Porcine TNF-alpha ELISA Kit

Sample

Cell culture supernates, serum, and plasma

Species Reactivity

Pig

Intended Use

For the quantitative determination of porcine Tumor Necrosis Factor alpha (TNF-α) concentrations in cell culture supernates, serum, and plasma.

Contents of Kit

1. Porcine TNF-α Microplate, 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody speciffc for porcine TNF-α. Return unused wells to the foil pouch containing the desiccant pack. Reseal along entire edge of the zip-seal. May be stored for up to 1 month at 2-8 °C.*
2. Porcine TNF-α Standard, 2 vials recombinant porcine TNF-α in a buffered protein base with preservatives;
lyophilized. Refer to the vial label for reconstitution volume. Discard within 8 hours of reconstitution. Use a new standard and control for each assay.
3. Porcine TNF-α Control, 2 vials of recombinant porcine TNF-α in a buffered protein base with preservatives; lyophilized. The assay value of the control should be within the range speciffed on the label. Discard within 8 hours of reconstitution. Use a new standard and control for each assay.
4. Porcine TNF-α Conjugate, 12 mL of a monoclonal antibody speciffc for porcine TNF-α conjugated to horseradish peroxidase with preservatives. May be stored for up to 1 month at 2-8 °C.*
5. Assay Diluent CD1-63, 12 mL of a buffered protein solution with preservatives. May be stored for up to 1 month at 2-8 °C.*
6. Calibrator Diluent CD5T, 21 mL of a buffered protein solution with preservatives. For cell culture supernate samples.May be stored for up to 1 month at 2-8 °C.*
7. Calibrator Diluent CD6-33, 21 mL of diluted animal serum with preservatives. For serum/plasma samples. May be stored for up to 1 month at 2-8 °C.*
8. Wash Buffer Concentrate, 21 mL of a 25-fold concentrated solution of buffered surfactant with preservative. May turn yellow over time. May be stored for up to 1 month at 2-8 °C.*
9. Color Reagent A, 12 mL of stabilized hydrogen peroxide. May be stored for up to 1 month at 2-8 °C.*
10. Color Reagent B, 12 mL of stabilized chromogen (tetramethylbenzidine). May be stored for up to 1 month at 2-8 °C.*
11. Stop Solution, 23 mL of diluted hydrochloric acid.
* Provided this is within the expiration date of the kit.

Storage

Store the unopened kit at 2-8 °C. Do not use past kit expiration date.

Precision

Intra-assay Precision (Precision within an assay)
Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-assay Precision (Precision between assays)
Three samples of known concentration were tested in twenty separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components.
CELL CULTURE SUPERNATE ASSAY

SERUM/PLASMA ASSAY

Sensitivity

Four assays were evaluated and the minimum detectable dose (MDD) of porcine TNF-α ranged from 2.8-5.0 pg/mL. The mean MDD was 3.7 pg/mL.
The MDD was determined by adding two standard deviations to the mean O.D. value of twenty zero standard replicates and calculating the corresponding concentration.

General Description

Tumor necrosis factor alpha (TNF-α), also known as cachectin, is a member of the TNF ligand superfamily and has been designated TNFSF1A. It binds to the same cell surface receptors, and shares some biological functions with TNF-β/TNFSF1B. TNF-α inhibits the growth of certain tumors. It also plays a critical role in normal host resistance to infection, serving as an immunomodulator and as a mediator of inffammatory responses. Over-production of TNF has been implicated in a number of pathological conditions, including cachexia, septic shock, and autoimmune disorders. TNF-α is produced primarily by activated macrophages. Various other porcine cell types, including NK cells, keratinocytes, vascular smooth muscle cells, and granulosa lutein cells are also known to produce TNF-α.
The porcine TNF-α gene product is a 232 amino acid (aa) residue type II membrane glycoprotein containing a 35 aa cytoplasmic domain, a 21 aa transmembrane domain and a 178 aa extracellular domain. The 156 aa residue soluble TNF-α is released from the C-terminus of the membrane protein by TNF-α converting enzyme (TACE, ADAM17), a member of the ADAM (a disintegrin and metalloprotease domain) family of metalloproteases. The biologically active TNF-α has been shown to exist as a trimer. Porcine TNF-α is active on mouse cells and shares 89% and 79% aa sequence identity with human and mouse TNF-α, respectively.
Two distinct TNF receptors, referred to as type I (type B, p55, or TNFRSF1A) and type II (type A, p75, or TNFRSF1B), that speciffcally bind TNF-α and TNF-β with equal afffnities are known. The two TNF receptors share aa sequence homology in their extracellular but not their cytoplasmic domains, suggesting that the two receptors employ different signal transduction pathways. Soluble forms of both types of receptors have been found in human and mouse serum. These soluble receptors are capable of neutralizing the biological activities of the TNFs and may serve to modulate the activities of TNF. Porcine TNF RI shares 79% and 72% aa homology with the human and mouse TNF RI, respectively.
The Porcine TNF-α Immunoassay is a 4.5 hour solid phase ELISA designed to measure porcine TNF-α in cell culture supernates, serum, and plasma samples. It contains E. coli-expressed recombinant porcine TNF-α and antibodies raised against the recombinant factor. This immunoassay has been shown to accurately measure recombinant porcine TNF-α. Results obtained using natural porcine TNF-α show dose response curves that are parallel to the standard curves obtained using the kit standards. These results indicate that this kit can be used to determine relative mass values for natural porcine TNF-α.

Standard Curve

These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed.