Plate protein-coated

Tsouloufis et al.8 used an ELISA to assess the refolding of a recombinant subunit of the extracellular domain of the human muscle acetylcholine receptor expressed in E. coli. The plates were coated with refolded or unfolded protein and then reacted with conformationally dependent MAbs. The use of specific... 

An indirect competitive ELISA has been also developed for the determination of streptomycin and dihydrosticptomyciri in milk (24). Prior to the analysis, the milk sample was skimmed and treated with oxalic acid. The antiserum was raised in rabbits using streptomycin linked to a bacterial protein as the antigen. To perform the test, microtiter plates were coated with streptomycin, and antiserum and milk samples were mixed to be added in the wells where they were incubated for 1 h. Depending on the amount of residues in the sample, more or less antibody remained available for binding to the streptomycin coat. A pig antirabbit antibody-enzyme conjugate was subsequently added and incubated for 90 min. Using a suitable substrate, streptomycin and dihydrostreptomycin could be detected down to 1.6 ppb, whereas quantification could be made possible up to 100 ppb when samples were used undiluted. 

Plates coated with purified soluble antigen. As an alternative to step 7 above, and providing purified soluble antigen is available, 96-well PS or polyvinyl chloride (PVC) plates can be coated with protein, recombinant protein, peptide, or peptide conjugated to a carrier protein Plates are coated as follows... 

Polystyrene microtiter plates are coated with purified target, washed to remove the excess of the protein, then saturated with a specific reagent, such as BSA, dried milk, or gelatin, to block the uncoated plastic surface. 

Alternatively, mass produced protein-coated plates can be purchased from commercial sources but may add significant cost to an assay. Advances in producing modified plastic surfaces to mimic the structure of collagen also have been used to provide an improved environment to culture cells. This approach can eliminate the use of animal-derived products that may contain undefined ingredients (Vukicevic et al. 1992) and provide a more consistent surface. These surfaces may prove to be a useful alternative for screening with cells that do not exhibit the desired responsiveness on a standard tissue culture treated polystyrene surface. 

Most immunochemically based sensors to date have been developed for liquid-phase measurements thus, the TSM resonator has been the device of choice. Of course, other plate-mode devices (SH-APM, FPW) would be equally well suited for liquid-phase detection and may have advantages in terms of sensitivity. A low-frequency (20 MHz) SAW liquid-phase immunoassay device has been reported [27], but operation of SAWs of higher frequencies in liquids is not feasible due to excessive attenuation of the SAW by the liquid. An alternative to in-situ detection is to expose a protein-coated AW device to a liquid-phase sample for a period of time, then dry it [226] the observed frequency shift is proportional to analyte concentration. When using this technique, it is crucial that careful control experiments in the absence of analyte be performed to obtain an accurate idea of the reproducibility of the baseline oscillation frequency throughout the procedure. 

The presence of these spontaneously adsorbed (i.e. adsorbed during minimum exposure times), thin films on polymeric substrates such as polystyrene culture dishes and glass plates usually cannot be demonstrated by other spectroscopic methods. For example, the modified internal reflection spectroscopic technique, in which an auxiliary salt prism (usually the malleable salt KRS-5) is pressed against the demonstrably (by other techniques) protein-coated substrates, always fails this technique is not sensitive enough for the near monolayer ranges required for this demonstration. The required sensitivity is achieved only when adsorption occurs directly on the face of a clean, or thin film-coated, internal reflection element. 

Fig. 6 Principle of the FlashPlate technology. The inner surfaces of the wells in a 96- or 384-well plate are coated with scintillant. After immobilization of the target of interest, the plate is incubated with radioligand and test compounds. Only the bound radioligands are in close enough proximity to the scintillant to elicit a signal. If the test compounds bind to the target protein then the displaced radioligand is freed into solution and no scintillation is produced.

The conjugate of biotinylation-anti-albumin was synthesized and purified as described in the paper. Biotinamidohexanoyl-6-aminohexanoic acid N-hydroxysuccinimide ester (BCNHS) with an extended spacer arm was applied to react with primary amines of the protein which can reduce the steric hindrance. Avidin-biotin chemiluminescence immunoassay procedure Immunoassay procedures are presented in Figure. 1. The wells of the microtiter plates were coated with 150 pL of avidin (3 mg/mL) in a phosphate buffered saline... 

Figure 1 Principle of the competitive lA. In the first format with immobilized Ab (a) the plates are coated with Ab. Analyte and enzyme-labeled analyte compete for the Ab binding sites. In the second format a hapten-protein conjugate is immobilized in the soUd phase (b). This protein conjugate and the free analyte compete for the binding sites of the Ab in solution. (Reproduced from Dankwardt and Hock with permission from Food Technology and Biotechnology.)...

Adsorption differences between ligand and binding protein (coated glass fiber filters, nitro-cellulose/PVDF membranes, polylysin-coated microtiter plates)... 

Microtiter plates were coated overnight with wild-type C. thermocellum cohesin samples (200 pl/well, 270 nM of Coh2CBD t). Plates were blocked for 2.5 h with the above-described blocking solution, and washed three times with TrisNC buffer. The cohesin-dockerin interaction was carried out by the addition of 100 pi of the desired competitor cohesin sample (i.e., wild-type or mutant Coh2CBD t at various concentrations, up to a maximum of 1.3 pM), immediately followed by the addition of dockerin solution (100 pi of XynDocS t to final concentration of 47 nM). Dilutions of the competitor cohesins were carried out in TrisNC buffer containing BSA, to maintain a constant protein concentration. After incubation for 2.5 h, the wells were washed five times, and the amount of dockerin bound to the coating cohesin was detected by means of the fiised-xylanase activity, as described above. 

If ffision protein is going to be directly used for panning, then the phage is initially incubated with tag protein (GST, MBP or other appropriate protein) for 15 min before adding to the protein coated polystyrene plates. 

Important insights into the structure of the avidine-biotin complex have been obtained by SPR. The technique was useful also in determining the absolute amount of protein molecules on metallic films. For that purpose, a gold film on glass plate was coated with a dextran layer exposed to a solution containing certain monoclonal antibodies in a flow-through cell. Optical quantities determined in this way were calibrated by means of radioactively marked antibodies. 

Figure 3. Immunochemical detection of lactose-protein Maillard products in several market and powdered milk samples. ELISA plates were coated with appropriately diluted milk samples and the lactose-derived antigens were detected with LlOl. Apparent antigen amount is shown as ELISA value (absorbance at 405 nm).

Figure 15.9 The results of capture ELISA on native RNase A and formalin-treated RNase A. Right panel, native RNase A (curve 1) and unfractionated formalin-treated RNase A (curve 2). Left panel, individual fractions of formalin-treated RNase A monomer (curve 3), dimmer (curve 4), trimer (curve 5), tetramer (curve 6), and a mixture of oligomers with >5 cross-linked proteins (curve 7). The ELISA plate wells were coated with monoclonal antibody against bovine pancreatic RNase A (1 pg/mL) overnight at 4°C and then blocked with bovine serum albumin. The wells were incubated for lh at 37°C in the presence of various concentrations of antigen in lOOpL of PBS. After washing, each plate well received a 1 4000 dilution of horseradish peroxidase conjugated rabbit polyclonal anti-RNase A antibody followed by incubation at ambient temperature for lh. After washing, detection was achieved using a mixture of 2,2,-azino-di-(3-ethylbenzthiazoline-6-sulphonate) and hydrogen peroxide. Absorbance was monitored at 405 nm. See Rait etal.11 for details.

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