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 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 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. 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.
Kalayci
O
O
Department of Pediatrics, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York, USA. okalayci@ato.org.tr
Moallem
H
H
Fikrig
S
S
Chice
S
S
Michl
J
J
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology* / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody; D002648:Child; D005557:Formaldehyde; D005656:Fungal Proteins / Q000276:immunology* / Q000378:metabolism; D005949:Glucose Oxidase / Q000276:immunology*; D006801:Humans; D007231:Infant, Newborn; D051379:Mice; D008807:Mice, Inbred BALB C; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism*; D016707:Tissue Fixation / Q000379:methods
D000911:Antibodies, Monoclonal; D005656:Fungal Proteins; D005557:Formaldehyde; D005949:Glucose Oxidase