Bovine TNF-alpha IQELISA™ Kit

RayBio® Bovine Immunoquantitative (PCR-Based) TNF alpha ELISA Kit for cell culture supernatants, plasma, and serum samples.

IQB-TNFa-1
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Antigen Information

Accession Number:
  • Q06599
Gene ID:
  • 280943
Gene Symbols:
  • TNF
  • TNFA
  • TNFSF2
Protein Name & Synonyms:
Tumor necrosis factor (Cachectin) (TNF-alpha) (Tumor necrosis factor ligand superfamily member 2) (TNF-a) [Cleaved into: Tumor necrosis factor, membrane form (N-terminal fragment) (NTF) Intracellular domain 1 (ICD1) Intracellular domain 2 (ICD2) C-domain 1 C-domain 2 Tumor necrosis factor, soluble form]
Target Species:
  • Bovine (cow)

Assay Format

Detection Range:
7.3 pg/ml - 30,000 pg/ml
Recommended Dilution (serum & plasma):
2 - 20 fold
Sensitivity:
29.2 pg/ml
Number of Targets Detected:
1
Species Detected:
  • Bovine (cow)
Compatible Sample Types:
  • Cell Culture Supernatants
  • Plasma
  • Serum
Design Principle:
  • Sandwich-based
Method of Detection:
qPCR
Quantitative/Semi-Quantitative:
Quantitative
Solid Support:
96-well Microplate

Product Specifications

Size:
1, 2, or 5 x 96-Well Strip Microplate Kit
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Introduction

The RayBio® Immuno Quantitative Enzyme Linked ImumunoSorbent Assay (IQELISA™) is an innovative new assay that combines the specificity and ease of use of an ELISA with the sensitivity of real-time PCR. This results in an assay that is simultaneously familiar and cutting edge and enables the use of lower sample volumes while also providing more sensitivity. The RayBio® Bovine TNF alpha IQELISA™ Kit is a modified ELISA assay with high sensitivity qPCR readout for the quantitative measurement of Bovine TNF alpha in serum, plasma, and cell culture supernatants. This assay employs an antibody specific for Bovine TNF alpha coated on a 96-well PCR plate. Standards and samples are pipetted into the wells and TNF alpha present in a sample is bound to the wells by the immobilized antibody. The wells are washed and a detection affinity molecule is added to the plates. After washing away unbound detection affinity molecule, primers and a PCR master mix are added to the wells and data is collected using qPCR. Ct values obtained from the qPCR are then used to calculate the amount of antigen contained in each sample, where lower Ct values indicate a higher concentration of antigen.

Comparison

Comparison

Standard Curve

Standard Curve

Typical Data

Application Notes

Kit Components
  • TNF alpha Microplate (Item A): 96 well PCR plate coated with anti-Bovine TNF alpha
  • Wash Buffer I Concentrate (20x) (Item B): 25 ml of 20x concentrated solution
  • Standards (Item C): 2 vials of recombinant Bovine TNF alpha
  • Assay Diluent A (Item D): 30 ml diluent buffer, 0.09% sodium azide as preservative. For Standard/Sample (serum/plasma) diluent
  • Assay Diluent B (Item E): 15 ml of 5x concentrated buffer. For Standard/Sample (cell culture medium/urine) diluent
  • Detection Affinity Reagent for TNF alpha (Item F): 2 vials of a 4x concentrated solution of anti-Bovine TNF alpha affinity reagent
  • IQELISA™ Detection Reagent (Item G): 1.4ml of a 10x concentrated stock
  • Primer Solution (Item I): 1.7 ml vial
  • PCR Master Mix (Item J): 1.2 ml vial
  • PCR Preparation buffer (Item K): 1ml vial of 10x concentrated buffer
  • Final Wash Buffer (Item L): 10ml vial of 10x concentrated buffer
Other Materials Required
  • Real-time PCR instrument, Bio-Rad recommended
  • Precision pipettes to deliver 2 µl to 1 ml volumes
  • Adjustable 1-25 ml pipettes for reagent preparation
  • 100 ml and 1 liter graduated cylinders
  • Absorbent paper
  • Distilled or deionized water
  • Log-log graph paper or computer and software for data analysis
  • Tubes to prepare standard or sample dilutions
  • Heating block or water bath capable of 80°C
Protocol Outline
  1. Prepare all reagents, samples and standards as instructed
  2. Add 25µl standard or sample to each well. Incubate for 2.5 hours at room temperature or overnight at 4°C
  3. Add 25 µl detection affinity reagent to each well. Incubate 1 hour at room temperature
  4. Add 25µL of IQELISA™ Detection Reagent to each well. Incubate 1 hour
  5. Add 15µL Primer solution 10µL of PCR master mix to each well
  6. Run real-time PCR

Storage/Stability

May be stored for up to 6 months at 2° to 8°C from the date of shipment. Standard (recombinant protein) should be stored at -20°C or -80°C (recommended at -80°C) after reconstitution. Opened PCR plate or reagents may be stored for up to 1 month at 2° to 8°C. Note: the kit can be used within one year if the whole kit is stored at -20°C. Avoid repeated freeze-thaw cycles.

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Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.

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