RBP4 (human) Competitive ELISA Kit
Contents
1. 1 800 1 82 1 95 107 a l 1 100 23 09 23 09 100 Omission of reagents prepared correctly and added in 1 200 11 55 9 81 85 Unexpected results the correct order 1 400 5 77 4 71 82 Diitomeitor Check pipetting technique and 1 800 2 89 2 33 81 double check calculations 1 100 31 03 31 03 100 3 1 200 15 52 13 55 87 1 400 7 76 6 69 86 1 800 3 88 3 26 84 FOR RESEARCH USE ONLY Not to be used on humans 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 22. 1X Diluent Dilute 5X Wash Buffer 1 4 with dH2O to obtain 1X Diluent Detection Antibody K4911 100 4 c 1X Detector Dilute 100X Detector 1 99 with 1X Diluent to obtain 1X Detector Detector 100X Hrp conjugated anti IgG K4911 100 5 d Substrate Solution Equal volumes of Substrate Solutions and II must be mixed K491 1 100 6 K4911 100 7 K4911 100 8 K4911 100 9 Human RBP4 Standard lyophilized 5 Lig Human RBP4 QC Sample lyophilized Substrate Solution TMB Substrate Solution II Peroxidase together Note The diluted Detector must be used within 1 hr of preparation Substrates and Il should be mixed within 15 min of use Protect Substrate Solution from light 3 Assay Protocol Stop Solution K4911 100 10 a Determine the number of 8 well strips needed for assay and insert them into the Plate Sealers K4911 100 11 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 Ill Storage Conditions b Add 50 ul of the Standards Samples and QC Sample into the appropriate wells in Reagents must be stored at 2 8 C when not in use Bring reagents to room temperature duplicate before use Do not expose reagents to temperatures greater than 25 C c Cover plate with plate sealer and incubate for 1 hr at 37 C IV Assay Procedure Read the ENTIRE Protocol Before Proceeding d Aspirate and wash x 3 with 300 ul of 1X Wash Buffer 1 Test Samples Stand
3. A 2 1914 070 368 4 Washes too stringent Use an automated plate washer if possible Incubation times should be Incubation times inadequate followed as indicated in the 3 z2 19 45 4 No signal or weak signal anal 25 31 P1384 TT 4A 5 Plate reader settings not Verify the wavelength and filter 2 Inter assay Precision 5 samples of known concentration were assayed in 4 separate optimal setting in the plate reader a ae 45 76 422 92 4 amp 4 k assays to test precision between assays Use recommended incubation Samples _ Mean ug ml SD CV if O a Incorrect assay temperature temperature Bring substrates to 15 39 room temperature before use pa ie 21 48 Abas 3795 I e 3 29 162 650 4 26 89 2st 858 Concentration of detector too Use recommended dilution high factor High background Ensure all wells are filling wash 3 Linearity Different human samples containing RBP4 were diluted several T 1 00 to Inadequate washing buffer and are aspirated 1 800 recoveries ranged from 81 to 110 completely er Expected Observed of Completely aspirate wells Samples Sample Dilution p p ug ml ug ml Expected aoa Seances Wells not completely aspirated between steps 1 R 1 o 1 s in Reagents poorly mixed Be sure that reagents are 1 400 3 65 4 01 110 Porong mixeg Be sure that reagents were
4. BioVision RBP4 human Competitive ELISA Kit _ Catalog K4911 100 100 assays Store kit at 4 C rev 2 14 For research use only Standards Reconstitute human RBP4 Standard with 1 ml of dH2O to produce a stock solution 24 ng 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 Description Retinol binding protein RBP 4 is the only specific transport protein for vitamin A in the circulation whose function is to deliver vitamin to target tissues In obesity and type 2 diabetes the expression of Glut4 is significantly impaired in adipocytes Glucose transport via Glut4 is the rate limiting step for glucose use by muscle and adipose tissue Adipocyte specific deletion of Gluts leads to notable elevation of mouse RBP4 causing systemic insulin resistance and that reduction of RBP4 improves insulin resistance This identified a novel role of RBP4 in regulating insulin action and RBP4 is recorded as an adipocyte derived hormone The RBP4 human ELISA Kit is to be used for the in vitro quantitative determination of human RBP4 in serum urine and cell culture supernatant This assay is a competitive ELISA which utilizies a 96 well microtiter plate which was pre coated with a human RBP4 A purified polyclonal which recognizing native human RBP4 reacts with a series of predetermined recombinant human RBP4 standard proteins or the test samples under compet
