Erythrocyte coating

Drugs frequently bind to blood elements directly (e.g. penicillin to erythrocytes) and the antibodies to the resultant complex react with, and damage, cells coated with the drug. Viruses, especially those that bud, become associated with the host cell surface antigens with the resultant generation of Tc cells. 

This flattened erythrocyte preparation has been used to study reversible nonspecific adsorption kinetics and surface diffusion of insulin on the external surface of erythrocytes.(123) The nonspecific adsorption of insulin to the polylysine-coated substrate is very large compared to the adsorption to the flattened membrane adhered to the substrate. Fortunately, this nonspecific background fluorescence can be very successfully quenched simply by preparing the polylysine coating on an aluminum-film-coated glass surface, rather than on bare glass. As discussed in Section 7.3, the aluminum abolishes the fluorescence of fluorophores adsorbed directly onto the polylysine substrate, but the fluorophores adsorbed to the erythrocyte surface are not substantially quenched, because they are spaced at least two membrane thicknesses away. 

The huffy coat is the top most layer of plasma after erythrocyte sedimentation. It has a higher leukocyteierythrocyte ratio than other regions of the plasma. Nevertheless, whole blood, without erythrocyte sedimentation, also may be used. 

Among the several experimental models available to evaluate the involvement of the complement system [31], the measurement of hemoglobin released from antibody-coated sheep erythrocytes (CH50 test) is frequently selected because of its simplicity. Briefly, the lower the CH50, the higher the activation of the complement system. The CH50 test has been extensively used to compare NS-CA [32]. 

Lysis of antibody-coated red cells through the products of the respiratory burst appears to be linked to phagocytosis Neutrophils from patients with chronic granulomatous disease lysed antibody-coated erythrocytes less effectively than PMNs from norma subjects in suspension, but when phagocytosis was prevented by attachment of the erythrocytes to plastic or by treatment with colchicine, PMNs from the patients with chronic granulomatous disease lysed the red cells normally. 

Principles to stabilize lipid bilayers by polymerization have been outlined schematically in Fig. 4a-d. Mother Nature — unfamiliar with the radically initiated polymerization of unsaturated compounds — uses other methods to-stabilize biomembranes. Polypeptides and polysaccharide derivatives act as a type of net which supports the biomembrane. Typical examples are spectrin, located at the inner surface of the erythrocyte membrane, clathrin, which is the major constituent of the coat structure in coated vesicles, and murein (peptidoglycan) a macromolecule coating the bacterial membrane as a component of the cell wall. Is it possible to mimic Nature and stabilize synthetic lipid bilayers by coating the liposome with a polymeric network without any covalent linkage between the vesicle and the polymer One can imagine different ways for the coating of liposomes with a polymer. This is illustrated below in Fig. 53. 

Figure 8.11 Electrophoretic mobility and zeta potentials of sheep erythrocyte. (Left) Relationship between true electrophoretic mobility in sheep erythrocyte and pH using NCE chips coated with BSA, gelatin, and MPC polymer. (Right) Relationship between sheep erythrocyte zeta potentials and pH using NCE chips coated with BSA, gelatin, and MPC polymer [39].

Freshly collected venous blood is anticoagulated with K-EDTA (1 mg/ml blood) or heparin (5 IU/ml heparin sodium) and centrifuged at 3000 rpm for 7 min. The supernatant (plasma) and the buffy coat are removed and discarded. The packed erythrocytes are resuspended in autologous plasma containing 0.25 % human albumin and the haematocrit value is fixed at 10 %. The red blood cells are altered by one or several of the stress factors mentioned above. 

Whole blood is centrifuged at 600 x g for 10 min at 4°C in graduated centrifuge tubes using a swing out rotor. The plasma and buffy coat (a layer of white blood cells over the erythrocytes) are removed by aspiration. Packed erythrocytes are washed twice in 10 vol. of isotonic 5 mM phosphate buffer pH 7.4, or other buffer as required, by centrifugation at 600 x g for 10 min at 4°C. 

If unfixed avidin-coated erythrocytes are used, lyse rosetted erythrocytes by resuspending the pellet in ammonium chloride soludon (1 mL for... 

In addition to the direct reaction of the antigen with antibody, techniques are available for performing indirect reactions. Antigens can be coupled chemically or via a tannic acid procedure to red blood cells. The antigen-coated erythrocytes in the presence of specific antibody then agglutinate (clump) as do red blood cells in the presence of antibody to the red blood cells (hemagglutination). 

Modifications are made also in order to use this reaction to conjugate nucleosides to erythrocyte surfaces, allowing use of the coated cells as targets for assays of antibody-forming splenic lymphocytes. Nucleoside, 10-20 mg, is oxidized in 1.5 ml of 0.1 M sodium periodate in 0.15M NaHCOa for 20 min at room temperature the reaction is stopped by the addition of 15 /zl of ethylene glycol. Sheep erythrocytes are washed twice with 0.15 M NaHCOs, and 0.5 ml of packed cells is then suspended in 2.0 ml of the bicarbonate solution in a40-ml centrifuge tube. The oxidized nucleoside is added dropwise to the cell suspension and the mixture is kept at room temperature for 15 min. fert-Butylamine borane (Aldrich Chemical Co.), 100 mg in 5 ml of 0.15 M NaHCOs, is added. The suspension is kept at room temperature for 3 min, and the tube is then quickly filled with bicarbonate solution and centrifuged at 1500 rpm for 10 min. 

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