Serum albumin assay screening

A test strip enzyme immunoassay that could not discriminate between the 17 - and 17 -stereoisomers but allowed on-site screening of urine samples within 45-60 min was also reported (118). In this assay, 17, 19-nortestosterone was coupled to bovine serum albumin through a hemisuccinate bridge at the 17-position (119). This conjugate was used to raise polyclonal antibodies that would... 

A generic enzyme immunoassay for the determination of several synthetic corticosteroids including dexamethasone, betamethasone, flumethasone, triamcinolone, prednisolone, and methylprednisolone in milk, liver, kidney, and muscle samples was recently developed (156). Antibodies raised against dexamethasone-21-hemisuccinate-bovine serum albumin were used in this assay, whereas dexa-methasone-horseradish peroxidase was the label conjugate. Skimmed milk could be directly screened for the presence of corticosteroids at limits of detection of 0.1 ppb for dexamethasone, betamethasone, and flumethasone, 0.3 ppb for triamcinolone and 0.5 ppb for prednisolone. Tissue samples were submitted, prior to the immunoassay, to an extraction/cleanup procedure involving liquid-liquid partitions with acetonitrile-water followed by hexane-chloroform. Background values for bovine liver, swine kidney, and calf muscle were determined to be 0.26, 0.26, and 0.07 ppb, respectively, of dexamethasone equivalents. 

Over 20,000 samples of microbial culture broths were subjected to our screening program for CETP inhibitors by method A. At first no BSA was added to the assay mixture, but many false-positive compounds such as fatty acids were isolated. To prevent this, the optimal concentration of BSA was tested and set up as 200 pM, resulting in a low hit rate in the primary screen. The serum albumin concentration in the assay is similar to that in human plasma. Finally, we discovered erabulenols from a fungal strain, and ferroverdins from an actinomycete strain, as novel CETP inhibitors (Fig. 5). 

Recently, it was shown that various nitrogen mustard-based cytostatics, for example, melphalan and cyclophosphamide, reacted with the cysteine 34 residue of human serum albumin in an analogous way. The tripeptide assay could be applied to samples of cancer patients treated with these cytostatics (28), which holds promise for optimization of chemotherapy with these agents by intensive screening of adduct levels in patients. 

Inert carrier proteins, such as bovine serum albumin, are commonly added to the assay media at high concentrations for screening of isolated targets in order to saturate the potential protein binding surfaces and reduce the loss of target proteins due to adsorption onto the surfaces of the containers, such as plates and vials. The added benefit of inert proteins is that they can bind to lipophilic compounds and increase the solubility of insoluble compounds in the assay buffer. The presence and absence of serum protein can cause difference in solubility and cause a discrepancies between the two assays. 

Due to the high cost of cell culture, Caco-2 assays are usually used as a follow-up to PAMPA in ADME screening [78], and as a result, the sample burden for bioanalysis is not as heavy as for some first-hne assays, such as metabolic stability. There have been a number of reports in the literature that use automated optimization and single LC-MS/MS for sample analysis for Caco-2 assay support [46,79-81]. Nevertheless, Caco-2 samples pose a unique bioanalytical challenge. Unlike plasma or microsomal samples rich in proteins that help solubilize compounds and prevent adsorptive loss, Caco-2 samples are essentially aqueous buffer samples with very little protein. As a result, compounds with low solubility and/ or adsorption problems tend to exhibit poor recoveries in the assay due to precipitation and adsorptive losses [82,83]. An effective solution to this problem is the use of organic solvent to catch compounds immediately after incubation, but prior to analysis, in order to maintain solubility and prevent adsorptive loss to container surfaces. Another approach involves the addition of some protein such as bovine serum albumin (BSA) to the assay buffer system, thus reducing compound loss/ precipitation and improving recoveries [84]. 

The separation or complexation of humic substances and/or heavy metals is successful in favorable cases, e.g., by the addition of bovine serum albumin (BSA) in a trinitrotoluene enzyme-linked immunosorbent assay (ELISA) [18]. In the. screening of soil samples for pesticides, persistent centrifugation at 17 000 rpm, and simple dilution with bidi.stilled water in the ratio 1 10 was sufficient [19]. 

Gu,C. Nikolic,D. Lai,J. Xu,X. VanBreemen,R.B. Assays of ligand-human serum albumin binding using pulsed ultrafiltration and liquid chromatography-mass spectrometry. Comb. Chem. High Throughput Screen. 1999,2, 353-359. 

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