ELISA albumin

Miyake et al reported an ELISA method for the determination of pesticide residues in the aquatic environment. The polyclonal antibody and three monoclonal antibodies of acifluorfen were prepared by immunization of rabbits and mice with acifluorfen-bovine serum albumin conjugates. The polyclonal antibody reacted with acifluorfen at concentrations of 1.5-800 mg L , while the monoclonal antibodies reacted with acifluorfen at concentrations of 1.5-144 mg L . Among three monoclonal antibodies, AF 75-144 reacted with chlornitrofen, which did not react with the other two antibodies. It seems that the ELISA method is effective for the determination of herbicide residues in the aquatic environment. 

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.

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.

Cattaneo, C., Gelsthorpe, K., Phillips, P and Sokol, R.J. (1992). Reliable identification of human albumin in ancient bone using ELISA and monoclonal antibodies. American Journal of Physical Anthropology 87 365-372. 

Braun and Alsenz6 used an ELISA to detect aggregates in interferon-alpha (IFN-a) formulations. They analyzed IFN-a formulations for possible aggregate formation because all marketed interferons are reported to induce antibodies to some extent. Because of its stabilizing effects, human serum albumin (HSA) is used in the formulation of marketed IFN-a at a great excess over IFN-a itself. HSA can also interact with other proteins. Braun and Alsenz developed an ELISA for the detection of both IFN-a-IFN-a and HSA-IFN-a aggregates. A MAb was used for the capture and detection of the IFN-a and a polyclonal for the detection of HSA. The assay is shown schematically in Figure 11.4. 

Cell Culture-Derived Media-Derived Protein Impurities. Immunoassays can detect low impurity levels (<1 ppm).4 The ELISA is probably one of the most sensitive analytical methods. If bovine serum is used as a media component, then testing should include ELISAs for bovine serum albumin (BSA), bovine transferrin, bovine fetuin, and bovine IgG. Often hormones and growth factors, such as insulin or insulinlike growth factor, are used as media components. ELISAs should be used to detect and quantitate these residuals in the various production steps as well as in the final product. There are commercially available antibodies to most commonly used media components. If proprietary media components are used, then the same investment in time and effort is required for the production of specific antibodies, as described above for host cell impurities. 

The optimum combination of all available methods, including the determination of albumin and IgG by laser nephelometry or by another suitable method (ELISA or particle-enhanced immunonephelometric determination if IgM and IgA), will help in the future to significantly increase the sensitivity of the assays in the CSF diagnostics of multiple sclerosis, thereby improving the correlations between individual examinations. 

Beyond polyclonal antibodies, monoclonal antibodies to isoxazolyl penicillins were recently produced by immunization of mice with a cloxacillin-human serum albumin conjugate prepared by a mixed anhydride procedure (35). Sensitivity and specificity of these antibodies were tested in an indirect ELISA in which a cloxacillin-glucose oxidase conjugate prepared by an activated ester procedure served as a coating agent. It was found diat die prepared antibodies could be... 

In another method for the determination of sulfamethazine in muscle and liver tissues (59), the extraction problem was successfully addressed by applying a matrix solid-phase dispersion procedure for rapid and efficient purification of the tissue extracts. Determination was made by ELISA on the basis of antibodies raised against sulfamethazine-diazo-bovine serum albumin conjugates. 

For lasalocid assay, polyclonal antibodies were raised in sheep (98). These antisera were applied in an ELISA validated for chicken serum, liver, and muscle. Bridge homology in the ELISA was overcome by absorbing unspecific antisera onto a conjugate between salinomycin and chicken serum albumin, which was immobilized onto Biosilon beads. The assay was highly specific for lasalocid and was capable of detecting it at concentrations less than 0.15 ppb. 

Bovine serum albumin (BSA) 1992 A Factor 100,000 (IPCR 9.6 x 10 22 mol ELISA 96 amol) Sano et al. [10], initial publication of the IPCR STV-protein A chimera for coupling biotinylated DNA with an antibody (see Fig. 2A and Section 2.1.1)... 

Plates are blocked with 300 xl/wcll of ELISA Block (ELISA Wash with 1% bovine serum albumin [BSA]) for a minimum of 1 h at room temperature. 

The ELISA procedure recently has been used for the analysis of parathion (31). Since this procedure has considerable potential a more detailed description of the analysis of parathion is in order. The conjugation procedure using amino parathion (AP) was described earlier (Fig. 3, Rn 9), and this conjugate was then administered to rabbits for development of a population of specific antibodies (Abi) against BSA or AP. Abi demonstrated immunological activity only for the hapten when AP was conjugated to rabbit serum albumin (RSA). This antigen (RSA-AP) was rendered insoluble via attachment to the polystyrene surface of microtiter plates under basic conditions (Fig. 6.1). 

Ascites Production. Anti-albumin clones HSA-1 and HSA-2 were thawed, subcloned, and reassayed by ELISA on human albumin, and a rapidly growing subclone was expanded for ascites production. Forty male BALB/c mice were primed with an intraperitoneal injection of 0.5 mL pristane on day 0. On day 14, 2 X 106 cells were injected into each mouse. Ascites fluid was collected over a 1-week period and pooled. Cells and other debris were removed by centrifugation. Approximately 180 mL of ascites fluid was obtained. 

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