CY-8068
Contents
1. c CircuLex Gees Manual For Research Use Only Not for use in diagnostic procedures S Anti CML human autoantibody ELISA Kit gt Summary of Procedure l Add 50 uL of diluted samples or standards to the wells SS 4 Incubate for 1 hour at room temp K Wash the wells 74 Add 50 uL of HRP conjugated anti human IgG antibody Q 4 Incubate for 1hour at room temp y Wash the wells RS N amp Add 50 uL of Substrate Reagent Ni t Y Add 50 uL of Stop Solution lt j S Measure absorbance at 450 nm CA Materials Provided a All samples and standards should be assayeM aplicate The following components are supplied and are sufficient for the one 96 well microplate CML BSA coated Microplate One gopa supplied ready to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a destecant pack Wells are coated with CML BSA BSA coated Microplate One micr plate supplied ready to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desicca ck Wells are coated with BSA Use for reference R 10X Wash Buffer One oR bottle of 10X buffer containing 270Tween 20 Dilution Buffer One begh containing each 50 mL of 1X buffer use for sample dilution Ready to use Anti CML Antibody Standard One vial containing 64 ng of Anti CML antibody HRP conjugat etection Antibody One vial containing 12 mL of HRP horseradish peroxidase conjugated anthffman IgG antibody Ready to use Su2. 450 540 nm Dual w ngths of 450 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a gi wavelength can be used Wells must be read within 30 minutes of adding the Stop Solution Note 1 Compig removal of liquid at each step is essential to good performance After the last wash rem any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it ag st clean paper towels Note 2 ge standard curves are obtained when either O D values do not exceed 0 35 units for the Q ank zero concentration or 2 0 units for the highest standard concentration The plate YA should be monitored at 5 minute intervals for approximately 30 minutes N VA Roa CY 8068 8 Version 140318 o AO Anti CML human autoantibody ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Calculations 3 Average the duplicate readings for each standard and sample in CML BSA coated plata BSA coated plate Subtract the average readings in BSA coated plate from those in CML BSA d plate Plot the optical density for the standards versus the concentration of the standards and the best curve The data can be linearized by using log log paper and regression analysis may b pplied to the log transformation To determine the anti CML adduct concentration of each sam first find the absorbance value on the y axis and extend a horizontal line to the standard curyg At t
3. Anti CML antibody ng ml 18 0 16 0 14 0 12 0 10 0 8 0 6 0 4 0 2 0 0 0 Anti CML Autoantibody Healthy volunteers Patients afflicted with lifestyle related disease ea Kor CY 8068 13 Version 140318 c Anti CML human autoantibody ELISA Kit CircuLex si Manual For Research Use Only Not for use in diagnostic procedures A Q O Q _ _ CS References A 1 Ikeda K Higashi T Sano H Jinnouchi Y Yoshida M Araki T Ueda S Horiuchi S Biok Ry 35 8075 8083 1996 RY 2 Reddy S Bichler J Wells Knecht KJ Thorpe SR Baynes JW Biochemistry 34 1087241 0878 1995 3 Makino H Shikata K Hironaka K Kushiro M Yamasaki Y Sugimoto H Ota aki N Horiuchi S Kidney Int 48 517 526 1995 Q 4 Suzuki D Yagame M Jinde K Naka R Yano N Endoh M Kaneshige WYNomoto Y Sakai H J Diabetes Complications 10 314 319 1996 Y Imai N Nishi S Suzuki Y Karasawa R Ueno M Shimada H Kawas Ni S Nakamaru T Miyakawa Y Araki N Horiuchi S Gejyo F Arakawa M Nephron 76 153 489 1997 n 6 Murata T Nagai R Ishibashi T Inomuta H Ikeda K fe Oers 40 764 769 1997 7 Kume S Takeya M Mori T Araki N Suzuki H Horiuchi g odama T Miyauchi Y Takahashi K Am J Pathol 147 654 667 1995 72 8 Sakata N Imanaga Y Meng J Tachikawa Y Takebayagbti S Nagai R Horiuchi S Itabe H Takano T Atherosclerosis 141 61 75 1998 Q
