Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).

Cordell, Jacqueline; Falini, Brunangelo; Erber, Wendy N.; Ghosh, Anna K; Abdulaziz, Z.; MacDonald, SM; Pulford, Karen; Stein, Harald; Mason, D. Y.

doi:10.1177/32.2.6198355

Cited by 2,999 publications

(1,015 citation statements)

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“…Sections were blocked with 10% normal rabbit serum for 30 min before adding monoclonal antibody against bcl-2 (MAb 124; DBA Italia, Milan, Italy) for 18-24 h at 1:20 dilution. The alkaline phosphatase-antialkaline phosphatase (APAAP) method (Cordell et al, 1984) was then used to amplify the primary antibody signal; the sections were incubated with rabbit anti-mouse antibody for 30 min, and then with mouse monoclonal APAAP for another 30 min. These two steps were then repeated once for 10 min each.…”

Section: Immunohistochemistrymentioning

confidence: 99%

Apoptosis and proliferation in thyroid carcinoma: correlation with bcl-2 and p53 protein expression

Basolo

Pollina²,

Fontanini³

et al. 1997

Br J Cancer

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Summary The aim of this study was to determine the apoptotic cell death in 92 thyroid carcinomas of different histotypes (42 papillary, PTC; 12 poorly differentiated, PDC; 21 undifferentiated, UC; and 17 medullary, MC) by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL). Apoptotic index (Al, evaluated as a percentage of TUNEL-positive cells of neoplastic cells) was calculated in each tumour. The Al was very low in all subtypes of thyroid carcinoma, ranging from a median value of 0.2 in PTC to 1.4 in UC. The proliferative activity was determined by immunohistochemistry using monoclonal antibody, MIB-1. The percentage of proliferating cells was significantly different among the histotypes, increasing with tumour aggressiveness (from the mean value of 3.1 for PTC to 5.6 for PDC and 51.8 for UC). In addition, the ratio between proliferative activity and apoptosis was significantly higher in UC than in the other histotypes. The expression of bcl-2 and p53 protein (important in the modulation of cell death) was correlated (bcl-2, inverse correlation, r2 = 0.1, P= 0.04; p53, direct correlation, r2= 0.11, P= 0.02) with apoptotic index in PTC.Keywords: apoptosis p53; bcl-2; thyroid cancer Cell numbers are regulated by a balance between proliferation, growth arrest and programmed cell death (apoptosis, PCD). Until recently, the majority of the studies on oncogenesis have focused on the regulation of cell proliferation. The finding that alterations of negative growth control, including growth arrest and PCD, play a key role in the development of human cancer has been demonstrated by the explosion of reports in this field (Barr and Tomei, 1994; Canman and Kastan, 1995;Katsikis and Leben, 1995;Salomon and Diaz-Cano, 1995; Stauton and Gaffeney, 1995). PCD is an active cellular process involved in a variety of physiological events in which rapid elimination of unwanted cells must occur. Its importance in the turnover of self-renewing tissues, morphogenesis, embryonic development, maturation of cells of the immune system, as well as in the development of cancer and other pathologies, is increasingly being recognized (Chandler et al, 1994; Cotman and Anderson, 1995;Isner et al, 1995;Osborne, 1995). Apoptotic cells can be identified both by nuclear morphology and by techniques that detect fragmentated DNA. The latter methodology could be applied for the in situ visualization of apoptosis by specific labelling of DNA fragmentation. By using Gavrieli's method (Gavrieli et al, 1992), apoptosis can be detected at singlecell level in formalin-fixed, paraffin-embedded tissue sections, thereby allowing the study of retrospective cases.In the present study, we analysed apoptosis in thyroid carcinoma, focusing on its association with the proliferation rate of the tumours themselves. In addition, since apoptosis can be regulated by the p53 tumour-suppressor gene and by bcl-2, we analysed the p53 and bcl-2 expression on the tumour. WHO (Hedinger, 1988) and subsequent updating (Sakamoto et ...

