CY-8075 Mouse AIM/CD5L/Spα ELISA Kit
Contents
1. E UI MERE se DEN Serum 1 gt Serum 2 EE Cp CU Eee See See uii SSeS See p k Serum 3 AA MALI LL Lr Sa E o o x TQ ere M M dI LLLDLR C C c o o c c6 ue a cael a ae a 0 0 0 2 4 6 8 Serum Dilution Ratio x 1 10 000 Cat CY 8075 12 Version 140318 Mouse AIM CDSL Spa ELISA Kit t C ircuLex User s Manual AN For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Concentrations of free form mouse AIM CDSL Spa in Balb C and A J strain mice n 52 S c net o0 3 Ss o c o o o o 3 o E E Fe o qe o o x qe C CY 8075 13 Versions 140318 Mouse AIM CDSL Spa ELISA Kit t C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 2 Correlation between concentration of total AIM CDSL SPoa measured by western blotting those of free form AIM CDSL SPa measured by AIM CDSL Spa ELISA Kit Correlation between mouse AIM concentrations e measured by WB and by ELISA J mouse AIM conc measured by ELISA ug ml N 1 20 1 5 1 0 s R 0 5782 0 0 0 50 100 150 200 250 300 mouse AIM conc measured by WB ug ml C CY 8075 14 Versions 140318 Tii Mouse AIM CDSL Spa ELISA Kit t C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures References 1 Gebe J A Kiener P A R2. Tii Mouse AIM CD5L Spa ELISA Kit t C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures 4 ELISA Kit for Measuring Free Form Mouse AIM CDSL Spa SN CircuLex Mouse AIM CD5L Spa ELISA KA Cat CY 8075 Q Intended Mr T 1 ANOT aT M 1 Tr OG GU OI S suavi qur pte tta acne 2 Qy Principle of the Assay eerte 2 3 Materials Provided erect 3 Materials Required but not Provided 4 Precautions and Recommendations 5 Sample Collection and Storage 6 Detailed Protoool ie sess enter rrt matte etn ocins 7 8 Cl OULU ONG T S 9 Measurement Range 9 Troubleshooting iui osse reiner eo eene 9 Reagent SEDI uitia deett tbe peers 9 Assay Characteristios see ceste e pnus 10 Example of Test Results ioi Related PROGUCtS isivssscancassscndssinesavasyarcncnnesdss 15 Intended Use The CycLex Research Product CircuLe ouse AIM CDSL Spa ELISA Kit is used for the quantitative measurement of free form Q CD5L Spa in serum plasma and other biological media This assay kit is for research use o d not for use in diagnostic or therapeutic procedures Storage Upon receipt store all co e Don t expose reagents A C CY 8075 1 Versions 140318 Tii Mouse AIM CDSL Spa ELISA Kit t C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction A AIM also called
3. 300 uL 0 1 ng mL Std 7 300 uL of Std 6 0 1 ng mL 300 uL 0 05 ng mL Blank 300 uL 0 ng mL Note Do not use a Repeating pipette Change tips for every dilution Wet tip with Dilution Buffer before dispensing Unusedgportions of Master Standard should be aliquoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Sample Preparation e Serum and plasmasamples require a 1 600 fold dilution e g First Make40 fold dilution 2 uL of sample 78 uL of Dilution Buffer Second Make 40 fold dilution 6 uL of 40 fold diluted sample 234 uL Dilution Buffer Other biological samples require neat to appropriate dilution Cat CY 8075 T Version 140318 Tii Mouse AIM CDSL Spa ELISA Kit t C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay Procedure for free form mouse AIM CD5L Spa Remove the appropriate number of microtiter 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 2 Dilute samples with Dilution Buffer See Sample Preparation above o2 Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted samples in duplicates into the appropriate wells 4 Incubate the plate 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 wit
4. Wash Buffer by aspirating or decanting Invert the plate and blot it against Clean pap r towels Note 2 Reliable standard curves are obtained when either O D values do not exceed 0 25 units for the blank Zero concentration or 3 0 units for the highest standard concentration The plate shouldbe monitored at 5 minute intervals for approximately 30 minutes Note 3 1f the miicfoplate reader is not capable of reading absorbance greater than the absorbance of the highestystandard perform a second reading at 405 nm A new standard curve constructed using the values measured at 405 nm is used to determine mouse AIM CDSL Spa concentration of off scale samples The readings at 405 nm should not replace the on scale feadings at 450 nm Cat CY 8075 8 Version 140318 Tii Mouse AIM CDSL Spa ELISA Kit t C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Calculations Average the duplicate readings for each standard control and sample and subtract the average zero standard optical density 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 mouse AIM CDSL Spo concentration of each sample first find the absorbance value on the y axis and extend a horizontal line to the standard curve At the point of intersection ex
