CY-8069
Contents
1. For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Immunoreactivity of plasma of diabetic rat to CML BSA and BSA 600 800 600 800 S 3 5 3 600 amp amp 600 2 g 2 E g 4 E 400 3 2 S 400 3 5 400 2200 z 200 a a 200 a 200 0 0 0 0 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 weeks weeks weeks weeks 0 7 p SA 07 r STZ injected rat No 1 El CML BSA STZ injected rat No 2 El CML BSA BSA BSA 06 F 0 6 F 0 5 F 0 5 r o 0 4 T o 04 F N N 0 3 r 03 F 02r 0 2 F 0 1 F 01r o0 oo m E E 0 10 14 23 0 10 14 23 Time weeks after STZ injection Time weeks after STZ injection 600 800 600 800 3 2 S a I 2 600 400 go 400 E E 2 2 3 2400 400 3 200 n 200 a 200 a 200 0 0 0 0 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 weeks weeks weeks weeks 07 r 07 r El CML BSA Sham rat No 2 WA Sham rat No 1 RN ek E CML BSA BSA 0 5 F 0 5 r 2 04 F 2 04 r st lt lt o3 F 0 3 r 02r 02r oo oo 0 10 14 23 0 10 14 23 Time weeks after vehicle injection Time weeks after vehicle injection CY 8069 12 Version 140318 ii Anti CML rat autoantibody ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures References 1 Ikeda K Higashi T Sano2. Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cyclex co jp CycLex CircuLex products are Supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY 8069 14 Version 140318
3. ii Anti CML rat autoantibody ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Anti CML N Carboxymethyl lysine rat autoantibody CircuLex Anti CML rat autoantibody ELISA Kit Cat CY 8069 Intended Mr s 1 Be 1 Tii tO OUI OI a erscasesnsexintanslentanaratoteciacncevedeads 2 Principle of the Assay wmmse 2 3 Materials Provided lt 3 Materials Required but not Provided 4 Precautions and Recommendations 3 Sample Collection and Stage 6 Detailed Protocol i iesseses esset retten 7 9 CHIC M LATIONS 9 Measurement Range m 9 TroubleshoOOBmp ue etn ritas inen eben 9 Reagent Stability swmmmmmmsa 10 Assay Characteristics eroe ron rn 10 14 Example of Test Results 12 Referen ai 13 Related Products eiat 14 Intended Use The CycLex Research Product CircuLez Anti CML rat autoantibody ELISA Kit is used for the semi quantitative measurement of IgG Class anti CML N Carboxymethyl lysine rat autoantibody in rat serum and plasma This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage Upon receipt store allpgomponents at 4 C e Don t expose eagents to excessive light Cat CY 8069 1 Version 140318 ii A
4. H Jinnouchi Y Yoshida M Araki T Ueda S Horiuchi S Biochemistry 5 8075 8083 1996 2 Reddy S Bichler J Wells Knecht KJ Thorpe SR Baynes JW Biochemistry 34 10872 40878 1995 3 Makino H Shikata K Hironaka K Kushiro M Yamasaki Y Sugimoto H Ota Z Araki N Horiuchi S Kidney Int 48 517 526 1995 4 Suzuki D Yagame M Jinde K Naka R Yano N Endoh M Kaneshige H Noifoto Y Sakai H J Diabetes Complications 10 314 319 1996 5 Imai N Nishi S Suzuki Y Karasawa R Ueno M Shimada H Kawashima S NaKamaru T Miyakawa Y Araki N Horiuchi S Gejyo F Arakawa M Nephron 76 153 160 1997 6 Murata T Nagai R Ishibashi T Inomuta H Ikeda K Horiuchi S Diab tologia 40 764 769 1997 7 Kume S Takeya M Mori T Araki N Suzuki H Horiuchi S Kodama T Miyauchi Y Takahashi K Am J Pathol 147 654 667 1995 8 Sakata N Imanaga Y Meng J Tachikawa Y Takebayashi S Nagai R Horiuchi S Itabe H Takano T Atherosclerosis 141 61 75 1998 9 Sakata N Imanaga Y Meng J Tachikawa Y Takebayashi S Nagai R Horiuchi S Atherosclerosis 142 67 77 1999 10 Kislinger T Fu C Huber B Qu W Taguichi A Du Yan S Hofmann M Yan SF Pischetsrieder M Stern D Schmidt AM J Biol Chem 274 31740 31749 1999 11 Shibayama R Araki N Nag i R Horiuchi S Autoantibody against N epsilon carboxymethyl lysine anadwanced glycation end product of the Maillard reaction Diabetes 1999 Sep 48 9 1842 9 1
5. as the high standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 150 uL of Master Standard 450 uL 20 ng mL Std 2 300 uL of Std 1 20 ng ml 300 uL 10 ng mL Std 3 300 uL of Std 210 ng ml 300 uL 5 ng mL Std 4 300 uL of Std43 5 ng ml 300 uL 2 5 ng mL Std 5 300 UP of Std 4 2 5 ng ml 300 uL 1 25 ng mL Std 6 300 uB of Std 5 1 25 ng ml 300 uL 0 63ng mL Std 7 300 XL of Std 6 0 63 ng ml 300 uL 0 31 ng mL Blank s 300 uL 0 ng mL Note Do not use a Repeating pipette Change tips for every dilution Wet tip with Dilution Buffer befor dispensing Unused portions of Master Standards should be aliquoted and stored at below 270 G immediately Avoid multiple freeze and thaw cycles Cat CY 8069 7 Version 140318 ii Anti CML rat autoantibody ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Dilution Serum and plasma samples require 100 fold dilution e g 2 uL sample 198 uL Dilution Buffer Assay Procedure Remove the appropriate number of both CML BSA coated Microplate and BSA coated Microplate wells from the foil pouch and place them into the well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C n2 Dilute samples with Dilution Buffer See Sample Preparation above w Pipette 50 uL of Stand
6. procedures Sample Collection Dilution and Storage Serum Use a serum separator tube and allow samples to clot for 60 30 minutes Centrifug samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or store samp ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 C fore ed periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma by use of EDTA Na as the anticoagulant If possible collect the toa mixture of EDTA Na and Futhan5 to stabilize the sample against spontaneous in vitro lement store samples on ice for up to 6 hours before assaying Aliquots of plasma may als ored at below 70 C for extended periods of time Avoid repeated freeze thaw cycles Note Citrate plasma has not been validated for use in this assay G Other biological samples Remove any particulates by centrifugation an immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x g i immediately or C CY 8069 6 Version 140318 ii Anti CML rat autoantibody ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex Anti CML rat autoantibody ELISA Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number
7. 2 Vay D Vidali M Allochis G usaro C Rolla R Mottaran E Bellomo G Albano E Antibodies against advanced glycation efd product Nepsilon carboxymethyl lysine in healthy controls and diabetic patients Diabetologia 2000 Nov 43 11 1385 8 Cat CY 8069 13 Version 140318 ii Anti CML rat autoantibody ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Related Products 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 CircuLex Anti CML rat autoantibody ELISA Kit Cat CY 8069 CML BSA N Carboxymethyl lysine BSA Cat CY R2052 CML OVA N Carboxymethyl lysine OVA Cat CY R2053 CEL BSA N Carboxyethyl lysine BSA Cat CY R2054 CEL OVA N Carboxyethyl lysine OVA Cat CY R2055 Glucose AGE BSA Cat CY R2056 Glucose AGE OVA Cat CY R2057 Glyceraldehyde AGE BSA Cat CY R2058 Glyceraldehyde AGE OVA Cat CY R2059 Glycolaldehyde AGE BSA Cat CY R2060 Glycolaldehyde AGE OVA Cat CY R2061 Methylglyoxal AGE BSA Cat CY R2062 Methylglyoxal AGE OVA Cat CY R2063 Glyoxal AGE BSA Cat CY R2064 Glyoxal AGE OVA Cat CY R2065 CML HSA N Carboxymethyl lysine HSA Cat CY R2066 CEL HSA N Carboxyethyllysine HSA Cat CY R2067 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002
8. andard curve Standard courve A450 0 5 10 15 20 Anti CML Ab ng ml 2 Specificity The antibodies mbthe CircuLex Anti CML rat autoantibody ELISA Kit are highly specific of CML adduct with no detectable cross reactivity to non CML protein that may be present in rat serum plasma Cat CY 8069 10 Version 140318 Anti CML rat autoantibody ELISA Kit C ircuLex User s Manual A For Research Use Only Not for use in diagnostic procedures 3 Precision Intra assay Precision Precision within an assay Three samples of known concentration were tested eight times on one plate to assess intra precision Intra assay Within Run n 8 Inter assay Precision Precision between assays Three samples of known concentration were tested in four separate assays to assess inter assay precision nter assay Run to Run n 4 4 Spiking Recover Serum samples were spiked with different amounts of anti CML antibodyja The recovery of anti CML antibody spiked to levels throughou be Y ge of the assay was evaluated Sample Average Recovery Range Sera n 4 90 78 93 121 5 Linearity To assess the linearity of the assay samples containing iked with high concentrations of anti CML antibody were serially diluted with the Dilution r to produce samples with values within the dynamic range of the assay Q C CY 8069 11 Version 140318 Anti CML rat autoantibody ELISA Kit User s Manual CircuLex
9. ard Solutions Std1 Std7 Blank and diluted samplesdn duplicates into the appropriate wells of both CML BSA coated Microplate and BSA coated Microplate A Incubate the wells at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker Nn Wash 4 times by filling each well with Wash Buffer 350 ulo using a squirt bottle multi channel pipette manifold dispenser or microplate washer 6 Add 50 uL of HRP conjugated Detection Antibody into each well l Incubate the plate at room temperature ca 25 for 1 hour shaking at ca 300 rpm on an orbital microplate shaker 8 Wash 4 times by filling each well with Wash Buffer 350 uL using a squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 50 uL of Substrate Reagent Avoid exposing the microtiter plate to direct sunlight Covering the plate with e g aluminum foil is recommend d Return Substrate Reagent to 4 C immediately after the necessary volume is removed 10 Incubate the plate at room temperature ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The 4ncubation time may be extended up to 30 minutes if the reaction temperature is below than 202 11 Add 50 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 12 Measure absorbanc in each well using a spectrophotometric microplate reader at dual wavelengt
10. ated kit expiration date Use only the microtiter wells provided with the kit Rinse all detergent residue from glassware Use deionized water of the highest quality Do not mix reagents from different kits The buffers and reagents in this kit may contain preservatives or other chemicals Care should be taken to avoid direct contact with these reagents Do not mouth pipette or ingest any of the reagents Do not smoke eat or drink when performing the assay or in areas where samples or reagents are handled Dispose of tetra methylbenzidine TMB containifig solutions in compliance with local regulations Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide Wear gloves and eye protection when handliftietmmunodiagnostic materials and samples of rat origin and these reagents In case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention when necessary Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly CAUTION Sulfuric Acid is a Strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 8069 5 Version 140318 Anti CML rat autoantibody ELISA Kit C ircuLex User s Manual 4 For Research Use Only Not for use in diagnostic
11. hs of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single wavelength can be used Wells must be read within 30 minutes of adding the Stop Solution Note 1 Complete removal of liquid at each step is essential to good performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against clean paper towels Note 2 Reliable standard curves are obtained when either O D values do not exceed 2 0 units for the Cat CY 8069 8 Versions 140318 Anti CML rat autoantibody ELISA Kit C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures blank zero concentrations or 0 35 units for the highest standard concentration The plate should be monitored at 5 minute intervals for approximately 30 minutes Calculations Average the duplicate readings for each standard and sample in CML BSA coated plate and BSA coated plate Subtract the average readings in BSA coated plate from those in CML BSA coated plate Plot the optical density for the standards versus the concentration of the standards and draw the best curve The data can be linearized by using log log paper and regression analysis may be applied to the log transformation To determine the anti CML adduct concentration of each sample first find the absorbance value on the y axis and extend a horizontal line to the standard curve At the p
12. ining 20 mL of 1 N H2SO Ready to use Cat CY 8069 3 Versions 140318 Anti CML rat autoantibody ELISA Kit C ircuLex User s Manual AN For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor Orbital microplate shaker Microcentrifuge and tubes for sample preparation Plate reader capable of measuring absorbance in 96 well plates at dual wa s of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used n also be read at a single wavelength of 450 nm which will give a somewhat higher readi Vortex mixer Qy e Microplate washer optional Manual washing is possible but not preferable Software package facilitating data generation and analysis opti 500 or 1000 mL graduated cylinder Reagent reservoirs Deionized water of the highest quality Disposable paper towels C CY 8069 4 Version 140318 ii Anti CML rat autoantibody ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations Allow all the components to come to room temperature before use All microplate strips that are not immediately required should be returned to the zip lock pouch which must be carefully resealed to avoid moisture absorption Do not use kit components beyond the indic
13. lure than in normal subjects or diabetic patients without renal failure 11 These results suggest that autoantibody against CML might play a possible role in the development of diabetic nephropathy or chronic renal failure Principle of the Assay The CircuLex Anti CML rat autoantibody ELISA Kit employs the semi quantitative enzyme immunoassay technique CML BSA or BSA has been pre coated onto a microplate Standards or samples are pipetted into the wells Any anti CML adduct autoantibody present is bound by the immobilized CML BSA but not by the immobiliz d BSA After washing away any unbound substances an HRP conjugated antibody specific for rat IgG is added to the wells Following a wash to remove any unbound antibody HRP conjugate the remaining conjugate is allowed to react with the substrate H O gt tetramethylbenzidine The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured at 450 um Mhe absorbance is proportional to the concentration of anti CML adduct antibody A standard curve is constructed by plotting absorbance values versus anti CML adduct autoantibody concentrations of calibrators and concentrations of unknown samples are determined using this standard curve Cat CY 8069 2 Version 140318 ii Anti CML rat autoantibody ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 50 uL of diluted sample
14. nti CML rat autoantibody ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Reducing sugars react with protein amino groups to form a diverse group of protein bound moieties with fluorescent and cross linking properties These compounds called advanced glycosylation end products AGEs have been implicated in the structural and functional alterations of proteins that oecur during aging and long term diabetes Although several AGE structures have been reported 1 2 it was demonstrated that N Carboxymethyl lysine CML is a major antigenic AGE structure CML concentration i also increased in patients who have diabetes with complications including nephropathy 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 expression 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 as an inifriunological epitope to raise autoantibodies against AGE structures particularly CML Shibayama et al showed the presence of autoantibodies against AGE structures particularly those against CML adduct in streptozotocin STZ induced diabetic rats and patients with several diseases 11 12 The autoantibody against CML adduct was higher in patients with renal fai
