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Technical Briefs |
1-Antitrypsin Deficiency
1 Wieslab AB, Ideon, Science Park, Lund, Sweden
2 Department of Medicine, University Hospital Malmö, Malmö, Sweden
aaddress correspondence to this author at: Wieslab AB, Ideon, Science Park, Sölvegatan 41, 22370 Lund, Sweden; fax 46-46-14-08-90, e-mail sg{at}wieslab.se
1-Antitrypsin (AAT) deficiency is a hereditary autosomal disorder resulting from a variety of mutations in the AAT gene. The most common severe deficiency variant of AAT is Z, which has been identified in most populations but occurs most frequently in northwest Europe. The frequency of the Z allele in the US population of European descent is between 0.01 and 0.02, with the homozygous deficiency affecting 1 in every 2000 to 7000 individuals. In Scandinavia, the frequency of the Z allele is considerably higher: at birth, 1 of every 1600 babies is homozygous for the Z allele. Individuals homozygous for the AAT Z allele have a high risk for developing early-onset pulmonary emphysema and/or abnormal liver function in infancy that may lead to complete liver failure. The Z allele is also suspected in patients with Wegener granulomatosis and panniculitis. Here we briefly describe a simple and accurate new ELISA-based test for identifying carriers of the AAT Z allele.
AAT is the main circulating and tissue serine protease inhibitor in humans (1). The mean concentration of AAT in serum or plasma in healthy individuals is estimated to be 1.3–1.7 g/L, with a half-life of 3–5 days (2)(3). Circulating AAT increases rapidly to concentrations exceeding 2 g/L in response to a wide range of inflammatory conditions, infections, cancer, liver disease, or pregnancy (4)(5)(6). When the AAT concentration in plasma decreases to <0.7 g/L, the individual is considered AAT-deficient (7). To date, more than 75 alleles have been identified, of which at least 20 affect either the amount or function of the AAT molecule (8). A protein inhibitor (Pi) system has been developed to describe the various allelic variants; this system is based on the migration of the protein in an electric field (9). The position of the migrated protein is given a letter designation. The most common variant, Pi M (i.e., AAT migrates in the middle), is present in 
95% of the Caucasian US population and is regarded as the variant associated with normal serum concentrations of functional AAT. The concentration of circulating AAT in the MM phenotype is therefore assigned a relative value of 100%. Heterozygous or homozygous combinations have AAT serum concentrations corresponding to 50% (MZ), 37.5% (SZ), 65% (SS), and 15% (ZZ) of this MM value, respectively (10)(11). More than 90% of clinical cases of severe AAT deficiency are caused by the homozygous Z variant (11)(12). The clinical role of intermediate deficiency (MZ and SZ) of AAT is less clear.
A single amino acid substitution in the Z AAT molecule (Glu342Lys) makes it susceptible to spontaneous polymerization. The accumulation of Z AAT polymers within the endoplasmic reticulum of hepatocytes causes protein overload, which may lead to neonatal hepatitis, cirrhosis, and hepatocellular carcinoma (13). Individuals homozygous for the AAT Z allele have a markedly increased risk of developing lung emphysema that is linked to the lack of proteinase inhibitor and uncontrolled proteolysis (14)(15).
Immunologic measurements of the plasma concentration of AAT and isoelectric focusing (IEF) have been the standard clinic diagnostic tests for AAT alleles for more than 20 years in the United States and Europe. Measurement of serum AAT by quantitative immunoprecipitation is insufficient for the diagnosis of AAT deficiency because the concentration of this protein is known to increase during the acute-phase response, pregnancy, cancer, or other conditions, and thus can mask a partial AAT deficiency. Quantitative measurements of AAT must be combined with phenotypic analysis performed by IEF or agarose gel electrophoresis with immunofixation, a method proposed by Jeppsson and Franzen (16). These tests are, however, fairly cumbersome to perform, and interpretation of the gel pattern requires special training and skills.
The molecular genetics tools for defining the defect in the nucleotide coding sequence for each of the defective alleles have recently been developed (17)(18), but these methods are not routinely available in diagnostic laboratories because they are expensive, require special skills, and are not well suited for screening purposes.
In patients with manifestations of liver disease, liver biopsy for light microscopy and histochemistry and possible electron microscopy is valuable for staging liver disease and for identification of periodic acid-Schiff (PAS)-positive/diastase-resistant globules within the hepatocytes (19). Confirmation of the nature of the globules may be provided by immunohistochemical techniques using antibodies specific against AAT. Wallmark et al. (20) developed a monoclonal antibody (ATZ11) by immunizing mice with globular inclusions purified from a homozygous Z AAT liver. Using immunohistochemical techniques, Callea et al. (21) found that this antibody specifically labeled the PiZ AAT protein in hepatocytes. In an ELISA, ATZ11 antibody recognized sera from PiZ but not from PiM, PiS, or PiF individuals (20). This antibody was therefore considered of great value for identification of carriers of the PiZ gene.
