BT Lab ELISA Test Kit Sandwich Assay Principle For Cell Lysates BMP - 4 Detection

High Specificity and Sensitivity Rat Bone Morphogenetic Protein 4 ELISA Kit 96 Wells

Cat.No E0266Ra

Standard Curve Range: 2ng/L - 600ng/L

Sensitivity: 1.01ng/L

Size: 96 wells

*This product is for research use only, not for use in diagnosis procedures. It’s highly recommend to read this instruction entirely before use.

Precision

Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision.

Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision.

CV(%) = SD/mean x 100

Intra-Assay: CV<8%

Inter-Assay: CV<10%

Intended Use

This sandwich kit is for the accurate quantitative detection of Rat Bone Morphogenetic Protein 4 (also known as BMP-4) in serum, plasma, cell culture supernates, cell lysates, tissue homogenates.

Assay Principle

This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Rat BMP-4 antibody. BMP-4 present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Rat BMP-4 Antibody is added and binds to BMP-4 in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated BMP-4 antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of Rat BMP-4. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.

Specimen Collection

Serum Allow serum to clot for 10-20 minutes at room temperature. Centrifuge at 2000-3000 RPM for 20 minutes.

Plasma Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 2000-3000 RPM at 2 - 8°C within 30 minutes of collection.

Urine Collect by sterile tube. Centrifuge at 2000-3000 RPM for approximately 20 minutes. When collecting pleuroperitoneal fluid and cerebrospinal fluid, please follow the procedures above-mentioned.

Cell Culture Supernatant Collect by sterile tubes when examining secrete components. Centrifuge at 2000-3000 RPM for approximately 20 minutes. Collect the supernatants carefully. When examining the components within the cell, use PBS (pH 7.2-7.4) to dilute cell suspension to the cell concentration of approximately 1 million/ml. Damage cells through repeated freeze-thaw cycles to let out the inside components. Centrifuge at 2000-3000 RPM for approximately 20 minutes.

Tissue Rinse tissues in PBS (pH 7.4) to remove excess blood thoroughly and weigh before homogenization. Mince tissues and homogenize them in PBS (pH7.4) with a glass homogenizer on ice. Thaw at 2-8°C or freeze at -20°C. Centrifuge at 2000-3000 RPM for approximately 20 minutes.

*Sample can't be diluted with this kit. Owing to the the material we use to prepare the kit, the sample matrix interference may falsely depress the specificity and accuracy of the assay.

Reagent Preparation

All reagents should be brought to room temperature before use.

Standard Reconstitute the 120μl of the standard (640ng/L) with 120μl of standard diluent to generate a 320ng/L standard stock solution. Allow the standard to sit for 15 mins with gentle agitation prior to making dilutions. Prepare duplicate standard points by serially diluting the standard stock solution (320ng/L) 1:2 with standard diluent to produce 160ng/L, 80ng/L, 40ng/L and 20ng/L solutions. Standard diluent serves as the zero standard(0 ng/L). Any remaining solution should be frozen at -20°C and used within one month. Dilution of standard solutions suggested are as follows:

Wash Buffer Dilute 20ml of Wash Buffer Concentrate 30x into deionized or distilled water to yield 500 ml of 1x Wash Buffer. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved.

Calculation of Result

Construct a standard curve by plotting the average OD for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis and draw a best fit curve through the points on the graph. These calculations can be best performed with computer-based curve-fitting software and the best fit line can be determined by regression analysis.

Referances

"Expression and purification of biologically active rat bone morphogenetic protein-4 produced as inclusion bodies in recombinant Escherichia coli."
Klosch B., Furst W., Kneidinger R., Schuller M., Rupp B., Banerjee A., Redl H.
Biotechnol. Lett. 27:1559-1564(2005)