15. of strips required for the particular determination Since experimental conditions may vary an aliquot of the Anti CML antibody Standard within the kit should be included in each assay as a calibrator Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay reagents are supplied ready to use with the exception of 10X Wash Buffer 20X HRP conjugated Detection Antibody and Anti CML Antibody Standard n Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer to 900 mL of deionized distilled water ddH5O Mix well Store at 4 C for two weeksjor 20 C for long term storage 2 Prepare HRP conjugated Detection Antibody by 20 fold diluting the 20X HRP conjugated Detection Antibody with Conjugate Dilution Buffer at the time of assays Prepare appropriate volume for your assay Discard dmy unused HRP conjugated Detection Antibody after diluted 3 Reconstitute Anti CML Antibody Standard with 0 8 mL of ddH O The concentration of the anti CML antibody in vial should be 80 ng mL which is referred as a Master Standard of anti CML antibody Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series below Mix each tube thoroughly before the next transfer The 20 ng mL standard Std 1 serves
16. oint of intersection extend a vertical line to the x axis and read the corresponding wanti CML adduct concentration If the samples have been diluted the concentration read from thefSfandard curve must be multiplied by the dilution factor 1 The dose response curve of this assay fits best to a sigmoidal 5 par meter logistic equation The results of unknown samples can be calculated with any computer program having a 5 parameter logistic function It is important to make an appropriate mathematicabadjustment to accommodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations f analyte concentration The calibration curve is constructed by plotting the absorbance Y of calibrators versus log of the known concentration X of calibrators using the four parameterfunction Alternatively the logit log function can be used to linearize the calibration curve be 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 20 ng mL 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 the anti 4CML rat autoantibody concentration Troubleshooting The anti CML antibody Standard should be run in duplicate using the protocol described in the Detailed Protocol Incubation time
17. s or standards to the wells 4 Incubate for 1 hour at room temp Wash the wells t Add 50 uL of HRP conjugated anti rat IgG antibody 1 Incubate for 1hour at room temp Wash the wells t Add 50 uL of Substrate Reagent t Add 50 uL of Stop Solution t Measure absorbance at 450 nm Materials Provided All samples and standards should be assayed in d plicate The following components are supplied and are sufficient for the one 96 well microplate kit CML BSA coated Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant4paek Wells are coated with CML BSA BSA coated Microplate One microplate supplied eady to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated with BSA Use for reference 10X Wash Buffer One 100 mL bottl amp jof 10X buffer containing 2 Tween 20 Dilution Buffer One bottle containing 50 mL of 1X buffer use for sample dilution Ready to use Anti CML Antibody Standard Orie vials containing 64 ng of Anti CML antibody 20X HRP conjugated Detection Antibody One vial containing 0 6 mL of HRP horseradish peroxidase conjugated nti rat IgG antibody Conjugate Dilution Buffer One bottle containing 12 mL of Conjugate Dilution Buffer Substrate Reagent One bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready tose Stop Solution One bottle conta
18. s or temperatures significantly different from those specified may give erroneous results 2 Poor duplicates accompanied by elevated values for wells containing no sample indicate insufficient washing If all instructions in the Detailed Protocol were followed accurately such results indicate a need for washer maintenance 3 Overall low signal mayjindicate that desiccation of the plate has occurred between the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wash Cat CY 8069 9 Version 140318 ii Anti CML rat autoantibody ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Reagent Stability All of the reagents included in the CycLex Research Product CircuLex Anti CML rat autoantibody ELISA Kit have been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt kit reagents should be stored at 4 C Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack For research use only not for use in diagnostic or therapeutic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of anti CML antibody giving absorbance lower than mean absorbance plus three standard deviations of the absorbance of Blank Blank 3 SD Blank is better than 0 44 ng mL of sample Typical st
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