Human IL-3 ELISA Kit

*: [96T/48T] 
#: It’s OK to keep the kit in 4℃, if the kit is scheduled to be used up in one week. Please keep the 
reagent in -20℃ for long-term storage or repeated use. 
The reagent in each vial is slightly more that its volume written on label, please take out the required 
volume by certain tools (such as transferpettor, measuring cylinder), rather than pouring directly. 
 Human IL-3 ELISA Kit
Test principle 
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has 
been pre-coated with an antibody specific to Human IL-3. Standards or samples are then added to the 
appropriate micro ELISA plate wells and combined to the specific antibody. Then a biotinylated 
detection antibody specific for Human IL-3 and Avidin-Horseradish Peroxidase (HRP) conjugate is 
added to each micro plate well and incubated. Free components are washed away. The substrate 
solution is added to each well. Only those wells that contain Human IL-3, biotinylated detection 
antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is 
terminated by the addition of a sulphuric acid solution and the color turn yellow. The optical density 
(OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is  
Human IL-3 ELISA Kit
9 
proportional to the concentration of Human IL-3. You can calculate the concentration of IL-3 in the 
samples by comparing the OD of the samples to the standard curve. 
 
Sample collection and storage 
Serum - Allow samples to clot for 2 hours at room temperature or overnight at 4°Cbefore 
centrifugation for 20 minutes at approximay 1000×g. Collect the supernatant and carry out the 
assay immediay. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin. 
 
Plasma - Collect plasma using EDTA-Na2 or heparin as an anticoagulant. Centrifuge samples for 15 
minutes at 1000×g at 2 - 8°C within 30 minutes of collection. Collect the supernatant and carry out 
the assay immediay. Avoid hemolysis, high cholesterol samples. 
 
Tissue homogenates– For general information, hemolysis blood may affect the result, so you should 
rinse the tissues with ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue 
pieces should be weighed and then minced to small pieces which will be homogenized in PBS (the 
volume depends on the weight of the tissue. 9mL PBS would be appropriate to 1 gram tissue pieces. 
Some protease inhibitor is recommended to add into the PBS.) with a glass homogenizer on ice. To 
further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to 
freeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000×g to get the 
supernate. 
 
Cell culture supernate – Centrifuge supernate for 20 minutes to remove insoluble impurity and cell 
debris at 1000×g at 2 - 8°C. Collect the clear supernate and carry out the assay immediay. 
 
Other biological fluids –Centrifuge samples for 20 minutes at 1000×g at 2 - 8°C. Collect the 
supernatant and carry out the assay immediay. (You can refer to our website for detailed processing 

Sample preparation – Samples should be clear and transparent and be centrifuged to remove 
suspended solids. 
Note: Serum and plasma to be used within 7 days when stored at 2-8°C, otherwise samples must be 
divided and stored at -20°C (≤1 month) or -80°C (≤6 months) to avoid loss of bioactivity and 
contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room 
temperature. If the sample concentration is higher than the maximum standard value, please dilute it 
with appropriate factor according to the actual situation. (A pre-test is recommended to determine the 
dilute factor). 
 
Other supplies required  

Microplate reader with 450nm wavelength filter 
High-precision transferpettor, EP tubes and disposable pipette tips 
37°C Incubator, Deionized or distilled water 
Absorbent paper 
 
Reagent preparation 
Bring all reagents to room temperature before use. 
Wash Buffer - Dilute 30mL of Concentrated Wash Buffer into 750 mL of Wash Buffer with deionized 
or distilled water. Put unused solution back at 4°C. If crystals have formed in the concentrate, you can 
warm it with 40°Cwater bath (Heating temperature should not exceed 50°C) and mix it gently until the 
crystals have compley dissolved. The solution should be cooled to room temperature before use. 
Standard- Centrifuge at 10,000×g for 1 minute, and reconstitute the Standard with 1.0mL of 
Reference Standard &Sample Diluent. Tighten the lid, let it stand for 10 minutes and turn it upside 
down for several times. After it dissolves fully, mix it thoroughly with a pipette. This reconstitution 
produces a stock solution of 1000pg/mL. Then make serial dilutions as needed (Making serial 
dilution in the wells directly is not permitted). The recommended concentrations are as follows:1000、
500、250、125、62.5、31.25、15.625、0pg/mL . As if you want to make standard solution at the 
concentration of 500pg/mL, you can take 0.5mL the standard at 1000pg/mL, add it to an EP tube with 
0.5mL Reference Standard &Sample Diluent, and mix it. The procedures of making the remaining 
concentrations are all the same. The undiluted standard serves as the highest standard (1000pg/mL). 
The Reference Standard &Sample Diluent serves as the zero (0pg/mL). 
(500μL/tube，for example. Can also be diluted according to the actual amount，such as 200μL/tube)