We recently reevaluated the monoclonal ATZ11 antibody and found that the epitope recognized by this antibody does not involve the mutation per se, but is exclusively exposed on the polymerized form of AAT (22). Application of the ATZ11 antibody in Western blot analysis provides clear evidence that in PiZ carriers, a significant fraction of plasma AAT is in a polymeric form (22). Moreover, the ATZ11 antibody did not react with polymers formed by two other AAT deficiency variants, namely Siiyama (Ser53Phe in exon II) and Mmalton (deleted 52Phe in exon II), and with polymers formed by cleaved AAT or C-sheet polymers formed in vitro by heating AAT in citrate and at low pH (23). These findings further confirmed that the ATZ11 antibody is specific for Z AAT polymers and can be used to identify Z allele heterozygosity (MZ and SZ) or homozygosity in either tissue samples or serum.
We evaluated the newly developed Wielisa® PiZ test in ELISA format (Wieslab®) for rapid and specific qualitative identification of PiZ AAT carriers. In brief, the microtiter strips are coated with anti-AAT polyclonal antibodies. Serum samples are diluted 1:80 with a buffer containing antibody ATZ11 and applied to the wells (100 µL/well). After incubation for 30 min at room temperature, wells are washed with 0.15 mol/L NaCl containing 0.5 mL/L Tween 20, and alkaline phosphatase-labeled rabbit antibody to mouse IgG (100 µL/well) is applied for 30 min. After another washing step, the AAT–ATZ11 antibody complex is detected by addition of substrate containing p-nitrophenyl phosphate for 30 min. The amount of bound ATZ11 correlates to the absorbance at 405 nm. According to the recommendations in the Wielisa assay instructions, the ratio of the absorbance of a patient sample to that of a positive control is calculated; samples with an absorbance ratio <1 are considered as negative, and those with a ratio 
1 are considered as positive.
We determined the presence of Z AAT allele in serum and plasma samples obtained from two, well-characterized clinical materials. The first studied material consisted of 24 PiM individuals (14 healthy individuals and 10 patients with chronic obstructive pulmonary disease) and 20 individuals homozygous for PiZ AAT (10 asymptomatic and 10 patients with chronic obstructive pulmonary disease) (22). AAT phenotyping was performed by IEF, and the AAT concentration was measured by nephelometry at the Department of Clinical Chemistry, University Hospital, Malmö. The mean concentrations of AAT in PiZ and PiM cases were 0.25 g/L (range, 0.182–0.456 g/L) and 1.47 g/L (1.28–1.88 g/L), respectively. Using the Wielisa PiZ assay, we correctly identified the AAT Z allele for all 20 PiZ AAT individuals. All PiM individuals were also correctly categorized as having no inherited Z mutation (Fig. 1A
).
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The second study group included 220 individuals belonging to the Malmö Diet and Cancer (MDC) study cohort (24). The AAT concentration in individual plasma samples was measured by rocket immunoelectrophoresis (25). Using the Wielisa ELISA test, we identified 12 cases who were AAT PiZ heterozygotes (among them 1 Z AAT homozygote; Fig. 1B
). Our findings correlated completely with results obtained by IEF. The mean plasma concentration in PiZ heterozygotes was 0.84 g/L (range, 0.27–0.7 g/L), and that in PiM homozygotes was 1.57 g/L (1.2–1.6 g/L). The relative sensitivity and specificity of the Wielisa test were both 100% compared with IEF.
Compared with existing methodologies, the Wielisa PiZ AAT ELISA is highly specific, accurate, rapid, and simple to perform; it appears to be well suited for routine analysis and screening applications in a clinical laboratory setting. The test could be of considerable use for evaluating health risks associated with expression of the Z allele of AAT.
References
1-antitrypsin and 1-antichymotrypsin. Biochem Soc Trans 2002;30:93-98.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
1-antitrypsin in cancer development and progression. Lancet Oncol 2004;5:182-190.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
1-antitrypsin deficiency gene and its deficiency states. Trends Genet 1989;5:411-417.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
1-Antitrypsin deficiency. 1: epidemiology of 1-antitrypsin deficiency. Thorax 2004;59:164-169.-1-globulin pattern of serum in -1-antitrypsin deficiency. Scand J Clin Lab Invest 1963;15:132-140.[Web of Science]
1-Antitrypsin deficiency. N Engl J Med 2002;346:45-53.1-Antitrypsin genotypes and phenotypes. Crystal RG eds. 1-Antitrypsin deficiency 1996:45-59 Marcel Dekker New York. .
1-antitrypsin screening: parents’ views and reactions 20 years after the identification of the deficiency state. Acta Paediatr 1999;88:315-318.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
1-antitrypsin deficiency associated liver disease. J Gastroenterol Hepatol 1997;12:404-416.[Web of Science][Medline]
[Order article via Infotrieve]
1-antitrypsin by electrofocusing. Clin Chem 1982;28:219-225.1-antitrypsin genotype analysis. Eur Respir J 2003;21:215-219.-1-antitrypsin variants by duplex PCR assay. Mol Cell Probes 1999;13:389-391.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
-1-antitrypsin (AAT) immunohistochemistry in paraffin-embedded liver tissue specimens. J Hepatol 1986;2:389-401.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
1-antitrypsin and its application in an ELISA procedure for identification of PiZ gene carriers. Proc Natl Acad Sci U S A 1984;8:5690-5693.
1-antitrypsin. J Hepatol 1991;12:372-376.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
1-antitrypsin by a monoclonal antibody. J Biol Chem 2002;277:26540-26546.1-antitrypsin. Hepatology 2004;40:1203-1210.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
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