Immunoassays standard

In spite of the great success of the spline functions"Tor radio-immunoassay standard curves caveats are voiced primarily concerning the conscientious choice of the smoothing parameters (25) and the overfitting (26). Both aspects deserve attention in other applications as weTT. ... 

The Variability Associated with Each Standard Point on the Analytical Curve. The reliability of immunoassay standard curves is not uniform across the entire dynamic range of the curve. The least analytical variability is usually observed in the central regions of the curve in the vicinity of 50% ligand displacement, with variability increasing at the extremes of the curve. 

Table II. Intraassay Variability of Picloram in Four Fortified Plant Extract Samples from Enzyme Immunoassay Standard Curve using Polyclonal or Monoclonal Antibodies...

The FDA bioanalytical guidance document [27] recommends a minimum of six nonzero calibrators for immunoassay standard curves. The 3rd AAPS/FDA... 

The ideal PK immunoassay standard curve, which is nonlinear and heteroscedastic, is derived from solutions of well-characterized macromolecules added to a relatively nonreactive sample matrix. However, rarely does one encounter an ideal situation when describing bioanalytical methodology, thus developing and validating analytical methods for macromolecules, and analyzing samples from preclinical or clinical trials must include an evaluation of these variables and possibly many others. Careful consideration must be given to the topics described in this chapter to achieve the goal of accurate and reproducible quantification of biotherapeutics necessary for pharmacokinetic analysis. 

Immunoassay standard curves. Immunosensors were prepared as described. Sample, standards, and conjugate were added sequentially to the respective cells to start the competitive assay. After a 10 min incubation, the solution was discarded and a proprietary buffer was added to each cell. A sufficient concentration of glucose was then added to each cell to drive the enzyme reaction to maximum velocity. As in the qualification tests, the hydrogen... 

The capacitance measurement technique applied to the antibody-coated sensor in our model system yielded a sensitivity to atrazine concentrations over the ppb to sub-ppb range. The immunoassay standard curve showed that the sensor is capable of the resolution needed for a semiquantitative assay [39]. Scatter in the data is sufficiently small to allow for a positive or negative result within... 

Table I. Optical density of immunoassay standards expressed as a percent of the negative control...

Immunological techniques For production of monoclonal antibodies and immunoassays standard techniques were applied. 

Figure 6-13. McGuigan s immunoassay standard plot of the C-terminal tetrapeptide amine of gastrin (A) and human gastrin I ( ). Radioactivity unadsorbed to charcoal (antibody bound) is plotted against the logarithm of the amount of unlabeled peptide in the reaction mixture. (From McGuidan JE. Antibodies to the carboxy-terminal tetrapeptide of gastrin in guinea pig. I Lab Clin Med 71 964-970, 1968.)...

Immunodiffusion and immunoprecipitation, developed ia the 1940s as a means to identify and semiquantitate specific proteias, were the direct precursors to the development ia 1953 of Immunoelectrophoresis, a method used ia many clinical laboratories (5). Single- and double-gel immunodiffusion and immunoelectrophoresis were, ia effect, the first standardized and routinely used immunoassay methods (see Electroseparations, electrophoresis). 

Biomedical Applications. TRIS AMINO is used for a number of purposes in its pure form, it is an acidimetric standard the USP grade can be utilized intraveneously for therapeutic control of blood acidosis TRIS AMINO also is useful in genetic engineering as a buffering agent for enzyme systems, industrial protein purification, and electrophoresis. AMP has found use as a reagent in enzyme-linked immunoassays. The primary appHcation is for alkaline phosphatase assays. 

Example 6-2 The following standard addition plot was obtained for a competitive electrochemical enzyme immunoassay of the pesticide 2,4-D. A ground water sample (diluted 1 20 was subsequently assayed by the same protocol to yield a current signal of 65 nA. Calculate the concentration of 2,4-D in the original sample. 

GC = gas chromatography ECD = electron capture detector EIA = enzyme-immunoassay GPC = gel permeation chromatography HPLC = high-performance liquid chromatography ITMS = ion trap mass spectrometer LSE = liquid solid extraction MS = mass spectrometry RSD = relative standard deviation SPE = solid phase extraction... 

The microplate ELISA testis conducted in standard 96-well microplates. A microplate consists of a 12 X 8 grid of wells for test solutions. The three most widely used ELISA formats are immobilized antigen competitive immunoassay, immobilized antibody competitive immunoassay and sandwich immunoassay. " ... 

Alternatively, competitive ELISA can be used to estimate the hapten density if an antibody that specitically recognizes the hapten is available. At first observation this approach seems circular because the immunoassay developed is used to determine hapten density on proteins used for immunization. However, if a small molecule mimic of the protein conjugate is used as a standard, the method can be accurate. For example, a hapten containing a carboxylic acid can be coupled to phenethylamine or tyramine, its structure confirmed and the material used to generate a calibratron curve to estimate hapten density. 

