Albumin assays

That conclusion is supported and extended by the virtually simultaneous publication of Teale and Benjamin (1976). These investigators studied the oxidative regeneration of bovine serum albumin, assaying the extent of refolding immunochemically. Their results showed clearly that some parts of the molecule fold faster than others. Two fragments of albumin were tested for oxidative regeneration in the same way. Substantial return of native structure was seen in both fragments. 

Bilirubin effects depend on the method used for analysis. Interferences in direct serum protein methods are observed at bilirubin levels greater than 5 mg/100 ml (K7). A sample containing 20 mg of bilirubin per 100 ml increased the apparent total protein by 0.2 g/100 ml. Concentrations of bilirubin as high as 20 mg/ml do not effect albumin assays using bromocresol green binding (D12), but have a marked effect on these assays when [2-(p-hydroxyphenylazo)-benzoic acid] (HABA) dye is used (A7b). 

E41 Sanford, K.J., Eikenberty, J.N. (1980). Albumin assay in a multilayered analytical element using bromcresol green. Clin. Chem. 26, 1059, Abstr. 526. 

Using the results of quantitative albumin assays, the following ratio is calculated ... 

In 1932, Race discovered that plasma albumin after precipitation with TCA was soluble in alcohol and, further, that horse, human, sheep, and ox albumin were completely soluble, while their globulins were totally insoluble, in a solution containing 20 g TCA, 80 ml acetone, and 15 ml water. At the same time Race (Rl) devised a method for determining the albumin in plasma by first extracting it from TCA-precipitated proteins with 80% aqueous acetone containing 2-3 ml 20% TCA. Albumin nitrogen was then determined in the extract. With this acid-acetone fractionation technique, plasma from patients with rheumatoid arthritis gave lower albumin levels than those found by Howe s method, and, indeed, these were probably the first really accurate albumin assays on blood to be reported. 

In the context of case-control studies of liver cancer the limitation is more prominently the lack of information on past (i.e. 20-30 years) exposure. This again may be circumvented by examining end-points other than cancer. Precancerous lesions or markers (e.g., alphafoetoprotein) could be used as end-points with which the AF-albumin assay could be compared in a case-control approach. In addition, a case-control approach could be employed to investigate short-term effects of AF exposure such as aflatoxicosis. 

Biological Applications Albumin assays " detecting gamma-hydroxybutyric acid (GHB) treating cancer, cerebrovascular disorder, brain degenerative disease, demyelinating disease, muscle degenerative disease ... 

This experiment describes the adaptation of the bicinchoninic acid (BCA) protein assay to a flow injection analysis. The assay is based on the reduction of Cu + to Cu+ by the protein, followed by the reaction of Cu+ with bicinchoninic acid to form a purple complex that absorbs at 562 nm. Directions are provided for the analysis of bovine serum albumin and rabbit immunoglobulin G, and suggestions are provided for additional analyses. 

Medroxyprog esteroneAcetate. Accurate pharmacokinetic and metaboHsm studies on MPA have been difficult because the radioimmunoassays employed caimot differentiate between MPA and its metaboHtes (346). Comparison of MPA plasma levels assayed by hplc and radioimmunoassay show that radioimmunoassay may overestimate intact MPA concentrations by about fivefold (347). However, values of the mean elimination half-life of MPA were similar, being 33.8 and 39.7 h when measured by hplc and radioimmunoassay, respectively (347). Approximately 94% of MPA in the blood is bound to albumin (348). When taken orally, MPA is rapidly absorbed with Htde or no first-pass metaboHsm (13). Peak semm levels ate reached after 3 h. Steady state occurs after three days of daily adininistration (349). The pharmacokinetics of MPA when adininistered in a depot formulation have been described (350). 

The separation of proteins and peptides mixtures is the objective of protein biochemisdy. Albumin (Mr 66 000) concentration in a biological fluid (seaim, urine or cerebrbrospinal fluid) is assayed as markers for a series disease, such as nephritic syndrome or chronic glomuleronephritis. In diabetic patients the progression of microalbuminuria is accompanied by an increase in urinary concentrations of human semm albumen. In normal the excretion of albumin is 20 (tg/ml, in pathology - 20-200 p.g/ml. 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

The protein content was determined using a commercial assay kit (Bio-Rad Protein Assay kit) with Bovine Serum Albumin as standard, following the procedure described by Bradford [13]. 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Preparations of PEG-modified proteins. A. SC-PEG (1 g, 0.2 mmol) was added to a stirred solution of Bovine Serum Albumin (BSA) (100 mg, 1.5 x 10 6 mol) in 0.1 M sodium phosphate, pH 7.8 (60 mL). Sodium hydroxide (0.5 N) was used to maintain pH 7.8 for 30 min. The excess of free PEG was removed by diafiltration using 50 mM phosphate buffered saline. Approximately 30 amino groups of the native protein were modified as determined by trinitrobenzenesulfonate (TNBS) assay (28). The same degree of modification was obtained when the experiment was repeated under identical conditions using SS-PEG instead of SC-PEG. 

Fig. 8.7 CTB-GM1-ganglioside binding ELISA assay. Plates, coated first with GMrganglioside and bovine serum albumin (BSA), respectively, were irrigated with total soluble plant protein from chloroplast transgenic lines (3 and 7) and 300 ng of purified bacterial CTB. The absorbance of the GM1-ganglioside-CTB-antibody complex in each case was measured at 405 nm. Total soluble protein from untransformed plants was used as the negative control.

---