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Catalog # HK335

SP-D, Human, ELISA kit

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Surfactant protein D (SP-D) belongs to the collectin familiy. These proteins are oligomeric proteins composed of carbohydrate-recognition domains (CRD) attached to collagenous regions. Collectins are structurally similar to the ficolins although they make use of different CRD structures: C-type lectin domain for the collectins. The lung is the major site of synthesis of SP-D, where the molecules are produced and secreted onto the epithelial surface by alveolar type II cells and unciliated bronchial epithelial cells. SP-D is also found in different epithelial cells of the gastrointestinal tract and in epithelial cells of various exocrine glands. SP-D is an important factor in the pulmonary anti-microbial defense. The anti-microbial defense mechanisms of SP-D are direct opsonization, neutralization and agglutination. This results in limiting the infection and concurrently orchestrating the subsequent adaptive immune response. SP-D synthesis and secretion increase significantly during inflammatory stress. In inflamed lungs increased amounts of SP-D in bronchial lavage fluid and tissue are found, particularly in type II pneumocytes, Clara cells and hyperplastic goblet cells are found in inflamed lungs. The localization of SP-D in endocytic vesicles and in lysosomal granules of alveolar macrophages suggests a receptor-mediated uptake. SP-D is also involved the apoptotic process. SP-D binds to apoptotic neutrophils and enhances their clearance by alveolar macrophages. Although complete deficiency of SP-D has not been described in man, basal levels are highly variable and are largely genetically determined. SP-D levels in healthy individuals vary from 200 ng/ml to 5200 ng/ml. Mean SP-D levels increase from 700 ng/ml in children to 1100 ng/ml in adults. Bronchoalveolar fluid SP-D control levels range from 7 ng/ml to 28 µg/ml. SP-D levels can rise dramatically during acute pulmonary infections and are raised in certain non-infectious lung diseases.
The ELISA is not influenced by the presence of lipopolysaccharides.

Application
The human SP-D ELISA kit is to be used for the in vitro quantitative determination of human SP-D in plasma, bronchoalveolar lavage fluid, and cell culture supernatant samples.

Principle
The human SP-D 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 2 plates with twelve disposable 8-well strips allows free choice of batch size for the assay. Samples and standards are incubated in microtiter wells coated with antibodies recognizing human SP-D. Biotinylated tracer antibody will bind to captured human SP-D. 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 SP-D standards (log). The human SP-D concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.

Linearity
The linearity of the assay was determined by serially diluting a sample containing 250 ng/ml human SP-D. The diluted samples were measured in the assay. The line obtained a slope of 1.08 and a correlation coefficient of 0.999.

Recovery
Normal human blood samples (plasma), were spiked with human SP-D in concentrations of 5 and 100 ng/ml. Samples with and without human SP-D were incubated for 1 hour at room temperature. Samples were measured using the ELISA. Values for human SP-D ranged between 83% and 133% (mean 108%).

Product type
Assays
Quantity
1 x 96 det., 2 x 96 det.
Standard range
6.3 to 400 ng/ml
Detection level
6.3 ng/ml
Working volume
100 µl/well
Species
Human
Cross reactivity
Rat – Weak
Storage and stability
Product should be stored at 4 °C. Under recommended storage conditions, product is stable for at least six months.
Precautions
For research use only. Not for use in or on humans or animals or for diagnostics. It is the responsibility of the user to comply with all local/state and Federal rules in the use of this product. Hycult Biotech is not responsible for any patent infringements that might result with the use of or derivation of this product.
Disease
Pulmonology
Safety Data Sheet Assay Manual
HK335
455 kb

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