Unlike GC/MS methods, internai standards are not appropriate for immunoassays. Internal standards that would react with the antibody but would not interfere with the assay are nonexistent. In the place of internal standards, external QC must be maintained. 

Figure 18 Flow chart of the automated on-line flow injection immunoassay (FllA). Six steps are involved in each cycle (1) addition of antibody and incubation (2) addition of analyte (or standard) and incubation (3) addition of enzyme-tracer and incubation (4) addition of substrate and incubation (5) downstream measurement of fluorescence (6) regeneration of affinity column...

The analytical response generated by an immunoassay is caused by the interaction of the analyte with the antibody. Although immunoassays have greater specificity than many other analytical procedures, they are also subject to significant interference problems. Interference is defined as any alteration in the assay signal different from the signal produced by the assay under standard conditions. Specific (cross-reactivity) and nonspecific (matrix) interferences may be major sources of immunoassay error and should be controlled to the greatest extent possible. Because of their different impacts on analyses, different approaches to minimize matrix effects and antibody cross-reactivity will be discussed separately. 

Beasley et al. developed a panel of immunoassays to monitor DDT, its metabolites, and structurally related compounds, but they found that milk has a severe effect on the assay performance. They found that when directly utilizing whole milk, color development was completely inhibited. Even when using 1 100 dilutions of whole milk, the assay sensitivity was reduced by 90% (based on the IC50 shift, not simply the dilution factor). A number of procedures were evaluated to eliminate the interferences from the fat-soluble analytes. However, many of the procedures that removed interferences also removed the analytes. Extraction with a mixture of solvents and the use of similarly processed blank milk to prepare the standards ultimately yielded more accurate results. This article demonstrates the difficulties encountered in analyzing lipid-soluble analytes. 

An immunoassay was developed to determine the penicillinase stable isoxazolyl penicillins cloxacillin and dicloxacillin in milk by Usleber et alJ The assay detected lOpgkg" of cloxacillin and 30pgkg of dicloxacillin with recoveries of 102% and 84%, respectively. The calibration curve was prepared by fortifying skimmed milk powder (lOOgL ) with standards. Fortified samples were prepared in pasteurized milk and analyzed directly after decreaming by centrifugation. This immunoassay was performed with minimal sample preparation, probably because the extensive water solubility of the penicillins prevents problems associated with more lipid-soluble analytes. 

Third, the bulk of the items in Table 1 address method performance. These requirements must be satisfied on a substrate-by-substrate basis to address substrate-specific interferences. As discussed above, interferences are best dealt with by application of conventional sample preparation techniques use of blank substrate to account for background interferences is not permitted. The analyst must establish a limit of detection (LOD), the lowest standard concentration that yields a signal that can be differentiated from background, and an LOQ (the reader is referred to Brady for a discussion of different techniques used to determine the LOD for immunoassays). For example, analysis of a variety of corn fractions requires the generation of LOD and LOQ data for each fraction. Procedural recoveries must accompany each analytical set and be based on fresh fortification of substrate prior to extraction. Recovery samples serve to confirm that the extraction and cleanup procedures were conducted correctly for all samples in each set of analyses. Carrying control substrate through the analytical procedure is good practice if practicable. 

Differences in the relative proportion of f-PSA and PSA-ACT can affect the result obtained for t-PSA because of the differences in the nature of calibration and the molar response, sensitivity, and specificity of antibodies used in various immunoassays. The efficiency of these immunoassays has been evaluated by several investigators. Because the proportion of free and complexed PSA varies in benign and malignant diseases, these immunoassays measure one form or the other, giving rise to different results for different patient groups. It is very important that data from clinical studies support the proposed intended uses of these assays, since as many as 5 percent of men with a negative free PSA test (free PSA values >25 percent) will have cancer and not be recommended for biopsy. Therefore, a goal for standardization is to detect total and free PSA accurately in equimolar fractions. 

The application of PSA measurements for clinical monitoring of prostatic carcinoma requires fine tuning of PSA assays. One important aspect of this tuning is to have well-defined standards (primary calibrators). Calibrators or primary reference materials consisting of PSA complexed with ACT have been prepared and are available to sponsors of commercial immunoassays. As a result of this, some sponsors have studied calibration stability and have shown that calibration did not change within 14 to 90 days. Primary references of 90 percent PSA-ACT and 10 percent f-PS A have been shown to minimize differences in PSA measurements between different assays [NCCLS Document—Primary Reference Preparations Used to Standardize Calibration of Immunochemical Assays for Serum Prostate... 

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