ALPHA 1 ANTITRYPSIN ELISA KIT
INTENDED USE
The Alpha 1 Antitrypsin ELISA kit is intended for the quantitative determination of alpha-1-Antitrypsin in stool. It is for in vitro diagnostic use only.
INTRODUCTION
The alpha 1-antitrypsin acts as a primary inhibitor of elastase from polymorph nuclear neutrophilic granulocytes (PMN) and is released during inflammatory processes in order to reduce the proteolytic activity of the PMN-elastase in the inflammation region. In addition, it inhibits via complex formation a series of serine proteinases, like blood-clotting proteinases, trypsin, chymotrypsin, etc. Thus, the alpha 1-antitrypsin plays an important regulatory as well as anti-inflammatory role.
The alpha 1-antitrypsin is a linear glycoprotein with a molecular weight of ca. 52 kDa (394 amino acid residues), a free cysteine residue and three carbohydrate side chains. It is predominantly synthesized in the liver but also by intestinal macrophages, monocytes and epithelial cells. Although alpha 1-antitrypsin is the main serine proteinase inhibitor in human plasma, the proof of fecal alpha 1-antitrypsin has become an important marker for intestinal protein loss and permeability, as it is able to resist degradation in the gut due to its anti-proteolytic activity. It will therefore stay intact and it is possible to detect it in the faeces using an immunoassay. Moreover, the measurement of fecal alpha 1-antitrypsin-concentration is used to evaluate and monitor chronic inflammatory intestinal diseases. In the clinical routine, the alpha 1-antitrypsin-clearence (ratio of the alpha 1-antitrypsin-ELISA-values of stool and serum samples) has been established along with the sole determination of the 24h- alpha 1-antitrypsin-secretion in stool. Thus, the group of J. S. Fordtran (Strygler et al. 1990) reports, that the sole determination of the alpha 1-antitrypsin-concentration in stool yielded false positive or false negative results in 21% of the patients compared to the alpha 1-antitrypsin-clearencemeasurements.
In a comparative study with the radial immundiffusion (RID), routinely used in the clinical diagnostics, the alpha 1-antitrypsin-ELISA Kit demonstrated significant advantages in the analysis of serum, stool and Caco-2-cell culture supernatants (Faust et al. 2001):
1) The alpha 1-antitrypsin-concentrations obtained by the ELISA were on average about 30 % higher than the corresponding values from the radial immunodiffusion measurements.
2) Only the ELISA-system detected alpha 1-antitrypsin in the cell culture supernatants.
The results clearly demonstrate that our alpha 1-antitrypsin-ELISA-test is more sensitive than other routinely used methods and that it recognizes the hepatic as well as the enteral alpha 1-antitrypsin form. This newly developed test represents a promising alternative to the use of current clinical routine methods. It is superior to the radial immundiffusion especially in cases with extremely high protein loss through the gut. The combination of two specific antibodies eliminates to a large extent the possibility of false negative results guaranteeing reliable diagnoses. This newly developed test is a non-invasive, simple test for the detection of protein loss through the gut.
PRINCIPLE OF THE TEST
Alpha 1 Antitrypsin ELISA kit
The Alpha 1 Antitrypsin ELISA kit is intended for the quantitative determination of alpha-1-Antitrypsin in stool. The Alpha 1 Antitrypsin ELISA kit assay utilizes the “sandwich” technique with two selected polyclonal antibodies that bind to human alpha -1-antitrypsin.
Standards, controls and prediluted samples which are assayed for human alpha -1-antitrypsin are added into the wells of a micro plate coated with a high affine polyclonal anti-human alpha -1-antitrypsin antibody. During the first incubation step, alpha -1-antitrypsin is bound by the immobilized antibody. Then a peroxidase-conjugated polyclonal anti-human alpha -1 antitrypsin antibody is added into each microtiter well and a “sandwich” of capture antibody - human alpha -1-antitrypsin – peroxidase-conjugate is formed. Tetramethylbenzidine (TMB) is used as peroxidase substrate. Finally, an acidic stop solution is added to terminate the reaction. The colour changes from blue to yellow. The intensity of the yellow colour is directly proportional to the concentration of alpha -1-antitrypsin. A dose response curve of the absorbance unit (optical density, OD at 450 nm) vs. concentration is generated, using the values obtained from the standard. alpha -1-antitrypsin present in the samples, is determined directly from this curve.
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