Antibody gating

Autoantibodies are directed against nicotinic acetylcholine receptors in myasthenia gravis, resulting in receptor loss, skeletal muscle paralysis, and dysfunction (100). In addition, antibodies directed against voltage-gated Ca " channels produce similar neuromuscular dysfunction of Lambert-Eaton... 

Coating the antigen or antibody directly on appropriately modified metal substrates has yielded electrodes that respond potentiometrically to antigen-antibody reactions An immunoelectrode has been prepared by coating the gate of a... 

Secondary antibodies incubate sections for 60 min at room temperature with a secondary HRP-conjugated antibody raised against the corresponding IgG of the primary antibody. Using DAKO HRP-EnVision System instead of HRP-conju-gated antibody at this step will contribute for further powerful signal amplification. Wash sections in PBS for 3x3 min. 

Internalization The toxins diffuse laterally in the membrane, bind to protein receptors and are internalized by receptor-mediated endocyto-sis. After this stage is completed, the toxins are protected against antibody inactivation. The internalized HC forms aggregates and induces pores in endosomal membrane. The pores serve as gates through which the LC enters a cytoplasm (Ledoux et al., 1994). 

Secondary antibody (affinity-purified fluorescent antiglobulin conjugate reactive with the species globulin of the first step, e.g., affinity-purified rhodamine-conju-gated goat antimouse IgG (H + L chains) (Jackson ImmunoResearch, West Grove, PA) diluted in NGG-sap-PBS (25 pg/mL). This can be stored in this form frozen, harvested back from the dish, and reused the same as the first antibody solution. Minimum volume = 1 mL for each dish to be incubated. 

In typical labelling protocols, antibody or antibody fragment immunoconju-gates with fluorophores are synthesized by incubating a buffered solution (pH... 

After incubation with antibody and second antibody HRP-conju-gate, wash the blot three times with about 1 ml/cm of Soln. A for 5 min each. Be sure that no buffer used after the incubation with conjugate contains sodium azide or another inhibitor of HRP. 

Analyze a control sample (FITC/PE/PE-Cy5 control antibody incubation) using the scatter gate to restrict the analysis to lymphocytes... 

Fig. 3. Three-color analysis of peripheral blood lymphocytes for CD45RA, CD45RO, and CD4. Lymphocytes were stained with antibodies directly labeled with FITC, PE, and PECy5, respectively. The distribution of control labeled cells is shown in A and B, and labeled cells in C—F. The relationship between CD45RA and CD45RO in CD4-positive cells is shown in (F), which is gated for PECy5-positive cells.

For detection of myc-tagged antibody fragments, use 9E10 MAb and HRP-conju-gated monoclonal antimouse IgG Fc secondary antibody. 

This method was modified by Lisi et al. (L8) using antibody-coated microspheres [Ab— -]. Added unlabeled antigen [Ag] and fluorescein-labeled antibody [F-Ab] bind to the antibody-coated microspheres as in the conventional sandwich immunoassay [F-Ab Ag Ab - -]. After the reaction is complete the suspension is introduced into a flow cytometer with a laser light source. By gating fluorescence light accumulation on scattered light pulses,... 

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