5. different from those specified may give erroneous results 2 Poor duplicates accompanied byyelevated 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 may Andicate 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 Reagent Stability All of the reagents included in the CycLex Research Product CircuLex Mouse AIM CDSL Spa ELISA Kit hay been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt kit reagents should be stored at 4 C except the reconstituted Mouse AIM CD5L Spa Standard must be stored at below 70 C Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack Cat CY 8075 9 Version 140318 Mouse AIM CDSL Spa ELISA Kit t C ircuLex User s Manual AN For Research Use Only Not for use in diagnostic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of mouse AIM CDS5L Spa giving absorban higher than mean absorbance of blank plus three standard deviations of the absorbance of blank A blank 3SD blank is better than 26 6 pg ml of sample Dilution Buffer is p
6. CD5L SPa is a soluble 38 or 40 kDa glycoprotein expressed by macrophages present in lymphoid tissues spleen lymph node thymus and bone marrow for which little functional and structural information is available 1 It belongs to the group B of the scavenger receptor cysteine rich SRCR superfamily that includes the lymphocyte surface receptors CD5 and D6 among others AIM is able to bind to not only myelomonocytic and lymphoid cells 2 but also the surface of several Gram positive and Gram negative bacterial strains 3 which suggests that it may play an important role in the regulation of the innate and adaptive immune systems It has been reported that AIM is present in IgM fraction but not IgG or IgA fractions from human serum 4 AIM is a circulating protein that may play a role in the homeostasis of IgM antibodies In addition it has been reported that AIM expression is induced in mouse macrophages in response to loading with highly oxidized low density lipoprotein 5 and that AIM is expressed in foam cellsowithin atherosclerotic lesions AIM may be feasible as a potential biomarker in the diagnosis and treatment of atopic dermatitis 6 although AIM was reported as candidate marker in asthma 7 and liver cirrhosis 8 9 Principle of the Assay The CycLex Research Product CircuLex Mouse AIM CDSL Spa ELISA Kit employs the quantitative sandwich enzyme immunoassay technique An antibody specific for mouse AIM CDSL Spa is pre coated
7. CED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyele amp cojp URL http wwweeyclex co jp CycLex CircuLex products are supplied for research use only CycLex CircuLex products and compon nts 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 8075 15 Version 140318
8. Lex User s Manual 4 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 Vortex mixer s of 450 540 nm e read at a single Plate reader capable of measuring absorbance in 96 well plates at dual wa Microplate washer optional Manual washing is possible but not preferable i Dual wavelengths of 450 550 or 450 595 nm can also be used The O b wavelength of 450 nm which will give a somewhat higher reading 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 8075 4 Versions 140318 Tii Mouse AIM CDSL Spa ELISA Kit t 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 indicated kit expiration date Use only the microtiter wells provided with the kit Rinse all detergent residue from glassw
9. are 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 handling immunodiagnostic materials and samples of human origin and these reagents In case of contact witli 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 8075 5 Version 140318 Mouse AIM CDSL Spa ELISA Kit t C ircuLex User s Manual 4 For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Serum Use a serum separator tube and allow samples to clot for 60 30 minu
