Enzyme linked immunosorbent assay standardization

In each of the assays of potency the amount of the immunoglobulin and the amount of a corresponding standard preparation that are required to neutralize the infectivity or other biological activity of a defined amount of virus or to neutralize a defined amount of a bacterial toxin are determined. The two determined amounts and the assigned unitage of the standard preparation are then used to calculate the potency of the immunoglobulin in International Units (lU). ELISA, enzyme-linked immunosorbent assay. 

In order to measure the exact amount of a specific protein (analyte) by IHC signal intensity, a critical requirement is the availability of a standard reference material (present in a known amount by weight) that can be used to calibrate the assay (IHC stain). It is then possible to determine the amount of test analyte (protein) by a translation process from the intensity of IHC signals. In this respect it is helpful to consider the IHC stain as a tissue based ELISA assay (Enzyme Linked ImmunoSorbent Assay), noting that ELISA is used in the clinical laboratory as a standard quantitative method for measuring protein by weight in fluids, by reference to a calibrating reference standard. 

Immune detection is a key utility of antibodies in biotechnology [3, 5]. Antiden-drimer sera efficiently detect dendrimers in multiple assay formats, including enzyme-linked immunosorbent assays (ELISA), and in Western and dot blots [3, 5], ELISA assays are commonly used to quantitate proteins, and a quantitative ELISA could be developed for dendrimers using our sera, though doing so would require development of dendrimer standards of known concentration that could be used for calibration. 

After incubation, the supernatants of the ceU cultures can be used for the cytokine assay by capture enzyme-linked immunosorbent assay (ELISA) using specific antibody pairs and recombinant cytokine standards and ELISA plates. 

Currently there are few methods for specific investigation of immunotoxic effects, which are regarded as sufficiently validated for routine use (EC 2003). The plaque forming assay or the equivalent using the ELISA method (Enzyme-linked Immunosorbent Assay) are recommended to identify altered T-cell-dependent humoral responses. Of particular value for hazard assessment are the so-called host resistance models, in which the clinical relevance of immunotoxicity can be evaluated. Other methods may also be of value to provide information on the mode of immunotoxic action, e.g., mitogen stimulation tests and leucocyte phenotyping. However, further work is needed on standardization and validation of these test methods. 

The CML is the most characterized AGE and is referred to as a glycoxidation product. The inhibitory effects of C-glycosylflavones on the CML formation were tested by enzyme-linked immunosorbent assay in kidney diabetic subjects. The results showed that the percent inhibition was about 53% for chrysoeriol 6-C-boivinosyl 7-0-glucoside, 64% for chrysoeriol 6-C-boivinosyl, 80%i chrysoeriol 6-C-fucosyl, and only 2% for 4"-OH-3 -methox-ymaysin versus 60%i for the standard glycation inhibitor, aminoguanidine. 

Apart from radioimmunoassays, various enzyme-linked immunosorbent assays have been described as well. Campbell et al. (42) first reported a sensitive and specific ELISA using polystyrene tubes and a polyclonal antibody. However, the performance of this method was not evaluated with real samples but only with standards and aqueous muscle tissue extracts. Sensitive ELISAs were also developed for the determination of chloramphenicol in milk (43) and eggs (44) the results drawn by the latter assay correlated well with those obtained by application of a radioimmunoassay. 

A standard double-sandwich enzyme-linked immunosorbent assay (ELISA) or Western blot analysis is used. As the concentration of factor is very low in normal plasma (approximately 0.1 mg/1 for factor VIII), it is necessary to subject plasma samples to cryoprecipitation in order to concentrate the sample, prior to Western blot analysis. The cryoprecipitation protocol described by Bi et al. is as follows (Bi et al., 1996 Sarkar et al., 2000 Mah et al., 2003). Plasma samples are collected as described above for the Coatest assay. Plasma is then frozen at -80 °C overnight. Frozen samples are then subject to centrifugation at 7000 x g for 20 min at 4°C. The precipitate is washed with... 

TIR-based techniques that use fluorescence transduction (TIRF) have been used for the majority of the applications discussed above. These platforms, while not currently the gold standard for measurement of many target analytes, do offer advantages over current technologies, such as cell culture, chromatography, and the enzyme-linked immunosorbant assay (ELISA). Advantages include the ability to perform faster, more sensitive, multiple analyte, and real-time measurements. 

Radioactivity, however, is still a very sensitive means of measuring the presence or absence of a given material. Assay methodology has now come full circle, to the development of an ultrasensitive enzyme RIA. In this technique, an antigen is bound to a solid phase. Antibody will bind to the antigen, which could be a drug-protein conjugate, and the presence of bound antibody is detected by means of a second antibody coupled to alkaline phosphatase. So far this is the standard enzyme-linked immunosorbent assay (ELISA). However, if the substrate is tritium-labeled adenosine monophosphate, it is converted by the enzyme to tritium-labeled adenosine, which may be readily separated and measured. The detection limit for this assay for cholera toxin is approximately 600 molecules of the toxin (22). 

The use of immunoassays in the field of agricultural research has increased dramatically in recent years, and has become a reliable analytical tool that possesses numerous advantages over standard, chemical extraction and analytical methods. A few Of these advantages (described in several review articles (1,2)), include its greater sensitivity and specificity, the increased speed of the assay, which allows greater sample thrdugh-put, the requirement for smaller samples for extraction, and the assay s improved cost effectiveness. Enzyme-linked immunosorbent assays (ELISA) have been... 

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for maduramicin in poultry feed. The assay utilized polyclonal anti-maduramicin antibody raised in rabbits, maduramicin monoamide with 1,6-hexane diamine-conjugated ovalbumin as the coating antigen, horseradish peroxidase conjugated goat anti-rabbit IgG and 2,2 azinobis(3-ethylbenzthiazoline) sulfonic acid (ABTS) for quantitation. Standard curves ranging from 0 to 80 ng/mL maduramicin were constructed. The assay did not cross-react with monensin, lasalocid, salinomycin, lincomycin, narasin, chlortetracycline or roxarsone. Broiler feed fortified at 4 to 7 ppm maduramicin were shown to be quantifiable by ELISA at an average recovery of 98.1%. This ELISA method for maduramicin in poultry feed is comparable to the established HPLC-F method. 

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