Selective binding of a monoclonal antibody to Aspergillus niger glucose oxidase by formaldehyde fixed human polymorphonuclear leukocytes
✍ Scribed by Omer Kalayci; Hamid Moallem; Senih Fikrig; Seto Chice; Josef Michl
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 142 KB
- Volume
- 39
- Category
- Article
- ISSN
- 0196-4763
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✦ Synopsis
Background: Many of the procedures used in handling neutrophils may affect the expression of surface antigens, and hence their quantitation by flow cytometry. 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 in a study involving the normal expression of neutrophil specific BH2-Ag in different age groups. Results: When peripheral blood leukocytes (PBL) were freshly prepared, processed 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 median fluorescent intensity (MFI) and per cent of positively stained polymorphonuclear leukocytes (PMN) were similar to that obtained with a background sample without any antibody. However, when PBL were fixed after isolation with different concentrations of formalde-hyde 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. Saturation was achieved at a finite concentration of the antibody. In a competition 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 specifically recognized by the mAb GO.