Human albumin ELISA

Cattaneo, C., Gelsthorpe, K., Phillips, P and Sokol, R.J. (1992). Reliable identification of human albumin in ancient bone using ELISA and monoclonal antibodies. American Journal of Physical Anthropology 87 365-372. 

Ascites Production. Anti-albumin clones HSA-1 and HSA-2 were thawed, subcloned, and reassayed by ELISA on human albumin, and a rapidly growing subclone was expanded for ascites production. Forty male BALB/c mice were primed with an intraperitoneal injection of 0.5 mL pristane on day 0. On day 14, 2 X 106 cells were injected into each mouse. Ascites fluid was collected over a 1-week period and pooled. Cells and other debris were removed by centrifugation. Approximately 180 mL of ascites fluid was obtained. 

Figure 6. Summary of the steps of the ELISA for detecting human albumin.

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. 

Beyond polyclonal antibodies, monoclonal antibodies to isoxazolyl penicillins were recently produced by immunization of mice with a cloxacillin-human serum albumin conjugate prepared by a mixed anhydride procedure (35). Sensitivity and specificity of these antibodies were tested in an indirect ELISA in which a cloxacillin-glucose oxidase conjugate prepared by an activated ester procedure served as a coating agent. It was found diat die prepared antibodies could be... 

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. 

Pilot human dosimetry studies have demonstrated the usefulness of adduct measurements in exposure assessments. Higher adduct levels in heavily exposed coke oven workers compared to those with lower exposures or controls have been reported using USERIA (3 )> ELISA (36) or the P-post1abe1ing assay (41). Tobacco consumption is also positively correlated with adduct formation (49). Chemotherapeutic trials are among the best evidence that adduct levels are related to exposure because in these cases, negative controls are truly negative. The development of immunoassays for cisplatin-DNA adducts by Poirier et a1., have led to internal dosimetry estimates and predictions for prognosis (35). Levels of aflatoxin B -adducts have also been identified in human liver and breast with ELISA (40.871. in serum albumin with RIA (88) and ELISA (38)- Multiple adduct assessments are also feasible (Table III) (89). 

The primary alternative to the instrumental methods summarized above is immunodetection of EA with specific antibodies. Antiserum raised in response to immunization with lysergol (6a) coupled to human serum albumin recognizes several EA (100). Used for competitive enzyme-linked immunosorbent assay (ELISA), this broad-spectrum antiserum allows rapid detection of total EA in many samples concurrently. However, it does not separately quantify the different EA that contribute to the profile, and different EA show different reactivities to the antiserum. Monoclonal antibodies specific for individual EA species, such as ergotamine, also have been raised and provide an effective means for detection and quantification of the particular EA, as well as a valuable tool for physiological studies (101). 

One of the original clones secreted anti-forskolin antibody which was purified by protein A affinity column chromatography and its purity was confirmed by MALDl mass spectrometry. The MAh was bound to polystyrene microtitration plates precoated with a forskolin-human serum albumin (HAS). The full measuring range of the assay extended from 6 ng to 200 ng mP of forskolin [68, 76]. The ELISA system established in this study was more sensitive compared to TEC [47], GLC [74] or HPLC [47, 77]. 

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