10. h Wash Buffer 350 uL using a sq irt4bottle multi channel pipette manifold dispenser or microplate washer 6 Add 100 uL of HRP conjugated Detection Antibody into each well 7 Incubate the plate at room temperature ca 25 C 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 uD using a squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent Avoid exposifig the microtiter plate to direct sunlight Covering the plate with e g aluminum foil is recommended 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 incubation 4im may be extended up to 30 minutes if the reaction temperature is below than 20 C 11 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 12 Measure absorbance in each well using aspectrophotometric microplate reader at dual wavelengths 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 wayelength 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
11. ing H Z Li X Francke U Aruffo A J Biol Chem 272 6151 6158 1997 2 John A Gebe Peter A Kiener Huijun Z Ring Xu Li Uta Francke and Alejandro Atuffo J Biol Chem 272 6151 1997 3 Maria Rosa Sarrias Sandra Rosell Fernando S nchez Barbero Josep M Sierra Jordi Vila Jos Y lamos Jordi Vives Cristina Casals and Francisco Lozano J Biol Chem 280 35391 35398 2005 4 Tissot J D Sanchez J C Vuadens F Scherl A Schifferli J A Hochstrasser D F j Schneider P and Duchosal M A Electrophoresis 23 1203 1206 2002 5 Arai S Shelton J M Chen M Bradley M N Castrillo A Bookout A E Mak P A Edwards P A Mangelsdorf D J Tontonoz P Miyazaki T Cell Metab 1 201 213 2005 6 WK Kim HR Hwang do H Kim PY Lee YJ In HY Ryu SG Parks KH Bae and SC Lee Exp Mol Med 40 677 685 2008 7 Wu J Kobayashi M Sousa EA Liu W Cai J Goldman SJ Dorner AJ Projan SJ Kavuru MS Qiu Y Thomassen MJ Mol Cell Proteomics 4 1251 64 2005 8 Gangadharan B Antrobus R Dwek RA Zitzmann N 4Novel serum biomarker candidates for liver fibrosis in hepatitis C patients Clin Chem 53 179241799 2007 9 Joe Gray et al BMC Cancer 9 271 2009 Related Products CircuLex Mouse AIM CDSL Spo ELISA Kit Cat CY 8075 CircuLex Human AIM CDSL Spo ELASA Kit Cat CY 8080 Anti Human AIM CDS5L Spa polyclonal antibody Cat CY P1036 Human AIM CDSL Spao Cat CY R2270 PRODU
12. ipetted into blank wells Typical Standard Curve Mouse AIM Standard Curve 2D c LM 2 0 A450 0 1 2 3 4 Mouse AIM conc ng ml CY 8075 10 Versions 140318 Mouse AIM CDSL Spa ELISA Kit t C ircuLex User s Manual AN For Research Use Only Not for use in diagnostic procedures 2 Precision Intra assay Precision Precision within an assay Three samples of known concentration were tested sixteen times on one plate to assess intra precision Intra assay Within Run n 16 CV 1 4 2 5 96 mouse AIM CDSL conc ug ml C CY 8075 11 Versions 140318 ii Mouse AIM CDSL Spa ELISA Kit t C 1rcu Lex User s 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 five separate assays to assess inter assay precision e Inter assay Run to Run n 5 CV 5 7 7 6 96 mouse AIM CDSL conc ug ml Serum 1 Serum 2 Serum 3 1 3 63 2 44 0 87 2 3 79 2 46 0 90 3 3 63 2 57 0 91 4 3 83 2 44 0 97 5 4 16 2 90 1 02 MAX 4 16 2 90 1 02 MIN 3 63 2 44 0 87 MEAN 3 81 2 56 0 93 S D 0 215 0 195 0 063 C V 5 7 7 6 6 8 3 Linearity To assess the linearity of the assay samples containifig and or spiked with high concentrations of free form mouse AIM CDS5L Spa were serially diluted with the Dilution Buffer to produce samples with values within the dynamic range of the assay
13. n the kit should be included in each assay asfa calibrato 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 and Mouse AIM CDSL Spo Standard 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer to 900 mL of deionized distilled water Mix well 2 Reconstitute Mouse AIM CDSL Spao Standard with 1 0 mL of Dilution Buffer and transfer to Microcentrifuge tube immediately Keep on ice The concentration of the mouse AIM CDSL Spa should be 64 ng mL which is referred as a Master Standard of mouse AIM CD5L Spa Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series below MiX each tube thoroughly before the next transfer The 3 2 ng mL standard Std 1 serves as the highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 50 uL of Master Standard 64 ng mL 950 uL 3 2 ng mL Std 2 300 uL of Std 1 3 2 ng mL 300 uL 1 6 ng mL Std 3 300 uL of Std 2 1 6 ng mL 300 uL 0 8 ng mL Std 4 300 uL of Std 3 0 8 ng mL 300 uL 0 4 ng mL Std 5 300 uL of Std 4 0 4 ng mL 300 uL 0 2 ng mL Std 6 300 uL of Std 5 0 2 ng mb