4. 9 Sakata N Imanaga Y Meng J Tachikawa Y MRebayashi S Nagai R Horiuchi S Atherosclerosis 142 67 77 1999 x i A Du Yan S Hofmann M Yan SF Pischetsrieder M 740 31749 1999 10 Kislinger T Fu C Huber B Qu W T Stern D Schmidt AM J Biol Chem K 11 Shibayama R Araki 4 Nagai R Horiuchi S Autoantibody against N epsilon carbozymethyl lysi advanced glycation end product of the Maillard reaction Diabetes 1999 Sep 48 9 184 12 Vay D Vidali M Mo Cusaro C Rolla R Mottaran E Bellomo G Albano E Antibodies against advanced gly end product Nepsilon carboxymethyl lysine in healthy controls and diabetic patients me ogia 2000 Nov 43 11 1385 8 N oO B O RS Q A Roa CY 8068 14 Version 140318 o AO P ihi Anti CML human autoantibody ELISA Kit C 1rcu Lex User s Manual AS For Research Use Only Not for use in diagnostic procedures Z Related Products 3 CircuLex CML N Carboxymethyl lysine ELISA Kit Cat CY 8066 CircuLex Anti CML mouse autoantibody ELISA Kit Cat CY 8067 CircuLex Anti CML human autoantibody ELISA Kit Cat CY 8068 KY CircuLex Anti CML rat autoantibody ELISA Kit Cat CY 8069 Q CML BSA N Carboxymethy lysine BSA Cat CY R2052 A CML OVA N Carboxymethy lysine OVA Cat CY R2053 CEL BSA N Carboxyethyllysine BSA Cat CY R2054 A CEL OVA N Carboxyethyl lysine OVA Cat CY R2055 Yon Glucose AGE BSA Cat CY R2056 ne Glucose AGE OVA Ca
5. CML human autoantibody ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Detailed Protocol 3 The CycLex Research Product CircuLex Anti CML human autoantibody ELISA Kit i vided with removable strips of wells so the assay can be carried out on separate occasions usi only the number of strips required for the particular determination Since experimental See vary an aliquot of the Anti CML human antibody Standard within the kit should be included in gch assay as a calibrator Disposable pipette tips and reagent troughs should be used for all liquid sfers to avoid cross contamination of reagents or samples Y Q Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay reagents are supplied ready to use with the exception of 10X Wash Buffer and Anti CML Antikody Standard deionized distilled water ddH20 Mix well Store at 4 C for weeks or 20 C for long term 1 Prepare a working solution of Wash Buffer by adding 100 mL of Ss Wash Buffer to 900 mL of storage C 2 Reconstitute Anti CML Antibody Standard with 0 8 mir ddH20 The concentration of the anti CML antibody in vial should be 80 ng mL which is reffgrred as a Master Standard of anti CML antibody gt Prepare Standard Solutions as follows gt Use the Master Standard to produce a dilutio Q ries below Mix each tube thoroughly before the next transfer T
6. uL of Standard Solutions Std1 Std7 Blank and diluted samples O ians into the appropriate wells of both CML BSA coated Microplate and BSA coated Microplate Y 4 Incubate the wells at room temperature ca 25 C for 1 hour shaking aQ 300 rpm on an orbital microplate shaker 5 Wash 4 times by filling each well with Wash Buffer 350 uL RSA squirt bottle multi channel pipette manifold dispenser or microplate washer 6 Add 50 uL of HRP conjugated Detection Antibody into eac 11 7 Incubate the plate at room temperature ca 25 C for Lar shaking at ca 300 rpm on an orbital microplate shaker 4 8 Wash 4 times by filling each well with Wash Bulgyy 350 uL using a squirt bottle multi channel pipette manifold dispenser or microplate washeragy Ww 9 Add 50 uL of Substrate Reagent Avoid expOpsing the microtiter plate to direct sunlight Covering the plate with e g aluminum foil is recommeftded Return Substrate Reagent to 4 C immediately after the necessary volume is removed ro Incubate the plate at room temperat ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubation time may be extended up to 30 minutes if the reaction temperature is below than 20 C oe Add 50 pL of Stop Solutio each well in the same order as the previously added Substrate Reagent aY 12 Measure absorbance S well using a spectrophotometric microplate reader at dual wavelengths of
