Antibodies plate coating

Fig. 8.7 CTB-GM1-ganglioside binding ELISA assay. Plates, coated first with GMrganglioside and bovine serum albumin (BSA), respectively, were irrigated with total soluble plant protein from chloroplast transgenic lines (3 and 7) and 300 ng of purified bacterial CTB. The absorbance of the GM1-ganglioside-CTB-antibody complex in each case was measured at 405 nm. Total soluble protein from untransformed plants was used as the negative control.

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]. 

Double-antibody sandwich. Antibody is coated on or adsorbed to the plastic plate. The sample to be assayed containing the antigen (antibiotic) is added followed by a second antibody that is conjugated to the enz)rme (horseradish peroxidase, alkaline phosphatase, or 3 galactosidase). The substrate is added and the intensity of the color produced is directly proportional to the antigen in the test sample. 

Antibody inhibition. Antibody is preincubated with the sample being assayed. If any antigen is present in the sample it will bind with antibody. When the assay mixture is added to a microtiter plate coated with antigen, there is a decrease in the intensity of the color produced. 

We (K1) attempted to develop a noncompetitive assay based on the anti-idiotype antibodies for a conjugated bile acid metabolite, ursodeoxycholic acid 7-A-acetyl-glucosaminide (UDCA 7-NAG), which is expected to serve as a diagnostic index for an autoimmune disease, primary biliary cirrhosis. In our assay, the hapten UDCA 7-NAG, a /3-type antibody, and a biotin-labeled a-type antibody were simultaneously added to a microtiter plate coated with an F(ab )2 fragment of a specific anti-UDCA 7-NAG antibody, then incubated at room temperature for 8 h. Bound biotin was then detected with HRP-labeled streptavidin, whose enzyme activity was measured using o-phenylenediamine/H202 as a substrate. This noncompetitive assay system provided a subfemtomole-order sensitivity (detection limit 118 amol) that was 7 times lower than the competitive immunoassay using the same anti-hapten antibody (K2), even with a common colorimetric detection (Fig. 13). Somewhat improved specificity was also obtained namely, better... 

The following protocols are not optimized procedures for EIA, but they are suitable for screening, e.g., for antibody titers of sera or mAb cell culture supernatants. A high-performance EIA has to be evaluated with respect to selection of type of microtiter plates, coating concentration, coating conditions, analyte dilution, sample buffer, washing buffer, incubation times and temperatures, conjugate dilution, and substrate composition. 

Another format to test for newly expressed proteins is provided through different ELISA assays. Typically, one antibody is coated on a microtiter plate and serves as a capture antibody while a second antibody (added later in the process) is labeled with a reporter molecule allowing the read-out with optical devices. These ELISAs can be operated in a quantitative manner, but need to be calibrated. The measurement unit can be traced back to the amount of protein present in the calibrator, independent of whether the calibrator consists of purified proteins or other biological materials (e.g., seeds, leaves). The amount of proteins within a plant-derived matrix (leaves, seeds, grains), however, depends on several factors, including environmental conditions and can thus not directly be related to thresholds expressed in weight-%. 

Enzygnost HBe monoclonal (test plate) microtitration plate coated with monoclonal antibodies to HBe antigen... 

Figure 5. Sensitivity of antigen-trapping assay with monoclonal antibodies HSA-1 and HSA-2. HSA-2 (type B antibody) was coated on the plate, and then human albumin (A) or serum (B) was diluted as indicated and added to the wells. Biotin-conjugated HSA-1 served as the specific probe. The optical density at 410 nm (OD410) was measured after 15 min.

In analogy with constrained peptide libraries, several reports have described the use of small proteins, protein domains, or antibodies as scaffolds for the display of random polypeptide sequences to obtain novel binding proteins or antibodies. Koide et al. (81) used the tenth FN3 sequence, a 94-amino-acid fibronectin domain (82, 83) known to be involved in molecular recognition, as a scaffold to build a phagemid 3+3 library L7 (Fig. 10.16) where less than a copy of modified FN3 was present on each phage capsid. The 10 -member library was screened using plates coated with ubiquitin, a small protein for which native FN3 does not have any affinity. The library was made by randomizing five amino acids in positions 26-30 (BC) and five amino acids in... 

These are useful for mapping epitopes, determining whether or not two antibodies crossreact, and for comparing antibody affinities. They are the basis of quantitative assays (RIA) and use multiwell plates coated either with antigen or antibody, as described in Section 2.3. above. The principle is to either ... 

