Research Use Cases for Mass Spect Products: MSG4000

Paper: Characterizing Antibody Cross-reactivity for Immunoaffinity Purification of Analytes prior to Multiplexed Liquid Chromatography–Tandem Mass Spectrometry

- Full paper on Clinical Chemistry Journal found here.

Summary

This paper describes the development of a new method for quantifying five vitamin D metabolites in a single assay using multiplexed liquid chromatography–tandem mass spectrometry (LC-MS/MS). The method was evaluated using the MSG4000 product, a vitamin D–depleted human serum matrix. The authors found that the MSG4000 product was useful for assessing the recovery of vitamin D metabolites from immunoaffinity enrichment.

Overall, this study highlights the importance of understanding antibody cross-reactivity and provides insights into the chemical features of vitamin D metabolites that influence their binding to antibodies. It also demonstrates the utility of LC-MS/MS in improving the specificity of immunoassays for accurate measurement of these metabolites.

Significance of MSG4000 Product:

The MSG4000 product, a vitamin D–depleted human serum matrix, played a crucial role in the study. It served as a matrix for testing the antibody's extraction efficiency (analytical recovery) for different vitamin D metabolites. By adding each metabolite to MSG4000, extracting it with the immunoaffinity reagent, and then quantifying it with LC-MS/MS, the researchers determined how well the antibody captured each metabolite.

The significance of MSG4000 lies in its utility as a standardized matrix for assessing the performance of the antibody-based immunoaffinity method. It allowed researchers to evaluate the antibody's affinity for different vitamin D metabolites and to identify specific features that affected binding. This information was crucial for improving the accuracy and specificity of immunoassays for 1α,25(OH)2D and other vitamin D metabolites.

In summary, MSG4000 served as a vital component in evaluating the antibody's performance in capturing vitamin D metabolites, leading to a better understanding of antibody cross-reactivity and ultimately improving the accuracy of vitamin D measurements in clinical settings.The MSG4000 product is also a good source of other matrix components that may affect the performance of immunoaffinity enrichment methods, such as proteins and lipids.

Key findings and methods of the study include:

1. Background: Immunoassays for 1α,25(OH)2D lack specificity, which can be problematic for accurate measurements. The study aims to understand the cross-reactivity of an anti-1α,25(OH)2D antibody with various vitamin D metabolites and identify the chemical features of 1α,25(OH)2D important for antibody binding.

2. Methods: The researchers employed immunoaffinity enrichment with a solid-phase anti-1α,25(OH)2D antibody and derivatization as the sample preparation process. The analytes were then quantified using LC-MS/MS. The study involved supplementation and recovery studies for 11 vitamin D metabolites, and a method was developed to simultaneously quantify 25(OH)D2, 25(OH)D3, 24,25(OH)2D3, 1α,25(OH)2D2, and 1α,25(OH)2D3 with deuterated internal standards for each analyte.

3. Results: The study found that several chemical features of vitamin D metabolites are important for binding to the antibody. These features include the native orientation of the hydroxyl group on carbon C3 in the A ring, the lack of substitution at carbon C4 in the A ring, and the overall polarity of the metabolite. The multiplexed assay developed in the study had lower limits of quantification for different vitamin D metabolites.

4. Conclusions: The research concludes that LC-MS/MS can be effectively used to characterize antibody cross-reactivity. The study's findings support the use of immunoenrichment in combination with LC-MS/MS for the targeted analysis of multiple vitamin D metabolites in a clinical setting.

Authors:

Thomas J. Laha(1) 

Frederick G. Strathmann(2,3) 

Zhican Wang(4) 

Ian H. de Boer(5)

Kenneth E. Thummel(4)  

Andrew N. Hoofnagle(1,5)

Author Affiliations

1 Department of Laboratory Medicine, University of Washington, Seattle, WA;

2 Department of Pathology, University of Utah, Salt Lake City, UT;

3 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT;

4 Departments of 4 Pharmaceutics and

5 Medicine, University of Washington, Seattle, WA

Citation:

Thomas J Laha, Frederick G Strathmann, Zhican Wang, Ian H de Boer, Kenneth E Thummel, Andrew N Hoofnagle, Characterizing Antibody Cross-reactivity for Immunoaffinity Purification of Analytes prior to Multiplexed Liquid Chromatography–Tandem Mass Spectrometry, Clinical Chemistry, Volume 58, Issue 12, 1 December 2012, Pages 1711–1716, https://doi.org/10.1373/clinchem.2012.185827