Bovine Flotillin-2 (FLOT2) ELISA Kit

This assay employs a two-site sandwich ELISA to quantitate FLOT2 in samples. An antibody specific for FLOT2 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any FLOT2 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for FLOT2 is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of FLOT2 bound in the initial step. The color development is stopped and the intensity of the color is measured.
 

Caveolae are small domains on the inner cell membrane involved in vesicular trafficking and signal transduction. Flotillin-2 is a caveolae-associated, integral membrane protein, which is thought to function in neuronal signaling. Human epidermal surface antigen-1 was identified as a 35-kD antigen by means of a mouse monoclonal antibody raised against human keratinocytes. The monoclonal antibody was found to cause keratinocyte dissociation in vitro; hence, the conclusion that the native protein is involved in cell adhesion. The mouse and human 5-prime- and 3-prime untranslated sequences are conserved. Similar RNA folding patterns of the 5-prime untranslated region are predicted despite a 91-bp insertion in the mouse.
 

Reagents	Quantity	Reagents	Quantity
Assay plate (96 Wells)	1	Instruction manual	1
Standard (lyophilized)	2	Sample Diluent	1 x 20 mL
Biotin-Conjugate (concentrate 100 x)	1 x 120 μL	Biotin-Conjugate Diluent	1 x 12 mL
Streptavidin-HRP  (concentrate 100 x)	1 x 120 μL	Streptavidin-HRP Diluent	1 x 12 mL
Wash Buffer (concentrate 25 x)	1 x 20 mL	Substrate Solution	1 x 10 mL
Stop Solution	1 x 6 mL	Adhesive Films	4

 

This assay has high sensitivity and excellent specificity for detection of Bovine FLOT2. No significant cross-reactivity or interference between Bovine FLOT2 and analogues was observed.
 

Matrices listed below were spiked with certain level of recombinant Bovine FLOT2 and the recovery rates were calculated by comparing the measured value to the expected amount of Bovine FLOT2 in samples.
 

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 forty separate assays to assess inter-assay precision. CV (%) = SD/meanX100 Intra-Assay: CV<8% Inter-Assay: CV<12%
 

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Bovine FLOT2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
 

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).
 

Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
 

Store at 2-8°C. Please refer to Instruction Manual.