Competitive antibody binding to soluble CD16B antigen and CD16B antigen on neutrophils in whole blood by flow cytometry

Abstract

Background Shed receptors from the surface of white blood cells in whole blood have been quantitated using the long and tedious enzyme‐linked immunosorbent assay (ELISA) method. A simple rapid flow cytometric method of analysis for shed antigen in the presence of cell‐bound antigen can be advantageous. Methods Magnetic bead depletion of neutrophils in whole blood with CD16b antibody‐conjugated beads as measured by flow cytometric analysis of the remaining cell suspension was inhibited by the presence of soluble CD16b antigen in the blood plasma of normal donors. We describe a competitive binding assay between labeled and unlabeled CD16b antibody for receptors shed from the surface of formed bodies (cells) into solution. Also presented is a new method of obtaining the amount of soluble antigen in a sample. We determine the total unlabeled and labeled ligand concentration at which the fluorescence intensity of the labeled ligand matches the fluorescence intensity in a control run with only the labeled ligand. Results Normal blood donors showed serum concentration levels of shed CD16b antigen in the range of 1–50 nM as determined by a flow cytometric competitive binding assay. These figures compared favorably with parallel determinations using magnetic bead depletion of targeted neutrophils for washed and unwashed whole blood samples to evaluate the concentration of shed CD16b antigen. Conclusions The competitive antibody binding assay for shed and cell‐bound CD16b antigen can be applied to similar GPI‐linked antigens, for which purified antibody and fluorescent antibody against the same antigenic receptor are available. Cytometry 44:30–37, 2001. © 2001 Wiley‐Liss, Inc.

Preparation of Aminodextran−CdS Nanoparticle Complexes and Biologically Active Antibody−Aminodextran−CdS Nanoparticle Conjugates

“…The nanoparticles of 2−3 nm in diameter are substantially smaller than the 5X-Amdex, coil of 9 to 25 nm in mean square end-to-end distance and MW = 26 to 170 kDa, that was used subsequently in conjugations of monoclonal antibody. Thus, it is possible for more than one nanoparticle to be attached to a single polymer molecule, which is similar to the conjugation of multiple phycoerythrin (PE) molecules together with monoclonal antibody to Amdex. , PE is monodisperse with a molecular weight of 270 kDa and a diameter of 27.7 nm. The following calculations are useful in order to compare the molecular weight and the size of CdS nanoparticles and PE attached to Amdex.…”

“…In the titration of whole blood with CD4−5X-Amdex−CdS/SAM-PE and analysis by flow cytometry, 10 000 gated lymphocytes were analyzed for a blood donor containing 54% CD4+ cells in the lymphocyte population as determined with CD4-FITC. Since normal CD4+ cells contain about 6 × 10 4 receptors per cell, − the analyzed blood sample contained about 3.2 × 10 8 CD4+ receptors. The latter figure is about 20-fold lower than the binding sites on the above beads.…”

“…Using the same level of activation of 5X-Amdex with sulfo-SMCC as in previous conjugations of this polymer with CD3 antibody 42 or with phycoerythrin and monoclonal antibody , would require 0.45 cm 3 of 10 mg cm -3 sulfo-SMCC solution or 1 × 10 -5 mol sulfo-SMCC (MW 436.4 g mol -1 ) per 25 mg of 5X-Amdex. For a two diaminopropane group repeating unit of 4 × 355 Da and 2 units of primary amine per repeating unit, there would be 0.035 mmol of amine groups per 25 mg of 5X-Amdex.The amine-to-sulfo-SMCC molar ratio was 3.5 (activation level = 1/3.5), thus providing enough sulfo-SMCC to activate about 30% of the primary amine groups in 5X-Amdex.…”