Cathelicidins are a family of antimicrobial proteins predominantly found in the peroxidase-negative granules of neutrophils. The cathelicidins are synthesized as preproproteins. Within the neutrophils, they are stored in granules as inactive proforms after removal of the signal peptide. The active biologic domains of the cathelicidins generally reside in the C-terminus. The C-terminal antimicrobial peptides are activated when cleaved from the proforms of the cathelicidins by serine proteases from azurophil granules. Human cationic antimicrobial protein (hCAP)18 is the only human cathelicidin identified to date. The antibacterial C-terminus of hCAP-18, LL37 (37 amino acids), has been shown to exert broad antimicrobial activity towards gram-negative as well as gram-positive bacteria and to have synergistic antibacterial effects with the defensins. For instance, deficiency in saliva LL37 accords with occurrence of periodontal disease in patients with morbus Kostmann. Moreover, it functions as a chemotactic agent for neutrophils, monocytes and T cells. LL-37 is markedly resistant to proteolytic degradation and to a limited extent also cytotoxic towards mammalian cells. LL-37 was demonstrated to be present in plasma in levels from 25 – 250 ng/ml and to be enhanced in infectious diseases.
The human LL-37 ELISA kit is to be used for the in vitro quantitative determination of human LL-37 in plasma and cell culture supernatant samples.
The human LL-37 ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 3½ hours.
The efficient format of a plate with twelve disposable 8-well strips allows free choice of batch size for the assay.
Samples and standards are captured by a solid bound specific antibody.
Biotinylated tracer antibody will bind to captured human LL-37.
Streptavidin-peroxidase conjugate will bind to the biotinylated tracer antibody.
Streptavidin-peroxidase conjugate will react with the substrate, tetramethylbenzidine (TMB).
The enzyme reaction is stopped by the addition of oxalic acid.
The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the human LL-37 standards (log).
The human LL-37 concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
The linearity of the assay was determined by serially diluting a sample containing 35 ng/ml human LL-37. The diluted samples were measured in the assay. The line obtained a slope of 1.185 and a correlation coefficient of 0.999.