Human serum albumin, assay

The helium-neon (HeNe) laser immediately comes to mind, having a very useful spectral line at 633 nm for steady-state red/near-IR fluorescence studies. Kessler and Wolfbeis have demonstrated the fluorescence assay of the protein human serum albumin using the probe albumin blue excited with a red HeNe laser.(71) Another useful wavelength available from the green HeNe laser is at 543.5 nm and this has been used with phase-modulation fluorometry by Lakowicz etal. to study probes such as carboxy seminaphtorhodafluor-6 (SNARF-6) as a means of measuring pH.(72)... 

Braun and Alsenz6 used an ELISA to detect aggregates in interferon-alpha (IFN-a) formulations. They analyzed IFN-a formulations for possible aggregate formation because all marketed interferons are reported to induce antibodies to some extent. Because of its stabilizing effects, human serum albumin (HSA) is used in the formulation of marketed IFN-a at a great excess over IFN-a itself. HSA can also interact with other proteins. Braun and Alsenz developed an ELISA for the detection of both IFN-a-IFN-a and HSA-IFN-a aggregates. A MAb was used for the capture and detection of the IFN-a and a polyclonal for the detection of HSA. The assay is shown schematically in Figure 11.4. 

Lazaro, E., Lowe, P.J., Briand, X. and faller, B. (2008) New approach to measure protein binding based on a parallel artificial membrane assay and human serum albumin. Journal of Medicinal Chemistry, 51, 2009-2017. 

Bhagavan NV, Lai EM, Rios PA, et al. Evaluation of human serum albumin cobalt binding assay for the assessment of myocardial ischemia and myocardial infarction. Clin Chem 2003 49 581-585. 

Sample preparation procedures for GC are generially more involved. For example, for methadone hydrochloride, 0.5jV sodium hydroxide is added to give the free base, followed by extraction with methylene chloride. An internal standard is added after the extract is dried with anhydrous sodium sulfate [3, p. 970]. The assay of interleukin-la formulated with human serum albumin does not require any sample treatment prior to analysis by capillary electrophoresis [44]. 

L7. Lim, C. S., Miller, J. N., and Bridges, J. W., Energy-transfer immunoassay A study of the experimental parameters in an assay for human serum albumin. Anal. Biochem. 108, 176-184 (1980). 

N. Ahmed, and P. J. Thornalley, Chromatographic assay of glycation adducts in human serum albumin glycated in vitro by derivatization with 6-aminoquinolyl-iV-hydroxysuc-cinimidyl-carbamate and intrinsic fluorescence, Biochem. J., 2002, 364, 15-24. 

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. 

An enzyme immunoelectrode suitable for the assay of human serum albumin and insulin uses an oxygen electrode covered with an antibody-containing nylon net kept in place with an O-ring. From 1 to 25 ng/L of albumin and 5 to 100 ng/L of insulin can be assayed (306). A specific sensor for the tumor antigen a-fetoprotein (AFP) is prepared by immobilizing anti-AFP antibody covalendy on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyl octane, and GA (307). The sensor is applied to an EIA based on competitive Ab/... 

The authors of this review have used I-human serum albumin for determining proteolytic activity in various types of foodstuffs, enzymatic preparations (determination of the contaminating proteolytic activity), pharmaceuticals and other materials Proteolytic activity assays using radioactively labeled substrates... 

One of the advantages of an intrinsically eiectroactive label is that there is no need for substrate, cofactor or the special incubation steps for an enzyme label. However a consequence is the loss of enzyme signal amplification which increases the demands on detection sensitivity. Human serum albumin has been detected in a competitive heterogeneous assay using indium ions. ... 

An antiserum was raised by immunization of rabbits with an immunogen prepared by coupling hyoscyamine to human serum albumin and using [3H]-atropine as tracer. Atropine and hyoscyamine reacted to equal extents with the antibodies. Some structurally related drugs e.g. homatropine or scopolamine as well as atropine hydrolysis products (tropine and tropic acid) did not interfere in the assay. 

One of the earliest efforts of qualitative measurement of a protein (human serum albumin) in a microchip-based device was based on bead agglutination in a microchamber (approximately lOjaL). Subsequently, several quantitative immunoassays have been performed using microchip electrophoretic systems that permit separation and quantitation of free- and bound-labeled antigens in competitive assays (see Chapter 5). Most are carried out in channels micro-machined into fused silica substrates. Early work on quantitative assays achieved measurement of cortisol in serum.The assay used cortisol labeled with fluorescein and an argon laser detector at 488 nm and required only 80 pL of a 40x dilution of serum as the sample. Other capillary electrophoresis-based assays for a variety of antibodies have also been developed that include immunoglobulins (IgG, IgA, and IgM), antibovine serum albumin, and antiestradiol. ... 

The IRMM is currently preparing a serum-based reference material for serum total protein assays, referenced to highly purified human serum albumin. 

Carraro et al. (1997) developed an indirect competitive immunoenzyme assay to detect serum IgG antibodies against formaldehyde conjugated to human serum albumin. This technique was used to compare the presence or absence of the antibodies in 219 healthy subjects who differed in smoking habits (tobacco smoke is a significant source of fonnaldehyde exposure) and occupational exposure to formaldehyde. The indirect competitive immunoenzyme assay was developed and applied as a qualitative method. Additional research is needed to detennine if the method can be modified to provide a reliable and precise measure to quantify exposure level or exposure duration. 

Peripheral blood mononuclear cells were obtained by density gradient separation through Ficol-Hypaque technology 17). Thymidine incorporation proliferation assays were set up as previously described using human serum albumin (HSA) or STn-HSA as stimulant (77). A stimulation index of > 2 SD compared to normal donors mononuclear cells was used as evidence of positive response. Fifty seven percent of tested vaccinated patients had evidence of specific T cell proliferation against STn. 

In homogeneous competitive assays the electrode-active group of the labeled antigen is masked by the bound antibody. Such electrochemical immunoassays have been developed for human serum albumin (HSA) (Alam and Christian, 1982,1985). HSA was labeled with Pd2+ or Zn2+, and the bound metal ion was measured by differential pulse polarogra-phy at a mercury electrode. Binding of antibody caused a drop of the peak current. 

Optoelectronic biosensors based on immobilized dyes have been developed for the determination of glucose, urea, penicillin, and human serum albumin (Lowe et al., 1983). Other promising approaches use immobilized luciferase or horseradish peroxidase to assay ATP or NADH or, when coupled with oxidases, to measure uric acid or cholesterol. These principles have not yet been generally accepted for use in routine analysis. Most probably, the first commercial optical biosensors will be those for immunological assays. 

The specificity of the ELISA method for Mn-SOD was examined. None of the major proteins found in human sera or erythrocytes (such as human serum albumin human a-, (3-, or y-globulin Cu,Zn-SOD or hemoglobin) showed a reaction with this method. To test the possibility of assaying the enzyme in human sera, three different concentrations of Mn-SOD were added to normal human serum and the effect of dilution on the ELISA was examined. A linear relationship was observed between the immunoreactive Mn-SOD level and the serum dilution in the range of 8- to 128-fold (Fig. 13). The ELISA was performed as described in Section 3,4,2,1, wherein a serum sample is diluted 10- or 11-fold and used for the assay. 

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