Swine TNF-α ELISA Kit

Sample

cell culture supernates, serum, plasma

Species Reactivity

Pig

Detection Method

Sandwich ELISA

Intended Use

For the quantitative determination of swine tumor necrosis factor(TNF-α) concentrations in cell culture supernates, serum, and plasma.This package insert must be read in its entirety before using this product.

Contents of Kit

1. Aluminium pouches with a Microwell Plate coated with antibody to swine TNF-α (8 x 12)
2. 2 vials swine TNF-α Standard lyophilized, 1000 pg/mL upon reconstitution
3. 2 vials concentrated Biotin-Conjugate anti-swine TNF-α antibody
4. 2 vials Streptavidin-HRP solution,
5. 1 bottle Standard/sample Diluent
6. 1 bottle Biotin-Conjugate antibody Diluent
7. 1 bottle Streptavidin-HRP Diluent
8. 1 bottle Wash Buffer Concentrate 20X (PBS with 1% Tween-20)
9. 1 vial Substrate Solution
10. 1 vial Stop Solution

Storage

For more detailed information, please download the following document on our website.

Performance Characteristics

1. When mixing or reconstituting protein solutions, always avoid foaming.
2. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
3. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
4. Substrate Solution should remain colorless until added to the plate. Stop Solution should be added to the plate in the same order as the Substrate Solution. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue.
5. A standard curve should be generated for each set of samples assayed. According to the content of tested factors in the sample, appropriate diluted or concentrated samples, it is best to do pre-experiment.

1. REPEATABILITY: The coefficient of variation of both intra-assay and inter-assay were less than 10%.
2. SENSITIVITY: The minimum detectable dose was 7pg/mL.
3. SPECIFICITY: This assay recognizes both recombinant and natural porcine TNF-α. The factors listed below were prepared at 50 ng/mL in Calibrator Diluent RD6-33 and assayed for cross-reactivity. Preparations of the following factors at the same concentrations in a mid-range porcine TNF-α control were assayed for interference. No significant cross-reactivity or interference was observed.

Factors assayed for cross-reactivity

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 inflammatory responses. Over-production of TNF has been implicated in a number of pathological conditions, including cachexia, septic shock, and autoimmune disorders (1-4). TNF-α is produced primarily by activated macrophages (1-6). Various other porcine cell types, including NK cells (6), keratinocytes (7), vascular smooth muscle cells (8), and granulosa lutein cells (9) 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 (10-13). 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(10, 11, 14). The biologically active TNF-α has been shown to exist as a trimer (1-4). Porcine TNF-α is active on mouse cells and shares 89% and 79% aa sequence identity with human and mouse TNF-α, respectively (10, 14).Two distinct TNF receptors, referred to as type I (type B, p55, or TNFRSF1A) and type II (type A, p75, or TNFRSF1B), that specifically bind TNF-α and TNF-β with equal affinities are known (15-17). 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 (18-20).
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 (21-23).