Nicolai,

 

Your approach seems reasonable if you have a priori defined  acceptance criteria that meet the needs of your assay.   You say that matrix components may be left within the sample after TCA stipping and column, so in that case, selectivity may be a good parameter to assess.  So, you would want to know whether your assay works in multiple different lots (individuals) of matrix rather than just a single lot.  Blank, low QC and high QC spikes in 6 to 10 individual lots of matrix is usual.  Also, you might consider inter assay precision and accuracy as you may just get lucky with the first plate.

 

hope it works!

So you presumably have a capture and detection reagent specific for your protein of interest and you are building a curve in a matrix (diluent) that isn't human serum and perhaps less complex....so the serum has a blocking property and blocks non-specific binding sites for the assay reagents so you get higher background in the curve matrix (buffer or something that doesn't have the blocking property).  Let us know what reagents you are using for the assay and what you are trying to detect and we'll try to advise.

So I am presuming that you wish to estimate the quantity of antibodies specific to your protein x, which is either a pathogen/ vaccine / biological therapeutic. Each individual will mount an immune response to protein X in his/her own individual way so the antibodies from each individual will bind to protein X with varying affinity/avidity, so your results will always be relative to whatever standard you adopt and not truly quantitative.

It may be possible to affinity purify specific antibodies from positive serum samples using a protein X affinity matrix followed by acid elution of the specific antibodies and quantify the eluted antibodies and create a standard curve from that. You could use this as your own reference which you will always refer back to. Alternatively, you could generate a polyclonal anti protein X antibody in a different species (and this may be acceptable in certain disciplines but your detection antibody would have to cross react with human and poly species IgG) or generate an engineered human mAb or panel of human mAbs specific for protein x. Several providers can do this for you, one you might look into is HuCal antibodies from Abd Serotec if funds permit.

With that said, there are published guidelines for immunogenicity testing of therapeutics/vaccines etc, which take into consideration many factors including antigen tolerance (your assay won't work if you have antigen X in the sample binding to the anti-antigen X antibodies). Relevant guidelines for anti-therapeutic antibodies can be found by googling 'Immunogenicity testing guidelines - Millipore', I am sure you can find similar guidelines for vaccines/pathogens/or other...time spent becoming familiar with the specific approaches for your particular discipline would be very useful before moving forward.

I think all here are agreed that the human IgG quantification kit is not helpful in this endeavour.

good luck

As both Missile and I are suggesting, you are running two assays on one plate...it makes little sense to compare the results of your assay with the specific protein coat to a curve with a different coat for the reasons we have both indicated. If you require only relative results...create a pool of serum containing your antibodies for your specific protein, use this to prepare your curve with arbitrary units, and use this as a reference for moving forward.

When ODs fall, what is 'slower than expected'? Each assay format will behave differently depending on the binding affinities and avidities of the antibody/antigen interaction and the curve will fall slower with one format than another. When you say high CVs, do you mean high CVs between replicates of the same sample, or CVs generated from back calculated results at different dilutions of the same sample? If your replicate wells give the same ODs when loaded with the same sample at the same dilution, there is hope for the assay, just stick with the specific protein coat and find a way to standardize the curve so you can compare like with like.

For TMB, as Missile suggests, 30 minutes before stopping TMB is a little on the long side from my experience too.

Good luck

I am having difficulty in understanding what you are trying to measure and what the format of the assay is.

In order to compare quantities of unknown against your human IgG standard curve, the entire plate should be coated with the same thing (i.e bethyl anti-IgG Fc or your specific protein, but not both). It seems that you are running two different assays on one plate:

assay 1 (curve): capture with anti-human IgG Fc, human IgG standard, detect with another anti-human IgG-HRP (or other conjugate)

assay 2 (samples): capture with specific protein (presumably some viral or vaccine antigen), samples containing unknown titres/quantities of anti-antigen antibodies, detect the antibodies with the anti-human IgG-HRP

As the two formats rely on different coating efficiencies and binding kinetics, it is not surprising that the two curves exhibit lack of parallelism (can not be super-imposed).

Unless I am missing something, the entire plate should be coated with your specific protein, and you need a reference serum (or purified standard) containing or consisting of human anti-antigen IgG to use as your standard.

Let us know how you get on, or if you need any more help with this assay

good luck!

I understand your issues with kits from non-reputable manufacturers. There are two categories of kit supplier: those who are experts in the field who rely on scientific niche application and reputation, and those who rely on providing a kit for any molecule with minimal scientific justification and make their living from single no-repeat sales.


It seems that the assay you have works to detect the OSM in manufactured buffer (TC supt etc) , so playing with incubation times etc is icing on the cake, but does not address fundamental questions of work/work not in serum. Similarly with human anti animal antibodies, this may be a problem in some serum lots but not all.

I agree, there is probably a high abundance binder of OSM in serum, and the litmus test for success is consistent spike recovery in multiple donor lots of human serum/plasma. An alternative is that there may be a blood born protease degrading the protein.

If its a protease, you could add a broad spectrum protease inhibitor (Sigma P8340 for example) to the serum at 1% V/V and repeat the spike in experiments. Although that will give a reason why you can't measure the protein, it doesn't offer a solution, because the protein will be degraded by the protease in serum/plasma samples prior to adding the inhibitor.

If it's a binding protein, you need to find a way to kill the binder, but maintain the eptiopes to which the antibodies bind on the OSM intact. Various armaments can be deployed, but it's a process of trial and error.

1) 1 Modify pH. Combine serum with equal volume of 1 M acetic acid (to hit ~ pH 3) or ~ 250 mM HCl (to hit lower pH) and neutralize in diluent on the coated plate with 0.5 M Tris at an appropriate pH (to neutralize). Don't be afraid to leave the sample to cook in the acid for a while (do a time course) as the OSM is reportably pH stable and the binders may not be. Also, you can play around with the pH of the capture antibody incubation (don't have a neutralization step after acidification for example) as the binders may stay unbound but the antibody-OSM binding may work over a wide pH range (I have seen monoclonals bind well at < pH 2).

2) Modify salt concentration of the sample diluent. 1 M NaCl for example

3) 10% DMSO to the sample diluent

4) Heat the sample to denature the binders but leave the OSM intact. OSM is reportedly quite heat stable. It will be necessary to dilute the serum to prevent it solidifying in the heat, so a 3 fold dilution in diluent (1% casein-PBS from Pierce for example or the R+D systems sample diluent) and a 10 minute heat treatment at 70 degrees may well work for you. It's a case of trial and error with the temperature and the duration.

I have used all of these strategies multiple times with different molecules (with success). My best bet would be heat and second acid.

Good luck!

It certainly would not be a recommended approach to re-use an ELISA plate from a kit. If the reagents are stripped off using acid (1 M acetic acid for example) there may be some binding activity left, but the assay performance is likely to be changed so that the dynamic range is likely to be shifted and precision is likely to be widened. If the kit is obtainded from a reputable manufacturer the cost of the kit is a reflection of the manufacturer's research and effort put into developing the kit so that you can provide quality research data. The dollars that you might save by trying to reuse these plates will work against the quality of your research.

Good luck!