Assays general serum

Quantitative LC/MS assays generally involve four steps (1) sample preparation, (2) assay calibration, (3) sample analysis, and (4) data management. In this method, the human serum samples are prepared with a liquid-liquid extraction procedure. The internal... 

In a general serum stability assay, the peptide is subjected to human serum (see Note 3) at realistic temperature conditions and incubated for various time intervals, comparable to traditional protease assays. The reactions are stopped by TCA or ethanol, precipitating larger serum proteins while leaving peptides... 

Adherence to sound principles of the assay of serum enzymes in general (F5-F7) is required for both manual and automatic techniques. Thus, one should insure that the release of product that is being measured is a linear function of the amount of alkaline phosphatase. The reaction must be carried out at the optimal pH characteristic of the enzyme with that particular buffer and substrate. The period of hydrolysis and substrate concentration should be adjusted in such a way that an initial zero-order rate of hydrolysis is obtained. The standard curve should obey Lambert-Beer s law. Arbitrary and variable dilutions of high-titer sera are not recommended. 

Some examples of problems found in the assay of serum enzyme are probably quite general Many enzymes are not substrate specific but give interfering reactions with analytes present in serum. A few instances of nonspecificity are given as follows. 

In general, serum levels exceeding 450 mg/L are associated with drowsiness or obtundation, and levels greater than 850 mg/L are associated with coma, respiratory depression, and metabolic perturbations. However, there appears to be poor correlation of serum levels with outcome. Moreover, assays may or may not include metabolites. 

Assays for Serum Antibodies - Serum antibodies appear to be present in rather low concentrations in drug hypersensitivity in man. For example, in penicillin hypersensitivity, the serum concentrations of BPO-specific antibody protein is under 20-30 pg per ml. and most commonly under 5 pg/ml. Thus the quantitative precipitin analysis, which cannot accurately assay less than about 50 pg/ml, cannot generally be used for drug hypersensitivity. However,there are rare reports of precipitating antibody in drug allergy. 

Unfortunately there is no commercial assay for routine measurement of serum levels of RCM-specific IgE antibodies. The reliability of other in vitro tests, such as the basophil activation test, has not yet been established. Results from individual patients indicate that the basophil activation test may be helpful [36]. However, at the moment it is only added on an experimental scientific basis. Provocation is not generally recommended, as intravenous applications of as low as 0.5-1 ml RCM have led to severe anaphylaxis [41]. 

If serum protein or surfactant is added to the acceptor wells, then, in general, p[A l> and P r> are not the same, even under iso-pH conditions. The acceptor-to-donor permeability needs to be solved by performing a separate iso-pH assay, where the serum protein or surfactant is added to the donor side, instead of the acceptor side. The value of Pe is determined, using Eq. (7.20), and used in gradient-pH cases in place of P A /) , as described in the preceding section. The gradient-pH calculation procedure is iterative as well. 

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