7. CircuLex Gees Manual For Research Use Only Not for use in diagnostic procedures ww S Anti CML human autoantibody ELISA Kit S ELISA Kit for Measuring Anti CML N Carboxymethyl lysine human ante CircuLex Anti CML human autoantib ELISA Kit g Cat CY 8068 2 OF Intended US 0 cssssscsscesceosnivsssasadarcaadacveanienatands 1 ne Be sat AAA 1 a TPO CTO MGs sasasissscenighoghings aaintebscinsiaaysaulesad 2 Yon Principle Of the Assay wmse 3 gt Materials Provided 3 o Materials Required but not Provided 4 lt Precautions and Recommendations 5 4 Sample Collection and Storage 6 e2 Detailed Protocol cccceccccceccceeceesseeeseeeeees 7 8 4 CIC MLAUIOIIS ccscannmactesascenaasetesianeesesaesciataens 9 gt Measurement Range 9 O THOU DIeSHO GUIS si cnissrnsasesanndcnsstarnnasientenss a gt Reagent Stability w mse Key Assay CharacteristiCS lt lt lt syers 0 Example of Test Results zp4 gr 1213 RELCLCH CES anaanza Weess 14 Related ProductsS lt s s 15 Intended Use The CycLex Research Produ CircuLex Anti CML human autoantibody ELISA Kit is used for the semi quantitative nen of IgG class anti CML N Carboxymethyl lysine human autoantibody in human seru d plasma This assay kit is for reah use only and not fo
8. bstrate Quent One bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB y to use sia aaa One bottle supplied ready to use containing 20 mL of 1 N H2S04 e Roa CY 8068 3 Version 140318 o c A CircuLex DaMan S Anti CML human autoantibody ELISA Kit For Research Use Only Not for use in diagnostic procedures 4 Materials Required but not Provided 3 e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips RS N e Precision repeating pipettor N e Orbital microplate shaker g e Microcentrifuge and tubes for sample preparation 2 e Vortex mixer y e Microplate washer optional Manual washing is possible but not prefett e Plate reader capable of measuring absorbance in 96 well plates Gi wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be us The plate can also be read at a single wavelength of 450 nm which will give a somewhat a e Software package facilitating data generation and analysig o tional e 500 or 1000 mL graduated cylinder I e Reagent reservoirs amp Y e Deionized water of the highest quality wW O e Disposable paper towels VA Roa CY 8068 4 Version 140318 o c S Anti CML human autoantibody ELISA Kit 9 CircuLex DaMan For Research Use Only Not for use in diagnostic procedures Z Precautions and Recommendations 3 e Allow all the components to come to room tem
9. he 20 ng mL standard serves as the highest standard The Dilution Buffer serves as the zero standard Blank O Volume of Standard Dilution Buffer Concentration Std 1 150 uL of Master S d 450 uL 20 ng mL Std 2 300 uL of Std 1 ieee 300 uL 10 ng mL Std 3 300 uL of Std 2 10 ng ml 300 uL 5 ng mL Std 4 300 uL of ses ng ml 300 uL 2 5 ng mL Std 5 300 uL of Stig 4 2 5 ng ml 300 uL 1 25 ng mL Std 6 300 u d 5 1 25 ng ml 300 uL 0 63ng mL Std 7 300 4L f Std 6 0 63 ng ml 300 uL 0 31 ng mL Blank aw 300 uL 0 ng mL N Note Do not u Kane pipette Change tips for every dilution Wet tip with Dilution Buffer before disp nsing Unused portions of Standards should be aliquoted and stored at below 70 C geal Avoid multiple freeze and thaw cycles Sample Pregitation e Ser d plasma samples require 100 fold dilution e UL sample 4 198 uL Dilution Buffer S 72 Roa CY 8068 7 Version 140318 o AO Assay Procedure ay 2 Dilute samples with Dilution Buffer See Sample Preparation above amp ies 10 Anti CML human autoantibody ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Remove the appropriate number of both CML BSA coated Microplate and BSA coated plate wells from the foil pouch and place them into the well holder Return any unused well the foil pouch refold seal with tape and store at 4 C Pipette 50
10. he point of intersection extend a vertical line to the x axis and read the correspondifig anti CML adduct concentration If the samples have been diluted the concentration read from theygtandard curve must be multiplied by the dilution factor Yon 1 The dose response curve of this assay fits best to a sigmoidal 5 pagghiter logistic equation The results of unknown samples can be calculated with any computer gram having a 5 parameter logistic function It is important to make an appropriate mathematica justment to accommodate for the dilution factor amp 2 Most microtiter plate readers perform automatic calculations Hiart concentration The calibration curve is constructed by plotting the absorbance Y calibrators versus log of the known concentration X of calibrators using the four parami function Alternatively the logit log function can be used to linearize the calibration curve logit of absorbance Y is plotted versus log of the known concentration X of calibrators Measurement Range The measurement range is 0 31 ng mL to Wem Any sample reading higher than the highest standard should be diluted with Dilution Buffer in higher dilution and re assayed Dilution factors need to be taken into consideration in calculating anti CML human autoantibody concentration va Troubleshooting r2 1 The anti CML antibody Sta should be run in duplicate using the protocol described in the Detailed Protocol Incub imes
11. k is better than 0 16 ng mL of sample Typical standard curve aw 1 6 r Standard courve A450 0 5 10 15 20 Anti CML Ab ng ml 2 Specificity The antibodies he CircuLex Anti CML human autoantibody ELISA Kit are highly specific of CML adduct no detectable cross reactivity to non CML protein that may be present in human ka piaanas 3 Precision Intra say Precision Precision within an assay io samples of known concentration were tested eight times on one plate to assess intra assay pe s ion Qy Intra assay Within Run n 8 CV 5 6 Jo Roa CY 8068 10 Version 140318 o c Anti CML human autoantibody ELISA Kit CircuLex Gees Manual For Research Use Only Not for use in diagnostic procedures Inter assay Precision Precision between assays Three samples of known concentration were tested in four separate assays to assess in sassay precision ww e Inter assay Run to Run n 4 CV 8 7 eA 4 Spiking Recover Serum samples were spiked with different amounts of anti CML antibody and ycd The recovery of anti CML antibody spiked to levels throughout the re of the assay was evaluated Y Sample Average Recovery Range a gt Sera n 4 79 83 90 123 C 5 Linearity To assess the linearity of the assay samples containing and or ed with high concentrations of anti CML antibody were serially diluted with the Dilution Bu to produce samples with values within the dynamic ra
12. nge of the assay lt eA Roa CY 8068 11 Version 140318 o c A CircuLex si Manual For Research Use Only Not for use in diagnostic procedures 4 Example of Test Results 3 Fig 1 Immunoreactivity of sera from 40 patient afflicted lifestyle related disease to CML B SAQKTB SA WI RS Anti CML human autoantibody ELISA Kit Ma Sera from 40 patients afflicted with lifestyle related diseases 0 6 r E CML BSA ABSA AN wa A f f f d Pl P2 P3 P4 P5 P P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 P25 P26 P27 P28 P29 P30 P31 P32 P33 P34 P35 P36 P37 P38 P39 P40 0 5 f A450 Sample No JJ SS Fig 2 Immunoreactivity of sera from 40 healthy YSlunteers to CML BSA and BSA N AS Sera from 40 healthy volunteers 0 6 p HCML BSA ABSA 0 5 H 0 4 H gt mn 0 3 H PASO NNN SSS S 0 2 7 0 1 YA 0 0 Ni N2 N3 N4 N5 N6 N7 N8 N9 N10 N11 N12 N13 N14 N15 N16 N17 N18 N19 N20 N21 N22 N23 N24 N25 N26 N27 N28 N29 N30 N31 N32 N33 N34 N35 N36 N37 N38 N39 N40 ea Roa CY 8068 12 Version 140318 ya c Sample No CircuLex For Research Use Only Not for use in diagnostic procedures Anti CML human autoantibody ELISA Kit User s Manual A G gt oO Q S Fig 3 Comparison of anti CML autoantibody concentration in sera from healthy volunteers and pa Ets afflicted with lifestyle related disease aN o gL
13. or temperatures significantly different from those specified may give erroneous results 2 Poor duplicates acc ied by elevated values for wells containing no sample indicate insufficient washing If all instrdCtons in the Detailed Protocol were followed accurately such results indicate a need for washer tenance 3 Overall low Cu may indicate that desiccation of the plate has occurred between the final wash and addition of fu strate Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wasia gt g Roa CY 8068 9 Version 140318 o AO A gt P ihi Anti CML human autoantibody ELISA Kit O L 1rcu Lex User s Manual rey For Research Use Only Not for use in diagnostic procedures 4 Reagent Stability 3 All of the reagents included in the CycLex Research Product CircuLex Anti C y man autoantibody ELISA Kit have been tested for stability Reagents should not be used beyo e stated expiration date Upon receipt kit reagents should be stored at 4 C Coated assay plates sh be stored in the original foil bag sealed by the zip lock and containing a desiccant pack WA For research use only not for use in diagnostic or therapeutic procedures Ka Assay Characteristics AR 1 Sensitivity S The limit of detection defined as such a concentration of anti CMfantibody giving absorbance lower than mean absorbance plus three standard deviations of the abs nce of Blank Blank 3 SD Blan
14. perature before use eS All microplate strips that are not immediately required should be returned to the zip lock Xa which must be carefully resealed to avoid moisture absorption WA e Do not use kit components beyond the indicated kit expiration date e Use only the microtiter wells provided with the kit Meg e Rinse all detergent residue from glassware Rd e Use deionized water of the highest quality gt e Do not mix reagents from different kits Ni The buffers and reagents in this kit may contain preservatives V chemicals Care should be taken to avoid direct contact with these reagents 4 Q e Do not smoke eat or drink when performing the yay or in areas where samples or reagents are handled aS e Dispose of tetra methylbenzidine TMB contgiGhe solutions in compliance with local regulations e Do not mouth pipette or ingest any of the reagents e Avoid contact with the acidic Stop Song d Substrate Solution which contains hydrogen peroxide e Wear gloves and eye protection whey Fandling immunodiagnostic materials and samples of human origin and these reagents In case gf contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and se edical attention when necessary Biological samples may be EL minated with infectious agents Do not ingest expose to open wounds or breathe aeros gt Wear protective gloves and dispose of biological samples properly handlin Solu
15. r use in diagnostic or therapeutic procedures Storage e Upon receipt Rre all components at 4 C e Don t expos feagents to excessive light A Roa CY 8068 1 Version 140318 o AO Anti CML human autoantibody ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Introduction 3 Reducing sugars react with protein amino groups to form a diverse group of protein bour ieties with fluorescent and cross linking properties These compounds called advanced glycosyfation end products AGEs have been implicated in the structural and functional alterations of protes that occur during aging and long term diabetes amp Although several AGE structures have been reported 1 2 it was nstrated that N Carboxymethyl lysine CML is a major antigenic AGE structure CML cen is also increased in patients who have diabetes with complications including nephropa 3 5 retinopathy 6 and atherosclerosis 7 9 CML is also recognized by receptor for AGE RAGE and CML RAGE interaction activates cell signaling pathways such as NF B and enhances the eXPyession of vascular cell adhesion molecule 1 in human umbilical vein endothelial cells 10 It has been postulated that AGE structures present in vivo could serve YY immunological epitope to raise autoantibodies against AGE structures particularly CML Shibaya t al showed the presence of autoantibodies against AGE structures partic