In this collaborative study fourteen laboratories analyzed six different commodities containing aflatoxin B, at two concentration levels as blind duplicates. Two standards (15 and 50 ng B,/g) were provided, and collaborators were asked to report their results as <15 or >15 ppb. Laboratories with microtiter well readers were asked to determine aflatoxin concentrations both visually and spectrophotometrically. In this kit aflatoxin B,-antibodies are coated onto plastic microtiter wells. The aflatoxin-containing sample is extracted with Me0H-H,0 (55+45). The extract is defatted with hexane, and the MeOH extract mixed with the aflatoxin-enzyme conjugate and added to the well of the antibody-coated microtiter plate. The aflatoxin in the extract and the aflatoxin-enzyme complex compete for the antibody binding sites. The enzyme substrate(ABTS) and HjOj solution are then added, the reaction leading to a colored product in the presence of enzyme. The intensity of color is determined visually or spectrophotometrically at 580nm. 

Several ELISA formats have been considered, and the one currently being tested is called the "hapten tracer" method. First introduced to this project by Dr. Freya Jung while a postdoctoral fellow in Dr. Hammock s laboratory, this method uses microtiter plates coated with a commercial preparation of goat anti-mouse antibody. Enzyme-labeled hapten competes with analyte for receptor sites on a mouse monoclonal antibody specific for the analyte. The amount of enzyme left after washing is inversely proportional to the... 

Table I. Specificities of Monoclonal Antibodies for Various Avermectins. The values are the amount of the indicated compound giving half-maximal inhibition in a competition EIA on plates coated with ivermectin 4 -hemisuccinate-conalbumin (coating antigen 1) or ivermectin-Cg-hemisuccinate-BSA (coating antigen 2) at 25 ng of carrier protein per well. All analogs were approximately 80 20 mixtures of the respective isomers, e.g., Avermectin A2 was a mixture of avermectin A2a avermectin Agb 80 20. Stock solutions of all compounds in methanol were standardized by spectrophotometry (see refs. 14-17 for extinction coefficients and chemical data), and dilutions for assay were made in PBS-Tween-5% methanol. Results are the mean of 8 replicates, and standard errors were approximately 5% of the means. NR = not recognized in amounts up to 10 ppm...

In the stool test, specific H. pylori antigens are detected in microtiter plates coated with polyclonal antibodies. Debate continues regarding its accuracy for assessing treatment outcome some of the variability in results between different studies appears to be caused by lack of standardization of the interval between completion of eradication therapy and stool testing.However, the test is currently recommended for posteradication testing if the urea breath test is not available. 

One of the laboratory tests used in the identification of patients with heparin-induced thrombocytopenia, employs a micro-ELISA plate coated with purified PF4-heparin complex. Upon incubation of patient s plasma, if antibody is present, it binds to the PF4-heparin complex. The bound antibody is detected with goat antihuman antibody coupled to peroxidase, followed by incubation with substrates o-phenylenediamine/H202 (Chapter 8). 

The active caspase ELISAs quantify the events closely following the release of cytochrome c from mitochondria. The active caspase ELISA quantifies a single active caspase family member (19). The procedure uses the covalent modification of the active site cysteine with a biotinylated inhibitor to label active caspases and selected capture on a plate coated with a caspase-specific monoclonal antibody (MAb). In this chapter, a typical characterization of an active caspase ELISA is included as a demonstration of proof that the ELISA quantifies a specific active caspase. The ability of the assay to... 

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.

Specificity of the active caspase-7 ELISA was determined by analyzing captured polypeptides. Capture antibody is the caspase-7-specific antibody coated in the ELISA 96-well plates and is different from the anti-caspase-7 antibodies used for western blotting. Polypeptides that are bound by the capture antibody are referred to as captured. Jurkat cells were incubated with STS for 0-4 h and then with bzVKD-fmk for an additional 1 h. Cell extracts were incubated in 6-well plates coated with caspase-7 capture antibody. After washing, polypeptides captured on the plate were solubilized in SDS sample buffer and Western-blotted. Captured polypeptides were blotted with anti-caspase-7 LSU and anti-caspase-7 SSU to detect polypeptides derived from caspase-7. Captured polypeptides were blotted with HRP-streptavidin to detect the polypeptides covalently modified with the bzVKD-fmk inhibitor. Captured polypeptides covalently modified with bzVKD-fmk is the material that the ELISA quantifies. 

If weak color development causes flattening at the low end of the curve resulting in a loss of sensitivity or failure of the LLOQ calibrator level, the enzyme conjugate may be too dilute, expired, or calibrator dilutions are in error. If this is a bridging assay with an Ig analyte, the concentration of the capture antibody used in plate coating could be too high or too low and require reoptimization. 

---