14. onto a microplate Standards and samples are pip tted into the wells and the immobilized antibody binds any mouse AIM CD5L Spa present After w shing away any unbound substances an HRP conjugated antibody specific for mouse AIM CD5L Spa 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 H5O tetramethylbenzidine The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured at 450 nm The absorbance is proportional to the concentration of mouse AIM CDSL Spa A standard curve is constructed by plotting absorbance values versus mouse AIM CD5L Spa concentrations of calibratons and concentrations of unknown samples are determined using this standard curve The CycLex Research Product CircuLex Mouse AIM CDSL Spa ELISA Kit is designed to measure the concentration of free form mouse AIM CD5L Spa in mouse serum plasma or conditioned medium Most of mouse AIM CD5L Spa associates with IgM and other serum proteins in the circulation CircuLex Mouse AIM CDSL Spa ELISA Kit can measure only free form mouse AIM CD5L Spa concentration using standard assay procedure The concentration of free form mouse AIM CD5L Spa showed a clear correlation with one of total mouse AIM CD5L Spa See Fig 2 page 14 Cat CY 8075 2 Version 140318 Mouse AIM CDSL Spa ELISA Kit t C ircuLex User s Manual AN For Research Use Only N
15. ot for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted samples to the wells 4 Incubate for 1 hour at room temp Wash the wells Q Add 100 uL of HRP conjugated anti mouse AIM CD5L Spa antibody 4 Incubate for 1 hour at room temp Wash the wells Add 100 uL of Substrate Reagent g t Add 100 uL of Stop Solution Measure absorbance at 450 nm Materials Provided All samples and standards should be assayed in The following components are supplied and are sufficient for the one 96 well microplate kit Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coate ti mouse AIM CDSL Spa antibody as a capture antibody 10X Wash Buffer One bottle containing 100 of 10X buffer containing 206 Tween 20 Dilution Buffer One bottle co mL of 1X buffer use for reconstitution of mouse AIM CDSL Spa Standard and sample dilution Ready to use Mouse AIM CDSL Spa St ne vial containing 64 ng of lyophilized recombinant mouse AIM CDSL Spa HRP conjugated Detecti tibody One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti mous CD5L Spa antibody Ready to use Substrate Reag TMB Ready ttle containing 20 mL of the chromogenic substrate tetra methylbenzidine Stop S ottle containing 20 mL of 1 N H5SO Ready to use Versions 140318 Mouse AIM CDSL Spa ELISA Kit t C ircu
16. tend a vertical line to the x axis and read the corresponding mouse AIM CDSL Spa concentration If the samples have been diluted the concentration read from the standard curve must be multiplied by the dilution factor 1 The dose response curve of this assay fits best to a sigmoidal 4 parameter logistic equation The results of unknown samples can be calculated with any computer programzhaving 4 parameter logistic function It is important to make an appropriate mathematical adjustinent to accommodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations of analyte concentration The calibration curve is constructed by plotting the absorbance Y of calibrators Wersus log of the known concentration X of calibrators using the four parameter function Alternatively the logit log function can be used to linearize the calibration curve i e logit of absorbance Y is plotted versus log of the known concentration X of calibrators Measurement Range The measurement range is 0 05 ng mL to 3 2 ng mL LA Any ample 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 mouse AIM CDSL Spa concentration Troubleshooting 1 The Mouse AIM CD5L Spa Standard should be run in duplicate using the protocol described in the Detailed Protocol Incubation times or temperatures significantly
17. tes 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 using EDTA Nay as the anticoagulant If possible collect the amp oa 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 8075 6 Versions 140318 Tii Mouse AIM CDSL Spa ELISA Kit t C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex Mouse AIM CD5L Spa ELISA Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination Since experimental conditions may vary an aliquot of the mouse AIM CDSL Spa Standard withi
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