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What is it that you are quantifying with your ELISA? Is it a therapeutic human IgG that binds to a target and your coating peptide is a portion of that target? Just trying to understand your ELISA.
The combination of your biotinylated antibody and streptavidin-HRP in a pre-incubation/complexing step may be unrobust and will need titering for each lot used or perhaps you should consider avoiding the complex thing completely. If each lot of biotinylated antibody has a varaible free biotin component (and the free component may increase with inappropriate storage), it will variably reduce the capacity of the streptavidin to bind the antibody. Also, the proportion of streptavidin in your streptavidin-HRP prep and number of available biotin binding sites on each streptavidin molecule (it's usually 4, but some will be blocked by the HRP), and the extent of biotinylation of your anti-human IgG (one or multiple biotins on each molecule), all will contribute the the size of your anti-human IgG-biotin-streptavidin-HRP complex and will ultimately affect the number of HRP molecules attached (indirectly) to your human IgG. If you manage to create a complex of 20 HRPs for example, then you will get a signal 20 x higher than if you don't generate a complex at all.
Someone probably once titered the streptavidin-HRP and anti-human IgG-bioin and came to the conclusion that the pre-incubation complex route gave greater sensitivity for THOSE LOTS OF REAGENTS. It may be beneficial to make things simple, and add the anti-human IgG biotin and streptavidin-HRP sequentially with a wash step between. In this way, there is no possibilty of free biotin left in the biotinylated antibody blocking the streptavidin, and no unreproducible complexes of biotinylated anti-human IgG/streptavidin-HRP to deal with.
So the critical question is: has there been a lot change in either the streptavidin-HRP or the anti-human IgG? Has the biotinylated antibody been stored incorrectly and released some of it's biotin?
More details of your assay may allow us to give additional suggestions,
good luck!
#144963 Human IgG Elisa variability problems
Posted
on 09 November 2012 - 08:35 PM
#142167 ELISA SETUP,negative control OD is higher than tests
Posted
on 24 September 2012 - 04:26 PM
So, if I have understood correctly, you are coating the vaccine antigen on the plate, and then capturing specific mouse anti vaccine antibodies on the immobilized vaccine antigen, and then detecting with a specific species (goat for example) anti-mouse enzyme conjugate. All mice will have an abundance of mouse IgG in their serum, and your vaccinated mice have an abundance of mouse IgG in their serum, a portion of which is specific for the vaccine.
So, the secondary antibody (anti-mouse conjugate) is binding to the contents of all wells somehow. This could be because the reagent has gone off and is binding nonspecifically to the contents of the wells, or alternatively the IgG within the samples is binding to the well contents and the binding is happening by some process independent of the vaccine antigen on the plate.
In assays to detect human IgGs in various species including human (my experience) the sample quality is often very important in getting a clean signal. Several freeze thaws and a few months at -20 will be enough to result in binding of non-specific sample IgG to the plate making it impossible to differentiate signal from noise.
Another possibility could relate to the blocking (and diluent) Has the blocking solution been stored for a while without preservative? If the blocker has gone off, then you might expect the IgG within the samples to bind to the incompletely blocked plastic surface.
So suggestions:
Test fresh blank matrix (mouse serum)
Test fresh blocker and different lot
Test fresh anti mouse detection reagent and different lot
Test diluent only instead of anti mouse detection reagent (to make sure there isn't something else in the assay affecting the chromogen)
Prepare fresh coating buffer in disposable plastic as IgG does have a habit of getting into coating solutions especially when reusable glass is used to prepare them
Please let us know how you get on
good luck
So, the secondary antibody (anti-mouse conjugate) is binding to the contents of all wells somehow. This could be because the reagent has gone off and is binding nonspecifically to the contents of the wells, or alternatively the IgG within the samples is binding to the well contents and the binding is happening by some process independent of the vaccine antigen on the plate.
In assays to detect human IgGs in various species including human (my experience) the sample quality is often very important in getting a clean signal. Several freeze thaws and a few months at -20 will be enough to result in binding of non-specific sample IgG to the plate making it impossible to differentiate signal from noise.
Another possibility could relate to the blocking (and diluent) Has the blocking solution been stored for a while without preservative? If the blocker has gone off, then you might expect the IgG within the samples to bind to the incompletely blocked plastic surface.
So suggestions:
Test fresh blank matrix (mouse serum)
Test fresh blocker and different lot
Test fresh anti mouse detection reagent and different lot
Test diluent only instead of anti mouse detection reagent (to make sure there isn't something else in the assay affecting the chromogen)
Prepare fresh coating buffer in disposable plastic as IgG does have a habit of getting into coating solutions especially when reusable glass is used to prepare them
Please let us know how you get on
good luck
#139397 Plasma samples with lower OD than blank
Posted
on 13 August 2012 - 02:59 PM
The composition of your samples is entirely different from the composition of your curve and it would not be unexpected to have results as you describe due to matrix bulk effect differences.
