ELISA Absorbance Interpretation

[susidoo]

Hi,

I'm a new member to this site and also a lab technologist student. I have a question about ELISA and am looking at absorbance results for the first time. I'm not clear on how to interpret these results.

Please correct me if I'm wrong in what I'm about to follow with.

Antigen: RSA
Primary Antibody: Anti-RSA
Secondary Antibody: Goat-anti-mouse IgG-HRP conjugate
Substrate: OPD

2 wells with Positive control: mouse anti-RSA polyclonal serum AU=0.923 and 0.922

?Negative control: Preimmune mouse serum
(is this considered a non-specific binding control because it would not have the antibodies for RSA, and any interaction between it and the RSA would be nonspecific? Also, the Fc portion where the secondary antibody (with the HRP) binds to this primary antibody (if bound to RSA), should be the same for all mouse IgG shouldn't it? So if the primary antibody has nonspecific interaction with RSA (which it seems it did because of AU) and the secondary antibody binds to it, than is this what is considered background (nonspecific interactions)? Ultimately, I'm not sure how to interpret the negative control in terms of what needs to be considered in terms of what is binding or not.) I've tried to sketch/draw it out, and I can't figure out what is bound to the RSA.

I have a bunch of wells unused coated with RSA and coating buffer only with absorbances of 0.045 - 0.069.

The 2 negative control wells compare to these with readings of 0.404 and 0.410.

Or is this the nonspecific binding control (I'm not sure what the term means)?I have 2 wells that are coated with RSA, coating buffer and blocking buffer, it has absorbances of 0.428 and 0.404.

So something must be binding in the last two controls mentioned above, because of the absorbance reading.

I hope I haven't confused you, and hope the questions in there were not inconspicuous. I'm having trouble asking what I need to understand, and maybe it's because I'm not really sure what it is I'm understanding or not.

Thanks a million.

[danfive]

Originally Posted by susidoo

Hi,

Antigen: RSA
Primary Antibody: Anti-RSA
Secondary Antibody: Goat-anti-mouse IgG-HRP conjugate
Substrate: OPD

You are detecting endogenous antibody against RSA, right? But is your primary antibody a competing antibody or did you misname it above.

I'll assume you are producing anti-RSA antibody in mice and looking to detect mouse anti-RSA antibody.

?Negative control: Preimmune mouse serum
(is this considered a non-specific binding control because it would not have the antibodies for RSA, and any interaction between it and the RSA would be nonspecific?

YES, this is negative control, or negative sera control.

Also, the Fc portion where the secondary antibody (with the HRP) binds to this primary antibody (if bound to RSA), should be the same for all mouse IgG shouldn't it?

Yes, since you know that negative sera produces a high signal (0.4+ is high) you need to consider that the secondary antibody is binding to all this nonspecific mouse antibody.

So if the primary antibody has nonspecific interaction with RSA (which it seems it did because of AU) and the secondary antibody binds to it, than is this what is considered background (nonspecific interactions)?

NO, big problem here. You don't really have primary antibody, do you? You have anti-RSA sera. This is a big point, please make sure.
If you have "primary antibody" that means purified anti-RSA and no nonspecific antibodies. If you have sera you have a mix of anti-RSA and nonspecific anti-RSA.
You can call it background because the empty wells are so low (below 0.1Abs). But it really is nonspecific signal or sera background.

Ultimately, I'm not sure how to interpret the negative control in terms of what needs to be considered in terms of what is binding or not.) I've tried to sketch/draw it out, and I can't figure out what is bound to the RSA.

You need the sera neg. ctrl and blank (no anti-RSA sera applied).
your values for the sera neg ctrl say you have a big nonspecific binding problem, under current ELISA conditions. Further optimization may improve this or it may not and you may need a new supply.

I have a bunch of wells unused coated with RSA and coating buffer only with absorbances of 0.045 - 0.069.

These are the "blanks" I just mentioned. This is low plate background and doesn't need optimization, good news.

The 2 negative control wells compare to these with readings of 0.404 and 0.410.

These I suppose are the negative control sera (preimmunized sera).

Or is this the nonspecific binding control (I'm not sure what the term means)?I have 2 wells that are coated with RSA, coating buffer and blocking buffer, it has absorbances of 0.428 and 0.404.

So something must be binding in the last two controls mentioned above, because of the absorbance reading.

I hope I haven't confused you, and hope the questions in there were not inconspicuous. I'm having trouble asking what I need to understand, and maybe it's because I'm not really sure what it is I'm understanding or not.

Thanks a million.

This last part is confusing.

[susidoo]

Hi danfive,

I'm reposting my data combined with clarification from you reply, many points you made were very helpful and sensible. However, I compared my first post with your reply and got confused again.

Data
Antigen: RSA
Primary Antibody (B1-E12): Endogenous mouse anti-RSA polyclonal serum
Secondary Antibody (A1-E12): Goat-anti-mouse IgG-HRP conjugate
Substrate (A1-E12): OPD
2 wells: +Control: Mouse-anti-RSA polyclonal serum AU= 0.923, 0.922
2 wells: -Control: Preimmune mouse serum AU= 0.404, 0.410
2 wells: RSA,coating buffer, blocking buffer, Goat anti-mouse IgG-HRP conjugate AU= 0.428, 0.404

Test sample (Primary antibody) Rows:
B- (saline injections) 0 Ring ID mouse serum in blocking buffer
C- (RSA immunized) 1 Ring ID mouse serum in blocking buffer
D- (RSA immunized) 2 Ring mouse serum in blocking buffer
E- (RSA immunized) 3 Ring mouse ID serum in blocking buffer

Last 3 rows F,G,and H: RSA, coating buffer, blocking buffer only

Questions:
From the above data:
1. What is considered the non-specific binding control? What defines this control?

2. What is the blank? (everything but the anti-RSA polyclonal serum right? Meaning Goat-anti-mouse IgG-HRP conjugate + OPD is included?) I know blanks are everything except for what is being assayed.

I'm sorry for re-asking the same question again, I realized that I left out and mixed up the given data previously. When you replied back (so amazingly), I couldn't decipher between the above two questions.

By the way, thank you so much for your reply; it's cleared up so much of the confusion!!!!!!!!!!!!!!!!!!!!!!!!

PS: If I can get my scanner working, I'll send a file your way as a direct message, in case my question is unclear.

[danfive]

Originally Posted by susidoo

Hi danfive,

I'm reposting my data combined with clarification from you reply, many points you made were very helpful and sensible. However, I compared my first post with your reply and got confused again.

Data
Antigen: RSA
Primary Antibody (B1-E12): Endogenous mouse anti-RSA polyclonal serum
Secondary Antibody (A1-E12): Goat-anti-mouse IgG-HRP conjugate
Substrate (A1-E12): OPD

Serum Positive Control (should keep track of dilution used2 wells: +Control: Mouse-anti-RSA polyclonal serum AU= 0.923, 0.922
Neg. Serum controls/pre-immunization sera 2 wells: -Control: Preimmune mouse serum AU= 0.404, 0.410
Blanks: 2 wells: RSA,coating buffer, blocking buffer, Goat anti-mouse IgG-HRP conjugate AU= 0.428, 0.404

Test sample (Primary antibody) Rows:
B- (saline injections) 0 Ring ID mouse serum in blocking buffer
C- (RSA immunized) 1 Ring ID mouse serum in blocking buffer
D- (RSA immunized) 2 Ring mouse serum in blocking buffer
E- (RSA immunized) 3 Ring mouse ID serum in blocking buffer

Last 3 rows F,G,and H: RSA, coating buffer, blocking buffer only

Questions:
From the above data:
1. What is considered the non-specific binding control? What defines this control?
Blanks (no sera of any kind) are background caused by secondary Ab binding; Non-specific binding is the pre-immunized sera

2. What is the blank? (everything but the anti-RSA polyclonal serum right? Meaning Goat-anti-mouse IgG-HRP conjugate + OPD is included?) I know blanks are everything except for what is being assayed.
The Blank is the plate (coated with antigen in your case) treated by the incubation times, block/wash buffers, secondary Ab and color reagent.....this is all just background noise.
I'm sorry for re-asking the same question again, I realized that I left out and mixed up the given data previously. When you replied back (so amazingly), I couldn't decipher between the above two questions.

BTW what does RSA stand for?

[danfive]

Originally Posted by susidoo

2 wells: +Control: Mouse-anti-RSA polyclonal serum AU= 0.923, 0.922
2 wells: -Control: Preimmune mouse serum AU= 0.404, 0.410
2 wells: RSA,coating buffer, blocking buffer, Goat anti-mouse IgG-HRP conjugate AU= 0.428, 0.404

Test sample (Primary antibody) Rows:
B- (saline injections) 0 Ring ID mouse serum in blocking buffer
C- (RSA immunized) 1 Ring ID mouse serum in blocking buffer
D- (RSA immunized) 2 Ring mouse serum in blocking buffer
E- (RSA immunized) 3 Ring mouse ID serum in blocking buffer

Does the above imply you have multiple mice and thus multiple sera.
When this happens, which is most of the time as it is standard to immunize several animals; then each mouse is tracked for immune response (via the methods you are doing now). But that means mouse 1 is compared as "mouse 1 pre-immunized sera vs mouse 1 post-immunized sera (30,60,90 day mark)."

If desired the sera can then be pooled and retested as "pooled sera pre-immunized vs pooled sera post immunized (30,60,90day mark)."

