I am currently doing a comparative study between Becton Dickinson Cam 5.2 and Ventana Cam 5.2. However I had some unexpected results on my control sections. Some inflammatory cells that lined the stomach epithelium, basal cells of tonsil crypts and muscle cells of large bowel were positive for Ventana Cam 5.2. Does anyone have any idea? I've done research through literature but unfortunately there is little information.
How good are your negative control stainings - are the same cells really negative ?
I am not an expert for your question, but did you check with both companies on their latest description on those antibodies - it seems that changed (according to my preliminary check on the internet); so, maybe the companies have some explanation not published anywhere.
Hi, they are supposed to be negative. However, some previous researches for BD Cam 5.2 mentioned that there is staining on muscle cells due to K8 presence and some websites mentioned about staining of reticulum cells within tonsilar crypts. However, nowhere is mentioned about the Ventana Cam 5.2 antibody. The Ventana inserts and website only says that tonsil sections should be negative but does not mention anything about smooth muscle only about skeletal. No information found about staining of inflammatory cells of stomach from either manufacturers or previous researches.
also muscle cells of large bowel (and muscle cells of a case of colonic adenocarcinoma ) are really intensively stained with Ventana Cam 5.2 but not with BD. However muscle of appendix showed to be negative. Which does not make much sense. If you know anything please let me know
Hi Zina, I wonder, why there should be a major difference between the two companies providing that antibody. I could be totally wrong, but isn't it the name for the same clone of antibody, which could be provided by a number of companies? Or are they really two very different antibodies against an epitope called CAM5.2 (I don't expect that, but you should know that better). So, maybe the one (staining) antibody is just a little more concentrated and some less specific or unspecific binding occurs, which needs more washing away? There are so many ways a perfect looking staining could slightly go wrong and show wonderfully stained cells. But if your negative controls are really negative, then you can already exclude many things not related to the antibody.
The clone for both of the Cam 5.2 antibodies is the same. The thing is that BD Cam 5.2 is diluted in 1:60 and was okay as was proven from previous validation studies. However, the Ventana is concentrated and ready to use without any dilutions. The only thing that I could think off is that the composition of the antibody was changed from the manufacturer over time. The negative controls are really negative for both. However the problem is with the non-specific background staining we are experiencing with Ventana Cam 5.2. Based on manufacturers instructions tonsil sections should be negative, however basal cells show positivity and as they differentiating into stratified squamous epithelium the stain faint (which I guess I could call it as negative if that makes sense). I am not completely sure about though.
The companies may purify, dilute and store their antibodies differently; but the results should be the same. I could think of slightly different concentrations of your antibody resulting in a difference in sensitivity and specificity. There are typically several options of blocking unspecific binding, and increasing (multiplying) specific binding - mixed with some washing steps - so it may be easy for a pathologist to shortly look through your slides and make a recommendation, it should be a routine evaluation of what is going on. Not sure if there are pathologists around your research area.
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