Lysozyme ELISA for Human Stool (ImmuChrom)

Lysozyme ELISA for Human Stool (ImmuChrom)

IC6900
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Intended Use

The ImmuChrom Lysozyme ELISA Kit is an immunoassay intended for the quantitative determination of lysozyme in human stool samples. For research use only.

Description

Lysozyme is a relatively small enzyme consisting of a 129 amino acid polypeptide chain with a molecular weight of 14.6 kDa.

Lysozyme is a glycosidase that, as a component of the innate immune system, is primarily directed against the murein-containing cell wall of gram-positive bacteria. Therefore, it is also called muramidase because it can cleave the murein (peptidoglycan) from bacterial walls by hydrosylating the beta-1,4-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid.

Lysozyme is found in many secretions of the human body, including tear fluid, saliva, blood serum and cerebrospinal fluid. It is produced in the tissues of the respiratory tract, kidneys, and intestinal mucosa, as well as by neutrophil granulocytes and macrophages. [2;3]

Under physiological conditions, about 80% of the lysozyme in blood plasma is due to neutrophil granulocyte degradation. [4]

Lysozyme is normally undetectable or detectable in small amounts in the intestinal contents. However, fecal lysozyme may occur through intestinal granulocytes. Lysozyme can be detected in all cells of the inflammatory infiltrate in Crohn's disease. Furthermore, lysozyme can be actively secreted into the intestinal lumen by monocytes and macrophages. [1;] Applications • Inflammatory processes in the intestine • Detection of a disturbed immunological barrier at the intestinal mucosa

Type

Sandwich ELISA, HRP-labelled antibody

Other names

Muramidase, N-acetylmuramide glycanhydrolase, EC 3.2.1.17, LYZL1 gene product

Principle of Method

The lysozyme-ELISA test determines human lysozyme according to the “sandwich”- principle. Lysozyme in sample, standard and controls binds to antibodies, which are coated to the microtiter plate. After a washing step a peroxidase labeled detection antibody is added. A second washing step is followed by the addition of the substrate which is converted to a colored product by the peroxidase. The reaction is terminated by the addition of an acidic stop solution. The optical densities are read at 450 nm (against the reference wavelength 620 nm) in a microtiter plate reader. The lysozyme concentration can be calculated from the standard curve

Sample Types

Stool

Sample Volume

Extraction in Stool extraction vials

In a stool sample extraction vial mix 15 mg stool with 0.75 ml diluted wash buffer, then vortex it until the mixture is homogenous. Transfer the resulting slurry to a plastic vial and centrifuge it for 10 min at 3000xg. The supernatant is diluted 1:10 in SAMPLEBUF. We recommend 20 µl supernatant to mix with 180 µl SAMPLEBUF. 100 µl of the dilution are used in the test per well. Please use only plastic vials and no glass vials.

Standard Curve

Calibration Range:  0.55 ng/ml - 5 ng/ml

Limit of Detection

Stool 150 ng/g

For the determination the zero-standard was measured 20 times. The 3-fold standard deviation was added to the mean value of the optical density. The respective concentration was read from the standard curve.

Intra-assay (Within-Run) C.V.

5.3%

Inter-assay (Run-to-Run) C.V.

6.4%

Resources

Ilex Life Sciences LLC is an official distributor of Immuchrom GmbH.

References to Summary

1 - Johan Brouwer, Trudi van Leeuwen-Herberts und Marjo Otting-van de Ruit: Determination of lysozyme in serum, urine, cerebrospinal fluid and feces by enzyme immunoassay. In: Clinica Chimica Acta. 142, Nr.1, 15.September 1984, S.21-30.

2 - Lien Callewaert, Chris W. Michiels: Lysozymes in the animal kingdom. In: Journal of Biosciences. 35, Nr.1, März 2010, S.127–160.

3 - H. A. McKenzie und F.H. White (Jr.): Lysozyme and α-lactalbumin: structure, function and interrelationships. In: Advances in Protein Chemistry. 41, 1991, S.173- 315.

4 - J. P. van de Merwe, J. Lindemans und G. J. Mol: Plasma lysozyme levels and decay of neutrophilic granulocytes in patients with Crohn's disease. In: Hepatogastroenterology. 27, Nr.2, April 1980, S.130-134.