The figure above shows chemiluminescent staining of Der p 1 allergen exposure of an entire disk. The 12-hour exposure was performed in an exposure chamber using high mite-content dust from culture; it is not a natural domestic sample.
We were not able to get press-blotting working as well as well as we had hoped and more work is required. The supplementary data from the 2016 PLoS paper discussing press-blotting can be found here, (link)
While for some applications we prefer Halogen as it allows visualisation of the individual particles that are the origin of the allergen, it also has some technical issues. These include that it is slow, (18-24 hours for entire run), there can be an annoying background, the immunostaining can be weak and difficult to resolve among other particles and non-specific rubbish and it is difficult to quantify the intensity of the staining, at least in our hands.
The long time required for incubations and the multiple washes is because all reagents must enter and leave via diffusion through the protein-binding membrane (PBM) sandwich created by the PBM-(particles)-impermeable adhesive film. To get good halos, it is better to use 0.2 micron PBM rather than the more porous 0.45. This slow diffusion can also result in some granular background colouration. Most (but not all) of our work has used coloured substrates generated by enzyme labels, and these are difficult to quantify the density and interpret as quantity of allergen.
We rationalised that we were not particularly interested in particles per-se, and could get better quantification if we used press-blotting. This was originally described by Schumacher and has been extensively practiced by Takahashi and others.
That is, the particles are collected onto an adhesive, then press-blotted onto a damp PB membrane. This is then removed leaving an unlaminated PBM carrying the allergen for analysis. This unlaminated membrane would be quicker and cleaner to wash. We did this with chemiluminescence in order to attempt quantification (light emitted is proportional to allergen quantity). We did not find any of the adhesives used in the literature gave good results and we used iPad protective film adhesive (as described elsewhere here).
In the unpublished preliminaries, we found that while this worked, it was much better if the blotting membrane was pre-coated with the capture antibody (from the pair in the ELISA kit), blocked, press-blotting performed and then binding of allergen detected using the biotin-labelled second antibody followed by chemiluminescence, using standard protocols and equipment as used for Western Blotting. More details are provided here.
To quantify allergen there should (in theory) also be standards printed onto the membrane. While we tried to microdot these on, this was not particularly successful, and again it is a skill that requires development.
The other technical skill required here would be more expertise in getting chemiluminescent detection working on a microscopic scale, (we used a gel scanner).
The overall methods has a lot of promise and would whole 12-hour samples to be processed as a single entity.
However it inevitably risks being biased towards the detection of the particles carrying the most allergen, and the presence of small particle carrying low amounts of allergen might be missed.
While for some applications we prefer Halogen as it allows visualisation of the individual particles that are the origin of the allergen, it also has some technical issues. These include that it is slow, (18-24 hours for entire run), there can be an annoying background, the immunostaining can be weak and difficult to resolve among other particles and non-specific rubbish and it is difficult to quantify the intensity of the staining, at least in our hands.
The long time required for incubations and the multiple washes is because all reagents must enter and leave via diffusion through the protein-binding membrane (PBM) sandwich created by the PBM-(particles)-impermeable adhesive film. To get good halos, it is better to use 0.2 micron PBM rather than the more porous 0.45. This slow diffusion can also result in some granular background colouration. Most (but not all) of our work has used coloured substrates generated by enzyme labels, and these are difficult to quantify the density and interpret as quantity of allergen.
We rationalised that we were not particularly interested in particles per-se, and could get better quantification if we used press-blotting. This was originally described by Schumacher and has been extensively practiced by Takahashi and others.
That is, the particles are collected onto an adhesive, then press-blotted onto a damp PB membrane. This is then removed leaving an unlaminated PBM carrying the allergen for analysis. This unlaminated membrane would be quicker and cleaner to wash. We did this with chemiluminescence in order to attempt quantification (light emitted is proportional to allergen quantity). We did not find any of the adhesives used in the literature gave good results and we used iPad protective film adhesive (as described elsewhere here).
In the unpublished preliminaries, we found that while this worked, it was much better if the blotting membrane was pre-coated with the capture antibody (from the pair in the ELISA kit), blocked, press-blotting performed and then binding of allergen detected using the biotin-labelled second antibody followed by chemiluminescence, using standard protocols and equipment as used for Western Blotting. More details are provided here.
To quantify allergen there should (in theory) also be standards printed onto the membrane. While we tried to microdot these on, this was not particularly successful, and again it is a skill that requires development.
The other technical skill required here would be more expertise in getting chemiluminescent detection working on a microscopic scale, (we used a gel scanner).
The overall methods has a lot of promise and would whole 12-hour samples to be processed as a single entity.
However it inevitably risks being biased towards the detection of the particles carrying the most allergen, and the presence of small particle carrying low amounts of allergen might be missed.
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