Inhibition of nonspecific binding of fluorescent-labelled antibodies to human eosinophils.
Eosinophils and their products play a major role in inflammatory reactions associated with asthma and allergic diseases. There is a growing body of evidence that eosinophils synthesize, store, and release bioactive cytokines and chemokines with the potential to contribute to local inflammatory changes. Fluorescein isothiocyanate (FITC) has been widely used as an immunofluorescent conjugate for antibodies specific for detection of these molecules. However, FITC is an ionic fluorochrome (negatively charged) which binds strongly to positively charged eosinophil granule proteins. We developed new methods to prevent charge-based interactions of ionic fluorochromes with granule proteins, and optimised immunofluorescent staining techniques for eosinophils. An antibody to interleukin-6 (IL-6) was used to optimise this procedure for eosinophil-derived granule proteins. We attempted to block nonspecific binding of FITC-labelled anti-IL-6 using normal human IgG, foetal calf serum (FCS), bovine serum albumin (BSA), and goat, horse, and normal human sera at concentrations ranging between 1-10%. Only human IgG (2%; 20 mg/ml) was able to reduce background fluorescence. These results were confirmed using Texas Red conjugates. We also used antibodies conjugated to a nonionic fluorochrome, BODIPY FL, to detect IL-6 in eosinophils. Unlike FITC, BODIPY FL-conjugated antibodies did not require strong blocking conditions (2% BSA). We recommend that a neutral fluorochrome (BODIPY FL) should be used for immunofluorescence studies in eosinophils. Alternatively, strong blocking conditions may be used to decrease background binding of FITC-conjugated antibodies.
Mahmudi-Azer
S
S
Pulmonary Research Group, University of Alberta, Edmonton, Canada.
Lacy
P
P
Bablitz
B
B
Moqbel
R
R
D000818:Animals; D000937:Antigen-Antibody Reactions; D016477:Artifacts; D001249:Asthma / Q000097:blood; D001798:Blood Proteins / Q000378:metabolism; D001896:Boron Compounds; D002417:Cattle; D003594:Cytoplasmic Granules / Q000378:metabolism; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000276:immunology* / Q000378:metabolism; D005189:False Positive Reactions; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism*; D006041:Goats / Q000097:blood; D006736:Horses / Q000097:blood; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology*; D015850:Interleukin-6 / Q000032:analysis / Q000276:immunology; D018613:Microscopy, Confocal; D011485:Protein Binding; D012260:Ribonucleases; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014966:Xanthenes
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001798:Blood Proteins; D001896:Boron Compounds; D047091:Eosinophil Granule Proteins; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D015850:Interleukin-6; D014966:Xanthenes; D012710:Serum Albumin, Bovine; C034657:Texas red; D012260:Ribonucleases; D016650:Fluorescein-5-isothiocyanate