Phenotyping Polyclonal Kappa and Lambda Light Chain Molecular Mass Distributions in Patient Serum Using Mass Spectrometry

Abstract

We previously described a microLC-ESI-Q-TOF MS method for identifying monoclonal immunoglobulins in serum and then tracking them over time using their accurate molecular mass. Here we demonstrate how the same methodology can be used to identify and characterize polyclonal immunoglobulins in serum. We establish that two molecular mass distributions observed by microLC-ESI-Q-TOF MS are from polyclonal kappa and lambda light chains using a combination of theoretical molecular masses from gene sequence data and the analysis of commercially available purified polyclonal IgG kappa and IgG lambda from normal human serum. A linear regression comparison of kappa/lambda ratios for 74 serum samples (25 hypergammaglobulinemia, 24 hypogammaglobulinemia, 25 normal) determined by microflowLC-ESI-Q-TOF MS and immunonephelometry had a slope of 1.37 and a correlation coefficient of 0.639. In addition to providing kappa/lambda ratios, the same microLC-ESI-Q-TOF MS analysis can determine the molecular mass for oligoclonal light chains observed above the polyclonal background in patient samples. In 2 patients with immune disorders and hypergammaglobulinemia, we observed a skewed polyclonal molecular mass distribution which translated into biased kappa/lambda ratios. Mass spectrometry provides a rapid and simple way to combine the polyclonal kappa/lambda light chain abundance ratios with the identification of dominant monoclonal as well as oligoclonal light chain immunoglobulins. We anticipate that this approach to evaluating immunoglobulin light chains will lead to improved understanding of immune deficiencies, autoimmune diseases, and antibody responses.

Challenges of measuring monoclonal proteins in serum

“…A strong hint of what testing for M-proteins will be in the near future has come from a team at the Mayo Clinic in the past 2 years [88][89][90]. They have shown that mass spectrometry is an exquisitely sensitive and specific technique that offers great promise to change the entire manner in which M-proteins are characterized and measured [88][89][90].…”

“…They have shown that mass spectrometry is an exquisitely sensitive and specific technique that offers great promise to change the entire manner in which M-proteins are characterized and measured [88][89][90]. Mills et al reported using immunochemical techniques to purify immunoglobulins from other proteins and reduction to separate the heavy and The method to purify and prepare immunoglobulins, followed by microflow liquid chromatography and electrospray ionization with quadropole time of flight mass spectrometry.…”

Pseudo-monoclonal gammopathy: a report of four cases

“…The miRAMM LC mass distribution ( Figure 1B) Patient 1) revealed a markedly skewed polyclonal κ to l light chain ratio (~8) and a polyclonal background seen in other hypergammaglobulinemia patients. 6 Typical IgG κ to l ratios for normal patients is between 1.17-3.61.…”

Mass Spectrometry Approaches for Identification and Quantitation of Therapeutic Monoclonal Antibodies in the Clinical Laboratory

“…Methods to overcome this interference in the laboratory have been published but are not yet widely available. MS-based methods have the potential to be utilized (8–10), and incubation with anti-idiotypic antibodies against the therapeutic MAb changes its electrophoretic migration, allowing it to be discriminated from the original endogenous clone when they comigrate by SPEP and IFE (11). Immunoassays may also be impacted by the presence of therapies such as DARA, which causes panreactivity in antibody screenings in transfusion medicine, as recently reported by blood banking services (12).…”