產(chǎn)品類型： ELISA Kit
 
 產(chǎn)品名稱： 犬透明質(zhì)酸酶4（HAase 4）ELISA Kit
 
 英文名稱： Canine Hyaluronidase 4（HAase 4）ELISA Kit
 
 別名： CSHY, chondroitin sulfate hydrolase|hyaluronidase 4
 
 規(guī)格： 96T
 
 種屬： Dog
 
 待測物名稱： Hyaluronidase 4（HAase 4）
 
 縮寫： HAase 4
 
 檢測范圍： Request Information
 
 靈敏度： Request Information
 
 反應(yīng)時間： 1-5h
 
 所需樣本體積： 50-100ul
 
 檢測波長： 450 nm
 
 用途： For research use only. Not for diagnostic use.
 
 原理 ： This assay employs the quantitative sandwich enzyme immunoassay technique. Antibody specific for HAase 4 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any HAase 4 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for HAase 4 is added to the wells. After washing, avidin conjugated Horseradish Peroxidase （HRP） is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of HAase 4 bound in the initial step. The color development is stopped and the intensity of the color is measured. 
 特異性： This assay has high sensitivity and excellent specificity for detection of Dog HAase 4. No significant cross-reactivity or interference between Dog HAase 4 and analogues was observed. 
 精密度： Intra-assay Precision （Precision within an assay）: CV%<8%
Three samples of known concentration were tested twenty times on one plate to assess.
Inter-assay Precision （Precision between assays）: CV%<10%
Three samples of known concentration were tested in twenty assays to assess.
 
 樣本搜集及儲存： Serum: Use a serum separator tube （SST） and allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 15 minutes at 1000 ×g. Remove serum and assay immediay or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.
Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 ×g at 2-8°C within 30 minutes of collection. Assay immediay or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.
 
 檢測步驟： Bring all reagents and samples to room temperature before use. Centrifuge the sample again after thawing before the assay. It is recommended that all samples and standards be assayed in duplicate.
1. Prepare all reagents, working standards, and samples as directed in the previous sections.
2. Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 4°C.
3. Add 100μl of standard and sample per well. Cover with the adhesive strip provided. Incubate for 2 hours at 37°C. A plate layout is provided to record standards and samples assayed.
4. Remove the liquid of each well, don't wash.
5. Add 100μl of Biotin-antibody （1x） to each well. Cover with a new adhesive strip. Incubate for 1 hour at 37°C. （Biotin-antibody （1x） may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform.）
6. Aspirate each well and wash, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer （200μl） using a squirt bottle, multi-channel pipette, manifold dispenser, or autowasher, and let it stand for 2 minutes, complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining wash Buffer by aspirating ordecanting. Invert the plate and blot it against clean paper towels.
7. Add 100μl of HRP-avidin （1x） to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C.
8. Repeat the aspiration/wash process for five times as in step 6.
9. Add 90μl of TMB Substrate to each well. Incubate for 15-30 minutes at 37°C. Protect from light.
10. Add 50μl of Stop Solution to each well, gently tap the plate to ensure thorough mixing.
11. Determine the optical density of each well within 5 minutes, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. Subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
 
 結(jié)果計算： Using the professional soft "Curve Expert 1.3" to make a standard curve is recommended, which can be downloaded from our web.
Average the duplicate readings for each standard and sample and subtract the average zero standard optical density.
Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic （4-PL） curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the HAase 4 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data.
If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.