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|抗原||Complement Component 5a (C5a) ELISA试剂盒|
Kits with alternative reactivity to:
|检测范围||78 pg/mL - 5000 pg/mL|
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C5AELISA Kit详情目标详细情况 使用细节 贮存及处理 References for C5A Kit (ABIN414444) 图像
|原理||The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of C5a in Serum,Plasma,Tissue Homogenate,Cell Lysate,Cell Culture Supernatant,Biological Fluids|
|样品类型||Serum, Plasma, Tissue Homogenate, Cell Lysate, Cell Culture Supernatant, Biological Fluids|
This assay has high sensitivity and excellent specificity for detection of Complement Component 5a (C5a).
|交叉反应 （详细）||No significant cross-reactivity or interference between Complement Component 5a (C5a) and analogues was observed.|
目标详细情况C5AELISA Kit详情 使用细节 贮存及处理 References for C5A Kit (ABIN414444) 图像 回页首
|别名||C5a (C5a ELISA Kit 摘要)|
|途径||Complement System, Carbohydrate Homeostasis|
使用细节C5AELISA Kit详情 目标详细情况 贮存及处理 References for C5A Kit (ABIN414444) 图像 回页首
Information on standard material:
|实验流程||The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Complement Component 5a (C5a). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Complement Component 5a (C5a). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Complement Component 5a (C5a), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Complement Component 5a (C5a) in the samples is then determined by comparing the O.D. of the samples to the standard curve.|
Serum: Allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000 × g. Assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
Tissue Homogenates: The preparation of tissue homogenates will vary depending upon tissue type. For this assay, rinse tissues in ice-cold PBS (0.02mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weigh before homogenization. Mince the tissues to small pieces and homogenize them in 5-10 mL of PBS with a glass homogenizer on ice (Micro Tissue Grinders work, too). Sonicate the resulting suspension with an ultrasonic cell disrupter or subject it to two freeze-thaw cycles to further break the cell membranes. Centrifugate the homogenates for 5 minutes at 5000 × g. Remove the supernate and assay immediately or aliquot and store at -20°C
Cell Lysate: Cells must be lysed before assaying according to the following directions. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly). Wash cells three times in cold PBS. Resuspend cells in PBS (1×) and subject them to ultrasonication for 4 times (or Freeze cells at -20 °C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.) Centrifuge at 1500 × g for 10 minutes at 2 - 8°C to remove cellular debris.
Cell Culture Supernatant: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
Biological Fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
Average the duplicate readings for each standard, control, and samples and subtract the average zero standard optical density. Construct a standard curve by plotting the mean O.D. and concentration for each standard and draw a best fit curve through the points on the graph or create a standard curve on log-log graph paper with C5a concentration on the y-axis and absorbance on the x-axis. Using some plot software, for instance, curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the known concentration of the standard (Y-axis), although concentration is the independent variable and O.D. value is the dependent variable. However, the O.D. values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), plotting log of the data to establish standard curve for each test is recommended. Typical standard curve below is provided for reference only.
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Complement Component 5a (C5a) were tested 20 times on one plate, respectively.
贮存及处理C5AELISA Kit详情 目标详细情况 使用细节 References for C5A Kit (ABIN414444) 图像 回页首
|注意事项||The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.|
The stability of 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.
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.
References for C5A Kit (ABIN414444)C5AELISA Kit详情 目标详细情况 使用细节 贮存及处理 图像 回页首
Ye, Kong, Zhang: "Complement Split Products C3a/C5a and Receptors: Are They Regulated by Circulating Angiotensin II Type 1 Receptor Autoantibody in Severe Preeclampsia?" in: Gynecologic and obstetric investigation, Vol. 81, Issue 1, pp. 28-33, 2016
Eriksson, Studahl, Bergström: "Acute and prolonged complement activation in the central nervous system during herpes simplex encephalitis." in: Journal of neuroimmunology, Vol. 295-296, pp. 130-8, 2016
Wirstlein, Miko?ajczyk, Jasi?ski, Skrzypczak: "Evaluation of the markers of inflammation in the umbilical cord blood of newborns of mothers with thrombophilia." in: American journal of reproductive immunology (New York, N.Y. : 1989), Vol. 72, Issue 6, pp. 561-70, 2014
Denny, Coulthard, Finnell, Callaway, Taylor, Woodruff: "Elevated complement factor C5a in maternal and umbilical cord plasma in preeclampsia." in: Journal of reproductive immunology, Vol. 97, Issue 2, pp. 211-6, 2013
Huber-Lang, Denk, Fulda, Erler, Kalbitz, Weckbach, Schneider, Weiss, Kanse, Perl: "Cathepsin D is released after severe tissue trauma in vivo and is capable of generating C5a in vitro." in: Molecular immunology, Vol. 50, Issue 1-2, pp. 60-5, 2012
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