Beta Defensin 4 Mouse ELISA Kit , 96 Wells DEFB4 ELISA Assay Kit

96 wells Mouse High Sensitivie DEFB4 ELISA Kit
 
Cat.No E1204Mo
Standard Curve Range: 0.05ng/ml - 10ng/ml
Sensitivity: 0.021ng/ml
Size: 96 wells
Storage: Store the reagents at 2-8°C. For over 6-month storage refer to the expiration date keep it at -20°C. Avoid repeated thaw cycles. If individual reagents are opened it is recommended that the kit be used within 1 month.
* This product is for research use only, not for use in diagnosis procedures. It’s highly recommend to read this instruction entirely before use.
 
Intended Use
This sandwich kit is for the accurate quantitative detection of Mouse Beta-defensin 4 (also known as DEFB4) 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 Mouse DEFB4 antibody. DEFB4 present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Mouse DEFB4 Antibody is added and binds to DEFB4 in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated DEFB4 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 Mouse DEFB4. 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.
 
Note

• Sample concentrations should be predicted before being used in the assay. If the sample concentration is not within the range of the standard curve, users must contact us to determine the optimal sample for their particular experiments.

• Samples to be used within 5 days should be stored at 2-8°C. Samples should be aliquoted or must be stored at -20°C within 1 month or -80°C within 6 months. Avoid repeated freeze thaw cycles.

• Samples should be brought to room temperature before starting the assay.

• Centrifuge to collect sample before use.

• Samples containing NaN3 can’t be tested as it inhibits the activity of Horse Radish Peroxidase (HRP).

• Collect the supernatants carefully. When sediments occurred during storage, centrifugation should be performed again.

* 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 (12.8ng/ml) with 120μl of standard diluent to generate a 6.4ng/ml 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 (6.4ng/ml) 1:2 with standard diluent to produce 3.2ng/ml, 1.6ng/ml, 0.8ng/ml and 0.4ng/ml solutions. Standard diluent serves as the zero standard(0 ng/ml). 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.
 
Summary

1. Prepare all reagents, samples and standards.
2. Add ample and ELISA reagent into each well. Incubate for 1 hour at 37°C.

3. Wash the plate 5 times.

4. Add substrate solution A and B. Incubate for 10 minutes at 37°C.

5. Add stop solution and color develops.

6. Read the OD value within 10 minutes.

References
Rivas-Santiago B., Sada E., Tsutsumi V., Aguilar-Leon D., Contreras J.L., Hernandez-Pando R.
J. Infect. Dis. 194:697-701(2006)