A chemiluminescence assay for erythrophagocytosis

Abstract

A luminol-dependent chemiluminescence assay for the assessment of the phagocytosis of erythrocytes sensitized with anti-D IgG immunoglobulin by mononuclear leukocytes is described. The mononuclear leukocytes were obtained by apheresis enriched by centrifugation through a density gradient and stored in liquid nitrogen before use. The total reaction mixture, consisting of mononuclear leukocytes-luminol-erythrocytes (either anti-D IgG sensitized or unsensitized controls) was 500 microliters, light detection was by an LKB 1251 luminometer. Peak luminescence was seen between 35-45 minutes, the reaction being exhausted by 120 minutes. Determination of the reproducibility of the assay gave intra- and inter-assay coefficients of variation of 5% and 13% respectively. We found the chemiluminescent response to be affected by the number of erythrocytes used in the assay and by the composition of the medium in which the cells were resuspended, particularly the pH at the initiation of the assay. We also compared the chemiluminescence assay to a microscopic phagocytic assay and found the results virtually identical. However, the former chemiluminescence assay was much easier to perform, marginally more sensitive, less laborious and eliminated any possibility of subjective error.

Tetramolecular immune complexes are more efficient than IVIg to prevent antibody‐dependent in vitro and in vivo phagocytosis of blood cells

“…The sensitized red blood cells were washed four times in PBS and suspended in Hank's balanced salt solution (HBSS; Sigma, Oakville, ON, Canada) containing 1% bovine serum albumin (BSA) (Fraction V, Fisher Scientific Ville St‐Laurent, QC, Canada), at a final concentration of 1%. The erythrophagocytosis assay was carried out as described by Downing et al (1990) with a few modifications. Briefly, an aliquot of PBMC was rapidly thawed at 37°C, washed with PBS containing 1% BSA and incubated at 15 × 10 6 cells/ml in 67 μ l HBSS containing 1% BSA and 1% depleted autologous human serum, in the wells of white flat‐bottom 96‐well microplates (Greiner, MJS Biolynx, Brockville, ON, Canada), for 1 h at 37°C in a humid atmosphere containing 10% CO 2 .…”

CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display

“…This test, performed essentially as described previously (Downing et al, 1990), was used to measure the activation of mononuclear cells by RBCs sensitized with MDJ8s anti-Rh-D antibody. This test, performed essentially as described previously (Downing et al, 1990), was used to measure the activation of mononuclear cells by RBCs sensitized with MDJ8s anti-Rh-D antibody.…”

Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis

“…The clinical significance of antibodies in vivo involves active phagocytosis by macrophages found in the spleen and/ or the liver; thus, this method does not provide a relevant readout of phagocytosis. The CLT uses luminol to monitor the oxidative burst during monocyte phagocytosis of RBC, since luminol fluoresces blue when oxidized in the phagosome 15 . This method is good, but contamination by neutrophils can confound the readout.…”