Endogenous monoclonal immunoglobulins analyzed using the EXENT® solution and LC-MS.

Introduction: The EXENT® Solution, a fully automated system, is a recent advancement for identifying and quantifying monoclonal immunoglobulins in serum. It combines immunoprecipitation with MALDI-TOF mass spectrometry. Compared to gel-based methods, like SPEP and IFE, it has demonstrated the ability to detect monoclonal immunoglobulins in serum at lower levels. In this study, samples that tested negative using EXENT® were reflexed to LC-MS to determine if the more sensitive LC-MS method could identify monoclonal immunoglobulins missed by EXENT®. Objectives: To assess whether monoclonal immunoglobulins that are not detected by EXENT® can be detected by LC-MS using a low flow LC system coupled to a Q-TOF mass spectrometer. Methods: Samples obtained from patients confirmed to have multiple myeloma (MM) were diluted with pooled polyclonal human serum and analyzed using EXENT®. If a specific monoclonal immunoglobulin was not detected by EXENT®, the sample was then subjected to analysis by LC-MS. For the LC-MS analysis, the sample eluate, obtained after the MALDI-TOF MS spotting step, was collected and transferred to an autosampler tray for subsequent analysis using LC-MS. Conclusion: LC-MS has the capability to detect monoclonal immunoglobulins that are no longer detected by EXENT®. Reflexing samples to LC-MS for analysis does not involve additional sample handling, allowing for a faster time-to-result compared to current approaches, such as Next-Generation Sequencing, Next-Generation Flow, and clonotypic peptide methods. Notably, LC-MS offers equivalent sensitivity in detecting these specific monoclonal immunoglobulins.

Determination of IgG1 and IgG3 SARS-CoV-2 Spike Protein and Nucleocapsid Binding-Who Is Binding Who and Why?

The involvement of immunoglobulin (Ig) G3 in the humoral immune response to SARS-CoV-2 infection has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) in COVID-19. The exact molecular mechanism is unknown, but it is thought to involve this IgG subtype's differential ability to fix, complement and stimulate cytokine release. We examined the binding of convalescent patient antibodies to immobilized nucleocapsids and spike proteins by matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry. IgG3 was a major immunoglobulin found in all samples. Differential analysis of the spectral signatures found for the nucleocapsid versus the spike protein demonstrated that the predominant humoral immune response to the nucleocapsid was IgG3, whilst for the spike protein it was IgG1. However, the spike protein displayed a strong affinity for IgG3 itself, as it would bind from control plasma samples, as well as from those previously infected with SARS-CoV-2, similar to the way protein G binds IgG1. Furthermore, detailed spectral analysis indicated that a mass shift consistent with hyper-glycosylation or glycation was a characteristic of the IgG3 captured by the spike protein.

Quantification of human complement C2 protein using an automated turbidimetric immunoassay.

BACKGROUND: The measurement of complement components is clinically useful where a deficiency is suspected, or where excessive activation and consumption are present in disease. C2 deficiency carries an increased risk of developing systemic lupus erythematosus, recurrent infections and atherosclerosis. In this study, we have evaluated The Binding Site's Human Complement C2 SPAPLUS® assay. METHODS: Linearity was tested using 13 sample dilutions covering the standard measuring range. Within- and between-assay variabilities were calculated using five samples with different C2 concentrations. The correlation between C2 concentrations in EDTA-plasma and serum was assessed, as was the correlation between C2 measurements by the automated assay and radial immunodiffusion. C2 concentrations were compared with CH50 activity, and quantified in individuals with homozygous or heterozygous C2 deficiency, acquired angioedema and patients with chronic inflammatory conditions. RESULTS: The assay was linear across the measuring range (3.8-42.3 mg/L). Intra- and interassay variability were 2.3%-3.8% and 0%-3.3%, respectively. Comparison between C2 measurements in EDTA-plasma and serum provided a strong correlation (p<0.0001, R2=0.82, slope 0.92), as did the correlation between the automated and radial immunodiffusion methods (p<0.0001, R2=0.89, slope 1.07). A positive correlation between C2 concentration and CH50 activity was demonstrated (p<0.0001, R2=0.48). Significant differences were observed between the median C2 concentrations obtained in healthy controls and the patient clinical samples, with homozygous C2-deficient patients giving below detectable results. CONCLUSIONS: This C2 SPAPLUS® assay allows the automated, rapid and precice quantification of complement C2 protein and could therefore be considered as a replacement for older, more time-consuming methods.

Measurement of the IgG2 response to Pneumococcal capsular polysaccharides may identify an antibody deficiency in individuals referred for immunological investigation.

