Human Lactoferrin ELISA Assay
The Human Lactoferrin ELISA Assay is For Research Use Only
Size: 1 × 96 wells, 2 x 96 wells
Sensitivity: 0.4 ng/ml
Dynamic Range: 0.4 ng/ml – 100 ng/ml
Incubation Time: 4 hours 30 minutes
Sample Type: Plasma, Urine, Feces, Breast Milk, Cell Culture Supernatant
Sample Size: 100 μL
Assay Principle
The Human lactoferrin ELISA kit is a solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 4½ hours. Samples and standards are captured by a solid bound specific antibody. Biotinylated tracer antibody will bind to captured human lactoferrin. 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 lactoferrin standards (log). The human lactoferrin concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
Assay Background
Human lactoferrin (LF) is an 80 kDa glycoprotein found concentrated in the secondary granules of the neutrophils. In addition, lactoferrin can be found in epithelia and most body fluids and secretions. Lactoferrin was first isolated from human milk and plays an important part in the immune system by helping to fight infections. It has the ability to bind iron and possesses five different enzyme activities: DNase, RNase, ATPase, phosphatase, and malto-oligosaccharide hydrolysis. Lactoferrin is a natural anti-bacterial, anti-fungal and anti-viral protein, it is an antioxidant and also possesses immunomodulatory properties.
Furthermore, lactoferrin promotes the health of the gastro-intestinal system by improving the intestinal microbalance. Lactoferrin is secreted in plasma by neutrophils. Plasma of healthy individuals contains ~190-500 ng/ml LF. The lactoferrin plasma concentration represents a positive relation to the total pool of neutrophils and the rate of neutrophil turnover. Upon inflammation, lactoferrin is released from the secondary neutrophil granules into the extracellular medium. Therefore, the extracellular lactoferrin concentration can be used as an index for neutrophil activation. The iron binding property of lactoferrin is considered to be an important antimicrobial function. Human lactoferrin binds via its highly positively charged amino-terminus to bacterial products. It kills various bacteria, most probably by inducing intracellular changes in these bacteria without affecting the membrane permeability.
Cleavage by pepsin of lactoferrin leads to the release of lactoferricin H. This 47 amino acid peptide has more antimicrobial activity than its precursor and it can inhibit the classical but not the alternative complement pathway. Urine or breast milk of healthy persons contain ~30 ng/ml and ~500 µg/ml LF, respectively. During infection, the LF concentration can raise 10-100-fold. In feces of healthy persons, ~1 µg/g LF can be detected, whereas in feces derived from colon cancer or inflammatory bowel disease (IBD) patients, LF levels range from ~75-310 µg/g. Fecal lactoferrin is useful as a sensitive and specific marker in identifying intestinal inflammation such as Crohn’s disease and IBD.
Combination of several markers, such as calprotectin, defensin, elastase, MPO, I-FABP and MAdCAM, may be useful for classifying IBD, as well as for identifying tumor grade and to confirm remission/response to treatment. Therefore, the Human Lactoferrin ELISA is a sensitive, non-invasive tool for monitoring disease activity.
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