Hydrasys

To detect and quantify the presence of an M-protein, agarose gel electrophoresis and immunofixation were performed on the Hydrasys (Sebia, Evry, France) according to the manufacturer’s protocol. Serum free light-chain analysis was performed on a BNII analyzer (Siemens, Marburg, Germany) using Freelite reagents (The Binding Site Ltd, Birmingham, UK) according to the manufacturer’s protocol.

Serum samples (n = 97) were submitted to a commercial reference laboratory (Gribbles Veterinary Pathology, Clayton, VIC) for determination of total protein and protein fraction (albumin, total globulins and α-1, α-2, β and γ globulin) concentrations. Electrophoresis was conducted according to the manufacturer's recommendations using the semi-automated agarose gel electrophoresis system (Hydrasys, Sebia Inc., Norcross, GA) and the split protein β1 and β2 gel reagent (Hydragel 30, Sebia Inc., Norcross, GA). The resultant gel was fixed, stained and scanned using the same equipment. Densitometer laser tracings were used to measure protein fraction percentages [49] , and absolute values were determined on the basis of biuret total protein measurement. The albumin-globulin (A–G) ratio was calculated by dividing the albumin value by the sum of the globulin fraction values.

AAT protein phenotyping and glycosylation analysis in patient sera were performed by means of the immunofixation of serum glycoforms via isoelectric focusing gel electrophoresis using HYDRASYS (Sebia, Lisses, France). Phenotypes were confirmed by means of paired allele genotyping (Supplementary Figure S2).

IgG, IgA, IgM values were determined by immunoturbidimetry (Roche COBAS 6000, Roche Diagnostics International Ltd, CH-6343, Rotkreuz, Switzerland). IgG reference values were based on Adeli et al. (23 (link)). Serum protein electrophoresis (SEP) was performed using Hydrasys (Sebia, Paris, France) instruments and Hydragel Protein (E) gels (Sebia, Paris, France). The visualization of the gel provided qualitative analysis, while reading of the agarose gels on a Sebia reader provided protein profiles for relative quantitative analysis by Hydrasys 2 Scan (Sebia, Paris, France) scanning system. CG values were obtained by subtracting the albumin levels from total protein values. The gamma globulin fraction was directly determined by protein electrophoresis.

Blood was collected in Eppendorf by retro-orbital sampling. Semiautomated electrophoresis was performed on the Hydrasys instrument (Sebia, Lissex, France). According to the manufacturer’s instructions, 10 μl of undiluted serum were manually applied to the Hydragel agarose gels (Sebia). The subsequent steps, electrophoresis (pH 9.2, 20 W constant current at 20°C), drying, amidoblack staining, de-staining, and final drying, were carried out automatically. The use of Hydrasys densitometer and Phoresis software (Sebia) for scanning resulting profiles provided accurate relative concentrations (percentage) of individual protein zones. M-spike levels were calculated as total gamma globulins/albumin ratio (G/A).

Mouse blood was periodically collected in Eppendorf by retro-orbital sampling. Semi-automated electrophoresis was performed on the Hydrasys instrument (Sebia, Lissex, France). According to the manufacturer’s instructions, 10 µL of undiluted serum were manually applied to the Hydragel agarose gels (Sebia). The subsequent steps: electrophoresis (pH 9.2, 20 W constant current at 20 °C), drying, amidoblack staining, de-staining and final drying were carried out automatically. The use of Hydrasys densitometer and Phoresis software (Sebia) for scanning resulting profiles provided accurate relative concentrations (percentage) of individual protein zones. M-spike levels were calculated as total gamma globulins/albumin ratio (G/A)^{17 (link)}.