Serum, plasma, cell culture supernatant and other biological samples
Species Reactivity
Mouse
Detection Method
Sandwich-ELISA
Intended Use
In vitro quantitative determination of AAT concentrations in serum, plasma, cell culture supernatant and other biological samples.
Contents of Kit
No.
Components
Size
Storage Conditions
1
ELISA Microplate(Dismountable)
96T (8×12)
2-8°C/-20°C
2
Lyophilized Standard
2 vials
2-8°C/-20°C
3
Biotin-labeled Antibody
120ul
2-8°C
4
HRP-labeled Conjugate (SABC, 100X)
120ul
2-8°C (Avoid Direct Light)
5
TMB Substrate
10ml
2-8°C (Avoid Direct Light)
6
Sample Dilution Buffer
20ml
2-8°C
7
Antibody Dilution Buffer
10ml
2-8°C
8
SABC Dilution Buffer
10ml
2-8°C
9
Stop Solution
10ml
2-8°C
10
Wash Buffer(25X)
30ml
2-8°C
11
Plate Sealer
5 pieces
Storage
2-8°C (for sealed box), please do not freeze! See kit label for expiry date.
Performance Characteristics
Stability: Perform the stability test for the sealed kit at 37°C and 2-8°C and get relevant data.
Precision
Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on same plate. Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.
Detection Range
31.25-2000ng/ml
Sensitivity
18.75ng/ml
Standard Curve
Citations
Publication ()
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Background
Alpha-1 antitrypsin (AAT) is an ordinary serine protease inhibitor superfamily member: a serpin A member. It is the highest concentration of serine protease inhibitor in human plasma. AAT is coded by the gene SERPINA1 that has seven exons on the long arm of chromosome 14 (14q31–32.3). It's a single-chain glycoprotein with a 52 kDa molecular weight. AAT has a polypeptide chain with 394 amino acid residues: one free cysteine and three side chains of carbohydrate linked to asparagine. Because they share a disulfide bond with two cysteine residues, the cysteine residues oxidise and give rise to dimers. In its location, there are three beta sheets and eight or nine alpha helices for alpha-1 antitrypsin.
The bulk of AAT is released by hepatocytes, of course, but so can alveolar epithelial cells, intestinal epithelial cells, monocytes, macrophages and bronchial epithelial cells. AAT is an acute-phase response protein; it's the one we use in serum (0.9 to 2.3 g/L). If inflammation is extreme, AAT flows down the blood vessels to tissue fluid and settles in concentrated amounts at the site of inflammation to modulate the immune system. AAT can also undergo oxidation, cleavage, or binding with other molecules, which may affect its biological activity. Since AAT is a serine protease inhibitor, it inhibits not only neutrophil NE, proteinase 3 and cathepsin G but also trypsin, chymotrypsin, hyaluronidase and plasmin (making it a broad-spectrum protease inhibitor). AAT normally attaches to NE at 1:1 molar ratio to create an AAT-protease complex that macrophages recognise, phagocytose and clear away in the blood. The primary function of AAT is to protect the proteins of the alveolar matrix from degradation by neutrophil elastase. Low circulating concentrations of AAT in plasma can increase the risk of liver disease and lung disease.
AAT deficiency is a metabolic genetic condition in which the AAT gene is mutated in different ways: point mutations, insertions, deletions. The most common point mutation swaps glutamic acid for lysine in 342 of the peptide chain, changing the charge and tertiary structure of the enzyme and possibly causing AAT to aggregate. In hepatocytes, the mutated AAT may misfold or even aggregate within the endoplasmic reticulum, preventing it from entering circulation. These retained mutant forms of AAT in the liver can cause liver damage and lead to diseases such as liver cirrhosis.
Figure 1. Clinical implications of AAT deficiency (Source: O'Brien ME, et al. 2022)
1. O'Brien ME, et al. A Review of Alpha-1 Antitrypsin Binding Partners for Immune Regulation and Potential Therapeutic Application. Int J Mol Sci. 2022 Feb 23;23(5):2441.
