Immunoenzymatic assays

We have developed chemiluminescent immunoenzymatic assays for (3-ago-nist drugs in the 96-well-microtiter-plate format. Such competitive assays have been used for determination of clenbuterol and of the overall content of p-agonist drugs in the sample. They matched the standard requirements of precision and accuracy, and were more sensitive compared to the conventional colorimetric methods. Moreover, CL detection was very rapid, making these assays suitable for screening analysis. 

Depending on the isotype (i.e. class/subclass and kind of light chain), immunoglobulin molecules will display a particular biological property and will require an appropriate method for purification. The identification of mAb isotypes generally employs the culture supernatants of hybridomas and commercially available kits for the specific immunoenzymatic assays. This knowledge about the specific isotype facilitates the selection of the purification process in the next step. 

Gosling, J.P. (1980) In Immunoenzymatic assay techniques (R. Malvano, ed.) p. 259. Martinus Nijhoff, The Hague. 

At the moment, validation (through the IRC, Association of Official Analytical Chemists, or multiple laboratory performance test) has not been performed on these kits. Our group has experience with the Soy Protein Residue Assay (Enhanced Assay) (ELISA Systems), which is used routinely for soy detection. This kit was one of the first available commercially for the detection and quantification of soy traces. We verified the applicability of the kit to a wide range of raw and processed matrices with good values for recoveries and reproducibility. Sensitivity is adequate to allergen detection in food (2.5 mg/kg as soy proteins). The extraction phase and subsequent steps of the immunoenzymatic assay are simple and fast. So far we have not found any problem... 

Various immunoenzymatic assays have been developed and several kits are now commercially available [64]. The aim of these techniques is to achieve a simpler, faster, and less costly determination of these compounds than is possible with GLC, while preserving the high degree of sensitivity and specificity that such analyses require. They are particularly useful for conducting a preliminary screening in laboratories where a larger number of samples must be analyzed [65]. 

Intense efforts are being undertaken both to overcome detection limits and to increase the number of determinable compounds, also for the purpose of developing alternative analytical methods that are simpler, cheaper, and require less solvent. Researchers are currently experimenting new methods for purifying samples and new extraction systems specially tailored to the bee s body that rely on techniques such as gel permeation [68] and solid phase extraction. A particular focus is placed on the study of enzymatic and immunoenzymatic assays for classes of compounds these methods may allow samples to be passed through a fast prehminary qualitative screening and thus drastically reduce the number of analyses to be performed. 

E. Boschetti, Immunoenzymatic assay of dyes currently used in affinity chromatography for protein purification, J. Chromatogr. 597 (1992), 315-322. 

The quantification of p2-M concentration in human urine and serum can be performed with immunoassays, e.g., enzyme-linked immunosorbent assay, radio immunoassay [62], latex immunoassay [63,64], immunoenzymatic assay with chemiluminescence detection, and immunoturbidimetric assay [65]. 

The results of fibronectin binding onto pol5mrethanes, as evaluated by I-labelled fibronectin and immunoenzymatic assay (ELISA), are reported in Figure 2 and 3 respectively, and in Table 3. 

On what refers to immunization, antibodies titers, assayed by ELISA were similar to those obtained when immunizing the animals with native venom (data not shown). However, local signs following injection,when present, were discrete, suggesting once again attenuation of venom activity. Immunoenzymatic assay indicates similar immunoreactivity for all venoms assayed excepting Micrurus venom which presented lower reactivity. 

Despite the fact that biological and predominantly immunoenzymatic methods have received increasing attention during the last decade for the determination of aflatoxins, chemical and immunochemical assays have to be preferred for their characteristics, including a lower limit of detection and high specificity. 

For cell surface antigens, the most frequently used assays are immunofluorescence or immunoenzymatic, with their numerous variations. For soluble protein or peptide antigens, the most common assays are immunoenzymatic, enzyme-linked immunosorbent assays (ELISA) or Western blot tests. 

Immunoenzymatic Techniques, INSERM Symposium No. 2 (G. Feldman, P. Dniet, J. Bignon and S. Avrameas, eds.). North-HoUand/Elsevier, Amsterdam, 1975. Enzyme-linked Immunosorbent Assay (ELISA) for Infectious Agents (J. L. Sever and D. L. Madden, eds.). J. Infect. Dis. 136, Suppl. (1977). 

Even though we consider it a good immunoenzymatic kit, some inconveniences can affect analytical determinations using this system. The assay does not always apply to matrices that have undergone thermal processing, hydrolysis, or fermentation, as the protein structure could be destroyed by these treatments. In few cases we obtained doubtful results, probably due to interferences from other food components. In addition to this, as the assay is temperature and time sensitive, it is necessary to perform the analysis complying strictly with the indication reported in the kit instructions. General kit characteristics ... 

Only limited development of new methodologies has taken place for immunochemical analysis of nucleic acids. Most published methods rely on modifications to classical DNA probe hybridization or immunoassay methods, with considerable blending of the two. For example, some methods employ immobilized oligonucleotide probes to capture the analyte DNA followed by immunoenzymatic detection. Other methods use immunocapture followed by detection with an enzyme-labeled DNA probe. Distinctly new methodologies mostly impact on assay formats (e.g., DNA microarrays and in situ hybridization) and detection reagents (e.g., chemiluminescent enzyme substrates). 

Further enhancement in detection sensitivity of reporter enzymes is achievable by enzyme amplification or cascade reactions often termed enzyme cycling assays. One approach is to use alkaline phosphatase as the reporter enzyme. Phosphatase cleavage of NADP forms NAD, which enters cyclic reactions catalyzed by alcohol dehydrogenase and diaphorase. Each turnover of phosphatase substrate initiates a cascade resulting in numerous detectable product molecules. Such approaches, perhaps incorporating chemiluminophores as the terminal product, hold promise for further extension of the sensitivity of immunoenzymatic methods. 

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