ELISA well coating

Where the protein microarray differed from the classic ELISA was in the much smaller quantities of proteins deposited on the substrate. While micrograms ( 1(P g) of protein are employed in the coating of an ELISA well, microarray spots may contain only picograms ( 10" g) or less of protein. The accurate delivery of small volumes [picoliters (pL) or nanoliters (nL)] containing small amormts of protein can be problematic. A certain amount of protein is likely to adsorb onto the quills or capillaries used for printing. The degree at which adsorption takes place is dependent upon a number of factors such as buffer composition, quill surface features and, more importantly, the physical and chemical nature of fhe protein itself. 

Coat each of four ELISA wells with 100 pL antigen (1—10 pg/mL in O.lMsodium hydrogen carbonate) overnight at 4°C. 

Make a dilution of the capture antibody in coating buffer from 0.5 to 10 pg/mL and use to coat ELISA wells. The binding capacity of ELISA wells may be reduced if the concentration of antibody is not optimal. Overdilution, and paradoxically underdilution may lead to poor well binding. In some competition ELISA assays up to 100 pg/mL may be required. 

A nonisotopic ELISA method in which serum specimens are added to microtiter wells coated with human Tg is also available. In this method, antibody binding is assessed using a peroxidase-conjugated anti-IgG/o-phenylenedi-amine system. An automated two-step fluorescent enzyme immunoassay has been reported. In this assay, Tg is immobilized on magnetic beads, and anti-human IgG mouse monoclonal antibody is labeled with alkafine phosphatase 4-methyiumbelliferyl phosphate is used as the substrate. IRMA and ELISA both have similar detection limits (approximately 3 to 5 U/mL). A considerably more sensitive radioassay has been reported in which diluted serum is incubated with T-labeled Tg to allow formation of antigen-antibody complexes these complexes are then precipitated by adding solid-phase protein A. Its detection limit is reported to be approximately 0.2 U/mL. 

Fig. 4. Detection of cytochrome c release from mitochondria in the short format cytochrome c release assay. (A) Assay buffer, HRP-detection antibody, and 52 nM caspase-8-cleaved human Bid (solid bars), 52 nM human cleaved Bid and 156 nM mouse Bcl-xL (open bars), no additions (light gray bars), or Triton X-100 (stippled bars), were added to a 96-well ELISA plate coated with cytochrome c capture antibody. Mitochondria were added and the plate was incubated at 30°C for 30 min (A) or 10 min (C). The wells were washed for 2 min and color developing reagent was added for 3 min. (B) shows a 35-min assay in which the concentration of human cleaved Bid was varied. Open triangle indicates cytochrome c detected when Triton X-100 was added to mitochondria and open diamond indicates cytochrome c detected when no Bcl-2 family proteins were added to mitochondria.

By coating the ELISA wells with individual peptides containing the sequences of the SLP, ELP and FCB blocks, we were able to determine where the immunoreactivity of the SELPF polymer was directed. The final sera from each of the animals exhibiting elevated reactivities reacted only with the SLP peptide with a reactivity score of 108. No significant reactivity was directed at the ELP or FCB peptides. 

The anti-TSST-1 titers of immunized mice were determined by an ELISA for each injection group with pooled sera taken 5 days after the final boost. Pooled sera were diluted serially in PBSTG and added to ELISA wells previously coated with a 1 pg TSST-l/ml of carbonate buffer solution. Data are presented as the mean reading of triplicate samples SD. 

Figure 1 Schematic sequence of the direct and indirect competitive ELISA. The principle difference is that for direct competitive immunoassay, the well is coated with primary antibody directly, and for indirect competitive immunoassay, the well is coated with antigen. Primary antibody (Y), blocking protein (Y), analyte (T), analyte-tracer ( ), enzyme labeled secondary antibody ), color development ( J)...

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.

Although other assay containers can be used (e.g., coated tubes or beads), the most popular one is the 96-well microtiter plate. ELISAs that are detected in a microplate luminometer must be performed in opaque white or black plates. 

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

Membranes cast upon glass slides also fall into the 3D surface category. Most notable are the nitrocellulose-coated FAST slides offered by Schleicher Schuell (S S) BioScience. A cytokine micro-ELISA product xmder the trade name Provision has been introduced in single-slide and 96-well spacing (64 usable wells) formats. It is called FAST Quant (Harvey, 2003). Historically, nitrocellulose membrane has been used for the sequestering of both proteins and nucleic acids. The adaptation by S S of this microporous (0.2-p pores) material cast into a microarray format has been relatively straightforward. 

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