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References
Microarray Analysis of NF-kappa B-dependent Genes in Chronic Rhinosinusitis with Nasal Polyps
Background. The inflammatory process underlying nasal polyposis is induced and perpetuated by the enhanced activity of several agents including transcription factors. It has recently been demonstrated that one of them, named nuclear factor-kappa B (NF-kappa B), is implicated in the regulation of multiple pro-inflammatory genes. Objectives. The aim of the study was to identify using microarray technology which NF-kappa B-dependent genes are activated in nasal polyp (NP) samples compared to the control mucosa. Material and Methods. The transcriptional activity of genes was analyzed using an oligonucleotide microarray on 15 NPs and 8 cases of normal nasal mucosa. Results. Gene expression patterns obtained in NPs were significantly different from those in normal mucosa. NPs and control cases clustered separately, each of them with large homogeneity in gene expression. Among 582 human NF-kappa B-dependent genes 25 showed a significantly higher expression in NPs compared to the control. The largest increase focused on gene encoding TFF3 (a 5-fold higher expression) followed by NOS2A (5x), SERPINA1 (4x), UCP2 (4x), OXTR (4x) and IL8 (3x) (p < 0.05). In healthy mucosa 19 genes presented increased transcription activity compared to NPs. The most significantly enhanced levels were shown in case of LTF gene (20 fold) followed by KRT6B (7x), LYZ (7x), SD11B2 (5x) and MMP3 (4x) (p <0.05). Conclusions. DNA microarray technology highlights the involvement of many unsuspected pathologic pathways which could be involved in NP growth. The identification of novel disease-related genes may help to understand the biology of NPs and elaborate new targeted therapy
Alpha-1-Antitrypsin in Pathogenesis of Hepatocellular Carcinoma
Context: Alpha-1-antitrypsin (A1AT) is the most abundant liver-derived, highly polymorphic, glycoprotein in plasma. Hereditary deficiency of alpha-1-antitrypsin in plasma (A1ATD) is a consequence of accumulation of polymers of A1AT mutants in endoplasmic reticulum of hepatocytes and other A1AT-producing cells. One of the clinical manifestations of A1ATD is liver disease in childhood and cirrhosis and/or hepatocellular carcinoma (HCC) in adulthood. Epidemiology and pathophysiology of liver failure in early childhood caused by A1ATD are well known, but the association with hepatocellular carcinoma is not clarified. The aim of this article is to review different aspects of association between A1AT variants and hepatocellular carcinoma, with emphasis on the epidemiology and molecular pathogenesis. The significance of A1AT as a biomarker in the diagnosis of HCC is also discussed. Evidence Acquisitions: Search for relevant articles were performed through Pub Med, HighWire, and Science Direct using the keywords "alpha-1-antitrypsin", "liver diseases", "hepatocellular carcinoma", "SERPINA1". Articles published until 2011 were reviewed. Results: Epidemiology studies revealed that severe A1ATD is a significant risk factor for cirrhosis and HCC unrelated to the presence of HBV or HCV infections. However, predisposition to HCC in moderate A1ATD is rare, and probably happens in combination with HBV and/or HCV infections or other unknown risk factors. It is assumed that accumulation of polymers of A1ATD variants in endoplasmic reticulum of hepatocytes leads to damage of hepatocytes by gain-of-function mechanism. Also, increased level of A1AT was recognized as diagnostic and prognostic marker of HCC. Conclusions: Clarification of a carcinogenic role for A1ATD and identification of pro-inflammatory or some still unknown factors that lead to increased susceptibility to HCC associated with A1ATD may contribute to a better understanding of hepatic carcinogenesis and to the development of new drugs. Published by Kowsar Corp, 2012. cc 3.0.
COA
Publication 
Figure 1. Clinical implications of AAT deficiency
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