5. andard Curve regression curve as shown below in the form of a 4 parameter logistic e Do not combine leftover reagents with those reserved for additional wells e Reagents from the kit with a volume less than 100 ul should be centrifuged J equation Residual wash liquid should be drained from the wells after last wash by tapping the d Ifthe Test Samples were diluted multiply the interpolated values by the dilution plate on absorbent paper factor to calculate the corrected human RBP4 concentrations e Crystals could appear in the 10X solution due to high salt concentration in the stock solutions Crystals are readily dissolved at room temperature or at 37 C before 25 dilution of the buffer solutions e Once reagents have been added to the 8 well strips DO NOT let the strips DRY at any time during the assay e Keep 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 Dh OD at 450 nm bad tn Troubleshooting 0 0 001 001 01 1 10 PROBLEM POSSIBLE CAUSES SOLUTIONS Cone pg ml vi Performance Characteristics Omission of key reagent Check that all reagents have 1 Intra assay Precision 5 samples of known concentration were assayed in replicates 4 been added in the correct order times to test precision within an assay Samples Mean ug ml BEER cv ae 16 42 043 2
6. ards QC Sample We recommend these be run in duplicate e Warm Detection Antibody to room temperature Add 100 ul to each well and tap a Serum Use a serum separator tube Let samples clot at room temperature for 30 min gently on the side of the plate to mix before centrifugation for 20 min at 1000 x g Assay freshly prepared serum or store serum f Cover plate with plate sealer and incubate for 1 hr at 37 C in aliquots at 20 C for future use Avoid repeated freeze thaw cycles Serum should be g Aspirate and wash x 3 with 300 ul of 1X Wash Buffer diluted in Diluent 1X Samples containing visible precipitates must be clarified before use h Add 100 ul of the 1X Detector to each well As a starting point 1 100 dilution is suggested If samples fall outside the assay range a i Cover plate with plate sealer and incubate for 1 hr at 37 C lower or higher dilution may be required j Remove plate from 37 C aspirate and wash x 5 with 300 ul of 1X Wash Buffer b Urine Aseptically collect the urine voided directly into a sterile container Assay k After last wash tap inverted plate on a stack of paper towels Complete removal of immediately or aliquot and store at 20 C Avoid repeated freeze thaw cycles Urine should liquid is essential for good performance be diluted in Diluent 1X Samples containing visible precipitates must be clarified before I Add 100 ul of the mixed Substrate Solution to each well use m Allow the color to develop at roo
7. ition in the human RBP4 coated plate Their relative reactivity is plotted with that of the standard proteins This ELISA is specific for the e f 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 e 300 ul 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 measurement of natural and recombinant human RBP4 It does not cross react with mouse 300 ul 200 ul 300 ul 3300p 200ul 5Opl SO ul RBP4 rat RBP4 human adiponectin rat adiponectin human resistin human vaspin human clusterin human leptin human IL 23 human IL 33 human GPX3 human Nampt human ANG1 Z 4 M Y Y 4 A N human ANG2 human ANGPTL3 human ANGPTL4 human ANGPTL6 human FABP4 human RELM B rat RELM a mouse Nampt human PAI 1 The assay range is 0 001 5 ug RBP4 ml Er and a detection limit of 1 ng ml based on adding two standard deviations to the mean value of STD the 50 zero standards ti Il Kit Contents 5 2 5 1 0 5 0 25 0 1 0 01 0 001 ug ml ug ml ug ml pg ml ug ml pg ml pg ml pg ml Pre coated Microtiter Plate K4911 100 1 2 Reagent Preparation Prepare just the appropriate amounts for the assay Wash Buffer 10X K4911 100 2 a 1X Wash Buffer Dilute 10X Wash Buffer 1 9 with dH2O to obtain 1X Wash Buffer Diluent 5X K4911 100 3 b
8. m temperature in the dark for 20 min Note Serum Plasma Urine or Cell Culture Supernatant have to be diluted in Diluent n Stop the reaction by adding 100 ul of Stop Solution to each well 1X Samples containing visible precipitates must be clarified before use o Tap the plate gently to ensure thorough mixing The substrate reaction yields a blue c QC Sample Reconstitute human RBP4 QC Sample with 1 ml of dH2O Mix the QC Sample a ges he hi P at ena to ensure complete reconstitution Allow to sit for a minimum of 15 min The QC Sample is 5 Measure the OD 4t450 nm ian ELISA plate reader within Samin ready to use do not dilute it refer to the C of A for current QC Sample concentration BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA Fax 408 493 1801 tech biovision com Tel 408 493 1800 www biovision com Page 1 of 2 BioVision ao 3 Calculations Expected values RBP4 levels in plasma amp serum range from 10 to gt 70 mg ml healthy a Average the duplicate readings for each Standard QC Sample and Test Sample l l donors ee b Generate a Standard Curve by plotting the average absorbance on the vertical Y Technical Hints and Limitations axis vs the corresponding concentration ug ml on the horizontal X axis See e Itis recommended that all standards QC sample and samples be run in duplicate Typical Data below c Calculate the Test Sample RBP4 concentrations by interpolation of the St
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