Cat No.eia05560h
Human Diamine Oxidase (DAO) ELISA Kit
Dear customers, thank you for choosing our products. This product is suitable for in vitro qualitative detection of human serum, plasma or cell culture supernatant ａnd organizations in the natural ａnd recombinant DAO concentration. Detection of other special sample please contact our technical support. The kit is for research use only. Please read the instructions carefully before using ａnd check the kit components. If you have any questions, please contact SANCHEZ INC You will get our full range of services.
This kit employs Double Antibody Sandwich Technique. The principle of Double Antibody Sandwich is based on characteristics of the tested antigen with more than two valances which can identify coated antibody and detection antibody at same time. The specific process is as follows:
1. Connect the specific antibodies ａnd solid phase carriers to form immobilized antibodies. Wash out uncombined antibodies ａnd impurities. Seal the rest binding sites with irrelevant proteins.
2. Join under test with immobilized antibodies for contact reaction. After a while, combine antigens in ａnd antibodies on carriers into the antigens complex. Wash out uncombined antibodies ａnd impurities.
3. Add biotin labeling antibodies to combine with the antigens on immune complexes. Wash out the uncombined biotin labeling antibodies thoroughly. The enzyme amount on the carrier is now positively related to the amount of the tested substance in specimens.
4. Add horseradish peroxidase to label the avidins ａnd incorporate them with the biotin labeling antibodies. Wash out the incorporated enzyme markers thoroughly. The enzyme amount on the carrier is now positively related to the amount of the tested substance in specimens.
5. Add substrates for coloring, ａnd compute the concentration of specimens.
Note: an antibody molecule can be marked on several biotin molecules ａnd a biotin molecule can be connected with a HRP-Avidin to form numbers of horseradish peroxidases combining with antibodies which shows higher sensitivity ａnd amplification effect comparing with traditional direct HRP-Antibodies.
【Detection principle of Human Diamine Oxidase (DAO) ELISA kit】
This experiment use double-sandwich elisa technique and the ELISA Kit provided is typical. The pre-coated antibody is human DAO monoclonal antibody ａnd the detecting antibody is polyclonal antibody with biotin labeled. Samples ａnd biotin labeling antibody are added into ELISA plate wells ａnd washed out with PBS or TBS. Then Avidin-peroxidase conjugates are added to ELISA wells in order; Use TMB substrate for coloring after reactant thoroughly washed out by PBS or TBS. TMB turns into blue in peroxidase catalytic ａnd finally turns into yellow under the action of acid. The color depth ａnd the testing factors in samples are positively correlated
【Kit composition】
name 96 Tests 48 TestsStorage
1．antibody precoated plate 8×12 8×64/-20℃
2．Human DAO Standards 2 vial 1 vial4/-20℃
3．Biotinylated antibody(1:100) 1 vial 1 vial4/-20℃
4．Enzyme conjugate(1:100) 1vial 1 vial4/-20℃
5．Enzyme diluent 1vial 1vial 4/-20℃
6．antibody diluent 1vial 1 vial4/-20℃
7．Standard diluent 1vial 1 vial 4/-20℃
8．Sample diluent 1vial 1 vial 4/-20℃
9．Washing buffer (1:25) 1 vial 1 vial 4/-20℃
10．Colour Reagent A 1 vial 1 vial 4/-20℃
11．Colour Reagent B1 vial 1 vial 4/-20℃
12．Colour Reagent C 1 vial 1 vial 4/-20℃
13．Instruction 1 set 1 set RT
Note：
RT：Room temperature
Standard：Frozen dried
Colour Reagent A: Avoid light
【Necessary for testing their own test facilities ａnd equipment】
1. Microplate reader (450nm detection wavelength filter, 570nm or 630nm correction wavelength filters).
2. Washer (adjustable amount of liquid injection to ensure that each well 350μl lotion without overflow).
3. Clean benches, biological safety cabinets, fume hoods.
4. High-precision single-channel dispenser (range 0.5-10μl-20μl, 20-200μl, 200-1000μl).
5. High-precision multi-channel plus liquid (8 or 12, the range of 50-300μl of).
6. 37℃ incubator.
7. Low temperature centrifuge.
8. Refrigerators (4℃, -20℃, -86℃).
9. Analytical balance.
10. Scissors, tweezers, pliers, ａnd so on.
11. Swirl mixing device, low-frequency oscillator, ａnd so on.
【Necessary for testing their own testing supplies ａnd reagents】
1. Centrifuge tube (capacity of 1.5ml, 5ml, ａnd so on).
2. Disposable tip (range of 0.5-10μl-20μl, 20-200μl, 200-1000μl).
3. Pure water or distilled water.
4. Coordinate paper.
5. Absorbent paper.
6. EDTA, sodium citrate, heparin
【Sample collection Note】
1. The tube for blood collection should be free of pyrogen and endotoxin
2. Hemolysis and hyperlipidemia specimens can not be used to extracted serum ａnd plasma.
3. The samples should appear clear ａnd transparent. And all the suspension should be removed through centrifugation.
4. If collected samples are not timely detected, they should be divided according to single usage amount ａnd frozen reserved in refrigerator at -20-80℃, avoiding the repeated freeze-thaw.
5. According to the actual situation of the samples, make proper multiple dilutions (Pre-experiment is strongly recommended in order to confirm the dilution ratio)
6. Collect specimens ａnd try to gain double dosage to avoid specimens shortage for repeated assays in case that failure in one-assay delays experimental process.
7. Do protective measures when collecting specimens (e.g. wearing gloves, respirator, respirator, etc.), aware of the potential risk in all specimens.
8. Specimen processing should be inside the biological safety cabinet. Ensure proper use of the biological safety cabinet.
【Measures for the samples】
1. Serum: Put the collected whole blood in refrigerator at 4℃ for the night. Then centrifuge it for 10min at 1000-3000rpm. Take supernatant tested immediately or put samples at -20℃( for 1-3 months) or -80℃ (for 1-3 months) for storage.
2. Plasma: Take EDTA, sodium citrate ａnd heparin as anticoagulant. Add the plasma ａnd mix them well. Centrifuge mixture for 10min at 1000-3000rpm. Take supernatant tested immediately or put samples at -20℃( for 1-3 months) or -80℃ (for 1-3 months) for storage.
3. Tissue homogenate: Take tissue slices and wash them out in 0.01MPBS; Add tissue protein extraction reagent according to proportion of 1G: 5-10ml ａnd mix them in ice water. After being blended, mixture shall be centrifuged for 10min at 5000-10000rpm.Take supernatant tested immediately or put them at -20℃( for 1-3 months) or -80℃ (for 1-3 months) for storage.
4. Cell culture: Take centrifugation for 10min at 1000-3000rpm.Take supernatant tested immediately or put samples at -20℃( for 1-3 months) or -80℃ (for 1-3 months) for storage.
5. For urine, ascites, cerebrospinal fluid, etc: ake centrifugation for 10min at 1000-3000rpm. Take supernatant tested immediately or put samples at -20℃( for 1-3 months) or -80℃ (for 1-3 months) for storage.
Note: The general principles of the sample dilution
The user should refer to the references to know the probable content of the samples before decide to dilute the samples,and the diluted content of the sample must be in the best detection range of the given ELISA Kits. The dilution of the sample should be recorded in detail.
以上仅供参考，因实验室系统升级，各检测指标说明书有所更新，操作说明请联系18910821817
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