HPLC-ELISA

A comparison of ELISA, HPLC, and HPLC-ELISA methods was published for the determination of SAL in chicken liver tissue. Samples were homogenized with MeOH and extracted with methylenechloride. Some samples were analyzed by HPLC using the isocratic solvent system and postcolumn derivatization (vanillin in MeOH containing sulphuric acid), with the eluent monitoring at 520 nm. The HPLC-ELISA system was used to characterize nonspecific effects analyzing column fractions by ELISA, since this detection is over 1000 times more sensitive than the spectrophotometric one. This alleviated the need to derivatize the drug prior to the detection (104). 

Brunn, H. 1994. Identification and quantification of dexamethasone and related xenobiotic corticosteroids in cattle urine with ELISA and HPLC/ELISA. Archiv Lebensmittelhyg 45 96. 

Assays and reagents Dose retain analysis HPLC ELISA... 

Coupling of two (separation) techniques (hyphenated or orthogonal techniques, multidimensional chromatography) e.g. HPLC-GC, HPLC-TLC. HPLC-CE, HPLC-Elisa, HPLC-Westemblott. 

Carbonyl groups apo B Spectrophotometric HPLC ELISA Westem/Dot blot... 

The data from animal models and from human samples reported above supports the rationale of measuring 7-medG in peripheral blood cells and the use of hplc-ELISA together with other complementary techniques appears feasible. 

Aflatoxins Deoxynivalenol Fumonisins Monilifomin Ochratoxin Zearalenone TLC, HPLC, ELISA, immunoaffinity column GC, HPLC, ELISA, immunoaffinity column HPLC, ELISA, immunoaffinity column HPLC TLC, HPLC, ELISA, immunoaffinity column TLC, HPLC, ELISA, immunoaffinity coluum... 

Hagedom, H.-W. Schulz, R. Jaeschke, G. Identification and verification of the anabolic steroid boldenone in equine blood and urine by HPLC/ELISA, Biomed.Chromatogr., 1994, 8, 63-68. 

Kramer HJ, Stevens J, Seeger W. Analysis of 2- and 3-series prostanoids by post-HPLC ELISA. Anal Biochem 1993 214 535-543. 

HPLC, high-petfomtance liquid chromatogtaphy ELISA, enzyme-linked immunosorbent assay method RIA, radioimmunoassay method. 

There are many proteins in the human body. A few hundreds of these compounds can be identified in urine. The qualitative determination of one or a series of proteins is performed by one of the electrophoresis techniques. Capillary electrophoresis can be automated and thus more quantified (Oda et al. 1997). Newer techniques also enable quantitative determination of proteins by gel electrophoresis (Wiedeman and Umbreit 1999). For quantitative determinations, the former method of decomposition into the constituent amino acids was followed by an automated spectropho-tometric measurement of the ninhydrin-amino add complex. Currently, a number of methods are available, induding spectrophotometry (Doumas and Peters 1997) and, most frequently, ELISAs. Small proteins can be detected by techniques such as electrophoresis, isoelectric focusing, and chromatography (Waller et al. 1989). These methods have the advantage of low detection limits. Sometimes, these methods have a lack of specifidty (cross-over reactions) and HPLC techniques are increasingly used to assess different proteins. The state-of-the-art of protein determination was mentioned by Walker (1996). 

A monoclonal antibody-based ELISA has been utilized to determine ceftiofur levels in milk. The authors noted that matrix interference occurred, but a 1 100 dilution lowered the interference, and a 1 1000 dilution eliminated the matrix interference. Because of the high dilution, samples could not be measured below l.Opgkg The assay measured ceftiofur, its major metabolite desfuroylceftiofur, and ceftiofur protein conjugates and has been utilized to measure residues in milk from cows treated with therapeutic doses of the drug. The results from the incurred residue correlated well with a previous study using radiolabeled ceftiofur, confirming the detection of a metabolite that was not detected by HPLC. 

The most commonly measured product of DNA oxidation is 8-hydroxy-deoxyguanosine (8-OHdG). This product is usually measured by HPLC methods however, the recent development of commercial ELISA kits is expanding research in this area. 

Other attempts have been made to detect BPA at a low concentration range. Thus Kodaira et al. [274] analyzed BPA in urine samples with an assay that showed a working range between 0.5 and 5 pg L The assay was validated by HPLC. DeMeulenaer et al. [275] developed an indirect competitive ELISA using PAbs obtained from chicken egg yolk, but the assay achieved an IC50 value of only 570 pg L-1. 

Immunoaffinity procedures have also been developed to selectively extract corticosteroids from different sample matrices. Thus, Seymour et al. demonstrated the higher efficiency of the immunoaffinity methods compared with the conventional extraction procedures using organic solvents [177]. Immunosorbents have also been used for online procedures followed by HLPC-UV [178, 179], HPLC-APCI-MS [179,180], GC-MS [176,181], or capillary electrophoresis [182]. Poly(hydroxyethyl methacrylate) (HEMA) was evaluated as a support material for the anti-dexamethasone antibodies used in IAC. The online IAC-HPLC-MS allowed determination of dexamethasone and flumethasone in equine urine with LODs in the range 3-4 ng mL-1 [180]. The cross-reactivity values obtained in the ELISA and the recoveries of an IAC-HPLC procedure are presented in Table 7. Bagnati et al. developed an immunoaffinity extraction... 

The ELISA can be used as one component of a battery of analyses. Rarely is only one method used in isolation. Other tests include chromatographic methods such as reversed-phase high-performance liquid chromatography (HPLC), size exclusion chromatography, and physical structure analytical methods such as UV spectral analysis, mass spectroscopy, etc. 

If the product is an antibody, then it is essential to distinguish the immunoglobulin product, e.g., mouse IgG, from any media immunoglobulin components, e.g., bovine IgG. Lucas et al.16 developed an immunoassay to measure nanogram quantities of bovine IgG in the presence of a large excess of a structurally homologous protein, mouse MAb. The bovine IgG was a contaminant that copurified with the product from a protein A column. For the bovine IgG assay, whole IgG and protein A-purified IgG reacted differently in the assay. It is important to evaluate these types of assays for cross-reactivity. For other media components, such as chemicals or antibiotics, ELISA is probably not the most appropriate method due to the low immunogenicity of chemicals. Techniques such as HPLC would be better to detect these chemical components. 

A variety of analytical methods, such as ELISA and HPLC, can be used to evaluate the effect of excipients or lyophilization on the stability of the biophar-maceutical product. Some parameters the analytical methods should evaluate are degradation, chemical and physical changes, aggregation, adsorption, and loss of biological activity. 

Because of antibody-based selectivity, ELISAs are capable of handling samples that are impure or only semipurihed. It is possible to perform ELISAs in a variety of matrices. This is in contrast to other methods such as HPLC that require relatively pure material. During the development and validation of the ELISA method, it needs to be demonstrated that the ELISA is not affected by interfering substances that could be in the test sample, such as buffers, salts, contaminating proteins, and excipients. It also needs to be demonstrated that the conjugated antibody does not bind nonspecihcally to the coated solid phase. 

Over the years, in addition to developments with ELISA reagents such as labels, there have been improvements in automation. This has enabled ELISA to be utilized as a high-throughput tool. Typically, ELISAs can be performed in several hours to days. The most common practice is to precoat the microtiter plate for an overnight incubation period, with the remainder of the steps performed the following day. While ELISAs are fast when compared to other assays such as bioassays, which can take days to weeks, they might be considered slow when compared to methods like HPLC, in which the time from sample injection to chromatogram is a matter of minutes. 

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