ELISA blocking agents and statistics - (Aug/12/2010 )

Hi EVERYONE

newbie reader with first post.

Basically iv been running an ELISA with a monoclonal antibody with a known cncetration testing to see if it binds.
But instead of 2% BSA as a blocking buffer iv been using skimmed milk.
Now i know that i can use the milk as a blcker but the catch is that the antigen i should be using is not available
its the Lewis a sialyted antigen on pancreatic cells. So my supervisor said i should try milk since it too has sialyted sugars.
Now my first question how can i use the milk as my blocker and my antigen?
Second i used only 9 wells with A1-3 as sample B1-3 as positive control with no antibody only HRP + seconadry Ab with TMB and C1-3 as negative control.

I only added 110ul of TMB and the well was not full so my readings were kinda low but the A1-3readings were highest followed by B1-3 and then C1-3.
what i what to find out now is, if the milk issue doesnt screw the validity of the test, if there is significance in the OD readings between the controls and sample ie stats.
What test should i use and where can i find a run through so i can do it myself.

Thanks in advance guys

Hello,
The blocking agent (to block uncoated plastic surface) should not react with your capture ab in the test you are running. Thus, with your true antigen used for coating you would use a blocking agent that would not react with your ab. Blocking with a 'structurally similar' material would be ok IF the ab is so specific it would not recognize the blocking agent. Such an ab would have to be very well characterized OR the level of cross reactivity would have to be negligible.

You should be coating all the screening antigens at the same concentrations, volumes, and buffer in the wells and blocking with a completely different protein. You will have to clarify what the difference is between your sample and positive are as you indicated you are not using a true positive in your screening process.

sgt4boston on Thu Aug 12 14:37:25 2010 said:

Hi
thanks for the reply
the difference betwen the sample and positive cntrol is that the control lacks the recombinant antibody but contains everything else ie the secondary Ab with HRP and the TMB plus the washe of course. the sample has the recombinant Ab added extra.
Hope thats clear
oh and what about the other question?

Thanks a million

hello,
I thought you were coating your antigen in the wells and screening for Mab binding...is this not correct?

sgt4boston on Thu Aug 12 18:47:18 2010 said:

thanks for the reply

basically i coated the wells with 10ug/ml of skimmed milk in PBS.
then wrapped in cling film and waited for 24 hrs. that was the antigen prep stage. then discarded any liqued in morning and washed with wash buffer.
then i applied a blocker of 2% skimmed milk in PBS and after a hour druied out over tissue.
then i added a solution of 1:100 of purified ntibody to PBS. again wait an hour then dry and rinse again and add
secondary antibody with HRP conjugate and dry and wash then finally add TMB and read colour change.

Now what im asking is how can i use the skimmed milk as the antigen coating the well and then as the blocker then add the Ab? Surely i would not be blocking the well but incraesing the antibodies chances of binding.
Furthermore the antibody was disgned to target the lewis a antigen so its not even the right antigen.
All i could think off was that the ELISA would show non specific binding of a similar structure (i.e the sialy sugars in milk)

Now if the basis of the test can be said to be valid (which im seriuosly doubting) what tests would i run to check the stat significance of the results (WELLS A1 A2 A3 were as described above, while WELLS B1 B2 B3 lacked the purified antibody (positive control since HRP-Ab reacts with TMB) and WELLS C1 C2 C3 were negative control)
Someone suggested an ANOVA test


thanks for any replies
much appreciated

Just a few technique issues first:

1. "added a solution of 1:100 of purified ntibody to PBS. again wait an hour then dry" There should be no "drying" of the primary ab binding stage. Seal the plate and incubate then wash several times with blotting.
2. "add secondary antibody with HRP conjugate and dry" Also, no 'drying' of the conjugate step. Add the conjugate, seal the plate, incubate, wash several times with blotting.

Now the question of controls coating etc. You are using skim milk as antigen and coating at 10 ug/ml and blocking at 20,000 ug/ml. You should have a completely different type of 'blocker'. At this point you have no 'negative' wells. You are calling wells that do not have primary ab only the conjugate as 'positive control' and those that do not as 'negative'.

You need
1. negative wells (blocked only) to make sure the ab is binding specifically to the antigen.(you should have source of BSA or fish protein). And, as you are doing a 'screening' test, it would be a good idea to coat some proteins that are structurally similar but very different than your target to insure your ab is indeed binding specifically or is just cross reactive.
2. Non-specific ab to use in parallel to your primary which would be your negative ab control. (use at the same concentration as your primary ab)
3. If possible a true positive reactive ab (perhaps commercial source) to use in parallel with your ab.