Antibody beads

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

Both IgG and Csb were found to stimulate the respiratory burst separately and independently in one study but in another was active only in the presence of IgG. The divergent findings may be attributable to differing methods. In one study serum was mixed with agarose beads which fix complement with the binding of Csb- One set of beads was then heated at 50° for 30 min to remove 35 and the other was boiled in 2 M NaCl to remove IgG. The completeness of the removal of each component was verified by the loss of reactivity of the beads with a fluorescent monospecific antibody. Beads prepared in this way with either IgG or with Cjb attached elicited the formation of O by PMNs whereas the release of lysosomal contents occurred only with beads containing both IgG and Cab. The effects of the IgG-agarose were blocked by Ffab lj and those of Cjb were blocked by antiserum to C,. 

The principle approach to immunoassay is illustrated in Figure 1, which shows a basic sandwich immunoassay. In this type of assay, an antibody to the analyte to be measured is immobilized onto a solid surface, such as a bead or a plastic (microtiter) plate. The test sample suspected of containing the analyte is mixed with the antibody beads or placed in the plastic plate, resulting in the formation of the antibody—analyte complex. A second antibody which carries an indicator reagent is then added to the mixture. This indicator may be a radioisotope, for RIA an enzyme, for EIA or a fluorophore, for fluorescence immunoassay (FIA). The antibody-indicator binds to the first antibody—analyte complex, free second antibody-indicator is washed away, and the two-antibody—analyte complex is quantified using a method compatible with the indicator reagent, such as quantifying radioactivity or enzyme-mediated color formation (see Automated instrumentation, clinical chemistry). 

An innovative immunochemo-proteomics method was developed that allowed the identification of an inhibitor of the heme-binding protein (83). This method is based on the coupling of a small molecule, bis-indolylmaleimide-III, a known inhibitor of protein kinase C-a, to the FLAG peptidic epitope. This leads to the isolation of binding proteins from a cell lysate via the reaction of the FLAG epitope with anti-FLAG antibody beads. 

The following example taken from our unpublished work [O. Siiman and A. Burshteyn] on CDS antibody-beads illustrates the strategies involved in obtaining optimum antibody-bead performance in bead-blood assays. The main factors... 

CD3 obtained with lot no. 1 antibody-beads was consistently closer to the %CD3 measured by flow cytometry. This was consistent with CD3/CD3-PE (PE = R-phycoerythrin) competitive binding experiments performed by flow cytometry, which showed that lot no. 1 CD3 antibody had a higher affinity for its antigenic receptors on T-cells, A assoc = b9 x 10 than lot no. 2 CD3 antibody, which had A assoc = 1.1. 5 x The binding constant of CD3-PE for the same... 

Various titers, 1-50 pL, of lot no. 1, 4.2% w/v solids, CDS antibody beads were mixed in duplicate for 8 min with 4(X)pL of whole blood from three donors to establish the dependence of %CD3 (Table 3) in the Coulter STKS2B hematology analyzer assay on bead titer. 

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