Ceruloplasmin (Cp) is an enzyme belonging to the multi-copper oxidase family and contains six copper atoms [1, 2]. In human plasma from healthy subjects, more than 95% of the total copper is bound to Cp. Serum Cp levels of less than 200 mg/L are considered to be a diagnostic criterion for Wilson’s disease , an autosomal recessive inherited disorder of copper metabolism, which can be fatal, if not treated properly. In addition, the Menkes disease [4, 5] (“kinky hair syndrome”) can be confirmed by determination of Cp.
The diagnostic determination of Cp in serum or plasma is usually performed by turbidimetric or nephelometric  and other immunoassays . In addition, some other methods have been published, such as SEC–ICP-MS , where a depletion cartridge was used to remove highly abundant proteins, such as albumin. In any case, standardization of Cp analysis turned out to be quite difficult and left some serious questions unanswered largely due to the lack of Cp reference materials. To resolve these issues, we explored the feasibility of an approach based on an immunocapture step, which was performed as clean-up and enrichment, followed by hetero-element detection by use of inductively coupled plasma mass spectrometry (ICP-MS).
Immunocapture, which is a variant of affinity extraction or affinity chromatography, can be regarded as one of the most powerful separation techniques available [9,10,11]. This approach is particularly valuable when the analyte is present in low concentrations, and the matrix is complex, such as in food analysis or human diagnostics. In the field of high-sensitivity protein analysis, usually only affinity-based techniques are feasible, e.g. enzyme-linked immunosorbent assay (ELISA) . Unfortunately, the calibration of immunoassays is not trivial and many inexperienced users have problems to interpret the results properly. Particularly, the existence of “cross-reactivity” often leads to some confusion, although this is nothing else as an analytical interference, which is occurring in any instrumental method. Affinity extraction is often combined with instrumental analytical techniques, such as mass spectrometry [13, 14] and hence does not need much rethinking in relation to a more traditional analytical workflow. However, the most selective affinity techniques are commonly based on antibody/analyte interactions, requiring sufficient amounts of high-quality antibodies directed against the respective analyte. The availability of such antibodies may be limited and almost always the cost of a sufficient amount of antibodies is high. On the other hand, in the field of protein analysis, even researchers with many years of experience sometimes seem to underestimate the complexity of their samples and therefore do not sufficiently appreciate the importance of extensive sample-preparation steps.
In this work, we used an uncommon approach to produce sufficient amounts of antibodies in chicken eggs at a reasonable cost in an animal-friendly way. The developed antibodies were used for the enrichment and isolation of Cp, followed by its quantitative determination using ICP-MS.