INTENDED USE
The kit is for in vitro quantitative measurement of this index in serum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids.

MATERIALS REQUIRED BUT NOT SUPPLIED
1.Microplate reader with 450 ± 10nm filter.
2.Precision single or multi-channel pipettes and disposable tips.
3.Eppendorf Tubes for diluting samples.
4.Deionized or distilled water.
5.Absorbent paper for blotting the microtiter plate.
6.Container for Wash Solution.

STORAGE OF THE KITS
1.For unopened kit: All the reagents should be kept according to the labels on vials. The TMB Substrate, Wash Buffer (30 × concentrate) and the Stop Solution should be stored at 4oC upon receipt while the others should be at -20oC.
2.For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.

Note:
It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit. For the expiration date of the kit, please refer to the label on the kit box. All components are stable until this expiration date.

SAMPLE COLLECTION AND STORAGE
?Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4oC before centrifugation for 20 minutes at approximately 1000×g. Assay freshly prepared serum immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.
?Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2-8oC within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.
?Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed in ice-cold PBS(0.01mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weighed before homogenization. Minced the tissues to small pieces and homogenized them in 5-10 mL of PBS with a glass homogenizer on ice(Micro Tissue Grinders woks, too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifugated for 5 minutes at 5000×g. Remove the supernate and assay immediately or aliquot and store at ≤-20oC.
?Cell Lysates - Cells must be lysed before assaying according to the following directions.
1.Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly).
2.Wash cells three times in cold PBS.
3.Resuspend cells in PBS (1×) and the cells was subject to ultrasonication for 4 times (or Freeze cells at ≤-20oC. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.).
4.Centrifuge at 1500×g for 10 minutes at 2-8oC to remove cellular debris.
?Cell culture supernates and other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquot at -20oC or -80oC. Avoid repeated freeze/thaw cycles.

Note:
1.Samples to be used within 5 days may be stored at 4oC, otherwise samples must be stored at -20oC (≤1 month) or -80oC (≤2 months) to avoid loss of bioactivity and contamination.
2.Sample hemolysis will influence the result, so hemolytic specimen can not be detected.
3.When performing the assay, bring samples to room temperature.


Note:
1.Making serial dilution in the wells directly is not permitted.
2.Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37oC directly.
3.Detection Reagent A and B are sticky solutions, therefore, slowly pipette them to reduce the volume errors.
4.Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μL for once pipetting.
5.The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
6.If crystals have formed in the Wash Solution concentrate (30×), warm to room temperature and mix gently until the crystals are completely dissolved.
7.Contaminated water or container for reagent preparation will influence the detection result.

SAMPLE PREPARATION
1.DL Sci & Tech Development Co.,Ltd. is only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.
2.Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments. Sample should be diluted by 0.01mol/L PBS(pH=7.0-7.2).
3.If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
4.Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals.
5.Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products.
6.Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture supernatant may not be detected by the kit.
7.Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.

SPECIFICITY
This assay has high sensitivity and excellent specificity for detection of BDNF.
No significant cross-reactivity or interference between this index and analogues was observed.
Note:
Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between this index and all the analogues, therefore, cross reaction may still exist.

PRECISION
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level BDNF were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level BDNF were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

STABILITY
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.
Note:
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

IMPORTANT NOTE
1.Limited by the current condition and scientific technology, we can't completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit.
2.The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available.
3.Kits from different batches may be a little different in detection range, sensitivity and color developing time. Please perform the experiment exactly according to the instruction attached in kit while electronic ones from our website is only for information.
4.Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
5.Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism.
6.There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed.
7.Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment.
8.Even the same operator might get different results in two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before assay for each batch is recommended.
9.Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay variance among kits from different batches might arise from above factors, too.
10.Kits from different manufacturers with the same item might produce different results, since we haven’t compared our products with other manufacturers.
11.Valid period: 12 months.

PRECAUTION
The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

Wuxi Donglin Sci & Tech Development Co.,Ltd., a manufacturer and supplier of biological reagents for life science, is located in the scenic area beside Taihu Lake. At the inception in 1997, our company focused on providing OEM service of ELISA kits across the world. In 2013, we launched our own brand Dldevelop.

We mainly specialize in manufacturing ELISA kits, distributed to Asia, Oceania, Africa, Europe and America. Headquartered in Wuxi, China, we have an overseas branch in Canada, a domestic branch in Shanghai, China.

Selected raw materials and state-of-the- art technologies help ensure the superior quality of our products with a one-year shelf life, which is six months longer than other manufacturers.
We have more than 6000 ElISA kits available. With the advance of our technology , more kinds of and more specific ELISA kits will be developed and manufactured to meet scientists and researchers’ need.

we strictly obey national and international QC requirements. Our company is a ISO9001:2008 and CE certificated enterprise.

Based on our strong innovative R&D teams, we are always committed to providing global scientists and researchers with the high quality ELISA kits and the best customer experience.