CTRP5 (human) Competitive ELISA Kit
Contents
1. Do not combine leftover reagents with those reserved for additional wells b Generate a Standard Curve by plotting the average absorbance on the vertical Y e Reagents from the kit with a volume less than 100 ul should be centrifuged axis vs the corresponding concentration ug ml on the horizontal X axis See e Residual wash liquid should be drained from the wells after last wash by tapping the Typical Data below plate on absorbent paper c Calculate the Test Sample CTRPS5 concentrations by interpolation of the Standard e Crystals could appear in the 10X solution due to high salt concentration in the stock Curve regression curve as shown below in the form of a 4 parameter logistic solutions Crystals are readily dissolved at room temperature or at 37 C before PUAN dilution of the buffer solutions If the T i Itiply the i he diluti ee ee ee ane ENON e Once reagents have been added to the 8 well strips DO NOT let the strips DRY at 25 any time during the assay e Keep TMB Substrate Solution protected from light e The Stop Solution consists of phosphoric acid Although diluted the Stop Solution should be handled with gloves eye protection and protective clothing uw Troubleshooting PROBLEM POSSIBLE CAUSES SOLUTIONS Siccin mopkeweaaent Check that all reagents have Teag been added in the correct order g ow 0 01 24 ic Washes too stringent Use an automated plate washer Cors ugi if possible Vi Perfo2. BioVision IV 1 CTRP5 human Competitive ELISA Kit Catalog K4925 100 100 assays Store kit at 4 C Description CTRP5 a 25 kD secretory protein is a member of the Ciq and tumor necrosis factor superfamily whose structure resembles adiponectin A RT PCR study demonstrated CTRP5 expression in RPE liver lung placenta and brain and it has been proposed that a CTRP mutation S163R plays a critical role in affecting its higher order protein structure potentially leading to a cause of abnormal adhesion between the RPE and Bruch membrane Recent data indicates that CTRP is one of the genes highly induced by elimination of mitochondria and able to activate AMPK in a rat myotube cell line L6 Stimulation of L6 with recombinant CTRP5 full length as well as globular domain enhanced glucose uptake and fatty acid oxidation These biochemical events did not seem to be mediated via AdipoR1 or AdipoR2 suggesting that a novel receptor s may exist for CTRP5 in this muscle cell line Some CTRP members can physically interact with adiponectin forming various multimeric structures Therefore measuring serum or plasma CTRP5 may provide important information on its involvement in novel metabolism This assay is a competitive Enzyme Linked Immunosorbent Assay ELISA for quantitative determination of human CTRP5 in biological fluids A polyclonal antibody recognizing native human CTRP5 reacts with a series of predetermined recombinant human CTRP5 sta
3. into the appropriate wells in duplicate Add 50 ul of the Detection Antibody to each well and tap gently on the side of the plate to mix Cover plate with plate sealer and incubate for 1 hr at 37 C Aspirate and wash x 3 with 300 ul of 1X Wash Buffer After last wash tap inverted plate on a stack of paper towels Complete removal of liquid is essential for good performance Add 100 ul of the 1X Detector to each well Cover plate with plate sealer and incubate for 1 hr at 37 C Remove plate from 37 C aspirate and wash x 5 with 300 ul of 1X Wash Buffer After last wash tap inverted plate on a stack of paper towels Complete removal of liquid is essential for good performance Add 100 ul of the TMB Substrate Solution to each well Allow the color to develop at room temperature in the dark for 10 min Stop the reaction by adding 100 ul of Stop Solution to each well Tap the plate gently to ensure thorough mixing The substrate reaction yields a blue solution that turns yellow when Stop Solution is added Caution Stop Solution is a Corrosive Solution Measure the OD at 450 nm in an ELISA plate reader within 30 min Tel 408 493 1800 www biovision com Fax 408 493 1801 tech biovision com Page 1 of 2 BioVision AA Technical Hints and Limitations 3 Calculations e It is recommended that all standards QC sample and samples be run in duplicate a Average the duplicate readings for each Standard QC Sample and Test Sample
4. ndard proteins or samples under competition in the human CTRP5 coated plate Their relative reactivity is plotted with that of the standard proteins This ELISA is specific for the measurement of natural and recombinant human CTRP5 It does not cross react with mouse CTRP5 human CTRP2 globular human CTRP6 human CTRP7 globular human CTRP9 globular human CTRP10 globular mouse CTRP2 globular mouse CTRP9 globular human adiponectin human adiponectin globular mouse adiponectin mouse adiponectin globular rat adiponectin rat adiponectin globular human RBP4 human Nampt human vaspin human GPX3 human ANGPTL8 human progranulin human leptin The assay range is 0 001 5 ug CTRP5 ml and a detection limit of 1 ng ml based on adding two standard deviations to the mean value of the 50 zero standards Kit Contents Pre coated Microtiter Plate 1 ea 12 x 8 well strips K4912 100 1 Wash Buffer 10X 50 ml K4912 100 2 Diluent 5X 50 ml K4912 100 3 Detection Antibody 12 ml K4912 100 4 150 ul K4912 100 5 1 vial K4912 100 6 1 vial K4912 100 7 12 ml K4912 100 8 12 ml K4912 100 10 3 each K4912 100 11 Detector 100X Hrp conjugated anti IgG Human CTRP5 Standard lyophilized 5 Og Human CTRP5 QC Sample lyophilized TMB Substrate Solution Stop Solution Plate Sealers Storage Conditions Reagents must be stored at 2 8 C when not in use Bring reagents to room temperature before use Do not expose reagents to temperat
