Method Description

Alpha-1-Antitrypsin Proteotype S/Z

The method detects the differences in mass to charge ratios of alpha-1-antitrypsin (A1A) tryptic peptides containing the S and Z point mutations. Serum from a patient suspected of having an A1A S or Z mutation is prepared for digestion and then digested using trypsin to generate tryptic peptides from the proteins. The peptides are then subjected to liquid chromatography tandem mass spectrometry to interrogate the mixture for peptides consistent with wild type (Non S, Non Z) or mutated (S, Z) peptides from the S and Z A1A phenotypes. Reverse phase liquid chromatography is used to separate the peptides based on hydrophobicity while the eluate is scanned for the desired peptides using a triple quadrupole mass spectrometer operating under Standard Reaction Monitoring principles. Labeled internal standards, which have identical chemistry to the desired peptides but have slightly different masses, are used to assure the detected peptides from the sample are consistent with the internal standard elution times. By detecting the presence or absence of these peptides, the phenotype status for the S and Z alleles can be determined. It should be kept in mind that this method does not detect other phenotypes. Discrepancies between A1A quantitation levels and the results of this test need be followed up with isoelectric focusing testing.(Unpublished Mayo Method)

Alpha-1-Anitrypsin

In this Siemens Nephelometer II method, the light scattered onto the antigen-antibody complexes is measured. The intensity of the measured scattered light is proportional to the amount of antigen-antibody complexes in the sample under certain conditions. If the antibody volume is kept constant, the signal behaves proportionally to the antigen volume.

A reference curve is generated by a standard with a known antigen content on which the scattered light signals of the samples can be evaluated and calculated as an antigen concentration. Antigen-antibody complexes are formed when a sample containing antigen and the corresponding antiserum are put into a cuvette. A light beam is generated with a light emitting diode, which is transmitted through the cuvette. The light is scattered onto the immuno-complexes that are present. Antigen and antibody are mixed in the initial measurement, but no complex is formed yet. An antigen-antibody complex is formed in the final measurement.

The result is calculated by subtracting value of the final measurement from the initial measurement. The distribution of intensity of the scattered light depends on the ratio of the particle size of the antigen-antibody complexes to the radiated wavelength.(Instruction manual: Siemens Nephelometer II. Siemens, Inc; Version 2.3, 2008; Addendum to the Instruction Manual 2.3, 08/2017)