The point is don't use 1 mouse's pre-immune sera for all the mice; one may be bonkers and the rest may be good producers, but you won't be able to tell if you aren't comparing matching sera.

[danfive]

Originally Posted by susidoo

Data

2 wells: +Control: Mouse-anti-RSA polyclonal serum AU= 0.923, 0.922
2 wells: -Control: Preimmune mouse serum AU= 0.404, 0.410
2 wells: RSA,coating buffer, blocking buffer, Goat anti-mouse IgG-HRP conjugate AU= 0.428, 0.404

Here, make sure you notice that the Blank is avg 0.416 and neg sera is 0.407 (approx). This is a big difference from before when I assumed blank was 0.04.

First thing to note is that they are basically equivalent (neg sera and blank). That is good because these kind of mice shouldn't really be producing any antibody until immunization. But on the other hand it is bad that the background or Blank signal is 0.4. It is kinda high and I think your assay is not yet optimized. You should look for a quick fix like:
1) reasonable secondary antibody concentration like 1-4 ug/ml.
2) Badass blocking buffers: My personal list is topped by StartingBlock and Superblock both from Pierce.

Originally Posted by susidoo

Last 3 rows F,G,and H: RSA, coating buffer, blocking buffer only

I assume these are the wells with 0.04-0.06 OD values. If so, running these wells really not very useful unless you had doubts about reagents spilling over (cross contamination) or reactivity from blocking buffer (some are made with serum proteins, not the ones I mentioned from Pierce though).

Anyway since blanks and pre-immunization sera are equivalent, you are kind of safe.
Are the mice still producing sera; the production usually peaks and plateaus, so very early "draws or bleeds" may actually be low in the antibody concentration.
But the later ones have higher concentrations and thus would create higher signal.

Always good to know that your signal to noise ratio (0.9/0.4, currently) is 4-8X, you are currently about 2X.

[susidoo]

Thanks Dan,

I ended up having to submit my report before hearing from you so I actually didn't mention anything about a blank in my report. Upon speaking to my peers, they explained that a blank is not necessary with an ELISA because of the subtraction of background (I don't know if this is correct or not, maybe you can clarify this assumption).

For the nonspecific binding control... oh man, I ended up saying that was what you were referring to as a blank!

I'm still learning, so I suppose I won't forget now, once I have it all sorted out in my head. BTW, I couldn't send you the file I wanted to... I need more posts before I can directly send messages to other users.

Thanks again,

S

[susidoo]

Originally Posted by danfive

Does the above imply you have multiple mice and thus multiple sera.
When this happens, which is most of the time as it is standard to immunize several animals; then each mouse is tracked for immune response (via the methods you are doing now). But that means mouse 1 is compared as "mouse 1 pre-immunized sera vs mouse 1 post-immunized sera (30,60,90 day mark)."

If desired the sera can then be pooled and retested as "pooled sera pre-immunized vs pooled sera post immunized (30,60,90day mark)."

The point is don't use 1 mouse's pre-immune sera for all the mice; one may be bonkers and the rest may be good producers, but you won't be able to tell if you aren't comparing matching sera.

three mice with RSA (Rabbit serum albumin) 3 injections each, and one mouse with 3 saline injections. For the controls we used catalogue ordered (2nd antibody)Goat-anti mouse IgG-HRP conjugate, postitive control (mouse anti-RSA), and the negative control preimmune serum (I'm not sure, but I think my teacher prepared both herself).

[susidoo]

Originally Posted by danfive

Here, make sure you notice that the Blank is avg 0.416 and neg sera is 0.407 (approx). This is a big difference from before when I assumed blank was 0.04.

First thing to note is that they are basically equivalent (neg sera and blank). That is good because these kind of mice shouldn't really be producing any antibody until immunization. But on the other hand it is bad that the background or Blank signal is 0.4. It is kinda high and I think your assay is not yet optimized. You should look for a quick fix like:
1) reasonable secondary antibody concentration like 1-4 ug/ml.
2) Badass blocking buffers: My personal list is topped by StartingBlock and Superblock both from Pierce.


I assume these are the wells with 0.04-0.06 OD values. If so, running these wells really not very useful unless you had doubts about reagents spilling over (cross contamination) or reactivity from blocking buffer (some are made with serum proteins, not the ones I mentioned from Pierce though).

Anyway since blanks and pre-immunization sera are equivalent, you are kind of safe.
Are the mice still producing sera; the production usually peaks and plateaus, so very early "draws or bleeds" may actually be low in the antibody concentration.
But the later ones have higher concentrations and thus would create higher signal.

Always good to know that your signal to noise ratio (0.9/0.4, currently) is 4-8X, you are currently about 2X.

We actually did a terminal cardiac puncture on all 4 mice, I was very sad... but it's a requirement to the course, and I suppose I should learn the procedure as well...