5. rmance Characteristics Incubation times should be 1 Intra assay Precision 4 samples of known concentration were assayed in replicates 9 Incubation times inadequate followed as indicated in the times to test precision within an assay manual Samples Mean ug ml Sb CV on 0 351 0 02 67 0 293 10 00 9 Incorrect assay temperature temperature Bring substrates to 2 inter assay Precision 4 samples of iowa concentration were assayed in 3 separate room temperature before use t precision bet n ASSAYS 10 ee MEOW eee ASSAYS Concentration of detector too Use recommended dilution high factor Samples _ Mean g ml SD CV m OoOo n 1 0348 0 02 F686 FS i T a 19279 i Lii a Ensure all wells are filing wash Inadequate washing buffer and are aspirated completely 0 253 4 oie Wells not completely aspirated Completely aspirate wells between steps Poor standard curve Reaaents poorly mixed Be sure that reagents are ii thoroughly mixed 3 Linearity Human serum samples containing CTRP5 were diluted several fold and the Be sure that reagents were measured recoveries ranged from 79 to 130 Omission of reagents prepared correctly and added in Samples _ Sample Dilution Expected ug ml Observed ug ml of Expected Unexpected results the correct order 0 260 0 260 Pilutisaenae Check pipetting technique and double check calculations a 0 130 0 065 0 071 Expected Values CTRP5 le
6. tock solution 5 ug ml Mix the Stock solution to ensure complete reconstitution Allow to sit for a minimum of 15 min The reconstituted standard should be aliquoted and stored at 20 C Prepare 1X Diluent Dilute 5X Diluent 1 4 with dH2O Prepare Standard Curve using 2 fold serial dilutions with 1X Diluent Toobtain Ad gmt ET 300 pl of 1X Diluent 300 ul of 1X Diluent 300 ul of 1X Diluent 300 ul of 1X Diluent 300 ul of 1X Diluent 450 ul of 1X Diluent 450 ul of 1X Diluent 300 ul 200 ul 300 ul 300 ul 200 ul 50 ul 50 ul a Vh Y IvANANA YAY zy ug ml 5 a 200 ul of CTRPS 2 5 ug ml 0 5 ug ml 300 pl of CTRPS5 1 ug ml 0 1 2 5 1 0 5 0 25 0 1 0 01 ug ml ug ml pg ml ug ml ug ml pg ml 0 001 ug ml 2 Reagent Preparation Prepare just the appropriate amounts for the assay a b C 1X Wash Buffer Dilute 10X Wash Buffer 1 9 with dH20O to obtain 1X Wash Buffer 1X Diluent Dilute 5X Wash Buffer 1 4 with dH20 to obtain 1X Diluent 1X Detector Dilute 100X Detector 1 99 with 1X Diluent to obtain 1X Detector Note The diluted Detector must be used within 1 hr of preparation 3 Assay Protocol a IN I JIQ xr a3 _ 2 Determine the number of 8 well strips needed for assay and insert them into the frame for current use The extra strips should be resealed in the foil pouch and can be stored at 4 C for up to 1 month Add 50 ul of the Standards Samples and QC Sample
7. ures greater than 25 C Assay Procedure Read the ENTIRE Protocol Before Proceeding Test Samples Standards QC Sample We recommend these be run in duplicate a Serum Use a serum separator tube Let samples clot at room temperature for 30 min before centrifugation for 20 min at 1 000 x g Assay freshly prepared serum or store serum in aliquots at lt 20 C for later use Avoid repeated freeze thaw cycles b Plasma Collect plasma using heparin EDTA or citrate as an anticoagulant Centrifuge for 15 minutes at 1000xg within 30 minutes of collection Assay freshly prepared plasma or store plasma sample in aliquot at lt 20 C for later use Avoid repeated freeze thaw cycles Note Serum Plasma or Cell Culture Supernatant have to be diluted in Diluent 1X Samples containing visible precipitates must be clarified before use Note As a starting point 1 2 dilution of serum or plasma is recommended If samples fall the outside range of assay a lower or higher dilution may be required BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 01 12 For research use only QC Sample Reconstitute the human CTRP5 QC Sample with 1 ml of dH20 Mix the QC Sample to ensure complete reconstitution Allow to sit for a minimum of 15 min The QC Sample is ready to use do not dilute it refer to the C of A for current QC Sample concentration Standards Reconstitute the human CTRP5 Standard with 1 ml of dH2O to produce a s
8. vels range in plasma and serum from 0 05 to gt 0 5 pg ml r Healthy donors FOR RESEARCH USE ONLY Not to be used on humans Of 21450 om No signal or weak signal Plate reader settings not Verify the wavelength and filter optimal setting in the plate reader a E 0 174 0 174 12 0 087 0 082 SE tnt E BioVision Incorporated Tel 408 493 1800 Fax 408 493 1801 155 S Milpitas Boulevard Milpitas CA 95035 USA www biovision com tech biovision com Page 2 of 2
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