16. t CY R2057 a Glyceraldehyde AGE BSA Cat CY R2058 Y Glyceraldehyde AGE OVA Cat CY R2059 wy Glycolaldehyde AGE BSA Cat CY R2060 amp Glycolaldehyde AGE OVA Cat CY R2061 Yon Methylglyoxal AGE BSA Cat CY R2062 lt Methylglyoxal AGE OVA Cat CY R2063 rq Glyoxal AGE BSA Cat CY R2064 amp Glyoxal AGE OVA Cat CY R2065 CML HSA N Carboxymethyllysine HSA Cat CR 2066 CEL HSA N Carboxyethyllysine HSA Cat C R2067 PRODUCED BY amp CycLex Co Ltd O 1063 103 Terasawaoka Ina Nagano 396 0002 ge Japan Fax 81 265 76 7618 Q e mail info cyclex co jpy e URL http www cycl j CycLex CircuLex frddlucts are supplied for research use only CycLex CircuLex products and components theregf may not be resold modified for resale or used to manufacture commercial products witho rior written approval from CycLex Co Ltd To inquire about licensing for such commergfafuse please contact us via email amp N gL A Roa CY 8068 15 Version 140318 o c
17. tion O nw a Qe amp A Roa CY 8068 5 Version 140318 e CAUTION Sulfuric y is a strong acid Wear disposable gloves and eye protection when e ee A CircuLex si Manual For Research Use Only Not for use in diagnostic procedures 4 Sample Collection and Storage 5 Serum Use a serum separator tube and allow samples to clot for 60 30 minutes Cen Qe the samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or store ples on ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 Gaior extended periods of time Avoid repeated freeze thaw cycles Q y Anti CML human autoantibody ELISA Kit Plasma Collect plasma by use of EDTA Naz as the anticoagulant If possible cle plasma into a mixture of EDTA Naz and FuthanS to stabilize the sample against spontaneous ig vitro complement activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x gg Assay immediately or store samples on ice for up to 6 hours before assaying Aliquots of plasma wami be stored at below 70 C for extended periods of time Avoid repeated freeze thaw cycles gt gt Other biological samples Remove any particulates by centrifugatioGand assay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cyc ay Note Citrate plasma has not been validated for use in this assay VA Roa CY 8068 6 Version 140318 amp lt C Anti
18. ularly those against L adduct in streptozotocin STZ induced diabetic rats and patients with several diseases 11 10 The autoantibody against CML adduct was higher in patients with renal failure than in normal subjQQs or diabetic patients without renal failure 11 These results suggest that autoantibody against might play a possible role in the development of diabetic nephropathy or chronic renal failure lt Principle of the Assa Q The CircuLex Anti CML human autoantibody gea Kit employs the semi quantitative enzyme immunoassay technique CML BSA or BSA hagen pre coated onto a microplate Standards or samples are pipetted into the wells Any anti adduct autoantibody present is bound by the immobilized CML BSA but not by the imo BSA After washing away any unbound substances an HRP conjugated antibody specific for human IgG is added to the wells Following a wash to remove any unbound antibody HRP conjugate gerne conjugate is allowed to react with the substrate H2O2 tetramethylbenzidine The reaction gg Stopped by addition of acidic solution and absorbance of the resulting yellow product is measured nt nm The absorbance is proportional to the concentration of anti CML adduct antibody A stan curve is constructed by plotting absorbance values versus anti CML adduct autoantibody con gntrations of calibrators and concentrations of unknown samples are determined using this standard irve 3 o oe Roa CY 8068 2 Version 140318 o
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