Immunoassay detection levels

Several considerations influence the suitability of the immunoassay as a qualitative or quantitative tool for the determination of tissue residues. These include the assay format, the end user (on-farm or laboratory use), effects of sample matrix on the analysis, cross-reactivity considerations, detection levels required of the assay, target tissues to be used in the assay, and the use of incurred or fortified tissues for validation of the immunoassay against accepted instrumental methods. Although these variables are often interrelated, each topic will be discussed in further detail below. 

Immunochemical methods have been developed and placed on the market to analyze tetracycline residues (see Table 4). Thus, a qualitative EIA has been developed and used to analyze tetracyclines in honey samples with a detection level of 20 pg/kg-1 [96]. A microplate-based indirect ELISA has been developed to analyze tetracyclines using polyclonal antibodies. The assay could measure tetracycline in the range between 0.1 and 6 ng mL L Other tetracycline antibiotics such as chlortetracycline, rolitetracycline, or minocycline are also highly recognized in this assay [98]. Several immunoassay kits are commercially available for the analysis of tetracyclines although, to our knowledge, none of them... 

Immunoassay Methods. Radioimmunoassay (RIA) allows measurement of biologically active materials which are not detectable by traditional cold chemistry techniques. RIAs can be used to measure molecules that cannot be radiolabeled to detectable levels in vivo. They also are used for molecules unable to fix complement when bound to antibodies, or they can be used to identify cross-reacting antigens that compete and bind with the antibody. 

In 1982, the first enzyme immunoassay of clenbuterol was described (134). It was used to determine clenbuterol levels in plasma of human patients treated by oral route with this drug. It was a highly sensitive double-antibody and heterologous immunoassay based on a competition for binding to a clenbuterol-specific antibody between a diazotized clenbuterol analogue labeled with -galactosidase and unlabeled standard or sample clenbuterol. The antibody-bound enzyme hapten was separated from free hapten by anti-rabbit IgG immobilized to a polystyrene ball. The assay could detect levels as low as 0.5 pg clenbuterol per tube. 

The need for shortening the time and increasing the sensitivity for detection of antigens has lead to development of different amplification systems. Some of the initial efforts focused on use of more pure antibodies (59, 60), more specific monoclonal over less specific polyclonal antibodies (61) and use of a combination of monoclonal antibodies (62). The next phase saw the incorporation of labels as discussed in the previous section. The use of labels does increase the sensitivity however, there is a need to go down in detection levels to enable faster turnaround time for immunoassays. This can mean significant savings in the food industry. In the case of Salmonella, the assay time is being reduced from several days to less than a day (63, 64). 

PCBs can be conveniently determined by most of the common analytical techniques which include GC-ECD, GC-HECD, GC-FID, GC/MS, HPLC, NMR, and enzyme immunoassay. Among these, GC-ECD and GC/MS are by far the most widely used techniques for the determination of PCBs in the environmental samples at a very low level of detection. While the former can detect the PCBs at subnanogram range, the mass selective detector (GC/MS) identifies the components relatively at a higher detection range, 10 to 50 times higher than the ECD detection level. GC/MS, however, is the best confirmatory method to positively confirm the presence of PCBs, especially in heavily contaminated samples. Aqueous and nonaqueous samples must be extracted into a suitable solvent prior to their analysis. 

Several qualitative and quantitative immunochemical methods and their application to the analysis of environmental samples have been described for OP insecticides, a family that includes widely used pesticides such as azinphos-ethyl/methyl, dichlorvos, fenitrothion or fenthion, malathion, mevinphos, and parathion. Mercader and Montoya202 produced monoclonal antibodies against azinphos-methyl and developed an ELISA that was used for the analysis of water samples from different sources, reaching detectability levels near 0.05 pg I. Watanabe et al.203 reported the production of polyclonal antibodies and ELISA procedures to analyze fenitrothion in river, tap, and mineral water (LOD = 0.3 pg L ). Banks et al.204 produced polyclonal antibodies against dichlorvos, an organophosphate insecticide used for stored grain, which also cross-reacts with fenitrothion. Nishi et al.205 reported the first immunoassay for malathion. Residues of this insecticide have... 