IgG2 is the most efficient subclass for providing protection against pneumococcal pathogens. We hypothesised that some individuals may be unable to mount an effective pneumococcal capsular polysaccharide (PCP) IgG2 response despite having a normal PCP IgG concentration (PCP IgG2 deficient). The median pre-vaccination PCP IgG2 concentration was significantly lower in individuals referred for immunological investigation compared to healthy controls (2.8 mg/L range, 95% CI 1.1-88 vs. 29.5mg/L, 95% CI 13.5-90, p = 0.0002). PCP IgG:IgG2 ratios were significantly higher for the referral population than for healthy controls suggesting the increased production of PCP specific subclasses other than IgG2. The percentage of individuals with PCP IgG2 deficiency was significantly higher in referral groups compared to controls (31% vs. 5%; p = 0.0009) and in an individual with PCP IgG2 deficiency, the balance of PCP specific IgG subclass antibodies post vaccination changed from IgG2>IgG1>IgG3>IgG4 to IgG1>IgG3>IgG2>IgG4. The median PCP IgG2 concentration in those with PCP IgG2 deficiency was significantly lower in the referral groups compared to controls (7.8 mg/L, 95% CI 1.1-12 vs. 12.7 mg/L, 95% CI 11.8-13.1; p = 0.006). The data suggests a defect in the production PCP IgG2 may be present in individuals with normal PCP IgG referred for immunological investigation.

Purification and characterisation of anti-pneumococcal capsular polysaccharide IgG immunoglobulins.

OBJECTIVES: The production of reference materials for quantifying pneumococcal antibody concentrations relies upon large scale vaccination. An alternative simple, reproducible protocol has been developed for the affinity purification of 23 serotype anti-pneumococcal capsular polysaccharide (PCP) IgG immunoglobulins. DESIGN & METHODS: The purification protocol utilised IgG fractionation, capsular polysaccharide (CPS) adsorption, and affinity chromatography using Pneumovax®-Sepharose. Purification efficiency and method reproducibility were assessed by comparison of 4 batches of anti-PCP IgG. Immunoglobulin composition was determined using nephelometry and functionality was evaluated using VaccZyme™ ELISAs. RESULTS: Anti-PCP IgG preparations were ≥95% pure by SDS-PAGE analysis with no contaminating IgA or IgM immunoglobulins or IgG antigen specific antibodies towards haemophilus influenzae b, diphtheria toxoid or tetanus toxoid. The predominant IgG subclass in the preparation was IgG2. CONCLUSIONS: This novel purification procedure produced highly specific anti-PCP IgG preparations that compared well to both Lot 89SF and 007sp international serum standards and could be used as an alternative method for the production of reference materials.

Estimation of polyclonal IgG4 hybrids in normal human serum.

The in vivo or in vitro formation of IgG4 hybrid molecules, wherein the immunoglobulins have exchanged half molecules, has previously been reported under experimental conditions. Here we estimate the incidence of polyclonal IgG4 hybrids in normal human serum and comment on the existence of IgG4 molecules with different immunoglobulin light chains. Polyclonal IgG4 was purified from pooled or individual donor human sera and sequentially fractionated using light-chain affinity and size exclusion chromatography. Fractions were analysed by SDS-PAGE, immunoblotting, ELISA, immunodiffusion and matrix-assisted laser-desorption mass spectrometry. Polyclonal IgG4 purified from normal serum contained IgG4κ, IgG4λ and IgG4κ/λ molecules. Size exclusion chromatography showed that IgG4 was principally present in monomeric form (150 000 MW). SDS-PAGE, immunoblotting and ELISA showed the purity of the three IgG4 samples. Immunodiffusion, light-chain sandwich ELISA and mass spectrometry demonstrated that both κ and λ light chains were present on only the IgG4κ/λ molecules. The amounts of IgG4κ/λ hybrid molecules ranged from 21 to 33% from the five sera analysed. Based on the molecular weight these molecules were formed of two IgG4 heavy chains plus one κ and one λ light chain. Polyclonal IgG (IgG4-depleted) was similarly fractionated according to light-chain specificity. No evidence of hybrid IgG κ/λ antibodies was observed. These results indicate that hybrid IgG4κ/λ antibodies compose a substantial portion of IgG4 from normal human serum.

Assessment of monoclonal gammopathies by nephelometric measurement of individual immunoglobulin kappa/lambda ratios.

BACKGROUND: Currently, monoclonal immunoglobulins are identified and quantified from bands on electrophoretic gels. As an alternative, clonality might be determined by measuring the separate light chain types of each Ig class to allow numerical assessment of Ig'kappa/Ig'lambda ratios, analogous to free light chain kappa/lambda ratios. METHODS: Using immunization, tolerization, and adsorption procedures, we prepared sheep antibodies against each of the 6 separate molecules, IgGkappa, IgGlambda, IgAkappa, IgAlambda, IgMkappa, and IgMlambda. Antibody targets comprised the junctional epitopes between the heavy chain and light chain domains. After purification, we assessed the antisera on a Siemens Dade-Behring BN II nephelometer for analytical quality and clinical utility. RESULTS: High-avidity, specific antibodies allowed the production of automated nephelometric immunoassays for each Ig light chain type. Laboratory comparison with serum protein electrophoresis, using dilution experiments, showed lower analytical sensitivity for monoclonal IgG detection but similar or greater sensitivity for IgA and IgM, particularly when the monoclonal bands overlaid transferrin. Results obtained from typing of monoclonal proteins into IgG, A, or M types were comparable with results obtained by immunofixation-electrophoresis methods. Initial clinical studies, in multiple myeloma patients, indicated that Ig'kappa/Ig'lambda ratios were sometimes more sensitive than immunofixation electrophoresis, provided numerical results, and correlated with changes in disease. CONCLUSIONS: Immunoassays for intact Ig kappa/lambda pairs are possible and should assist in the management of patients with monoclonal gammopathies.