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Section: Immunohistochemistrymentioning

confidence: 99%

Apoptosis and proliferation in thyroid carcinoma: correlation with bcl-2 and p53 protein expression

Basolo

Pollina²,

Fontanini³

et al. 1997

Br J Cancer

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“…Cytochemical stains such as myeloperoxidase (MPO), Sudan Black B (SBB) were always utilised. Immunophenotyping was carried out on routinely prepared blood and bone marrow smears using the alkaline phosphatase antialkaline phosphatase (APAAP) technique [6,7]. The protocol followed was : …”

Section: Methodsmentioning

confidence: 99%

Immunophenotyping of Acute Leukaemias : A Critical Analysis of 35 Cases

Kumar

Sivadas³

et al. 1995

Medical Journal Armed Forces India

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Accurate classification of acute leukaemias is essential for proper case management. The utility of monoclonal antibodies in the diagnosis and classification of acute leukaemias is well established. This diagnostic utility relates primarily to two poiuts : firstly the distiuction between acute myeloid leukaemia and acute lymphoblastic leukaemia and secondly to subtypes of acute lymphoblastic leukaemia. Leukaemic cells were immunophenotyped using the. alkaline phosphatase antialkaline phosphatase techniques. The monoclonal antibodies were .very useful in distinguishing cases of acute myeloid leukaemia from acute lymphoblastic leukaemia. Cases of acute lymphoblastic leukaemia with CDI0 positivity showed a better prognosis. T-cell acute lymphoblastic leukaemia was uncommon and was associated with unfavourable prognosis. The alkaline phosphatase antialkaline phosphatase technique served as a reliable and convenient method for immunophenotyping of Ieukaemias, MJAFI 1995; 51 : 165-169

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“…NeoR -based PCR was performed as described above. Immunohistological studies with a polyclonal antihuman CD3 antibody ( DAKO, Glostrup, Denmark ) followed by alkaline phosphatase and monoclonal anti -alkaline phosphatase ( APAAP ) APAAP staining 11 were also performed on spleens and livers.…”

Section: In Vivo Studiesmentioning

confidence: 99%

“…NeoR -based PCR was performed as described above. Immunohistological studies with a polyclonal antihuman CD3 antibody followed by APAAP staining 11 were also performed on spleens and livers. We also tested a different schedule of GCV administration, starting on day +8 ( 10 mg / kg daily) until day +22.…”

Section: In Vivo Studiesmentioning

confidence: 99%

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Homing and survival of thymidine kinase-transduced human T cells in NOD/SCID mice

et al. 2002

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The herpes simplex virus thymidine kinase ( HSV -tk ) gene conferring ganciclovir ( GCV ) -specific sensitivity to transduced cells might control Graft -versus -Leukemia ( GvL ) / Graft -versus -Host Disease ( GvHD ). Human T lymphocytes were engineered with an LSN -tk retroviral vector encoding tk and neomycin resistance ( NeoR ) genes. A total of 80Â10 6 tk + lymphocytes were injected intraperitoneally in NOD -SCID mice. Engraftment was evaluated by human CD45 + / CD3 + cytofluorimetric analysis and NeoRbased polymerase chain reaction ( PCR ) on peripheral blood, bone marrow, liver, thymus, and spleen on day + 5. After 14 days, GCV ( 10 mg / kg daily ) cytofluorimetric analysis and PCR were repeated ( day + 19 ). Immunohistological studies with anti -CD3 monoclonal antibody followed by alkaline phosphatase and monoclonal anti -alkaline phosphatase staining were performed on spleen and liver at the same time points. Human CD45 + / CD3 + cells were engrafted in all tissues on day + 5 according to cytofluorimetry, immunohistology, and PCR. Lymphocytes ''homed'' to the white pulp T -cell area and to the red pulp; liver localization is prevalently at the periportal area. After GCV ( day + 19 ), cytofluorimetry and immunohistology showed very few CD3 + cells. PCR identified the transgene in 22% tissue samples ( positive only in thymus and spleen ). GvHD did not occur in any animal. These data demonstrate elevated doses of human -transduced CD3 + cells engraft in NOD / SCID mice; after GCV, very few CD3 + cells can be detected and those that escape treatment can be found in the thymus and in the spleen on day + 19. Lack of full response to GCV may account for cases of GvHD in patients receiving tk -transduced T lymphocytes.

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Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).

Cited by 2,999 publications

References 12 publications

Apoptosis and proliferation in thyroid carcinoma: correlation with bcl-2 and p53 protein expression

Apoptosis and proliferation in thyroid carcinoma: correlation with bcl-2 and p53 protein expression

Immunophenotyping of Acute Leukaemias : A Critical Analysis of 35 Cases

Homing and survival of thymidine kinase-transduced human T cells in NOD/SCID mice

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