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which elisa should i plan to determine the antibody concentration and specificit - (Aug/31/2010 )

after immunization of my rabbit i collected its serum after 21 day of the booster. now b4 purifying the antibody i need to do an elisa to check its specificity and sensitivity. i have never done an elisa before and im really confused as to what should i coat my wells aith and which type of elisa i need to do. please enlighten me.

-eurisko-

Briefly; you need to coat the plates with the same protein you immunised the rabbits with. Also remember to leave some uncoated wells as blanks. Then you incubate the plate with the rabbit sera and controls, use an anti-rabbit IgG HRP or AP conjugated as secondary antibody, and the appropriate substrate (ABTS/TMB for HRP, or PNPP for AP).

A bit more detailed protocol will be:
1. coat plates with 1g/ml of protein in carbonate buffer (pH ~9.9) leave at 4C overnight I use to leave some blanks wells (uncoated) to test for nonspecific binding of the antibodies to the plastic.
2. block plates for 1-2h with BSA or milk in TBST at 37C
3. Add sera and controls. Ideally your control should be the pre-immunisation sera. If you dont have that, just sera from a rabbit that has not been immunised with the protein of interest, or a completely naive rabbit. You will need to dilute your
sera, I think 1:200 and 1:1000 are good points to start. Leave some wells without sera at all (blanks).
4. Incubate plates with the sera for 1-2h at 37C
5. wash plates 3-5 times with TBST
6. add your secondary antibody at the recommended dilution. This should be an anti-Rabbit-IgG - HRP or AP
7. incubate plates with secondary antibody for 1h at 37C
8. wash plates 3-5 times with TBST
9. wash plates 2-3 times with ddH2O (normally the tween in TBST interferes with the enzyme substrate)
10. add substrate (I recommend ABTS or TMB for HRP, and PNPP for AP) and incubate plates until you see colour change
11. Read plates

You can either stop the reaction and red the plates once. Or, read every 15-30min while colour is still developing. Again here is best you follow manufacturer instructions.


I've just written this protocol of the top of my head, and I'm probably missing loads of details, but I hope it helps you to get an idea where to start.
Good luck!

-almost a doctor-

For specificity: you need to compare binding of your antibody to structurally similar antigens. For large molecules (ie hormones) you can vary their concentrations in the wells (bound to surface) and determine if your ab binding is specific.
If your immunogen was a small molecule coupled to carrier protein you would need to run a competative test. The antibody would be bound in the wells and your would react your molecule-enzyme conjugate with samples containing varying concentrations of the molecule(analyte) and structurally similar agents. (ie steriods, small molecule drugs, etc).

For the above 2 methods individuals usually report the specificity (cross reactivity) at the 50% binding point in the assay. That concentration which produces a signal = to 50% of the test analyte.

For sensitivity: this is detection of the least concentration of analyte present in a sample. You would run serial dilutions of the analyte in a working system and determine that concentration which can be discriminated from "0" OR run multiple replicates of "0" and use 2 standard deviations away. (there are also other calculations that can be used...these are the easy ones...

-sgt4boston-

almost a doctor on Wed Sep 1 08:33:09 2010 said:


Briefly; you need to coat the plates with the same protein you immunised the rabbits with. Also remember to leave some uncoated wells as blanks. Then you incubate the plate with the rabbit sera and controls, use an anti-rabbit IgG HRP or AP conjugated as secondary antibody, and the appropriate substrate (ABTS/TMB for HRP, or PNPP for AP).

A bit more detailed protocol will be:
1. coat plates with 1µg/ml of protein in carbonate buffer (pH ~9.9) leave at 4C overnight I use to leave some blanks wells (uncoated) to test for nonspecific binding of the antibodies to the plastic.
2. block plates for 1-2h with BSA or milk in TBST at 37C
3. Add sera and controls. Ideally your control should be the pre-immunisation sera. If you dont have that, just sera from a rabbit that has not been immunised with the protein of interest, or a completely naive rabbit. You will need to dilute your
sera, I think 1:200 and 1:1000 are good points to start. Leave some wells without sera at all (blanks).
4. Incubate plates with the sera for 1-2h at 37C
5. wash plates 3-5 times with TBST
6. add your secondary antibody at the recommended dilution. This should be an anti-Rabbit-IgG - HRP or AP
7. incubate plates with secondary antibody for 1h at 37C
8. wash plates 3-5 times with TBST
9. wash plates 2-3 times with ddH2O (normally the tween in TBST interferes with the enzyme substrate)
10. add substrate (I recommend ABTS or TMB for HRP, and PNPP for AP) and incubate plates until you see colour change
11. Read plates

You can either stop the reaction and red the plates once. Or, read every 15-30min while colour is still developing. Again here is best you follow manufacturer instructions.


I've just written this protocol of the top of my head, and I'm probably missing loads of details, but I hope it helps you to get an idea where to start.
Good luck!

thanks a lot
did the direct elisa assay
started with a dilution of 1: 100 but had to continue diluting
finally got my titre at ~60,000. used a protocol very similar to the one u suggested
thanks again
now going to start with my competitive elisas.
wish me luck

-eurisko-

sgt4boston on Wed Sep 1 10:57:43 2010 said:


For specificity: you need to compare binding of your antibody to structurally similar antigens. For large molecules (ie hormones) you can vary their concentrations in the wells (bound to surface) and determine if your ab binding is specific.
If your immunogen was a small molecule coupled to carrier protein you would need to run a competative test. The antibody would be bound in the wells and your would react your molecule-enzyme conjugate with samples containing varying concentrations of the molecule(analyte) and structurally similar agents. (ie steriods, small molecule drugs, etc).

For the above 2 methods individuals usually report the specificity (cross reactivity) at the 50% binding point in the assay. That concentration which produces a signal = to 50% of the test analyte.

For sensitivity: this is detection of the least concentration of analyte present in a sample. You would run serial dilutions of the analyte in a working system and determine that concentration which can be discriminated from "0" OR run multiple replicates of "0" and use 2 standard deviations away. (there are also other calculations that can be used...these are the easy ones...


hey thanks a lot
just got done with my direct elisas
going to plan competitive elisas very soon
ill definitely take ur suggestion into considerations
thank a lot once again
bye

-eurisko-