Selective binding of a monoclonal antibody to aspergillus niger glucose oxidase by formaldehyde fixed human polymorphonuclear leukocytes

Background: Many of the procedures used in handling neutrophils may affect the expression of surface antigens, and hence their quantitation by flow cy tometry. Methods: Because the enzyme glucose oxidase of Aspergillus niger is absent in human tissues, an IgM against it (mAb GO) was used as negative control i n a study involving the normal expression of neutrophil specific BH2-Ag in different age groups. Results: When peripheral blood leukocytes (PBL) were freshly prepared, proc essed and stained with FITC-mab GO without fixation or when the cells were stained with FITC-mAb GO prior to fixation with 2% formaldehyde, both media n fluorescent intensity (MFI) and per cent of positively stained polymorpho nuclear leukocytes (PMN) were similar to that obtained with a background sa mple without any antibody. However, when PBL were fixed after isolation wit h different concentrations of formaldehyde and for varying: durations, MFI and per cent of positively stained PMN but not of monocytes or lymphocytes with FITC-mAb GO increased in a time and concentration dependent manner. Sa turation was achieved at a finite concentration of the antibody. In a compe tition assay unlabelled mAb GO reduced binding of FITC-mAb GO to PMN by 79% and 95% at concentrations 100 and 200 times that of FITC labelled antibody , respectively. Conclusions:These observations strongly suggest that formaldehyde fixation causes the expression or accessibility of an epitope on PMN that is specifi cally recognized by the mAb GO. Cytometry 39:260-265, 2000. (C) 2000 Wiley- Liss, Inc.