Immunoassays detect and quantify unknown levels of antibody or antigen and may be competitive or noncompetitive and heterogeneous or homogeneous. 

The Immunoassay submitted for evaluation must be "mature," because expensive evaluation studies cannot be undertaken on unoptimized methods. The developer must first optimize and evaluate the immunoassay in-house, preferably by individuals most familiar with the assay system. Parameters addressed should include bias, precision (repeatability and reproducibility at the detection levels of interest), and rate of false positive and false negative results. The Agency should be supplied the raw data documenting assay performance. When possible, this information should be submitted on an IBM compatible floppy disk. A review of the data must be conducted before undertaking the evaluation study to verify interpretations of the data made the developer. 

The staphylococcal enterotoxins are moderately stable proteins therefore, immunological evaluation should be possible on samples collected in either deployed or fixed medical treatment facilities. Immunoassays can detect picogram quantities of toxins in environmental samples. For comparison, 440 pg/mL was reported as the mean concentration of TSST-1 in human sera from patients with toxic shock syndrome.25 Anti-TSST-1 antibody titers are either suppressed or depleted in patients with toxic shock syndrome26,27 and the levels only recover during convalescence. In addition, most normal human serum samples contain detectable levels of antibody reacting with several different bacterial pyrogenic toxins, including... 

Immunoassay results for samples collected during the fall low-streamflow period were similar to the pre-application results (Table IV). Ninety percent of the samples had triazine concentrations of 1.1 ug/L or less and only one sample had triazine concentrations larger than 3 ug/L. This sample was determined by GC/MS to contain 3.1 ug/L of atrazlne. However, about 60 percent of the fall low-flow samples contained detectable levels of triazines by Immunoassay (0.2 ug/L or more) compared to 45 percent of the pre-application samples. Additional analysis will be made... 

An unusual example concerns the presence of a PTH-like factor in the circulation of cancer patients. The clinical condition indicated a considerable concentration of PTH-like activity, associated with loss of bone and hypercalcemia (the hypercalcemia of malignancy). Immunoassay detected no elevated PTH levels only the cytochemical bioassay was able to show high concentrations of PTH-like activity, although such activity did not show true parallelism. The material causing this effect has now been isolated and purified it is known as the PTH-related peptide. 

Quantitative immunoassays have also been used as screening devices to determine whether drug residues exceed established maximum residue limits (MRLs) or tolerances in edible tissues. " For these applications, a cut-off value is set at the tolerance or MRL samples detected above this level are positive , and samples below this level are negative. ... 

Van Emon et al. ° developed an immunoassay for paraquat and applied this assay to beef tissue and milk samples. Milk was diluted with a Tween 20-sodium phosphate buffer (pH 7.4), fortified with paraquat, and analyzed directly. Fortified paraquat was detected in milk at less than 1 pgkg , a concentration which is considerably below the tolerance level of 10 pg kg Ground beef was extracted with 6 N HCl and sonication. Radiolabeled paraquat was extracted from ground beef with recoveries of 60-70% under these conditions. The correlation coefficient of ELISA and LSC results for the ground beef sample was excellent, with = 0.99, although the slope was 0.86, indicating a significant but reproducible difference between the assays. 

Matsumoto et al. developed an immunoassay for the determination of clenbuterol in bovine and equine tissues and in bovine milk. The LOD of clenbuterol in milk, muscle, liver, kidney, small intestine, and adipose tissues was 0.1 qgkg Bovine tissue samples fortified wifh 1 qg kg of clenbuterol had recoveries that varied from 75 to 96%, but recoveries from milk samples were 99%. The authors utilized this method to estimate the clenbuterol withdrawal periods for cattle and horses. Cattle were treated with a bolus dose of either 0.3 or 0.6 qg kg body weight, by intravenous injection, and three animals were slaughtered at days 1, 6, and 9. Tissue clenbuterol levels were detectable only on day 1. Clenbuterol in milk was not detectable after a 2.5-day withdrawal period. Liver contained the highest clenbuterol concentration of the tissues measured, but this group did not measure eye tissues. 

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