If you don't have endogenous protein in normal human plasma, then prepare your curve in normal human plasma and you'll be comparing like with like. You may have an anti-rodent antibody effect, but I would expect this to elevate your response in matrix rather than to decrease it. Perhaps consider a 2 fold minimum required dilution (MRD) of your standards and samples (both in human plasma) in something like 1% casein-PBS (Pierce) containing 5% mouse plasma to be safe.
If you don't have a source of human plasma without endogenous analyte, then consider mouse plasma to build the curve (and it shouldn't contain immunoreactive analyte because your capture antibody was made in mouse). Alternatively, try a range of commercial diluents. ICT has a sample pack. It is important to demonstrate parallelism between your curve, and analyte spiked into the study sample matrix.
When you have figured out a suitable curve diluent (human or animal plasma, or commercial diluent) and the need for an MRD or not, perform selectivity experiments in multiple lots of plasma from donors matching your study samples and the blanks should be blank, and the spikes should recover consistently against the curve. If there is endogenous, then the unspiked measured concentrations + spiked measured concentrations should add up.
Is your milk grocery store stuff? If it is, then perhaps move to a commercial vendor casein which has a lot #/expiry data assocated with it.
Also, drop the 37 degree incubations, it usually results in edge effects better to keep everything at lab ambient temperature, go for longer if you need to.
Also, don't know the rationale for the final water wash, especially as your HRP has been happily sitting in physiological buffer since preparation and the brief exposure to zero salt concentration may result in your immuno sandwich to fall apart and wash off.
good luck!
If you don't have endogenous protein in normal human plasma, then prepare your curve in normal human plasma and you'll be comparing like with like. You may have an anti-rodent antibody effect, but I would expect this to elevate your response in matrix rather than to decrease it. Perhaps consider a 2 fold minimum required dilution (MRD) of your standards and samples (both in human plasma) in something like 1% casein-PBS (Pierce) containing 5% mouse plasma to be safe.
If you don't have a source of human plasma without endogenous analyte, then consider mouse plasma to build the curve (and it shouldn't contain immunoreactive analyte because your capture antibody was made in mouse). Alternatively, try a range of commercial diluents. ICT has a sample pack. It is important to demonstrate parallelism between your curve, and analyte spiked into the study sample matrix.
When you have figured out a suitable curve diluent (human or animal plasma, or commercial diluent) and the need for an MRD or not, perform selectivity experiments in multiple lots of plasma from donors matching your study samples and the blanks should be blank, and the spikes should recover consistently against the curve. If there is endogenous, then the unspiked measured concentrations + spiked measured concentrations should add up.
Is your milk grocery store stuff? If it is, then perhaps move to a commercial vendor casein which has a lot #/expiry data assocated with it.
Also, drop the 37 degree incubations, it usually results in edge effects better to keep everything at lab ambient temperature, go for longer if you need to.
Also, don't know the rationale for the final water wash, especially as your HRP has been happily sitting in physiological buffer since preparation and the brief exposure to zero salt concentration may result in your immuno sandwich to fall apart and wash off.
good luck!
#126702 Possible to calculate avidity/affinity from an ELISA?
Posted
on 08 January 2012 - 05:58 AM
OK, so I'm presuming that you have data from indirect ELISAs where the antigen is on the plate and you are generating a curves by varying the concentration of the test antibodies which are then detected using labelled anti antibodies.
We know that the experiments were designed not with the goal of quantfying and differentiating differences between affinity and avidity and we know that the data will not yeild accurate assessments of either.
I suspect what the reviewer is suggesting is that you do some non linear regression approach to derive affinity parameters from the data you have. This is fine to do, but if you do that, you will have to present the analysis highlighting the limitations of the approach which is the possibility that one antibody binds with one arm, the other both, or both antibodies with both, and that the antigen may not be presented as it would be in solution, and that the binding interaction was not stopped and the results not assessed with the minimum of delay because no cold wash was performed and a subsequent incubation which allowed an undefined level of dissociation to occur. Additionally, if one of your antibodies is IgG1 and the other IgG2 or some other variant for example, you have the considerations that the binding of the detection antibody to your test antibodies may also vary.
good luck!
We know that the experiments were designed not with the goal of quantfying and differentiating differences between affinity and avidity and we know that the data will not yeild accurate assessments of either.
I suspect what the reviewer is suggesting is that you do some non linear regression approach to derive affinity parameters from the data you have. This is fine to do, but if you do that, you will have to present the analysis highlighting the limitations of the approach which is the possibility that one antibody binds with one arm, the other both, or both antibodies with both, and that the antigen may not be presented as it would be in solution, and that the binding interaction was not stopped and the results not assessed with the minimum of delay because no cold wash was performed and a subsequent incubation which allowed an undefined level of dissociation to occur. Additionally, if one of your antibodies is IgG1 and the other IgG2 or some other variant for example, you have the considerations that the binding of the detection antibody to your test antibodies may also vary.
good luck!
