Quantitation of IgG Subclasses in Serum Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Abstract

Serum IgG subclasses (IgGSC) are measured for a number of indications, but the most common are the identification of selective immunodeficiency disease and the diagnosis of IgG4-related disease (IgG4RD). Traditional nephelometric (IN) assays can suffer from two issues impacting the accuracy of the results: (1) hook effect and (2) antibody cross-reactivity between the subclasses. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is not vulnerable to these modes of interference and therefore serves as an excellent and relatively inexpensive means of diagnosing and/or monitoring the relevant clinical conditions.We describe a semiautomated and simple method for the accurate and precise measurement of IgGSC from 20μL of serum using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method following digestion of serum proteins in 96-well plate format. Due to the high abundance of the target proteins, no specialized sample preparation (such as solid phase extraction) is required. Twenty microliters are injected to the LC-MS/MS system. Quantitation is performed against a five-point duplicate linear calibration curve prepared in blank matrix. The assay calibration range is 0.38-7.74g/L for IgG1, 0.24-4.46g/L for IgG2, 0.038-0.752g/L for IgG3, 0.025-0.435g/L for IgG4, and 0.62-15.5g/L for total IgG. Total IgG is used as an internal quality control marker and is compared to the sum of the four subclass results. Total imprecision in clinical production has been observed to be 5.1-10.6% for in-house prepared control materials having IgGSC mean values in the range of 0.38-8.43g/L for IgG1, 0.22-3.76g/L for IgG2, 0.0387-0.721g/L for Ig3, and 0.0279-1.46g/L for IgG4. Limit of quantitation (LoQ) was determined to be 0.29g/L for IgG1, 0.22g/L for IgG2, 0.019g/L for IgG3, and 0.0067g/L for IgG4.