flask to flask. Also some antibodies are stable at room temperature, others loose activity pretty rapidly especially in buffers without protectants such as BSA.
Just my 2 cents.
Regards,
Miha
there are a number of possibilities, which you would probably have to check out one by one. Starting from the plastic plates - did you change your supplier, have they been exposed to any weird conditions, etc.
Otherwise my best bet would be that your serum concentration is lower than it used to be. You didn't say what kind of serum you're using, so it's difficult to say. I can say that our hybridoma cell production varies quite a bit from flask to flask. Also some antibodies are stable at room temperature, others loose activity pretty rapidly especially in buffers without protectants such as BSA.
Just my 2 cents.
Regards,
Miha
Hi Miha,
We are using mouse, rat or rabbit serum. The thing is, we went back to old samples that have been stored at -20C and are now getting the same funky results. Also, our plate supplier has not changed....immulon plates from VWR. Thanks for your suggestion though.
there are a number of possibilities, which you would probably have to check out one by one. Starting from the plastic plates - did you change your supplier, have they been exposed to any weird conditions, etc.
Otherwise my best bet would be that your serum concentration is lower than it used to be. You didn't say what kind of serum you're using, so it's difficult to say. I can say that our hybridoma cell production varies quite a bit from flask to flask. Also some antibodies are stable at room temperature, others loose activity pretty rapidly especially in buffers without protectants such as BSA.
Just my 2 cents.
Regards,
Miha
Hi Miha,
We are using mouse, rat or rabbit serum. The thing is, we went back to old samples that have been stored at -20C and are now getting the same funky results. Also, our plate supplier has not changed....immulon plates from VWR. Thanks for your suggestion though.
Serum samples stored at -20 deg. should remain intact for a long time. Perhaps the fridge you stored them in is used a lot (opened and closed often) in which case the temperature fluctuates a lot and can cause decay in the sera or if you had a melt-down.