Epitope enhancement for immunohistochemical demonstration of tartrate-resistant acid phosphatase.
We have developed a monoclonal antibody (9C5) for immunohistochemical localization of tartrate-resistant acid phosphatase (TRAcP). This antibody reacts with a denatured epitope of TRAcP and requires enhancement methods to promote antigenicity in paraffin-embedded tissues. We used this antibody to systematically examine proteolytic digestion and heat denaturation conditions for epitope enhancement in both paraffin sections and fixed smears. The goal was to increase the sensitivity of the immunohistochemical stain for TRAcP. Optimal conditions for proteolytic digestion were established. Denaturation in a conventional boiling water bath was compared to microwave irradiation in several commonly used solutions. Immunohistochemistry was compared directly to TRAcP cytochemistry in fixed smears from hairy cell leukemia specimens to gauge the level of sensitivity of our improved method. Attempts were made to "retrieve" the 9C5 epitope from overfixed tissues and aged smears. Maximal immunoreactivity of TRAcP was achieved by microwave irradiation in a citrate or Tris buffer of pH 6.0-8.0 without the need for a subsequent protease digestion step. With this method of epitope enhancement, immunohistochemistry with antibody 9C5 was as sensitive as direct cytochemical staining of TRAcP activity. However, once a tissue specimen had been overfixed or a smear stored for a year or more, the 9C5 epitope was no longer retrievable. The key element in epitope enhancement for 9C5 immunohistochemistry is heat denaturation of the target epitope. Immunohistochemistry of TRAcP in paraffin sections would be a great asset to the study of specialized forms of the monocyte/macrophage lineage and to the process of macrophage activation. It would also provide another means for more precise evaluation of residual disease in bone marrow of patients treated for hairy cell leukemia.
Janckila
A J
AJ
Medical Service, Department of Veterans Affairs Medical Center, Louisville, Kentucky 40206, USA.
Lear
S C
SC
Martin
A W
AW
Yam
L T
LT
D000135:Acid Phosphatase / Q000032:analysis* / Q000276:immunology; D014408:Biomarkers, Tumor / Q000032:analysis*; D000939:Epitopes; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007527:Isoenzymes / Q000032:analysis* / Q000276:immunology; D007943:Leukemia, Hairy Cell / Q000201:enzymology*; D012680:Sensitivity and Specificity; D000071681:Tartrate-Resistant Acid Phosphatase
D014408:Biomarkers, Tumor; D000939:Epitopes; D007527:Isoenzymes; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase