User Manual
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1. esRAGE which can capture AGE and neutralize the effects of AGE on cells Endogenous secretory RAGE has been found circulating in blood and in extracellular fluids of our bodies The B Bridge esRAGE ELISA Kit is designed to measure the concentration of human esRAGE in serum plasma and tissue culture medium The principle of the assay is shown in Figure 1 Standards or samples and detection antibody esRAGE antibody horseradish peroxidase conjugated are incubated in an anti RAGE antibody coated 96 well microtiter plate After 16 24 hour incubation and four washing substrate is added to each well The plate is incubated for 30 minutes The enzymatic reaction is stopped by the addition of stop solution and absorbance of the resulting yellow product is measured at 450 nm The absorbance is proportional to the concentration of esRAGE A standard curve is constructed by plotting absorbance values versus esRAGE concentrations of standards and concentrations of unknown samples are determined using this standard curve B Bridge International Inc www b bridge com esRAGE ELISA Kit User Manual Figure 1 Assay Principle esRAGE antibody Substrate H O esRAGE HRP conjugated tetramethlybenzidine RR Color Development pr iS ey 1st Reaction 2nd Reaction 000 SL vey YS Anti RAGE antibody coated plate B Bridge International Inc 4 www b bridge com esRAGE ELISA Kit User Manual ll List of Components e Sto2. wash solution Aspirate the wash solution Repeat steps 6 and 7 three times for a total of 4 washes Invert the plate and gently tap on a clean absorbent towel to remove residual droplets of wash solution Dispense 100 ul of substrate into each well Avoid exposing the microtiter plate to direct sunlight Covering the plate with aluminum foil is recommended Incubate at 20 30 C for 30 minutes Add 100 ul of stop solution to each well Read the plate within 30 minutes after stopping the reaction The plates are measured at wavelength 450 nm The recommended reference wavelength is 630 nm www b bridge com esRAGE ELISA Kit User Manual Figure 2 Flow Chart of Assay Anti RAGE Antibody Coated Plate Standard and Sample Dilutions esRAGE Antibody HRP Conjugated 100 ul Standards or Samples 20 ul Incubate 4 109C 16 24 hours 4 x Substrate 100 ul Color development Wash 350 ul 20 30 C 30 minutes Stop Solution 100 ul Read Absorbance at 450 nm B Bridge International Inc www b bridge com esRAGE ELISA Kit User Manual VII Calculation of Results 1 Subtract the mean absorbance value of the 0 ng ml blank from each mean absorbance value of the standard series and samples tested Net Absorbance 2 Plot the log of known concentrations of each standard and the calculated Net Absorbance on the X axis and Y axis respectively Fit an appropriate regression curve to the plotted points 3 Determine the
3. 997 1992 7 Neeper M Schmidt A M Brett J Yan S D Wang F Pan Y C E Elliston K Stern D and Shaw A Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins J Biol Chem 267 14998 15004 1992 8 Brett J Schmidt A M Yan S D Zou Y S Weidman E Pinsky D Nowygrod R Neeper M Przysiecki C Shaw A Migheli A and Stern D Survey of the distribution of newly characterized receptor for advanced glycation end products in tissues Am J Pathol 143 1699 1712 1993 9 Yan S D Schmidt A M Anderson G M Zhang J Brett J Zou Y S Pinsky D and Stern D Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors binding proteins J Biol Chem 269 9889 9897 1994 10 Lander H M Tauras J M Ogiste J S Hori O Moss R A and Schmidt A M Activation of the receptor for advanced glycation end products triggers a p21 dependent mitogen activated protein kinase pathway regulated by oxidant stress J Biol Chem 272 17810 17814 1997 11 Schmidt A M and Stern D M RAGE a new target for the prevention and treatment of the vascular and inflammatory complications of diabetes Trends Endocrinol Metab 11 368 375 2000 12 Tanaka N Yonekura H Yamagishi S Fujimori H Yamamoto Y and Yamamoto H The receptor for advanced glycation end products is induced by the glycation products themselves and tumor necr
4. B Bridge Intemational Inc N esRAGE ELISA Kit User Manual UM 100901 Published 01 January 2005 Catalog K1009 1 Manufactured by Daiichi Fine Chemical Co LTD Distributed by B Bridge International Inc See List of Components for Storage Conditions FOR RESEARCH USE ONLY esRAGE ELISA Kit User Manual Table of Contents l Introduction and Protocol Overview 3 Il List of Components 5 Ill Additional Materials Required 6 IV Reagent Preparation and Storage 7 V Sample Preparation 7 VI Human esRAGE ELISA Protocol 8 VII Calculation of Results 10 VIII Troubleshooting Guide 11 IX References 12 Notice to Purchaser This product is to be used for Research Purposes Only It is not to be used for Drug or Diagnostic Purposes nor is it intended for Human Use B Bridge products may not be resold modified for resale or used to manufacture commercial products without the express written consent of B Bridge International Inc EXCEPT AS OTHERWISE EXPRESSLY SET FORTH IN THIS USER MANUAL B BRIDGE DOES NOT MAKE ANY REPRESENTATION OR WARRANTIES OR CONDITIONS OF ANY KIND EITHER EXPRESS OR IMPLIED WITH RESPECT TO THE PRODUCTS OR INFORMATION DISCLOSED HEREUNDER INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY FIT FOR A PARTICULAR PURPOSE OR NONINFRINGEMENT OF THE INTELLECTUAL PROPERTY RIGHTS OF THIRD PARTIES 2002 B Bridge International Inc All Rights Reserved B Bridge International Inc www b bridge
5. Manual NOTES B Bridge International Inc www b bridge com 15 B Bridge International Inc 639 N Pastoria Avenue Sunnyvale CA 94085 USA Tel 408 735 7727 Fax 40 8 735 7737 customersupport b bridge com B Bridge International Inc esRAGE ELISA Kit User Manual www b bridge com
6. References 1 Brownlee M Cerami A and Vlassara H Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications N Engl J Med 318 1315 1321 1988 2 Harding J J Nonenzymatic covalent posttranslational modification of proteins in vivo Adv Protein Chem 37 248 334 1985 3 Sakurai S Yonekura H Yamamoto Y Watanabe T Tanaka N Li H Azadur Rahman A K M Myint K M Kim C H and Yamamoto H The AGE RAGE system and diabetic nephropathy J Am Soc Nephrol 14 5259 5263 2003 4 Wautier J L Wautier M P Schmidt A M Anderson G M Hori O Zoukourian C Capron LO Chappey O Yan S D Brett J et al Advanced glycation end products AGEs on the surface of diabetic erythrocytes bind to the vessel wall via a specific receptor inducing oxidant stress in the vasculature a link between surface associated AGEs and diabetic complications Proc Natl Acad Sci USA Aug 2 91 16 7742 7746 1994 5 M nch G Kuhla B L th H J Arendt T and Robinson S R Anti AGEing defences against Alzheimer s disease Biochem Soc Trans 31 1397 1399 2003 6 Schmidt A M Vianna M Gerlach M Brett J Ryan J Kao J Esposito C Hegarty H Hurley W Clause M Wang F Pan Y C E Tsang T C and Stern D Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial surface J Biol Chem 267 14987 14
7. com esRAGE ELISA Kit User Manual Il Introduction and Protocol Overview In multicellular organisms most cellular and plasma proteins undergo a nonenzymatic glycation by the covalent binding of reduced sugars to the amino groups on proteins balance exists between early glycation products and non glycated proteins during homeostasis Under the condition of prolonged hyperglycemia as in diabetes these early stage glycation products undergo additional chemical rearrangements to form irreversibly bound advanced glycation end products AGE AGE has been shown to be a major factor in vascular cell derangement in diabetic patients AGE has also been implicated in Alzheimer s Disease by the glycation of amyloid peptide and microtubule associated protein tau The receptor for AGE RAGE a 45kDa protein and a member of the immunoglobulin superfamily of cell surface molecules is found in mononuclear phagocytes endothelium vascular smooth muscle and the central nervous system RAGE is a multi ligand receptor The most pathological consequence of the AGE RAGE engagement is induction of oxidative stress activation of nuclear factor kB NF kB resulting in disruption of homoeostatic functions in the vasculature 11 AGE has also been shown to up regulate the RAGE expression in endothelial cells Translation of RAGE mRNA isolated from human endothelial cells revealed a novel splice variant named endogenous secretory receptor
8. ed with buffer if quantitation of the sample protein is above the 3 2 ng ml data point on the standard curve B Bridge International Inc 7 www b bridge com esRAGE ELISA Kit User Manual VI Human esRAGE ELISA Protocol Note Allow all reagents to come to room temperature 20 30 C prior to the start of the assay and prepare 1X Wash Solution and esRAGE Standards as described in the previous sections 1 10 11 12 13 B Bridge International Inc Remove Primary Antibody Coated Plate from its foil pouch Remove any unneeded strips from the plate frame reseal them in the foil pouch and return the foil pouch to 2 8 C If a 96 well plate washer is used the plate frame should be completely filled with wells by adding as many null strips as necessary Identify well position s for each sample on a data sheet or plate map Add 100 ul of esRAGE antibody HRP conjugate to the appropriate number of antibody coated wells Add 20 ul of assay standards 0 0 05 0 1 0 2 0 4 0 8 1 6 3 2 ng ml or sample to the plate and mix well by pipetting All assay standards and samples should be run in duplicates Every plate must include the assay standards to properly correlate the sample readings Cover plate s securely with a plate sealer and incubate at 4 10 C for 16 24 hours The plates should not be agitated or rocked during the incubation period Aspirate the reaction mixture from each well Wash each well with 350 ul of 1x
9. esRAGE concentrations of the samples by interpolation of the regression curve formula 4 If samples were diluted then concentration calculations must be multiplied by the appropriate dilution factor to obtain the correct results for the undiluted samples Figure 3 Typical Standard Curve Standard Curve 3 E 2 oO wo w Q 3 1 0 0 11 0 23 0 45 0 9 1 8 3 6 MMP 1 ng ml B Bridge International Inc www b bridge com esRAGE ELISA Kit User Manual Vill Troubleshooting Guide Troubleshooting Guide 1 Lack of signal or weak signal in all wells Possible explanations e Omission of a reagent or a step e Improper preparation or storage of a reagent e Assay performed before reagents were allowed to come to 20 30 C e Plate reader did not perform well 2 High signal and background in all wells Possible explanations e Improper or inadequate washing be certain that all wash volumes and repetitions were correct e Improper dilution of detection antibody e Overdeveloping decrease the incubation time before the Stop Solution is added 3 High background in sample wells only Possible explanations e Sample concentration was too high e Improper dilution of detection antibody 4 Weak signal in sample wells only Possible explanations e Sample concentration was too low e Improper dilution of detection antibody B Bridge International Inc 11 www b bridge com esRAGE ELISA Kit User Manual IX
10. osis factor a through nuclear factor KB and by 17b estradiol through Sp 1 in human vascular endothelial cells J Biol Chem 275 25781 25790 2000 13 Yonekura H Yamamoto Y Sakurai S Petrova R G Abedin J Li H Yasui K Takeuchi M Makita Z Takasawa S Okamoto H Watanabe T and Yamamoto H Novel splice variant of the receptor for advanced glycation end products expressed in human vascular endothelial cells and pericytes and their putative roles in diabetes induced vascular injury Biochem J 370 1097 1109 2003 B Bridge International Inc www b bridge com esRAGE ELISA Kit User Manual B Bridge Products amp Partnering Opportunities If you would like to receive information from B Bridge about our products and or partnering opportunities please contact us at Telephone 408 735 7727 Fax 408 735 7737 customersupport b bridge com B Bridge product line for life science researchers Apoptosis Kits ELISA Kits for adipokines esRAGE and TAFI Transfection reagents Molecular biology reagents Electrophoresis apparatus siRNA products and design service STREX mechanical cell stress Custom services Antibody production Cloning Protein expression Protein protein interaction screening Expression profiling Tissue section Immunohistochemistry B Bridge International Inc www b bridge com esRAGE ELISA Kit User Manual NOTES B Bridge International Inc www b bridge com 14 esRAGE ELISA Kit User
11. re 20 30 C prior to the start of reagent preparation 1 1X Wash Solution If crystals are observed in the 5X Wash Solution bottle warm the bottle in a 37 C water bath until the crystals disappear Prepare 1X Wash Solution by mixing 50 ml of the 5X Wash Solution with 200 ml of deionized water or equivalent Mix well After preparation store 1X Wash Solution at 2 8 C The 1X Wash Solution is stable for 1 month at 2 8 C 2 esRAGE Standard Reconstitute esRAGE standard bottle with 0 5 ml of distilled water Prepare each esRAGE assay standard 1 6 ng ml 0 8 ng ml 0 4 ng ml 0 2 ng ml 0 1 ng ml 0 05 ng ml by serially diluting the reconstituted standard with buffer bottle Use the undiluted esRAGE Standard 3 2 ng ml and buffer for the 3 2 ng ml and 0 ng ml assay standards respectively Assay standards should be prepared prior to use and any unused assay standards should be discarded Reconstituted esRAGE standard is stable for 1 month at 2 8 C 3 Other kit reagents Anti RAGE antibody coated plate esRAGE antibody horseradish peroxidase HRP conjugated substrate stop solution and buffer are ready for use Once opened these reagents are stable for 3 months at 2 8 C Note The unopened reagents if properly maintained at 2 8 C are stable until the expiration date printed on the box label Do not mix reagents from different kits unless they have the same lot number V Sample Preparation Samples may be dilut
12. re all components at 2 8 C DO NOT FREEZE 1 PRIMARY ANTIBODY COATED PLATE 1 Plate One 96 well plate with adsorbed anti RAGE antibody Plate is provided in a resealable foil pouch with desiccant 2 DETECTION ANTIBODY 1 Bottle 11 ml esRAGE antibody horseradish peroxidase HRP conjugated 3 SUBSTRATE 1 Bottle 15 ml 3 3 5 5 tetramethylbenzidine and hydrogen peroxide 4 STOP SOLUTION 1 Bottle 15 ml 1 M sulfuric acid 5 BUFFER 1 Bottle 5 ml Phosphate buffered saline containing bovine serum albumin 6 esRAGE STANDARD 1 Bottle lyophilized Human esRAGE 1 6 ng 7 5X WASH SOLUTION 2 Bottle 50 ml each Phosphate buffered saline with Tween 20 MSDS forms are available on our website please visit www b bridge com B Bridge International Inc www b bridge com esRAGE ELISA Kit User Manual lll Additional Materials Required The following materials are required but not supplied e Graduated cylinder e Micropipette s and disposable pipette tips e Null strips for 96 well plate e 96 well plate or manual strip washer e Paper towels or absorbent paper e Plate reader capable of measuring absorbance at a wavelength of 450 nm reference filter at 650 nm optional e Well closed containers such as microtubes 1 5 ml or more in capacity e Refrigerator 2 8 C B Bridge International Inc www b bridge com esRAGE ELISA Kit User Manual IV Reagent Preparation and Storage Allow all the reagents to come to room temperatu
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